Endothelin and calcium dynamics in vascular smooth muscle.

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Annu. Rev. Physiol. 1992.54:257-77 Copyright © 1992 by Annual Reviews

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ENDOTHELIN AND CALCIUM

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DYNAMICS IN VASCULAR SMOOTH MUSCLE Robert F. Highsmith, Karen Blackburn, and David J. Schmidt Department of Physiology and Biophysics, University of Cincinnati College of

Medicine, Cincinnati, Ohio 45267--0576 KEY WORDS:

vascular smooth muscle, calcium channels, signal transduction, sensitization, vasoconstriction

INTRODUCTION AND BACKGROUND The vascular endothelium is a widely dispersed organ system composed of a continuous monolayer of endothelial cells (ECs) that partition blood from underlying vascular smooth muscle

(YSM).

This intriguing structure is

uniquely positioned to play a variety of key roles in modulating vasomotor tone. ECs provide a structural and functional barrier to the interaction of circulating vasoactive molecules with

VSM

and are both a source of and a

sink for vasoactive material. Thus ECs actively participate in the regulation of vascular resistance by binding and catabolizing blood-borne vasoactive sub stances, converting inactive precursors to vasoactive products, and directly synthesizing and releasing molecules that alter vasomotor tone. I,Intense investigations over the past 15 years have confirmed that vasoactive products of ECs are potent modulators of VSM contractility. Following description of the two unstable vasodilators, prostacyclin

(80)

and the en

dothelium-derived relaxing factor (EDRF) or nitric oxide (27), Hickey and colleagues

(40)

reported the discovery and characterization of a potent

and stable peptidergic vasoconstrictor produced by cultured ECs, originally termed endotensin or endothelium-derived constricting factor (EDCF). After confirmation

of

these

findings

(29, 89),

the

peptide

vasoconstrictor 257

0066-4278/92/0315-0257$02.00


258

HIGHSMITH, BLACKBURN & SCHMIDT

was purified, the cDNA cloned, and the molecule renamed endothelin (ET) ( 140). ET shares little structural similarity to other mammalian vasoactive peptides, but regional homology does exist between ET and the a-scorpion toxins, a group of Na+ channel-directed molecules (140), and considerable homology is apparent with the sarafotoxins, cardiotoxic molecules present in the venom of the burrowing asp (62, 71). Analysis of human genomic DNA (50) revealed the existence of three distinct genes encoding three ET isopeptides (ET- 1, ET-2, and ET-3), differing slightly in sequence but signifi cantly in properties. ET-l is apparently the only isoform produced and released from ECs. A fourth isopeptide, ET-{3, has been similarly identified from mouse genomic DNA and has been termed vasoactive intestinal con stricting peptide, or VIC, because of its expression in intestinal tissue ( 104). The production of mature ET- 1 by ECs is novel among vasoactive peptides in that a preproform (203 amino acids) is first synthesized, cleaved to yield a 39-residue intermediate proform termed big-ET-l, which in tum is acted upon by a putative endothelin-converting enzyme (ECE) (Figure 1). The first proteolytic cleavage is mediated by dibasic pair-specific endopeptidases and carboxypeptidases, while the final cleavage by ECE occurs at an unusual Trp-Val sequence. The latter event appears to be essential for the vasoactive role of ET-I since big-ET-I is approximately IOO-fold less potent than ET-I in eliciting vasoconstriction in isolated vessels (59). Also, infusion of big ET -1 into rats elicited a pressor response equal in magnitude to ET-1 infusion, which suggests in vivo conversion of the proform to the mature ET-l ( 109). Furthermore, a crude inhibitor of ECE, phosphoramidon, not only prevented the conversion of big-ET-l to ET-I, but also blocked the pressor response occurring in vivo following infusion of big ET -1 (76). ECE may be a neutral metalloproteinase bound to the EC membrane because it has a neutral and narrow pH optimum, is inhibited by the neutral protease inhibitor phosphor amidon and EDTA, and is largely unaffected by inhibitors directed at cys teine-, serine-, and aspartic-proteases (49, 9 1). ET- 1 is the most potent endogenous vasoconstrictor that has been characte rized to date. In our experience, contraction of isolated VSM in response to ET-1 generally begins at a threshold dose of about 100 pM. The role of ET-I as an important endocrine substance may be questionable, however, because circulating levels of immunoreactive ET-1 under normal circumstances are at least 20-fold less «5 pM) (3, 38, 105, 106, 124, 125). This observation does not preclude an autocrine or paracrine role for ET-l . For example, ET-l receptors have been identified on ECs, and ET -1 stimulation is known to release the vasodilators, EDRF (NO) (19, 135) and prostacyclin (39, 94), perhaps in an autocrine-feedback fashion. Also, local abluminal release of ET- 1 into the subendothelial space at concentrations exceeding threshold values could allow a key role for ET-I as a paracrine contractile substance.

ENDOTHELIN AND Ca2+ DYNAMICS

Lys5:

N

� 1

20

Arg5,

Arg!l� Arg 93

1 } 73

N- iUg 53

203

91

74

Trp -Val


�c

81

dibasic-palr-specific endopeptidase carboxypeptidase

74

}

f-C

91

}

}

endothelin converting enzyme

}

259

Preproendothelin (203aa)

1

Proendothelin "Big Endothelin" (39aa)

1

Mature Endothelin (ET-1) (21aa)

Figure 1 Proteolytic processing of preproendothelin and proendothelin in the biosynthesis of mature endothelin-l.

ET-l may also have potentiating or sensitizing effects on VSM contractility (142), as well as effects on VSM cell growth (66). Little infonnation is available concerning the factors underlying the sensitization phenomenon, but recent evidence for additional mechanisms affecting VSM contractility (60, 61, 63, 86, 87) suggests possible effects on Ca2+ homeostasis. Despite a flood of publications dealing with ETs, relatively little is known about their physiological role or pathological significance. Thus much speculation has emerged proposing autocrine, paracrine, and endocrine roles for the peptide as well as for its potential involvement in hypertension, coronary and cerebral vasospasm, and other vaso-occlusive disorders. Resolving these issues will likely require advanced techniques for quantifying the peptides, as well as for more specific inhibitors of ECE and ET receptor antagonists. In contrast, the contractile mechanism of action of the ETs, particularly of ET-l in VSM, has been intensely investigated since the commercial availabil ity of the peptides( . in VSM with emphasis on Ca2+ dynamics, including the events controlling entry of extracellular Ca2+, the signal transduction pathways involved in the mobilization of intracellular Ca2+, and other cellular mechanisms affected by ET-l that may influence Ca2+ signaling in VSM. References selected for the

260


review are representative publications; space limitations prohibited a com prehensive literature citation. ENDOTHELIN AND EXTRACELLULAR CALCIUM


Calcium Channels, Membrane Potential, and Sodium-Calcium Exchange

Usually, the entry of extracellular Ca2+ into VSM cells via membrane channels is regulated by either voltage-operated channels (VOC) or receptor operated channels (ROC). The gating of these channels is initiated by either direct binding of an agonist to a receptor located at or near the channel, or indirectly through an intracellularly released second messenger. Early clues as to the mechanism of action of ET in VSM and the role of extracellular Ca2+ were first provided in studies by Hickey et al (40), who demonstrated that EDCF-induced contractions of isolated vessels were dependent on extracellu lar Ca2+ and were attenuated by the L-type Ca2+ antagonist, verapamil. The structural homology of ET - 1 with a-scorpion toxin and the observed attenua tion of ET- 1-induced contractions by dihydropyridine (DHP) Ca2+ channel blockers led Yanagisawa et al ( 140) to speculate that ET's requirement for extracellular Ca2+ most likely reflected its action as an endogenous agonist of the DHP-sensitive, L-type Ca2+ channel. Evidence that ET- 1 may trigger VOC comes from studies directly measuring Ca2+ conductances with the patch-clamp technique (32, 131) and from findings that DHP-type blockers attenuate ET-1-induced contractions of isolated vessels, as well as Ca2+ uptake and changes in [Ca2+]i in ET-stimulated VSM cells ( 14, 20, 2 1, 22, 3 1, 42, 79, 90, 107, 119, 129, 130, 134, 136, 141). It should be noted however, that these results are highly dependent on the dose of ET- 1 as well as the source of the vessel or VSM cells (75, 1 10). Several lines of evidence point to an indirect gating of VOC after ET-l simulation that is mediated by diffusible second messengers. For example, ET-1 does not compete or dis place the binding of several different L-type Ca2+ channel ligands (36), nor does it activate isolated L-channel activity (83), yet the peptide increases the activity of an L-type Ca2+ channel when applied to cells clamped in the cell-attached mode (5 1, 112). ET-1 induces transient hyperpolarization followed by a sustained, yet modest, depolarization of smooth muscle membranes (41, 68, 70, 84, 131, 134). Relatively little data are available concerning the ionic basis of the depolarization, although Nakao et al (84) suggest that Na+ may be the underlying ion. Conflicting hypotheses have been presented concerning the relative importance of depolarization in mediating ET-l-induced Ca2+ influx. For example, Van Renterghem et al ( 13 1) provided data to support activation of a cation channel by ET- 1 and proposed that L-channel activation was



261

secondary to depolarization. On the other hand, Silberberg et al ( 112) re ported that ET-l mediates an increase in single VOC channel activity and hypothesized an as yet unspecified second messenger system. In addition, the relationship between depolarization and Ca2+ influx via non-L-type pathways has not been resolved. As discussed below, the existence of a non-DHP sensitive Ca2+ influx pathway could underlie the depolarization response and serve as the pathway for Ca2+ entry. Alternatively, two or more separate events could underlie depolarization and non-L-channel-mediated Ca2+ entry. The results of several studies (119, 134) indicate that at lower contractile doses of ET- 1 removal of extracellular Ca2+ has a more profound inhibitory effect on force development in VSM than does treatment with L-type Ca2+ channel blockers. This discrepancy, coupled with recent descriptions of Ca2+ permeant channels in VSM that are distinct from the L-type channel, suggest that activation of additional non-DHP-sensitive Ca2+ influx pathway(s) may be important in the early mechanism of action of ET-l . This possibility was proposed by Van Renterghem et al (13 1) and Wallnofer et al (134) and has received experimental support from studies in our laboratory (10). We evalu ated the effects of Ni2+, a Ca2+ channel antagonist with clearly documented potency toward cation channels in non-excitable tissues (69), on ET-1mediated contractions of VSM. In a dose- and Ca2+-dependent fashion, Ni2+ markedly inhibited ET- 1 induced contractions of VSM at concentrations that did not affect the response to L-type Ca2+ channel activation by K+-induced depolarization or by agents known to mobilize intracellular Ca2+ stores. In more recent studies using single VSM cells and Ca2+ -induced Fura-2 fluorescence, we found that Ni2+ does not act by altering basal cytoplasmic Ca2+ or by significantly disrupting intracellular Ca2+ release. Utilizing Mn2+ quench of the Fura-2 fluorescence signal as an indicator of cation flux from the extracellular to the intracellular space, we also found an increase in Mn2+ quench following ET-I stimulation (R. Highsmith et aI, unpublished observa tions). Since Mn2+ blocked voltage-gated Ca2+ channels and was used as a probe for Ca2+ -permeant cation channels (ROC) (37, 103), these data support the existence of an ET-1-activatable Ca2+-permeant pathway that is distinct from the L-type Ca2+ channel (VOC). Our preliminary data suggest that this pathway may be inhibited by Ni2+. In recent work by Inoue et al (51), using patch-clamped cells to directly measure Ca2+ conductances, ET-l increased nifedipine-resistant Ca2+ channel activity, which further suggests alternative pathways for Ca2+ entry independent of L-type VOc. Definitive studies have not been done to address the potential role of Na+ICa2+ exchange at the VSM membrane as a mechanism responsible for the entry of extracellular Ca2+ induced by ET- 1. Although some investigators have postulated that ET- 1 might activate this ion exchanger (12, 17), the inability to control for non-specific effects of purported inhibitors of Na+I


262


Ca2+· exchange, or for the consequences of removal or replacement of ex tracellular Na+, considerably cloud the interpretation of these studies. Thus the potential contribution of Na+/Ca2 + exchange as a target of action of ET-l remains speCUlative. In summary, the entry of extracellular Ca2+ through membrane channels is a critical step in the mechanism underlying ET- I-induced contractions of YSM. In addition to affecting membrane depolarization and indirect modula tion of voltage-sensitive Ca2+ entry through YOC, ET-l also promotes Ca2+ entry through voltage-insensitive pathways (ROC). However, the lack of detailed knowledge of the specific properties of YOC, and particularly those of ROC in YSM, impedes further description of the precise pathways mediat ing ET-l-induced Ca2+ influx. ENDOTHELIN AND INTRACELLULAR CALCIUM

Recent evidence confirms the presence of at least two different receptors for ET, each coupled to G proteins, yet probably serving different functions. One receptor, termed ETA, is most likely the YSM receptor, has high specificity for ET-l, and its mRNA is widely distributed in the central nervous system, heart, and lungs (5). The other receptor, isolated from rat lung and termed ETB, is probably of EC origin, is nonspecific in that it does not discriminate between ET isoforms, and its mRNA is not found in YSM cells ( 108). Several studies (6, 99, 100, 1 14) indicate that ET-l induces stimulation of phospholipase C, most likely via receptor-ligand coupling by a G protein. Pretreatment of YSM or mesangial cells with pertussis toxin markedly attenu ated the increase in phosphatidylinositide (PI) hydrolysis normally obtained with ET-l stimulation, and treatment of mesangial cells with GTPyS potenti ated the effect ( 102, 1 13). Pertussis toxin treatment has also been shown to attenuate ET- I-induced increases in contractility in ventricular myocytes (57). However, other studies using either YSM, a smooth muscle-like cell line (A-IO), or fibroblasts, have shown no evidence of a pertussis toxin-sensitive G protein (82, 127). Similarly, Boonen & DeMey ( 1 1) observed little effect of the toxin on ET-l-induced force development in rat mesenteric arteries, and Yigne et al ( 132) demonstrated no effect on inositol phosphate production in atrial myocytes. These findings suggest that multiple G proteins may couple the ET-I receptor to phospholipase C activation in a cell specific fashion and/or that PI hydrolysis, in some instances, may be secondary to other events. Activation of Phospholipase C and Phosphatidylinositol Turnover

The binding of ET-l to receptors on YSM results in the rapid activation of phospholipase C and subsequent hydrolysis of membrane PI (6, 4 1, 42, 45,



263

48, 56, 74, 77, 82, 90, 93, 95, 100, 120, 139). The products fonned, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), represent two important intracellular signals that result in release of Ca2+ from the sarcoplasmic reticulum or calciosome (8, 43, 53, 77, 115) and activation of protein kinase C (see below), respectively. Enhancement of PI turnover in VSM appears to be an important early event in the mechanism of action of ET-I since a significant elevation of IP3 occurs within 1 0 sec following ET-1 stimulation (95). As a result of ET-l stimulation, various additional inositol phosphate derivatives may be formed, depending on the specific target cell and its relative kinase and phosphatase activities. For example, in mesangial cells, ET stimulates formation of inositoll ,3,4,5-tetrakisphosphate (IP4) which, in synergism with IP3, may mediate the elevation in [Ca2+]j. As noted by Simonson & Dunn (11 3), because of the sensitivity of various kinases to [Ca2+]j, it is likely that the magnitude of the initial increase in [Ca2+ )j elicited by ET-1 may be an important determinant of the pathway by which the trisphosphate is metabolized. The subsequent generation of secondary inositol phosphates may have further effects on [Ca2+k The formation of the second messenger IP3 not only results in the liberation of intracellular stores of Ca2+ , but also may be involved in stimulating further influx of extracellular Ca2+. For example, Vilven & Coronado (133) demon strated that IP3 can induce opening of DHP-sensitive Ca2+ channels in vesicular preparations of skeletal muscle transverse tubules. Also, Ca2+ influx following vasopressin stimulation of rat VSM may be mediated by IP3, based on the finding that a phospholipase inhibitor prevented vasopressin induced Ca2+ influx as well as IP3 formation (67). On the other hand, Kobayashi et al (64) concluded that IP3 does not modify Ca2+ influx in smooth muscle, based on the finding that intracellularly applied heparin, an IP3 receptor antagonist, had no effect on agonist-induced Ca2+ influx. This suggestion presumes, however, that the same receptor mediates IP3 ef fects at both the sarcoplasmic reticulum and plasmalemmal sites and that the modulation of L-channel function by this intermediate would require a heparin-sensitive IP3-receptor interaction. There appears to be no di rect evidence of activation of L-type Ca2+ channels by inositol phosphates in VSM. Thus substantial evidence indicates that ET -1 stimulation of VSM results in activation of phopholipase C, an increase in PI turnover, and the rapid release of intracellular Caz+ from the sarcoplasmic reticulum. In most preparations this process appears to be mediated by IP3. Although not confirmed for ET- l stimulation of VSM, Ca2+ -induced ca2+ release from the sarcoplasmic re ticulum could also play an additional role. Other evidence suggests that inositol phosphates may have secondary effects on modulating Ca2+ influx pathways in VSM.

264

.



ENDOTHELIN AND ACTIVATION OF PROTEIN KINASE C Agonist-induced increases in PI turnover in VSM result in release of DAG, which in tum activates protein kinase C (PKC). Although the precise function of PKC activation in ET-I-induced responses is unclear, this kinase may have diverse effects on both Ca2+ dynamics and contraction. Interest in a role for PKC in ET-l induced contractions ofVSM was sparked by early observations indicating a similarity of ET-l-induced contractions of VSM with those elicited by PKC activators (7). In a fashion similar to ET-l, DAG analogues, which activate PKC, induce smooth muscle contractions that are slow in onset and highly resistant to washout ( 15, 18, 52, 78). The remaining sections briefly review the activation of PKC in VSM in general, followed by a more specific discussion of ET - 1-induced formation of DAG and activation of PKC, as well as the potential role for ET-l in the sensitization of the contractile apparatus to Ca2+.

Protein Kinase C Activation in Vascular Smooth Muscle The role of DAG-induced PKC activation in contraction of VSM is con troversial (26, 58, 96-98). PKC activation may specifically affectVSM Ca2+ dynamics in numerous, yet seemingly disparate, ways. PKC activation has been shown to reduce plasmalemmal activation via negative feedback on PI hydrolysis and/or receptor down-regulation in some tissues, while the op posite effect has been noted in other tissues (24). PKC activators likely induce bidirectional plasmalemmal Ca2+ flux (26) with variable net effects on in tracellular Ca2+ levels and may induce alterations in the sensitivity of con tractile proteins to Ca2+ (52). Reports concerning the effect of PKC on Ca2+ influx in VSM through voltage-activated Ca2+ channels are contradictory. Galazzi et al (28) found that DHP-sensitive Ca2+ influx stimulated by depolarization with K+ was inhibited by DAG analogues or phorbol esters. In contrast, enhancement of L-type Ca2+ currents was obtained under similar conditions using a smooth muscle-like cell line patch-clamped in the whole cell configuration (25). Mori et al (81) reported that in porcine coronary arteries net increases or decreases in intracellular Ca2+ levels could be obtained depending on the dose of phorbol ester and that this PKC activator also produced a sensitization of contractile proteins to Ca2+. Recent literature (60, 6 1, 63, 86, 87) validates that a leftward shift of the Ca2+ -force relationship, or sensitization of the contractile apparatus to Ca2+ , can occur in smooth muse}; and that DAG activation of PKC may stimulate this process (52, 8 1, 87). Activation of PKC may lead to multiple phosphory lations at different loci of the contractile machinery, including the heavy and light chains of myosin (54, 85, 1 16, 123), a putative phosphatase inhibitor protein ( 1 17), and/or the proposed actin filament regulatory proteins, caldes-

ENDOTHELlN AND Ca2+ DYNAMICS

265

mon and calponin (1, 46, 128, 137). G protein-dependent Ca2+ sensitization by agonists or GTPyS enhances the phosphorylation of myosin light chains at a given [Ca2+]j, thus supporting the concept of phosphatase control (60). However, the kinase specificity of the phosphorylation sites has not been determined. Other recent reports (128, 137) indicate that caldesmon and calponin are phosphorylated by PKC, which results in a decrease in actin affinity and reduces inhibition of actomyosin ATPase activity in vitro.


Endothelin and the Production of Diacylglycerol ET- 1 may elicit PKC-mediated effects on Ca2+ reponses in VSM similar to

those described above for the phorbol esters. Elevations in DAG and in membrane-bound PKC activity have been documented following VSM stimulation with ET-l (35, 72, 82, 121, 122). ET-l elicits a biphasic increase in DAG that is well sustained for 20 min or longer (35, 72, 82). The precise source of DAG formed in response to ET-l is not known. However, in addition to that derived from PI, DAG may also be provided from phospholi pase C hydrolysis of phosphatidylcholine (PC), or from degradation of phosphatidic acid produced by phospholipase D action on PC (9, 23, 111). DAG production from PC is thought to occur later and to be maintained much longer than DAG derived from PI hydrolysis (9, 23). It was proposed that initial transient increases in DAG are derived from PI and are likely responsi ble for the down-regulatory plasmalemmal effects of PKC activation, whereas PC-derivedDAG is long lasting and maintains PKC-dependent cell activation (9). ET-l treatment has been reported to affect the catabolism of PC (73, 100). Nearly all of these studies indicate that the DAG response to ET-l has a late, prolonged component, which suggests that PC breakdown may be an impor tant source of DAG. Other processes that increase or maintain DAG produc tion may also be involved in the response to ET-1. For example, a prolonged DAG production coupled to clathrin-mediated receptor-ligand internalization has recently been described for angiotensin II (33, 34). It is possible that ET-l may produce similar effects onDAG production since receptor-bound ET-1 is internalized (4, 44). Studies attempting to define a role for PKC activation in contractions induced by ET- 1 are somewhat difficult to interpret because of the use of putative PKC inhibitors, which lack specificity, or their use at doses that are likely to affect other processes required for contraction. In addition, PKC exists in at least seven isoforms (88) with differing intracellular localizations and activation properties such as Ca2+ sensitivity (24). Cells contain multiple isoforms (24), and each may vary in its susceptibility to exogenous PKC activators or inhibitors. Nonetheless, compounds thought to inhibit PKC, such as staurosporine and phloretin, inhibit both phorbol ester- and ET-I induced contractions (7, 120). H-7, another kinase inhibitor, has also been


266


reported to block contractions in response to ET-I (120). However, Auguet et al (7) demonstrated that H-7 inhibited phenylephrine- or KCl-induced con tractions more potently than those elicited by ET-1 and that the inhibitor did not block the contractile response to phorbol ester. Several other lines of evidence support a role for PKC activation in ET-I-induced contractions of VSM. For example, ET- I stimulates phosphorylation of a 76-kd protein that is a known substrate for PKC (35). Down-regulation of PKC by preincubation of VSM with phorbol esters markedly attenuates phosphorylation of the 76-kd protein by ET-1 (35). Also indicative of PKC activation are the findings of Lee et al (72) that PKC activity appears to translocate from the cytosol to plasmalemma following stimulation of isolated VSM cells with ET-1. Endothelin, Protein Kinase C, and Calcium Dynamics

The relationship of ET-l-induced activation of PKC to Caz+ dynamics in VSM is poorly understood. PKC activation promotes ET-l receptor down regulation and blunts ET- I-induced PI hydrolysis (35, 101, 102). Silberberg et al (112) proposed that activated PKC may be the second messenger involved in ET-1 stimulation of L-type Caz+ current. According to Simonson & Dunn, PKC can either inhibit or enhance the phosphoinositide-linked Caz+ signal (113). For example, in mesangial cells, PKC activation results in attenuation of ET-I-stimulated Caz+ signaling. Thus pretreatment with phor bol esters orDAG analogues reduces the increase in [Caz+]j elicited by ET-1, while down-regulation of PKC amplifies ET-l-induced increases in [Ca2+]j. These authors propose three potential mechanisms to account for the in hibitory effect of PKC on Ca2+ signaling: (a) inhibition of the hydrolysis of phosphatidylinositol 4,5-bisphosphate with subsequent reduction in the amount of the second messenger I P3 ; (b) activation of Caz+ pumps to remove Ca2+ from the cytosolic compartment; and (c) stimulation of IP3 phosphatase resulting in dephosphorylation and inactivation of the second messenger. ENDOTHELIN AND SENSITIZATION OF VASCULAR SMOOTH MUSCLE

Certain qualitative features of ET- I-induced contractions suggest that sensitization or amplification processes may be operative and may predomi nate at low doses of the peptide. For example, the biphasic nature and poor reversibility of ET-l-induced contractions and the ability of ET-I to elicit weak, yet tonic, contractions even at very low extracellular Caz+ levels (13, 47, 65, 90, 92, 107, 134) suggest a sensitization phenomenon. In addition, a growing body of evidence suggests that at low doses, ET-1 may also sensitize smooth muscle to activation by other agonists (118, 126, 138). For example, Yang et al (142) described potentiation of the contractile responses to seroto-


ENDOTHELlN AND

Ca2+

DYNAMICS

267

nin and norepinephrine in human arteries pretreated with threshold doses of ET-1. The potentiation was dependent on a Ca2+ influx mechanism that was blocked by a Ca2+ channel antagonist. Consigny (16) confirmed ET-l induced arterial sensitization to serotonin. Low-dose ET- I also potentiated the contractile responses to an agonist of the L-type Ca2+ channel, Bay K 8644, and to an O'-adrenoreceptor agonist, clonidine (30, 31). Application of low doses of ET-1 to isolated coronary vessels resulted in a leftward shift of the dose-response curve to KCl (55) and, similarly, ET-l enhanced KC1-induced contractions of tracheal smooth muscle, as well as pulmonary artery and vein (13). In preliminary studies we note that the endothelium-dependent contrac tion of coronary vessels that occurs upon transition from normoxic to hypoxic conditions is greatly amplified by prior exposure of the vessels to sub threshold doses of ET-l (R. Highsmith et aI, unpublished observations). Essentially no information is available as to the mechanisms underlying the sensitization phenomenon accompanying ET-1 stimulation of VSM. Obvious possibilities include the generally well characterized cellular processes known to be involved in regulation of VSM contraction: (a) depolarization of the cell membrane to potentials near threshold for Ca2+ channel activation; (b) change in degree of basal PI turnover or pathways of inositol phosphate metabolism; and (c) change in intracellular Caz+ concentrations or Caz+ handling. In addition, however, ET-1 stimulation of VSM and subsequent PKC activation may induce sensitization of contractile proteins to Ca2+. Sunako et al (122) suggest that PKC may modulate the sustained phase of ET-1-induced contrac tion by increasing the Ca2+ sensitivity of the contractile apparatus. ET-l has been shown to stimulate PKC-dependent phosphorylation of myosin light chain and caldesmon (2). Thus a disinhibition of the contractile apparatus via PKC-induced phosphorylation of these regulatory proteins is a possible out come of ET-1 stimulation of VSM that needs further study. In summary, ET-I stimulation of VSM is clearly associated with a phos pholipase-mediated increase in the formation of DAG and subsequent activa tion of PKC. A variety of consequences ensue from the diverse effects of PKC on intracellular functions, which could contribute to the sustained phase of ET-l-induced contraction of VSM. Because of general uncertainty about the sequelae of DAG-coupled PKC activation in VSM, however, the significance of these events to changes in intracellular Caz+ dynamics induced by ET-l cannot'be currently ascertained. ENDOTHELIN AND INTEGRATED CALCIUM SIGNALING IN VASCULAR SMOOTH MUSCLE: A MODEL AND SUMMARY

ET- 1 stimulation of VSM results in a dose-dependent elevation in [Caz+]i that occurs in two phases representing the net effect of activation of different Caz+

268


signaling pathways. Within a few seconds and prior to development of force, 2+ a rapid transient increase in [Ca ]j occurs that is immediately followed by a somewhat slower decrease to a new steady state level that is slightly higher 2+ than basal values and is well maintained. The absolute value of [Ca ]i following ET-1 stimulation is highly variable and is dependent on the method of quantitation, the source and passage number of the cell, and the con 2 centrations of [Ca +]o and ET-l . In general, most VSM cells with resting

[Ca2+]j levels between 50-100 nM demonstrate a peak transient value ranging

150 nM to nearly 1 p,M when stimulated with 1-20 nM ET-l. The 2+ sustained increase in [Ca ]i also varies and ranges from 20--50 nM above the


from

unstimulated level.

2+ The elevation in cytosolic [Ca ]j after ET-1 stimulation is the net result of

the temporal interplay of several processes: increases in the influx of ex 2+ tracellular Ca and release of Ca2+ from intracellular storage sites, as well as 2+ 2+ Ca efflux and sequestration. Evidence clearly indicates that both Ca 2+ influx and intracellular release contribute to the overall ET-l -induced Ca signal, with each contributing to varying degrees depending on the time after ET-1 stimulation. Immediately after exposure to ET- 1, the initial transient increase in [Ca2+]j is primarily mediated by IP3-triggered intracellular release 2+

, with some contribution from the influx of extracellular Ca2+. In contrast, the later sustained phase of ET-l-induced increases in [Ca2+]i is predominantly fueled by Ca2+ influx. The precise role of activated PKC in the

of Ca

2+ ET-l-induced increase in [Ca ]i is not known, but most likely it contributes to the later sustained phase in which Ca2+ influx predominates.

Figure 2 presents a hypothetical model of the primary actions of ET-1 that 2+ affect Ca dynamics in VSM. Not illustrated are other proposed actions of 2+ ET-1 in VSM such as initial membrane hyperpolarization and transient Ca + + efflux, activation of phospholipase A2, modulation of Na -K ATPase,

2 + stimulation of electroneutral Na+-H+ antiport and Na -Ca + exchange, as well as longer term actions of ET-1, such as mitogenesis, which require changes in gene expression. It should be noted that not all of the mechanisms shown are demonstrable in all VSM cells and, for several reasons stated earlier, a great deal of variability is inherent in ET-1-induced Ca2+ responses. Similarly, the relevance of these processes must be viewed with some skepti cism because in many instances the responses obtained or mechanisms evoked are highly dependent on the dose of ET-1 which may or may not prove to be physiological. The primary mechanisms by which ET- 1 induces a rapid and sustained

increase in [Ca2+]i most likely involve the following events: (a) binding of ET- 1 to a specific high affinity receptor on the VSM membrane that is coupled to phospholipase C activation via a G protein. The receptor for ET-l is distinct from the DHP-sensitive Ca2+ channel. (b) The receptor-ET-l

ENDOTHELIN AND CaH DYNAMICS


t

269

Ca�'

t

}Sensl�;atlon

P.MYOSI"

Myosin

'-1 Figure 2

�

Contractile ""PO"""

Other PKC Olitpondent Actions

:::�

Hypothetical model of the primary actions of endothelin·l that affect calcium dyna

mics in vascular smooth muscle. See text for abbreviations.

complex interacts with a specific G protein and produces a rapid phopholipase C-mediated increase in PI turnover and subsequent formation of IP3. While this second messenger is most likely the trigger for release of intracellular Ca2+ stores, Ca2+ -induced release of Ca2+ may also occur in VSM. (c) In response to ET-I, the initial transient burst in [Ca2+]i probably initiates several Ca2+ -dependent processes that are poorly understood. For example, the sudden increase in [Ca2+]i may underlie both the brief Ca2+ efflux response as well as the transient membrane hyperpolarization that may be mediated by a Ca2+ -activated K+ channel. The hyperpolarization is quickly followed by a modest, yet sustained, depolarization of the plasma membrane. The precise ionic basis of the depolarization response is not yet known.

(d)

in a well sustained net increase in the uptake of extracellular Ca2+ through membrane channels, both of the ROC and VOC type. ET-l gating of the ROC is distinct from activation of the L-typ e , DHP-sensitive VOC. Whether ET-I acts directly or indirectly in

After a brief net efflux of Ca2+ , ET-I results

gating the ROC is not known. Similar uncertainty exists in regard to the

spatial and temporal relationship between the gating of ROC by ET-l and binding to the high affinity receptor. Opening of the VOC appears to be indirect and a consequence of membrane depolarization, or perhaps stimula tion by other second messengers such as the inositol phosphates, PKC, or products of PKC-dependent actions.

(e) As a result of the activation of

phospholipase C and perhaps phospholipaseD, a biphasic production ofDAG ensues that contributes to activation of PKC. Although clouded by lack of


270


specific pharmacological probes, data suggest that PKC activation may con tribute in many ways to the overall cellular response to ET-1. Initially, PI-derived DAG may modulate the gating of VOC and may serve in a negative feedback fashion to blunt the increase in [Ca2+]j perhaps by uncou pling the receptor-mediated activation of phospholipase C. In addition, PKC dependent actions may enhance the responsiveness of VSM to other agonists. Possible mechanisms for this effect include a sensitization of the contractile apparatus to Ca2+. Other effects of ET-1 mediated by PKC include protein phosphorylation, which in part stimulates Na +-H+ exchange, as well as longer term effects on gene transcription. (j) The final common pathway underlying ET-I-induced contractions of VSM following elevation of [Ca2+]j involves interaction of Ca2+ with calmodulin and myosin light chain kinase, which results in the phosphorylation of mYOSin, actin-myosin crossbridge formation, and the development of force. With continued occupation of the receptor by ET-1 , latch bridge formation may then serve to maintain tone despite decreasing levels of [Caz+Ji, The speculative processes involving sensitization to Ca2+ and thin filament regulation may affect force after ET-l receptor occupation subsides, or following internalization of the receptor ligand complex. In conclusion, we have attempted to review the major events underlying ET-l-induced changes in Ca2+ dynamics in VSM. This task is formidable because of the lack of clear understanding of agonist-induced effects on VSM intracellular Caz+ , in general, as well as the preliminary nature of many of the rapid publications dealing with the mechanism of action of ET-l in VSM. Although our knowledge about this interesting vl!-soactive peptide has acceler ated at a dramatic pace, there remain many gaps yet to be closed. We hope that this review will serve as a framework upon which more detailed ex periments can build. ACKNOWLEDGMENTS This work was supported by National Institutes of Health research grant HL 31543 and training grant HL 07571. Literature Cited . 1. Abe, M . , Takahashi, K . , Hiwada, K . 1990. Effect of calponin o n actin activated myosin ATPase activity. 1 . Biochem. 108:835--38 2. Adam, L. P., Milio, L . , Brengle, B . , Hathaway, D . R. 1990. Myosin light chain and caldesmon phosphorylation in arterial muscle stimulated with endothe lin-I. 1. Mol. Cell Cardiol. 22:1017-23 3. Ando, K . , Hirata, Y., Shichiri, M . , Emori, T . , Marumo, F . 1989. Presence of immunoreactive endothelin in human plasma. FEBS Lett. 245:164-66

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32

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NOTE ADDED IN PROOF

Subsequent to the submission of this manuscript, several reports have been published that provide further information concerning the mechanism 9f action of ET-1 in VSM. These articles provide additional insights that are particularly relevant to the concepts presented in our review. Two reports suggest that the membrane depolarization induced by ET may be mediated by a chloride permeant channel (143, 144). A recent report provides evidence that ET stimulates the Na+ -K+ -ATPase (145). Finally another paper indicates that ET activates a Ca2+ -dependent, nonspecific cation channel in cultured rat VSM cells that is NiH -sensitive, yet resistant to inhibition by nifedipine

(146).

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145. Gupta, S . , Ruderman, N . , Cragoe, E . , Sussman, I . 1 99 1 . Endothelin stimulates Na+-K+-ATPase activity by a protein kinase C-dependent pathway . Am. 1 . Physiol. 261 :H38--45 146. Chen, c . , Wagoner, P. 1 99 1 . Endothe lin induces a nonselective cation current in vascular smooth muscle cells. eire. Res. 69:447-54

Endothelin-induced calcium responses in human vascular smooth muscle cells.

Endothelin-1 and endothelin-3 stimulate calcium mobilization by different mechanisms in vascular smooth muscle.

Effect of endothelin-3 on cytosolic calcium level in vascular endothelium and on smooth muscle contraction.

Regulation of endothelin-mediated calcium mobilization in vascular smooth muscle cells by isoproterenol.

Endothelin ETA and ETB receptors mediate vascular smooth muscle contraction.

Calcium antagonists and vascular smooth muscle cells in atherogenesis.

Tris(hydroxymethyl)aminomethane inhibits calcium uptake in vascular smooth muscle.

Electron microscopic localization of calcium in vascular smooth muscle.

Mechanisms of calcium mobilization and phosphoinositide hydrolysis in human bronchial smooth muscle cells by endothelin 1.

Stimulation of endothelin mRNA and secretion in rat vascular smooth muscle cells: a novel autocrine function.

Inducible endothelin mRNA expression and peptide secretion in cultured human vascular smooth muscle cells.

Vascular Smooth Muscle Cells.

Cloning and functional expression of a vascular smooth muscle endothelin 1 receptor.

Dexamethasone potentiates production of inositol trisphosphate evoked by endothelin-1 in vascular smooth muscle cells.

Conversion of big endothelin-1 to endothelin-1 by two-types of metalloproteinases of cultured porcine vascular smooth muscle cells.

Effects of endothelin-1 on vascular smooth muscle cell phenotypic differentiation.

Sodium and calcium interactions in vascular smooth muscle cells of the rabbit ear artery.

Calcium exchange in vascular smooth muscle, action of noradrenaline and lanthanum.

Enhanced proliferation and altered calcium handling in RGS2-deficient vascular smooth muscle cells.

Feasibility of simultaneous measurement of cytosolic calcium and hydrogen peroxide in vascular smooth muscle cells.

Magnesium regulates intracellular free ionized calcium concentration and cell geometry in vascular smooth muscle cells.

Specific pharmacology of calcium in myocardium, cardiac pacemakers, and vascular smooth muscle.

Cholesterol enrichment increases basal and agonist-stimulated calcium influx in rat vascular smooth muscle cells.

Influence of magnesium on calcium- and potassium-related responses in vascular smooth muscle.