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.
Department of Pharmacology, University of Nevada School of Medicine, Reno 89557-0046, USA. yambo@med.unr.edu
I A
IA
Yamboliev
K M
KM
Wiesmann
C A
CA
Singer
J C
JC
Hedges
W T
WT
Gerthoffer
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