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.
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.
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