BOTH DINITROPHENOL AND BA2+ REDUCE KCL AND FLUID SECRETION IN MALPIGHIAN TUBULES OF FORMICA-POLYCTENA - THE ROLE OF THE APICAL H+ AND K+ CONCENTRATION GRADIENT

Abstract

In the present study, further evidence is presented for the close relationship between fluid secretion and the ratio of the apical H+ over K+ concentration gradient. Two agents with a fast and reversible inhibitory effect on fluid secretion were tested on intracellular and luminal H+ and K+ concentrations and on transepithelial and transmembrane potential differences: 6 mM Ba2+, a K+ channel blocker, and 2.10(-4) M, 2,4-dinitrophenol (DNP), a well-known protonophore and uncoupler of oxidative phosphorylation. Ba2+ hyperpolarized the apical membrane potential difference (V-ap, lumen reference) from -59 +/- 3 to -90 +/- 6 mV (n = 6) and the basal membrane potential difference (V-bl) from -23 +/- 3 to -74 +/- 7 mV (n = 6). At the same time, the cell acidified (H-c increased from 18 +/- 4 to 50 +/- 21 nM and the lumen alkalinized (H-1 decreased from 117 +/- 23 to 74 +/- 11 nM); H-l/H-c was reduced from 7.7 +/- 2.4 to 2.8 +/- 0.8 (n = 6). On the other hand, K-c dropped from 85 +/- 6 to 73 +/- 6 mM (n = 9) and K-1 from 143 +/- 3 to 121 +/- 1 mM (n = 5); consequently K-l/K-c remained unchanged (i.e. 1.7 +/- 0.1). As a result, the H-l/H-c over K-l/K-c ratio decreased from 4.5 to 1.7. DNP depolarized V-ap from -63 +/- 7 to -26 +/- 3 mV (n = 8); V-bl slightly depolarized from -21 +/- 1 to 20 +/- 1 mV; In the presence of 6 mM Ba2+, V-ap and the basal membrane potential difference (V-bl) (bath reference) depolarized from -81 +/- 5 to 1 +/- 2 mV and from -68 +/- 6 to 1 +/- 1 mV, respectively (n = 5) when applying DNP. Like Ba2+, the addition of 2.10(-4) M DNP to the control solution caused an acidification of the cytosol (H-c rose from 24 +/- 5 to 81 +/- 9 nM); H-1 was not significantly changed (i.e. 80 +/- 12 and 80 +/- 9 nM in the absence and presence of DNP, respectively). Consequently, H-l/H-c dropped from 3.0 +/- 0.7 to 1.0 +/- 0.2 (n = 8). K-c diminished from 104 +/- 9 to 80 +/- 5 mM and K-l from 141 +/- 8 to 93 +/- 6 mM after the addition of DNP; K-l/K-c was not significantly changed (i.e. 1.4 +/- 0.1 and 1.2 +/- 0.2 in the absence and presence of DNP, respectively, n = 6). The overall result was a reduction of the ratio (H-l/H-c over K-l/K-c) from 2.1 to 0.8. On the other hand, the sensitivity of V-bl and V-ap to a change in bath K+ from 5 to 51 mM was virtually unchanged from control in the presence of 2.10(-4) MDNP: V-bl depolarized with 33 +/- 1 and 32 +/- 1 mV, and V-ap with 19 +/- 3 and 22 +/- 1 mV, in the absence and presence of DNP, respectively. Furthermore, the transient changes of V-bl on varying the bath K+, suggesting a change in K-c, were comparable whether DNP was present or absent. These findings are consistent with the hypothesis of an apical K+/H+ antiporter. Ba2+ and DNP reduce the driving force for K+ secretion to the lumen by slowing down the K+/H+ antiporter. Ba2+ increases the electrical component of the proton motive force of the electrogenic H+ pump, thereby decreasing the free energy for building up a proton concentration gradient. DNP inhibits the realization of an apical lumen to cell directed H+ concentration gradient by a double action mechanism. It depletes the