Inhibition Of Calcium Channel Currents Research Articles

Effect of gingerol on colonic motilityviainhibition of calcium channel currents in rats

To investigate the effect of gingerol on colonic motility and the action of L-type calcium channel currents in this process. The distal colon was cut along the mesenteric border and cleaned with Ca(2+)-free physiological saline solution. Muscle strips were removed and placed in Ca(2+)-free physiological saline solution, which was oxygenated continuously. Longitudinal smooth muscle samples were prepared by cutting along the muscle strips and were then placed in a chamber. Mechanical contractile activities of isolated colonic segments in rats were recorded by a 4-channel physiograph. Colon smooth muscle cells were dissociated by enzymatic digestion. L-type calcium currents were recorded using the conventional whole-cell patch-clamp technique. Gingerol inhibited the spontaneous contraction of colonic longitudinal smooth muscle in a dose-dependent manner with inhibition percentages of 13.3% ± 4.1%, 43.4% ± 3.9%, 78.2% ± 3.6% and 80.5% ± 4.5% at 25 μmol/L, 50 μmol/L, 75 μmol/L and 100 μmol/L, respectively (P < 0.01). Nifedipine, an L-type calcium channel blocker, diminished the inhibition of colonic motility by gingerol. Gingerol inhibited L-type calcium channel currents in colonic longitudinal myocytes of rats. At a 75 μmol/L concentration of gingerol, the percentage of gingerol-induced inhibition was diminished by nifedipine from 77.1% ± 4.2% to 42.6% ± 3.6% (P < 0.01). Gingerol suppressed IBa in a dose-dependent manner, and the inhibition rates were 22.7% ± 2.38%, 35.77% ± 3.14%, 49.78% ± 3.48% and 53.78% ± 4.16% of control at 0 mV, respectively, at concentrations of 25 μmol/L, 50 μmol/L, 75 μmol/L and 100 μmol/L (P < 0.01). The steady-state activation curve was shifted to the right by treatment with gingerol. The value of half activation was -14.23 ± 1.12 mV in the control group and -10.56 ± 1.04 mV in the 75 μmol/L group (P < 0.05) with slope factors, Ks, of 7.16 ± 0.84 and 7.02 ± 0.93 (P < 0.05) in the control and 75 μmol/L groups, respectively. However, the steady-state inactivation curve was not changed, with a half-inactivation voltage, 0.5 V, of -27.43 ± 1.26 mV in the control group and -26.56 ± 1.53 mV in the 75 μmol/L gingerol group (P > 0.05), and a slope factor, K, of 13.24 ± 1.62 in the control group and 13.45 ± 1.68 (P > 0.05) in the 75 μmol/L gingerol group. Gingerol inhibits colonic motility by preventing Ca(2+) influx through L-type calcium channels.

Opioid Agonists Have Different Efficacy Profiles for G Protein Activation, Rapid Desensitization, and Endocytosis of Mu-opioid Receptors

The differential ability of various mu-opioid receptor (MOP) agonists to induce rapid receptor desensitization and endocytosis of MOP could arise simply from differences in their efficacy to activate G proteins or, alternatively, be due to differential capacity for activation of other signaling processes. We used AtT20 cells stably expressing a low density of FLAG-tagged MOP to compare the efficacies of a range of agonists to 1) activate G proteins using inhibition of calcium channel currents (ICa) as a reporter before and after inactivation of a fraction of receptors by beta-chlornaltrexamine, 2) produce rapid, homologous desensitization of ICa inhibition, and 3) internalize receptors. Relative efficacies determined for G protein coupling were [Tyr-D-Ala-Gly-MePhe-Glyol]enkephalin (DAMGO) (1) > or = methadone (0.98) > morphine (0.58) > pentazocine (0.15). The same rank order of efficacies for rapid desensitization of MOP was observed, but greater concentrations of agonist were required than for G protein activation. By contrast, relative efficacies for promoting endocytosis of MOP were DAMGO (1) > methadone (0.59) >> morphine (0.07) > or = pentazocine (0.03). These results indicate that the efficacy of opioids to produce activation of G proteins and rapid desensitization is distinct from their capacity to internalize mu-opioid receptors but that, contrary to some previous reports, morphine can produce rapid, homologous desensitization of MOP.

Sigma receptors inhibit high-voltage-activated calcium channels in rat sympathetic and parasympathetic neurons.

Studies on the expression and cellular function of sigma receptors in autonomic neurons were conducted in neonatal rat intracardiac and superior cervical (SCG) ganglia. Individual neurons from SCG and intracardiac ganglia were shown to express transcripts encoding the sigma-1 receptor using single-cell RT-PCR techniques. The relationship between sigma receptors and calcium channels was studied in isolated neurons of these ganglia under voltage-clamp mode using the perforated-patch configuration of the whole cell patch-clamp recording technique. Bath application of sigma receptor agonists was shown to rapidly depress peak calcium channel currents in a reversible manner in both SCG and intracardiac ganglion neurons. The inhibition of barium (I(Ba)) currents was dose-dependent, and half-maximal inhibitory concentration (IC50) values for haloperidol, ibogaine, (+)-pentazocine, and 1,3-Di-O-tolylguanidin (DTG) were 6, 31, 61, and 133 microM, respectively. The rank order potency of haloperidol > ibogaine > (+)-pentazocine > DTG is consistent with the effects on calcium channels being mediated by a sigma-2 receptor. Preincubation of neurons with the irreversible sigma receptor antagonist, metaphit, blocked DTG-mediated inhibition of Ca2+ channel currents. Maximum inhibition of calcium channel currents was > or =95%, suggesting that sigma receptors block all calcium channel subtypes found on the cell body of these neurons, which includes N-, L-, P/Q-, and R-type calcium channels. In addition to depressing peak Ca2+ channel current, sigma receptors altered the biophysical properties of these channels. Following sigma receptor activation, Ca2+ channel inactivation rate was accelerated, and the voltage dependence of both steady-state inactivation and activation shifted toward more negative potentials. Experiments on the signal transduction cascade coupling sigma receptors and Ca2+ channels demonstrated that neither cell dialysis nor intracellular application of 100 microM guanosine 5'-O-(2-thiodiphosphate) trilithium salt (GDP-beta-S) abolished the modulation of I(Ba) by sigma receptor agonists. These data suggest that neither a diffusible cytosolic second messenger nor a G protein is involved in this pathway. Activation of sigma receptors on sympathetic and parasympathetic neurons is likely to modulate cell-to-cell signaling in autonomic ganglia and thus the regulation of cardiac function by the peripheral nervous system.

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.

Regulation of Cloned Cardiac L-type Calcium Channels by cGMP-dependent Protein Kinase

We have studied the effect of 8-bromo-cyclic GMP (8-Br-cGMP) on cloned cardiac L-type calcium channel currents to determine the site and mechanism of action underlying the functional effect. Rabbit cardiac alpha(1C) subunit, in the presence or absence of beta(1) subunit (rabbit skeletal muscle) or beta(2) subunit (rat cardiac/brain), was expressed in Xenopus oocytes, and two-electrode voltage-clamp recordings were made 2 or 3 days later. Application of 8-Br-cGMP caused decreases in calcium channel currents in cells expressing the alpha(1C) subunit, whether or not a beta subunit was co-expressed. No inhibition of currents by 8-Br-cGMP was observed in the presence of the protein kinase G inhibitor KT5823. Substitutions of serine residues by alanine were made at residues Ser(533) and Ser(1371) on the alpha(1C) subunit. As for wild type, the mutant S1371A exhibited inhibition of calcium channel currents by 8-Br-cGMP, whereas no effect of 8-Br-cGMP was observed for mutant S533A. Inhibition of calcium currents by 8-Br-cGMP was also observed in the additional presence of the alpha(2)delta subunit for wild type channels but not for the mutant S533A. These results indicate that cGMP causes inhibition of L-type calcium channel currents by phosphorylation of the alpha(1C) subunit at position Ser(533) via the action of protein kinase G.

Acute desensitization of nociceptin/orphanin FQ inhibition of voltage-gated calcium channels in freshly dissociated hippocampal neurons.

Nociceptin/orphanin FQ (N/OFQ), an endogenous ligand for opioid receptor-like receptor, has been shown to inhibit high-voltage-gated calcium channels (VGCCs) in acutely dissociated rat hippocampal pyramidal cells [Knoflach, F., Reinscheid, R.K., Civelli, O. & Kemp, J.A. (1996), J. Neurosci., 16, 6657]. In this study, it was further demonstrated that N/OFQ inhibition of calcium channel current was blocked by its specific antagonist PGN, [Phe1-psi(CH2-NH)-Gly2]nociceptin (1-13)-NH2, and the EC50 of the N/OFQ inhibition was approximately 10 nM, indicating that this effect was really mediated via the opioid receptor-like receptor. The N/OFQ inhibition of the calcium channel current was significantly reduced, as the maximal inhibition decreased from 36 to 23%, by 1-min pretreatment of freshly dissociated hippocampal neurons with the same peptide. The inhibition completely recovered from this acute desensitization in less than 20 min. The N/OFQ inhibition was also greatly attenuated by pretreatment of the neurons with the GABAB (gamma-aminobutyric acid) agonist baclofen while the baclofen inhibition of the calcium channel current was significantly reduced by N/OFQ pretreatment, revealing the agonist-induced desensitization was heterologous in nature. This desensitization was blocked by pretreating the neurons with the sodium channel blocker, tetrodotoxin (TTX), or by removing the extracellular calcium, which indicates the necessity of membrane depolarization and extracellular calcium influx in the process. Furthermore, pretreatment of the neurons with the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), attenuated the N/OFQ inhibition of the calcium channel current whereas the cAMP-dependent kinase A activator, forskolin, showed no effect, suggesting the probable involvement of PKC in the N/OFQ-induced desensitization.

Muscarinic M2-receptors in rat thoracic dorsal root ganglia

The occurrence and distribution of the muscarinic M2-receptor subtype (M2R) was investigated in rat thoracic dorsal root ganglia (DRG). Messenger RNA for M2R was demonstrated by RT-PCR in total RNA from DRG. Immunoreactivity to M2R-protein was localized to 26% of sensory neurons, the majority of them (85%) belonging to the size class of 25–40 μm in diameter. Double-labeling (immuno)histochemistry revealed that all M2R-immunoreactive neurons bind the lectin, I-B4, whereas they are generally devoid of substance P-immunoreactivity. These data show the presence of M2R on a subpopulation of presumably nociceptive primary afferent neurons, thereby extending previous pharmacological and electrophysiological studies that indicated a role of M2R and/or M4R in inhibition of calcium channel currents in rat sensory neurons (Wanke, E., Bianchi, L., Mantegazza, M., Guatteo, E., Macinelli, E. and Ferroni, A., Muscarinic regulation of Ca 2+ currents in rat sensory neurons: channel and receptor types, dose-response relationships and cross-talk pathways. Eur. J. Neurosci., 6 (1994) 381–391).

Receptors couple to L-type calcium channels via distinct Go proteins in rat neuroendocrine cell lines.

1. The present study examines the hypothesis of G protein subtype selectivity in receptor-induced inhibition of calcium channel currents (ICa) in the insulin-secreting RINm5F and pituitary GH3 rat cell lines. Specificity of receptor coupling to G proteins was studied by infusion of purified G alpha isoforms into cells via a patch pipette. 2. In RINm5F cells, the neuropeptide galanin inhibited dihydropyridine (DHP)- and omega-conotoxin-sensitive components of ICa and slowed down their activation kinetics. In GH3 cells, DHP-sensitive ICa was inhibited by galanin, as well as by somatostatin and carbachol. Agonist-induced ICa inhibition was suppressed by pertussis toxin (PTX) pretreatment of the cells. In PTX-pretreated cells of either cell line, the response to galanin was restored only by the G alpha o1 subunit. Following PTX treatment of GH3 cells, only the G alpha o1 subunit restored carbachol-induced inhibition of ICa, whereas only the G alpha o2 subunit restored somatostatin-induced inhibition of ICa. G(i) subtypes had no effect on ICa inhibition. 3. Both cell lines expressed two distinct immunoreactive Go proteins. Whereas in RINm5F cell membranes Go1 was found to be the predominant isoform, we detected more Go2 than Go1 in GH3 cell membranes. Nevertheless, all agonists stimulated incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into both G(o) isoforms. 4. The results indicate that the same Go subtype, i.e. Go1, mediates galanin-induced inhibition of ICa in both cell lines and that the Go subtype specificity of receptor-G protein coupling is confined to intact cells.

Variation in serotonergic inhibition of calcium channel currents in four types of rat sensory neurons differentiated by membrane properties

1. Rat dorsal root ganglion (DRG) cell bodies were screened according to action potential (AP) duration, capsaicin sensitivity, expression of IH, IA, and N-, L-, and T-type Ca2+ channel currents. AP duration was measured at half of total amplitude at a membrane potential of -60 mV. Sensitivity to capsaicin was defined as production of an inward current at a holding potential (HP) of -60 mV by 1 microM capsaicin. IH was evoked by a 787-ms hyperpolarization to -110 mV from an HP of -60 mV. IA was evoked by repolarization to -60 mV after a 787-ms hyperpolarization to -110 mV. High-threshold Ca2+ channel current was evoked by a depolarization to -10 or 0 mV from an HP of -60 mV, and L- and N-type Ca2+ channel current was fractionated using selective Ca2+ channel blockers (nimodipine and omega-conotoxin GVIA). T-type Ca2+ channel current was evoked by a depolarization to -40 mV from an HP of -90 mV. Ninety-seven of the 116 DRG cells studied fit closely into one of four categories based on expression of the above characteristics. These four categories, referred to as types 1-4, are described below. 2. Type 1 DRG cells (soma diameter 24.6 +/- 0.5 microns, mean +/- SE; n = 34) had long-duration APs (average = 9.8 ms) with a prominent shoulder on the falling limb and were capsaicin sensitive. Significant IH or IA was not expressed. High-threshold Ca2+ channel current was on average 28% omega-conotoxin GVIA sensitive (N-type) and 46% nimodipine sensitive (L-type); 26% was resistant to both blockers (resistant). T-type Ca2+ channel currents averaged 245 pA. 3. Type 2 DRG cells (soma diameter 25.2 +/- 0.9 microns, n = 19) had short-duration APs (average = 2.9 ms) with a small shoulder on the falling limb and were capsaicin sensitive. IH was negligible but IA averaged 184 pA. High-threshold Ca2+ channel current averaged 42% N-type, 23% L-type, and 35% resistant. T-type Ca2+ channel currents averaged 47 pA. 4. Type 3 DRG cells (soma diameter 18.6 +/- 0.8 microns, n = 21) had short-duration APs (average = 1.8 ms) and were insensitive to capsaicin. IA was not expressed but IH averaged 147 pA. High-threshold Ca2+ channel current averaged 27% N-type, 44% L-type, and 29% resistant. T-type Ca2+ channel currents averaged 306 pA. 5. Type 4 DRG cells (soma diameter 33.9 +/- 0.4 microns, n = 23) had short-duration APs (average = 1.1 ms) and were capsaicin insensitive. IA was not expressed but IH averaged 810 pA. High-threshold Ca2+ channel current was 16% N-type, 4% L-type, and 80% resistant. T-type Ca2+ channel currents averaged 4,031 pA. 6. There was a large variation in the inhibition of high-threshold Ca2+ channel currents by serotonin (5-HT) and (+)8-OH-DPAT in type 1 DRG cells versus types 2-4. On average, 5-HT (10 microM) inhibited high-threshold Ca2+ channel current by an average of 42% in type 1 DRG cells, compared with 15%, 18%, and 7% inhibition in types 2-4, respectively. Similarly, (+)8-OH-DPAT (1 microM) inhibited high-threshold Ca2+ channel current by an average of 35% in type 1 DRG cells, compared with 5%, 8%, and 3% inhibition in types 2-4, respectively. 7. It is possible that DRG cells that vary in their expression of membrane properties may represent sensory neurons that transmit different types of sensory information. Thus the variation in inhibition of Ca2+ channel current by 5-HT and (+)8-OH-DPAT in the above categories of DRG cells may indicate that 5-HT1A receptor activation inhibits Ca2+ entry into some types of DRG sensory neurons more than others.

P21-ras is involved in regulation of voltage-dependent calcium channels in cultured rat dorsal root ganglion cells.

P21-ras in combination with ras-GAP has been found to block activation of atrial K+ channels by M2 muscarinic receptors (mAChRs), an effect which was suggested to involve interference with receptor-G-protein coupling [l]. Since both M2 and M4 mAChRs are known to inhibit Ca2' channels directly via the pertussis toxin (PTX)-sensitive G-protein, Go, p21-ras may also be involved in regulation of neuronal Ca2+ channels [2, 31. Here we report the results from a study in which the whole cell patch clamp technique has been used to examine the involvement of endogenous p21-ras in regulation of Ca channels in primary cultured rat dorsal root ganglion cells (DRGs). In preliminary ex eriments, the presence of the muscarinic agonist, carbachol(10' M) in the external medium caused a 32.0 * 4.0 YO (mean i s.e.m.) inhibition of calcium channel current, IBa. Use of the M 1 M4-selective muscarinic antagonist, pirenzepine ( M), the M4-selective antagonist, himbacine ( M), and PTX (230 ng/ml for 15 h) suggested the involvement of a PTXsensitive M4 receptor in rat DRGs. In DRGs microinjected with the p21-ras neutralising antibody (Y13-259, 0.1 mg/ml, 50 pl/cell) 2 h prior to experiments, a 43.7 f 4.0 % inhibition of IBa was observed in the presence of carbachol. This result was not significantly different from control cells injected with 1 mg/ml of mouse IgG (36.2 5 4.0 %) or from noninjected cells (42.9 * 4.0 %). The voltage-dependence of both activation and inactivation of IB, shifted between 5-10 mV in a depolarising direction in all three treatments following application of carbachol, although these shifts were not significant either within, or, between treatments. Thus, microinjection of Y 13-259 had no effect on the ability of the muscarinic agonist to inhibit I B ~ Importantly, however, there was a significant reduction in both peak amplitude of maximum IBa (36.0 * 3.0 %, p < 0.05) and the maximum tail current amplitude (31.0 f 3.0 %, p < 0.05) in antibody-injected cells versus controls. No significant differences in the voltage-dependence of either activation or inactivation of maximum peak amplitude of IB, were observed between treatments. Thus, acute blockade of a pathway involving endogenous p21 -ras reduces calcium currents. Intracellular application of phosphopeptides (10-1 5 amino acids) with the sequences of tyrosine autophosphorylation sites on protein tyrosine kinases involved in growth factor activation of p21-ras (NGFR, TRK490; EGFR1068; and CRK adaptor protein, CRK221; at 0.1 mg/ml) also reduced both the peak amplitude of IBa, and maximum tail current amplitude. Effects of these phosphopeptides are thought to be due to the blockade of productive SH2-SH3 interactions which are initiated during the activation of p21-ras by growth factors. Inclusion of TRK490 in the patch pipette for up to 10 min caused a significant reduction of both peak IB, (1 5.8 f 2 %, p < 0.05) and tail current (34.0 f 6.4 %, p < 0.05). Application of EGFR1068 for 10 min resulted in a 19.8 f 4.0 % reduction of IB, (significant at p < 0.05) , and reduced the tail current by 12.8 f 3.0 %, which was not significant. CRK221 caused a significant reduction of peak I B ~ by 18.3 f 2.0 % (p < 0.05), and maximum tail current by 29.2 f 2.0 % (p < 0.01), with time. In control cells, neither the peak amplitude of Is,, nor the tail current amplitude changed significantly with time (up to 10 min). Similarly, internal application of a control (scrambled) phosphopeptide, also had no ! . significant effects on either the maximum peak amplitude of IB,, or the maximum tail current amplitude. No significant changes in voltage-dependence of either current activation or inactivation were observed either within, or, between any of the above treatments. Thus, blocking a pathway possibly involving growth factor activation of p21-ras also reduced calcium current amplitude in these cells. In contrast, microinjection of oncogenic p21-K-ras (2 mg/ml, 24 h prior to experiments) caused a significant increase in the peak amplitude of maximum IBa (61 f 3.0 %, p < 0.01) when compared with control cells. Similarly, the maximum tail current amplitude also increased significantly by 59.7 * 3.0 % (p < 0.01) The voltagedependence of both activation and inactivation of the currents exhibited no significant differences in p21-K-ras-injected, versus control cells. Thus, we have provided evidence that endogenous p2 1 -ras, possibly activated via a protein tyrosine kinase pathway, is involved in the regulation of Ca2+ channels in rat DRGs, although unlike in cardiac myocytes, it is not involved in muscarinic inhibition of these Ca currents. These results tie in with other studies in which the injection of oncogenic p21-ras has been found to increase Ca2+ current in neuronal cells [4,5,6].

Calcium channel currents in bovine adrenal chromaffin cells and their modulation by anaesthetic agents.

1. The calcium channel currents of bovine adrenal chromaffin cells were characterized using a variety of voltage pulse protocols and selective channel blockers before examination of their modulation by anaesthetic agents. 2. All the anaesthetics studied (halothane, methoxyflurane, etomidate and methohexitone) inhibited the calcium channel currents in a concentration-dependent manner and increased the rate of current decay. 3. The anaesthetics did not shift the current-voltage relation nor did they change the voltage for half-maximal channel activation derived from analysis of the voltage dependence of the tail currents. None of the anaesthetics appeared to alter the time constant of tail current decay. 4. To complement earlier studies of the inhibitory actions of anaesthetics on K(+)-evoked catecholamine secretion and the associated Ca2+ uptake, the IC50 values for etomidate and methohexitone were determined using a biochemical assay. The IC50 values for anaesthetic inhibition of calcium channel currents corresponded closely with those for inhibition of K(+)-evoked calcium uptake and catecholamine secretion. 5. The inhibitory effect of the volatile anaesthetics and etomidate is best explained by dual action: a reduction in the probability of channel opening coupled with an increase in the rate of channel inactivation. Methohexitone appeared to inhibit the currents by a use-dependent slow block.

The action of amlodipine on voltage-operated calcium channels in vascular smooth muscle.

1. Amlodipine, a dihydropyridine derivative largely ionized at physiological pH, inhibited calcium channel currents in single vascular smooth muscle cells isolated from rabbit ear artery in a concentration-dependent manner. 2. Amlodipine inhibited the current-voltage relationship for calcium channel currents across the range of test potentials used. However, the effect of amlodipine was more marked on more depolarized test potentials. Amlodipine also shifted the steady-state inactivation curve for calcium channel currents in a hyperpolarized direction. 3. The potency of amlodipine as determined from the steady-state inhibition of calcium channel current induced by the drug was dependent on the holding potential of the cells. Use of a more depolarized holding potential increased the potency of amlodipine. 4. Onset of amlodipine-induced inhibition was relatively rapid at both -60 mV and -40 mV holding potential. The use of a more depolarized holding potential increased the rate of association of amlodipine. No recovery from amlodipine-induced inhibition was seen over a 20 min period following washout of the drug. 5. In addition to voltage-dependence, the action of amlodipine showed use-dependence, in that the effect of amlodipine was more marked when calcium channel currents were evoked frequently. Increasing the frequency of activation of calcium channel currents did not alter the apparent onset rate of amlodipine-induced inhibition, but increased the degree of inhibition achieved by the drug. 6. The electrophysiological properties of amlodipine, particularly its voltage-dependence are probably important determinants of its action in vivo.

Intracellular ATP and GTP are both required to preserve modulation of N-type calcium channel current by norepinephrine.

Norepinephrine (NE) inhibits voltage-dependent calcium channels of sympathetic neurons. We investigated the role of intracellular nucleotides in this inhibition for clues to receptor-channel coupling mechanisms. Both ATP and GTP are required to preserve NE responsiveness during whole-cell dialysis. The response to NE was gradually lost in bullfrog sympathetic neurons dialyzed with GTP as the only nucleotide, ATP only, or no nucleotides. Replacing ATP with ATP[gamma-S] resulted in spontaneous modulation of calcium channel current, possibly because of production of GTP[gamma-S]. The nonhydrolyzable ATP analog p[NH]ppA could substitute for ATP to preserve NE responsiveness. The protein phosphatase inhibitors okadaic acid and calyculin-A did not affect NE inhibition of calcium channel current, or recovery from that inhibition. These results suggest protein phosphorylation is not involved in the inhibition of calcium channel current, but binding of ATP to some intracellular site is required for the coupling of adrenergic receptors to calcium channels.

Fenoverine inhibition of calcium channel currents in single smooth muscle cells from rat portal vein and myometrium

1. The effects of fenoverine, an antispasmodic drug, have been studied on the Ca2+ channel currents of isolated cells from rat portal vein and pregnant myometrium by the patch-clamp technique (whole-cell configuration). 2. Fenoverine inhibited both fast and slow Ca2+ channel currents in a concentration-dependent manner. Half-inhibition of fast Ca2+ channel current (holding potential of -70 mV) and slow Ca2+ channel current (holding potential of -40 mV) in portal vein smooth muscle were obtained at concentrations of 7.5 and 1.9 microM, respectively. In myometrium, the fenoverine concentration which blocked 50% of the slow Ca2+ channel current (holding potential of -70 mV) was 2.3 microM. 3. Administration of fenoverine at rest reduced both Ca2+ channel currents. Currents activated repetitively, at a rate between 0.05 and 0.1 Hz, were inhibited equally which indicates an absence of use-dependent inhibition. 4. When cells held at depolarized membrane potentials at which fast or slow Ca2+ channel currents were strongly inactivated, the inhibitory effects of fenoverine were enhanced on both Ca2+ channel currents which indicates that the fenoverine-induced inhibition was voltage-dependent. The fenoverine concentrations which blocked the inactivated Ca2+ channels were 5-7 times lower than those which blocked the resting Ca2+ channels. 5. Our results show that fenoverine depresses inward currents through fast and slow Ca2+ channels. This effect may be explained by the preferential binding of fenoverine to resting Ca2+ channels. In addition, fenoverine has a higher affinity for inactivated Ca2+ channels than for resting channels.

Caffeine‐induced inhibition of calcium channel current in cultured smooth muscle cells from pregnant rat myometrium

1. The inhibitory effect of caffeine on the calcium channel current was investigated in cultured myometrial cells isolated from pregnant rats. 2. Caffeine inhibited the calcium channel current elicited from a holding potential of -70 mV in a concentration-dependent manner. The IC50 was estimated to be 35 mM. 3. The caffeine inhibition was not enhanced when calcium channels were opened by a conditioning depolarizing pulse sequence or when the number of inactivated calcium channels was increased at depolarized holding potentials. 4. Caffeine antagonized the specific binding of (+)-[3H]-isradipine to myometrial membranes. The IC50 value found in binding experiments was similar to the IC50 value for half-maximal inhibition of calcium channel current. Caffeine decreased the maximal binding capacity of (+)-[3H]-isradipine to myometrial membranes without any significant change in the dissociation constant. 5. The results indicate that caffeine interacts with a site closely associated with the voltage-dependent calcium channels in myometrial cells and, in turn, inhibits calcium influx.

An investigation into the mechanisms of inhibition of calcium channel currents in cultured sensory neurones of the rat by guanine nucleotide analogues and (−)− baclofen

1. The mechanism of inhibition of calcium channel currents by the guanine nucleotide analogue guanosine 5'-O-3 thiotriphosphate (GTP-gamma-S) and by the GABAB agonist (-)-baclofen has been studied in cultured dorsal root ganglion neurones of the rat. The inhibition by GTP-gamma-S is particularly characterized by an abolition of the transient component of calcium channel currents carried either by Ba2+ (IBa) or by Ca2+ (ICa). 2. The effect of agents increasing intracellular cyclic AMP levels has been examined. Neither internal cyclic AMP nor forskolin prevented the inhibition of IBa by baclofen. Neither forskolin nor pretreatment of cells with cholera toxin prevented the inhibition of the transient component of IBa by GTP-gamma-S. However, both these treatments increased the amplitude of the sustained IBa in the presence of GTP-gamma-S. The ATP analogue adenosine imido-diphosphate which inhibits many ATP requiring enzymes did not prevent the effect of GTP-gamma-S although it reduced the amplitude of IBa. 3. Baclofen (100 microM) produced a 22 +/- 2% increase in inositol phosphate production in 30 s, whereas the increase produced by bradykinin (1 microM) was 70 +/- 14%. However, unlike baclofen, bradykinin did not inhibit IBa or ICa in these cells. 4. The effect of protein kinase C inhibitors was examined. Polymixin B (20 microM in patch pipette) had no effect on the inhibition of IBa by baclofen or GTP-gamma-S. A higher concentration (100 microM) alone inhibited IBa and no further inhibition by baclofen was observed. Neither H7 (50 microM) nor staurosporine (100 nM), applied extracellularly, prevented the response to GTP-gamma-S. 5. The protein kinase C activator di-octanoyl glycerol (20 microM) did not inhibit IBa. Arachidonic acid (100 microM) also produced no inhibition of IBa. 6. In conclusion we have obtained no evidence that a second messenger system mediates the inhibition of calcium channel currents by GTP-gamma-S or baclofen in dorsal root ganglion neurones. These results support the hypothesis that GABAB receptors are directly coupled to calcium channels by G proteins.

Photoalteration of calcium channel blockade in the cardiac Purkinje fiber

Organic compounds that block calcium channel current (calcium antagonists) are important tools for the characterization of this channel. However, the practically irreversible nature of this block restricts the usefulness of this group of drugs. In this paper, we investigate the influence of light on calcium channel blockade by several organic compounds. Our results show that inhibition of calcium channel current by two dihydropyridine derivatives that contain an o-nitro moiety (nisoldipine and nifedipine) can be rapidly reversed by illumination. The energy range important to this reaction is for light wavelengths between 320 and 450 nm. Calcium channel inhibition by two other dihydropyridine derivatives (nicardipine and nitrendipine) as well as by D600, is not modulated by illumination. These results indicate that the photosensitivity of certain dihydropyridine calcium channel blockers make these compounds useful as reversible blockers of this channel.