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.     

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.     

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.     

Differential Stimulation of Noradrenaline Release by Reversal of the Na+/Ca2+ Exchanger and Depolarization in Chromaffin Cells

We compared the effectiveness of Ca²⁺ entering by Na⁺/Ca²⁺ exchange with that of Ca²⁺ entering by channels produced by membrane depolarization with K⁺ in inducing catecholamine release from bovine adrenal chromaffin cells. The Ca²⁺ influx through the Na⁺/Ca²⁺ exchanger was promoted by reversing the normal inward gradient of Na⁺ by preincubating the cells with ouabain to increase the intracellular Na⁺ and then removing Na⁺ from the external medium. In this way we were able to increase the cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) by Na⁺/Ca²⁺ exchange to 325 ± 14 nM, which was similar to the rise in [Ca²⁺]_c observed upon depolarization with 35 mM K⁺ of cells not treated with ouabain. After incubating the cells with ouabain, K⁺ depolarization raised the [Ca²⁺]_c to 398 ± 31 nM, and the recovery of [Ca²⁺]_c to resting levels was significantly slower. Reversal of the Na⁺ gradient caused an −6-fold increase in the release of noradrenaline or adrenaline, whereas K⁺ depolarization induced a 12-fold increase in noradrenaline release but only a 9-fold increase in adrenaline release. The ratio of noradrenaline to adrenaline release was 1.24 ± 0.23 upon reversal of the Na⁺/Ca²⁺ exchange, whereas it was 1.83 ± 0.19 for K⁺ depolarization. Reversal of the Na⁺/Ca²⁺ exchange appeared to be as efficient as membrane depolarization in inducing adrenaline release, in that the relation of [Ca²⁺]_c to adrenaline release was the same in both cases. In contrast, we found that for the same average [Ca²⁺]_c, the Ca²⁺ influx through voltage-gated channels was much more efficient than the Ca²⁺ entering through the Na⁺/Ca²⁺ exchanger in inducing noradrenaline release from chromaffin ceils. This greater effectiveness of membrane depolarization in stimulating noradrenaline release suggests that there is a pool of noradrenaline vesicles which is more accessible to Ca²⁺ entering through voltage-gated Ca²⁺ channels than to Ca²⁺ entering through the Na⁺/Ca²⁺ exchanger, whereas the adrenaline vesicles do not distinguish between the source of Ca²⁺.     

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.     

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.     

Seletracetam (ucb 44212) inhibits high-voltage–activated Ca2+ currents and intracellular Ca2+ increase in rat cortical neurons in vitro

Purpose: We analyzed the effects of seletracetam (ucb 44212; SEL), a new antiepileptic drug candidate, in an in vitro model of epileptic activity. The activity of SEL was compared to the effects of levetiracetam (LEV; Keppra), in the same assays. Methods: Combined electrophysiologic and microfluorometric recordings were performed from layer V pyramidal neurons in rat cortical slices to study the effects of SEL on the paroxysmal depolarization shifts (PDSs), and the simultaneous elevations of intracellular Ca²⁺ concentration [Ca²⁺]_i. Moreover, the involvement of high‐voltage activated Ca²⁺ currents (HVACCs) was investigated by means of patch‐clamp recordings from acutely dissociated pyramidal neurons. Results: SEL significantly reduced both the duration of PDSs (IC₅₀ = 241.0 ± 21.7 nm ) as well as the number of action potentials per PDS (IC₅₀ = 82.7 ± 9.7 nm ). In addition, SEL largely decreased the [Ca²⁺]_i rise accompanying PDSs (up to 75% of control values, IC₅₀ = 345.0 ± 15.0 nm ). Furthermore, SEL significantly reduced HVACCs in pyramidal neurons. This effect was mimicked by ω‐conotoxin GVIA and, to a lesser extent, by ω‐conotoxin MVIIC, blockers of N‐ and Q‐type HVACC, respectively. The combination of these two toxins occluded the action of SEL, suggesting that N‐type Ca²⁺ channels, and partly Q‐type subtypes are preferentially targeted. Conclusions: These results demonstrate a powerful inhibitory effect of SEL on epileptiform events in vitro. SEL showed a higher potency than LEV. The effective limitation of [Ca²⁺]_i influx might be relevant for its antiepileptic efficacy and, more broadly, for pathologic processes involving neuronal [Ca²⁺]_i overload.     

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.     

Ethanol and nerve growth factor effects on calcium homeostasis in cultured embryonic rat medial septal neurons before and during depolarization

Ethanol and nerve growth factor (NGF) affect the survival of cholinergic neurons in the rat medial septum. To investigate whether calcium (Ca²⁺) homeostasis in these neurons is affected by ethanol or NGF treatment, changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) were studied in embryonic (E21) cultured medial septal neurons before stimulation (basal) and during stimulation with high potassium (K⁺). Changes in [Ca²⁺]; across time were measured in cultures of neurons treated without ethanol or with 100, 2110, 400, or 800 mg% ethanol with NGF (+NGF) or without NGF (-NGF). Changes in [Ca²⁺]_i were analyzed from fluorescence images, using indo-1. The effect of ethanol or NGF treatment was to reduce the rise in basal [Ca²⁺]_i. The combination of ethanol and NGF treatment in +NGF neurons led to increases in basal [Ca²⁺]_i with the greatest increase in basal [Ca²⁺]_i occurring with 200 mg% ethanol. The effect of ethanol or NGF was to increase [Ca²⁺]_i; during stimulation with high K⁺. The greatest increases in [Ca⁺]_i occurred with 100 and 800 mg% ethanol. Together, ethanol and NGF treatment in +NGF-treated neurons led to significantly greater increases or decreases in K⁺ stimulated changes in [Ca²⁺]_i compared to similarly treated -NGF neurons. We conclude that in medial septal neurons (before and during depolarization) changes in Ca²⁺ homeostasis occur in the presence of ethanol or NGF. The changes in [Ca²⁺]_i, following ethanol treatment are greater when NGF is present.     

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.     

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⁺].     

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.     

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.     

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.     

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.     

Stimulation of Ca2+ Entry in Lactotrophs and Somatotrophs from Immature Rat Pituitary by N-Terminal Fragments of Proopiomelanocortin

We have previously shown that 10–12 kDa N-terminal fragments of rat proopiomelanocortin (POMC) and human POMC_1–76 stimulate mitosis and/or differentiation in lactotrophs of early postnatal rat pituitary. A truncated form, POMC_1–26, mimics the differentiation-inducing but not the mitogenic action of the former peptides. To further characterize these two biological responses, the present study compared changes in the intracellular free calcium concentration ([Ca²⁺]_i) in response to POMC_1–76 and POMC_1–26 in isolated pituitary cells from 14-day-old female rats. Calcium (Ca²⁺) responses were also used as a guide to determine whether the responsive cells belong to the lactosomatotroph lineage. Application of POMC_1–76 or POMC_1–26 induced a maintained oscillating [Ca²⁺]_i increase in a small population of cells. Increasing doses of the peptides did not affect the magnitude and the frequency of [Ca²⁺]_i oscillations but clearly augmented the number of responding cells. Approximately 2% of the cells responded at 0.1 nM POMC_1–76 or 5 nM POMC_1–26, and 11–13% of the cells responded at 10 nM and 500 nM of the respective peptides. About one-third of the cells responsive to these peptides also showed a [Ca²⁺]_i increase in response to growth hormone-releasing peptide-6 (GHRP-6) while, in a small number of responsive cells, [Ca²⁺]_i was depressed by dopamine, suggesting that the former cells are somatotrophs and the latter lactotrophs. This interpretation was confirmed by immunocytochemical identification of prolactin and growth hormone (GH) in the cells. Of the cells showing Ca²⁺ response to POMC_1–76, approximately one-third contained GH and another third prolactin. The remainder contained neither GH nor prolactin. Comparable results were obtained with POMC_1–26. The rise of [Ca²⁺]_i induced by the N-terminal POMC peptides persisted after depletion of intracellular Ca²⁺ stores by thapsigargin. Removal of Ca²⁺ from the extracellular medium or addition of cadmium completely abolished both the POMC_1–76- and POMC_1–26-induced [Ca²⁺]_i increase. Nifedipine inhibited the Ca²⁺ response to both peptides, although only in 55% of the responsive cells. Depletion of some isoforms of protein kinase C by preincubation with the phorbol ester PMA for 24 h did not modify the Ca²⁺ responses. In contrast, blockade of the protein kinase A pathway with Rp-cAMPs partially inhibited the POMC_1–76- or POMC_1–26-induced [Ca²⁺]_i increase. The present data show that, in immature pituitary cells, POMC_1–76 induces an increase in [Ca²⁺]_i through extracellular Ca²⁺ influx, possibly mediated in part by protein kinase A activation. The active domain of POMC_1–76 seems to comprise its N-terminal moiety. The data support the hypothesis that POMC_1–76 exerts a specific function in the development of different members of the lactosomatotroph lineage and that the peptide mobilizes different subsets of cells within this lineage, by a mechanism determined by its concentration.     

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.     

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.     

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.     

K-current modulated by PO2 in type I cells in rat carotid body is not a chemosensor

According to the membrane channel hypothesis of carotid body O₂ chemoreception, hypoxia suppresses K⁺ currents leading to cell depolarization, [Ca²⁺]_i rise, neurosecretion, increased neural discharge from the carotid body. We show here that tetraethylammonium (TEA) plus 4-aminopyridine (4-AP) which suppressed the Ca²⁺ sensitive and other K⁺ currents in rat carotid body type I cells, with and without low [Ca²⁺]_o plus high [Mg²⁺]_o, did not essentially influence low

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effects on [Ca²⁺]_i and chemosensory discharge. Thus, hypoxia may suppress the K⁺ currents in glomus cells but K⁺ current suppression of itself does not lead to chemosensory excitation. Therefore, the hypothesis that K⁺–O₂ current is linked to events in chemoreception is not substantiated. K⁺–O₂ current is an epiphemenon which is not directly linked with O₂ chemoreception.