Cation transport by sweat ducts in primary culture. Ionic mechanism of cholinergically evoked current oscillations.

摘要

1. The coiled reabsorptive segment of human sweat ducts was cultured in vitro. Cells were then harvested and plated onto a dialysis membrane which was glued over a hole in a small disc. Cultures were maintained in a low serum, hormone-supplemented medium that allowed the cells to grow to confluency. The disc was then placed as a partition between two compartments of a miniature Ussing chamber. The chamber was mounted on the stage of an inverted microscope and intracellular potentials were recorded under transepithelial open-circuit or voltage clamp conditions. All values are given as means +/- S.E.M. and n refers to the number of preparations or duct cells. 2. Under control conditions, the cultured epithelia developed mucosa-negative transepithelial potentials (Vt) ranging from -2.5 to -38 mV (-13.5 +/- 1.5 mV, n = 36). The basolateral membrane potential (Vb) was -39.4 +/- 0.7 mV (n = 50 cells), and the apical membrane potential (Va) was linearly correlated with Vt:Va = 1.0 Vt -39.3 mV (r = -0.78, n = 50). 3. The epithelium generated inwardly directed short-circuit currents (Isc) of 12-95 microA cm-2 (45 +/- 4 microA cm-2, n = 36) with a steady-state intracellular potential. Vc = -31.1 +/- 0.6 mV and a fractional resistance of the apical membrane, fR = 0.59 +/- 0.01 (n = 115 cells). 4. The Na+ channel blocker amiloride (mucosal bath, 10 microM) abolished Isc -0.8 +/- 0.6 microA cm-2), the cells hyperpolarized to -61.0 +/- 1.2 mV, and fR increased to 0.85 +/- 0.01 (n = 44). These effects were fully reversible. 5. During initial stimulation with the cholinergic agonist, methacholine (serosa, 5 or 10 microM), the short-circuit current increased to 80 +/- 10 microA cm-2, the cells hyperpolarized to -55.8 +/- 1.2 mV, and fR increased to 0.82 +/- 0.01 (n = 35). 6. In short-circuited preparations stimulated with methacholine an increase in mucosal potassium concentration ([K+]m) from 5 to 25 mM had no significant effect, while a similar increase in the serosal K+ concentration ([K+]s) produced a change in Vc of 44 +/- 3 mV per log10[K+]s (n = 9). In non-stimulated preparations this change was only 16 +/- 2 mV per log10[K+]s (n = 13). After blocking the apical Na+ channels with amiloride the slope was 24 +/- 5 mV per log10[K+]s in unstimulated preparations.(ABSTRACT TRUNCATED AT 400 WORDS)