ryanodine and indo-1

The effect of micromolar intracellular levels of ryanodine was tested on the myoplasmic free calcium concentration ([Ca(2+)](i)) measured from a portion of isolated mouse skeletal muscle fibers voltage-clamped at -80 mV. When ryanodine-injected fibers were transiently depolarized to 0 mV, the early decay phase of [Ca(2+)](i) upon membrane repolarization was followed by a steady elevated [Ca(2+)](i) level. This effect could be qualitatively well simulated, assuming that ryanodine binds to release channels that open during depolarization and that ryanodine-bound channels do not close upon repolarization. The amplitude of the postpulse [Ca(2+)](i) elevation depended on the duration of the depolarization, being hardly detectable for pulses shorter than 100 ms, and very prominent for duration pulses of seconds. Within a series of consecutive pulses of the same duration, the effect of ryanodine produced a staircase increase in resting [Ca(2+)](i), the slope of which was approximately twice larger for depolarizations to 0 or +10 mV than to -30 or -20 mV. Overall results are consistent with the "open-locked" state because of ryanodine binding to calcium release channels that open during depolarization. Within the voltage-sensitive range of calcium release, increasing either the amplitude or the duration of the depolarization seems to enhance the fraction of release channels accessible to ryanodine.

Topics: Animals; Biophysical Phenomena; Biophysics; Calcium; Calibration; Cell Membrane; Electrophysiology; Fluorescent Dyes; Indoles; Mice; Microscopy, Fluorescence; Muscle, Skeletal; Ryanodine; Time Factors

In the cat ventricle angiotensin II exerts a positive inotropic effect produced by an increase in intracellular calcium associated with a prolongation of relaxation. The signaling cascades involved in these effects as well as the subcellular mechanisms of the negative lusitropic effect are still not clearly defined. The present study was directed to investigate these issues in cat papillary muscles and isolated myocytes. The functional suppression of the sarcoplasmic reticulum (SR) with either 0.5 microm ryanodine or 0.5 microm ryanodine plus 1 microm thapsigargin or the preincubation of the myocytes with the specific inhibitor of the inositol 1,4,5-triphosphate (IP3) receptors [diphenylborinic acid, ethanolamine ester (2-APB), 5-50 microm] did not prevent the positive inotropic effect and the increment in Ca2+ transient produced by 1 microm angiotensin II. In contrast, protein kinase C (PKC) inhibitors, chelerythrine (20 microm) and calphostin C (1 microm) completely inhibited both, the angiotensin II-induced increase in L-type calcium current and positive inotropic effect. The prolongation of half relaxation time produced by 0.5 microm angiotensin II [207+/-15.4 msec (control) to 235+/-19.98 msec (angiotensin II), P<0.05] was completely blunted by PKC inhibition. This antirelaxant effect, which was independent of intracellular pH changes, was associated with a prolongation of the action potential duration and was preserved after either the inhibition of the SR and the SR Ca2+ ATPase (ryanodine plus thapsigargin) or of the reverse mode of the Na+/Ca2+ exchanger (KB-R7943, 5 microm). We conclude that in feline myocardium the positive inotropic and negative lusitropic effects of angiotensin II are both entirely mediated by PKC without any significant participation of the IP3 limb of the phosphatidylinositol/phospholipase C cascade. The results suggest that the antirelaxant effect of angiotensin II might be determined by the decrease in Ca2+ efflux through the Na+/Ca2+ exchanger produced by the angiotensin II-induced prolongation of the action potential duration.

Topics: Angiotensin II; Animals; Calcium; Calcium Channels; Cardiotonic Agents; Cats; Collagenases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Indoles; Inositol 1,4,5-Trisphosphate Receptors; Microscopy, Fluorescence; Myocardium; Naphthalenes; Papillary Muscles; Patch-Clamp Techniques; Phosphorylation; Protein Kinase C; Receptors, Cytoplasmic and Nuclear; Ryanodine; Sarcoplasmic Reticulum; Signal Transduction; Thapsigargin; Time Factors

Gonadotropin releasing hormone (GnRH) stimulates both transcription and secretion of the alpha subunit of the gonadotropins in a Ca2+-dependent fashion. In this study, we examined the role of Ca2+ as the signal coupling agonist occupancy of GnRH receptors to hormone secretion using the gonadotropic cell line alphaT3-1. Treatment of alphaT3-1 cells for 60 min with GnRH (0.1-100 nM), veratridine (50 microM) or high K+ (56 mM) was completely ineffective in stimulating secretion. The lack of effect occurred in spite of a robust, specific, and dose-dependent biphasic [Ca2+]i response consisting of a rapid peak sensitive to thapsigargin (200 nM) followed by a smaller plateau sensitive to the extracellular application of EGTA (5 mM). On the other hand, treatment of alphaT3-1 cells with the Ca2+ ionophore ionomycin resulted in a significant dose-dependent stimulation of secretion and [Ca2+]i responses comparable to those elicited by GnRH. Binding assays revealed the presence of Ins(1,4,5)P3 receptors (Kd = 3.2 nM, Bmax = 50.5 fmol/mg protein) but not ryanodine receptors in alphaT3-1 cell membranes. Together, these results show a functional uncoupling between the [Ca2+]i response and secretion in this cell line, suggesting that the increase in [Ca2+]i triggered by GnRH and depolarization may be necessary but not sufficient to stimulate exocytosis.

Topics: Calcium; Calcium Channel Blockers; Calcium Signaling; Cell Line; Cell Survival; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Exocytosis; Fluorescent Dyes; Gadolinium; Glycoprotein Hormones, alpha Subunit; Glycoproteins; Gonadotropin-Releasing Hormone; Indoles; Inositol 1,4,5-Trisphosphate; Ionomycin; Ionophores; L-Lactate Dehydrogenase; Membrane Potentials; Nifedipine; Pituitary Gland; Receptors, LHRH; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sulfur Radioisotopes; Thapsigargin; Tritium

1. Single pacemaker cells were isolated from the sinus venosus of cane toad (Bufo marinus) in order to study the mechanisms involved in the spontaneous firing rate of action potentials. Intracellular calcium concentration ([Ca2+]i) was measured with indo-1 to determine whether [Ca2+]i influenced firing rate. A rapid transient rise of [Ca2+]i was recorded together with each spontaneous action potential. [Ca2+]i at the peak of systole was 655 +/- 64 nM and the minimum at the end of diastole was 195 +/- 15 nM. 2. Reduction of extracellular Ca2+ concentration from 2 to 0.5 mM caused a reduction in both systolic and diastolic [Ca2+]i and the spontaneous firing rate also gradually declined. 3. Application of the acetoxymethyl (AM) ester of BAPTA (10 microM), in order to increase intracellular calcium buffering, caused a decline in systolic and diastolic [Ca2+]i. The firing rate declined progressively until the cells stopped firing after 10-15 min. At the time that firing ceased, the diastolic [Ca2+]i had declined by 141 +/- 38 nM. 4. In the presence of ryanodine (2 microM), which interferes with Ca2+ release from the sarcoplasmic reticulum, the systolic and diastolic [Ca2+]i both declined and the firing rate decreased until the cells stopped firing. At quiescence diastolic [Ca2+]i had declined by 93 +/- 20 nM. 5. Exposure of the cells to Na+-free solution caused a rise in [Ca2+]i which exceeded the systolic level after 4.8 +/- 0.3 s. This rise is consistent with Ca2+ entry on a Na+-Ca2+ exchanger. 6. Rapid application of caffeine (10-20 mM) to cells clamped at -60 mV caused a rapid increase in [Ca2+]i which then spontaneously declined. An inward current with a similar time course to that of [Ca2+]i was also generated. Application of Ni2+ (5 mM) or 2,4-dichlorobenzamil (25 microM) reduced the amplitude of the inward current produced by caffeine by 96 +/- 1 % and 74 +/- 10 %, respectively. In a Na+-free solution the caffeine-induced current was reduced by 93 +/- 7 %. 7. Under a variety of circumstances the diastolic [Ca2+]i showed a close association with pacemaker firing rate. The existence of a Na+-Ca2+ exchanger and its estimated contribution to inward current during the pacemaker potential suggest that the Na+-Ca2+ exchange current makes a contribution to pacemaker activity.

Topics: Action Potentials; Animals; Bufo marinus; Caffeine; Calcium; Calibration; Diastole; Egtazic Acid; In Vitro Techniques; Indoles; Nickel; Regression Analysis; Ryanodine; Sarcoplasmic Reticulum; Sinoatrial Node; Sodium-Calcium Exchanger; Spectrometry, Fluorescence; Systole; Time Factors

We studied myocardial Ca2+ cycling during cardiopulmonary bypass and cold-blood cardioplegia (CPB/CBC) in patients with coronary heart disease undergoing coronary artery bypass grafting. Right atrial biopsies were taken from 13 patients before and after CPB/CBC: after pericardiotomy, immediately after aortic cross-clamp removal, and following termination of CPB/CBC. Changes in ionized Ca2+ concentration (nM) were monitored with indo 1 during Ca2+ uptake and Ca2+ release by sarcoplasmic reticulum in a medium containing 1% homogenized myocardium. Ryanodine inhibition was used to estimate Ca2+ release channel activity. With CPB/CBC, the initial Ca2+ concentration of reaction media increased 33%, (962 +/- 150 to 1262 +/- 106 nM; mean +/- SD). Ca2+ cycling increased asymmetrically, 108% for Ca2+ uptake (3.91 +/- 1.32 to 8.15 +/- 3.17 nM/s), 197% for Ca2+ release (0.90 +/- 0.80 to 2.73 +/- 1.13 nM/s), and 68% for the ratio of Ca(2+)-release to Ca(2+)-uptake activities (0.22 +/- 0.14 to 0.37 +/- 0.13). The dissociation constant of the Ca2+ pump for Ca2+ was unaltered by CPB/CBC (289 +/- 76 nM). During the time period that was studied post-bypass, Ca(2+)-pump activity remained increased, although the Ca(2+)-channel activity returned to pre-bypass values (all p < 0.05). We conclude that CPB/CBC produces increased myocardial Ca2+ load, twofold increased Ca2+ uptake, and threefold increased Ca2+ release by sarcoplasmic reticulum.

Topics: Calcium; Calcium Channels; Calcium-Transporting ATPases; Cardiopulmonary Bypass; Coronary Disease; Heart Arrest, Induced; Heart Atria; Humans; Indoles; Myocardium; Ryanodine; Sarcoplasmic Reticulum

Ca2+ is an essential activator of motility in the obligate intracellular parasite Toxoplasma gondii. Ca2+ ionophore A23187 and intracellular microinjection of Ca2+ initiate motility of parasites residing in parasitophorous vacuoles (PV). The source of Ca2+ and the mechanism by which it activates motility in vivo remain uncertain. Exposure of the parasites to dithiothreitol (DTT) can activate egress of previously nonmotile intravacuolar parasites within 60 sec. DTT is also known to activate both isoforms of the highly concentrated nucleoside triphosphate hydrolase (NTPase) produced by T. gondii. Using an adherent cell analysis system (ACAS) for Ca2+ imaging, a brief 15-50% increase in intra-PV fluorescence ratio was observed after exposure of infected fibroblasts to 5 mM DTT. Chelation of intracellular Ca2+ with BAPTA-AM and extracellular Ca2+ with EGTA blocked the DTT effect; however, this chelation did not prevent the activation of parasites nor the Ca2+ response to the Ca2+ ionophore ionomycin, suggesting that the Ca2+ that activates motility may reside near or within the parasite itself. This result demonstrates that an increase in Ca2+ within the vacuole precedes the onset of motility and the correlation of the DTT effect on motility and tachyzoite NTPase suggests that NTPase activation may be involved in the Ca2+ flux.

Topics: Animals; Calcium; Cells, Cultured; Chelating Agents; Dithiothreitol; Drug Interactions; Egtazic Acid; Fibroblasts; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Indoles; Ionomycin; Ionophores; Microscopy, Confocal; Reproducibility of Results; Ryanodine; Sulfhydryl Reagents; Thapsigargin; Toxoplasma; Vacuoles

We used cultured rat chromaffin cells to test the hypothesis that Ca2+ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca2+ sources: (a) Ca2+ surrounding single cells was transiently displaced by applying agonist with or without Ca2+ from an ejection pipette. (b) Intracellular stores of Ca2+ were depleted by soaking cells in Ca2+ -free plus 1 mM EGTA solution before transient exposure to agonist plus Ca2+. Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca2+ concentration ([Ca2+]i) was measured by indo-1 fluorescence. KCl (35 mM) and nicotine (10 microM) caused an immediate increase in [Ca2+]i and secretion in cells with or without internal Ca2+ stores, but only when applied with Ca2+ in the ejection pipette. Caffeine (10 mM) and muscarine (30 microM) evoked exocytosis whether or not Ca2+ was included in the pipette, but neither produced responses in cells depleted of internal Ca2+ stores. Pretreatment with ryanodine (0.1 microM) inhibited caffeine- but not muscarine-stimulated responses. Elevated [Ca2+]i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 +/- 0.38 s) or muscarine (2.2 +/- 0.25 s). However, the duration of caffeine-evoked exocytosis (7.1 +/- 0.8 s) was significantly shorter than that evoked by muscarine (33.1 +/- 3.5 s). The duration of caffeine-evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An approximately 20-s refractory period was found between repeated caffeine-evoked exocytosis bursts even though [Ca2+]i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca2+ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca2+. These results and conclusions are consistent with our original observations in the perfused adrenal gland.

Topics: Adrenal Glands; Animals; Caffeine; Calcium; Cells, Cultured; Chromaffin System; Electrophysiology; Exocytosis; Fluorescent Dyes; Indoles; Kinetics; Muscarine; Potassium Chloride; Rats; Rats, Sprague-Dawley; Ryanodine

We performed experiments using the calcium indicator Indo-1 to determine the relative roles of the sarcolemmal mechanisms involved in the regulation of diastolic intracellular calcium concentration ([Ca2+]i) in trabeculae from the rat heart. Ryanodine was used to eliminate sarcoplasmic reticulum (SR) function. In the functional absence of the SR, 76.8 +/- 3.9% of the calcium was extruded by the Na-Ca exchange carrier in the [Ca2+]i range of diastolic concentration +/- 200-400 nM. This was assessed by measuring the recovery of [Ca2+]i from small perturbations in the presence and absence of extracellular sodium. The steady-state relationship between [Ca2+]o and [Ca2+]i was linear over the range of 1-40 mM, a 20-fold increase of [Ca2+]o produced a 1.97-fold +/- 0.13-fold increase in [Ca2+]i (n = 5). In the absence of extracellular sodium raising [Ca2+]o had a variable effect. In some preparations there was little change of [Ca2+]i while in others the response was almost as large as in control conditions. We conclude that the Na-Ca exchanger contributes approximately 77% of sarcolemmal calcium extrusion following small perturbations in [Ca2+]i and that this fraction does not diminish as the [Ca2+]i declines. In addition we have shown a sodium-independent entry of calcium into quiescent cardiac muscle under resting conditions.

Topics: Animals; Calcium; Carrier Proteins; Diastole; Electric Stimulation; Enzyme Inhibitors; Fluorescent Dyes; Indoles; Male; Myocardium; Rats; Rats, Wistar; Ryanodine; Sarcolemma; Sarcoplasmic Reticulum; Sodium; Sodium-Calcium Exchanger; Sodium-Potassium-Exchanging ATPase

To investigate the effect of altering internal Na and cell Ca load on the voltage-dependence of the intracellular Ca transient.. Ventricular myocytes were isolated enzymatically from the guinea-pig heart. They were patch clamped and dialysed internally with pipette solutions which contained either 0 or 10 or 20 mM Na. Intracellular Ca was monitored with Indo-1 and experiments were carried out at 36 degrees C. A standard level of Ca loading was established before each test pulse by applying a train of conditioning pulses. The voltage-dependence of the Cai (Indo-1) transient provided information about the mechanisms which trigger Ca release from the sarcoplasmic reticulum (SR).. The voltage-dependence of L-type Ca current (ICa.L) was assessed in separate experiments by dialysing myocytes with a Cs-based solution. ICa.L had a maximum amplitude at 0 mV, declined at more positive potentials and there was little net inward ICa.L at +100 mV. The rapid initial phasic component of the Indo-1 transient was abolished by ryanodine/thapsigargin; therefore, this component reflected the magnitude of SR Ca release. In cells dialysed with 10 mM Na, the voltage-dependence of the Indo-1 transient was different from ICa.L. The Indo-1 transient became maximal at +20 mV, and the decline of the Indo-1 transient at positive potentials was less steep than the decline of ICa.L. A large proportion of the phasic Indo-1 transient could remain at positive potentials where there was no detectable ICa.L. Increasing dialysing Na from 10 to 20 mM led to a marked change in voltage-dependence of the Indo-1 transient. With 20 mM Na, the amplitude of the phasic Indo-1 transient remained large between +20 and +100 mV. Removing Na from the pipette dialysis solution had the opposite effect on voltage-dependence of the transient. For each dialysing [Na], the level of cellular and SR Ca content was altered by varying the potential of conditioning pulses applied before each test pulse. There was no significant effect on voltage-dependence of the Indo-1 transient of either increasing or reducing the cellular Ca content.. These data are consistent with the hypothesis that the voltage-dependence of the Cai transient results from the sum of the voltage-dependencies of the two main trigger mechanisms--Ica.L and reverse Na/Ca exchange. When a myocyte was dialysed with Na-free solution, the voltage-dependence of the Cai transient became more similar (but not identical) to that for ICa.L. With 20 mM Na dialysis, the altered voltage-dependence of the Cai transient may reflect an increased trigger influence of reverse Na/Ca exchange.

Topics: Animals; Anti-Arrhythmia Agents; Calcium; Calcium-Transporting ATPases; Guinea Pigs; In Vitro Techniques; Indoles; Membrane Potentials; Myocardium; Patch-Clamp Techniques; Ryanodine; Sarcoplasmic Reticulum; Sodium; Thapsigargin

Intracellular Ca2+ pools contribute to changes in cytosolic [Ca2+] ([Ca2+]i), which play an important role in endothelial cell signaling. Recently, endothelial ryanodine-sensitive Ca2+ stores were shown to regulate agonist-sensitive intracellular Ca2+ pools. Since caffeine binds the ryanodine Ca2+ release channel on the endoplasmic reticulum in a variety of cell types, we examined the effect of caffeine on [Ca2+]i in human aortic endothelial cell monolayers loaded with the fluorescent probe indo 1. Under baseline conditions, 10 mmol/L caffeine induced a small increase in [Ca2+]i from 86 +/- 10 to 115 +/- 17 nmol/L (mean +/- SEM); this effect was similar to that of 5 mumol/L ryanodine and was unaffected by buffer Ca2+ removal. After depletion of an intracellular Ca2+ store by the irreversible endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin (1 mumol/L), ryanodine did not affect [Ca2+]i. In contrast, caffeine induced a large rapid increase in [Ca2+]i (176 +/- 19 to 338 +/- 35 nmol/L, P < .001) after thapsigargin exposure; this effect of caffeine was only observed when extracellular Ca2+ was present. A similar increase in [Ca2+]i was induced by caffeine after depletion of ryanodine- and histamine-sensitive Ca2+ stores or after pretreatment with the endoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid (10 mumol/L). Thus, under baseline conditions the effect of caffeine on [Ca2+]i is similar to that of ryanodine and appears to be due to the release of an intracellular store. However, after depletion of an endoplasmic reticulum Ca2+ store, caffeine, but not ryanodine, stimulates Ca2+ influx, resulting in a large increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aorta; Buffers; Caffeine; Calcium; Calcium Channels; Calcium-Transporting ATPases; Cells, Cultured; Chelating Agents; Endoplasmic Reticulum; Endothelium, Vascular; Enzyme Inhibitors; Fluorescent Dyes; Humans; Indoles; Ryanodine; Terpenes; Thapsigargin

The ryanodine (R)-induced loss of sarcoplasmic reticulum (SR) Ca2+ and the abilities of the SR to accumulate Ca2+ and participate in contractile activation after R were studied in rat ventricular myocytes. Indo 1 fluorescence (IF) indexed cytosolic Ca2+, and caffeine assayed SR Ca2+. Before R, there was a negative staircase, and the SR accumulated Ca2+ at rest. During stimulation (0.5 Hz), R decreased IF and contraction, converting the negative staircase to positive. When R was pulsed onto resting cells, IF increased and cells shortened, subsequently behaving as if stimulated in R. After R, there was no caffeine-releasable Ca2+ at rest, and little accumulated during 0.5-Hz stimulation. At high rates, caffeine-releasable Ca2+ and diastolic IF increased. In isoproterenol and R, IF transients and contractions recovered at 0.5 Hz with a marked positive staircase and little diastolic IF increase. Within 10 beats, SR Ca2+ accumulated to pre-R levels. R eliminated the positive inotropic effect of paired-pulse stimulation, but isoproterenol temporarily restored it. Twitch contractions in thapsigargin, an SR Ca2+ pump blocker, and isoproterenol were slow compared with control or R + isoproterenol. R leaks SR Ca2+ into the cytosol. SR Ca2+ can be repleted in R by high-rate stimulation or by low-rate stimulation with a beta-adrenergic agonist. SR Ca2+ release in R can be temporarily restored if Ca2+ influx and SR Ca2+ pumping are increased enough to overcome the SR Ca2+ leak.

Topics: Animals; Caffeine; Calcium; Calcium Channel Blockers; Electric Stimulation; Fluorescent Dyes; Heart Ventricles; Indoles; Isoproterenol; Myocardial Contraction; Myocardium; Rats; Ryanodine; Sarcoplasmic Reticulum; Terpenes; Thapsigargin