cyclic-gmp and ethylisopropylamiloride

The inner medullary collecting duct cell line, mIMCD-K2, absorbs Na+ by an amiloride-sensitive, electrogenic mechanism. The goal of the present study was to characterize the amiloride-sensitive, Na+ -conducting channels responsible for electrogenic Na+ absorption. To this end, we measured Na+ currents in single cells with the patch-clamp technique and Na+ currents across monolayers mounted in Ussing-type chambers. In whole cell patch-clamp experiments, amiloride-sensitive, inward Na+ currents were mediated by nonselective cation channels. In single-channel patch-clamp experiments, amiloride- and guanosine 3',5'-cyclic monophosphate (cGMP)-sensitive, 20-pS nonselective cation channels (i.e., CNG channels) were identified in the apical membrane. CNG channels were inhibited by amiloride, diltiazem, ethylisopropylamiloride (EIPA), and 8-bromo-cGMP and were permeable to Ca2+ and Mg2+. Epithelial Na+ channels were never observed in whole cell or single-channel recordings. Na+ absorption across confluent monolayers was inhibited with a rank order potency of benzamil > amiloride > phenamil >> EIPA > diltiazem. Our data are most consistent with the view that CNG channels mediate electrogenic Na+ absorption across mIMCD-K2 cells.

Topics: Amiloride; Amino Acid Sequence; Animals; Biological Transport, Active; Calcium; Clone Cells; Cyclic GMP; Diltiazem; Ion Channel Gating; Ion Channels; Kidney Medulla; Kidney Tubules, Collecting; Magnesium; Membrane Potentials; Mice; Molecular Sequence Data; Nucleotides, Cyclic; Patch-Clamp Techniques; Sodium; Sodium Channels

We have used the patch clamp technique to study the effects of inhibiting the apical Na+ transport on the basolateral small-conductance K+ channel (SK) in cell-attached patches in cortical collecting duct (CCD) of the rat kidney. Application of 50 microM amiloride decreased the activity of SK, defined as nPo (a product of channel open probability and channel number), to 61% of the control value. Application of 1 microM benzamil, a specific Na+ channel blocker, mimicked the effects of amiloride and decreased the activity of the SK to 62% of the control value. In addition, benzamil reduced intracellular Na+ concentration from 15 to 11 mM. The effect of amiloride was not the result of a decrease in intracellular pH, since addition 50 microM 5-(n-ethyl-n-isopropyl) amiloride (EIPA), an agent that specifically blocks the Na/H exchanger, did not alter the channel activity. The inhibitory effect of amiloride depends on extracellular Ca2+ because removal of Ca2+ from the bath abolished the effect. Using Fura-2 AM to measure the intracellular Ca2+, we observed that amiloride and benzamil significantly decreased intracellular Ca2+ in the Ca2+-containing solution but had no effect in a Ca2+-free bath. Furthermore, raising intracellular Ca2+ from 10 to 50 and 100 nM with ionomycin increased the activity of the SK in cell-attached patches but not in excised patches, suggesting that changes in intracellular Ca2+ are responsible for the effects on SK activity of inhibition of the Na+ transport. Since the neuronal form of nitric oxide synthase (nNOS) is expressed in the CCD and the function of the nNOS is Ca2+ dependent, we examined whether the effects of amiloride or benzamil were mediated by the NO-cGMP-dependent pathways. Addition of 10 microM S-nitroso-n-acetyl-penicillamine (SNAP) or 100 microM 8-bromoguanosine 3':5'-cyclic monophosphate (8Br-cGMP) completely restored channel activity when it had been decreased by either amiloride or benzamil. Finally, addition of SNAP caused a significant increase in channel activity in the Ca2+-free bath solution. We conclude that Ca2+-dependent NO generation mediates the effect of inhibiting the apical Na+ transport on the basolateral SK in the rat CCD.

Topics: Amiloride; Animals; Anti-Arrhythmia Agents; Biological Transport; Calcium; Cyclic GMP; Diuretics; Enzyme Inhibitors; Kidney Tubules, Collecting; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Patch-Clamp Techniques; Penicillamine; Potassium; Potassium Channels; Rats; Rats, Sprague-Dawley; S-Nitroso-N-Acetylpenicillamine; Sodium; Sodium Channels; Specific Pathogen-Free Organisms

An endothelium-derived factor with the properties of nitric oxide (NO) has been implicated in the regulation of Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity in vascular smooth muscle. To examine this phenomenon further and to explore its modulation by guanosine 3',5'-cyclic monophosphate (cGMP), studies were carried out in the isolated rabbit aorta. Incubation of endothelium-denuded rings with NO (1 microM) or sodium nitroprusside (SNP, 10 microM) caused a time-dependent increase in ouabain-sensitive (OS) 86Rb uptake with the maximal stimulation (approximately 170%) seen after 20 min. In contrast, increases in cGMP concentration caused by NO and SNP (40- and 20-fold increases, respectively) were transient, with peak values observed after 2 min and significantly lower values by 10 min. The ability of NO or SNP to increase OS Rb uptake in endothelium-denuded rings was not mimicked by incubation with 8-bromo- or dibutyryl-cGMP or increases in cGMP caused by treatment with the phosphodiesterase inhibitor isobutylmethylxanthine. Depletion of intracellular cGMP levels by the guanylate cyclase inhibitor LY83583 also did not alter OS Rb uptake. SNP-stimulated OS Rb uptake was not inhibited by LY83583 in endothelium-denuded rings; however, it was completely prevented by the Na(+)-H+ exchange inhibitors amiloride and ethylisopropylamiloride. The results suggest that NO stimulates Na(+)-K(+)-ATPase activity in rabbit aorta by a mechanism independent of its ability to increase the intracellular cGMP concentration. They also suggest that NO may stimulate Na(+)-K(+)-ATPase activity secondary to increases in Na(+)-H+ exchange.

Topics: Amiloride; Aminoquinolines; Animals; Aorta, Thoracic; Biological Transport; Cyclic GMP; Endothelium, Vascular; Guanylate Cyclase; In Vitro Techniques; Kinetics; Male; Muscle, Smooth, Vascular; Nitric Oxide; Nitroprusside; Ouabain; Rabbits; Rubidium; Sodium-Potassium-Exchanging ATPase

Because atrial natriuretic factor (Atriopeptin II, ANF) exerts potent effects on Na+ transport in a number of tissues, we examined its influence on 22Na+-uptake in isolated rabbit aorta segments and the possible relation to ANF-induced vasorelaxation. ANF increased 22Na+-uptake by 44% with an EC50 of 12 nM whereas sodium nitroprusside was without effect. The increase was blocked by the selective inhibitor of Na+/H+ exchange 5-(N-ethyl-N-isopropyl)amiloride (EIPA) but was not sensitive to the guanylate cyclase inhibitor LY 83583, indicating ANF-induced activation of Na+/H+ exchange via a cyclic GMP (cGMP) independent pathway. The ability of ANF to relax phenylephrine-induced contractions of rabbit aorta was inhibited by EIPA at concentrations which produced inhibition of Na+/H+ exchange whereas sodium nitroprusside-induced relaxation was only marginally affected. EIPA caused a 90% decrease in the ability of ANF to increase cGMP, but did not interfere with the sodium nitroprusside-induced increase. LY 83583 blocked the ability of ANF to increase cGMP formation but failed to reduce ANF-induced vasorelaxation. These results suggest that ANF activation of EIPA-sensitive 22Na+-uptake occurs before cGMP formation perhaps at the level of the ANF receptor itself. The vasorelaxant effects of ANF involve a significant cGMP-independent component which is EIPA sensitive.

Topics: Amiloride; Animals; Aorta, Thoracic; Atrial Natriuretic Factor; Cyclic GMP; Hydrogen-Ion Concentration; In Vitro Techniques; Male; Nitroprusside; Phorbol 12,13-Dibutyrate; Rabbits; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface; Sodium; Vasodilation