title: Adenylyl cyclase in SH-SY5Y human neuroblastoma cells is regulated by intra- and extracellular calcium.
abstract: Adenylyl cyclase exists as a family of closely related subtypes which differ in their tissue distribution and regulatory properties. Submicromolar rises in [Ca2+]i produced via activation of phospholipase C (PLC) or Ca2+ channel opening, provide a mechanism by which Ca2+/calmodulin (CaM) or protein kinase C (PKC)-sensitive isoforms of adenylyl cyclase can be regulated. In this study we have examined, in detail, the muscarinic (M3) regulation of adenylyl cyclase in SH-SY5Y cells and report a role for both [Ca2+]e and [Ca2+]i. Carbachol (1 mM) and potassium (100 mM) caused a time (T1/2 = 3 and 4 min, respectively) and dose (EC50 = 6.95 microM and 34.7 mM respectively) related increase in cAMP formation. This amounted to an approximate two-fold increase over basal levels. Carbachol and potassium also caused a biphasic increase in [Ca2+]i with basal, peak and plateau values of 118.4 nM, 697.6 nM, 253.0 nM and 104.0 nM, 351.6 nM, 181.5 nM, respectively. Calcium channel blockade with nickel (2.5 mM) abolished potassium-stimulated cAMP formation and rises in [Ca2+]i. However, carbachol-stimulated cAMP formation was significantly decreased only at the later time points, where rises in [Ca2+]i were also essentially abolished. Further evidence for a role for [Ca2+]e and [Ca2+]i is provided by the stimulation of cAMP formation by carbachol in the absence of added Ca2+, followed by a further increase on its re-addition. Carbachol- and potassium-stimulated cAMP formation were inhibited by the CaM antagonist trifluoperazine (100 microM). The mu-opiate agonists, morphine and fentanyl also inhibited carbachol-stimulated cAMP formation. In addition, cAMP formation in SH-SY5Y cell membranes was significantly increased in the presence of Ca2+ (1.46 microM), CaM (200 nM) and forskolin (1 microM). PKC inhibition with Ro 31 8220 did not affect carbachol-stimulated cAMP formation. Taken collectively, these data suggest that SH-SY5Y cells express type 1, and possibly type 8 isoforms of adenylyl cyclase, which can be regulated by intra- and extracellular Ca2+.
authors.affiliation: University Department of Anaesthesia, Leicester Royal Infirmary, U.K.
authors.forename: R A
authors.initials: RA
authors.lastname: Hirst
authors.forename: D G
authors.initials: DG
authors.lastname: Lambert
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D001285:Atropine / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D009116:Muscarine / Q000494:pharmacology; D009447:Neuroblastoma / Q000201:enzymology*; D011188:Potassium / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000187:drug effects; D014268:Trifluoperazine / Q000494:pharmacology
chemical_list: D011976:Receptors, Muscarinic; D014268:Trifluoperazine; D001285:Atropine; D009116:Muscarine; D002217:Carbachol; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D011188:Potassium; D002118:Calcium