Isoproterenol and cyclic AMP increase intracellular free [Ca] in MDCK cells.
We examined the relationship between cyclic AMP and the intracellular free calcium concentration ([Ca]i) in MDCK cells, a hormonally responsive and chloride-secreting cell line. We measured [Ca]i using the calcium-sensitive dye fura-2, fluorescence microscopy, and a silicon intensifier target camera to amplify the signal. Isoproterenol, known to stimulate chloride transport via cyclic AMP, increased [Ca]i from 112 +/- 17 to 373 +/- 53 nM. The rise appeared due to an increase in cyclic AMP: isobutylmethylxanthine enhanced the effect of a submaximal dose of isoproterenol on [Ca]i, the cyclic AMP analogue 8-bromo-cyclic AMP caused [Ca]i to increase from 100 +/- 8 to 278 +/- 40 nM, and direct activation of adenylate cyclase with forskolin increased [Ca]i from 192 +/- 29 to 279 +/- 35 nM. The rise in [Ca]i after cyclic AMP may be due to an influx of calcium from the outside: the cyclic AMP-induced increase in [Ca]i was prevented either by lowering extracellular [Ca] or by addition of 1 mM lanthanum. The mechanism by which calcium enters the cell may not be a calcium channel because neither verapamil nor nitrendipine prevented cyclic AMP from increasing [Ca]i. Cyclic AMP also does not appear to act directly on sodium-calcium exchange. [Ca]i increased as well in low [Na] as in high [Na] following addition of the nucleotide. Thus, isoproterenol, acting through an increase in [cyclic AMP] causes an increase in [Ca]i in MDCK cells. The source and route of entry of calcium into the cytoplasm remain uncertain.
Chase
H S
HS
Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Wong
S M
SM
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology*; D009994:Osmolar Concentration; D019831:Sodium-Calcium Exchanger
D002352:Carrier Proteins; D019831:Sodium-Calcium Exchanger; D000242:Cyclic AMP; D007545:Isoproterenol; D002118:Calcium