Hyposmotic activation hyperpolarizes outer hair cells of guinea pig cochlea

The electrophysiological responses of isolated guinea pig outer hair cells (OHCs) to hyposmotic activation were studied using the whole-cell patch-clamp technique. The cell swelling by hyposmotic activation hyperpolarized OHCs by 6.6 ± 2.3 mV from the resting membrane potential of −58.5 ± 5.9 mV (n = 48). This hyperpolarization was associated with an outward current ( 97.7 ± 22.2, pA, n = 15). The hyperpolarization was inhibited by 300 μM quinine, 5 mN Ba²⁺ and increasing the extracellular K⁺ to 30 mM from 5 mM. In the absence of extracellular Ca²⁺ (1 mM EGTA), the hyperpolarization during hyposmotic activation was also abolished while the following depolarization was preserved. 50 μM GdCl₃, which is known to block strecch-activated non-specific cation channels, inhibited the hyperpolarization reversibly. 50 μM GdCl₃ also inhibited [Ca²⁺]_i increase during hyposmotic activation as shown by the calcium-sensitive dye fura-2. Simultaneously, the [Ca²⁺]_i increase and the hyperpolarization during hyposmotic activation could be observed using the combined method of whole-cell patch clamp and fura-2 technique. It is concluded that the cell swelling by hyposmotic activation may activate the stretch-activated non-specific cation channels in the OHCs which allow a Ca²⁺ influx. In turn, this [Ca²⁺]_i increase leads to an activation of the Ca²⁺-activated K⁺ channels at the basolateral membrane of OHCs which results finally in a reversible hyperpolarization of OHCs by K⁺ efflux.     

Inhibition of [Ca2+]i Transients in Rat Adrenal Chromaffin Cells by Neuropeptide Y Role for a cGMP-dependent Protein Kinase-activated K+ Conductance

The effects of neuropeptide Y on the intracellular level of Ca²⁺ ([Ca²⁺]_i) were studied in cultured rat adrenal chromaffin cells loaded with fura-2. A proportion (16%) of cells exhibited spontaneous rhythmic [Ca²⁺]_i oscillations. In silent cells, oscillations could be induced by forskolin and 1,9–dideoxyforskolin. This action of forskolin was not modified by H-89, an inhibitor of protein kinase A. Spontaneous [Ca²⁺_i fluctuations and [Ca²⁺]_i fluctuations induced by forskolin- and 1,9-dideoxyforskolin were inhibited by neuropeptide Y. Increases in [Ca²⁺]_i induced by 10 and 20 mM KCI but not by 50 mM KCI were diminished by neuropeptide Y. However, neuropeptide Y had no effect on [Ca²⁺]_i increases evoked by (-)BAY K8644 and the inhibitory effect of neuropeptide Y on responses induced by 20 mM KCI was not modified by o-conotoxin GVIA, consistent with neither L- nor N-type voltage-sensitive Ca²⁺ channels being affected by neuropeptide Y. Rises in [Ca²⁺]_i provoked by 10 mM tetraethylammonium were not decreased by neuropeptide Y, suggesting that K⁺ channel blockade reduces the effect of neuropeptide Y. However, [Ca²⁺]_i transients induced by 1 mM tetraethylammonium and charybdotoxin were still inhibited by neuropeptide Y, as were those to 20 mM KCI in the presence of apamin. The actions of neuropeptide Y on [Ca²⁺]_i transients provoked by 20 and 50 mM KCI, 1 mM tetraethylammonium, (-)BAY K8644 and charybdotoxin were mimicked by 8–bromo-cGMP. In contrast, 8–bromo-CAMP did not modify responses to 20 mM KCI or 1 mM tetraethylammonium. The inhibitory effects of neuropeptide Y and 8–bromo-cGMP on increases in [Ca²⁺]_i induced by 1 mM tetraethylammonium were abolished by the Rp-8–pCPT-cGMPS, an inhibitor of protein kinase G, but not by H-89. A rapid, transient increase in cGMP level was found in rat adrenal medullary tissues stimulated with 1 μM neuropeptide Y. Rises in [Ca²⁺]_i produced by DMPP, a nicotinic agonist, but not by muscarine, were decreased by neuropeptide Y. Our data suggest that neuropeptide Y activates a K⁺ conductance via a protein kinase G-dependent pathway, thereby opposing the depolarizing action of K⁺ channel blocking agents and the associated rise in [Ca²⁺]_i.     

Calcium Signalling in Mouse Bergmann Glial Cells Mediated by α1-adrenoreceptors and H1 Histamine - Receptors

The presence of adrenergic and histaminergic receptors in Bergmann glial cells from cerebellar slices from mice aged 20–25 days was determined using fura-2 Ca²⁺ microfluorimetry. To measure the cytoplasmic concentration of Ca²⁺ ([Ca²⁺]_i), either individual cells were loaded with the Ca²⁺-sensitive probe fura-2 using the whole-cell patch-clamp technique or slices were incubated with a membrane-permeable form of the dye (fura-2/AM) and the microfluorimetric system was focused on individual cells. The monoamines adrenalin and noradrenalin (0.1-10 μM) and histamine (10-100 μM) triggered a transient increase in [Ca²⁺]_i. The involvement of the α₁-adrenoreceptor was inferred from the observations that monoamine-triggered [Ca²⁺]_i responses were blocked by the selective α¹-adreno-antagonist prazosin and were mimicked by the α¹-adreno-agonist phenylephrine. The monoamine-induced [Ca²⁺]_i signals were not affected by β- and α₂-adrenoreceptor antagonists (propranolol and yohimbine), and were not mimicked by β- and α₂-adrenoreceptor agonists (isoproterenol and clonidine). Histamine-induced [Ca²⁺]_i responses demonstrated specific sensitivity to only H₁ histamine receptor modulators. [Ca²⁺]_i responses to monoamines and histamine did not require the presence of extracellular Ca²⁺ and they were blocked by preincubation of slices with thapsigargin (500 nM), indicating that the [Ca²⁺]_i increase is due to release from intracellular pools. No [Ca²⁺]_i responses were recorded after application of aspartate, bradykinin, dopamine, GABA, glycine, oxytocin, serotonin, somatostatin, substance P, taurine or vasopressin. We conclude that cerebellar Bergmann glial cells are endowed with α₁ -adrenoreceptors and H₁ histamine receptors which induce the generation of intracellular [Ca²⁺]_i signals via activation of Ca²⁺ release from inositol-l,4,5-trisphosphate-sensitive intracellular stores.     

Role of Voltage-gated Ca2+ Channels and Intracellular Ca2+ in Rat Sympathetic Neuron Survival and Function Promoted by High K+ and Cyclic AMP in the Presence or Absence of NGF

We have examined how NGF-dependent rat sympathetic neurons maintain Ca²⁺ homeostasis when challenged with high K⁺ or 8-(4-chlorophenylthio)cyclic AMP (CPTcAMP), two survival factors. In the presence of NGF, high K⁺ (55 mM) caused a stable, 65% reduction in the density of cell soma voltage-sensitive Ca²⁺ channels within 2 days. Although resting [Ca²⁺]_i was elevated by 1.6-fold, this was 50% less than the rise in [Ca²⁺]_i measured before down-regulation occurred, suggesting that down-regulation may help prevent the toxic effects of persistently elevated [Ca²⁺]_i. Inhibition of protein synthesis by cycloheximide blocked recovery from down-regulation. Moreover, treatment with cycloheximide or actinomycin-D caused a 2-fold rise in the peak Ca²⁺ current, suggesting that voltage-sensitive Ca²⁺ channel activity may be tonically attenuated during normal growth. In the absence of NGF, neurons survived for several days in high K⁺ medium with no significant rise in resting [Ca²⁺]_i, although neurites did not grow. Neither Ca²⁺ channel density nor resting [Ca²⁺]_i were altered in neurons surviving with CPTcAMP. Moreover, CPTcAMP lowered the dependence on extracellular Ca²⁺. However, the dihydropyridine antagonist nitrendipine blocked both high K⁺- and CPTcAMP-dependent survival although it had no effect in the presence of NGF. Thus, in the absence of NGF, sympathetic neurons do not require elevation of [Ca²⁺]_i above resting levels to survive with either high K⁺ or CPTcAMP, but dihydropyridine-sensitive Ca²⁺ channel activity may be essential for their survival promoting actions.     

Relationship between resting cytosolic Ca and responses induced byN-methyl-d-aspartate in hippocampal neurons

Cytosolic calcium concentrations ([Ca²⁺]_i) in cultured hippocampal neurons from rat embryos were measured using fura-2. Neurons with higher resting [Ca²⁺]_i showed greater [Ca²⁺]_i responses toN-methyl-d-aspartate (NMDA) and K⁺ depolarization. There was a strong relationship between resting [Ca²⁺]_i and the maximal changes in [Ca²⁺]_i (Δ[Ca²⁺]_i), which fit the our proposed equation to describe this relationship.     

Developmental Loss of GABA- and Glycine-induced Depolarization and Ca2+ Transients in Embryonic Rat Dorsal Horn Neurons in Culture

More than 90% of dorsal horn neurons from embryonic day 15–16 rats responded to the inhibitory amino acids GABA and glycine by a transient elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) when maintained in culture for <1 week. This [Ca²⁺]_i response has previously been shown to be due to depolarization and subsequent Ca²⁺ entry through voltage-gated Ca²⁺ channels following activation of bicuculline-sensitive GABA_A receptors and strychnine-sensitive glycine receptors. Both the number of cells responding to GABA and glycine and the amplitude of the [Ca²⁺]_i response diminished over time in culture. By 30 days in culture, none of the cells responded to GABA, muscimol or glycine by elevation of [Ca²⁺]_i. The loss of the [Ca²⁺]_i response was not due to a change in the abundance or the properties of voltage-gated Ca²⁺ channels, since over the same period of time dorsal horn neurons showed a large increase in the amplitude of the [Ca²⁺]_i transient in response to 30 mM K⁺. Nor was the loss of the [Ca²⁺]_i response due to a loss of GABA and glycine receptors. Instead, the decrease in the [Ca²⁺]_i response over time paralleled a similar change in the electrophysiological responses. More than 90% of the neurons tested were depolarized in response to inhibitory amino acids during the first week in culture. After 30 days, all neurons tested responded to GABA and glycine with a hyperpolarization. These observations add support to the suggestion that GABA and glycine may excite dorsal horn neurons earlyin development and play a role in postmitotic differentiation.     

Gonadotropin releasing hormone modulates γ-aminobutyric acid-evoked intracellular calcium increase in immortalized hypothalamic gonadotropin releasing hormone neurons

To examine the functional role of calcium signaling in the interactive modulation of gonadotropin releasing hormone (GnRH) neurons by γ-aminobutyric acid (GABA) and GnRH itself, we analyzed the intracellular calcium level ([Ca²⁺]_i), using fura-2AM fluorescent dye in immortalized hypothalamic GT1-1 cells. GT1-1 cells showed spontaneous [Ca²⁺]_i oscillations, which were dependent on extracellular Ca²⁺ level, L-type Ca²⁺ channel and SK-type K⁺ channel. When GABA or a specific GABA_A type receptor agonist, muscimol was applied to the media, [Ca²⁺]_i rapidly increased through L-type Ca²⁺ channel in a dose-dependent manner, and subsequently decreased below the basal level without any oscillation. However, a specific GABA_B type receptor agonist, baclofen showed no effect. On the other hand, application of GnRH or its potent agonist buserelin, rapidly abolished the spontaneous [Ca²⁺]_i oscillations. Interestingly, a prior treatment with buserelin abolished GABA-evoked increase in [Ca²⁺]_i in a noncompetitive manner. Since buserelin also blocked K⁺-evoked increase in [Ca²⁺]_i, we suggest that GnRH may block spontaneous [Ca²⁺]_i oscillation through modulating the L-type [Ca²⁺]_i channel activity. These results show that GABAergic agents may exert both stimulatory and inhibitory controls over the GnRH neuronal activity, and GnRH can block the stimulatory effect of GABA, implicating the possible existence of an ultrashort feedback circuit.     

Potassium depolarization of mammalian vestibular sensory cells increases [Ca2+]i through voltage‐sensitive calcium channels

The existence of voltage-sensitive Ca²⁺ channels in type I vestibular hair cells of mammals has not been conclusively proven. Furthermore, Ca²⁺ channels present in type II vestibular hair cells of mammals have not been pharmacologically identified. Fura-2 fluorescence was used to estimate, in both cell types, intracellular Ca²⁺ concentration ([Ca²⁺]_i) variations induced by K⁺ depolarization and modified by specific Ca²⁺ channel agonists and antagonists. At rest, [Ca²⁺]_i was 90 ± 20 nm in both cell types. Microperifusion of high-K⁺ solution (50 mm ) for 1 s increased [Ca²⁺]_i to 290 ± 50 nm in type I (n = 20) and to 440 ± 50 nm in type II cells (n = 10). In Ca²⁺-free medium, K⁺ did not alter [Ca²⁺]_i. The specific L-type Ca²⁺ channel agonist, Bay K, and antagonist, nitrendipine, modified in a dose-dependent manner the K⁺-induced [Ca²⁺]_i increase in both cell types with maximum effect at 2 μm and 400 nm , respectively. Ni²⁺, a T-type Ca²⁺ channel blocker, reduced K⁺-evoked Ca²⁺ responses in a dose-dependent manner. For elevated Ni²⁺ concentrations, the response was differently affected by Ni²⁺ alone, or combined to nitrendipine (500 nm ). In optimal conditions, nitrendipine and Ni²⁺ strongly depressed by 95% the [Ca²⁺]_i increases. By contrast, neither ω-agatoxin IVA (1 μm ), a specific P- and Q-type blocker, nor ω-conotoxin GVIA (1 μm ), a specific N-type blocker, affected K⁺-evoked Ca²⁺_i responses. These results provide the first direct evidence that L- and probably T-type channels control the K⁺-induced Ca²⁺ influx in both types of sensory cells.     

Relation Between Dendritic Ca2+ Levels and the Polarity of Synaptic Long-term Modifications in Rat Visual Cortex Neurons

Long-term changes of synaptic efficacy, in particular when they are use-dependent, are candidate mechanisms for the storage of information in the nervous system. In a variety of brain structures, including the neocortex and hippocampus, synapses are susceptible to long-term potentiation (LTP) and long-term depression (LTD). It has been hypothesized that the polarity of the synaptic gain change depends on the amplitude of the postsynaptic [Ca²⁺]_i rise, the threshold for the induction of LTD being lower than that for the induction of LTP. To test this assumption, we characterized Ca²⁺ signals in layer II/III pyramidal cells of rat visual cortex slices, using the fluorescent Ca²⁺ indicator fura-2, during application of stimulation protocols that had been adjusted to reliably induce either LTP or LTD in cells not loaded with fura-2. At dendritic sites activated by the stimulated afferents the intracellular [Ca²⁺] concentration ([Ca²⁺]_i) reached higher amplitudes and decayed more slowly with stimuli inducing LTP than with those inducing LTD. To directly analyse the functional significance of the observed difference in the Ca²⁺ signal amplitude, we examined whether a tetanization protocol suitable for the induction of LTP can be converted into a protocol inducing LTD by injecting the postsynaptic cells with Ca²⁺ chelators that reduce the concentration of effective free Ca²⁺. In the presence of fura-2 or BAPTA [bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetate], the stimulation protocol that would normally produce LTP induced either LTD or failed to induce synaptic modifications altogether. These results support the hypothesis that the amplitude of the postsynaptic rise in [Ca²⁺]_i is a key factor in the determination of the polarity of synaptic gain change.     

Mobilization of intracellular Ca and suppression of inward currents in a neuronal hybrid cell line triggered by bradykinin

Bradykinin triggered intracellular Ca mobilizations and ionic conductance changes were studied in the neuroblastoma × glioma hybrid cell line NG108-15 using Ca-sensitive fluorescent indicator fura-2 under patch pipette whole cell voltage clamp condition. The time course of outward current induced by bradykinin was closely related to the time-course of [Ca²⁺]_i change. Following application of bradykinin, [Ca²⁺]_i increased transiently and then decreased below the basal level before bradykinin application. The inward currents activated by step-depolarization were suppressed after bradykinin application, but the time-course of the suppression did not go in parallel with the [Ca²⁺]_i changes: the suppression started before the [Ca²⁺]_i change emerged and outlasted the phase of [Ca²⁺]_i increase. Both transient type and long-lasting type Ca current were suppressed by bradykinin. [Ca²⁺]_i increase induced by high potassium depolarization was suppressed by bradykinin. Pertussis toxin did not affect the Ca transient nor the suppression of Ca channel induced by bradykinin. Our results suggest that the modifications of ionic channels by bradykinin could be through the other mechanisms than the well established activation of the G-protein leading to the IP₃ mechanisms and that the bradykinin receptor might couple with the pertussis toxin-insensitive G protein which regulates the calcium channels.     

Divalent Cation Entry in Cultured Rat Cerebellar Granule Cells Measured Using Mn2+ Quench of Fura 2 Fluorescence

In this study the rate of Mn²⁺ quench of fura-2 fluorescence evoked by glutamatergic and cholinergic agonists, depolarization and Ca²⁺ store modulators was measured in cultured cerebellar granule cells, in order to study their effects on Ca²⁺ entry in isolation from effects on Ca²⁺ store release. The rate of fluorescence quench by 0.1 mM Mn²⁺ was markedly increased by 25 mM K⁺- evoked depolarization or by 200 μM N-methyl-D-aspartate (NMDA), with a significantly greater increase occurring during the rapid-onset peak phase compared to the plateau phase of the K⁺- or NMDA-evoked [Ca²⁺]_i response. The stimulatory effect of NMDA on Mn²⁺ quench was abolished by dizocilpine (10 μM), but nitrendipine (2 μM), while decreasing the rate of basal quench, did not affect NMDA-stimulated Mn²⁺ entry. This suggests that nitrendipine may not act on NMDA channels in granule cells, at least under these conditions, and that voltage-operated Ca²⁺ channels are involved in control quench whereas the NMDA-evoked quench is dependent on entry through the receptor channel. The t_1/2 of quench was unaffected by α-amino-hydroxyisoxazole propionic acid (200 μM) and carbamyl choline (1 mM). Neither thapsigargin (10 μM) nor dantrolene (30 μM) significantly affected the rate of quench under control or NMDA- or K⁺-stimulated conditions, which confirms that the previously reported inhibitory effects on [Ca²⁺]_i elevations evoked by these agents are due to actions on Ca²⁺ stores. However, thapsigargin elevated [Ca²⁺Ii in the presence of normal [Ca²⁺]_i, but not in nominally Ca²⁺-free medium, indicating that it evokes Ca²⁺ entry in cerebellar granule cells, probably subsequent to store depletion, which appears to be either too small to be detected by Mn²⁺ quench or to occur via Mn²⁺-impermeant channels.     

Spatiotemporal Distribution of Ca2+ Following Axotomy and Throughout the Recovery Process of Cultured Aplysia Neurons

This study investigates the alterations in the spatiotemporal distribution pattern of the free intracellular Ca²⁺ concentration ([Ca²⁺]_i) during axotomy and throughout the recovery process of cultured Aplysia neurons, and correlates these alterations with changes in the neurons input resistance and trans-membrane potential. For the experiments, the axons were transected while imaging the changes in [Ca²⁺]_i with fura-2, and monitoring the neurons’resting potential and input resistance (R_i) with an intracellular microelectrode inserted into the cell body. The alterations in the spatiotemporal distribution pattern of [Ca²⁺]_i were essentially the same in the proximal and the distal segments, and occurred in two distinct steps: concomitantly with the rupturing of the axolemma, as evidenced by membrane depolarization and a decrease in the input resistance, [Ca²⁺]_i increased from resting levels of 0.05 – 0.1 μM to 1 – 1.5 μM along the entire axon. This is followed by a slower process in which a [Ca²⁺]_i front propagates at a rate of 11 – 16 μm/s from the point of transection towards the intact ends, elevating [Ca²⁺]_i to 3 – 18 μM. Following the resealing of the cut end 0.5 – 2 min post-axotomy, [Ca²⁺]_i recovers in a typical pattern of a retreating front, travelling from the intact ends towards the cut regions. The [Ca²⁺]_i recovers to the control level 7 – 10 min post-axotomy. In Ca²⁺-free artificial sea water (2.5 mM EGTA) axotomy does not lead to increased [Ca²⁺]_i and a membrane seal is not formed over the cut end. Upon reperfusion with normal artificial sea water, [Ca²⁺]_i is elevated at the tip of the cut axon and a membrane seal is formed. This experiment, together with the observations that injections of Ca²⁺, Mg²⁺ and Na⁺ into intact axons do not induce the release of Ca²⁺ from intracellular stores, indicates that Ca²⁺ influx through voltage gated Ca²⁺ channels and through the cut end are the primary sources of [Ca²⁺]_i following axotomy. However, examination of the spatiotemporal distribution pattern of [Ca²⁺]_i following axotomy and during the recovery process indicates that diffusion is not the dominating process in shaping the [Ca²⁺]_i gradients. Other Ca²⁺ regulatory mechanisms seem to be very effective in limiting these gradients, thus enabling the neuron to survive the injury.     

The ouabain-induced [Ca]i increase in leech Retzius neurones is mediated by voltage-dependent Ca channels

In leech Retzius neurones the inhibition of the Na⁺–K⁺ pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca²⁺]_i). To elucidate the mechanism of this increase we investigated the changes in [Ca²⁺]_i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na⁺–K⁺ pump in bathing solutions of different ionic composition. The results show that Na⁺–K⁺ pump inhibition induced a [Ca²⁺]_i increase only if the cells depolarized sufficiently in the presence of extracellular Ca²⁺. Specifically, the relationship between [Ca²⁺]_i and the membrane potential upon Na⁺–K⁺ pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca²⁺ channels by raising the extracellular K⁺ concentration. It is concluded that the [Ca²⁺]_i increase caused by inhibiting the Na⁺–K⁺ pump in leech Retzius neurones is exclusively due to Ca²⁺ influx through voltage-dependent Ca²⁺ channels.     

Emergence of excitotoxicity in cultured forebrain neurons coincides with larger glutamate-stimulated [Ca]i increases and NMDA receptor mRNA levels

We examined several factors related to the increase in susceptibility to excitotoxicity that occurs in embryonic forebrain neurons over time in culture. Neuronal cultures were resistant to a 5-min exposure to 100 μM glutamate/10 μM glycine at 5 days in vitro (DIV), but became vulnerable to the same stimulus by 14 DIV. We used the fluorescent indicators, fura-2 and magfura-2, which have high and low affinity for Ca²⁺, respectively, to measure changes in [Ca²⁺]_i. Glutamate-stimulated increases in the fura-2 and magfura-2 ratio reached maximum values by 10 DIV. Fura-2 reported similar [Ca²⁺]_i changes with exposure to 3 or 100 μM glutamate for 5 min, whereas magfura-2 reported larger [Ca²⁺]_i increases with 5-min exposure to 100 μM glutamate than with exposure to 3 μM glutamate, 100 μM kainate or 50 mM K⁺ from 10 DIV onward. This suggests that the magnitude of the [Ca²⁺]_i changes correlated with the excitotoxicity potential of a stimulus when magfura-2, but not fura-2, was used to measure Ca²⁺. We also used RNase protection assays to measure NMDA receptor subunit mRNA levels. NR1 and NR2A mRNA increased continuously over time in culture, whereas NR2B mRNA increased dramatically during the first 10 days and subsequently remained stable. The time course of the increase in NR2B mRNA most closely followed the increase in glutamate-stimulated changes in the magfura-2 signal and neuronal injury. Therefore, the increases in the glutamate-stimulated [Ca²⁺]_i responses and NMDA receptor subunit mRNA levels (especially NR2B) are likely involved in the development of susceptibility to excitotoxicity in cultured rat forebrain neurons.     

Secretion from rat neurohypophysial nerve terminals (neurosecretosomes) rapidly inactivates despite continued elevation of intracellular Ca

Cytoplasmic calcium concentration was measured in neurosecretory nerve terminals (neurosecretosomes) isolated from rat neurohypophyses by fura-2 fluorescence measurements and digital video microscopy. Hormone release and cytoplasmic calcium concentration were measured during depolarizations induced by elevated extracellular potassium concentration. During prolonged depolarizations with 55 mM [K⁺]₀, the cytoplasmic calcium concentration remained elevated as long as depolarization persisted, while secretion inactivated after the initial sharp rise. The amplitude and duration of the increase in [Ca²⁺]_i was dependent on the degree of depolarization such that upon low levels of depolarizations (12.5 mM or 25 mM [K⁺]₀), the calcium responses were smaller and relatively transient, and with higher levels of depolarization (55 mM [K⁺]₀) the responses were sustained and were higher in amplitude. Responses to low levels of depolarization were less sensitive to the dihydropyridine calcium channel blocker, nimodipine, while the increase in [Ca²⁺]_i induced by 55 mM [K⁺]₀ became transient, and was significantly smaller. These observations suggest that these peptidergic nerve terminals possess at least two different types of voltage-gated calcium channels. Removal of extracellular sodium resulted in a significant increase in [Ca²⁺]_i and secretion in the absence of depolarizing stimulus, suggesting that sodium-calcium exchange mechanism is operative in these nerve terminals. Although the [Ca²⁺]_i increase was of similar magnitude to the depolarization-induced changes, the resultant secretion was 10-fold lower, but the rate of inactivation of secretion, however, was comparable.     

Ionotropic and Metabotropic Glutamate Receptor Agonist-Induced [Ca2+]i Increase in Isolated Rat Supraoptic Neurons

In the present study, the effects of glutamate and of agonists for ionotropic and metabotropic glutamate receptors on intracellular Ca²⁺ concentration ([Ca²⁺]_i) were investigated in neurons of the rat supraoptic nucleus (SON). We used the intracellular Ca²⁺ imaging technique with fura-2, in single magnocellular neurons dissociated from the SON of rats. Glutamate (10^?6?10^?4 M) evoked a dose-dependent increase in [Ca²⁺]_i. The glutamate agonists exerted similar effects, although with some differences in the characteristics of their responses. The [Ca²⁺]_i response to NMDA was smaller than those of glutamate or the non-NMDA receptor agonists, AMPA and kainate, but was significantly enhanced by the removal of extracellular Mg²⁺. Glutamate, as well as quisqualate, an agonist for both ionotropic and metabotropic glutamate receptors, evoked a [Ca²⁺]_i increase in a Ca²⁺-free condition, suggesting Ca²⁺ release from intracellular Ca²⁺ stores. This was further evidenced by [Ca²⁺]_i increases in response to a more selective metabotropic glutamate receptor agonist, t-ACPD, in the absence of extracellular Ca²⁺. Furthermore, the quisqualate-induced Ca²⁺ release was abolished by the selective metabotropic glutamate receptor antagonist, (S)-4-carboxyphenylglycine. The results suggest that metabotropic glutamate receptors as well as non-NMDA and NMDA receptors are present in the SON neurons, and that activation of the first leads to Ca²⁺ release from intracellular Ca²⁺ stores and the activation of the latter two types induces Ca²⁺ entry. These dual mechanisms of Ca²⁺ signalling may play a role in the regulation of SON neurosecretory cells by glutamate.     

Measurement of intracellular Ca in the bullfrog sympathetic ganglion cells using fura-2 fluorescence

The intracellular free ([Ca²⁺]_i) of the bullfrog sympathetic ganglion cell was measured with fura-2 fluorescence under various conditions, and compared with changes in membrane potential recorded with an intracellular electrode. The [Ca²⁺]_i was 109 nM on average under the resting condition and increased by raising the extracellular K⁺, stimulating repetitively the pre- or post-ganglionic nerve, or by applying acetylcholine or muscarine. Since all these procedures depolarized the cell membrane, most of the rise in [Ca²⁺]_i could be the result of opening of voltage-dependent Ca²⁺ channels. However, Ca²⁺ entries through nicotinic acetylcholine receptor channels and the channel activated by the muscarinic acetylcholine receptor were also indicated by considering the threshold for the opening of voltage-dependent Ca²⁺ channels (for both entries) or a limited number of the cells showing the latter response.     

Properties of the Ryanodine-sensitive Release Channels that Underlie Caffeine-induced Ca2+ Mobilization from Intracellular Stores in Mammalian Sympathetic Neurons

The most compelling evidence for a functional role of caffeine-sensitive intracellular Ca²⁺ reservoirs in nerve cells derives from experiments on peripheral neurons. However, the properties of their ryanodine receptor calcium release channels have not been studied. This work combines single-cell fura-2 microfluorometry, [³ H]ryanodine binding and recording of Ca²⁺ release channels to examine calcium release from these intracellular stores in rat sympathetic neurons from the superior cervical ganglion. Intracellular Ca²⁺ measurements showed that these cells possess caffeine-sensitive intracellular Ca²⁺ stores capable of releasing the equivalent of 40% of the calcium that enters through voltage-gated calcium channels. The efficiency of caffeine in releasing Ca²⁺ showed a complex dependence on [Ca²⁺]_i. Transient elevations of [Ca²⁺]_i by 50–500 nM were facilitatory, but they became less facilitatory or depressing when [Ca²⁺]_i reached higher levels. The caffeine-induced Ca²⁺ release and its dependence on [Ca²⁺]_i was further examined by [³ H]ryanodine binding to ganglionic microsomal membranes. These membranes showed a high-affinity binding site for ryanodine with a dissociation constant (K_D= 10 nM) similar to that previously reported for brain microsomes. However, the density of [³H]ryanodine binding sites (B_max= 2.06 pmol/mg protein) was at least three-fold larger than the highest reported for brain tissue. [³ H]Ryanodine binding showed a sigmoidal dependence on [Ca²⁺] in the range 0.1–10 μM that was further increased by caffeine. Caffeine-dependent enhancement of [³ H]ryanodine binding increased and then decreased as [Ca²⁺] rose, with an optimum at [Ca²⁺] between 100 and 500 nM and a 50% decrease between 1 and 10 μM. At 100 μM [Ca²⁺], caffeine and ATP enhanced [³ H]ryanodine binding by 35 and 170% respectively, while binding was reduced by >90% with ruthenium red and MgCl₂. High-conductance (240 pS) Ca²⁺ release channels present in ganglionic microsomal membranes were incorporated into planar phospholipid bilayers. These channels were activated by caffeine and by micromolar concentrations of Ca²⁺ from the cytosolic side, and were blocked by Mg²⁺ and ruthenium red. Ryanodine (2 μM) slowed channel gating and elicited a long-lasting subconductance state while 10 mM ryanodine closed the channel with infrequent opening to the subconductance level. These results show that the properties of the ryanodine receptor/Ca²⁺ release channels present in mammalian peripheral neurons can account for the properties of caffeine-induced Ca²⁺ release. Our data also suggest that the release of Ca²⁺ by caffeine has a bell-shaped dependence on Ca²⁺ in the physiological range of cytoplasmic [Ca²⁺].     

Mastoparan-induced apoptosis of cultured cerebellar granule neurons is initiated by calcium release from intracellular stores

We have recently reported that mastoparan, a peptide toxin isolated from wasp venom, induces apoptosis in cultured cerebellar granule neurons that can be blocked by cholera toxin, an activator of G_s. Measurements of intracellular free calcium concentration ([Ca²⁺]_i) reveal that mastoparan induces a dramatic elevation of [Ca²⁺]_i that is frequently followed by enhanced leakage of fura-2 out of the neurons, suggesting that this rise in [Ca²⁺]_i may be due to a more generalized change in membrane permeability. However, the mastoparan-induced initial elevation of [Ca²⁺]_i is maintained in the absence of extracellular Ca²⁺, suggesting that the rise of [Ca²⁺]_i is from intracellular stores. This conclusion is supported by the observation that depletion of [Ca²⁺]_i stores by pretreatment with either caffeine or thapsigargin attenuates both the rise in [Ca²⁺]_i and cell death induced by mastoparan. Phospholipase C (PLC) inhibitors, neomycin and U73122 block mastoparan-induced increases of [Ca²⁺]_i and protect against neuronal death. Pretreatment with cholera toxin, but not pertussis toxin, reduced the mastoparan-induced rise in [Ca²⁺]_i. Taken together, our data suggest that mastoparan initiates cell death in cerebellar granule neurons by inducing Ca²⁺ release from intracellular stores, probably via activation of PLC and IP₃. A secondary or parallel process results in disruption of plasma membrane integrity and may be ultimately responsible for the death of these neurons by mastoparan.     

Selective enhancement by basic fibroblast growth factor of NMDA receptor-mediated increase of intracellular Ca concentration in hippocampal neurons

The short-term effect of bFGF on intracellular Ca²⁺ concentration ([Ca²⁺]_i) of hippocampal neurons was investigated using dissociated cell cultures. Changes in [Ca²⁺]_i were measured by microfluorometrically monitoring the fluorescence intesities from indivudual neurons loaded with fura-2. Perfusion of bFGF (20 ng/ml) alone did not affect the basal level of [Ca²⁺]_i in hippocampal neurons, but clearly enhanced the [Ca²⁺]_i increase induced by NMDA. Quisqualate or KCl-induced [Ca²⁺]_i increase was not influenced by bFGF. These results suggest that bFGF selectively enhances the NMDA receptor-mediated response in hippocampal neurons.