Effects of adrenocorticotropin on action potential and calcium currents in cultured rat and bovine glomerulosa cells.

Action potentials (APs) and ionic currents were recorded in primary cultured rat and bovine glomerulosa cells by using the whole-cell recording technique. Switching from the current-clamp mode to the voltage-clamp mode allowed recordings of APs and currents in the same cell. APs can be elicited by appropriate stimulation in conditions where the excitability of the cell is increased by blocking a transient outward current. A T-current or a N-current was always present in cells in which APs were recorded; an L-current could also be recorded, but a cell presenting only an L-current was not able to fire an AP. The addition of Bay K 8644 (10(-8) M) induced a dramatic increase in the action potential duration. In the same cells, the analysis of the currents showed that the L-current was increased, whereas the T-current was not significantly affected. The effects of ACTH (10(-8) M) were analysed on APs and currents. On APs, at least two phases could be distinguished, the first corresponded to the reduction of the action potential duration, whereas the second was a huge increase of the plateau duration. The T-current was strongly affected by ACTH as a great inhibition took place in the first seconds after the superfusion with a 10(-8) M ACTH medium. Then a partial recovery of the T-current appeared. The effects of ACTh were reversible on washing. On the contrary, the L-current was increased by ACTH, but this effect was not reversible. The effects of ACTH were mimicked by 8 Bromo cAMP (10(-3) M). Similar results were found in rat and bovine glomerulosa cells. These results suggest that second messengers generated by ACTH would regulate Ca2+ entrance by nondetermined phosphorylation process in the sense of an increase in intracellular Ca2+.     

Ca channels in adrenal glomerulosa cells: K+ and angiotensin II increase T-type Ca channel current.

Ca channel currents were studied in freshly dispersed bovine adrenal glomerulosa cells to better understand the control of aldosterone secretion by extracellular K concentration (Ko) and angiotensin II (AII). The whole-cell variation of the patch voltage clamp technique was used. Two types of Ca channels were found. One type is similar to the "T-type" Ca channels found in many excitable cells. These channels deactivate slowly (tau approximately equal to 7 ms at -75 mV) and inactivate rapidly during strong depolarizations. The second channel type activates and inactivates at more positive potentials than the T-type Ca channels and deactivates rapidly. These channels are similar to the "L-type" Ca channels found in muscle and nerve. Our studies provide three reasons for concluding that T-type Ca channels have an important role in mediating stimulus-secretion coupling in response to high K+ or AII: (i) aldosterone secretion and steady-state current through T-type Ca channels are biphasic functions of Ko and both increase in parallel for Ko = 2-10 mM; (ii) nitrendipine blocks the T-type Ca channels and the stimulation of aldosterone secretion by high K+ or AII with similar potency; (iii) AII increases Ca entry through the T-type Ca channels.     

Excitation-secretion coupling: involvement of potassium channels in ACTH-stimulated rat adrenocortical cells

The mechanism by which ACTH stimulates calcium influx and steroid secretion was studied using rat adrenal glomerulosa cells, which were either freshly isolated or maintained in primary culture for 3 days. The potassium channel blocker tetraethylammonium chloride (TEA) stimulated twofold both corticosterone and aldosterone secretion; this stimulation was lower than that induced by ACTH at low concentrations (10 pmol/l). However, TEA and ACTH induced similar increases in Ca2+ influx and inositol phosphate accumulation. The three responses (steroid secretion, calcium influx and inositol phosphate accumulation) induced by TEA or low concentrations of ACTH were blocked by CoCl2. The greater stimulatory effect on steroid secretion of 10 nmol ACTH/l was decreased but not blocked by CoCl2. These data further document the complex mechanism of action of ACTH. It is postulated that, at low concentrations, ACTH binds preferentially to the high-affinity site of its receptor, leading to calcium influx by depolarization of the membrane potential, and to steroid secretion predominantly through an inositol phosphate- and Ca2+-stimulated pathway and also a cyclic AMP pathway. At higher concentrations, the hormone also binds to the low-affinity site of its receptor, largely stimulating cyclic AMP production and further increasing steroid secretion.     

Effects of Ca^2＋ channel blockers on store-operated Ca^2＋ channel currents of Kupffer cells after hepatic ischemia/reperfusion injury in rats

AIM: To study the effects of hepatic ischemia/reperfusion (I/R) injury on store-operated calcium channel (SOC) currents (I(SOC)) in freshly isolated rat Kupffer cells, and the effects of Ca(2+) channel blockers, 2-aminoethoxydiphenyl borate (2-APB), SK and F96365, econazole and miconazole, on I(SOC) in isolated rat Kupffer cells after hepatic I/R injury. METHODS: The model of rat hepatic I/R injury was established. Whole-cell patch-clamp techniques were performed to investigate the effects of 2-APB, SK and F96365, econazole and miconazole on I(SOC) in isolated rat Kupffer cells after hepatic I /R injury. RESULTS: I/R injury significantly increased I(SOC) from -80.4 +/- 25.2pA to -159.5 +/- 34.5pA ((b)P < 0.01, n = 30). 2-APB (20, 40, 60, 80, 100 micromol/L), SK and F96365 (5, 10, 20, 40, 50 micromol/L), econazole (0.1, 0.3, 1, 3, 10 micromol/L) and miconazole (0.1, 0.3, 1, 3, 10 micromol/L) inhibited I(SOC) in a concentration-dependent manner with IC50 of 37.41 micromol/L (n = 8), 5.89 micromol/L (n = 11), 0.21 micromol/L (n = 13), and 0.28 micromol/L (n = 10). The peak value of I(SOC) in the I-V relationship was decreased by the blockers in different concentrations, but the reverse potential of I(SOC) was not transformed. CONCLUSION: SOC is the main channel for the influx of Ca(2+) during hepatic I/R injuries. Calcium channel blockers, 2-APB, SK and F96365, econazole and miconazole, have obviously protective effects on I/R injury, probably by inhibiting I(SOC) in Kupffer cells and preventing the activation of Kupffer cells.     

Early down-regulation of K+ channel genes and currents in the postinfarction heart

INTRODUCTION: Down-regulation of key K+ channel subunit gene expression and K+ currents is a universal response to cardiac hypertrophy, whatever the cause, including the postmyocardial infarction (post-MI) remodeled heart. METHODS AND RESULTS: We investigated the hypothesis that down-regulation of K+ channel genes and currents post-MI occurs early and before significant remodeled hypertrophy of the noninfarcted myocardium could be detected. We investigated (1) the incidence of induced ventricular tachyarrhythmias (VT) in 3-day post-MI rat heart; (2) action potential (AP) characteristics of isolated left ventricular (LV) myocytes from sham-operated and 3-day post-MI heart; (3) time course of changes in outward K+ currents Ito-fast(f) and I(K) in isolated myocytes from 3-day and 4-week post-MI noninfarcted LV and compared the changes with sham-operated animals; and (4) changes in the messenger and protein levels of Kv2.1, Kv4.2, and Kv4.3 in the LV and right ventricle of 3-day post-MI heart. Sustained VT was induced in 6 of 10 3-day post-MI rats and in none of 8 sham rats. The membrane capacitance of myocytes isolated from 3-day post-MI noninfarcted LV was not significantly different from control, whereas membrane capacitance 4-week post-MI was significantly higher, reflecting the development of hypertrophy. AP duration was increased and the density of Ito-f and I(K) were significantly decreased in 3-day post-MI LV myocytes compared with sham. The reduced density of Ito did not significantly differ in 4-week post-MI LV myocytes, whereas the density of I(K) was decreased further at 4 weeks post-MI. The changes in Ito-f and I(K) correlated with decreased messenger and protein levels of Kv4.2/Kv4.3 and Kv2.1, respectively. CONCLUSION: These results support the hypothesis that down-regulation of K+ channel gene expression and current in the post-MI LV occurs early and may be dissociated from the slower time course of post-MI remodeled hypertrophy. These changes may contribute to early arrhythmogenesis of the post-MI heart.     

Effects of K+ channel blockers on vascular tone in the perfused rat lung

To learn more of the role of K+ channel activity in the regulation of pulmonary vascular tone, we compared the pressor effects of the differential blockers of numerous K+ channels, tetraethylammonium chloride and 4-aminopyridine, and the inhibitor of ATP-sensitive K+ channels glibenclamide in meclofenamate-treated salt solution-perfused rat lungs. Tetraethylammonium (1 to 20 mM) and 4-aminopyridine (1 to 10 mM), but not glibenclamide (1 to 20 microM) caused vasoconstriction in the normoxic lung. The Ca++ channel blocker nifedipine (0.1 microM) and the alpha adrenoceptor antagonist phentolamine (10 microM) inhibited the 4-aminopyridine response by about 50% and reduced slightly the smaller tetraethylammonium response. 4-Aminopyridine and, to a lesser extent, tetraethylammonium, but not glibenclamide, also potentiated peak vasoconstriction to angiotensin II and airway hypoxia. Nifedipine, but not phentolamine, inhibited hypoxic vasoconstriction and prevented the potentiation by 4-aminopyridine. These results suggest that Ca(++)- and/or voltage-activated (not ATP-sensitive) K+ channels may be important in maintaining low pulmonary vascular tone.     

Single K+ channel currents of anomalous rectification in cultured rat myotubes.

The currents through single K+ channels of the anomalous (or inward) rectifier were recorded in tissue cultured rat myotubes by using the "gigohm seal" patch clamp technique developed by Sigworth and Neher. These unitary currents were detected as current fluctuations due to the blocking and unblocking of channels by Ba2+. The single-channel conductance was obtained from the slope of the linear relationship between unitary current amplitude and membrane potential. When the external solution contained 155 mM K+, the single-channel conductance was 10.4 +/- 2.6 pS (+/- SD; n = 6). This value was independent of the the concentration of blocking ions but increased with increasing external K+ concentration. The behavior of the unitary current agreed with that expected from the blocking kinetics of Ba2+ on the macroscopic K+ current of the anomalous rectifier. The density of the channel is likely to be small and may even be less than 1/micrometers 2.     

Gating currents of the cloned delayed-rectifier K+ channel DRK1.

Gating currents of the cloned delayed-rectifier K+ channel DRK1 expressed in Xenopus oocytes were measured with the open-oocyte Vaseline-gap voltage-clamp technique. DRK1 gating charge had the following salient properties: (i) gating-charge amplitude correlated positively with size of the expressed ionic K+ currents; (ii) the time integral of ON and OFF gating currents was similar, indicating charge conservation and lack of charge immobilization; (iii) the gating-charge activation curve was shallower and had a half-activation potential 15 mV more negative than the activation curve for K+ conductance; (iv) effective valence for the gating current was about two electronic charges per gating subunit; (v) for large depolarizations (to > 0 mV) prominent rising phases were observed during the ON and OFF gating charge, which appeared as shoulders in unsubtracted traces; (vi) for small depolarizing pulses (to < 0 mV) ionic-current activation and deactivation had time constants similar to ON and OFF gating-current decay, respectively; (vii) negative prepulses made more prominent the ON rising phase and delayed ionic and gating currents. The results are consistent with a model for K+ channel activation that has an early slow and/or weakly voltage-dependent transition between early closed states followed by more voltage-dependent transitions between later closed states and a final voltage-independent closed-open transition.     

Role of cell volume in K+-induced Ca2+ signaling by rat adrenal glomerulosa cells

The involvement of cell volume in the K+-evoked Ca2+ signaling was studied in cultured rat glomerulosa cells. Previously we reported that hyposmosis (250 mOsm) increased the amplitude of T-type Ca2+ current and, accordingly, enhanced the Ca2+ response of cultured rat glomerulosa cells to K+. In the present study we found that this enhancement is not influenced by the cytoskeleton-disrupting drugs cytochalasin-D (20 microM) and colchicine (100 microM). Elevation of extracellular potassium concentration ([K+]e) from 3.6 to 4.6-8.6 mM induced cell swelling, which had slower kinetics than the Ca2+ signal. Cytoplasmic Ca2+ signal measured in single glomerulosa cells in response to stimulation with 5 mm K+ for 2 min showed two phases: after a rapid rise reaching a plateau within 20-30 sec, [Ca2+]c increased further slowly by approximately one third. When 5 mM K+ was coapplied with elevation of extracellular osmolarity from 290 to 320 mOsm, the second phase was prevented. These results indicate that cell swelling evoked by physiological elevation of [K+]e may contribute to the generation of sustained Ca2+ signals by enhancing voltage-activated Ca2+ influx.     

Antiatherosclerotic effects of calcium channel blockers

Over the past 30 years, a considerable body of experimental and clinical evidence has accumulated to support the suggestion that calcium channel blockers (CCBs) have significant antiatherosclerotic effects that are independent of their hypotensive effects. Early research using animal models of atherosclerosis and CCBs in concentrations that exceeded the normal therapeutic dose range showed definite antiatherosclerotic effects, especially in the development of new lesions. Investigations of these effects in humans have used quantitative coronary angiography and B-mode ultrasonography and have demonstrated some antiatherosclerotic effects. This article reviews the currently available evidence of antiatherosclerotic effects of CCBs in animal models and in clinical trials.     

A Ras-dependent chloride current activated by adrenocorticotropin in rat adrenal zona glomerulosa cells

In the present study, we report that ACTH induces a transient chloride current. The lack of correlation between ACTH-induced cAMP production and amplitude of the Cl- current, as well as the absence of stimulation by forskolin or 8Br-cAMP indicated that the ACTH-induced current was not cAMP-dependent. We explored the possibility that one or several elements of the Ras/Raf MAPK cascade were involved. Indeed, we found that ACTH at 10(-10) M induced activation of Ras. Inhibition of the current by QEHA peptide, a Gbetagamma sequestrant, demonstrated that Gbetagamma subunits transduced the message. Blockage of the Ras activation using an inhibitor of farnesyl transferase (BZA-5B) or the monoclonal antibody H-Ras(259) abrogated the current. Moreover, the addition of Ras-GTPyS in the pipette medium gave rise to the Cl- current. Treatment of the cells with BZA decreased the aldosterone secretion induced by 10(-10) M ACTH but not that induced by 10(-8) M ACTH, confirming the involvement of Ras in steroid secretion. We conclude that ACTH triggers a Cl- current through the activation of the Ras protein by Gbetagamma subunits. This current, activated at physiological ACTH concentrations (1 to 100 pM) where cAMP production is very low, could play a significant role in aldosterone production.     

Inhibitory effects of endothelial cells and calcium channel blockers on platelet aggregation

This study examined the effects of human umbilical vein endothelial cells (ECs) and calcium channel blockers (Ca2+ blockers), diltiazem, verapamil, and nicardipine, on platelet aggregation in vitro. ECs markedly inhibited the platelet aggregation induced by ADP, collagen, thromboxane A2 (TXA2), or thrombin. When platelets were incubated with ECs, the antiaggregatory activity reached a plateau within 5 to 10 min. As the number of ECs added to the platelet-rich plasma was increased, platelet aggregation declined progressively. The antiaggregatory activity of ECs was attenuated considerably by the addition of aspirin. All three Ca2+ blockers inhibited platelet aggregation in a dose-dependent manner. The combination of ECs and a Ca2+ blocker resulted in more potent inhibition of platelet aggregation than either alone, but the effects were not synergistic. Both ECs and Ca2+ blockers inhibited the synthesis of TXA2 during platelet aggregation. However, Ca2+ blockers did not significantly influence the production of prostaglandin I2 (PGI2) by ECs during incubation and aggregation. These results suggest that PGI2 is an important factor in endothelial antiaggregatory activity. Ca2+ blockers directly inhibit platelet aggregation by suppressing TXA2 formation, but do not appear to enhance the antiaggregatory activity of ECs.     

钙激活性钾通道是人小肠上皮细胞的容积调节性通道(英文)

目的 :研究细胞容积调节机制 ,探讨人类小肠上皮细胞调节性容积减小 （RVD）过程中离子通道的作用及其种类。方法 :膜片钳全细胞记录和单通道记录法记录培养的人类小肠上皮细胞 RVD过程中电流的变化 ,细胞容积测定法观察 RVD过程中特异性钙激活性钾通道阻断剂 （clotrimazole）的作用。结果 :全细胞记录法证实细胞 RVD过程中 K+通道和 Cl-通道电流同时被激活 ,该 K+通道电流具有明显的钙依赖性并可被 clotrim azole阻断。单通道记录法进一步证实 RVD过程中激活的 K+通道为 Interm ediate- conductance钙激活性钾通道。结论 :人类小肠上皮细胞在低渗溶液作用下具有 RVD过程 ,钙激活性钾通道在 RVD过程中具有十分重要的作用。     

Renal effects of calcium channel blockers in vivo

Daily output of urine was measured in three groups of five rabbits each, before and during seven days of oral therapy with nifedipine, verapamil and diltiazem. All the drugs caused significant (P less than 0.001) increases in mean daily output. All the drugs decreased levels of serum sodium but serum potassium levels were lowered only by diltiazem. Inhibition of tubular transport appears to be the important mechanism.     

Central sympathoinhibitory effects of calcium channel blockers

It is generally assumed that the arterial vasodilation induced by inhibition of Ca²⁺ influx into vascular smooth muscle cells represents the main mechanism for the hypotensive effect of dihydropyridine calcium channel blockers. Increases in sympathetic tone have been related to activation of the arterial baroreflex by rapid lowering of blood pressure. This review highlights new findings in two areas. First, in animal studies, direct central administration of dihydropyridines such as nifedipine or amlodipine lowers sympathetic nerve activity and thereby blood pressure. Peripheral administration of nifedipine or amlodipine at low rates appears to result in gradual accumulation of drug in the central nervous system, and also causes lowering of sympathetic nerve activity and thereby lowering of blood pressure (rather than by arterial vasodilation). Second, in hypertensive humans treated with long-acting dihydropyridines and presumably little activation of the arterial baroreflex, some studies have demonstrated lowering of sympathetic activity (as assessed by plasma norepinephrine), but others reported increases (as assessed by plasma norepinephrine or microneurography). This sympathoexcitatory response may be due to activation of the reninangiotensin system, particularly at higher doses.     

Thyrotropin-releasing hormone induces opposite effects on Ca2+ channel currents in pituitary cells by two pathways.

Thyrotropin-releasing hormone (TRH) stimulates pituitary secretion by steps involving a cytosolic Ca2+ rise. We examined various pathways of Ca2+ elevation in pituitary GH3 cells. By using the patch clamp technique in the whole-cell configuration and Ba2+ as divalent charge carrier through Ca2+ channels, TRH (1 microM) reversibly reduced the current by about 55%. This hormonal effect was prevented by infusing guanine 5'-[beta-thio]diphosphate (GDP[beta S]) intracellularly but not by pretreating the cell with pertussis toxin (PT). Since PT-insensitive guanine nucleotide-binding regulatory (G) proteins are known to mediate a hormone-stimulated inositol trisphosphate-mediated Ca2+ release from intracellular stores, we assume that the inhibitory effect of TRH on Ba2+ currents through Ca2+ channels is caused by the increased intracellular Ca2+. To prevent a Ca(2+)-release-dependent inhibition of Ca2+ channels, we preincubated GH3 cells in a medium free of divalent charge carriers and measured the Na+ current through Ca2+ channels. When fura-2 was used as indicator for the cytosolic Ca2+, TRH induced a release from intracellular stores only once and had no effect on the intracellular Ca2+ concentration during further applications. In line with this observation, TRH initially reduced the Na+ current through Ca2+ channels but stimulated it during subsequent applications. The stimulation was sensitive to GDP[beta S] and was abolished by pretreatment with PT, suggesting that the stimulatory action of TRH is mediated by a G protein different from the one that functionally couples the receptor to phosphatidylinositol 4,5-bisphosphate hydrolysis. In conclusion, the present data suggest that TRH increases the intracellular Ca2+ concentration by two interacting pathways, that release from intracellular stores causes a secondary blockage of Ca2+ channels, and that, especially with empty intracellular Ca2+ stores, Ca2+ channels are stimulated by a PT-sensitive G protein.     

Comparison of K+ currents in cardiac Purkinje cells isolated from rabbit and dog

The repolarization reserve determines the ability of drugs to prolong the cardiac action potential duration. Differences in K(+) currents between rabbit and dog cardiac Purkinje cells were studied by recording the transient outward K(+) current (I(to)) as well as the delayed rectifier K(+) currents (I(Ks) and I(Kr)) during repolarization. Purkinje fibers were dissected from dog and rabbit hearts and exposed to enzymatic digestion until isolated cells were obtained. Whole cell voltage clamp methods were used to measure K(+) currents in both cell types. Action potential (AP) recordings from Purkinje cells displayed a rapid phase 1 repolarization due to a prominent I(to) with densities of 13.3+/-2.3 and 9.6+/-0.6 pA/pF at +40 mV in dog and rabbit respectively. I(Ks) tail currents were significantly larger in dog Purkinje cells. I(Kr) tail current densities were comparable in Purkinje cell from both species. Rabbit ventricular and Purkinje cell AP waveforms were used for action potential clamp experiments in TSA201 cells expressing human ether a go-go related gene (HERG). HERG currents elicited by the ventricular waveform reached its maximum amplitude during phase 3 repolarization. In contrast, Purkinje cell AP waveform elicited markedly smaller HERG currents even though the action potential duration was longer. The observations suggest that the fast phase 1 and negative plateau of the Purkinje cell AP limits the contribution of I(Kr) to repolarization. These results provide evidence that rabbit Purkinje cells have a smaller repolarization reserve and provide a biophysical explanation for a previously observed higher sensitivity to QT prolonging drugs in rabbit than dog Purkinje fibers.     

K+ currents of glomus cells and chemosensory functions of carotid body.

The mechanism by which the carotid body senses hypoxia is not resolved, but the glomus cell, a secretory cell apposed to the afferent nerve endings, is believed to play an essential role. It is proposed that hypoxia causes glomus cell depolarization, leading to activation of voltage-gated calcium influx and enhanced secretion of an excitatory transmitter. The initial step, hypoxia induced depolarization, may be mediated by several candidate K+ channels which are sensitive to hypoxia, including: (1) a transient, voltage-dependent current; (2) a calcium and voltage dependent current; and (3) a non-voltage dependent, leak K+ current. If these channels represent the initial step in the hypoxia transduction cascade then it would be expected that K+ channel blocking agents would mimic the hypoxia response, leading to glomus cell secretion and increased nerve activity. This has been tested for the first two channels which are sensitive to classical K+ channel blocking agents, and, in general, results have not borne out this prediction. At present, the pharmacology of the leak K+ channel is not determined, and the experiment has not been undertaken. Thus, at present, hypoxic inhibition to a K+ channel in the glomus cell may initiate chemotransduction but there are many unanswered questions, especially the failure of K+ channel blocking agents to emulate the hypoxic response.