Effects of osmotic stresses on isolated rat hepatocytes I. Ionic mechanisms of cell volume regulation JAMES G. CORASANTI, DERMOT GLEESON, AND JAMES L. BOYER Department of Medicine and Liver Center, Yale University School of Medicine, New Haven, Connecticut 06510

CORASANTI, JAMES G., DERMOT GLEESON, AND JAMES L. BOYER. Effects of osmotic stresses on isolated rat hepatocytes. I. Ionic mechanisms of cell volume regulation. Am. J. Physiol. 258

(Gastrointest. Liver Physiol. 21): G290-G298, 1990.-Isolated hepatocyte suspensions were exposed to hypotonic and hypertonic stresses and serial cell volume measurements were made with an electronic particle size analyzer. With the exposure to hypotonic (160 mosM) buffer, hepatocytes swelled within 3060 s as osmometers [relative volume (RV) = 1.44 t 0.081 and subsequently underwent regulatory volume decrease (RVD) back toward the resting (isotonic) level (1.16 t 0.05). This volume recovery was blocked by 65 mM extracellular K+ concentration and inhibited by barium (1 mM) and quinine (0.5 mM) but not by bumetanide (0.1 mM). Chloride depletion inhibited RVD by -40% while 0.5 mM 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS) blocked the recovery by almost 90%. Calcium deprivation had no effect on RVD, nor did ouabain, amiloride, or sodium replacement. When exposed to buffer made hypertonic by addition of 200 mM sucrose, cells shrunk as osmometers (RV = 0.74 & 0.02) but did not exhibit regulatory volume increase (RVI). However, when cells that had first undergone RVD were reexposed to isotonic medium (relative hypertonic stress) RVI could be demonstrated from RV 0.77 t 0.17 to 0.91 t 0.20. This response was dependent on sodium, partially dependent on bicarbonate and chloride, and inhibited by the Na’-H’ exchange inhibitor amiloride (1 mM) but not by DIDS. Our findings suggest that RVD in rat hepatocytes is mediated by quinine- and barium-sensitive K+ conductance and DIDS-sensitive anion conductance, which is partly accounted for by Cl-; RVI is mediated by activation of Na’-H’ exchange coupled with a bicarbonateand chloridedependent but DIDS-insensitive process. isolated cells; hypertonic solutions; hypotonic solutions; potassium channels; ion transport; regulatory volume decrease; regulatory volume increase; sodium-hydrogen exchange; pH

WHEN EXPOSED TO ANISOTONIC CONDITIONS, most Cells behave initially as osmometers, changing their volumes in accordance with the tonicity of the surrounding medium. Many cell types, however, exhibit an ability to return toward their resting (isotonic) volumes despite continued osmotic perturbation by activating various ion transport processes (for review see Refs. 10, 33, 38, 49). Regulatory volume decrease (RVD) and regulatory volume increase (RVI) are the terms used to describe these restorations of cell volume after hypotonic swelling and hypertonic shrinkage, respectively. Studies of cell volume regulation, originally performed on free-floating blood elements, but since then extended G290

0193-1857/90

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to many cell types including epithelia, have defined a number of ion transport systems that are activated when cells are incubated in anisosmotic medium. In most cells RVD is effected by net extrusion of K+ and Cl- with the accompanying loss of cellular water. The mechanisms responsible for RVD include 1) activation of barium- and quinine-sensitive (Ca2+ -dependent) K+ channels (19, 28, 29, 32, 36, 37, 47, 54), 2) stimulation of furosemide- or bumetanide-sensitive K+-Cl- cotransport processes (7, 33, 35, 44), and 3) parallel activation of K+-H+ and Cl--HCO; exchangers (4, 5). Transport of amino acids such as taurine, polyglycols such as sorbitol, and quaternary amines such as tetramethylamine have also been demonstrated as volume regulatory mechanisms, particularly in lower vertebrate species. However roles of these substances in mammals are not well defined (14). In contrast, RVI occurs in response to hypertonic shrinkage and is generally mediated by salt and water entry into cells. However, as with RVD, the transport mechanisms responsible for the net ion uptake differ among cell types. The two most common mechanisms responsible for RVI are 1) activation of a ouabain-insensitive but furosemide- and bumetanide-sensitive Na+K+-2Cl- cotransport system (9, 13, 33, 42-44) and 2) coupled activation of Na+-H+ and Cl--HCOy exchangers (4, 5, 8, 11, 20, 23, 26, 39, 45). Each of these processes results in a net gain of Na+ and Cl- with eventual exchange of Na+ for K+ by the Na+-K+-ATPase. Recently a K+-independent Na+-2Cl- cotransport process has been described as a mediator of RVI in Ehrlich ascites tumor cells (30); however, the possibility that this actually represents Na+-K+-2Clcotransport with altered K+ affinity has yet to be addressed. Most studies of volume regulation in liver have assessed osmotically induced changes in net ion fluxes, membrane potential, or hepatic histology or have used indirect methods of determining hepatocyte volumes. Van Rossum and Russo (51, 52), using morphometric analyses, described an ouabain-insensitive component of RVD in rat liver tissue slices at 38°C after a period of swelling at 1°C that was Na+ and Cl- dependent but furosemide insensitive (2 mM) and that was accompanied by the formation of pericanalicular vesicles. Berthon (2) and Bakker-Grunwald (1) used isolated rat hepatocyte suspensions and demonstrated a net K+ efflux in response to cell swelling that was Ca2+ independent and bumetanide insensitive. Consistent with this observation, Haddad et al. (24) found barium- and qui-

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MECHANISMS

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nine-sensitive increases in perfusate K+ efflux in the isolated perfused rat liver, in response to hypotonic perfusions. Similarly, Howard and Wondergem (31) have shown that mouse liver slices exposed to hyposmotic medium exhibit membrane hyperpolarization that is blocked by quinine or by increases in extracellular K’, again suggesting a role for K+ efflux in RVD in hepatocytes. RVI in liver has not been studied extensively. BakkerGrunwald (1) failed to demonstrate a regulatory response to hypertonic stress in isolated rat hepatocyte suspensions and specifically could find no evidence for activation of a bumetanide-sensitive K+ transport system. Haddad et al. (25) and Graf et al. (17) demonstrated a ouabain-sensitive net K+ influx in an isolated perfused rat liver in response to a lo-min sucrose exposure, but the cellular mechani .sm( s) of this phenomenon has yet to be characterized. In the present study we employed a Coulter counter technique modified from that of Grinstein et al. (22) to measure directly the changes in volumes of isolated rat hepatocytes in suspension in response to hypotonic and hypertonic stresses. We also used ion substitutions and inhibitor profiles to define more accurately the mechanisms of both RVD and RVI. In the accompanying paper (15) we examine the effects of osmotic stresses on the hepatocyte Na+-H+ exchanger using the pH-sensitive fluorescent dye 2’-7’-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). MATERIALS

AND

METHODS

Animals and reagents. Hepatocytes were isolated from male Sprague-Dawley rats (X0-200 g) obtained from Camm Laboratories, Wayne, NJ. Collagenase (type l), 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS), amiloride, barium chloride, ouabain, quinine, tetramethylammonium (TMA), and ethylene glycol-bis( p-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) were obtained from Sigma Chemical (St. Louis, MO). Bumetanide was purchased from Hoffmann-LaRoche (Nutley, NJ). Leibovitz-15 (L-15) culture medium was obtained from GIBCO (Grand Island, NY). All other chemicals were reagent grade or the highest purity available. Hepatocyte isolation. Isolated hepatocytes were prepared by in situ perfusion with Ca’+- and Mg2+-free Hanks’ medium (in mM, 120 NaCl, 5.0 KCl, 25 NaHC03, 0.66 KH2P04, 0.33 Na2HP04, 0.1% D-ghCOSe) followed by 0.05% collagenase in Ca2+- and Mg2+-containing Hanks’ buffer as previously described by this laboratory (12, 16). This technique was modified slightly to assure required for Coulter a uniformity of the cell suspensions counter measurements. Briefly, after collagenase perfusion, the liver was removed from the carcass, grasped at the hilum by forceps, and the hepatocytes combed into L-15 medium at 0°C with a rubber policeman. This cell suspension was filtered sequentially through 80- and 45pm mesh and then centrifuged at 50 g. The supernatant was discarded, and the cells were washed with and resuspended in cold L-15 medium. The addition of these two steps to the isolation procedure eliminated most of the nonparenchymal cells and debris from the final suspen-

IN

HEPATOCYTES

G291

sion and also resulted in a predominantly single-cell population. Cell viability was assessed with each preparation using trypan blue exclusion and ranged from 80 to 94%. Cell volume determination. Cell volume measurements were made with a Coulter model ZF electronic particle analyzer (loo-pm aperture diameter) and loo-channel Channelyzer attachment (Coulter Electronics, Hialeah, FL). Calibrations were performed with lo- and 20-pm latex beads. Optimum settings for measurement of hepatocyte volumes were found to be l/amplification = 4; l/aperture current = 16; and base channel threshold = 5. At these settings the calculated threshold factor of 175 pm3/channel (-l/35 of hepatocyte size) allows detection of relatively small changes in hepatocyte volume given the reported accuracy within one channel on either side of a peak (Coulter Electronics). Median cell volume of the suspensions was determined according to the method of Segel et al. (48) from the Channelyzer frequency distribution with manual delimiting of the major hepatocyte peak. Cell volumes are expressed as relative volume (measured volume/isotonic volume for each experiment). ExperimentaL protocol. Freshly isolated hepatocytes were maintained on ice in L-15 medium for O-3 h before institution of experimental protocols. At the beginning of each experiment, hepatocytes were resuspended in isotonic N-2-hydroxyethylpiperazine-2\‘-2-ethanesulfonic acid (HEPES) or Krebs-Ringer bicarbonate (KRB) buffer (see Table 1) at 37°C for 20-30 min to allow recovery from cold-induced swelling. Hypotonic stress. As shown in Table 1, hypotonic solutions were prepared by removing 70 mM choline chloride (or TMA gluconate in Cl--free experiments) from the original isotonic buffer; this was done to maintain constant extracellular Na’ concentration. (In preliminary experiments substitution of choline for sodium in the isotonic buffer had no effect on basal cell volume or on volume recovery from a hypotonic stress.) After the aforementioned recovery period in isotonic medium, aliquots (200-300 ~1) of the suspension, concentrated by gravity sedimentation and removal of most of the supernatant, were diluted in 25 ml of hypotonic HEPES or KRB (Table 1) in a plastic vial (Sarstedt, Princeton, NJ) in a 37°C water bath at t = 0 min and maintained adjacent to the Coulter counter. After gentle agitation, cell volume determinations were made at l-, 5-, lo-, and 20-min intervals. For each ion substitution or inhibitor experiment a paired control was performed using the same initial cell preparation. As the degree (%) of volume recovery, RVD, was essentially the same in both hypotonic HEPES and KRB (cf. Figs. 2 and 7), all subsequent experiments were performed in HEPES for convenience. Table 1 shows the ion substitutions used. In all cases except that of KC1 replacement for NaCl, the substitutions were made in isotonic buffer 15 min prior to exposure to the substituted hypotonic media (KC1 was substituted for NaCl only in the hypotonic medium). Inhibitors were added to the hypotonic buffers alone unless otherwise stated. Hypertonic stress. Hepatocytes were exposed to hyper-

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G292 TABLE

MECHANISMS

OF

VOLUME

REGULATION

IN

HEPATOCYTES

1. Ion composition of isotonic, hypotonic, and hypertonic buffers Buffer

Na’

K’

Cl-

Choline

Ca2+

MgSOs,

PO:-

HCO:

HEPES

Gluconate

Other

Isotonic Control HEPES* KRB* Cl- free HEPES KRB Na’ free HEPES KRB

65 65

4.2 4.2

65 65

4.2 4.2 4.2 4.2

141 141

141 141

70 70

1.25 1.25

1.0 1.0

1.67 1.67

25

20

25

1.25 1.25

1.0 1.0

1.67 1.67

25

135 135

1.25 1.25

1.0 1.0

1.67 1.67

25

20

141 141

70 mM 45 mM

TMA TMA

20

Hypotonic Control HEPES KRB Cl- free HEPES KRB Na’ free HEPES KRB

65 65

4.2 4.2

65 65

4.2 4.2

71 71

1.25 1.25

1.0 1.0

1.67 1.67

25

1.25 1.25

1.0 1.0

1.67 1.67

25

1.0 1.0

1.67 1.67

25

1.67 1.67

25

4.2 4.2

71 71

65 65

1.25 1.25

4.2 4.2

141 141

70 70

1.25 1.25

20

20

71 71

20

Hypertonic HEPES KRB Values are expressed in bicarbonate-buffered Ringer bicarbonate.

65 65

1.0 1.0

20

200 mM 200 mM

in mM. pH was adjusted to 7.40 by addition of NaOH in HEPES-buffered solution or by gassing solutions. TMA, tetramethylammonium; HEPES, IV-2-hydroxyethylpiperazine-IV’-2-ethanesulfonic

tonic stress in one of two separate methods. 1) In a manner analogous to that described for hypotonic stress (seeHypotonic stress), aliquots of cells were resuspended in HEPES or KRB rendered hypertonic by addition of 200 mM sucrose to isotonic medium (Table l), and cell volume measurements were made at l-, 5, lo-, 20-, and 30-min intervals. In selected experiments additional measurements were made at 45 and 60 min. 2) As certain other cell types exhibit RVI only after reexposure to isotonic medium after hypotonically induced RVD (21, 30, 42, 46), cell volumes in hepatocytes treated in this fashion were also serially measured. In these experiments the initial isotonic suspension of hepatocytes was allowed to gravity sediment, an aliquot was then placed into hypotonic medium for subsequent serial volume measurements to assure that the cells indeed underwent RVD, and the remaining cell pellet was then resuspended in hypotonic buffer. After a 20-min incubation in hypotonic buffer, concentrated aliquots (200-300 ~1) were resuspended in isotonic buffer, and volume measurements were made at t = 1, 5, 10, 20, and 30 min. As RVI was dependent on HCO: (Fig. 7; Table 3), all subsequent ion substitution and inhibitor experiments were performed in KRB. Table 1 also depicts the various ion substitutions used in RVI experiments. Choline was substituted for Na+ in the isotonic buffer used for reexposure (relative hypertonic stress), while gluconate was substituted for Cl- in hypotonic KRB for an additional 15 min of RVD before exposure to Cl--free isotonic buffer (to deplete hepatocytes of intracellular Cl-). Inhibitors were added to the isotonic reexposure buffer alone. Statistical analysis. Data were analyzed using non-

sucrose sucrose

with 95% Oz-5% CO* acid; KRB, Krebs-

paired, two-tailed Student’s t tests. Data from Fig. 1 (Boyle-van’t Hoff relationship) were assessed by linear regression analysis. RESULTS

Cell uolume. Median volumes of hepatocyte suspensions were 6,168 t 588 pm3 (relative volume = 1.00, n = 63) in isotonic buffer (HEPES and KRB); 9,179 t 892 pm3 (relative volume = 1.48 t 0.11, n = 51 pairs) in hypotonic buffer; and 4,654 t 614 pm3 (relative volume 2.00 slope = 183.3 y-int = .375 ;= .998 1.50 -

0.50

0)

I I

0.00

0.000

0.005

0.010

1 /OSMOLALIW FIG. 1. Boyle-van’t Hoff relationship for isolated rat hepatocytes. Rat hepatocytes in suspension were maintained on ice in L-15 medium for O-3 h after isolation and then resuspended in isotonic HEPES or KRB buffer at 37°C for 30 min to allow recovery from cold-induced swelling. Aliquots of cells were transferred to hypotonic (160 mosM) or hypertonic (510 mosM) buffer and median cell volumes measured 30-60 s later. Results are expressed as relative volume (measured volume/isotonic volume for that experiment). Values are means t SD of no. of experiments noted at each point.

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MECHANISMS

OF VOLUME

REGULATION

= 0.74 t 0.02, n = 10 pairs) in hypertonic buffer. Hepatocyte volumes in hypotonic and hypertonic media are those recorded 30-60 s after exposure to the anisotonic buffer because maximal osmotically induced changes were observed in this period. Figure 1 demonstrates the linearity of the Boyle-van’t Hoff relationship (volume vs. l/osmolality) for these values. Extrapolation to the yintercept yields a nonsolvent volume of -38%. RVD. Figure 2 demonstrates the effect of continued hypotonic stress on hepatocyte suspensions for all combined samples in HEPES buffer (n = 53). Cells swelled rapidly (within 1 min) as near-perfect osmometers (relative volume = 1.44 t 0.08), and then over a period of -20 min they underwent RVD back toward the resting (isotonic) level (1.16 t 0.05, 64% RVD). Figures 3-5 and 6B depict the effects of various ion substitutions and inhibitor administrations on the volume regulatory responses of hepatocyte suspensions during continued hypotonic stresses; each experimental manipulation is compared with controls performed on the same suspension. Addition of HCOZ to the medium (i.e., KRB) had no effect on RVD (Fig. 6B). However, replacement of NaCl with KC1 (65 mM) in the hypotonic buffer

l.O&

B

0

.I 10

! 1.0 20 T'."

l.ST

-1.4 1.3 1.2

1.2-e

1.1 l.Ob

1.6 HEPES

G293

IN HEPATOCYTES

0

(n-53)

C

1.5

1. 70

!- 1.0 20

T

T

1.4

-1.4

1.3

Control

1.2

1.06 0

TIME (minutes)

2. Effect of hypotonic stress on rat hepatocyte volumes. Suspensions of rat hepatocytes were exposed to hypotonic HEPES buffer at t = 0 min and median cell volumes measured at t = 1, 5, 10, and 20 min after exposure (n = 53 experiments). FIG.

1.6

TIME (minutes)

3. Replacement of NaCl by KC1 in hypotonic buffer. NaCl in hypotonic buffer was replaced by equimolar (65 mM) KCl, and serial volume measurements were made. Results are compared with control cells from the same suspension that were resuspended in NaCl-containing hvnotonic HEPES. Values are means t SD of 9 experiments. FIG.

1.5

I. 10

-4.3

, ,

1.2

! 1.0 20

TIME (minutes) FIG. 4. Effects of K+ transport inhibitors on regulatory volume decrease (RVD). Hepatocyte suspensions were exposed to hypotonic HEPES buffer containing 0.5 mM quinine (A), 1.0 mM barium (B), or 0.1 mM bumetanide (C). Inhibitors were incorporated into the hypotonic buffers only. Control experiments were those in hypotonic buffer without inhibitors. Values are means t, SD of 6 experiments each for barium and quinine and 4 experiments for bumetanide and controls.

completely abolished this regulatory volume change (Fig. 3). Incorporation of the K+-channel blockers, barium (1 mM) or quinine (0.5 mM), into the hypotonic buffer significantly inhibited RVD at all measured time points after the initial swelling, whereas the loop diuretic bumetanide (0.1 mM) had no significant effect on the regulatory volume response (Fig. 4). When added to the isotonic suspensions, none of these agents affected basal cell volume measured over a 20-min period (results not shown). The anion transport inhibitor DIDS had an apparent dose-dependent effect on RVD (Fig. 5). Compared with controls at t = 20 min, 0.05 mM DIDS inhibited the regulatory response to hypotonic shock by -25% (although this did not reach statistical significance),

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MECHANISMS

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A .5 mM DIDS

IN

HEPATOCYTES

HYPERTONIC

1.20-

-1.6 0

W

0.90

E 4

0.80

k! 0.70 10 0.60

6I

!

*1

TIME (minutes) FIG. 5. Effect of DIDS on regulatory volume decrease (RVD). Hepatocyte suspensions were exposed to the anion transport inhibitor DIDS at two different concentrations (0.05 and 0.5 mM) in isotonic HEPES buffer for 15 min prior to resuspension in DIDS-containing hypotonic HEPES. Controls were exposed to an additional 15 min in isotonic HEPES prior to resuspension in hypotonic HEPES without addition of inhibitors. Values are means & SD for 3 experiments each.

whereas 0.5 mM DIDS caused almost complete (-90%) inhibition of RVD (Table 2). DIDS had no effect on basal cell volume over a lo-min period. To determine the contribution of Cl- to the anion dependency of RVD, hepatocytes were depleted of intracellular Cl- by incubation for 20 min before hypotonic shock in Cl-free isotonic HEPES. Chloride depletion had a number of effects on hepatocyte volume. 1) Cells incubated in Cl--free isotonic HEPES buffer for 20 min shrank to 80% of control values (5,849 t 261 vs. 7,078 t 431 pm3; P < 0.01). 2) With hypotonic shock, Cl-depleted cells swelled to 1.56 t 0.05 times their isotonic size compared with a factor of 1.39 t 0.06 in controls (P < 0.01). 3) Cl- depletion resulted in a partial (40%) inhibition of RVD (Table 2). To assess the Ca2+ dependence of RVD, hepatocytes were suspended in Ca2+- free isotonic HEPES buffer containing 1 mM EGTA for 10 min prior to hypotonic shock. As shown in Table 2, Ca2’ deprivation had no effect on the degree of cell swelling or on RVD. Table 2 summarizes the effects of the aforementioned interventions expressed as percent inhibition of control RVD at 20 min of exposure to hypotonic buffer. Table 2 also demonstrates that substitution for Na+ by choline, ouabain (1 mM) treatment, and amiloride (1 mM) incorporation had no significant effects on RVD when added to the hypotonic buffers. RVI. Exposure of hepatocyte suspensions to hypertonic HEPES or KRB resulted in shrinkage within 1 min in accordance with the medium osmolality (Figs. 1 and 6A). However, no RVI was observed for periods up to 60 min of incubation in the medium made hypertonic with ZOO mM sucrose. In contrast, cells that had first undergone hypotonically induced RVD for 20 min and were then reexposed to isotonic KRB (relative hypertonic stress) did exhibit a significant increase in volume from 5,121 t 461 to 6,004 t 420 pm3 (relative volume 0.77 t 0.17 to 0.91 t 0.20, Fig. 6B). RVI was inhibited by the Na+-H+ exchange inhibitor amiloride (1 mM) or when choline was substituted for Na’. The volume re-

TIME

B

(minutes)

2.0

W

z

1.5

3 9

0.5

7-

0

I I

10

I 1

1 I

20

30

TIME

1 I

40

50

(minutes)

6. Effect of absolute and relative hypertonic stresses on rat hepatocyte volumes. A: absolute hypertonic stress; hepatocyte suspensions were maintained in isotonic KRB buffer at 37°C for 30 min and then resuspended in KRB made hypertonic by the addition of 200 mM sucrose to isotonic medium. Values are means t SD of 8 experiments. B: relative hypertonic stress; hepatocytes were exposed to hypotonic KRB at t = 0 min and serial volume measurements made as in prior RVD experiments; at t = 20 min the medium was changed back to isotonic (relatively hypertonic) KRB and serial volume measurements were made for an additional 30 min. Values are means t SD of 16 experiments. FIG.

covery was also inhibited, but to a lesser degree, when experiments were performed in the absence of HCO: or when cells were depleted of Cl- (Fig. 7; Table 3). Table 3 also demonstrates that the anion exchange inhibitor DIDS did not inhibit RVI. In fact, RVI at 30 min after relative hypertonic stress was augmented by ~80% in the presence of 0.5 mM DIDS (see NOTE ADDED IN PROOF). Ouabain had no significant effect on RVI. DISCUSSION

The present study demonstrates that isolated rat hepatocytes undergo a spontaneous RVD when exposed to continued hypotonic stress. RVD is prevented by increasing extracellular K+ concentration ([K+]) and is blocked by barium and quinine but not by bumetanide. These findings suggest that RVD is mediated, in part, by efflux of K+ from the liver cell via a conductive pathway rather than by a cotransport mechanism. The cellular loss of K+ in response to hypotonic stress is necessarily

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MECHANISMS

OF

VOLUME

REGULATION

2. Effects of ion substitutions and inhibitors of ion transport on maximal regulatory volume decrease TABLE

Condition HCO; containing 65 mM K,,, DIDS (0.5 mM) Quinine (0.5 mM) Cl- free Barium (0.1 mM) DIDS (0.05 mM) Ouabain (1 mM) Na’ free Amiloride (1 mM) Bumetanide (0.1 mM) Ca2+ free

% RVD,,,

n

16 59.9k8.6 9 -7.5t24 3 6.3t16.2 6 21.329.6 4 38.7k4.2 6 50.0t8.7 4 45.4k8.6 3 51.6t8.4 3 55.7tlO.O 3 64.9tl.O 4 54.0t8.8 9

64.2t7.7

% RVLmtro~ 64.1k9.0 55.3t8.4 6O.lk4.2 60.6k2.9 62.5k6.1 72.3t7.2 58.825.3 60.5t2.4 61.7210.3 68.026.5 56.Ok8.0 65.3t9.0

%Inhibition 113.7 89.5 64.8 37.6 30.3 22.3 14.7 8.1 3.3 1.9 1.7

P

Effects of osmotic stresses on isolated rat hepatocytes. I. Ionic mechanisms of cell volume regulation.

Isolated hepatocyte suspensions were exposed to hypotonic and hypertonic stresses and serial cell volume measurements were made with an electronic par...
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