Properties of the caffeine-sensitive intracellular calcium stores in mammalian neurons

Using indo-1- and fura-2-based microfluorometry for measuring the cytoplasmic free calcium concentration ([Ca²⁺] _in ), the properties of caffeine-induced Ca²⁺ release from internal stores were studied in rat cultured central and peripheral neurons, including dorsal root ganglion (DRG) neurons, neurons from then. cuneatus, CA1 and CA3 hippocampal regions, and pyramidal neocortical neurons. Under resting conditions, the Ca²⁺ content of internal stores in DRG neurons was high enough to produce caffeine-triggered [Ca²⁺] _in transients. Prolonged exposure of caffeine depleted the caffeine-sensitive stores of releasable Ca²⁺; the degree of this depletion depended on caffeine concentration. The depletion of the caffeine-sensitive internal stores to some extent was linked to calcium extrusion via La³⁺-sensitive plasmalemmal Ca²⁺-ATPases. Caffeine-induced Ca²⁺ release deprived internal stores in DRG neurons, but they refilled themselves spontaneously within 10 min. Pharmacological manipulation with caffeine-sensitive stores interferred with the depolarization-induced [Ca²⁺] _in transients. In the presence of low caffeine concentration (0.5–1.0 mM) in the extracellular solution, the rate of rise of the depolarization-triggered [Ca²⁺] _in transients significantly increased (by a factor of 2.15 ± 0.29) suggesting the occurrence of Ca²⁺-induced Ca²⁺ release. When the caffeine-sensitive stores were emptied by prolonged application of caffeine, the amplitude and rate of rise of the depolarization-induced [Ca²⁺] _in transients decreased. These findings suggest the involvement of internal caffeine-sensitive calcium stores in generation of calcium signal in sensory neurons. In contrast, in all types of central neurons tested the resting Ca²⁺ content of internal stores was low, but the stores could be charged by transmembrane Ca²⁺ entry through voltage-operated calcium channels. After charging, the stores in central neurons spontaneously lost releasable calcium content and within 10 min they became completely empty again. We suggest that internal Ca²⁺ stores in peripheral and central neurons, although having similar pharmacological characteristics, handle Ca²⁺ ions in a different manner. Calcium stores in sensory neurons are continuously filled by releasable calcium and after discharging they can be spontaneously refilled, whereas in central neurons internal calcium stores can be charged by releasable calcium only transiently. Caffeine-evoked [Ca²⁺] _in transients in all types of neurons were effectively blocked by 10 mM ryanodine, 5 mM procaine, 10 mM dantrolene, or 0.5 mM Ba²⁺, thus sharing the basic properties of the Ca²⁺-induced Ca²⁺ release from endoplasmic reticulum.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 16–25, January–February, 1994.     

Altered Ca2+ signaling in skeletal muscle fibers of the R6/2 mouse,a model of Huntington’s disease

Huntington’s disease (HD) is caused by an expanded CAG trinucleotide repeat within the gene encoding the protein huntingtin. The resulting elongated glutamine (poly-Q) sequence of mutant huntingtin (mhtt) affects both central neurons and skeletal muscle. Recent reports suggest that ryanodine receptor–based Ca²⁺ signaling, which is crucial for skeletal muscle excitation–contraction coupling (ECC), is changed by mhtt in HD neurons. Consequently, we searched for alterations of ECC in muscle fibers of the R6/2 mouse, a mouse model of HD. We performed fluorometric recordings of action potentials (APs) and cellular Ca²⁺ transients on intact isolated toe muscle fibers (musculi interossei), and measured L-type Ca²⁺ inward currents on internally dialyzed fibers under voltage-clamp conditions. Both APs and AP-triggered Ca²⁺ transients showed slower kinetics in R6/2 fibers than in fibers from wild-type mice. Ca²⁺ removal from the myoplasm and Ca²⁺ release flux from the sarcoplasmic reticulum were characterized using a Ca²⁺ binding and transport model, which indicated a significant reduction in slow Ca²⁺ removal activity and Ca²⁺ release flux both after APs and under voltage-clamp conditions. In addition, the voltage-clamp experiments showed a highly significant decrease in L-type Ca²⁺ channel conductance. These results indicate profound changes of Ca²⁺ turnover in skeletal muscle of R6/2 mice and suggest that these changes may be associated with muscle pathology in HD.     

Calcium signalling in cortical and thalamic neurons of rats with streptozotocin-induced diabetes

Intracellular Ca²⁺ transients were measured with the use of a Ca²⁺-sensitive fluorescent indicator, fura-2, in neocortical and thalamic neurons in brain slices from control rats and rats with uncompensated streptozotocin-induced diabetes. The transients were evoked by high-potassium (50 mM)-induced membrane depolarization. The amplitude of depolarization-induced Ca²⁺ transients demonstrated a tendency to increase under diabetic conditions, beeing more expressed in cortical neurons compared with thalamic ones. The transients in cortical neurons from diabetic animals became also more susceptible to the blocking action of nifedipine (100μM) and less sensitive to Ni²⁺ (50μM), indicating that diabetic changes affect mostly Ca²⁺ transients triggered by high-voltage activated (L-type) calcium channels. The duration of a statistically significant increase was observed in the residual elevation of intracellular Ca²⁺ changes. However, a statistically significant increase was observed in the residual elevation of intracellular Ca²⁺ measured 60 sec after termination of membrane depolarization in both cortical and thalamic neurons, indicating alterations in the mechanisms that restore the resting level of Ca²⁺ in the cytosol. It is concluded that uncomensated insulin-dependent diabetes, which according to earlier data substantially alters calcium signalling in primary sensory neurons, also affects such signalling in the neurons of higher brain structures including the thalamus and cortex.     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

RyR2 Modulates a Ca2+-Activated K+ Current in Mouse Cardiac Myocytes

In cardiomyocytes, Ca²⁺ entry through voltage-dependent Ca²⁺ channels (VDCCs) binds to and activates RyR2 channels, resulting in subsequent Ca²⁺ release from the sarcoplasmic reticulum (SR) and cardiac contraction. Previous research has documented the molecular coupling of small-conductance Ca²⁺-activated K⁺ channels (SK channels) to VDCCs in mouse cardiac muscle. Little is known regarding the role of RyRs-sensitive Ca²⁺ release in the SK channels in cardiac muscle. In this study, using whole-cell patch clamp techniques, we observed that a Ca²⁺-activated K+ current (I_K,Ca) recorded from isolated adult C57B/L mouse atrial myocytes was significantly decreased by ryanodine, an inhibitor of ryanodine receptor type 2 (RyR2), or by the co-application of ryanodine and thapsigargin, an inhibitor of the sarcoplasmic reticulum calcium ATPase (SERCA) (p<0.05, p<0.01, respectively). The activation of RyR2 by caffeine increased the I_K,Ca in the cardiac cells (p<0.05, p<0.01, respectively). We further analyzed the effect of RyR2 knockdown on I_K,Ca and Ca²⁺ in isolated adult mouse cardiomyocytes using a whole-cell patch clamp technique and confocal imaging. RyR2 knockdown in mouse atrial cells transduced with lentivirus-mediated small hairpin interference RNA (shRNA) exhibited a significant decrease in I_K,Ca (p<0.05) and [Ca²⁺]i fluorescence intensity (p<0.01). An immunoprecipitated complex of SK2 and RyR2 was identified in native cardiac tissue by co-immunoprecipitation assays. Our findings indicate that RyR2-mediated Ca²⁺ release is responsible for the activation and modulation of SK channels in cardiac myocytes.     

Changes produced in intracellular calcium concentration and transmembrane currents by iontophoretic injection of cAMP in Helix pomatia neurons

Transmembrane currents and changed [Ca²⁺]_in produced by iontophoretic injection of cAMP were investigated in voltage clampedHelix pomatia neurons. The Fura-2 fluorescence probe technique was used to measure [Ca²⁺]_in. Injection of cAMP was found to produce a protracted rise in the latter at a membrane potential range of –40 to –100 mV in conjunction with transmembrane inward current. Duration of the changes in [Ca²⁺]_in largely dependent on neuronal size and varied between 50 and 500 sec (parameters for neurons with somata of around 100 and 40 µm respectively). In a medium with Ca²⁺ replaced by Mg²⁺ (as well as after addition of EDTA, a calcium chelator) both transmembrane current and the pattern of increase in [Ca²⁺]_in remained unchanged. Inward current usually declined substantially but degree of change in [Ca²⁺]_in remained the same when Na⁺ was eliminated from the solution by replacing its Tris⁺. Addition of 2 mM Cd²⁺ to the external medium hardly affected current level and increase in [Ca²⁺]_in. Neither procaine, a local anesthetic, nor ryanodine (which inhibits release of calcium from the intracellular store) changed the cAMP effects observed. A concentration of 1 mM La³⁺ depressed both inward current and the [Ca²⁺]_in increase. Findings would imply the occurrence of cAMP-dependent release of calcium from the intracellular store in the neurons tested.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 3, pp. 396–402, May–June, 1989.     

A Calcium Signaling Cascade Essential for Myosin Thick Filament Assembly in Xenopus Myocytes

Spontaneous calcium release from intracellular stores occurs during myofibrillogenesis, the process of sarcomeric protein assembly in striated muscle. Preventing these Ca²⁺ transients disrupts sarcomere formation, but the signal transduction cascade has not been identified. Here we report that specific blockade of Ca²⁺ release from the ryanodine receptor (RyR) activated Ca²⁺ store blocks transients and disrupts myosin thick filament (A band) assembly. Inhibition of an embryonic Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) by blocking the ATP-binding site, by allosteric phosphorylation, or by intracellular delivery of a pseudosubstrate peptide, also disrupts sarcomeric organization. The results indicate that both RyRs and MLCK, which have well-described calcium signaling roles in mature muscle contraction, have essential developmental roles during construction of the contractile apparatus.     

Similar Ca dependences of [H]ryanodine binding to α- and β-ryanodine receptors purified from bullfrog skeletal muscle in an isotonic medium

To understand the functions of the two ryanodine receptor isoforms (α- and β-RyRs) in nonmammalian skeletal muscles, we determined [³H]ryanodine binding to these isoforms purified from bullfrog skeletal muscle. In 0.17 M-NaCl medium, both isoforms demonstrated similar Ca²⁺ dependent ryanodine binding activities, while the Ca²⁺ sensitivity for activation of β-RyR was increased in 1 M-NaCl medium. This enhancement in Ca²⁺ sensitivity depended on the kind of salts used. These results imply that α- and β-RyRs may have similar properties as Ca²⁺-induced Ca²⁺ release channels in bullfrog skeletal muscle.     

Voltage-Induced Ca2+ Release in Postganglionic Sympathetic Neurons in Adult Mice

Recent studies have provided evidence that depolarization in the absence of extracellular Ca²⁺ can trigger Ca²⁺ release from internal stores in a variety of neuron subtypes. Here we examine whether postganglionic sympathetic neurons are able to mobilize Ca²⁺ from intracellular stores in response to depolarization, independent of Ca²⁺ influx. We measured changes in cytosolic ΔF/F₀ in individual fluo-4 –loaded sympathetic ganglion neurons in response to maintained K⁺ depolarization in the presence (2 mM) and absence of extracellular Ca²⁺ ([Ca²⁺]_e). Progressive elevations in extracellular [K⁺]_e caused increasing membrane depolarizations that were of similar magnitude in 0 and 2 mM [Ca²⁺]_e. Peak amplitude of ΔF/F₀ transients in 2 mM [Ca²⁺]_e increased in a linear fashion as the membrane become more depolarized. Peak elevations of ΔF/F₀ in 0 mM [Ca²⁺]_e were ~5–10% of those evoked at the same membrane potential in 2 mM [Ca²⁺]_e and exhibited an inverse U-shaped dependence on voltage. Both the rise and decay of ΔF/F₀ transients in 0 mM [Ca²⁺]_e were slower than those of ΔF/F₀ transients evoked in 2 mM [Ca²⁺]_e. Rises in ΔF/F₀ evoked by high [K⁺]_e in the absence of extracellular Ca²⁺ were blocked by thapsigargin, an inhibitor of endoplasmic reticulum Ca²⁺ ATPase, or the inositol 1,4,5-triphosphate (IP₃) receptor antagonists 2-aminoethoxydiphenyl borate and xestospongin C, but not by extracellular Cd²⁺, the dihydropyridine antagonist nifedipine, or by ryanodine at concentrations that caused depletion of ryanodine-sensitive Ca²⁺ stores. These results support the notion that postganglionic sympathetic neurons possess the ability to release Ca²⁺ from IP₃-sensitive internal stores in response to membrane depolarization, independent of Ca²⁺ influx.     

Differential Activation of Cultured Neonatal Cardiomyocytes by Plasmalemmal Versus Intracellular G Protein-coupled Receptor 55

The L-α-lysophosphatidylinositol (LPI)-sensitive receptor GPR55 is coupled to Ca²⁺ signaling. Low levels of GPR55 expression in the heart have been reported. Similar to other G protein-coupled receptors involved in cardiac function, GPR55 may be expressed both at the sarcolemma and intracellularly. Thus, to explore the role of GPR55 in cardiomyocytes, we used calcium and voltage imaging and extracellular administration or intracellular microinjection of GPR55 ligands. We provide the first evidence that, in cultured neonatal ventricular myocytes, LPI triggers distinct signaling pathways via GPR55, depending on receptor localization. GPR55 activation at the sarcolemma elicits, on one hand, Ca²⁺ entry via L-type Ca²⁺ channels and, on the other, inositol 1,4,5-trisphosphate-dependent Ca²⁺ release. The latter signal is further amplified by Ca²⁺-induced Ca²⁺ release via ryanodine receptors. Conversely, activation of GPR55 at the membrane of intracellular organelles promotes Ca²⁺ release from acidic-like Ca²⁺ stores via the endolysosomal NAADP-sensitive two-pore channels. This response is similarly enhanced by Ca²⁺-induced Ca²⁺ release via ryanodine receptors. Extracellularly applied LPI produces Ca²⁺-independent membrane depolarization, whereas the Ca²⁺ signal induced by intracellular microinjection of LPI converges to hyperpolarization of the sarcolemma. Collectively, our findings point to GPR55 as a novel G protein-coupled receptor regulating cardiac function at two cellular sites. This work may serve as a platform for future studies exploring the potential of GPR55 as a therapeutic target in cardiac disorders.     

Characterisation of the Role of Calcium in AlF4-Induced Long-Term Potentiation in Area CA1 of Rat Hippocampus

We investigated calcium influx in the long lasting potentiation induced in area CA1 of rat hippocampus by brief bath application of the G-protein activator AlF₄ ^–(NaF/AlCl₃). Brief (10 min) bath application of AlF₄ ^– in standard saline (with 2mM Ca²⁺) consistently induced a long lasting potentiation which was not observed if AlF₄ ^– was bath-applied in nominally calcium free saline. Increasing the potential calcium influx, either by raising extracellular calcium concentration to 3.5 mM or by addition of the voltage operated calcium channel (VOCC) agonist BayK8644. failed to increase the number of slices exhibiting potentiation or the mean level of potentiation. Bath application of AlF₄ ^– in the presence of the VOCC antagonist failed to block the potentiation and AlF₄ ^– readily induced a long lasting potentiation under voltage clamp conditions, strongly suggesting that the calcium influx required for AlF₄-induced potentiation is not through NMDA receptors or VOCC channels. It is suggested that the calcium required may be provided by an ongoing recharging and emptying of IP3 sensitive intracellular Ca²⁺ stores.     

Taurine prevents intracellular calcium overload during calcium paradox of cultured cardiomyocytes

Summary The effect of taurine on the cellular distribution of [Ca²⁺]_i, during the calcium paradox was examined by digital imaging of a single fura-2-loaded cell. Cardiomyocytes superfused with control medium containing 2mM Ca²⁺ exhibited typical transients associated with spontaneous beating. When the cells were exposed to Ca²⁺-free buffer, immediate cessation of both spontaneous contractions and calcium transients was observed as [Ca²⁺]; rapidly fell to a level of 3–6 × 10^–8M. Subsequent restoration of medium calcium increased [Ca²⁺]_i to level 4–7 times normal. Large increases in [Ca²⁺]_i were observed in most cells and were associated with the development of contracture and bleb formation.Taurine pretreatment (20mM) caused no significant effect on [Ca²⁺]_i during Ca²⁺ depletion. However, it inhibited excessive accumulation of [Ca²⁺]_i during the Ca²⁺ repletion. Moreover, taurine treated cells recovered their Ca²⁺-transients and beating pattern earlier than non-treated cells. Finally morphological abnormalities commonly associated with calcium overload were attenuated by taurine treatment.     

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-mediated Calcium Signaling and Arrhythmias in the Heart Evoked by β-Adrenergic Stimulation

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca²⁺-releasing second messenger known to date. Here, we report a new role for NAADP in arrhythmogenic Ca²⁺ release in cardiac myocytes evoked by β-adrenergic stimulation. Infusion of NAADP into intact cardiac myocytes induced global Ca²⁺ signals sensitive to inhibitors of both acidic Ca²⁺ stores and ryanodine receptors and to NAADP antagonist BZ194. Furthermore, in electrically paced cardiac myocytes BZ194 blocked spontaneous diastolic Ca²⁺ transients caused by high concentrations of the β-adrenergic agonist isoproterenol. Ca²⁺ transients were recorded both as increases of the free cytosolic Ca²⁺ concentration and as decreases of the sarcoplasmic luminal Ca²⁺ concentration. Importantly, NAADP antagonist BZ194 largely ameliorated isoproterenol-induced arrhythmias in awake mice. We provide strong evidence that NAADP-mediated modulation of couplon activity plays a role for triggering spontaneous diastolic Ca²⁺ transients in isolated cardiac myocytes and arrhythmias in the intact animal. Thus, NAADP signaling appears an attractive novel target for antiarrhythmic therapy.     

Cytoplasmic Ca2+ does not inhibit the cardiac muscle sarcoplasmic reticulum ryanodine receptor Ca2+ channel,although Ca2+-induced Ca2+ inactivation of Ca2+ release is observed in native vesicles

Single channel properties of cardiac and fast-twitch skeletal muscle sarcoplasmic reticulum (SR) release channels were compared in a planar bilayer by fusing SR membranes in a Cs⁺-conducting medium. We found that the pharmacology, Cs⁺ conductance and selectivity to monovalent and divalent cations of the two channels were similar. The cardiac SR channel exhibited multiple kinetic states. The open and closed lifetimes were not altered from a range of 10^–7 to 10^–3 M Ca²⁺, but the proportion of closed and open states shifted to shorter closings and openings, respectively.However, while the single channel activity of the skeletal SR channel was activated and inactivated by micromolar and millimolar Ca²⁺, respectively, the cardiac SR channel remained activated in the presence of high [Ca²⁺]. In correlation to these studies, [³H]ryanodine binding by the receptors of the two channel receptors was inhibited by high [Ca²⁺] in skeletal but not in cardiac membranes in the presence of adenine nucleotides. There is, however, a minor inhibition of [³H]ryanodine binding of cardiac SR at millimolar Ca²⁺ in the absence of adenine nucleotides.When Ca²⁺-induced Ca²⁺ release was examined from preloaded native SR vesicles, the release rates followed a normal biphasic curve, with Ca²⁺-induced inactivation at high [Ca²⁺] for both cardiac and skeletal SR. Our data suggest that the molecular basis of regulation of the SR Ca²⁺ release channel in cardiac and skeletal muscle is different, and that the cardiac SR channel isoform lacks a Ca²⁺-inactivated site.This work was supported by research grants from the National Institutes of Health HL13870 and AR38970, and the Texas Affiliate of the American Heart Association, 91A-188. M. Fill was the recipient of an NIH fellowship AR01834.     

Calcium Signaling in <Emphasis Type="Italic">Carassius</Emphasis> Cerebellar Neurons: Role of the Mitochondria

We studied the involvement of the mitochondria playing the role of a calcium store in the control of calcium exchange in cerebellar neurons of a fish species tolerant to hypoxia, crucian (Carassius gibelio). In our experiments we used an ionophore, CCCP, that blocked accumulation of calcium by the above organelles. The intracellular concentration of free Ca²⁺ ([Ca²⁺] _і ) was measured using a calcium-sensitive dye, Fura-2AM, and the microfluorescent technique. We found that cerebellar neurons of Carassius gibelio possess a well-expressed system clearing the cytoplasm from excessive Ca²⁺, and the mitochondria are actively involved in this process. Under conditions of suppression of the process of accumulation of calcium by the mitochondria under the action of CCCP, the amplitude of calcium transients increased by about 50%. In addition, the decay phase of depolarization-induced intracellular calcium transients was slowed down considerably. Therefore, our experiments are indicative of the significant role of the mitochondria in the control of calcium dynamics in cerebellar neurons of Carassius gibelio in the course of functional activity of these cells.     

Ca2+-induced Ca2+ Release Phenomena in Mammalian Sympathetic Neurons Are Critically Dependent on the Rate of Rise of Trigger Ca2+

The role of ryanodine-sensitive intracellular Ca²⁺ stores present in nonmuscular cells is not yet completely understood. Here we examine the physiological parameters determining the dynamics of caffeine-induced Ca²⁺ release in individual fura-2–loaded sympathetic neurons. Two ryanodine-sensitive release components were distinguished: an early, transient release (TR) and a delayed, persistent release (PR). The TR component shows refractoriness, depends on the filling status of the store, and requires caffeine concentrations ≥10 mM. Furthermore, it is selectively suppressed by tetracaine and intracellular BAPTA, which interfere with Ca²⁺-mediated feedback loops, suggesting that it constitutes a Ca²⁺-induced Ca²⁺-release phenomenon. The dynamics of release is markedly affected when Sr²⁺ substitutes for Ca²⁺, indicating that Sr²⁺ release may operate with lower feedback gain than Ca²⁺ release. Our data indicate that when the initial release occurs at an adequately fast rate, Ca²⁺ triggers further release, producing a regenerative response, which is interrupted by depletion of releasable Ca²⁺ and Ca²⁺-dependent inactivation. A compartmentalized linear diffusion model can reproduce caffeine responses: When the Ca²⁺ reservoir is full, the rapid initial Ca²⁺ rise determines a faster occupation of the ryanodine receptor Ca²⁺ activation site giving rise to a regenerative release. With the store only partially loaded, the slower initial Ca²⁺ rise allows the inactivating site of the release channel to become occupied nearly as quickly as the activating site, thereby suppressing the initial fast release. The PR component is less dependent on the store''s Ca²⁺ content. This study suggests that transmembrane Ca²⁺ influx in rat sympathetic neurons does not evoke widespread amplification by CICR because of its inability to raise [Ca²⁺] near the Ca²⁺ release channels sufficiently fast to overcome their Ca²⁺-dependent inactivation. Conversely, caffeine-induced Ca²⁺ release can undergo considerable amplification especially when Ca²⁺ stores are full. We propose that the primary function of ryanodine-sensitive stores in neurons and perhaps in other nonmuscular cells, is to emphasize subcellular Ca²⁺ gradients resulting from agonist-induced intracellular release. The amplification gain is dependent both on the agonist concentration and on the filling status of intracellular Ca²⁺ stores.     

Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas

Summary We have previously shown that inositol-1,4,5-trisphosphate (IP₃) releases Ca²⁺ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983,Nature (London) 306:67–69). This observation suggests that IP₃ might provide the missing link between activation of the muscarinic receptor and Ca²⁺ release from intracellular stores during stimulation. In order to localize the intracellular IP₃-sensitive calcium pool, IP₃-induced Ca²⁺ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl₂. IP₃-induced Ca²⁺ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000×g, sucrose-density centrifugation, or MgCl₂ precipitation there was a close correlation of IP₃-induced Ca²⁺ release with the endoplasmic reticulum markers ribonucleic acid (r=0.96, 1.00, 0.91, respectively) and NADPH cytochromec reductase (r=0.63, 0.98, 090, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochromec oxidase (r=–0.64) and glutamate dehydrogenase (r=–0.75) and with the plasma membrane markers (Na⁺+K⁺)-ATPase (r=–0.81) and alkaline phosphatase (r=–0.77) in all fractions analyzed. IP₃-induced Ca²⁺ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca²⁺ from endoplasmic reticulum in pancreatic acinar cells.     

Sensory neurons derived from diabetic rats have diminished internal Ca2+ stores linked to impaired re-uptake by the endoplasmic reticulum

Distal symmetrical sensory neuropathy in diabetes involves the dying back of axons, and the pathology equates with axonal dystrophy generated under conditions of aberrant Ca²⁺ signalling. Previous work has described abnormalities in Ca²⁺ homoeostasis in sensory and dorsal horn neurons acutely isolated from diabetic rodents. We extended this work by testing the hypothesis that sensory neurons exposed to long-term Type 1 diabetes in vivo would exhibit abnormal axonal Ca²⁺ homoeostasis and focused on the role of SERCA (sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase). DRG (dorsal root ganglia) sensory neurons from age-matched normal and 3–5-month-old STZ (streptozotocin)-diabetic rats (an experimental model of Type 1 diabetes) were cultured. At 1–2 days in vitro an array of parameters were measured to investigate Ca²⁺ homoeostasis including (i) axonal levels of intracellular Ca²⁺, (ii) Ca²⁺ uptake by the ER (endoplasmic reticulum), (iii) assessment of Ca²⁺ signalling following a long-term thapsigargin-induced blockade of SERCA and (iv) determination of expression of ER mass and stress markers using immunocytochemistry and Western blotting. KCl- and caffeine-induced Ca²⁺ transients in axons were 2-fold lower in cultures of diabetic neurons compared with normal neurons indicative of reduced ER calcium loading. The rate of uptake of Ca²⁺ into the ER was reduced by 2-fold (P<0.05) in diabetic neurons, while markers for ER mass and ER stress were unchanged. Abnormalities in Ca²⁺ homoeostasis in diabetic neurons could be mimicked via long-term inhibition of SERCA in normal neurons. In summary, axons of neurons from diabetic rats exhibited aberrant Ca²⁺ homoeo<1?show=[fo]?>stasis possibly triggered by sub-optimal SERCA activity that could contribute to the distal axonopathy observed in diabetes.     

Effect of insulin and nimodipin on calcium signals in murine primary afferent neurons with experimental diabetes

The effect of insulin injections and long-term administration of the calcium channel blocker nimodipin on depolarization-induced calcium signals was studied in neurons isolated from the dorsal root ganglia of mice with streptozotocin-induced diabetes. Induction of diabetes in mice was followed by slowing of the calcium signal decay kinetics in small neurons (mainly related to the transmission of nociceptive signals). Subcutaneous insulin injections (1 U/kg) tended to normalize the parameters of calcium signals modified by diabetes. Preliminary 3-week-long peroral administration of nimodipin (40 mg/kg) increased the peak amplitude of depolarization-induced calcium signals in isolated neurons and caused spontaneous activity usually absent in the cells under control conditions. Kinetics of calcium transients in this case remained slow. It was concluded that hyperglycemia and related impairments of the surplus Ca²⁺ extrusion mechanisms play an essential role in genesis of the changes in calcium signals caused by streptozotocin-induced diabetes.Neirofiziologiya/Neurophysiology, Vol. 27, No. 5/6, pp. 342–349, September–December, 1995.     

Extracellular ATP-induced nuclear Ca transient is mediated by inositol 1,4,5-trisphosphate receptors in mouse pancreatic β-cells

Extracellular ATP (eATP) induces an intracellular Ca²⁺ transient by activating phospholipase C (PLC)-associated P2X4 purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent Ca²⁺ release from intracellular stores in mouse pancreatic β-cells. Using laser scanning confocal microscopy, Ca²⁺ indicator fluo-4 AM, and the cell permeable nuclear indicator Hoechst 33342, we examined the properties of eATP-induced Ca²⁺ release in pancreatic β-cell nuclei. eATP induced a higher nuclear Ca²⁺ transient in pancreatic β-cell nuclei than in the cytosol. After pretreatment with thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) pumps, the amplitude of eATP-induced Ca²⁺ transients in the nucleus was still much higher than those in the cytosol. This effect of eATP was not altered by inhibition of either the plasma membrane Ca²⁺-ATPase (PMCA) or the plasma membrane Na⁺/Ca²⁺ exchanger (NCX) by LaCl₃ or by replacement of Na⁺ with N-Methyl-Glucosamine. eATP-induced nuclear Ca²⁺ transients were abolished by a cell-permeable IP3R inhibitor, 2-aminoethoxydiphenyl borate (2-APB), but were not blocked by the ryanodine receptor (RyR) antagonist ryanodine. Immunofluorescence studies showed that IP3Rs are expressed on the nuclear envelope of pancreatic β-cells. These results indicate that eATP triggers nuclear Ca²⁺ transients by mobilizing a nuclear Ca²⁺ store via nuclear IP3Rs.