Muscarinic inhibition of adenylyl cyclase regulates intracellular calcium in single airway smooth muscle cells.
To determine whether muscarinic agonists attenuated isoproterenol-stimulated decreases in intracellular calcium concentration ([Ca2+]i), changes in [Ca2+]i were measured in single airway smooth muscle cells using ratiometric analysis of fura 2 fluorescence. Isoproterenol (10(-5) M) and 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) decreased [Ca2+]i by 24 +/- 3% (P < 0.05, n= 28) and 17 +/- 1% (P < 0.05, n = 6), respectively. The decreased [Ca2+]i in response to isoproterenol was inhibited by propranolol (10(-6) M) and Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) (10-40 microM). In subsequent experiments assessing the effects of muscarinic agonists, isoproterenol did not decrease [Ca2+]i in the presence of carbachol (5 x 10(-8) M) (6 +/- 8% increase; NS, n = 8). To determine the mechanism underlying this inhibitory effect of carbachol, cells were loaded with 4,5-dimethoxy-2-nitrobenzyl adenosine-3',5'-cyclic monophosphate (caged cAMP). For cells loaded with 20 microM caged cAMP, photolysis of caged cAMP decreased basal [Ca2+]i by 28 +/- 3% (P < 0.05, n = 12). In the presence of carbachol (5 x 10(-8) M), photolysis of caged cAMP still induced a 27 +/- 4% decrease in [Ca2+]i (P < 0.05, n = 12). We concluded that a low concentration of carbachol did attenuate isoproterenol-stimulated decreases in [Ca2+]i. Because low concentrations of carbachol attenuated the decreases in [Ca2+]i stimulated by isoproterenol but not the comparable decreases stimulated by cAMP directly, we concluded that the inhibition of adenylyl cyclase activity by muscarinic agonists contributed to the regulation of [Ca2+]i in airway smooth muscle cells. The findings suggested that physiological levels of cholinergic stimulation inhibit adenylyl cyclase, thereby attenuating the effects that beta-adrenergic agonists have on [Ca2+]i.
Madison
J M
JM
Department of Medicine, University of Massachusetts Medical School, Worcester 01655, USA.
Yamaguchi
H
H
D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D018678:Cholinergic Agents / Q000494:pharmacology*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors; D007425:Intracellular Membranes / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D009994:Osmolar Concentration; D014132:Trachea / Q000166:cytology / Q000378:metabolism*
C089981:(2-nitrophenyl)ethyl-cyclic adenosine monophosphate; D000067956:Adenylyl Cyclase Inhibitors; D002121:Calcium Channel Blockers; D018678:Cholinergic Agents; D002217:Carbachol; D000242:Cyclic AMP; D007545:Isoproterenol; D002118:Calcium