FCCP depolarizes plasma membrane potential by activating proton and Na+ currents in bovine aortic endothelial cells

We investigated the effects of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore and uncoupler of mitochondrial oxidative phosphorylation in mitochondria, on plasma membrane potential and ionic currents in bovine aortic endothelial cells (BAECs). The membrane potential and ionic currents of BAECs were recorded using the patch-clamp technique in current-clamp and voltage-clamp modes, respectively. FCCP activated ionic currents and depolarized the plasma membrane potential in a dose-dependent manner. Neither the removal of extracellular Ca2+ nor pretreatment with BAPTA/AM affected the FCCP-induced currents, implying that the currents are not associated with the FCCP-induced intracellular [Ca2+]i increase. FCCP-induced currents were significantly influenced by the changes in extracellular or intracellular pH; the increased proton gradient produced by lowering the extracellular pH or intracellular alkalinization augmented the changes in membrane potential and ionic currents caused by FCCP. FCCP-induced currents were significantly reduced under extracellular Na+-free conditions. The reversal potentials of FCCP-induced currents under Na+-free conditions were well fitted to the calculated equilibrium potential for protons. Interestingly, FCCP-induced Na+ transport (subtracted currents, Icontrol-INa+-free) was closely dependent on extracellular pH, whereas FCCP-induced H+ transport was not significantly affected by the absence of Na+. These results suggest that the FCCP-induced ionic currents and depolarization, which are strongly dependent on the plasmalemmal proton gradient, are likely to be mediated by both H+ and Na+ currents across the plasma membrane. The relationship between H+ and Na+ transport still needs to be determined

Na+-K+ pump activation inhibits endothelium-dependent relaxation by activating the forward mode of Na+/Ca2+ exchanger in mouse aorta

The effect of Na+-K+ pump activation on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) was examined in mouse aorta and mouse aortic endothelial cells (MAECs). The Na+-K+ pump was activated by increasing extracellular K+ concentration ([K +]o) from 6 to 12 mM. In aortic rings, the Na+ ionophore monensin evoked EDR, and this EDR was inhibited by the Na +/Ca2+ exchanger (NCX; reverse mode) inhibitor KB-R7943. Monensin-induced Na+ loading or extracellular Na+ depletion (Na+ replaced by Li+) increased [Ca 2+]i in MAECs, and this increase was inhibited by KB-R7943. Na+-K+ pump activation inhibited EDR and [Ca2+]i increase (K+-induced inhibition of EDR and [Ca2+]i increase). The Na+-K+ pump inhibitor ouabain inhibited K+-induced inhibition of EDR. Monensin (>0.1 μM) and the NCX (forward and reverse mode) inhibitors 2′4′-dichlorobenzamil (> 10 μM) or Ni2+ (>100 μM) inhibited K+-induced inhibition of EDR and [Ca 2+]i increase. KB-R7943 did not inhibit K +-induced inhibition at up to 10 μM but did at 30 μM. In current-clamped MAECs, an increase in [K+]o from 6 to 12 mM depolarized the membrane potential, which was inhibited by ouabain, Ni 2+, or KB-R7943. In aortic rings, the concentration of cGMP was significantly increased by acetylcholine and decreased on increasing [K +]o from 6 to 12 mM. This decrease in cGMP was significantly inhibited by pretreating with ouabain (100 μM), Ni2+ (300 μM), or KB-R7943 (30 μM). These results suggest that activation of the forward mode of NCX after Na+-K+ pump activation inhibits Ca2+ mobilization in endothelial cells, thereby modulating vasomotor tone. Copyright © 2005 the American Physiological Society

Suppression of the carbachol-activated nonselective cationic current by antibody against alpha subunit of G(o) protein in guinea-pig gastric myocytes

In this study, we investigated which subtype of GTP-binding protein (G protein) is related to muscarinic activation of nonselective cation (NSC) channels in gastric smooth muscle. Inward cationic current was activated by the application of 50 pM carbachol (I(CCh)) at a holding potential of -60 mV with the same CsCl-rich solution in both pipette and bath. The same cationic current as I(CCh) was slowly activated by the dialysis of guanosine 5'-O-(3- thiotriphosphate) (GTP[γ-S]) through the pipette. Since it is known that pertussis toxin pretreatment can block I(CCh), antibodies (Abs) against G(α,i) (anti-G(α,i)) or G(α,o) (anti-G(α,o)) were tested. Activation of I(CCh) was blocked by the addition of anti-G(α,o). However, anti-G(α,i) Abs had no significant effect on I(CCh). The expression of G(α,o) in guinea-pig gastric smooth muscle was confirmed by Western immunoblot analysis. These results suggest that G(o)-type protein may mediate signals from the muscarinic receptor to NSC channel in guinea-pig gastric myocytes

TRPC4 is an essential component of the nonselective cation channel activated by muscarinic stimulation in mouse visceral smooth muscle cells

Classical transient receptor potential channels (TRPCs) are thought to be candidates for the nonselective cation channels (NSCCs) involved in pacemaker activity and its neuromodulation in murine stomach smooth muscle. We aimed to determine the role of TRPC4 in the formation of NSCCs and in the generation of slow waves. At a holding potential of -60 mV, 50 μM carbachol (CCh) induced INSCC of amplitude [500.8 ± 161.8 pA (n = 8)] at -60 mV in mouse gastric smooth muscle cells. We investigated the effects of commercially available antibodies to TRPC4 on recombinant TRPC4 expressed in HEK cells and CCh-induced NSCCs in gastric smooth muscle cells. TRPC4 currents in HEK cells were reduced from 1525.6 ±414.4 pA (n = 8) to 146.4 ± 83.3 pA (n = 10) by anti-TRPC4 antibody and INSCC amplitudes were reduced from 230.9 ± 36.3 pA (n = 15) to 49.8 ± 11.8 pA (n = 9). Furthermore, INScc in the gastric smooth muscle cells of TRPC4 knockout mice was only 34.4 ± 10.4 pA (n = 8) at -60 mV. However, slow waves were still present in the knockout mice. Our data suggest that TRPC4 is an essential component of the NSCC activated by muscarinic stimulation in the murine stomach. ©KSMCB 2005

Hypoxia-induced EDNO release is further augmented by previous hypoxia and reoxygenation in rabbit aortic endothelium

The present study was designed: (1) to determine whether or not hypoxia stimulates the release of endothelium-derived relaxing factors (EDRFs) from endothelial cells, and (2) to examine whether or not the hypoxia-induced EDRFs release is further augmented by pervious hypoxia-reoxygenation, using bioassay system. In the bioasssay experiment, rabbit aorta with endothelium was used as EDRFs donor vessel and rabbit carotid artery without endothelium as a bioassay test ring. The test ring was contracted by prostaglandin F(2a) (3X10-6 M/L), which was added to the solution perfusing through the aortic segment. Hypoxia was evoked by switching the solution aerated with 95% O2/5% CO2 mixed gas to one aerated with 95 N2/5% CO2 mixed gas. When the contraction induced by prostaglandin F(2a) reached a steady state, the solution was exchanged for hypoxic one. And then, hypoxia and reoxygenation were interchanged at intervals of 2 minutes (intermittent hypoxi). The endothelial cells were also exposed to single 10-minute hypoxia (continuous hypoxia). When the bioassay ring was superfused with the perfusate through intact aorta, hypoxia relaxed the precontracted bioassay test ring markedly. Whereas, when bioassay ring was superfused with the perfusate through denuded aorta or polyethylene tubing, hypoxia relaxed the precontracted ring slightly. The relaxation was not inhibited by indomethacin but by nitro-L- arginine or methylene blue. The hypoxia-induced relaxation was further augmented by previous hypoxia-reoxygenation and the magnitude of the relaxation by intermittent hypoxia was significantly greater than that of the relaxation by continuous hypoxia. The results suggest that hypoxia stimulates EDNO release from endothelial cells and that the hypoxia-induced EDNO release is further augmented by previous hypoxia-reoxygenation

Lyso-globotriaosylceramide downregulates KCa3.1 channel expression to inhibit collagen synthesis in fibroblasts

Fabry disease is an X-linked lysosomal storage disorder that is caused by a deficiency of a-galactosidase A. The disease ultimately manifests as multiple organ dysfunctions owing to excessive accumulation of globotriaosylceramide (Gb3). Among the several complications of Fabry disease, ascending thoracic aortic aneurysm is relatively common, which is classically associated with connective tissue disorders characterized by abnormal defects or deficiencies in structural proteins such as collagen and elastin. Although an elevated Gb3 level is regarded as a prerequisite for the manifestations of Fabry disease, only this excess accumulation cannot explain the pathophysiology of these complications. Recently, an increased plasma level of Iyso-Gb3 was suggested as a new biomarker in Fabry disease. Therefore, the aim of this study was to assess the effects of Iyso-Gb3 on the pathogenesis of thoracic ascending aortic aneurysms in Fabry disease, with a particular focus on the responses related to aortic remodeling by fibroblasts. We found that Iyso-Gb3 inhibited the growth of fibroblasts, as well as their differentiation into myofibroblasts, and collagen expression. Moreover, all of these compromised responses could be attributed to the effects of Iyso-Gb3 on downregulation of KCa3.1 channel expression, and these impairments could be rescued when activating the KCa3.1 channel or increasing intracellular Ca2+ concentration. This study provides new evidence that Iyso-Gb3 inhibits the differentiation into myofibroblasts and collagen synthesis of fibroblasts owing to decreased Ca2+ levels by KCa3.1 channel dysfunction. These findings suggest that the KCa3.1 channel can serve as a new target to attenuate and prevent development of ascending thoracic aortic aneurysm in Fabry disease. (C) 2015 Elsevier Inc. All rights reserved

ATP and nitric oxide modulate a Ca2+-activated non-selective cation current in macrovascular endothelial cells

We have studied the properties of a non-selective cation current (NSCCa) in macrovascular endothelial cells derived from human umbilical vein (EA cells) that is activated by an increase of intracellular Ca2+ concentration, [Ca2+]i. Current-voltage relationships are linear and the kinetics of the current is time-independent. Current-[Ca2+]i relationships were fitted to a Ca2+ binding site model with a concentration for half-maximal activation of 417±76 nM, a Hill coefficient of 2.3±0.8 and a maximum current of -23.9±2.7 pA/pF at -50 mV. The Ca2+-activated channel is more permeable to Na+ than for Cs+ (PCs/PNa=0.58, n=7), but virtually impermeable to Ca2+. Current activation was transient if ATP was omitted from the pipette solution. The maximal currents at 300 and 500 nM [Ca2+]i were smaller than in the absence of ATP, but were not significantly different at 2 μM. The intracellular Ca2+ concentration for half-maximal activation of the Ca2+-activated current was shifted to 811±12 nM in the absence of ATP. Substitution of ATP by the non-hydrolysable ATP analogue adenylylim-idodiphosphate (AMP-PNP) did not affect current activation. Sodium nitroprusside (SNP) decreased NSCCa in a concentration-dependent manner. The nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine (SIN-1) also inhibited NSCCa. In contrast, nitro-L-arginine (NLA), which inhibits all NO-synthases, potentiated NSCCa, whereas superoxide dismutase (SOD), which inhibits the breakdown of NO, inhibited NSCCa. It is concluded that the Ca2+-activated non-selective action channel in EA cells is modulated by the metabolic state of the cell and by NO

Capsaicin inhibits the voltage-operated calcium channels intracellularly in the antral circular myocytes of guinea-pig stomach

Studies of the effect of capsaicin (CAP) on the smooth muscle contractions have shown both contraction and relaxation in various preparations. The direct effect of CAP on gastric smooth muscle itself has not yet been reported, though CAP was reported to relax the isolated guinea-pig stomach by releasing nitric oxide from the CAP-sensitive sensory neurons. Here we showed an evidence that CAP evokes a prolonged relaxation of gastric antral circular smooth muscle(CAP-induced relaxation) by blocking the voltage-operated Ca2+ channels (VOCC) from inside of the cell. CAP suppressed dose-dependently the spontaneous contractions of guinea-pig gastric circular muscle strip under the condition without neural influence (IC50 = 5.8 μM). The inhibitory effects of CAP both on the high K+ contracture induced by 50 mM K+ Tyrode solution and on the slow waves recorded using a conventional intracellular microelectrode technique were similar to those of Ca2+ channel antagonists, indicating that Ca2+ influx through the VOCC is decreased by CAP. Ca2+ channel current (IBa) decreased in a concentration-dependent manner on superfusing the physiological salt solution containing various concentrations of CAP. The steady-state activation and inactivation curves of IBa were not affected by the treatment with CAP. The experiment using a synthetic water-soluble analog of CAP, DA-5018·HCl, suggested that the acting site of CAP is present in the intracellular side. Spontaneous transient outward K+ currents(STOCs) recorded at a holding potential of 0 mV were also inhibited by CAP and verapamil, Ca channel blocker. Taken together, these results indicate that CAP-induced relaxation is associated with the direct inhibitory action on the VOCC from inside of the cell. © 2001 Elsevier Science Inc

Protein kinase C mediates the desensitization of CCh-activated nonselective cationic current in guinea-pig gastric myocytes

The possibility of the protein kinase C (PKC) pathway being a mechanism underlying the desensitization of carbachol- (CCh-)activated nonselective cationic current (I(CCh)) was investigated in a study of guinea-pig gastric myocytes. Using the conventional whole-cell patch-clamp technique with symmetrical CsCl-rich solution in pipette and bath, I(CCh) was induced by bath application of 50 μM CCh. With 0.5 mM EGTA [ethyleneglycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid] in the pipette solution (0.5 mM [EGTA](i)), I(CCh) decayed spontaneously (desensitization of I(CCh)) to around 20% within 10 min. Desensitization of I(CCh) was significantly attenuated with 2 mM [EGTA](i). At a concentration of 20 μM OAG (1-oleoyl-2-acetyl-sn-glycerol), a PKC activator, inhibited I(CCh) at 0.5 mM [EGTA](i) but far less at 2 mM [EGTA](i) (18% and 81% of control, respectively). The same cationic current induced by intracellular guanosine-5'-O-(3-thiotriphosphate) (GTP[γ-S]) was not inhibited by OAG with 0.5 mM [EGTA](i). The pretreatment of gastric myocytes with PKC inhibitors, either 1 μM chelerythrine or 1 μM peptide inhibitor, attenuated the desensitization of I(CCh). [Ca2+](i) was also measured by single cell microfluorometry using fura-2. Under CCh stimulation with 2 mM [EGTA](i), [Ca2+](i) did not increase above 100 nM while it increased to around 260 nM with 0.5 mM [EGTA](i). These results suggest that the desensitization of I(CCh) is partly due to the Ca2+-dependent PKC pathway in guinea-pig gastric myocytes