IDENTIFICATION AND EXPRESSION OF THE NA+ H+ EXCHANGER IN MAMMALIAN CEREBROVASCULAR AND CHOROIDAL TISSUES - CHARACTERIZATION BY AMILORIDE-SENSITIVE [H-3]MIA BINDING AND RT-PCR ANALYSIS/

We report the initial characterization of [H-3]S-(N-methyl-N-isobutyl) amiloride (MIA) binding to the Na+/H+ exchanger (NHE) and expression o f its gene in mammalian cerebrovascular, choroidal and neocortical tis sues. [H-3]MIA bound reversibly to particulate fractions of rat, pig a nd human cerebral microvessels, choroid plexus and cerebral cortex. Sc atchard analyses revealed binding to a single amiloride-sensitive site with dissociation constants (K-d) ranging from 20 to 90 nM for the va rious tissue preparations. The maximal binding capacities (B-max) were between 2 to 17 pmol/mg protein and were several-fold greater in cere bral microvessels compared to the cerebral cortex. Amiloride, MIA, 5-( N, N-hexamethylene)amiloride (HMA), 5-(N,N-dimethyl)amiloride (DMA) an d 5-(N-methyl-N-isopropyl)amiloride (IPA) variably displaced [H-3]MIA binding to the microvessels in the following rank order: MIA > HMA gre ater than or equal to IPA > DMA > amiloride. Benzamil, a potent ligand of the Na+/Ca+ transporter was the least sensitive. These binding res ults were most compatible with the existence of the amiloride-sensitiv e NHE type 1 in the brain vascular and choroidal tissues. To substanti ate this, we utilized reverse transcription polymerase chain reaction (RT-PCR) techniques to search for NHE-1 mRNA. Using primers correspond ing to conserved sequences of the human growth factor-activatable NHE gene, RT-PCR revealed strong expression of NHE-1 mRNA in cerebral micr ovessels, choroid plexus, pial vessels and vascular smooth muscle cell s relative to neocortical tissues from several species including rat, pig, cow, monkey and human subjects. Further confirmation of NHE-1 iso form mRNA expression in the cerebrovascular tissues was obtained by Hp aII restriction digestion analysis and by subcloning and sequencing of the PCR amplified products. Our study suggests that mammalian cerebro vascular and choroidal tissues contain high amounts of the ubiquitous amiloride-sensitive [H-3]MIA binding proteins consistent with the expr ession of NHE type 1 mRNA. (C) 1998 Elsevier Science B.V. All rights r eserved.