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 [Ca 2+] i produced via activation of phospholipase C (PLC) or Ca 2+ channel opening, provide a mechanism by which Ca 2+/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 (M 3) regulation of adenylyl cyclase in SH-SY5Y cells and report a role for both [Ca 2+] e and [Ca 2+] i. Carbachol (1 mM) and potassium (100 mM) caused a time ( T 1 2 = 3 and 4 min , respectively) and dose (EC 50 = 6.95 μM 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 [Ca 2+] 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 [Ca 2+] i. However, carbachol-stimulated cAMP formation was significantly decreased only at the later time points, where rises in [Ca 2+] i were also essentially abolished. Further evidence for a role for [Ca 2+] e and [Ca 2+] i is provided by the stimulation of cAMP formation by carbachol in the absence of added Ca 2+, followed by a further increase on its re-addition. Carbachol- and potassium-stimulated cAMP formation were inhibited by the CaM antagonist trifluoperazine (100 μM). The μ-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 Ca 2+ (1.46 μM), CaM (200 nM) and forskolin (1 μM). 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 Ca 2+.