Activation of p42/p44 mitogen-activated protein kinase and contraction by prostaglandin F2alpha, ionomycin, and thapsigargin in cat iris sphincter smooth muscle: inhibition by PD98059, KN-93, and isoproterenol.
In the present study we investigated the cross talk between the Ca2+ mobilization pathway and the mitogen-activated protein (MAP) kinase pathway and contraction in the cat iris sphincter smooth muscle. Three Ca2+-mobilizing agonists, namely, prostaglandin F2alpha (PGF2alpha), ionomycin, and thapsigargin, and three specific inhibitors, PD98059, a p42/p44 MAP kinase inhibitor; KN-93, a Ca2+-calmodulin-dependent protein kinase II (CaMKII) blocker; and isoproterenol, a cAMP-elevating agent, were used. Changes in tension in response to the agonists were recorded isometrically and MAP kinase phosphorylation and activation were monitored by Western blotting and by in situ myelin basic protein phosphorylation, respectively. We found that 1) stimulation of the sphincter muscle with PGF2alpha, ionomycin, or thapsigargin resulted in rapid phosphorylation and activation of p42/p44 MAP kinase and contraction; and 2) treatment of the muscles with PD98059, KN-93, or isoproterenol resulted in inhibition of the Ca2+-mobilizing agonist-induced responses. The contractile responses induced by PGF2alpha, ionomycin, and thapsigargin were (mg of tension/mg of wet weight tissue) 15.2, 15.4, and 16.2, respectively; the increases in MAP kinase phosphorylation by these agonists were 228, 203, and 190%, respectively; and the increases in MAP kinase activation by the agonists were 212, 191, and 162%, respectively. The stimulatory effects of the agonists on contraction and on MAP kinase phosphorylation and activation were blocked by preincubation of the muscle with PD98059, KN-93, or isoproterenol. These data demonstrate that in the iris sphincter phosphorylation and activation of p42/p44 MAP kinases by PGF2alpha, ionomycin, or thapsigargin require intracellular Ca2+ either from extracellular sources or from internal stores, that CaMKII plays an important role in the regulation of contraction, that CaMKII acts upstream of MAP kinase to control its activation, and that the MAP kinase signaling pathway can play a significant role in mediating the cellular effects of these Ca2+-mobilizing agonists.
Ansari
H R
HR
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912-2100, USA.
Husain
S
S
Abdel-Latif
A A
AA
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002415:Cats; D003720:Densitometry; D015237:Dinoprost / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology; D066298:In Vitro Techniques; D015759:Ionomycin / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology*; D007498:Iris / Q000187:drug effects; D007545:Isoproterenol / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects*; D013449:Sulfonamides / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology*; D000096926:Benzenesulfonamides
D000318:Adrenergic beta-Agonists; D001596:Benzylamines; D000252:Calcium-Transporting ATPases; D015237:Dinoprost; D004791:Enzyme Inhibitors; D005419:Flavonoids; D015759:Ionomycin; D007476:Ionophores; D007545:Isoproterenol; D019950:Mitogen-Activated Protein Kinase 1; D013449:Sulfonamides; D019284:Thapsigargin; C072105:KN 93; D000096926:Benzenesulfonamides; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one