Comparison of three tetramic acids and their ability to alter membrane function in cultured skeletal muscle cells and sarcoplasmic reticulum vesicles.

Cyclopiazonic acid is a potent inhibitor of calcium uptake and Ca(2+)-ATPase activity in sarcoplasmic and endoplasmic reticulum. In L6 muscle myoblasts, cyclopiazonic acid stimulates the uptake of tetraphenylphosphonium, a lipophilic membrane potential probe, and has antioxidant properties. The purpose of the present study was to investigate the structural requirements necessary for causing the surface charge alterations, and the antioxidant activity in L6 skeletal muscle myoblasts, and for inhibition of calcium transport by rat skeletal muscle sarcoplasmic reticulum vesicles. This was accomplished by comparing the effects of two structurally related tetramic acids, cyclopiazonic acid imine and tenuazonic acid, with cyclopiazonic acid. Cyclopiazonic acid imine inhibited oxalate-assisted 45Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles and stimulated tetraphenylphosphonium accumulation by L6 muscle myoblasts. However, these effects required an approximately fourfold higher concentration than that of cyclopiazonic acid. Tenuazonic acid, up to 1 mM, had no effect on oxalate-assisted 45Ca2+ uptake or Ca(2+)-ATPase activity in sarcoplasmic reticulum vesicles and did not stimulate tetraphenylphosphonium accumulation by L6 muscle myoblasts. Cyclopiazonic acid was only slightly more effective than cyclopiazonic acid imine at preventing the patulin-induced increase in thiobarbituric acid positive substance (used to estimate lipid peroxidation); tenuazonic acid was totally ineffective. Previously, it was shown that cyclopiazonic acid was twice as effective as cyclopiazonic acid imine at preventing increases in thiobarbituric acid positive substance in cultured renal cells, LLC-PK1. Thus, the indole nucleus of cyclopiazonic acid is essential for the membrane-associated biological activity; however, modification of the acetyl group reduces the potency of the activity.     

Superoxide anion radical selectively increases Ca2+ release from cardiac sarcoplasmic reticulum through ryanodine receptor Ca2+ channel

Because the net Ca2+ uptake in the sarcoplasmic reticulum (SR) of cardiac muscle is a result of the activity of Ca(2+)-ATPase and of the SR Ca(2+)-release channel, an abnormal Ca2+ uptake may be the result of the dysfunction of either or both structures. The site or sites of action for oxygen-derived free radicals (OFR) damage are unknown, although previous studies on the SR have focused on damage to the Ca2+ pump. Direct effects of OFR on SR Ca(2+)-release channels may be important in understanding their potential contribution to myocardial ischemia/reperfusion injury. We confirmed that superoxide anion radical (O2.-) generated from hypoxanthine-xanthine oxidase reaction decreases calmodulin content and increases 45Ca2+ efflux from the heavy fraction of canine cardiac SR vesicles. Electron spin resonance study showed that hydroxyl radicals are generated in addition to O2.- from hypoxanthine-xanthine oxidase reaction, and data indicate that O2.- is responsible for the observed effect. Current fluctuations through single Ca(2+)-release channels have been also monitored after incorporation into planar phospholipid bilayers. We directly demonstrate that activation of the channel by O2.- stimulates Ca2+ release from heavy SR vesicles and suggest the importance of accessory proteins such as calmodulin in modulating the effect of O2.-.     

Ryanodine induces persistent inactivation of the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.

Junctional sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle were pretreated with 0.1-500 microM ryanodine under equilibrium conditions optimal for receptor binding, followed by the removal of bound alkaloid by several washes in Ca(2+)- and ryanodine-free buffer. Pretreatment with > 100 nM ryanodine results in a concentration-dependent decrease in the Bmax of the high affinity sites and a complete loss of measurable low affinity binding sites that persist for > 48 hr. Quantitative analysis of residual ryanodine using three different methods demonstrates that the inhibition is not the result of residual ryanodine bound to its receptor. Ca2+ transport measurements made with antipyrylazo III show that actively loaded ryanodine-pretreated SR exhibits a persistent insensitivity to ryanodine- and daunomycin-induced Ca2+ release that is not seen with washed control vesicles. Lipid bilayer membranes fused with SR vesicles exhibit rapidly fluctuating single-channel events with a conductance of 468 pS in asymmetric CsCl solutions. Ryanodine (10 microM) produces a unidirectional transition to a slowly fluctuating half-conductance state that is not reversed by perfusion of the bilayer with Ca(2+)-free buffer and subsequent addition of dithiothreitol. However, dithiothreitol added in the ryanodine pretreatment medium offers marked protection against ryanodine-induced loss of binding sites and allows complete restoration of native gating behavior of single channels in bilayer lipid membrane. Using three different experimental approaches, the data demonstrate that the alkaloid at micromolar concentration persistently alters SR Ca2+ release channel function, perhaps by uncoupling four negatively cooperative binding sites. The oxidation of critical receptor thiols is implicated in the process.     

Long-chain acylcarnitine induces Ca2+ efflux from the sarcoplasmic reticulum

Long-chain acylcarnitines increase intracellular Ca2+ (Ca2+i) and induce electrophysiologic alterations that likely contribute to the genesis of malignant ventricular arrhythmias induced during myocardial ischemia. The mechanisms by which long-chain acylcarnitines increase Ca2+i are not known, although it occurs in the presence of Ca2+ channel blockade and inhibition of Na+/Ca2+ exchange. Long-chain acylcarnitines activate Ca2+ release channels from skeletal muscle sarcoplasmic reticulum (SR), but their effect on cardiac SR is unclear. To test the hypothesis that long-chain acylcarnitines increase Ca2+i from the SR, SR-enriched membrane fractions were prepared from rabbit left ventricular myocardium using sucrose density-gradient centrifugation and characterized by marker enzyme analysis. 45Ca2+ efflux was assessed in the presence or absence of long-chain acylcarnitines. Palmitoylcarnitine and stearoylcarnitine produced concentration-dependent efflux of 45Ca2+, whereas shorter chain acylcarnitines, palmitate, and palmitoyl-coenzyme A did not. Pretreatment of cardiac SR vesicles with ryanodine did not prevent palmitoylcarnitine-induced Ca2+ release. In addition, palmitoylcarnitine did not influence specific [3H]ryanodine binding, suggesting a mechanism independent of alterations in ryanodine receptor/Ca2+ release channel binding. In summary, long-chain acylcarnitines enhance Ca2+ release from cardiac SR vesicles and may thereby mobilize Ca2+i to induce electrophysiologic derangements under conditions, such as ischemia, in which these amphiphiles accumulate.     

Induction of calcium release from sarcoplasmic reticulum of skeletal muscle by xanthone and norathyriol.

1. Effects of xanthone and its derivative, 1,3,6,7-tetrahydroxyxanthone (norathyriol), on Ca2+ release and ryanodine binding were studied in isolated sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle. 2. Both xanthone and norathyriol dose-dependently induced Ca2+ release from the actively loaded SR vesicles which was blocked by ruthenium red, a specific Ca2+ release inhibitor, and Mg2+. 3. Xanthone and norathyriol also dose-dependently increased apparent [3H]-ryanodine binding. Norathyriol, but not xanthone, produced a synergistic effect on binding activation when added concurrently with caffeine. 4. In the presence of Mg2+, which inhibits ryanodine binding, both caffeine and norathyriol, but not xanthone, could restore the binding to the level observed in the absence of Mg2+. 5. Xanthone activated the Ca(2+)-ATPase activity of isolated SR vesicles dose-dependently reaching 70% activation at 300 microM. 6. When tested in mouse diaphragm, norathyriol potentiated the muscle contraction followed by twitch depression and contracture in either a Ca(2+) -free bathing solution or one containing 2.5 mM Ca2+. These norathyriol-induced effects on muscle were inhibited by pretreatment with ruthenium red or ryanodine. 7. These data suggest that xanthone and norathyriol can induce Ca2+ release from the SR of skeletal muscle through a direct interaction with the Ca2+ release channel, also known as the ryanodine receptor.     

Anthraquinone-sensitized Ca2+ release channel from rat cardiac sarcoplasmic reticulum: possible receptor-mediated mechanism of doxorubicin cardiomyopathy

Rat cardiac membrane vesicles enriched in biochemical markers of the junctional region of sarcoplasmic reticulum (SR) and exhibiting ruthenium red-sensitive rapid Ca2+ release have been prepared. Doxorubicin and seven congeners are shown to enhance the binding of [3H]ryanodine to the ryanodine receptor with a strong structural requirement. Doxorubicin enhances the binding of [3H]ryanodine to SR membranes and soluble receptor preparations and induces Ca2+ release from SR vesicles in a highly Ca2(+)-dependent manner, suggesting that anthraquinones promote the open state of the junctional Ca2+ release channel by increasing the affinity of the Ca2+ activator site for Ca2+. Doxorubicin reduces the Kd of [3H]ryanodine binding solely by enhancing the rat of association. Caffeine competes for the same site with anthraquinones, because the caffeine-activated binding of [3H]ryanodine is inhibited by doxorubicin and vice versa. The acute effect of doxorubicin on the cardiac Ca2+ release channel is fully reversible; however, long term treatment (up to 24 hr) with doxorubicin increases the sensitivity of the preparation to subsequent acute challenge with doxorubicin. The thiol-reductive agent dithiothreitol enhances, whereas the reactive disulfide 4,4'-dithiodipyridine reduces, the doxorubicin-enhanced binding of [3H]ryanodine. These results demonstrate that the acute and chronic cardiotoxicity of anthraquinones may be accounted for by a receptor-mediated mechanism. Our findings suggest that the chronic effects observed with the clinical use of anthraquinones may be the result of a receptor-mediated shift in the redox equilibrium of allosteric thiols at the ryanodine receptor complex, which in turn leads to long term sensitization of the Ca2+ release channel.     

3,5-di-t-butyl-4-hydroxyanisole (DTBHA) activation of rat skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase.

3,5-Di-t-butyl-4-hydroxyanisole (DTBHA) increased in a concentration-dependent manner (calculated pEC(50) = 4.55 +/- 0.18 M) the oxalate-stimulated Ca(2+)-pumping rate of rat skeletal muscle sarcoplasmic reticulum (SR) vesicles. Kinetic analysis of this effect suggested that the activation of SR Ca(2+)-ATPase operated by (DTBHA) was of both mixed and non-competitive type with respect to ATP in the range of concentrations 0.1-0.5 mM and above 1 mM, respectively; furthermore, it was independent of the free Ca(2+) concentrations. This indicated that the enzyme activation took place through the acceleration of the enzyme-substrate complex breakdown. Moreover, it appeared that its target site was cyclopiazonic acid sensitive. The uncommon ability of (DTBHA) to upregulate SR Ca(2+) uptake is of interest in view of its possible use for treating pathological conditions characterised by cell Ca(2+) overload as well as genetic disorders where SR Ca(2+) homeostasis is altered.     

Digoxin activates sarcoplasmic reticulum Ca(2+)-release channels: a possible role in cardiac inotropy.

1. The effect of digoxin on rapid 45Ca2+ efflux from cardiac and skeletal sarcoplasmic reticulum (SR) vesicles was investigated. Additionally the interaction of digoxin with single cardiac and skeletal muscle SR Ca(2+)-release channels incorporated into planar phospholipid bilayers and held under voltage clamp was determined. 2. Digoxin (1 nM) increased the initial rate and amount of Ca(2+)-induced release of 45Ca2+ from cardiac SR vesicles, passively loaded with 45CaCl2, at an extravesicular [Ca2+] of 0.1 microM. The efflux in the presence and absence of digoxin was inhibited at pM extravesicular Ca2+ and blocked by 5 mM Mg2+. 3. To elucidate the mechanism of action of digoxin, single-channel recording was used. Digoxin (1-20 nM) increased single-channel open probability (Po) when added to the cytosolic but not the luminal face of the cardiac channel in the presence of sub-maximally activating Ca2+ (0.1 microM-10 microM) with an EC50 of 0.91 nM at 10 microM Ca2+. The mechanisms underlying the action of digoxin appear to be concentration-dependent. The activation observed at 1 nM digoxin appears to be consistent with the sensitization of the channel to the effects of Ca2+. At higher concentrations the drug appears to interact synergistically with Ca2+ to produce values of Po considerably greater than those seen with Ca2+ as the sole activating ligand. 4. Digoxin had no effect on single-channel conductance or the Ca2+/Tris permeability ratio. In channels activated by digoxin the Po was decreased by Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-activated ryanodine binding: mechanisms of sensitivity and intensity modulation by Mg2+, caffeine, and adenine nucleotides

The Ca2+-ryanodine receptor complex is a functional unit at the terminal cisternae (TC) of the sarcoplasmic reticulum (SR) whose proteins comprise the Ca2+ release channels which may be involved in excitation-contraction coupling. Ca2+, Mg2+, caffeine, and adenine nucleotides, but not inositol 1,4,5-trisphosphate, may exert their inotropic effects on skeletal muscle SR by direct allosteric modulation of the [3H]ryanodine-binding site. Micromolar Ca2+ is primarily responsible for activating [3H]ryanodine binding by regulating receptor site density, affinity, and cooperativity. Mg2+ reduces the sensitivity to Ca2+ activation by directly competing with Ca2+ for the activator site. However, inhibition by Mg2+ is overcome in the presence of beta,gamma-methyleneadenosine 5'-triphosphate (AMP-PCP; 1 mM) or caffeine (20 mM). Caffeine dramatically increases the affinity of the Ca2+ activator site for Ca2+, whereas AMP-PCP or cAMP enhances the gating efficiency or the lifetime of the open state of the TC SR channel. A kinetic model is proposed for four functional domains of the Ca2+-ryanodine receptor complex: the Ca2+-regulatory domain which binds Ca2+ with microM affinity is primarily responsible for gating the Ca2+ channel of the TC SR in a cooperative manner, and is inhibited by mM Mg2+ by direct competition for the activator site which appears to contain critical sulfhydryl groups; a Ca2+-activate alkaloid binding domain in close proximity to the channel which binds ryanodine with nM affinity and rapidly occludes upon complex formation; a domain which binds caffeine with low (greater than mM) affinity and directly influences the sensitivity of the Ca2+-regulatory site; and a domain which binds adenine nucleotides with intermediate affinity (less than mM), does not require phosphorylation, and intensifies the Ca2+ signal which triggers opening of the Ca2+-release channel.     

Endogenous cannabinoid anandamide directly inhibits voltage-dependent Ca(2+) fluxes in rabbit T-tubule membranes

The effect of the endogenous cannabinoid, anandamide on Ca(2+) flux responses mediated by voltage-dependent Ca(2+) channels was studied in transverse tubule membrane vesicles from rabbit skeletal muscle. Vesicles were loaded with 45Ca(2+) and membrane potentials were generated by establishing K(+) gradients across the vesicle using the ionophore, valinomycin. Anandamide, in the range of 1-100 microM, inhibited depolarization-induced efflux responses. Anandamide also functionally modulated the effects of nifedipine (1-10 microM) and Bay K 8644 (1 microM) on Ca(2+) flux responses. Pretreatment with the specific cannabinoid receptor antagonist, SR141716A (1 microM), pertussis toxin (5 microg/ml), the amidohydrolase inhibitor, phenylmethylsulfonyl fluoride (0.2 mM) or the cyclooxygenase inhibitor, indomethacin (5 microM) did not alter the inhibition of efflux responses by anandamide. Arachidonic acid (10-100 microM) also effectively inhibited 45Ca(2+) efflux from membrane vesicles. In radioligand binding studies, it was found that both anandamide and arachidonic acid inhibited the specific binding of [3H]PN 200-110 to transverse tubule membranes with IC(50) values of 4.4+/-0. 7 and 13.4+/-3.5 microM, respectively. These results indicate that anandamide, independent of cannabinoid receptor activation, directly inhibits the function of voltage-dependent calcium channels and modulates the specific binding of calcium channel ligands of the dihydropyridine class.     

Effect of carticaine on the sarcoplasmic reticulum Ca2+-dependent adenosine triphosphatase

The sarcoplasmic reticulum Ca2+-ATPase (calcium-dependent adenosine triphosphatase) transports Ca2+ from the myoplasm to the reticulum lumen at the expense of free energy from ATP hydrolysis. Carticaine is a local anesthetic of frequent use in dentistry which is now entering other clinical fields. We studied the action of carticaine on the sarcoplasmic reticulum (SR) skeletal muscle Ca2+-ATPase. SR vesicles from rabbit fast skeletal muscle were used. Carticaine inhibits the enzymatic activity. The inhibition of the enzymatic activity depends on pH, [Ca2+] and the presence of calcimycin. Half-maximal carticaine concentration that inhibits the ATPase activity tends to a maximal value upon increasing [Ca2+]. Carticaine concentrations required to inhibit the enzymatic activity at myoplasmic calcium concentration are lower than usual clinical doses: Ki=6.0+/-1.4 mM carticaine (n=5) for 0.1 microM [Ca2+]. ATP-dependent calcium uptake is also inhibited by the local anesthetic: Ki=30.5+/-3.4 mM (n=4). Besides, carticaine inhibits the phosphorylation of the enzyme by inorganic phosphate (Pi): Ki=20.0+/-3.4 (n=5) - 33.2+/-4.6 (n=4) mM, for [Pi] 1-4 mM. Carticaine increases the membrane permeability to Ca2+. Ca2+ efflux from preloaded vesicles is prevented by Ca2+ and Mg2+. Our results suggest that the diffusion of the local anesthetic into muscle fibers might trigger undesired effects such as sustained contraction of the masticatory muscles.     

Interaction of phenylglyoxal with the human erythrocyte (Ca2+ + Mg2+)-ATPase. Evidence for the presence of an essential arginyl residue

Incubation of human erythrocyte membranes with phenylglyoxal irreversibly inhibited (Ca2+ + Mg2+)-ATPase activity in a pseudo-first order manner, but followed overall second order kinetics. The enzyme exhibited a low affinity ATP-binding site with a Km of approximately 125 microM. The effects of the inhibitor could be markedly diminished if ATP was also present during phenylglyoxalation. This indicated that phenylglyoxal and ATP were binding at the same site on the enzyme. The concentration-dependent inactivation reduced both the Vmax and the Km of the enzyme, but did not change the apparent affinity for Ca2+. Because ATP could protect against inactivation in the absence of Ca2+ and Mg2+, we suggest that free ATP can bind at the low affinity site. The modified enzyme was still capable of being activated by calmodulin. Our data indicate that only the ATP site was affected by the inhibitor, whereas the Ca2+ sites were not. Since it is established that phenylglyoxal can react with arginyl residues, we conclude that the binding of the ATP to the low affinity site on the human erythrocyte (Ca2+ + Mg2+)-ATPase involves such a residue.     

Effects of ethanol and barbiturates on Ca2+-ATPase activity of erythrocyte and brain membranes

Exposure to ethanol or pentobarbital in vitro stimulated the ATP-dependent efflux of calcium from human red blood cells (RBC) and the Ca2+-ATPase activity of RBC and rat brain synaptic plasma membranes (SPM). These effects were obtained with concentrations of ethanol (50 mM) and pentobarbital (60 microM) associated with intoxication in vivo. The enhancement of SPM Ca2+-ATPase by ethanol was due to an increase in the apparent affinity of the enzyme for calcium with no change in the maximum velocity. SPM Ca2+-ATPase was also stimulated by an unsaturated fatty acid, cis-vaccenic acid methyl ester (cis-VAME). The membrane-disordering effects of ethanol, four barbiturates and cis-VAME were evaluated in SPM using the fluorescent probe molecule 1,6-diphenyl-1,3,5-hexatriene (DPH). All the compounds decreased the fluorescence polarization of DPH, and these decreases were proportional to the increase in Ca2+-ATPase produced by these drugs. These findings suggest that the increase in Ca2+-ATPase and calcium efflux produced by ethanol and pentobarbital results from the membrane-disordering effects of these drugs.     

Interaction of Ca2+ with cardiolipin-containing liposomes and its inhibition by adriamycin

The interaction of cardiolipin-containing, unilamellar liposomes with Ca2+ was assessed by flow dialysis in the presence of 2-100 microM 45Ca2+, using vesicles formed from phosphatidylcholine (PC) and from PC and cardiolipin in mole ratios from 16:1 to 1:1. Control (PC only) vesicles bound no detectable Ca2+. In contrast, Ca2+ binding to cardiolipin-containing vesicles was substantial and dependent on vesicle concentration. Scatchard plots for the binding were concave upward. Resolution of the data, assuming the presence of two independent classes of binding sites, indicated a high-affinity site with apparent KD = 5.57 +/- 0.48 microM (S.D.) and a second site with KD in the millimolar range. Interaction of cardiolipin-containing liposomes with Ca2+ was insensitive to monovalent cations (Na+, K+, Rb+), but was inhibited by ruthenium red much greater than La3+ greater than Mn2+ greater than Mg2+. Progressive increases in the PC: cardiolipin ratio markedly increased the apparent KD for Ca2+ at the high-affinity site. Stoichiometry of Ca2+ binding at the site passed through a maximum at a PC: cardiolipin ratio of 4:1. The potent antineoplastic agent adriamycin also inhibited the interaction of Ca2+ with cardiolipin-containing liposomes in a dose-dependent manner; effects were detected at 10 microM antibiotic. Unlike PC, adriamycin altered the stoichiometry of the high-affinity interaction but not the apparent KD. Adriamycin effects increased with pH in the range of the pKA of its amino group. These results suggest that inhibition by adriamycin may result from a mechanism other than simple competition for the charged head group of cardiolipin.     

Characterization of multiple [3H]ryanodine binding sites on the Ca2+ release channel of sarcoplasmic reticulum from skeletal and cardiac muscle: evidence for a sequential mechanism in ryanodine action.

Kinetic and equilibrium measurements of [3H]ryanodine binding to the Ca2+ release channel of rabbit skeletal and rat cardiac sarcoplasmic reticulum (SR) are examined to ascertain the nature of cooperative interactions among high and low affinity binding sites and to quantitate their distribution. Equilibrium studies reveal affinities of 1-4 nM for the highest affinity binding site and of 30-50 nM, 500-800 nM, and 2-4 microM for the lower affinity sites in both preparations, with Hill coefficients of significantly less than 1, and initial rates of association and dissociation increase with increasing concentrations of ryanodine. SR vesicles are actively loaded in the presence of pyrophosphate, and fluctuations in extravesicular Ca2+ are measured by the absorbance change of antipyrylazo III. The data demonstrate a biphasic, time- and concentration-dependent action of ryanodine on the release of Ca2+, with an initial activation and a subsequent inactivation phase. Kinetic analysis of the activation of Ca2+ release by ryanodine, in consonance with the binding data, demonstrates the existence of multiple binding sites for the alkaloid on the channel complex, with nanomolar to micromolar affinities. Based on the present findings obtained by receptor binding analysis and Ca2+ transport measurements, we suggest a model that describes four, most plausibly negatively cooperative, binding sites on the Ca2+ release channel. Occupation of ryanodine binding sites produces sequential activation followed by inactivation of the SR channel, revealing the strong possibility of an irreversible uncoupling of the native function of the receptor/channel complex by high concentrations of ryanodine. A model relating ryanodine receptor occupancy with SR Ca2+ release stresses two important new findings regarding the interaction of ryanodine with its receptor. First, ryanodine binds to four sites on the oligomeric channel complex with decreasing affinities, which can be best described by allosteric negative cooperativity. Second, binding of ryanodine to its receptor activates the Ca2+ release channel in a concentration-dependent and saturable manner in the range of 20 nM to 1 mM and produces a kinetically limited and sequential inactivation of the Ca2+ channel, with the concomitant attainment of full negative cooperativity. The results presented suggest that driving of the complex toward full negative cooperativity with high concentrations of ryanodine promotes a long-lived conformational state in which ryanodine is physically occluded and hindered from free diffusion from its binding site.     

Effects of cyclopiazonic acid on contraction and intracellular Ca2+ in oesophageal striated muscle of normotensive and spontaneously hypertensive rats

1. The effects of cyclopiazonic acid (CPA), a selective inhibitor of sarcoplasmic reticulum (SR) Ca2+-ATPase, on twitch contraction and on the resting state of tension and intracellular Ca2+ level ([Ca2+]i) of the oesophageal striated muscle of stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar Kyoto rats (WKY) were compared. 2. CPA (10 micronM) augmented the twitch contraction of oesophageal striated muscle preparations from both SHRSP and WKY, reducing the rate of relaxation (-dT/dt), and thus resulting in the prolongation of the time to 80% relaxation. The effect was significantly smaller in the SHRSP preparations. 3. In the resting state, CPA caused a sustained elevation of [Ca2+]i. The elevation was greater in the WKY preparations. Tension development accompanied by the elevation was observed in WKY preparations, but not in SHRSP preparations. 4. The sustained elevation of [Ca2+]i induced by CPA was eliminated by the removal of extracellular Ca2+. Both the elevated [Ca2+]i and tension in the preparations from WKY were reduced by flufenamic acid (100 micronM), mefenamic acid (100 micronM), lanthanum (La3+, 100 micronM), gadolinium (Gd3+, 100 micronM) and SK&F 96365 (100 micronM) but not by verapamil (10 micronM). 5. Thapsigargin (3 micronM), another SR Ca2+-ATPase inhibitor, produced similar effects on basal tension to those of CPA, although it reduced the amplitude of twitch contraction. 6. These results suggest that in the rat oesophageal striated muscle, (1) CPA extends the sequestrating time of Ca2+ into the SR, (2) CPA induces a Ca2+ influx mediated through verapamil-insensitive pathways, possibly nonselective cation channels, and (3) the mechanism of [Ca2+](i) modulation due to CPA-sensitive SR Ca2+-ATPase is deteriorated in the oesophageal striated muscle from SHRSP as compared with WKY preparations.     

Ca2+ entry activated by emptying of intracellular Ca2+ stores in ileal smooth muscle of the rat.

1. The effects of depletion of intracellular Ca2+ stores on muscle tension and the intracellular Ca2+ concentration ([Ca2+])i were studied in fura-2 loaded longitudinal smooth muscle cells of the rat ileum. 2. After exposure to a Ca(2+)-free solution, application of Ca2+ caused a small contraction and a rise in [Ca2+]i, both of which were potentiated when the muscle was challenged with carbachol or caffeine before the addition of Ca2+. 3. Cyclopiazonic acid (CPA), a specific inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, dose-dependently decreased tension development and the rises in [Ca2+]i induced by carbachol and caffeine in the Ca(2+)-free solution, but conversely increased the Ca(2+)-induced responses even in the presence of the voltage-dependent Ca2+ channel blockers, methoxyverapamil and nifedipine. 4. The contraction and rise in [Ca2+]i evoked by Ca2+ gradually declined with time after removal of CPA, while the reverse was the case for the responses to carbachol and caffeine. 5. The Ca(2+)-induced contraction and rise in [Ca2+]i in the presence of CPA were inhibited by the replacement of Na+ with K+ or Cs+, and by the addition of Cd2+, Ba2+, Ni2+ or La3+. 6. The influx of Mn2+ was much greater in extent in the presence of CPA than in its absence. 7. These results suggest that the emptying of intracellular Ca2+ stores may activate Ca2+ influx not associated with voltage-dependent Ca2+ channels in the rat ileal smooth muscle.     

Vasopressin-evoked [Ca2+]i responses in neonatal rat cardiomyocytes.

The presence of arginine vasopressin (AVP) V1 receptors on neonatal rat cardiomyocytes (NRCs) linked to processes capable of elevating intracellular free calcium ([Ca2+]i) is now firmly established. This study examined the sources and signaling involved in [Ca2+]i elevations evoked by AVP in NRCs. AVP promoted increases in both [Ca2+]i and 1,4,5-inositoltrisphosphate (IP3) levels in NRCs. The degree of [Ca2+]i elevation was less than that of angiotensin II, but greater than that of endothelin-1. Extracellular Mg2+ depletion led to diminution of the maximal [Ca2+]i response, with a rightward shift in the concentration-response curves to AVP. The phospholipase C inhibitors, D-609, NCDC, or U73122, and the IP3 receptor blocker, heparin, abolished the [Ca2+]i response to AVP. Neither cyclooxygenase inhibition with indomethacin nor PKC inhibition with staurosporine had any effect. Neither ryanodine nor caffeine, which deplete sarcoplasmic reticulum (SR) Ca2+ stores, nor ruthenium red, which inhibits both SR and mitochondrial Ca2+ stores, affected [Ca2+]i responses to AVP. The SR Ca2+ pump inhibitor, cyclopiazonic acid, abolished, and removal of extracellular Ca2+ attenuated, the response to AVP. These data indicate that activation of cardiac V1 receptors by AVP results in mobilization of Ca2+ from a distinct, non-SR, nonmitochondrial, intracellular Ca2+ pool that is Ca2+ pump replenished and IP3 sensitive. This process occurs secondary to phospholipase C (PLC)-mediated generation of IP3, requires the presence of Mg2+ and extracellular Ca2+, and occurs in a manner independent of PKC and cyclooxygenase activation. Such mechanisms of Ca2+ mobilization might indicate a distinct role for AVP in cardiac physiology and disease.     

Ca2+ buffering function of sarcoplasmic reticulum in rat tail arteries: comparison in normotensive and spontaneously hypertensive rats

The superficial buffer barrier function of the sarcoplasmic reticulum (SR) during rest and that during stimulation with Bay k 8644, an agonist of L-type Ca2+ channels, were compared in endothelium-denuded strips of tail arteries from 13-week-old normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), by measuring the effects of cyclopiazonic acid (CPA) and thapsigargin that inhibit SR Ca2+-ATPase and the effect of ryanodine that depletes SR Ca2+. The addition of 10 microM CPA induced a transient contraction that was not significantly different between WKY and SHR. The CPA-induced contraction was strongly inhibited by 100 nM nifedipine and was abolished by Ca2+-free solution in both strains. Thapsigargin (100 nM) or ryanodine (10 microM) induced similar, small transient contractions in the two strains. The addition of Bay k 8644 (1-100 nM) almost failed to induce a contraction in both WKY and SHR. When the strips were preincubated with 10 microM CPA, 100 nM thapsigargin or 10 microM ryanodine, Bay k 8644 induced similar concentration-dependent contractions in the two strains. The amount of Ca2+ stored in the SR, as estimated from the 20 mM caffeine-induced contraction, was not significantly different between WKY and SHR. Our results suggest that the SR of rat tail arteries can buffer a large amount of Ca2+ that enters the cell during the rest and the Bay k 8644 stimulation, and these functions are not altered in SHR.     

Ca2+ stores regulate ryanodine receptor Ca2+ release channels via luminal and cytosolic Ca2+ sites

1. In muscle, intracellular calcium concentration, hence skeletal muscle force and cardiac output, is regulated by uptake and release of calcium from the sarcoplasmic reticulum. The ryanodine receptor (RyR) forms the calcium release channel in the sarcoplasmic reticulum. 2. The free [Ca2+] in the sarcoplasmic reticulum regulates the excitability of this store by stimulating the Ca2+ release channels in its membrane. This process involves Ca2+-sensing mechanisms on both the luminal and cytoplasmic sides of the RyR. In the cardiac RyR, these have been shown to be a luminal Ca2+ activation site (L-site; 60 micromol/L affinity), a cytoplasmic activation site (A-site; 0.9 micromol/L affinity) and a cytoplasmic Ca2+ inactivation site (I2-site; 1.2 micromol/L affinity). 3. Cardiac RyR activation by luminal Ca2+ occurs by a multistep process dubbed 'luminal-triggered Ca2+ feed-through'. Binding of Ca2+ to the L-site initiates brief (1 msec) openings at a rate of up to 10/s. Once the pore is open, luminal Ca2+ has access to the A-site (producing up to 30-fold prolongation of openings) and to the I2-site (causing inactivation at high levels of Ca2+ feed-through). 4. The present paper reviews the evidence for the principal aspects of the 'luminal-triggered Ca2+ feed-through' model, the properties of the various Ca2+-dependent gating mechanisms and their likely role in controlling sarcoplasmic reticulum (SR) Ca2+ release in cardiac muscle. 5. The model makes the following important predictions: (i) there will be a close link between luminal and cytoplasmic regulation of RyRs and any cofactor that prolongs channel openings triggered by cytoplasmic Ca2+ will also promote RyR activation by luminal Ca2+; (ii) luminal Mg2+ (1 mmol/L) is essential for the control of SR excitability in cardiac muscle by luminal Ca2+; and (iii) the different RyR isoforms in skeletal and cardiac muscle will be controlled quite differently by the luminal milieu. For example, Mg2+ in the SR lumen (approximately 1 mmol/L) can strongly inhibit RyR2 by competing with Ca2+ for the L-site, whereas RyR1 is not affected by luminal Mg2+.