Inhibition of M3 muscarinic acetylcholine receptor-mediated Ca2+ influx and intracellular Ca2+ mobilization in neuroblastoma cells by the Ca2+/calmodulin-dependent protein kinase inhibitor 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-trosyl]-4-phenylpiperazin e (KN-62).
The role of Ca2+/calmodulin-dependent protein kinase (CaM kinase; EC 2.7.1.123) in the generation of Ca2+ signals by muscarinic acetylcholine receptors (mAChR) was studied. Changes in intracellular Ca2+ concentrations ([Ca2+]i) induced by mAChR activation were monitored in SK-N-SH human neuroblastoma cells using the dye Fura-2. SK-N-SH cells express M3 mAChR, as well as CaM kinase types II and IV, which are specifically inhibited by the CaM kinase antagonist KN-62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne). Carbamylcholine (100 microM) elicited an initial transient peak in [Ca2+]i due to mobilization of Ca2+ from internal stores, followed by a sustained elevation in [Ca2+]i that depended on the influx of extracellular Ca2+ and which was inhibited by EGTA and Ni2+. These mAChR-induced Ca2+ signals were diminished to an equal extent by preincubating the cells with 0.01 to 100 microM KN-62. KN-62 inhibited mAChR-induced Ca2+ influx and mobilization from internal stores by about 25-30%, producing a half-maximal effect at approximately 1 microM. In contrast, KN-62 (25 microM) almost completely abolished carbamylcholine-stimulated entry of divalent cations through Mn2+-permeant channels, as revealed by Mn2+ quenching of Fura-2 fluorescence. KN-62 also almost completely abolished Ca2+ influx induced by depolarization of the cells with 25 mM K+ (IC50 = 3 microM). These results suggest that CaM kinases regulate both the mobilization of intracellular Ca2+ and the stimulation of Ca2+ influx that are induced by mAChR activation, and indicate that the mAChR-induced influx of Ca2+ occurs through Ca2+ channels other than, or in addition to, the voltage-gated calcium channels or Mn2+-permeant channels which are inhibited by KN-62.
Puhl
H L
HL
Laboratory of Neurobiology, Guthrie Research Institute, Sayre, PA 18840, U.S.A.
Raman
P S
PS
Williams
C L
CL
Aronstam
R S
RS
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors*; D002217:Carbachol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D009447:Neuroblastoma; D010880:Piperidines / Q000494:pharmacology; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000187:drug effects*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured / Q000378:metabolism
D004791:Enzyme Inhibitors; D010880:Piperidines; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; C063302:KN 62; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002217:Carbachol; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium