Calcium buffering activity of mitochondria controls basal growth hormone secretion and modulates specific neuropeptide signaling

Goldfish somatotropes contain multiple functionally distinct classes of non-mitochondrial intracellular Ca(2+) stores. In this study, we investigated the role of mitochondrial Ca(2+) handling in the control of hormone secretion. Inhibition of mitochondrial Ca(2+) uptake with 10 microM ruthenium red (RR) and 10 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP) caused a small and reversible increase in cytosolic [Ca(2+)]. Despite relatively modest global Ca(2+) signals, RR and CCCP stimulated robust GH secretion under basal culture conditions. CCCP-stimulated hormone release was abolished in cells pre-incubated with 50 microM BAPTA-AM, suggesting that elevations in cytosolic [Ca(2+)] mediate this release of GH. Both caffeine-sensitive intracellular Ca(2+) stores and L-type Ca(2+) channels can be the source of the Ca(2+) buffered by mitochondria in somatotropes. The stimulatory effect of RR on caffeine-stimulated GH release was enhanced dramatically in the presence of ryanodine, pointing to a complex interaction between these three Ca(2+) stores. Inhibition of mitochondrial Ca(2+) uptake with RR augmented GH release evoked by only one of the two endogenous gonadotropin-releasing hormones. Thus, we provide the first evidence that mitochondrial Ca(2+) buffering is differentially involved in specific agonist Ca(2+) signaling pathways and plays an important role in the control of basal GH release.     

Lysosome-sarcoplasmic reticulum junctions. A trigger zone for calcium signaling by nicotinic acid adenine dinucleotide phosphate and endothelin-1

Previous studies on pulmonary arterial smooth muscle cells have shown that nicotinic acid adenine dinucleotide phosphate (NAADP) evokes highly localized intracellular Ca(2+) signals by mobilizing thapsigargin-insensitive stores. Such localized Ca(2+) signals may initiate global Ca(2+) waves and contraction of the myocytes through the recruitment of ryanodine receptors on the sarcoplasmic reticulum via Ca(2+)-induced Ca(2+) release. Here we show that NAADP evokes localized Ca(2+) signals by mobilizing a bafilomycin A1-sensitive, lysosome-related Ca(2+) store. These lysosomal stores facilitate this process by co-localizing with a portion of the sarcoplasmic reticulum expressing ryanodine receptors to comprise a highly specialized trigger zone for NAADP-dependent Ca(2+) signaling by the vasoconstrictor hormone, endothelin-1. These findings further advance our understanding of how the spatial organization of discrete, organellar Ca(2+) stores may underpin the generation of differential Ca(2+) signaling patterns by different Ca(2+)-mobilizing messengers.     

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.     

Function-specific calcium stores selectively regulate growth hormone secretion, storage, and mRNA level

Ca(+) stores may regulate multiple components of the secretory pathway. We examined the roles of biochemically independent intracellular Ca(2+) stores on acute and long-term growth hormone (GH) release, storage, and mRNA levels in goldfish somatotropes. Thapsigargin-evoked intracellular Ca(2+) concentration ([Ca(2+)](i)) signal amplitude was similar to the Ca(2+)-mobilizing agonist gonadotropin-releasing hormone, but thapsigargin (2 microM) did not acutely increase GH release, suggesting uncoupling between [Ca(2+)](i) and exocytosis. However, 2 microM thapsigargin affected long-term secretory function. Thapsigargin-treated cells displayed a steady secretion of GH (2, 12, and 24 h), which decreased GH content (12 and 24 h), but not GH mRNA/production (24 h). In contrast to the results with thapsigargin, activating the ryanodine (Ry) receptor (RyR) with 1 nM Ry transiently increased GH release (2 h). Prolonged activation of RyR (24 h) reduced GH release, contents and apparent production, without changing GH mRNA levels. Inhibiting RyR with 10 microM Ry increased GH mRNA levels, production, and storage (2 h). Increasing [Ca(2+)](i) independently of Ca(2+) stores with the use of 30 mM KCl decreased GH mRNA. Collectively, these results suggest that parts of the secretory pathway can be controlled independently by function-specific Ca(2+) stores.     

Nicotinic acid adenine dinucleotide phosphate potentiates neurite outgrowth

Ca(2+) regulates a spectrum of cellular processes including many aspects of neuronal function. Ca(2+)-sensitive events such as neurite extension and axonal guidance are driven by Ca(2+) signals that are precisely organized in both time and space. These complex cues result from both Ca(2+) influx across the plasma membrane and the mobilization of intracellular Ca(2+) stores. In the present study, using rat cortical neurons, we have examined the effects of the novel intracellular Ca(2+)-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) on neurite length and cytosolic Ca(2+) levels. We show that NAADP potentiates neurite extension in response to serum and nerve growth factor and stimulates increases in cytosolic Ca(2+) from bafilomycin-sensitive Ca(2+) stores. Simultaneous blockade of inositol trisphosphate and ryanodine receptors abolished the effects of NAADP on neurite length and reduced the magnitude of NAADP-mediated Ca(2+) signals. This is the first report demonstrating functional NAADP receptors in a mammalian neuron. Interplay between NAADP receptors and more established intracellular Ca(2+) channels may therefore play important signaling roles in the nervous system.     

Impairment of Ca(2+) mobilization in circular muscle cells of the inflamed colon

This study investigated whether inflammation modulates the mobilization of Ca(2+) in canine colonic circular muscle cells. The contractile response of single cells from the inflamed colon was significantly suppressed in response to ACh, KCl, and BAY K8644. Methoxyverapamil and reduction in extracellular Ca(2+) concentration dose-dependently blocked the response in both normal and inflamed cells. The increase in intracellular Ca(2+) concentration in response to ACh and KCl was significantly reduced in the inflamed cells. However, Ca(2+) efflux from the ryanodine- and inositol 1,4, 5-trisphosphate (IP(3))-sensitive stores, as well as the decrease of cell length in response to ryanodine and IP(3), were not affected. Heparin significantly blocked Ca(2+) efflux and contraction in response to ACh in both conditions. ACh-stimulated accumulation of IP(3) and the binding of [(3)H]ryanodine to its receptors were not altered by inflammation. Ruthenium red partially inhibited the response to ACh in normal and inflamed states. We conclude that the canine colonic circular muscle cells utilize Ca(2+) influx through L-type channels as well as Ca(2+) release from the ryanodine- and IP(3)-sensitive stores to contract. Inflammation impairs Ca(2+) influx through L-type channels, but it may not affect intracellular Ca(2+) release. The impairment of Ca(2+) influx may contribute to the suppression of circular muscle contractility in the inflamed state.     

Caffeine-stimulated GTH-II release involves Ca(2+) stores with novel properties

Modulation of Ca(2+) stores with 10 mM caffeine stimulates robust secretion of gonadotropin (GTH-II) from goldfish gonadotropes. Although both endogenous forms of gonadotropin-releasing hormone (GnRH) utilize a common intracellular Ca(2+) store, sGnRH, but not cGnRH-II, uses an additional caffeine-sensitive mechanism. We examined caffeine signaling by using Ca(2+) imaging, electrophysiology, and cell-column perifusion. Although caffeine inhibited K+ channels, this action appeared to be unrelated to caffeine-induced GTH-II release, because the latter was insensitive to tetraethylammonium. The effects of caffeine also were not mediated by the cAMP/protein kinase A pathway. Instead, caffeine-evoked GTH-II responses were Ca(2+) signal dependent because they were abolished by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid loading. Caffeine generated localized Ca(2+) signals that began near secretory granules. Surprisingly, caffeine-stimulated GTH-II release was insensitive to 100 microM ryanodine and, unlike GnRH action, was unaffected by inhibitors of voltage-gated Ca(2+) channels or sarco(endo)plasmic reticulum Ca(2+)-ATPases. Collectively, these data indicate that caffeine-stimulated GTH-II release is not mediated by typical agonist-sensitive Ca(2+) stores found in endoplasmic reticulum.     

Role of Ca(2+)-ATPase in spontaneous oscillations of cytosolic free Ca2+ in GH3 rat pituitary cells.

The relative contribution of voltage-sensitive Ca2+ channels, Ca(2+)-ATPases, and Ca2+ release from intracellular stores to spontaneous oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) observed in secretory cells is not well characterized owing to a lack of specific inhibitors for a novel thapsigargin (Tg)-insensitive Ca(2+)-ATPase expressed in these cells. We show that spontaneous [Ca2+]i oscillations in GH3 cells were unaffected by Ca2+ depletion in inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores by the treatment of Tg, but could be initiated by application of caffeine. Moreover, we demonstrate for the first time that these spontaneous [Ca2+]i oscillations were highly temperature dependent. Decreasing the temperature from 22 to 17 degrees C resulted in an increase in the frequency, a reduction in the amplitude, and large inhibition of [Ca2+]i oscillations. Furthermore, the rate of ATP-dependent 45Ca2+ uptake into GH3-derived microsomes was greatly reduced at 17 degrees C. The effect of decreased temperatures on extracellular Ca2+ influx was minor because the frequency and amplitude of spontaneous action potentials, which activate L-type Ca2+ channels, was relatively unchanged at 17 degrees C. These results suggest that in GH3 secretory cells, Ca2+ influx via L-type Ca2+ channels initiates spontaneous [Ca2+]i oscillations, which are then maintained by the combined activity of Ca(2+)-ATPase and Ca(2+)-induced Ca2+ release from Tg/IP3-insensitive intracellular stores.     

Hydrogen peroxide-induced Ca2+ mobilization in pulmonary arterial smooth muscle cells

Reactive oxygen species (ROS) generated from NADPH oxidases and mitochondria have been implicated as key messengers for pulmonary vasoconstriction and vascular remodeling induced by agonists and hypoxia. Since Ca(2+) mobilization is essential for vasoconstriction and cell proliferation, we sought to characterize the Ca(2+) response and to delineate the Ca(2+) pathways activated by hydrogen peroxide (H(2)O(2)) in rat intralobar pulmonary arterial smooth muscle cells (PASMCs). Exogenous application of 10 microM to 1 mM H(2)O(2) elicited concentration-dependent increase in intracellular Ca(2+) concentration in PASMCs, with an initial rise followed by a plateau or slow secondary increase. The initial phase was related to intracellular release. It was attenuated by the inositol trisphosphate (IP(3)) receptor antagonist 2-aminoethyl diphenylborate, ryanodine, or thapsigargin, but was unaffected by the removal of Ca(2+) in external solution. The secondary phase was dependent on extracellular Ca(2+) influx. It was unaffected by the voltage-gated Ca(2+) channel blocker nifedipine or the nonselective cation channel blockers SKF-96365 and La(3+), but inhibited concentration dependently by millimolar Ni(2+), and potentiated by the Na(+)/Ca(2+) exchange inhibitor KB-R 7943. H(2)O(2) did not alter the rate of Mn(2+) quenching of fura 2, suggesting store- and receptor-operated Ca(2+) channels were not involved. By contrast, H(2)O(2) elicited a sustained inward current carried by Na(+) at -70 mV, and the current was inhibited by Ni(2+). These results suggest that H(2)O(2) mobilizes intracellular Ca(2+) through multiple pathways, including the IP(3)- and ryanodine receptor-gated Ca(2+) stores, and Ni(2+)-sensitive cation channels. Activation of these Ca(2+) pathways may play important roles in ROS signaling in PASMCs.     

Coactivation of capacitative calcium entry and L-type calcium channels in guinea pig gallbladder

We have evaluated the presence of capacitative Ca(2+) entry (CCE) in guinea pig gallbladder smooth muscle (GBSM), including a possible relation with activation of L-type Ca(2+) channels. Changes in cytosolic Ca(2+) concentration induced by Ca(2+) entry were assessed by digital microfluorometry in isolated, fura 2-loaded GBSM cells. Application of thapsigargin, a specific inhibitor of the Ca(2+) store pump, induced a transient Ca(2+) release followed by sustained entry of extracellular Ca(2+). Depletion of the stores with thapsigargin, cyclopiazonic acid, ryanodine and caffeine, high levels of the Ca(2+)-mobilizing hormone cholecystokinin octapeptide, or simple removal of external Ca(2+) resulted in a sustained increase in Ca(2+) entry on subsequent reapplication of Ca(2+). This entry was attenuated by 2-aminoethoxydiphenylborane, L-type Ca(2+) channel blockade, pinacidil, and Gd(3+). Accumulation of the voltage-sensitive dye 3,3'-dipentylcarbocyanine and direct intracellular recordings showed that depletion of the stores is sufficient for depolarization of the plasma membrane. Contractility studies in intact gallbladder muscle strips showed that CCE induced contractions. The CCE-evoked contraction was sensitive to 2-aminoethoxydiphenylborane, L-type Ca(2+) channel blockers, and Gd(3+). We conclude that, in GBSM, release of Ca(2+) from internal stores activates a CCE pathway and depolarizes plasma membrane, allowing coactivation of voltage-operated L-type Ca(2+) channels. This process may play a role in excitation-contraction coupling in GBSM.     

Using total fluorescence increase (signal mass) to determine the Ca2+ current underlying localized Ca2+ events

The feasibility of determining localized Ca(2+) influx using only wide-field fluorescence images was explored by imaging (using fluo-3) single channel Ca(2+) fluorescence transients (SCCaFTs), due to Ca(2+) entry through single openings of Ca(2+)-permeable ion channels, while recording unitary channel currents. Since the image obtained with wide-field optics is an integration of both in-focus and out-of-focus light, the total fluorescence increase (DeltaF(total) or "signal mass") associated with a SCCaFT can be measured directly from the image by adding together the fluorescence increase due to Ca(2+) influx in all of the pixels. The assumptions necessary for obtaining the signal mass from confocal linescan images are not required. Two- and three-dimensional imaging was used to show that DeltaF(total) is essentially independent of the position of the channel with respect to the focal plane of the microscope. The relationship between Ca(2+) influx and DeltaF(total) was obtained using SCCaFTs from plasma membrane caffeine-activated cation channels when Ca(2+) was the only charge carrier of the inward current. This relationship was found to be linear, with the value of the slope (or converting factor) affected by the particular imaging system set-up, the experimental conditions, and the properties of the fluorescent indicator, including its binding capacity with respect to other cellular buffers. The converting factor was used to estimate the Ca(2+) current passing through caffeine-activated channels in near physiological saline and to estimate the endogenous buffer binding capacity. In addition, it allowed a more accurate estimate of the Ca(2+) current underlying Ca(2+) sparks resulting from Ca(2+) release from intracellular stores via ryanodine receptors in the same preparation.     

Imaging calcium entering the cytosol through a single opening of plasma membrane ion channels: SCCaFTs--fundamental calcium events

Recently, it has become possible to record the localized fluorescence transient associated with the opening of a single plasma membrane Ca(2+) permeable ion channel using Ca(2+) indicators like fluo-3. These Single Channel Ca(2+) Fluorescence Transients (SCCaFTs) share some of the characteristics of such elementary events as Ca(2+) sparks and Ca(2+) puffs caused by Ca(2+) release from intracellular stores (due to the opening of ryanodine receptors and IP(3) receptors, respectively). In contrast to intracellular Ca(2+) release events, SCCaFTs can be observed while simultaneously recording the unitary channel currents using patch-clamp techniques to verify the channel openings. Imaging SCCaFTs provides a way to examine localized Ca(2+) handling in the vicinity of a channel with a known Ca(2+) influx, to obtain the Ca(2+) current passing through plasma membrane cation channels in near physiological solutions, to localize Ca(2+) permeable ion channels on the plasma membrane, and to estimate the Ca(2+) currents underlying those elementary events where the Ca(2+) currents cannot be recorded. Here we review studies of these fluorescence transients associated with caffeine-activated channels, L-type Ca(2+) channels, and stretch-activated channels. For the L-type Ca(2+) channel, SCCaFTs have been termed sparklets. In addition, we discuss how SCCaFTs have been used to estimate Ca(2+) currents using the rate of rise of the fluorescence transient as well as the signal mass associated with the total fluorescence increase.     

Internal Ca(2+) release in yeast is triggered by hypertonic shock and mediated by a TRP channel homologue.

Calcium ions, present inside all eukaryotic cells, are important second messengers in the transduction of biological signals. In mammalian cells, the release of Ca(2+) from intracellular compartments is required for signaling and involves the regulated opening of ryanodine and inositol-1,4,5-trisphosphate (IP3) receptors. However, in budding yeast, no signaling pathway has been shown to involve Ca(2+) release from internal stores, and no homologues of ryanodine or IP3 receptors exist in the genome. Here we show that hyperosmotic shock provokes a transient increase in cytosolic Ca(2+) in vivo. Vacuolar Ca(2+), which is the major intracellular Ca(2+) store in yeast, is required for this response, whereas extracellular Ca(2+) is not. We aimed to identify the channel responsible for this regulated vacuolar Ca(2+) release. Here we report that Yvc1p, a vacuolar membrane protein with homology to transient receptor potential (TRP) channels, mediates the hyperosmolarity induced Ca(2+) release. After this release, low cytosolic Ca(2+) is restored and vacuolar Ca(2+) is replenished through the activity of Vcx1p, a Ca(2+)/H(+) exchanger. These studies reveal a novel mechanism of internal Ca(2+) release and establish a new function for TRP channels.     

Release of Ca(2+) from intracellular stores and entry of extracellular Ca(2+) are involved in sea squirt sperm activation.

A rise in intracellular free Ca(2+) concentration ([Ca(2+)](i)) is required to activate sperm of all organisms studied. Such elevation of [Ca(2+)](i) can occur either by influx of extracellular Ca(2+) or by release of Ca(2+) from intracellular stores. We have examined these sources of Ca(2+) in sperm from the sea squirt Ascidia ceratodes using mitochondrial translocation to evaluate activation and the Ca(2+)-sensitive dye fura-2 to monitor [Ca(2+)](i) by bulk spectrofluorometry. Sperm activation artificially evoked by incubation in high-pH seawater was inhibited by reducing seawater [Ca(2+)], as well as by the presence of high [K(+)](o) or the Ca channel blockers pimozide, penfluridol, or Ni(2+), but not nifedipine or Co(2+). The accompanying rise in [Ca(2+)](i) was also blocked by pimozide or penfluridol. These results indicate that activation produced by alkaline incubation involves opening of plasmalemmal voltage-dependent Ca channels and Ca(2+) entry to initiate mitochondrial translocation. Incubation in thimerosal or thapsigargin, but not ryanodine (even if combined with caffeine pretreatment), evoked sperm activation. Activation by thimerosal was insensitive to reduced external calcium and to Ca channel blockers. Sperm [Ca(2+)](i) increased upon incubation in high-pH or thimerosal-containing seawater, but only the high-pH-dependent elevation in [Ca(2+)](i) could be inhibited by pimozide or penfluridol. Treatment with the protonophore CCCP indicated that only a small percentage of sperm could release enough Ca(2+) from mitochondria to cause activation. Inositol 1,4,5-trisphosphate (IP(3)) delivered by liposomes or by permeabilization increased sperm activation. Both of these effects were blocked by heparin. We conclude that high external pH induces intracellular alkalization that directly or indirectly activates plasma membrane voltage-dependent Ca channels allowing entry of external Ca(2+) and that thimerosal stimulates release of Ca(2+) from IP(3)-sensitive intracellular stores.     

Calcium-induced calcium release in neurosecretory insect neurons: fast and slow responses

The dynamics of intracellular free Ca(2+)([Ca(2+)](i)) changes were investigated in dorsal unpaired median (DUM) neurons of the cockroach Periplaneta americana. Activation of voltage-gated Ca(2+) channels caused a steep increase in [Ca(2+)](i). Depolarizations lasting for < 100ms led to Ca(2+) release from intracellular stores as is indicated by the finding that the rise of [Ca(2+)](i) was greatly reduced by the antagonists of ryanodine receptors, ryanodine and ruthenium red. There is a resting Ca(2+)current which is potentiated on application of a neuropeptide, Neurohormone D (NHD), a member of the adipokinetic hormone family. Ca(2+) influx enhanced in this way again caused a rise of [Ca(2+)](i) sensitive to ryanodine and ruthenium red. Such rises developed and relaxed much more slowly than the depolarization-induced signals. Ca(2+)responses similar to those induced by NHD were obtained with the ryanodine receptor agonists caffeine (20mM) and cADP-ribose (cADPR, 100nM). These Ca(2+) responses, however, varied considerably in size and kinetics, and part of the cells did not respond at all to caffeine or cADPR. Such cells, however, produced Ca(2+) rises after having been treated with NHD. Thus, the variability of Ca(2+) signals might be caused by different filling states of Ca(2+) stores, and the resting Ca(2+) current seems to represent a source to fill empty Ca(2+) stores. In line with this notion, block of the endoplasmic Ca(2+) pump by thapsigargin (1 microM) produced either no or largely varying Ca(2+) responses. The Ca(2+) signals induced by caffeine and cADPR displayed different sensitivity to ryanodine receptor blockers. cADPR failed to elicit any response when ryanodine or ruthenium red were present. By contrast, the response to caffeine, in the presence of ryanodine, was only reduced by about 50% and, in the presence of ruthenium red, it was not at all reduced. Thus, there may be different types of Ca(2+) release channels. Block of mitochondrial Ca(2+) uptake with carbonyl cyanide m -chlorophenylhydrazone (CCCP, 1 microM) completely abolished cADPR-induced Ca(2+) signals, but it did not affect the caffeine-induced signals. Taken together our findings seem to indicate that there are different stores using different Ca(2+) uptake pathways and that some of these pathways involve mitochondria.     

Role of intracellular Ca(2+) release in histamine-induced depolarization in rabbit middle cerebral artery

The role of Ca(2+) mobilization from intracellular stores and Ca(2+)-activated Cl(-) channels in caffeine- and histamine-induced depolarization and contraction of the rabbit middle cerebral artery has been studied by recording membrane potential and isometric force. Caffeine induced a transient contraction and a transient followed by sustained depolarization. The transient depolarization was abolished by ryanodine, DIDS, and niflumic acid, suggesting involvement of Ca(2+)-activated Cl(-) channels. Histamine-evoked transient contraction in Ca(2+)-free solution was abolished by ryanodine or by caffeine-induced depletion of Ca(2+) stores. Ryanodine slowed the development of depolarization induced by histamine in Ca(2+)-containing solution but did not affect its magnitude. In arteries treated with 1 mM Co(2+), histamine elicited a transient depolarization and contraction, which was abolished by ryanodine. DIDS and niflumic acid reduced histamine-evoked depolarization and contraction. Histamine caused a sustained depolarization and contraction in low-Cl(-) solution. These results suggest that Ca(2+) mobilization from ryanodine-sensitive stores is involved in histamine-induced initial, but not sustained, depolarization and contraction. Ca(2+)-activated Cl(-) channels contribute mainly to histamine-induced initial depolarization and less importantly to sustained depolarization, which is most likely dependent on activation of nonselective cation channels.     

Calcium release in HSY cells conforms to a steady-state mechanism involving regulation of the inositol 1,4,5-trisphosphate receptor Ca2+ channel by luminal [Ca2+]

In many cell types, low concentrations of inositol 1,4,5-trisphosphate (IP3) release only a portion of the intracellular IP3-sensitive Ca2+ store, a phenomenon known as "quantal" Ca2+ release. It has been suggested that this effect is a result of reduced activity of the IP3- dependent Ca2+ channel with decreasing calcium concentration within the IP3-sensitive store ([Ca2+]s). To test this hypothesis, the properties of IP3-dependent Ca2+ release in single saponin-permeabilized HSY cells were studied by monitoring [Ca2+]s using the Ca(2+)-sensitive fluorescent dye mag-fura-2. In permeabilized cells, blockade of the sarco/ER Ca(2+)-ATPase pump in stores partially depleted by IP3 induced further Ca2+ release via an IP3-dependent route, indicating that Ca2+ entry via the sarco/ER Ca(2+)-ATPase pump had been balanced by Ca2+ loss via the IP3-sensitive channel before pump inhibition. IP3- dependent Mn2+ entry, monitored via quenching of luminal mag-fura-2 fluorescence, was readily apparent in filled stores but undetectable in Ca(2+)-depleted stores, indicating markedly reduced IP3-sensitive channel activity in the latter. Also consistent with reduced responsiveness of Ca(2+)-depleted stores to IP3, the initial rate of refilling of these stores was unaffected by the presence of 0.3 microM IP3, a concentration that was clearly effective in eliciting Ca2+ release from filled stores. Analysis of the rate of Ca2+ release at various IP3 concentrations indicated a significant shift of the IP3 dose response toward higher [IP3] with decreasing [Ca2+]s. We conclude that IP3-dependent Ca2+ release in HSY cells is a steady-state process wherein Ca2+ efflux via the IP3 receptor Ca2+ channel is regulated by [Ca2+]s, apparently via changes in the sensitivity of the channel to IP3.     

Arachidonic acid is a physiological activator of the ryanodine receptor in pancreatic beta-cells

Pancreatic beta-cells have ryanodine receptors but little is known about their physiological regulation. Previous studies have shown that arachidonic acid releases Ca(2+) from intracellular stores in beta-cells but the identity of the channels involved in the Ca(2+) release has not been elucidated. We studied the mechanism by which arachidonic acid induces Ca(2+) concentration changes in pancreatic beta-cells. Cytosolic free Ca(2+) concentration was measured in fura-2-loaded INS-1E cells and in primary beta-cells from Wistar rats. The increase of cytosolic Ca(2+) concentration induced by arachidonic acid (150microM) was due to both Ca(2+) release from intracellular stores and influx of Ca(2+) from extracellular medium. 5,8,11,14-Eicosatetraynoic acid, a non-metabolizable analogue of arachidonic acid, mimicked the effect of arachidonic acid, indicating that arachidonic acid itself mediated Ca(2+) increase. The Ca(2+) release induced by arachidonic acid was from the endoplasmic reticulum since it was blocked by thapsigargin. 2-Aminoethyl diphenylborinate (50microM), which is known to inhibit 1,4,5-inositol-triphosphate-receptors, did not block Ca(2+) release by arachidonic acid. However, ryanodine (100microM), a blocker of ryanodine receptors, abolished the effect of arachidonic acid on Ca(2+) release in both types of cells. These observations indicate that arachidonic acid is a physiological activator of ryanodine receptors in beta-cells.     

Depolarization-induced Ca2+ release in ischemic spinal cord white matter involves L-type Ca2+ channel activation of ryanodine receptors

The mechanisms of Ca(2+) release from intracellular stores in CNS white matter remain undefined. In rat dorsal columns, electrophysiological recordings showed that in vitro ischemia caused severe injury, which persisted after removal of extracellular Ca(2+); Ca(2+) imaging confirmed that an axoplasmic Ca(2+) rise persisted in Ca(2+)-free perfusate. However, depletion of Ca(2+) stores or reduction of ischemic depolarization (low Na(+), TTX) were protective, but only in Ca(2+)-free bath. Ryanodine or blockers of L-type Ca(2+) channel voltage sensors (nimodipine, diltiazem, but not Cd(2+)) were also protective in zero Ca(2+), but their effects were not additive with ryanodine. Immunoprecipitation revealed an association between L-type Ca(2+) channels and RyRs, and immunohistochemistry confirmed colocalization of Ca(2+) channels and RyR clusters on axons. Similar to "excitation-contraction coupling" in skeletal muscle, these results indicate a functional coupling whereby depolarization sensed by L-type Ca(2+) channels activates RyRs, thus releasing damaging amounts of Ca(2+) under pathological conditions in white matter.