Yamaki, M.

We have studied localization of mRNAs coding isozymes of rat plasma membrane Ca(2+)-adenosinetriphosphatase pump (rPMCA) in the rat kidney, with use of reverse transcription (RT) with subsequent amplification by polymerase chain reaction (PCR). When zones of the kidney were separated by macrodissection, a large amount of mRNA coding isozyme rPMCA1 was found in all zones; mRNA for isozyme rPMCA2 was abundant in cortex and in outer medulla, and mRNA for isozyme rPMCA3 was prominent in outer medulla. The mRNAs were analyzed in microdissected cortical nephron segments by use of RT-PCR approach described previously [T. Moriyama, H. R. Murphy, B. M. Martin, and A. Garcia-Perez. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F1470-F1474, 1990]. We detected mRNA for isozyme rPMCA2 in microdissected distal convoluted tubules (DCT) and in cortical thick ascending limbs (CTAL) and, less consistently, also in proximal convoluted tubule and in glomeruli. The mRNA for isozyme rPMCA1 was abundant in glomeruli but was absent in all examined cortical tubular segments. Our results document that mRNAs for all three major isozymes of rPMCA are present and show a unique distribution in the three major zones of rat renal parenchyma. Specific mRNA coding for rPMCA2 was detected in cortical tubules, namely in CTAL and DCT, whereas mRNA coding isozyme rPMCA1 was found in glomeruli. We suggest that isozyme rPMCA2 might be specifically related to epithelial cells and their function, whereas rPMCA1 is probably a component of nonepithelial cells including these in glomeruli.

We studied cyclic 3',5'-nucleotide phosphodiesterase (PDE) isozymes and their role in adenosine 3',5'-cyclic monophosphate (cAMP) and cGMP metabolism in a rat inner medullary collecting duct (IMCD) cell line. The homogenized and fractionated IMCD cells of cAMP-PDE and all of cGMP-PDE activity were found in the cytosol. The majority of cytosolic cAMP-PDE (greater than 50%) was isozyme PDE-IV; the Ca(2+)-calmodulin-sensitive PDE-I was present only in cytosol. Preincubation of IMCD cells with PDE-IV inhibitor rolipram markedly (5x) enhanced levels of cAMP both basal and in the presence of [Arg8]vasopressin (AVP). Cilostamide (for PDE-III) or vinpocetine had no effect, whereas PDE-I inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MeoM-IBMX) enhanced AVP-dependent cAMP levels. Exposure of IMCD cells to 2 microM ionomycin decreased both basal and AVP-stimulated cAMP. Depletion of Ca2+ by preincubation of IMCD cells in the Ca(2+)-free medium with ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid markedly enhanced the stimulatory response of cAMP to AVP, and addition of 8-MeoM-IBMX further enhanced the AVP response. The levels of cGMP, basal or in response to atriopeptin (ANP), were not affected by PDE-V inhibitor zaprinast, but both inhibitors of PDE-I, 8-MeoM-IBMX and vinpocetine, increased basal cGMP, and 8-MeoM-IBMX also increased cGMP levels enhanced by ANP. The depletion of Ca2+ from IMCD cells alone had no effect on cGMP levels, but effects of 8-MeoM-IBMX and vinpocetine on the ANP-stimulated cGMP levels were enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)

In our previous study of plasma membrane Ca2+ pump (PMCA) isozymes in the rat kidney, we found that the mRNA coding isoform PMCA3 was detected primarily in the outer medullary zone of rat kidney tissue. We now investigated the location of the mRNAs coding for isoforms PMCA3 and PMCA4 of Ca2+ pump in the nephron segments that are present in outer medullary parenchyma using the method of reverse transcription and polymerase chain reaction. In mRNA extracted from whole dissected outer medulla we detected mRNA encoding three splice variants (a, b, and c) of isoform PMCA3; isoform PMCA4 was found in the outer medulla almost exclusively as variant b. Analysis of mRNA from microdissected tubule segments show that proximal straight tubules (PST), medullary thick ascending limbs, outer medullary collecting ducts, and descending thin limb of Henle's loop (DTL) all contained mRNA for isoform 4b. In contrast, the mRNA encoding isoform 3 was detected exclusively in DTL and not in other nephron segments. The unique presence of isoform 3 in DTL is rather surprising, since the specific role of this nephron segment in vectorial Ca2+ transport or in intracellular Ca2+ signaling is not known. The data suggest that isoform 3 in cells of the DTL may have a hitherto unrecognized specific role in Ca2+ signaling or transport of Ca2+, which is distinct from the role of the isoforms of the PMCA in all other nephron segments.