M-rk_et_al-1995-Cell_Biology_International

increases in cellular chloride and potassium permeability that underlie secretion (petersen, 1992; Wilson et al., 1994). Adrenergic agonists evoke secretion in human sweat glands by interacting both with a- and fj-adrenoceptors, although these responses are generally smaller than the response to acetylcholine. o-Adrenoceptor agonists act by evoking phoshatidylinositol-4,5-biphosphate(pIP,) hydrolysis and so increasing intracellular free calcium ([Ca2 +]J , whereas fj-adrenergic agonists activate adenylate cyclase and hence the effects of these agents are thought to be mediated by cyclic AMP (Sato et al., 1991). ATP can evoke secretion from isolated sweat glands, although the pharmacology of this response is not known (Sato et al., 1991; Wilson et al., 1994). ATP can increase [Ca2 +);, the release of calcium from intracellular stores and subsequently sustained calcium influx. In equine sweat glands, it has been shown that extracellular ATP (at 100 JtM) can also
Department of HU11Uln Anatomy, University of Uppsala, Box 571, 5-75123 Uppsala, Sweden.
*Corresponding author.
ABSfRACT
Chloride secretion in primary cultures of cells originating from the secretory coil of human sweat glands was investigated by electron probe X-ray microanalysis. The total intracellular a concentration was lowered by muscarinic agonists (carbachol and acetylcholine), as well as by the calcium ionophore A23I87. The muscarinic agonists also lowered the cellular K concentration. a- secretion induced by these agonists could be inhibited by the chloride channel blocker NPPB. After cAMP stimulation, the frequency distribution of the Cl concentration changed from Gaussian to bimodal, indicating that cAMP induces Ct secretion only from a subpopulation of the cells. Also ATP stimulated Ct secretion, indicating the presence ofpurinergic receptors. The results suggest that some ofthe cells in addition to Ca"-regulated a- channels also possess cAMP-activated Ct channels. Hence, the primary cultures still possess the Ct transport mechanisms known to be present in intact glands. It can, however, not be excluded that some coil cells have acquired ductal characteristics during culture.
INTRODUCTION Ion transport in human sweat glands has recently received relatively much attention, mainly because sweat glands provide an easily accessible model organ for the study of epithelial chloride transport. This is of special relevance in the disease cystic fibrosis, where the activation of chloride transport by cAMP is defective, which results in elevated concentrations of NaCI in the sweat of these patients (Quinton, 1990). The secretory coil cells of the human sweat gland secrete an isotonic NaCI solution into the lumen. This primary sweat flows along the length of the duct where part of the salt is reabsorbed before the sweat reaches the skin surface. Sweating is primarily regulated by acetylcholine, which acts via muscarinic receptors that are functionally coupled to phospholipase C. This evokes the release of calcium from intracellular stores. This increase in free intracellular calcium is sustained by calcium influx from the extracellular compartment and results in
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raise the cellular cAMP content (Ko et al., 1994). Secretion of primary sweat generally conforms to a secondary active chloride (Cl) transport mechanism, where chloride enters the cell due to the combined activities of three basolateral membrane transport mechanisms: potassium (K+) channels, Na+-K+-2CIcotransporters and Nat/Ktpumps, ClIeaves the cell across the luminal membrane, presumably via CIchannels, down its electrochemical potential gradient. In the duct, chloride is reabsorbed via the so-called cystic fibrosis transmembrane conductance regulator (CFTR), a channel that is regulated by cAMp-{jependent phoshorylation and by intracellular ATP, and possibly, Ca2+ ions. Human sweat glands can be grown in primary cultures, either from whole sweat glands or from isolated ducts or coils (pedersen, 1984; Pedersen et al., 1985; Brayden and Cuthbert, 1990). Coil cells appear to have a pluripotential capacity which is revealed in culture since both whole-gland and secretory coil cell cultures exhibit some properties usually associated in vivo with duct cells (Brayden et al., 1991). By infection with SV40 virus an immortalized cell line (NCL-SG3) has been constructed where CI- transport can be activated by l3-adrenergic agonists (Lee and Dessi, 1989). Lee and Dessi (1989) found no activation by cholinergic agonists, in contrast to recent measurements in our laboratory (Ring, Mork and Roomans, submitted for publication) where cholinergic stimulation of Cl transport could be observed in cells grown on a permeable substrate. X-ray microanalysis can be used for the study of ion transport in cultured cells. The method has the advantage that changes in several different ions can be studied simultaneously, and that inhomogeneities in cell cultures can be easily revealed (von Euler et al., 1993). Most work of this kind has been carried out on cancer cells or transformed cell lines, but a few studies have been carried out on primary cultures (Sagstrorn et aI., 1992; Warley et al., 1994). Recently, we have studied ion transport in NCL-SG3 cells, and demonstrated that CI- transport can be activated both by cAMP analogues (Mork and