Search

In the present study, the authors have examined the direct actions of angiotensin II (AII) in rabbit renal brush border membrane (BBM) where binding sites for AII exist. Addition of AII (10(-11)-10(-7) M) was found to stimulate 22Na+ uptake by the isolated BBM vesicles directly. All did not affect the Na(+)-dependent BBM glucose uptake, and the effect of AII on BBM 22Na+ uptake was inhibited by amiloride, suggesting the involvement of Na+/H+ exchange mechanism. BBM proton permeability as assessed by acridine orange quenching was not affected by AII, indicating the direct effect of AII on Na+/H+ antiport system. In search of the signal transduction mechanism, it was found that AII activated BBM phospholipase A2 (PLA) and that BBM contains a 42-kDa guanine nucleotide-binding regulatory protein (G-protein) that underwent pertussis toxin (PTX)-catalyzed ADP-ribosylation. Addition of GTP potentiated, while GDP-beta S or PTX abolished, the effects of AII on BBM PLA and 22Na+ uptake, suggesting the involvement of G-protein in AII's actions. On the other hand, inhibition of PLA by mepacrine prevented AII's effect on BBM 22Na+ uptake, and activation of PLA by mellitin or addition of arachidonic acid similarly enhanced BBM 22Na+ uptake, suggesting the role of PLA activation in mediatingmore » AII's effect on BBM 22Na+ uptake. In summary, results of the present study show a direct stimulatory effect of AII on BBM Na+/H+ antiport system, and suggest the presence of a local signal transduction system involving G-protein mediated PLA activation.« less

Increased Na+/H+ antiport activity in the renal brush border membrane of SHR. Defect in renal salt excretion may play an important role in the pathogenesis of hypertension. We examined sodium (Na+) uptake by brush border membrane (BBM) vesicles of young (6 week old) spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) of the same age. SHR had lower urinary Na+ excretion (223.1 ± 9.3 vs. 266.3 ± 3.7 µEq/day/lOO g, N = 8, P < 0.01) and higher systolic blood pressure (98.9 ± 1.2 vs. 82.9 ± 1.8mm Hg, N = 8, P < 0.01) than WKY. BBM vesicle Na+ uptake, measured by rapid filtration technique, was higher in SHR when compared to WKY (1.44 ± 0.03 vs. 1.01 ± 0.06 nmol/mg/5 sec, N = 4, N < 0.01). This increase in Na+ influx was apparent only in the present of an outward-directed proton (H+) gradient and was abolished by 1mM amiloride. BBM permeability to H+ as assessed by acridine orange quenching was not different between SHR and WKY. Kinetic analyses of the amiloride-sensitive BBM Na+ uptake revealed a higher Vmax (2.13 ± 0.27 vs. 0.70 ± 0.30 nmol/mg/5 sec, N = 4, P < 0.01) and a higher km for Na+ (3.55 ± 0.32 vs. 1.23 ± 0.14mM, N = 4, P < 0.05) in SHR. These findings thus demonstrate an intrinsic derangement in BBM Na+ transport in young SHR which is characterized by increased Na+/H+ antiport activity. This alteration in antiport activity is not attributable to changes in membrane permeability to H+, and is characterized by higher Vmax and km. Similar reports of increased Na+/H+ antiport activity in other tissues of SHR suggest that a generalized membrane transport disorder may exist in this model of genetic hypertension.

Intracellular Ca2+ has been suggested to play an important role in the regulation of epithelial Na+ transport. Previous studies showed that preincubation of toad urinary bladder, a tight epithelium, in Ca2+-free medium enhanced Na+ uptake by the subsequently isolated apical membrane vesicles, suggesting the downregulation of Na+ entry across the apical membrane by intracellular Ca2+. In the present study, we have examined the effect of Ca2+-free preincubation on apical membrane Na+ transport in a leaky epithelium, i.e., brush border membrane (BBM) of rabbit renal proximal tubule. In contrast to toad urinary bladder, it was found that BBM vesicles derived from proximal tubules incubated in 1 mM Ca2+ medium exhibited higher Na+ uptake than those derived from proximal tubules incubated in Ca2+-free EGTA medium. Such effect of Ca2+ in the preincubation medium was temperature dependent and could not be replaced by another divalent cation. Ba2+ (1 mM). Ca2+ in the preincubation medium did not affect Na+-dependent BBM glucose uptake, and its effect on BBM Na+ uptake was pH gradient dependent and amiloride (10(-3) M) sensitive, suggesting the involvement of Na+/H+ antiport system. Addition of verapamil (10(-4) M) to 1 mM Ca2+ preincubation medium abolished while ionomycin (10(-6) M) potentiated the effect of Ca2+ in the preincubation medium is likely to be mediated by Ca2+-dependent cellular pathways and not due to a direct effect of extracellular Ca2+ on BBM.(ABSTRACT TRUNCATED AT 250 WORDS)