Effects of magnesium on45Ca uptake and release at different sites in rabbit aortic smooth muscle

Effects of 1.5 mM Mg²⁺ on muscle tension and on⁴⁵Ca uptake and release at different sites in the rabbit aortic media-intimal layer were investigated. The sustained contraction induced by either 10^?6 M norepinephrine (NE) or 60 mM K⁺ was not affected by 1.5 mM Mg²⁺ in the presence of 1.5 mM Ca²⁺. However, the contractions elicited with NE or K⁺ in 0.03 mM Ca²⁺-containing solution were inhibited by 1.5 mM Mg²⁺ by 67% and 27%, respectively. Total⁴⁵Ca uptake measured in the presence of either 1.5 mM or 0.03 mM Ca²⁺ was not affected by 1.5 mM Mg²⁺. The rate of residual⁴⁵Ca uptake (⁴⁵Ca uptake followed by a wash in La³⁺-containing solution at low temperature) measured in the presence of 1.5 mM Ca²⁺ was slightly lower in the presence of 1.5 mM Mg²⁺. However, the increase in rate of residual⁴⁵Ca uptake induced by NE or the net increase in the residual⁴⁵Ca uptake induced by K⁺ was not decreased by 1.5 mM Mg²⁺. The residual⁴⁵Ca uptake measured in the presence of 0.03 mM Ca²⁺ was reduced to 64% and 24% of controls by addition of 1.5 mM Mg²⁺ or Sr²⁺, respectively. A part of the residual⁴⁵Ca was released by NE. Uptake of⁴⁵Ca at this NE-affected Ca²⁺ site did not take place in the presence of 1.5 mM Mg²⁺ when the Ca²⁺ concentration of the medium was 0.03 mM. However, this⁴⁵Ca uptake component was only partially inhibited when the Ca²⁺ concentration of the medium was 1.5 mM. The NE-induced increase in⁴⁵Ca efflux was not inhibited by 1.5 mM Mg²⁺. From these results, Mg²⁺ appears to be a weak antagonist for both Ca²⁺ entry into the vascular smooth muscle cell and Ca²⁺ binding at a high affinity intracellular site.     

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 μM glutamate/10 μM 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 μM). 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.     

Increased calcium influx in the presence of ethanol in mouse pancreatic acinar cells

The effects of alcohol on Ca²⁺ signalling remains poorly understood. Here we have investigated the effects of acute ethanol exposure on Ca²⁺ influx in mouse pancreatic acinar cells. Cells were loaded with fura‐2 and the changes in fluorescence were monitored by spectrofluorimetry and imaging analysis. Stimulation of cells with 20 pM cholecystokinin evoked an oscillatory pattern in [Ca²⁺]_c, both in the presence and in the absence of extracellular Ca²⁺. Stimulation of cells with cholecystokinin in the presence of 50 mM ethanol led to a transformation of physiological oscillations into a single transient increase in [Ca²⁺]_c. This effect was observed when Ca²⁺ was present in the extracellular medium, and did not appear in its absence. Addition of 1 mM CaCl₂ to the extracellular medium, following release of Ca²⁺ from intracellular stores by stimulation of cells with 1 nM cholecystokinin or 1 μM thapsigargin in the absence of extracellular Ca²⁺, was followed by an increase in [Ca²⁺]_c. Ca²⁺ influx was increased in the presence of 50 mM ethanol. The anti‐oxidant cinnamtannin B‐1 (10 μM) or inhibition of alcohol dehydrogenase by 4‐MP (1 mM), significantly reduced Ca²⁺ influx evoked by cholecystokinin in the presence of ethanol. In summary, intoxicating concentrations of ethanol may lead to over stimulation of pancreatic acinar cells by cholecystokinin. This might be partially explained by the generation of reactive oxygen species and an increased Ca²⁺ entry in the presence of ethanol. Potentially ethanol might lead to Ca²⁺ overload, which is a common pathological precursor that is implicated in pancreatitis.     

The phosphodiesterase III inhibitor olprinone inhibits hippocampal glutamate release via a cGMP/PKG pathway

Olprinone, an inhibitor of cyclic nucleotide phosphodiesterase III, inhibited an increase in intracellular Ca²⁺ concentrations for acutely dissociated rat hippocampal pyramidal neurons induced by extracellular high K⁺ (35 mM) depolarization. Olprinone (100 μM) significantly reduced spontaneous glutamate release from rat hippocampal slices. Furthermore, olprinone significantly decreased the rate of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated miniature excitatory postsynaptic currents (AMPA-mEPSCs) monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was blocked by KT5823, an inhibitor of protein kinase G (PKG), but not by H-89, an inhibitor of protein kinase A (PKA). In the PKA assay using PC-12 cells, olprinone did not activate PKA. Taken together, the results of the present study show that olprinone attenuates intracellular Ca²⁺ rise through voltage-sensitive Ca²⁺ channels and inhibits presynaptic glutamate release via a cGMP/PKG pathway.     

The use of antioxidants to prevent glutamate-induced derangement of calcium ion metabolism in rat cerebral cortex synaptosomes

Glutamate is shown to induce increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i), increases in⁴⁵Ca²⁺ influx, decreases in the activity of Na⁺, K⁺,-ATPase activity, and activation of the Na⁺/Ca²⁺ exchanger in rat cerebral cortex synaptosomes. NMDA receptor antagonists virtually prevented these effects. Preincubation of synaptosomes with α-tocopherol, superoxide dismutase, and ganglioside GM₁ normalized [Ca²⁺]_i,⁴⁵Ca²⁺, influx, and Na⁺, K⁺-ATPase activity in rat cerebral cortex synaptosomes exposed to glutamate. Glutamate and GM₁ activated the Na⁺/K⁺ exchanger, and their effects were additive. Calcium ions entering cerebral cortex nerve cells via NMDA receptors during exposure to high glutamate concentrations appeared to be only the trigger for the processes activating free-radical reactions. Activation of these reactions led to increases in Ca²⁺ influx into cells, decreases in Na⁺, K⁺-ATPase activity, and significant increases in [Ca²⁺]_i, though this could be prevented by antioxidants and gangliosides. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 85, No. 4, pp. 488–496, April. 1999.     

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres

The influence of myoplasmic Mg²⁺ (0.05–10 mM) on Ca²⁺ accumulation (net Ca²⁺ flux) and Ca²⁺ uptake (pump-driven Ca²⁺ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca²⁺ accumulation was optimal between 1 and 3 mM Mg²⁺ in toad fibres and reached a plateau between 1 and 10 mM Mg²⁺ in the rat EDL fibres and between 3 and 10 mM Mg²⁺ in the rat cardiac fibres. In soleus fibres, optimal Ca²⁺ accumulation occurred at 10 mM Mg²⁺. The same trend was obtained with all preparations at 0.3 and 1 M Ca²⁺. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca²⁺ pump, revealed a marked Ca²⁺ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 M Ca²⁺ and 1 mM Mg²⁺. Further experiments indicated that the SR Ca²⁺ leak could be blocked by 10 M ruthenium red without affecting the SR Ca²⁺ pump and this allowed separation between SR Ca²⁺ uptake and SR Ca²⁺ accumulation. At 0.3 M Ca²⁺, Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg²⁺ in the rat soleus and trabeculae preparations. At higher [Ca²⁺] (1 M), Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the iliofibularis fibres and between 1 and 3 mM Mg²⁺ in the EDL fibres. In the soleus and cardiac preparations Ca²⁺ uptake was optimal between 1 and 10 mM Mg²⁺. The results of this study demonstrate that SR Ca²⁺ accumulation is different from SR Ca²⁺ uptake and that these two important determinants of muscle function are differently affected by Mg²⁺ in different muscle fibre types.     

Differences in magnesium and calcium effects on N-methyl-D-aspartate- and quisqualate-induced decreases in extracellular sodium concentration in rat hippocampal slices

Summary Decreases in extracellular sodium concentration ([Na⁺]o) and associated slow negative field potentials (fp's) were monitored with double barreled sodium sensitive/reference microelectrodes in area CA1 of rat hippocampal slices during iontophoretic application of the glutamate receptor agonists N-methyl-D-aspartate (NMDA) and quisqualate (quis). The effects of lowering [Ca²⁺]o on these signals were compared to those of lowering [Mg²⁺]o. Both NMDA- and quis-induced decreases in [Na⁺]o of up to 60 mM and in the fp's of up to 8 mV. Decreasing [Mg²⁺]o enhanced NMDA-induced signals, whereas quis-induced signals were unaffected. Lowering [Ca²⁺]o also enhanced NMDA signals, although somewhat less than lowering [Mg²⁺]o. This effect was still present, even when voltage dependent Na⁺ currents were blocked by 10^-7 tetrodotoxin. Interestingly, quis-induced signals could be enhanced in a low Ca²⁺ medium as well, but only when high quis concentrations were used. The results suggest that, during the sorts of large decreases of [Ca²⁺]o observed during seizure activity, activation of NMDA receptors is facilitated.Supported by a grant from the Deutsche Forschungsgemeinschaft (He 1128/2-4)     

Different effects of the ionophore A-23187 and D-glucose on 45Ca2+ fluxes in isolated islets of ob/ob-mice

Fluxes of ⁴⁵Ca²⁺ were studied in β-cell rich islets of non-inbred ob/ob-mice, using LaCl₃ to wash out extracellular and superficially bound ⁴⁵Ca²⁺. The ionophore A-23187 (10,μM) increased the ⁴⁵Ca²⁺ uptake in islets both at 3 and 20 mM D-glucose, the effect being more pronounced after 10 min than after 120 min of incubation. In incubations for 120 min, 20 mM D-glucose induced a higher uptake of ⁴⁵Ca²⁺ than did A-23187. The ionophore enhanced the unidirectional efflux of ⁴⁵Ca^a+ from preloaded islets. Pretreatment of islets with 20 mM D-glucose in non-radioactive medium inhibited the subsequent D-glucose-induced ⁴⁵Ca²⁺ uptake. Similar pretreatment with A-23187 increased the subsequent ionophore-induced ⁴⁵Ca²⁺ uptake. The results suggest that A-23187 acts by catalyzing Ca²⁺ fluxes across the β-cell plasma membrane. The different effects of D-glucose and A-23187 on ⁴⁵Ca²⁺ fluxes suggest that the two agents act through different mechanisms in the β-cells.     

Inhibition of sigma-1 receptor reduces N-methyl-d-aspartate induced neuronal injury in methamphetamine-exposed and -naive hippocampi

Acute and prolonged methamphetamine (METH) exposure has been reported to moderate the function of N-methyl-d-aspartate type glutamate receptors (NMDAr) in the hippocampus. These effects have been found to be associated with enhanced NMDAr-dependent release of Ca²⁺ from IP₃-sensitive intracellular stores. The present studies were designed to extend these findings and examine the role of the endoplasmic membrane (ER) bound orphan receptor, the sigma-1 receptor, in NMDA-induced neuronal injury and METH withdrawal-potentiated NMDA-induced neuronal injury. Organotypic hippocampal slice cultures were exposed to METH (0 or 100 μM) for 6 days and withdrawn for 7 days, then exposed to NMDA (0 or 5 μM) for 24 h. Additional cultures were also exposed to this regimen and were co-incubated with BD1047 (100 μM), a specific inhibitor of ER-bound sigma-1 receptors, for the 24 h NMDA exposure. Cytotoxicity was assessed by analysis of propidium iodide uptake. These studies demonstrated that protracted METH exposure and withdrawal significantly potentiated the neuronal injury produced by NMDA exposure. Further, co-exposure to BD1047 with NMDA markedly attenuated neuronal injury in METH-naïve and METH-withdrawn organotypic cultures. As a whole, these data demonstrate that prolonged METH exposure, even at non-toxic concentrations, significantly alters glutamate receptor signaling. Inhibition of sigma-1 receptor-dependent Ca²⁺ release from the ER entirely prevented NMDA-induced toxicity in METH-naïve cultures and markedly reduced METH-potentiated toxicity. These findings demonstrate the importance of Ca²⁺-induced intracellular Ca²⁺ release in excitotoxic insult and suggest that blockade of glutamatergic overactivity may represent a therapeutic target in the treatment of METH withdrawal.     

Calcium receptor message, expression and function decrease in differentiating keratinocytes

Calcium-sensing receptor (CaSR) expression and function were studied in proliferating and differentiating cultured human gingival keratinocytes (HGKs). CaSR mRNA and protein were present in proliferating HGKs cultured in 0.03 mM [Ca²⁺] and decreased in cells induced to differentiate by culturing in 1.2 mM [Ca²⁺] for 2 days. CaSR protein was also detected in gingival tissue. Exposure to 10 mM extracellular [Ca²⁺] activated two sequential whole-cell currents. The first was a small, transient calcium release activated calcium current I_CRAC-like current with an inwardly rectifying I-V curve. The second current was larger with a linear I-V curve. Both currents were significantly decreased in differentiating cells. Neither neomycin nor gadolinium induced changes in whole cell currents nor in intracellular [Ca²⁺], but neomycin inhibited the late large current. Extracellular Ca²⁺ increased intracellular [Ca²⁺] of proliferating HGKs in a dose-dependent fashion. Comparison of the time-courses of the whole-cell currents and the intracellular [Ca²⁺] responses indicated both induced currents supported a Ca²⁺ influx. Extracellular [Mg²⁺] changes did not affect intracellular [Ca²⁺]. La³⁺ and 2-APB inhibited the whole cell current and intracellular [Ca²⁺] changes. The results indicate that the CaSR signaling response likely plays a major role in initiating Ca²⁺ induced differentiation responses in HGKs.     

Toxic effect of glutamate on granular cells of the cerebellum reduces cell ATP content. The role of calcium ions

The content of ATP in cultured cerebellar granular cells during and after glutamate intoxication is determined using the luciferin-luciferase method. A 15-min exposure to glutamate (100 μM in a Mg²⁺-free medium) reduces the content of ATP to 63.8±3.01% of the initial value, followed by further drop of ATP during the postglutamate period. In a Ca²⁺-free medium, the content of ATP during incubation with glutamate and in the postglutamate period drops to 89.2±4.8 and 74.7±3.6% of the initial level, respectively, while subsequent washout with a medium containing 1.8 mM Ca²⁺ results in a further decrease in the ATP content. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 123, No. 2, pp. 162–164, February, 1997     

Effects of Na+, K+ and Mg2+ on45Ca2+ uptake by pancreatic islets

Microdissected pancreatic islets of noninbredob/ob-mice were used to study ionic effects on the lanthanum-nondisplaceable⁴⁵Ca²⁺ uptake by islet cells. Omission of Mg²⁺ from the incubation medium had no effect, but the⁴⁵Ca²⁺ uptake was increased by omission of Na⁺ and decreased by omission of K⁺. Excess Mg²⁺ (1.2–15 mM) inhibited and excess K⁺ (4.7–25 mM) stimulated the⁴⁵Ca²⁺ uptake in a concentration-dependent manner. Stimulation of⁴⁵Ca²⁺ uptake in Na⁺-deficient islets was associated with an enhancement of the basal insulin release. Total abolishment of glucose-stimulated⁴⁵Ca²⁺ uptake in K⁺-deficient islets did not preclude a significant secretory response to glucose. It is concluded that the lanthanum-nondisplaceable⁴⁵Ca²⁺ uptake shows a partial correlation to insulin release.     

Intracellular nucleotides and polyamines inhibit the Ca2+-activated cation channel TRPM4b

TRPM4b (in contrast to the short splice variant TRPM4a) is a Ca²⁺-activated but Ca²⁺-impermeable cation channel. We have studied TRPM4 currents in inside-out patches. Supramicromolar Ca²⁺ concentrations applied at the inner side, [Ca²⁺]_i, activated TRPM4 with an EC₅₀ value of 0.37 mM, a value that is much higher than that of whole-cell currents. Current amplitudes decreased above 1 mM [Ca²⁺]_i, (IC₅₀ 9.3 mM). Sr²⁺ but not Ba²⁺could partially substitute for Ca²⁺. ATP, ADP, AMP and AMP-PNP all quickly and reversibly inhibited TRPM4 with IC₅₀ values between 2 and 19 M (at +100 mV). Adenosine also blocked TRPM4 at 630 M. The block at high ATP concentrations was incomplete and was not affected by the presence of free Mg²⁺. ADP induced the most sensitive block with an IC₅₀ of 2.2 M. For inhibition of TRPM4 by free ATP^4–, an IC₅₀ value of 1.7±0.3 M was calculated. GTP, UTP and CTP at concentrations up to 1 mM did not induce a similar block. Spermine blocked TRPM4 currents with an IC₅₀ of 61 M. In conclusion, TRPM4 is a channel that can be effectively modulated by intracellular nucleotides and polyamines.     

Adenosine Triphosphate Dependent Calcium Uptake by Subcellular Fractions from Bovine Neurohypophyses

Bovine neurohypophyses were fractionated by differential and density gradient ultracentrifugation and the Ca²⁺ uptake and ATPase activities in the microsomal, mitochondrial and secretory granule fractions were studied. The microsomal and mitochondrial fractions accumulated Ca²⁺ in the presence of ATP. The accumulation by the latter per mg protein was at least twice as large as by the former. This Ca²⁺ accumulation was accompanied by liberation of inorganic phosphate (P_i). In the presence of sodium azide (2 mM) Ca²⁺ uptake and P_i liberation were inhibited in the mitochondrial, but not in the microsomal fraction. Further studies of the microsomal fraction revealed that the ATP-dependent Ca²⁺ uptake and P_i liberation activities were temperature and pH-dependent and required Mg²⁺. Both activities were stimulated by very low concentrations of Ca²⁺ (1–10 μM) and were inhibited by EGTA (2 mM). N-ethyl-maleimide (2 mM) inhibited both the Ca²⁺ uptake and ATPase activities of the microsomal fraction. These results suggest the presence of a membrane ATPase that is stimulated by both Ca²⁺ and Mg²⁺. It is suggested that the observed Ca²⁺ uptake activities are involved in maintaining a low axoplasmic free Ca²⁺ concentration, thus playing an important role in the release mechanism of vasopressin by the neuro-secretory terminals.     

Immunocytochemical evidence for in vitro release of glutamate and GABA from separate nerve terminal populations in the rat pontine nuclei

Summary A quantitative electron microscopic immunocytochemical method was used to study the synaptic handling of glutamate and GABA in slice preparations from the rat pontine nuclei. Slices were subjected to a depolarizing stimulus (55 mM K⁺, 20 min) in the presence of a physiological or low Ca²⁺concentration. Depolarization at physiological [Ca²⁺] evoked a depletion of glutamate-like immunoreactivity from nerve terminals that contain round vesicles and establish asymmetric synaptic contacts. When depolarization was induced in the presence of only 0.1 mM Ca²⁺ (10 mM Mg²⁺ added), the loss of glutamate was significantly reduced or abolished, indicative of a Ca²⁺dependent component of glutamate release. By means of a double labeling immunocytochemical method we could identify a population of nerve terminals that displayed strong GABA-like immunoreactivity, and a level of glutamate like immunoreactivity that was low but yet clearly above background level. This type of terminal contains elongated or pleomorphic vesicles and establishes symmetric synaptic contacts. In these terminals, depolarization evoked a Ca²⁺-dependent depletion of GABA like immunoreactivity, but failed to change the level of glutamate like immunoreactivity. The present study demonstrates that two different types of nerve terminal in the rat pontine nuclei contain releasable pools of glutamate and GABA, respectively, and that the GABA releasing terminals also contain a non releasable pool of glutamate. The glutamate of the latter pool could act as precursor of GABA.     

Magnesium dependent contraction of glycerinated smooth muscle

The effect of Mg²⁺ on the contraction of glycerinated smooth and skeletal muscles was examined. In the absence of Ca²⁺, glycerinated smooth muscle contracted slowly when exposed to 5 mM adenosine triphosphate (ATP) and Mg²⁺. The magnitude of this contraction depended on the concentration of Mg²⁺ in a range of 1–20 mM. When 20 mM Mg²⁺ was used with ATP, glycerinated smooth muscle contracted almost maximally in a Ca²⁺ free environment, and it did not contract further on subsequent addition of 0.1 mM of free Ca²⁺ (EGTA-CaEGTA buffer). In contrast, in skeletal muscle a Ca²⁺ dependent contraction could always be elicited in the presence of 1–20 mM Mg²⁺ and 5 mM ATP. Tension development in smooth muscle induced by Mg²⁺ and ATP was not due to a contamination by Ca²⁺ or the removal of a Ca²⁺-sensitive protein. In the present experimental conditions, glycerinated smooth muscle developed a Mg²⁺-dependent contraction in a Ca²⁺ free medium, but glycerinated skeletal muscle did not.     

Releasing Effect of Calcium and Phosphate on Catechol-amines,ATP, and Protein from Chromaffin Cell Granules

Addition of Ca²⁺3 mM to isolated bovine adrenal niedullary granules incubated in 130 mM K-phosphate buffer pH 6.8–7.2 causes a rapid initial release of noradrenaline (NA), adrenaline (A), ATP and soluble protein. The effect is increased by addition of 1–6 mg/ml of RNA and inhibited by 1–6 mM Mg²⁺. The effect of Ca²⁺is dependent on the presence of phosphate ions in the incubation medium and does not occur in sucrose, RCI or NaCl. Addition of freshly precipitated calcium phosphate is also effective in inducing release, but addition of CaHPO₄ or Ca₂P₂O₇ had no effect. CaCl₂ 0.3–1 mM did not increase the release but was effective together with 1–6 mg/ml of RNA. In the absence of phosphate ions Ca²⁺and Mg²⁺as well as the divalent ions Ba²⁺and Sr²⁺inhibited the release. The catecholamine releasing effect of Ca²⁺in the presence of phosphate is reduced by 2 mM ATP and almost completely prevented by ATP-Mg²⁺. It is suggested that the increased outflow of granular contents is due to membrane alterations induced by Ca-phosphate.     

Role of K+ efflux in apoptosis induced by AMPA and kainate in mouse cortical neurons

Activation of ionotropic glutamate receptors can induce neuronal apoptosis in vitro and in vivo. We showed previously that activation of the N-methyl-D-aspartic acid (NMDA) subtype of glutamate receptors in a low Ca²⁺ and low Na⁺ condition induced apoptotic neuronal death, and that the K⁺ efflux via NMDA receptor channels was likely a key event in NMDA-induced apoptosis. Since non-NMDA receptors, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and kainate receptors, are also permeable to K⁺, we tested the hypothesis that stimulating K⁺ efflux via non-NMDA receptor channels could induce apoptosis in cultured cortical neurons.Using a Ca²⁺-free and Na⁺-free external solution, application of kainate revealed outward membrane currents carried by K⁺ efflux. In a low Ca²⁺/low Na⁺ medium, a 5-h exposure to 50–500 μM AMPA in the presence of the NMDA receptor antagonist MK801 induced dose-dependent neuronal death 24 h after the onset of the insult, accompanied by intracellular K⁺ reduction and caspase-3 activation. The AMPA-induced cell death was attenuated by the caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) and by the protein synthesis inhibitor cycloheximide. Reducing K⁺ efflux by raising extracellular K⁺ concentration from 5 to 25 mM attenuated AMPA-triggered cell death, the Ca²⁺ channel antagonist nifedipine showed no effect on the AMPA toxicity. Kainate induced similar neuronal death sensitive to attenuation by Z-VAD-FMK or elevated extracellular K⁺.We suggest that the non-NMDA receptor-mediated K⁺ efflux may participate in apoptotic process and that blocking excessive K⁺ efflux mediated by NMDA and non-NMDA receptors may selectively prevent neuronal apoptosis under certain pathological conditions.     

Effects of stimulation and inhibition of protein kinase C on the cytosolic calcium concentration in rabbit afferent arterioles

The effect of the protein kinase C (PKC) inhibitor chelerytrine (Ch) and the PKC activator 12–0-tetradecanoyl-phorbol-13-acetate (TPA) on the cytosolic calcium concentration ([ Ca²⁺]_i) in isolated intact rabbit afferent arterioles was investigated. [Ca²⁺]_i was measured in the proximal and distal parts of the arteriole. Administration of 1 μM Ch gave rise to a peak followed by an elevated level of [Ca²⁺]_i in both these parts. Neither the peak nor the elevated level of [Ca²⁺]_i was significantly reduced by 1 μM nifedipine. The relative peak increase in [Ca²⁺]_i in response to 1 μM noradrenaline (NA) or to 10 nM angiotensin II (AII) was significantly blunted in both parts after preincubation with 1 μM Ch. Depolarization with 25 mM K⁺ increased [Ca²⁺]_i in both parts. Preincubation with Ch did not affect the increase in [Ca²⁺]_i induced by 25 mM K⁺. TPA (10 and 100 nM ) did not significantly affect the basal [Ca²⁺]_i in the afferent arteriole. The [Ca²⁺]_i response to NA or 25 mM K⁺ was not affected by TPA. We conclude that blockade of PKC increases [Ca²⁺]_i in afferent arteriolar smooth muscle by a mechanism independent of L-type voltage-sensitive calcium channels. Inhibition of PKC blunts the relative increase in [Ca²⁺]_i in response to AII and, to a lesser extent, that induced by NA. We conclude that PKC might be important in modulating the calcium changes that occur in response to these vasoconstrictors.     

Glutamate receptor activation in cultured cerebellar granule cells increases cytosolic free Ca2+ by mobilization of cellular Ca2+ and activation of Ca2+ influx

Summary The Ca²⁺ sensitive fluorescent probe, fura-2 has been used to monitor cytosolic free calcium levels in mature primary cultures of cerebellar granule cells during exposure to L-glutamate and other excitatory amino acids: quisqualate (QA), kainate (KA) and N-methyl-D-aspartate (NMDA). Glutamate at micromolar concentrations produced a prompt and dose-related increase in the intracellular concentration of free Ca²⁺, ([Ca²⁺]_i), whereas QA, KA and NMDA had no effect. This increase was also seen in the absence of extracellular Ca²⁺, suggesting that L-glutamate promotes mobilization of Ca²⁺ from intracellular stores. In the presence of extracellular calcium, the elevation of [Ca²⁺]_i was, in part, mediated by an increase in the plasma membrane permeability to Ca²⁺. This Ca²⁺ influx was not affected by the Ca²⁺-channel antagonist 1-Verapamil. However, L-Verapamil did block the increase in [Ca²⁺]_i seen after depolarization of the cells with potassium. The Ca²⁺ response elicited by glutamate was partially blocked by the excitatory amino acid antagonist glutamate diethyl ester (GDEE). Furthermore, glutamate stimulated the formation of inositol mono-, bis-, tris and tetrakisphosphates (IP1, IP2, IP3, and IP4) suggesting a role for these compounds for the increase in [Ca²⁺]_i.