title: Intracellular application of calmidazolium increases Ca2+ current through activation of protein kinase A in cultured vascular smooth muscle cells.
abstract: In the present study, we investigated the actions of calmodulin (CaM) and CaM-dependent protein kinase II (CaMK-II) on the L-type Ca2+ currents (ICa(L)) of cultured vascular smooth muscle (VSM) cells (A7r5 cell line), using the whole-cell voltage clamp method. The peak IBa (Ca2+ channel using 5 mM Ba2+ as charge carrier) was evoked every 15 s by a test potential to +10 mV from a holding potential of -60 mV. To test the effect of CaM on IBa, 1 microM calmidazolium (CMZ), an inhibitor of CaM, was added to the pipette solution (pCa of 6.5 or 300 nM [Ca]i). The amplitude of maximally activated IBa was -4.3 +/- 0.5 pA/pF (n = 13) for control and -8.1 +/- 0.9 pA/pF (n = 14) in the presence of CMZ. This difference was statistically significant (p = 0.016). The CMZ stimulation of IBa was not abolished when 5 microM KN-62, a specific inhibitor of CaMK-II, was included in the pipette (-9.5 +/- 1.1 pA/pF; n = 10). Introduction of CaMK-II itself intracellularly had no effect on the basal IBa. On the other hand, the CMZ stimulation of IBa was prevented by both H-7, a nonspecific protein kinase inhibitor, and H-89, a specific inhibitor of protein kinase A (PK-A). Since CMZ is a strong inhibitor of Ca2+/CaM-dependent phosphodiesterase (type I PDE), we studied the effect of 8-methoxymethyl-3-isobutyl-1-methylxanthine (MIBMX), another specific inhibitor of the PDE. MIBMX, like CMZ, stimulated IBa: control, -4.6 +/- 0.4 pA/pF (n = 10); MIBMX, -9.6 +/- 1.2 (n = 8), and CMZ, -7.9 +/- 0.9 (n = 15). 0.1 mM 8Br-cAMP, a membrane permeable cAMP analogue, stimulated IBa by +42%: before, -3.7 +/- 0. 7 pA/pF; after, -5.2 +/- 1.0 (n = 6). In conclusion, Ca2+ channels of VSM cells might not be directly regulated by the CaM/CaMK-II pathway. Therefore, the CMZ stimulation of IBa might occur due to the increase in intracellular concentration of cAMP produced by inhibition of CaM-dependent PDE.
authors.affiliation: Department of Molecular and Cellular Physiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio, USA. sunagam@email.uc.edu
authors.forename: M
authors.initials: M
authors.lastname: Sunagawa
authors.forename: H
authors.initials: H
authors.lastname: Yokoshiki
authors.forename: T
authors.initials: T
authors.lastname: Seki
authors.forename: N
authors.initials: N
authors.lastname: Sperelakis
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007093:Imidazoles / Q000008:administration & dosage*; D007424:Intracellular Fluid / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D051381:Rats; D013449:Sulfonamides / Q000494:pharmacology; D000096926:Benzenesulfonamides
chemical_list: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D001596:Benzylamines; D002118:Calcium; D015220:Calcium Channels; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D007093:Imidazoles; D007546:Isoquinolines; D013449:Sulfonamides; C072105:KN 93; D000096926:Benzenesulfonamides; C031938:calmidazolium; C063302:KN 62; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide