Reduced intracellular pH in lymphocytes from the spontaneously hypertensive rat.

Abstract

This study was designed to determine the cytoplasmic pH (pH,) profile of lymphocytes from a rat model of genetic hypertension that is well suited for study before and after the development of spontaneous hypertension. For this purpose, pHj was measured in thymic lymphocytes obtained from spontaneously hypertensive rats (SHR) and from age-matched Wistar-Kyoto (WKY) control rats using 2',7'-bis carboxyethyl-5,6-carboxyfluorescein (BCECF), a pHsensitive fluorescence probe. At the age of 16-20 weeks, pH, of lymphocytes suspended in a HCOj-free HEPES-buffered solution, was markedly lower in the SHR than in the WKY rats (7.07±0.02,7i=16 and 7.22±0.01, n=15, respectively,p<0.001), whereas systolic blood pressure was higher in SHR than in WKY rats (175±5.0 and 105±3.0 mm Hg, respectively,p<0.001). In rats less than 5 weeks of age, pH, was also lower in SHR than in WKY rat lymphocytes (7.12±0.04, n = l l and 7.23±0.04, n = l l , respectively, p<0.05), although at this age systolic blood pressure was not different between the two groups (87±4.0 and 85±3.0 mm Hg, respectively). In lymphocytes suspended in a more physiological HCO 3 /CO 2 -buffered solution, pHj was again lower in the adult SHR than in the WKY rat (7.18±0.02, n=U and 7.31 ±0.02, n=16, respectively,p<0.001). Under this condition, blockade of Na + -H + exchange by a specific inhibitor, ethyl-isopropyl-amiloride (EIPA), or by exposure to Na + -free media resulted in intracellular acidification in both SHR and WKY rat lymphocytes. The difference in pH ; between SHR and WKY rat lymphocytes persisted after blockade of the Na + -H + exchanger by either EIPA (7.09 ±0.04 vs. 7.20+0.03, p< 0.05) or by removal of external Na + (7.04 ±0.05 vs. 7.19±0.02,/;<0.005). Our data show that reduced pH, is a feature of lymphocytes from the SHR that is apparent before and after the development of hypertension. Persistence of reduced pHj in SHR cells, as compared with WKY rat cells, after Na + -H + blockade suggests that a primary abnormality in the activity of the Na + -H + exchanger is unlikely to account for the observed reduction in steady-state pH : of SHR cells. This alteration in pHj regulation persisted after removal of external Na + and was seen in the presence and in the absence of external HCO 3 , indicating that a sodium-independent, bicarbonate-independent mechanism of cytoplasmic acid accumulation accounts for the observed difference in pH,. (Hypertension 1990;15:97-103)

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

“…As we have previously shown, in studies conducted in the absence of HCO 3 -CO 2 , 3 ' 4 -45 the rate of proton extrusion after cell acidification in lymphocytes is virtually identical between SHR and WKY rats. In the presence of HCO 3 , like in its absence, the Na + -H + exchanger has a steep pH| dependency, and there are no significant differences in the activity of this exchanger between SHR and WKY rats (Fig 3). As we have previously shown 34 the steadystate pH, of lymphocytes from SHR was lower than that from WKY rats.…”

“…The procedure of lymphocyte separation was as previously described. 3 The thymus gland was removed from anesthetized rats (5 mg/100 g body wt IP phenobarbital) and placed in RPMI 1640. The thymus was then cleared of blood vessels, minced, pipetted, and filtered through gauze to remove extraneous tissue.…”

“…After Cl" readdition pH, fell rapidly in the first minute, reaching steady state in 4 to 5 minutes (see "Results"). The stimulated activity of the Na + -independent C1"-HCO 3 " exchanger was derived from data obtained in the first 40 seconds of the pH, decline. This pH, recovery was DIDS sensitive and external Cl" dependent but unaffected by either EIPA or Na + removal (see "Results").…”

“…2433 The latter exchanger, like the Na + -H + antiporter, provides a mechanism of defense against cell acidification. Whereas the Na + -H + antiporter extrudes protons in exchange for extracellular sodium, the Na + -dependent d"-HCO 3 " exchanger defends against cell acidification by acting as a pathway for HCO 3 " entry into the cell in exchange for internal chloride. 25 - 29 Both transporters thus regulate pH, and sodium entry into the cell.…”

“…22 Overactrvity of either one or both of these transporters, particularly if accompanied by reduced sodium extrusion via the plasma membrane Na + ,K + -ATPase pump, would explain the elevated levels of intracellular sodium recently reported by us in lymphocytes from the spontaneously hypertensive rat (SHR) 34 and by others in red blood cells of subjects with primary hypertension. 35 In the present study we examined the activity of the Na + -H + antiporter in the presence of HCO 3 -CO 2 as well as the activity and kinetic properties of the Na + -dependent and Na + -independent C1"-HCO 3 " exchangers using freshly isolated lymphocytes from SHR and WistarKyoto (WKY) rats.…”