Calcium signaling in cell volume regulation.

PHYSIOLOGICAL REVIEWS Vol. 72, No. 4, October 1992 Printed in U.S.A.

Calcium Signaling in Cell Volume Regulation NAEL Division

of Biology,

A. MCCARTY

AND

ROGER

G. O’NEIL

California Institute of Technology, Pasadena, California; and Department and Cell Biology, University of Texas Medical School, Houston, Texas

of Physiology

I. Introduction ......................................................................................... II. Phenomenological Overview of Cell Volume Regulation ........................................... A. Control of normal cell volume ................................................................... B. Consequences of swelling and shrinking ........................................................ III. Mechanisms of Cell Volume Regulation ............................................................ A. Transport mechanisms in regulatory volume increase .......................................... B. Transport mechanisms in regulatory volume decrease ......................................... IV. Signaling Pathways in Regulatory Volume Increase ............................................... V. Signaling Pathways in Regulatory Volume Decrease .............................................. A. Role of protein kinases and phosphatases ....................................................... B. Role of calcium ................................................................................... C. Calcium dependence of volume regulation ...................................................... D. Calcium entry or calcium release ................................................................ E. Nature of calcium entry pathway ............................................................... VI. Controversy in Calcium-Dependent Control of Regulatory Volume Decrease ..................... A. Lymphocytes ..................................................................................... B. Ehrlich ascites tumor cells ...................................................................... C. Correlation between swelling- and ionophore-induced activation .............................. VII. Is There a Calcium Threshold for Regulatory Volume Decrease? .................................. VIII. Model of Calcium-Dependent Regulatory Volume Decrease Mechanisms: Renal Proximal Tubule A. Proximal straight tubule regulatory volume decrease response ................................ B. Calcium dependence of regulatory volume decrease: entry and release ........................ C. Calcium entry pathways under isotonic and hypotonic conditions: calcium channels ......... D. Calcium window: temporal dependence of regulatory volume decrease ........................ IX. Summary ............................................................................................

I. INTRODUCTION

In 1733, the Reverend Stephen Hales first observed that injection of a large amount of water into an animal’s blood led to considerable swelling of the muscles, liver, kidneys, and other organs and that this swelling could be reversed by injection of a common salt solution (cited in Ref. 163). Since then, the control of cell volume has been under continued investigation. It is now generally agreed that most mammalian cells are capable of maintaining some control of their intrinsic volume following exposure to anisosmotic media. Although the transport mechanisms by which many cells achieve volume regulation have been elucidated, it is still unclear how these transport processes are controlled. A prominent controlling role has been ascribed to Ca2+ in a wide variety of cells. The purpose of this review is to relate the current understanding of 1) the control of normal cell volume and the transport mechanisms underlying volume regulation following volume perturbation; 2) the cellular signaling and regulatory pathways underlying solute and 0031-9333/92 $2.00 Copyright 0 1992 the American Physiological

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volume regulation, with special emphasis on the role of Ca2’; 3) the controversy surrounding the role of Ca2+ in modulating hypotonic cell volume regulation; and 4) a cellular model of Ca2’ signaling as a key component of hypotonic cell volume regulation. Several reviews on the general mechanisms and diversity of cell volume regulation have recently been published (17, 38, 75, 77, 109, 140, 154). The present review focuses on the role of Ca2+ as a controlling factor in cell volume regulation in vertebrate and invertebrate animal cells. Furthermore, because available evidence indicates that the involvement of Ca2’ in controlling volume regulation is related more to regulation under hypotonic conditions, the discussion is biased toward this case and away from regulation under hypertonic conditions. Although Ca2+ appears to play a central role in the control of hypotonic cell volume regulation in most cells, our understanding of the factors controlling intracellular Ca2’ levels in hypotonic cell volume regulation is only beginning to emerge. Furthermore, the mechanism(s) by which Ca2+ controls or modulates volume regulatory processes remains poorly understood.

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1038

NAEL

A. MCCARTY

A lsotonic Medium

0 j

Ax=0

J, = L, (A P-Ax) Ci > Co, .‘. A?c is positive J,= Lp(A.-Ax) Co > Cj, :. An is negative

Hypertonic Medium

B Entry

= Ex/f

Entry

z ExM

Solute

Entry

c ExM

FIG. 1. Phenomena that lead to volume perturbations. A: anisotonic cell swelling and shrinking. When bathed by a medium that is isotonic to cytoplasm (top), cells maintain their volume by balancing solute content of cell such that there is no osmotic pressure gradient (AII) across plasma membrane. Under hypotonic conditions (middle), concentration of osmolytes is greater inside (Ci) than outside (C,), and therefore activity of water inside cell is less than outside, which leads to a positive osmotic pressure gradient and subsequent water entry. Rate of water flow (J..) is a function of hvdraulic permeability (~5,) and difference between Ail and the hydrostatic pressure gradient(iP), if any. Water entry causes cells to swell. In contrast, AII and J, are oriented in opposite direction under hypertonic conditions (bottom), leading to cell shrinkage. B: solute-induced volume perturbations. In cells, in general, volume is maintained by balancing rates of solute entry and exit (top). When this condition is not met, volume perturbations occur due to movement of osmotically obliged water. When solute entry exceeds exit, cells swell due to water entry (middle). When solute exit exceeds entry, cells shrink (bottom). This process of isotonic cell swelling is particularly important in epithelia, since transmural solute movement must be balanced at apical and basolateral membranes.

II. PHENOMENOLOGICAL OVERVIEW OF CELL VOLUME REGULATION

A. Control of Normal

Cell Volume

When cells are bathed by an isotonic medium, they maintain a certain volume by balancing the rates of solute entry and exit (102,184). In this way, a steady-state condition is achieved that maintains equality between water chemical potentials inside and outside the cell, avoiding the Donnan equilibrium (Fig. 1A). The intracellular osmotic pressure generated by the presence of charged intracellular macromolecules is offset by efflux

AND ROGER G. O’NEIL

Volume 72

of osmolytes (smaller osmotically active molecules, e.g., ions and amino acids), thereby maintaining cell volume without requiring the presence of a significant hydrostatic pressure gradient. Thus extracellular osmolytes, such as Na+, provide an osmotic force to offset the intracellular osmotic force by remaining effectively impermeant (102,120). Conversely, the volume of a cell at any given time may be considered to be a function of the permeability of the plasma membrane to all pertinent osmolytes and the driving forces for entry/exit of these osmolytes. With few exceptions, the water permeability of most cell membranes is high while the solute permeability is low. Because water moves across cell membranes predominantly due to osmotic gradients, uncompensated alterations in the net transmembrane solute transport activity of any osmolyte would be expected to lead to changes in cell volume. For this reason, when solute entry is stimulated, for instance, by activation of Na+dependent cotransport mechanisms, cells swell due to the increased entry of Na+-substrate and osmotically obliged water (5, 16, 80, 176). In this case, swelling is induced by making the intracellular fluid slightly hypertonic relative to the bath (Fig. 1B). This swelling process may be studied by an alternative approach where the bath is made hypotonic relative to the intracellular milieu (Fig. 1A). In both cases, water entry causes extensive cell swelling. In the absence of compensatory mechanisms, cells exposed to hypotonic conditions will swell and could lyse unless they establish a hydrostatic pressure to counteract the osmotic pressure. Because animal cell membranes are fragile and cannot support a significant hydrostatic pressure difference, potent mechanisms evolved to provide net solute transport to counteract cell swelling, providing for cell volume regulation. B. Consequences of Swelling and Shrinking Cell swelling has a variety of consequences, including alterations in morphology, ion content, metabolic state, and water content (and therefore hydration status of internal macromolecules). Hypotonic shock causes water to accumulate in cells by osmosis due to the high osmotic permeability of cell membranes (79). Gilles et al. (47) and Kleinzeller et al. (90) have shown that rabbit kidney cortex slices gain water to 40% over isotonic water content on exposure to 45% hypotonic solutions. It is expected that such gains in intracellular water content would result in significant dilution of cell contents. The most obvious effect of swelling is an increase in cell size, which leads to distortion (and possibly distension) of the plasma membrane. Swelling is also associated with a loss of microvilli in villated cells (91). The expansion of the plasma membrane surface area by the microvilli (and therefore increased surface-to-volume ratio) may allow villated cells to accomodate a greater extent of swelling without lysis than nonvillated cells,


October

1992

CALCIUM CONTROL OF CELL VOLUME

such as red blood cells (RBCs). Other ultrastructural effects of cell swelling include clumping of chromatin, dilatation of endoplasmic reticulum, irregularity of mitochondrial appearance, and elongation of the mitochondrial intramembrane space (120). Swelling has also been shown to induce a rearrangement of the cytoskeletal network in T2 mouse fibrosarcoma cells and PC12 rat pheochromocytoma cells (22). Interestingly, only the microfilament network was modified, not the microtubular network. The rate of cell swelling varies drastically between cell types. This may be due to real differences in water permeability (25, 79, 198), artifactual differences in water permeability due to damage of attached cells during dissection, or the greater geometrical surface areato-volume ratio of attached cells, which retain microvilli and membrane infolds (31). These surface features are often lost on dissection of attached cells from their substrates. For a given cell type with a certain water permeability, however, the rate of swelling on acute exposure to hypotonic medium is a function of the magnitude of the osmolarity difference across the cell membrane (Fig. lA), since the rate of water flow [which is the sum of osmotic, hydraulic, and electrosmotic flows (79)] is dominated by osmotic flow. Shrinkage under hyperosmotic conditions leads to changes in cellular osmolyte concentrations (El), changes in cell shape, disturbance of cellular metabolism (116), and, by way of changes in hydration status, alterations in the osmotic contribution of cellular macromolecules (163). Thus these changes can lead, directly or indirectly, to alterations in electrical and/or chemical driving forces. This can have detrimental effects in many cells. In epithelia, for example, where vectorial ion transport depends on the maintenance of transcellular ionic concentration gradients within certain limits, and in nerve and muscle, where intracellular ion activities determine the crucial electrical excitability, these changes in cellular hydration status may have serious effects on cell function if uncompensated. The effect of hyperosmolality depends on the permeability of the cell membrane to the added solute, i.e., the reflection coefficient (121). Relatively permeable solutes, such as urea, equilibrate throughout the intracellular and extracellular compartments and have only transient effects on cell volume. In contrast, when hyperosmolality is produced by addition of exogenous impermeant solutes, such as mannitol, cells respond to the persistence of an osmotic pressure difference by losing water and shrinking. When cell volume has been disturbed, what are the mechanisms by which cells can respond to this challenge? Cells appear to have at least two mechanisms for controlling cell volume in response to swelling (47). One is initiated rapidly and is partially responsible for limiting the degree of swelling on acute exposure to hypotonic solutions. Because this phase is so rapid (milliseconds to seconds, depending on the rate of swelling), it is difficult to study and is, therefore, largely unknown. It has been proposed that the cytoskeleton serves a passive role in this capacitv (47. 113). Cvtoskeletal elements

1039

have also been implicated in the response to swelling in several tissues (41,46,209). Disruption of the microfilament network with cytochalasin B led to a significant decrease in cell volume in PC12 cells even in isotonic conditions (2.2). Linshaw et al. (111) recently detailed the effects of cytoskeletal disrupters on various stages of the response to swelling in rabbit proximal convoluted tubules (PCT). In this case, in contrast to that observed in PC12 cells, treatment with cytochalasin B led to an increase in cell volume under isosmotic conditions and reduced the fractional volume recovery in response to subsequent swelling in hypotonic medium. Interestingly, however, cytochalasin B did not lead to an increase in the extent of swelling on exposure to dilute medium, measured as the change above the cytochalasin-treated isosmotic volume. These data indicate that the cytoskeleton plays less of a role in limiting swelling but rather serves a regulatory role in subsequent volume recovery. Furthermore, because microtubule inhibitors alone were without effect on cell volume but operated synergistically with cytochalasin B, a secondary role of microtubules in cell volume regulation was indicated. Knauf and co-workers (61) recently showed that although F-actin content was reduced during swelling in HL-60 cells, there was little influence on regulatory volume decrease (RVD). In some cells, such as renal tubular cells, the establishment of a small hydrostatic pressure gradient in response to swelling has been postulated (28, 112). These studies relied on the presence of the basal lamina, the elastic collagenous mesh that surrounds all tubular segments. It was proposed that the basal lamina may limit the extent of swelling and may even allow the generation of a hydrostatic pressure, which could aid water removal by nonosmotic forces (ultrafiltration). However, collagenase treatment indicated that the basal lamina was only important for volume regulation in tubules where the Na+-K+-ATPase was inhibited with ouabain. Thus the generation of a hydrostatic pressure may not be important for cell volume regulation in intact cells. In contrast to the poorly characterized early phase of anisosmotic volume control, our understanding of volume regulation is more advanced for the slower second phase of regulation, namely the phase in which cell volume returns toward control levels after swelling or shrinking. The processes underlying volume control after swelling or shrinking invariably require redistribution of intracellular osmolytes, allowing water to follow by osmosis. Among mammalian cells, initial volume regulation is achieved predominantly by regulating the fluxes of inorganic ions across the plasma membrane, although a contribution from rapid efflux of organic osmolytes has also been seen in some cells (3, 101, 135). Additionally, free amino acids and other organic solutes can serve as important osmolytes, which may allow continued volume adjustment even after ionic gradients approach an equilibrium state such that net passive ion movements are abolished (43, 66, 72, 73, 134, 136). For instance, if cells are swollen in high bath K+ concentra-


NAEL

post-RVD

;

A. MCCARTY

RVI

h


Volume

72

osmolyte content and volume (Fig. ZB). Although our knowledge of the solute transport mechanisms involved in cell volume regulation is assuredly not complete, several common threads have been found among tissues and across phyla. The two phenomenological processes in anisosmotic cell volume regulation, i.e., RVD and RVI, are addressed by cells utilizing specific solute transporters that do not appear to operate in both modes of volume regulation. The ion fluxes involved in achieving both hypotonic and hypertonic volume readjustment have been studied extensively (for reviews see Refs. 17, 71, 75, 109, 120, 178). In general, the principal inorganic ions involved in these processes are Na+, K+, and Cl-. The fluxes may occur via a variety of transporters, including pumps, channels, cotransporters, and exchangers. Table 1 contains a general description of the ionic transport mechanisms, and associated ions, used by different cell types to achieve RVI and RVD. It is important to note, however, that many of the distinctions among types of transport mechanisms have been made on the basis of inhibitor studies or ion substitution studies alone, without electrophysiological evidence or other forms of support. Hence, in some cases, the categorization may be considered only tentative. For instance, KC1 exit during RVD in gallbladder epithelial cells was originally ascribed to K+-Cl- cotransport (98) on the basis of inhibition by bumetanide. However, more recent experiments indicate that RVD is supported by nitrate or thiocyanate substitutions for chloride, which is in direct contrast to the operation of a K+-dependent, Cl--specific cotransporter (42), implicating a role for separate K+ and Clionic channels.

__-----post-WI

I I

I I

1

2

RVD

Time FIG. 2. Schematic representations of responses to swelling and shrinking. A: swelling and regulatory volume decrease (RVD). At time 1, cells either were exposed to hypotonic solution or solute entry was stimulated (e.g., via activation of Na’-glucose cotransport). In response, cells swelled and then underwent RVD, extruding osmolytes and losing water, to reach a new plateau volume approximating control volume. When, at time 2, cells were returned to isotonic medium, bath was slightly hypertonic with respect to cytoplasm due to loss of intracellular osmolytes in RVD, and cells shrank rapidly below normal volume (undershoot). Cells then regained osmolytes and volume through a process termed post-RVD regulatory volume increase (RVI). B: shrinking and RVI. At time 1, cells either were exposed to hypertonic conditions or solute exit was stimulated (e.g., by catecholaminergic stimulation of K+ efflux). In response, cells shrank and then exhibited RVI, gaining osmolytes and water, to return cell volume to normal levels. At time 2, cells were returned to isotonic solution or were relieved of stimulation for solute exit. As a result, cells swelled above control volume due to new direction of imbalance in water activities (overshoot) and underwent a process similar to RVD (post-RVI RVD). Note short-term RVI process is emphasized, whereas long-term RVI may not cause acute changes in cell volume (see text).

tion, passive K+ exit and therefore RVD will be blocked. Under these conditions, volume recovery may proceed by extrusion of organic solutes (24, 73, 153). The transport mechanisms by which intracellular osmolytes are redistributed in response to swelling or shrinking are covered in the next section. III.

MECHANISMS

OF CELL

VOLUME

REGULATION

The process whereby swollen cells shrink toward control volume is termed RVD, since it involves both a decrease in cell osmolyte content and a decrease in cell volume (Fig. ZA). Similarly, the process whereby shrunken cells swell toward control volume is termed regulatory volume increase (RVI), since it involves increases in

A. Transport Mechanisms in Regulatory Volume Increase

Short-term RVI (minutes) in most cells relies on the accumulation of Na+ and Cl- and in some cases K+ (Table 1). Three modes of transport have been indicated: I) Na+-H+ exchange linked to Cl--HCO, exchange (lo), 2) Na+-K+-Xlcotransport (60), and 3) Na+-Cl- cotransport (68), with the accumulated Na+ being partially exchanged for K+ by the Na+-K+-ATPase. Long-term osmolyte accumulation may be mediated by amino acids, urea, or polyols (for review see Refs. 17,43,109). Amino acids may also have a protective function against acute changes in the osmotic environment, as well as play a part in osmotic adjustment (101). Some cells undergoing RVI also indicate a decrease in the passive solute leak pathways, particularly for K+ and Cl-, such that K+ and Cl- that are brought into the cell during RVI will stay in the cell (see Ref. 109). In some cells, such as Ehrlich ascites tumor cells (lo@, osmotically induced shrinkage is followed by maintenance of a constant volume (rather than continued shrinking) by activation of Na+-H’ exchange, Na’-K+ active countertransport (Na’ pump), and Na+-K+-ZClcotransport. It is proposed that the cotransport mechanism does not increase cell volume


October

TABLE

CALCIUM

1992

CONTROL

1. Ionic mechanisms of R VI and R VD

Transport

Mechanism

Cell

Types

References

RVI N a’-K’-2Cl cotransport

Na’-H’ exchange linked to Cl-HCO, exchange

Na’-Cl-

cotransport

Na’-H’ exchange, anion uncertain Accumulation of organic compounds (e.g., sugars, polyols, amino acids, methylamines, and urea)

Flounder RBCs Duck RBCs Ehrlich ascites tumor cells Dog RBCs Rabbit urinary bladder Mouse medullary thick ascending limb Human lymphocytes Rabbit renal PST Ehrlich ascites tumor cells CHO cells

9, 11 92,93,1X5 44,107,108

Many cells, including plants, animals, bacteria, fungi, and protists

17

147,148 32 65 53,167 117 69,106 171

RVD K’

K’

and Clconductances

conductance, with HCO, dependence

K’

conductance, anion unknown K+-Clcotransport

K’-H’ exchange linked to Cl--HCO, exchange Kt efflux, anion unclear Na’-Ca2’ exchange Supplemented by amino acid efflux

Human

lymphocytes

Ehrlich cells Rabbit

ascites renal

tumor PT cells

52, 54, 55, 172, 173 69, 74, 76, 81 33, 85, 87, 199, 200,202 122 26,27

Frog skin Amphibian urinary bladder Intestine 407 cells Necturus enterocytes MDCK renal cells HeLa epi thelioid carcinoma cells Necturus gallbladder CHO cells Human platelets Mouse renal PST Necturus early renal PT MDCK cells Rabbit renal PCT Opossum kidney cells Avian RBCs Fish RBCs Dog RBCs Human RBCs Low-K+ sheep RBCs Amph,iuma RBCs

86, 162 5 186,187 92 9, 11, 100 146,147 143 34 10,13

Rat hepatocytes

21,97

Dog RBCs MDCK cells Ehrlich ascites tumor cells Mollusc RBCs Elasmobranch RBCs

147 169 73

62 48,99 169 183 42 171 114 190,191 118

.

131, 132, 153 105

CHO, Chinese hamster ovary; MDCK, Madin-Darby canine kidney; PCT, proximal convoluted tubule; PST, proximal straight tubule; PT, proximal tubule; RBC, red blood cell; RVD, regulatory volume decrease; RVI, regulatory volume increase.

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toward isotonic levels following NaCl-induced hyperosmolality due to regulation of this pathway by the distribution ratio of Cl- concentration from cell to medium, which would not change significantly as intracellular Cl- concentration ([Cl-Ii) would follow extracellular Clconcentration ([Cl-],). In both the rabbit cortical collecting duct (CCD) (137) and the proximal tubule (117,164), cell volume regulation under hypertonic conditions is regulated by the availability of metabolizable fatty acids in the medium. Acetate, butyrate, and valerate enhance the ability of proximal tubule cells to maintain a constant cell volume under mildly hypertonic conditions (117, 164). Grantham and colleagues (117,164) suggested that nonionic acids entering the cell may support Na+-H+ exchange by supplying intracellular protons. Furthermore, acetate anion may be exchanged for bath Cl- or may be metabolized to CO,, hydrated, and then exchanged via the Cl--HCO, antiporter. In CCD cells, RVI is not seen in the absence of short-chain fatty acids but is enabled by butyrate (137). Consistent with a role in supplying protons to a Na+-H+ exchanger, the effect of butyrate in CCD cells is blocked by amiloride, an inhibitor of Na+-H+ exchange. Although most mammalian cells are capable of RVD, many cells do not appear to undergo RVI in hypertonic conditions. However, when these cells are swollen in hypotonic medium, allowed to undergo RVD, and returned to isotonic medium, they shrink below the control volume, since the originally isotonic medium is now effectively hypertonic to the cytoplasm due to loss of cellular osmolytes during RVD (Fig. ZA). These cells are then capable of regaining osmolytes to allow them to swell back up to control volume. This RVI-like process has been given many names but probably the most common term is “post-RVD RVI.” It is unknown whether the mechanisms involved differ from those in actual RVI (109, 137). B. Transport Mechanisms in Regulatory Volume Decrease

In vertebrates, RVD is achieved primarily by the extrusion of Kf and Cl-, either by the activation of electroneutral K+-Cl- cotransport or separate, conductive K+ and Cl- pathways. The RBCs of the freshwater urodele Amphiuma appear to be unique in that KC1 efflux occurs by K+-H+ exchange functionally linked to Cl--HCO, exchange (12, 13), whereas all other RBCs studied utilize K+-Cl- cotransport (Table 1). Among epithelial cells, virtually all tissues studied make use of K+ and Cl- channels, which may be separately controlled. Although Cl- is the dominant anion released in RVD in most tissues, in a few cases, e.g., the mouse proximal tubule (190, 191) and Madin-Darby canine kidney (MDCK) cells (86), HCO, may be the dominant anion. Bicarbonate may be necessary in other cells as well, serving to resupply intracellular Cl- by Cl--HCO, exchange. Interestingly, the RVD process also appears to


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A. MCCARTY

AND

involve the operation of amiloride-sensitive Na+-H+ exchange (186). This observation may be somewhat controversial, since amiloride treatment during RVD has been shown to have both inhibiting and enhancing effects on volume control, depending on the tissue. Livne and Hoffmann (115) have recently shown that RVD in Ehrlich ascites tumor cells is associated with cytoplasmic acidification and that this results in activation of Na’-H’ exchange. The resulting influx of Na+ in the Ehrlich cells may be responsible for the incomplete volume recovery by swollen cells, such that inhibition of the Na’-H+ exchanger with amiloride enhances the capacity for RVD. In contrast, Gilles and collaborators (29) have shown that amiloride inhibits RVD in PC12 cells. Similarly, amiloride inhibited RVD in frog urinary bladder cells (26). It may be important to note that Ehrlich cells were preincubated with amiloride for only 1 min, whereas frog bladder cells were incubated for 15 min and PC12 cells for 45 min. In the cells that exhibited amiloride inhibition of RVD, therefore, it is likely that prolonged treatment led to alterations in isotonic cell volume and in ion con .tent of the cytop lasm. In inve rtebrates, RVD appears to be m ore heavily reliant on the extrusion of organic compounds than it is in vertebrates (18, 132, 156, 165). For instance, it has been shown that glycine, taurine, alanine, and the quaternary ammonium compound, glycine betaine, are the principal osmolytes lost in the RVD response of rock crab muscle (131), molluscan RBCs (179), and horseshoe crab myocardium (194). Skate RBCs may export intracellular taurine via the band 3 anion exchanger (49). However, invertebrate cells only use organic osmolyte TABLE

Volume

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efflux to support a secondary phase of RVD, which is preceded by the loss of inorganic ions as in their vertebrate counterparts. The secondary late phase of loss of organics in invertebrate cells may serve more of an acclimation function. Thus the volume regulatory strategies between vertebrates and invertebrates may not be as different as originally thought, but rather the contributions of organic and inorganic osmolytes to the process may differ through time (153). IV.

SIGNALING VOLUME

PATHWAYS

IN REGULATORY

INCREASE

As our understanding of the transport mechanisms involved in achieving anisosmotic cell volume regulation has improved, many investigators have begun asking how these transport processes are controlled in the response to swelling or shrinking. It is still not known what transmembrane signals or biochemical pathways are involved in activating the ion transporters used to reestablish normal cell volume and whether these transporters are subsequently inactivated. In attempts to characterize the pathways involved in signal transduction, evidence has been put forth for the involvement of many factors, including the prominent second messengers Ca2+ and adenosine 3’,5’-cyclic monophosphate (CAMP) (Table 2). Our understanding of the signals for activation of the RVI machinery is very limited. Cyclic AMP has been implicated in activation of the Na+-H+ and Cl--HCO, pathways in the mouse medullary thick ascending limb

2. Proposed effecters $0~ controlling the volume regulatory apparatus Cell

Frog

ROGER

urinary

Type

bladder

Effecters

Human

lymphocytes

Ca2’ Ca2+, CaM Ca2’, CaM,

Rabbit

renal

Ca2’, CaM

A~/@~ iu ))((I RBCs PST

Vm, membrane

Ca2+ Ca2’, membrane Ca2+ Ca2+ IP ca2+’ 3

PC12 cells T2 cells Goldfish retinal axon Rabbit, human RBCs Human lymphocytes Mouse mTALH Duck RBCs

Microfilaments Microfilaments CaM, not cytoskeleton Dephosphorylation Ca2’, unknown protein CAMP Phosphorylation

CaM, calmodulin; DAG, diacyglycerol; IP,, Henle; PC12, rat pheochromocytoma cell; PKC, performed to show that effecters listed were efflux. $D a t a f or elasmobranch RBCs pertain

CaM, CaM PKC, CaM,

RVD RVD RVD

stretch

or RVI

(not

RVI)*

(not

RVI)*

RVD

Rabbit renal mTALH Intestine 407 cells Osteosarcoma cells Cultured renal PCT cells Toad urinary bladder NW~WUS gallbladder Molluscan RBCst Elasmobranch RBCs$ Ehrlich ascites tumor cells

Ca2’, Ca2’, Ca2’, Ca2+,

RVD

RVD RVD RVD RVD RVD RVD RVD RVD RVD

stretch

microfilaments not CaM LTD4

kinase,

DAG,

IP,

inositol1,4,5-trisphosphate; LTD,, leukotriene protein kinase C; T2, mouse fibrosarcoma cells; not involved in RVI. t Data for molluscan to taurine efflux.

RVD RVD RVD RVD RVI RVI RVI

References

26,27 12,14 54, 172,173 57 125-127 139 130 62, 64, 141 206 180 204, 205 41 155,179 105,133 44, 74, 76 95,96 22 22 35 83,84 54,56 65 152

D,; mTALH,

medullary thick ascending limb of voltage. * Experiments were pertain to taurine efflux only, not KC1

Vm, membrane RBCs


October

1992

CALCIUM

CONTROL

RVI response (65). Regulatory volume increase was strictly dependent on treatment with antidiuretic hormone, which leads to activation of adenylate cyclase. This effect of antidiuretic hormone was mimicked by treatment with a membrane-permeant analogue of CAMP, dibutyryl-CAMP. The shrinkage-induced stimulation of Na+-H+ exchange activity in human lymphocytes can be mimicked by treatment with 1%0-tetradecanoylphorbol 13-acetate, a stimulator of protein kinase C (56, 197). The responses to shrinkage and to 1%0-tetradecanoylphorbol 13-acetate both induce phosphorylation of membrane proteins, with one substrate being common between the two treatments (56). These findings seemed to indicate a role for protein kinase C-mediated activation of RVI. However, neither diacylglycerol (DAG) nor inositol l&%trisphosphate (IP,) was liberated in shrunken lymphocytes, nor was a redistribution of cytoplasmic protein kinase C associated with shrinkage. The authors concluded that activation of the Na’-H’ exchanger in lymphocyte RVI is due to phosphorylation by a protein kinase other than protein kinase C (56). Recent studies by Pewitt et al. (152) shed considerable light on the signaling pathways leading to activation of RVI in duck RBCs. They show that activation of bumetanide-inhibitable Na+-K+-2Clcotransport on shrinkage is mediated by phosphorylation from both CAMP-dependent and -independent kinases. It is to be noted that hypertonicity does not raise CAMP levels in these cells (144). Protein kinase inhibitors blocked activation of the cotransporter, as modeled in Figure 3. Okadaic acid, an inhibitor of serine-threonine protein phosphatase types 1 and 2A, stimulated both cotransport activity and bumetanide binding to duck RBC membranes, even in the absence of CAMP-mediated or shrinkage-induced stimulation, probably by blockade of dephosphorylation of the transporter itself and/or an associated regulatory protein. Thus RVI in duck RBCs is regulated via a dynamic equilibrium between phosphorylation and dephosphorylation of a regulatory site on some component(s) of the signal transduction pathway. Because Ca2+ does not generally appear to be involved in the control of RVI (38), the mechanisms controlling activation of RVI are not discussed further in this review.

OF

CELL

v.

SIGNALING

PATHWAYS

VOLUME

IN REGULATORY

DECREASE

Several signaling pathways have been implicated in the regulation of RVD in various cell types. Calcium signaling has been of particular focus and, indeed, has been demonstrated to be involved in RVD mechanisms of many cells (see sect. vC). However, in some cells Ca2+-independent signaling appears to underlie RVD. It may be that RVD is not regulated solely by any one pathway but that it involves the interaction of several signaling pathways and possibly cofactors, each of which may exert variable control of the different modulator pathways involved in RVD. A. Role of Protein

Kinases

and Phosphatases

The mode of activation of RVD in Ca2+-independent systems has only recently begun to emerge. Studies by Jennings and Schulz (83) and shortly thereafter by Kaji and Tsukitani (84) and Parker et al. (149), independently investigating the activation of K+-Cl- cotransport in swollen mammalian RBCs, have now indicated that protein dephosphorylation is involved in stimulation of volume-dependent KC1 efflux (Fig. 4). Inhibition of dephosphorylation by okadaic acid led to inhibition of volume-activated K+ transport and, hence, blockade of RVD. The authors suggest that swelling operates via inhibition of a protein kinase, thereby pushing the equilibrium toward dephosphorylation. This is not likely protein kinase A or C: Jennings and Schulz (83) propose the term “protein kinase V” for this kinase involved in volume regulation. This conclusion was based on the following evidence. First, preincubation of the cells with CAMP (to stimulate protein kinase A) did not inhibit volume-activated K+ flux. Second, phorbol ester-induced stimulation of protein kinase C also led to no change in volume-activated K+ flux. Thus the inhibition of the K+-Cl- cotransporter under nonswelling conditions appears to be due to phosphorylation by an unknown kinase (83). The similarity of the above pathway with the pathway for shrinkage-induced activation of Na+-K+-2Cl cotransport in duck RBCs (see sect. IV) is staggering and

r (4

Shrinking

FIG. 3. Shrinking-induced activation of Na+K ‘-Xl cotransport in duck red blood cells (RBCs) (152). Exposure to hypertonic conditions stimulates phosphorylation of Na’-K+-2Cll cotransporter itself or a regulatory protein (R) by activation of a kinase, thereby activating cotransporter. Okadaic acid inhibits deactivation of transport mechanism by blocking dephosphorylation of R.

1043

VOLUME

Okadaic

Acid

Normal Volume

Pi

NaM2CI l l

l

Active Cotransport

Solute influx High affinity bumetanide binding RVI

*

Inactive Na/K/2CI Cotransport

R-P

AdP

ATP Kinase

Inhibitors


NAEL

1044


Volume

72

Swelling

Normal Volume

Okadaic

+

Acid

(9 pi

1 Inactive KCI Cotransport

A. MCCARTY

R-P

R,

Active KCI Cotransport l l

l

Kinase

Solute efflux Low affinity bumetanide binding RVD

Inhibitors

makes one wonder whether the transduction pathways share common components (23). It seems possible that the two models may be superimposed, as shown in Figure 5. Thus there may be a regulatory component in common between the RVI pathway and the RVD pathway. Furthermore, both the Na+-K+-ZCl- cotransporter and the K+-Cl- cotransporter bind bumetanide, although the former binds it 100 times as well (178). Duck RBCs, for which the data for Na+-K+-2Clcotransport apply, do undergo RVD via a K+-Cl- cotransporter (93). Similarly, mammalian RBCs may undergo RVI via a Na+-K+-2Clcotransporter (44). Might these transporter functions be provided by one and the same complex of proteins (see Refs. 37,70,178), where phosphorylation leads to a conformational change resulting in the presentation of a high-affinity bumetanide binding site and Na+ dependency for influx, whereas dephosphorylation removes this dependence and shifts the predominant driving force to that for K+ exit? Clearly, these notions require further experimentation. One possible avenue of investigation would be to ask whether covalently labeling shrinkage-activated Na+-K+-2Clcotransporters with a photoreactive analogue of bumetanide leads to subsequent inhibition of K+-Cl- cotransport on swelling. Shrinking

B. Role of Calcium

In 1933, Ellis (36) indicated that the annelid worm Nereis gained water when exposed to 17% dilute seawater medium and then lost water through the subsequent several hours. In Ca2+-free dilute medium, Nereis continued to gain water (swell) for hours until returned to full-strength seawater. Thus an involvement of Ca2’ in controlling cell volume has been postulated for decades (94). It is now known that Ca2’ plays a dominant role in the activation of RVD in many cells (Table 2). It is interesting to note that, with the exception of Amphiuma RBCs wherein RVD occurs by the Ca2+-dependent activation of a K+-H+ exchanger, the majority of cells shown to have a Ca2+-dependent RVD process are cells in which osmolyte efflux occurs predominantly via K+ and Cl- conductances (cf. Table 2 to Table 1). This relationship has led many investigators to conclude that the ion conductances in these cells are directly regulated by Ca2+. Indeed, Ca2+-activated K+ and/or Cl- conductances have been shown in many tissues, including several tissues that indicated Ca2+-dependent RVD (for references see Table 2). It is obviously tempting to infer that these conductive pathways constitute the locus of Swelling

a. . . Acla 1.I Wadalc

Active Cotransport

NaM2CI

FIG. 4. Swelling-induced activation of the K+Cl- cotransporter in mammalian RBCs (83, 84). Cell swelling inhibits a kinase that thereby favors dephosphorylation of K+-Cl- cotransporter or a regulatory site (R) and hence activation of cotransporter. Okadaic acid stimulates activation of K+-Cl- cotransporter by blocking dephosphorylation of R.

R

Active KCI Cotransport

l

Solute influx

l

l

High

l

l

RVI

affinity

bumetanide

binding l

Kinase

Solute efflux Low affinity bumetanide binding RVD

Inhibitors

FIG. 5. Proposed synthesis of control of Na’-K+-2Clcotransport and K+-Cl- cotransport by cell volume. Cotransport functions by a common protein or complex of proteins (R, R-P) are regulated as a function of cell volume by a dynamic equilibrium between phosphorylation and dephosphorylation. At normal volumes, both cotransport functions are nominally expressed. Cell shrinking stimulates kinase to activate solute influx via Na+-K+-2Cl- cotransporter, leading to RVI. Conversely, cell swelling inhibits kinase, causing a shift in equilibrium toward dephosphorylation and leading to activation of solute efflux via K+-Cl- cotransporter, resulting in RVD. This model accounts for directions of solute flux and volume change and affinity of bumetanide binding as a function of volume (see text). However, note that evidence of commonality between RVI and RVD transporter molecules has not been shown.


October

1992

CALCIUM

CONTROL

the Ca2+ dependence of RVD. It is not clear, however, how Ca2’ performs its function to modulate RVD. For instance, it may be that the K+ and/or Cl- conductances are not directly activated by Ca2+ but indirectly activated through the operation of another Ca2+-dependent process. The biological actions of Ca2’ can be mediated through several biochemical pathways. Calmodulin has been implicated in the pathway between Ca2+ and the control of RVD in many tissues (Table 2). However, all of these conclusions are based on pharmacological inhibition by calmodulin antagonists, such as trifluoperazine, chlorpromazine, or pimozide. Other Ca2+-dependent processes may be involved in the control of RVD, such as production of secondary intracellular messengers (i.e., DAG), the exocytic insertion into the plasma membrane of vesicles containing ion channels, and phosphorylation or dephosphorylation of existent transporters. Of particular note in this regard is the Ca2’-dependent protein kinase C. Leite and Goldstein (105) have shown that taurine efflux from hypotonically stressed skate RBCs, but not KC1 efflux, is stimulated by both Ca2+ ionophore treatment and phorbol ester treatment, implicating protein kinase C in the RVD process. C. Calcium Dependence of Volume Regulation A dependence on [Ca”‘], of the ability to undergo RVD has been shown in Amphiuma RBCs, frog urinary bladder, Necturus gallbladder, cultured PCT cells, toad urinary bladder, osteosarcoma cells, isolated rabbit proximal straight tubules (PST), clam RBCs, skate RBCs, and Intestine 407 cells (for references see Table 2). Extracellular Ca2’ apparently has a stabilizing effect on cell membranes such that in the prolonged absence of extracellular Ca2’, membrane function and cell morphology may be altered, perhaps irreversibly (41,88,89). Accordingly, it is important to note that most studies on the role of Ca2’ in RVD have not addressed the involvement of extracellular Ca2+ in maintaining isotonic cell volume. Thus, in many cases, we do not know what the effects are on cell volume of reducing [Ca”‘], in isotonic conditions. Foskett and Spring (41) presented conclusive evidence that gallbladder cell morphology was grossly altered by exposure to a nominally Ca2+-free solution. In hypotonic conditions, Ca2+-free solutions had destabilizing effects on cell volume, other than reduced RVD, in toad urinary bladder cells (204) and in killifish PCT (unpublished observations). In contrast, a Ca2+-free isotonic solution was without effect on cell volume in frog urinary bladder (27), human lymphocytes (54), and cultured medullary thick ascending limb cells (130). In clam RBCs, isotonic [Ca”‘], reduction did not change cellular K+ or Cl- contents (179). Effects of isotonic [Ca”‘], reduction cannot be inferred from other studies, since most investigators either reported cell volumes as relative volumes, rather than absolute volumes, or did not report the cell volumes before [Ca”‘], reduction. Does Ca2’ serve its controlling function from inside

OF

CELL

1045

VOLUME

or outside the cell? Because no one has reported a mechanism for control of RVD by Ca2’ at the outer face of the membrane, it is widely assumed that extracellular Ca2+ serves to supply intracellular Ca2+ and that the regulatory role of Ca2’ is asserted at an intracellular site. Despite this generalization and the profusion of cases ascribing control of RVD to Ca2+, measurements of [Ca”‘], during hypotonic shock are relatively few. Cala et al. (14) first showed that swelling caused an increase in [Ca2+]i in Amphiuma RBCs using arsenazo III absorption in a null-point experiment. The swelling-induced increase in [Ca2+]i was postulated to control the K+-H+ and Na+-H+ exchange systems in these cells. With the use of the fluorescent Ca2’ chelator quin2, cell swelling was also shown to cause a modest increase in [Ca2+]i in isolated toad urinary bladder cells (204). This reported increase (-35 nM over baseline) was supported by radioisotope measurements, indicating increased 45Ca2+ uptake in swollen cells. These findings were confirmed recently, using the second-generation Ca2’ indicator probe, fura- (205). In this study, 15% swelling caused an average increase in [Ca2+]i of ~260 nM. Pierce et al. (155) reported that swelling induced a rapid influx of 45Ca2f in invertebrate RBCs. Osteosarcoma cells indicated an increase in [Ca2+]i by -350 nM in response to swelling (206). In S49 mouse lymphoma cells, hypotonicity induced a rise in [Ca2+]i that was accompanied by, but not coupled to, accumulation of CAMP (195). Later studies indicated that the increase in cellular CAMP concentration was not involved in RVD (196). A slow rise in [Ca2+]i was shown in fura-2-loaded PCT primary cultures (180), whereas McCarty and O’Neil(l25,126) demonstrated a more dramatic, rapid rise in [Ca2+]i in isolated hypotonically swollen PSTs, as discussed in section VIIIB. Beck et al. (4) recently indicated that [Ca2+]i in isolated rabbit PCT increased relatively transiently on swelling. These authors suggest that neither the basolateral K+ nor Cl- conductances are regulated directly by Ca2’ but that the K+ conductance is activated by Ca2+ indirectly. With the use of indirect methods, involving patch-clamp measurements of the activity of Ca2+-activated K+ channels, increases in [Ca2+]i during swelling were also shown in Necturus choroid plexus epithelium (19), cultured opossum kidney (OK) cells (186-188), primary cultures of rabbit PCT (33), and Intestine 407 epithelial cells (62). In this latter case, the response of [Ca2+]i to swelling was biphasic, rising from 83 to 160 nM, dipping near baseline, and rising again to about the same level (64). D. Calcium

Entry

or Calcium

Release

Because swelling-induced elevation of [Ca”‘], has been found in several of the cell types shown to have an RVD mechanism dependent on [Ca”‘],, it is generally assumed that the source of Ca2+ is extracellular. However, in some cases, evidence has been presented that Ca2’ entry across the plasma membrane may be supplemented bv. or even replaced bv. Ca2’ released from in-


1046

NAEL

A. MCCARTY

AND

tracellular stores. In fact, if Ca2’ released from internal stores was sufficient to support activation of the ion conductances in a given tissue, RVD in that tissue may not appear to be Ca2’ dependent by the usual defin ition. storage sites for swelling-activated Ca2+ Intracellular release may be the mitochondria (in pathophysiological states; see sect. VIB), the endoplasmic reticulum, or an associated organelle, the calciosome (166,192). How cell swelling is transduced into a release of Ca2’ from these storage sites is unknown at present but may be related to than .ges in voltage across the organel lar membrane, by stretch of the organellar membrane, or stimulation inositol polyphosphates. Inositol polyphosphates have also been proposed to play a role in regulating Ca2’ entry during swelling. Phosphoinositide metabolites have been shown to be important messengers in a wide range of signal transduction systems (6, 7, 177). In this pathway, a minor component of the plasma membrane, phosphatidylinositol4,5-bisphosphate (PIP,), is metabolized to at least three important second messengers: DAG, IP,, and inositol 1,3,4,5-tetrakisphosphate (IP,). Inositol1,4,5-trisphosphate causes release of intracellular Ca2’ stores, IP, may be linked to enhanced Ca2’ entry at the plasma membrane, and DAG is a stimulator of protein kinase C. Hoffmann and co-workers (20) have given preliminary evidence that the levels of PIP2 are decreased and that the levels of IP, are increased in response to swelling in Ehrlich cells. Suzuki et al. (180) have shown that the IP, plus IP, levels are tripled within the first minute of hypotonic shock in cultured PCT cells. Preliminary evidence indicates that cell swelling is accompanied by increased turnover of inositol phosphates in PST as well (O’Neil, unpublished observations). Rat hepatocytes, which can undergo RVD in Ca2+-free media, also show increased inositol phosphate turnover on swelling (193). Chase and co-workers (205) showed that the swelling-induced rise in [Ca”‘], in toad urinary bladder cells in monolayers was absent in low-[Ca2+] serosal solutions, indicating no involvement of Ca2’ release. Similarly, strict dependence on Ca2’ entry was found in osteosarcoma cells (206), Amphiuma RBCs (14), PCT cultured cells (180), choroid plexus (19), and Intestine 407 cells (62). In contrast, evidence for a role of Ca2’ release in supporting Ca2+- dependent RVD has been given in Ehrlich ascites tumor cells (74; see sect. VIB) and OK cells (186), where the authors investigated swelling-induced activation of Ca2+-dependent K+ channels. In Intestine 407 cells, Ca2+-induced Ca2’ release appears to be important for RVD (63). As discussed in detail in section VIIIB, evidence for both Ca2’ entry and Ca2’ release has been shown by McCarty and O’Neil in rabbit PST (125-127). The [Ca2+]i response to swelling in this tissue was complex, involving both transient Ca2’ release and sustained increased rates of Ca2’ entry. However, Ca2’ released from intracellular stores in response to swelling was not sufficient to support RVD. Sustained Ca2’ entry is responsible for activation of the Ca2+-dependent cell volume regulatory apparatus.

ROGER

E. Nature

G. O’NEIL

Volume

of Calcium

Entry

72

Pathway

The nature of the pathway for swelling-induced Ca2+ entry has been investigated only in a few tissues. Several alternative Ca2+ permeation pathways have been proposed, including Ca2’-selective channels, voltage-activated Ca2’ channels, and stretch-activated Ca2+-permeating channels. In Necturus gallbladder (41), RVD was not disturbed by the Ca2’ entry blocker D-600 (methoxyverapamil). Calcium ion entry in response to swelling in osteosarcoma cells was inhibited by verapamil, lanthanum, and the dihydropyridine (DHP) nicardipine, in increasing order of potency (206). In toad urinary bladder cells, the pharmacology is a bit confusing. Although swelling-induced 45Ca2+ influx was not inhibited by verapamil or diltiazem in an early report (204), the later measurements of [Ca2+]i indicated a significant inhibition of the rise in [Ca2+]i by lanthanum, verapamil, and nitrendipine, another DHP (205). However, in this study, the tissues were exposed to these agents (and their vehicles) for 15 min before swelling, which may have altered parts of the RVD machinery other than the Ca2’ entry pathway. Interestingly, preexposure to lanthanum, verapamil, or nitrendipine did not lead to a decrease in [Ca2+]i under isotonic conditions, although preexposure to low [Ca”‘], (200 nM) did reduce [Ca2+]i considerably. Effects of these maneuvers on cell volume were not reported. In rabbit medullary thick ascending limb cells, swelling-induced Ca2’ entry was inhibited by verapamil and nifedipine (another DHP), although there were no effects of these blockers on isotonic baseline [Ca2+]i. As expanded on in section VIIIC, swelling-induced Ca2’ entry in PST cells was inhibited by verapamil and nifedipine, whereas only verapamil reduced isotonic baseline [Ca”‘], (126, 127). The current favorite mechanism for control of the Ca2+ entry pathway during swelling is th .e distortion of the plasma membrane due to stretching (19,39,141, 181, 195). It has been proposed that channel proteins are tethered in the plasma membrane by cytoskeletal components that run parallel to the inner surface of the membrane. When cells swell, increased tension on cytoskeletal elements in the membrane is transmitted to the channel, causing it to open (58, 170, 208). Such stretchactivated channels could conceivably allow a considerable quantity of Ca2’ to enter cells. This notion is supported by findings that RVD is inhibited both by disruption of the microfilament network with cytochalasin B (22,41) and by bath application of lanthanides (e.g., see Ref. 125), which are potent blockers of stretch-activated channels (208). VI.

CONTROVERSY OF REGULATORY

IN CALCIUM-DEPENDENT VOLUME

CONTROL

DECREASE

As summarized, it appears that a swelling-induced increase in [Ca2+]i is a general phenomenon present in most cell types that undergo RVD by separate K+ and Cl- conductances. All evidence in these cells points to


Oc f oht?rw 1992


Ca2+-dependent control of RVD. However, this apparent trend is confounded by the lack of a swelling-induced increase in [Ca”‘], in human lymphocytes, either as measured with quin2 (161) or indo-l (57), despite early expectations that Caf’ controlled the volume-dependent ion conductances in this cell type (54). This dichotomy, and the possibility of alternative interpretations for the role of Ca2’ in data from other cells, has led some to question as to how Ca2+ really works to control RVD in cells where KC1 efflux occurs via conductive pathways (57). Because the basis for our current understanding of the control of RVD comes predominantly from studies in lymphocytes and Ehrlich ascites tumor cells, these studies can be used to illustrate the development of evidence for and against direct control of RVD by Ca2’. A. Lymph,ocytes

In 1973, Roti-Roti and Rothstein (168) demonstrated that mouse lymphoid cells exhibit RVD in hypotonic media and that RVD was associated with a large loss of cell K+. Shortly thereafter the prevalence of Ca2+-activated K+ channels became apparent (43a; for review see Refs. 124,151). This prompted Grinstein et al. (54) to speculate that “changes in cytoplasmic Ca2+ levels might also be involved in the regulation of the volume-induced K+ pathway.” Since then the quest for understanding the role of Ca2’ in RVD has led to a burgeoning of interest in the field. Grinstein et al. (54) initially approached the question of the role of Ca2+ in RVD using primarily pharmacological strategies. First, by electronic cell sizing, Grinstein et al. showed that swelling in high-K+ media not only led to a loss of RVD but in fact led to secondary swelling after the initial hypotonic shock. This implicated a conductive K+ permeability in the volume response, since cell volume was affected by the direction of the driving force for passive K+ movements, a conclusion supported by increased rates of *‘Rb+ efflux under hypotonic conditions. Treatment of cells with the Ca2’ ionophore A.23187 under isotonic conditions also caused a dramatic loss of “Rb+, even in Ca2+-free media, implicating release of Ca2+ from internal stores. Furthermore, treatment with A23187 in isotonic, Ca2+-containing media caused a dramatic cell shrinkage. Both the swelling-induced K+ loss and RVD were inhibited by quinine, a purported blocker of Ca2’-induced K+ movements in RBCs (Z), which had previously been shown to cause a partial depolarization of lymphocyte resting membrane potential (160). These findings indicated an apparent Ca2+ -dependent K+ channel, as well as a presumed Ca2+-activated Cl- channe!, to effect KC1 efflux. Supporting evidence for a role of Ca2’ came from studies in which trifluoperazine, a calmodulin antagonist, was shown to inhibit RVD and K+ loss in the same manner as quinine. Other experiments indicated that swelling increased an anion permeability in lymphocytes (52). It was at this point that the authors speculated that the effects of ionophore plus Ca2’ treatment on the K+ per-

1047

meability were parallel to those of hypotonic shock. Fortunately, the unexpected insensitivity of lymphocyte RVD to extracellular Ca2’ kept Grinstein et al. from concluding that the volume-induced and A23187 plus Ca2’-induced K+ conductances were the same. Although lymphocytes did show a redistribution of intracellular 45Ca2+ during swelling as Ca2+ was released from stores and then lost to the medium, the authors noted that RVD was not affected by removal of bath Ca2’ and was only partially inhibited by depletion of intracellular Ca2+ stores (54). The advent of fluorescent intracellular Ca2’ chelators allowed the reinvestigation of the involvement of Ca2+ in lymphocyte RVD using quin2. However, Rink et al. (161) found no change in [Ca2+]i associated with swelling or RVD in human peripheral blood lymphocytes (an observation that disputes the ubiquity of Ca2+-permeable stretch-activated channels), results that were later corroborated using indo-l (57). This led Sarkadi et al. (173) to reinvestigate the role of Ca2’ in control of the K+ and Cl- permeabilities. As shown previously (52), treatment of resting lymphocytes with gramicidin, a cation-selective pore-forming antibiotic, led to no change in cell volume, indicating that the resting anion permeability, in this case the Clpermeability (Pcl), was normally extremely low, preventing isotonic cell shrinkage (173). However, when cells were swollen in the presence of quinine to block the endogenous K+ permeability &), gramicidin easily overcame the inhibition of RVD, demonstrating that swelling also activated a P,,. In fact, RVD and K+ loss were faster in the presence of gramicidin, suggesting that the antibiotic-induced P, was much greater than the swelling-induced P,. Thus swelling in lymphocytes activated a P,, and a P,, but the P, was rate limiting for the achievement of RVD. A role for Ca2’ in RVD was supported by the demonstration that Ca2’-depleted lymphocytes showed very slow rates of RVD and of K+-induced secondary swelling (173; Fig. 6). Both the rates of RVD and secondary swelling were increased in Ca2’ -depleted cells by readdition of Ca2’ plus A23187. Although not shown, *‘Rb+ fluxes were reportedly reduced by Ca2’ depletion, whereas 36C1- fluxes were not affected. These results led the authors to conclude that Ca2’ depletion specifically reduced the volume-induced P, without affecting the volume-induced PCl. However, it should be noted that A23187 plus Ca2’ treatment in Ca2+-depleted cells did not lead to full volume recovery compared with normal Ca2+-containing cells where recovery was complete in IO min. Furthermore, gramicidin treatment may have been a poor substitute for volume-induced K+ transport on which to base measurements of anion transport, since volume recovery and secondary swelling were both faster and more extensive in the presence of gramicidin than in normal untreated cells and in cells that were depleted of Ca2’ and then returned to Ca2+-containing medium. In this regard, it would have been interesting to see the additive effects of gramicidin plus A23187 plus Ca2’ on rates of RVD and secondary swelling. If the


1048

NAEL

A. MCCARTY

B

A

1.6

5

B 1.4 a .&

i.,

1.0

.._..._......

.(J.

.

Gramicidin

T

I 1

a

I .^

1”

0

TFme (min)

FIG. 6. CazC dependence of RVD and secondary swelling . A. . effect of Ca” depletion on volume decrease in hypotonically shocked human peripheral blood lymphocytes. Media contained choline-Cl as A 23187 + Ca++ predominant salt. Isotonic media with or without 0.5 PM gramicidin (0), 0.7X isotonic control (o), 0.7X isotonic plus 1 PM A23187 and 1 mM Ca2’ (o), or 0.7x isotonic plus 0.5 pM gramicidin (A). B: effect of Ca’+ Control depletion on secondary swelling of hypotonically shocked human peripheral blood lymphocytes. Media contained KC1 as predominant salt. Isotonic media with or without 0.5 PM gramicidin (0), 0.7~ isotonic control (o), 0.7x isotonic plus 1 pM A23187 and 1 mM Ca2’ (o), or 0.7~ isotonic plus 0.5 PM gramicidin (A). [From Sarkadi et al. (173), by copyright permission of Rockefeller University Press.]

,:

$1.2

*

g

72

~&mG

1.3

+ Ca++

Volume

Gramicidin

1.8

A 23187


1.0

I r

Time

(min)

effects were additive, one might speculate that activation of the anion transport pathway was, in fact, somewhat Ca2+ dependent. An accompanying study by Sarkadi et al. (172) indicated that the swelling-induced PC,in lymphocytes was activated in an all-or-none fashion once a relative volume of 1.15 was reached (115% of control volume). The swelling-induced PC1 underwent volume-dependent inactivation to stabilize cell volume as it approached isotonic levels, as shown by the ability to avoid K+-induced secondary swelling by rapidly returning to control volume with the addition of extra salt. However, these experiments were performed in the presence of gramicidin, which likely affected the electrical properties of the cells. Also, it is unclear how gramicidin treatment in normal hypotonic medium can lead to RVD below the isotonic volume (173) if the counterion pathway (the volume-induced PC,) is inactivated at volumes near isotonic levels. Time-dependent inactivation of the volume-induced PC, was shown in lymphocytes, using a protocol similar to what we have called a “volume clamp” (125). By swelling lymphocytes in the presence of quinine or Ca2+-depleting conditions, conditions under which RVD does not occur, cells were clamped in the swollen state (Fig. 7). Subsequent additions of gramicidin over the next several minutes (to induce a known PK) led to less and less RVD as the delay time between swelling and gramicidin addition was prolonged. This inactivation was not due to a reduced driving force for Cl- efflux, since nystatin treatment at the end of the experiment led to rapid RVD. From these data, it was concluded that the volume-activated Cl- conductance underwent time-dependent inactivation, with an apparent half time of -8-9 min. The activation of the volume-induced PK, on the other hand, was characterized by a graded response depending on the magnitude of swelling, and in this case inactivation appeared to be mostly time dependent and only partially volume dependent (172). Qualitatively similar RVD mechanisms were found in related studies using Chinese hamster ovary (CHO) cells (171). The studies on RVD in lymphocytes may be summa-

rized as follows. Regulatory volume decrease is achieved by the activation of separate, conductive K’ and Clpathways. Even though the isotonic PC, is very low, the Ca2+ ionophore caused shrinkage under isotonic conditions. Both RVD and the volume-induced PK were inhibited by calmodulin antagonists. Swelling did not cause a rise in [Ca2+li, and RVD was not affected by bath Ca2+ removal and was only partially slowed by depletion of intracellular Ca2’. Depletion of Ca2+ reduced volume-activated K+ transport (Rb+ efflux) but did not effect Cltransport. Swelling activated a PC, in an all-or-none fashion, whereas the PK was activated in a graded response proportional to the difference in osmolality. The PK was rate limiting for the RVD process. The volumeactivated PC, exhibited both volume-dependent and time-dependent inactivation, whereas inactivation of the volume-induced PK was mostly time dependent and

, 0

10

20 fime

30

e

(min)

FIG. 7. Effect of gramicidin on shrinkage of hypotonically shocked human peripheral blood lymphocytes. Isotonic media (0.7~) contained choline-Cl as a predominant salt plus 100 PM quinine. At times indicated by arrows, 1 pM gramicidin was added to media. Open arrow indicates addition of 50 pg/ml nystatin to media. [From Sarkadi et al. (172), by copyright permission of the Rockefeller University Press.]


October

1992

CALCIUM

CONTROL

volume independent. The activation of the P, was coneluded to be mediated by Ca2+, whereas cell size (volume) was considered the activator of the pcl. B. Ehrlich Ascites Tumor Cells

Shortly after the above-noted reports on lymphocyte RVD, Hoffmann et al. (76) reported similar investigations into the mechanism of RVD in Ehrlich ascites tumor cells. The techniques used were similar in that cell volume measurements were made by electronic sizing in the presence or absence of inhibitors, ionophores, and Ca2’. Hoffmann et al. (76) first showed that Ehrlich cells exposed to 50% hypotonicity undergo partial RVD within 7-10 min, which is accompanied by substantial losses of K+, Cl-, and water. The KC1 losses and RVD were unaffected by anion-exchanger inhibitors [4,4’diisothiocyanostilbene-2,2’-disulfonic acid (DIDS) and bumetanide], suggesting that efflux occurs via conductive pathways. Regulatory volume decrease was partially inhibited by quinine and quinidine (76) but only at concentrations lo-fold higher than needed in lymphocytes [(54); it should be noted that Parker (145) initially showed that quinidine also inhibited volume-dependent Na+ flux at low concentrations and thus may have problematic secondary actions]. Calmodulin antagonists inhibited RVD at concentrations similar to those required in lymphocytes. Given these pharmacological effects and the reported presence of Ca2+-dependent K+ channels in Ehrlich cells (189), Hoffmann et al. concluded that RVD involved the activation of a Ca2+-dependent K+ channel in the plasma membrane. In Ehrlich cells RVD appeared to be independent of [Ca2’],, as the magnitude of volume recovery was the same at 0 and 0.5 mM [Ca”‘], (76). However, the rate of RVD in Ca2+-containing solutions was dramatically improved by addition of A23187 during the peak of swelling (Fig, 8). Similarly, RVD in Ca2+-free media, already occurring at a rate similar to that in Ca2+-containing

1049

OF CELL VOLUME

media, was potentiated by abruptly increasing [Ca”‘],, indicating that [Ca”‘],, in fact, influences RVD. To determine if Ca2+ release from intracellular stores was responsible for activation of RVD, Hoffmann et al. investigated the effect of depleting Ca2’ stores on the ability of Ehrlich cells to volume regulate. Prolonged preincubation in the presence of hypotonic solutions with ethylene glycol-bis(P-aminoethyl ether)-N,N,hr,W-tetraacetic acid (EGTA) and A23187 (to release intracellular Ca2+ stores) led to only a 43% reduction in the extent of RVD when cells were subsequently returned to an isotonic solution and reswollen in low-Ca2+ medium. This result may be consistent with dependence of RVD on [Ca”‘], being indicated only when [Ca2+]i is already low, as has been suggested for medullary thick ascending limb cells (130; see sect. VII). Despite the apparent low sensitivity of Ehrlich cell RVD to the availability of Ca2+, it was concluded that RVD involved the activation of Ca2+-dependent channels (76). It would have been beneficial if the cell volume regulation studies utilizing Ehrlich cells were supported by measurements of [Ca2+]i under similar conditions. Nonetheless, in the absence of these data, Hoffmann et al. (76) and others (14) proposed that swelling causes a change in the Ca2’ affinity of the unknown regulatory sites, without requiring a rise in [Ca2+]i. An equally viable alternative explanation is that the remaining capacity of Ehrlich cells to volume regulate in Ca2+-depleting conditions reflects the loss of amino acids and taurine, which were previously shown to serve as osmolytes in the Ehrlich cell RVD process (73). Hoffmann et al. (74) extended the proposed parallels between volume-activated and A23187 plus Ca2+-activated conductances in Ehrlich cells using gramicidin with and/or without inhibitors in a similar fashion as Sarkadi et al. (172, 173). In Ca2+-containing hypotonic medium RVD was potentiated by addition of gramicidin during the peak of swelling. However, if the addition of gramicidin was delayed in the presence of quinine (to block the endogenous K+ conductance), its ability to enhance the rate of RVD was diminished over time. HoffB

A23187,2pM

Ca*+, 1mM

2.0

+ 8. Effect of Ca2’ ionophore A23187 and of external Ca2’ on RVD in Ehrlich ascites cells. A: cells were preincubated for 15 min in 300 mosM medium with 1 mM Ca2’ and 0.1 mM M8+ and transferred to 150 mosM medium with one-half the Ca2’ and M$+ concentration. In experimental group, A23187 (2 PM) was added to hypotonic medium 1 min after transfer (0). B: cells were preincubated (15 min) in 300 mosM medium with 0.1 mM EGTA and 0.15 mM M$+ and transferred to 150 mosM medium with one-half the M$+ concentration without EGTA. In experimental group, 1 mM Ca2’ was added to hypotonic medium 1.2 min after transfer (0). Experiments are representative of 15 experiments with addition of A23187 and 4 with addition of external Ca2+. [From Hoffmann et al. (76).] FIG.

z 2 a, ’

zj

1.5

k f 2 .$ 0z I 5

I

I

I

I

I

I

10

15

0

5

10

15

Time After Change in Osmolarity (min)


1050

NAEL

A. MCCARTY


mann et al. concluded that the swelling-induced PK is rate limiting and that the swelling-induced P,, undergoes inactivation over time, possibly due to the presumed (not measured) transience of the swelling-induced increase in [Ca2+]i. Thus the volume-activated PC1 in Ehrlich cells undergoes time-dependent inactivation, much like its lymphocyte counterpart. However, it should be noted that the data on which these conclusions were based, wherein gramicidin enhancement of RVD diminished over time, show a significant volume recovery in the presence of quinine, even before gramicidin addition (74). This probably signifies a substantial loss of the driving force for Cl- exit. Hence an alternative explanation may be that the PC, is volume dependent and turns off as partial RVD is achieved, as has been shown in lymphocytes (172). The Hoffmann group has presented considerable evidence for the presence of separate Ca2+-activated K+ and Cl- transport pathways in Ehrlich cells. Treatment with A23187 under isotonic conditions in the presence of Ca2+ caused a substantial loss of ions, water (76), and volume (74). Calmodulin inhibitors (e.g., pimozide) blocked the response to ionophore plus Ca2+. The data presented, however, seem to indicate that the ability of pimozide to block the A23187 plus Ca2+-induced cell shrinkage was strongly dependent on the predominant anion in the cytoplasm, even at high pimozide concentrations. Pimozide block of RVD, in contrast, was independent of anion composition (74), even at low pimozide concentrations, indicating that swelling-induced shrinkage and A23187 plus Ca2+-induced shrinkage may occur by different mechanisms. Parallel studies were not performed to show that pimozide blocked A23187 plus Ca2+induced shrinkage with the same potency as that observed for the inhibition of RVD (76). In contrast to lymphocytes, Ehrlich ascites cells have an appreciable Cl- conductance in isotonic conditions, as evidenced by the ability of gramicidin to induce isotonic cell shrinkage (74). The A23187 plus Ca2+-induced PC, supported rapid 36C1- entry by a mechanism insensitive to DIDS and bumetanide (74). In two other experiments, Hoffmann and co-workers used the dual antibiotic approach to show that the activation of the A23187 plus Ca2+-induced PC, is transient, similar to the volume-induced PC,. Under isotonic conditions, with quinine in the medium, cells were treated with A23187 followed by gramicidin treatment at various times (Fig. 9). In Ca2+-containing isotonic media, gramicidin addition caused immediate cell shrinkage even 10 min after A23187 treatment. In Ca2+-free medium, however, the extent of gramicidin-induced shrinkage was diminished as the delay between ionophores was increased above 1 min. Thus the A23187 plus Ca2’-induced PC, appeared to inactivate, which was ascribed to the presumed transient nature of ionophore-induced increase in [Ca2+]i. It should be pointed out that the experiments demonstrating a transient nature of the PC, involved prolonged incubation with EGTA and A23187, a carboxylic acid ionophore. This molecule serves as a Ca2+-H+ exchanger and therefore is capable of rapidly uncoupling

Volume

72

mitochondria, leading to a fast rundown of cellular ATP levels (1, 159), which is potentiated in the absence of extracellular Ca2+ (158) and is associated with the spontaneous release of mitochondrial Ca2+ in Ehrlich ascites tumor cells (40), presumably as well as release of Ca2+ from more physiological stores. Hence, it is doubtful whether A23187-induced Ca2+ release can serve as a good model of swelling-induced Ca2+ release, since the mitochondria are not considered to be the physiological store of Ca2+ that is releasable on stimulation. The studies on RVD in Ehrlich ascites tumor cells may be summarized as follows. Regulatory volume decrease is achieved by separate, conductive PK and P,,. In contrast to lymphocytes, there is an appreciable PC, under isotonic conditions. Similarly, however, Ca2+ ionophore caused cell shrinkage under isotonic conditions. Regulatory volume decrease was inhibited by calmodulin antagonists. As in lymphocytes, RVD was independent of bath Ca2+ concentration, unless cells were previously incubated in low-Ca2+ medium. Also similar was A

A 23187

cu-c E8 2cn ’0 a, 0.91~ 2 1=- iij oa A

. Ca*+-free

0.8

I B

0 A 23187

2

4 Gramicidin

I’ ++

,

6

8 I

+

Time (min) FIG. 9. Time dependence of Cl- net permeability following addition of ionophore A23187 in Ca’+-free and Cazf-containing choline medium. Media contained quinine to block Ca’+-dependent K’ channels. Gramicidin was added at times indicated by arrows to impose a high cation permeability. Choline medium was nominally Ca2+ free, containing 0.5 mM EGTA (A), or contained 1 mM Ca2+ (B). In both media 0.8 mM choline was replaced by Na+ to provide an equilibrium potential for Na+ near that for K”. Experiment is representative of 2 and 3 experiments in Ca’+-free and Ca’+-containing medium, respectively. Cell volume is given relative to value measured before addition of A23187 and gramicidin (open symbols). Curves shown in A are compiled from 2 experiments marked individually. [From Hoffmann et al. (74).]


, l!Mi2 Odobur,


the finding that the volume-activated P, was rate limiting for RVD. Although the swelling-induced P,, undergoes time-dependent, and possibly volume-dependent, inactivation as in lymphocytes, the time dependence in Ehrlich cells was attributed to inactivation of Ca2’ release and/or entry. Thus it was concluded that swelling causes the activation of a Ca2+-dependent P, and a Ca2+dependent PC1 and that activation is predominantly via Ca2’ release, although Ca2’ entry also contributes somewhat to the Ca2’ dependency of RVD. Bet/wee% Swelling- and lon~~~)~~,(~~e-I~ducedActivation

C. Corrzlution

The above-discussed parallelism between the volume-activated P, and P,, and the A23187 plus Ca2+-activated P, and P,, was drawn based on similarities in Ca2+ dependence, time dependence, sensitivity to quinine, and inhibition by calmodulin antagonists. It should be noted that, contrary to inferences propagated in the most recent reviews (17,38,109,154), the permeabilities activated by swelling in either lymphocytes or Ehrlich cells were not shown to be the same as those activated by ionophore plus Ca2’. As pointed out in a recent review by Hoffmann and Simonsen (75), the relative magnitudes of increased P, and P,, in Ehrlich cells are exactly opposite between swelling activation and ionophore plus Ca2+ activation. The ionophore A23187 plus Ca2+ caused a calculated increase of PC1 by only U-fold and of P, by Zl-fold. In contrast, swelling caused an increase of P,, by 60-fold and increased P, less than A23187 plus Ca2’ did. This indicates that the swellinginduced activation of P,, involves more than just Ca2’ (or Ca2’-calmodulin) and that the swelling-induced P, relies less on Ca2+ activation than previously thought but may involve other K+ transport pathways. Recent studies by Grinstein and Smith (57) shed light on the question of Ca2+-dependent regulation of cell volume. Lymphocytes were shown to undergo RVD even when intracellular Ca2’ was depleted (by preincubation with EGTA plus ionomycin) or buffered [by loading with the Ca2’ chelator, I,%bis(2-aminophenoxy)ethane-N,N,N,hr-tetraacetic acid (BAPTA)], arguing against the involvement of Ca2+-dependent K+ channels in RVD. The authors presented evidence that, although Ca2+-activated K+ channels are found in lymphoid cells (8, 57), their involvement in RVD may be minimal. Rather, swelling-induced K+ loss may occur via Ca2’-independent K+ channels that may be voltage activated (57). Thus Grinstein and Smith proposed that RVD may occur through the operation of voltage-gated K+ channels, which may be opened due to the initial depolarization arising from the activation of Cl-selective channels. These latter channels may be opened by stretch activation during swelling (8, 30, 103, 104, 157). However, if RVD occurs by the activation of voltage-activated K+ channels and stretch-activated Cl- channels, thus discounting a role for Ca2’, one must wonder what caused the partial inhibition of RVD originally indicated in Ca2+-depleted lymphocytes (see next section).

1051

The above findings make it clear that the Ca2’ dependency of mechanisms involved in RVD cannot be studied simply by application of ionophores under isotonic conditions. Not only do these ionophores often uncouple cellular respiration, resulting in metabolic imbalances and their consequential effects on cytoplasmic composition, ionophore-induced conductances serve as poor substitutes of the original conductances, since they are unregulated leak pathways. All conclusions based on studying one volume regulatory pathway in the presence of an ionophore-induced counterion pathway or second messenger pathway must be interpreted with caution, since the investigator can never be certain what conditions prevail within the cell. VII.

IS THERE VOLUME

A CALCIUM

THRESHOLD

FOR

REGULATORY

DECREASE?

It is apparent from the above discussion that RVD mechanisms in many cells are Ca2’ dependent to variable degrees. Many cells require that [Ca2+]i rises during swelling, either by release from intracellular stores or by activation of Ca2’ influx pathways, to activate the RVD machinery. In a few cases, however, the Ca2’ dependency of RVD is not readily evident, as the normal [Ca2+]i levels appear to be sufficient to activate the RVD mechanisms during swelling without a rise in [Ca2+]i (e.g., see Refs. 4, 57, 130). In this latter case, a Ca2’ dependency of RVD, if it exists, may only be apparent when [Ca2+]i levels are reduced from normal levels. This was particularly evident in a recent study of renal thick ascending limb cells (130). In these cells, hypotonic swelling activated Ca2’ entry and resulted in a rise in [Ca2+]i. In the absence of extracellular Ca2’, the rise in [Ca”‘], was reduced from the normal value of 150 nM to -50 nM; while RVD persisted, it was modestly reduced. As [Ca2+]i levels were experimentally reduced below the 50 nM range, RVD was dramatically inhibited. It would appear, therefore, that in these cells, RVD was Ca2’ dependent but that an apparent “Ca2+ threshold” for RVD existed that was poised well below the normal [Ca2+]i levels. Hence, RVD would appear to be Ca2’ independent in the presence of normal [Ca2+]i levels but only because these levels were greater than the apparent Ca2’ threshold. The presence of an apparent Ca2’ threshold for RVD in a few cells suggests the possibility that a Ca2’ threshold may exist in most cells. The presence of such a threshold may be difficult to assess, since it may be masked by continued Ca2’ influx across the plasma membrane. Indeed, recent evidence indicates that receptor-mediated Ca2’ influx in nonexcitable cells may be modulated by [Ca2+]i such that appreciable levels of Ca2’ entry are measured only when [Ca2+]i is buffered to low values (see Ref. 138). Nonetheless, the notion of a Ca2’ threshold for RVD has appeal because it provides a unifying hypothesis as to the apparent variable role of Ca2’ in RVD. The Ca2’ threshold itself could be variable among cell types and possibly among differing func-


1052

NAEL

A. MCCARTY


tional states of the cell, reflecting different levels of endogenous Ca2’ buffering systems (150), which may be unknowingly altered by experimental manipulations. Hence, in cells with a Ca2’ threshold for RVD that was less than the normal [Ca2+]i levels, the RVD following swelling would not appear to be Ca2’ dependent unless intracellular Ca2’ stores were depleted sufficiently so as to reduce [Ca”‘], below this threshold. Under these conditions, intracellular Ca2’ would be acting more as a cofactor so that swelling-activated Ca2’ signaling would not be required to induce RVD. Alternatively, in cells where the Ca2’ threshold was greater than normal Ca2’ levels, Ca2+ signaling would be required, as RVD would not be activated unless intracellular Ca2’ levels were elevated above the threshold during swelling as a result of Ca2’ release and/or increased Ca2+ entry. Hence, in some cells, such as Ehrlich ascites tumor cells, swellinginduced Ca2’ release alone would appear to be sufficient to raise intracellular Ca2’ levels above the Ca2’ threshold and activate RVD. In other cells, such as the renal PST cells (125, 126), increased Ca2’ entry appears to be required to raise intracellular levels above the Ca2’ threshold to activate RVD. Finally, in cells in which the RVD would appear to be Ca2’ independent, such as the renal thick ascending limb cells (130), or possibly the lymphocyte (57), the Ca2’ threshold for RVD may be less than the normal intracellular levels so that the Ca2’ dependency of RVD would only be apparent after sufficient depletion of intracellular Ca2’ stores. The site at which Ca2’ is limiting or regulating RVD may be variable among cells and conceivably could exist at any point in the underlying regulatory biochemical pathways, such as a phosphorylation event, to direct the control of one or more ion channels or transporters. Hence, the concept of a Ca2’ threshold for RVD could explain much of the apparent variability among tissues and cells in their Ca2’ dependency of hypotonic cell volume regulation. VIII.

MODEL VOLUME RENAL

OF CALCIUM-DEPENDENT DECREASE PROXIMAL

REGULATORY

MECHANISMS: TUBULE

The RVD behavior of the mammalian renal PST has been studied extensively for many years (for reviews see Refs. 50,203). In terms of its response to hypotonicity, PST appears to be a very good model of the types of cells that achieve RVD by separate K+ and Clconductances. In this tissue, similar to lymphocytes and CHO cells, the plasma membrane (basolateral membrane) in the isotonic state is characterized by a dominant barium-sensitive K+ conductance and a minimal Cl- conductance (45, 201). Grantham et al. (51) originally showed that the PST loses K+ when undergoing RVD. Several investigators then showed evidence for a conductive nature of the volume-activated K+ efflux pathway by virtue of inhibition of RVD or the K+ conductance by barium and quinine (87,200,202). Exposure to hypotonic medium results in an increase in PK, as evidenced through electrophssiological measurements

Volume

7.2

and through increased sensitivity of cell volume to alterations in peritubular K+ concentration (28,87,202). The anion that accompanies K+ was long assumed to be either Cl- or HCO, (119), until it was shown conclusively by Welling and Linshaw (199) that RVD supports Clefflux. This was recently confirmed by Schild et al. (174), who showed that Cl- exits across the basolateral membrane in response to swelling via a conductive pathway that is inhibitable by diphenylamine-2-carboxylate. A. Proximal Straight Tubule Regulatory Volume Decrease Response

It has recently been shown that RVD in rabbit PST is HCO; independent and is characterized by an increase in both the K+ and Cl- conductances of the basolateral membrane (202). The patterns of cell volume and membrane voltage changes are very similar to those of human lymphocytes (57), Ehrlich cells (76), and CHO cells (171). On exposure of isolated lumen-collapsed, nonperfused PSTs to hypotonic medium, the cells swell very rapidly to a peak volume, usually within the initial 30 s (125-127, 202; Fig. 10). Concomitantly, the voltage across the basolateral membrane (I&) becomes transiently hyperpolarized by 10 mV. This hyperpolarization is toward the Nernst equilibrium potential for K+ (&) and likely reflects an increase in the K+ conductance. Following the brief hyperpolarization is a prolonged depolarization, as Vbl approaches &i, indicating that part of the depolarization reflects activation of a Cl- conductance (202). Over the next several minutes, Hypotonic

Ringer (150 mOsm/Kg)

z

- -30 5 t -40

I

I

-50

Q) 1.6~ E 22 3 1.23 .s l.O3 ’ Oa8

I -1

I 0

I 1

I 2

I

3

I

I

4 5 Minutes

I

I

I

I

&

6

7

8

9

10

FIG. 10. Effect of hypotonic challenge on proximal straight tubule. Typical experiment showing effects of a reduction in bathing medium osmolality from 290 to 150 mosmol/kgH,O by a NaCl dilution. Simultaneous measurements of relative tubule volume (indicative of relative cell volume) and basolateral membrane potential difference (I&) within a single tubule are plotted with respect to time. [From Welling and O’Neil (202).1


October 1992

CALCIUM

CONTROL

PST cells undergo RVD to return to normal cell volumes. The PST cell is similar to other epithelial cells in terms of membrane permeabilities and in the control of [Ca2+]i, as discussed in section VIIIB. Furthermore, the relative ease of isolation of the proximal tubular epithelium away from any underlying mucosal tissue allows the volume regulation process to be studied in cells that are undergoing normal transmural solute and water movements. Finally, the general characteristics of PST cell RVD behavior, in terms of time course of volume changes and control of intracellular Ca2+, are preserved in primary cultures of rabbit PST cells, allowing the RVD process to be studied at the single cell level (O’Neil, unpublished observations).

1053

OF CELL VOLUME

‘0‘ 2 L

1.6 1.5 1.4 -

3

1.3 1.2 -

B. Calcium Dependence of Regulatory Decrease: Entry and Release

Volume

Although there was much prior background work on the regulation of [Ca2+]i in PST cells (82,110,123,182, 207), RVD in the rabbit PST was first shown to be Ca2’ dependent by McCarty and O’Neil(125; for preliminary report see Ref. 139). In these studies, it was found that

1.1

-

1.0

-

0.9

J b

-120

-60

0

60

120

180

240

4

300

Time (seconds) FIG. 12. Simultaneous measurements of [Ca2+]i (A) and cell volume (B) during exposure of an isolated proximal straight tubule to hypotonic solution with 1 PM Ca2+. Following a transient increase, [Ca2+]i fell below original baseline. RVD was inhibited under these conditions. [From McCarty and O’Neil (126).]

A 400

-

350

-

z

300.

r_ &

250-

pj -

150.

200

h J

-

100

-

50

-

B -

1.6

2> -

1.5

Q) E =I

1.4.

1

1.3.

s

1.2-

!E c, s Q) CT

11 * 1.0.

0.9'

1

*-

@ -120

,

-

*

'

-60

0

60

120

Time

4 180

240

300

(seconds)

FIG. 11. Response of isolated proximal (straight) tubule to hypotonicity in presence of normal extracellular Ca2+ concentration ([Ca”‘J,,) in a typical experiment. At time 0, superfusate (bath perfusion solution) was switched from isotonic (Iso) to hypotonic with 1 mM [Ca”‘], (Hypo), followed by return to isotonic solution (Iso) at 180 s. A: intracellular free Ca2’ concentration ([Ca”‘],) was determined from background-corrected fluorescence of fura- using a dual-wavelength microspectrofluorimeter. Individual points are not shown but were generated every 2-3 s throughout duration of experiment. When exposed to hypotonic solution, [Ca2+]i rose rapidly to a peak and then fell to an elevated plateau. On return to isotonic solution, [Ca”‘], returned slowly to original baseline. B: simultaneous measurement of cell volume during same time period as in A. Cell volume is expressed as relative volume (V/V,). When exposed to hypotonic solution, cells swelled to a peak volume and then underwent RVD. On return to isotonic solution, cell volume dipped below isotonic volume due to loss of osmolytes during RVD. [From McCarty and O’Neil (126).]

RVD was highly dependent on extracellular Ca2’, since the magnitude of the RVD response was inhibited half maximally by modest reduction in [Ca”‘], from 1 to 0.1 mM. Subsequent measurements of [Ca”‘]i during swelling and RVD (126) indicated that the response of intracellular Ca2’ to swelling consisted of two phases: a fast transient phase wherein [Ca2+]i increased by 276 nM above baseline, followed by a prolonged, sustained phase wherein [Ca2+]i was maintained at 132 nM above baseline (Fig. 11). When the hypotonic solution contained only l-10 PM Ca2+, the transient phase of increased [Ca2+]i remained present, albeit diminished, while the sustained phase was abolished, and RVD was blocked (Fig. 12). These swelling-induced changes in [Ca2+]i were shown to occur via two mechanisms: one that predominantly occurs via the release of Ca2’ from intracellular stores, leading to a transient peak response regardless of the presence of Ca2’ in the bath , and one that is strictly dependent on Ca2’ entry from the m.edium, leading to the sustained increase in Ca2’ entry. C. Calcium Entry Pathways Under Isotonic and Hypotonic Conditions: Calcium Channels

The Ca2’ permeability pathways of the rabbit renal PST basolateral membrane have also been investigated (126,127). Under isotonic conditions, reduction of [Ca”‘], from 1 mM to 1 PM or the addition of 10 PM verapamil led to an immediate fall in [Ca2+]i, which was associated with destabilization of cell volume, leading to a trend for cell swelling when [Ca2+]i had been lowered significantly (Fig. 13). However, treatment with the DHP Ca2’ channel blocker nifedipine in isotonic conditions did not af*


1054

NAEL

A. MCCARTY AND ROGER G. O’NEIL

Volume Nifedipine

1pM Ca2+

Verapamil

D

1pM Ca2+

‘75 1.05 > 2

72

Nifedipine

1.05

E 3 =>

1.00

4

1.00.

1.00

:

v-w*

.-F 5 z 0.95

b

-120

4

-60

0

60

120

Time (seconds)

180

0.95

.

-120

1

-60

0

60

120

Time (seconds)

180

0.95d

b -120

-60

0

60

120

4 180

Time (seconds)

FIG. 13. Composite showing effects of bath [Ca”‘] reduction and Ca2’ channel blocker administration on [Ca2’]i (A, C, E) and cell volume (B, D, F) that were measured simultaneously in a proximal tubule in isotonic steady state. Isotonic exposure to 1 PM Ca2’ (A, B) led to a rapid drop in [Ca2+]i that was associated with induction of fluctuations in cell volume. Administration of 10 PM verapamil (C, 0) had similar effects. Administration of 10 PM nifedipine (E, F), a dihydropyridine (DHP), was largely without effects on [Ca2+]i and volume. [From McCarty and O’Neil (X27).]

feet either [Ca”‘], or cell volume. Thus the PST basolatera1 membrane is characterized under isotonic conditions by an apparent Ca2’ channel that is verapamil sensitive but DHP insensitive. The permeability pathways present under hypotonic conditions in the PST appear to differ from those present under isotonic conditions (127). When tubules were swollen in hypotonic solution with low Ca2’ or verapamil, the initial transient increase in [Ca2+]i was still present but was reduced. However, the plateau phase was completely abolished such that [Ca2+]i fell below the baseline level immediately following the peak in the transient phase. In contrast, when tubules were swollen in the presence of nifedipine, the [Ca2+]i transient was, again, only partially reduced, but the plateau phase concentration was only lowered to the baseline concentration or slightly above. Under nifedipine treatment, with or without preincubation, the [Ca”‘], plateau phase of the response to swelling did not fall below the baseline, in contrast to that observed with verapamil treatment, indicating a different mode of action of nifedipine compared with verapamil. Thus the swelling-induced Ca2+ enty pathway is both DHP sensitive and verapamil sensitive. It appears, therefore, that PST cells have two separate Ca2’ entry pathways (channels) in the basolateral plasma membrane (Fig. 14). One is active during the isotonic state and continues to operate during swelling and RVD. This baseline, or constitutive, permeability is

verapamil sensitive but DHP insensitive. During isotonic, steady-state conditions, this permeability plays a partial role in establishing the baseline [Ca2+]i, since, when it is inhibited, baseline [Ca2+]i falls rapidly (see Fig. 13). Cell swelling induces a second Ca2+ channel that is quiescent in the isotonic state. This swelling-activated Ca2’ channel is DHP sensitive and verapamil sensitive. It is this channel that supplies the Ca2’ for the observed rise in [Ca2+]i, which is a prerequisite for the initiation of RVD. Figure 15 summarizes the relative contributions of each of these Ca2’ entry/release pathways to [Ca2+]i as a generalized response. The DHP sensitivity of the swelling-induced Ca2+ channel of the renal PST may indicate that this channel has properties of L-type Ca2’ channels. The L-type Ca2’ channels have been extensively studied in excitable tissues and are characterized by a DHP-sensitive blockade (l&67,78,129,185). Indeed, the abolishment of RVD in PST cells by the three Ca2’ channel blockers used in these studies (125) (representing different classes of blockers), namely lanthanum, verapamil, and DHPs, would be consistent with blockade of L-type Ca2’ channels. Preliminary evidence has shown that rabbit PST cells possess a high-affinity DHP receptor, consistent with the presence of L-type Ca2’ channels in this tissue (142). Furthermore, these same studies demonstrate that the PST cells possess significant levels of L-type Ca2+ channel messenger RNA &-subunit), which has a high degree of similarity to the cardiac muscle L-type


October 1992

CALCIUM

Ca*+

CONTROL

1055

OF CELL VOLUME

Ca*+

Quiescent Swelling-activated Ca2+ Channel Basolateral Membrane

Luminal Membrane

FIG. 14. Schematic representation of Ca ‘+ transport functions in proximal straight tubule that are active in isotonic state (A) and hypotonic state (B). A: under isotonic conditions, Ca2+ cycling across intracellular storage site (depicted as endoplasmic reticulum) is accompanied by extrusion from cell via Ca2’-ATPase and Na+-Caa+ exchanger. A baseline level of Caa” entry occurs through a verapamil-sensitive, DHP-insensitive channel. B: under hypotonic conditions, Ca2” release is stimulated, as is Ca*+ entry through a DHP- and verapamil-sensitive Ca2+ channel that opens during swelling. Resultant Ca*+ influx activates K’ and/or Cl- channels through an unknown pathway. During swelling and RVD, Ca2+ entry through baseline channel continues but at a low rate that does not support Ca2+-dependent cell volume regulation. Ver, verapamil; Nif, nifedipine.

Ca2+ channel. Hence renal PST cells appear to express an L-type Ca2+ channel. Even though it is likely that this is the swelling-activated Ca2+ channel in PST cells, further evidence in support of this view is still forthcoming. D. Calcium Window: Temporal Dependence of Regulatory Volume Decrease The ability of Ca2+ to stimulate RVD showed a distinct time dependence, which did not appear to be related to modulation of the Ca2+ entry pathway (125127). It was shown that Ca2+ entry from the extracellular medium was required to occur within the first 30-60 s after exposure to hypotonic conditions. Beyond this time period, the RVD mechanism was inactivated and could not be reactivated by addition of Ca2+ to the me-

dium or introduction of Ca2+ to the cytoplasm, using the Ca2+ ionophore ionomycin. A very similar response was seen in PCT cells of the killifish, Fundulus (128). The term “Ca’+ window” was used to describe this phenomenon. At this point, the nature of the time-dependent component is unknown. However, two possibilities may be put forth. First, the time-dependent component may be related to the inactivation of either the K+ or the Clchannel. Because both pathways are required to be active to achieve RVD, inhibition of one or the other, and not necessarily the Ca2+-dependent one, would lead to inhibition of RVD. Alternatively, it may be that the Ca2+ signal itself, although prolonged in nature, must be transduced to the next component of the pathway in a certain amount of time before that signal is shunted off in a direction inappropriate to activating RVD (125,

500 -

400 FIG. 15. Relative contributions of 3 sources of Ca2+ to increase in [Ca’+J in idealized response. Thick top solid line depicts average time course of changes in [Ca2+li during swelling and RVD under normal conditions and is sum of individual components listed below. Proportional contributions from Ca2+ release, verapamil-sensitive baseline Ca2+ entry, and swelling-activated DHP-sensitive Ca2+ entry are shown.

1 $= 300F J o %l -

200

_

Swelling-activated

Ca*+ Release

Resting [Ca**li

t 100 -

3

_-_____-------------------------------,

0

I

I

I

I

0

60

120

180

Verapamil-sensitive Baseline Ca2* Entry

Time (seconds)


1056

NAEL

A. MCCARTY

127). Thus the control of proximal tubule RVD is complex, involving Ca2+ entry, Ca2’ release, and a temporal dependence in the action of Ca2+ on the cell volume regulatory machinery.


Volume

72

REFERENCES 1. ALLAN, D., AND R. H. MICHEL. A comparison of the effects of phytohemagglutinin and of calcium ionophore A23187 on the metabolism of glycerolipids in small lymphocytes. Biochem. J. 166: 495-499,1977.

ARMANDO-HARDY, M., C. J. ELLORY, H. G. FERREIRA, S. FLEMINGER, AND V. L. LEW. Inhibition of the calcium-induced increase in the potassium permeability of human red blood cells by quinine (Abstract). J. Physiol. Lond. 250: 32P-33P, 1975. 3. BAGNASCO, S. M., H. R. MURPHY, J. J. BEDFORD, AND M. B. BURG. Osmoregulation by slow changes in aldose reductase and rapid changes in sorbitol flux. Am. J. Physiol. 254 (CeZZ Physiol 2.

IX.

SUMMARY

It is evident from the present analysis that although a role for Ca2’ in controlling hypertonic cell volume regulation and RVI mechanisms has not been shown, Ca2’ plays a central role in activating and controlling hypotonic cell volume regulation and RVD mechanisms in most cells. However, this Ca2’ dependency is highly variable among cell types and tissues. Cells can be grouped into three general categories based on the relative dependency of RVD on Ca2+: I) cells that are highly dependent on extracellular Ca2’ and the activation of Ca2’ influx, supposedly reflecting activation of Ca2’ channels, such as observed for the renal PST cells and osteosarcoma cells; 2) cells that are not dependent on extracellular Ca2’ and Ca2+ influx but that require at least a certain basal intracellular Ca2’ level or transient release of Ca2’ from internal stores, such as observed for the Ehrlich ascites tumor cells and medullary thick ascending limb cells; and 3) cells that display little if any Ca2’ dependency, such as the lymphocytes. There is initial evidence that this variable dependency of RVD on Ca2’ may reflect, in large part, a variable Ca2’ threshold of RVD processes, although this notion has not been fully investigated. The site and mechanism of Ca2+ dependency of RVD are poorly understood. Initial studies pointed to a possible direct control of K+ and/or Cl- channels by Ca2+ to modulate KC1 efflux and, hence, RVD. This view appears to be too simplistic, however, as it is increasingly evident that the ion channels involved in RVD may not be directly Ca2+ dependent and that some other regulatory process controlling the channels, perhaps a phosphorylation step, may be the Ca2+-dependent event. Given the added complexity of the time-dependent variability of the action of Ca2’, i.e., the Ca2+ window, coupled with the variability of the RVD mechanisms among cell and tissue types, it is likely that the RVD mechanism is a highly complex process involving events and biochemical pathways throughout the cell rather than events simply localized to the inner face of the plasma membrane. It remains for future studies to determine the exact biochemical events that underly the RVD mechanism and its control, and the Ca2’ dependency of each step, before a full understanding will be attained of the role of Ca2’ in modulating RVD. The authors thank Dr. Robert Roer for critically reading the manuscript andJim Pastore and Linda Eshelman for preparation of the figures. During the preparation of this manuscript, N. A. McCarty was supported by National Institute of Diabetes and Digestive and Kidney DiseasesPostdoctoral Fellowship DK08559-02.This work was supported by National Institute of Diabetesand Digestive and Kidney DiseasesGrant DK-40545.

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