The norepinephrine-sensitive Ca2+-storage site differs from the caffeine-sensitive site in vascular smooth muscle of the rat aorta

Using microfluorometry of quin 2, a Ca2+-sensitive dye, we characterized the release and uptake of Ca2+ by the norepinephrine-sensitive Ca2+-storage site and the caffeine-sensitive one. The norepinephrine-sensitive Ca2+-storage site was readily depleted in Ca2+-free medium and almost completely replenished by loading with 1.0 mM Ca2+ solution for 3 min, whereas the caffeine-sensitive site was scarcely affected. Furthermore, norepinephrine has little effect on the caffeine-sensitive Ca2+-storage site in Ca2+-free medium, and vice versa. We conclude that the location and mechanisms of release and uptake of Ca2+ of these two Ca2+-storage sites differ in the case of rat aortic vascular smooth muscle cells in primary culture.     

Endothelin-sensitive intracellular Ca2+ store overlaps with caffeine-sensitive one in rat aortic smooth muscle cells in primary cultures

We made use of quin2 microfluorometry to determine the effects of endothelin (ET) on cytosolic free Ca2+ concentrations [Ca2+]i) in rat aortic smooth muscle cells in primary culture. In Ca2+-containing medium, ET induced a rapid and sustained elevation of [Ca2+]i. In the latter component, in particular, the elevation of [Ca2+]i was inhibited by diltiazem. In Ca2+-free medium, ET induced a rapid and transient [Ca2+]i elevation, which was not inhibited by diltiazem. When the caffeine-sensitive intracellular Ca2+ store was practically depleted by repeated treatment with caffeine in Ca2+-free media, ET did not elevate [Ca2+]i. Thus, it was suggested that ET induces [Ca2+]i elevation not only by extracellular Ca2+-dependent, mechanisms but also by releasing Ca2+ from the intracellular store, and that the ET-sensitive Ca2+ store may overlap with the caffeine-sensitive one, in cultured vascular smooth muscle cells.     

Inhibition of calcium transients in cultured vascular smooth muscle cells by pertussis toxin

Effects of pertussis toxin on Ca2+ transients in rat arterial smooth muscle cells in primary culture were monitored, using quin 2-microfluorometry. In the presence or the absence of extracellular Ca2+, norepinephrine, histamine, caffeine and high extracellular K+ induced elevations in cytosolic Ca2+ concentration. Cytosolic Ca2+ elevations induced by norepinephrine and histamine were inhibited by pretreatment of the cells with pertussis toxin, time- and dose-dependently. However, elevations induced by caffeine and K+-depolarization were unaffected by the pretreatment with this toxin. Thus, it is suggested that GTP binding protein, a pertussis toxin substrate and involved in the receptor-mediated cytosolic Ca2+ transients, is not involved in transient elevations in cytosolic Ca2+ induced by caffeine and K+-depolarization in cultured vascular smooth muscle cells.     

Ca2+ compartments in saponin-skinned cultured vascular smooth muscle cells

A method for saponin skinning of primary cultured rat aortic smooth muscle cells was established. The saponin-treated cells could be stained with trypan blue and incorporated more 45Ca2+ than the nontreated cells under the same conditions. At low free Ca2+ concentration, greater than 85% of 45Ca2+ uptake into the skinned cells was dependent on the extracellularly supplied MgATP. In the intact cells, both caffeine and norepinephrine increased 45Ca2+ efflux. In the skinned cells, caffeine increased 45Ca2+ efflux, whereas norepinephrine did not. The caffeine-releasable 45Ca2+ uptake fraction in the skinned cells appeared at 3 X 10(-7) M Ca2+, increased gradually with the increase in free Ca2+ concentration, and reached a plateau at 1 X 10(-5) M Ca2+. The 45Ca2+ uptake fraction, which was significantly suppressed by sodium azide, appeared at 1 X 10(-5) M Ca2+ and increased monotonically with increasing free Ca2+ concentration. The results suggest that the caffeine-sensitive Ca2+ store, presumably the sarcoplasmic reticulum, plays a physiological role by releasing Ca2+ in response to norepinephrine or caffeine and by buffering excessive Ca2+. The 45Ca2+ uptake by mitochondria appears too insensitive to be important under physiological conditions.     

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.     

K+-depolarization induces a direct release of Ca2+ from intracellular storage sites in cultured vascular smooth muscle cells from rat aorta

Using an intracellularly trapped dye, quin 2, effects of K+-depolarization on cytosolic free calcium concentrations were recorded microfluorometrically in rat aorta vascular smooth muscle cells in primary culture. When the cells were exposed to high extracellular K+ in Ca+-free media containing 2mM EGTA, there was a transient and dose-dependent elevation of cytosolic Ca2+ concentrations. However, the concentration of the cytosolic Ca2+ was not elevated when the intracellularly stored Ca2+ was depleted by the repetitive treatment with caffeine prior to the application of high K+. Thus depolarization of plasma membrane, per se, directly induces a release of Ca2+ from intracellular storage sites in vascular smooth muscle cells, and the main fraction of this released Ca2+ is derived from the caffeine sensitive storage sites; perhaps from the sarcoplasmic reticulum.     

Subcellular mechanism of desensitization of the ATP-induced Ca2+ mobilization in human umbilical vein endothelial cells: role of intracellular Ca2+ stores

Confluent monolayers of human umbilical vein endothelial cells subcultured on glass coverslips were loaded with the fluorescent Ca2+ indicator, fura-2. Changes in fura-2 fluorescence were detected by means of a fluorescence spectrophotometer. Both ATP and ADP (0.3-100 microM) caused a concentration-dependent transient peak response of the intracellular free calcium concentration ([Ca2+]i), followed by a lower sustained response. AMP and adenosine did not induce detectable changes in [Ca2+]i. The sustained response to ATP was abolished by superfusion with the Ca2(+)-free solution (with 1 mM EGTA), while the transient peak response was uninfluenced. The transient peak response to ATP (30 microM) was inhibited by pre-exposure to ATP in a graded manner depending on the concentration of ATP. The response to ATP recovered after washout for 20 min with the solution containing Ca2+, but not with the Ca2(+)-free solution. The transient peak response to ATP was markedly reduced by preceding exposure to histamine, while the response to histamine was not influenced by pre-exposure to ATP. These findings indicate that depletion and refilling of the ATP-sensitive intracellular Ca2+ store may be responsible for the desensitization and recovery of the ATP-induced [Ca2+]i response. The pharmacological characteristics of the ATP-sensitive intracellular Ca2+ store seem different from those of the histamine-sensitive store.     

Depolarization promotes caffeine induced [3H]-noradrenaline release in calcium-free solution from peripheral sympathetic nerves

The transmitter releasing action of caffeine was studied in the absence of extracellular Ca2+ from the peripheral sympathetic nerves of the rabbit main pulmonary artery. Caffeine (10 mM) increased the release of [3H]-noradrenaline moderately, but not significantly in Ca2(+)-free (+1 mM EGTA) Krebs solution. When peripheral nerve endings/varicosities were depolarized by elevating extracellular K+ to 47.2 mM and 70.8 mM in Ca2(+)-free solution, the transmitter releasing effect of 10 mM caffeine became significant. Ca2+ removal itself transiently increased the [3H]-noradrenaline outflow. In the individual experiments the amount of the caffeine evoked transmitter release at 47.2 mM and 70.8 mM K(+)-depolarization was inversely correlated to the release evoked by Ca2(+)-removal. Our results suggest that caffeine-sensitive calcium stores are present in peripheral nerve terminals of rabbit pulmonary artery, and part of the caffeine sensitive calcium stores may discharge during Ca2(+)-removal from the extracellular solution.     

Characterisation of Distinct Inositol 1,4,5-Trisphosphate-Sensitive and Caffeine-Sensitive Calcium Stores in Digitonin-Permeabilised Adrenal Chromaffin Cells

The effect of inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3] and caffeine on Ca2+ release from digitonin-permeabilised bovine adrenal chromaffin cells was examined by using the Ca2+ indicator fura-2 to monitor [Ca2+]. Permeabilised cells accumulated Ca2+ in the presence of ATP and addition of either Ins(1,4,5)P3 or caffeine released 17% or 40-50%, respectively, of the accumulated Ca2+, indicated by sustained rises in [Ca2+] in the cell suspension. Prior addition of Ins(1,4,5)P3 had no effect on the magnitude of the response to a subsequent addition of caffeine. The response to Ins(1,4,5)P3 was prevented by prior addition of caffeine or CaCl2, indicating that the Ins(1,4,5)P3 response was blocked by elevated [Ca2+]. The responses were essentially identical in the presence of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, indicating that the Ca2+ release was not from mitochondria or secretory granules and that a proton gradient was not required for Ca2+ accumulation into the Ins(1,4,5)P3- or caffeine-sensitive stores. Ca2+ release from the caffeine-sensitive store was selectively blocked by ryanodine. The Ins(1,4,5)P3-sensitive store was emptied by thapsigargin, which had no effect on caffeine responses. These data suggest that permeabilised chromaffin cells possess two distinct nonoverlapping Ca2+ stores sensitive to either Ins(1,4,5)P3 or caffeine and support previous conclusions that these stores possess different Ca2(+)-ATPases.     

Intracellular Ca2+ pools in PC12 cells. A unique, rapidly exchanging pool is sensitive to both inositol 1,4,5-trisphosphate and caffeine-ryanodine.

Release of Ca2+ from intracellular stores was studied in the parent PC12 cell line and in recently isolated clones sensitive or insensitive to caffeine. In the caffeine-sensitive cells the cytosolic free Ca2+ concentration ([Ca2+]i) responses by the xanthine drug and by stimulants of receptors coupled to inositol 1,4,5-trisphosphate (Ins-P3) generation (bradykinin, ATP) depend on separate pathways because 1) caffeine does not stimulate the hydrolysis of phosphatidylinositol 4,5-bisphosphate and 2) Ca(2+)-induced Ca2+ release, the process activated by caffeine, plays no major role in the Ins-P3-induced Ca2+ mobilization. Although distinct, these two mechanisms converge onto the same Ca2+ store. In fact 1) the [Ca2+]i responses by receptor agonists and caffeine were not additive; 2) either type of agent reduced (up to complete inhibition) the response to a subsequent administration of the same or the other agent; 3) all these responses were prevented by selective Ca2+ ATPase blockers; 4) ryanodine, which affects the intracellular Ca2+ channel sensitive to caffeine, also induced depletion of the receptor-sensitive Ca2+ pool; 5) in the 10 PC12 clones tested, sensitivity to caffeine paralleled ryanodine sensitivity. Therefore, PC12 cells, similar to some smooth muscle fibers but at variance with neurons and other secretory cells, express a single, rapidly exchanging Ca2+ store in which two distinct intracellular Ca2+ channels, i.e. the receptors for caffeine-ryanodine and Ins-P3, appear to be colocalized.     

The caffeine-sensitive Ca2+ store in bovine adrenal chromaffin cells; an examination of its role in triggering secretion and Ca2+ homeostasis

The effect of caffeine on catecholamine secretion and intracellular free Ca2+ concentration [( Ca2+]i) in bovine adrenal chromaffin cells was examined using single fura-2-loaded cells and cell populations. In cell populations caffeine elicited a large (approximately 200 nM) transient rise in [Ca2+]i that was independent of external Ca2+. This rise in [Ca2+]i triggered little secretion. Single cell measurements of [Ca2+]i showed that most cells responded with a large (greater than 200 nM) rise in [Ca2+]i, whereas a minority failed to respond. The latter, whose caffeine-sensitive store was empty, buffered a Ca2+ load induced by a depolarizing stimulus more effectively than those whose store was full. The caffeine-sensitive store in bovine chromaffin cells may be involved in Ca2+ homeostasis rather than in triggering exocytosis.     

The inositol 1,4,5-trisphosphate-forming agonist histamine activates a ryanodine-sensitive Ca2+ release mechanism in bovine adrenal chromaffin cells

The role of a Ca(2+)-induced Ca2+ release (CICR) mechanism in the generation of agonist-induced increases of intracellular free Ca2+ concentration ([Ca2+]i) was studied in bovine adrenal chromaffin cells. In single cells, repetitive stimulations with caffeine at 200-s intervals evoked reproducible spikes of [Ca2+]i. Ryanodine, an agent that interacts with the CICR channel of muscle, inhibited the caffeine-induced spikes of [Ca2+]i in a "use-dependent" way. High affinity binding sites for [3H]ryanodine (Kd 3.3 nM, Bmax 26 fmol/mg protein) were also detected in membranes from chromaffin cells, supporting the presence of a caffeine- and ryanodine-sensitive CICR channel. Pretreatment of single cells with caffeine + ryanodine to reduce the size of the caffeine-sensitive Ca2+ compartment inhibited a subsequent spike of [Ca2+]i evoked by histamine, a D-myo-inositol 1,4,5-trisphosphate-forming agonist. This demonstrates that a significant portion of the Ca2+ released by histamine comes from a caffeine- and ryanodine-sensitive pool. Ryanodine inhibited by 50% the size of [Ca2+]i spikes evoked by repetitive stimulation with histamine and did so in a use-dependent manner. These data suggest that, in addition to D-myoinositol 1,4,5-trisphosphate, activation of a caffeine- and ryanodine-sensitive CICR channel participates in the generation of histamine-induced release of intracellular Ca2+.     

Changes in functional properties of the caffeine-sensitive Ca2+ store during differentiation of human SH-SY5Y neuroblastoma cells

We have used single cell fluorescence imaging techniques to examine how functional properties of the caffeine-sensitive Ca(2+) store change during differentiation of a sub-population of caffeine-sensitive SH-SY5Y cells. Application of caffeine (30 mM) 1-10.5 min after a 'priming' depolarisation pulse of 55 mM K(+) revealed that the caffeine-sensitive store in undifferentiated cells remained replete, whereas that in 9-cis retinoic acid (9cRA)-differentiated cells spontaneously dissipated with a t(1/2) of 2.8 min, and was essentially completely depleted approximately 10 min after priming. In 9cRA-differentiated cells that were stimulated with methacholine (10 microM) 1 min after priming, the amplitude, rate of rise and propagation velocity of the Ca(2+) wave in the neurites were all constant, whereas these kinetic parameters all progressively decreased as the wave travelled along the neurites in cells that were stimulated 10 min after priming. Use-dependent block with ryanodine inhibited the global Ca(2+) signal in 9cRA-differentiated cells stimulated with methacholine 1 min after priming (71+/-8%) but not 10 min after priming. Depolarisation was more effective at priming the caffeine-sensitive Ca(2+) store in 9cRA-differentiated cells, which lack a functional store-operated Ca(2+) entry pathway. We conclude that differentiation of caffeine-sensitive SH-SY5Y cells is accompanied by an increase in lability of the caffeine-sensitive Ca(2+) store, and that spontaneous dissipation of Ca(2+) from the store limits the time course of its molecular 'memory' during which it can amplify the hormone-induced Ca(2+) signal by Ca(2+)-induced Ca(2+) release.     

Ca2+-dependent membrane currents in vascular smooth muscle cells of the rabbit.

The membrane potential in vascular smooth muscle cells contributes to the regulation of cytosolic [Ca2+], which in turn regulates membrane potential by means of Ca2+i-dependent ionic currents. We investigated the characteristics of Ca2+i-dependent currents in rabbit coronary and pulmonary arterial smooth muscle cells. Ca2+i-dependent currents were recorded using the whole-cell patch-clamp technique while cytosolic [Ca2+] was increased by caffeine. The reversal potentials of caffeine-induced currents were between -80 and -10 mV under normal ionic conditions, whereas they were about 0 mV when K+-free NaCl solutions were used both in pipette and bath. The total substitution of extracellular Na+ with membrane-impermeable cation N-Methyl-D-glucamine did not affect caffeine-induced currents, implying no significant contribution of Na+ as a permeant ion to the currents. The substitution of extracellular NaCl with sucrose reduced outward component of the currents and shifted the reversal potentials according to the change in Cl- equilibrium potential. Upon application of the niflumic acid under K+-free conditions, most of the current induced by caffeine was inhibited. Taken together, the results of the present study indicate that K+ and Cl- currents are major components of Ca2+i-dependent currents in vascular smooth muscles isolated from coronary and pulmonary arteries of the rabbit, and the relative contribution of each type of current to total currents are not different between the two arteries.     

Functional receptor-channel coupling compared in contractile and proliferative human vascular smooth muscle

We have previously identified a human vascular smooth muscle clone that can reversibly convert between proliferative and contractile phenotypes. Here we compared receptor-channel coupling in these cells using fura-2 to monitor [Ca(2+)](i) and patch-clamp to record currents. Histamine elevated [Ca(2+)](i) in all cells and caused contraction of cells exhibiting the contractile phenotype. The rise of [Ca(2+)](i) persisted in Ca(2+)-free solution and was abolished by thapsigargin, indicating involvement of stores. Whole cell electrophysiological recording revealed that histamine evoked transient outward K(+) current, indicating functional receptor-channel coupling. The time-course and amplitude of the histamine-activated current were similar in cells of the proliferative and contractile phenotypes. Moreover, a large conductance K(+) channel was recorded in cell-attached patches and was activated by histamine as well as the Ca(2+) ionophore A-23187, identifying it as the large conductance Ca(2+)-dependent K(+) channel. This K(+) channel showed similar characteristics and activation in both proliferative and contractile phenotypes, indicating that expression was independent of phenotype. In contrast, histamine also elicited an inward Cl(-) current in some contractile cells, suggesting differential regulation of this current depending on phenotype. These studies demonstrate the usefulness of this human vascular cell clone for studying functional plasticity of smooth muscle, while avoiding complications arising from extended times in culture.     

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.     

Histamine activates H1-receptors to induce cytosolic free calcium transients in cultured vascular smooth muscle cells from rat aorta

Using an intracellularly trapped dye, quin 2, the effects of histamine on cytosolic free calcium concentrations in rat aortic vascular smooth muscle cells in primary culture were recorded, microfluorometrically. When the cells were exposed to histamine, both in the presence and the absence of extracellular Ca2+, there was a rapid, transient and dose-dependent elevation of cytosolic Ca2+ concentrations, with a similar time course. This elevation of cytosolic Ca2+ was dose-dependently inhibited by mepyramine, but not by cimetidine. Thus, histamine activates H1- but not H2- receptors to mediate a release of Ca2+ from the store sites, and there is a rapid and transient elevation of cytosolic Ca2+.     

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.     

Ca2+ responses of pulmonary arterial myocytes to acute hypoxia require release from ryanodine and inositol trisphosphate receptors in sarcoplasmic reticulum

In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) release from the sarcoplasmic reticulum (SR) and Ca(2+) influx through store- and voltage-operated Ca(2+) channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca(2+) release, we measured [Ca(2+)](i) with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca(2+)-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 μM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP(3)R), respectively, or 4% O(2) caused rapid transient increases in [Ca(2+)](i), indicating intracellular Ca(2+) release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM) blocked subsequent Ca(2+) release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 μM) blocked Ca(2+) release to caffeine and hypoxia but not norepinephrine. The IP(3)R antagonist xestospongin C (XeC, 0.1 μM) blocked Ca(2+) release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca(2+)](i) that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca(2+)](i) induced by hypoxia, as well as the subsequent Ca(2+) influx that sustained this increase, required release of Ca(2+) from both RyR and IP(3)R, and 2) the SR Ca(2+) stores accessed by RyR, IP(3)R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca(2+)-ATPase.     

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.