Ca2+-induced Ca2+ desensitization of myosin light chain phosphorylation and contraction in phasic smooth muscle

The temporal relationship between Ca2+-induced contraction and phosphorylation of 20 kDa myosin light chain (MLC) during a step increase in Ca2+ was investigated using permeabilized phasic smooth muscle from rabbit portal vein and guinea-pig ileum at 25°C. We describe here a Ca2+-induced Ca2+ desensitization phenomenon in which a transient rise in MLC phosphorylation is followed by a transient rise in contractile force. During and after the peak contraction, the force to phosphorylation ratio remained constant. Further treatment with cytochalasin D, an actin fragmenting agent, did not affect the transient increase in phosphorylation, but blocked force development. Together, these results indicate that the transient phosphorylation causes the transient contraction and that neither inhomogeneous contractility nor reduced thin filament integrity effects the transient phosphorylation. Lastly, we show that known inhibitors to MLC kinase kinases and to a Ca2+-dependent protein phosphatase did not eliminate the desensitized contractile force. This study suggests that the Ca2+-induced Ca2+ desensitization phenomenon in phasic smooth muscle does not result from any of the known intrinsic mechanisms involved with other aspects of smooth muscle contractility.     

Cyclic AMP modulation of adrenoreceptor-mediated arterial smooth muscle contraction

We examined the effects of cyclic AMP (cAMP) on the intracellular Ca2+ release in both the intact and skinned arterial smooth muscle. The amount of Ca2+ in the sarcoplasmic reticulum (SR) was estimated indirectly by caffeine-induced contraction of the skinned preparation and directly by caffeine-stimulated 45Ca efflux from the previously labeled skinned preparation. The norepinephrine-induced release contraction was markedly enhanced by dibutyryl cAMP (dbcAMP) and reduced by propranolol. The stimulatory effect of dbcAMP was best observed when the muscle was exposed to 10(-5) M dbcAMP and 2 X 10(-6) M norepinephrine was used to induce the release contraction. 10(-5) M cAMP had no effect on the Ca2+-induced contraction or on the pCa-tension relationship in the skinned preparation. This concentration of cAMP increased Ca2+ uptake into the SR of the skinned preparation when the Ca2+ in the SR was first depleted. 10(-5) M cAMP stimulated Ca2+-induced Ca2+ release from the SR after optimal Ca2+ accumulation by the SR. The results indicate that the stimulatory effect of cAMP on the norepinephrine-induced release contraction could be due to enhancement of the Ca2+-induced Ca2+ release from the SR in arterial smooth muscle.     

Effects of doxorubicin and ruthenium red on intracellular Ca2+ stores in skinned rabbit mesenteric smooth-muscle fibres

Several agents are known to influence the contraction of skeletal and cardiac muscle via a modification of the Ca2+ release mechanism of the sarcoplasmic reticulum, e.g. caffeine, ryanodine, ruthenium red and doxorubicin. Of these substances, only the effects of caffeine and ryanodine have been described in smooth muscle. In this paper we describe the action of ruthenium red and doxorubicin on saponin-skinned mesenteric arteries of the rabbit. A high concentration (20 microM) of ruthenium red inhibited the Ca2+ release induced by low concentrations of caffeine, but had little effect on Ca2+ release induced by high concentrations (20 mM) of caffeine. This result indicates that the Ca2+ release channel of the internal Ca2+ store of smooth muscle cells is less sensitive to inhibition by ruthenium red than that of striated muscle. Doxorubicin in the micromolar range elicited a Ca2+ release and a concomitant contraction, essentially similar to its effect on skinned skeletal muscle cells. This work reveals further similarities between the Ca2+ release mechanisms of smooth and striated muscle, but the results also indicate that important differences between both systems may exist.     

8-Bromo-cAMP decreases the Ca2+ sensitivity of airway smooth muscle contraction through a mechanism distinct from inhibition of Rho-kinase

To clarify whether cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA) activation and Rho-kinase inhibition share a common mechanism to decrease the Ca2+ sensitivity of airway smooth muscle contraction, we examined the effects of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), a stable cAMP analog, and (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide dihydrochloride, monohydrate (Y-27632), a Rho-kinase inhibitor, on carbachol (CCh)-, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-, 4beta-phorbol 12,13-dibutyrate (PDBu)-, and leukotriene D4 (LTD4)-induced Ca2+ sensitization in alpha-toxin-permeabilized rabbit tracheal and human bronchial smooth muscle. In rabbit trachea, CCh-induced smooth muscle contraction was inhibited by 8-BrcAMP and Y-27632 to a similar extent. However, GTPgammaS-induced smooth muscle contraction was resistant to 8-BrcAMP. In the presence of a saturating concentration of Y-27632, PDBu-induced smooth muscle contraction was completely reversed by 8-BrcAMP. Conversely, PDBu-induced smooth muscle contraction was resistant to Y-27632. In the presence of a saturating concentration of 8-BrcAMP, GTPgammaS-induced Ca2+ sensitization was also reversed by Y-27632. The 8-BrcAMP had no effect on the ATP-triggered contraction of tracheal smooth muscle that had been treated with calyculin A in rigor solutions. The 8-BrcAMP and Y-27632 additively accelerated the relaxation rate of PDBu- and GTPgammaS-treated smooth muscle under myosin light chain kinase-inhibited conditions. In human bronchus, LTD4-induced smooth muscle contraction was inhibited by both 8-BrcAMP and Y-27632. We conclude that cAMP/PKA-induced Ca2+ desensitization contains at least two mechanisms: 1) inhibition of the muscarinic receptor signaling upstream from Rho activation and 2) cAMP/PKA's preferential reversal of PKC-mediated Ca2+ sensitization in airway smooth muscle.     

Differentiation of mechanisms for mobilization of calcium in smooth muscle

Techniques to dissociate different sites or stores important for Ca2+ entry or release in smooth muscle include washouts of 45Ca in cold La3+ -substituted solutions. Scatchard-coordinate plots of Ca2+ uptake, substitution of Sr2+ for Ca2+, and both desaturation and rate coefficient plots. Rabbit aortic smooth muscle is particularly useful because Ca2+ mobilization components can be clearly separated. Other vascular preparations investigated (e.g., renal vessels, coronary arteries) appear to have similar components, but their relative importance varies. Respiratory smooth muscle also has similar Ca2+ mobilization components, but they are less readily dissociated by techniques employed in vascular smooth muscles. In guinea pig trachea, cold La3+ washouts do not retain cellular Ca2+ as well as in other preparations: use of other experimental approaches including the Ca2+ channel entry stimulator, CGP 28392, can demonstrate different Ca2+ uptake mechanisms for K+ -stimulated and agonist-induced Ca2+ uptake. In rabbit aorta, CGP 28392 potentiates tension increases elicited with lower concentrations of added K+ but has no effect on norepinephrine-induced contraction. A general model illustrating different Ca2+ entry mechanisms present in three types of smooth muscle provides examples drawn from a spectrum of possible variations in smooth muscle specificity for Ca2+ mobilization.     

RU486对兔输卵管平滑肌的收缩效应

应用肌肉机械－电换能器和Ｇｉｌｓｏｎ生理记录仪,观察ＲＵ４８６对假孕４ｄ兔离体输卵管平滑肌的收缩效应。结果显示：（１）ＲＵ４８６可直接作用输卵管平滑肌,使其收缩频率增加,而未明显改变收缩张力及振幅,与在体肌内注射ＲＵ４８６观察到的结果相似;（２）ＲＵ４８６部分抑制ｃａ~（２＋）诱发的平滑肌收缩活动,它还与Ｖｅｒａｐａｍｉｌ诱发的抑制效应有协同作用,与ＮＥ诱发的收缩张力有拮抗作用,而对Ｆｏｒｓｋｏｌｉｎ诱发的效应未产生任何影响。以上结果表明,ＲＵ４８６对输卵管平滑肌的作用似乎是改变细胞内游离Ｃａ（２＋）的结果,可能干扰Ｃａ（２＋）的流入、或／和内质网Ｃａ（２＋）释放以及Ｃａ（２＋）－Ｉｐ３信息传递机制。     

Changes in responsiveness of the canine basilar artery to endothelin-1 after subarachnoid hemorrhage

The effect of endothelin-1 (ET-1) on the basilar arteries from control and subarachnoid hemorrhage (SAH) dogs were examined. The maximal contraction of the basilar artery in response to ET-1 was markedly decreased in the SAH group. Treatment with 10(-8)M phorbol 12-myristate 13-acetate (PMA) reduced the contractile responses to ET-1 in the basilar arteries from control dogs. ET-1-induced contractions of the basilar arteries from control dogs were similar to those in strips from SAH dogs by the treatment with 10(-8) M PMA. Ca(2+)-induced contraction of the basilar arteries which were depolarized with isotonic K+ (64 mM) were significantly attenuated in SAH dogs. Treatment with PMA also reduced the contractile responses to Ca2+ in the basilar arteries from control dogs. These results indicate that decreased contractile responses of the basilar arteries to ET-1 and Ca2+ in the SAH group may be related to changes in the activity of the protein kinase C in vascular smooth muscle.     

Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction.

In the rabbit mesenteric arterial smooth muscle skinned by saponin, Ca2+ induced contraction in a concentration-dependent manner. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), a non-hydrolyzable GTP analogue, lowered the Ca2+ concentrations required for this contraction and increased the Ca2+ sensitivity of the skinned smooth muscle contraction. GTP gamma S alone did not induce the contraction in the absence of Ca2+. This GTP gamma S-enhanced Ca2+ sensitivity was completely abolished by an exoenzyme of Staphylococcus aureus, named EDIN, and an exoenzyme of Clostridium botulinum, named C3, both of which are known to ADP-ribosylate the rho p21 family that belongs to the ras p21-like small GTP-binding protein superfamily. The GTP gamma S-bound form of rhoA p21 overcame the inhibitory action of EDIN. smg p21B, another small GTP-binding protein, was inactive. EDIN ADP-ribosylated a protein, which was most likely to be rho p21, in the skinned smooth muscle. The GTP gamma S-bound form of rhoA p21, but not the GDP-bound form, substituted for GTP gamma S and enhanced the Ca2+ sensitivity of the skinned smooth muscle contraction. smg p21B was inactive. These results indicate that rhoA p21 is involved in the GTP gamma S-enhanced Ca2+ sensitivity of the smooth muscle contraction.     

Impaired endothelin-induced vasoconstriction in coronary arteries of Zucker obese rats is associated with uncoupling of [Ca2+]i signaling

Although insulin resistance (IR) is a major risk factor for coronary artery disease, little is known about the regulation of coronary vascular tone in IR by endothelin-1 (ET-1). We examined ET-1 and PGF(2alpha)-induced vasoconstriction in isolated small coronary arteries (SCAs; approximately 250 microM) of Zucker obese (ZO) rats and control Zucker lean (ZL) rats. ET-1 response was assessed in the absence and presence of endothelin type A (ET(A); BQ-123), type B (ET(B); BQ-788), or both receptor inhibitors. ZO arteries displayed reduced contraction to ET-1 compared with ZL arteries. In contrast, PGF(2alpha) elicited similar vasoconstriction in both groups. ET(A) inhibition diminished the ET-1 response in both groups. ET(B) inhibition alone or in combination with ET(A) blockade, however, restored the ET-1 response in ZO arteries to the level of ZL arteries. Similarly, inhibition of endothelial nitric oxide (NO) synthase with N(omega)-nitro-l-arginine methyl ester (l-NAME) enhanced the contraction to ET-1 and abolished the difference between ZO and ZL arteries. In vascular smooth muscle cells from ZO, ET-1-induced elevation of myoplasmic intracellular free calcium concentration ([Ca2+]i) (measured by fluo-4 AM fluorescence), and maximal contractions were diminished compared with ZL, both in the presence and absence of l-NAME. However, increases in [Ca2+]i elicited similar contractions of the vascular smooth muscle cells in both groups. Analysis of protein and total RNA from SCA of ZO and ZL revealed equal expression of ET-1 and the ET(A) and ET(B) receptors. Thus coronary arteries from ZO rats exhibit reduced ET-1-induced vasoconstriction resulting from increased ET(B)-mediated generation of NO and diminished elevation of myoplasmic [Ca2+]i.     

Inhibition of iloprost of the contractile effect of noradrenaline in mesenteric artery rings: evidence for a possible calcium-dependent mechanism.

Iloprost caused a concentration-dependent decrease in the response to noradrenaline in the rabbit isolated endothelium denuded rings from superior mesenteric artery but not thoracic aorta. Similar inhibition was obtained by verapamil using identical concentrations. In Ca(2+)-free EGTA containing medium noradrenaline both at lower and higher concentrations elicited a reduced contractile response and further addition of Ca2+ (2.5 mM) to the medium produced a second contraction in both mesenteric artery and aortic rings which was significantly and equally inhibited by iloprost and verapamil using identical concentrations in mesenteric artery but not in aortic rings. Prior addition of iloprost to the medium did not protect the inhibitory effect of phenoxybenzamine against noradrenaline-induced contraction. These results were taken as an evidence for the possible Ca2+ entry reducing effect of iloprost in mesenteric artery but not thoracic aorta. These results were also taken as an indirect evidence supporting the hypothesis that increased synthesis of prostacyclin by noradrenaline in the vascular wall may inhibit the contractile effect of the agonist by a (-) feedback mechanism mediated by Ca2+ entry into the vascular smooth muscle.     

Zipper-interacting protein kinase induces Ca(2+)-free smooth muscle contraction via myosin light chain phosphorylation

The inhibition of myosin phosphatase evokes smooth muscle contraction in the absence of Ca(2+), yet the underlying mechanisms are not understood. To this end, we have cloned smooth muscle zipper-interacting protein (ZIP) kinase cDNA. ZIP kinase is present in various smooth muscle tissues including arteries. Triton X-100 skinning did not diminish ZIP kinase content, suggesting that ZIP kinase associates with the filamentous component in smooth muscle. Smooth muscle ZIP kinase phosphorylated smooth muscle myosin as well as the isolated 20-kDa myosin light chain in a Ca(2+)/calmodulin-independent manner. ZIP kinase phosphorylated myosin light chain at both Ser(19) and Thr(18) residues with the same rate constant. The actin-activated ATPase activity of myosin increased significantly following ZIP kinase-induced phosphorylation. Introduction of ZIP kinase into Triton X-100-permeabilized rabbit mesenteric artery provoked a Ca(2+)-free contraction. A protein phosphatase inhibitor, microcystin LR, also induced contraction in the absence of Ca(2+), which was accompanied by an increase in both mono- and diphosphorylation of myosin light chain. The observed sensitivity of the microcystin-induced contraction to various protein kinase inhibitors was identical to the sensitivity of isolated ZIP kinase to these inhibitors. These results suggest that ZIP kinase is responsible for Ca(2+) independent myosin phosphorylation and contraction in smooth muscle.     

Ca(2+)](i) signaling in renal arterial smooth muscle cells of pregnant rat is enhanced during inhibition of NOS

Vascular resistance and arterial pressure are reduced during normal pregnancy, but dangerously elevated during pregnancy-induced hypertension (PIH), and changes in nitric oxide (NO) synthesis have been hypothesized as one potential cause. In support of this hypothesis, chronic inhibition of NO synthesis in pregnant rats has been shown to cause significant increases in renal vascular resistance and hypertension; however, the cellular mechanisms involved are unclear. We tested the hypothesis that the pregnancy-associated changes in renal vascular resistance reflect changes in contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) of renal arterial smooth muscle. Smooth muscle cells were isolated from renal interlobular arteries of virgin and pregnant Sprague-Dawley rats untreated or treated with the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 4 mg. kg(-1). day(-1) for 5 days), then loaded with fura 2. In cells of virgin rats incubated in Hanks' solution (1 mM Ca(2+)), the basal [Ca(2+)](i) was 86 +/- 6 nM. Phenylephrine (Phe, 10(-5) M) caused a transient increase in [Ca(2+)](i) to 417 +/- 11 nM and maintained an increase to 183 +/- 8 nM and 32 +/- 3% cell contraction. Membrane depolarization by 51 mM KCl, which stimulates Ca(2+) entry from the extracellular space, caused maintained increase in [Ca(2+)](i) to 292 +/- 12 nM and 31 +/- 2% contraction. The maintained Phe- and KCl-induced [Ca(2+)](i) and contractions were reduced in pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. Phe- and KCl-induced contraction and [Ca(2+)](i) were not significantly different between untreated and L-NAME-treated virgin rats or between untreated and L-NAME + L-arginine treated pregnant rats. In Ca(2+)-free Hanks', application of Phe or caffeine (10 mM), to stimulate Ca(2+) release from the intracellular stores, caused a transient increase in [Ca(2+)](i) and a small cell contraction that were not significantly different among the different groups. Thus renal interlobular smooth muscle of normal pregnant rats exhibits reduction in [Ca(2+)](i) signaling that involves Ca(2+) entry from the extracellular space but not Ca(2+) release from the intracellular stores. The reduced renal smooth muscle cell contraction and [Ca(2+)](i) in pregnant rats may explain the decreased renal vascular resistance associated with normal pregnancy, whereas the enhanced cell contraction and [Ca(2+)](i) during inhibition of NO synthesis in pregnant rats may, in part, explain the increased renal vascular resistance associated with PIH.     

The role of Ca2+ in the contractility of rabbit small intestine in vitro.

This study evaluated the role of Ca2+ in spontaneous and ACh- and KCl-induced contractions in longitudinal and circular smooth muscle from rabbit small intestine in vitro. In the first experiment, the amplitude, frequency and tone of spontaneous contractions in longitudinal and circular smooth muscle of small intestine were determined and, in the second experiment, the ACh- and KCl-induced responses of longitudinal and circular smooth muscle were measured. Atropine and guanethidine reduced the amplitude and tone of contractions in longitudinal and circular muscle, but reduced the frequency of contractions in circular muscle, only. TTX attenuated the amplitude of contractions and decreased the tone of contractions in longitudinal muscle, but increased the tone in circular muscle. Ca2+-free solutions, verapamil, nifedipine and caffeine diminished the three parameters of spontaneous contractions. Thapsigargin and cyclopiazonic acid increased the amplitude and tone of contractions in ileum longitudinal muscle, only, and cyclopiazonic acid increased the amplitude of contractions in circular muscle. Ca2+-free solutions, verapamil, nifedipine, thapsigargin, cyclopiazonic acid, and caffeine diminished ACh- and KCl-induced contractions. Those results suggest that extracellular Ca2+ plays a role in spontaneous contractions, and extracellular and intracellular Ca2+ participate in the ACh- and KCl-induced contractions of rabbit small intestine.     

Tianeptine's effects on spontaneous and Ca2+-induced uterine smooth muscle contraction

Tianeptine is a novel anti-depressant with an efficacy equivalent to that of classical anti-depressants. Additional beneficial effects include neuroprotection, anti-stress and anti-ulcer properties whose molecular mechanisms are still not completely understood but may involve changes in the anti-oxidant defence system. Herein, we have studied the effects of tianeptine on both contractile activity of isolated rat uteri and components of the endogenous anti-oxidative defence system. Tianeptine-induced dose-dependent inhibition of both spontaneous and Ca2+-induced contraction of uterine smooth muscle. The effect was more pronounced in the latter. Tianeptine treatment increased glutathione peroxidase (GSH-Px) and catalase (CAT) activities in spontaneous and Ca2+-stimulated uteri. A significant decrease in glutathione-reductase (GR) activity in both spontaneous and Ca2+-induced uterine contractions after tianeptine treatment indicated a reduction in reduced glutathione and consequently a shift toward a more oxidised state in the treated uteri. In spontaneously contracting uteri, tianeptine caused a decrease in copper-zinc SOD (CuZnSOD) activity. Tianeptine's anti-depressant effects may be accomplished by triggering a cascade of cellular adaptations including inhibition of smooth muscle contractility and an adequate anti-oxidative protection response.     

Novel role for K+-dependent Na+/Ca2+ exchangers in regulation of cytoplasmic free Ca2+ and contractility in arterial smooth muscle

Cytoplasmic free Ca2+ ([Ca2+]cyt) is essential for the contraction and relaxation of blood vessels. The role of plasma membrane Na+/Ca2+ exchange (NCX) activity in the regulation of vascular Ca2+ homeostasis was previously ascribed to the NCX1 protein. However, recent studies suggest that a relatively newly discovered K+-dependent Na+/Ca2+ exchanger, NCKX (gene family SLC24), is also present in vascular smooth muscle. The purpose of the present study was to identify the expression and function of NCKX in arteries. mRNA encoding NCKX3 and NCKX4 was demonstrated by RT-PCR and Northern blot in both rat mesenteric and aortic smooth muscle. NCXK3 and NCKX4 proteins were also demonstrated by immunoblot and immunofluorescence. After voltage-gated Ca2+ channels, store-operated Ca2+ channels, and Na+ pump were pharmacologically blocked, when the extracellular Na+ was replaced with Li+ (0 Na+) to induce reverse mode (Ca2+ entry) activity of Na+/Ca2+ exchangers, a large increase in [Ca2+]cyt signal was observed in primary cultured aortic smooth muscle cells. About one-half of this [Ca2+]cyt signal depended on the extracellular K+. In addition, after the activity of NCX was inhibited by KB-R7943, Na+ replacement-induced Ca2+ entry was absolutely dependent on extracellular K+. In arterial rings denuded of endothelium, a significant fraction of the phenylephrine-induced and nifedipine-resistant aortic or mesenteric contraction could be prevented by removal of extracellular K+. Taken together, these data provide strong evidence for the expression of NCKX proteins in the vascular smooth muscle and their novel role in mediating agonist-stimulated [Ca2+]cyt and thereby vascular tone.     

Mechanisms of the Ba2+-induced contraction in smooth muscle cells of the rabbit mesenteric artery

The mechanism of the Ba2+-induced contraction was investigated using intact and saponin-treated skinned smooth muscle (skinned muscle) strips of the rabbit mesenteric artery. After depletion of Ca2+ stored in the caffeine-sensitive site, greater than 0.65 mM Ba2+ evoked contraction in muscle strips depolarized with 128 mM K+ in Ca2+-free solution in a dose-dependent fashion, and the ED50 values for Ca2+ and Ba2+ were 0.5 mM and 1.2 mM in intact muscle strips, respectively. Nisoldipine (10 nM) blocked the contraction evoked by high K+ or 10 microM norepinephrine (NE) in the presence of 2.6 mM Ba2+, but did not block the contraction evoked in the presence of 2.6 mM Ca2+. These results may indicate that Ba2+ permeates the voltage-dependent Ca2+ channel. In skinned muscle strips, the ED50 values for Ca2+ and Ba2+ were 0.34 and 90 microM, respectively, as estimated from the pCa- and pBa-tension relationships. Calmodulin enhanced and trifluoperazine inhibited the Ba2+- and Ca2+-induced contractions. After the application of Ba2+ or Ca2+ with ATP gamma S in rigor solution, myosin light chain (MLC) was irreversibly thiophosphorylated, as estimated from the Ba2+- or Ca2+-independent contraction. Furthermore, both divalent cations phosphorylated MLC, as measured using two-dimensional gel electrophoresis, to the extent expected from the amplitudes of the contraction evoked by these cations. Thus, Ba2+ is capable of activating the contractile proteins as Ca2+ does. The amount of Ca2+ or Ba2+ stored in cells was estimated from the caffeine response evoked in Ca2+-free solution in intact and skinned muscle strips. After the application of 0.3 microM Ca2+ or 0.1 mM Ba2+ for 60 s to skinned muscle strips after the depletion of Ca2+ stored in cells, caffeine produced a contraction only upon pretreatment with Ca2+ but not with Ba2+. When Ba2+ was applied successively just after the application of Ca2+, the subsequently evoked caffeine-induced contraction was much smaller than that evoked by pretreatment with Ca2+ alone. The above results indicate that Ba2+ permeates the voltage-dependent Ca2+ channel but may not permeate the receptor-operated Ca2+ channel, it releases Ca2+ from store sites but is not accumulated into the store site, and it directly activates the contractile proteins via formation of a Ba2+-calmodulin complex.     

Calcium release in smooth muscle

In smooth muscle, maintenance of the contractile response is due to Ca2+ influx through two types of Ca2+ channel, a voltage-dependent Ca2+ channel and a receptor-linked Ca2+ channel. However, a more transient contraction can be obtained by release of Ca2+ from a cellular store, possibly the sarcoplasmic reticulum. In spike generating smooth muscle (e.g., guinea-pig taenia caeci), spike discharges may trigger the release of cellular Ca2+ by activating a Ca2+-induced Ca2+ release mechanism. Caffeine directly activates this mechanism in the absence of a triggered Ca2+ influx. In contrast to this, maintained depolarization may not only release but also refill the Ca2+ store. Drug-receptor interactions also release Ca2+ from a cellular store. This release may be elicited with inositol trisphosphate produced by receptor-linked phosphoinositide turnover. In non-spike generating smooth muscle (e.g., rabbit thoracic aorta), maintained membrane depolarization does not release but, instead, fills the Ca2+ store. However, caffeine and receptor-agonists release the Ca2+ store - possibly by activating the Ca2+-induced Ca2+ release mechanism and phosphoinositide turnover, respectively. The Ca2+ store in smooth muscle is filled by Ca2+ entry through voltage dependent Ca2+ channels and also by resting Ca2+ influx in the absence of receptor-agonists. The Ca2+ entering the cells through these pathways may be accumulated by the Ca2+ store and may activate the contractile filaments.     

Interstitial cells in the vasculature

Interstitial cells of Cajal are believed to play an important role in gastrointestinal tissues by generating and propagating electrical slow waves to gastrointestinal muscles and/or mediating signals from the enteric nervous system. Recently cells with similar morphological characteristics have been found in the wall of blood vessels such as rabbit portal vein and guinea pig mesenteric artery. These non-contractile cells are characterised by the presence of numerous processes and were easily detected in the wall of the rabbit portal vein by staining with methylene blue or by antibodies to the marker of Interstitial Cells of Cajal c-kit. These vascular cells have been termed "interstitial cells" by analogy with interstitial cells found in the gastrointestinal tract. Freshly dispersed interstitial cells from rabbit portal vein and guinea pig mesenteric artery displayed various Ca2+-release events from endo/sarcoplasmic reticulum including fast localised Ca2+ transients (Ca2+ sparks) and longer and slower Ca2+ events. Single interstitial cells from the rabbit portal vein, which is a spontaneously active vessel, also demonstrated rhythmical Ca2+ oscillations associated with membrane depolarisations, which suggests that in this vessel interstitial cells may act as pacemakers for smooth muscle cells. The function of interstitial cells from the mesenteric arteries is yet unknown. This article reviews some of the recent findings regarding interstitial cells from blood vessels obtained by our laboratory using electron microscopy, immunohistochemistry, tight-seal patch-clamp recording, and fluorescence confocal imaging techniques.     

Role of Cl- current in endothelin-1-induced contraction in rabbit basilar artery

Cl- efflux induces depolarization and contraction of smooth muscle cells. This study was undertaken to explore the role of Cl- channels in endothelin-1 (ET-1)-induced contraction in rabbit basilar artery. Male New Zealand White rabbits (n = 26), weighing 1.8-2.5 kg, were euthanized by an overdose of pentobarbital. The basilar arteries were removed for isometric tension recording. ET-1 produced a concentration-dependent contraction of the rabbit basilar artery in the normal Cl- Krebs-Henseleit bicarbonate buffer (123 mM Cl-). The ET-1-induced contraction was reduced by the following manipulations: 1) inhibition of Na+-K+-2Cl- cotransporter with bumetanide (3 x 10(-5) and 10(-4) M), 2) bicarbonate-free solution to disable Cl-/HCO exchanger, and 3) preincubation of rings with the Cl- channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and indanyloxyacetic acid 94. The ET-1-induced contraction was enhanced by substitution of extracellular Cl- (10 mM) with methanesulfonic acid (113 mM). Cl- channels are involved in ET-1-induced contraction in the rabbit basilar artery.