Antimigraine dotarizine blocks P/Q Ca2+ channels and exocytosis in a voltage-dependent manner in chromaffin cells

The mechanism of blockade of P/Q Ca(2+) channels by antimigraine, dotarizine, was studied in voltage-clamped bovine adrenal chromaffin cells. Inward currents through P/Q channels were pharmacologically isolated by superfusing the cells with omega-conotoxin GVIA (1 microM) plus nifedipine (3 microM). Dotarizine (10-30 microM) blocked the P/Q fraction of I(Ba) and promoted current inactivation. Thus, dotarizine caused a greater blockade of the late I(Ba), compared with blockade of the early peak I(Ba). This effect was more prominent, the longer was the duration of the depolarising pulse. The blockade of I(Ba) was also greater at more depolarising holding potentials (i.e. -60 mV), than was the blockade produced at more hyperpolarising holding potentials (i.e. -80 or -110 mV). Catecholamine secretory responses to brief pulses (2 s) of a Krebs-HEPES solution containing 100 mM K(+) and 2 mM Ca(2+) was blocked by 3 microM dotarizine. Blockade was faster and greater when dotarizine was applied on cells that were previously depolarised with Krebs-HEPES deprived of Ca(2+) and containing increasing concentrations of K(+). This voltage-dependent blockade of P/Q channels and exocytosis might be the underlying mechanism explaining the dotarizine prophylaxis of migraine attacks.     

KCl-induced insulin secretion from RINm5F cells is mediated through Ca2+ influx along L-type Ca2+ channels

Summary In order to characterize the voltage-dependent Ca²⁺ channels of insulin secretory RINm 5F cells, we have studied the binding of the dihydropyridine (DHP) type Ca²⁺ antagonist PN 200-110 and its effect on insulin release. In the membrane preparation from RINm 5F cells [³H]-(+)-PN 200-110 bound to a high affinity binding site in a stereoselective manner (KD: 7.0 nM, B_max: 858 fmol/mg protein). The benzothiazepine type Ca²⁺ antagonist D-cis-diltiazem increased the binding of [³H]-(+)-PN 200-110 in a temperature-dependent manner. The phenylalkylamine-type Ca²⁺ antagonist verapamil decreased PN binding with an IC₅₀ of 100 M. (+)-PN 200-110 inhibited KCl-(25 mM)-induced insulin release (IC₅₀ = 10 nM), Effects on binding and hormone release occurred over comparable concentration ranges: 1 M PN 200-110 produced 100% displacement and totally abolished depolarization-mediated insulin release. The N-type Ca²⁺-antagonist -conotoxin showed no effect on KCl-induced insulin release. The data suggest that in RINm 5 F cells only l-type Ca²⁺ channels are involved in the mechanism of depolarization-mediated insulin release.Some of the data reported here were presented at the 27th Annual Meeting of the European Association for the Study of Diabetes, Dublin, 10–14 September 1991 Correspondence to: H. Safayhi at the above address     

SNX482 selectively blocks P/Q Ca2+ channels and delays the inactivation of Na+ channels of chromaffin cells

The effects of the toxin SXN482 on Ca2+ channel currents (ICa), Na+ currents (INa), and K+ currents (IK) have been studied in bovine adrenal medullary chromaffin cells voltage-clamped at -80 mV. Currents were elicited by depolarising pulses to 0-10 mV (ICa and INa) or to +60 mV (IK). SNX482 blocked ICa in a concentration-dependent manner. The inhibition curve exhibited two phases. The first high-affinity phase comprised 28% of the whole-cell current and exhibited an IC50 of 30.2 nM. The second low-affinity phase comprised over 70% of ICa and had an IC50 of 758.6 nM. Blockade was rapid and fully reversible upon washout of the toxin. Occlusion experiments showed additivity of blockade exerted by nifedipine plus SNX482 (0.3 microM) and by omega-conotoxin GVIA plus SNX482. In contrast, blockade exerted by combined omega-agatoxin IVA plus SNX482 (about 50% of the whole cell) did not show additivity. At 0.3 microM and higher concentrations, SNX482 delayed the inactivation of INa. The time constant (tau) for inactivation of INa in control conditions doubled in the presence of 0.5 microM SNX482. At 0.3 microM, SNX482 did not affect IK. Our data demonstrate that: (i) SNX482 selectively blocks P/Q Ca2+ channels at submicromolar concentrations; (ii) the toxin partially blocks Na+ channels; (iii) SNX482 delays the inactivation of Na+ channels. These results reveal novel properties of SNX482 and cast doubts on the claimed selectivity and specificity of the toxin to block the R-type Ca2+ channel.     

Neomycin blocks dihydropyridine-insensitive Ca2+ influx in bovine adrenal chromaffin cells

There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca²⁺ influx pathways. Although recent electrophysiological work indicates that the dihydropyridine-resistant pathway is partially mediated by w-conotoxin-sensitive and -insensitive Ca²⁺ channels, the pharmacological sensitivity of the latter channels remains elusive. We have now found that combined incubations with nitrendipine (1 μM) and neomycin (0.5 mM) reduced high K⁺ (50 mM)-evoked intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients to a larger extent than each drug separately. [Ca²⁺]_i was measured using the fluorescent intracellular Ca²⁺ indicator fura-2. Neomycin (0.05−2 mM) reduced high K⁺-evoked ⁴⁵Ca²⁺ uptake in a dose-dependent manner (IC₅₀ = 0.09 mM). In the presence of nitrendipine (1 μM), the minimal neomycin concentration necessary for total blockade of ⁴⁵Ca²⁺ uptake was reduced to 0.3 mM. Moreover, in the absence of nitrendipine the ⁴⁵Ca²⁺ uptake remaining in 0.3 mM neomycin (26% of maximum) was similar to the fractional inhibition by nitrendipine alone (29%). Neomycin (0.05−2 mM) inhibited the [Ca²⁺]_i transient induced by the L-type Ca²⁺ channel agonist Bay K 8644 (1 μM) much more extensively at 2 mM than at 0.3 mM (percent inhibition = 59% and 15%, respectively). Neomycin (0.05−2 mM) blocked high K⁺-evoked noradrenaline and adrenaline release in a dose-dependent fashion (IC₅₀ = 0.8−1.1 mM), the blockade efficiency being enhanced in the presence of 1 μM nitrendipine (IC₅₀ = 0.17−0.19 mM). It is concluded that neomycin (≤ 0.3 mM) blocks preferentially the dihydropyridine-insensitive Ca²⁺ influx pathway of the chromaffin cell. Moreover, both the dihydropyridine-sensitive and the dihydropyridine-resistant, neomycin-sensitive Ca²⁺ influx pathways contribute strongly to depolarization-evoked catecholamine secretion.     

Inhibition of voltage-gated Ca2+ channels and insulin secretion in HIT cells by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62: comparison with antagonists of calmodulin and L-type Ca2+ channels.

To probe for the involvement of Ca2+/calmodulin-dependent protein kinase II in the regulation of insulin secretion, the effects of a specific inhibitor of this enzyme, KN-62, on secretagogue-stimulated insulin secretion, cytosolic Ca2+ concentration ([Ca2+]i) rise, membrane depolarization, and nutrient metabolism were examined in HIT-T15 cells. KN-62 dose-dependently inhibited insulin secretion induced by a nutrient mixture (10 mM glucose, 5 mM leucine, and 5 mM glutamine) alone or combined with either the Ca(2+)-mobilizing receptor agonist bombesin or the cAMP-raising agent forskolin in intact cells. KN-62 did not affect Ca(2+)- or GTP analogue-induced insulin secretion from permeabilized cells, indicating an action at a step before exocytosis. The stimulating effects of nutrients on insulin secretion, [Ca2+]i, and membrane depolarization were potentiated by bombesin. Similarly, bombesin promoted a larger depolarization and [Ca2+]i rise in the presence of nutrients. This was associated with enhanced Ca2+ mobilization and the appearance of sustained [Ca2+]i elevation. The bombesin-induced membrane depolarization, like the nutrient effect, was inhibited by diazoxide, suggesting that this is due to closure of ATP-sensitive K+ channels. Bombesin elicited Ca2+ influx by both membrane potential-sensitive and -insensitive conductance pathways. KN-62 did not affect Ca2+ mobilization and only partially reduced Ca2+ entry during the sustained [Ca2+]i rise in bombesin-stimulated cells. When added before or during the stimulation, KN-62 dose-dependently inhibited nutrient- and KCl-stimulated [Ca2+]i elevation and Mn2+ influx (reflecting Ca2+ entry). The calmodulin antagonist CGS 9343B and the L-type Ca2+ channel blocker SR-7037 mimicked the inhibitory effect of KN-62 on stimulated insulin secretion and [Ca2+]i elevation. Membrane depolarization and nutrient metabolism (reduction of a tetrazolium derivative), however, were not altered by KN-62 treatment, indicating that the early coupling events from nutrient metabolism to closure of ATP-sensitive K+ channels remain operative. These results suggest that KN-62 and the calmodulin antagonist CGS 9343B inhibit Ca2+ influx by means of direct interaction with L-type Ca2+ channels, which, in turn, causes inhibition of stimulated insulin secretion. Thus, it appears that Ca2+/calmodulin-dependent protein kinase II is not involved in the regulation of insulin secretion.     

Hormone-stimulated redistribution of gonadotrope protein kinase C in vivo: dependence on Ca2+ influx

In the present study we show that natural sequence gonadotropin-releasing hormone (GnRH) and a high affinity, metabolically stable agonist (Buserelin) promote redistribution of protein kinase C (PKC) activity to a particulate fraction prepared from anterior pituitary. The action of the agonists, administered in vivo to ovariectomized rats, is both time and dose dependent. GnRH antagonist alone does not measurably alter distribution of this enzymatic activity but inhibits the ability of GnRH to do so and to stimulate luteinizing hormone release. This finding indicates that receptor occupancy alone is insufficient to cause PKC redistribution. Redistribution of PKC in response to Buserelin is inhibited by the calcium ion channel antagonist methoxyverapamil (D600), suggesting that redistribution of PKC activity, like GnRH-stimulated gonadotropin release, requires the influx of extracellular calcium.     

Ethanol inhibition of L-type Ca2+ channels in PC12 cells: role of permeant ions

The effects of acute ethanol exposure on voltage-activated Ca²⁺ channels in undifferentiated pheochromocytoma (PC12) cells were investigated using whole-cell patch clamp techniques. Concentrations of ethanol (5, 25 and 50 mM), at or below blood alcohol levels which constitute legal intoxication significantly reduced Ca²⁺ currents evoked from a holding potential of −30 mV. Ethanol-induced inhibition of current was voltage-dependent in some cases, but this was not consistently observed. Inhibition of currents was reversible and was not due to an osmotic effect. The non-inactivating nature of the current, the inhibition of the current by nifedipine, and the lack of inhibition by ω-conotoxin, indicated that the current was carried through high-voltage activated, L-type Ca²⁺ channels. Since intracellular Ca²⁺ levels were highly buffered by exchange with the contents of the patch pipet, ethanol-induced inhibition of currents in PC12 cells is not likely to involve either a change in driving force due to a change in intracellular Ca²⁺ levels or potentiation of Ca²⁺-dependent Ca²⁺ channel inactivation by the influx of Ca²⁺. The degree of inhibition by 25 mM ethanol was the same when either Ca²⁺, Ba²⁺ or Na⁺ was used as the current-carrying ion. This equivalency suggests that the channel's ion selectivity filter is not a site of action for ethanol.     

蛋白酪氨酸激酶抑制剂对脑血管平滑肌细胞Ca~(2+)池操纵性Ca~(2+)内流的影响

目的　研究蛋白酪氨酸激酶和蛋白酪氨酸磷酸酶抑制剂对牛脑血管平滑肌细胞 (CSMC)Ca2 + 池操纵性Ca2 + 内流的影响。方法　采用培养的CSMC ,在生物荧光双波长影像分析系统用Fura 2 /Am荧光探针测定单个细胞内游离Ca2 + 浓度。结果　 (1)蛋白酪氨酸激酶抑制剂 (genistein ,2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低内皮素 1(ET 1,10 -7mol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为5 6%± 2 .9%、2 5 6%± 3 9%、48 9%± 3 7% ;蛋白酪氨酸磷酸酶抑制剂 (vanadate ,2 ,4,8μmol·L-1)能浓度依赖性升高CPA刺激引起的CSMCCa2 + 内流 ,增加比率分别为8 2 %± 3 9%、18 8%± 4 9%、46 6%± 6 9% ;(2 ) genistein(2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低ATP(10 μmol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为 6 7%±2 6%、2 4 6%± 6 5 %、5 1 3 %± 6 9% ;vanadate (2 ,4,8μmol·L-1)能浓度依赖性升高ATP刺激引起的CSMCCa2 +内流 ,增加比率分别为 4 8%± 2 0 %、2 8 5 %± 4 6%、49 6%± 3 3 % ;(3 ) genistein (2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低环匹阿尼酸 (Cyclopiazonicacid ,CPA ,10 μmol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为 6 5 %± 3 0 %、2 2 5 %± 5 2 %、     

蛋白激酶C与血管平滑肌α_1肾上腺素受体触发Ca~（2＋）内流的关系

在酶新鲜分离的犬肠系膜上动脉平滑肌细胞，蛋白激酶Ｃ（ＰＫＣ）的激活剂佛波二丁酯（ＰＤＢ）引起的胞内Ｃａ２＋增高作用可被ＰＫＣ抑制剂１－（５－异喹啉磺酰）－２－甲基哌嗪（Ｈ７）所阻断，而哌唑嗪和普萘洛尔则不能阻断ＰＤＢ的这一作用；１０μｍｏｌ·Ｌ－１苯福林引起的胞内Ｃａ２＋增高作用可被１０和２０μｍｏｌ·Ｌ－１Ｈ７部分阻断；在无Ｃａ２＋液，Ｈ７可部分抑制苯福林引起的内Ｃａ２＋释放和外Ｃａ２＋内流，两者分别被抑制了３３±３％和５８±６％；ＫＣｌ（２０－１００ｍｍｏｌ·Ｌ－１）可浓度依赖性地引起胞内钙升高，这一作用可被１０和２０μｍｏｌ·Ｌ－１Ｈ７不同程度地阻断；ＰＤＢ引起的胞内Ｃａ２＋增高也可分别被１．２５和２．５μｍｏｌ·Ｌ－１维拉帕米部分和全部阻断。上述结果提示ＰＫＣ参与苯福林引起的部分内Ｃａ２＋释放和外Ｃａ２＋内流，但以参与外Ｃａ２＋内流为主；这一作用可能与ＰＫＣ激活，引起电压依赖性Ｃａ２＋通道开放有关。     

Potentiation by endothelin-1 of Ca2+ sensitivity of contractile elements depends on Ca2+ influx through L-type Ca2+ channels in the canine cerebral artery

• 1.1. Endothelin-1 (ET-1) contracted canine cerebral artery in a concentration-dependent manner with an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), and at higher concentrations it produced a greater contraction with a smaller increase in [Ca²⁺]_i.
• 2.2. Ca²⁺ channel antagonist such as d-cis-diltiazem inhibited the tension more effectively than the [Ca²⁺]_i increased by ET-1.
• 3.3. In Ca²⁺-free solution containing 0.2 mM EGTA, ET-1 elicited a transient increase in [Ca²⁺]_i and tension.
• 4.4. In the Staphylococcus aureus α-toxin-permeabilized artery, ET-1 shifted the pCa-tension relationship leftwards in the presence of GTP.
• 5.5. These findings suggest that ET-I contracts the canine cerebral artery by increasing not only the Ca²⁺ influx through L-type Ca²⁺ channels, but also Ca²⁺ release from the intracellular storage sites, and also Ca²⁺ sensitivity of contractile elements. The degree of Ca²⁺ sensitivity is strongly affected by [Ca²⁺]_i which is increased by the Ca²⁺ influx through L-type Ca²⁺ channels.

     

Regulation of OX1 orexin/hypocretin receptor-coupling to phospholipase C by Ca2+ influx

BACKGROUND AND PURPOSE: Orexin (OX) receptors induce Ca2+ elevations via both receptor-operated Ca2+ channels (ROCs) and the "conventional" phospholipase C (PLC)-Ca2+ release-store-operated Ca2+ channel (SOC) pathways. In this study we assessed the ability of these different Ca2+ influx pathways to amplify OX1 receptor signalling to PLC in response to stimulation with the physiological ligand orexin-A. EXPERIMENTAL APPROACH: PLC activity was assessed in CHO cells stably expressing human OX1 receptors. KEY RESULTS: Inhibition of total Ca2+ influx by reduction of the extracellular [Ca2+] to 1 microM effectively inhibited the receptor-stimulated PLC activity at low orexin-A concentrations (by 93% at 1 nM), and this effect was gradually reduced by higher orexin-A concentrations. A similar but weaker inhibitory effect (84% at 1 nM) was obtained on depolarization to approximately 0 mV, which disrupts most of the driving force for Ca2+ entry. The inhibitor of the OX1 receptor-activated ROCs, tetraethylammonium chloride (TEA), was somewhat less effective than the reduction in extracellular [Ca2+] at inhibiting PLC activation, probably because it only partially blocks ROCs. The partial inhibitor of both ROCs and SOCs, Mg2+, and the SOC inhibitors, dextromethorphan, SKF-96365 (1-[beta-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole HCL) and 2-APB (2-aminoethoxydiphenyl borate), inhibited PLC activity at low concentrations of orexin-A, but were not as effective as TEA. CONCLUSIONS AND IMPLICATIONS: Both ROCs and SOCs markedly amplify the OX(1) receptor-induced PLC response, but ROCs are more central for this response. These data indicate the crucial role of ROCs in orexin receptor signalling.     

Ginsenoside Rg1 protects neurons from hypoxic-ischemic injury possibly by inhibiting Ca2+ influx through NMDA receptors and L-type voltage-dependent Ca2+ channels

The purpose of this study is to assess the neuroprotective effect of Rg1, a ginsenoside. We measured cell viability and lactate dehydrogenase (LDH) release from primary culture of rat hippocampal neurons and electrical activities in hippocampal slices of rats, before and after the neurons were deprived of oxygen and glucose. In addition, cerebral damage was evaluated with magnetic resonance imaging after middle cerebral artery was occluded transiently. Nissl staining was used for histological observation and immunohistochemistry analysis for activated caspase-3 expression of the brain. Furthermore, calcium influx was measured with laser confocal microscopy in neurons perfused with KCl (50 mM) or N-methyl-d-aspartate (NMDA, 1 mM), or deprived of oxygen and glucose. The influences of ginsenoside Rg1 on these parameters were determined simultaneously. We found that treatment of Rg1: 1) increased the neuronal viability; 2) promoted the recovery of electrical activity in hippocampal slices; 3) reduced the release of LDH, cerebral damage area, neuronal loss and expression of caspase-3; and 4) inhibited calcium influx induced by NMDA, KCl or oxygen/glucose deprivation. However, the protective effect of Rg1 was blocked by mifepristone, an antagonist of glucocorticoid receptors. Taken together, these results suggest that ginsenoside Rg1 can reduce neuronal death, including apoptotic cell death, induced by hypoxic-ischemic insults. This neuroprotective effect is probably mediated by the activation of glucocorticoid receptors, and by the inhibition of calcium influx through NMDA receptors and L-type voltage-dependent Ca2+ channels and the resultant reduction of intracellular free Ca2+.     

Chlorpromazine-induced inhibition of catecholamine secretion by a differential blockade of nicotinic receptors and L-type Ca2+ channels in rat pheochromocytoma cells.

We investigated the effect of chlorpromazine (CPZ), a phenothiazine neuroleptic, on catecholamine secretion in rat pheochromocytoma (PC12) cells. CPZ inhibited [3H]norepinephrine ([3H]NE) secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), an agonist of nicotinic acetylcholine receptors (nAChRs) with an IC50 value of 1.0 +/- 0.2 microM. The DMPP-induced rise in cytosolic free Ca2+ concentration [Ca2+]i was inhibited by CPZ with an IC50 of 1.9 +/- 0.1 microM. The DMPP-induced increase in cytosolic free Na+ concentration [Na+]i was also inhibited by CPZ with a similar potency. Furthermore, the binding of [3H]nicotine to PC12 cells was inhibited by CPZ with an IC50 value of 2.7 +/- 0.6 microM, suggesting that the nAChRs themselves are inhibited by CPZ. In addition, both 70 mM K+-induced [3H]NE secretion and [Ca2+]i increase were inhibited by CPZ with IC50 of 7.9 +/- 1.1 and 6.2 +/- 0.3 microM, respectively. Experiments with Ca2+ channel antagonists suggest that L-type Ca2+ channels are mainly responsible for the inhibition. We conclude that CPZ inhibits catecholamine secretion by blocking nAChRs and L-type Ca2+ channels, with the former being more sensitive to CPZ.     

Mefenamic acid as a novel activator of L-type voltage-dependent Ca2+ channels in smooth muscle cells from pig proximal urethra

The effects of mefenamic acid and Bay K 8644 on voltage-dependent nifedipine-sensitive inward Ba2+ currents in pig urethral myocytes were investigated by use of conventional whole-cell configuration patch clamp. Mefenamic acid increased the peak amplitude of voltage-dependent nifedipine-sensitive inward Ba2+ current without shifting the position of the current-voltage relationship. Mefenamic acid (300 microM) caused little shift in the activation curve although the voltage dependence of the steady-state inactivation was shifted to more positive potentials by 11 mV in the presence of mefenamic acid. Bay K 8644 (> or = 100 nM) enhanced voltage-dependent nifedipine-sensitive inward Ba2+ currents in a concentration- and voltage-dependent manner, shifting the maximum of the current-voltage relationship by 10 mV in the hyperpolarizing direction. Bay K 8644 (1 microM) significantly shifted the voltage dependence of the activation curve to more negative potentials by approximately 9 mV although Bay K 8644 caused little shift in the steady-state inactivation curve. These results indicate that mefenamic acid increased voltage-dependent nifedipine-sensitive inward Ba2+ currents through the activation of L-type Ca2+ channels with different kinetics from those of Bay K 8644 in pig urethral myocytes.     

Acidosis-induced protein tyrosine phosphorylation depends on Ca2+ influx via voltage-dependent Ca2+ channels in SHR aorta

The contractile response to acidosis in isolated aorta from spontaneously hypertensive rat (SHR) depends upon tyrosine phosphorylation of phosphatidylinositol 3 kinase (PI3-kinase) and Ca2+ influx via voltage-dependent Ca2+ channels (VDCC). In this study, verapamil, a VDCC inhibitor, was shown to markedly inhibit acidic pH-induced contraction, whereas the residual contraction in the presence of verapamil was unaffected by the PI3-kinase inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY-294002). Interestingly, the LY-294002-insensitive component of contraction was further inhibited by verapamil in the presence of LY-294002. Western blotting revealed that acidosis stimulated tyrosine phosphorylation of p85, which was abolished when tissues were pretreated with tyrphostin 23, a tyrosine kinase inhibitor, verapamil or EGTA. In fura-2-loaded aortic strips, acidosis induced a rise in intracellular Ca2+ ([Ca2+]i) that was partially inhibited by LY-294002. The residual increase in [Ca2+]i caused by acidosis in the presence of LY-294002 was abolished by verapamil. These findings suggest that acidosis-induced Ca2+ influx through VDCC is the upstream event leading to the tyrosine phosphorylation of PI3-kinase, which in turn contributes to the enhancement of Ca2+ entry to some extent in SHR aorta.     

Inhibition in L-type Ca2+ channel by stimulation of protein kinase C in isolated guinea pig ventricular cardiomyocytes.

1. Electrophysiological effects of phorbol esters on the L-type Ca2+ current (ICa(L)) in isolated single ventricular cells from guinea pig hearts were investigated. 2. In whole-cell voltage-clamped myocytes, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) at 10(-7) M inhibited ICa(L). An antagonist of protein kinase C (PK-C), H-7, at 10(-5) M did not modify the TPA-induced inhibition. The time-course of inactivation process for ICa(L) was greatly slowed. 3. In cell-attached patch-clamp experiments, TPA (10(-7) M) also markedly decreased the opening of L-type Ca2+ channels. The conductance was unaffected. 4. Even H-7 (10(-5) M) alone inhibited the opening of the channels. Addition of TPA (10(-7)-10(-8) M) caused further decrease in the opening. 5. On the other hand, 4-alpha-phorbol-12,13-didecanoate (not a PK-C activator) had no effect on the Ca2+ channels. 6. These results indicate that the PK-C activation induced by TPA greatly depresses the opening of L-type Ca2+ channels in ventricular cell membranes.     

Regulation of brain capillary endothelial cells by P2Y receptors coupled to Ca2+, phospholipase C and mitogen-activated protein kinase

1. The blood-brain barrier is formed by capillary endothelial cells and is regulated by cell-surface receptors, such as the G protein-coupled P2Y receptors for nucleotides. Here we investigated some of the characteristics of control of brain endothelial cells by these receptors, characterizing the phospholipase C and Ca²⁺ response and investigating the possible involvement of mitogen-activated protein kinases (MAPK).
2. Using an unpassaged primary culture of rat brain capillary endothelial cells we showed that ATP, UTP and 2-methylthio ATP (2MeSATP) give similar and substantial increases in cytosolic Ca²⁺, with a rapid rise to peak followed by a slower decline towards basal or to a sustained plateau. Removal of extracellular Ca²⁺ had little effect on the peak Ca²⁺-response, but resulted in a more rapid decline to basal. There was no response to α,β-MethylATP (α,βMeATP) in these unpassaged cells, but a response to this P2X agonist was seen after a single passage.
3. ATP (log EC₅₀ −5.1±0.2) also caused an increase in the total [³H]-inositol (poly)phosphates ([³H]-InsP_x) in the presence of lithium with a rank order of agonist potency of ATP=UTP=UDP>ADP, with 2MeSATP and α,βMeATP giving no detectable response.
4. Stimulating the cells with ATP or UTP gave a rapid rise in the level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃), with a peak at 10 s followed by a decline to a sustained plateau phase. 2MeSATP gave no detectable increase in the level of Ins(1,4,5)P₃.
5. None of the nucleotides tested affected basal cyclic AMP, while ATP and ATPγS, but not 2MeSATP, stimulated cyclic AMP levels in the presence of 5 μM forskolin.
6. Both UTP and ATP stimulated tyrosine phosphorylation of p42 and p44 mitogen-activated protein kinase (MAPK), while 2MeSATP gave a smaller increase in this index of MAPK activation. By use of a peptide kinase assay, UTP gave a substantial increase in MAPK activity with a concentration-dependency consistent with activation at P2Y₂ receptors. 2MeSATP gave a much smaller response with a lower potency than UTP.
7. These results are consistent with brain endothelial regulation by P2Y₂ receptors coupled to phospholipase C, Ca²⁺ and MAPK; and by P2Y₁-like (2MeSATP-sensitive) receptors which are linked to Ca²⁺ mobilization by a mechanism apparently independent of agonist stimulated Ins (1,4,5)P₃ levels. A further response to ATP, acting at an undefined receptor, caused an increase in cyclic AMP levels in the presence of forskolin. The differential MAPK coupling of these receptors suggests that they exert fundamentally distinct influences over brain endothelial function.

     

Characterization of Ca2+ influx through recombinant P2X receptor in C6BU-1 cells

1. The effects of exogenous adenosine 5′-triphosphate (ATP) and α,β-methylene ATP (α,βmeATP) on C6BU-1 cells transfected with P2X₂ and P2X₃ subtypes, separately or together (P2X₂₊₃), were investigated using fura-2 fluorescence recording and whole-cell patch clamp recording methods.
2. Untransfected C6BU-1 cells showed no intracellular Ca²⁺ ([Ca²⁺]_i) increase in response to depolarizing stimulation with high K⁺ or stimulation with ATP. There was no current induced by ATP under voltage clamp conditions in untransfected C6BU-1 cells. ATP caused Ca²⁺ influx only from extracellular sources in C6BU-1 cells transfected with the P2X subtypes, suggesting that the C6BU-1 cell line is suitable for the characterization of Ca²⁺ influx through the P2X subtypes.
3. In C6BU-1 cells transfected with the P2X₂ subtype, ATP (more than 10 μM) but not α,βmeATP (up to 100 μM) evoked a rise in [Ca²⁺]_i.
4. In the cells transfected with the P2X₃ subtype, current responses under voltage clamp conditions were observed at ATP concentrations higher than 0.1 μM of α,βmeATP were required. This discrepancy in the concentration dependence of the agonist responses with respect to the [Ca²⁺]_i rise and the current response was seen only with the P2X₃ subtype. In addition, the agonist-induced rise in [Ca²⁺]_i was observed only after the first application because of desensitization of this subtype.
5. In C6BU-1 cells co-transfected with P2X₂ and P2X₃, ATP at 1 μM evoked a [Ca²⁺]_i rise. This responsiveness was higher than that of the other subtype combinations tested. The efficiency of expression was improved by co-transfection with P2X₂ and P2X₃, when compared to transfection with the P2X₃ subtype alone. The desensitization of the P2X₂₊₃ was apparently slower than that of the P2X₃ subtype alone. Therefore, this combination could respond to the repeated application of agonists each time with a [Ca²⁺]_i rise.
6. These results suggest that the P2X₂ and P2X₃ subtypes assemble a heteromultimer and that this heterogeneous expression acquires more effective Ca²⁺ dynamics than that by homogenously expressed P2X₂ or P2X₃

     

Quercetin as a novel activator of L-type Ca(2+) channels in rat tail artery smooth muscle cells

1. The aim of this study was to investigate the effects of quercetin, a natural polyphenolic flavonoid, on voltage-dependent Ca(2+) channels of smooth muscle cells freshly isolated from the rat tail artery, using either the conventional or the amphotericin B-perforated whole-cell patch-clamp method. 2. Quercetin increased L-type Ca(2+) current [I(Ca(L))] in a concentration- (pEC(50)=5.09+/-0.05) and voltage-dependent manner and shifted the maximum of the current-voltage relationship by 10 mV in the hyperpolarizing direction, without, however, modifying the threshold and the equilibrium potential for Ca(2+). 3. Quercetin-induced I(Ca(L)) stimulation was reversible upon wash-out. T-type Ca(2+) current was not affected by quercetin. Quercetin shifted the voltage dependence of the steady-state inactivation and activation curves to more negative potentials by about 5.5 and 7.5 mV respectively, in the mid-potential of the curves as well as increasing the slope of activation. Quercetin slowed both the activation and the deactivation kinetics of the I(Ca(L)). The inactivation time course was also slowed but only at voltages higher than 10 mV. Moreover quercetin slowed the rate of recovery from inactivation. 4. These results prove quercetin to be a naturally-occurring L-type Ca(2+) channel activator.     

Inhibition of voltage-gated L-type calcium channels by labedipinedilol-A involves protein kinase C in rat cerebrovascular smooth muscle cells

Labedipinedilol-A, a novel calcium channel blocker with α/β-adrenoceptor blockade properties, inhibits L-type calcium channels (LTCCs) in rat cerebrovascular smooth muscle cells (CSMCs). We used conventional whole cell patch-clamp electrophysiology to investigate Ba²⁺ currents (I_Ba) through LTCCs in rat CSMCs enzymatically dissociated from rat cerebral arteries. Labedipinedilol-A (1, 10 µM) reversibly inhibited I_Ba in a voltage-dependent manner without modifying the I_Ba current–voltage relationship. The I_Ba was also abolished by the LTCC blocker nifedipine (1 µM), but enhanced by the LTCC activator Bay K8644 (100 nM). Labedipinedilol-A shifted the steady-state inactivation curve of I_Ba to more negative potentials. Additionally, labedipinedilol-A had greater inhibitory activity on I_Ba holding at − 40 mV than at − 80 mV. This might contribute to labedipinedilol-A's more selective effect on vascular muscles compared to cardiac muscles. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and norepinephrine-enhanced I_Ba were also inhibited by labedipinedilol-A. Pretreatment with the PKC inhibitor chelerythrine (5 µM) attenuated labedipinedilol-A-mediated I_Ba inhibition. However, the Rho kinase inhibitor Y-27632 (30 µM) had little effect on labedipinedilol-A inhibition of I_Ba. Labedipinedilol-A inhibition of voltage-dependent LTCCs may be, at least in part, due to its modulation of the PKC pathway.