Involvement of multiple intracellular release channels in calcium sparks of skeletal muscle

In many types of muscle, intracellular Ca(2+) release for contraction consists of brief Ca(2+) sparks. Whether these result from the opening of one or many channels in the sarcoplasmic reticulum is not known. Examining massive numbers of sparks from frog skeletal muscle and evaluating their Ca(2+) release current, we provide evidence that they are generated by multiple channels. A mode is demonstrated in the distribution of spark rise times in the presence of the channel activator caffeine. This finding contradicts expectations for single channels evolving reversibly, but not for channels in a group, which collectively could give rise to a stereotyped spark. The release channel agonists imperatoxin A, ryanodine, and bastadin 10 elicit fluorescence events that start with a spark, then decay to steady levels roughly proportional to the unitary conductances of 35%, 50%, and 100% that the agonists, respectively, promote in bilayer experiments. This correspondence indicates that the steady phase is produced by one open channel. Calculated Ca(2+) release current decays 10- to 20-fold from spark to steady phase, which requires that six or more channels be open during the spark.     

Neuronal calcium sparks and intracellular calcium "noise"

Intracellular calcium ions are involved in many forms of cellular function. To accommodate so many control functions, a complex spatiotemporal organization of calcium signaling has developed. In both excitable and nonexcitable cells, calcium signaling was found to fluctuate. Sudden localized increases in the intracellular calcium concentration-or calcium sparks-were found in heart, striated and smooth muscle, Xenopus Laevis oocytes, and HeLa and P12 cells. In the nervous system, intracellular calcium ions were found important in key processes such as transmitter release, repetitive firing, and gene expression. Hence, we examined whether calcium sparks also exist in neurons. Using confocal laser-scanning microscopy and fluorescent probes, we found that calcium sparks exist in two types of neuronal preparations: the presynaptic boutons of the lizard neuromuscular junction and rat hippocampal neurons in cell culture. Control experiments exclude the possibility that these calcium sparks originate from instrumental or biological artifacts. Calcium sparks seem to be just the tip of the iceberg of a more general phenomenon of intracellular calcium "noise." We speculate that calcium sparks and calcium noise may be of key importance in calcium signaling in the nervous system.     

Hypersensitivity of BKCa to Ca2+ sparks underlies hyporeactivity of arterial smooth muscle in shock

Large conductance Ca(2+)-activated K(+) channels (BK(Ca)) play a critical role in blood pressure regulation by tuning the vascular smooth muscle tone, and hyposensitivity of BK(Ca) to Ca(2+) sparks resulting from its altered beta1 subunit stoichiometry underlies vasoconstriction in animal models of hypertension. Here we demonstrate hypersensitivity of BK(Ca) to Ca(2+) sparks that contributes to hypotension and blunted vasoreactivity in acute hemorrhagic shock. In arterial smooth muscle cells under voltage-clamp conditions (0 mV), the amplitude and duration, but not the frequency, of spontaneous transient outward currents of BK(Ca) origin were markedly enhanced in hemorrhagic shock, resulting in a 265% greater hyperpolarizing current. Concomitantly, subsurface Ca(2+) spark frequency was either unaltered (at 0 mV) or decreased in hyperpolarized resting cells. Examining the relationship between spark and spontaneous transient outward current amplitudes revealed a hypersensitive BK(Ca) activity to Ca(2+) spark in hemorrhagic shock, whereas the spark-spontaneous transient outward current coupling fidelity was near unity in both groups. Importantly, we found an acute upregulation of the beta1 subunit of the channel, and single-channel recording substantiated BK(Ca) hypersensitivity at micromolar Ca(2+), which promotes the alpha and beta1 subunit interaction. Treatment of shock animals with the BK(Ca) inhibitors iberiotoxin and charybdotoxin partially restored vascular membrane potential and vasoreactivity to norepinephrine and blood reinfusion. Thus, the results underscore a dynamic regulation of the BK(Ca)-Ca(2+) spark coupling and its therapeutic potential in hemorrhagic shock-associated vascular disorders.     

Integrative analysis of calcium cycling in cardiac muscle

The control of intracellular calcium is central to regulation of contractile force in cardiac muscle. This review illustrates how analysis of the control of calcium requires an integrated approach in which several systems are considered. Thus, the calcium content of the sarcoplasmic reticulum (SR) is a major determinant of the amount of Ca(2+) released from the SR and the amplitude of the Ca(2+) transient. The amplitude of the transient, in turn, controls Ca(2+) fluxes across the sarcolemma and thence SR content. This control of SR content influences the response to maneuvers that modify, for example, the properties of the SR Ca(2+) release channel or ryanodine receptor. Specifically, modulation of the open probability of the ryanodine receptor produces only transient effects on the Ca(2+) transient as a result of changes of SR content. These interactions between various Ca(2+) fluxes are modified by the Ca(2+) buffering properties of the cell. Finally, we predict that, under some conditions, the above interactions can result in instability (such as alternans) rather than ordered control of contractility.     

Cholinergic regulation of calcium sensitivity in cardiac muscle

The range of calcium concentrations over which activation of cardiac contractile proteins occurs (calcium sensitivity) is regulated by a cAMP controlled phosphorylation of the inhibitory subunit of troponin (TNI) [6, 8]. Phosphorylation of TNI increases the concentration of calcium required for activating myofibrillar ATPase [8] and for generating active tension in hyperpermeable fibers [6]. As TNI goes from a completely dephosphorylated to a completely phosphorylated state, there is a decrease in calcium sensitivity that, in hyperpermeable fibers, may produce as much as a five fold increase in the concentration of calcium required for 50% of maximum activation ([Ca]₅₀) [6]. In both hyperpermeable [6] and intact [2, 11] cells, catecholamines appear to promote cAMP production and TNI phosphorylation. Dephosphorylation of TNI is promoted by cGMP in the hyperpermeable trabecula [6] and by acetylcholine in the perfused heart [2]. Therefore calcium sensitivity may be regulated not only by catecholamine control of protein kinase but in addition by cholinergic control of phosphatase through guanylate cyclase and cGMP. In the present study, the effects of muscarinic cholinergic stimulation on the calcium sensitivity of the contractile system have been examined in the hyperpermeable rat trabecula. Exposure to 10 μm methacholine increases calcium sensitivity in all fibers that initially require 3.3 μm Ca²⁺ or greater for 50% activation. The extent of the change is inversely and linearly related to the initial calcium sensitivity. The effect of methacholine on calcium sensitivity is prevented by 10 μm atropine, implicating the muscarinic receptor in the action of the cholinergic agent.     

Role of calcium in endothelium-dependent relaxation of arterial smooth muscle

Endothelium-dependent relaxation was studied in rings of rabbit thoracic aorta. Relaxation responses were induced with methacholine, the calcium ionophore A23187 and maitotoxin before and after removal of Ca++ from the external medium; in the presence of calcium-channel entry blockers (verapamil and nifedipine); or with trifluoperazine. Deletion of Ca++ greatly impaired responses to all 3 agonists while trifluoperazine only blocked cholinergic-induced relaxation. The calcium-channel blockers had effects that were concentration- and time-dependent, but their action included blockade of A23187. Cytosolic-free Ca++ concentrations were measured in cultured endothelial cells after incubation of the cells with 10 microM Fura-2/AM or 50 microM Quin 2/AM. Bradykinin (1 X 10(-10) to 1 X 10(-7) M) and melittin (0.5 to 5 micrograms/ml) caused dose-dependent increases in intracellular Ca++ with maximal responses at 3 X 10(-8) M and 3 micrograms/ml, respectively. Both agents were able to induce an increase in cytosolic-free Ca++ in the presence of EGTA (1.5 X 10(-3) M) or verapamil (1 X 10(-5) M). The plateau phase of the Ca++ transient appeared to be modified slightly by verapamil, while the peak responses and plateau were attenuated by '0' Ca++/EGTA. To assess a function of the endothelium, production of endothelium-derived relaxing factor (EDRF) was studied in cells grown on microcarrier beads superfused in a column, and the column effluent was bioassayed on aortic rings.(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage-dependent calcium release in guinea-pig cardiac ventricular muscle as antagonized by magnesium and calcium

Summary An increase in extracellular potassium concentration from 4 to 16 mmol/l caused a decrease in membrane potential from –92 to –59 mV and selectively diminished the earlier of two contraction components of guinea-pig papillary muscles at 0.2 Hz stimulation frequency in the presence of noradrenaline. The influence on the early contraction component had a threshold of 8 mmol/l K⁺, corresponding to a membrane potential of –77 mV. However, test contractions elicited 800 ms after the 5 s stimulation interval exhibited an unimpaired early component. Since the activator calcium responsible for the early contraction component is derived, in mammalian ventricular muscle, from the junctional sarcoplasmic reticulum (20), it is assumed that the release site of the reticulum was filled with calcium shortly (800 ms) after a regular contraction, and lost its calcium at 16 mmol/l extracellular K⁺ during the 5 s stimulation interval. The potassium-induced depolarization determined the rate of calcium leakage during rest from the intracellular store. The depolarization-induced decline of the early contraction component was equally well antagonized by Mg²⁺ or Ca²⁺ without influencing the measured transmembrane potential. Both divalent cations shifted the relation between potassium concentration or membrane potential and the strength of the early contraction component to less negative membrane potentials. In order to reduce the early contraction component by 25% in the presence of 9.6 instead of 1.2 mmol/l Mg²⁺, the potassium concentration had to be increased from 9.6 to 22.0 mmol/l, with a respective decrease in resting membrane potential from –72.6 to –51.1 mV. The antagonistic effect of both divalent cations is thought to result from the neutralization of negative charges outside the sarcolemma with a respective decrease in the outside surface potential.     

Prevention by hypothermia of paradoxical calcium necrosis in cardiac muscle

Isolated rat hearts were perfused with calcium-free medium at either 37° or 4°C, and electrical and mechanical phenomena recorded. The calcium-free perfusion of the isolated rat heart at 37°C for periods in excess of 3 min, caused quick cessation of contractile activity with electro-mechanical dissociation. When calcium was restored to the perfusate at 37°C irreversible electro-mechanical dissociation and myocytolysis occurred, accompanied by a massive efflux of intracellular constituents into the perfusate. When the isolated rat heart was perfused with calcium-free medium at 4°C, both the electrocardiogram and contractility disappeared within 15–20 s. The restoration of calcium to these hearts perfused with calcium-free medium at 4°C, permitted rapid recovery of cardiac function upon warming to 37°C. Electron microscopy of hearts perfused with calcium after calcium-free perfusion at 37°C and 4°C showed that at 37°C extensive alteration and lysis of the cardiac organelles occurred, whereas at 4° they were well protected. Uptake of ⁴⁵Ca²⁺ by the myocardium subsequent to calcium-free perfusion at 37°C was approximately 20 times greater than that observed in hearts subjected to calcium deprivation at 4°C. These findings lend support to the view that the paradoxical calcium necrosis of cardiac muscle at 37°C is due to the massive influx of calcium into the cardiac muscle cells following damage to the sarcolemma. It is suggested that the damage to the sarcolemma observed at 37° consists of loss of membrane-bound proteins responsible for the physiologic “calcium channel”. At 4°C their escape from the bilayer appears to beretarded by a more sluggish shift in conformation from the calcium-bound to the calcium-free state, and a more crystalline membrane.     

Involvement of extracellularly bound calcium in the activation of arterial smooth muscle

The role of extracellularly bound Ca in the activation of rabbit aorta by the PGH2 analogue U-44069 was assessed using 45Ca flux determinations. The 45Ca influx process, which is activated by U-44069, was found to exhibit two phases: an initial rapid phase which was transient and a slower phase which was sustained. The 45Ca-40Ca exchange labeling rate of the Ca pool which enters the cell during the initial phase of Ca influx was found to be much slower than that of the free extracellular Ca2+, but quite similar to that of an extracellularly bound Ca pool. These findings suggest that an extracellularly bound Ca pool, possibly located on the outer surface of the plasmalemma, is involved in the U-44069-stimulated Ca influx process.     

Involvement of lipid rafts and caveolae in cardiac ion channel function

A variety of lipid microdomains, including caveolae, have been shown to play an important role in both protein targetting and in controlling protein-protein interactions. There is increasing evidence for significant ion channel localization in lipid rafts. Cardiac channel subunits known to localize in lipid rafts include Kv1.4, Kv1.5, Kv2.1, Kv4, Kir2, Kir3, K(ATP), Nav and Cav subunits. This article reviews what is known about the occurrence and functional significance of cardiac ion channel/lipid raft interactions. Much remains to be learned about this area of potentially enormous importance to cardiac function in health and disease.     

Cholesterol depletion disrupts caveolae and differentially impairs agonist-induced arterial contraction

OBJECTIVE: This study assessed the role of cholesterol-rich membrane regions, including caveolae, in the regulation of arterial contractility. Methods and Results- Rat tail artery devoid of endothelium was treated with the cholesterol acceptor methyl-beta-cyclodextrin, and the effects on force and Ca2+ handling were evaluated. In cholesterol-depleted preparations, the force responses to alpha1-adrenergic receptors, membrane depolarization, inhibition of myosin light chain phosphatase, and activation of G proteins with a mixture of 20 mmol/L NaF and 60 micro mol/L AlCl3 were unaffected. In contrast, responses to 5-hydroxytryptamine (5-HT), vasopressin, and endothelin were reduced by >50%. The rise in global intracellular free Ca2+ concentration in response to 5-HT was attenuated, as was the generation of Ca2+ waves at the cellular level. By electron microscopy, cholesterol depletion was found to disrupt caveolae. The 5-HT response could be restored by exogenous cholesterol, which also restored caveolae. Western blots showed that the levels of 5-HT2A receptor and of caveolin-1 were unaffected by cholesterol extraction. Sucrose gradient centrifugation showed enrichment of 5-HT2A receptors, but not alpha1-adrenergic receptors, in the caveolin-1-containing fractions, suggesting localization of the former to caveolae. CONCLUSIONS: These results show that a subset of signaling pathways that regulate smooth muscle contraction depends specifically on cholesterol. Furthermore, the cholesterol-dependent step in serotonergic signaling occurs early in the pathway and depends on the integrity of caveolae.     

The calcium antagonist nifedipine inhibits arterial smooth muscle cell proliferation

Migration of smooth muscle cells from the media to the intima of the arterial wall and proliferation of intimal smooth muscle are major early events in the formation of an atherosclerotic lesion. The start of proliferation requires that the cells have passed through a modulation from contractile to synthetic phenotype and that they are stimulated with growth factors. Here, we have examined the effects of the calcium antagonist nifedipine on phenotypic modulation and growth of isolated rat arterial smooth muscle cells cultivated in vitro. The results indicate that micromolar concentrations of nifedipine slow down the rate of transformation of the cells from a contractile to a synthetic phenotype and inhibit initiation of DNA synthesis as well as cellular proliferation. The inhibitory effect on DNA synthesis was seen both in cells stimulated with whole blood serum and with purified platelet-derived growth factor. The results raise the possibility that nifedipine may be used to prevent atherogenesis and to inhibit progression of fibromuscular lesions by interfering with the proliferation of arterial smooth muscle cells.     

Effects of increase in extracellular calcium on frequency-dependent inotropism in cardiac muscle

Frequency-dependent effects on contractility in isolated rabbit papillary muscles were compared at different calcium concentrations ranging between 1 and 12.5 mM. Increase in extracellular calcium up to 3 mM resulted in an increase in the frequency-dependent positive inotropy. A further increase in extracellular calcium, up to 5 mM, brought about a quantitative decrease in the frequency-dependent positive inotropy. Still higher concentrations of extracellular calcium, 7.5 and 12.5 mM, altogether abolished the frequency-dependent increase in contractility. The latter at these concentrations of calcium was rather depressed in response to frequency increases. These characteristic changes in the frequency-dependent inotropism at different extracellular calcium concentrations may have been due to the occurrence of a moderate to severe intracellular calcium overload. It is suggested that heart function in certain cardiac diseases, associated with intracellular calcium overload, may not improve with an increase in heart rate.     

Interaction between propranolol and calcium channel blockers in cardiac and vascular smooth muscle

The ability of the beta-receptor antagonist propranolol to influence the response of isolated cardiac and vascular smooth muscle to several classes of calcium channel blockers was examined. For comparison, the interactions between propranolol and other classes of negative inotropic and vasorelaxant agents was also evaluated. The results of these studies demonstrate that propranolol pre-treatment significantly enhances the in vitro response to the dihydropyridine calcium channel blocker nifedipine, but not the thiazapine calcium channel blocker diltiazem. This enhancement was unrelated to the negative inotropic or vasorelaxant properties of these agents. In addition, propranolol pre-treatment of rat cortical membranes also enhanced the affinity of nifedipine for the 3H-nitrendipine binding site, but did not alter the effect of diltiazem on 3H-nitrendipine binding. These observations suggest that a direct interaction may exist between beta-receptor antagonists and dihydropyrine-type calcium channel blockers. This interaction may be an important factor in selecting drug therapy for conditions such as hypertension and angina.     

Heat and fluorescence changes in cardiac muscle: effects of substrate and calcium.

A combined myothermic and fluorescent investigation into the effects of substrate and calcium ions on the contractility of paired rabbit papillary muscles has shown that, (i) as shown in earlier studies pyruvate, when substituted for glucose, increases contractility, increases the levels of fluorescence and resting heat production and speeds the kinetics of fluorescent transients and the time cours of heat production, (ii) reducing the extracellular calcium level from 2.5mm to 1.25mm, decreases contractility without altering the resting fluorescence level and without changing the kinetics of fluorescent transients or the time course of active heat production. Neither treatment (substrate or calcium) altered the isometric heat coefficient. Evidence is presented showing that the fluorescence-time integral is linearly related to the energy expended in a cardiac contraction.     

Rhythm-dependent role of different calcium stores in cardiac muscle: X-ray microanalysis

Under extremely different stimulation patterns such as paired-pulse stimulation, or stimulation after prolonged rest, papillary muscles of guinea-pig show differences in contraction. An early high contraction (360 ms to peak tension) results from paired stimulation at short basic stimulus interval, whereas a small “late” contraction normally occurs (550 ms to peak tension) at a stimulus interval of several minutes (rested state contraction) [Figure 1(a)]. The marked differences in the rate of rise and time to peak of tension in the two cases suggest that different calcium stores releasing calcium in a different mode could be responsible for the different pattern of contraction [1, 7].We have examined this question by looking for calcium stores involved in such states of contraction with the aid of crytotechniques and electron probe microanalysis of chemically untreated shock frozen preparations of guinea-pig papillary muscle.     

Association of smooth muscle cell tissue factor with caveolae

There is still no satisfactory explanation for the low catalytic activity of tissue factor (TF)/factor VII(a) complexes towards coagulation factor X, as found on the apical surface side of cell layers. It has been hypothesized that TF exists in a latent form. Layers of cultured human smooth muscle cells, constitutively expressing TF, were immunogold-labeled for TF in situ and processed for electron microscopy. We showed that, besides internalization and accumulation in lysosomal-like structures, TF remained associated with noncoated, flask- shaped microinvaginations of the plasma membrane. These invaginations were identified as caveolae. In regions in which intercellular contacts were interrupted, more TF-positive caveolae were observed. Enzymatically detached smooth muscle cells exhibited a similar enlargement of caveolar structures. Concomitantly, an increase of catalytic activity of apically formed TF/VIIa complexes towards factor X was found on the suspended cells. We speculate that caveolae- associated TF may function as a latent pool of procoagulant activity, which can rapidly be activated at sites in which vessel wall integrity is lost.     

Compartmentalisation of cAMP-dependent signalling by caveolae in the adult cardiac myocyte

Cyclic AMP exhibits local (sarcolemmal) and global (cytosolic) patterns of signalling, allowing receptor-specific signals to be generated by a single second messenger. Here we determine whether caveolae, invaginated lipid rafts, are responsible for confining the β₂ adrenoceptor (AR) cAMP signal to the sarcolemmal compartment. Myocytes were treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MβC) to disrupt caveolae. Caveolae-containing membrane fractions were isolated by detergent-free sucrose gradient fractionation. Cell shortening and phosphorylation of the sarcoplasmic reticular protein phospholamban (PLB) and the myofilament protein troponin I (TnI) were measured in response to β₂ AR stimulation (with salbutamol in the presence of 1 μM atenolol). Ser¹⁶ phosphorylation of PLB (pPLB), Ser^22,23 phosphorylation of TnI (pTnI), and positive lusitropy were used as indices of global cAMP signals. The ability of MβC to disrupt caveolae was confirmed by selective depletion of the buoyant membrane fractions of cholesterol and caveolin 3, the 2 essential components of caveolae. In control cells, no change in pPLB, pTnI or time to half relaxation was recorded with β₂ AR stimulation (P > 0.05), but following caveolar disruption a 60–70% increase in phosphorylation of both proteins was seen, accompanied by positive lusitropy (P < 0.05). These data show for the first time that disrupting caveolae converts the sarcolemmal-confined cAMP signal associated with β₂ AR stimulation to a global signal that targets proteins of the sarcoplasmic reticulum and myofilaments, with functional sequelae. The role of caveolae in spatial control of cAMP may be relevant to perturbation of β AR signalling in cardiovascular disease.