Single cells and cell clusters isolated from the swimbladder epithelium of the European eel Anguilla anguilla attached to collagen S-coated petri dishes and proliferated in a modified Dulbecco's modified Eagle's medium, supplemented with 0.5% fetal calf serum. At a temperature of 20-22 degrees C, the growing colonies reached confluence typically within 6-8 days. Activities of glycolytic and pentose phosphate shunt enzymes remained stable or increased only slightly during the first 10 days of primary culture. Incubated in a defined medium providing glucose as a fuel, gas gland cells in primary culture produced and released lactic acid. The rate of acid secretion of cultured gas gland cells measured with a cytosensor microphysiometer was not influenced by cholinergic stimulation. Similarly, the Ca2+ ionophore A-23187 had no effect. Adrenergic stimulation with epinephrine or the beta-agonist isoproterenol also did not increase the rate of acid secretion, indicating that in gas gland cells the metabolic activity cannot be stimulated via beta-adrenergic stimulation followed by an increase in adenosine 3',5'-cyclic monophosphate (cAMP). Artificially increasing the intracellular concentration of cAMP by incubation with forskolin or the cAMP analogue 8-(4-chlorophenylthio)-cAMP even resulted in a marked reduction in the rate of acid secretion. The results demonstrate that primary cell culture provides a useful means for the analysis of metabolic control and of ion transfer processes in swimbladder gas gland cells.
The effects of isoproterenol on the growth of human salivary gland cells in primary culture.
