Increased Na+/H+ exchanger isoform 1 activity in spontaneously hypertensive rats: lack of mutations within the coding region of NHE1

Enhanced Na+/H+ exchange, measured as amiloride derivative-sensitive Na+ an d H+ fluxes in cells with a preliminary acidified cytoplasm (Delta mu(H+)-i nduced Na+/H+ exchange), is one of the most prominent intermediate phenotyp es of altered Vascular smooth muscle cell (VSMC) function in spontaneously hypertensive rats (SHR). Analysis of Na+/H+ exchange in F-2 hybrids of SHR and normotensive rats seems to be the most appropriate approach in the sear ch for the genetic determinants of abnormal activity of this carrier. Howev er, the measurement of Delta mu(H+)-induced Na+/H+ exchange is hardly appro priate for precise analysis of the carrier's activity in VSMC derived from several hundred F-2 hybrids. To overcome this problem, we compared the rate of Na-22 influx under baseline conditions and in Na+-loaded (ouabain-treat ed) VSMC. The dose-dependency of the rate of Delta mu(H+)-induced H+ efflux as well as of Na-22 influx in control and ouabain-treated cells on ethylis opropylamiloride (EIPA) concentration were not different (K(0.5)similar to 0.3 mu M), suggesting that these ion transport pathways are mediated by the same carrier. EIPA-sensitive Na-22 influx in Na+-loaded cells was similar to 6-fold higher than in ouabain-untreated VSMC and was increased by 50-70% in two different substrains of SHR. About the same increment of EIPA-sensi tive Na-22 influx in Na+-loaded VSMC was observed in 5- to 6-week-old SHR ( an age at which hypertension has not yet developed) as well as in stroke-pr one SHR (SHRSP) with severe hypertension, indicating that the heightened ac tivity of Na+/H+ exchange is not a consequence of long-term blood pressure elevation. To examine whether or not the augmented activity of Na+/H+ excha nge in SHR is caused by mutation of NHE1, i.e. the only isoform of this car rier expressed in VSMC, we undertook single-stranded conformational polymor phism analysis of 23 NHE1 cDNA fragments from SHR and SHRSP and sequencing of the 456-2421 NHE1 cDNA fragment. This study did not reveal any mutation in the entire coding region of NHE1. The lack of mutation in the coding reg ion of NHE1 indicates that the augmented activity of the ubiquitous Na+/Hexchanger in primary hypertension is caused by altered regulation of carrie r turnover number or/and its plasma membrane content. (C) 2000 Elsevier Sci ence B.V. All rights reserved.