Calcium channels in smooth muscle cells

In smooth muscle cells, the electrophysiological properties of potential-dependent calcium channels are similar to those described in other excitable cells. The calcium current is dependent on the extracellular calcium concentration; it is insensitive to external sodium removal and tetrodotoxin application. Other ions (Ba2+, Sr2+, Na+) can flow through the calcium channel. This channel is blocked by Mn2+, Co2+, Cd2+ and by organic inhibitors. The inactivation mechanism is mediated by both the membrane potential and the calcium influx. Ca2+ ions can also penetrate into the cell through receptor-operated channels. These channels show a low ionic selectivity and are generally less sensitive to organic Ca-blockers than the potential-dependent calcium channels. The finding of specific channel inhibitors as well as the study of the biochemical pathways between receptor activation and channel opening are prerequisites to further characterization of receptor-operated channels.     

Voltage-dependent anion-selective channels in cultured smooth muscle cells of the rat aorta

1. Properties of the voltage-dependent anion-selective channel in cultured smooth muscle cells of the rat aorta were studied using the patch-clamp technique. 2. The channel had a single channel conductance of 346 +/- 4 pS (n = 43, mean +/- SEM) with symmetrical 142 mM-Cl- solution in inside-out patch configurations. 3. The channel was activated spontaneously at a potential range -20 approximately +20 mV and inactivated more rapidly with increases to more positive or negative potentials. 4. The channel was selective for anions and the permeability ratio for monovalent anion was Br-:Cl-:HCOO-:CH3COO-:propionate-:aspartate- = 1.1:1:0.7:0.4: less than 0.02: less than 0.02. 5. The openings of the channels were observed more frequently in inside-out membrane patches than in cell-attached ones, and were independent of intracellular free Ca concentrations. 6. The density of this channel was estimated to be 1.3/micron2. 7. Physiological roles of the channel were discussed.     

Calcium influx factor directly activates store-operated cation channels in vascular smooth muscle cells

Recently, we described a novel 3-pS Ca(2+)-conducting channel that is activated by BAPTA and thapsigargin-induced passive depletion of intracellular Ca(2+) stores and likely to be a native store-operated channel in vascular smooth muscle cells (SMC). Neither Ca(2+) nor inositol 1,4,5-trisphosphate or other second messengers tested activated this channel in membrane patches excised from resting SMC. Here we report that these 3-pS channels are activated in inside-out membrane patches from SMC immediately upon application of Ca(2+) influx factor (CIF) extracted from mutant yeast, which has been previously shown to activate Ca(2+) influx in Xenopus oocytes and Ca(2+) release-activated Ca(2+) current in Jurkat cells. In bioassay experiments depletion of Ca(2+) stores in permeabilized human platelets resulted in the release of endogenous factor, which activated 3-pS channels in isolated inside-out membrane patches excised from SMC and exposed to permeabilized platelets. The same 3-pS channels in excised membrane patches were also activated by acid extracts of CIF derived from human platelets with depleted Ca(2+) stores, which also stimulated Ca(2+) influx upon injection into Xenopus oocytes. Specific high pressure liquid chromatography fractions of platelet extracts were found to have CIF activity when injected into oocytes and activate 3-pS channels in excised membrane patches. These data show for the first time that CIF produced by mammalian cells and yeast with depleted Ca(2+) stores directly activates native 3-pS cation channels, which in intact SMC are activated by Ca(2+) store depletion.     

平滑肌细胞上的钙库操纵性通道

钙库操纵性通道(SOC)是目前研究较热门的一种离子通道,其开放与关闭受内质网中Ca2 贮量调控。SOC参与机体许多重要生理功能的调节,尤其在平滑肌紧张性变化的调节中起重要作用。果蝇瞬时受体电位(transient receptor potential,TRP)蛋白在光信号传递中发挥重要作用,在哺乳动物中,发现TRP蛋白的同系物TRPC1蛋白是SOC的组成成分。研究并深入了解SOC的特性对于开发一类新的钙通道拮抗剂具有重要的理论意义。     

Calcium channel modulation by the dihydropyridine derivative Bay K 8644 in mammalian visceral smooth muscle cells

1. Modulation of Ca channels by the dihydropyridine Ca agonist Bay K 8644 in guinea-pig taenia coli smooth muscle cells was investigated using the patch clamp technique. 2. Single Ca channel activity was obtained from cell-attached patch recordings with the use of pipettes filled with 50 mM Ba. Bath application of the drug markedly increased the opening probability of Ca channels. 3. The effect was found to be due to an increase in the mean opening times of Ca channels. Due to this increase, the mean current reconstructed by averaging individual current trace responses was markedly increased in the presence of Bay K 8644.     

Distribution of vimentin and desmin filaments in smooth muscle tissue of mammalian and avian aorta

The presence of intermediate filament proteins in vascular tissue cells has been examined by immunofluorescence microscopy on frozen sections of the aortic wall of diverse vertebrates (rat, cow, human and chicken) and by gel electrophoresis of cytoskeletal proteins from whole aortic tissue or from stripped tunica media of cow and man. Most cells of the aortic wall in these species contain vimentin filaments, including smoooth muscle cells of the tunica media. In addition, we have observed aortic cells that are positively stained by antibodies to desmin. The presence of desmin in aortic tissue has also been demonstrated by gel electrophoresis for rat, cow and chicken. In aortic tissue some smooth muscle cells contain both types of intermediate filament proteins, vimentin and desmin. Bovine aorta contains, besides cells in which vimentin and desmin seem to co-exist, distinct bundles of smooth muscle cells, located in outer regions of the tunica media, which contain only desmin. The results suggest that (i) intermediate-sized filaments of both kinds, desmin and vimentin, can occur in vascular smooth muscle in situ and (ii) smooth muscle cells of the vascular system are heterogeneous and can be distinguished by their intermediate filament proteins. The finding of different vascular smooth muscle cells is discussed in relation to development and differentiation of the vascular system.     

Calcium incorporation by smooth muscle microsomes.

The purpose of the present work was to study the factors influencing calcium incorporation into a microsomal fraction prepared from the longitudinal smooth muscle of the guinea-pig ileum. Calcium incorporation required the presence of both ATP and Mg2+ and was unaffected by azide. It was enhanced by oxalate; this effect was pH dependent and it was maximal at pH 6.6. The relation between calcium uptake with oxalate and free Ca2+ concentration in the medium was represented by a curve with an optimum for Ca2+ equal to 3-10-5 M. The threshold concentration was comprised between 5-10-7 and 10-6 7. The optimum calcium uptake rate was 4.5 nmol Ca2+/mg protein per min. In the absence of oxalate, two distinct groups of binding sites were identified. Low affinity sites had a binding constant of 7-104 M-1 and a maximum binding capacity of 0.6-106 M-1 and a binding capacity of 33 nmol Ca2+/mg protein; their capacity was sensitive to pH changes. In the absence of oxalate, Ca2+ binding was depressed by Na+ with respect to K+ or choline. When the medium was supplemented with oxalate, the stimulation of 45Ca incorporation was barely detectable in the presence of choline+ and it was lower in a medium containing Na+ instead of K+. The subcellular distribution profiles of calcium incorporation with and without oxalate indicate the microsomal location of both activities. However, the oxalate-stimulated calcium uptake activity sedimented faster than the calcium binding activity. The subcellular distribution of marker enzyme actvities has been examined. The present results indicate that Ca2+ incorporations with and without oxalate are the result of two processes likely related to two different structures. The role of microsomal calcium uptake in excitation-contraction coupling and its modification by the activity of the sodium pump is discussed.     

Store-operated Ca-permeable non-selective cation channels in smooth muscle cells

Over twenty years ago it was shown that depletion of the intracellular Ca²⁺ store in smooth muscle triggered a Ca²⁺ influx mechanism. The purpose of this review it to describe recent electrophysiological data which indicate that Ca²⁺ influx occurs through discrete ion channels in the plasmalemma of smooth muscle cells. The effect of external Ca²⁺ on the amplitude and reversal potential of whole-cell and single channel currents suggests that there are at least two, and probably more, distinct store-operated channels (SOCs) which have markedly different permeabilities to Ca²⁺ ions. Two activation mechanisms have been identified which involve Ca²⁺ influx factor and protein kinase C (PKC) activation via diacylglycerol. In addition, in rabbit portal vein cells there is evidence that stimulation of α-adrenoceptors can stimulate SOC opening via PKC in a store-independent manner. There is at present little knowledge on the molecular identity of SOCs but it has been proposed that TRPC1 may be a component of the functional channel. We also summarise the data showing that SOCs may be involved in contraction and cell proliferation of smooth muscle. Finally, we highlight the similarities and differences of SOCs and receptor-operated cation channels that are present in native rabbit portal vein myocytes.     

Muscarinic effects on ion channels in smooth muscle cells

Acetylcholine is the most important excitatory neurotransmitter providing depolarization of the membrane and contraction of different smooth muscle cells due to activation of the muscarinic receptors. In our review, we analyze and summarize the published data on the effects of activation of acetylcholine muscarinic receptors on ion channels expressed in smooth muscle cells of different organs and the results of our own studies of this topic. Special attention is paid to the mechanisms of depolarizing effects of acetylcholine mediated by activation of non-selective cationic channels. Intracellular mechanisms underlying modulating influences on calcium, potassium, and chloride channels are also analyzed. Physiological roles of activation and regulation of different ion channels and possible interactions within this complicated system are discussed.     

Calcium and contractions of isolated smooth muscle cells from rat myometrium

Smooth muscle cells were isolated from estrogenized rat myometrium by collagenase digestion. Electron microscopic examination and measurement of cell lengths by image-splitting micrometry were carried out after fixation with acrolein. Mean lengths of cells before and after isolation were 81.7 and 66.9 micron, respectively. Responses of cells were compared with contractions of isolated strips recorded isometrically. Effects of carbachol and KCl were examined in 2 mM Ca, 2 mM Ca + 4 mM EGTA, and 2 mM Ca + 10(-8) M nitrendipine solution. Carbachol and KCl produced concentration-dependent shortening of isolated cells maximal at 30 s after addition. The concentrations of carbachol required to produce shortenings were about 100-fold less than those required to produce isometric contractions; but no major difference was observed in the concentration dependence of cell shortening and isometric contraction produced by potassium-induced depolarization. In 2 mM Ca solution, there was a phasic response, followed by a tonic response such that more than 50% of maximum cell shortening was maintained for 10 min. However, in 2 mM Ca + 4 mM EGTA or 10(-8) M nitrendipine, the tonic contraction was abolished and cells rapidly relaxed after 30 s. If carbachol was added to cells after varying times in the EGTA-containing solution, the ability to initiate a contraction declined exponentially with a half-time of 160 s. Effects of depolarization by KCl were examined in 2 mM Ca plus nitrendipine and 2 mM Ca + 4 mM EGTA solution. Shortening occurred in 2 mM Ca solution by depolarization but not if nitrendipine was added. Though shortening was not observed in 2 mM Ca + 4 mM EGTA solution by KCl, subsequent addition of carbachol induced shortening. These results suggested that there was an intracellular Ca store site from which Ca was released by carbachol and which was not affected by depolarization in the absence of external Ca. No evidence was obtained that the contraction persists in Ca2+-free medium in isolated cells, which is in agreement with previous findings in small muscle strips in which only a similar transient response was obtained.     

Metabolism of glycosaminoglycans in cultured smooth muscle cells from pig aorta

Arterial wall smooth muscle cells, originating from the inner layer (media) of pig aortas, were grown in culture. The synthesis and secretion of proteoglycans by these cells were investigated. These cells were incubated in the presence of [³⁵S] sulfate or [¹⁴C] glucosamine and these precursors incorporation into glycosaminoglycans was followed.Proteoglycans synthesized by media cells exhibit different glycosaminoglycan distribution patterns according to their localization. The glycosaminoglycan components are largely confined to the medium (80 per cent) and exhibit a distribution pattern that ressembles closely that found in pig aorta tissue. In comparison with the extracellular and intracellular pools, the pericellular pool (trypsin released material) contains proportionally more heparan sulfate.Isotopic chase experiments demonstrated that glycosaminoglycans leave the intracellular and pericellular compartments with initial half-lives of 7 – 8 h and 13 – 14 h, respectively.About half of the labelled glycosaminoglycans was released into the medium, in an apparently undegraded form, while the rest was degraded.The production of proteoglycans is not affected by modifying the exogenous concentration of hyaluronic acid or chondroitin sulfate present in the culture medium. The synthesis of proteoglycans, but not their secretion is inhibited with cytochalasin-B, a microfilament modifying agent. The secretion of proteoglycans and also — in part — their synthesis is inhibited by antimicrotubular agents: colchicine and vinblastine, with observed intracellular accumulation of proteoglycans.These data suggest that, in arterial cells, the intracellular movement of proteoglycans during the secretory process is mediated by microtubular elements.In conclusion, our results provide evidence for the responsiveness of cultured mediacytes to antimicrotubular and antimicrofilamentar drugs, the utilization of which allows modification in the metabolism and secretion of arterial proteoglycans.     

Calcium sparks in smooth muscle

Local intracellular Ca²⁺transients, termed Ca²⁺ sparks, are caused by thecoordinated opening of a cluster of ryanodine-sensitive Ca²⁺ release channels in the sarcoplasmic reticulum ofsmooth muscle cells. Ca²⁺ sparks are activated byCa²⁺ entry through dihydropyridine-sensitivevoltage-dependent Ca²⁺ channels, although the precisemechanisms of communication of Ca²⁺ entry toCa²⁺ spark activation are not clear in smooth muscle.Ca²⁺ sparks act as a positive-feedback element to increasesmooth muscle contractility, directly by contributing to the globalcytoplasmic Ca²⁺ concentration([Ca²⁺]) and indirectly by increasingCa²⁺ entry through membrane potential depolarization,caused by activation of Ca²⁺ spark-activatedCl channels. Ca²⁺ sparks also have aprofound negative-feedback effect on contractility by decreasingCa²⁺ entry through membrane potential hyperpolarization,caused by activation of large-conductance, Ca²⁺-sensitiveK⁺ channels. In this review, the roles of Ca²⁺sparks in positive- and negative-feedback regulation of smooth musclefunction are explored. We also propose that frequency and amplitudemodulation of Ca²⁺ sparks by contractile and relaxantagents is an important mechanism to regulate smooth muscle function.