Kinetic properties of the Na+/H+ antiporter of lymphocytes from the spontaneously hypertensive rat: role of intracellular pH.

Abstract

Enhanced activity of the Na+/H' antiporter is increasingly reported as a feature of cells from hypertensive subjects but the intracellular pH (ipH) dependency of its activity has not been examined. This study was designed to characterize the kinetic properties of the Na+/H' antiporter in lymphocytes from adult spontaneously hypertensive rats (SHR) and in those from age-matched normotensive Wistar-Kyoto (WKY) controls. Steady-state ipH, estimated from the measurement of BCECF fluorescence, was significantly lower in lymphocytes from the SHR than in those from WKY rats (7.09±0.02, n = 17 and 7.17±0.03, n = 19, respectively, P < 0.025). The velocity of the antiporter determined from the product of the We conclude that while the ipH of SHR lymphocytes is reduced, the kinetic properties of the Na+/H' antiporter are virtually identical in SHR and WKY lymphocytes. Consequently, a primary abnormality in the activity of this antiporter is not an inherent feature of lymphocytes from the SHR model of genetic hypertension. We propose that the activity of the

Regulation of intracellular pH in the spontaneously hypertensive rat. Role of bicarbonate-dependent transporters.

“…The 500:440 ratio of BCECF-AM was calibrated to pH, with a calibration curve generated in a KCI buffer containing 120 mmol/L K + using the nigericin technique. 4 All calibration curves had a correlation coefficient of .99 or higher. BCECF-AM leakage in our lymphocyte preparation was minimal (less than 5%/h).…”

“…The HCO 3s olution was constantly bubbled with 5% CO 2 -95% O 2 and capped with parafilm to minimize CO 2 diffusion. For experiments using Na + -free solutions, choline chloride was substituted for NaCl, choline bicarbonate for NaHCO 3 , and KH 2 PO 4 for NaHPO 4 , and KCI was adjusted appropriately. Chloride-free solutions were prepared by substituting the sodium and potassium salts of aspartic acid for NaCl and KCI; MgSO 4 was substituted for MgCl 2 ; and calcium gluconate was substituted for CaCI 2 .…”

“…Cells were placed into a spectrofluorometer cuvette that was tightly capped with an air line run through a pin hole in the cap to maintain the atmosphere at 95% 0^5 % CO 2 . The dye was alternately excited at 500 and 440 nm, and emission was measured at 520 4 CI pulse is not shown. pH recovery from cell acidification was monitored while cells were kept in an HCO 3 -COj-containing solution under control conditions (Na + , 140 mmol/L) in the presence of ethyiisopropylamilorlde (EIPA) (20 jtmol/L) or 4,4'-dilsothiocyanatostjlbene-2,2'-dlsulfonic acid (DIDS) (400 /imol/L) or In an Na + -free HCCVCOr-containing solution.…”

“…39 - 40 The buffering power was calculated as the ratio of ANH, + and ApH,, where ANH/ equals the rise in [NH 4 + ], produced by the alkaline pulse, and ApH| is the difference between the pH, prevailing before and after the pulse. The ApH, range used for these calculations was 0.10 to 0.25 pH units and was achieved using an NH 4 C1 range of 1 to 5 mmol/L (1 mmol/L when the departing pH| was acid and 5 mmol/L when the departing pH, was at steady state or higher).…”

“…In these studies we preset pH| at various levels using the nigericin technique. 4 Resuspension in a chloride-free medium containing EIPA, to ensure that the Na + -H + antiporter could not contribute to cell alkalinization, resulted in a rapid and progressive pH, increase. The pH, increase was maximal near steady-state pH, (approximately 7.0 to 7.2) and was absent when pH| was very high (>7.5) or very low (<6.4) (Fig 5A and 5B).…”