Increase in cytoplasmic free Ca2+ elicited by noradrenalin and serotonin in cultured local interneurons of mouse olfactory bulb.

Effects of noradrenalin and serotonin on cytoplasmic free Ca2+ concentrations ([Ca2+]i) were studied by using the fluorescent indicator fura-2 in cultured local interneurons of mouse olfactory bulb. Application of noradrenalin (0.1-100 microM) caused a rapid and concentration-dependent rise in [Ca2+]i, while isoproterenol was ineffective at concentrations up to 100 microM. The noradrenalin (1 microM)-induced increase in [Ca2+]i was completely inhibited by pretreatment with alpha 1-antagonist, prazosin (100 nM), whereas the inhibitory effect of alpha 2-antagonist, yohimbine, was about 100-times less potent. Serotonin (0.1-100 microM) also caused the dose-dependent rise in [Ca2+]i, which was inhibited by serotonin2 antagonist, ketanserin. Even in the absence of the extracellular calcium, the noradrenalin- or serotonin-induced increase in [Ca2+]i was observed. These results indicate that both noradrenalin and serotonin elicit the rise in [Ca2+]i in local interneurons of the olfactory bulb. They also suggest that the rise in [Ca2+]i is mediated by alpha 1-adrenergic and serotonin2 receptors, and that the increased calcium is mainly derived from intracellular calcium storage sites. The above results provide evidence to suggest that in the olfactory bulb, noradrenergic and serotonergic centrifugal fibers exert modulatory influences on synaptic interactions between mitral/tufted cells and local interneurons by increasing cytoplasmic Ca2+ in local interneurons.     

Calcium release induced by high K+ and caffeine in cultured skeletal muscle cells of embryonic chicken

To further understand the function of excitation-contraction coupling in skeletal muscle cells developing in vitro, Ca2+ transients elicited by high-K+ depolarization in the presence and absence of extracellular Ca2+ were compared with Ca2+ release induced by caffeine in cultured skeletal muscle cells isolated from 9-day-old chicken embryos (E9). Almost all myoblasts and myotubes cultured for 1 (E9I1) to 8 (E9I8) days responded to 80 mM [K+]O with an elevation of [Ca2+]i. Although all myotubes cultured for more than 4 days exhibited Ca2+ release independent of extracellular Ca2+, only about 50% of E9I1 and E9I2 cells maintained their response to Ca(2+)-free high-[K+]O solution. Strikingly, a considerable proportion of cells of short-term culture were insensitive to 10 mM caffeine. Moreover, 46.8% of the caffeine-insensitive E9I1 and E9I2 cells, 29 out of 62, was still responsive to 80 mM [K+]O in the absence of extracellular Ca2+. Western blot and immunocytochemistry showed that ryanodine receptor (RyRs) expression increases with culture. The Ca2+ release from caffeine-insensitive cells induced by Ca(2+)-free high-[K+]O solution could be blocked by 100-200 microM ryanodine, which suggests the involvement of RyRs. Evidence is presented to show that a low resting [Ca2+]i may be one factor responsible for the caffeine insensitivity of RyRs in cells of short-term culture.     

Muscarinic stimulation of guinea pig adrenal chromaffin cells stimulates catecholamine secretion without significant increase in Ca2+ uptake

In isolated guinea pig adrenal chromaffin cells, not only nicotine, but also muscarine stimulated catecholamine (CA) secretion, the stimulation by muscarine being the greater. The secretions of CA by muscarine and nicotine were both dependent on the presence of Ca2+ in the medium, but only the latter was associated with a rapid increase in 45Ca2+ uptake. Experiments with the fluorescent Ca2+ indicator quin 2, showed that muscarine caused an increase in cytoplasmic free Ca2+ concentration [( Ca2+]i). Moreover, the intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) inhibited both CA secretion and increase in [Ca2+]i induced by muscarine. These results indicate that in isolated guinea pig adrenal chromaffin cells, nicotine stimulated CA secretion by increasing Ca2+ uptake by the cells, whereas muscarine stimulated CA secretion by mobilizing Ca2+ from the intracellular pool.     

Muscarinic and nicotinic ACh receptor activation differentially mobilize Ca2+ in rat intracardiac ganglion neurons

The origin of intracellular Ca2+ concentration ([Ca2+]i) transients stimulated by nicotinic (nAChR) and muscarinic (mAChR) receptor activation was investigated in fura-2-loaded neonatal rat intracardiac neurons. ACh evoked [Ca2+]i increases that were reduced to approximately 60% of control in the presence of either atropine (1 microM) or mecamylamine (3 microM) and to <20% in the presence of both antagonists. Removal of external Ca2+ reduced ACh-induced responses to 58% of control, which was unchanged in the presence of mecamylamine but reduced to 5% of control by atropine. The nAChR-induced [Ca2+]i response was reduced to 50% by 10 microM ryanodine, whereas the mAChR-induced response was unaffected by ryanodine, suggesting that Ca2+ release from ryanodine-sensitive Ca2+ stores may only contribute to the nAChR-induced [Ca2+]i responses. Perforated-patch whole cell recording at -60 mV shows that the rise in [Ca2+]i is concomitant with slow outward currents on mAChR activation and with rapid inward currents after nAChR activation. In conclusion, different signaling pathways mediate the rise in [Ca2+]i and membrane currents evoked by ACh binding to nicotinic and muscarinic receptors in rat intracardiac neurons.     

Acute reduction of extracellular sodium differentially affects receptor-mediated and K+-induced calcium uptake in PC12 pheochromocytoma cells

Using a rapid-quench method to measure 45Ca2+ uptake into PC12 cells in suspension, we have studied basal, carbachol-stimulated and K+-induced Ca2+ uptake under control conditions [( Na+]o = 130 mM) and in the presence of acutely lowered extracellular sodium concentration [( Na+]o = 65 mM). Acute reduction of [Na+]o stimulates basal and K+-evoked Ca2+ uptake, but reduces net carbachol-stimulated uptake. Since total Ca2+ uptake measured in the presence of carbachol under control and low [Na+]o conditions is unchanged, the reduction in carbachol-stimulated uptake is due to the increase in basal uptake induced by low [Na+]o. These results reconcile apparently conflicting data regarding a specific Na+ requirement for nicotinic acetylcholine receptor-mediated responses in PC12 cells and adrenal chromaffin cells and suggest a mechanism for loss of nicotinic acetylcholine receptor (nAChR) responsiveness to agonists under low Na+ conditions.     

ATP acting on P2Y receptors triggers calcium mobilization in primary cultures of rat neurohypophysial astrocytes (pituicytes)

 The effect of adenosine triphosphate (ATP) on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) of cultured neurohypophysial astrocytes (pituicytes) was studied by fluorescence videomicroscopy. ATP evoked a [Ca²⁺]_i increase, which was dose dependent in the 2.5–50 μM range (EC₅₀=4.3 μM). The ATP-evoked [Ca²⁺]_i rise was not modified during the first minute following the removal of external Ca²⁺. Application of 500 nM thapsigargin inhibited the ATP-dependent [Ca²⁺]_i increase. Caffeine (10 mM) and ryanodine (1 μM) did not affect the ATP-induced [Ca²⁺]_i rise. The pituicytes responded to various P₂ purinoceptor agonists with the following order of potency: ATP=ATP[γ-S]=2-MeSATP≥ADP, where ATP[γ-S] is adenosine 5′-O-(3-thiotriphosphate) and 2-MeSATP is 2-methylthio-adenosine-5′-triphosphate. Adenosine, AMP, α,β-methylene adenosine-5′-triphosphate (α,β-MeATP), β,γ methylene adenosine-5′-triphosphate (β,γ-MeATP) and uridine 5′-triphosphate (UTP) were ineffective. The P₂ purinoceptor antagonists blocked the ATP-evoked [Ca²⁺]_i increase with the following selectivity: RB-2>suramin>PPADS, where RB-2 is Reactive Blue 2 and PPADS is pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid. The ATP-evoked [Ca²⁺]_i increase was substantially blocked by pertussis toxin treatment, suggesting that it might be mediated by a pertussis-toxin-sensitive G protein. The phospholipase C (PLC) inhibitor U-73122 (0.5 μM) abolished the ATP-evoked [Ca²⁺]_i rise, whereas its inactive stereoisomer U-73343 (0.5 μM) remained ineffective. Our results indicate that, in rat cultured pituicytes, ATP stimulation induces an increase in [Ca²⁺]_i due to PLC-mediated release from intracellular stores through activation of a pertussis-toxin-sensitive, G-protein-linked P_2Y receptor. Received: 24 September 1998 / Received after revision: 10 December 1998 / Accepted: 18 December 1998     

Trifluoperazine, a calmodulin inhibitor, blocks secretion in cultured chromaffin cells at a step distal from calcium entry

Trifluoperazine, a calmodulin antagonist, inhibited the secretory response of cultured bovine adrenal medullary chromaffin cells to acetylcholine (10(-4) M) or a depolarizing concentration of [K+] (56 mM KCl) in a dose-related fashion. The ID50s of this effect were 2 x 10(-7) M and 2.2 x 10(-6) M for acetylcholine and high [K+], respectively. A decrease in external [Ca2+] concentration of the incubation medium from 4.4 to 0.275 mM resulted in an increase in the percentage of inhibition produced by trifluoperazine on the acetylcholine-evoked secretory response from 20.7 to 96.5%, respectively. However, trifluoperazine inhibited the acetylcholine-evoked catecholamine output by a similar absolute magnitude for all [Ca2+] concentrations tested with the exception of 4.4 mM [Ca2+]. Trifluoperazine, unlike the [Ca2+] channel blocker Ni2+, in concentrations (10(-6)-10(-5) M) that were found to inhibit significantly [K+]-induced amine output did not modify [K+]-induced 45Ca uptake or 45Ca efflux. However, trifluoperazine at a concentration of 2.5 x 10(-5) M was found to produce a small decrease in the 45Ca efflux curve and a decrease in the [K+]-evoked 45Ca uptake of 30 +/- 14% (n = 6). In addition, 2.5 x 10(-6) M trifluoperazine, a concentration which was found to suppress high [K+]-induced amine release by 64 +/- 5%, did not inhibit the 45Ca2+-Ca2+ exchange mechanism. These results demonstrate that trifluoperazine, an antipsychotic agent with anticalmodulin activity, blocks catecholamine release from cultured chromaffin cells at a step distal from calcium entry and, consequently, suggests a role for calmodulin in the secretory process of these cells.     

ATP-induced Ca2+ response mediated by P2U and P2Y purinoceptors in human macrophages: signalling from dying cells to macrophages

The activation of macrophages by various stimuli leading to chemotactic migration and phagocytosis is known to be mediated by an increase in intracellular Ca2+ concentration ([Ca2+]i). We measured changes in [Ca2+]i using a Ca2+ imaging method in individual human macrophages differentiated from freshly prepared peripheral blood monocytes during culture of 1-2 days. A transient rise in [Ca2+]i (duration 3-4 min) occurred in 10-15 macrophages in the vicinity of a single tumour cell that was attacked and permeabilized by a natural killer cell in a dish. Similar Ca2+ transients were produced in 90% of macrophages by application of supernatant obtained after inducing the lysis of tumour cells with hypo-osmotic treatment. Ca2+ transients were also evoked by ATP in a dose-dependent manner between 0.1 and 100 microm. The ATP-induced [Ca2+]i rise was reduced to less than one-quarter in Ca2+-free medium, indicating that it is mainly due to Ca2+ entry and partly due to intracellular Ca2+ release. UTP (P2U purinoceptor agonist) was more potent than ATP or 2-chloro-ATP (P2Y agonist). Oxidized ATP (P2Z antagonist) had no inhibitory effect. Both cell lysate- and ATP-induced Ca2+ responses were inhibited by Reactive Blue 2 (P2Y and P2U antagonist) to the same extent, but were not affected by PPADS (P2X antagonist). Sequential stimuli by cell lysate and ATP underwent long-lasting desensitization in the Ca2+ response to the second stimulation. The present study supports the view that macrophages respond to signal messengers discharged from damaged or dying cells to be ingested, and ATP is at least one of the messengers and causes a [Ca2+]i rise via P2U and P2Y receptors.     

Inhibitory effect of polymyxin B on catecholamine secretion from cultured bovine adrenal medullary cells

Incubation of cultured bovine adrenal medullary cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), was associated with increased secretion of catecholamine (CA) from the cells. Polymyxin B (PMB, 30-300 microM), a preferential inhibitor of protein kinase C, inhibited the TPA-induced secretion of CA. PMB also inhibited CA secretion induced by other secretagogues, the Ca2+ ionophore ionomycin (10 microM), 56 mM K+ or acetylcholine (ACh). Ionomycin, 56 mM K+ or ACh increased the concentration of intracellular free Ca2+ ([Ca2+]i) (measured using the fluorescent calcium indicator quin2), whereas TPA did not increase [Ca2+]i. PMB blocked the increase in [Ca2+]i induced by 56 mM K+ or ACh at concentrations similar to those inhibiting the secretion of CA. In contrast, PMB did not affect ionomycin-induced increase in [Ca2+]i. These results strongly suggest that CA secretion induced by TPA or ionomycin is mediated via activation of protein kinase C. The results further indicate that in 56 mM K+- or ACh-evoked CA secretion, PMB inhibits the secretion by blocking Ca2+ influx into the cells.     

Metabotropic receptor-mediated Ca2+ signaling elevates mitochondrial Ca2+ and stimulates oxidative metabolism in hippocampal slice cultures

Metabotropic receptors modulate numerous cellular processes by intracellular Ca2+ signaling, but less is known about their role in regulating mitochondrial metabolic function within the CNS. In this study, we demonstrate in area CA3 of rat organotypic hippocampal slice cultures that glutamatergic, serotonergic, and muscarinic metabotropic receptor ligands, namely trans-azetidine-2,4-dicarboxylic acid, alpha-methyl-5-hydroxytryptamine, and carbachol, transiently increase mitochondrial Ca2+ concentration ([Ca2+]m) as recorded by changes in Rhod-2 fluorescence, stimulate mitochondrial oxidative metabolism as revealed by elevations in NAD(P)H fluorescence, and induce K+ outward currents as monitored by rapid increases in extracellular K+ concentration ([K+]o). Carbachol (1-1,000 microM) elevated NAD(P)H fluorescence by 相似文献   

Pregnenolone sulfate augments NMDA receptor mediated increases in intracellular Ca2+ in cultured rat hippocampal neurons.

The ability of the neuroactive steroid pregnenolone sulfate to alter N-methyl-D-aspartate (NMDA) receptor-mediated elevations in intracellular Ca2+ ([Ca2+]i) was studied in cultured fetal rat hippocampal neurons using microspectrofluorimetry and the Ca2+ sensitive indicator fura-2. Pregnenolone sulfate (5-250 microM) caused a concentration-dependent and reversible potentiation of the rise (up to approximately 800%) in [Ca2+]i induced by NMDA. In contrast, the steroid failed to alter basal (unstimulated) [Ca2+]i or to modify the rise in [Ca2+]i that occurs when hippocampal neurons are depolarized by high K+ in the presence of the NMDA receptor antagonist CPP. These data suggest that the previously reported excitatory properties of pregnenolone sulfate may be due, in part, to an augmentation of the action of glutamic acid at the NMDA receptor.     

Stimulatory effect of vasoactive intestinal polypeptide on catecholamine secretion from isolated guinea pig adrenal chromaffin cells

The stimulatory effect of vasoactive intestinal polypeptide (VIP) on catecholamine (CA) secretion from isolated guinea pig adrenal chromaffin cell was studied. VIP (1-10 microM) induced dose-dependent CA secretion, which was slow and continued for at least 30 min. This VIP-induced CA secretion was dependent on the presence of Ca2+ in the medium, but no significant increase in Ca2+ uptake by the cells was observed during their stimulation with VIP. Studies on the intracellular free Ca2+ level ([Ca2+]i) using fura-2 showed that acetylcholine and muscarine induced a marked increase in the [Ca2+]i, but that VIP induced only a slight increase. Thus VIP may induce CA secretion by increasing the sensitivity of the secretion of CA to Ca2+.     

Characterization of platelet-activating factor-induced cytosolic calcium mobilization in human eosinophils.

BACKGROUND: Activated eosinophils play an important role in the pathogenesis of bronchial asthma and other allergic diseases, and platelet-activating factor (PAF) is a potent activator of eosinophils. OBJECTIVE: To characterize the cytosolic Ca2+ ([Ca2+]i) mobilization in human eosinophils in response to PAF. METHODS: [Ca2+]i responses to PAF were examined in human eosinophils using a microscopic fura-2 fluorescence-ratio imaging system. RESULTS: PAF caused a significant and dose-dependent increase in (Ca2+)i, which consisted of an initial rapid rise followed by a sustained elevation. This PAF-induced (Ca2+)i rise was inhibited by WEB 2086, a specific PAF receptor antagonist. The addition of 5 mM EGTA or 1 mM Ni2+ to a nominally Ca2+-free solution did not appreciably reduce the initial rise but significantly inhibited the sustained rise. The application of a protein kinase C inhibitor, Ro31-8220, augmented the sustained increase by PAF. Thapsigargin, a microsomal Ca2+ ATPase inhibitor, induced no appreciable change in a nominally Ca2+-free solution but induced a marked increase in (Ca2+)i when changed to a Ca2+-containing solution. CONCLUSIONS: The initial rapid rise and the following sustained rise in (Ca2+)i by PAF depends on Ca2+ release from the intracellular Ca2+ stores and Ca2+ influx, respectively, which are regulated by protein kinase C in human eosinophils. Furthermore, the so called Ca2+-capacitative entry is possibly involved in the Ca2+ influx from the extracellular solution in human eosinophils.     

Voltage-sensitive calcium flux into bovine chromaffin cells occurs through dihydropyridine-sensitive and dihydropyridine- and omega-conotoxin-insensitive pathways

The fluorescent Ca2+ indicator FURA-2 was used to characterize the depolarization-related intracellular Ca2+ signalling process in bovine adrenal chromaffin cells. Depolarization with high K+ (10-65 mM) gave rise to a very rapid increase in intracellular free Ca2+ concentration, which subsequently decayed slowly towards a "plateau". The size of this initial increase varied sigmoidally with the calculated membrane potential, the relationship being described well by a Boltzmann distribution function for a transition between two states (transition potential, -23 mV). A dihydropyridine calcium channel agonist [(+)202-791, 1 microM] raised intracellular free Ca2+ concentration further in the presence of 30 mM K+, and it enhanced the initial intracellular Ca2+ response to depolarization. Voltage-sensitive calcium channels in chromaffin cells are believed to include the L-type. Several dihydropyridine calcium channel antagonists [(-)202-791, nifedipine, nitrendipine; 1-5 microM], known to be active on L-type channels, caused only modest inhibition of K+ -induced increase in intracellular free Ca2+ concentration: c. 50% (at 30 mM K+) and 25% (at 40-70 mM K+). In addition, omega-conotoxin GVIA (1-10 microM), a blocker of neuronal N- and L-type calcium channels, reduced the initial increase in intracellular free Ca2+ concentration only slightly at 55 mM K+. Further, the dihydropyridine-insensitive component of the intracellular Ca2+ signal was also insensitive to omega-conotoxin, which was otherwise quite active in a central nervous rat in vivo preparation Gd3+ (40 microM), a potent calcium antagonist in the chromaffin cell, blocked the intracellular Ca2+ response to depolarization. When added at different times after K+ stimulation, however, Gd3+ reduced intracellular free Ca2+ concentration to control levels along a slow time course of several minutes. Similar results were obtained when EGTA was added to reduce extracellular Ca2+ concentration to sub-nanomolar levels, in the presence of high K+. We conclude that bovine chromaffin cells are equipped with at least two different classes of voltage-dependent calcium channels, only one of which is likely to be the L-type channel. We also propose that depolarization, in addition to stimulating Ca2+ influx, may also lead to enhancement of Ca2+ release from an intracellular store.     

Facilitation of calcium influx by propylene glycol through the voltage-dependent calcium channels in PC12 cells

Propylene glycol (PG) raises an intracellular calcium concentration ([Ca2+]i) in PC12 cells. The present study has been undertaken to examine whether or not the voltage-dependent Ca2+ channels are involved in the PG-induced rise in [Ca2+]i and, if so, to determine which types participate in it. CdCl2 (50 micro M) and the Ca2+ -free saline depressed the action of PG (0.5 - 10 %v/v)-induced [Ca2+]i rise. Although NiCl2 (50 micro M) at the same concentration as CdCl2, and omega-agatoxin (50 and 300 nM) had no effect on the PG-induced [Ca2+]i rise, each of omega-conotoxin (1 micro M), nifedipine (10 micro M), nicardipine (10 micro M), varapamil (10 micro M) and diltiazem (10 micro M) significantly decreased it. Electrical stimulation and Bay K 8644 (1 micro M) enhanced the PG-induced [Ca2+]i rise. The second phase of the [Ca2]i rise was fallen fast by nicardipine (10 micro M), but not by omega-conotoxin (1 micro M). The results obtained suggested that the Ca2+ influx through the L- and N-type Ca2+ channels are involved in the PG-induced [Ca2+]i rise.     

Cytoplasmic free concentrations of Ca2+ and Mg2+ in skeletal muscle fibers at rest and during contraction

This review summarizes estimates for cytoplasmic-free concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) at rest and during contraction of skeletal muscles, from which substantial quantitative information about them has been accumulated. Although the estimates of resting [Ca2+]i in the literature widely differ, which is because of the variety of difficulties related to different methodologies used, recent studies suggest that estimates of resting [Ca2+]i of approximately 0.05-0.1 microM are likely to be correct. Following action potential propagation, the Ca2+ release from the sarcoplasmic reticulum causes a transient rise of [Ca2+]i (Ca2+ transient). The large peak amplitude and brief time course of the Ca2+ transients have been established only recently by studies with low-affinity Ca2+ indicators developed in the past decade. These technical improvements in [Ca2+]i measurements have made it possible to study relationships between [Ca2+]i and force in intact muscle fibers. In the second part of this review, various estimates of [Mg2+]i in the resting muscle are discussed. Relatively recent estimates of the [Mg2+]i level appear to be about 1.0 mM. Using the current knowledge of concentrations and reaction properties of intracellular Ca2+-Mg2+ binding sites, we constructed a model for dynamic Mg2+ movement following Ca2+ transients. The model predicts that with a train of action potentials, the sustained rise of [Ca2+]i produces an elevation of [Mg2+]i of about 200 microM.     

Cyclic AMP-mediated inhibition of noradrenaline-induced contraction and Ca2+ influx in guinea-pig vas deferens

The relaxation effects of forskolin and methylxanthines on noradrenaline (NA)-induced contractions were investigated by measuring isotonic contraction and intracellular calcium concentration ([Ca2+]i) in the epididymal side of guinea-pig vas deferens. NA (100 microM) and high K+ (55 mM) induced a biphasic contraction; fast, transient (phasic) and slow, sustained (tonic) phases. Both phases in either NA or high K+ stimulation were abolished in Ca2+-free solution. Pretreatment with 10 microM nifedipine, an L-type Ca2+ channel blocker, reduced both phasic and tonic contractions induced by high K+. In the case of NA-induced contraction, however, nifedipine reduced the phasic contraction but not the tonic contraction. The nifedipine-insensitive tonic contraction was relaxed by the application of polyvalent cations (Mn2+, Co2+, Cd2+ and La3+). These findings indicate that NA-induced biphasic contraction is mainly due to nifedipine-insensitive Ca2+ influx, especially in the tonic phase. Cyclic AMP-increasing agents such as forskolin (0.5-10 microM), IBMX (5-500 microM) and caffeine (1-20 mM) relaxed the NA-induced contraction extensively in a concentration-dependent manner. However, these agents only partially relaxed the high K+-induced contraction. Forskolin (10 microM) and IBMX (100 microM) reduced the [Ca2+]i response to NA, but had no effect on the [Ca2+]i response to high K+. These results suggest that an increase in intracellular cAMP may relax the NA-induced contraction by attenuating a nifedipine-insensitive Ca2+ influx and by a mechanism independent of a reduction in [Ca2+]i.     

Effects of hypoxia on membrane potential and intracellular calcium in rat neonatal carotid body type I cells.

1. We have studied the effects of hypoxia on membrane potential and [Ca2+]i in enzymically isolated type I cells of the neonatal rat carotid body (the principal respiratory O2 chemosensor). Isolated cells were maintained in short term culture (3-36 h) before use. [Ca2+]i was measured using the Ca(2+)-sensitive fluoroprobe indo-1. Indo-1 was loaded into cells using the esterified form indo-1 AM. Membrane potential was measured (and clamped) in single isolated type I cells using the perforated-patch (amphotericin B) whole-cell recording technique. 2. Graded reductions in PO2 from 160 Torr to 38, 19, 8, 5 and 0 Torr induced a graded rise of [Ca2+]i in both single and clumps of type I cells. 3. The rise of [Ca2+]i in response to anoxia was 98% inhibited by removal of external Ca2+ (+1 mM EGTA), indicating the probable involvement of Ca2+ influx from the external medium in mediating the anoxic [Ca2+]i response. 4. The L-type Ca2+ channel antagonist nicardipine (10 microM) inhibited the anoxic [Ca2+]i response by 67%, and the non-selective Ca2+ channel antagonist Ni2+ (2 mM) inhibited the response by 77%. 5. Under voltage recording conditions, anoxia induced a reversible membrane depolarization (or receptor potential) accompanied, in many cases, by trains of action potentials. These electrical events were coincident with a rapid rise of [Ca2+]i. When cells were voltage clamped close to their resting potential (-40 to -60 mV), the [Ca2+]i response to anoxia was greatly reduced and its onset was much slower.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subcellular localization of glutamate-stimulated intracellular magnesium concentration changes in cultured rat forebrain neurons using confocal microscopy

Glutamate can stimulate increases in intracellular magnesium concentration ([Mg2+]i) and induce neurotoxicity, both independent of Ca2+ changes. Although Mg2+ is essential within the cell, very little is known about how it is regulated, especially in neurons. Therefore we used the fluorescent indicator, magindo-1 and confocal microscopy to examine possible intracellular pools of Mg2+ in cultured neurons that can be dynamically regulated by glutamate. The magindo-1 fluorescence signal was present throughout the cell body and extends into the neuronal processes. The magindo-1 405 nm/490 nm ratio signal was similar in the cytoplasm and nucleus, suggesting that resting [Mg2+]i is uniform across the neuron. The addition of 100 microM glutamate/10 microM glycine in an extracellular Ca2+- and Na+-free buffer stimulated an increase in [Mg2+]i in both the nuclear and cytoplasmic regions of similar magnitude and duration. This glutamate exposure also stimulated a [Mg2+]i increase in neuronal processes which was inhibited by the N-methyl-D-aspartate receptor antagonist, MK-801 (10 microM). The glutamate-stimulated [Mg2+]i increase in both the cell body and neuronal processes was dependent on the extracellular Mg2+ concentration. These findings suggest glutamate-stimulated [Mg2+]i changes may not only impact cytoplasmic processes, but also directly trigger nuclear events involved, for example, in neuronal injury.     

Effects of magnesium on prostacyclin synthesis and intracellular free calcium concentration in vascular cells.

This study investigated the effects of extracellular magnesium concentration ([Mg2+]e; 0.3-3 mM) on intracellular free calcium concentration ([Ca2+]i) and prostacyclin (PGI2) production in cultured human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells from rats (VSMC) under basal and agonist-stimulated conditions. We used histamine as agonist which increases [Ca2+]i and PGI2 production in HUVEC, norepinephrine in VSMC. [Mg2+]e dose-dependently increased basal and agonist-stimulated PGI2 production in both cells. [Mg2+]e dose-dependently reduced basal [Ca2+]i in VSMC, but did not influence in HUVEC. In both cells, increasing [Mg2+]e reduced agonist-stimulated [Ca2+]i responses. Furthermore, [Mg2+]e dose-dependently reduced agonist-stimulated [Ca2+]i in Ca(2+)-free buffer, indicating intracellular Ca2+ release. In VSMC, 10(-6) M diltiazem and 10(-7) M nifedipine, Ca2+ channel blockers, reduced agonist-stimulated [Ca2+]i as well as 3 mM Mg2+, but did not affect PGI2 production. [Mg2+]e amplified dose-dependently arachidonic acid-induced PGI2 production in both cells, suggesting the activation of cyclooxygenase and/or PGI2 synthetase. Our results suggest that [Mg2+]e influences intracellular Ca2+ mobilization of not only vascular smooth muscle cells but also endothelial cells by inhibiting both Ca2+ influx and intracellular Ca2+ release. [Mg2+]e enhances PGI2 production in both types of cells, although the mechanism is likely to be independent from Ca2+ mobilization.