Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.     

The pathway for refilling intracellular Ca2+ stores passes through the cytosol in human leukaemia cells

The pathway for refilling the intracellular Ca²⁺ stores of HL60 and U937 human leukaemia cells loaded with fura-2 has been investigated. On addition of external Ca²⁺ to cells with empty stores there was an increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) which preceded the refilling of the stores. The increase in [Ca²⁺]_i was faster than the refilling, by 3-to 15-fold, depending on the cell type. In measurements in single HL60 cells we found that the refilling of the stores correlated with the extent of the [Ca²⁺]_i increase on addition of external Ca²⁺. The cells showing no [Ca²⁺]_i increase were unable to refill their stores. The addition of Ni²⁺ to the extracellular medium prevented both the [Ca²⁺]_i increase and the refilling of the stores. These results indicate that the limiting step for store refilling is the entry of Ca²⁺ from the extracellular medium to the cytosol. Hence, we conclude that extracellular Ca²⁺ cannot gain access directly to the intracellular Ca²⁺ stores in these cells, but must first enter the cytosol and be taken up from there into the stores.     

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.     

Effect of thapsigargin and caffeine on Ca2+ homeostasis in HeLa cells: implications for histamine-induced Ca2+ oscillations

Several studies have already established that the stimulation of H1 receptors by exogenous histamine induces intracellular Ca²⁺ oscillations in HeLa cells. The molecular mechanism underlying this oscillatory process remains, however, unclear. A series of fura-2 experiments was undertaken in which the nature of the Ca²⁺ pools involved in the histamine-induced Ca²⁺ oscillations was investigated using the tumour promoter agent thapsigargin (TG) and the Ca²⁺-induced Ca²⁺-release promoter, caffeine. The results obtained indicate first that TG causes a gradual increase in cytosolic Ca²⁺ without inducing internal Ca²⁺ oscillations, and second that TG and histamine share common internal Ca²⁺ storage sites. The latter conclusion was derived from experiments performed in the absence of external Ca²⁺, where the addition of TG before histamine resulted in a total inhibition of the Ca²⁺ response linked to H1 receptor stimulation, whereas the addition of histamine before TG decreased by more than 90% the TG-induced Ca²⁺ release. Finally, TG was found to inhibit irreversibly histamine-induced Ca²⁺ oscillations when added to the bathing medium during the oscillatory process. The effect of caffeine at concentrations ranging from 1 mM to 10 mM on intracellular Ca²⁺ homeostasis was also investigated. The results obtained show that caffeine does not affect systematically the internal Ca²⁺ concentration in resting and TG-stimulated HeLa cells, but increases the Ca²⁺ sequestration ability of inositol-trisphosphate (InsP ₃)-related Ca²⁺ stores. These results suggest either that TG acts on InsP ₃-sensitive as well as InsP ₃-insensitive Ca²⁺ pools, so that no final conclusion on the nature of the pools involved in Ca²⁺ spike generation can be currently drawn, or that the contribution of an InsP ₃-insensitive Ca²⁺-induced Ca²⁺-release process is not essential to the Ca²⁺ oscillation machinery in these cells. It is also concluded that a release of Ca²⁺ by caffeine may not be directly accessible to fura-2 measurements in this cellular preparation, but that the inhibitory effect of caffeine on the Ca²⁺ mobilization process triggered by InsP ₃ can be clearly documented using this experimental approach.     

Comparison of Ca2+-dependent K+ channels in the membrane of smooth muscle cells isolated from adult and foetal human aorta

Ca²⁺-activated K⁺ ionic currents in the membrane of cultured smooth muscle cells isolated from foetal and adult human aorta were studied using whole cell and single-channel patch-clamp techniques. Whole cell currents in adult smooth muscle cells were 3–8 times larger than in foetal cells of similar sizes. The elementary conductance and ionic selectivity of single Ca²⁺-activated K⁺ were identical for both types of cells. Channel openings occurred in burst, the duration of which was 3–5-fold longer in adult than in foetal cells. The voltage dependency of the channel activating mechanism and the dependency of the mean open time on the Ca²⁺ concentration on the inner side of the membrane were similar for both types of cells. These results suggest that the main reason for the increase in potassium conductance during development is an alteration in the open time of the Ca²⁺-activated K⁺ channels.     

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.     

Caffeine and histamine-induced oscillations of K(Ca) current in single smooth muscle cells of rabbit cerebral artery

In the present experiment, we characterized the intracellular Ca²⁺ oscillations induced by caffeine (1 mM) or histamine (1–3 M) in voltage-clamped single smooth muscle cells of rabbit cerebral (basilar) artery. Superfusion of caffeine or histamine induced periodic oscillations of large whole-cell K⁺ current with fairly uniform amplitudes and intervals. The oscillatory K⁺ current was abolished by inclusion of ethylenebis(oxonitrilo)tetraacetate (EGTA, 5 mM) in the pipette solution. Caffeine- and histamine-induced periodic activation of the large-conductance Ca²⁺-activated K⁺ [K_(Ca)] channel was recorded in the cell-attached patch mode. These results suggest that the oscillations of K⁺ current are carried by the K_(Ca) channel and reflect the oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Ryanodine (1–10 M) abolished both caffeine- and histamine-induced oscillations. Caffeine- induced oscillations were abolished by the sarcoplasmic reticulum Ca²⁺-adenosine 5-triphosphatase (Ca²⁺-ATPase) inhibitor, cyclopiazonic acid (10 M), and a high concentration of caffeine (10 mM). Inclusion of heparin (3 mg/ml) in the pipette solution blocked histamine-induced oscillations, but did not block caffeine-induced oscillations. By the removal of extracellular Ca²⁺, but not by the addition of verapamil and Cd²⁺, the caffeine-induced oscillations were abolished. Increasing Ca²⁺ influx rate increased the frequencies of caffeine-induced oscillations. Spontaneous oscillations were also observed in cells that were not superfused with agonists, and had similar characteristics to the caffeine-induced oscillations. From the above results, it is concluded, that in smooth muscle cells of the rabbit cerebral (basilar) artery, ryanodine-sensitive Ca²⁺-induced Ca²⁺ release pools play key roles in the generation of caffeine- and histamine-induced intracellular Ca²⁺ oscillations.     

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.     

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K _o ⁺ (80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca _i ²⁺ of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca _i ²⁺ to a mean of 266±83 nM (n=15). Reduction of Na _p ⁺ (96% Li⁺ replaced) significantly increased Ca _i ²⁺ to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na _o ⁺ replacement (Li⁺) increased Ca _i ²⁺ at a significantly faster rate [3.6 nM min^–1 (control) cf. 19.8 nM min^–1 (ouabain)].     

ATP-induced intracellular Ca2+ signals in isolated human insulin-secreting cells

Using isolated -cells from human islets of Langerhans we have demonstrated that purinergic receptor agonists are functionally coupled to rises in the intracellular calcium ion concentration ([Ca²⁺]_i). The effects of ATP, ADP and AMP have been examined over a range of concentrations, 0.5 to 500M. The actions of ATP were more potent than those of either ADP or AMP suggesting that a P₂-type of purinergic receptor operates in these cells. Responses to ATP were concentration-related, but exhibited marked desensitisation at high concentrations (>100M). Purinergic receptor agonists elevate [Ca²⁺]_i by mechanisms that involve both Ca²⁺ influx and Ca²⁺ mobilisation from intracellular stores. The physiological significance of our data has been discussed, and related to previous studies carried out upon rodent and clonal insulin-secreting cells.     

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.     

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.     

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.     

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.     

Regulation of membrane excitability by intracellular pH (pHi) changers through Ca2+-activated K+ current (BK channel) in single smooth muscle cells from rabbit basilar artery

Employing microfluorometric system and patch clamp technique in rabbit basilar arterial myocytes, regulation mechanisms of vascular excitability were investigated by applying intracellular pH (pH_i) changers such as sodium acetate (SA) and NH₄Cl. Applications of caffeine produced transient phasic contractions in a reversible manner. These caffeine-induced contractions were significantly enhanced by SA and suppressed by NH₄Cl. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was monitored in a single isolated myocyte and based the ratio of fluorescence using Fura-2 AM (R _340/380). SA (20 mM) increased and NH₄Cl (20 mM) decreased R _340/380 by 0.2 ± 0.03 and 0.1 ± 0.02, respectively, in a reversible manner. Caffeine (10 mM) transiently increased R _340/380 by 0.9 ± 0.07, and the ratio increment was significantly enhanced by SA and suppressed by NH₄Cl, implying that SA and NH₄Cl may affect [Ca²⁺]_i (p < 0.05). Accordingly, we studied the effects of SA and NH₄Cl on Ca²⁺-activated K⁺ current (IK_Ca) under patch clamp technique. Caffeine produced transient outward current at holding potential (V _h) of 0 mV, caffeine induced transient outward K⁺ current, and the spontaneous transient outward currents were significantly enhanced by SA and suppressed by NH₄Cl. In addition, IK_Ca was significantly increased by acidotic condition when pH_i was lowered by altering the NH₄Cl gradient across the cell membrane. Finally, the effects of SA and NH₄Cl on the membrane excitability and basal tension were studied: Under current clamp mode, resting membrane potential (RMP) was −28 ± 2.3 mV in a single cell level and was depolarized by 13 ± 2.4 mV with 2 mM tetraethylammonium (TEA). SA hyperpolarized and NH₄Cl depolarized RMP by 10 ± 1.9 and 16 ± 4.7 mV, respectively. SA-induced hyperpolarization and relaxation of basal tension was significantly inhibited by TEA. These results suggest that SA and NH₄Cl might regulate vascular tone by altering membrane excitability through modulation of [Ca²⁺]_i and Ca²⁺-activated K channels in rabbit basilar artery.     

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.     

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.     

High-frequency fatigue of skeletal muscle: role of extracellular Ca2+

The present study evaluated whether Ca(2+) entry operates during fatigue of skeletal muscle. The involvement of different skeletal muscle membrane calcium channels and of the Na(+)/Ca(2+) exchanger (NCX) has been examined. The decline of force was analysed in vitro in mouse soleus and EDL muscles submitted to 60 and 110 Hz continuous stimulation, respectively. Stimulation with this high-frequency fatigue (HFF) protocol, in Ca(2+)-free conditions, caused in soleus muscle a dramatic increase of fatigue, while in the presence of high Ca(2+) fatigue was reduced. In EDL muscle, HFF was not affected by external Ca(2+) levels either way, suggesting that external Ca(2+) plays a general protective role only in soleus. Calciseptine, a specific antagonist of the cardiac isoform (alpha1C) of the dihydropyridine receptor, gadolinium, a blocker of both stretch-activated and store-operated Ca(2+) channels, as well as inhibitors of P2X receptors did not affect the development of HFF. Conversely, the Ca(2+) ionophore A23187 increased the protective action of extracellular Ca(2+). KB-R7943, a selective inhibitor of the reverse mode of NCX, produced an effect similar to that of Ca(2+)-free solution. These results indicate that a transmembrane Ca(2+) influx, mainly through NCX, may play a protective role during HFF development in soleus muscle.     

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⁺.     

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.