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1 Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT, USA; Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA
2 Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT, USA
* To whom correspondence should be addressed. E-mail: larry.renfro{at}uconn.edu.
Active transpeithelial sulfate secretion rate by winter flounder renal proximal tubule epithelium in primary culture (fPTC) is dependent on intracellular carbonic anhydrase (CA) and enhanced by cortisol. To further evaluate this relationship a partial cDNA clone (327 bp) of carbonic anhydrase II (CAII) with high sequence similarity to CAII from numerous species including fish, chicken, and human was obtained from fPTC's. The majority of CA activity and CAII protein was present in the cytosol of fPTC's; however, significant amounts of both (in addition to SDS-resistant CA activity, i.e., CAIV-like isoform) were present in concentrated plasma membranes. CAII from concentrated membranes migrated differently than purified CAII on non-denaturing PAGE gels, suggesting that CAII associates with another membrane component. Treatment of fPTC's with the cell soluble CA inhibitor methazolamide (100 µM) caused a 58% reduction in active transepithelial SO42- secretion. fPTCs that were previously cultured under high cortisol concentrations, when subjected to 5 days of low physiological levels of cortisol, had decreased CA activity (28%), CAII protein abundance (65%), and net active SO42- secretion (28%), with no effect on epithelial differentiation. Methazolamide and low cortisol treatment in combination inhibited net active SO42- secretion 56%, which was not different than the effect of methazolamide treatment alone. These data indicate that cortisol directly increases renal CA activity, CAII protein abundance, and CA-dependent SO42- secretion in the marine teleost renal proximal tubule.
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