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Primary cultures of human reabsorptive sweat duct cells were grown in MCDB 170 medium buffered with either HEPES, bicarbonate, or a mixture of HEPES and bicarbonate buffers. Cultures grown in MCDB media containing bicarbonate seemed to differentiate into a multilayered, keratinized epithelium and began senescing after 1 wk in culture. In contrast, cultures grown in media containing HEPES as the only buffer seemed to undergo a selection process, resulting in the outgrowth of cells that did not multilayer or keratinize extensively for up to 3 or 4 wk in culture. Despite marked differences in growth, cells grown in both bicarbonate and HEPES-buffered media retained electrophysiologic characteristics appropriate to the progenitor. Mean resting potentials were -21.8 +/- 0.8 mV (n = 82), -23.3 +/- 1.3 mV (n = 70) and -18.2 +/- 0.8 mV (n = 82) for duct cells grown in HEPES, bicarbonate, and HEPES-bicarbonate media, respectively. Substitution of Cl- with the impermeant anion gluconate in the bathing medium caused membrane potential depolarization in all media, revealing the presence of a Cl- conductance. Administration of the Na+ conductance inhibitor amiloride hyperpolarized the mean resting potential of cells grown in HEPES medium (-6.8 +/- 0.6 mV, n = 68), bicarbonate medium (-6.9 +/- 0.5 mV, n = 60), and HEPES-bicarbonate medium (-5.9 +/- 0.6 mV, n = 69), demonstrating expression of a Na+ conductance. We observed some but minimal variation with age in any of these conditions.

The present investigation was undertaken to examine the usefulness of cultured human sweat duct cells for ion transport and related studies in the genetic disease, cystic fibrosis. Electrical properties of cultured duct (CD) cells were compared with electrical properties of microperfused duct (MPD) cells. The resting apical membrane potential (Va) of the CD cells was -26.4 +/- 0.9 mV, n = 158 cells as compared to -24.3 +/- 0.6 mV, n = 105 of MPD cells. The Na+-K+ pump inhibitor ouabain, when applied to the apical surface of the CD cells and basolateral surface of MPD cells, depolarized both CD cells (from -28.6 +/- 3.6 to -16.8 +/- 2.4 mV, n = 5) and MPD cells (from -23.8 +/- 0.5 mV to -19.5 +/- 1.8 mV, n = 6). The Na+ conductance inhibitor amiloride applied to the apical surface hyperpolarized the apical membrane potentials (Va) of CD cells and MPD cells by -13.2 +/- 1.4 mV, n = 43 and -34.3 +/- 3.1 mV, n = 19), respectively, indicating the presence of amiloride sensitive Na+ channels in both groups of cells. However, the amiloride sensitivity of CD cells was dependent on the age of the culture. Cl- substitution at the apical side by the impermeant anion gluconate depolarized the Va of CD cells and MPD cells by 12.2 +/- 0.9 mV, n = 32 and 37.9 +/- 4.3 mV, n = 12, respectively. The effect of beta-adrenergic agonist isoproterenol (IPR), was inconsistent. In CD cells, IPR either hyperpolarized (delta Va = -8.3 +/- 1.2 mV, n = 5) or depolarized (delta Va = 8.2 +/- 2.3 mV, n = 4) or had no effect, n = 2. In contrast, most of the MPD cells did not respond to IPR, but three cells had a varied response to IPR. Our results suggest that CD cells, like MPD cells, retain significant Na+ and Cl- conductances. CD cells seem to have developed a higher sensitivity to beta-adrenergic stimulation in tissue culture as compared to MPD cells.