Phosphatidylinositol 3-kinases regulate ERK and p38 MAP kinases in canine colonic smooth muscle.
In canine colon, M2/M3 muscarinic receptors are coupled to extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases. We tested the hypothesis that this coupling is mediated by enzymes of the phosphatidylinositol (PI) 3-kinase family. RT-PCR and Western blotting demonstrated expression of two isoforms, PI 3-kinase-alpha and PI 3-kinase-gamma. Muscarinic stimulation of intact muscle strips (10 microM ACh) activated PI 3-kinase-gamma, ERK and p38 MAP kinases, and MAP kinase-activated protein kinase-2, whereas PI 3-kinase-alpha activation was not detected. Wortmannin (25 microM) abolished the activation of PI 3-kinase-gamma, ERK, and p38 MAP kinases. MAP kinase inhibition was a PI 3-kinase-gamma-specific effect, since wortmannin did not inhibit recombinant activated murine ERK2 MAP kinase, protein kinase C, Raf-1, or MAP kinase kinase. In cultured muscle cells, newborn calf serum (3%) activated PI 3-kinase-alpha and PI 3-kinase-gamma isoforms, ERK and p38 MAP kinases, and stimulated chemotactic cell migration. Using wortmannin and LY-294002 to inhibit PI 3-kinase activity and PD-098059 and SB-203580 to inhibit ERK and p38 MAP kinases, we established that these enzymes are functionally important for regulation of chemotactic migration of colonic myocytes.
Yamboliev
I A
IA
Department of Pharmacology, University of Nevada School of Medicine, Reno 89557-0046, USA. yambo@med.unr.edu
Wiesmann
K M
KM
Singer
C A
CA
Hedges
J C
JC
Gerthoffer
W T
WT
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D003106:Colon / Q000187:drug effects / Q000201:enzymology*; D004285:Dogs; D005260:Female; D006801:Humans; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism; D014665:Vasodilator Agents / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases
D000109:Acetylcholine; D020928:Mitogen-Activated Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011976:Receptors, Muscarinic; D014665:Vasodilator Agents; D048051:p38 Mitogen-Activated Protein Kinases