Contraction coupling in colonic smooth muscle.

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1992. 54:395-414

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CONTRACTION COUPLING IN COLONIC SMOOTH MUSCLE E. A. Mayer and X. P. Sun Department of Physiology and Medicine, UCLA and VA Wadsworth Medical Center, Los Angeles, California 90024 R. F. Willenbucher Department of Medicine, Harbor-UCLA Medical Center, Torrance, California 90509 KEY WORDS:

patch clamp, ion transport, Ca2+ signaling, sarcoplasmic reticulum

INTRODUCTION Contracation coupling in smooth muscle refers primarily to the membrane and intracellular events that mediate a change in the intracellular calcium concen tration ([Ca2+]j) sufficient to modulate the activity of contractile proteins. Extracellular stimuli, which result in contraction coupling, can be divided into receptor activation, membrane depolarization, and stretch of the plasma membrane. Receptor activation results from interaction of plasma membrane receptors with neuromessengers released from myenteric motor neurons, or with paracrine or endocrine substances. Receptor-independent membrane depolarization can result via electrotonic spread from neighboring myocytes (l08, 109) or from interstitial cells of Cajal (13, 109). Stretch of the plasma membrane can result in the activation of stretch-activated membrane chan nels, which results in membrane depolarization (77). Increases in free [Ca2+]j in response to any of these stimuli cause force generation by stimulating myosin light chain phosphorylation and by directly increasing the myosin ATPase activity (120, 121). Alternative mechanisms of force generation, such as latch-bridge formation, are not a subject of this discussion and have been reviewed elsewhere (4 2). Similar to other excitable cell types, the 395

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MAYER, SUN & WILLENBUCHER

excitation of smooth muscle can be divided into mechanisms that mediate influx of extracellular Ca2+ through membrane pathways and release of Ca2+ from intracellular stores (60, 120, 121). The former, electro-mechanical coupling (EMC), is associated with changes in membrane potential. The latter, pharmaco-mechanical coupling (PMC), does not involve potential changes. The contribution of EMC and PMC to activation of smooth muscle differs between agonists and between different types of smooth muscle (16, 26). In both EMC and PMC, the temporo-spatial changes in [Ca2+]j, and therefore the duration of contraction, is crucially dependent on the activity of Ca2+ pumps in the SR membrane and the sarcolemma (25, 121). The use of the patch-clamp technique to study ion channels and their regulation in single, isolated myocytes has made it possible to characterize the individual plasma membrane conductances involved in EMC and PMC. Imaging techniques using Ca2+ -sensitive dyes to monitor spatio-temporal patterns of [Ca2+]j have revealed the relative contribution of extra- and intracellular Ca2+ pools and of different intracellular Ca2+ compartments to contraction coupling. In this review, we draw on published reports from all types of smooth muscle, but emphasize the results obtained in myocytes from the colon. We discuss (a) mechanisms of EMC and PMC, (b) regulation of [Ca2+]j, (c) mechanisms of inhibition of excitation-contraction (EC) coupling, and (d) interactions be tween a, b, and c in the intact tissue. ELECTRICAL ACTIVITY OF COLONIC SMOOTH MUSCLE Colonic smooth muscle displays rhythmic variations in membrane potential in the form of slow waves (109), which are similar to intact smooth muscle from other parts of the gastrointestinal tract. In circular mammalian colonic muscle from different species, the configuration of the slow wave is similar to spontaneous electrical activity generated by the gastric muscle, which in volves an upstroke potential and a plateau potential (28, 109, 127). As in gastric muscle, EC coupling occurs during the plateau phase of the slow wave in the absence of superimposed spike potentials. Agonists such as acetylcho line or substance P stimulate contractile activity by increasing the size (pri marily the duration) of the plateau potential (109). The longitudinal muscle layer of the mammalian colon generates bursts of membrane potential oscilla tions that are superimposed on slow membrane depolarizations (44, 55, 62). Oscillations within a burst increase in amplitude, and spike potentials develop at the peak of oscillations (44, 55, 62). EC coupling occurs during bursts of membrane potential oscillations. Agonists of contraction increase the frequen cy of bursts (44, 62), but not the configuration of the burst. Based on the difference in slow wave pattern and the effect of agonists on slow wave

SMOOTH MUSCLE EXCITATION

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activity, one would predict significant differences between the two muscle layers in receptor-mediated EC coupling.


ELECTRO-MECHANICAL COUPLING The membrane mechanisms regulating [Ca2+]j in gastrointestinal smooth muscle in response to membrane depolarization involve voltage-sensitive Ca2+ channels (VSCC) (32, 88, 94, 116, 135, 147), Ca2+-activated K+ channels (10, 31, 35, 94, 116, 118, 135), and slow voltage-sensitive K+ channels (7, 84). The mechanisms by which receptor occupation results in membrane depolarization sufficient to activate VSCCs remain controversial (20). The closing of a resting K+ conductance (the M current) (115, 117), the activation of nonselective cation currents (9, 66), the opening of fast Na + channels (100), and the activation of Cl- channels (2, 37, 48, 126) have all been described in different types of smooth muscle. Currently it remains

unclear if these proposed mechanisms for initiation of cell depolarization reflect differences in species, smooth muscle type, region within the gastroin testinal tract, or experimental conditions. Influx of Ca2+ through plasma membrane pathways could play a role in EC coupling if Ca2+ fluxes during slow waves raise [Ca2+]j to a level sufficient to activate contractile proteins, or agonists increase [Ca2+]j currents to such levels. Agonists could increase Ca2+ currents by either directly modulating the Ca2+ channel or by inhibiting channels involved in repolarizing the plasma membrane.

Membrane Pathways Mediating

ccl+

Influx

In smooth muscle, the principal membrane pathways mediating Ca2+ flow from the extracellular space to the cytosol include VSCCs and possibly receptor-operated Ca2+ channels (ROCs). Currents through VSCCs have been described in myocytes from the mammalian esophagus (116), stomach (94), small intestine (147), from the toad stomach (32, 135), and from both muscle layers of the colon (82, 83, 147). The properties of macroscopic currents and the sensitivity to dihydropyridines and other organic and inorgan ic blockers indicate that the reported currents are similar and appear to flow primarily through L type VSCCs. Several reports using whole cell recordings suggest the presence of different types of Ca2+ channels in gastric (32, 135), small intestine (147), and circular (82), but not longitudinal, colonic smooth muscle (88, 136). In myocytes from taenia caeci, both L and T types of VSSCs were described in single channel recordings (147). Reported activa tion thresholds for currents through VSCCs are -40 mV, and peak currents have been obtained around 0 mV (83, 88). Both voltage- and Ca2+ -mediated inactivation of Ca2+ currents have been reported (135, 147). Similar to �


398


reported properties of VSCCs in other excitable tissues (93),currents through T type Ca2+ channels are only available from resting membrane potential more negative than -80 mV ( 147). Based on the reported resting membrane potentials for colonic muscle and its regional variations within muscle layers (109),it can be concluded that Ca2+ currents may not be fully available at the resting membrane potential (20). For example, resting membrane potential values between -50 mV and -63 mV have been reported for the longitudinal muscle layer (54, 1 19). The value for the circular muscle layer varied between -78 mV and -43 mV (119) with the distance from the submucosal surface. These findings suggest that currents through T type Ca2+ channels may only play a role in certain parts of the mammalian colon (see below). Reported overlap of the activation-inactivation curve in the potential range between -40 and -20 mV in the form of a window current ( 137) is consistent with Ca2+ influx during the plateau phase of the slow wave in the absence of Ca2+ spikes. Similar to reports from other excitable tissues, currents through VSCCs in smooth muscle can be modulated via receptor-mediated mech anisms (5, 12,27,32,49,61,88,93, 146). Reported mechanisms for such modulation include a change in the open probability of channels at a given voltage, andlor a change in the voltage-dependence of the channel. In am phibian gastric muscle, acetylcholine, substance P, and analogues of di acylglycerol were found to increase the current and slow the decay of the current through L type Ca2+ channels (32). In the longitudinal muscle layer of the rabbit colon, the NK-l receptor agonist, substance P methylester, was also found to have a stimulatory effect on dihydropyridine-sensitive Ca2+ currents (88),whereas no effect of acetylcholine on VSCCs in small intestinal muscle was seen (8). It remains to be determined if VSCCs in colonic smooth muscle are modulated by protein kinases and by direct G protein coupling, as reported in other excitable tissues (27, 58, 146). The presence of receptor-operated calcium channels (ROCs) in smooth muscle has been postulated on the basis of whole tissue studies (19,92,132, 133, 145). To qualify as a ROC, a channel must exhibit a high permeability for Ca2+ and the ability to be activated without second messenger mediation or voltage changes ( 19). In vascular smooth muscle, a receptor-operated Ca2+-permeable channel, which is activated by external ATP in cell free patches, has been described (11). This 5 pS channel has a selectivity for Ca2+ over Na+ of 3: 1. Currently, no single channel with similar properties has been identified in colonic smooth muscle. In different types of smooth muscle, an ATP-activated membrane nonselective cation conductance, which results in membrane depolarization in response to purinergic receptor activa tion, has been described (2,11,48,65, 98). In myocytes from the longitudi nal muscle layer of the guinea pig ileum, an acetylcholine-activated cation channel with a single channel conductance of 25 pS and poor discrimination


399

between Na+ and K+ has been described (67). Even though it is clear that the ATP- and acetylcholine-activated channels represent different conductances, it remains to be determined if the nonselective cation channels are the basis for earlier findings in intact tissues that lead to the concept of ROCs (see below).


Membrane Pathways Resulting in Initial Depolarization In toad gastric muscle, both substance P and acetylcholine depolarize the cell by suppressing a resting K+ conductance, the M current (32, 115, 117). Muscarinic agonists have been shown to suppress this current, whereas J3-adrenergic agonists, which inhibit gastric smooth muscle contraction, in creased the current (115). These findings could not be reproduced in other types of smooth muscle and may be unique to non-mammalian smooth muscle (20). It remains to be determined if the resting K+ conductanc(; reported in different types of visceral smooth muscle is related to the M current. The single channel conductance underlying both currents has not been character ized and is probably too small to be detected in patch-clamp recordings (20). Suppression of spontaneous transient outward currents by muscarinic ago nists in esophageal (116) and intestinal (8) myocytes has been suggested as a mechanism for initial cell depolarization. These outward currents (different from the M current), which have been observed only in partially depolarized visceral myocytes during whole cell recordings, appear to represent the synchronous opening of several Ca2+-activated K+ channels (8). However, Ca2+-activated K+ channel activity is not observed in cell-attached recordings of unstimulated myocytes at the resting potential (10, 87, 88) so that it is unlikely that these channels play a physiological role in initial cell depolariza tion. Nonetheless, the reported transient initial activation of these channels following receptor-mediated cell activation (8, 34, 87,116) could play a role in Ca2+ channel regulation. As discussed above, Ca2+ currents, in particular those through T type channels, are only partially available at the resting potential and may be inactivated at the low resting potential reported for the myenteric border of circular muscle (119). The transient initial hyperpolariza tion associated with activation of Ca2+-activated K+ channels by agonists of contraction could make the cell potential sufficiently negative to increase the availability of Ca2+ current. If inward and outward currents overlap, such initial hyperpolarization may not be seen during the action potential. The possible role of Ca2+ -activated K+ channels in the regulation of the slow wave is discussed below. Activation of nonselective cation conductances has been reported in rat vas deferens (48), guinea pig urinary bladder (65), guinea pig ileum (66), and longitudinal colonic muscle (89). Whole cell recordings have shown that in guinea pig ileum, the acetylcholine-activated current results in membrane


400


depolarization. The underlying channel is coupled to a G protein (9, 66, 97). The properties of this conductance include a bell-shaped voltage activation curve, with increasing activity from the resting potential to 0 mV, and decreasing activity at membrane potentials more positive than 0 mV and more negative than the resting potential (65). After activation by a G protein, this type of voltage-dependence would result in a positive feedback effect on membrane depolarization within the physiological range of membrane poten tials. Because of the high intracellular chloride concentration ([Cl-h 30-40 mM) of smooth muscle (24), activation of Cl- channels at the cell resting potential would result in membrane depolarization. Cl- currents have been described in vascular smooth muscle (2, 30), anococcygeus muscle (29), and more recently in esophageal and colonic muscle (126). In rabbit ear artery, noradrenaline depolarizes myocytes by activating both a cation conductance and a Cl- conductance (2). In the longitudinal colonic muscle, the con ductance is a Ca2+-insensitive maxi Cl- channel with a bell-shaped voltage activation curve, which is activated by substance P methyl ester and GTPyS in cell free membrane patches (126). =

Membrane Pathways Regulating ccl+ Influx In addition to Ca2+-induced inhibition of Ca2+ channels and Ca2+ channel modulation by receptor-mediated mechanisms, membrane repolarization plays a major role in regulating voltage-sensitive Ca2+ influx (27 , 32, 49, 53, 61). Membrane repolarization results primarily from the activation of K+ channels by voltage- and/or cytosolic calcium. 2+ CA -ACTIVATED K+ CHANNELS

Ca2+-activated K+ currents and delayed rectifier currents have been characterized in myocytes from different regions ofthe gastrointestinal tract (94, 116, 118, 135), including the circular (31,35) and longitudinal (87) layers of the mammalian colon. In all types of gastroin testinal smooth muscle, including colonic muscle, the dominant current regulating the duration of voltage-sensitive Ca2+ influx appears to be outward current through Ca2+-and voltage-sensitive K+ channels (31, 35, 87, 88, 94, 116, 118, 135). The characteristics of these channels reported from several tissues are similar and include a single channel conductance around 200 pS in high symmetric K+ concentrations, low opening probability in response to voltage at cytosolic [Ca2+] below 1O-6M, and regulation by agonists of contraction (8, 34, 87, 88, 136). Studies using whole cell recordings in myocytes from the mammalian circular and longitudinal muscle layers sug gest a difference in the potential threshold for activation of Ca2+-activated K+ currents between the two muscle layers (35, 90, 136). The more positive



401

activation threshold found in longitudinal muscle, and therefore the lesser degree of feedback inhibition on Ca2+ currents, could explain the presence of spike potentials in longitudinal myocytes (136). There is currently no evi dence for Ca2+-activated K+ channels of smaller conductance in colonic smooth muscle (31, 89). The synchronous opening of multiple Ca2+-activated K+ channels may underlie the spontaneous transient outward currents (STOCs) observed in a variety of visceral smooth muscles (8, 116). Syn chronous periodic opening of several Ca2+-activated K+ channels in cell attached recordings in response to threshold doses of substance P have been observed in myocytes from the longitudinal muscle layer of the colon (87). Higher doses of the peptide resulted in inhibition of the channels, similar to the muscarinic suppression of STOCs in esophageal (116), small intestinal (8), and circular colonic cells (34) following transient stimulation. Suppres sion of Ca2+-activated K+ currents may be a common mechanism by which agonists of smooth muscle can increase the duration of the plateau phase of the action potential ( l09). If agonists only increased voltage-sensitive Ca2+ currents, the resulting more rapid activation of Ca2+-activated K+ currents would shorten the plateau phase. The intracellular pathways responsible for receptor-mediated modulation of these channels in colonic muscle are not known, but may involve direct G protein coupling (36, 46, 136), phosphorylation by PKC (32, 75), or a combination of both mechanisms. It is currently unclear if antagonists of colonic smooth muscle contraction, such as ,B-adrenergic agents, can increase the open probability of these channels under physiological conditions (see below).

CA2+ -INSENSITIVE K+ CHANNELS Other K+ currents reported in visceral smooth muscle include a 50 pS, voltage-sensitive channel with slow kinetics (7), a 23 pS outwardly rectifying channel activated by fatty acids (101), a TEA- and Ba2+-insensitive current with fast kinetics, and an apamin-sensitive channel that is activated by ATP and the nonadrenergic, noncholinergic inhibitory neurotransmitter (20). Since the precise role of these conductances in EC coupling in gastrointestinal muscle is not known, the properties of these channels will not be discussed here and the interested reader is referred to the references listed above. A model summarizing the interactions of plasma membrane ion channels in the regulation of Ca2+ entry is shown in Figure IA. This model emphasizes the central role of Ca2+ flux through VSCCs in electro-mechanical coupling in colonic smooth muscle. Voltage-sensitive Ca2+ influx is initiated by opening of G protein-coupled cation and/or anion channels. It is limited by feedback mechanisms involving direct Ca2+ inhibition of VSCCs and via activation of Ca2+- and voltage-activated K+ channels, which result in repolarization.

402


AGONIST


RECEPTOR

CCCHANNEL

AGONIST RECEPTOR

Figure 1

Mechanisms involved in electromechanical coupling

spatio-temporal patterns of [Ca2+],

(1B). A.

(1A),

and in the generation of

Ca2+ influx through L and T type voltage-sensitive

calcium channels (VSCCs) plays a central role in EC coupling. The right side shows the proposed mechanisms involved in initial activation of VSCCs (G protein-coupled anion and/or cation channels). The left side shows the main mechanisms involved in feedback inhibition of current through VSCCs: direct Ca2+ inhibition, Ca2+ -activated K+ (Kca) channels, and slow, de polarization-activated K+ (K,low) channels. temporal [Ca2+]; patterns.

Upper part:

B.

Proposed model for the gel'''ration of spatio

IPrsensitive regions of the SR in one cell pole release

Ca2+ in response to plasma membrane receptor activation. The released CaH diffuses to Ca2+ -sensitive SR regions located along the plasma membrane, which results in propagated, regenerative Ca2+-induced Ca2+

release (14, 15). Lower part:

Sub-plasmalemmal [CaH],

gradients arise from spontaneous SR Ca2 + release and reuptake (121). For further detail, see text.


403


PHARMACO-MECHANICAL COUPLING PMC was first postulated based on the observation that a variety of intact smooth muscle preparations can generate force in depolarizing solutions and in the absence of extracellular Ca2+ (1, 21, 23, 40, 81, 123). Two basic mechanisms are responsible for PMC: (a) release of Ca2+ from intracellular stores by receptor- or voltage-mediated mechanisms, and (b) modulation of the Ca2+ sensitivity of enzymes crucial to the activation of the contractile apparatus.

Ca2+ Release from Intracellular Stores The intracellular organelles identified as the primary Ca2+ storage and release sites in gastrointestinal smooth muscle are the junctional and central sarcoplasmic reticulum (SR) (40, 120, 12 1). The total [Ca2+] within these stores has estimated to be 30-50 mmollkg dry weight, several orders of magnitude higher than the reported values of 80-200 nM for the cytosolic [Ca2+] (121). The [Ca2+] gradient between SR and cytosol is maintained by active uptake of Ca2+ from the cytosol by the SR Ca2+-ATPase and by buffering of SR Ca2+ with high affinity Ca2+-binding proteins such as calsequestrin (124, 142). At least four mechanisms have been described by which cell activation can result in Ca2+ release from intracellular stores in smooth muscle: activation of the SR Ca2+ release channel by receptor induced inositol 1,4,5 trisphosphate (IP3) generation (16, 104, 143); 2 Ca2+ release by GTPI'S (79); Ca2+ -induced CA2+ release (64); and voltage induced Ca2+ release (74, 78, 120). Recently, direct evidence for a ryanodine receptor-Ca2+ release channel in toad gastric muscle was reported (143). This channel appears to have properties similar to the Ca2+ release channel in skeletal and cardiac muscle (47) and, when incorporated into lipid bilayers, can be activated by Ca2+, ATP, and IP3 (143). Even though all the above listed mechanisms for receptor-mediated SR Ca2+ release have been de scribed in different smooth muscle preparations, their role in the receptor mediated activation of colonic muscle is incompletely understood. Based on the small fraction of the cell that contains SR structures (0.5-7.5%) and the non-homogeneous distribution of the SR (junctional and central), one would expect non-homogeneous distribution of free [Ca2+]i following cell activa tion. Such non-homogeneities of free Ca2+ distribution have been reported from a variety of cell types, including myocytes from the toad stomach (140, 141) and the rabbit colon (79, 90). In the rabbit colon, regions of increased levels of free [Ca2+h (at least tenfold higher than elsewhere in the cytosol) were found not only in the sub-plasmalemma! space, but also preferentially in one cell pole (85, 90). Two different hypothesis have been proposed to explain these sub-plasmalemmal gradients: spontaneous release and reuptake


404


from the junctional SR (1 21, 1 25), or cycling of Ca2+ through the plasma membrane via influx through VSCC and extrusion by the Ca2+-ATPase (106). In longitudinal colonic muscle, sub-plasmalemmal [Ca2+]j gradients were maintained in depolarizing solutions and in the presence of La3+, arguing against the latter hypothesis (85, 90). Subcellular [Ca2+]j gradients have significant implications for excitation coupling in smooth muscle. Given the different chemical pathways that can mediate SR Ca2+ release and the topographically different intracellular stor age and release sites, site-specific patterns of [Ca2+]j changes and contraction could be generated by differ�nt agonists. Indeed, substance P and acetylcho line, both of which stimulate colonic muscle contraction, were found to generate different spatial patterns of [Ca2+]j changes in colonic myocytes (85, 90). Furthermore, isolated contraction of cell poles in the absence of general ized contraction has been described (85, 90, 1 40). Subcellular [Ca2+]j gra dients have been suggested as an alternative model to the latch-bridge theory for maintained force generation in the absence of elevated average [Ca2+]j (106). The specific spatial arrangement of IP3 and Ca2+ -sensitive release sites along the length of the cell may enable the cell to generate propagated Ca2+ waves (4, 15). For example, if Ca2+i release is initiated by receptor-mediated IP3 generation in one cell pole ( 15), the Ca2+ signal could be propagated along the length of the cell by a regenerative mechanism involving diffusion and Ca2+-induced Ca2+ release (4, 45). A model summarizing the proposed mechanisms for the generation of sub-plasmalemmal [Ca2+]j gradients and of Ca2+ waves is shown in Figure l B. The finding of regional gradients of high [Ca2+]j, such as the sub-plasmalemmal space, may have important im plications for the regulation of Ca2+ -sensitive pathways in the plasma mem brane. These implications may differ considerably from conclusions based on averaged cytosolic [Ca2+]. For example, the cytosolic side of the plasma membrane and sub-plasmalemmal organelles may be exposed to [Ca2+]j of up to several milimolar, concentrations that have been considered unphysiolog ical in the past. These considerations are pertinent to the regulation of Ca2+ -activated K+ channels, Ca2+ -dependent inhibition of Ca2+ channels, and to the process of Ca2+-induced Ca2+ release. Correlations of Ca2+ fluxes to changes in [Ca2+]j and cell activation (6, 83 ) may have to be reassessed since averaged calcium signals obtained from the whole cell do not take into account subcellular [Ca2+]j gradients (see below). In a variety of cell types, including colonic smooth muscle, spatial and temporal variations of [Ca2+Jj gradients have been reported (14, 33). Similar to recent reports of distinct spatio-temporal [Ca2+]j patterns in cardiac myocytes (128), neurons and glial cells (38, 59), and secretory cells (107, 13 1), spontaneous [Ca2+]j oscillations and propagation of [Ca2+]j gradients along the longitudinal axis of the cell in the form of Ca2+ waves have been

SMOOTH MUSCLE EXCITATION reported in longitudinal colonic muscle

405

(90). The oscillations in colonic 10 and 20 cpm, are present in

myocytes have a frequency varying between

depolarized cells, and are only partially dependent on influx of extracellular Ca2+ through VSCCs. Agonists such as carbachol and substance P increase the amplitude of oscillations without affecting their frequency. In these cells,

[Ca2+]i gradients were found to propagate along the longitudinal cell axis in the form of Ca2+ waves, with a propagation speed of

20 /Lm per sec. Given


that the subcellular [Ca2+]i during the peaks of oscillations is in the micromo lar range, both activation of Ca2+ -sensitive membrane pathways and EC coupling could occur during spontaneous Ca2+ oscillations. Thus in longitu dinal colonic muscle, [Ca2+]i oscillations generated by a cytosolic oscillator

(85, 90) could function as a pacemaker for slow wave events. It is intriguing to speculate that propagation of these Ca2+ waves through gap junctions, located preferentially at the poles of longitudinal myocytes (56), could represent the underlying mechanisms for slow wave propagation in longitu dinal smooth muscle. The intracellular mechanisms responsible for [Ca2+]i oscillations in colonic smooth muscle remain to be determined (5 7).

Ca2+ -Uptake Mechanisms and Modulators of Ca2+ Sensitivity In addition to the mechanisms that lead to an increase in [Ca2+]i by increased influx or increased release, other receptor-mediated factors play a role in contraction coupling. These can be broken down into mechanisms related to

Ca2+ uptake and sequestration and mechanisms modulating the sensitivity of contractile proteins to a given [Ca2+]i ( 25, 71, 1 21). Cytosolic [Ca2+] is maintained between 80 and 200 nM by the activity of the Ca2+ -ATPase of the SR and plasma membrane and by the Na+-Ca2+ exchanger ( 25, 1ll, 1 21). Increased [Ca2+]i levels increase the rate of Ca2+ uptake (6), in part mediated by the stimulatory effect of the Ca2+-calmodulin complex on Ca2+-ATPase activity ( 25, Il l ). In addition to regulation by protein kinases, the Ca2+ pump in smooth muscle may also be regulated by membrane potential (5 2). The observation that smooth muscle may generate different amounts of force at the same [Ca2+L suggests that Ca2+-sensitizing and desensitizing mechanisms may exist (71, 121). Indirect evidence suggests a role for PKC in this process, possibly via phosphorylation of a protein phosphatase inhibitor

(1 2 2).

INHIBITION OF EC COUPLING Inhibition EC coupling, i.e. smooth muscle relaxation, occurs under three circumstances: relaxation resulting from withdrawal of the contractile stimu lus; relaxation despite the continued presence of the contractile agonist, as in the case of phasically contracting muscle; and relaxation mediated by an

inhibitor of contraction in the continued presence of the contractile agonist,


406


involving the generation of an intracellular second messenger (i.e. cyclic nucleotide, arachidonic acid metabolite). The first two mechanisms involve intracellular mechanisms for Ca2+ extrusion and for the generation of tempo ral variations in [Ca2+]i' and were discussed above. Even though other protein kinases are likely to be involved in the regulation of smooth muscle relaxation (110), the following section will focus on cAMP-mediated mech anisms mediating relaxation in the presence of agonists. Cyclic AMP is thought to be a major second messenger mediating relaxation in a variety of gastrointestinal smooth muscles (17, 1 29) including colonic muscle (86, 138), and may mediate the inhibitory effect of VIP (17,1 29,138) and CGRP (70, 86) in these tissues. In contrast to vascular and tracheal smooth muscle (63, 69, 96, 114), there is currently little evidence that guanylate cyclase/cGMP plays an important role in gastrointestinal smooth muscle (95, 1 29), and to date no known endogenous mediator of relaxation in the gastrointestinal tract has been shown to act through the activation of guanylate cyclase. Even though the physiological significance remains to be defined, reported effects of cAMP on smooth muscle can be divided into three levels of regulation: regulation of [Ca2+];, modulation of the sensitivity of the contractile appara tus to existing [Ca2+]io and inhibition of phosphoinositol hydrolysis. Mech anisms by which cAMP could lower [Ca2+]i are a decrease in influx of extracellular Ca2+ and an increase in sarcolemmal transport and/or in tracellular sequestration (11 2, 113, 144). As discussed above, channel phosphorylation by protein kinase A (PKA) could result in a decreased open probability of Ca2+ channels or an increased open probability of K+ channels. However, the effects of cAMP on membrane potential have been inconsistent (144) and the role of PKA-mediated modulation of membrane pathways regulating Ca2+ influx in the inhibition of colonic smooth muscle remains to be defined. In toad gastric muscle, adrenergic agonists increase the open probability of the K+ conductance underlying the M current (115). In longitu dinal colonic muscle, no effect of cAMP on dihydropyridine-sensitive current through VSCCs, or on Ca2+ -activated K+ channels was observed (80). An alternative mechanism to lower [Ca2+]; in smooth muscle involves PKA mediated activation of Ca2+ transport mechanisms in the plasma membrane or in the SR membrane. Evidence has been presented to suggest that cAMP stimulates the sarcolemmal Na+-K+-ATPase and the Na+-Ca2+ exchanger (11 2, 113). The relative contribution of different protein kinases to the physiological regulation of the Ca2+ pump remains to be established. Evi dence from studies performed in vascular muscle suggests a role for cGMP, but not for cAMP in the regulation of this pump (51). In addition to its effect on the plasma membrane, PKA has also been shown to promote SR uptake of Ca2+ via phosphorylation of phospholamban, an SR membrane regulatory protein of the SR Ca2+ -ATPase (43,105). PKA activation may also result in a



407

modulation of the sensItIvIty of contractile proteins to a given [Ca2+]i. PKA-mediated phosphorylation of myosin light chain kinase (MLCK) may inhibit action-myosin interactions presumably by impairing the phosphoryla tion of the myosin regulatory subunit (39). Phosphorylation at the A site decreases the affinity of MLCK for calcium-calmodulin (39, 73). The physi ological role of PKA-mediated MLCK phosphorylation of smooth muscle remains to be determined and may differ between different types of smooth muscle. In the circular muscle of the rabbit colon, VIP released by electrical field stimulation and intracellularly generated cAMP cause rapid relaxation of contracted circular muscle (139). Preliminary work using caged cAMP in intact tissue suggests that modulation of the Ca2+ pumps of sarcolemma and possibly the SR membrane plays an important role in cAMP-induced relaxa tion (139). In contrast, PKA-mediated modulation of the Na+ pump (139), sarcolemmal K+ conductances (80), or modulation of the MLCK (139) do not seem to play a critical role. INTERACTION OF ELECTRO-MECHANICAL AND PHARMACO-MECHANICAL COUPLING MECHANISMS Several questions regarding the relative contributions of EMC and PMC to receptor-mediated contraction and the interactions of these mechanisms re main unresolved. Contrary to earlier assumptions that the small fractional volume of the SR in smooth muscle cells limits the amount of releasable Ca2+, guinea pig portal vein, which contains only 2% SR, can contract maximally in Ca2+-free solution (22). In contrast to vascular smooth muscle, tissue from the circular and longitudinal muscle layers of the mammalian colon cannot generate full contractions in the absence of voltage-sensitive Ca2+ influx, even though the dependence of contraction coupling on Ca2+ influx appears to vary between muscle from the longitudinal and circular layer (16, 26). In gastric and colonic myocytes, calculated Ca2+ fluxes through VSCCs during depolarization have been compared to the simultaneously measured average increase in [Ca2+]i (6, 83). In gastric myocytes, the estimated influx during a 50 ms depolarization to 0 mV was 40- to 150-fold greater than the observed change in free [Ca2+]i, whereas in circular colonic muscle a 5 sec depolarization to -40 mV was estimated to increase [Ca2+]i by 6.4 JLM (83). Even though both studies did not take into consideration the non-homogeneities of [Ca2+]i distribution, they indicate that voltage-sensitive Ca2+ influx plays a dominant role in contraction coupling, and thus confirm results obtained in whole tissue (86, 91). Using video imaging techniques, substance P and carbachol were found to stimulate sub-plasmalemmal [Ca2+]i in longitudinal colonic muscle in the presence of the dihydropyridine an-


408


tagonist nifedipine, while the average cell [Ca2+]j was markedly decreased from control (130). These results are similar to reports from chromaffin cells, where receptor-mediated increases in [Ca2+]j of similar magnitude, resulting from either extra- or intracellular compartments, have different effects on excitation-secretion coupling (76). Based on reports from other tissues, several possibilities for interaction between EMC and PMC have to be considered. Mechanisms related to PMC could influence voltage-sensitive Ca2+ influx by the following mechanisms: (a) Ca2+ release from the junctional SR may inhibit voltage-sensitive Ca2+ flux (99) and activate Ca2+-sensitive plasma membrane pathways. Similarly, PKC may alter the kinetics of plasma membrane ion pathways (3, 50, 110), and the Ca2+-calmodulin complex can modulate Ca2+ extrusion and reuptake mechanisms (25, 111). Thus by altering membrane potential, Ca2+ release from the SR and activation of protein kinases could modulate EMC. (b) IP3 alone or in combination with IP4 could modulate Ca2+ influx across the plasma membrane via receptor-operated Ca2+ channels (68, 72, 102). Mech anisms related to EMC could influence SR Ca2+ release by the following mechanisms: (a) The increase in Ca2+ from voltage-sensitive influx may inhibit the binding of IP3 to the Ca2+ release channel on the SR membrane and alter the degradation rate of IP3 (103). (b) Voltage-sensitive Ca2+ influx may be involved in loading of the SR, thereby determining the amplitude of IP3 or Ca2+ -mediated Ca2+ release (134). It remains to be determined which of these mechanisms plays a role in excitation-contraction coupling in colonic smooth muscle. FUTURE PERSPECTIVES The recent explosive development of imaging techniques and fluorescent dye indicators will allow the temporo-spatial characterization of subcellular changes in [Ca2+]j ionic composition, intracellular pH, and even second messengers. Important questions to be addressed by these approaches are the mechanisms generating [Ca2+]j oscillations and Ca2+ waves and the correla tion of regional changes in [Ca2+] with cell function. Confocal microscopy will provide three-dimensional characterization of these processes within an individual cell as well as illuminating the mechanisms for intercellular com munication within intact tissues. Characterization of mechanisms underlying contraction coupling in intact tissues will eliminate many of the artifacts introduced by cell isolation techniques and clarify the role of cell-cell com munication, such as the propagation of Ca2+ waves in EC coupling.Com bined with non-invasive electrophysiological techniques, such as the use of voltage-sensitive dyes or the perforated patch technique, advanced imaging techniques will elucidate the correlation of channel regulation and associated


409

subcellular changes in intracellular messengers. Based on the recent reports from different cell types, it is likely that much of the complexity of possible interactions between EMC and PMC can be resolved on the basis of regional regulation at the subcellular level. ACKNOWLEDGMENTS


This work was supported by National Institutes of Health Grant DK 40919.

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Flash photolysis studies of excitation-contraction coupling, regulation, and contraction in smooth muscle.

Defect in colonic smooth muscle contraction in patients with ulcerative colitis.

Developmental changes in agonist-mediated colonic smooth muscle contraction in the rabbit.

Evidence that unphosphorylated smooth muscle myosin supports smooth muscle contraction.

Negative-feedback regulation of excitation-contraction coupling in gastric smooth muscle.

Excitation-contraction coupling in cardiac muscle.

Smooth muscle contraction bands in intestinal infarction.

Excitation-contraction coupling in multiunit tracheal smooth muscle during metabolic depletion: induction of rhythmicity.

Cyclic AMP-mediated regulation of excitation-contraction coupling in canine gastric smooth muscle.

Effects of calcium and calcium antagonists on the excitation-contraction coupling in striated and smooth muscle.

Action of indapamide on excitation-contraction coupling in vascular smooth muscle.

Excitation-contraction coupling in the smooth muscle cells of the rabbit main pulmonary artery.

Effects of cocaine on excitation-contraction coupling of aortic smooth muscle from the ferret.

Excitation-contraction coupling and the mechanism of muscle contraction.

Mechanics of vascular smooth muscle contraction.

Urethane and contraction of vascular smooth muscle.

Cellular basis of detrusor smooth muscle contraction.

Contraction of detergent-treated smooth muscle.

Pentobarbital and contraction of vascular smooth muscle.

Coupling mechanisms in airway smooth muscle.

[Cellular mechanism of muscle contraction of bronchial smooth muscle].

Structural changes in smooth muscle cells during isotonic contraction.

Rem uncouples excitation-contraction coupling in adult skeletal muscle fibers.

Theory of excitation-contraction coupling in cardiac muscle.