Beta-adrenergic control of cell volume and chloride transport in an established rat submandibular cell line

Rat submandibular cells treated with methylcholanthrene are able to be propagated in continuous culture while retaining beta-adrenergic responsiveness. A specific clone, RSMT-A5, has been isolated and studied in detail. RSMT-A5 cells possess beta-adrenergic receptors (BARS) as judged by [3H]-dihydroalprenolol ([3H]-DHA) binding studies. [3H]-DHA binds to RSMT-A5 membranes in a specific and saturable manner with respect to time and [3H]-DHA concentration. Specific binding is saturable within three min of incubation, and a Scatchard analysis reveals a single class of high affinity binding sites with an equilibrium dissociation constant of 0.62 +/- 0.03 nM and a receptor density of 101 +/- 4 fmole/mg protein. Antagonist competition studies indicate that the BARs are primarily of the beta 2-subtype. The BARs are functional since isoproterenol stimulation results in an increased intracellular cAMP content, marked morphological change, and decreased cell volume and chloride content. These same responses can be evoked by treating RSMT-A5 cells with 8-bromo-cAMP. Ion transport inhibitors such as bumetanide (an inhibitor of Na/K/Cl cotransport), SITS and DIDS (inhibitors of chloride-bicarbonate exchange), amiloride (an inhibitor of Na-H exchange), ouabain (an inhibitor of Na/K-ATPase), and dipyridamole and 9-anthracene carboxylic acid (chloride channel blockers) fail to inhibit the isoproterenol-stimulated change in chloride content. The effects of either isoproterenol or 8-bromo-cAMP on both chloride content and cell volume can be inhibited by the chloride channel blocker N-phenylanthranilic acid, however. Taken together, our results indicate that RSMT-A5 cells possess a beta-adrenergic receptor system which controls intracellular volume and chloride content by modulating transport processes that are 1) cAMP-responsive and 2) inhibitable by the putative chloride channel blocker N-phenylanthranilic acid.