Polarization of adenosine effects on intracellular pH in A6 renal epithelial cells

Abstract

The effect of adenosine on Na+/H+ exchange activity was examined in cultured A6 renal epithelial cells. Adenosine and its analogue N6-cyclopentyladenosine (CPA) had different effects on Na+/H+ exchange activity depending on the side of addition. Basolateral CPA induced a stimulation of Na+/H+ exchange activity that was completely prevented by preincubation with an A2A-selective antagonist, 8-(3-chlorostyryl)caffeine, whereas apical CPA induced a slight but significant inhibition of Na+/H+ exchange activity that was significantly reduced by the A1-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine. Protein kinase C activation may be involved in mediating the apical CPA inhibition of Na+/H+ exchange activity; this inhibition was prevented by the protein kinase C inhibitor calphostin C. Treatment with either forskolin or 8-bromo-cAMP significantly stimulated Na+/H+ exchange activity; only basolateral CPA addition induced an increase in cAMP level. These observations together with the finding that the CPA-dependent stimulation of exchange activity was prevented by the protein kinase A inhibitor H-89 support the hypothesis that basolateral CPA stimulates Na+/H+ exchange via adenylate cyclase/protein kinase A activation. Basolateral CPA also increased transepithelial Na+ transport, and this stimulation was prevented by the Na+/H+ exchange inhibitor HOE-694, suggesting that changes in pHi during hormone action can act as an intermediate in the second-messenger cascade.

Fig. 1.

Representative traces depicting the effect of 10⁻⁶ m CPA on Na⁺/H⁺ exchange activity in A₆ monolayers. Na⁺/H⁺ exchange activity was assayed as the initial rate of Na⁺-dependent pH_i recovery induced by the basolateral addition of 22 mM Na⁺ after intracellular acidification via superfusion with NH₄⁺ medium followed by Na⁺-free TMA medium (see Experimental Procedures). First trace, absence of CPA. Second trace, obtained in the same monolayer after a 20-min preincubation with CPA. A, CPA was added only to the apical side. B, CPA application was only to the basolateral side of the monolayer. pH_i corresponds to the nigericin calibration performed as described in Experimental Procedures.

Fig. 2.

Effect of apical or basolateral CPA addition on the Na⁺/H⁺ exchange activity. Experiments were performed according to the protocol described in the legend to Fig. 1. Data are presented as percentage of control values. Columns, mean ± standard error of five experiments. ***, p < 0.001.

Fig. 3.

Effect of increasing concentration of CPA on Na⁺/H⁺ exchange activity. Experiments were performed at increasing concentrations of CPA according to the protocol described in the legend to Fig. 1. The basolateral side of A₆ monolayers was exposed to CPA for 20 min before analysis of pH_i recovery from an acid load. Columns, mean ± standard error of the experiments made at different CPA concentrations (10⁻⁶ M, seven experiments; 10⁻⁷ M, three experiments; 10⁻⁸ M, three experiments; 10⁻⁹ M, four experiments). **, p < 0.01; ***, p < 0.001.

Fig. 4.

Effect of various Na concentrations on CPA-dependent stimulation of Na⁺/H⁺ exchange. Experiments were performed according to the protocol described in the legend to Fig. 1 at different sodium concentrations (11 mM, seven experiments; 22 mM, five experiments; 37 mM, three experiments; 55 mM, seven experiments; 110 mM, six experiments) in the absence (□) or presence (○) of 10⁻⁶ m CPA. Na⁺ activation of basolateral Na⁺/H⁺ exchange followed simple Michaelis-Menten kinetics. The apparent K_m values were 40 ± 5.6 mM Na⁺ in the absence and 12 ± 3.9 mM Na⁺ in the presence of CPA.

Fig. 5.

Effect of the A₁-selective antagonist CPX and the A_2A-selective antagonist CSC on basolateral CPA stimulation of the Na⁺/H⁺ exchanger. Experiments were performed according to the protocol described in the legend to Fig. 1. The A₁ adenosine-selective antagonist CPX (10⁻⁷ M) or the A_2A antagonist CSC (10⁻⁷ M) was added to the basal solution 5 min before stimulation with CPA (10⁻⁶ M). Columns, mean ± standard error of five experiments. ***, p < 0.001, compared with the value for CPA alone.

Fig. 6.

Effect of the the pharmacological activation of several regulatory pathways on Na⁺/H⁺ exchange activity. Paired measurements of initial rate of pH_i increase induced by the basolateral superfusion of 22 mM Na⁺ medium to acid-loaded cells in the absence (control; open columns) or presence of the indicated agents were performed as described in the legend to Fig. 1. Cell monolayers were exposed to various agents from both the apical and basolateral side for different times: forskolin (FSK) and 8-bromo-cAMP for 10 min, ionomycin (Iono) for 2 min, and TPA for 5 min. Values are mean ± standard error of five experiments for the forskolin, four experiments for 8-bromo-cAMP, three experiments for TPA, and four experiments for ionomycin determinations. **, p < 0.01; ***, p < 0.001.

Fig. 7.

Effect of preincubation with the antagonists CPX and CSC on the basolateral CPA-induced cAMP generation. A₆ monolayers were preincubated with antagonists 5 min before the addition of CPA (10⁻⁶ M) to the basolateral side of the monolayers. Levels of intracellular cAMP was determined as described in Experimental Procedures. Columns, mean ± standard error of three experiments. ***, p < 0.001, compared with the value for CPA alone.