The proton/sodium antiporter (exchanger) in the oocyte membrane of Dysdercus intermedius is electrogenic (2H +/Na +) and causes perioocytic proton accumulation

Abstract

Vitellogenic follicles of Dysdercus intermedius (Heteroptera: Pyrrhocoridae) were treated with sodium azide (NaN 3) or 2,4 dinitrophenol (DNP) in order to load the ooplasm with protons along their electrochemical gradient. Ooplasmic pH ( pH OOC ) was recorded using proton-specific microelectrodes. Treatment for six min with 0.5 mM of DNP (dissolved in physiological saline solution; PSS) resulted in acidification of the ooplasm from 7.41±0.05 in PSS to pH OOC(DNP) =7.09±0.04. Immersing follicles in PSS after DNP treatment resulted in reactivation of a proton/sodium antiporter and recovery of the initial pH OOC . Additionally, the proton-specific microelectrodes were placed at a distance of ≈10 μm from the surface of the vitellogenic follicle. The extracellular pH ( pH EX ) was measured before ( pH EX(PSS) ), during ( pH EX(DNP) ) and after ( pH EX(PSS) ) DNP treatment. Along the lateral surface of the follicle, the recorded pH EX(PSS) was initially 6.79±0.02, similar to the pH of the medium ( pH MED =6.80; recorded at a distance of 300 μm from the surface of the follicle) and higher than the pH EX(PSS) of 6.52±0.03 measured in the interfollicular constriction between individual vitellogenic follicles (interfollicular region). During DNP treatment, values changed to 6.80±0.03 in the constriction and 6.80±0.01 along the lateral surface. After removal of DNP the initial control pH values were reestablished. These extrafollicular H + distributions fit into a model of extrafollicular currents reported earlier for D. intermedius. Proton distribution between the ooplasm and the medium was also affected in the presence of 5 mM NaN 3, resulting in a drop in ooplasmic pH from 7.40±0.05 down to pH OOC(NaN3) =7.07±0.03. Changes in cytosolic proton activities after DNP or NaN 3 treatment were evidenced by monitoring both the increase in ooplasmic pH (Δ pH) and, simultaneously, the change in the resting potential (Δ Em). Recovery of the ooplasmic pH depended on the transfer of ≈6×10 9 H +/ oocyte (after DNP treatment) or ≈3×10 9 H +/ oocyte (after NaN 3 treatment), whereas recovery of Em by charging the capacitance of the oocyte membrane could be attributed to a net efflux of ≈3×10 9 H +/ oocyte (after DNP treatment) or ≈1.7×10 9 H +/ oocyte (after NaN 3 treatment). In the light of previous reports on the monensin-sensitive proton/sodium antiporter (external Na + for ooplasmic H +), the operating efficiency of this antiporter is 2H +/Na +. Vitellogenesis during and after DNP treatment was demonstrated by the accumulation of fluorescence labelled hemolymph proteins in yolk spheres in the cortex of the oocyte: vitellogenesis came to a halt in PSS containing DNP when the ooplasm was acidified and no H + accumulation around the follicle was detectable. Vitellogenesis stopped under the condition of DNP MED =0.5 mM, but resumed again by exchanging the medium for PSS without DNP. Simultaneously with the appearance of the regular pH OOC(PSS) =7.40±0.03 (efflux of H + out of the ooplasm), extrafollicular proton accumulation by H + influx into the constriction reappeared within 10 minutes. The results obtained with proton-specific microelectrodes and the in vitro assay to detect vitellogenesis indicate that electrogenic H + extrusion out of the ooplasm plays an important role in both maintaining the ooplasmic pH 0.6 units above pH MED =6.8 and in the generation of the external current pattern. A model is discussed explaining the acidification of endosomes as a prerequisite for endosomal processing leading to yolk spheres.