Effects of hirsutine, an antihypertensive indole alkaloid from Uncaria rhynchophylla, on intracellular calcium in rat thoracic aorta.

The effects of hirsutine, an indole alkaloid from Uncaria rhynchophylla (MIQ.) Jackson, on cytosolic Ca2+ level ([Ca2+]cyt) were studied by using fura-2-Ca2+ fluorescence in smooth muscle of the isolated rat aorta. Noradrenaline and high K+ solution produced a sustained increase in [Ca2+]cyt. Application of hirsutine after the increases in [Ca2+]cyt induced by noradrenaline and high K+ notably decreased [Ca2+]cyt, suggesting that hirsutine inhibits Ca2+ influx mainly through a voltage-dependent Ca2+ channel. Furthermore, the effect of hirsutine on intracellular Ca2+ store was studied by using contractile responses to caffeine under the Ca(2+)-free nutrient condition in the rat aorta. When hirsutine was added at 30 microM before caffeine treatment, the agent slightly but significantly reduced the caffeine-induced contraction. When added during Ca2+ loading, hirsutine definitely augmented the contractile response to caffeine. These results suggest that hirsutine inhibits Ca2+ release from the Ca2+ store and increases Ca2+ uptake into the Ca2+ store, leading to a reduction of intracellular Ca2+ level. It is concluded that hirsutine reduces intracellular Ca2+ level through its effect on the Ca2+ store as well as through its effect on the voltage-dependent Ca2+ channel.     

Ca2+ influx mediates enhanced alpha2-adrenergic contraction in aortas from rats treated with NOS inhibitor

Previously, we reported that aortic segments from rats made hypertensive with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NNA) exhibit enhanced contractile sensitivity to both alpha2-adrenergic receptor (alpha2-AR) stimulation and to KCl-induced depolarization. We hypothesized that increased contractile responses to these agents was due to a change in the common effector L-type voltage-dependent calcium channel (VDCC). In aortic segments from control and L-NNA-treated rats, contraction to the alpha2-AR agonist UK-14304 stimulated Ca2+ influx but released intracellular Ca2+ only in control arteries. UK-14304-induced contraction was blocked by the VDCC antagonist nifedipine in both control and L-NNA aortas but contraction of aortas from L-NNA-treated rats was blocked by lower concentrations. Calcium imaging studies in fura 2-loaded freshly isolated aortic vascular smooth muscle cells also demonstrated UK-14304-stimulated Ca2+ influx sensitive to nifedipine only in cells from L-NNA-treated rats. We conclude that alpha2-AR contraction in the rat aorta is mediated primarily by Ca2+ influx and that L-NNA-induced hypertension increases the dependence of this contraction on VDCCs.     

Ca2+ release and contraction induced by IP3 and contractile agonists in mammalian gastric smooth muscle

The source, time course and stoichiometry of cytosolic free Ca²⁺ ([Ca²⁺]_i) during contraction were examined in smooth muscle cells isolated from the guinea pig and human stomach. Contraction by receptor-linked agonists (eg, acetylcholine, cholecystokinin octapeptide and Met-enkephalin) was preceded by stoichiometric increases in [Ca²⁺]_i and net ⁴⁵Ca²⁺ efflux that were maintained in the absence of extracellular Ca²⁺ or in the presence of a Ca²⁺ channel blocker (13600). The intracellular Ca²⁺ store could be depleted by repeated stimulation with all agonists in Ca²⁺-free medium or in the presence of 13600 resulting in loss of contractile response; response was restored by re-exposure of the cells to Ca²⁺.The source of intracellular Ca²⁺ an the signal for its release were examined in saponin-permeabilized muscle cells. The cells retained their ability to contract in response to receptor-linked agonists and developed an ability to contract in response to inositol trisphosphate (IP₃). The cells accumulated Ca²⁺ to the same extent as intact muscle cells, but only in the presence of ATP. IP₃ caused a prompt, concentration-dependent increase in contraction, [Ca²⁺]_i and net ⁴⁵Ca²⁺ efflux. These effects were maximally similar to those produced by CCK-8 alone or in combination with IP₃: Depletion of the Ca²⁺ store by repeated stimulation of single muscle cells in Ca²⁺-free medium with IP₃, acetylcholine or CCK-8 separately resulted in loss of contractile response to all three agents; the response was restored by re-exposure of the muscle cell to a cytosol-like perfusate (Ca²⁺ 180 nM).The studies demonstrate that a product of membrane phosphoinositide hydrolysis is capable of mobilizing Ca²⁺ from a depletable, non-mitochondrial intracellular store that is utilized by receptor-linked agonists. The magnitude of IP₃-induced Ca²⁺ release is correlated with contraction.     

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.     

Stoichiometry of contraction and Ca2+ mobilization by inositol 1,4,5-trisphosphate in isolated gastric smooth muscle cells

Smooth muscle cells were isolated from the circular muscle layer of guinea pig stomach and permeabilized by brief exposure to saponin. Both permeabilized and intact muscle cells contracted in response to cholecystokinin octapeptide (CCK-8) and acetylcholine, but only permeabilized muscle cells contracted in response to inositol 1,4,5-trisphosphate (InsP3). The contractile response to InsP3 was prompt (peak less than 5 s), concentration-dependent (EC50-0.3 microM), and insensitive to antimycin or oligomycin. Contraction induced by either InsP3 or CCK-8 was accompanied by a concentration-dependent increase in free Ca2+ that was directly correlated with the magnitude of contraction. Both InsP3 and CCK-8 caused rapid net efflux of Ca2+ from cells preloaded with 45Ca2+. Contraction, increase in free Ca2+ concentration, and net 45Ca2+ efflux elicited by a combination of maximal concentrations of InsP3 and CCK-8 were not significantly different from those elicited by maximal concentrations of either agent alone. Repeated stimulation of single muscle cells with either InsP3 or CCK-8 in Ca2+-free medium caused eventual loss of the contractile response to all agents. The response to all agents was restored upon re-exposure of the cell to a cytosol-like concentration of Ca2+, implying equal access of InsP3 and receptor-linked agonists to the same intracellular Ca2+ store. The results demonstrate that InsP3 mimics the effects of receptor-linked agonists on contraction and mobilization of intracellular Ca2+ in permeabilized smooth muscle cells that retain the functional properties of intact smooth muscle cells and support a role for InsP3 as membrane-derived messenger responsible for mobilization of intracellular Ca2+ in smooth muscle cells.     

Role of calcium on the contraction induced by potassium and norepinephrine in the sheep rumen

The effect of calcium on the contractile responses induced by high K+ solutions and noradrenaline has been investigated Ca2+-free-solutions and two selective antagonists of calcium channels (verapamil and sodium nitroprusside) have been used. Both types of responses were inhibited by Ca2+-free-solutions. Contractions induced by high K+ solutions were inhibited by verapamil, but not by sodium nitroprusside. However, the responses to noradrenaline were specifically inhibited by sodium nitroprusside. These results suggest that in rumen circular smooth muscle of the sheep there are two types of calcium channels, a voltage-dependent Ca2+ channel and receptor-linked Ca2+ channel.     

Origin and mechanisms of Ca2+ waves in smooth muscle as revealed by localized photolysis of caged inositol 1,4,5-trisphosphate

The cytosolic Ca(2+) concentration ([Ca(2+)](c)) controls diverse cellular events via various Ca(2+) signaling patterns; the latter are influenced by the method of cell activation. Here, in single-voltage clamped smooth muscle cells, sarcolemma depolarization generated uniform increases in [Ca(2+)](c) throughout the cell entirely by Ca(2+) influx. On the other hand, the Ca(2+) signal produced by InsP(3)-generating agonists was a propagated wave. Using localized uncaged InsP(3), the forward movement of the Ca(2+) wave arose from Ca(2+)-induced Ca(2+) release at the InsP(3) receptor (InsP(3)R) without ryanodine receptor involvement. The decline in [Ca(2+)](c) (the back of the wave) occurred from a functional compartmentalization of the store, which rendered the site of InsP(3)-mediated Ca(2+) release, and only this site, refractory to the phosphoinositide. The functional compartmentalization arose by a localized feedback deactivation of InsP(3) receptors produced by an increased [Ca(2+)](c) rather than a reduced luminal [Ca(2+)] or an increased cytoplasmic [InsP(3)]. The deactivation of the InsP(3) receptor was delayed in onset, compared with the time of the rise in [Ca(2+)](c), persisted (>30 s) even when [Ca(2+)](c) had regained resting levels, and was not prevented by kinase or phosphatase inhibitors. Thus different forms of cell activation generate distinct Ca(2+) signaling patterns in smooth muscle. Sarcolemma Ca(2+) entry increases [Ca(2+)](c) uniformly; agonists activate InsP(3)R and produce Ca(2+) waves. Waves progress by Ca(2+)-induced Ca(2+) release at InsP(3)R, and persistent Ca(2+)-dependent inhibition of InsP(3)R accounts for the decline in [Ca(2+)](c) at the back of the wave.     

Agonist-induced calcium transients in cultured smooth muscle cells: measurements with fura-2 loaded monolayers

Elevation of cytosolic Ca2+ in response to depolarization and various receptor agonists was measured in several types of cultured smooth muscle cells (DDT1, A10, rabbit aorta) loaded with the either quin-2 or fura-2, and assayed either in suspension or in monolayer cultures attached to plastic cover slips. Agonists (norepinephrine, vasopressin) induced both the release of intracellular Ca2+ and the influx of extracellular Ca2+. Agonist-induced Ca2+ influx was not blocked by dihydropyridines, and depolarization did not induce Ca2+ influx. However, in fura-2 loaded monolayers of PC12 cells, depolarization did induce dihydropyridine-sensitive Ca2+ influx. Thus cultured smooth muscle cells appear to express receptor-operated Ca2+ channels, but not functional voltage-operated Ca2+ channels.     

Subtype specificity of the ryanodine receptor for Ca2+ signal amplification in excitation-contraction coupling.

In excitable cells membrane depolarization is translated into intracellular Ca2+ signals. The ryanodine receptor (RyR) amplifies the Ca2+ signal by releasing Ca2+ from the intracellular Ca2+ store upon receipt of a message from the dihydropyridine receptor (DHPR) on the plasma membrane in striated muscle. There are two distinct mechanisms for the amplification of Ca2+ signalling. In cardiac cells depolarization-dependent Ca2+ influx through DHPR triggers Ca2+-induced Ca2+ release via RyR, while in skeletal muscle cells a voltage-induced change in DHPR is thought to be mechanically transmitted, without a requirement for Ca2+ influx, to RyR to cause it to open. In expression experiments using mutant skeletal myocytes lacking an intrinsic subtype of RyR (RyR-1), we demonstrate that RyR-1, but not the cardiac subtype (RyR-2), is capable of supporting skeletal muscle-type coupling. Furthermore, when RyR-2 was expressed in skeletal myocytes, we observed depolarization-independent spontaneous Ca2+ waves and oscillations, which suggests that RyR-2 is prone to regenerative Ca2+ release responses. These results demonstrate functional diversity among RyR subtypes and indicate that the subtype of RyR is the key to Ca2+ signal amplification.     

Vasopressin increases 45Ca2+ influx in rat aortic smooth muscle cells

[Arg8]Vasopressin (AVP)-induced 45Ca2+ influx was examined in vascular smooth muscle cells derived from rat aorta. AVP stimulated the 45Ca2+ influx in a concentration-dependent manner. The effect was abolished in the presence of La3+. The dihydropyridine calcium channel antagonist darodipine did not affect the AVP-induced influx of 45Ca2+. These data suggest that AVP stimulates in these cultured aortic smooth muscle cells a receptor-operated channel (ROC) that is permeable to Ca2+.     

Ca2+ influx stimulated by vasopressin is mediated by phosphoinositide hydrolysis in rat smooth muscle cells

The mechanism of Ca2+ influx stimulated by arginine vasopressin (AVP) was studied in cultured rat smooth muscle cells. AVP stimulated 45Ca2+ influx even in the presence of nifedipine, a Ca2+ antagonist that inhibits voltage-dependent Ca2+ channel. NaF, a GTP-binding protein activator, mimicked the AVP-stimulated 45Ca2+ influx. The 45Ca2+ influx stimulated by a combination of AVP and NaF was not additive. The affinity of AVP receptor was decreased by guanosine 5'-O-(3-thiotriphosphate). Pertussis toxin failed to affect the AVP-stimulated 45Ca2+ influx. AVP did not stimulate cAMP production, but increased inositol trisphosphate generation. Both AVP-stimulated 45Ca2+ influx and inositol trisphosphate generation were inhibited by neomycin, a phospholipase C inhibitor, in a dose-dependent manner, and the patterns of both inhibitions were similar. These results suggest that, in rat smooth muscle cells, AVP-stimulated Ca2+ influx is mediated exclusively through phosphoinositide hydrolysis.     

Mechanisms of integrin-mediated calcium signaling in MDCK cells: regulation of adhesion by IP3- and store-independent calcium influx.

Peptides containing Arg-Gly-Asp (RGD) immobilized on beads bind to integrins and trigger biphasic, transient increases in intracellular free Ca2+ ([Ca2+]i) in Madin-Darby canine kidney epithelial cells. The [Ca2+]i increase participates in feedback regulation of integrin-mediated adhesion in these cells. We examined influx pathways and inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ store release as possible sources of the [Ca2+]i rise. The RGD-induced [Ca2+]i response requires external Ca2+ (threshold approximately 150 microM), and its magnitude is proportional to extracellular calcium. RGD-induced transients were attenuated by Ca2+ channel inhibitors (Ni2+ and carboxy-amidotriazole) or by plasma membrane depolarization, indicating that Ca2+ influx contributes to the response. Loading cells with heparin reduced the size of RGD-induced [Ca2+]i transients, indicating that IP3-mediated release of Ca2+ from stores may also contribute to the RGD response. Depletion of Ca2+ stores with thapsigargin activated Ni(2+)-sensitive Ca2+ influx that might also be expected to occur after IP3-mediated depletion of stored Ca2-. However, RGD elicited a Ni(2+)-sensitive Ca2+ influx even after pretreatment with thapsigargin, indicating that Ca2+ influx is controlled by a mechanism independent of IP3-mediated store depletion. We conclude that RGD-induced [Ca2+]i transients in Madin-Darby canine kidney cells result primarily from the combination of two distinct mechanisms: 1) IP3-mediated release of intracellular stores, and 2) activation of a Ca2+ influx pathway regulated independently of IP3 and Ca2+ store release. Because Ni2+ and carboxy-amidotriazole inhibited adhesion, whereas store depletion with thapsigargin had little effect, we suggest that the Ca2+ influx mechanism is most important for feedback regulation of integrin-mediated adhesion by increased [Ca2+]i.     

Modulation of electrical activity in gastrointestinal smooth muscle by peptides.

A rise in intracellular calcium is the predominant signal that leads to the activation of the contractile machinery in gastrointestinal smooth muscle. The primary sources of activating calcium are illustrated in Fig. 2. Voltage- and peptide-mediated release of intracellular calcium contribute to activation of some gastrointestinal smooth muscles. However, the primary source of activating calcium appears to be an influx of calcium across the plasma membrane. The degree of modulation of electrical activity by peptides varies depending upon the region of the gastrointestinal tract studied. Second messenger systems are undoubtly involved in the transduction pathway for receptor-mediated changes in ion channel activity in gastrointestinal smooth muscle. However, in comparison to other excitable cell types, little is known about the coupling mechanisms whereby peptide-receptor binding alters ion channel activity in gastrointestinal smooth muscle. This represents one of the challenging areas to be studied in the field of gastrointestinal smooth muscle. One disease in which a better appreciation of the regulation of ion channel activity could lead to therapeutic benefit is irritable bowel syndrome. A coupling of smooth muscle electrical activity to hypermotility in irritable bowel syndrome has been reported. CCK increases the level of spike activity which triggers hypermotility [40]. It would follow that inhibition of calcium influx should reduce spiking and, therefore, hypermotility. In fact, the calcium channel blockers nifedipine and nicardipine have been shown to decrease colonic motility in irritable bowel syndrome patients [62-64]. As our understanding of gastrointestinal smooth muscle ion channels expands, development of a gastrointestinal selective calcium channel blocker may be possible. This class of agents would be effective in the treatment of irritable bowel syndrome and potentially other peptide-related spastic smooth muscle disorders.     

Postsynaptic alpha adrenoceptors on vascular smooth muscle

A heterogeneous population of alpha adrenoceptors mediates vasoconstriction in the canine saphenous vein (CSV). Studies with isolated strips of venous smooth muscle incubated with selective alpha-adrenoceptor agonists and antagonists revealed that both alpha 1 and alpha 2 adrenoceptors exist independently in this tissue and both subtypes mediate a contractile response. Measurement of contractile responses in reduced or zero external calcium conditions indicates that stimulation of alpha 1 adrenoceptors induces contractions by influx of extracellular calcium and release of calcium from internal stores. In contrast, 45Ca uptake studies suggest that activation of the postsynaptic alpha 2 adrenoceptor produces vasoconstriction dependent only on influx of extracellular calcium. The influx of calcium produced by the selective alpha 2-adrenoceptor agonist BHT-920 is inhibited by calcium entry blockers. Measurements of transmembrane potentials from smooth muscle cells of the CSV suggest that alpha 1-adrenoceptor activation produces depolarization and contraction (electromechanical coupling) whereas alpha 2-adrenoceptor stimulation does not result in concentration-dependent depolarization of the smooth muscle cells (pharmacomechanical coupling).     

Resting calcium influx in airway smooth muscle

Plasma membrane Ca2+ leak remains the most uncertain of the cellular Ca2+ regulation pathways. During passive Ca2+ influx in non-stimulated smooth muscle cells, basal activity of constitutive Ca2+ channels seems to be involved. In vascular smooth muscle, the 3 following Ca2+ entry pathways contribute to this phenomenon: (i) via voltage-dependent Ca2+ channels, (ii) receptor gated Ca2+ channels, and (iii) store operated Ca2+ channels, although, in airway smooth muscle it seems only 2 passive Ca2+ influx pathways are implicated, one sensitive to SKF 96365 (receptor gated Ca2+ channels) and the other to Ni2+ (store operated Ca2+ channels). Resting Ca2+ entry could provide a sufficient amount of Ca2+ and contribute to resting intracellular Ca2+ concentration ([Ca2+]i), maintenance of the resting membrane potential, myogenic tone, and sarcoplasmic reticulum-Ca2+ refilling. However, further research, especially in airway smooth muscle, is required to better explore the physiological role of this passive Ca2+ influx pathway as it could be involved in airway hyperresponsiveness.     

Ion channels in smooth muscle: regulators of intracellular calcium and contractility

Smooth muscle (SM) is essential to all aspects of human physiology and, therefore, key to the maintenance of life. Ion channels expressed within SM cells regulate the membrane potential, intracellular Ca2+ concentration, and contractility of SM. Excitatory ion channels function to depolarize the membrane potential. These include nonselective cation channels that allow Na+ and Ca2+ to permeate into SM cells. The nonselective cation channel family includes tonically active channels (Icat), as well as channels activated by agonists, pressure-stretch, and intracellular Ca2+ store depletion. Cl--selective channels, activated by intracellular Ca2+ or stretch, also mediate SM depolarization. Plasma membrane depolarization in SM activates voltage-dependent Ca2+ channels that demonstrate a high Ca2+ selectivity and provide influx of contractile Ca2+. Ca2+ is also released from SM intracellular Ca2+ stores of the sarcoplasmic reticulum (SR) through ryanodine and inositol trisphosphate receptor Ca2+ channels. This is part of a negative feedback mechanism limiting contraction that occurs by the Ca2+-dependent activation of large-conductance K+ channels, which hyper polarize the plasma membrane. Unlike the well-defined contractile role of SR-released Ca2+ in skeletal and cardiac muscle, the literature suggests that in SM Ca2+ released from the SR functions to limit contractility. Depolarization-activated K+ chan nels, ATP-sensitive K+ channels, and inward rectifier K+ channels also hyperpolarize SM, favouring relaxation. The expression pattern, density, and biophysical properties of ion channels vary among SM types and are key determinants of electrical activity, contractility, and SM function.     

Store-operated Ca2+-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 alpha-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.     

Calcium signaling in restricted diffusion spaces.

One- and two-dimensional models of Ca2+ diffusion and regulation were developed and used to study the magnitudes and the spatial and temporal characteristics of the Ca2+ transients that are likely to develop in smooth muscle cells in restricted diffusion spaces between the plasma membrane and intracellular organelles. Simulations with the models showed that high [Ca2+] (on the order of several microM) can develop in such spaces and persist for 100-200 ms. These Ca2+ transients could: 1) facilitate the coupling of Ca2+ influx to intracellular Ca2+ release; 2) provide a mechanism for the regulation of stored Ca2+ that does not affect the contractile state of smooth muscle; 3) locally activate specific signal transduction pathways, before, or without activating other Ca2+ dependent pathways in the central cytoplasm of the cell. The latter possibility suggests that independent enzymatic processes in cells could be differentially regulated by the same intracellular second messenger.     

Excitation-contraction coupling in cardiac muscle revisited.

According to the current views the direct and indispensable source of Ca2+ activating contraction is sarcoplasmic reticulum (SR). Ca2+ is released from the SR when its release channels (ryanodine receptors) are activated by Ca2+ influx through the L-type Ca2+ channels (dihydropyridine receptors). In contrast, ryanodine receptors of skeletal muscles are activated by conformational changes in dihydropyridine receptors induced by sarcolemmal voltage. Ca2+ influx is not necessary for their activation. In this review the papers not quite conforming with the current views are referred to and discussed. Their results suggest that SR is not an indispensable source of contractile Ca2+ at least in some mammalian species, and that cardiac ryanodine receptors may be activated by conformational changes in dihydropyridine receptors without Ca2+ influx (like in skeletal muscle). This may be a mechanism parallel to or accessory to the Ca2+ induced release of Ca2+ (CIRC).     

Effects of Cl- channel blockers on endothelin-1-induced proliferation of rat vascular smooth muscle cells

The effects of Cl- channel blockers on endothelin-1 (ET-1)-induced proliferation of rat aortic vascular smooth muscle cells (VSMC) were examined. We found ET-1 concentration-dependently increased cell count and [3H]-thymidine incorporation into VSMC, with EC50 values of 24.8 and 11.4 nM, respectively. Both nifedipine and SK&F96365 inhibited 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC with the maximal inhibitory concentrations of 1 and 10 microM, respectively. DIDS inhibited 10 nM ET-1-induced increase in cell count and [3H]-thymidine incorporation into VSMC in a concentration-dependent manner, whereas other Cl- channel blockers including IAA-94, NPPB, DPC, SITS and furosemide did not produce these effects. 3 microM DIDS reduced 10 nM ET-1-induced sustained increase in cytoplasmic Ca2+ concentration ([Ca2+]) by 52%. Pretreatment of VSMC with 1 microM nifedipine completely inhibited the DIDS effect on 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC and sustained increase in [Ca2+]i, whereas pretreatment with 10 microM SK&F96365 did not completely block these effects of DIDS. DIDS did not affect ET-1-induced Ca2+ release and 30 mM KCl-induced increase in [Ca2+]i. Our data suggest that DIDS-sensitive Cl- channels mediate VSMC proliferation induced by ET-1 by mechanisms related to membrane depolarization and Ca2+ influx through voltage-dependent Ca2+ channels.