Proton transport and membrane shuttling in turtle bladder epithelium

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Abstract
Summary Proton secretion in the urinary bladder of the freshwater turtle is mediated by proton pumps located in the apical membrane of carbonic-anhydrase (CA)-rich cells. It has been proposed that the rate of proton transport is regulated by endocytotic and exocytotic fusion processes which alter the apical membrane area, and hence number of exposed pumps. Three techniques were used to study this process. Analyses of transepithelial impedance provided estimates of transport-associated changes in net membrane area, as well as other electrical parameters. Electron microscopy allowed visualization of the endocytotic vesicles thought to be involved in the process. Finally, uptake of a florescent fluid-phase markerprovided measurements of the rates of endocytosis. We report the following: (i) endocytotic and exocytotic processes occur primarily in the CA-rich cells; (ii) inhibition of proton transport resulting from 0.5mm acetazolamide (AZ) results in a decrease in the apical membrane area of approximately 0.47 cm2/cm2 tissue; (iii) the apical membrane specific conductance of the CA-rich cells is approximately 220 μS/μF, and possibly represents a Cl− conductance that may function in counter-ion flow; (iv) the decline in transport following AZ is not directly proportional to the decline in apical membrane area, suggesting that changes in pump kinetics are also involved in the regulation of transport; (v) the CA-rich cells exhibit a high rate of constitutive pinocytosis, and hence membrane shuttling, which appears to be independent of the rate of transport; (vi) AZ induces a transient increase in the rates of endocytosis and shuttling; and (vii) the transport-associated changes in apical membrane area may reflect an effect of AZ on a regulated endocytotic pathway which is distinct from the pinocytotic process.