Action of hypoxia on different types of calcium channels in hippocampal neurons

Whole-cell patch clamp and polarographic oxygen partial pressure (pO2) measurements were used to establish the sensitivity of high-voltage-activated (HVA) Ca2+ channel subtypes of CA1 hippocampal neurons of rats to hypoxic conditions. Decrease of pO2 to 15-30 mm Hg induced a potentiation of HVA Ca2+ currents by 94%. Using selective blockers of N- and L-types of calcium channels, we found that inhibition of L-type channels decreased the effect by 54%, whereas N-type blocker attenuated the effect by 30%. Taking into account the ratio of currents mediated by these channel subtypes in CA1 hippocampal neurons, we concluded that both types of HVA Ca2+ channels are sensitive to hypoxia, however, L-type was about 3.5 times more sensitive to oxygen reduction.     

R-type calcium channels in myenteric neurons of guinea pig small intestine

Currents carried by L-, N-, and P/Q-type calcium channels do not account for the total calcium current in myenteric neurons. This study identified all calcium channels expressed by guinea pig small intestinal myenteric neurons maintained in primary culture. Calcium currents were recorded using whole cell techniques. Depolarizations (holding potential = -70 mV) elicited inward currents that were blocked by CdCl(2) (100 microM). Combined application of nifedipine (blocks L-type channels), Omega-conotoxin GVIA (blocks N-type channels), and Omega-agatoxin IVA (blocks P/Q-type channels) inhibited calcium currents by 56%. Subsequent addition of the R-type calcium channel antagonists, NiCl(2) (50 microM) or SNX-482 (0.1 microM), abolished the residual calcium current. NiCl(2) or SNX-482 alone inhibited calcium currents by 46%. The activation threshold for R-type calcium currents was -30 mV, the half-activation voltage was -5.2 +/- 5 mV, and the voltage sensitivity was 17 +/- 3 mV. R-type currents activated fully in 10 ms at 10 mV. R-type calcium currents inactivated in 1 s at 10 mV, and they inactivated (voltage sensitivity of 16 +/- 1 mV) with a half-inactivation voltage of -76 +/- 5 mV. These studies have accounted for all of the calcium channels in myenteric neurons. The data indicate that R-type calcium channels make the largest contribution to the total calcium current in myenteric neurons. The relatively positive half-activation voltage and rapid activation kinetics suggest that R-type channels could contribute to calcium entry during somal action potentials or during action potential-induced neurotransmitter release.     

Classification of voltage-dependent Ca2+ channels in trigeminal ganglion neurons from neonatal rats

We examined the subtypes and characteristics of the Ca(2+) channel in small (diameter < 30 microm) trigeminal ganglion (TG) neurons from neonatal rats by means of whole cell patch clamp techniques. There were two current components, low-voltage activated (LVA) and high-voltage activated (HVA) I(Ba), with different activation ranges and waveforms. LVA I(Ba) elicited from a depolarizing step pulse at a holding potential (HP) of -80 mV was inhibited by 0.25 mM amiloride (62%), which did not produce any significant inhibition of the peak amplitude of HVA I(Ba). The application of 0.5 mM amiloride inhibited 10% of the HVA I(Ba). The LVA I(Ba) was also reduced by changing the HP from -80 to -60 mV (61%), and under these conditions the peak amplitude of HVA I(Ba) did not change significantly. In addition, HVA I(Ba) and LVA I(Ba) showed marked differences in their inactivation properties. Experiments with several Ca(2+) channel blockers revealed that on average, 26% of the HVA I(Ba) was nifedipine (10 microM) sensitive, 55% was sensitive to omega-conotoxinGVIA (1 microM), 4% was blocked by omega-agatoxinIVA (1 microM), and the remainder of the current that was resistant to the co-application of all three Ca(2+) channel blockers was 15% of the total current. These results suggest that the application of amiloride and the alteration of the holding potential level can discriminate between HVA and LVA Ba(2+) currents in TG neurons, and that TG neurons expressed T-, L-, N-, P-/Q- and R-type Ca(2+) channels.     

Influence of external pH on two types of low-voltage-activated calcium currents in primary sensory neurons of rats

The influence of extracellular pH (pH(o)) on low-voltage-activated calcium channels of acutely isolated DRG neurons of rats was examined using the whole cell patch-clamp technique. It has been found that in the neurons of middle size with capacitance C=60+/-4.8 pF (mean+/-S.E., n=8) extracellular acidification from pH(o) 7.35 to pH(o) 6.0 significantly and reversibly decreased LVA calcium current densities by 75+/-3.7%, shifted potential for half-maximal activation to more positive voltages by 18.7+/-0.6 mV with significant reduction of its voltage dependence. The half-maximal potential of steady-state inactivation shifted to more positive voltages by 12.1+/-1.7 mV (n=8) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of middle size have midpoint pK(a)=6.6+/-0.02 and hill coefficient h=0.94+/-0.04 (n=5). In small cells with capacitance C=26+/-3.6 pF (n=5), acidosis decreased LVA calcium current densities only by 15.3+/-1.3% and shifted potential for half-maximal activation by 5.5+/-1.0 mV with reduction of its voltage dependence. Half-maximal potential of steady-state inactivation shifted to more positive voltages by 10+/-1.6 mV (n=4) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of small size have midpoint pK(a)=7.9+/-0.04 and hill coefficient h=0.25+/-0.1 (n=4). These two identified types of LVA currents besides different pH sensitivity demonstrated different kinetic properties. The deactivation of LVA currents with weak pH sensitivity after switching off depolarization to -30 mV had substantially longer decay time than do currents with strong pH sensitivity (tau(d) approximately 5 ms vs. 2 ms respectively). It was found that the prolongation of depolarization steps slows the subsequent deactivation of T-type currents in small DRG neurons. Deactivation traces in these neurons were better described by the sum of two exponentials. Thus, we suppose that T-type channels in small DRG neurons are presented mostly by alpha1I subunit. We suggest that these two types of LVA calcium channels with different sensitivity to external pH can be differently involved in the origin of neuropathic changes.     

Selectivity of omega-CgTx-MVIIC toxin from Conus magus on calcium currents in enteric neurons.

Whole-cell perforated patch clamp recordings were used to analyze selectivity of omega-CgTx-MVIIC toxin for voltage-dependent calcium currents in cultured myenteric neurons from guinea-pig small intestine. Omega-CgTx-MVIIC (300 nM) blocked 37 +/- 9% of the peak current activated from -80 mV in 15 neurons by mostly affecting the plateau phase of the current. The toxin suppressed peak current activated from -30 mV dose-dependently with an IC50 of 70 +/- 8 nM. The blockade was complete at toxin concentrations of 1 microM. Thus, it appears that omega-CgTx-MVIIC blocks high voltage activated (HVA) calcium channels in the myenteric neurons unselectively as well as other types of HVA Ca2+ channels including P and Q channels.     

Prenatal stress on the kinetic properties of Ca2+ and K+ channels in offspring hippocampal CA3 pyramidal neurons

Prenatal stress is known to cause neuronal loss and oxidative damage in the hippocampus of offspring rats. To further understand the mechanisms, the present study was undertaken to investigate the effects of prenatal stress on the kinetic properties of high-voltage-activated (HVA) Ca(2+) and K(+) channels in freshly isolated hippocampal CA3 pyramidal neurons of offspring rats. Pregnant rats in the prenatal stress group were exposed to restraint stress on days 14-20 of pregnancy three times daily for 45 min. The patch clamp technique was employed to record HVA Ca(2+) and K(+) channel currents. Prenatal stress significantly increased HVA Ca(2+) channel disturbance including the maximal average HVA calcium peak current amplitude (-576.52+/-7.03 pA in control group and -702.05+/-6.82 pA in prenatal stress group, p<0.01), the maximal average HVA Ca(2+) current density (-40.89+/-0.31 pA/pF in control group and -49.44+/-0.37 pA/pF in prenatal stress group, p<0.01), and the maximal average integral current of the HVA Ca(2+) channel (106.81+/-4.20 nA ms in control group and 133.49+/-4.59 nA ms in prenatal stress group, p<0.01). The current-voltage relationship and conductance--voltage relationship of HVA Ca(2+) channels and potassium channels in offspring CA3 neurons were not affected by prenatal stress. These data suggest that exposure of animals to stressful experience during pregnancy can exert effects on calcium ion channels of offspring hippocampal neurons and that the calcium channel disturbance may play a role in prenatal stress-induced neuronal loss and oxidative damage in offspring brain.     

Voltage-sensitive calcium currents and their role in regulating phrenic motoneuron electrical excitability during the perinatal period

This study examined the ontogeny of voltage-sensitive calcium conductances in rat phrenic motoneurons (PMNs) and their role in regulating electrical excitability during the perinatal period. Specifically, we studied the period spanning from embryonic day (E)16 through postnatal day (P)1, when PMNs undergo fundamental transformation in their morphology, passive properties, ionic channel composition, synaptic inputs, and electrical excitability. Low voltage-activated (LVA) and high voltage-activated (HVA) conductances were measured using whole cell patch recordings utilizing a cervical slice-phrenic nerve preparation from perinatal rats. Changes between E16 and P0-1 included the following: an approximately 2-fold increase in the density of total calcium conductances, an approximately 2-fold decrease in the density of LVA calcium conductances, and an approximately 3-fold increase in the density of HVA conductances. The elevated expression of T-type calcium channels during the embryonic period lengthened the action potential and enhanced electrical excitability as evidenced by a hyperpolarization-evoked rebound depolarization. The reduction of LVA current density coupled to the presence of a hyperpolarizing outward A-type potassium current had a critical effect in diminishing the rebound depolarization in neonatal PMNs. The increase in HVA current density was concomitant with the emergence of a calcium-dependent "hump-like" afterdepolarization (ADP) and burst-like firing. Neonatal PMNs develop a prominent medium-duration afterhyperpolarization (mAHP) as the result of coupling between N-type calcium channels and small conductance, calcium-activated potassium channels. These data demonstrate that changes in calcium channel expression contribute to the maturation of PMN electrophysiological properties during the time from the commencement of fetal inspiratory drive to the onset of continuous breathing at birth.     

Intra and extracellular surface charges near Ca2+ channels in neurons and neuroblastoma cells.

The properties of low (LVA) and high (HVA) voltage-activated calcium currents were investigated in rat sensory neurons and a murine neuroblastoma cell line exposed to various concentrations of intra- or extracellular monovalent ([c+]i/o) and trivalent ([c3+]i/o) cations. In neurons, when [c+]i was changed from 150 to 20 mM, positive shifts of 18-28 mV were observed in activation curves of both LVA and HVA currents, as well as in LVA inactivation curves. Extracellularly, in divalent-free solutions, [c+]o of 20-50 mM produced medium (12-22 mV) negative shifts of the LVA channel properties. These data were used to estimate, by a "screening" model, a negative surface charge density around neuron's calcium channels of 1/1,000 and 1/1,325 eA-2 at the outside or inside face, respectively. In the presence of physiological concentrations of divalent cations, [c+]o of 20-60 mM caused smaller (4-11 mV) negative shifts of the activation and inactivation curves, which can be explained by assuming a partial neutralization of negative charges by divalent cations. By applying the above procedure to LVA channels of neuroblastoma cells, the ratio of extra- to intracellular surface charge density turned out to be more than tenfold higher than in neurons. Effects produced by [c3+]i/o were not in agreement with expectations based on screening or binding models.     

Low voltage-activated calcium channels in vascular smooth muscle: T-type channels and AVP-stimulated calcium spiking

An important path of extracellular calcium influx in vascular smooth muscle (VSM) cells is through voltage-activated Ca2+ channels of the plasma membrane. Both high (HVA)- and low (LVA)-voltage-activated Ca2+ currents are present in VSM cells, yet little is known about the relevance of the LVA T-type channels. In this report, we provide molecular evidence for T-type Ca2+ channels in rat arterial VSM and characterize endogenous LVA Ca2+ currents in the aortic smooth muscle-derived cell line A7r5. AVP is a vasoconstrictor hormone that, at physiological concentrations, stimulates Ca2+ oscillations (spiking) in monolayer cultures of A7r5 cells. The present study investigated the role of T-type Ca2+ channels in this response with a combination of pharmacological and molecular approaches. We demonstrate that AVP-stimulated Ca2+ spiking can be abolished by mibefradil at low concentrations (<1 microM) that should not inhibit L-type currents. Infection of A7r5 cells with an adenovirus containing the Cav3.2 T-type channel resulted in robust LVA Ca2+ currents but did not alter the AVP-stimulated Ca2+ spiking response. Together these data suggest that T-type Ca2+ channels are necessary for the onset of AVP-stimulated calcium oscillations; however, LVA Ca2+ entry through these channels is not limiting for repetitive Ca2+ spiking observed in A7r5 cells.     

Role of high-voltage-activated calcium channels in glucose-regulated beta-cell calcium homeostasis and insulin release

High-voltage-activated (HVA) calcium channels are known to be the primary source of calcium for glucose-stimulated insulin secretion. However, few studies have investigated how these channels can be regulated by chronically elevated levels of glucose. In the present study, we determined the level of expression of the four major HVA calcium channels (N-type, P/Q-type, L(C)-type, and L(D)-type) in rat pancreatic beta-cells. Using quantitative real-time PCR (QRT-PCR), we found the expression of all four HVA genes in rat insulinoma cells (INS-1) and in primary isolated rat islet cells. We then determined the role of each channel in insulin secretion by using channel-selective antagonists. Insulin secretion analysis revealed that N- and L-type channels are both involved in immediate glucose-induced insulin secretion. However, L-type was preferentially coupled to secretion at later time points. P/Q-type channels were not found to play a role in insulin secretion at any stage. It was also found that long-term exposure to elevated glucose increases basal calcium in these cells. Interestingly, chronically elevated glucose decreased the mRNA expression of the channels involved with insulin secretion and diminished the level of stimulated calcium influx in these cells. Using whole cell patch clamp, we found that N- and L-type channel currents increase gradually subsequent to lower intracellular calcium perfusion, suggesting that these channels may be regulated by glucose-induced changes in calcium.     

Modified sympathetic nerve system activity with overexpression of the voltage-dependent calcium channel beta3 subunit

N-type voltage-dependent calcium channels (VDCCs) play determining roles in calcium entry at sympathetic nerve terminals and trigger the release of the neurotransmitter norepinephrine. The accessory beta3 subunit of these channels preferentially forms N-type channels with a pore-forming CaV2.2 subunit. To examine its role in sympathetic nerve regulation, we established a beta3-overexpressing transgenic (beta3-Tg) mouse line. In these mice, we analyzed cardiovascular functions such as electrocardiography, blood pressure, echocardiography, and isovolumic contraction of the left ventricle with a Langendorff apparatus. Furthermore, we compared the cardiac function with that of beta3-null and CaV2.2 (alpha1B)-null mice. The beta3-Tg mice showed increased expression of the beta3 subunit, resulting in increased amounts of CaV2.2 in supracervical ganglion (SCG) neurons. The beta3-Tg mice had increased heart rate and enhanced sensitivity to N-type channel-specific blockers in electrocardiography, blood pressure, and echocardiography. In contrast, cardiac atria of the beta3-Tg mice revealed normal contractility to isoproterenol. Furthermore, their cardiac myocytes showed normal calcium channel currents, indicating unchanged calcium influx through VDCCs. Langendorff heart perfusion analysis revealed enhanced sensitivity to electric field stimulation in the beta3-Tg mice, whereas beta3-null and Cav2.2-null showed decreased responsiveness. The plasma epinephrine and norepinephrine levels in the beta3-Tg mice were significantly increased in the basal state, indicating enhanced sympathetic tone. Electrophysiological analysis in SCG neurons of beta3-Tg mice revealed increased calcium channel currents, especially N- and L-type currents. These results identify a determining role for the beta3 subunit in the N-type channel population in SCG and a major role in sympathetic nerve regulation.     

AGAP,a new recombinant neurotoxic polypeptide,targets the voltage-gated calcium channels in rat small diameter DRG neurons

A previous study showed that antitumor-analgesic peptide (AGAP), a novel recombinant polypeptide, which had been expressed in Escherichia coli, exhibits analgesic and antitumor effects in mice. In the present study, we investigated the underlying analgesic mechanism of AGAP. The effect of AGAP on voltage-gated calcium channels (VGCCs) was assessed in acutely isolated rat dorsal root ganglia (DRG) neurons using the whole-cell patch clamp technique. The results showed that AGAP potently inhibited VGCCs, especially high-voltage activated (HVA) calcium channels. AGAP inhibited HVA and T-type calcium currents in a dose-dependent manner, but had no significant effect on their dynamic functions in rat small-diameter DRG neurons. AGAP inhibited N- and L-type calcium currents at 78.2% and 57.3%, respectively. Thus, the present study demonstrates that AGAP affects calcium currents through the inhibition of N-, L- and T-type channels in DRG neurons, explaining the potential mechanisms of antinociception.     

Down-regulation of endogenous KLHL1 decreases voltage-gated calcium current density

The actin-binding protein Kelch-like 1 (KLHL1) can modulate voltage-gated calcium channels in vitro. KLHL1 interacts with actin and with the pore-forming subunits of Cav2.1 and CaV3.2 calcium channels, resulting in up-regulation of P/Q and T-type current density. Here we tested whether endogenous KLHL1 modulates voltage gated calcium currents in cultured hippocampal neurons by down-regulating the expression of KLHL1 via adenoviral delivery of shRNA targeted against KLHL1 (shKLHL1). Control adenoviruses did not affect any of the neuronal properties measured, yet down-regulation of KLHL1 resulted in HVA current densities ∼68% smaller and LVA current densities 44% smaller than uninfected controls, with a concomitant reduction in α_1A and α_1H protein levels. Biophysical analysis and western blot experiments suggest Ca_V3.1 and 3.3 currents are also present in shKLHL1-infected neurons. Synapsin I levels, miniature postsynaptic current frequency, and excitatory and inhibitory synapse number were reduced in KLHL1 knockdown. This study corroborates the physiological role of KLHL1 as a calcium channel modulator and demonstrates a novel, presynaptic role.     

Novel ω-Conotoxins Block Dihydropyridine-Insensitive High Voltage-Activated Calcium Channels in Molluscan Neurons

Abstract: We have identified two novel peptide toxins from molluscivorous Conus species that discriminate subtypes of high voltage-activated (HVA) calcium currents in molluscan neurons. The toxins were purified using assays on HVA calcium currents in the caudodorsal cells (CDCs) of the snail Lymnaea stagnalis . The CDC HVA current consists of a rapidly inactivating, transient current that is relatively insensitive to dihydropyridines (DHPs) and a slowly inactivating, DHP-sensitive L-current. The novel toxins, designated ω-conotoxins PnVIA and PnVIB, completely and selectively block the transient HVA current in CDCs with little (PnVIA) or no (PnVIB) effect on the sustained L-type current. The block is rapid and completely reversible. It is noteworthy that both PnVIA and PnVIB reveal very steep dose dependences of the block, which may imply cooperativity in toxin action. The amino acid sequences of PnVIA (GCLEVDYFCGIPFANNGLCCSGNCVFVCTPQ) and of PnVIB (DDDCEPPGNFCGMIKIGPPCCSGWCFFACA) show very little homology to previously described ω-conotoxins, although both toxins share the typical ω-conotoxin cysteine framework but have an unusual high content of hydrophobic residues and net negative charge. These novel ω-conotoxins will facilitate selective analysis of the functions of HVA calcium channels and may enable the rational design of drugs that are selective for relevant subtypes.     

产前应激对子代大鼠海马CA3区神经元Ca2+和K+通道的影响

本研究的目的在于探讨产前应激对子代大鼠海马CA3神经元高电压激活(HVA)钙通道、延迟整流钾电流(delayedrectifierpotassiumcurrents,IKD)的影响。产前应激(prenatalstress,PNS)组孕鼠孕晚期给予束缚应激,应用全细胞膜片钳技术进行研究。结果显示产前应激增加了子代海马CA3神经元HVA钙通道峰电流幅值,对照组和产前应激组子代CA3神经元平均最大HVA钙电流峰值分别为-576.52±7.03pA和-702.05±6.82pA(P<0.01)。同时未改变其电导-电压关系,也未改变延迟整流钾通道电流-电压关系、电导-电压关系。结果提示,在胎儿发育的关键时期,给予母体产前应激,引起子代海马神经元HVA钙电流增加,其机制一方面PNS导致皮质酮升高,从而可能增加HVA钙通道mRNA表达;另一方面PNS所致反应性氧化产物(reactiveoxygenspecies,ROS)增多,后者可能通过磷酸化HVACa2 通道亚单位,从而提高HVA钙电流幅值。     

Voltage-dependent calcium channels in cultivated neurons of the rat hippocampus

Calcium currents through the somatic membrane of cultivated (a low-density culture) hippocampal neurons of rats were studied with the use of a patch-clamp technique in the whole-cell configuration. Low- and high-threshold components of calcium currents were found in the somata of all studied cells. Low-threshold currents were activated at a membrane potential of about−75 mV and reached the maximum amplitude at −45±4 mV, while the maximum amplitude of high-threshold currents was observed at 17±6 mV. Low-threshold calcium currents differed from high-threshold current in weak suppression by low Cd²⁺ concentration (10–20 μM), while Ni²⁺ inhibited both types of calcium currents to an equal extent. Experiments with organic channel blockers showed that in most neurons at least four channel types were expressed: these were L, N, P, and channels insensitive to the used blockers (presumably, R-type). A blocker of L-type calcium channels, nifedipine (10 μM), blocked, on the average, 22.7±5.2%; a blocker of N-type channels, ω-CTx-GVIA (1.0 μM), blocked 30.0±5.0% and a blocker of P/Q channels, ω-Aga-IVA (200 nM), blocked 37.2±13.3% of the integral high-threshold current. A resistive component equalled 15.7±5.1% of the latter current. It is concluded that hippocampal neurons cultivated with a low density express a pharmacologically heterogeneous population of calcium channels, and the relative proportions of different type channels are close to the earlier described channel type composition in rat hippocampal slices. Our study shows that the low-density culture can be used as an adequate model for studying calcium channels in the somatic membrane of hippocampal neurons.     

Adenosine inhibits calcium channel currents via A1 receptors on salamander retinal ganglion cells in a mini-slice preparation

The effects of adenosine on high-voltage-activated calcium channel currents in tiger salamander retinal ganglion cells were investigated in a mini-slice preparation. Adenosine produced a concentration-dependent decrease in the amplitude of calcium channel current with a maximum inhibition of 26%. The effects of adenosine on calcium channel current were both time- and voltage-dependent. In cells dialyzed with GTP-gamma-s, adenosine caused a sustained and irreversible inhibition of calcium channel current, suggesting involvement of a GTP-binding protein. The inhibitory effect of adenosine on calcium channel current was blocked by the A1 antagonist 8-cyclopentyltheophylline (DPCPX, 1-10 microm), but not by the A2 antagonist 3-7-dimethyl-1-propargylxanthine (DMPX, 10 microm), and was mimicked by the A1 agonist N6-cyclohexyladenosine (CHA, 1 microm) but not by the A2 agonist 5'-(N-cyclopropyl) carbox-amidoadenosine (CPCA, 1 microm). Adenosine's inhibition of calcium channel current was not affected by the L-type calcium channel blocker nifedipine (5 microm). However, adenosine's inhibition of calcium channel current was reduced to approximately 10% after application of omega-conotoxin GVIA (1 microm), suggesting that adenosine inhibits N-type calcium channels. These results show that adenosine acts on an A1 adenosine receptor subtype via a G protein-coupled pathway to inhibit the component of calcium channel current carried in N-type calcium channels.     

Effects of divalent cations on voltage-gated Ca2+ channels and depolarization-induced [Ca2+], transients of freshly isolated pyramidal cells of the rat dorsal cochlear nucleus

The effects of divalent cations on voltage-activated Ca2+ channels and depolarization-evoked cytoplasmic [Ca2+] elevations were studied in pyramidal neurones isolated from the dorsal cochlear nucleus of the rat. Ca2+ currents were recorded using the whole-cell configuration of the patch-clamp technique. 10 micromol x l(-1) Cd2+ exerted a greater blocking effect on the high-voltage activated (HVA) currents than on the low-voltage activated (LVA) ones (decrease to 26.6+/-2.5% and to 87.8+/-2.1%, respectively). The blocking effect of 200 micromol x l(-1) Cd2+ was more pronounced and the difference between the effect on the HVA and LVA currents became smaller (decrease to 11.7+/-2.1% and to 32.4+/-2.7%, respectively). 200 micromol x l(-1) Ni2+ reduced the LVA component more effectively (to 77.6+/-5.4%) than the HVA one (to 86.9+/-2.6%). Cytoplasmic [Ca2+] changes were measured applying a fluorimetric technique (Fura-2). 10 micromol x l(-1) Cd2+ decreased the peak values of 50 mmol x l(-1) K+ depolarization-induced [Ca2]+i transients to 30.4+/-1.4% while 200 micromol x l(-1) Cd2+ caused a drop to 2.5+/-0.2%. 200 micromol x l(-1) Ni2+ decreased the peak of the transients to 69.6+/-2.9%. Comparison of the blocking effects of divalent cations on Ca2+ currents and [Ca2+]i transients supports further the conclusion that the depolarization-induced [Ca2+]i changes are produced mainly by the activation of the HVA Ca2+ channels.     

Different effects of L-, N- and T-type calcium channel blockers on striatal dopamine release measured by microdialysis in freely moving rats.

Using a microdialysis method, we have investigated effects of the voltage-dependent calcium channel blockers, verapamil, nicardipine, omega-conotoxin and flunarizine on the dopamine release and metabolism in the striatum of freely moving rat. Perfusion of verapamil (1-300 microM) and nicardipine (1-100 microM), an L-type calcium channel blocker, into the striatum through the dialysis membrane showed a dose-dependent decrease of dopamine release in the dialysate and slight increase of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels. Treatment of omega-conotoxin (0.1, 1 microM), an N-type channel blocker, decreased about 50% basal dopamine release and slightly decreased DOPAC and HVA levels. Treatment with flunarizine (10 microM), an T-type channel blocker, did not affect the dopamine release and metabolism. From these data, it appears that treatments of the L- and N-type voltage-dependent calcium channel blockers in rat striatum suppress basal dopamine release, but T-type blocker does not suppress it, suggesting that L-, N- and T-type calcium channels regulate in vivo dopamine release in a different mechanism.