EPYLRFamide-mediated reduction of acetylcholine-induced inward currents in Helix lucorum-identified neurones: role of NAADP-dependent and IP3-dependent Ca2+ release from internal stores,calmodulin and Ca2+/calmodulin-dependent protein kinase II

The effect of seven compounds intracellularly applied by spontaneous diffusion were investigated on the EPYLRFamide-induced reduction of acetylcholine-induced inward current (ACh-current) recorded from identified neurones from Helix lucorum. Inward currents were recorded from neurones LPa2, LPa3, RPa3 and RPa2 in isolated ganglia preparations using two-electrode voltage clamp technique. ACh was applied ionophoretically. Heparin, an antagonist of IP(3) receptors (IP(3)Rs), and IP(3), the agonist of IP(3)Rs, decreased the effect of EPYLRFamide. Thio-NADP, a blocker of NAADP-induced Ca(2+) release, beta-NAADP, Ca(2+) releaser, R24571, W-7 (both calmodulin antagonists), and KN-62, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase II, did not change the modulatory effect of EPYLRFamide. These data suggest that EPYLRFamide decreases ACh-current through elevation of the basal intracellular level of the putative endogenous agonist of IP(3)Rs which activates release of Ca(2+) from intracellular stores. It is concluded that intracellular free Ca(2+) acts on ACh receptor/ionic channel without activation of calmodulin and Ca(2+)/calmodulin-dependent protein kinase II.     

Effects of SEPYLRFamide on acetylcholine-induced currents of Helix aspersa neurones: role of ryanodine receptors

The possible participation of ryanodine receptors in the modulatory effects of the endogenous Helix heptapeptide, SEPYLRFamide, on the acetylcholine-induced currents (ACh-currents) of Helix aspersa neurones was studied using the two-electrode voltage clamp technique. SEPYLRFamide (bath application) caused a reduction of the ACh-currents of D1, D2, F1, F2, F76 and F77 neurones. Ryanodine (10 microM; bath application), which modifies ryanodine-controlled Ca(2+) channels, potentiated the inhibitory effect of SEPYLRFamide on the ACh-current. An antagonist of cyclic adenosine diphosphate ribose (cADPR) and ryanodine receptors, ruthenium red (1 mM; intracellular injection), reduced the inhibitory effects of SEPYLRFamide on the ACh-current. Ryanodine (10 microM) did not change the inhibitory effect of SEPYLRFamide on the ACh-current after intracellular injection of ruthenium red. An agonist of ryanodine receptors, caffeine (5 mM; bath application), reduced the ACh-current. Ryanodine (10 microM) did not change the reduction of ACh-currents induced by the first application of caffeine but decreased the reduction of ACh-currents induced by subsequent applications of caffeine. It is proposed that ryanodine receptors are involved in the inhibitory modulatory effects of SEPYLRFamide on somatic cholinergic receptors of Helix aspersa neurones.     

Effects of SEPYLRFamide on acetylcholine-induced currents of Helix aspersa neurones: role of ryanodine receptors

The possible participation of ryanodine receptors in the modulatory effects of the endogenous Helix heptapeptide, SEPYLRFamide, on the acetylcholine-induced currents (ACh-currents) of Helix aspersa neurones was studied using the two-electrode voltage clamp technique. SEPYLRFamide (bath application) caused a reduction of the ACh-currents of D1, D2, F1, F2, F76 and F77 neurones. Ryanodine (10 μM; bath application), which modifies ryanodine-controlled Ca²⁺ channels, potentiated the inhibitory effect of SEPYLRFamide on the ACh-current. An antagonist of cyclic adenosine diphosphate ribose (cADPR) and ryanodine receptors, ruthenium red (1 mM; intracellular injection), reduced the inhibitory effects of SEPYLRFamide on the ACh-current. Ryanodine (10 μM) did not change the inhibitory effect of SEPYLRFamide on the ACh-current after intracellular injection of ruthenium red. An agonist of ryanodine receptors, caffeine (5 mM; bath application), reduced the ACh-current. Ryanodine (10 μM) did not change the reduction of ACh-currents induced by the first application of caffeine but decreased the reduction of ACh-currents induced by subsequent applications of caffeine. It is proposed that ryanodine receptors are involved in the inhibitory modulatory effects of SEPYLRFamide on somatic cholinergic receptors of Helix aspersa neurones. Accepted: 1 July 1998     

Participation of Na/Ca-exchange and intracellular mobilized Ca2+ in regulating depression of cholino-sensitive Helix lucorum neurons to a cellular analog of habituation

The role the Na/Ca-exchange and intracellular Ca2+ released from Ca(2+)-depots in the modulatory action of Na,K-pump inhibitor ouabain on cholinosensitivity in the command neurons of Helix lucorum was studied in a cellular analogue of habituation. The integral transmembrane inward currents in LPa2, LPa3, RPa3, and RPa2 neurons were recorded in Helix lucorum ganglia preparation using two-electrode voltage clamp technique. The reduction of cholinosensitivity of a neuron was estimated as a depth of the depression of the acetylcholine-induced inward currents during the rhythmic local acetylcholine applications (with the interstimulus interval of 2-4 min) on a somatic membrane. The inhibitor of the Na/Ca-exchange benzamil (the extracellular action, 15-35 mcM) and two specific inhibitors of Ca-ATPase in the sarcoplasmic and endoplasmic reticulum, cyclopiazonic acid and thapsigargin (intracellular injection by spontaneous diffusion, 0.1 mM) prevented the modification of the depression of acetylcholine-induced current by ouabain (100 mcM) during the rhythmic application of acetylcholine. A conclusion is drawn that the inhibitor of the Na,K-pump ouabain modifies the depression of neuron cholinosensitivity in the cellular analogue of habituation via the Na/Ca-exchange and intracellular Ca2+ released from Ca2+ depots.     

Involvement of Na,K-pump in SEPYLRFamide-mediated reduction of cholinosensitivity in Helix neurons

SEPYLRFamide acts as an inhibitory modulator of acetylcholine (ACh) receptors in Helix lucorum neurones. Ouabain, a specific inhibitor of Na,K-pump, (0.1 mM, bath application) decreased the ACh-induced inward current (ACh-current) and increased the leak current. Ouabain decreased the modulatory SEPYLRFamide effect on the ACh-current. There was a correlation between the effects of ouabain on the amplitude of the ACh-current and on the modulatory peptide effect. Ouabain and SEPYLRFamide inhibited the activity of Helix aspersa brain Na,K-ATPase. Activation of Na,K-pump by intracellular injection of 3 M Na acetate or 3 M NaCl reduced the modulatory peptide effect on the ACh-current. An inhibitor of Na/Ca-exchange, benzamil (25 muM, bath application), and an inhibitor of Ca(2+)-pump in the endoplasmic reticulum, thapsigargin (TG, applied intracellularly), both prevented the effect of ouabain on SEPYLRFamide-mediated modulatory effect. Another inhibitor of Ca(2+)-pump in the endoplasmic reticulum, cyclopiazonic acid (applied intracellularly), did not prevent the effect of ouabain on SEPYLRFamide-mediated modulatory effect. These results indicate that Na,K-pump is responsible for the SEPYLRFamide-mediated inhibition of ACh receptors in Helix neurons. Na/Ca-exchange and intracellular Ca(2+) released from internal pools containing TG-sensitive Ca(2+)-pump are involved in the Na,K-pump pathway for the SEPYLRFamide-mediated inhibition of ACh receptors.     

Inositoltriphosphate receptors and ryanodine receptors in regulation of cholinosensitivity of Helix lucorum neurones by Na,K-pump during habituation

Influence of ouabain, the inhibitor of Na,K-pump, on habituation of Helix to tactile stimulation was identical to the ouabain-induced modification of cholinosensitivity reduction in command neurones of defensive behaviour of Helix lucorum in cellular model of habituation. Effects of intracellularly injected ligands of two types of Ca2+ -depot receptors, inositoltrisphosphate (IP3) and ryanodine receptors, on ouabain-induced changes were studied in cellular model of habituation. The antagonist of IP3 receptors heparin (0.1 mM), their agonist IP3 (0.1 mM) and inhibitor of ryanodine-dependent Ca2+ mobilization dantrolen (0.1 mM) prevented the depression of acetylcholine-induced current from the ouabain-evoked modification. The agonist/antagonist of ryanodine receptors ryanodine at two tested concentrations (0.1 mM and 1 mM) did not change the ouabain effect. It is concluded that Ca2+ released from intracellular Ca2+ -depots via IP3 receptors is involved into neuronal mechanism of Na,K-pump regulation of habituation in Helix lucorum to tactile stimulation.     

The Na,K pump regulates a decrease in the cholinosensitivity of the neurons in the snail Helix lucorum taurica Kryn in a cellular analog of habituation: its dependence on intracellular calcium

In Helix lucorum snail we studied the effects of ouabain, inhibitor of Na,K-pump, on the depression of cholinosensitivity in command neurons of withdrawal behavior and the role of the intracellular free Ca2+. The cellular analog of the negative learning (habituation) was used Transmembrane integral inward currents were recorded from the identified LPa2, LPa3, RPa3, and RPa2 neurons in ganglia preparation using two-electrode voltage clamp technique. Acetylcholine (ACh) was locally applied iontophoretically. Reduction of neuronal cholinosensitivity was estimated as a depth of depression of the ACh-induced inward current during rhythmic local application of ACh (interstimulus interval of 1-3 min) onto the somatic membrane. Bath application of ouabain (0.1 mM) produced an increase in depression in one group of neurons and its decrease in another group. After 60-150 min of spontaneous diffusion of a calcium ion chelator BAPTA (1 mM) from the intracellular microelectrode, ouabain produced only the increase in depression. If CaCl2 (100 mM) was added to the solution of the voltage-recording intracellular microelectrode, 60 min later ouabain produced only the reduction of the depression of the ACh current. The conclusion is drawn that the inhibition of the Na,K-pump by ouabain modifies the depression of neuronal cholinosensitivity in the cellular analog of habituation. The direction of the modulatory effect depends on the basal concentration of the intracellular free Ca2+.     

Ca-dependent regulation by Na, K-pump of post-tetanic sensitization of extrasynaptic choline receptors in Helix lucorum neurons

Posttetanic potentiation (by orthodromic stimulation) of cholinosensitivity in LPa3 and RPa3 Helix lucorum neurons that are command in respect to withdrawal behavior was shown earlier (Pivovarov et al., 1999). Now we studied the regulatory role of the Na,K-pump and intracellular free Ga2+ in the posttetanic potentiation (PTP) of cholinosensitivity in command neurons. Semiintact Helix preparation "CNS-visceral bag" was used in experiments. Acetylcholine-induced inward currents were recorded using two-electrode voltage clamp technique. Acetylcholine was applied to somata of the identified LPa3 and RPa3 neurons with a 10-min interval before and after electrical tetanic stimulation of the n. intestinalis (10.5 mA; 0.1 s; 2/s; 2 min). Ouabain (extracellular application, 70 mcM) blocked the PTP. Intracellular injection of BAPTA (1 mM), chelator of Ca2+ ions, prevented the PTP. The PTP was absent after the ouabain application against the background of preliminary intracellular injection of BAPTA. A conclusion war drawn about Ca-dependent participation of Na,K-pump in posttetanic potentiation of cholinosensitivity in command Helix lucorum neurons of withdrawal behavior.     

Store depletion by caffeine/ryanodine activates capacitative Ca(2+) entry in nonexcitable A549 cells

Capacitative Ca(2+) entry is essential for refilling intracellular Ca(2+) stores and is thought to be regulated primarily by inositol 1, 4,5-trisphosphate (IP(3))-sensitive stores in nonexcitable cells. In nonexcitable A549 cells, the application of caffeine or ryanodine induces Ca(2+) release in the absence of extracellular Ca(2+) similar to that induced by thapsigargin (Tg), and Ca(2+) entry occurs upon the readdition of extracellular Ca(2+). The channels thus activated are also permeable to Mn(2+). The channels responsible for this effect appear to be activated by the depletion of caffeine/ryanodine-sensitive stores per se, as evidenced by the activation even in the absence of increased intracellular Ca(2+) concentration. Tg pretreatment abrogates the response to caffeine/ryanodine, whereas Tg application subsequent to caffeine/ryanodine treatment induces further Ca(2+) release. The response to caffeine/ryanodine is also abolished by initial ATP application, whereas ATP added subsequent to caffeine/ryanodine induces additional Ca(2+) release. RT-PCR analyses showed the expression of a type 1 ryanodine receptor, two human homologues of transient receptor potential protein (hTrp1 and hTrp6), as well as all three types of the IP(3) receptor. These results suggest that in A549 cells, (i) capacitative Ca(2+) entry can also be regulated by caffeine/ryanodine-sensitive stores, and (ii) the RyR-gated stores interact functionally with those sensitive to IP(3), probably via Ca(2+)-induced Ca(2+) release.     

Ca2+ responses of pulmonary arterial myocytes to acute hypoxia require release from ryanodine and inositol trisphosphate receptors in sarcoplasmic reticulum

In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) release from the sarcoplasmic reticulum (SR) and Ca(2+) influx through store- and voltage-operated Ca(2+) channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca(2+) release, we measured [Ca(2+)](i) with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca(2+)-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 μM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP(3)R), respectively, or 4% O(2) caused rapid transient increases in [Ca(2+)](i), indicating intracellular Ca(2+) release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM) blocked subsequent Ca(2+) release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 μM) blocked Ca(2+) release to caffeine and hypoxia but not norepinephrine. The IP(3)R antagonist xestospongin C (XeC, 0.1 μM) blocked Ca(2+) release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca(2+)](i) that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca(2+)](i) induced by hypoxia, as well as the subsequent Ca(2+) influx that sustained this increase, required release of Ca(2+) from both RyR and IP(3)R, and 2) the SR Ca(2+) stores accessed by RyR, IP(3)R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca(2+)-ATPase.     

Calcium regulation of the short-term plasticity of the cholinoreceptors in neurons RPa3 and LPa3 of the edible snail]

Pharmacological influences, changing intracellular content of Ca2+, reversibly change the speed and depth of extinction of the input current of the Helix RPa3 and LPa3 neurones, elicited by a repeated iontophoretic application of acetylcholine to the soma. Suppression by extracellular medium, devoid of Ca2+ and by verapamyl (100-150 mumol/l) of Ca2+ input to the cell, induced by cholinoreceptors activation, reversibly weakens the extinction. Raise of intracellular Ca2+ level by blockade with ruthenium red (5-10 mumol/l) of specific Ca2+ transport by mitochondria and by mobilization with caffeine (1-4 mmol/l) of Ca2+, deposited by endoplasmic reticulum, accelerates and intensifies the extinction. The obtained results testify that the short-term cholinoreceptors plasticity of the above neurones is positively controlled by Ca2+ entering the cell by chemically controlled ion channels and mobilized from intracellular Ca-depot.     

Protein kinase C is not involved in regulating the short-term cholinoreceptor plasticity of the PPa3 and LPa3 neurons of the edible snail

A voltage clamp technique on identified Helix lucorum's RPa3 and LPa3 neurons has been used to negate the effect of protein kinase C on extinction of response to repeated iontophoretic applications of acetylcholine to soma. Extracellular influence of phorbol ether, protein kinase C activator (12-O-tetradecanoylphorbol-13-acetate, 0.1-10 mumol/l), or polymyxin B, its blocker (100-500 mumol/l), do not affect the extinction of acetylcholine-induced neuronal response. The data show that protein kinase C is not involved into molecular mechanisms responsible for the regulation of short-term plasticity of RPa3 and LPa3 neuronal cholinoreceptors in Helix lucorum.     

Interplay between ryanodine and IP3 receptors in ATP-stimulated mouse luteinized-granulosa cells

In mouse luteinized-granulosa cells (MGLC), ATP induces an increase in intracellular Ca2+ concentration by stimulating phospholipase C (PLC) associated with purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent release of Ca2+ from intracellular stores. In this study, we examined the cross-talk between the ryanodine receptors (RyR) and IP3 receptors (IP3R) in response to ATP in MGLC. Specifically, the effect of RyR modulators on ATP response was examined. The results showed that ATP-induced intracellular calcium elevation was abolished by inhibitors of the RyR, such as dantrolene (25 microM) and ryanodine (80 microM). When the MGLC were stimulated with activators of RyR, 2 microM ryanodine and 10 mM caffeine, the ATP-elicited response was decreased. These actions were independent of IP3 production stimulated by ATP. Hence, ATP-induced intracellular Ca2+ mobilization involves the coordinated action of both types of calcium release channels (CRCs). Using fluorescent probes, it was shown that IP3R is uniformly distributed throughout the cell; in contrast, RyR is mainly found around the nuclei. It is concluded that the IP3R and the RyR are functionally associated, and both play a role in the pattern of Ca2+ increase observed during purinergic stimulation of MGLC. This coupling may provide a highly efficient amplification mechanism for ATP stimulation of Ca2+ mobilization.     

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.     

Ryanodine Inhibits Caffeine-Evoked Ca2+ Mobilization and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells

The effects of ryanodine, a selective inhibitor of the Ca(2+)-induced Ca2+ release mechanism, on caffeine-evoked changes in cytosolic Ca2+ concentration ([Ca2+]i) and catecholamine secretion were investigated using cultured bovine adrenal chromaffin cells. Caffeine (5-40 mM) caused a concentration-dependent transient rise in [Ca2+]i and catecholamine secretion in Ca2+/Mg(2+)-free medium containing 0.2 mM EGTA. Ryanodine (5 x 10(-5) M) alone had no effect on either [Ca2+]i or catecholamine secretion. Although the application of ryanodine plus caffeine caused the same increase in both [Ca2+]i and catecholamine secretion as those induced by caffeine alone, ryanodine (4 x 10(-7) - 5 x 10(-5) M) irreversibly prevented the increase in both [Ca2+]i and catecholamine secretion resulting from subsequent caffeine application over a range of concentrations. The secretory response to caffeine was markedly enhanced by replacement of Na+ with sucrose in Ca2+/Mg(2+)-free medium, and this enhanced response was also blocked by ryanodine. Caffeine was found to decrease the susceptibility of the secretory apparatus to Ca2+ in digitonin-permeabilized cells. These results indicate that caffeine mobilizes Ca2+ from intracellular stores, the function of which is irreversibly blocked by ryanodine, resulting in the increase in catecholamine secretion in the bovine adrenal chromaffin cell.     

Heterogeneous function of ryanodine receptors, but not IP3 receptors, in hamster cremaster muscle feed arteries and arterioles

The roles played by ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP?Rs) in vascular smooth muscle in the microcirculation remain unclear. Therefore, the function of both RyRs and IP?Rs in Ca(2+) signals and myogenic tone in hamster cremaster muscle feed arteries and downstream arterioles were assessed using confocal imaging and pressure myography. Feed artery vascular smooth muscle displayed Ca(2+) sparks and Ca(2+) waves, which were inhibited by the RyR antagonists ryanodine (10 μM) or tetracaine (100 μM). Despite the inhibition of sparks and waves, ryanodine or tetracaine increased global intracellular Ca(2+) and constricted the arteries. The blockade of IP?Rs with xestospongin D (5 μM) or 2-aminoethoxydiphenyl borate (100 μM) or the inhibition of phospholipase C using U-73122 (10 μM) also attenuated Ca(2+) waves without affecting Ca(2+) sparks. Importantly, the IP?Rs and phospholipase C antagonists decreased global intracellular Ca(2+) and dilated the arteries. In contrast, cremaster arterioles displayed only Ca(2+) waves: Ca(2+) sparks were not observed, and neither ryanodine (10-50 μM) nor tetracaine (100 μM) affected either Ca(2+) signals or arteriolar tone despite the presence of functional RyRs as assessed by responses to the RyR agonist caffeine (10 mM). As in feed arteries, arteriolar Ca(2+) waves were attenuated by xestospongin D (5 μM), 2-aminoethoxydiphenyl borate (100 μM), and U-73122 (10 μM), accompanied by decreased global intracellular Ca(2+) and vasodilation. These findings highlight the contrasting roles played by RyRs and IP?Rs in Ca(2+) signals and myogenic tone in feed arteries and demonstrate important differences in the function of RyRs between feed arteries and downstream arterioles.     

Release and sequestration of Ca2+ by a caffeine- and ryanodine-sensitive store in a sub-population of human SH-SY5Y neuroblastoma cells

We have used single cell fluorescence imaging techniques to examine the role that ryanodine receptors play in the stimulus-induced Ca(2+) responses of SH-SY5Y cells. The muscarinic agonist methacholine (1mM) resulted in a Ca(2+) signal in 95% of all cells. Caffeine (30 mM) however stimulated a Ca(2+) signal in only 1-7% of N-type (neuroblastic) cells within any given field. The caffeine response was independent of extracellular Ca(2+), regenerative in nature, and abolished in a use-dependent fashion by ryanodine. In caffeine-responsive cells, the magnitude of the methacholine-induced Ca(2+) signal was inhibited by 75.07 +/- 5.51% by pretreatment with caffeine and ryanodine, suggesting that the caffeine-sensitive store may act as a Ca(2+) source after muscarinic stimulation. When these data were combined with equivalent data from non-caffeine-responsive cells, the degree of apparent inhibition was significantly reduced. In contrast, after store depletion by caffeine, the Ca(2+) signal induced by 55 mM K(+) was potentiated 2.5-fold in the presence of ryanodine, suggesting that the store may act a Ca(2+) sink after depolarisation. We conclude that a caffeine- and ryanodine-sensitive store can act as a Ca(2+) source and sink in SH-SY5Y cells, and that effects of the store can become obscured if data from caffeine-insensitive cells are not excluded.     

Vectorial Ca2+ release via ryanodine receptors contributes to Ca2+ extrusion from freshly isolated rabbit aortic endothelial cells

In this study, we identified ryanodine receptors (RyRs) as a component of a cytosolic Ca(2+) removal pathway in freshly isolated rabbit aortic endothelial cells. In an earlier article, we reported that the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and Na(+)/Ca(2+) exchanger (NCX) function in series to extrude cytosolic Ca(2+) to the extracellular space. Here we employed caffeine and ryanodine as modulators of RyR and showed that they act as the linkage between SERCA and NCX in removing Ca(2+) from the cytoplasm. Our data indicate that both 15 mM caffeine and 1 microM ryanodine facilitated Ca(2+) extrusion by activating RyRs while 100 microM ryanodine had the opposite effect by blocking RyRs. A further attempt to investigate RyR pharmacology revealed that in the absence of extracellular Ca(2+), ryanodine at 1 microM, but not 100 microM, stimulated Ca(2+) loss from the endoplasmic reticulum (ER). Blockade of RyR had no effect on the Ca(2+) removal rate when NCX had been previously blocked. In addition, the localization of RyR was determined using confocal microscopy of BODIPY TR-X fluorescent staining. Taken together, our findings suggest that in freshly isolated endothelial cells Ca(2+) is removed in part by transport through SERCA, RyR, and eventually NCX, and that RyR and NCX are in close functional proximity near the plasma membrane. After blockade of this component, Ca(2+) extrusion could be further inhibited by carboxyeosin, indicating a parallel contribution by the plasmalemmal Ca(2+)-ATPase (PMCA).     

Regulation of ryanodine receptor opening by lumenal Ca(2+) underlies quantal Ca(2+) release in PC12 cells

Graded or "quantal" Ca(2+) release from intracellular stores has been observed in various cell types following activation of either ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate receptors (InsP(3)R). The mechanism causing the release of Ca(2+) stores in direct proportion to the strength of stimulation is unresolved. We investigated the properties of quantal Ca(2+) release evoked by activation of RyR in PC12 cells, and in particular whether the sensitivity of RyR to the agonist caffeine was altered by lumenal Ca(2+). Quantal Ca(2+) release was observed in cells stimulated with 1 to 40 mM caffeine, a range of caffeine concentrations giving a >10-fold change in lumenal Ca(2+) content. The Ca(2+) load of the caffeine-sensitive stores was modulated by allowing them to refill for varying times after complete discharge with maximal caffeine, or by depolarizing the cells with K(+) to enhance their normal steady-state loading. The threshold for RyR activation was sensitized approximately 10-fold as the Ca(2+) load increased from a minimal to a maximal loading. In addition, the fraction of Ca(2+) released by low caffeine concentrations increased. Our data suggest that RyR are sensitive to lumenal Ca(2+) over the full range of Ca(2+) loads that can be achieved in an intact PC12 cell, and that changes in RyR sensitivity may be responsible for the termination of Ca(2+) release underlying the quantal effect.