In freshly dissociated rhesus palm eccrine clear cells, regulatory volume increase (RVI) was studied using image analysis as a measure of Na-K-2Cl cotransport activity. Pseudo-RVIs, as well as RVI during methacholine (MCh)-induced cell shrinkage, were observed in clear cells and were inhibited by 100 microM bumetanide or in Na-free medium, but were not inhibited by amiloride or ouabain. RVI in hypertonic medium and RVI after MCh-induced cell shrinkage were accelerated by adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents (forskolin+isoproterenol) and inhibited by phorbol ester. RVI in hypertonic medium was enhanced by a phosphatase inhibitor, okadaic acid. mRNA for Na-K-2Cl cotransporter (NaKCC) was demonstrated in freshly isolated rhesus sweat secretory coils by polymerase chain reaction (PCR) after reverse transcription using a set of primers derived from the published human NaKCC (hNaKCC) 1 sequence, i.e., nucleotides 2,043-2,810. The deduced amino acid sequence of the PCR-amplified 767-base pair segment was identical to that of hNaKCC 1 from a human colon cell line (T84). The data are interpreted to indicate that NaKCC, showing strong homology to secretory type hNaKCC 1, is present in rhesus eccrine secretory coils and may participate in the cotransport component of eccrine sweat secretion and cell volume regulation, especially during cholinergic stimulation. The data also raise the possibility that sweat gland NaKCC may be upregulated by cAMP-mediated protein phosphorylation and downregulated by protein kinase C.

No abstract