Activation Of Voltage-sensitive Calcium Channels Research Articles

Role of voltage-gated calcium channels on striatal dopamine release induced by inorganic mercury in freely moving rats

The possible role of voltage-sensitive calcium channels (VSCC) activation on the HgCl2-induced dopamine release was investigated using selective VSCC blockers and the dopamine levels were measured by HPLC from samples obtained by in vivo brain microdialysis. Infusion of HgCl2 in nicardipine (10 or 100 μM) or flunaricine (10 μM) pretreated animals had no significant effect on dopamine release induced by HgCl2. Pretreatment with 100 μM flunaricine, 20 μM ω-conotoxin MVIIC, or ω-conotoxin GVIA significantly decreased the HgCl2-induced dopamine release over 61%, 88%, and 99%, respectively. HgCl2-induced dopamine release could be produced, at least in part, by activation of VSCC at dopaminergic terminals, especially N- and P/Q-type.

Relaxant effect of tetrazepam on rat uterine smooth muscle: role of calcium movement.

Tetrazepam is a benzodiazepine derivative clinically used as a muscle relaxant. The aim of the present work was to examine its effect on uterine smooth muscle of the rat in estrus. Tetrazepam required micromolar concentrations to relax contractile responses induced by KCl and acetylcholine in Ca2+ solution, but not oxytocin-induced contraction. In Ca(2+)-free solution, tetrazepam inhibited Ca(2+)-induced contractions in depolarized uterus and vanadate-induced contractions. We suggest that tetrazepam relaxes contractile responses induced by activation of voltage-sensitive calcium channels and receptor-operated calcium channels with little selectivity or that it antagonizes the effect of calcium at subsequent steps, possibly intracellular stores sensitive to vanadate but not sensitive to oxytocin. The inhibition of contraction of rat uterus is not related to high-affinity peripheral benzodiazepine binding sites.

Cell type‐specific and activity‐dependent dynamics of action potential‐evoked Ca2+ signals in dendrites of hippocampal inhibitory interneurons

In most central neurons, action potentials (APs), generated in the initial axon segment, propagate back into dendrites and trigger considerable Ca(2+) entry via activation of voltage-sensitive calcium channels (VSCCs). Despite the similarity in its underlying mechanisms, however, AP-evoked dendritic Ca(2+) signalling often demonstrates a cell type-specific profile that is determined by the neuron dendritic properties. Using two-photon Ca(2+) imaging in combination with patch-clamp whole-cell recordings,we found that in distinct types of hippocampal inhibitory interneurons Ca(2+) transients evoked by backpropagating APs not only were shaped by the interneuron-specific properties of dendritic Ca(2+) handling but also involved specific Ca(2+) mechanisms that were regulated dynamically by distinct activity patterns. In dendrites of regularly spiking basket cells, AP-evoked Ca(2+) rises were of large amplitude and fast kinetics; however, they decreased with membrane hyperpolarization or following high-frequency firing episodes. In contrast, AP-evoked Ca(2+) elevations in dendrites of Schaffer collateral-associated cells exhibited significantly smaller amplitude and slower kinetics, but increased with membrane hyperpolarization. These cell type-specific properties of AP-evoked dendritic Ca(2+) signalling were determined by distinct endogenous buffer capacities of the interneurons examined and by specific types of VSCCs recruited by APs during different patterns of activity. Furthermore, AP-evoked Ca(2+) transients summated efficiently during theta-like bursting and were associated with the induction of long-term potentiation at inhibitory synapses onto both types of interneurons. Therefore, the cell type-specific profile of AP-evoked dendritic Ca(2+) signalling is shaped in an activity-dependent manner, such that the same pattern of hippocampal activity can be differentially translated into dendritic Ca(2+) signals in different cell types. However, Cell type-specific differences in Ca(2+) signals can be 'smoothed out' by changes in neuronal activity, providing a means for common, cell-type-independent forms of synaptic plasticity.

Elevated Glucose Changes the Expression of Ionotropic Glutamate Receptor Subunits and Impairs Calcium Homeostasis in Retinal Neural Cells

Altered glutamatergic neurotransmission and calcium homeostasis may contribute to retinal neural cell dysfunction and apoptosis in diabetic retinopathy (DR). The purpose of this study was to determine the effect of high glucose on the protein content of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate glutamate receptor subunits, particularly the GluR2 subunit, because it controls Ca2+ permeability of AMPA receptor-associated channels. The effect of high glucose on the concentration of cytosolic free calcium ([Ca2+]i) was also investigated. The protein content of GluR1, GluR2, GluR6/7, and KA2 subunits was assessed by Western blot. Cobalt staining was used to identify cells containing calcium/cobalt-permeable AMPA receptors. The [Ca2+]i changes evoked by KCl or kainate were recorded by live-cell confocal microscopy in R28 cells and in primary cultures of rat retina, loaded with fluo-4. In primary cultures, high glucose significantly decreased the protein content of GluR1 and GluR6/7 subunits and increased the protein content of GluR2 and KA2 subunits. High glucose decreased the number of cobalt-positive cells, suggesting a decrease in calcium permeability through AMPA receptor-associated channels. In high-glucose-treated cells, changes in [Ca2+]i were greater than in control cells, and the recovery to basal levels was delayed. However, in the absence of Na+, to prevent the activation of voltage-sensitive calcium channels, the [Ca2+]i changes evoked by kainate in the presence of cyclothiazide, which inhibits AMPA receptor desensitization, were significantly lower in high-glucose-treated cells than in control cultures, further indicating that AMPA receptors were less permeable to calcium. Mannitol, used as an osmotic control, did not cause significant changes compared with the control. The results suggest that elevated glucose may alter glutamate neurotransmission and calcium homeostasis in the retina, which may have implications for the mechanisms of vision loss in DR.

CRF-induced calcium signaling in guinea pig small intestine myenteric neurons involves CRF-1 receptors and activation of voltage-sensitive calcium channels

Corticotropin-releasing factor (CRF) is a 41-amino acid peptide with distinct effects on gastrointestinal motility involving both CRF-1 and CRF-2 receptor-mediated mechanisms that are generally claimed to be centrally mediated. Evidence for a direct peripheral effect is rather limited. Electrophysiological studies showed a cAMP-dependent prolonged depolarization of guinea pig myenteric neurons on application of CRF. The current study aimed to test the direct effect of CRF on myenteric neurons and to identify the receptor subtype and the possible mechanisms involved. Longitudinal muscle myenteric plexus preparations and myenteric neuron cultures of guinea pig small intestine were incubated with the calcium indicator Fluo-4. Confocal Ca(2+) imaging was used to visualize activation of neurons on application of CRF. All in situ experiments were performed in the presence of nicardipine 10(-6) M to reduce tissue movement. Images were analyzed using Scion image and a specifically developed macro to correct for residual minimal movements. A 75 mM K(+)-Krebs solution identified 1,076 neurons in 46 myenteric ganglia (16 animals). Administration of CRF 10(-6) M and CRF 10(-7) M during 30 s induced a Ca(2+) response in 22.4% of the myenteric neurons (n = 303). Responses were completely abolished in the presence of the nonselective CRF antagonist astressin (n = 55). The selective CRF-1 receptor antagonist CP 154,526 (n = 187) reduced the response significantly to 2.1%. Stresscopin, a CRF-2 receptor agonist, could not activate neurons at 10(-7) M, and its effect at 10(-6) M (15.3%, n = 59) was completely blocked by CP 154,526. TTX 10(-6) M (n = 70) could not block the CRF-induced Ca(2+) transients but reduced the amplitude of the signals significantly. Removal of extracellular Ca(2+) blocked all responses to CRF (n = 47). L-type channels did not contribute to the CRF-induced Ca(2+) transients. Blocking N- or P/Q-type Ca(2+) channels did not reduce the responses significantly. Combined L- and R-type Ca(2+) channel blocking (SNX-482 10(-8) M, n = 64) abolished nearly all responses in situ. Combined L-, N-, and P/Q-type channel blocking also significantly reduced the response to 8.6%. Immunohistochemical staining for CRF-1 receptors clearly labeled individual cell bodies in the ganglia, whereas the CRF-2 receptor staining was barely above background. CRF induces Ca(2+) transients in myenteric neurons via a CRF-1 receptor-dependent mechanism. These Ca(2+) transients highly depend on somatic calcium influx through voltage-operated Ca(2+) channels, in particular R-type channels. Action potential firing through voltage-sensitive sodium channels increases the amplitude of the Ca(2+) signals. Besides centrally mediated effects, CRF is likely to modulate gastrointestinal motility on the myenteric neuronal level.

Characterization of the intracellular mechanisms mediating somatostatin and lanreotide inhibition of DNA synthesis and growth hormone release from dispersed human GH-secreting pituitary adenoma cells in vitro.

Somatostatin is an endogenous inhibitor of hormone secretion and cell proliferation. Treatment with somatostatin analogues in humans causes a reduction in size and secretory activity of endocrine tumours, including GH-secreting pituitary adenomas. This study was aimed to characterize the intracellular mechanisms mediating the in vitro antiproliferative and antisecretory effects of somatostatin and its analogue lanreotide, on primary cultures of GH-secreting pituitary adenoma cells. Thirteen GH-secreting pituitary adenoma postsurgical specimens were analysed for somatostatin receptor (SSTR) mRNA expression and a subset of them was analysed in vitro for the effect of somatostatin on cell proliferation, assessed by means of [3H]-thymidine uptake, and GH release, using an immunoradiometric assay. Moreover, the intracellular signalling involved in such effects has been studied. All the adenomas analysed expressed at least one somatostatin receptor subtype mRNA. SSTR2 mRNA was identified in 77% of the adenomas, SSTR1 and SSTR3 in 69% and SSTR5 in 60%. Somatostatin and lanreotide inhibited cell proliferation in phorbol ester (PMA)-stimulated conditions (10/13 adenomas), as well as after fetal calf serum (3/3 adenomas) or IGF-I stimulation (2/2 adenomas). Conversely, GHRH or forskolin treatments did not significantly affect DNA synthesis in adenoma cells in the presence or absence of somatostatin (2/2 and 4/4 adenomas, respectively). Vanadate pretreatment reversed somatostatin inhibition of PMA-induced DNA synthesis suggesting an involvement of tyrosine phosphatase in this effect (2/2 adenomas); this was confirmed by the direct induction of tyrosine phosphatase activity in two adenomas after somatostatin treatment. Somatostatin and also lanreotide caused significant inhibition of phorbol ester, forskolin, GHRH and KCl-dependent increase of GH secretion in the culture medium. Moreover, voltage-sensitive calcium channel activity induced by 40 mm KCl depolarization in microfluorimetric analysis, was significantly reduced (5/5 adenomas). These data show that somatostatin and lanreotide inhibit human GH-secreting pituitary adenoma cell proliferation and hormone release in vitro, and suggest that the activation of tyrosine phosphatases may represent intracellular signals mediating the antiproliferative effects and that the inhibition of the voltage-dependent calcium channels and adenylyl cyclase activities may control GH secretion.

Role of the extracellular signal-regulated kinase (Erk) signal transduction cascade in alpha(2) adrenoceptor-mediated vasoconstriction in porcine palmar lateral vein.

The mechanism of alpha(2) adrenoceptor-mediated vasoconstriction is unknown, but may involve activation of voltage-sensitive calcium channels, and/or a protein tyrosine kinase. Recently the extracellular signal-regulated kinase (Erk) cascade, often an event downstream of tyrosine kinase activation, has been shown to mediate vasoconstriction to a variety of agents. The aim of this present study was to determine the involvement of the Erk signal transduction cascade in alpha(2) adrenoceptor-mediated vasoconstriction, and to confirm the involvement of activation of voltage-sensitive calcium channels, and protein tyrosine kinase. Contractions to the alpha(2) adrenoceptor agonist UK14304 in the porcine palmar lateral vein in vitro were reduced 70 - 80% by the MEK inhibitors PD98059 (10 - 50 microM) and U0126 (10 - 50 microM), indicating the involvement of the Erk signal transduction cascade. Immunoblots also demonstrated an increase in the phosphorylated (activated) form of Erk in palmar lateral vein segments after contraction with UK14304, which was inhibited by PD98059 and U0126. The calcium channel blockers nifedipine and verapamil, or removal of extracellular calcium inhibited UK14304-induced contractions and phosphorylation of Erk, demonstrating the importance of an influx of extracellular calcium. UK14304-induced contractions were inhibited by PP2 (1 - 10 microM), a selective inhibitor of Src tyrosine kinases, but not by PP3, an inactive analogue. PP2 also prevented the phosphorylation of Erk by UK14304. These data demonstrate that alpha(2) adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon activation of the Erk signal transduction cascade, which is downstream of an influx of extracellular calcium, and activation of Src tyrosine kinases.

GABA inhibition of immortalized gonadotropin-releasing hormone neuronal excitability involves GABA(A) receptors negatively coupled to cyclic adenosine monophosphate formation.

Gamma-aminobutyric acid (GABA) has been implicated in the regulation of reproduction, particularly in the developmental modulation of gonadotropin-releasing hormone (GnRH) secretion. GnRH neurons are innervated by GABA-containing processes, and the administration of GABA stimulates and inhibits GnRH secretion in vivo and in vitro. We have previously shown that GABA can exert both of these actions in sequence, by acting directly on immortalized GnRH neurons. While the stimulation is the result of a GABA(A) receptor-mediated depolarization of the plasma membrane, the mechanism involved in the delayed inhibition is the subject of the present investigation. GABA (1 nM-10 microM) decreased the intracellular concentration of cyclic adenosine monophosphate (cAMP) in a dose- and time-dependent fashion. This effect was blocked by bicuculline and mimicked by muscimol but not by baclofen. To analyze the effect of GABA on cellular excitability, we used fura-2 loaded GT1-7 cells. Activation of voltage-sensitive calcium channels by high K+-induced depolarization (35 mM) increased [Ca2+]i. GABA (10 microM) and muscimol (10 microM) reduced the amplitude of K+-induced [Ca2+]i transients. This inhibition was blocked by forskolin (20 microM) or 8-Br-cAMP (1 mM). Altogether, these results show that GABA(A) receptors mediate a sustained inhibitory effect of GABA on GnRH neurons, and suggest the involvement of the cAMP pathway decreasing cellular excitability.

Apoptotic Cell Death and Impairment of L-Type Voltage-Sensitive Calcium Channel Activity in Rat Cerebellar Granule Cells Treated with the Prion Protein Fragment 106–126

Prion diseases are neurodegenerative pathologies characterized by the accumulation, in the brain, of altered forms of the prion protein (PrP), named PrPSc. A synthetic peptide homologous to residues 106–126 of PrP (PrP106–126) was reported to maintain the neurodegenerative characteristics of PrPSc. We investigated the intracellular mechanisms involved in PrP106–126-dependent degeneration of primary cultures of cerebellar granule neurons. Prolonged exposure of such neurons to PrP106–126 induced apoptotic cell death. The L-type voltage-sensitive calcium channel blocker nicardipine reproduced this effect, suggesting that blockade of Ca2+ entry through this class of calcium channels may be responsible for the granule cell degeneration. Microfluorometric analysis showed that PrP106–126 caused a reduction in cytosolic calcium levels, elicited by depolarizing K+ concentrations in these neurons. Electrophysiological studies demonstrated that PrP106–126 and nicardipine selectively reduce the L-type calcium channel current. These data demonstrate that PrP106–126 alters the activity of L-type voltage-sensitive calcium channels in rat cerebellar granule cells and suggest that this phenomenon is related to the cell death induced by the peptide.

Activation of the sodium pump blocks the growth hormone-induced increase in cytosolic free calcium in rat adipocytes.

GH promptly increases cytosolic free calcium ([Ca2+]i) in freshly isolated rat adipocytes. Adipocytes deprived of GH for 3 h or longer are incapable of increasing [Ca2+]i in response to GH, but instead respond in an insulin-like manner. Insulin blocks the GH-induced increase in [Ca2+]i in GH-replete cells and stimulates the sodium pump (i.e. Na+/K+-ATPase), thereby hyperpolarizing the cell membrane. Blockade of the Na+/K+-ATPase with 100 microM ouabain reversed these effects of insulin and enabled GH to increase [Ca2+]i in GH-deprived adipocytes. Both insulin and GH activated the sodium pump in GH-deprived adipocytes, as indicated by increased uptake of 86Rb+. Decreasing availability of intracellular Na+ by blockade of Na+/K+/ 2Cl- symporters or Na+/H+ antiporters abolished the effects of both hormones on 86Rb+ uptake and enabled both GH and insulin to increase [Ca2+]i in GH-deprived adipocytes. The data suggest that hormonal stimulation of Na+/K+-ATPase activity interferes with activation of voltage-sensitive calcium channels by either membrane hyperpolarization or some unknown interaction between the sodium pump and calcium channels.

Somatostatin and its analog lanreotide inhibit the proliferation of dispersed human non-functioning pituitary adenoma cells in vitro.

Somatostatin is a powerful inhibitor of hormone secretion and cell proliferation. Treatment with somatostatin analogs in humans causes a reduction in size and secretory activity of some endocrine tumors, including somatotropic pituitary adenomas. Less studied are the effects of somatostatin agonists on non-functioning pituitary adenomas (NFPAs). In this study we characterized the effects of somatostatin and its analog lanreotide on the proliferation of NFPAs in vitro and the intracellular mechanisms involved. Twenty-three NFPA post-surgical specimens were analyzed for somatostatin receptor (SSTR) expression and 12 of them were cultured in vitro to study somatostatin's effects on cell proliferation, assessed by means of [(3)H]thymidine uptake, and the intracellular signaling. One or more SSTR subtypes were expressed in 90% of the adenomas tested. Somatostatin and lanreotide treatment inhibited phorbol myristate acetate (PMA)-induced cell proliferation. Vanadate pretreatment reversed somatostatin and lanreotide inhibition of PMA-induced DNA synthesis suggesting an involvement of tyrosine phosphatase in this effect. In the only adenoma tested, somatostatin directly induced a tyrosine phosphatase activity. Somatostatin and lanreotide caused also a significant inhibition of voltage-sensitive calcium channel activity induced by 40mmol/l K(+) depolarization in microfluorimetric analysis. These data show that somatostatin and lanreotide inhibit human NFPA cell proliferation in vitro, and suggest that activation of tyrosine phosphatases and inhibition of the activity of voltage-dependent calcium channels may represent intracellular signals mediating this effect.

Prion protein fragment 106-126 induces apoptotic cell death and impairment of L-type voltage-sensitive calcium channel activity in the GH3 cell line.

The prion diseases are transmissible neurodegenerative pathologies characterized by the accumulation of altered forms of the prion protein (PrP), termed PrP(Sc), in the brain. Previous studies have shown that a synthetic peptide homologous to residues 106-126 of PrP (PrP 106-126) maintains many characteristics of PrP(Sc), i.e., the ability to form amyloid fibrils and to induce apoptosis in neurons. We have investigated the intracellular mechanisms involved in the cellular degeneration induced by PrP 106-126, using the GH3 cells as a model of excitable cells. When assayed in serum-deprived conditions (48 hr), PrP 106-126 (50 microM) induced cell death time-dependently, and this process showed the characteristics of the apoptosis. This effect was specific because a peptide with a scrambled sequence of PrP 106-126 was not effective. Then we performed microfluorimetric analysis of single cells to monitor intracellular calcium concentrations and showed that PrP 106-126 caused a complete blockade of the increase in the cytosolic calcium levels induced by K+ (40 mM) depolarization. Conversely, the scrambled peptide was ineffective. The L-type voltage-sensitive calcium channel blocker nicardipine (1 microM) also induced apoptosis in GH3 cells, suggesting that the blockade of Ca2+ entry through this class of calcium channels may cause GH3 apoptotic cell death. We thus analyzed, by means of electrophysiological studies, whether Prp 106-126 modulate L-type calcium channels activity and demonstrated that the apoptotic effect of PrP 106-126 was due to a dose-dependent inactivation of the L-type calcium channels. These data demonstrate that the prion protein fragment 106-126 induces a GH3 apoptotic cell death inducing a selective inhibition of the activity of the L-type voltage-sensitive calcium channels.

Calcium regulation of gene expression: isn't that spatial?

Calcium regulation of gene expression: isn't that spatial?

Attenuation of beta-amyloid neurotoxicity in vitro by potassium-induced depolarization.

The cell death of cultured neurons triggered by beta-amyloid peptides has been theorized to model, at least in part, the neurodegeneration associated with Alzheimer's disease. To investigate potential strategies to interrupt beta-amyloid neurotoxicity in vitro, we examined the effects of potassium-induced membrane depolarization, a treatment previously demonstrated to reduce development-related apoptosis in cultured neurons. We report here that cultured rat hippocampal neurons pretreated for several hours with 30 mM KCl exhibit significantly reduced vulnerability to aggregated beta-amyloid peptides. The potassium-mediated neuroprotection was mimicked by activation of voltage-sensitive calcium channels using S(-)-Bay K8644 and was attenuated by R(+)-Bay K 8644, a blocker of voltage-dependent calcium channels, and KN-82, an inhibitor of calcium/calmodulin-dependent protein kinase II. The protein synthesis inhibitor cycloheximide also attenuated beta-amyloid neurotoxicity. Addition of cycloheximide following 30 mM KCl significantly increased protection offered by membrane depolarization, whereas cycloheximide addition during membrane depolarization blocked the protective effect. These data suggest that one cellular pathway that can inhibit neuronal death induced by beta-amyloid involves calcium influx through voltage-sensitive channels followed by stimulation of calcium/calmodulin-dependent protein kinase activity and synthesis of new protein(s).

Stimulation of growth factor receptor signal transduction by activation of voltage-sensitive calcium channels.

To understand the mechanisms by which electrical activity may generate long-term responses in the nervous system, we examined how activation of voltage-sensitive calcium channels (VSCCs) can stimulate the Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Calcium influx through L-type VSCCs leads to tyrosine phosphorylation of the adaptor protein Shc and its association with the adaptor protein Grb2, which is bound to the guanine nucleotide exchange factor Sos1. In response to calcium influx, Shc, Grb2, and Sos1 inducibly associate with a 180-kDa tyrosine-phosphorylated protein, which was determined to be the epidermal growth factor receptor (EGFR). Calcium influx induces tyrosine phosphorylation of the EGFR to levels that can activate the MAPK signaling pathway. Thus, ion channel activation stimulates growth factor receptor signal transduction.

Specialization of tachykinin NK 1 and NK 2 receptors in producing fast and slow atropine-resistant neurotransmission to the circular muscle of the guinea-pig colon

We studied the relative contribution of tachykinin NK 1 and NK 2 receptors in producing nonadrenergic noncholinergic excitation of the circular muscle of the guinea-pig proximal colon in response to electrical field stimulation. All experiments were performed in the presence of atropine, guanethidine, indomethacin, apamin and l-nitroarginine. In organ bath experiments, electrical stimulation produced a tetrodotoxin-sensitive frequency-dependent contraction. The NK 1 receptor antagonists, FK 888 (1–10 μM) and GR 82,334 (0.3–3 μM) markedly reduced but did not abolish the nonadrenergic noncholinergic response. The NK 2 receptor antagonist, GR 94,800 (0.3–3 μM) was partly effective at 3 μM. The combined administration of FK 888 (10 μM) and GR 94,800 (3 μM) or GR 82,334 and GR 94,800 abolished the nonadrenergic noncholinergic contraction. The response to a prolonged period of stimulation (3 Hz for 5 min) was evenly depressed by FK 888 or GR 82,334, while GR 94,800 was more effective in inhibiting the late (87% inhibition) than the peak response (25% inhibition). In the presence of nifedipine (1 μM) a marked inhibition of the nonadrenergic noncholinergic contraction was observed and a time lag was evident between stimulus application and onset of contraction, which showed slow onset and offset kinetics. The nifedipine-resistant nonadrenergic noncholinergic contraction was unaffected by FK 888 or GR 82,334 but was suppressed by GR 94,800. Submaximally effective (1–3 nM) concentrations of substance P and neurokinin A produced distinct patterns of contraction: the response to substance P was fast and declined rapidly toward baseline; the response to neurokinin A was slow and sustained. In the presence of nifedipine, the response to substance P was greatly depressed and became slower in onset; nifedipine did not affect the contraction to neurokinin A but slowed its time-course. In sucrose gap experiments, either a short (10 Hz for 1 s) or a prolonged period of electrical stimulation (3 Hz for 3 min) evoked membrane depolarization, action potentials and contraction: in response to the “prolonged” stimulation, distinct phasic and tonic component of contraction were observed. Nifedipine abolished action potentials and the phasic contraction produced by a short period of stimulation, reduced by about 50% the maximal contraction developed during the prolonged stimulation without affecting the amplitude of the tonic response. In the presence of nifedipine, GR 82,334 (3 μM) blocked the membrane depolarization but did not affect contraction; GR 94,800 (0.1 μM) did not affect deporalization but abolished contraction. The present findings demonstrate that both NK 1 and NK 2 receptors mediate the nonadrenergic noncholinergic excitation of the circular muscle of the guinea-pig colon to nerve stimulation and indicate a remarkable specialization of the two receptors in producing a “fast” neurotransmission, NK 1 receptor-mediated, which relies upon the activation of voltage-sensitive calcium channels, versus a “slow” type of neurotransmission, NK 2 receptor-mediated, which is largely independent from activation of voltage-sensitive calcium channels.

Requirement for BDNF in Activity-Dependent Survival of Cortical Neurons

Cultured embryonic cortical neurons from rats were used to explore mechanisms of activity-dependent neuronal survival. Cell survival was increased by the activation of voltage-sensitive calcium channels (VSCCs) but not by activation of N-methyl-D-aspartate receptors. These effects correlated with the expression of brain-derived neurotrophic factor (BDNF) induced by these two classes of calcium channels. Antibodies to BDNF (which block intracellular signaling by BDNF, but not by nerve growth factor, NT3, or NT4/5) reduced the survival of cortical neurons and reversed the VSCC-mediated increase in survival. Thus, endogenous BDNF is a trophic factor for cortical neurons whose expression is VSCC-regulated and that functions in the VSCC-dependent survival of these neurons.

Calcium modulatory properties of 2,6-dibutylbenzylamine (B25) in rat isolated vas deferens, cardiac and smooth muscle preparations.

1. In rat isolated vas deferens the new compound 2,6-dibutylbenzylamine (B25) evoked a series of repeating rhythmic contractions. Concentration-response curves constructed for this effect were bell-shaped, indicating a biphasic effect for this compound. By contrast, B25 depressed heart contractility without any visible positive inotropic or chronotropic activity. 2. Experiments with tetrodotoxin, reserpine, capsaicin, alpha-adrenoceptor blocking compounds and other agents permit us to exclude a release of neuromediators or a direct stimulation of post-synaptic receptors to account for the rhythmic effect of B25 in the rat vas deferens. 3. In the same tissue, the increase in 45Ca2+ uptake, the voltage-dependency as well as the dependence of the B25-induced rhythmic activity upon the external calcium concentration indicate a direct activation of voltage-sensitive calcium channels (VSCC). 4. Verapamil paradoxically stimulated the rhythmic effect of B25 in the rat vas deferens. La3+ was inactive while nifedipine was a weak inhibitor. By contrast Ni2+ and Mn2+ ions were good inhibitors (IC50 < 10(-4) M), suggesting that a possible opening of T-type VSCC underlies rhythmic effect of B25. 5. In radioligand binding studies competition experiments with [3H]-nitrendipine indicated that only at high concentrations was B25 able to interact with dihydropyridine-sensitive binding sites of heart and vas deferens smooth muscle. 6. B25 (3-30 microM) counteracted the inhibitory effects of omega-conotoxin GVIA in field-stimulated rat vas deferens.

Voltage sensitive calcium channels mark a critical period in mouse neurodevelopment

Voltage sensitive calcium channel (VSCC) probes 125I-ω-GVIA Conotoxin (ω-GVIA), (+)-[5-methyl-3H]-PN200-110 (3H-PN200), and 3H-Nimodipine were bound to developing Swiss Webster mouse whole brain from postnatal days 3 to 24. 125I-ω-GVIA binding, thought to be presynaptic, showed a 50% increase between days 11 and 14. 3H-dihydropyridine binding, thought to be postsynaptic, showed spike patterns when measured developmentally. 3H-PN200 binding showed a >150% increase between days 11 and 15. 3H-Nimodipine binding showed a >100% increase between days 11 and 14. Depolarization-induced 45Ca fluxes also increased between days 8 and 16 by >500%. The dramatic increases indicated by these binding data correspond to a critical period described by Himwich (Int. Rev. Neurobiol. 4, 117, 1962) between postnatal days 11 and 14 in Swiss Webster mice; during this critical period, dendrites exhibit rapid outgrowth, sensory modalities come on line, EEG patterns mature, and the cortex reaches adult proportions. We conclude from these data that the increase in VSCC activity parallels a critical period in the development of the central nervous system in Swiss Webster mice.

Maitotoxin-induced intracellular calcium rise in PC12 cells: involvement of dihydropyridine-sensitive and omega-conotoxin-sensitive calcium channels and phosphoinositide breakdown.

The biological activities of maitotoxin are strictly dependent on the extracellular calcium concentration and are always associated with an increase of the free cytosolic calcium level. We tested the effects of voltage-sensitive calcium channel blockers (nicardipine and omega-conotoxin) on maitotoxin-induced intracellular calcium increase, membrane depolarization, and inositol phosphate production in PC12 cells. Maitotoxin dose dependently increased the cytosolic calcium level, as measured by the fluorescent probe fura 2. This effect disappeared in a calcium-free medium; it was still observed in the absence of extracellular sodium and was enhanced by the dihydropyridine calcium agonist Bay K 8644. Nicardipine inhibited the effect of maitotoxin on intracellular calcium concentration in a dose-dependent manner. The maitotoxin-induced calcium rise was also reduced by pretreating cells with omega-conotoxin. Pretreatment of cells with maitotoxin did not modify 125I-omega-conotoxin and [3H]PN 200-110 binding to PC12 membranes. Nicardipine and omega-conotoxin inhibition of maitotoxin-evoked calcium increase was reduced by pertussis toxin pretreatment. Maitotoxin caused a substantial membrane depolarization of PC12 cells as assessed by the fluorescent dye bisoxonol. This effect was reduced by pretreating the cells with either nicardipine or omega-conotoxin and was almost completely abolished by the simultaneous pretreatment with both calcium antagonists. Maitotoxin stimulated inositol phosphate production in a dose-dependent manner. This effect was reduced by pretreating the cells with 1 microM nicardipine and was completely abolished in a calcium-free EGTA-containing medium. The findings on maitotoxin-induced cytosolic calcium rise and membrane depolarization suggest that maitotoxin exerts its action primarily through the activation of voltage-sensitive calcium channels, the increase of inositol phosphate production likely being an effect dependent on calcium influx. The ability of nicardipine and omega-conotoxin to inhibit the effect of maitotoxin on both calcium homeostasis and membrane potential suggests that L- and N-type calcium channel activation is responsible for the influx of calcium following exposure to maitotoxin, and not that a depolarization of unknown nature causes the opening of calcium channels.