Frogs are faced with various osmoregulatory problems, such as compensation of salt and water loss or metabolic acidification. Being exposed both to air and to pond water of low salinity in their natural habitat, the epithelium of the frog skin serves as one of the major organs for body fluid homeostasis. For years, the frog skin has been the guiding model for ion transport processes in animal cells energized by a Na(+)-motive force. Meanwhile, however, it was demonstrated that under natural conditions Na+ uptake is electrically coupled to active H+ secretion, mediated by an electrogenic H+ pump. A proton-motive force generated at the apical membrane of the mitochondria-rich cells (MR cells) energizes Na+ entry via apical Na+ channels. The basolateral Na+/K+ P-ATPase then pumps Na+ out of the cell into the body fluid. Thus, there are two pumps functioning in series, both involved in transepithelial Na+ transport. Our recent investigations provided conclusive evidence that the H+ pump of the frog skin is an H+ V-ATPase. In transport studies, Na+ absorption and H+ secretion were blocked by micromolar concentrations of bafilomycin A1 or concanamycin A, two highly specific inhibitors of H(+)-V-ATPases. Using immunofluorescence microscopy, H(+)-V-ATPase-like immunoreactivity was found in MR cells in the region of their apical membrane foldings and intracellularly in the apical portion of the cell at so far unidentified locations. Besides the definition of its molecular nature, these results also confirmed the localization of the H+ pump in the apical membrane of the MR cells. These cells were already candidates for H(+)-V-ATPase localization mostly from correlations between their morphological features and their epithelial H+ secretion capacity. So far, there is evidence for only one type of MR cell serving both H+ and HCO3- secretion through an apical Cl-/HCO3- antiporter. H(+)-V-ATPase-mediated H+ secretion and thus Na+ absorption can be modulated by complementary mechanisms. Changes in intracellular H+ concentration linked to the animal's acid-base status will directly influence H+ V-ATPase activity. Acute acidification increases H+ current, probably as a result of the insertion of H(+)-V-ATPase-bearing vesicles by exocytotic processes, while alkalization causes the reverse effects. Chronic metabolic acidosis induces an increase in MR cell number in response to hormonal signals.

The spatial and temporal distribution of the plasma membrane V-ATPase and its encoding mRNA in the midgut of Manduca sexta were investigated during the moult from the fourth to the fifth larval instar. Digoxigenin-labelled RNA probes were used for in situ hybridization of V-ATPase mRNA of both peripheral and integrated subunits; monoclonal antibodies to subunits of the peripheral sector of the purified plasma membrane V-ATPase were used for immunocytochemistry. Extensive mRNA labelling was found in both mature columnar and goblet cells of intermoult and moulting larvae. Hybridization screening in several tissues suggested that only cells with increased V-ATPase biosynthesis were labelled by our hybridization method. Mature goblet cells contain a large amount of V-ATPase in the apical plasma membrane and were therefore expected to contain V-ATPase mRNA. The intense mRNA signal found in mature columnar cells was unexpected. However, after refining the techniques of tissue preparation, immunolabelling in apical blebs of columnar cells was demonstrated. Since this immunoreactivity did not appear to be membrane-associated, it suggested a cytosolic localization of peripheral V1 subunits. The mRNA encoding subunit A of the peripheral V1 sector was distributed unevenly in columnar cells with a strong apical preference, whereas the mRNA for the proteolipid of the integral V0 sector was evenly distributed in the cytosol. This spatial pattern reflected the distribution of free ribosomes and rough endoplasmic reticulum in the cell, supporting the view that V1 subunits are synthesized at free ribosomes, whereas the V0 subunits are synthesized at the rough endoplasmic reticulum. All undifferentiated cells exhibited intense mRNA signals for V-ATPase subunits of both holoenzyme sectors from the start of proliferation and thus precursors of columnar and goblet cells could not be distinguished.

Active electrogenic K+ transport in insects serves as the energy source for secretion or absorption in gastrointestinal epithelia or for the receptor current in sensory epithelia. In the larval midgut of the tobacco hornworm Manduca sexta, a vacuolar-type proton pump (V-ATPase) and a K+/nH+ antiport represent the functional elements of the potassium pump. Several immunological findings support the hypothesis that active K+ transport in other insect epithelia may also be energized by a V-ATPase. In immunoblots, crude homogenates of sensilla-rich antennae and Malpighian tubules of M. sexta cross reacted with an immune serum directed to the purified plasma membrane V-ATPase from the midgut; the M. sexta midgut V-ATPase cross reacted with polyclonal antibodies to endomembrane V-ATPases from xenic origin. In immunocytochemical investigations of larvae of M. sexta and adults of Antheraea pernyi, monoclonal antibodies to defined subunits of the purified midgut V-ATPase or polyclonal antibodies to xenic endomembrane V-ATPase labelled the sites of active K+ transport: the goblet cell apical membrane in the midgut, the brush border of Malpighian tubules and the apical projections of the auxiliary cells in antennal sensilla. The functional mechanism of a primary H+-pumping V-ATPase and a secondary H+-dependent K+ transport postulated for K+-transporting insect epithelia may be further applicable to active Na+ or Cl- transport and would provide a unifying concept for all ouabain-insensitive electrogenic ion transport in insects. The findings from the midgut investigations, however, are the first instance in which a V-ATPase provides an alternative to the Na+/K+-ATPase in energizing secondary active transport in animal plasma membranes.

In immunobiochemical blots, polyclonal antibodies against subunits of plant and mammalian vacuolar-type ATPases (V-ATPases) cross-react strongly with corresponding subunits of larval Manduca sexta midgut plasma membrane V-ATPase. Thus, rabbit antiserum against Kalanchoe daigremontiana tonoplast V-ATPase holoenzyme cross-reacts with the 67, 56, 40, 28 and 20 kDa subunits of midgut V-ATPase separated by SDS-PAGE. Antisera against bovine chromaffin granule 72 and 39 kDa V-ATPase subunits cross-react with the corresponding 67 and 43 kDa subunits of midgut V-ATPase. Antisera against the 57 kDa subunit of both beet root and oat root V-ATPase cross-react strongly with the midgut 56 kDa V-ATPase subunit. In immunocytochemical light micrographs, antiserum against the beet root 57 kDa V-ATPase subunit labels the goblet cell apical membrane of both posterior and anterior midgut in freeze-substituted and fixed sections. The plant antiserum also labels the apical brush-border plasma membrane of Malpighian tubules. The ability of antibodies against plant V-ATPase to label these insect membranes suggests a high sequence homology between V-ATPases from plants and insects. Both of the antibody-labelled insect membranes transport K+ and both membranes possess F1-like particles, portasomes, on their cytoplasmic surfaces. This immunolabelling by xenic V-ATPase antisera of two insect cation-transporting membranes suggests that the portasomes on these membranes may be V-ATPase particles, similar to those reported on V-ATPase-containing vacuolar membranes from various sources.