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

In the present study, further evidence is presented for the close rela tionship between fluid secretion and the ratio of the apical H+ over K + concentration gradient. Two agents with a fast and reversible inhibi tory effect on fluid secretion were tested on intracellular and lumina l H+ and K+ concentrations and on transepithelial and transmembrane po tential differences: 6 mM Ba2+, a K+ channel blocker, and 2.10(-4) M, 2,4-dinitrophenol (DNP), a well-known protonophore and uncoupler of ox idative phosphorylation. Ba2+ hyperpolarized the apical membrane poten tial 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 fro m 7.7 +/- 2.4 to 2.8 +/- 0.8 (n = 6). On the other hand, K-c dropped f rom 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 o f 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 aci dification 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 drop ped 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 ad dition 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 fr om 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, we re comparable whether DNP was present or absent. These findings are co nsistent with the hypothesis of an apical K+/H+ antiporter. Ba2+ and D NP reduce the driving force for K+ secretion to the lumen by slowing d own the K+/H+ antiporter. Ba2+ increases the electrical component of t he 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+ concen tration gradient by a double action mechanism. It depletes the