benzofurans and benzamil

Angiotensin II (AngII) helps to regulate overall renal tubular reabsorption of salt and water, yet its effects in the distal nephron have not been well studied. The purpose of these studies was to determine whether AngII stimulates luminal Na(+) transport in the cortical collecting duct (CCD). Intracellular Na(+) concentration ([Na(+)](i)), as a reflection of Na(+) transport across the apical membrane, was measured with fluorescence microscopy using sodium-binding benzofuran isophthalate (SBFI) in isolated, perfused CCD segments dissected from rabbit kidneys. Control [Na(+)](i), during perfusion with 25 mM NaCl and a Na(+)-free solution in the bath containing the Na(+)-ionophore monensin (10 microM, to eliminate basolateral membrane Na(+) transport) averaged 19.3 +/- 5.2 mM (n = 16). Increasing luminal [NaCl] to 150 mM elevated [Na(+)](i) by 9.87 +/- 1.5 mM (n = 7; P < 0.05). AngII (10(-9) M) added to the lumen significantly elevated baseline [Na(+)](i) by 6.3 +/- 1.0 mM and increased the magnitude (Delta = 25.2 +/- 3.7 mM) and initial rate ( approximately 5 fold) of change in [Na(+)](i) to increased luminal [NaCl]. AngII when added to the bath had similar stimulatory effects; however, AngII was much more effective from the lumen. Thus, AngII significantly increased the apical entry of Na(+) in the CCD. To determine if this apical entry step occurred via the epithelial Na(+) channel (ENaC), studies were performed using the specific ENaC blocker, benzamil hydrochloride (10(-6) M). When added to the perfusate, benzamil almost completely inhibited the elevations in [Na(+)](i) to increased luminal [NaCl] in both the presence and absence of AngII. These results suggest that AngII directly stimulates Na(+) channel activity in the CCD. AT(1) receptor blockade with candesartan or losartan (10(-6) M) prevented the stimulatory effects of AngII. Regulation of ENaC activity by AngII may play an important role in distal Na(+) reabsorption in health and disease.

Topics: Amiloride; Analysis of Variance; Angiotensin II; Angiotensin Receptor Antagonists; Animals; Antihypertensive Agents; Benzimidazoles; Benzofurans; Biphenyl Compounds; Hydrogen-Ion Concentration; Intracellular Membranes; Ionophores; Kidney Tubules, Collecting; Losartan; Mice; Microscopy, Fluorescence; Monensin; Rabbits; Receptor, Angiotensin, Type 1; Receptors, Angiotensin; Signal Transduction; Sodium Chloride; Tetrazoles

Treatment of Madin-Darby canine kidney (MDCK) cells with the peptide hormone angiotensin II (Ang II) results in an increase in the concentrations of cytosolic free calcium ([Ca(2+)](i)) and sodium ([Na(+)](i)) with a concomitant decrease in cytosolic free Mg(2+) concentration ([Mg(2+)](i)). In the present study we demonstrate that this hormone-induced decrease in [Mg(2+)](i) is independent of [Ca(2+)](i) but dependent on extracellular Na(+). [Mg(2+)](i), [Ca(2+)](i), and [Na(+)](i) were measured in Ang II-stimulated MDCK cells by fluorescence digital imaging using the selective fluoroprobes mag-fura-2AM, fura-2AM, and sodium-binding benzofuran isophthalate (acetoxymethyl ester), respectively. Ang II decreased [Mg(2+)](i) and increased [Na(+)](i) in a dose-dependent manner. These effects were inhibited by irbesartan (selective AT(1) receptor blocker) but not by PD123319 (selective AT(2) receptor blocker). Imipramine and quinidine (putative inhibitors of the Na(+)/Mg(2+) exchanger) and removal of extracellular Na(+) abrogated Ang II-mediated [Mg(2+)](i) effects. In cells pretreated with thapsigargin (reticular Ca(2+)-ATPase inhibitor), Ang II-stimulated [Ca(2+)](i) transients were attenuated (p < 0.01), whereas agonist-induced [Mg(2+)](i) responses were unchanged. Clamping the [Ca(2+)](i) near 50 nmol/liter with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) inhibited Ang II-induced [Ca(2+)](i) increases but failed to alter Ang II-induced [Mg(2+)](i) responses. Benzamil, a selective blocker of the Na(+)/Ca(2+) exchanger, inhibited [Na(+)](i) but not [Mg(2+)](i) responses. Our data demonstrate that in MDCK cells, AT(1) receptors modulate [Mg(2+)](i) via a Na(+)-dependent Mg(2+) transporter that is not directly related to [Ca(2+)](i). These data support the notion that rapid modulation of [Mg(2+)](i) is not simply a result of Mg(2+) redistribution from intracellular buffering sites by Ca(2+) and provide evidence for the existence of a Na(+)-dependent, hormonally regulated transporter for Mg(2+) in renally derived cells.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adrenergic alpha-Antagonists; Adrenergic Uptake Inhibitors; Amiloride; Angiotensin II; Animals; Antihypertensive Agents; Benzofurans; Biphenyl Compounds; Calcium; Calcium Channel Agonists; Cell Line; Cells, Cultured; Chelating Agents; Cytosol; Dogs; Dose-Response Relationship, Drug; Egtazic Acid; Ethers, Cyclic; Fluorescent Dyes; Fura-2; Imidazoles; Imipramine; Irbesartan; Kidney; Kinetics; Magnesium; Microscopy, Fluorescence; Peptides; Pyridines; Quinidine; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptors, Angiotensin; Sodium; Tetrazoles; Time Factors