The activity of the Na+/H+ exchanger was studied by measuring the effects of intracellular pH (pH(i)) and extracellular Na+ [(Na+)(o)] on pH(i) recovery and Na-22 uptake in rat adipocytes. The resting pH(i) was acidified from 7.30 +/- 0.02 to 6.99 +/- 0.01 with nigericin in the absence of(Na+)(o). pH(i) recovery induced by 30 mM NaCl was blocked by 100 mu M amiloride. The reversibility of the exchanger was studied by Na+ loading, which raised the py from 7.30 +/- 0.02 to 7.50 +/- 0.01, and by removing (Na+)(o), which decreased pH(i) to 6.97 +/- 0.01. Both functions of the exchanger, forward and backward, were inhibited by amiloride. The Na+/H+ exchanger was inactive at pH(i) higher than 7.1 and became increasingly active as pH(i) decreased to 6.2 (Na-22(+) uptake, 0.029 +/- 0.003 vs. 0.155 +/- 0.009 nmol/10(5) cells 2.5 min; P < 0.001); this 5-fold stimulation was largely abolished by amiloride (0.025 +/- 0.002; P < 0.001). Na+ influx was also increased as a function of (Na+)(o), with an apparent K-m of 35 mM. Respective 5- and 44-fold stimulations at 5 mM (0.135 +/- 0.007) and 140 mM (Na+)(o) (1.228 +/- 0.046 nmol/10(5) cells 2.5 min; P < 0.001) were inhibited by ethylisopropylamiloride. Isoproterenol (Iso; 100 nM) and agents that stimulate cAMP production, such as forskolin (10 mu M) and theophyline (1 mM), inhibited the activity of amiloride-sensitive Na-22(+) uptake by 85%. Iso inhibited the Na+/H+ exchanger, without affecting the Na+/K+-adenosine triphosphatase-dependent and the Na+/K+/Cl- cotransport mechanisms. (Bu)(2)cAMP (1 mM), a membrane-permeant cAMP analog, mimicked the effects of Iso on the exchanger. The inhibitory effect of Iso was blocked by propranolol, but not by metoprolol, a beta(1)-antagonist. In addition, the alpha-adrenergic agonists, phenylephrine (alpha(1)) and clonidine (alpha(2)), and the alpha-antagonists, prazocin (alpha(1)) and yohimbine (alpha(2)), did not prevent Iso-induced inhibition of the exchanger. In conclusion, rat adipocytes possess a reversible Na+/H+ exchange mechanism, which is activated by low pH(i) and normal (Na+)(o) and is inhibited by Iso via a beta(2)-adrenergic receptor stimulation and a cAMP-dependent mechanism.
