h-89 and 5-nitro-2-(3-phenylpropylamino)benzoic-acid

The effects of monocarboxylic acid-derived Cl(-) channel blockers on cardiac depolarization-activated K(+) currents were investigated. Membrane currents in rat ventricular myocytes were recorded using the whole-cell configuration of the patch-clamp technique. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and niflumic acid (NFA) induced an outward current at 0 mV. Both NPPB and NFA failed to induce any current when used intracellularly or after K(+) in the bath and pipette solutions was replaced by equimolar Cs(+). Voltage pulse protocols revealed that NPPB and NFA enhanced the steady-state K(+) current but inhibited the transient outward K(+) current. Genistein, a tyrosine kinase (PTK) inhibitor, inhibited NPPB- and NFA-induced outward current. Another PTK inhibitor, lavendustin A, produced a comparable effect. In contrast, the inactive analogue of genistein, daidzein, was ineffective. Orthovanadate, a tyrosine phosphatase inhibitor, markedly slowed the deactivation of the outward current induced by NPPB and NFA. The protein kinase A (PKA) inhibitor H-89 inhibited NPPB-induced outward current at 0 mV. In contrast, the protein kinase C (PKC) inhibitor H-7 was without significant effect on the action of NPPB. Pretreatment of the myocytes with genistein or H-89 prevented the enhancing effect of NPPB. Increasing intracellular Cl(-) from 22 to 132 mm slightly reduced NPPB-induced outward current at 0 mV. These results demonstrate that the monocarboxylic acid-derived Cl(-) channel blockers NPPB and NFA enhance cardiac steady-state K(+) current, and suggest that the enhancing effect of the Cl(-) channel blockers is mediated by stimulation of PKA and PTK signalling pathways.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Angiogenesis Inhibitors; Animals; Chloride Channels; Cyclooxygenase Inhibitors; Enzyme Inhibitors; Genistein; Isoquinolines; Myocytes, Cardiac; Niflumic Acid; Nitrobenzoates; Patch-Clamp Techniques; Phenols; Potassium Channels; Rats; Rats, Sprague-Dawley; Signal Transduction; Sulfonamides; Vanadates

1. The contribution of ClC-2 protein to the inwardly rectifying Cl- conductance in cultured porcine choroid plexus epithelial cells was investigated using Western analysis and whole-cell current recordings. 2. Inwardly rectifying currents were elicited by hyperpolarizing voltage at a potential more negative than -50 mV in the presence of intracellular protein kinase A (PKA). The relative halide selectivity estimated from the shift in the reversal potential (Erev) was I- > Br- > Cl- > F-. 3. Extracellular vasoactive intestinal peptide (VIP) activated the same currents in a dose-dependent manner with a half-maximal concentration of 167.3 nM. H-89 (a PKA inhibitor) interfered with the current activation by VIP. 4. The Cl- channel was inhibited by external Cd2+, Ba2+or H+, but only weakly inhibited by known Cl- channel blockers including glibenclamide, NPPB, DIDS and anthracene-9-carboxylic acid (9AC). 5. A specific antibody to ClC-2 detected a 79 kDa protein in porcine choroid plexus cells, which was reduced in cells treated with antisense oligodeoxynucleotide for ClC-2. Both PKA and VIP failed to activate the inwardly rectifying Cl- currents in cells transfected with the antisense oligodeoxynucleotide, while they activated the currents in cells transfected with GFP alone or the control oligodeoxynucleotide randomized from antisense oligonucleotide. 6. It is concluded that ClC-2 protein contributes to the inwardly rectifying Cl- conductance in porcine choroid plexus epithelial cells.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Animals; Barium; Biological Transport, Active; Blotting, Western; Cadmium; Cells, Cultured; Chloride Channels; Chlorides; Choroid Plexus; CLC-2 Chloride Channels; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Egtazic Acid; Enzyme Inhibitors; Epithelial Cells; Glyburide; Hydrogen-Ion Concentration; Hypoglycemic Agents; Ion Transport; Isoquinolines; Nitrobenzoates; Oligodeoxyribonucleotides, Antisense; Patch-Clamp Techniques; Sulfonamides; Swine; Transfection; Vasoactive Intestinal Peptide

We studied regulation of Cl- transport by cAMP and Ca2+ in renal epithelial A6 cells. Stimulation of A6 cells by 1 mM 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of phosphodiesterase), which increased cytosolic cAMP, elicited biphasic increases in short-circuit current (Isc), i.e., a transient phase followed by a sustained one. Apical application of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, a Cl- channel blocker) markedly and dose-dependently inhibited the IBMX-induced Isc. Pretreatment with nifedipine (100 microM, a Ca2+ channel blocker) or 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA/AM, 10 microM, a Ca2+ chelator) partially but markedly inhibited the Isc. On the other hand, a cAMP-dependent protein kinase inhibitor, H89 (0.5 microM for 1 h), also reduced the IBMX-induced Isc to a level similar to that following nifedipine or BAPTA pretreatment. Nifedipine had no synergistic effects on the IBMX-induced Isc in cells treated with H89. Ionomycin (a Ca2+ ionophore) could mimic the transient increase dose dependently, and H89 did not block the ionomycin-induced Isc. Taken together, our observations suggest that: (1) part of the IBMX-stimulated Cl- release is regulated by an increased cytosolic Ca2+ through nifedipine-sensitive Ca2+ influx; (2) cAMP-dependent phosphorylation may be required for elevation of the cytosolic Ca2+ concentration but not for activation of Cl- channels, which are directly activated by cytosolic Ca2+; and (3) the IBMX-induced sustained Cl- release requires cAMP elevation in addition to an increase in the cytosolic Ca2+ concentration.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 1-Methyl-3-isobutylxanthine; Animals; Calcium; Calcium Channel Blockers; Cell Line; Chloride Channels; Chlorides; Cyclic AMP; Cytosol; Enzyme Inhibitors; Epithelium; Ion Transport; Isoquinolines; Kidney; Kinetics; Nitrobenzoates; Phosphodiesterase Inhibitors; Protein Kinase Inhibitors; Sulfonamides; Xenopus laevis

To elucidate mechanisms of glucagon-induced bicarbonate-rich choleresis, we investigated the effect of glucagon on ion transport processes involved in the regulation of intracellular pH (pHi) in isolated rat hepatocyte couplets. It was found that glucagon (200 nM), without influencing resting pHi, significantly stimulates the Cl-/HCO3- exchange activity. The effect of glucagon was associated with a sevenfold increase in cAMP levels in rat hepatocytes. The activity of the Cl-/HCO3- exchanger was also stimulated by DBcAMP + forskolin. The effect of glucagon on the Cl-/HCO3- exchange was individually blocked by two specific and selective inhibitors of protein kinase A, Rp-cAMPs (10 microM) and H-89 (30 microM), the latter having no influence on the glucagon-induced cAMP accumulation in isolated rat hepatocytes. The Cl- channel blocker, NPPB (10 microM), showed no effect on either the basal or the glucagon-stimulated Cl-/HCO3 exchange. In contrast, the protein kinase C agonist, PMA (10 microM), completely blocked the glucagon stimulation of the Cl-/HCO3- exchange; however, this effect was achieved through a significant inhibition of the glucagon-stimulated cAMP accumulation in rat hepatocytes. Colchicine pretreatment inhibited the basal as well as the glucagon-stimulated Cl-/HCO3- exchange activity. The Na+/H+ exchanger was unaffected by glucagon either at basal pHi or at acid pHi values. In contrast, glucagon, at basal pHi, stimulated the Na(+)-HCO3- symport. The main findings of this study indicate that glucagon, through the cAMP-dependent protein kinase A pathway, stimulates the activity of the Cl-/HCO3- exchanger in isolated rat hepatocyte couplets, a mechanism which could account for the in vivo induced bicarbonate-rich choleresis.

Topics: Animals; Antiporters; Bicarbonates; Bile; Bucladesine; Cells, Cultured; Chloride-Bicarbonate Antiporters; Chlorides; Cholagogues and Choleretics; Colchicine; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Glucagon; Hydrogen-Ion Concentration; Intracellular Fluid; Isoquinolines; Liver; Male; Nitrobenzoates; Rats; Rats, Wistar; Sodium; Sodium-Hydrogen Exchangers; Sulfonamides; Tetradecanoylphorbol Acetate; Thionucleotides