Cyclic nucleotide-gated cation channels mediate sodium absorption by IMCD (mIMCD-K2) cells.

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.

Cell-specific expression of amiloride-sensitive, Na(+)-conducting ion channels in the kidney.

Amiloride-sensitive, electrogenic Na+ absorption across the distal nephron plays a vital role in regulating extracellular fluid volume and blood pressure. Recently, two amiloride-sensitive, Na(+)-conducting ion channel cDNAs were cloned. One, an epithelial Na(+)-selective channel (ENaC), is responsible for Na+ absorption throughout the distal nephron. The second, a guanosine 3',5'-cyclic monophosphate (cGMP)-inhibitable cation channel, is conductive to Na+ and Ca2+ and contributes to Na+ absorption across the inner medullary collecting duct (IMCD). As a first step toward understanding the segment-specific contributions(s) of cGMP-gated cation channels and ENaC to Na+ and Ca2+ uptake along the nephron, we used in situ reverse transcription-polymerase chain reaction (RT-PCR) hybridization, solution-phase RT-PCR, and Western blot analysis to examine the nephron and cell-specific expression of these channels in mouse kidney cell lines and/or dissected nephron segments. cGMP-gated cation channel mRNA was detected in proximal tubule, medullary thick ascending limb (mTAL), distal convoluted tubule (DCT), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and IMCD. cGMP-gated cation channel protein was detected in DCT, CCD, and IMCD cell lines. These observations suggest that hormones that modulate intracellular cGMP levels may regulate Na+, and perhaps Ca2+, uptake throughout the nephron. mRNA for alpha-mENaC, a subunit of the mouse ENaC, was detected in mTAL, DCT, CCD, OMCD, and IMCD. Coexpression of alpha-mENaC and cGMP-gated cation channel mRNAs in mTAL, DCT, CCD, OMCD, and IMCD suggests that both channels may contribute to Na+ absorption in these nephron segments.

Effects of diazoxide and glyburide on ATP-sensitive K+ channels from hypertrophied ventricular myocytes.

Hyperpolarizing vasodilators that specifically activate ATP-sensitive K+ currents (IK(ATP] in smooth muscle have been suggested as promising antihypertensive (if potentially arrhythmogenic and/or hyperglycemic) therapy. To date, however, the effects of agents presumed to influence these channels have not been characterized in hypertrophied cardiac muscle secondary to chronic hypertension. We used standard intracellular and patch clamp, single-channel recording techniques to study the effects of diazoxide, a presumed activator, as well as the sulfonylurea glyburide on IK(ATP) in cardiac muscle from control (WKY) and spontaneously hypertensive rats (SHR). Intracellular recordings were obtained from isolated left ventricles at 37 degrees C; unitary currents were recorded in excised, inside-out membrane patches with symmetrical transmembrane K+ at 21-23 degrees C. Diazoxide (5-100 microM) caused a decrease in action potential duration in both WKY and SHR ventricles. Glyburide (5-25 microM) produced dramatic dose-dependent increases in action potential duration approaching 100% in both groups. Action potential amplitude and resting membrane potential were unaffected by either agent. Before drug administration, unitary currents in hypertrophied myocytes exhibited a greater open state probability upon depolarization than those from control myocytes, although conductance, mean single-channel open time, and the number of channels per patch were not significantly different. Under patch clamp, both diazoxide (25 and 100 microM) and glyburide (50 microM) decreased IK(ATP) activity in cells from WKY and SHR in the absence of ATP. In both groups, the response reflected an overall decrease in open state probability. These data indicate that although IK(ATP) characteristics are altered in hypertension and myocardial hypertrophy, the effects of agents specific to this current are not significantly different in cells from SHR relative to control. On the other hand, the effects of diazoxide may be linked to temperature or to the metabolic state of the cell.