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.     

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.     

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.     

Intracellular Ca2+ during metabolic activation of KATP channels in spontaneously active dorsal vagal neurons in medullary slices

Intracellular Ca²⁺ ([Ca²⁺]_i) and membrane properties were measured in fura-2 dialysed dorsal vagal neurons (DVN) spontaneously active at a frequency of 0.5–5 Hz. [Ca²⁺]_i increased by about 30 nm upon rising spike frequency by more than 200% due to 20–50 pA current pulses or 10 μm serotonin. It fell by 30 nm upon block of spiking by current-injection, tetrodotoxin or Ni²⁺ and also during hyperpolarization due to γ-aminobutyric acid or opening of adenosine triphosphate (ATP) -sensitive K⁺ (K_ATP) channels with diazoxide. K_ATP channel-mediated hyperpolarizations during anoxia or cyanide produced an initial [Ca²⁺]_i decrease which reversed into a secondary Ca²⁺ rise by less than 100 nm . Similar moderate rises of [Ca²⁺]_i were observed during block of aerobic metabolism under voltage-clamp as well as in intact cells, loaded with fura-2 AM. The magnitude of the metabolism-related [Ca²⁺]_i transients did not correlate with the amplitude of the K_ATP channel-mediated outward current. [Ca²⁺]_i did not change during diazoxide-induced or spontaneous activation of K_ATP outward current observed in 10% of cells after establishing whole-cell recording. Increasing [Ca²⁺]_i with cyclopiazonic acid did not activate K_ATP channels. [Ca²⁺]_i was not affected upon block of outward current with sulphonylureas, but these K_ATP channel blockers were effective to reverse inhibition of spike discharge and, thus, the initial [Ca²⁺]_i fall upon spontaneous or diazoxide-, anoxia- and cyanide-induced K_ATP channel activation. A sulphonylurea-sensitive hyperpolarization and [Ca²⁺]_i fall was also revealed in the early phase of iodoacetate-induced metabolic arrest, whereas after about 20 min, occurrence of a progressive depolarization led to an irreversible rise of [Ca²⁺]_i to more than 1 μm . The results indicate that K_ATP channel activity in DVN is not affected by physiological changes of intracellular Ca²⁺ and the lack of a major perturbance of Ca²⁺ homeostasis contributes to their high tolerance to anoxia.     

Effects of the removal of extracellular Ca on [Ca]i responses to FCCP and acetate in carotid body glomus cells of adult rabbits

The effects of the removal of extracellular Ca²⁺ on the responses of cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) to acidic stimuli, a protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and an organic acid acetate, were examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2 microfluorometry. Application of FCCP (1 μM) induced an increase in [Ca²⁺]_i (mean±S.E.M., 108±14%). After withdrawal of the protonophore the increased [Ca²⁺]_i returned slowly to a resting level. The [Ca²⁺]_i response was attenuated by an inorganic Ca²⁺ channel antagonist Ni²⁺ (2 mM) by 81±4%, and by an L-type voltage-gated Ca²⁺ channel antagonist D600 (10 μM) by 53±13%. The removal of extracellular Ca²⁺ eliminated the [Ca²⁺]_i response in 71% of the tested cells (n=17), and depressed it by 68±6% in the rest. Recovery following stimulation with FCCP in the absence of Ca²⁺ reversibly produced a rapid and large rise in [Ca²⁺]_i, referred to as a [Ca²⁺]_i rise after Ca²⁺-free/FCCP. The magnitude of a [Ca²⁺]_i rise after Ca²⁺-free/FCCP (285±28%, P<0.05) was larger than that of an increase in [Ca²⁺]_i induced by FCCP in the presence of Ca²⁺ and had a correlation with the intensity of the suppression of the [Ca²⁺]_i response by Ca²⁺ removal. A [Ca²⁺]_i rise after Ca²⁺-free/FCCP was inhibited mostly by D600. Similarly, recovery following exposure to acetate in the absence of Ca²⁺ caused a rise in [Ca²⁺]_i, referred to as a [Ca²⁺]_i rise after Ca²⁺-free/acetate which was sensitive to D600. The magnitude of the [Ca²⁺]_i rise was larger than that of a change in [Ca²⁺]_i caused by acetate in the presence of Ca²⁺. These results suggest that FCCP-induced increase in [Ca²⁺]_i was, in most cells, due to Ca²⁺ influx via L-type voltage-gated Ca²⁺ channels and, in some cells, due to both Ca²⁺ influx and Ca²⁺ release from internal Ca²⁺ pool. The removal of extracellular Ca²⁺ might modify [Ca²⁺]_i responses to acidic stimuli, causing [Ca²⁺]_i rises after Ca²⁺-free/acidic stimuli which involve mostly L-type Ca²⁺ channels.     

Effects of hypoxia induced by Na2S2O4 on intracellular calcium and resting potential of mouse glomus cells

Isolated and cultured glomus cells, obtained from mouse carotid bodies, were superfused with Ham's F-12 equilibrated with air (mean PO₂, 119 Torr; altitude 1350 m). [Ca²⁺]_o was 3.0 mM. In one experimental series, dual cell penetrations with microelectrodes measured intracellular calcium ([Ca²⁺]_i) and the resting potential (E_m). In another series, [Ca²⁺]_i was measured with Indo-1/AM, dissolved in DMSO. Normoxic cells had a mean E_m of −42.4 mV and [Ca²⁺]_i was about 80 nM (measured with both methods). The calculated calcium equilibrium potential (E_Ca) was 137±0.74 mV. Hypoxia, induced by Na₂S₂O₄ 1 mM, reduced pO₂ to 10–14 Torr. This effect was accompanied by cell depolarization to −19.1 mV. Hypoxia increased [Ca²⁺]_i to 231 nM when detected with Ca-sensitive microelectrodes, but only to 130.2 nM when measured with Indo-1/AM. Calcium increases were preceded by decreases in [Ca²⁺]_i, which also were more pronounced with microelectrode measurements. CoCl₂ 1 mM blocked the hypoxic [Ca²⁺]_i increase and exaggerated the decreases in [Ca²⁺]_i. Correlations between ΔE_m and Δ[Ca²⁺]_i during hypoxia were significant (p<0.05) in 19% of the cells. But, in 29% of them significance was at the p<0.1 level. In the rest (52%), there was no correlation between these parameters. Thus, voltage-gated calcium channels are rare in mouse glomus cells. Their activation by depolarization cannot explain the two to threefold increase in [Ca²⁺]_i seen during hypoxia. More likely, [Ca²⁺]_i increase may be due to hypoxic inactivation of a Ca–Mg ATPase transport system across the cell membrane. The blunting of hypoxic [Ca²⁺]_i increase, seen in Indo-1/AM experiments, is probably due to its solvent (DMSO), which also depresses hypoxic cell depolarization.     

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.     

Mechanisms of intercellular calcium signaling in glial cells studied with dantrolene and thapsigargin

Mechanical stimulation of a single cell in a primary mixed glial cell culture induced a wave of increased intracellular calcium concentration ([Ca²⁺]_i) that was communicated to surrounding cells. Following propagation of the Ca²⁺ wave, many cells showed asynchronous oscillations in [Ca²⁺]_i. Dantrolene sodium (10 μM) inhibited the increase in [Ca²⁺]_i associated with this Ca²⁺ wave by 60-80%, and prevented subsequent Ca²⁺ oscillations. Despite the markedly decreased magnitude of the increase in [Ca²⁺]_i, the rate of propagation and the extent of communication of the Ca²⁺ wave were similar to those prior to the addition of dantrolene. Thapsigargin (10 nM to 1 μM) induced an initial increase in [Ca²⁺]_i ranging from 100 nM to 500 nM in all cells that was followed by a recovery of [Ca²⁺]_i to near resting levels in most cells. Transient exposure to thapsigargin for 2 min irreversibly blocked communication of a Ca²⁺ wave from the stimulated cell to adjacent cells. Glutamate (50 μM) induced an initial increase in [Ca²⁺]_i in most cells that was followed by sustained oscillations in [Ca²⁺]_i in some cells. Dantrolene (10 μM) inhibited this initial [Ca²⁺]_i increase caused by glutamate by 65-90% and abolished subsequent oscillations. Thapsigargin (10 nM to 1 μm) abolished the response to glutamate in over 99% of cells. These results suggest that while both dantrolene and thapsigargin inhibit intracellular Ca²⁺ release, only thapsigargin affects the mechanism that mediates intercellular communication of Ca²⁺ waves. These findings are consistent with the hypothesis that inositol trisphosphate (IP₃) mediates the propagation of Ca²⁺ waves whereas Ca²⁺ -induced Ca²⁺ release amplifies Ca²⁺ waves and generates subsequent Ca²⁺ oscillations.     

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⁺].     

Glutamate‐induced calcium increase mediates magnesium release from mitochondria in rat hippocampal neurons

Excess administration of glutamate is known to induce Ca²⁺ overload in neurons, which is the first step in excitotoxicity. Although some reports have suggested a role for Mg²⁺ in the excitotoxicity, little is known about its actual contribution. To investigate the role of Mg²⁺ in the excitotoxicity, we simultaneously measured intracellular Ca²⁺ and Mg²⁺, using fluorescent dyes, Fura red, a fluorescent Ca²⁺ probe, and KMG‐104, a highly selective fluorescent Mg²⁺ probe developed by our group, respectively. Administration of 100 μM glutamate supplemented with 10 μM glycine to rat hippocampal neurons induced an increase in intracellular Mg²⁺ concentration ([Mg²⁺]_i). Extracellular Mg²⁺ was not required for this glutamate‐induced increase in [Mg²⁺]_i, and no increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) or [Mg²⁺]_i was observed in neurons in nominally Ca²⁺‐free medium. Application of 5 μM carbonyl cyanide p‐(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of mitochondrial inner membrane potential, also elicited increases in [Ca²⁺]_i and [Mg²⁺]_i. Subsequent administration of glutamate and glycine following FCCP treatment did not induce a further increase in [Mg²⁺]_i but did induce an additive increase in [Ca²⁺]_i. Moreover, the glutamate‐induced increase in [Mg²⁺]_i was observed only in mitochondria localized areas. These results support the idea that glutamate is able to induced Mg²⁺ efflux from mitochondria to the cytosol. Furthermore, pretreatment with Ru360, an inhibitor of the mitochondrial Ca²⁺ uniporter, prevented this [Mg²⁺]_i increase. These results indicate that glutamate‐induced increases in [Mg²⁺]_i result from the Mg²⁺ release from mitochondria and that Ca²⁺ accumulation in the mitochondria is required for this Mg²⁺ release. © 2010 Wiley‐Liss, Inc.     

Acute action of rotenone on nigral dopaminergic neurons – involvement of reactive oxygen species and disruption of Ca2+ homeostasis

Rotenone is a toxin used to generate animal models of Parkinson’s disease; however, the mechanisms of toxicity in substantia nigra pars compacta (SNc) neurons have not been well characterized. We have investigated rotenone (0.05–1 μm ) effects on SNc neurons in acute rat midbrain slices, using whole‐cell patch‐clamp recording combined with microfluorometry. Rotenone evoked a tolbutamide‐sensitive outward current (94 ± 15 pA) associated with increases in intracellular [Ca²⁺] ([Ca²⁺]_i) (73.8 ± 7.7 nm ) and intracellular [Na⁺] (3.1 ± 0.6 mm ) (all with 1 μm ). The outward current was not affected by a high ATP level (10 mm ) in the patch pipette but was decreased by Trolox. The [Ca²⁺]_i rise was abolished by removing extracellular Ca²⁺, and attenuated by Trolox and a transient receptor potential M2 (TRPM2) channel blocker, N‐(p‐amylcinnamoyl) anthranilic acid. Other effects included mitochondrial depolarization (rhodamine‐123) and increased mitochondrial reactive oxygen species (ROS) production (MitoSox), which was also abolished by Trolox. A low concentration of rotenone (5 nm ) that, by itself, did not evoke a [Ca²⁺]_i rise resulted in a large (46.6 ± 25.3 nm ) Ca²⁺ response when baseline [Ca²⁺]_i was increased by a ‘priming’ protocol that activated voltage‐gated Ca²⁺ channels. There was also a positive correlation between ‘naturally’ occurring variations in baseline [Ca²⁺]_i and the rotenone‐induced [Ca²⁺]_i rise. This correlation was not seen in non‐dopaminergic neurons of the substantia nigra pars reticulata (SNr). Our results show that mitochondrial ROS production is a key element in the effect of rotenone on ATP‐gated K⁺ channels and TRPM2‐like channels in SNc neurons, and demonstrate, in these neurons (but not in the SNr), a large potentiation of rotenone‐induced [Ca²⁺]_i rise by a small increase in baseline [Ca²⁺]_i.     

Effect of the removal of extracellular Ca on the response of cytosolic concentrations of Ca to ouabain in carotid body glomus cells of adult rabbits

Effect of the removal of extracellular Ca²⁺ on the response of cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) to ouabain, an Na⁺/K⁺ exchanger antagonist, was examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2AM and microfluorometry. Application of ouabain (10 mM) induced a sustained increase in [Ca²⁺]_i (mean±S.E.M.; 38±5% increase, n=16) in 55% of tested cells (n=29). The ouabain-induced [Ca²⁺]_i increase was abolished by the removal of extracellular Na⁺. D600 (50 μM), an L-type voltage-gated Ca²⁺ channel antagonist, inhibited the [Ca²⁺]_i increase by 57±7% (n=4). Removal of extracellular Ca²⁺ eliminated the [Ca²⁺]_i increase, but subsequent washing out of ouabain in Ca²⁺-free solution produced a rise in [Ca²⁺]_i (62±8% increase, n=6, P<0.05), referred to as a [Ca²⁺]_i rise after Ca²⁺-free/ouabain. The magnitude of the [Ca²⁺]_i rise was larger than that of ouabain-induced [Ca²⁺]_i increase. D600 (5 μM) inhibited the [Ca²⁺]_i rise after Ca²⁺-free/ouabain by 83±10% (n=4). These results suggest that ouabain-induced [Ca²⁺]_i increase was due to Ca²⁺ entry involving L-type Ca²⁺ channels which could be activated by cytosolic Na⁺ accumulation. Ca²⁺ removal might modify the [Ca²⁺]_i response, resulting in the occurrence of a rise in [Ca²⁺]_i after Ca²⁺-free/ouabain which mostly involved L-type Ca²⁺ channels.     

Local calcium-dependent mechanisms determine whether a cut axonal end assembles a retarded endbulb or competent growth cone

The transformation of a cut axonal end into a growth cone (GC), after axotomy, is a critical event in the cascade leading to regeneration. In an earlier series of studies we analyzed the cellular cascades that transform a cut axonal end into a competent GC. We found that axotomy of cultured Aplysia neurons leads to a transient elevation of the free intracellular Ca²⁺ concentration ([Ca²⁺]_i), calpain activation and localized proteolysis of submembranal spectrin. These events are associated with the formation of distinct microtubule (MT) based vesicle traps that accumulate anterogradely transported vesicles that fuse with the spectrin free plasma membrane in support of the growth process (Erez, H., Malkinson, G., Prager-Khoutorsky, M., De Zeeuw, C.I., Hoogenraad, C.C., and Spira, M.E. 2007. Formation of microtubule-based traps controls the sorting and concentration of vesicles to restricted sites of regenerating neurons after axotomy. J. Cell Biol. 176: 497–507.; Erez, H., and Spira, M.E. 2008. Local self-assembly mechanisms underlie the differential transformation of the proximal and distal cut axonal ends into functional and aberrant growth cones. J. Comp. Neurol. 507: spc1.). Here we report that under conditions that limit calcium influx into the cut axonal end, axotomy leads to the formation of endbulbs (EBs) rather than to competent GCs. Under these conditions typical MT based vesicle traps are not formed, and Golgi derived vesicles concentrate at the very tip of the cut axon. Since under these conditions the spectrin barrier is not cleaved, vesicle fusion with the plasma membrane and actin polymerization are retarded and growth processes are impaired. We conclude that the immediate assembly of competent GC or an EB after axotomy is the outcome of autonomous local events that are shaped by the magnitudes of the [Ca²⁺]_i gradients at the site of injury.     

Calcium oscillations in a subpopulation of S-antigen-immunoreactive pinealocytes of the rainbow trout (Oncorhynchus mykiss)

By means of the fura-2 technique and image analysis the intracellular concentration of free calcium ions [Ca²⁺]_i was examined in isolated rainbow trout pinealocytes identified by S-antigen immunocytochemistry. Approximately 30% of the pinealocytes exhibited spontaneous [Ca²⁺]_i oscillations whose frequency differed from cell to cell. Neither illumination with bright light nor dark adaptation of the cells had an apparent effect on the oscillations. Removal of extracellular Ca²⁺ or application of 10 μM nifedipine caused a reversible breakdown of the [Ca²⁺]_i oscillations. Application of 60 mM KCl elevated [Ca²⁺]_i in 90% of the oscillating and 50% of the non-oscillating pinealocytes. The effect of KCl was blocked by 50 μM nifedipine. These results suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca²⁺]_i in trout pinealocytes. Experiments with thapsigargin (2 μM) revealed the presence of intracellular calcium stores in 80% of the trout pinealocytes, but their role for regulation of [Ca²⁺]_i remains elusive. Treatment with norepinephrine (100 pM–50 μM), previously shown to induce calcium release from intracellular calcium stores in rat pinealocytes, had no apparent effect on [Ca²⁺]_i in any trout pinealocyte. This finding conforms to the concept that noradrenergic mechanisms are not involved in signal transduction in the directly light-sensitive pineal organ of anamniotic vertebrates.     

Heterogeneity in cytosolic calcium responses to hypoxia in carotid body cells

Previous investigators have reported that intracellular pH responds to hypoxia with a heterogenous pattern in individual glomus cells of the carotid body. The aim of the present study was to examine whether hypoxia had similar effects on cytosolic calcium ([Ca²⁺]_i) in glomus cells, and if so, whether a heterogenous response pattern is also seen in other cell types. Experiments were performed on glomus cells from adult rat carotid bodies, rat pheochromocytoma (PC12) and vascular smooth muscle (A7r5) cells. Changes in [Ca²⁺]_i in individual cells were determined by fluorescence imaging using Fura-2. Glomus cells were identified by catecholamine fluorescence. [Ca²⁺]_i in glomus cells increased in response to hypoxia (pO₂ = 35 ± 8mmHg; 5 min), whereas hypoxia induced decreases in [Ca²⁺]_i were not seen. Increases in [Ca²⁺]_i were observed in 20% of the isolated cells and strings of cells, but clustered glomus cells never responded. The magnitude of the calcium change in responding cells was proportional to the hypoxic stimulus. Under a given hypoxic challenge, there were marked variations in the response pattern between glomus cells. The response pattern characteristic of any given cell was reproducible. At comparable levels of hypoxia, PC12 cells also responded with an increase in [Ca²⁺]_i with a heterogenous response pattern similar to that seen in glomus cells. In contrast, increases in [Ca²⁺]_i in A7r5 cells could be seen only with sustained hypoxia ( ∼ 20 min), and little heterogeneity in the response patterns was evident. These results demonstrate that: (a) hypoxia increases cytosolic calcium in glomus cells; (b) response patterns were heterogeneous in individual cells; and (c) the pattern of the hypoxia-induced changes in [Ca²⁺]_i is cell specific. These results suggest that hypoxia-induced increases in [Ca²⁺]_i are faster in secretory than in non-secretory cells.     

PACAP Increases the Cytosolic Ca2−/ Concentration and Stimulates Somatodendritic Vasopresson Release in Rat Supraoptic Neurons

Pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity and its receptor mRNA have been reported in the supraoptic and the paraventricular nucleus (SON and PVN, respectively) and PACAP has been implicated in the regulation of magnocellular neurosecretory cell function. To examine the site and the mechanism of the action of PACAP in the neurosecretory cells, we measured AVP release from SON slice preparations and the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) from single dissociated SON neurons. PACAP at concentrations from 10^?12 to 10^?7 M increased [Ca²⁺]_i in dissociated SON neurons in a dose-dependent manner. The patterns of the PACAP-induced [Ca²⁺]_i increase were either sustained increase or cytosolic Ca²⁺ oscillations. PACAP (10^?7 M) increased [Ca²⁺]_i in 27 of 27 neurons and glutamate (10^?4 M) increased [Ca²⁺]_i in 19 of 19 SON neurons examined, whereas angiotensin II (10^?7 M) increased [Ca²⁺]_i in only 15 of 60 SON neurons examined. PACAP at lower concentrations (10^?10 to 10^?8 M) increased [Ca²⁺]_i in 70–80% of neurons examined. Although the onset and recovery of the PACAP-induced [Ca²⁺]_i increase were slower than those observed with glutamate, the spatial distribution of the [Ca²⁺]_i increases in response to the two ligands were similar: [Ca²⁺]_i increase at the proximal dendrites was larger and faster and that at the center of the soma was smaller and slower. The PACAP-induced [Ca²⁺]_i responseswere abolished by extracellular Ca²⁺ removal, the l -type Ca²⁺-channel blocker, nicardipine, or by replacement of extracellular Na⁺ with N-methyl d-glucamine, and were partially inhibited by the Na⁺-channel blocker, tetrodotoxin. The N-type Ca²⁺-channel blocker, ω-conotoxin GVIA did not significantly inhibit the PACAP-induced [Ca²⁺]_i responses. Furthermore, PACAP (10^?7 M) as well as glutamate (10^?4 M) increased AVP release from SON slice preparations, and extracellular Ca²⁺ removal or nicardipine inhibited the AVP release in response to PACAP. These results indicate that PACAP enhances Ca²⁺ entry via voltage-gated Ca²⁺ channels and increases [Ca²⁺]_i, which, in turn, stimulates somatodendritic vasopressin release by directly activating PACAP receptors on SON neurons. The results also suggest that PACAP in the SON may play a pivotal role in the control of the neurohypophyseal function at the level of the soma or the dendrites.     

Opioid regulation of intracellular free calcium in cultured mouse dorsal root ganglion neurons

Opioid agonists induced an increase in the intracellular free calcium concentration ([Ca²⁺]_i) or an inhibition of K⁺ (25 mM)-stimulated increase in [Ca²⁺]_i in different subsets of mouse dorsal root ganglion (DRG) neurons. The total neuronal population was grouped into three classes according to somatic diameter and defined as small (<16 μm), intermediate (16–25 μm), or large (>25 μm) neurons. Substance P-like immunoreactivity was detected mainly in the small and intermediate neurons. The δ, κ, and μ opioid receptor agonists [D-Ser², Leu⁵]enkephalin-Thr (DSLET), U69593, and [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO) each induced a transient increase in [Ca²⁺]_i in a small fraction (<30%) of neurons. The increases in [Ca²⁺]_i were blocked by the opioid antagonist naloxone. The dihydropyridine-sensitive calcium channel blocker nifedipine also blocked the increase in [Ca²⁺]_i induced by 1 μM DSLET. The rank order of potency (percentage of cells responding to each opioid agonist) was DSLET > U69593 > DAMGO. The opioid-induced increase in [Ca²⁺]_i was observed mainly in large neurons, with a low incidence in small and intermediate neurons. Opioid agonists also caused inhibition of K⁺-stimulated increases in [Ca²⁺]_i, which were blocked by naloxone (1 μM). Inhibition of the K⁺-stimulated increase by 1 μM DSLET or U69593 was greater in small and intermediate neurons than in large neurons. © 1996 Wiley-Liss, Inc.     

Calcium dependence of the priming,activation and inactivation of ryanodine receptors in frog motor nerve terminals

We studied the effects of varying extracellular Ca²⁺ ([Ca²⁺]_o) and Ca²⁺ channel density and intracellular loading of Ca²⁺ chelators on stimulation‐induced rises in intracellular Ca²⁺ ([Ca²⁺]_i) in frog motor nerve terminals with Ca²⁺ imaging. The slowly waxing and waning components of rises in [Ca²⁺]_i induced by repetitive tetani were suppressed by blockers of Ca²⁺ pumps of the endoplasmic reticulum (thapsigargin and cyclopiazonic acid) and a blocker of ryanodine receptors [8‐(N,N‐diethylamino)octyl 3,4,5‐trimethoxybenzoate hydrochloride] without affecting the initial quickly‐rising component, thus reflecting the priming (and then subsequent rapid activation) and inactivation phases of Ca²⁺‐induced Ca²⁺ release (CICR) from the endoplasmic reticulum. A short tetanus‐induced rise in [Ca²⁺]_i was proportional to [Ca²⁺]_o, whereas the component of CICR was non‐linearly related to [Ca²⁺]_o with saturation at 0.9 mm . The progressive blockade of Ca²⁺ channels by ω‐conotoxin GVIA caused proportional decreases in CICR and short tetanus‐induced [Ca²⁺]_i rises. Intracellular loading of BAPTA and EGTA reduced the magnitude of CICR as well as short tetanus‐induced rises in [Ca²⁺]_i with a greater effect of BAPTA than EGTA on CICR. The time to peak and the half decay time of CICR were prolonged by a low [Ca²⁺]_o or Ca²⁺ channel blocker or [Ca²⁺]_i chelators. These results suggest that ryanodine receptors sense the high [Ca²⁺]_i transient following single action potentials for triggering CICR, whereas the priming and inactivation processes of CICR sense a slower, persisting rise in [Ca²⁺]_i during and after action potential trains. A model is presented that includes CICR activation in elementary units.     

Chronic lead exposure alters presynaptic calcium regulation and synaptic facilitation in Drosophila larvae

Prolonged exposure to inorganic lead (Pb²⁺) during development has been shown to influence activity-dependent synaptic plasticity in the mammalian brain, possibly by altering the regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). To explore this possibility, we studied the effect of Pb²⁺ exposure on [Ca²⁺]_i regulation and synaptic facilitation at the neuromuscular junction of larval Drosophila. Wild-type Drosophila (CS) were raised from egg stages through the third larval instar in media containing either 0 μM, 100 μM or 250 μM Pb²⁺ and identified motor terminals were examined in late third-instar larvae. To compare resting [Ca²⁺]_i and the changes in [Ca²⁺]_i produced by impulse activity, the motor terminals were loaded with a Ca²⁺ indicator, either Oregon Green 488 BAPTA-1 (OGB-1) or fura-2 conjugated to a dextran. We found that rearing in Pb²⁺ did not significantly change the resting [Ca²⁺]_i nor the Ca²⁺ transient produced in synaptic boutons by single action potentials (APs); however, the Ca²⁺ transients produced by 10 Hz and 20 Hz AP trains were larger in Pb²⁺-exposed boutons and decayed more slowly. For larvae raised in 250 μM Pb²⁺, the increase in [Ca²⁺]_i during an AP train (20 Hz) was 29% greater than in control larvae and the [Ca²⁺]_i decay τ was 69% greater. These differences appear to result from reduced activity of the plasma membrane Ca²⁺ ATPase (PMCA), which extrudes Ca²⁺ from these synaptic terminals. These findings are consistent with studies in mammals showing a Pb²⁺-dependent reduction in PMCA activity. We also observed a Pb²⁺-dependent enhancement of synaptic facilitation at these larval neuromuscular synapses. Facilitation of EPSP amplitude during AP trains (20 Hz) was 55% greater in Pb²⁺-reared larvae than in controls. These results showed that Pb²⁺ exposure produced changes in the regulation of [Ca²⁺]_i during impulse activity, which could affect various aspects of nervous system development. At the mature synapse, this altered [Ca²⁺]_i regulation produced changes in synaptic facilitation that are likely to influence the function of neural networks.     

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.