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⁺ and H⁺ fluxes in cells with a preliminary acidified cytoplasm (Δμ(H+)-induced Na⁺/H⁺ exchange), is one of the most prominent intermediate phenotypes of altered vascular smooth muscle cell (VSMC) function in spontaneously hypertensive rats (SHR). Analysis of Na⁺/H⁺ exchange in F₂ hybrids of SHR and normotensive rats seems to be the most appropriate approach in the search for the genetic determinants of abnormal activity of this carrier. However, the measurement of Δμ(H+)-induced Na⁺/H⁺ exchange is hardly appropriate for precise analysis of the carrier's activity in VSMC derived from several hundred F₂ hybrids. To overcome this problem, we compared the rate of ²²Na influx under baseline conditions and in Na⁺-loaded (ouabain-treated) VSMC. The dose-dependency of the rate of Δμ(H+)-induced H⁺ efflux as well as of ²²Na influx in control and ouabain-treated cells on ethylisopropylamiloride (EIPA) concentration were not different (K_0.5~0.3 μM), suggesting that these ion transport pathways are mediated by the same carrier. EIPA-sensitive ²²Na influx in Na⁺-loaded cells was ~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-sensitive ²²Na 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-prone SHR (SHRSP) with severe hypertension, indicating that the heightened activity 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⁺ exchange in SHR is caused by mutation of NHE1, i.e. the only isoform of this carrier expressed in VSMC, we undertook single-stranded conformational polymorphism 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 region of NHE1 indicates that the augmented activity of the ubiquitous Na⁺/H⁺ exchanger in primary hypertension is caused by altered regulation of carrier turnover number or/and its plasma membrane content. Copyright (C) 2000 Elsevier Science B.V.