Voltage-sensitive Calcium Channel Antagonists Research Articles

Activity regulates the expression of AMPA receptor subunit GluR4 in developing visual cortex

In the developing visual cortex, the expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit GluR4 precedes that of the other AMPAR subunits GluR1-3, and then declines to become almost absent in adults. The current study shows that the neuronal activity regulates the expression of GluR4 by a culture system in vitro and a dark-rearing (DR) system in vivo. Membrane depolarization by treatment of cultured neurons of the visual cortex with a high concentration of KCl (35 mm; HK) promoted a decline in the expression of GluR4. This effect of HK on the expression of GluR4 was significantly blocked by the addition of an N-methyl-d-aspartate receptor (NMDAR) antagonist, (D)-2-amino-5-phosphonovaleric acid (APV), but not by the voltage-sensitive calcium channel antagonist nifedipine. Moreover, the Ca(2+)-calmodulin-dependent kinase (CaMKII) inhibitor KN62 and the cAMP-dependent protein kinase A (PKA) inhibitor H-89 blocked this effect, which suggests the involvement of Ca(2+) influx via NMDAR and the subsequent activation of CaMKII and PKA. Conversely, the MAP kinase inhibitor PD98059 promoted the effect of HK on the expression of GluR4. Significantly, APV, KN62, H-89 and PD98059 either promoted or inhibited the expression of GluR4 even in normal KCl (5 mm) conditions. The developmental change in the expression of GluR4 was significantly attenuated in DR in vivo, and the results suggest that neuronal activity such as visual experience may be involved in the mechanism of the expression of GluR4, which is mediated by NMDAR and tuned by certain protein kinases at an early developmental stage in the visual cortex.

Frequency-Dependent ERK Phosphorylation in Spinal Neurons by Electric Stimulation of the Sciatic Nerve and the Role in Electrophysiological Activity

The phosphorylation of extracellular signal-regulated kinase (pERK) in DRG and dorsal horn neurons is induced by the C-fiber electrical stimulation to the peripheral nerve. The present study was designed to investigate the expression and modulation of pERK in the rat dorsal horn neurons produced by repetitive electrical stimulation, and its involvement in the electrophysiological activity of dorsal horn neurons. Electrical stimulation of C-fiber intensity at different frequencies was applied to the sciatic nerve; the stimuli-induced pERK expression and the activity in dorsal horn neurons were studied by immunohistochemistry and extracellular recording, respectively. Electrical stimulation of C-fibers (3 mA) induced pERK expression in dorsal horn neurons in a frequency-dependent manner, indicating that the frequency of electrical stimulation is an important factor which activates the intracellular signal pathway in the spinal cord. To demonstrate the underlying mechanism of this frequency-dependent pERK expression, an NMDA receptor antagonist, MK-801, and a voltage sensitive calcium channel antagonist, nifedipine, were administrated intrathecally before the stimulation. We found that high frequency (0.5 Hz and 10 Hz) but not low frequent (0.05 Hz) stimulus-evoked pERK was partially inhibited by MK-801. Both high and low frequency stimulus-evoked pERK were inhibited by the nifedipine treatment. The extracellular single unit activities were recorded from the laminae I-II and V of the L4-5 dorsal horn, and we found that blockage of the intracellular ERK signal suppressed the wind-up responses in a dose-dependent manner. In contrast, any change in the mechanically evoked responses was not observed following the administration of ERK inhibitor. These observations indicate that ERK activation plays an important role in the induction of the wind-up responses in dorsal horn nociceptive neurons.

Potent Neurotoxic Action of the Shellfish Biotoxin Yessotoxin on Cultured Cerebellar Neurons

Yessotoxin (YTX) and its analogues are disulphated polyether compounds of increasing occurrence in seafood. The biological effects of these algal toxins on mammals and the risk associated to their ingestion have not been clearly established. We have used primary cultures of rat cerebellar neurons to investigate whether YTX affected survival and functioning of central nervous system neurons. Exposure to YTX (> or =25 nM) caused first (approximately 8 h) weakening, granulation, and fragmentation of neuronal network, and later (approximately 48 h) complete disintegration of neurites and extensive neuronal death, with a significant decrease in the amount of filamentous actin. The concentration of YTX that reduced by 50% the maximum neuronal survival (EC50(48)) was approximately 20 nM. Lower toxin concentrations (approximately 15 nM) also caused visible signs of toxicity affecting neuronal network primarily. Removal of YTX after 5 h exposure delayed the onset of neurotoxicity but did not prevent neuronal degeneration and death. YTX induced a two-fold increase in cytosolic calcium that was prevented by the voltage-sensitive calcium channel antagonists nifedipine and verapamil. These antagonists were, however, completely ineffective in reducing neurotoxicity. Voltage-sensitive sodium channel antagonists saxitoxin and nefopam, and the NMDA receptor antagonist MK-801 also failed to prevent YTX neurotoxicity. Neuronal death by YTX involved typical hallmarks of apoptosis and required the synthesis of new proteins. Our data suggest neuronal tissue to be a vulnerable biological target for YTX. The potent neurotoxicity of YTX we report raises reasonable concern about the potential risk that exposure to YTX may represent for neuronal survival in vivo.

Effect of glutamate and extracellular calcium on uptake of inorganic lead (Pb 2+) in immortalized mouse hypothalamic GT1–7 neuronal cells

We have previously shown that although glutamate alone has no effects on viability of mouse hypothalamic GT1–7 cells, it clearly enhances Pb 2+-induced cytotoxicity. It is likely that Pb 2+ must enter cells to exert most of its toxic effects. Pb 2+ is known to substitute for Ca 2+ in many cellular processes. Therefore, we studied the uptake mechanisms of Pb 2+ into GT1–7 neuronal cells with a special focus on the role of extracellular calcium (Ca 2+), voltage-sensitive calcium channels (VSCCs) and glutamate. Basal uptake of Pb 2+ (1 μM or 10 μM), i.e. without any external stimulus, clearly increased in nominally Ca 2+-free buffer and was partially abolished by 13 mM Ca 2+ when compared to uptake in the presence of a physiological concentration of extracellular Ca 2+ (1.3 mM). Depolarization by 25 mM K +, or antagonists of VSCCs, verapamil (10 μM) or flunarizine (10 μM) had no clear effect on basal Pb 2+ uptake. Glutamate (1 mM) increased Pb 2+ uptake, but only when cells were treated with 1 μM Pb 2+ in the presence of 1.3 mM Ca 2+. Our data suggest that Pb 2+ competes for the same cellular uptake pathways with Ca 2+, although not via VSCCs. In addition, enhancement of Pb 2+-induced neurotoxicity by glutamate may be due to increased neuronal uptake of Pb 2+.

Glutamate increases toxicity of inorganic lead in GT1-7 neurons: partial protection induced by flunarizine.

Recent studies point to an interaction between the glutamatergic neurotransmitter system and inorganic lead (Pb) neurotoxicity. Pb (1-100 microM) evoked cytotoxicity over the period of 72 h in mouse hypothalamic GT1-7 neurons. Glutamate (0.1 or 1 mM) on its own did not have any effect on cell viability. However, 1 mM glutamate clearly increased Pb-induced cell death at 48 and 72 h. Although flunarizine (0.1-10 microM), an antagonist of L- and T-type voltage-sensitive calcium channels (VSCCs), partially protected from the cytotoxicity induced by co-exposure to Pb (10 or 100 micro M) and glutamate (1 mM), it had no protective effect on cytotoxicity induced by Pb alone. The flunarizine-induced protection was dependent on time and observed only at 48 h. Neither verapamil, an antagonist of L-type VSCCs, nor DIDS, an inhibitor of anion exchange, at non-toxic concentrations (0.1-10 microM) had any effect on cytotoxicity induced by Pb alone or together with glutamate at any studied time point. Co-exposure to Pb and glutamate also resulted in more prominent production of reactive oxygen species (ROS) than either of the compounds alone. Interestingly, we observed an increase in intracellular glutathione (GSH) levels in cells exposed to micromolar concentrations of Pb. Glutamate decreased the levels of intracellular GSH and also partially reduced the Pb-induced increase in GSH levels. These results suggest that the interaction of glutamate and Pb results in increased neuronal cell death via mechanisms that involve an increase in ROS production, a decrease in intracellular GSH defense against oxidative stress and probably T-type VSCCs.

Powerful antinociceptive effects of the cone snail venom-derived subtype-selective NMDA receptor antagonists conantokins G and T

Subunit non-selective N-methyl- d-aspartate (NMDA) receptor antagonists reduce injury-induced pain behavior, but generally produce unacceptable side effects. In this study, we examined the antinociceptive and motor effects of cone snail venom-derived peptides, conantokins G and T (conG and conT), which are selective inhibitors of the NR2B or NR2A and NR2B subtypes of the NMDA receptor, respectively. We tested the effects of conG and conT in models of tissue (formalin test), nerve injury (partial sciatic nerve ligation) and inflammation-induced (intraplantar Complete Freund's Adjuvant; CFA) pain in mice. In the formalin test, intrathecal (i.t.) conG or conT suppressed the ongoing pain behavior (ED 50 and 95% confidence intervals (CI), 11 (7–19) and 19 (11–33), respectively) at doses that were 17–27 times lower than those required to impair motor function (accelerating rotarod treadmill test: ED 50 and 95% CI, 300 (120–730) and 320 (190–540) pmol, respectively). By comparison, SNX-111, an N-type voltage-sensitive calcium channel antagonist that is also derived from cone snail venom, produced significant motor impairment at a dose (3.0 pmol, i.t.) that was only partially efficacious in the formalin test. Furthermore, conG reversed the allodynia produced by nerve injury, with greater potency on thermal (ED50 and 95% CI, 24 (10–55) pmol) than on mechanical allodynia (59 (33–105) pmol). Finally, a single dose of conG (100 pmol, i.t.) also reduced CFA-evoked thermal and mechanical allodynia. Taken together, these results demonstrate that conantokins exhibit potent antinociceptive effects in several models of injury-induced pain. The study supports the notion that drugs directed against subtypes of the NMDA receptor, by virtue of their reduced side-effect profile, hold promise as novel therapeutic agents for the control of pain.

Nicotine-induced phosphorylation of extracellular signal-regulated protein kinase and CREB in PC12h cells.

We have investigated mechanisms of nicotine-induced phosphorylation of extracellular signal-regulated protein kinase (p42/44 MAP kinase, ERK) and cAMP response element binding protein (CREB) in PC12h cells. Nicotine transiently induced ERK phosphorylation at more than 1 microM. The maximal level of nicotine-induced ERK phosphorylation was lower than that of the membrane depolarization induced and, to a great extent, the nerve growth factor (NGF)-induced ERK phosphorylation. Nicotinic acetylcholine receptor (nAChR) alpha7 subunit-selective inhibitors had no significant effect on nicotine-induced ERK phosphorylation. L-Type voltage-sensitive calcium channel antagonists inhibited nicotine-induced ERK phosphorylation. Calcium imaging experiments showed that alpha7-containing nAChR subtypes were functional at 1 microM of nicotine in the nicotine-induced calcium influx, and non-alpha7 nAChRs were prominent in the Ca(2+) influx at 50 microM of nicotine. An expression of dominant inhibitory Ras inhibited nicotine-induced ERK phosphorylation. A calmodulin antagonist, a CaM kinase inhibitor, a MAP kinase kinase inhibitor inhibited nicotine-induced ERK and CREB phosphorylation. The time course of the phosphorylation of CREB induced by nicotine was similar to that of ERK induced by nicotine. These results suggest that non-alpha7 nAChRs are involved in nicotine-induced ERK phosphorylation through CaM kinase and the Ras-MAP kinase cascade and most of the nicotine-induced CREB phosphorylation is mediated by the ERK phosphorylation in PC12h cells.

Characterization of electrically evoked [ 3H]- d-aspartate release from hippocampal slices

Electrical stimulation has certain advantages over chemical stimulation methods for the study of neurotransmitter release in brain slices. However, measuring detectable quantities of electrically evoked release of endogenous or radiolabeled markers of excitatory amino acid neurotransmitters has required current intensities or frequencies much higher than those usually required to study other transmitter systems. We demonstrate here that [ 3H]- d-aspartate ( d-ASP) release can be detected from hippocampal slices at lower stimulation intensities in the presence of a glutamate reuptake inhibitor. Subsequently, we optimized the electrical stimulus parameters for characterizing electrically evoked d-ASP release. Under the experimental conditions described, greater than 90% of electrically evoked d-ASP release is calcium-dependent. Evoked d-ASP release is markedly reduced by pre-treating slices with the synaptic vesicle toxin bafilomycin A1 (BAF A1) or in the presence of 10-mM magnesium. Evoked d-ASP release is also reduced to variable degrees by N- and P/Q type voltage-sensitive calcium channel antagonists. Neither spontaneous efflux nor evoked d-ASP release were affected by NMDA, AMPA or group I metabotropic glutamate receptor (mGluR) antagonists. Evoked d-ASP release was reduced in the presence of an adenosine A1 receptor agonist and potentiated by treatment with a group I mGluR5 agonist. Evoked [ 3H]- d-ASP release was similar in magnitude to evoked [ 3H]- l-glutamate ( l- GLU) release. Finally, in separate experiments using the same electrical stimulus parameters, more than 90% of electrically evoked endogenous l-GLU release was calcium dependent, a pattern similar to that observed for evoked [ 3H]- d-ASP release. Taken together, these results indicate that electrically evoked [ 3H]- d-ASP release mimics evoked glutamate release in brain slices under the experimental conditions employed in these studies.

The regulation of the retrograde axonal transport of [formula omitted]-β nerve growth factor is independent of calcium

Calcium has been shown to play a major role in the regulation of endocytosis and exocytosis of synaptic vesicles and retrograde axonal transport of proteins. The role of calcium in the regulation of neurotrophin retrograde axonal transport is unknown. This study aimed to determine if calcium plays a role in the uptake and retrograde axonal transport of 125 I -β nerve growth factor ( 125 I -βNGF) within sympathetic neurons innervating the iris by comparing it with 125 I -anti-dopamine beta hydroxylase (anti-DBH). The nonspecific voltage-sensitive calcium channel (VSCC) antagonists, cadmium (200 nmol/eye) and nickel (100 nmol/eye) reduced the amount of 125 I -anti-DBH retrograde axonal transport by 90 and 70%, respectively. In contrast, cadmium (200 nmol/eye) had no effect on 125 I -βNGF retrograde axonal transport, while nickel (100 nmol/eye) caused a significant increase in the amount transported to the ganglia. The L-type VSCC antagonist nifedipine (10 nmol/eye) and N-type VSCC antagonist omega-conotoxin (1.5 nmol/eye) both had no effect on 125 I -anti-DBH retrograde axonal transport which suggests that these types of calcium channels are not involved in the exocytosis/endocytosis of anti-DBH containing vesicles. Thapsigargin (0.2 nmol/eye), an inhibitor of sarcoplasmic reticulum Ca 2+-ATPases also significantly inhibited 125 I -anti-DBH transport but had no effect on 125 I -βNGF retrograde transport. This suggests that 125 I -anti-DBH and 125 I -βNGF are internalized into different vesicle types and that the endocytosis and retrograde axonal transport of 125 I -βNGF are not dependent upon calcium.

Cyclosporine-induced coronary artery constriction—dissociation between thromboxane release and coronary vasospasm

Cyclosporine influences vascular tone, including that of coronary arteries. But its effect on myocardial prostanoid release, which may contribute to a drug-induced coronary and/or myocardial dysfunction, remains unknown. We used the isolated perfused rat heart to study the effect of cyclosporine on both the mechanical function parameters and myocardial prostanoid release into the effluent by ELISA. Cyclosporine (5 μM) induced an increase of perfusion pressure from 40 ± 3 to 73 ± 4 mm Hg within 60 minutes ( p < 0.001), reflecting an increase of coronary tone. Cyclosporine did not affect heart rate but contractility (+dp/dt max) tended to decrease, although not significantly. The drug’s effect on coronary tone was rapidly reversible upon withdrawal. Cyclosporine perfusion resulted in an increase of thromboxane B 2 liberation from 236 ± 150 to 1321 ± 354 pg/ml effluent ( p < 0.001), whereas the 6-keto-prostaglandin F 1α release was unaffected. The vehicle cremophor did not change any of these parameters. Neither inhibition of myocardial prostanoid formation with acetylsalicylic acid nor thromboxane receptor blockade prevented the cyclosporine-induced increase of perfusion pressure. However, perfusion with nitroglycerin or the voltage-sensitive calcium channel antagonist nifedipine in addition to cyclosporine were able to prevent the increase of perfusion pressure. This is the first time it has been demonstrated that cyclosporine induces an acute release of the prostanoid thromboxane within the myocardium. Despite the resulting imbalance in favor of the vasoconstrictive prostanoid, a dependency of the cyclosporine-induced increase of coronary tone on this imbalance was excluded. Conversely, nitric oxide donation or calcium channel blockade were able to prevent the negative effect of the drug on coronary tone, supporting the concept of endothelium-dependent and/or myogenic mechanism of cyclosporine toxicity on the coronary vascular bed.

Long-term depression at the medial perforant path-granule cell synapse in developing rat dentate gyrus

Long-term depression (LTD) is a decrease in synaptic efficacy that may model the elimination of inappropriate synapses during brain development. LTD might therefore be expected to be prominent in the juvenile hippocampal dentate gyrus (DG), where the majority of neuronogenesis and excitatory synapse production and pruning occur in the first postnatal month. Thus far, however, LTD in immature DG remains unexplored. Low-frequency stimulus induced homosynaptic LTD was studied at the medial perforant path-granule cell synapse in rats 8–30 days of age. LTD was most consistent and was of greatest magnitude in the youngest animals, and was more robust in response to stimulation at 1 Hz than at 3 or 5 Hz. LTD was saturable by repetitive delivery of low-frequency stimulation, and reversible by tetanic stimulation that induced long-term potentiation (LTP). LTD of the field EPSP was not prevented by bath application of the NMDA receptor antagonist AP5, the mGluR antagonist MCPG, or the L-type voltage sensitive calcium channel antagonist nitrendipine. In whole cell recordings LTD induction was blocked by hyperpolarization of the postsynaptic neuron but not by calcium chelation with BAPTA. Calcium chelation blocked LTP and simultaneously unmasked tetanus induced LTD. These data demonstrate that LTD is prominent in immature DG, that LTP and LTD are complementary processes, and that LTD is likely to be induced postsynaptically because it is voltage dependent, although the mechanism of voltage dependence remains to be elucidated.

Activation of voltage-dependent sodium channels in cultured cerebellar poffule cells induces neurotoxicity that is not mediated by glutamate release.

Exposure of rat cerebellar granule cell cultures to neurotoxins that specifically enhance the open state probability of voltage-dependent Na+ channels, resulted in neuronal death as estimated by a cell viability assay based on fluorescent staining and 51Cr-uptake. Toxicity was detected within 1 h after addition of 100 microM veratridine and was complete within 10-18 h; it was dose-dependent and was found to be completely abolished by tetrodotoxin, an Na+ channel blocker. When veratridine was replaced by an alpha-scorpion toxin, similar observations were done. In contrast, when cultured neurons prepared ffom the cerebral hemisphere of fetal rat brain were exposed to either veratridine or alpha-scorpion toxin for 18 h or even for a longer time of incubation, no neuronal death was observed. DNA fragmentation analysis showed that the toxicity was not mediated by apoptosis. Neuronal death was neither prevented by glutamate receptor antagonists, nor by depletion of endogenous glutamate, nor by voltage sensitive calcium channel antagonists such as omega-Conotoxin-GVIA (N-type channels), omega-Agatoxin-IVA (P-type channels), nimodipine and nitrendipine (L-type channels). Our study indicates that prolonged opening of Na+ channels induced neuronal death of cerebellar granule cells which is not mediated by glutamate and reveals novel neurotoxic mechanism in addition to the well-established excitatory amino acid receptor pathway.

In Vitro Binding of [ 3H]Nimodipine and [ 3H]CGS-19755 to Rat Brain in Focal Cerebral Ischemia

Focal cerebral ischemia results in increased in vivo binding of calcium channel antagonists to both the L-type voltage sensitive calcium channel (VSCC) and the N-methyl- d-aspartate (NMDA) receptor-linked calcium channel. It was the aim of this study to investigate the effect of focal cerebral ischemia on the in vitro binding of calcium channel antagonists to rat brain. Quantitative autoradiography was used to measure regional in vitro binding of the L-type VSCC antagonist [ 3H]nimodipine and the competitive NMDA receptor antagonist [ 3H]CGS-19755 to rat brain following 4 h of irreversible focal cerebral ischemia. [ 3H]Nimodipine binding to the nonischemic hemisphere was characterized by one binding site with regional binding affinity ( K D ) estimates ranging from 221 to 482 p M and maximal binding site densities ( B max ) ranging from 13.2 (9.6-17.5) pmol/g tissue (estimate and 95% confidence interval) in CA1 to 32.5 (26.5-39.9) pmol/g tissue in dentate. [ 3H]CGS-19755 binding to the nonischemic hemisphere was characterized by K D estimates ranging from 59 to 97 n M and B max values ranging from 143 (108-192) pmol/g tissue in cortex to 569 (515-641) pmol/g tissue in CA1. For [ 3H]CGS-19755 a model of two binding sites was applicable in several brain regions. No difference in binding site densities or binding affinities between ischemic and paired nonischemic structures (cortex and striatum) was observed with either ligand. In vitro binding of [ 3H]nimodipine and [ 3H]CGS-19755 to ischemic brain failed to identify ischemic-induced changes in calcium channel function previously reported by in vivo binding methods.

Neuroanatomical distribution of receptors for a novel voltage-sensitive calcium-channel antagonist, SNX-230 (ω-conopeptide MVIIC)

Neuronal voltage-sensitive calcium channels (VSCCs) are a diverse family of proteins that regulate entry of Ca 2+ into neurons. Selective antagonists of VSCCs have proven to be powerful pharmacological tools for identifying and characterizing these channels. A new VSCC antagonist, SNX-230 (also known as ω-conopeptide MVIIC), binds with high affinity to receptors in rat brain and blocks one or more high-threshold VSCCs that are neither L- nor N-type. We have defined the neuroanatomical distribution of the high-affinity non-L, non-N VSCC receptors for SNX-230 using [ 125I]SNX-230 bound to rat brain sections and compared it with that of [ 125I]SNX-111, a reversible blocker of N-type VSCCs. Highest densities of binding for both ligands were seen in areas rich in synaptic connections, such as the oriens, radiatum and molecular layers of the hippocampus. In general, the density of [ 125I]SNX-230-binding was higher in cerebellum compared with that in forebrain. In contrast, this general distribution of density was reversed for [ 125I]SNX-111. In the glomeruli of the olfactory bulb, binding of [ 125I]SNX-230 was undetectable compared with the high density of [ 125I]SNX-111-binding. Differential localization of the two ligands was also seen in cervical spinal cord. The clearly different localization of [ 125I]SNX-230 compared with that of [ 125I]SNX-111 in the olfactory bulb and spinal cord suggested that the binding sites for [ 125I]SNX-230 in other brain regions, while co-localized macroscopically, are also distinct from those for [ 125I]SNX-111. This was confirmed when addition of saturating concentrations of SNX-111 did not affect the distribution pattern of [ 125I]SNX-230-binding. These observations demonstrate that the high-affinity [ 125I]SNX-230 receptors in the brain and those of the N-type VSCCs are distinct molecular entities. Thus, SNX-230 (ω-MVIIC) offers a useful ligand for investigating the distribution and functions of non-L-, non-N-type VSCCs in the brain.

L-type voltage-sensitive calcium channel antagonists block heterosynaptic long-term depression in the dentate gyrus of anaesthetized rats

We examined the role of dihydropyridine-sensitive Ca 2+ channels in long-term potentiation (LTP) and long-term depression (LTD) in the dentate gyrus of pentobarbital-anaesthetized rats. Intrahippocampal infusions (1.0 μl) of nimodipine (20 mM) and nifedipine (20 mM), two antagonists of L-type voltage-sensitive calcium channels (VSCC), 60 min prior to medial perforant path tetanization, completely blocked the appearance of LTD in the lateral perforant path, without significantly affecting the simultaneous induction of medial path LTP. Heterosynaptic LTD was not significantly different from control animals following infusions of the L-type calcium channel agonist BAY-K8644 (1 mM). These results suggest that L-type VSCC activity is necessary for the induction of heterosynaptic LTD in the dentate gyrus in vivo.

Selective neurotoxicity induced by lasalocid in dissociated cerebral cultures

Dispersed cerebral cells prepared from 15–16-day mouse foetuses were cultured for 7 days and exposed to lasalocid for periods of 4, 18 or 48 hr. Cultures were examined by phase contrast microscopy and processed for scanning electron microscopy. Lasalocid (1 and 2 μ m) induced neurotoxic effects that were evident already at 4 hr, including swelling of perikarya, followed by cytolysis of most neurons present in the cultures. During the following 18 hr virtually the entire neuronal population degenerated completely. Cultures exposed to a lower concentration of lasalocid (0.5 μ m) for 48 hr showed only mild damage to some neurons, and at 0.2 μ m no damage could be observed, even after several days of continuous exposure. Notably, glial and other non-neuronal cells were not damaged by exposure to 2 μ m-lasalocid, and cell division resumed on returning the cultures to regular growth media. Similarly, lasalocid (2 μ m) was not toxic to cultured rat astrocytes. These morphological observations were followed by measurements of 45Ca 2+ influx in the dissociated cerebral cultures. Lasalocid (1 μ m) induced 45Ca 2+ influx of 40% above dimethyl sulphoxide control values. The voltage-sensitive calcium channel antagonists nimodipine (10 μ m) and D-600 (50 μ m) did not inhibit lasalocid-mediated 45Ca 2+ influx, whereas MK-801 [10 μ m; a non-competitive N-methyl- d-aspartate (NMDA) receptor/channel antagonist] exclusively blocked this influx and prevented cytotoxic damage. Conversely, NMDA and glutamate, which by themselves mediated 45Ca 2+ influx in these cultures, both potentiated lasalocid-induced 45Ca 2+ influx and cellular damage. These observations clearly demonstrate the selective neurotoxicity of lasalocid to cultured cerebral neurons, and may imply involvement of the NMDA receptor/channel in lasalocid-mediated neurotoxicity.

The role of early Ca 2+ influx in the pathogenesis of delayed neuronal death after brief forebrain ischemia in gerbils

To examine the role of calcium influx in the early phase after brief forebrain ischemia and subsequent delayed neuronal cell death in the hippocampus, 45Ca autoradiography and electron microscopic cytochemistry, by a combined oxalate-pyroantimonate method, were carried out in gerbil brains after 5 min bilateral common carotid arterial occlusion. Further, neuronal during the ischemic and postischemic periods was determined by conventional or immunohistochemical staining for microtubule-associated protein 2 (MAP2) with and without calcium-entry blockers. 45Ca autoradiography showed a high peak of calcium in the hippocampus at 5 min of recirculation. Electron cytochemical microscopy also demonstrated accumulation of intracellular calcium pyroantimonate deposits in the neuronal cells in all regions. At 30 min of reperfusion, amounts of calcium in the hippocampus returned to the control levels, and intracellular dense calcium pyroantimonate deposits were reduced in these areas. Loss of the reaction for MAP2 was noted in the medial CA1 of the hippocampus immediately after 5 min ischemia and at 5 and 30 min after reperfusion. MK-801 (10 mg kg −1, an N-methyl- d-aspartate (NMDA) receptor antagonist, injected intraperitoneally 1 h before ischemia, suppressed the early increase of calcium in the forebrain and neuronal cell necrosis in the CA1. However, neither injection of MK-801 30 min after reperfusion nor preischemic treatment with 0.5 mg kg −1 Nicardipine, voltage-sensitive calcium channel antagonists, prevented neuronal death. In immunohistochemical staining for MAP2, the ischemic lesion in the medial CA1 maintained after 5 min ischemia and the subsequent early reperfusion period in the untreated brains was protected by the preischemic injection of 10 mg kg −1 MK-801, but was not restored by the injection of 0.5 mg kg −1 Nimodipine or 1 mg kg −1 Nicardipine. In conclusion, it is suggested that an early excess of calcium influx could be caused mainly by excitatory amino acid overload through NMDA receptor-mediated calcium channels during the ischemic and early postischemic periods.

The effect of the calcium antagonist nifedipine on peripheral nerve function in streptozotocin-diabetic rats.

Recent data suggests that reduced nerve blood flow is implicated in the aetiology of experimental diabetic neuropathy, which may be prevented by manipulations that reduce receptor-mediated vasoconstrictor activity. This investigation examines the effects of nifedipine, a voltage-sensitive calcium channel antagonist which has a direct vasodilatory effect on vessels, on nerve conduction, hypoxic resistance and capillary density in streptozotocin-induced diabetic rats. Treated and non-treated non-diabetic and diabetic groups were employed. Diabetes duration was 2 months. Treatment was preventive, groups received a nifedipine dietary supplement (40 mg.kg-1.day-1) for 2 months from the start of the study. Conduction was measured in sciatic motor branches supplying tibialis anterior and gastrocnemius muscles, and sensory saphenous nerve. Diabetes resulted in a 23-28% reduction in motor conduction velocity (p < 0.001), and a 15% deficit for sensory saphenous nerve (p < 0.001). In the nifedipine-treated diabetic group, motor and sensory conduction deficits were minimal compared with non-treated diabetes (p < 0.001). Nifedipine treatment had no significant effect on conduction velocity in non-diabetic rats. In vitro measurement of sciatic nerve hypoxic resistance revealed a 60% increase in the time taken for compound action potential amplitude to reach half its initial value with diabetes (p < 0.001). This was not significantly affected by nifedipine treatment. Experimental diabetes or nifedipine treatment did not significantly alter sciatic nerve endoneurial capillary density. We conclude that nifedipine, a vasodilator which acts directly on vascular smooth muscle, prevents nerve conduction deficits in experimental diabetes.

Calcium channel blockers in vivo inhibit serotonin N‐acetyltransferase (NAT) activity in chicken retina stimulated by darkness and not by agents elevating intracellular cyclic AMP level

The molecular mechanism underlying the role of calcium influx in the regulation of retinal serotonin N-acetyltransferase (NAT) activity was studied in vivo in chickens. Systemic administration of organic antagonists of voltage-sensitive calcium channels (VSCC), i.e., nimodipine and nifedipine, resulted in a marked suppression of the nocturnal increase of NAT activity in chicken retina. In contrast, NAT activity stimulated by nonhydrolysable analogs of cyclic AMP (dibutyryl-cyclic AMP and 8-bromo-cyclic AMP), forskolin, a direct activator of adenylate cyclase, and by phosphodiesterase inhibitors (aminophylline and 3-isobutyl-1-methylxanthine), was not significantly affected by various tested VSCC antagonists. The inhibitory effect of nimodipine on the dark-dependent increase in NAT activity of chicken retina was abolished by Bay K 8644, a selective VSCC agonist. The results presented in this paper indicate an important role of calcium influx through L-type of VSCC in the induction of NAT activity in chicken retina, and suggest that a requirement of calcium ions in the process of NAT induction in the retina may be primarily at the level of cyclic AMP production.

Actions of neomycin on neuronal L-, N-, and non-L/non-N-type voltage-sensitive calcium channel responses.

The effects of neomycin on neuronal voltage-sensitive calcium channel (VSCC) responses were investigated by evaluating its effects on calcium-dependent neuronal responses that are sensitive and insensitive to the N-type voltage-sensitive calcium channel antagonist omega-conotoxin GVIA and the L-type VSCC antagonist nitrendipine. Chick synaptosomal 45Ca2+ influx and K(+)-evoked release of [3H]norepinephrine from chick cortical brain slices were omega-conotoxin GVIA sensitive and nitrendipine insensitive, suggesting that these responses are mediated predominantly by N-type VSCC. The K(+)-evoked increase of intracellular calcium in cortical neurons and the K(+)-evoked release of [3H]norepinephrine from rat brain cortical slices was partially sensitive to omega-conotoxin GVIA and nitrendipine, suggesting that these responses are mediated by N-, L- and non-L/non-N-type VSCC. Rat synaptosomal 45Ca2+ influx and the K(+)-evoked release of [3H]D-aspartate from rat hippocampal slices were completely insensitive to omega-conotoxin GVIA and nitrendipine, suggesting that these responses were mediated predominantly by non-L/non-N-type VSCC. Neomycin caused a concentration-dependent and virtually complete inhibition of all response parameters, with IC50 values ranging from 90 to 400 microM. The results suggest that neomycin is a nonselective inhibitor of neuronal responses mediated by L-, N-, and non-L/non-N-type VSCC.