Effect of adrenergic agonists on Ca2+-channel currents in single vascular smooth muscle cells

Ca²⁺-channel currents have been measured in enzymatically dispersed single smooth muscle cells of the rabbit ear artery using the whole-cell patch clamp technique. Inward currents were elicited by depolarizing test pulses from a holding potential of–50 mV. These currents were activated from–30 mV onward and reached full activation around 0 mV. -Adrenergic agonists did not affect the background current measured at the holding potential, but markedly reduced the peak amplitude of the voltageactivated Ca²⁺-channel currents. This -adrenergic inhibition also occurred in cells which were internally perfused with solutions containing either 10 M cAMP, 10M cGMP or 0.1 mM GTP, but became irreversible when the pipette solution contained a non-hydrolyzable GTP-analog. The action of -agonists on the voltage-activated Ca²⁺-channel currents was variable, and ranged from no effect at all to a 50% reduction of the current. It is concluded that -agonists do not open receptor-operated Ca²⁺-channels in these smooth muscle cells. The inhibition of the voltageactivated Ca²⁺-currents does not seem to be mediated through changes in cyclic nucleotide levels, but might be mediated through G-proteins. Its physiological relevance remains however unclear. The action of -agonists is consistent with their relaxing effect, but the reason for the nonuniform response has not been elucidated.     

Dissociation of K+-induced tension and cellular Ca2+ retention in vascular and intestinal smooth muscle in normoxia and hypoxia

In experiments on smooth muscle preparations of rabbit aorta and guinea pig taenia coli, replacement of the external Na⁺ with K⁺ produced sustained contraction. When external K⁺ concentration was increased, cellular Ca²⁺ retention as measured by a modified lanthanum technique increased. However, when K⁺ concentration was above 80 mM, the tension decreased despite an increase in Ca²⁺ retention. Maximum amount of Ca²⁺ retained was 1280 nmol/g in aorta and 980 nmol/g in taenia coli while the control values for both tissues were approximately 430 nmol/g when the external Ca²⁺ concentration was 2.5 mM. Under hypoxia (N₂ aeration), sustained contraction was induced by 80 mM K⁺ in aorta and by 45.4 mM K⁺ (and 55 mM glucose) in taenia coli. However, no increase in the cellular Ca²⁺ retention was observed under these conditions. During the K⁺-induced sustained contraction in aorta, introduction of N₂ transiently increased, while readmission of O₂ transiently decreased the muscle tension. In taenia coli, the introduction of N₂ decreased the sustained contractile tension probably because of an ATP deficiency, while the readmission of O₂ further decreased the tension trasniently. From these results, it is concluded that, in the presence of a high concentration of K⁺, external Ca²⁺ enters the cell and activates the contractile machinery. A part of the cellular Ca²⁺ is taken up by mitochondria under normoxic but not under hypoxic conditions.     

Temperature-dependence of45Ca fluxes and contraction in vascular smooth muscle cells of rabbit ear artery

The effect of cooling from 35 to 20° C on the⁴⁵Ca-exchange and on the contractile response of rabbit ear artery has been investigated.The amplitude of the contraction induced by K-depolarization at 20° C is reduced to about 60% of its value at 35° C, whereas the response to noradrenaline is not significantly affected.Cooling induces a 2 to 4-fold reduction of the⁴⁵Ca-efflux rate. This effect also occurs in Ca-free medium and in solutions containing 1 mM La. It also occurs in Na-free medium and in tissues in which the transmembrane Na-gradient has been reduced.At 20° C, the⁴⁵Ca-influx in unstimulated tissues and in K-depolarized preparations is significantly lower than at 35° C. in Ca-depleted tissues, i.e. tissues in which the noradrenaline-sensitive Ca-store has been emptied by a stimulation with the agonist in Ca-free solution, the⁴⁵Ca-influx is not significantly affected by cooling.The gradual depletion of the noradrenaline-sensitive Ca-store in Ca-free solutions is at 20° C much slower than at 35° C. The amount of Ca released by noradrenaline is not affected by cooling, whereas for the same amount of Ca released the contractile response is higher at 20° C.These findings indicate that temperature affects the transmembrane Ca-extrusion and the Ca-influx through voltage-dependent channels. The properties of the noradrenaline-sensitive Ca-store are less sensitive to temperature.     

Functional characterization of the Ca2+-gated Ca2+ release channel of vascular smooth muscle sarcoplasmic reticulum

The Ca²⁺-gated Ca²⁺ release channel of aortic sarcoplasmic reticulum (SR) was partially purified and reconstituted into planar lipid bilayers. Canine and porcine aorta microsomal protein fractions were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) in the presence and absence of ³[H]-ryanodine and centrifuged through linear sucrose gradients. A single ³[H]-ryanodine receptor peak with an apparent sedimentation coefficient of 30 s was obtained. Upon reconstitution into planar lipid bilayers, the unlabelled 30 s protein fraction induced the formation of a Ca²⁺- and monovalent-ion-conducting channel (110 pS in 100 mM Ca²⁺, 360 pS in 250 mM K⁺). The channel was activated by micromolar Ca²⁺, modulated by millimolar adenosine triphosphate, Mg²⁺ and the Ca²⁺-releasing drug caffeine, and inhibited by micromolar ruthenium red. Micro- to millimolar concentrations of the plant alkaloid ryanodine induced a permanently closed state of the channel. Our results suggest that smooth muscle SR contains a Ca²⁺-gated Ca²⁺ release pathway, with properties similar to those observed for the skeletal and cardiac ryanodine receptor/Ca²⁺ release channel complexes.     

AlF 4 − induces Ca2+ oscillations in guinea-pig ileal smooth muscle

The effects of different compounds that inhibit the isolated plasma-membrane Ca²⁺/Mg²⁺-ATPase on the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and on the corresponding force development have been examined in smooth muscle of the longitudinal layer of the guinea-pig ileum. F^–, in the presence of Al³⁺, induced an increase of the resting force and of the amplitude of the superimposed phasic contractions. The increase of resting force was associated with an increased level of basal [Ca²⁺]_i while the phasic contractions were accompanied by concomitant oscillations in [Ca²⁺]_i. Comparable contractions could be induced by vanadate and the calmodulin antagonist calmidazolium. The oscillations of [Ca²⁺]_i and of force elicited by AlF ₄ ^– were not modified by adrenergic or cholinergic blocking agents but were inhibited by verapamil. These phasic contractions were not affected by depleting the intracellular Ca²⁺ stores with ryanodine. This finding excludes a cytosolic origin of these oscillations. However, hyperpolarization and complete depolarization of the cells inhibited the oscillations. It is concluded that AlF ₄ ^– , vanadate and calmidazolium induce cytoplasmic Ca²⁺ oscillations possibly by acting at the plasma membrane. Indeed all these substances affect by different mechanisms the isolated plasma-membrane Ca²⁺/Mg²⁺-ATPase. The generation of membrane-linked Ca²⁺ oscillations could therefore be related to an inhibition of the plasma-membrane Ca²⁺ pump resulting in an increase of [Ca²⁺]_i. This change in [Ca²⁺]_i could be responsible for the pronounced changes of the electrical and mechanical activity of this tissue.     

ATP-Dependent inhibition of Ca2+-activated K+ channels in vascular smooth muscle cells by neuropeptide Y

Neuropeptide Y(NPY) inhibits Ca²⁺-activated K⁺ channels reversibly in vascular smooth muscle cells from the rat tail artery. NPY (200 M) had no effect in the absence of intracellular adenosine 5triphosphate (ATP) and when the metabolic poison cyanide-M-chlorophenyl hydrozone (10 M) was included in the intracellular pipette solution. NPY was also not effective when ATP was substituted by the non-hydrolysable ATP analogue adenosine 5-[, -methylene]-triphosphate (AMP-PCP). NPY inhibited Ca²⁺-activated K⁺ channel activity when ATP was replaced by adenosine 5-O-(3-thiotriphosphate) (ATP [-S]) and the inhibition was not readily reversed upon washing. Protein kinase inhibitor (1 M), a specific inhibitor of adenosine 3, 5-cyclic monophosphatedependent protein kinase, had no significant effect on the inhibitory action of NPY. The effect of NPY on single-channel activity was inhibited by the tyrosine kinase inhibitor genistein (10 M) but not by daidzein, an inactive analogue of genistein. These observations suggest that the inhibition by NPY of Ca²⁺-activated K⁺ channels is mediated by ATP-dependent phosphorylation. The inhibitory effect of NPY was antagonized by the tyrosine kinase inhibitor genistein.     

Effects of ryanodine on acetylcholine-induced Ca2+ mobilization in single smooth muscle cells of the porcine coronary artery

To study the essential features of acetylcholine (ACh)-and caffeine-sensitive cellular Ca²⁺ storage sites in single vascular smooth muscle cells of the porcine coronary artery, the effects of ryanodine on both ACh- and caffeine-induced Ca²⁺ mobilization were investigated by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) using Fura 2 in Ca²⁺-containing or Ca²⁺-free solution. The resting [Ca²⁺]_i of the cells was 122 nM in normal physiological solution and no spontaneous activity was observed. In a solution containing 2.6 mM Ca²⁺, 10 M ACh or 128 mM K⁺ produced a phasic, followed by a tonic, increase in [Ca²⁺]_i but 20 mM caffeine produced only a phasic increase. In Ca²⁺-free solution containing 0.5 mM ethylenebis(oxonitrilo)tetraacetate (EGTA), the resting [Ca²⁺]_i rapidly decreased to 102 nM within 5 min, and 10 M ACh or 20 mM caffeine (but not 128 mM K⁺) transiently increased [Ca²⁺]_i. Ryanodine (50 M) greatly inhibited the phasic increase in [Ca²⁺]_i induced by 10 M ACh or 5 mM caffeine and increased the time to peak and to the half decay after the peak in the presence or absence of extracellular Ca²⁺. By contrast, ryanodine (50 M) enhanced the tonic increase in [Ca²⁺]_i induced by 128 mM K⁺ and also by 10 M ACh in Ca²⁺-containing solution. In Ca²⁺-free solution containing 0.5 mM EGTA, ACh (10 M) failed to increase [Ca²⁺]_i following application of 20 mM caffeine. The level of [Ca²⁺]_i induced by 20 mM caffeine was greatly reduced, but not abolished, following application of 10 M ACh in Ca²⁺-free solution. These results suggest that both ACh and caffeine release Ca²⁺ from the ryanodine-sensitive sarcoplasmic reticulum (SR) in smooth muscle cells of the porcine coronary artery. The finding that ryanodine significantly increased the resting [Ca²⁺]_i and inhibited the rate of decline of [Ca²⁺]_i following wasthout of high K⁺ or ACh in Ca²⁺-containing solution suggests that SR may negatively regulate the resting [Ca²⁺]_i in smooth muscle cells of the porcine coronary artery.     

Blocking actions of Ca2+ antagonists on the Ca2+ channels in the smooth muscle cell membrane of rabbit small intestine

Actions of Ca²⁺ antagonists, verapamil, nicardipine and diltiazem, were investigated on the Ca²⁺ inward current in the fragmented smooth muscle cell membrane (smooth muscle ball; SMB) obtained from the longitudinal muscle layer of the rabbit ileum, by enzymatic dispersion. All Ca²⁺ antagonists inhibited the inward current, in a dose-dependent manner. The ID₅₀ value on the maximum amplitude of the inward current of nicardipine was 24 nM, and this value was roughly 50 times lower than values obtained with verapamil and diltiazem, when the inward current was provoked by 0 mV command pulse from the holding potential of –60 mV. Lowering the holding potential to –80 mV shifted the dose-response curve to the right. When depolarizing pulses (100 ms, stepped up to 0 mV from –60 mV or –80 mV) were applied every 20 s, the peak amplitude of the inward current remained unchanged, but nicardipine immediately, and diltiazem and verapamil slowly reduced the peak amplitude. These slow inhibitions by the latter two drugs depended on the frequency or number of stimulations. Nicardipine but not diltiazem and verapamil shifted the voltage-dependent inactivation curve to the left (3 s duration of the conditioning pulse). However, with a longer conditioning pulse (10 s) verapamil and diltiazem shifted the voltage-dependent inactivation curves to the left. Therefore, the inhibitory actions of these Ca²⁺ antagonists differ. Namely, diltiazem and verapamil inhibit the Ca²⁺ channels, mainly in a frequency-or use-dependent manner while nicardipine does so in a voltage-dependent manner.     

Ca2+ and K+ current in cultured vascular smooth muscle cells from rat aorta

Ca²⁺ (I_Ca) and K⁺ (I_K) currents were recorded in single cultured cells from rat aorta using the whole cell clamp technique with patch electrodes. I_Ca was detected at–30 mV, and at 20 mV it reached a peak in about 10 ms and decayed with a t_1/2=50 ms. The mean maximum slope conductance (G_Ca) was 30 S/cm². I_K was detected at–10 mV and at 20 mV reached its maximum with a t_1/2=12 ms. For I_K, G_K=200 S/cm². These channels can be activated during action potentials and play a role in the excitation and contraction of vascular smooth muscle cells.Doctoral training program UAM-1     

Agonist concentration influences the pattern and time course of intracellular Ca2+ oscillations in human arterial smooth muscle cells

Oscillations in intracellular Ca²⁺ were recorded in cultured human uterine artery vascular smooth muscle cells. In the absence of external Ca²⁺, prolonged application of 3 M histamine activated a large transient increase in Ca²⁺ followed by a burst of Ca²⁺ spikes. The time course and frequency of the spikes were approximately constant until the last two to three spikes, when the inter-spike interval progressively increased. At 30 M histamine the response was different; the amplitude of the spikes decreased rapidly to zero, the rate of rise of successive transients fell and the time between spikes increased. The cessation of oscillatory activity was not associated with the depletion of intracellular Ca²⁺ stores, since increased doses of agonist or the sulphydryl reagent thimerosal could reactivate Ca²⁺ release. The changes in the pattern of intracellular Ca²⁺ spikes seen with increasing agonist concentration may reflect the involvement of different inactivation mechanisms in the termination of Ca²⁺ transients. In the presence of external Ca²⁺, histamine (3–30 M) activated regular Ca²⁺ oscillations. The frequency, but not the amplitude, of the oscillations was dependent on agonist concentration, the highest frequency of spiking was observed at 30 M histamine. In cells depolarised with 30 mM K⁺, histamine was still able to activate Ca²⁺ oscillations, but the dependence of spike frequency upon agonist concentration was abolished. Ca²⁺ oscillations could be activated in the presence of verapamil and nifedipine (10 M). These data suggest that in human uterine artery vascular smooth muscle cells histamine-induced Ca²⁺ oscillations are generated largely by a cytosolic oscillator and are modified by the influx of Ca²⁺ across the surface membrane.     

Ca2+ entry and vasoconstriction during osmotic swelling of vascular smooth muscle cells

Exposure of aortic strips from guinea-pigs to hypotonic extracellular fluid is followed by marked vasoconstriction, which is inhibited by D-600 (3 M), a blocker of voltage-sensitive Ca²⁺ channels. Conventional electrophysiology, patch-clamp studies, pH determination with 2, 7 bis(2-carboxyethyl)-5, 6-carboxyfluorescein (BCECF) and Ca²⁺ measurements with Fura-2 have been performed on smooth muscle cells cultured either from rat or human aorta to further elucidate the underlying mechanisms. Exposure of the cells to a 25% hypotonic extracellular fluid leads to a rapid and fully reversible depolarization, paralleled by an increase of the selectivity and conductance of the cell membrane to Cl^–, an acidification of the cytoplasm and an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The latter is inhibited by the Ca²⁺ channel blocker D-600 (1–3 M). It is concluded that osmotic cell swelling leads to the activation of an anion channel. The subsequent depolarization of the cell membrane activates voltage-sensitive Ca²⁺ channels which increases [Ca²⁺]_i, thus stimulating the contraction of vascular smooth muscle cells.     

Modulation of single slow (L-type) calcium channels by intracellular ATP in vascular smooth muscle cells

Involvement of ATP in the regulation of slow (L-type) Ca²⁺ channels of vascular smooth muscle cells was investigated by recording single Ca²⁺ channel currents (single-channel conductance of 18 pS) using a patch clamp technique. In the cell-attached configuration, intracellular composition was modified by permeabilizing the cell membrane with mechanical disruption at one end of the cell. Single cells were freshly isolated from guinea-pig portal vein by collagenase treatment. For the channel recordings, the pipette solution contained 100 mM Ba²⁺ and the bath contained K⁺-rich solution (with 5 mM EGTA) to depolarize the membrane to near 0 mV. The channel activity decreased usually within 3 min after permeabilizing the cell end and exposure to ATP-free bath solution. If ATP (1–5 mM) was applied to the bath (access to cell interior) before complete disappearance of channel activity, channel activity was partially recovered. ATP did not change the current amplitude (i) or the mean open time of the channels, whereas the number of channels available for opening and/or the probability of their being open (NP _o) were increased by ATP. A non-hydrolyzable analogue of ATP, AMP-PNP, did not exert an ATP-like effect; ATP--S had a weak effect. With 1 M Bay-K-8644 (Ca²⁺ channel agonist) in the pipette, the activity of the Ca²⁺ channel was high; such activity persisted for more than 10 min after permeabilizing the cell and exposting to ATP-free solution containing KCN (1 mM) and 2-deoxy-d-glucose (10 mM). These results indicate that activation of slow Ca²⁺ channels requires ATP. The effect of ATP may be exerted by phosphorylation and/or an energy-requiring step. Bay-K-8644 may change the nature of the slow Ca²⁺ channel, making it resistant to rundown.     

Agonist-dependent modulation of Ca2+ sensitivity in rabbit pulmonary artery smooth muscle

The effects of the stable thromboxane analogue U46619, the ₁-adrenergic agent phenylephrine and depolarization with high K⁺ on cytoplasmic Ca²⁺ ([Ca²⁺]_i) and force development were determined in rabbit pulmonary artery smooth muscle. Following stimulation with each of the excitatory agents, the time course of the [Ca²⁺]_i/force relationship described counter-clockwise hysteresis loops with the rise and fall in [Ca²⁺]_i leading, respectively, contraction and relaxation. The rank order of the force/[Ca²⁺]_i ratios evoked by the different methods of stimulation was: U46619 > phenylephrine high K⁺. The difference between the actions of U46619 and phenylephrine was due to the lesser Ca²⁺-releasing and greater Ca²⁺-sensitizing action of U46619. Both U46619 and phenylephrine also released intracellular Ca²⁺ in intact (non-permeabilized) preparations. The effects of the two agonists on force, at constant free cytoplasmic [Ca²⁺] maintained with EGTA, were also determined in preparations permeabilized with staphylococcal -toxin, in which intracellularly stored Ca²⁺ was eliminated with A23187. Sensitization of the contractile response to Ca²⁺ by agonists was indicated by the contractile responses of permeabilized muscles to U46619 and to phenylephrine, in the presence of constant, highly buffered [Ca²⁺]_i. These contractions were inhibited by GDP[S] and could also be elicited by GTP. We conclude that, in addition to changing [Ca²⁺]_i, pharmacomechanical coupling can also modulate contraction by altering the sensitivity of the regulatory/contractile apparatus of smooth muscle to [Ca²⁺]_i, through a G-protein-coupled mechanism.     

D600 blocks the Ca2+ channel from the outer surface of smooth muscle cell membrane of the rabbit intestine and portal vein

The voltage dependent Ca²⁺ inward current in single smooth muscle cells dispersed from the longitudinal muscle layer of the rabbit ileum and rabbit portal vein was recorded using the whole-cell voltage clamp technique. D600 added to the bathing solution inhibited the Ca²⁺ current, while the intracellular perfusion of this agent did not reduce the amplitude of this current. Thus, D600 probably acts from the outer surface of the membrane. The nature of the Ca²⁺ channel in smooth muscle cells seems to differ from that in cardiac muscle cells.     

Changes in cytosolic Ca2+ and contraction induced by various stimulants and relaxants in canine tracheal smooth muscle

Effects of stimulants and relaxants on the cytosolic Ca²⁺ level ([Ca²⁺]_cyt) and contraction were examined in isolated canine tracheal smooth muscle. High K⁺ and carbachol induced a sustained increase in [Ca²⁺]_cyt and muscle tension. Cumulative addition of KCl induced a graded increase in [Ca²⁺]_cyt and muscle tension. Cumulative addition of carbachol induced greater contraction than high K⁺ at a given [Ca²⁺]_cyt. 12-Deoxyphorbol 13-isobutyrate (DPB) (50 nmol/l) induced a small sustained contraction with little effect on [Ca²⁺]_cyt. A higher concentration (1 mol/l) of DPB induced a larger sustained contraction with a decrease in [Ca²⁺]_cyt. DPB (50 nmol/l) potentiated the KCl-induced contraction without or with only a small additional increase in [Ca²⁺]_cyt. By contrast, 1 mol/l DPB potentiated the high-K⁺-induced contraction with a decrease in [Ca²⁺]_cyt. Addition of 50 nmol/l or 1 mol/l DPB in the presence of carbachol inhibited both [Ca²⁺]_cyt and muscle tension. Verapamil, isoprenaline and forskolin did not change or slightly decreased [Ca²⁺]_cyt and muscle tension in resting trachea. Verapamil inhibited the contrction and [Ca²⁺]_cyt stimulated by high K⁺ and carbachol. Isoprenaline and forskolin inhibited the high-K⁺-induced contraction without changing [Ca²⁺]_cyt, whercas these inhibitors inhibited carbachol-induced contraction with a relatively small decrease in [Ca²⁺]_cyt. These results suggest that (a) sustained contractions induced by high K⁺ and carbachol are due to the sustained increase in [Ca²⁺]_cyt, (b) carbachol increases the sensitivity of contractile elements to Ca²⁺, and (c) isoprenaline and forskolin inhibit the contraction by the decrease in [Ca²⁺]_cyt and also by the decrease in the sensitivity of contractile elements to Ca²⁺. The carbachol-induced Ca²⁺ sensitization might be attributable to the activation of protein kinase C following the stimulation of the phosphatidylinositol turnover.     

The non-excitable smooth muscle: Calcium signaling and phenotypic switching during vascular disease

Calcium (Ca(2+)) is a highly versatile second messenger that controls vascular smooth muscle cell (VSMC) contraction, proliferation, and migration. By means of Ca(2+) permeable channels, Ca(2+) pumps and channels conducting other ions such as potassium and chloride, VSMC keep intracellular Ca(2+) levels under tight control. In healthy quiescent contractile VSMC, two important components of the Ca(2+) signaling pathways that regulate VSMC contraction are the plasma membrane voltage-operated Ca(2+) channel of the high voltage-activated type (L-type) and the sarcoplasmic reticulum Ca(2+) release channel, Ryanodine Receptor (RyR). Injury to the vessel wall is accompanied by VSMC phenotype switch from a contractile quiescent to a proliferative motile phenotype (synthetic phenotype) and by alteration of many components of VSMC Ca(2+) signaling pathways. Specifically, this switch that culminates in a VSMC phenotype reminiscent of a non-excitable cell is characterized by loss of L-type channels expression and increased expression of the low voltage-activated (T-type) Ca(2+) channels and the canonical transient receptor potential (TRPC) channels. The expression levels of intracellular Ca(2+) release channels, pumps and Ca(2+)-activated proteins are also altered: the proliferative VSMC lose the RyR3 and the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase isoform 2a pump and reciprocally regulate isoforms of the ca(2+)/calmodulin-dependent protein kinase II. This review focuses on the changes in expression of Ca(2+) signaling proteins associated with VSMC proliferation both in vitro and in vivo. The physiological implications of the altered expression of these Ca(2+) signaling molecules, their contribution to VSMC dysfunction during vascular disease and their potential as targets for drug therapy will be discussed.     

The time course of intracellular calcium movements in single human umbilical vein smooth muscle cells

Intracellular Ca²⁺ ([Ca²⁺]_i) was measured in single isolated human umbilical vein smooth muscle cells. Stimulation with histamine, in the absence of external Ca²⁺, mobilised Ca²⁺ from intracellular stores. When repeated brief applications of agonist were used, the time to onset, amplitude and rate of rise of the Ca²⁺ transients were found to change. Two components could often be discerned in the rising phase of the transients, an initial slow pacemaker and a second faster and larger component. Following the first histamine-activated transient the basal level of [Ca²⁺]_i was invariably lower than that prior to stimulation. This lower value was maintained whilst the cell remained in Ca²⁺-free solution, but could be returned to a higher level if the cell was exposed to external Ca²⁺. When the mobilisation of the intracellular store was reduced to undetectable levels, re-exposure to Ca²⁺-containing medium reactivated responses. In the absence of external Ca²⁺, continuous application of histamine activated a series of transient increases in intracellular Ca²⁺, which decreased progressively in amplitude and rate of rise. The interval between transients also increased. These findings are discussed in terms of the activation of inositol trisphosphate-sensitive intracellular Ca²⁺ stores and their sensitivity to cytoplasmic Ca²⁺ and intrasarcoplasmic reticulum Ca²⁺.     

A possible role of sarcoplasmic Ca2+ release in modulating the slow Ca2+ current of skeletal muscle

Ca²⁺ channels are regulated in a variety of different ways, one of which is modulation by the Ca²⁺ ion itself. In skeletal muscle, Ca²⁺ release sites are presumably located in the vicinity of the dihydropyridine-sensitive Ca²⁺ channel. In this study, we have tried to investigate the effects of Ca²⁺ release from the sarcoplasmic reticulum on the L-type Ca²⁺ channel in frog skeletal muscle, using the double Vaseline gap technique. We found an increase in Ca²⁺ current amplitude on application of caffeine, a well-known potentiator of Ca²⁺ release. Addition of the fast Ca²⁺ buffer BAPTA to the intracellular solution led to a gradual decline in Ca²⁺ current amplitude and eventually caused complete inhibition. Similar observations were made when the muscle fibre was perfused internally with the Ca²⁺ release channel blocker ruthenium red. The time course of Ca²⁺ current decline followed closely the increase in ruthenium red concentration. This suggests that Ca²⁺ release from the sarcoplasmic reticulum is involved in the regulation of L-type Ca²⁺ channels in frog skeletal muscle.     

Effect of Ca2+-independent myosin light chain kinase on different skinned smooth muscle fibers

In various skinned smooth muscle fiber preparations, (porcine carotid artery, rat tail artery, chicken gizzard and Taenia coli from guinea pig) a Ca²⁺-independent myosin light chain kinase (MLCK) initiated a contraction in absence of Ca²⁺. While the Ca²⁺ insensitive MLCK was effective on the vertebrate smooth muscles it did not act on the invertebrate skinned skeletal muscle preparation from Limulus and anterior byssus retractor muscle from Mytilus edulis. The results indicate that in vertebrate smooth muscles phosphorylation is sufficient for activation and that there is no obligatory role for an additional mechanism in initiation of contraction.     

Two distinct signaling pathways for regulation of spontaneous local Ca2+ release by phospholipase C in airway smooth muscle cells

Spontaneous local Ca²⁺ release events have been observed in airway smooth muscle cells (SMCs), but the underlying mechanisms are largely unknown. Considering that each type of SMCs may use its own mechanisms to regulate local Ca²⁺ release events, we sought to investigate the signaling pathway for spontaneous local Ca²⁺ release events in freshly isolated mouse airway SMCs using a laser scanning confocal microscope. Application of ryanodine to block ryanodine receptors (RyRs) abolished spontaneous local Ca²⁺ release events, indicating that these events are RyR-mediated Ca²⁺ sparks. Inhibition of inositol 1,4,5-triphosphate receptors (IP₃Rs) by 2-aminoethoxydiphenyl-borate (2-APB) or xestospongin-C significantly blocked the activity of Ca²⁺ sparks. Under patch clamp conditions, dialysis of IP₃ to activate IP₃Rs increased the activity of local Ca²⁺ events in control cells but had no effect in ryanodine-pretreated cells. The RyR agonist caffeine augmented the frequency of Ca²⁺ sparks in cells pretreated with and without 2-APB or xestospongin-C. The specific phospholipase C (PLC) blocker U73122 decreased the activity of Ca²⁺ sparks and prevented xestospongin-C from producing the inhibitory effect. The protein kinase C (PKC) activator 1-oleoyl-2-acetyl-glycerol or phorbol-12-myristate-13-acetate inhibited Ca²⁺ sparks, whereas the PKC inhibitor chelerythrine, PKCɛ inhibitory peptide, or PKCɛ gene knockout produced an opposite effect. Collectively, our data suggest that the basal activation of PLC regulates the activity of RyR-mediated, spontaneous Ca²⁺ sparks in airway SMCs through two distinct signaling pathways: a positive IP₃-IP₃R pathway and a negative diacylglycerol–PKCɛ pathway.