Concentrations Of Ryanodine Research Articles

Presynaptic calcium stores contribute to nicotine‐elicited potentiation of evoked synaptic transmission at CA3‐CA1 connections in the neonatal rat hippocampus

Nicotine acetylcholine (ACh) receptors (nAChRs) are ligand-gated ion channels that are widely expressed throughout the central nervous system. It is well established that presynaptic, alpha7-containing nAChRs modulate glutamate release in several brain areas, and that this modulation requires extracellular calcium. However, the intracellular mechanisms consecutive to nAChR opening are unclear. Recent studies have suggested a role for presynaptic calcium stores in the increase of neurotransmitter release following nAChR activation. Using the minimal stimulation protocol at low-probability Schaffer collateral synapses in acute hippocampal slices from neonatal rats, we show that nicotine acting on presynaptic alpha7 nAChRs persistently upregulates glutamate release. We tested the role of calcium stores in this potentiation. First, we examined the relationship between calcium stores and glutamate release. We found that bath application of SERCA pump inhibitors (cyclopiazonic acid and thapsigargin), as well as an agonist of ryanodine receptors (ryanodine 2 microM) increases the probability of glutamate release at CA3-CA1 synapses, decreases the coefficient of variation and the paired-pulse ratio, indicating that presynaptic activation of calcium-induced calcium release can modulate glutamatergic transmission. Next, we investigated whether blocking calcium release from internal stores could alter the effect of nicotine. Preincubation with thapsigargin (10 microM), cyclopiazonic acid (30 microM), or with a high (blocking) concentration of ryanodine (100 microM) for 30 min to 5 h failed to block the effect of nicotine. However, after preincubation in ryanodine, nicotine-elicited potentiation was significantly shortened. These results indicate that at immature Schaffer collateral-CA1 synapses, activation of presynaptic calcium stores is not necessary for but contributes to nicotine-elicited increase of neurotransmitter release.

Caffeine-induced calcium release from the endoplasmic reticulum of acinar cells of the submandibular salivary gland

Ryanodine receptors (RyRs) play a key role in the generalization and spreading of calcium waves in excitable cells; however, the question of the existence of functionally active RyRs in nonexcitable cells demonstrating the capacity for exocytosis (e.g., salivary gland acini) remains open. We studied changes in the total amount of calcium stored in the endoplasmic reticulum (ER) of acinar cells of the submandibular salivary gland of rats and changes in the concentration of ionized Ca2+ inside the ER ([Ca2+]ER) using, respectively, a metallochrome dye, arsenazo III, and a low-affinity fluorescent dye, mag-fura 2/AM. In permeabilized cells, caffeine caused dose-dependent decreases in the total amount of calcium and concentration of ionized calcium. The effective concentration of caffeine providing a 50% drop in the [Ca2+]ER (EC50) was, on average, 7.3 ± 1.1 mM. The caffeine-induced drop in the [Ca2+]ER was insensitive to heparin; in addition, it was blocked by high concentrations (100 µM) of ryanodine, potentiated by ryanodine applied in mild concentrations (10 µM), and also demonstrated a bell-shaped dependence on the concentration of cytoplasmic Ca2+. Such peculiarities are typical characteristics of the RyR-mediated reaction. Therefore, functional RyRs whose activation results in a transient release of calcium from the ER are present in acinar cells of the submandibular salivary gland.

Azumolene Inhibits a Component of Store-operated Calcium Entry Coupled to the Skeletal Muscle Ryanodine Receptor

Dantrolene reduces the elevated myoplasmic Ca(2+) generated during malignant hyperthermia, a pharmacogenetic crisis triggered by volatile anesthetics. Although specific binding of dantrolene to the type 1 ryanodine receptor (RyR1), the Ca(2+) release channel of skeletal muscle sarcoplasmic reticulum, has been demonstrated, there is little evidence for direct dantrolene inhibition of RyR1 channel function. Recent studies suggest store-operated Ca(2+) entry (SOCE) contributes to skeletal muscle function, but the effect of dantrolene on this pathway has not been examined. Here we show that azumolene, an equipotent dantrolene analog, inhibits a component of SOCE coupled to activation of RyR1 by caffeine and ryanodine, whereas the SOCE component induced by thapsigargin is not affected. Our data suggest that azumolene distinguishes between two mechanisms of cellular signaling to SOCE in skeletal muscle, one that is coupled to and one independent from RyR1.

Calcium‐induced calcium release in rod photoreceptor terminals boosts synaptic transmission during maintained depolarization

We examined the contribution of calcium-induced calcium release (CICR) to synaptic transmission from rod photoreceptor terminals. Whole-cell recording and confocal calcium imaging experiments were conducted on rods with intact synaptic terminals in a retinal slice preparation from salamander. Low concentrations of ryanodine stimulated calcium increases in rod terminals, consistent with the presence of ryanodine receptors. Application of strong depolarizing steps (-70 to -10 mV) exceeding 200 ms or longer in duration evoked a wave of calcium that spread across the synaptic terminals of voltage-clamped rods. This secondary calcium increase was blocked by high concentrations of ryanodine, indicating it was due to CICR. Ryanodine (50 microm) had no significant effect on rod calcium current (I(ca)) although it slightly diminished rod light-evoked voltage responses. Bath application of 50 microm ryanodine strongly inhibited light-evoked currents in horizontal cells. Whether applied extracellularly or delivered into the rod cell through the patch pipette, ryanodine (50 microm) also inhibited excitatory post-synaptic currents (EPSCs) evoked in horizontal cells by depolarizing steps applied to rods. Ryanodine caused a preferential reduction in the later portions of EPSCs evoked by depolarizing steps of 200 ms or longer. These results indicate that CICR enhances calcium increases in rod terminals evoked by sustained depolarization, which in turn acts to boost synaptic exocytosis from rods.

Effects of zonisamide on neurotransmitter exocytosis associated with ryanodine receptors

To clarify the antiepileptic and neuroprotective actions of zonisamide (ZNS), we determined acute effects of ZNS on exocytosis of GABA and glutamate associated with ryanodine-receptor (Ryr) in rat hippocampus using microdialysis. ZNS increased basal GABA release concentration-dependently without affecting basal glutamate release; however, K +-evoked glutamate and GABA releases were reduced by ZNS concentration-dependently. Inhibition of Ryr reduced K +-evoked GABA and glutamate releases without affecting their basal releases. Ryanodine affected GABA and glutamate releases biphasic concentration-dependently: lower concentration of ryanodine increased both basal and K +-evoked releases of GABA and glutamate, whereas higher concentration reduced them. The therapeutically relevant concentration of ZNS inhibited ryanodine-induced GABA and glutamate releases, and abolished the inflection point in concentration–response curve for ryanodine on neurotransmitter exocytosis. These data suggest that ZNS elevates seizure threshold via enhancement of GABAergic transmission during resting stage. ZNS inhibits propagation of epileptic hyperexcitability and Ryr-associated neuronal damage during neuronal hyperexcitable stage. These demonstrations indicate that the indirect inhibition of Ryr activities by ZNS during neuronal hyperexcitability appear to be involved in the mechanisms of action of antiepileptic and neuroprotective actions of ZNS.

Sphingosine releases Ca 2+ from intracellular stores via the ryanodine receptor in sea urchin egg homogenates

Various reports have demonstrated that the sphingolipids sphingosine and sphingosine-1-phosphate are able to induce Ca 2+ release from intracellular stores in a similar way to second messengers. Here, we have used the sea urchin egg homogenate, a model system for the study of intracellular Ca 2+ release mechanisms, to investigate the effect of these sphingolipids. While ceramide and sphingosine-1-phosphate did not display the ability to release Ca 2+, sphingosine stimulated transient Ca 2+ release from thapsigargin-sensitive intracellular stores. This release was inhibited by ryanodine receptor blockers (high concentrations of ryanodine, Mg 2+, and procaine) but not by pre-treatment of homogenates with cADPR, 8-bromo-cADPR or blockers of other intracellular Ca 2+ channels. However, sphingosine rendered the ryanodine receptor refractory to cADPR. We propose that, in the sea urchin egg, sphingosine is able to activate the ryanodine receptor via a mechanism distinct from that used by cADPR.

Disruption of endoplasmic reticulum calcium stores is involved in neuronal death induced by glycolysis inhibition in cultured hippocampal neurons

Disturbances in neuronal calcium homeostasis have been implicated in a variety of neuropathological conditions, including cerebral ischemia, hypoglycemia, and epilepsy, and possibly constitute part of the cell death process associated with chronic neurodegenerative disorders. We investigated if endoplasmic reticulum (ER) calcium stores participate in neuronal death triggered by moderate glycolysis inhibition induced by iodoacetate, an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, in cultured hippocampal neurons. Results show that exposure to iodoacetate leads to a slow partial decrease in cell survival, which is significantly prevented in the absence of Ca(2+) or in the presence of the calcium chelator BAPTA-AM. Treatment with caffeine and a low (1 microM) concentration of ryanodine, which activates the ryanodine receptor (RyR), exacerbates neuronal death, whereas dantrolene and 25 microM ryanodine, which antagonizes RyR, prevents damage. Xestospongin C (XeC), an antagonist of the inositol-3-phosphate (IP(3)) receptor (IP(3)R) also prevents neuronal damage. Inhibitors of the ER calcium ATPase (sarcoendoplasmic reticulum Ca(2+) ATPase; SERCA) have no effect. The decrease in ATP levels induced by iodoacetate is potentiated by caffeine and prevented by dantrolene. Although only a slight increase in glutamate extracellular levels is observed 3.5 hr after iodoacetate exposure, the N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, MK-801, efficiently prevents neuronal damage. Taken together, the data suggest that neuronal death induced during moderate glycolysis inhibition involves calcium influx through NMDA receptors and calcium release from intracellular ER stores. These results might be relevant to the understanding the mechanisms involved in neuronal damage related to aging and chronic neurodegenerative diseases, which have been associated with decreased glucose metabolism.

Ryanodine receptors and BK channels act as a presynaptic depressor of neurotransmission in cochlear inner hair cells

Ryanodine receptors (RyRs) are known to contribute to the regulation of free cytosolic calcium concentration. This family of intracellular calcium channels plays a significant role in calcium-induced-calcium-release (CICR), and have been implicated in calcium-dependent processes requiring exquisite spatio-temporal regulation. In order to characterize the importance of these intracellular calcium channels in cochlear physiology, we perfused the guinea pig cochlea with antagonistic concentrations of ryanodine. The distortion products of the cochlear microphonic and the compound action potential of the auditory nerve were reversibly inhibited by ryanodine (IC(50)=27.3 microm, Hill coefficient=1.9), indicating an action at the cochlear amplifier. Single auditory nerve fibre recordings showed that ryanodine slightly increased spontaneous firing rates by 22%, suggesting an excitatory effect of ryanodine. This paradoxical effect could be explained by an inhibitory action of ryanodine on presynaptic BK channels of inner hair cells (IHC). Indeed, perfusing iberiotoxin also increased the spontaneous firing activity of the auditory nerve fibres. Furthermore, whole-cell patch-clamp recordings demonstrated that ryanodine inhibits BK currents at the IHC level. Conversely, immunohistochemistry demonstrated a strong expression of RyR in IHCs and, more particularly, below the cuticular plate where membranous BK channels are highly expressed. Overall, the study demonstrated a key role for RyR and CICR in signal transduction at the IHCs. We therefore propose that coupled RyR--BK channels act to suppress the fast neurotransmission in IHCs.

Ryanodine Receptor-Mediated Rapid Increase in Intracellular Calcium Induced by 7,8-Benzo(a)Pyrene Quinone in Human and Murine Leukocytes

Benzo(a)pyrene (BaP) is an environmentally prevalent polycyclic aromatic hydrocarbon (PAH) known to produce immunotoxicity in murine and human lymphocytes. Previous studies by our lab have shown that certain BaP metabolites increase intracellular Ca(2+) in human and murine lymphocytes. The mechanism by which these BaP metabolites increase Ca(2+) may involve src kinase activation and mitochondrial oxidative stress. We have implicated a new pathway of Ca(2+) elevation in lymphocytes produced by a novel BaP metabolite, BaP-7,8-dione (7,8-BPQ). This ortho quinone is produced from BaP-7,8-dihydrodiol by aldoketoreductase 1C1 (AKR1C) isoforms in human cells. We have previously shown that 7,8-BPQ increases Ca(2+) levels in an in vitro rabbit skeletal muscle sarcoplasmic reticulum (SR) vesicle model via interaction with ryanodine receptors (RyR). In the present study, we found that 7,8-BPQ produced a RyR-dependent rapid increase in intracellular Ca(2+) in the Daudi human B cell line. However, other BP-diones including 1,6-, 3,6-, and 6,12-BPQs failed to produce a rapid increase in Ca(2+). Instead they produced a late increase in intracellular Ca(2+), presumably via a redox-cycling-dependent loss of Ca(2+) buffering capacity by mitochondria. Functional RyR were detected in Daudi using a (3)H-ryanodine binding assay. The studies were extended to normal human peripheral blood and murine spleen cells, where it was found that 7,8-BPQ rapidly elevated intracellular Ca(2+) in B cells and T cells in both species. The Ca(2+)-elevating effect of 7,8-BPQ was prevented by pretreatment with a high concentration of ryanodine (500 muM). Collectively, these results demonstrate a novel mechanism of Ca(2+) elevation by an environmentally relevant metabolite of BaP in murine and human lymphocytes.

Local recovery of Ca2+ release in rat ventricular myocytes

Excitation-contraction coupling in the heart depends on the positive feedback process of Ca2+-induced Ca2+ release (CICR). While CICR provides for robust triggering of Ca2+ sparks, the mechanisms underlying their termination remain unknown. At present, it is unclear how a cluster of Ca2+ release channels (ryanodine receptors or RyRs) can be made to turn off when their activity is sustained by the Ca2+ release itself. We use a novel experimental approach to investigate indirectly this issue by exploring restitution of Ca2+ sparks. We exploit the fact that ryanodine can bind, nearly irreversibly, to an RyR subunit (monomer) and increase the open probability of the homotetrameric channel. By applying low concentrations of ryanodine to rat ventricular myocytes, we observe repeated activations of individual Ca2+ spark sites. Examination of these repetitive Ca2+ sparks reveals that spark amplitude recovers with a time constant of 91 ms whereas the sigmoidal recovery of triggering probability lags behind amplitude recovery by approximately 80 ms. We conclude that restitution of Ca2+ sparks depends on local refilling of SR stores after depletion and may also depend on another time-dependent process such as recovery from inactivation or a slow conformational change after rebinding of Ca2+ to SR regulatory proteins.

Functional Ryanodine Receptor Expression Is Required for NAADP-mediated Local Ca2+ Signaling in T-lymphocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing nucleotide involved in T cell Ca2+ signaling (Berg, I., Potter, B. V. L., Mayr, G. W., and Guse, A. H. (2000) J. Cell Biol. 150, 581-588). The objective of this study was to analyze whether the first subcellular Ca2+ signals obtained upon NAADP stimulation of T-lymphocytes depend on the functional expression of ryanodine receptors. Using combined microinjection and high resolution confocal calcium imaging, we demonstrate here that subcellular Ca2+ signals, characterized by amplitudes between approximately 30 and 100 nM and diameters of approximately 0.5 microM, preceded global Ca2+ signals. Co-injection of the ryanodine receptor antagonists ruthenium red and ryanodine together with NAADP abolished the effects of NAADP, whereas the D-myo-inositol 1,4,5-trisphosphate antagonist heparin and the Ca2+ entry blocker SKF&96365 were without effect. This pharmacological approach was confirmed by a molecular knock-down approach. Jurkat T cell clones with largely reduced expression of ryanodine receptors did not respond to microinjections of NAADP. Taken together, our data suggest that the Ca2+ release channel sensitive to NAADP in T-lymphocytes is the ryanodine receptor.

ADP-ribosyl cyclase couples to cyclic AMP signaling in the cardiomyocytes

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca 2+-mobilizing second messenger cyclic ADP-ribose (cADPR) from β-NAD +. In this study, we examined the molecular basis of which β-adrenergic receptor (βAR) stimulation induces cADPR formation and characterized cardiac ADPR-cyclase. The results revealed that isoproterenol-mediated increase of [Ca 2+] i in rat cardiomyocytes was blocked by pretreatment with a cADPR antagonistic derivative 8-Br-cADPR, a PKA inhibitor H89 or high concentration of ryanodine. Moreover, incubation of ventricular lysates with isoproterenol, forskolin or cAMP resulted in activation of ADPR-cyclase that was inhibited by pretreatment with H89. Supporting the observations, the cADPR antagonist and H89 blocked 8-CPT-cAMP, a cell-permeant cAMP analog-induced increase in [Ca 2+] i but not cGMP-mediated increase. Characterization of partially purified cardiac ADPR-cyclase showed a molecular mass of approximately 42 kDa and no cross-activity with CD38 antibodies, and the enzyme activity was inhibited by Zn 2+ but not dithiothreitol. Microinjection of the enzyme into rat cardiomyocytes increased the level of [Ca 2+] i in a concentration-dependent manner. The enzyme-mediated increase of [Ca 2+] i was blocked by the cADPR antagonist. These findings suggest that βAR-mediated regulation of [Ca 2+] i in rat cardiomyocytes is primed by activation of cardiac ADPR-cyclase via cAMP/PKA signaling and that cardiac ADPR-cyclase differs from CD38 in biochemical and immunological properties.

Endothelin-1, superoxide and adeninediphosphate ribose cyclase in shark vascular smooth muscle

In vascular smooth muscle (VSM) of Squalus acanthias, endothelin-1 (ET-1) signals via the ET(B) receptor. In both shark and mammalian VSM, ET-1 induces a rise in cytosolic Ca(2+) concentration ([Ca(2+)](i)) via activation of the inositol trisphosphate (IP(3)) receptor (IP(3)R) and subsequent release of Ca(2+) from the sarcoplasmic reticulum (SR). IP(3)R-mediated release of SR Ca(2+) causes calcium-induced calcium release (CICR) via the ryanodine receptor (RyR), which can be sensitized by cyclic adeninediphosphate ribose (cADPR). cADPR is synthesized from NAD(+) by a membrane-bound bifunctional enzyme, ADPR cyclase. We have previously shown that the antagonists of the RyR, Ruthenium Red, high concentrations of ryanodine and 8-Br cADPR, diminish the [Ca(2+)](i) response to ET-1 in shark VSM. To investigate how ET-1 might influence the activity of the ADPR cyclase, we employed inhibitors of the cyclase. To explore the possibility that ET-1-induced production of superoxide (O(2)*-) might activate the cyclase, we used an inhibitor of NAD(P)H oxidase (NOX), DPI and a scavenger of O(2)*-, TEMPOL. Anterior mesenteric artery VSM was loaded with fura-2AM to measure [Ca(2+)](i). In Ca(2+)-free shark Ringers, ET-1 increased [Ca(2+)](i) by 104+/-8 nmol l(-1). The VSM ADPR cyclase inhibitors, nicotinamide and Zn(2+), diminished the response by 62% and 72%, respectively. Both DPI and TEMPOL reduced the response by 63%. The combination of the IP(3)R antagonists, 2-APB or TMB-8, with DPI or TEMPOL further reduced the response by 83%. We show for the first time that in shark VSM, inhibition of the ADPR cyclase reduces the [Ca(2+)](i) response to ET-1 and that superoxide may be involved in the activation of the cyclase.

Angiotensin II Ca2+signaling in rat afferent arterioles: stimulation of cyclic ADP ribose and IP3pathways

ANG II induces a rise in cytosolic Ca(2+) ([Ca(2+)](i)) in vascular smooth muscle (VSM) cells via inositol trisphosphate receptor (IP(3)R) activation and release of Ca(2+) from the sarcoplasmic reticulum (SR). The Ca(2+) signal is augmented by calcium-induced calcium release (CICR) and by cyclic adeninediphosphate ribose (cADPR), which sensitizes the ryanodine-sensitive receptor (RyR) to Ca(2+) to further amplify CICR. cADPR is synthesized from beta-nicotinamide adenine dinucleotide (NAD(+)) by a membrane-bound bifunctional enzyme, ADPR cyclase. To investigate the possibility that ANG II activates the ADPR cyclase of afferent arterioles, we used inhibitors of the IP(3)R, RyR, and ADPR cyclase. Afferent arterioles were isolated from rat kidney with the magnetized microsphere and sieving technique and loaded with fura-2 to measure [Ca(2+)](i). In Ca(2+)-containing buffer, ANG II increased [Ca(2+)](i) by 125 +/- 10 nM. In the presence of the IP(3)R antagonists TMB-8 and 2-APB, the peak responses to ANG II were reduced by 74 and 81%, respectively. The specific antagonist of cADPR 8-Br ADPR and a high concentration of ryanodine (100 microM) inhibited the ANG II-induced increases in [Ca(2+)](i) by 75 and 69%, respectively. Nicotinamide and Zn(2+) are known inhibitors of the VSM ADPR cyclase. Nicotinamide diminished the [Ca(2+)](i) response to ANG II by 66%. In calcium-free buffer, Zn(2+) reduced the ANG II response by 68%. Simultaneous blockade of the IP(3) and cADPR pathways diminished the [Ca(2+)](i) response to ANG II by 83%. We conclude that ANG II initiates Ca(2+) mobilization from the SR in afferent arterioles via the classic IP(3)R pathway and that ANG II may lead to activation of the ADPR cyclase to form cADPR, which, via its action on the RyR, substantially augments the Ca(2+) response.

Activation of ryanodine receptors in the nuclear envelope alters the conformation of the nuclear pore complex

Nuclear pore complexes (NPCs) are supramolecular protein pores that traverse the nuclear envelope and form the only known direct route of transport between the cytoplasmic and nuclear spaces. Detailed studies have identified both active and passive mechanisms of transport through the NPC and structural studies have revealed its three-dimensional architecture. Under certain conditions, structural studies have found evidence for a mass in the central pore of the NPC whose identity remains unclear. Some studies suggest this mass represents cargo caught in transit, while others suggest it is an integral component of the NPC, the position of which is sensitive to sample conditions. Regardless of its identity, previous studies have shown that the central mass location within the NPC pore is influenced by the presence of calcium in the cisternal spaces of the nuclear membrane. Specific depletion of these calcium stores through inositol 1,4,5-trisphosphate (IP 3) receptor activation leads to the apparent displacement of the central mass towards both the cytoplasmic and nucleoplasmic sides of the NPC. Whether the central mass is cargo or a NPC component, these observations may offer interesting insights linking transport and calcium signaling pathways. Here, we show that ryanodine (Ry) receptors are also present in the nuclear envelope of Xenopus laevis oocytes, and their specific activation can affect the conformational state of the NPC. Although previously undetected, Western blot analysis of isolated oocyte nuclei reveals the presence of Ry receptors in the nuclear envelope, albeit in low abundance. Extensive atomic force microscopy (AFM) studies at the single pore level of isolated, fixed nuclei reveal changes in the NPC conformational state following treatments that stimulate Ry receptor activity. At resting calcium levels (∼200 nM Ca 2+), the central mass within the lumen of the NPC is recessed 5.3 nm below the cytoplasmic rim of the NPC. Following treatment with 10 nM ryanodine, the central mass displaces towards the cytoplasmic face occupying a new position only 2.9 nm below the cytoplasmic rim. Interestingly, at high ryanodine concentrations (20 μM), which are reported to deactivate Ry receptors, the central mass is observed to return to the recessed position, 5.4 nm below the cytoplasmic rim. Treatments with caffeine also lead to large changes in the NPC conformation, confirming the link to specific activation of Ry receptors. These observations are consistent with a new mechanism of NPC regulation in which specific activation of Ry receptors located in the nuclear envelope can modulate cisternal calcium levels, leading to changes in the NPC conformation. Together with previous studies, it now appears that both IP 3 and Ry receptors are present in the nuclear envelope of Xenopus oocytes and are capable, through activation, of indirectly influencing the conformational state of the NPC.

RyR2 and Calpain-10 Delineate a Novel Apoptosis Pathway in Pancreatic Islets

Cells are programmed to die when critical signaling and metabolic pathways are disrupted. Inhibiting the type 2 ryanodine receptor (RyR2) in human and mouse pancreatic beta-cells markedly increased apoptosis. This mode of programmed cell death was not associated with robust caspase-3 activation prompting a search for an alternative mechanism. Increased calpain activity and calpain gene expression suggested a role for a calpain-dependent death pathway. Using a combination of pharmacological and genetic approaches, we demonstrated that the calpain-10 isoform mediated ryanodine-induced apoptosis. Apoptosis induced by the fatty acid palmitate and by low glucose also required calpain-10. Ryanodine-induced calpain activation and apoptosis were reversed by glucagon-like peptide or short-term exposure to high glucose. Thus RyR2 activity seems to play an essential role in beta-cell survival in vitro by suppressing a death pathway mediated by calpain-10, a type 2 diabetes susceptibility gene with previously unknown function.

Ryanodine receptors in human pancreatic beta cells: localization and effects on insulin secretion.

It is clear that pancreatic beta-cell dysfunction, including basal hyperinsulinemia and reduced insulin release in response to glucose, is a key determinant of disease progression in type 2 diabetes, but the underlying molecular defects are not known. In diabetes, the expression and function of ryanodine receptor (RyR) Ca2+ release channels are reduced. The present studies were undertaken to define the subcellular location and role of RyR in the control of stimulated and basal insulin release from human pancreatic beta cells. Using confocal microscopy, we observed RyR immunoreactivity in a vesicular pattern. RyRs did not colocalize with insulin secretory granules but partially colocalized with endosomes. Direct activation with nanomolar concentrations of ryanodine evoked increases in cytosolic Ca2+ that were coupled to transient insulin release. Insulin release stimulated by 1 nM ryanodine was sensitive to BAPTA-AM preincubation but independent of thapsigargin-sensitive endoplasmic reticulum (ER) Ca2+ pools. Blocking RyRs with micromolar concentrations of ryanodine led to BAPTA-resistant insulin release that was not associated with an increase in cytosolic Ca2+, which implicated alterations in luminal Ca2+. However, neither Ca2+ signals nor insulin release stimulated by glucose was blocked by 10-50 microM ryanodine, which suggests that the CD38/cyclic ADP-ribose/RyR pathway is not a primary mechanism of glucose action in nontransformed beta cells. We provide the first evidence that RyRs directly control insulin secretion in primary beta cells. Unexpectedly, stimulation of insulin secretion by ryanodine occurs independently of glucose and by two mechanisms, including a novel cytosolic Ca2+-independent mechanism likely involving changes in Ca2+ within the lumens of non-ER organelles, such as endosomes.

Signal transduction and gene expression regulated by calcium release from internal stores in excitable cells

Calcium regulation of several transcription factors involves different calcium-dependent signaling cascades and engages cytoplasmic as well as nuclear calcium signals. The study of the specific sources of calcium signals involved in regulation of gene expression in skeletal muscle has been addressed only recently. In this tissue, most cytoplasmic and nuclear calcium signals originate from calcium release from internal stores, mediated either by ryanodine receptor (RyR) or IP3 receptor (IP3R) channels. The latter are located both in the sarcoplasmic reticulum (SR) and in the nuclear membrane, and their activation results in long-lasting nuclear calcium increase. The calcium signals mediated by RyR and IP3R are very different in kinetics, amplitude and subcellular localization; an open question is whether these differences are differentially sensed by transcription factors. In neurons, it is well established that calcium entry through L-type calcium channels and NMDA receptors plays a role in the regulation of gene expression. Increasing evidence, however, points to a role for calcium release from intracellular stores in this process. In this article, we discuss how RyR-mediated calcium release contributes to the activation of the calcium-dependent transcription factor CREB and the subsequent LTP generation. We present novel results from our laboratory showing ERK-mediated CREB activation by hydrogen peroxide. This activation takes place in the absence of extracellular calcium and is blocked by inhibitory ryanodine concentrations, suggesting it is caused by redox activation of RyR-mediated calcium release.

Presynaptic Ryanodine-Sensitive Calcium Stores Contribute to Evoked Neurotransmitter Release at the Basket Cell-Purkinje Cell Synapse

Presynaptic terminals of cerebellar basket cells are known to contain ryanodine-sensitive calcium stores (RyCSs); recently, it has been shown that these stores control the frequency of miniature synaptic currents in the absence of presynaptic spiking. Here, using paired recordings of basket cell-Purkinje cell synapses, we show that blocking the RyCSs with high concentration of ryanodine decreases the mean amplitude of evoked IPSCs to 70% of the control value. The paired-pulse ratio and failure rate increase, indicating that the reduction stems from a decreased probability of evoked neurotransmitter release. Various control experiments eliminate the possibility of an indirect effect of ryanodine via activation of postsynaptic receptors. Prolonged application of cyclopiazonic acid, a blocker of the endoplasmic reticulum calcium pump, totally abolishes the ryanodine action. Our results indicate that calcium released from presynaptic RyCSs enhances the amplitude of evoked GABAergic synaptic currents. The precise mechanism by which calcium released from internal stores affect action potential-dependent release is unknown; however, our results suggest that these stores do not provide additional calcium for each presynaptic action potential; rather, they appear to enhance depolarization-induced calcium signals indirectly, perhaps by increasing the basal level of cytosolic calcium concentration in the vicinity of release sites.

Ca(2+)-induced Ca(2+) release in the pancreatic beta-cell: direct evidence of endoplasmic reticulum Ca(2+) release.

The role of the Ca(2+)-induced Ca(2+) release channel (ryanodine receptor) in MIN6 pancreatic beta-cells was investigated. An endoplasmic reticulum (ER)-targeted "cameleon" was used to report lumenal free Ca(2+). Depolarization of MIN6 cells with KCl led to release of Ca(2+) from the ER. This ER Ca(2+) release was mimicked by treatment with the ryanodine receptor agonists caffeine and 4-chloro-m-cresol, reversed by voltage-gated Ca(2+) channel antagonists and blocked by treatment with antagonistic concentrations of ryanodine. The depolarization-induced rise in cytoplasmic Ca(2+) was also inhibited by ryanodine, which did not alter voltage-gated Ca(2+) channel activation. Both ER and cytoplasmic Ca(2+) changes induced by depolarization occurred in a dose-dependent manner. Glucose caused a delayed rise in cytoplasmic Ca(2+) but no detectable change in ER Ca(2+). Carbamyl choline caused ER Ca(2+) release, a response that was not altered by ryanodine. Taken together, these results provide strong evidence that Ca(2+)-induced Ca(2+) release augments cytoplasmic Ca(2+) signals in pancreatic beta-cells.