Abstract
Using a guinea pig gastric longitudinal smooth muscle preparation, we have compared the contractile signaling pathways triggered by the thrombin receptor-activating peptide, TFLLR-NH2 (TF) and by epidermal growth factor-urogastrone (EGF). In addition to inhibitors of tyrosine kinase [tyrphostin 47/AG213, genistein and the src-selective inhibitor CP118,556/PP1], cyclooxygenase (indomethacin, INDO) and diacylglycerol lipase (U57, 908), we also used the signal pathway probe inhibitors of mitogen-activated protein-kinase-kinase (MEK:PD98059), phosphatidylinositol 3′-kinase [PI3K: Wortmannin (WM) and LY294002], protein kinase C [PKC: GF109203X (GF)], and of the EGF-receptor kinase (PD153035). We found that in addition to the inhibition of both TF and EGF-stimulated contractions by the inhibitors of tyrosine kinase, cyclooxygenase and diacylglycerol lipase, the actions of TF and EGF were also attenuated by PD98059, WM/LY294002 and GF. However, PD153035 blocked only EGF-triggered contractions. The contractile actions of both TF and EGF were dependent on extracellular calcium. In contrast, the contractile action of arachidonic acid, via a presumed cyclooxygenase product that mediated the contractions caused by both TF and EGF, was not blocked by any of the signal pathway probe inhibitors. The contractile actions of both TF and EGF were accompanied by increases in tissue phosphotyrosyl proteins and an increase in tissue c-src kinase activity. We conclude that protease-activated receptor no. 1- (thrombin receptor) mediated contractions in the logitudial muscle, like EGF receptor-activated responses, require the influx of extracellular calcium and use parallel signal pathways upstream of the cyclooxygenase step, involving MEK, PI3K, kinase C and possibly cellular src. The TF-induced response did not involve trans-activation of the EGF receptor kinase; but the converse (i.e.,trans-activation of protease-activated receptor no. 1 (thrombin receptor) by the EGF receptor kinase) could not be ruled out.
Footnotes
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Send reprint requests to: Dr. Morley D. Hollenberg, Department of Pharmacology & Therapeutics, University of Calgary, Faculty of Medicine, Calgary, Alberta, Canada T2N 4N1.
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↵1 This study was supported primarily by funds from the Medical Research Council of Canada with supplementary funds from the Heart & Stroke Foundation of Alberta. X.-L.Z. was supported in part by a William H. Davies Medical Research Scholarship and by a graduate studentship from the Canadian Hypertension Society in conjunction with Pfizer and the Medical Research Council of Canada.
- Abbreviations:
- Amino acids are abbreviated by their one-letter codes
- AA, arachidonic acid
- Cch
- carbachol
- CM
- circular muscle preparation
- EGF
- epidermal growth factor-urogastrone
- LM
- longitudinal muscle
- LPA
- lysophosphatidic acid
- MAPK
- mitogen-activated protein kinase
- MAPKK
- mitogen-activated protein kinase kinase, or MEK
- MEK
- mitogen-activated protein kinase kinase
- PAR1
- protease-activated receptor No. 1 (thrombin receptor)
- PAR2
- protease-activated receptor No. 2 (activated by trypsin)
- PAR1AP
- PAR1-activating peptide
- PAR2AP
- PAR2-activating peptide
- PI3K
- Phosphatidylinositol 3′-kinase
- PKC
- protein kinase C
- PMSF
- phenylmethylsulfoxyl fluoride
- TF
- TFLLR-NH2
- Received July 15, 1997.
- Accepted December 15, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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