Identification and expression of the Na+/H+ exchanger in mammalian cerebrovascular and choroidal tissues: Characterization by amiloride-sensitive [3H]MIA binding and RT-PCR analysis

Abstract We report the initial characterization of [ 3 H ]5-( N -methyl- N -isobutyl)amiloride (MIA) binding to the Na + /H + exchanger (NHE) and expression of its gene in mammalian cerebrovascular, choroidal and neocortical tissues. [ 3 H ]MIA bound reversibly to particulate fractions of rat, pig and human cerebral microvessels, choroid plexus and cerebral cortex. Scatchard analyses revealed binding to a single amiloride-sensitive site with dissociation constants ( K d ) ranging from 20 to 90 nM for the various tissue preparations. The maximal binding capacities ( B max ) were between 2 to 17 pmol/mg protein and were several-fold greater in cerebral microvessels compared to the cerebral cortex. Amiloride, MIA, 5-( N , N -hexamethylene)amiloride (HMA), 5-( N , N -dimethyl)amiloride (DMA) and 5-( N -methyl- N -isopropyl)amiloride (IPA) variably displaced [ 3 H ]MIA binding to the microvessels in the following rank order: MIA>HMA≥IPA>DMA>amiloride. Benzamil, a potent ligand of the Na + /Ca + transporter was the least sensitive. These binding results were most compatible with the existence of the amiloride-sensitive NHE type 1 in the brain vascular and choroidal tissues. To substantiate this, we utilized reverse transcription polymerase chain reaction (RT–PCR) techniques to search for NHE-1 mRNA. Using primers corresponding to conserved sequences of the human growth factor-activatable NHE gene, RT–PCR revealed strong expression of NHE-1 mRNA in cerebral microvessels, choroid plexus, pial vessels and vascular smooth muscle cells relative to neocortical tissues from several species including rat, pig, cow, monkey and human subjects. Further confirmation of NHE-1 isoform mRNA expression in the cerebrovascular tissues was obtained by Hpa II restriction digestion analysis and by subcloning and sequencing of the PCR amplified products. Our study suggests that mammalian cerebrovascular and choroidal tissues contain high amounts of the ubiquitous amiloride-sensitive [ 3 H ]MIA binding proteins consistent with the expression of NHE type 1 mRNA.