Mechanism of apical K+ channel modulation in principal renal tubule cells. Effect of inhibition of basolateral Na(+)-K(+)-ATPase

The effects of inhibition of the basolateral Na(+)-K(+)-ATPase (pump) on the apical low-conductance K+ channel of principal cells in rat cortical collecting duct (CCD) were studied with patch-clamp techniques. Inhibition of pump activity by removal of K+ from the bath solution or addition of strophanthidin reversibly reduced K+ channel activity in cell-attached patches to 36% of the control value. The effect of pump inhibition on K+ channel activity was dependent on the presence of extracellular Ca2+, since removal of Ca2+ in the bath solution abolished the inhibitory effect of 0 mM K+ bath. The intracellular [Ca2+] (measured with fura-2) was significantly increased, from 125 nM (control) to 335 nM (0 mM K+ bath) or 408 nM (0.2 mM strophanthidin), during inhibition of pump activity. In contrast, cell pH decreased only moderately, from 7.45 to 7.35. Raising intracellular Ca2+ by addition of 2 microM ionomycin mimicked the effect of pump inhibition on K+ channel activity. 0.1 mM amiloride also significantly reduced the inhibitory effect of the K+ removal. Because the apical low-conductance K channel in inside-out patches is not sensitive to Ca2+ (Wang, W., A. Schwab, and G. Giebisch, 1990. American Journal of Physiology. 259:F494-F502), it is suggested that the inhibitory effect of Ca2+ is mediated by a Ca(2+)-dependent signal transduction pathway. This view was supported in experiments in which application of 200 nM staurosporine, a potent inhibitor of Ca(2+)- dependent protein kinase C (PKC), markedly diminished the effect of the pump inhibition on channel activity. We conclude that a Ca(2+)- dependent protein kinase such as PKC plays a key role in the downregulation of apical low-conductance K+ channel activity during inhibition of the basolateral Na(+)-K(+)-ATPase.     

Relationship between extra and intracellular sources of calcium and the contractile effect of thiopental in rat aorta

To evaluate the relationship between the vasocontractile effect of thiopental and the extra and intracellular sources of Ca2+, we analyzed both the contractile effect of the barbiturate on rat aortic rings and its ability to modify the intracellular calcium concentration in cultured rat aorta smooth muscle cells. Thiopental (10-310 microg/mL) contracted aortic rings only in the presence of extracellular Ca2+, and this effect was not blocked by verapamil or diltiazem. On the contrary, Ca2+ (0.1-3.1 mM) evoked contractions only when thiopental (100 microg/mL) was present. Although in calcium-free solution thiopental (100 microg/mL) did not contract aortic rings, it abolished the contractile effect of either phenylephrine (10(-6) M) or caffeine (10 mM). Finally, thiopental augmented the intracellular calcium concentration in cultured smooth muscle cells incubated either in the presence or absence of calcium. In conclusion, thiopental's vasocontractile effect depends on extracellular calcium influx, which is independent of L-calcium channels. The increase in intracellular Ca2+ concentration elicited by thiopental in Ca2+-free solution and its ability to block the effect of phenylephrine and caffeine suggest that this barbiturate can deplete intracellular pools of calcium. Therefore, the calcium entry pathway associated with the contractile effect of thiopental may correspond to the capacitative calcium entry model.     

Effects of Cl- channel blockers on endothelin-1-induced proliferation of rat vascular smooth muscle cells

The effects of Cl- channel blockers on endothelin-1 (ET-1)-induced proliferation of rat aortic vascular smooth muscle cells (VSMC) were examined. We found ET-1 concentration-dependently increased cell count and [3H]-thymidine incorporation into VSMC, with EC50 values of 24.8 and 11.4 nM, respectively. Both nifedipine and SK&F96365 inhibited 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC with the maximal inhibitory concentrations of 1 and 10 microM, respectively. DIDS inhibited 10 nM ET-1-induced increase in cell count and [3H]-thymidine incorporation into VSMC in a concentration-dependent manner, whereas other Cl- channel blockers including IAA-94, NPPB, DPC, SITS and furosemide did not produce these effects. 3 microM DIDS reduced 10 nM ET-1-induced sustained increase in cytoplasmic Ca2+ concentration ([Ca2+]) by 52%. Pretreatment of VSMC with 1 microM nifedipine completely inhibited the DIDS effect on 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC and sustained increase in [Ca2+]i, whereas pretreatment with 10 microM SK&F96365 did not completely block these effects of DIDS. DIDS did not affect ET-1-induced Ca2+ release and 30 mM KCl-induced increase in [Ca2+]i. Our data suggest that DIDS-sensitive Cl- channels mediate VSMC proliferation induced by ET-1 by mechanisms related to membrane depolarization and Ca2+ influx through voltage-dependent Ca2+ channels.     

Influence of T-type Ca2+ (mibefradil) and Cl- (indanyloxyacetic acid 94) channel antagonists on alpha1-adrenoceptor mediated contractions in rat aorta

The effects of the T-type and L-type Ca2+ channel antagonists, mibefradil and nifedipine, respectively, and those of a Cl- channel antagonist, indanyloxyacetic acid 94, on mechanical responses elicited by selective activation of alpha1-adrenoceptors using cirazoline were examined in rat isolated aortic rings. The presence of mibefradil (300 nM), indanyloxyacetic acid, 94 (30 microM) and nifedipine (300 nM) alone inhibited mechanical responses elicited by cirazoline. The concentration-response curves to cirazoline were displaced to the right with significant increases in the EC50 and significant depressions of the maximal responses in the presence of the individual agents mibefradil, indanyloxyacetic acid 94, or nifedipine. A combination of mibefradil and indanyloxyacetic acid 94 further inhibited the mechanical activity produced by cirazoline. The further reduction in the maximal response to cirazoline, in the presence of mibefradil and nifedipine, was insignificant when compared with the effects of nifedipine alone. In addition, maximal mechanical responses produced by cirazoline were not significantly affected by a combination of nifedipine and indanyloxyacetic acid 94 when compared with either nifedipine alone or mibefradil and indanyloxyacetic acid 94 combined. Our current findings indicate that mibefradil, indanyloxyacetic acid 94, and nifedipine can inhibit cirazoline-induced contractions to a varying degree. Moreover, based on our present data it would be reasonable to suggest that the contribution of T-type versus L-type Ca2+ channels to contractile responses obtained with cirazoline are approximately 21% and 35%, respectively, of the Emax. It would appear that L-type Ca2+ channels play a greater role in processes that are involved in excitation-contraction coupling subsequent to stimulation of alpha1-adrenoceptors. In addition, Cl- channels also appear to be involved in the process of contraction following alpha1-adrenoceptor activation.     

Mechanisms of the vasorelaxant effect of 1, 5-dihydroxy-2, 3-dimethoxy-xanthone, an active metabolite of 1-hydroxy-2, 3, 5-trimethoxy-xanthone isolated from a Tibetan herb, Halenia elliptica, on rat coronary artery

1, 5-Dihydroxy-2, 3-dimethoxy-xanthone (HM-5) is one of the naturally-occurring xanthones of a Tibetan medicinal herb Halenia elliptica. Recently, it has been shown that HM-5 is one of the phase I metabolites of 1-hydroxy-2, 3, 5-trimethoxy-xanthone (HM-1), the major active component of H. elliptica with potent vasorelaxant actions. This study investigated the vasorelaxant effect of HM-5 and its mechanism(s). HM-5 (0.35-21.9 microM) produced a concentration-dependent relaxation in rat coronary artery rings pre-contracted with 1 microM 5-hydroxytryptamine (5-HT), with an EC(50) of 4.40+/-1.08 microM. Unlike HM-1, the effect of HM-5 was endothelial-independent such that removal of the endothelium did not affect its vasodilator potency. Nitric oxide synthase (NOS) inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME, 100 microM), the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-alpha] quinoxalin-1-one (ODQ, 10 microM) did not affect the vasodilatory effects of HM-5, thus confirming the non-involvement of endothelium related mechanisms. In endothelium-denuded coronary artery rings, the vasorelaxant effect of HM-5 was inhibited by a potassium channel blocker, TEA (10 mM), and 4-aminopyridine (4-AP, a K(v) blocker; 1 mM) but not by other K+ channel blockers such as iberiotoxin (100 nM), barium chloride (100 microM) and glibenclamide (10 microM). The involvement of Ca2+ channel was studied in artery rings pre-incubated with Ca2+-free buffer (intact endothelium or endothelium-denuded) and primed with 1 microM 5-HT or 60 mM KCl prior to the addition of CaCl2 to elicit contraction. In the 5-HT-primed preparations, HM-5 (34.7 microM) significantly inhibited the CaCl(2)-induced vasoconstriction (89.9% inhibition in intact endothelium artery rings; 83.3% inhibition in endothelium-denuded rings). In the KCl-primed preparations, HM-5 (34.7 microM) produced a 34% inhibition in endothelium-denuded rings. The same concentration of HM-5 inhibited (by 62.3%) the contractile response to 10 microM phorbol 12, 13-diacetate (PDA), a protein kinase C activator, in Ca2+-free solutions. Taken together, this study showed that the mechanisms of the vasorelaxant effects of HM-5 were distinctly different from those of its parent drug HM-1. The vasorelaxant effect of HM-5 was mediated through opening of potassium channel (4-AP) and altering intracellular calcium by partial inhibition of Ca2+ influx through L-type voltage-operated Ca2+ channels and intracellular Ca2+ stores.     

Calcium uptake studies of 1,4-dihydropyridine agonists into rabbit aortic smooth muscle cells in culture

The effects of the three dihydropyridine calcium channel agonists (+/-)BAY K 8644, (+)202-791 and (+/-)CGP 28392 on 45Ca++ uptake were studied in cultures of rabbit aortic smooth muscle cells. At 10(-7) M each agonist enhanced 45Ca++ uptake in 15-50 mM K+ but had no effect on the basal 45Ca++ uptake at 5 mM K+. At the uptake threshold of 15 mM K+ each agonist potentiated 45Ca++ uptake in a dose-dependent manner with half maximal effects at 2.4 nM for (+/-)BAY K 8644, 22 nM for (+)202-791 and 18 nM for (+/-)CGP 28392. The agonists showed no significant antagonistic activity. Responses were antagonized competitively by nifedipine and non-competitively by (+/-)D-600. The 45Ca++ uptake dose-response curves and the half maximal effects of the three agonists were over the same range of concentrations as their inhibition of [3H]nitrendipine binding to rat ventricular receptor membrane preparations. The data suggest that these cells mimic the calcium uptake by the intact aorta better than commercial vascular smooth muscle lines or cardiac cells.     

Vasorelaxant effect of the flavonoid galangin on isolated rat thoracic aorta

Here we investigated the effect of the flavonoid galangin in isolated rat thoracic aortic rings. Galangin (0.1-100 microM) induced relaxation in rings pre-contracted with phenylephrine (PE 1 microM) or with KCl (100 mM) or pre-treated with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 100 microM), the cyclooxygenase inhibitor indomethacin (10 microM) and the adenylate cyclase inhibitor, SQ 22,536 (100 microM). In another set of experiments, rat aortic rings were incubated with galangin (1-100 microM) and the contractile responses to PE (0.001-3 microM) or to KCl (60 mM) were evaluated. We also evaluated the effect of galangin (100 microM) on PE (10 microM)-induced contraction in a Ca2+-free medium. Galangin relaxed aortic rings with or without endothelium. Galangin effect was significantly inhibited by L-NAME. Galangin inhibited the contractile response to PE, either in presence or in absence of external calcium, and to KCl. In the end, we also found that galangin caused nitric oxide (NO) release from aortic rings and abolished the increase in [Ca2+]i triggered by PE or KCl in aortic smooth muscle cells, either in presence and in absence of external Ca2+. Our results suggest that galangin reduces the contractility of rat aortic rings through an endothelium-dependent mechanism, involving NO, and also through an endothelium-independent mechanism, inhibiting calcium movements through cell membranes.     

Ryanodine stabilizes multiple conformational states of the skeletal muscle calcium release channel.

Nanomolar to micromolar ryanodine alters the gating kinetics of the Ca2+ release channel from skeletal sarcoplasmic reticulum (SR) fused with bilayer lipid membranes (BLM). In the presence of asymmetric CsCl and 100 microM CaCl2 cis, ryanodine (RY) (5-40 nM) activates the channel, increasing the open probability (po; maximum 300% of control) without changing unitary conductance (468 picosiemens (pS)). Statistical analyses of gating kinetics reveal that open and closed dwell times exhibit biexponential distributions and are significantly modified by nanomolar RY. Altered channel gating kinetics with low nanomolar RY is fully reversible and correlates well with binding kinetics of nanomolar [3H]RY with its high affinity site (Kd1 = 0.7 nM) under identical experimental conditions. RY (20-50 nM) induces occasional 1/2 conductance fluctuations which correlate with [3H]RY binding to a second site having lower affinity (Kd2 = 23 nM). RY (5-50 nM) in the presence of 500 mM CsCl significantly enhances Ca(2+)-induced Ca2+ release from actively loaded SR vesicles. Ryanodine > or = 50 nM stabilizes the channel in a 234-pS subconductance which is not readily reversible. RY (> or = 70 microM) produces a unidirectional transition from the 1/2 to a 1/4 conductance fluctuation, whereas RY > or = 200 microM causes complete closure of the channel. The RY required for stabilizing 1/4 conductance transitions and channel closure do not quantitatively correlate with [3H]RY equilibrium binding constants and is attributed to significant reduction in association kinetics with > 200 nM [3H]RY in the presence of 500 mM CsCl. These results demonstrate that RY stabilizes four discrete states of the SR release channel and supports the existence of multiple interacting RY effector sites on the channel protein.     

Modulation of the epithelial calcium channel, ECaC, by intracellular Ca2+

We have studied the modulation by intracellular Ca2+ of the epithelial Ca2+ channel, ECaC, heterologously expressed in HEK 293 cells. Whole-cell and inside-out patch clamp current recordings were combined with FuraII-Ca2+ measurements:1. Currents through ECaC were dramatically inhibited if Ca2+ was the charge carrier. This inhibition was dependent on the extracellular Ca2+ concentration and occurred also in cells buffered intracellularly with 10 mM BAPTA.2. Application of 30 mM [Ca(2)]e induced in non-Ca2+] buffered HEK 293 cells at -80 m V an increase in intracellular Ca2+([Ca2]i) with a maximum rate of rise of 241 +/-15nM/s (n= 18 cells) and a peak value of 891 +/- 106 nM. The peak of the concomitant current with a density of 12.3 +/- 2.6 pA/pF was closely correlated with the peak of the first-time derivative of the Ca2+ transient, as expected if the Ca2+ transient is due to influx of Ca2+. Consequently, no Ca2+] signal was observed in cells transfected with the Ca2+ impermeable ECaC mutant, D542A, in which an aspartate in the pore region was neutralized.3. Increasing [Ca2+]i by dialyzing the cell with pipette solutions containing various Ca2+] concentrations, all buffered with 10 mM BAPTA, inhibited currents through ECaC carried by either Na+ or Ca2+] ions. Half maximal inhibition of Ca(2+)currents in the absence of monovalent cations occurred at 67 nM (n between 6 and 8), whereas Na+ currents in the absence of Ca2+] and Mg2+ were inhibited with an IC50 of 89 nM (n between 6 and 10). Currents through ECaC in the presence of 1 mM Ca2+ and Na+, which are mainly carried by Ca2+, are inhibited by [Ca2]i with an IC50of 82 nM (n between 6 and 8). Monovalent cation currents through the Ca2+impermeable D542A ECaC mutant were also inhibited by an elevation of [Ca2]i (IC50 = 123 nM, n between 7 and 18). 4. The sensitivity of ECaC currents in inside-out patches for [Ca2]i was slightly shifted to higher concentrations as compared with whole cell measurements. Half-maximal inhibition occurred at 169 nM if Na+ was the charge carrier (n between 4 and 11) and 228 nM at 1 mM [Ca2]e (n between 4 and 8).5. Recovery from inhibition upon washout of extracellular Ca2+ (whole-cell configuration) or removal of Ca2+ from the inner side of the channel (inside-out patches) was slow in both conditions. Half-maximal recovery was reached after 96 +/- 34 s (n= 15) in whole-cell mode and after 135 +/- 23 s (n = 17) in inside-out patches.6. We conclude that influx of Ca2+ through ECaC and [Ca2]i induce feedback inhibition of ECaC currents, which is controlled by the concentration of Ca2+ in a micro domain near the inner mouth of the channel. Slow recovery seems to depend on dissociation of Ca( 2+ from an internal Ca2+ binding site at ECaC.     

Inhibition of highly purified mammalian phospholipases A2 by non-steroidal anti-inflammatory agents. Modulation by calcium ions.

Highly purified Ca2+-dependent phospholipases A2 that were isolated from human platelets, rabbit alveolar macrophages and peritoneal polymorphonuclear leucocytes and were active in the neutral-to-alkaline pH range were inhibited 50% by 75 microM-indomethacin in the presence of 5.0 mM added Ca2+. Sodium meclofenamate and sodium flufenamate were also inhibitory; the sensitivity to inhibition was a function of Ca2+ concentration. The dose for 50% inhibition (ID50) with meclofenamate was 0.4 mM in the presence of 2.5 mM added Ca2+, but 50nM in the presence of 0.5 mM added Ca2+. Thus, inhibition of phospholipase A2 activity by non-steroidal anti-inflammatory agents via Ca2+ antagonism may significantly contribute to the mechanism of drug action.     

Antihypertensive mechanism of the dipeptide Val-Tyr in rat aorta

Antihypertensive peptides received much interest over the last decade. These peptides are known to be angiotensin converting enzyme (ACE) inhibitors in vitro, but the actual antihypertensive mechanisms in vivo are still unclear. In this research, we used rat aortic rings in organ bath experiments to investigate five potential vascular antihypertensive mechanisms of the dipeptide Val-Tyr. Only one significant effect was observed, namely preincubation of the aorta with Val-Tyr led to a significant shift of the concentration-response curve evoked by angiotensin I (Ang I). Val-Tyr had no effect on the angiotensin II receptor or the alpha-adrenergic receptor. Furthermore, it did not interact with voltage-operated Ca2+ channels, or with nitric oxide production/availability. In conclusion, our results show that Val-Tyr specifically inhibits Ang I-evoked contraction through ACE inhibition and that four other main mechanisms of vascular tone regulation are not affected.     

Involvement of ion channels in the induction of proenkephalin A gene expression by nicotine and cAMP in bovine chromaffin cells

The involvement of Na+ and Ca2+ channels in the stimulatory effect of nicotine and cAMP upon proenkephalin A mRNA (mRNA ENK) levels in primary cultures of bovine adrenal chromaffin cells was analyzed. Nicotine (10 microM) caused about a 2-3-fold increase in mRNA ENK which was abolished by the nicotinic receptor antagonist tubocurarine (4 X 10(-7) M), inhibited by the Ca2+ channel antagonist nifedipine (100 nM) abolished by the Ca2+ channel blocker D600 (10 microM), and augmented by the Ca2+ channel agonist BayK 8644 (100 nM). In contrast, blockade of the Na+ channel by tetrodotoxin (1 microM) did not modulate the nicotine-induced increase in mRNA ENK. Incubation of the cells with forskolin (25 microM) and 8-bromo-cAMP (1 mM) also resulted in an increase in mRNA ENK levels that was inhibited by the Ca2+ channel blocker verapamil (50 microM) and nifedipine (100 nM), whereas it was enhanced by BayK 8644 (100 nM). In addition, the effect of forskolin and 8-bromo-cAMP was decreased by the Na+ channel blocker tetrodotoxin (1 microM). These results suggest that the induction of proenkephalin A gene expression by cAMP and nicotine involves the modulation of ion channels. It appears that changes in Ca2+ flux are involved in mediating this induction. The dihydropyridines nifedipine and BayK 8644 and the Ca2+ channel blockers verapamil and D600 all modulate 45Ca uptake. In addition, we show that incubation of the cells with A23187 (10(-7) M), a Ca2+ ionophore, resulted in an increase in mRNA ENK, indicating that changes in intracellular Ca2+ levels may indeed modulate proenkephalin A gene expression. Although it appears that an elevation of mRNA ENK upon nicotinic receptor activation occurs rapidly (an increase could be detected after 2 h incubation), the findings that the rise in mRNA ENK could be abolished by the Ca2+ channel blocker D600 but not affected by tetrodotoxin (1 microM), and that agents such as KCl (20 mM) and veratridine (5 microM) that increase mRNA ENK by activation of voltage-dependent Ca2+ channels do not result in an increase in intracellular cAMP, provide no evidence for a major role of the adenylate cyclase system in the inducing effect of nicotine upon proenkephalin A gene expression.     

The relaxant effect of urocortin in rat pulmonary arteries

Urocortin is a potent vasodilator, which plays physiological or pathophysiological roles in systemic circulation. However, little is known about its action on pulmonary circulation. The present study was aimed to characterize some cellular mechanisms underlying the relaxant effect of urocortin in isolated rat pulmonary arteries. Changes in isometric tension were measured on small vessel myographs. Urocortin inhibited U46619-induced contraction with reduction of the maximal response. Urocortin-induced relaxation was independent of the presence of endothelium. Inhibitors of nitric oxide (NO)-dependent dilator, NG-nitro-L-arginine methyl ester or 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one, did not affect the relaxation. Astressin (100-500 nM), a corticotropin-releasing factor (CRF) receptor antagonist and KT5720, a protein kinase A (PKA) inhibitor reduced urocortin-induced relaxation. Urocortin produced less relaxant effect in 30 mM K+- than U46619-contracted arterial rings. Urocortin did not reduce CaCl2-induced contraction in 60 mM K+-containing solution. Ba2+ (100-500 microM) but not other K+ channel blockers reduced the relaxant responses to urocortin. Urocortin also relaxed the rings preconstricted by phorbol 12,13-diacetae in normal Krebs solution while this relaxation was less in a Ca2+-free solution. Our results show that urocortin relaxed rat pulmonary arteries via CRF receptor-mediated and PKA-dependent but endothelium/NO or voltage-gated Ca2+ channel-independent mechanisms. Stimulation of Ba2+-sensitive K+ channel may contribute to urocortin-induced relaxation. Finally, urocortin relaxed pulmonary arteries partly via inhibition of a PKC-dependent contractile mechanism.     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.     

State-dependent inhibition of L-type calcium channels: cell-based assay in high-throughput format

The current studies describe a new, robust cell-based functional assay useful to characterize L-type voltage-dependent calcium channels and their antagonists. The basis of this assay is measurement in plate format of Ca2+ influx through the L-type Ca2+ channel complex (alpha1C, alpha2delta, and beta2a subunits) in response to potassium-mediated depolarization; EC(50)=11 mM [K+](o). The Ca2+ influx was inhibited by the L-type Ca2+ channel antagonist, nimodipine; IC(50)=59 nM. These cells were also transfected with the Kir2.3 inward rectifier K(+) channel, which allows for changing the cell membrane potential by modulation of extracellular [K](o); -65 mV in physiological [K](o) and -28 mV in 30 mM [K](o) containing buffer. The conformational state of the voltage-sensitive Ca2+ channel is altered under these different conditions. Under the depolarized condition, nimodipine was a more potent antagonist, inhibiting Ca2+ influx with an IC(50) value of 3 nM. The results demonstrate that the interaction of nimodipine and other antagonists with the channel is modulated by changes in membrane potential and thus the state of the channel. Overall, this novel assay can be used to identify state-dependent calcium channel antagonists and should be useful for evaluating state-dependent inhibitory potency of a large number of samples.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle. Identification and characterization of an acid-stable phosphorylated intermediate of the Ca2+,Mg2+-ATPase

An acid-stable phosphoprotein was formed in a microsomal membrane fraction isolated from bovine aortic smooth muscle in the presence of Mg2+ + ATP and Ca2+. The microsomes also showed Ca2+ uptake activity. The Ca2+ dependence of phosphoprotein formation and of Ca2+ uptake occurred over the same range of Ca2+ concentration (1-10 microM), and resembled similar findings from rabbit skeletal microsomes. The molecular weight of the phosphorylated protein, estimated by SDS-gel electrophoresis, was approximately 105,000. The phosphoprotein was labile at alkaline pH, and its decomposition was accelerated by hydroxylamine. Half-maximum incorporation of 32P in the presence of 10 microM Ca2+ occurred at 60 nM ATP. The calcium-dependent phosphoprotein formation was not affected by 5 mM NaN3, but was inhibited in a dose-dependent fashion by ADP with a 50% inhibition occurring at 180 microM. Fifty mM MgCl2 was required for the maximal phosphorylation. The rate of phosphoprotein decomposition after adding 2 mM EGTA was accelerated by varying the Mg2+ concentration from 10 microM to 3 mM. Alkaline pH (9.0) slowed the rate of phosphoprotein decay. Optimal Ca2+-dependent phosphoprotein occurred at 15 degrees C over a broad pH range (6.4 to 9.0). The activation energy of EGTA-induced phosphoprotein decomposition was 25.6 kcal/mol between 0 and 16 degrees C and 14.6 kcal/mol between 16 and 30 degrees C. The phosphoprotein formed by aortic microsomes was thus quite similar to the acid-stable phosphorylated intermediate of the Ca2+-transport ATPase of sarcoplasmic reticulum from skeletal and cardiac muscle. These data suggest that the Ca2+-dependent phosphoprotein is a reaction intermediate of the Ca2+,Mg2+-ATPase of the aortic microsomes.     

Structural characteristics of the saxitoxin receptor on nerve.

The effects of uranyl ion (UO22+; at low concentrations binds specifically to phosphate groups) and the cationic dye methylene blue (MB+; binds strongly to carboxyl groups) on saxitoxin (STX) potency in crayfish axon has been studied by means of intracellular microelectrodes. At pH 6.00 +/- 0.05 and 13.5 mM Ca2+, addition of 10.0 muM UO22+ + 5.0 nM STX had only slightly, if any, less effect on the spike's maximum rate of rise [0.79 +/- 0.04 (viz., mean +/- SEM) of control value] than did addition of 5.0 nM STX alone (0.72 +/- 0.05). Under the same conditions of pH and Ca2+ concentration, 1.0 mM MB+ had approximately the same effect: 1.0 mM MB+ + 5.0 nM STX, 0.76 +/- 0.03; 5.0 nM STX alone, 0.70 +/- 0.04. However, at pH 7.00 +/- 0.05 and lower Ca2+ concentrations, 1.0 mM MB+ significantly reduced STX potency. Using 6.0 mM Ca2+: 1.0 mM MB+ + 5.0 nM STX, 0.92 +/- 0.01; 5.0 nM STX alone, 0.68 +/- 0.08. Using 3.0 mM Ca2+, the corresponding values were 0.94 +/- 0.03 and 0.67 +/- 0.04. It is concluded that: (1) In accord with previous suggestions, the ionized acidic group known to exist in the Na channel (and to which a guanidinium group of STX appears to bind) is very likely a carboxyl group and not a phosphate group. (2) The accessible part of the Na channel mouth serving as the saxitoxin receptor probably does not include phospholipid in its structure proper.     

Involvement of second messenger systems in stimulation of angiotensin converting enzyme of bovine endothelial cells.

We have demonstrated previously that a variety of agents including corticosteroids, thyroid hormone, cationophores, methylxanthines, and analogues of cAMP--all of which have diversified functions in various tissues--elevate cellular angiotensin converting enzyme (ACE) activity of bovine endothelial cells in culture. In addition to these agents, we have now found that direct and receptor-mediated stimulators of adenylate cyclase, i.e., forskolin and cholera toxin, increase cellular ACE activity after 48 h incubation in culture. In an attempt to search out a more unifying concept of these stimulatory effects, we have further investigated the roles of second messengers in the stimulatory actions. Ca2+ ionophore A23187 produced significant increases in both intracellular Ca2+ and ACE of endothelial cells. In contrast to Ca2+ ionophore, agents that transiently mobilize Ca2+ from intracellular reserves such as bradykinin, acetylcholine, and ATP have no effect on the level of cellular ACE. Representative agents that elevate cellular cAMP (e.g., isobutyl methylxanthine [IBMX] and dibutyryl cAMP) elevated cellular ACE, but the slightly increased [Ca2+]i produced by these agents did not reach statistical significance. While IBMX, cholera toxin, and forskolin elevated cellular cAMP, other ACE stimulatory agents (hormones and cationophores) had no effect on cAMP. Ca2+ ionophore and the agents that elevated intracellular cAMP potentiated the effect of dexamethasone, thyroid hormone, and aldosterone in elevating cellular ACE activity. Increases in ACE activity produced by all stimulants were inhibited by the presence of 10-50 nM ouabain in the culture medium. Inhibition of ACE elevation by oubain was reversed by increasing the extracellular [K+], thereby implicating Na+, K(+)-ATPase in the ACE regulatory mechanism. These results support the presence of multiple independent mechanisms for the regulation of cellular ACE. In addition to possible involvement of intracellular Ca(2+)- and cAMP-dependent pathways, ACE is also increased by corticosteroids and thyroid hormone through mechanisms unrelated to Ca2+ and cAMP.     

The dihydropyridine-sensitive calcium channel subtype in cone photoreceptors

High-voltage activated Ca channels in tiger salamander cone photoreceptors were studied with nystatin-permeabilized patch recordings in 3 mM Ca2+ and 10 mM Ba2+. The majority of Ca channel current was dihydropyridine sensitive, suggesting a preponderance of L- type Ca channels. However, voltage-dependent, incomplete block (maximum 60%) by nifedipine (0.1-100 microM) was evident in recordings of cones in tissue slice. In isolated cones, where the block was more potent, nifedipine (0.1-10 microM) or nisoldipine (0.5-5 microM) still failed to eliminate completely the Ca channel current. Nisoldipine was equally effective in blocking Ca channel current elicited in the presence of 10 mM Ba2+ (76% block) or 3 mM Ca2+ (88% block). 15% of the Ba2+ current was reversibly blocked by omega-conotoxin GVIA (1 microM). After enhancement with 1 microM Bay K 8644, omega-conotoxin GVIA blocked a greater proportion (22%) of Ba2+ current than in control. After achieving partial block of the Ba2+ current with nifedipine, concomitant application of omega-conotoxin GVIA produced no further block. The P-type Ca channel blocker, omega-agatoxin IVA (200 nM), had variable and insignificant effects. The current persisting in the presence of these blockers could be eliminated with Cd2+ (100 microM). These results indicate that photoreceptors express an L-type Ca channel having a distinguishing pharmacological profile similar to the alpha 1D Ca channel subtype. The presence of additional Ca channel subtypes, resistant to the widely used L-, N-, and P-type Ca channel blockers, cannot, however, be ruled out.     

Regulation of calcium channels in aortic muscle cells by protein kinase C activators (diacylglycerol and phorbol esters) and by peptides (vasopressin and bombesin) that stimulate phosphoinositide breakdown

Voltage-dependent Ca2+ channels of the aortic cell line A7r5 were studied using 45Ca2+ flux experiments. Ca2+ channels which have been studied belong to the L-type and are very sensitive to inhibitors and activators in the 1,4-dihydropyridine series as well as to (-)desmethoxyverapamil and d-cis-diltiazem. L-type Ca2+ channels in these smooth muscle cells are not affected by cyclic 8-bromo-AMP and dibutyryl cyclic AMP. However, the activity of these channels is strongly depressed after treatment with diacylglycerols (1-oleyl 2-acetylglycerol and 1,2-dioctanoylglycerol). Phorbol esters, which like diacylglycerols are well-known activators of protein kinase C (the Ca2+- and phospholipid-dependent enzyme), inhibit 70% of Ca2+ channel activity (K0.5 = 25 nM for phorbol 12-myristate 13-acetate and K0.5 = 200 nM for phorbol 12,13-dibutyrate). Phorbol esters that are inactive on kinase C are without effect on Ca2+ channel activity. [Arg8]Vasopressin and bombesin, two peptides that are well known for their action on polyphosphoinositide metabolism, inhibit Ca2+ channel activity to the same extent as active phorbol esters (65-70%). Oxytocin has the same type of effect presumably by acting at the V1-receptor. Both effects of [Arg8]vasopressin and oxytocin are suppressed by [1-(beta-mercapto-beta,beta-diethylpropionic acid)4-valine]arginine vasopressin, a specific vasopressin antagonist at the V1-receptor.