Voltage-gated Ca Channel Blocker Research Articles

Dual role played by Ca 2+ in the cytotoxicity of human metastatic breast cancer cells

Introduction Cancer cells exhibit enhanced potential to metastasize via increased acto-myosin cross bridges formation which requires increased Ca2+ influx via cell surface voltage-gated Ca2+ channels (VGCC) leading to activation of myosin light chain kinase (MLCK). The MLCK then phosphorylates myosin and forms acto-myosin cross-bridges. The role of MLCK, and calcium channel in cancer cell growth has not been extensively tested. Objective In this study, we tested the individual effects of blocking VGCC and MLCK on MCF-7 cell (human metastatic breast cancer cell) survival. We also treated the cells with high KCl to induce increased Ca2+ influx and test if the effects of KCl will be opposite to that of VGCC blocker. Methods The experimental design of this study is primarily focused on the administration of specific inhibitors to MCF-7 cells with an interest in the dose-dependent response of the cells to varying concentrations of the treatments as well as a thorough comparison of the cell morphology, viability, and proliferation to the effects of the 1% BME control. The three drugs that have been investigated so far are Nifedipine, a voltage-gated calcium blocker, Nifedipine in the presence of KCl [in order to artificially depolarize the cell membrane to open the VGCC], and ML-7, a myosin light chain kinase inhibitor. The cell viability was then assessed through an MTT colorimetric assay at 562 nm in order to visually and quantitatively assess the results. Additionally, transfection studies are planned to be carried out to substantiate pharmacological findings of inhibitors. Results & Conclusion Nifedipine dose-dependently inhibited MCF-7 cell viability. Following were the % absorbance values with increasing doses of nifedipine. All values are relative to control without nifediine considered as 100%; [0.625 um Nif – 43.05%, 1.25 um Nif – 42.19%, 2.5 um Nif – 40.15%, 5 um Nif -39.53%, 10 um Nif – 38.19%, 20 um Nif – 25.1%, 40 um Nif – 22.96%, 1% BME –39.26%]. The 1% BME (bitter melon extract) is the positive control in our studies as our lab have found BME to be toxic to MCF-7 cells. ML-7 also dose-dependently inhibited MCF-7 cell viability. Following were the % absorbance values with increasing doses of ML-7. All values are relative to control without ML-7 considered as 100%; (0.5 um ML7- 70.01%, 1 um ML7 – 65.74%, 2 um ML7 – 57.71%, 4 um ML7 – 49.36%, 8 um ML7 – 48.28%, 16 um ML7 – 34.13%, 1% BME – 36.17%]. When we stimulated cells with increasing doses of KCl, increasing toxicity was seen as reflected by the decreasing absorbance values. We tested the effects of KCl in the presence or absence of nifedipine. Following were the % absorbance values with increasing doses of KCl and/or nifedipine. All values are relative to control (neither KCl nor nifedipine) considered as 100%; [40 mm KCl – 29.55%, 20 mm KCl – 44.024%, 10 mm KCl –56.51%, 40 um Nif – 22.81%, 40 mm KCl + 40 um Nif – 13.69%, 20 mm KCl + 40 um Nif –15.87%, 10 mm KCl + 40 um Nif – 30.91%, EtOH – 86.85%, 1% BME – 35.27%.]. Thus, either inhibiting Ca2+ influx with nifedipine or opening VGCC with high KCl both exerted cytotoxic effects on MCF-7 cells. This suggests that Ca2+ is critical for the viability of these cells but at the same time too much Ca2+ is toxic to the cells.

Endothelial Nitric Oxide Suppresses Action-Potential-Like Transient Spikes and Vasospasm in Small Resistance Arteries.

Endothelial dysfunction in small arteries is a ubiquitous, early feature of cardiovascular disease, including hypertension. Dysfunction reflects reduced bioavailability of endothelium-derived nitric oxide (NO) and depressed endothelium-dependent hyperpolarization that enhances vasoreactivity. We measured smooth muscle membrane potential and tension, smooth muscle calcium, and used real-time quantitative polymerase chain reaction in small arteries and isolated tubes of endothelium to investigate how dysfunction enhances vasoreactivity. Rat nonmyogenic mesenteric resistance arteries developed vasomotion to micromolar phenylephrine (α1-adrenoceptor agonist); symmetrical vasoconstrictor oscillations mediated by L-type voltage-gated Ca2+ channels (VGCCs). Inhibiting NO synthesis abolished vasomotion so nanomolar phenylephrine now stimulated rapid, transient depolarizing spikes in the smooth muscle associated with chaotic vasomotion/vasospasm. Endothelium-dependent hyperpolarization block also enabled phenylephrine-vasospasm but without spikes or chaotic vasomotion. Depolarizing spikes were Ca2+-based and abolished by either T-type or L-type VGCCs blockers with depressed vasoconstriction. Removing NO also enabled transient spikes/vasoconstriction to Bay K-8644 (L-type VGCC activator). However, these were abolished by the L-type VGCC blocker nifedipine but not T-type VGCC block. Phenylephrine also initiated T-type VGCC-transient spikes and enhanced vasoconstriction after NO loss in nonmyogenic arteries from spontaneously hypertensive rats. In contrast to mesenteric arteries, myogenic coronary arteries displayed transient spikes and further vasoconstriction spontaneously on loss of NO. T-type VGCC block abolished these spikes and additional vasoconstriction but not myogenic tone. Therefore, in myogenic and nonmyogenic small arteries, reduced NO bioavailability engages T-type VGCCs, triggering transient depolarizing spikes in normally quiescent vascular smooth muscle to cause vasospasm. T-type block may offer a means to suppress vasospasm without inhibiting myogenic tone mediated by L-type VGCCs.

N- and L-type calcium channels blocker cilnidipine ameliorates neuropathic pain

Cilnidipine is a dihydropyridine derivative that inhibits N-type and L-type voltage-gated Ca2+ channels (VDCCs). We recently reported that a selective N-type VDCC blocker attenuated the spinal long-term potentiation (LTP) of C-fiber-evoked field potentials recorded in the spinal dorsal horn of rats, which served as a model for examining synaptic function during central pain sensitization. In this study, we investigated the effects of cilnidipine on the changes related to neuropathic pain induced by nerve injury. Mechanical allodynia and hyperalgesia were evaluated by von Frey test and pin prick test, respectively. Spinal LTP of C-fiber-evoked field potentials were evaluated by in vivo electrophysiology. Intrathecally administrated cilnidipine attenuated mechanical allodynia and hyperalgesia in the spared nerve injury mouse model. Using in vivo electrophysiology in rats, cilnidipine (10µm) administered spinally inhibited the induction and maintenance of high-frequency stimulation-induced LTP of C-fiber-evoked field potentials, while basal C-fiber-evoked field potentials in naïve rats were unaffected. The basal C-fiber-evoked field potentials in nerve-injured rats were strongly inhibited by cilnidipine. Treatment with a specific N-type VDCC blocker, ω-conotoxin GVIA, which reportedly attenuates C-fiber-evoked field potentials both before and after the induction of LTP, attenuated mechanical allodynia and hyperalgesia in nerve-injured mice. By contrast, an L-type VDCC blocker, nicardipine attenuated only mechanical hyperalgesia, but not mechanical allodynia in nerve-injured mice, and also attenuated the established LTP of C-fiber-evoked field potentials in rats. These results suggested that N-type and L-type VDCC blockers may effectively alleviate the hyperalgesia and allodynia associated with neuropathic pain without affecting normal pain perception.

Differential Regulation of Human Paired Associative Stimulation-Induced and Theta-Burst Stimulation-Induced Plasticity by L-type and T-type Ca2+ Channels.

Activity-dependent changes of postsynaptic Ca2+-concentration are influenced by a variety of different Ca2+-channels and play an important role in synaptic plasticity. Paired associative stimulation (PAS) and theta-burst stimulation (TBS) are noninvasive magnetic stimulation protocols used in human subjects to induce lasting corticospinal excitability changes that have been likened to synaptic long-term potentiation and long-term depression. To better characterize the Ca2+-related physiological mechanisms underlying PAS- and TBS-induced plasticity, we examined the impact of different Ca2+-sources. PAS-induced facilitation of corticospinal excitability was blocked by NMDA-receptor blocker dextromethorphan (DXM) and L-type voltage gated Ca2+ channels (VGCC) blocker nimodipine (NDP), but turned into depression by T-type VGCC blocker ethosuximide (ESM). Although, surprisingly, static corticospinal excitability was increased by the combination of DXM and NDP, PAS-induced facilitation was blocked. TBS-induced facilitation of corticospinal excitability, which has previously been shown to be turned into depression by L-type VGCC blocker NDP (Wankerl K, Weise D, Gentner R, Rumpf J, Classen J. 2010. L-type voltage-gated Ca2+ channels: a single molecular switch for long-term potentiation/long-term depression-like plasticity and activity-dependent metaplasticity in humans. J Neurosci. 30(18):6197-6204.), was blocked, but not reverted, by T-type VGCC blocker ESM. The different patterns of Ca2+-channel modulation of PAS- and TBS-induced plasticity may point to an important role of backpropagating action potentials in PAS-induced plasticity, similar as in spike-timing dependent synaptic plasticity, and to a requirement of dendritic Ca2+-dependent spikes in TBS-induced plasticity.

R‐Type Calcium Channels Contribute to Colonic Inhibitory Neuromuscular Transmission

The inhibitory neurotransmitters, ATP and nitric oxide (NO), relax the colonic smooth muscle which is important for propulsion of colonic content. Voltage‐gated Ca2+ channels (VGCC) control neurotransmitter release and regulate colonic motility. The α1E subunit of the R‐type VGCC is expressed by enteric neurons yet its role in regulating colonic motility is unclear. We investigated the role of R‐type VGCCs in colonic inhibitory neuromuscular transmission using alpha1E knockout (KO) mice. Intracellular recordings of inhibitory junction potentials (IJP) were obtained from circular muscle cells in distal colon. IJPs were evoked by trains of electrical stimulation (1s, 10 Hz, 40‐90 volts). IJPs have a fast ATP‐mediated component and a slow NO‐mediated component. Peak amplitude (ATP component) and area under the curve (AUC; both components) of the IJP were compared between wildtype (WT) and KO mice. Resting membrane potential (RMP) was measured as an index of ongoing inhibitory transmission. There was a voltage dependent increase in IJP amplitude and AUC in WT and KO mice. IJP amplitude was similar in WT and KO mice. The IJP AUC was decreased in KO compared to WT. Nifedipine (1 µM; L‐type VGCC blocker) had no effect on these responses. RMP in KO muscle cells was decreased compared with WT (‐44 ± 2 mV in WT and ‐38 ± 1 mV in KO; P < 0.05). Thus, there was a 5.4 mV RMP depolarization in KO mice. Nifedipine did not change RMP of WT or KO cells (‐39 ± 3 mV, WT and ‐40 ± 1 mV in KO). In conclusion, R‐type VGCCs contribute to colonic inhibitory neuromuscular transmission by controlling NO release. In addition, R‐type VGCCs contribute to ongoing release of NO and ATP which maintain colonic muscle tone.

Gating of dopamine transmission by calcium and axonal N-, Q-, T- and L-type voltage-gated calcium channels differs between striatal domains.

The voltage-gated Ca(2+) channels (VGCCs) that catalyse striatal dopamine transmission are critical to dopamine function and might prime subpopulations of neurons for parkinsonian degeneration. However, the VGCCs that operate on mesostriatal axons are incompletely defined; previous studies encompassed channels on striatal cholinergic interneurons that strongly influence dopamine transmission. We define that multiple types of axonal VGCCs operate that extend beyond classic presynaptic N/P/Q channels to include T- and L-types. We reveal differences in VGCC function between mouse axon types that in humans are vulnerable versus resistant to Parkinson's disease. We show for the first time that this is underpinned by different sensitivity of dopamine transmission to extracellular Ca(2+) and by different spatiotemporal intracellular Ca(2+) microdomains. These data define key principles of how Ca(2+) and VGCCs govern dopamine transmission in the healthy brain and reveal differences between neuron types that might contribute to vulnerability in disease. The axonal voltage-gated Ca(2+) channels (VGCCs) that catalyse dopamine (DA) transmission are incompletely defined. Yet, they are critical to DA function and might prime subpopulations of DA neurons for parkinsonian degeneration. Previous studies of VGCCs will have encompassed those on striatal cholinergic interneurons, which strongly influence DA transmission. We identify which VGCCs on DA axons govern DA transmission, we determine their dynamic properties and reveal an underlying basis for differences between the caudate putamen (CPu) and nucleus accumbens (NAc). We detected DA release evoked electrically during nicotinic receptor blockade or optogenetically by light activation of channel rhodopsin-expressing DA axons in mouse striatal slices. Subtype-specific VGCC blockers indicated that N-, Q-, T- and L-VGCCs govern DA release in CPu, but in NAc, T and L-channels are relatively silent. The roles of the most dominant channels were inversely frequency-dependent, due to low-pass filtering of DA release by Ca(2+)-dependent relationships between initial release probability and short-term plasticity. Ca(2+) concentration-response curves revealed that differences between CPu and NAc were due to greater underlying Ca(2+) sensitivity of DA transmission from CPu axons. Functions for 'silent' L- and T-channels in NAc could be unmasked by elevating extracellular [Ca(2+)]. Furthermore, we identified a greater coupling between BAPTA-sensitive, fast Ca(2+) transients and DA transmission in CPu axons, and evidence for endogenous fast buffering of Ca(2+) in NAc. These data reveal that a range of VGCCs operate dynamically on DA axons, depending on local driving forces. Furthermore, they reveal dramatic differences in Ca(2+) handling between axonal subpopulations that show different vulnerability to parkinsonian degeneration.

792 EFFECT OF BOTULINUM NEUROTOXIN-A ON T-TYPE VOLTAGE-GATED CA2+ CURRENT IN DETRUSOR SMOOTH MUSCLE

INTRODUCTION AND OBJECTIVES: Botulinum neurotoxin-A (BoNT/A) is one of novel therapeutic agents for urinary dysfunction including OAB. The inhibitory mechanism of BoNT/A in the bladder is considered mainly as that it cleaves synaptosome-associated protein of 25,000 daltons (SNAP-25), resulting in failure of acetylcholine exocytosis from efferent nerve terminals. On the other hand, we reported that BoNT/A had inhibitory effects on muscle contraction and L-type voltage-gated Ca channel (VGCC) activity in detrusor smooth muscle (AUA 2009). In this study, we further investigated the effects of BoNT/A on T-type VGCC in rat detrusor smooth muscle. METHODS: Bladder smooth muscle strips were obtained from female rats. Single myocytes were obtained by enzymatic digestion, and VGCC was investigated using barium as a charge carrier in whole-cell patch-clamp methods. We administrated BoNT/A light chain (BoNT/A-LC) or a peptide that designed as C-terminus 9 amino-acids of SNAP-25 (SNAP-25C) directory into the cytosol through a patch pipette. RESULTS: In control condition with normal pipette solution, T-type inward currents were evoked by depolarization to 40 mV from the holding potential of 90 mV, and the currents were not inhibited by nifedipine 5 M, a L-type VGCC blocker but abolished by NiCl2 500 M. We monitored the time course of peak amplitude of T-type inward currents. After 16 min intracellular perfusion of control pipette solution, the peak amplitude of T-type current did not change (relative value to control, 1.1 0.12, n 6). However, after 16 min perfusion of BoNT/ A-LC 100 nM, the peak amplitude of T-type current was significantly increased (relative value to control, 1.6 0.082, n 6). Similar result was demonstrated by intracellular perfusion of SNAP-25C 1 M (relative value to control, 1.4 0.16, n 6). Time to peak of T-type current was prolonged after intracellular perfusion of BoNT/A-LC or SNAP25C. In the analysis of steady-state inactivation of T-type VGCC in the presence of nifedipine 5 M, the Vh value in BoNT/A-LC 100 nM or SNAP-25C 1 M intracellular administrated group was significantly deeper than that in control group ( 52 2.5 mV (n 6) in control group, 62 0.66 mV (n 6) in BoNT/A-LC group and 66 3.6 mV (n 5) in SNAP-25C 1 M group). CONCLUSIONS: These results suggest that BoNT/A changes the ion channel kinetics of T-type VGCC in detrusor smooth muscle, and this effect seems to be mediated by cleaved SNAP-25 Cterminus. The negative shift of Vh values of T-type VGCC after BoNT/A treatment might contribute to a BoNT/A-induced reduction in detrusor muscle contractility in OAB patients.

Tumour necrosis factor α activates nuclear factor κB signalling to reduce N‐type voltage‐gated Ca2+ current in postganglionic sympathetic neurons

Inflammation has profound effects on the innervation of affected tissues, including altered neuronal excitability and neurotransmitter release. As Ca(2+) influx through voltage-gated Ca(2+) channels (VGCCs) is a critical determinant of excitation-secretion coupling in nerve terminals, the aim of this study was to characterize the effect of overnight incubation in the inflammatory mediator tumour necrosis factor alpha (TNFalpha; 1 nM) on VGCCs in dissociated neurons from mouse superior mesenteric ganglia (SMG). Voltage-gated Ca(2+) currents (I(Ca)) were measured using the perforated patch clamp technique and the VGCC subtypes present in SMG neurons were estimated based on inhibition by selective VGCC blockers: omega-conotoxin GVIA (300 nM; N-type), nifedipine (10 microM; L-type), and omega-conotoxin MVIIC (300 nM; N-, P/Q-type). We used intracellular Ca(2+) imaging with Fura-2 AM to compare Ca(2+) influx during depolarizations in control and TNFalpha-treated neurons. TNF receptor and VGCC mRNA expression were measured using PCR, and channel alpha subunit (CaV2.2) was localized with immunohistochemistry. Incubation in TNFalpha significantly decreased I(Ca) amplitude and depolarization-induced Ca(2+) influx. The reduction in I(Ca) was limited to omega-conotoxin GVIA-sensitive N-type Ca(2+) channels. Depletion of glial cells by incubation in cytosine arabinoside (5 microM) did not affect I(Ca) inhibition by TNFalpha. Preincubation of neurons with SC-514 (20 microM) or BAY 11-7082 (1 microM), which both inhibit nuclear factor kappaB signalling, prevented the reduction in I(Ca) by TNFalpha. Inhibition of N-type VGCCs following TNFalpha incubation was associated with a decrease in CaV2.2 mRNA and reduced membrane localization of CaV2.2 immunoreactivity. These data suggest that TNFalpha inhibits I(Ca) in SMG neurons and identify a novel role for NF-kappaB in the regulation of neurotransmitter release during inflammatory conditions with elevated circulating TNFalpha, such as Crohn's disease and Guillain-Barré syndrome.

Multiple Sources of Light-Evoked Intracellular Calcium Increases in Hermissenda Type B Photoreceptors

Previous research suggests that learning-produced changes in excitability and K+ currents of Hermissenda Type B photoreceptors are Ca2+-dependent phenomena. Little information is available concerning the sources and dynamics of Ca2+ in these cells. We have used Fura-2 dual-wavelength (340/380 nm) photometry to measure somatic [Ca2+]i in B cells. Thirty sec light steps (LS) produce a large increase in [Ca2+]i (∼ 246%). To determine the contribution of Ca2+-influx vs Ca2+-release, we measured [Ca2+]i throughout 5 consecutive LSs in either normal or Ca2+-free ASW (0 mM Ca2+, 30 mM EGTA). Cells exposed to Ca2+-free ASW had a basal [Ca2+]i much lower than when external Ca2+ was present, often below detection limits. Ca2+-free ASW abolished light-induced [Ca2+]i increases in all 7 cells tested. We next explored the role of voltage-gated Ca2+ channels (VGCCs) to Ca2+ influx with the use of cobalt (5mM), a VGCC blocker in B cells. Co2+ did not affect either basal [Ca2+]i or light-induced [Ca2+]i increases (n = 5). To assess the contributions of the ER to light-induced [Ca2+]i changes, B cells were incubated in the ryanodine receptor (RyR) blocker dantrolene (50 μM). Dantrolene reduced the [Ca2+]i response by ∼33% (n =5), and also produced a progressive reduction in basal [Ca2+]i (∼ 60%). Exposure of Ca2+-free ASW cells (n =3) to thapsigargin (TH; 100μM - 1mM, a blocker of the ER Ca2+-ATPase pump) increased basal [Ca2+]i , consistent with store depletion. Collectively, our results indicate that [Ca2+]o is necessary for normal basal [Ca2+]i and critical for light-induced [Ca2+]i increases; but little Ca2+ enters through VDCCs. This suggests that [Ca2+]o enters the cytosol via other routes (e.g. TRP channels) or that the contribution of Ca2+ through VGCCs is slight but serves to trigger Ca2+-induced Ca2+-release (CICR) from ER stores.

Ion-channel blocker sensitivity of voltage-gated calcium-channel homologue Cch1 in Saccharomyces cerevisiae

The Cch1 protein of the yeast Saccharomyces cerevisiae is a homologue of the pore-forming alpha1 subunit of mammalian voltage-gated Ca2+ channels (VGCCs), and it constitutes a high-affinity Ca2+-influx system with the Mid1 protein in this organism. Here, we characterized the kinetic property of a putative Cch1-Mid1 Ca2+ channel overexpressed in S. cerevisiae cells, and showed that the L-type VGCC blockers nifedipine and verapamil partially inhibited Cch1-Mid1 activity, but typical P/Q-, N-, R- and T-type VGCC blockers did not inhibit activity. In contrast, a third L-type VGCC blocker, diltiazem, increased Cch1-Mid1 activity. Diltiazem did not increase Ca2+ uptake in the cch1Delta and mid1Delta single mutants and the cch1Delta mid1Delta double mutant, indicating that the diltiazem-induced increase in Ca2+ uptake is completely dependent on Cch1-Mid1. These results suggest that Cch1 is pharmacologically similar to L-type VGCCs, but the interactions between Cch1 and the L-type VGCC blockers are more complicated than expected.

The inhibition of experimentally induced visceral hyperalgesia by nifedipine – A voltage-gated Ca 2+ channels blocker (VGCCs) in Sheep

Present study examined the effect of VGCC L-type blocker – nifedipine given i.c.v. (0.25, 0.5, 1 and/or 2 mg in toto) on the development of nociceptive behavior, clinical symptoms, plasma catecholamin concentration and reticulo-rumen motility following 5 min lasting mechanical duodenal distension (DD) in sheep. After 24 h of fasting, all animals received i.m. ketamine analgesia (20 mg kg −1 B.W) and anesthetized with pentobarbital (20 mg kg −1 B.W., i.v. infusion) The permanent stainless steel cannula 29 mm in length and 2 mm in diameter was inserted into the lateral cerebral ventricle (controlled by cerebro-spinal efflux) 10 mm above the bregma and 5 mm laterally from the midline sutures using stereotaxic method. Under the same general anesthesia/analgesia a T-shaped silicon cannula (inside diameter of 21 mm), was inserted into the duodenum (12 cm from pylorus). Second identical cannule was inserted into the dorsal sac of the rumen, a previously described. After surgery each animal was kept in individual boxes for 10 days prior to experiment and was treated i.m. with benzyl procaine penicillin 30,000 I.U kg −1 B.W.) + dihydrostreptomycine sulfate (10 g kg −1 B.W.) + prednisolone acetate (1.2 mg kg −1 B.W.) combination and i.m. ketamine (20 mg kg −1 B.W.) every day by seven consecutive days. Experimental DD was conducted by insertion and then distension of rubber balloon (containing 40 ml of warm water) inserted into sheep duodenum. Duodenal distension produced a significant increase in behavioral pain manifestations, tachycardia, hyperventilation, inhibition of reticulo-ruminal contractions rate (from 87.2 to 38.0% during 15–20 min), an increase of plasma catecholamine concentration (over 6.4-fold increase of epinephrine during 2 h following DD, 2-times norepinephrine and 84% increase of dopamine). Nifedipine infusion administered 10 min prior to DD decreased intensity of visceral pain manifestations such as: behavioral changes, hyperventilation, reticulo-rumen motility and efficiently prevent appearance of catecholamine release. These data demonstrated that the development and persistence of duodenal hyperalgesia depends on the activation of Ca 2+ ion flux leading to neurotransmitters release and modulation of membrane excitability. It seems that nifedipine given i.c.v. 10 min prior to DD (as a source of visceral pain), inhibited specific receptors 1 subunits of VGCCs in target tissues, prevented depolarization of cell membranes and release of neurotransmitters responsible for pain sensitivity in sheep. The observed antinociceptive action of VGCCs type L blockers suggest that these channels play a crucial role in the modulation of acute visceral hyperalgesia in sheep.

Involvement of T‐type Ca2+ channels in the potentiation of synaptic and visual responses during the critical period in rat visual cortex

Neocortical neuronal circuits are refined by experience during the critical period of early postnatal life. The shift of ocular dominance in the visual cortex following monocular deprivation has been intensively studied to unravel the mechanisms underlying the experience-dependent modification. Synaptic plasticity is considered to be involved in this process. We previously showed in layer 2/3 pyramidal neurons of rat visual cortex that low-frequency stimulation-induced long-term potentiation (LTP) at excitatory synapses, which requires the activation of Ni(2+)-sensitive (R-type or T-type) voltage-gated Ca(2+) channels (VGCCs) for induction, shared a similar age and experience dependence with ocular dominance plasticity. In this study, we examined whether this LTP is involved in ocular dominance plasticity. In visual cortical slices, LTP was blocked by mibefradil, kurtoxin and R-(-)-efonidipine, T-type VGCC blockers, but not by SNX-482, an R-type VGCC blocker, indicating that LTP induction requires T-type VGCC activation. Mibefradil did not affect synaptic transmission even at a dose about 30 times higher than that required for LTP blockade. Therefore, this drug was used to test the effect of T-type VGCC blockade on ocular dominance shift produced by 6 days of monocular deprivation during the critical period using visual evoked potentials (VEPs). Although this monocular deprivation commonly produced both depression of deprived eye responses and potentiation of nondeprived eye responses, only the former change occurred when mibefradil was infused into the visual cortex during monocular deprivation. Mibefradil infusion produced no acute effects on VEPs. These results suggest that T-type VGCC-dependent LTP contributes to the experience-dependent enhancement of visual responses.

Oxygen Tension and Normalisation Pressure Modulate Nifedipine-sensitive Relaxation of Human Placental Chorionic Plate Arteries

Fetoplacental blood vessel constriction in response to reduced oxygenation has been demonstrated in placenta perfused in vitro. In pulmonary vessels, hypoxic vasoconstriction involves Ca2+ influx into smooth muscle through membrane ion channels including voltage-gated Ca2+ channels (VGCCs). We hypothesised that VGCCs are involved in agonist-induced constriction of fetoplacental resistance vessels and that their contribution is modulated by oxygen. Chorionic plate small arteries were studied using wire myography. Arteries were normalised at high (0.9 of L(13.3 kPa)) or low (0.9 of L(5.1 kPa)) stretch and experiments performed at 156, 38 or 15 mmHg oxygen. At low stretch, U46619 (thromboxane-mimetic) or KCl (smooth muscle depolarisation) constriction was greater at 38 than 156 or 15 mmHg oxygen. An L-type VGCC blocker nifedipine, inhibited KCl constriction by >85% but was less effective in U46619 constrictions (43-67%). At high stretch, nifedipine inhibition of KCl- and U46619-induced constriction was less at 15 than 38 or 156 mmHg oxygen. Oxygen did not affect constriction to U46619 or nifedipine-induced relaxation when vessels were normalised at high stretch. In conclusion, oxygen modulates chorionic plate arterial constriction at low stretch but regulation is lost at high stretch. U46619 constriction is underlain by VGCCs and nifedipine-insensitive processes; their relative contribution is influenced by oxygen.

Voltage‐gated calcium channels play crucial roles in the glutamate‐induced phase shifts of the rat suprachiasmatic circadian clock

The resetting of the circadian clock based on photic cues delivered by the glutamatergic retinohypothalamic tract is an important process helping mammals to function adaptively to the daily light-dark cycle. To see if the photic resetting relies on voltage-gated Ca(2+) channels (VGCCs), we examined the effects of VGCC blockers on the glutamate-induced phase shifts of circadian firing activity rhythms of suprachiasmatic nucleus (SCN) neurons in hypothalamic slices. First, we found that a cocktail of amiloride, nimodipine and omega-conotoxin MVIIC (T-, L- and NPQ-type VGCC antagonists, respectively) completely blocked both phase delays and advances, which were, respectively, induced by glutamate application in early and late night. Next, we discovered that: (i) amiloride and another T-type VGCC antagonist, mibefradil, completely obstructed the delays without affecting the advances; (ii) nimodipine completely blocked the advances while having less impact on delays; and (iii) omega-conotoxin MVIIC blocked largely, if not entirely, both delays and advances. Subsequent whole-cell recordings revealed that T-type Ca(2+) currents in neurons in the ventrolateral, not dorsomedial, region of the SCN were larger during early than late night, whereas L-type Ca(2+) currents did not differ from early to late night in both regions. These results indicate that VGCCs play important roles in glutamate-induced phase shifts, T-type being more important for phase delays and L-type being so for phase advances. Moreover, the results point to the possibility that a nocturnal modulation of T-type Ca(2+) current in retinorecipient neurons is related to the differential involvement of T-type VGCC in phase delays and advances.

Functional interaction between neuropeptide Y receptors and modulation of calcium channels in the rat hippocampus

We investigated the functional interaction between neuropeptide Y (NPY) receptors using nerve terminals and cultured rat hippocampal neurons, and we evaluated the involvement of voltage-gated Ca 2+ channels (VGCCs) in NPY receptors-induced inhibition of Ca 2+ influx and glutamate release. The KCl-evoked release of glutamate from hippocampal synaptosomes was inhibited by 1 μM NPY and this effect was insensitive to either BIBP3226 (Y1 receptor antagonist) or L-152,804 (Y5 receptor antagonist), but was sensitive to BIIE0246 (Y2 receptor antagonist). We could also pharmacologically dissect the NPY receptors activity by using Y1, Y2 and Y5 receptor agonists ([Leu 31,Pro 34]NPY, NPY13-36, NPY (19-23)-(Gly 1,Ser 3,Gln 4,Thr 6,Ala 31,Aib 32,Gln 34)-pancreatic polypeptide (PP), respectively), and in all the cases we observed that these agonists could inhibited the KCl-induced release of glutamate. However, the selective and specific co-activation of both Y1 and Y2 or Y2 and Y5 receptors resulted in non-additive inhibition, and this effect was prevented in the presence of the Y2 antagonist, but was insensitive to the Y1 or Y5 receptor antagonist. Moreover, as we previously showed for Y1 receptors, we also observed that the activation of Y5 receptors inhibited the glutamate release in the dentate gyrus and CA3 subregion, without significant effect in the CA1 subregion of the hippocampus. The same qualitative results were obtained when we investigated the role of NPY Y1 and Y2 receptors in modulating the changes in [Ca 2+] i due to KCl depolarisation in cultured hippocampal neurons. The inhibitory effect of nitrendipine (L-type VGCC blocker) or ω-conotoxin GVIA (ω-CgTx; N-type VGCC blocker) was not potentiated by the simultaneous activation of Y1 or Y2 receptors. Moreover, the exocytotic release of glutamate was inhibited by ω-agatoxin IVA (ω-Aga; P-/Q-type VGCC blocker), and this VGCC blocker did not potentiate Y1, Y2 or Y5 receptor-mediated inhibition of glutamate release. Also, the effect of ionomycin in inducing the exocytotic release of glutamate from hippocampal synaptosomes was insensitive to the activation of NPY receptors. In the present paper, we identified a role for NPY Y1, Y2 and Y5 receptors in modulating the exocytotic release of glutamate and the [Ca 2+] i changes in the rat hippocampus. In conditions of co-activation, there appears to exist a physiological cross-talk between Y1 and Y2 and also between Y2 and Y5 receptors, in which Y2 receptors play a predominant role. Moreover, we also show that Y1 and Y2 receptors exert their inhibitory action by directly modulating L-, N-, and P-/Q-type VGCCs, whereas the inhibition of glutamate release mediated by the Y5 receptors seems to involve P-/Q-type VGCCs.

Fluorescence detection of plant extracts that affect neuronal voltage-gated Ca 2+ channels

Structurally novel compounds able to block voltage-gated Ca 2+ channels (VGCCs) are currently being sought for the development of new drugs directed at neurological disorders. Fluorescence techniques have recently been developed to facilitate the analysis of VGCC blockers in a multi-well format. By utilising the small cell lung carcinoma cell line, NCI-H146, we were able to detect changes in intracellular Ca 2+ concentration ([Ca 2+] i) using a fluorescence microplate reader. NCI-H146 cells have characteristics resembling those of neuronal cells and express multiple VGCC subtypes, including those of the L-, N- and P-type. We found that K +-depolarisation of fluo-3 loaded NCI-H146 cells causes a rapid and transient increase in fluorescence, which was readily detected in a 96-well plate. Extracts of Australian plants, including those used traditionally as headache or pain treatments, were tested in this study to identify those affecting Ca 2+ influx following membrane depolarisation of NCI-H146 cells. We found that E. bignoniiflora, A. symphyocarpa and E. vespertilio caused dose-dependent inhibition of K +-depolarised Ca 2+ influx, with IC 50 values calculated to be 234, 548 and 209 μg/ml, respectively. This data suggests an effect of these extracts on the function of VGCCs in these cells. Furthermore, we found similar effects using a fluorescence laser imaging plate reader (FLIPR) that allows simultaneous measurement of real-time fluorescence in a multi-well plate. Our results indicate that the dichloromethane extract of E. bignoniiflora and the methanolic extract of E. vespertilio show considerable promise as antagonists of neuronal VGCCs. Further analysis is required to characterise the function of the bioactive constituents in these extracts and determine their selectivity on VGCC subtypes.

Voltage-gated Ca 2+ channel blockers, ω-AgalVA and Ni 2+, suppress the induction of θ-burst induced long-term potentiation in guinea-pig hippocampal CA1 neurons

It is widely believed that a rise in post-synaptic calcium concentration ([Ca 2+];) is a necessary step in the induction of long-term potentiation (LTP) (Bliss and Collingridge, Nature, 361 (1993) 31–39). In this experiment, we examine the involvement of voltagegated Ca 2+ channels (VGCC) in the induction of AP5-sensitive LTP induced by θ-burst stimulation in guinea-pig hippocampal CA1 neurons. The VGCC blockers, Ni 2+, (25 μM, T -channel blocker) or ω-AgaIVA (60 nM, P-channel blocker), which have no effect on synaptic transmission, suppress 60% or 78% of the Θ-burst induced UP, respectively. This implies that Ca 2+ entry through VGCC is an important step in this form of LTP.

Primary action of endothelin on Ca release in bovine coronary artery smooth muscle cells

Intracellular free Ca concentrations (Cai) were determined by fura-2 microfluorometry in single freshly dispersed cells to differentiate endothelin (ET)-induced Ca release from Ca influx through voltage-gated Ca channels (VGCC). In physiological solution ET (10(-8) M) significantly (P less than 0.05) increased Cai 23 +/- 3% (+/- SE) above baseline; this increase was not significantly attenuated by 2 x 10(-4) M lanthanum, a blocker of VGCC, or Ca-free solution. When the sarcoplasmic reticulum was depleted of Ca by prolonged treatment with 5 x 10(-3) M caffeine, depolarization with 80 mM K (80K; or 30K) plus ET did not increase Cai above that induced by 80K (or 30K) in caffeine alone. In contrast, 10(-6) M BAY K 8644, instead of ET in the protocol, significantly (P less than 0.05) increased Cai above that induced by 80K (or 30K). ET released Ca from the caffeine-sensitive internal store but was not rapid and transient like caffeine-induced release, which elicited a peak Cai increase in less than 1 min; instead, release was more gradual and prolonged with Cai peaking in greater than 2 min, thus resembling the response to 10(-5) M ryanodine. With two ET exposures, either a transient nonrepeatable increase in Cai or a delayed, but sustained, increase in Cai resulted, similar to the response to ryanodine. These data indicate that in freshly dispersed bovine cells the predominant mechanism by which ET increases Cai is release of Ca from the sarcoplasmic reticulum; if any increase in L-type voltage-gated Ca influx occurred, it was minimal and matched by efflux.