Involvement of Na+–Ca2+ Exchanger in Reperfusion-induced Delayed Cell Death of Cultured Rat Astrocytes

In some cells, Ca²⁺ depletion induces an increase in intracellular Ca²⁺ ([Ca²⁺]_i) after reperfusion with Ca²⁺-containing solution, but the mechanism for the reperfusion injury is not fully elucidated. Using an antisense strategy we studied the role of the Na⁺-Ca²⁺ exchanger in reperfusion injury in cultured rat astrocytes. When astrocytes were perfused in Ca²⁺-free medium for 15–60 min, a persistent increase in [Ca²⁺]_i was observed immediately after reperfusion with Ca²⁺-containing medium, and the number of surviving cells decreased 3–5 days latter. The increase in [Ca²⁺]_i was enhanced by low extracellular Na⁺ ([Na⁺]_o) during reperfusion and blocked by the inhibitors of the Na⁺-Ca²⁺ exchanger amiloride and 3,4-dichlorobenzamil, but not by the Ca²⁺ channel antagonists nifedipine, Cd²⁺ and Ni²⁺. Treatment of astrocytes with antisense, but not sense, oligodeoxynucleotide to the Na⁺-Ca²⁺ exchanger decreased Na⁺–Ca²⁺ exchanger protein level and exchange activity. The antisense oligomer attenuated reperfusion-induced increase in [Ca²⁺]ⁱ and cell toxicity. The Na⁺-Ca²⁺ exchange inhibitors 3,4-dichlorobenzamil and ascorbic acid protected astrocytes from reperfusion injury partially, while the stimulators sodium nitroprusside and 8-bromo-cyclic GMP and low [Na⁺]_o exacerbated the injury. Pretreatment of astrocytes with ouabain and monensin caused similar delayed glial cell death. These findings suggest that Ca²⁺ entry via the Na⁺–Ca²⁺ exchanger plays an important role in reperfusion-induced delayed glial cell death.     

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.     

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.     

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.     

Protein kinase C modulates field-evoked transmitter release from cultured rat cerebellar granule cells via a dendrotoxin-sensitive K+ channel

The role of protein kinase C (PKC) in the control of neurotransmitter release from cultured rat cerebellar granule cells was investigated. Release of preloaded [³H]-d -aspartate which is incorporated into synaptic vesicles in this preparation was evoked by electrical field stimulation or elevated KCl. PKC activation by phorbol esters resulted in a large facilitation of field-evoked Ca²⁺-dependent [³H]-d -aspartate release and a lesser enhancement of KCl-stimulated release. Inhibition of PKC by Ro 31-8220 or staurosporine virtually abolished field-evoked release but had no effect on KCl-evoked release. Field-evoked, but not KCl-evoked, synaptic vesicle exocytosis monitored by the fluorescent vesicle probe FM2-10 was inhibited by staurosporine. PKC was not directly modulating neurite Ca²⁺ channels coupled to release, as Ro 31-8220 did not inhibit these channels. Activation or inhibition of PKC modulated field-evoked plasma membrane depolarization, but had no effect on KCl-evoked depolarization, consistent with a regulation of Na⁺ or K⁺ channels activated by field stimulation. No modulation of field-evoked neurite Na⁺ influx was seen using phorbol esters. Phorbol ester-induced facilitation of field-evoked [³H]-d -aspartate release and neurite Ca²⁺ entry was non-additive with that produced by the specific K⁺ channel antagonist dendrotoxin-1, suggesting that PKC modulates transmitter release from field-stimulated cerebellar granule cells by inhibiting a dendrotoxin-1-sensitive K⁺ channel.     

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.     

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.     

Intracellular Action of Spermine on Neuronal Ca2+ and K+ Currents

Intra-and extracellular effects of the polyamine spermine on electrical activity and membrane currents of identified neurons in the abdominal ganglion of Aplysia californica were studied under current-and voltage-clamp conditions. Lonophoretic injection of spermine reduced the amplitude of action potentials and altered their time course as well as spontaneous discharge activity. Investigation of membrane currents showed that intracellular spermine suppressed the total outward current but increased the inward rectifier current. After separation of ion currents it was found that the voltage-activated, delayed K⁺ outward current and the Ca²⁺ inward current were reduced by intracellular spermine in a dose- and voltage-dependent manner. The block of the K⁺ current can be described by a voltage-dependent reaction, where one spermine molecule binds to one channel. The binding constant K_b, at zero voltage, and the effective valency, zδ, had values of 176/M and 0.41 for cell R-15, 223/M and 0.64 for cell L-11, and 137/M and 0.42 for cell L-3. Apparently, more than one spermine cation is needed to block one Ca²⁺ channel, since the coefficient n, which absorbs the molecularity and cooperativity of the reaction, had non-integral values between 1.34 and 2.22. The binding constant K_b and the effective valency zδ had values of 265/M and 0.64 for cell R-15, 821M and 0.56 for cell L-4, and 263/M and 0.51 for cell L-6. Intracellular spermine also blocked the Ca²⁺-activated K⁺ current induced by ionophoretic Ca²⁺-injections, but increased the current at prolonged times after spermine injection. Extracellular spermine had no effect on electrical activity or on membrane currents. The results indicate that intracellular spermine affects the electrical discharge activity of neurons by acting as a blocker and/or modulator at voltage-dependent membrane conductances.     

Na+—Ca2+ Exchange Currents in Cortical Neurons: Concomitant Forward and Reverse Operation and Effect of Glutamate

Na⁺-Ca²⁺ exchanger-associated membrane currents were studied in cultured murine neocortical neurons, using whole-cell recording combined with intracellular perfusion. A net inward current specifically associated with forward (Na⁺_o-Ca²⁺_i) exchange was evoked at -40 mV by switching external 140 mM Li⁺ to 140 mM Na⁺. The voltage dependence of this current was consistent with that predicted for 3Na⁺:1Ca²⁺ exchange. As expected, the current depended on internal Ca²⁺, and could be blocked by intracellular application of the exchanger inhibitory peptide, XIP. Raising internal Na⁺ from 3 to 20 mM or switching the external solution from 140 mM Li⁺ to 30 mM Na⁺ activated outward currents, consistent with reverse (Na⁺,-Ca²⁺_o) exchange. An external Ca²⁺-sensitive current was also identified as associated with reverse Na⁺-Ca²⁺ exchange based on its internal Na⁺ dependence and sensitivity to XIP. Combined application of external Na⁺ and Ca²⁺ in the absence of internal Na⁺ triggered a 3.3–fold larger inward current than the current activated in the presence of 3 mM internal Na⁺, raising the intriguing possibility that Na⁺-Ca²⁺ exchangers might concurrently operate in both the forward and the reverse direction, perhaps in different subcellular locations. With this idea in mind, we examined the effect of excitotoxic glutamate receptor activation on exchanger operation. After 3–5 min of exposure to 100–200 μM glutamate, the forward exchanger current was significantly increased even when external Na⁺ was reduced to 100 mM, and the external Ca²⁺-activated reverse exchanger current was eliminated.     

Effects of Growth Hormone-Releasing Peptide-2 (GHRP-2) on Membrane Ca2+ Permeability in Cultured Ovine Somatotrophs

The newly synthesized GH-releasing peptide, GHRP-2 (D-Ala-D-βNal-Ala-Trp-D-Phe-Lys-NH₂), was studied in somatotroph-enriched populations of ovine pituitary cells in primary culture. Nystatin-perforated whole-cell recordings were made on identified somatotrophs after 4–14 days of culture. Using a standard bath solution (containing Na⁺, Ca²⁺) and an electrode solution containing K⁺ in current-clamp recordings, GHRP-2 (10 nM) depolarized the membrane potential of the cells triggering a burst of action potentials. Voltage-clamp recordings indicated that GHRP-2 produced a slowly inactivated inward current with a slight reduction in outward current. The inward current was blocked by the Ca²⁺ channel blocker, Co²⁺ (0.5 mM). Ca²⁺ currents were then isolated using tetraethylammonium bath solution and an electrode solution containing Cs⁺. Ovine somatotrophs possess transient (T type) and long lasting (L type) Ca²⁺ currents. The L type current was abolished by addition of nifedipine (3 μM) to the bath solution and T type current was isolated on this basis. Current-voltage relationships indicated an increase in both T and L type Ca²⁺ currents in response to GHRP-2. The voltage-dependent inactivation curve for T type Ca²⁺ current was shifted towards a less negative level by the peptide. Intracellular free Ca²⁺ concentration ([Ca²⁺]i) in somatotroph-enriched populations was specifically increased by GHRP-2 but this effect was totally abolished by blockade of membrane Ca²⁺ channels. These data show that GHRP-2 causes an influx of Ca²⁺ leading to an increase in [Ca²⁺]i in ovine somatotrophs. The Ca²⁺ currents were both L type and T type with a shift in the inactivation curve of the latter by the releasing peptide.     

Multiple Types of Ca2+ Channels in Mouse Motor Nerve Terminals

We measured neurotransmitter release and motor nerve terminal currents in mouse phrenic nerve-diaphragm and triangularis sterni preparations, to evaluate the role of Ca²⁺-channel subtypes in regulating transmitter release. Saturated concentrations of either ωagatoxin IVA [ω-Aga-IVA (0.3 μM), a blocker of P-type Ca²⁺channels] or ω-conotoxin MVIIC [ω-CTx-MVIIC (2 μM), a P-and Q-type Ca²⁺-channel blocker], inhibited nerve-evoked muscle contractions and the amplitude of endplate potentials respectively. In contrast, combined treatment with nifedipine (50 μM, a blocker of L-type Ca²⁺ channels) plus ω-conotoxin GVIA [ω-CTx-GVIA (2 μM), a blocker of N-type Ca²⁺ channels] did not elicit inhibitory effects on nerve-evoked muscle contractions, endplate potentials or nerve terminal waveforms. Because of the non-linear relationship between endplate potentials and Ca²⁺ signals, a small decrease in presynaptic Ca²⁺ entry can significantly reduce the amplitude of the endplate potential. Thus, we applied 3, 4-diaminopyridine (3, 4-DAP, a k⁺-channel blocker) or high Ca²⁺(10 mM) to accelerate and amplify the endplate potentials and Ca²⁺ currents. The endplate potentials amplified by 3, 4-DAP or by high Ca²⁺ correspondingly proved to be quite resistant to both ω-Aga-IVA and ω-CTx-MVIIC; ωAga-IVA exerted only a partial inhibitory effect on endplate potentials, and the ω-Aga-IVA-resistant component was further inhibited by ω-CTx-MVIIC. The component that was resistant to the two toxins could be completely blocked by the non-selective Ca²⁺ channel blocker Cd²⁺ (300 μM). A combination of the two toxins had no significant effects on either spontaneous transmitter release or postsynaptic resting membrane potentials of the diaphragm preparation and the Na⁺ and K⁺ waveforms of the triangularis sterni preparations. This finding suggests a preferential inhibitory effect at a presynaptic site. Measuring the Ca²⁺ currents in the triangularis sterni also revealed partial inhibition by ω-CTx-MVIIC with further incomplete inhibition by ω-Aga-IVA. Cd²⁺ (300 μM) abolished the toxin-resistant component of the Ca²⁺ current. In contrast, a combination of nifedipine (50 μM) with ω-CTx-GVIA (2 μM) was without inhibitory effect. We conclude that multiple types of Ca²⁺channels, i.e. ω-Aga-IVA-sensitive, ω-CTx-MVIIC-sensitive and toxin-resistant Ca²⁺ channels, coexist in mouse motor nerve terminals.     

Regulation of Intracellular Ca2+ in Response to Muscarinic and Glutamate Receptor Agonists During the Differentiation of NTERA2 Human Embryonal Carcinoma Cells into Neurons

Single cell microfluorimetry was used to study intracellular calcium ion signals ([Ca²⁺]_i) evoked by acetylcholine (ACh), glutamate receptor agonists and by KCI-induced membrane depolarization, during neuronal differentiation of the human embryonal carcinoma (EC) cell line, NTERA2. In undifferentiated NTERA2 EC cells, [Ca²⁺]_i) was elevated in response to ACh, but not to the glutamate receptor agonists NMDA, kainate or AMPA. The ACh-induced rise in [Ca²⁺]_i) was dependent upon both Ca²⁺ influx and Ca²⁺ mobilization from cytoplasmic calcium stores. Three other human EC cell lines responded similarly to ACh but not to glutamate or KCI-induced depolarization. In neurons derived from NTERA2 cells by retinoic acid induction, [Ca²⁺]_i) signals were evoked by ACh, NMDA, kainate and by an elevation of the extracellular KCI concentration. As in undifferentiated EC cells, the ACh-mediated increases in [Ca²⁺li were governed by both Ca²⁺ influx and Ca²⁺ mobilization. In contrast, the effects of NMDA, kainate and KCI did not involve intracellular Ca²⁺ mobilization. The appearance of glutamate and KCI responsiveness was not detected in non-neuronal differentiated derivatives of NTERA2 cells. Using a number of pharmacologically defined muscarinic receptor antagonists we found that NTERA2 EC cells express M₁, M₃, M₄ and possibly M₅ receptor subtypes linked to changes in [Ca²⁺]_i), whilst only M₃ and M₅ are present in NTERA2-derived neurons. The results were supported by PCR analysis of the muscarinic mRNA species expressed in the cells. The data demonstrate that differentiation of NTERA2 EC cells into neurons involves the induction of functional glutamate receptors coupled to rises in [Ca²⁺]_i), and changes in the expression of muscarinic ACh receptor subtypes.     

Cyclic AMP enhances acetylcholine (ACh)- induced ion fluxes and catecholamine release by inhibiting Na+, K+-ATPase and participates in the responses to ACh in cultured bovine adrenal medullary chromaffin cells

Summary The effects of cyclic AMP (cAMP) on intracellular Na⁺ concentration ([Na⁺]i), membrane depolarization and intracellular Ca²⁺ concentration ([Ca²⁺]i) and the involvement of cAMP in acetylcholine (ACh)-induced such cellular events and catecholamine (CA) release were studied in cultured bovine adrenal medullary chromaffin cells. 8-Bromo-cyclic AMP (8Br-cAMP) and forskolin caused a rise in [Na⁺]i, membrane depolarization and a rise in [Ca²⁺]i and potentiated these responses and CA release to ACh. The effects of 8Br-cAMP or forskolin on ACh-induced changes of but not on basal level of [Na⁺]i, membrane potential and [Ca²⁺]i were blocked by tetrodotoxin (TTX, 1 M). In Na⁺ deprivated medium, forskolin failed to produce an increase in basal [Ca²⁺]i level and to potentiate ACh-induced rise. The similar results as in 8Br-cAMP and forskolin were obtained using ouabain, and 8Br-cAMP or foskolin produced no further effects in the presence of ouabain. Inhibitors of cAMP-dependent protein kinase not only blocked the effects of 8Br-cAMP and forskolin on membrane depolarization, [Ca²⁺]i rise and CA release, but also reduced these responses to ACh. From the similarity between the effects of cAMP and those of ouabain on the cellular events and the counteraction of the effects of cAMP by ouabain, it may be suggested that cAMP produces its effects on ion fluxes and CA release probably via an inhibition of Na⁺, K⁺-ATPase in intact chromaffin and cAMP may participate in the responses to ACh.     

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.     

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.     

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.     

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.     

Potentiometric study of resting potential, contributing K+ channels and the onset of Na+ channel excitability in embryonic rat cortical cells

Resting membrane potential (RMP), K⁺ channel contribution to RMP and the development of excitability were investigated in the entire population of acutely dissociated embryonic (E) rat cortical cells over E11–22 using a voltage-sensitive fluorescent indicator dye and flow cytometry. During the period of intense proliferation (E11–13), two cell subpopulations with distinct estimated RMPs were recorded: one polarized at ∼–70 mV and the other relatively less-polarized at ∼–40 mV. Ca²⁺_o was critical in sustaining the RMP of the majority of less-polarized cells, while the well-polarized cells were characterized by membrane potentials exhibiting a ∼Nernstian relationship between RMP and [K⁺]_o. Analysis of these two subpopulations revealed that > 80% of less-polarized cells were proliferative, while > 90% of well-polarized cells were postmitotic. Throughout embryonic development, the disappearance of Ca²⁺_o-sensitive, less-polarized cells correlated with the disappearance of the proliferating population, while the appearance of the K⁺_o-sensitive, well-polarized population correlated with the appearance of terminally postmitotic neurons, immuno-identified as BrdU^–, tetanus toxin⁺ cells. Differentiating neurons were estimated to contain increased K⁺_i relative to less-polarized cells, coinciding with the developmental expression of Cs⁺/Ba²⁺-sensitive and Ca²⁺-dependent K⁺ channels. Both K⁺ channels contributed to the RMP of well-polarized cells, which became more negative toward the end of neurogenesis. Depolarizing effects of veratridine, first observed at E11, progressively changed from Ca²⁺_o-dependent and tetrodotoxin-insensitive to Na⁺_o-dependent and tetrodotoxin-sensitive response by E18. The results reveal a dynamic development of RMP, contributing K⁺ channels and voltage-dependent Na⁺ channels in the developing cortex as it transforms from proliferative to primarily differentiating tissue.     

Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular Ca and glycolysis

To elucidate the mechanism of pH_i changes induced by membrane depolarization, the variations in pH_i and [Ca²⁺]_i induced by a number of depolarizing agents, including high K⁺, veratridine, N-methyl-

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-aspartate (NMDA) and ouabain, were investigated in rat hippocampal slices by the fluorophotometrical technique using BCECF or fura-2. All of these depolarizing agents elicited a decrease in pH_i and an elevation of intracellular calcium ([Ca²⁺]_i) in the CA1 pyramidal cell layer. The increases in [Ca²⁺]_i caused by the depolarizing agents almost completely disappeared in the absence of Ca²⁺ (0 mM Ca²⁺ with 1 mM EGTA). In Ca²⁺ free media, pH_i acid shifts produced by high K⁺, veratridine or NMDA were attenuated by 10–25%, and those produced by ouabain decreased by 50%. Glucose-substitution with equimolar amounts of pyruvate suppressed by two-thirds the pH_i acid shifts induced by both high K⁺ and NMDA. Furthermore, lactate contents were significantly increased in hippocampal slices by exposure to high K⁺, veratridine or NMDA but not by ouabain. These results suggest that the intracellular acidification produced by these depolarizing agents, with the exception of ouabain, is mainly due to lactate accumulation which may occur as a result of accelerated glycolysis mediated by increased Na⁺–K⁺ ATPase activity. A Ca²⁺-dependent process may also contribute to the intracellular acidification induced by membrane depolarization. Since an increase in H⁺ concentration can attenuate neuronal activity, glycolytic acid production induced by membrane depolarization may contribute to the mechanism that prevents excessive neuronal excitation.     

Release of γ-[H]aminobutyric acid from synaptosomes: effect of external cations and of ouabain

In the present study we have investigated the effect of cations and ouabain on Ca²⁺-independent and Ca²⁺-dependent release of γ-[³H]aminobutyric acid ([³H]GABA) from sheep brain synaptosomes. The presence of Na⁺ in the external medium is essential for the Ca²⁺-independent release induced by K⁺ or ouabain. Thus, in the absence of Ca²⁺, ouabain of K⁺ causes the release of [³H]GABA provided that Na⁺ is present in the external medium. Under K⁺-depolarizing conditions, in a Na⁺ medium, either ouabain or Ca²⁺ further increases the [³H]GABA release induced by depolarization, but their effects are not additive. The presence of external Na⁺ is not required for the Ca²⁺-dependent release of [³H]GABA due to K⁺ depolarization, and this release, which occurs in a choline medium, is not modified by ouabain. Under these conditions (choline medium) K⁺-depolarization dependent release is absolutely dependent on external Ca²⁺, which suggests that this release of [³H]GABA occurs only by exocytosis, without the carrier-mediated efflux which normally co-exists with exocytosis due to K⁺-depolarization in a Na⁺ medium. It is likely that the release induced by ouabain or K⁺ involves the membrane carrier which responds to changes in membrane potential.