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.     

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.     

Re-evaluation of acute neurotoxic effects of Cd on mesencephalic trigeminal neurons of the adult rat

The mechanism of Cd²⁺ neurotoxicity, which is considered to be secondary to changes in blood vessels, was re-evaluated in dissociated mesencephalic trigeminal (Me5) neurons of the adult rat. Cd²⁺ induced morphological changes in Me5 neurons at 0.1 and 1 mM but not at 0.01 mM. The changes appeared predominantly in the cytoplasm: destruction of the cytoplasmic organelles, swelling and vacuolization of the cell body, and finally resulted in cell lysis. These observations indicate necrosis rather than apoptosis, and no sign of degraded nuclear DNA, characteristic to apoptosis, was detected by the TUNEL technique. Using a Ca²⁺-sensitive dye Indo-1, Cd²⁺ was found to elevate the intracellular Ca²⁺ concentration [Ca²⁺]_i (both in the cytoplasm and the nucleus). Both the elevation in [Ca²⁺]_i and the morphological alteration were inhibited either by removing Ca²⁺ from the bathing medium or by the application of BAPTA/AM (10 μM), a membrane-permeable intracellular Ca²⁺ chelator. Furthermore, neither morphological changes nor elevation in [Ca²⁺]_i by Cd²⁺ occurred in the presence of Zn²⁺. It is concluded that (1) Cd²⁺ can directly affect nerve cells, (2) toxicity of Cd²⁺ on Me5 neurons is mediated by continuous elevation in [Ca²⁺]_i, (3) Cd²⁺ induces necrotic cell death, and (4) Cd²⁺ neurotoxicity can be antagonized by Zn²⁺.     

Involvement of nitric oxide in the deregulation of cytosolic calcium in cerebellar neurons during combined glucose-oxygen deprivation

Nitric oxide (NO) has been proposed as a neuronal messenger molecule in hypoxic/ischemic cell injury (Nowicki et al., 1991; Trifiletti, 1992). We conducted studies in a model of combined glucose-oxygen deprivation using cultured rat cerebellar granule cells. Experiments were designed to test the hypothesis that sustained elevation of cytosolic calcium ([Ca²⁺]_i) and NO generation act in concert to trigger neuronal injury after anoxic insult. A hypoxic state was achieved by perfusing the cells with medium pre-equilibrated with argon gas. [Ca²⁺]_i was monitored using digital-imaging fluorescence microscopy in cells loaded with fura-2 AM. Under short-term hypoxic conditions, cells displayed a progressive and sustained, moderate increase of [Ca²⁺]_i, which returned to near basal levels on restoration of O₂-containing medium. Prolonged hypoxic conditions (>60 min) caused irreversible elevation of [Ca²⁺]_i followed by disruption of cell membrane integrity, as indicated by severe swelling, loss of regular cell shape and processes, leakage of dye fura-2, and propidium iodide uptake (“point of no return”). Pretreatment withN ^G-nitro-l-arginine methyl ester (l-NAME, 100 μM), a specific NO synthase inhibitor, markedly delayed the onset of intensity of the rise of [Ca²⁺]_i. The hypoxia-induced elevation of [Ca²⁺]_i was also greatly attenuated ifl-NAME (100 μM) was added to the argon-perfused medium before the cells demonstrated signs of irreversible injury. Prolonged or repeated hypoxic conditions, however, caused a rapid and intense increase of [Ca²⁺]_i, which could not be blocked by inhibition of NO synthase (NOS). In addition, reoxygenation after the “point of no return”, as characterized above, greatly potentiated [Ca²⁺]_i overload and facilitated the process of cell injury. The potentiation and facilitation of cell damage, as demonstrated by rapid massive increase of [Ca²⁺]_i and subsequent cell death, was not blocked by NOS inhibitor,l-NAME.     

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

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

Activation of alpha-2 adrenergic receptors stimulates GABA release by astrocytes

Norepinephrine is one of the key neurotransmitters in the hippocampus, but its role in the functioning of the neuroglial networks remains unclear. Here we show that norepinephrine suppresses NH₄Cl-induced oscillations of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in hippocampal neurons. We found that the inhibitory effect of norepinephrine against ammonium-induced [Ca²⁺]_i oscillations is mediated by activation of alpha-2 adrenergic receptors. Furthermore, UK 14,304, an agonist of alpha-2 adrenergic receptors, evokes a biphasic [Ca²⁺]_i elevation in a minor population of astrocytes. This elevation consists of an initial fast, peak-shaped [Ca²⁺]_i rise, mediated by G_iβγ subunit and subsequent PLC-induced mobilization of Ca²⁺ from internal stores, and a plateau phase, mediated by a Ca²⁺ influx from the extracellular medium through store-operated and TRPC3 channels. We show the correlation between the Ca²⁺ response in astrocytes and suppression of [Ca²⁺]_i oscillations in neurons. The inhibitory effect of UK 14,304 is abolished in the presence of gallein, an inhibitor of G_βγ-signaling. In turn, application of the agonist in the presence of the PLC inhibitor decreases the frequency and amplitude of [Ca²⁺]_i oscillations in neurons but does not suppress them. The same effect is observed in the presence of bicuculline, a GABA(A) receptor antagonist. We demonstrate that UK 14,304 application increases the frequency and amplitude of slow outward chloride currents in neurons, indicating the release of GABA by astrocytes. Thus, our findings indicate that the activation of astrocytic alpha-2 adrenergic receptors stimulates GABA release from astrocytes via G_iβγ subunit-associated signaling pathway, contributing to the suppression of neuronal activity.     

In vitro ischemia-induced intracellular Ca2+ elevation in cerebellar slices: a comparative study with the values found in hippocampal slices

Changes in levels of intracellular calcium ion ([Ca²⁺]_i) induced by in vitro ischemic conditions in gerbil cerebellar and hippocampal slices were investigated using a calcium imaging system and electron microscopy. When the cerebellar slice was perfused with a glucose-free physiological medium equilibrated with a 95% N₂/5% CO₂ gas mixture (in vitro ischemic medium), a large [Ca²⁺]_i elevation was region-specifically induced in the molecular laver of the cerebellar cortex (a dendritic field of Purkinje cells). When the hippocampal slice was perfused with in vitro ischemic medium, a large [Ca²⁺]_i elevation was region-specifically induced in CA1 field of the hippocampal slices. Electron microscopic examinations showed that the large [Ca²⁺]_i elevations occurred in Purkinje cells and CA1 pyramidal neurons. To isolate Ca²⁺ release from intracellular Ca²⁺ store sites, the slices were perfused with Ca²⁺-free in vitro ischemic medium. the increases in [Ca²⁺]_i in both cerebellar and hippocampal slices were significantly lower than those observed in the slices perfused with the Ca²⁺-containing in vitro ischemic medium. However, the suppression of the [Ca²⁺]_i-elevation in the molecular layer of the cerebellar slices was smaller than that in the CA1 field of the hippocampal slices. These results reinforce the hypothesis that calcium plays a pivotal role in the development of ischemia-induced neuronal death, and suggest that Ca²⁺ release from intracellular Ca²⁺ store sites may play an important role in the ischemia-induced [Ca²⁺]_i elevation in Purkinje cells.     

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

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

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.     

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.     

lntracellular ca2+ changes in cultured vascular smooth muscle cells by treatment with various spasmogens

Abstract

In order to evaluate various spasmogenswhich are candidates for cerebral vasospasm after subarachnoid haemorrhage, the intracellular calcium mobilizations were examined in cultured vascular smooth muscle cells preloaded with a fluorescent Ca²⁺ probe fura-2. Endothelin, oxyhaemoglobin, 5-hydroxytryptamine, norepinephrine, prostaglandin F₂α, leukotrienes C₄ and D₄ produced dose-dependent increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, bilirubin did not induce any significant [Ca²⁺]_i elevation. The maximal levels of [Ca²⁺]_i peak attained by endothelin or oxyhaemoglobin were higher than those of other compounds. Endothelin was the most potent in that it induced a high sustained [Ca²⁺]_i elevation at much lower concentrations compared with others. The combination of oxyhaemoglobin and endothelin induced a transient increase in [Ca²⁺]_i followed by a sustained lower plateau, then the [Ca²⁺]_i level was again increased slowly followed by a sustained higher plateau which lasted for more than 10 min after the exposure. These results suggest that endothelin and/or oxyhaemoglobin may play a crucial role in contraction of vascular smooth muscle after subarachnoid haemorrhage.     

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.     

Suppression of glomus cell K conductance by 4-aminopyridine is not related to [Ca]i, dopamine release and chemosensory discharge from carotid body

The hypothesis that suppression of O₂-sensitive K⁺ current is the initial event in hypoxic chemotransduction in the carotid body glomus cells was tested by using 4-aminopyridine (4-AP), a known suppressant of K⁺ current, on intracellular [Ca²⁺]_i, dopamine secretion and chemosensory discharge in cat carotid body (CB). In vitro experiments were performed with superfused–perfused cat CBs, measuring chemosensory discharge, monitoring dopamine release by microsensors without and with 4-AP (0.2, 1.0 and 2.0 mM in CO₂-HCO₃^- buffer) and recording [Ca²⁺]_i by ratio fluorometry in isolated cat and rat glomus cells. 4-AP decreased the chemosensory activities in normoxia but remained the same in hypoxia and in flow interruption. It decreased the tissue dopamine release in normoxia, and showed an additional inhibition with hypoxia. Also, 4-AP did not evoke any rise in [Ca²⁺]_i in glomus cells either during normoxia and hypoxia, although hypoxia stimulated it. Thus, the lack of stimulatory effect on chemosensory discharge, inhibition of dopamine release and unaltered [Ca²⁺]_i by 4-AP are not consistent with the implied meaning of the suppressant effect on K⁺ current of glomus cells.     

Heat shock protects cultured rat astrocytes in a model of reperfusion injury

We have previously found that incubation of cultured rat astrocytes in Ca²⁺-containing medium after exposure to Ca²⁺-free medium caused an increase in intracellular Ca²⁺ ([Ca²⁺_i) followed by delayed cell death. Here, we examined whether thermal stress protects astrocytes from cell death in this model system of reperfusion injury. Cultured astrocytes were preincubated at 40–44°C for 10–20 min in fetal calf serum-free medium, incubated at 37°C for 24 h in serum-containing medium, and subjected to the in vitro reperfusion experiment. Thermal stress attenuated reperfusion-induced cell toxicity. Furthermore, the stress increased cell viability after incubation with serum-free medium containing Ca²⁺. These effects of heat shock required incubation in serum-containing medium for at least 12 h after heat shock, and it was blocked by the protein synthesis inhibitor cycloheximide. Thermal stress increased synthesis of several proteins, and one of the inducible proteins was identified as the 72-kDa heat shock protein by an immunoblot analysis. Neither the increase in [Ca²⁺]_i nor the Na⁺-Ca²⁺ exchange activity in astrocytes induced in this model were affected by thermal stress. These findings suggest that heat shock proteins protect astrocytes from cell death in a model of reperfusion injury and they may affect processes down stream of the increase in [Ca²⁺]_i.     

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

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

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.     

Simultaneous Monitoring of Cytosolic Free Calcium and Exocytosis at the Single Cell Level

Quinacrine, a fluorescent basic molecule, accumulates in secretory granules of pituitary cells, as was revealed by its colocalization with immunoreactive prolactin. Thus quinacrine fluorescence may be used to monitor secretory activity at the single cell level. Rat pituitary cells in primary culture were loaded with quinacrine and stimulated with physiological secretagogues, such as thyrotrophin-releasing hormone or bradykinin, which induced a multiphasic lowering of fluorescence, corresponding to the loss of quinacrine contained in exocytosed granules. Quinacrine was further used in combination with the fluorescent calcium probe fura-2, in order to monitor simultaneously exocytosis and variations in the cytosolic free calcium concentration, [Ca²⁺]_i. With an appropriate selection of the excitation wavelengths, in dual excitation microfluorimetry experiments, it was possible to distinguish between fluorescence changes due to altered [Ca²⁺]_i versus quinacrine exocytosis. Transient elevations of [Ca²⁺]_i were provoked in individual pituitary cells by enhancing calcium influx through voltage gated channels. In part of the cells an initial increase in [Ca²⁺]_i coincided with stimulated quinacrine release. The approach was also applied to cells of the neuroblastoma line NCB20, where stimulation with bradykinin caused a transient rise in [Ca²⁺]_i, concomitantly with enhanced exocytosis. No increase in exocytosis was ever detected without an elevation of [Ca²⁺]_i, suggesting that in both cellular systems, an increase in [Ca²⁺]_i, is absolutely necessary, but not sufficient to induce secretion.     

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.     

NMDA-induced increase in [Ca]i andCa uptake in acutely dissociated brain cells derived from adult rats

A preparation of acutely dissociated brain cells derived from adult (3-month-old) rat has been developed under conditions preserving the metabolic integrity of the cells and the function of N-methyl-d-aspartate (NMDA) receptors. The effects of glutamate and NMDA on [Ca²⁺]_i measured with fluo3 and⁴⁵Ca²⁺ uptake have been studied on preparations derived from hippocampus and cerebral cortex. Glutamate (100 μM) and N-methyl-dl-aspartate (200 μM) increased [Ca²⁺]_i by 26-12 nM and 23-9 nM after 90 s in cerebral cortex and hippocampus, and stimulated⁴⁵Ca²⁺ uptake about 16–10% in the same regions. The increases in [Ca²⁺]_i and⁴⁵Ca²⁺ uptake were inhibited by 40% in the presence of 1 mM MgCl₂ and by 90–50% in the presence of MK-801. The results indicate (a) that a large fraction of the [Ca²⁺]_i response to glutamate in freshly dissociated brain cells from the adult rat involves NMDA receptors, (b) when compared with results in newborn rats, there is a substantial blunting of the [Ca²⁺]_i increase in adult age.