Ionomycin and 2,5'-di(tertbutyl)-1,4,-benzohydroquinone elicit Ca2+-induced Ca2+ release from intracellular pools in Physarum polycephalum

Calcium level in organelles of the slime mold Physarum polycephalum was monitored by chlortetracycline, a low-affinity calcium indicator. It was found that 2,5'-di(tertbutyl)-1,4,-benzohydroquinone (BHQ) at a concentration of 100 microM, but not the highly specific inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), thapsigargin (1-10 microM), elicited calcium release from the CTC-stained intracellular calcium pool. Ionomycin also caused a calcium release (23.7+/-5.1%), which was less than that induced by BHQ (30.1+/-6.0%). Procaine (10 mM), a blocker of ryanodine receptor, completely abolished the responses to BHQ and ionomycin. Another blocker, ryanodine (100 microM), only slightly diminished the responses to ionomycin and BHQ. Apparently, BHQ and ionomycin acting as a Ca2+-ATPase inhibitor and an ionophore, respectively, elicit an increase in [Ca2+]i, which in turn triggers a calcium-induced calcium release (CICR) via the ryanodine receptor. Caffeine, an activator of ryanodine receptor, at a concentration of 25-50 mM produced a Ca2+-release (5.6-16.0%), which was not similar in magnitude to CICR. The response to 25 mM caffeine was only moderately inhibited by 25 mM procaine, and almost completely abolished by 50 mM procaine and 100 microM ryanodine.     

Ca2+ entry via AMPA-type glutamate receptors triggers Ca2+-induced Ca2+ release from ryanodine receptors in rat spiral ganglion neurons

Ryanodine receptor (RyR)-gated Ca2+ stores have recently been identified in cochlear spiral ganglion neurons (SGN) and likely contribute to Ca2+ signalling associated with auditory neurotransmission. Here, we identify an ionotropic glutamate receptor signal transduction pathway which invokes RyR-gated Ca2+ stores in SGN via Ca2+-induced Ca2+ release (CICR). Ca2+ levels were recorded in SGN in situ within rat cochlear slices (postnatal day 0-17) using the Ca2+ indicator fluo-4. RyR-gated Ca2+ stores were confirmed by caffeine-induced increases in intracellular Ca2+ which were blocked by ryanodine (100 microM) and were independent of external Ca2+. Glutamate evoked comparable increases in intracellular Ca2+, but required the presence of external Ca2+. Ca2+ influx via the glutamate receptor was found to elicit CICR via RyR-gated Ca2+ stores, as shown by the inhibition of the response by prior depletion of the Ca2+ stores with caffeine, the SERCA inhibitor thapsigargin, or ryanodine. The glutamate analogue AMPA (alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid) elicited Ca2+ responses that could be inhibited by caffeine. Glutamate- and AMPA-mediated Ca2+ responses were eliminated with the AMPA/Kainate receptor antagonist DNQX (6,7-dinitroquinoxaline-2,3-dione). These data demonstrate functional coupling between somatic AMPA-type glutamate receptors and intracellular Ca(2+) stores via RyR-dependent CICR in primary auditory neurons.     

Intracellular calcium stores are involved in growth hormone-releasing hormone signal transduction in rat somatotrophs.

In rat pituitary somatotrophs, the stimulation of growth hormone secretion by growth hormone-releasing hormone (GHRH) is a Ca(2+)-dependent event involving Ca2+ influx. The presence of calcium-induced calcium release (CICR) Ca2+ stores has been suggested in these cells. The aim of our study was to demonstrate the presence of CICR stores in rat somatotrophs and to determine their function in GHRH Ca2+ signalling. To this end we measured cytosolic free Ca2+ concentration ([Ca2+]i), using indo-1 in purified rat somatotrophs in primary culture, while altering intracellular Ca2+ stores. Ionomycin (10 ttM) or 4-bromo-A23187 (10 ItM), used to mobilise organelle-bound Ca2+, raised [Ca2+]i in the absence of extracellular Ca2+. Caffeine (5 to 50 mM), used to mobilise Ca2+ from CICR stores, transiently raised [Ca2+]i in 65% of cells tested. The response to 40 mM caffeine was abolished when Ca2+ stores were depleted, with 1 microM thapsigargin or with 10 microM ryanodine. All cells that responded to 40 mM caffeine responded to 10 nM GHRH. The [Ca2+]i response to 10 nM GHRH was reversible and repeatable. However, the second response was 38% smaller than the first. Ryanodine treatment abolished the reduction in the second [Ca2+]i response, while thapsigargin increased the reduction by 67%. We conclude that rat somatotrophs possess CICR Ca2+ stores and that they account for 30-35% of the GHRH-induced increase in [Ca2+]i, and that their partial depletion is involved in somatotroph desensitization.     

Stimulation and inhibition of [3H]ryanodine binding to sarcoplasmic reticulum from malignant hyperthermia susceptible pigs

When compared to normal pig sarcoplasmic reticulum (SR), SR from malignant hyperthermia susceptible (MHS) porcine skeletal muscle has been shown to exhibit an increased rate of calcium release, as well as alterations in [3H]ryanodine-binding activity in the presence of microM Ca2+ (Mickelson et al., 1988, J. Biol. Chem. 263, 9310). In the present study, various stimulators (adenine nucleotides and caffeine) and inhibitors (ruthenium red and Mg2+) of the SR calcium release channel were examined for effects on MHS and normal SR [3H]ryanodine binding. The apparent affinity of the MHS SR receptor for ryanodine in the presence of 10 mM ATP (Kd = 6.0 nM) or 10 mM caffeine (Kd = 28 nM) was significantly greater than that of the normal SR (Kd = 8.5 and 65 nM in 10 mM ATP or caffeine, respectively), the Bmax (12-16 pmol/mg) was similar in all cases. The Ca2+(0.5) for inhibition of [3H]ryanodine binding in the presence of 5 mM AMPPNP (238 vs 74 microM for MHS and normal SR, respectively) and the Ca2+(0.5) for stimulation of [3H]ryanodine binding in the presence of 5 mM caffeine (0.049 vs 0.070 microM for MHS and normal SR, respectively) were also significantly different. Furthermore, in the presence of optimal Ca2+, MHS SR [3H]ryanodine binding was more sensitive to caffeine stimulation (C0.5 of 1.7 vs 3.4 mM) and was less sensitive to ruthenium red (C0.5 of 1.9 vs 1.2 microM) or Mg2+ inhibition (C0.5 of 0.34 vs 0.21 mM) than was normal SR. These results further support the hypothesis that differences in the ryanodine/receptor calcium release channel regulatory properties are responsible for the abnormal calcium releasing activity of MHS SR.     

Role of extracellular Ca2+ in diaphragmatic contraction: effects of ouabain, monensin, and ryanodine.

The effects of zero extracellular Ca2+ on the contractility of rat diaphragmatic strips in vitro were studied in conjunction with various pharmacological agents known to influence the intracellular Ca2+ concentration: the Na+ ionophore, monensin, and the Na(+)-K+ pump inhibitor, ouabain, which enhance [Ca2+]i, caffeine, which induces Ca2+ release from the sarcoplasmic reticulum (SR), and ryanodine, which prevents Ca2+ retention by the SR. The effect of increasing [Ca2+]i on diaphragmatic contraction was assessed by comparing contractions induced by 120 mM K+ in the small muscle strips before and after the addition of ouabain or monensin. Monensin (20 microM) and ouabain (1-100 microM) augmented contractions up to threefold. Treatment of diaphragm strips with 3 nM ryanodine increased baseline tension 360% above the original resting tension but only if the diaphragm was electrically stimulated concurrently; 100 microM ryanodine induced contracture in quiescent tissue. High K+ contractures were of greater magnitude in the presence of ryanodine compared with control, and relaxation time was prolonged by greater than 200%. Ca(2+)-free conditions ameliorated these actions of ryanodine. Ryanodine reduced contractions induced by 10 mM caffeine and nearly abolished them in Ca(2+)-free solution. The data demonstrate that extracellular Ca2+ is important in certain types of contractile responses of the diaphragm and suggest that the processes necessary to utilize extracellular Ca2+ are present in the diaphragm.     

Role of Ca2+ release from ryanodine stores in generation of NMDA-induced Ca2+ signal in rabbit hippocampus.

In vivo microdialysis combined with measurements of 45Ca efflux from pre-labelled rat hippocampus has been utilised in our laboratory to demonstrate NMDA-evoked 45Ca2+ release to dialysate, reflecting calcium-induced calcium release (CICR) via ryanodine receptors (RyR). In the present study we attempted to reproduce this phenomenon in the rabbit hippocampus. Application of 1 mM NMDA to dialysis medium induced a decrease in Ca2+ concentration in dialysate, as a result of extracellular Ca2+ influx to neurones. The release of 45Ca2+ was not observed, instead a decrease in 45Ca2+ efflux rate from the NMDA treated rabbit hippocampus was noted, along with release to dialysate of prostaglandin D2, taurine and phosphoethanolamine. All these effects, reflecting different steps of intracellular calcium signalling, were insensitive to 100 microM dantrolene and 50 microM ryanodine, RyR modulators known to interfere with NMDA-evoked 45Ca2+ release in the rat hippocampus. Thus, although the results of this study demonstrate the role of extracellular Ca2+ influx to neurones in NMDA-evoked generation of Ca2+ signal in the rabbit hippocampus, the activity of CICR was not detected.     

Calcium-induced calcium release mechanism in guinea pig taenia caeci

Fura-2 was used to measure the amount of Ca released from the intracellular Ca store of a saponin-skinned smooth muscle fiber bundle of the guinea pig taenia caeci (width, 150-250 microns) placed in a capillary cuvette at 20-22 degrees C. The amount of Ca actively loaded into the store was assayed when released by the application of 50 mM caffeine and/or 10 microM inositol 1,4,5-trisphosphate (IP3) in the absence of ATP, and was found to have a biphasic dependence on the loading [Ca2+] with a peak near pCa 6. After Ca loading at pCa 6, IP3 released almost all the releasable Ca, whereas caffeine discharged Ca from only approximately 40% of the store. The maximum amount of Ca in the store was some 220 mumol/liter cell water. Ca in the caffeine-releasable store was released approximately exponentially to zero with time when Ca2+ was applied in the absence of ATP, and the rate constant of the Ca-induced Ca release (CICR) increased steeply with the concentration of Ca2+ applied. Increase in [Mg2+] (0.5-5.0 mM) or decrease in pH (7.3-6.7) shifted the relation between pCa and the rate of CICR roughly in parallel toward the lower pCa. An adenine nucleotide increased the rate of the CICR, but it did not change the range of effective [Ca2+]. 5 mM caffeine greatly enhanced the CICR mechanism, making it approximately 30 times more sensitive to [Ca2+]. However the drug had no Ca-releasing action in the absence of Ca2+. Procaine in millimolar concentrations inhibited the rate of the CICR. These properties are similar to those of the skeletal muscle CICR and ryanodine receptor channels. Rates of the CICR under a physiological ionic milieu were estimated from the results, and a [Ca2+] greater than 1 microM was expected to be necessary for the activation of the Ca release. This Ca sensitivity seems too low for the CICR mechanism to play a primary physiological role in Ca mobilization, unless assisted by other mechanisms.     

Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic beta-cells

The effect of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibition on the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) was studied in primary insulin-releasing pancreatic beta-cells isolated from mice, rats and human subjects as well as in clonal rat insulinoma INS-1 cells. In Ca(2+)-deficient medium the individual primary beta-cells reacted to the SERCA inhibitor cyclopiazonic acid (CPA) with a slow rise of [Ca(2+)](i) followed by an explosive transient elevation. The [Ca(2+)](i) transients were preferentially observed at low intracellular concentrations of the Ca(2+) indicator fura-2 and were unaffected by pre-treatment with 100 microM ryanodine. Whereas 20mM caffeine had no effect on basal [Ca(2+)](i) or the slow rise in response to CPA, it completely prevented the CPA-induced [Ca(2+)](i) transients as well as inositol 1,4,5-trisphosphate-mediated [Ca(2+)](i) transients in response to carbachol. In striking contrast to the primary beta-cells, caffeine readily mobilized intracellular Ca(2+) in INS-1 cells under identical conditions, and such mobilization was prevented by ryanodine pre-treatment. The results indicate that leakage of Ca(2+) from the endoplasmic reticulum after SERCA inhibition is feedback-accelerated by Ca(2+)-induced Ca(2+) release (CICR). In primary pancreatic beta-cells this CICR is due to activation of inositol 1,4,5-trisphosphate receptors. CICR by ryanodine receptor activation may be restricted to clonal beta-cells.     

Calcium pools mobilized by calcium or inositol 1,4,5-trisphosphate are differentially localized in rat heart and brain.

Calcium-induced calcium release (CICR) pools have been demonstrated in brain and heart microsomes biochemically and autoradiographically by the sensitivity of 45Ca2+ accumulation to Mg2+, ATP, ruthenium red, caffeine, and tetracaine. The CICR pool colocalizes with [3H]ryanodine binding sites, supporting the notion that [3H]ryanodine labels CICR pools. Sites of CICR pools in the brain contrast with those of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools with reciprocal localizations between the two Ca2+ pools in several structures. Thus, in the hippocampus CA-1 is enriched in IP3-sensitive Ca2+ pools, whereas CICR pools are highest in CA-3 and the dentate gyrus. The corpus striatum and cerebellum are enriched in IP3 pools, whereas the medial septum and olfactory bulb have high CICR densities. In cardiac tissue, CICR is localized to atrial and ventricular muscle, whereas IP3 pools are concentrated in coronary vessels and cardiac conduction fibers. The reciprocal enrichment of IP3 and CICR Ca2+ pools implies differential regulation of Ca2+ hemostasis in these tissues.     

Ryanodine-sensitive Ca(2+) release mechanism of rat pancreatic acinar cells is modulated by calmodulin

The effects of calmodulin (CaM) and CaM antagonists on microsomal Ca(2+) release through a ryanodine-sensitive mechanism were investigated in rat pancreatic acinar cells. When caffeine (10 mM) was added after a steady state of ATP-dependent (45)Ca(2+) uptake into the microsomal vesicles, the caffeine-induced (45)Ca(2+) release was significantly increased by pretreatment with ryanodine (10 microM). The presence of W-7 (60 microM), a potent inhibitor of CaM, strongly inhibited the release, while W-5 (60 microM), an inactive CaM antagonist, showed no inhibition. Inhibition of the release by W-7 was observed at all caffeine concentrations (5-30 mM) tested. The presence of exogenously added CaM (10 microg/ml) markedly increased the caffeine (5-10 mM)-induced (45)Ca(2+) release and shifted the dose-response curve of caffeine-induced (45)Ca(2+) release to the left. Cyclic ADP-ribose (cADPR, 2 microM)-induced (45)Ca(2+) release was enhanced by the presence of ryanodine (10 microM). cADPR (2 microM)- or ryanodine (500 microM)-induced (45)Ca(2+) release was also inhibited by W-7 (60 microM), but not by W-5 (60 microM), and was stimulated by CaM (10 microg/ml). These results suggest that the ryanodine-sensitive Ca(2+) release mechanism of rat pancreatic acinar cells is modulated by CaM.     

Elucidation of the ryanodine-sensitive Ca2+ release mechanism of rat pancreatic acinar cells: modulation by cyclic ADP-ribose and FK506

The effects of cyclic ADP-ribose (cADPR) and the immunosuppressant drug FK506 on microsomal Ca2+ release through a ryanodine-sensitive mechanism were investigated in rat pancreatic acinar cells. After a steady state of 45Ca2+ uptake into the microsomal vesicles, ryanodine or caffeine was added. Preincubation of the vesicles with cADPR (0.5 microM) shifted the dose-response curve of ryanodine- or caffeine-induced 45Ca2+ release from the vesicles to the left. Preincubation with cADPR shifted the dose-response curve of the FK506-induced 45Ca2+ release upward. Preincubation with FK506 (3 microM) shifted the dose-response curve of the ryanodine- or caffeine-induced 45Ca2+ release to the left by the same extent as that in the case of cADPR. FK506 shifted the dose-response curve of the cADPR-induced 45Ca2+ release upward. The presence of both cADPR and FK506 enhanced the ryanodine (30 microM)- or caffeine (10 mM)-induced 45Ca2+ release by the same extent as that in the case of cADPR alone or FK506 alone. These results indicate that cADPR and FK506 modulate the ryanodine-sensitive Ca2+ release mechanism of rat pancreatic acinar cells by increasing the ryanodine or caffeine sensitivity to the mechanism. In addition, there is a possibility that the mechanisms of modulation by cADPR and FK506 are the same.     

Caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release from the endoplasmic reticulum in honeybee photoreceptors

Light stimulation of invertebrate microvillar photoreceptors causes a large rapid elevation in Cai, shown previously to modulate the adaptational state of the cells. Cai rises, at least in part, as a result of Ins(1,4,5)P3-induced Ca2+ release from the submicrovillar endoplasmic reticulum (ER). Here, we provide evidence for Ca(2+)- induced Ca2+ release (CICR) in an insect photoreceptor. In situ microphotometric measurements of Ca2+ fluxes across the ER membrane in permeabilized slices of drone bee retina show that (a) caffeine induces Ca2+ release from the ER; (b) caffeine and Ins(1,4,5)P3 open distinct Ca2+ release pathways because only caffeine-induced Ca2+ release is ryanodine sensitive and heparin insensitive, and because caffeine and Ins(1,4,5)P3 have additive effects on the rate of Ca2+ release; (c) Ca2+ itself stimulates release of Ca2+ via a ryanodine-sensitive pathway; and (d) cADPR is ineffective in releasing Ca2+. Microfluorometric intracellular Ca2+ measurements with fluo-3 indicate that caffeine induces a persistent elevation in Cai. Electrophysiological recordings demonstrate that caffeine mimics all aspects of Ca(2+)-mediated facilitation and adaptation in drone photoreceptors. We conclude that the ER in drone photoreceptors contains, in addition to the Ins(1,4,5)P3-sensitive release pathway, a CICR pathway that meets key pharmacological criteria for a ryanodine receptor. Coexpression of both release mechanisms could be required for the production of rapid light-induced Ca2+ elevations, because Ca2+ amplifies its own release through both pathways by a positive feedback. CICR may also mediate the spatial spread of Ca2+ release from the submicrovillar ER toward more remote ER subregions, thereby activating Ca(2+)-sensitive cell processes that are not directly involved in phototransduction.     

Agonist of dihydropyridine receptors, BayK8644 depresses excitation-contraction coupling in myocytes of guinea pig heart.

BayK8644(-)(BayK), an agonist of L-type Ca2+ channels has been recently shown to impair excitation-contraction coupling in cardiac myocytes by increasing Ca2+ leak from the sarcoplasmic reticulum (SR) and by decreasing the gain factor of calcium induced release of calcium. It has been proposed that BayK affects the properties of ryanodine receptors (RyRs) of SR by binding to the sarcolemmal dihydropyridine receptors (DHPRs). This would suggest that the linkage between these receptors is more direct than currently sought. However, it has been recently found that BayK may also directly affect the RyRs increasing their open probability. In this paper we tested the effect of BayK on excitation-contraction coupling in single ventricular myocytes of guinea-pig heart superfused with 5 mM Ni2+ which blocks the L-type Ca2+ current and Na+/Ca2+ exchange. We have previously shown that it is possible to activate in these cells nearly normal Ca2+ transients and contractions despite total inhibition of ICa. This eliminated the effect of ICa increased by BayK on excitation contraction coupling thus simplifying the studied system. 0.5 microM BayK increased the diastolic [Ca2+]i and decreased the diastolic length in stimulated or rested cells superfused with Ni2+, decreased by approximately 50% amplitude of Ca2+ transients and contractions and decreased by approximately 70% the responses of cells to rapid superfusion of 15mM caffeine used as an indirect index of the SR Ca2+ content. The effects on diastolic length and [Ca2+]i in rested cells were not affected by 20 microM nifedipine. We conclude that under our experimental conditions the dominating mechanism of suppression of excitation-contraction coupling by BayK was depletion of the SR Ca2+ by the direct effect on the RyRs.     

Physiology and excitation-contraction coupling in the intestinal muscle of the crayfish Procambarus clarkii

The intestinal muscles of Procambarus clarkii are striated and yet they are specialized to produce slow peristaltic waves of contraction, not unlike those seen in vertebrate visceral smooth muscle. These muscles cannot be tetanized either by repetitive stimulation or by elevated potassium saline. The excitation-contraction (E-C) coupling mechanism was explored and compared with that known in crustacean skeletal muscle. Contraction is dependent on external Ca2+ which triggers the release of intracellular calcium from the sarcoplasmic reticulum (SR) via calcium-induced calcium release (CICR). Whereas contraction force is proportional to [Ca2+]o up to that in normal saline (13.4 mM), higher levels of Ca2+ reduce force. Ryanodine, which blocks calcium release from the SR, abolishes electrically stimulated contractions and CICR. Relaxation is achieved by removal of calcium from the cytosol in at least two ways, first by the re-loading of calcium into the SR by Ca2+-ATPases and second by the movement of calcium out of the cell by extruding it across the sarcolemma via Na+/Ca2+-exchangers. It is hypothesized that the inability of this muscle to show tetanus arises from inactivation of the voltage-gated calcium channels by high calcium. This is supported by the result that caffeine application causes an increase in tonus and size of phasic contractions by circumventing the sarcolemma and dumping SR calcium stores.     

Functional expression of the calcium release channel from skeletal muscle ryanodine receptor cDNA

Combined patch-clamp and fura-2 measurements were performed to study the calcium release properties of Chinese hamster ovary (CHO) cells transfected with the rabbit skeletal muscle ryanodine receptor cDNA carried by an expression vector. Both caffeine (1-50 mM) and ryanodine (100 microM) induced release of calcium from intracellular stores of transformed CHO cells but not from control (non-transfected) CHO cells. The calcium responses to caffeine and ryanodine closely resembled those commonly observed in skeletal muscle. Repetitive applications of caffeine produced characteristic all-or-none rises in intracellular calcium. Inositol 1,4,5-trisphosphate (IP3) neither activated the ryanodine receptor channel nor interfered with the caffeine-elicited calcium release. These results indicate that functional calcium release channels are formed by expression of the ryanodine receptor cDNA.     

Ionic mechanisms of histamine-induced responses in guinea pig intracardiac neurons

Histamine, released from mast cells, can modulate the activity of intrinsic neurons in the guinea pig cardiac plexus. The present study examined the ionic mechanisms underlying the histamine-induced responses in these cells. Histamine evokes a small membrane depolarization and an increase in neuronal excitability. Using intracellular voltage recording from individual intracardiac neurons, we were able to demonstrate that removal of extracellular sodium reduced the membrane depolarization, whereas inhibition of K+ channels by 1 mM Ba2+, 2 mM Cs+, or 5 mM tetraethylammonium had no effect. The depolarization was also not inhibited by either 10 microM Gd3+ or a reduced Cl- solution. The histamine-induced increase in excitability was unaffected by K+ channel inhibitors; however, it was reduced by either blockage of voltage-gated Ca2+ channels with 200 microM Cd2+ or replacement of extracellular Ca2+ with Mg2+. Conversely, alterations in intracellular calcium with thapsigargin or caffeine did not inhibit the histamine-induced effects. However, in cells treated with both thapsigargin and caffeine to deplete internal calcium stores, the histamine-induced increase in excitability was decreased. Treatment with the phospholipase C inhibitor U73122 also prevented both the depolarization and the increase in excitability. From these data, we conclude that histamine, via activation of H1 receptors, activates phospholipase C, which results in 1) the opening of a nonspecific cation channel, such as a transient receptor potential channel 4 or 5; and 2) in combination with either the influx of Ca2+ through voltage-gated channels or the release of internal calcium stores leads to an increase in excitability.     

Biphasic effect of calcium on luteinizing hormone-stimulated cyclic adenosine 3',5'-monophosphate production in granulosa cells of the fowl (Gallus domesticus).

Both cAMP and Ca2+ play important roles in the steroidogenic action of LH in hen granulosa cells. However, the interaction of these intracellular messengers is not fully understood. In the present study we used two calcium ionophores (ionomycin and A23187), as well as trifluoperazine (TFP), an inhibitor of calmodulin, to investigate LH- and forskolin-induced cAMP production in granulosa cells isolated from the largest (F1) preovulatory follicle of White Leghorn laying hens. Between 0.1 and 1.0 microM, both ionophores significantly potentiated cAMP responses to LH in the presence of 0.1 mM extracellular Ca2+. When calcium was omitted from the medium, ionophores had no effect. When either calcium was raised above 1 mM, or the concentration of ionophores was increased above 1 microM, LH-induced cAMP production was drastically inhibited. In the presence of 0.5-2.0 mM calcium, A23187 inhibited forskolin-promoted cAMP synthesis. TFP, while having no effect on basal cAMP, suppressed LH-induced responses and the potentiating effect of ionomycin. It is concluded that for full activation of the adenylate cyclase/cAMP system by LH, Ca-calmodulin is required at a site upstream from the catalytic component of the enzyme. However, high intracellular Ca2+ and/or other effects of ionophores (such as uncoupling of oxidative phosphorylation) inhibit LH-induced cAMP production.     

Synergistic release of calcium in sea urchin eggs by caffeine and ryanodine.

A transient rise in intracellular Ca2+ during fertilization is necessary for activation of the quiescent sea urchin egg. Several mechanisms contribute to the rise in Ca2+ including influx across the egg plasma membrane and release from intracellular stores. The egg contains both IP3-sensitive and -insensitive Ca2+ release mechanisms and in this study we have used single-cell spectrofluorimetry to examine the effects of caffeine and ryanodine on Ca2+ release in eggs preloaded with fura 2. Caffeine induced a small Ca2+ release that was insensitive to heparin or ruthenium red. Ca2+ liberation by caffeine could be augmented by prior treatment with thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase. Variable Ca2+ releases were observed in response to microinjection of ryanodine. The action of ryanodine appeared to be enhanced by prior injection of heparin and partially inhibited by ruthenium red. The release of Ca2+ by caffeine or ryanodine was generally insufficient to trigger cortical granule exocytosis, thus these eggs could be fertilized and a second Ca2+ release during fertilization was measured. Unlike the caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release mechanism in somatic cells, the graded responses in eggs suggested this caffeine- and ryanodine-sensitive release mechanism is not sensitive to sudden changes in Ca2+. Thus we could examine the combined actions of caffeine and ryanodine on Ca2+ release, which were synergistic. Caffeine treatment of ryanodine-injected eggs or ryanodine injection of caffeine-treated eggs stimulated a Ca2+ release significantly larger than the release by either drug independently. The experiments presented here suggest that sea urchin eggs liberate Ca2+ in response to caffeine and ryanodine; however, the regulation of this release differs from that described for caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release of somatic cells.     

Nitric oxide affects sarcoplasmic calcium release in skeletal myotubes.

In the present study, we used real-time confocal microscopy to examine the effects of two nitric oxide (NO) donors on acetylcholine (ACh; 10 microM)- and caffeine (10 mM)-induced intracellular calcium concentration ([Ca2+]i) responses in C2C12 mouse skeletal myotubes. We hypothesized that NO reduces [Ca2+]i in activated skeletal myotubes through oxidation of thiols associated with the sarcoplasmic reticulum Ca2+-release channel. Exposure to diethylamine NONOate (DEA-NO) reversibly increased resting [Ca2+]i level and resulted in a dose-dependent reduction in the amplitude of ACh-induced [Ca2+]i responses (25 +/- 7% reduction with 10 microM DEA-NO and 78 +/- 14% reduction with 100 microM DEA-NO). These effects of DEA-NO were partly reversible after subsequent exposure to dithiothreitol (10 mM). Preexposure to DEA-NO (1, 10, and 50 microM) also reduced the amplitude of the caffeine-induced [Ca2+]i response. Similar data were obtained by using the chemically distinct NO donor S-nitroso-N-acetyl-penicillamine (100 microM). These results indicate that NO reduces sarcoplasmic reticulum Ca2+ release in skeletal myotubes, probably by a modification of hyperreactive thiols present on the ryanodine receptor channel.     

Regulation of a voltage-sensitive release mechanism by Ca(2+)-calmodulin-dependent kinase in cardiac myocytes

A role for Ca(2+)-calmodulin-dependent kinase (CamK) in regulation of the voltage-sensitive release mechanism (VSRM) was investigated in guinea pig ventricular myocytes. Voltage clamp was used to separate the VSRM from Ca(2+)-induced Ca(2+) release (CICR). VSRM contractions and Ca(2+) transients were absent in cells dialyzed with standard pipette solution but present when 2-5 microM calmodulin was included. Effects of calmodulin were blocked by KN-62 (CamK inhibitor), but not H-89, a protein kinase A (PKA) inhibitor. Ca(2+) current and caffeine contractures were not affected by calmodulin. Transient-voltage relations were bell-shaped without calmodulin, but they were sigmoidal and typical of the VSRM with calmodulin. Contractions with calmodulin exhibited inactivation typical of the VSRM. These contractions were inhibited by rapid application of 200 microM of tetracaine, but not 100 microM of Cd(2+), whereas CICR was inhibited by Cd(2+) but not tetracaine. In undialyzed myocytes (high-resistance microelectrodes), KN-62 or H-89 each reduced amplitudes of VSRM contractions by approximately 50%, but together they decreased VSRM contractions by 93%. Thus VSRM is facilitated by CamK or PKA, and both pathways regulate the VSRM in undialyzed cells.