ABSTRACT
The tonic smooth muscles of lower esophageal sphincter (LES) and internal anal sphincter (IAS) are subject to modulation by the neurohumoral agents. We report that angiotensin (Ang) II-induced contraction of rat IAS and LES smooth muscle cells (SMC) was inhibited by Clostridium botulinum C3 exozyme, HA 1077 and Y 27632, suggesting a role for Rho kinase and a Rho-associated kinase (ROK). Ang II-induced contraction of the SMC was also attenuated by genistein, antibodies to the pp60(c-src), p(190) RhoGTPase-activating protein (p190 RhoGAP), carboxyl terminus of Galpha13, carboxyl terminus peptide, and ADP ribosylation factor (ARF) antibody. Ang II-induced increase in p(190) RhoGAP tyrosine phosphorylation was attenuated by genistein. Furthermore, Ang II-induced increase in smooth muscle tone and phosphorylation of myosin light chain (MLC; 20 kDa; MLC20-P) were attenuated by Y 27632 and genistein. The results suggest an important role for Galpha13 and pp60(c-src) in the intracellular events responsible for the activation of RhoA/ROK in Ang II-induced contraction of LES and IAS SMC.
Subject(s)
Muscle Contraction/physiology , Myocytes, Smooth Muscle/physiology , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/physiology , rho GTP-Binding Proteins/metabolism , ADP Ribose Transferases , Anal Canal/physiology , Angiotensin II/pharmacology , Animals , Antibodies/pharmacology , Botulinum Toxins , Enzyme Inhibitors/pharmacology , Esophagogastric Junction/physiology , Intracellular Signaling Peptides and Proteins , Muscle Contraction/drug effects , Muscle, Smooth/drug effects , Muscle, Smooth/physiology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/ultrastructure , Myosin Light Chains/drug effects , Myosin Light Chains/metabolism , Phosphorylation , Rats , Signal Transduction/drug effects , Vasoconstrictor Agents/pharmacology , rho-Associated KinasesABSTRACT
We examined the role of mitogen-activated protein kinase (p(44/42) MAPK) in ANG II-induced contraction of lower esophageal sphincter (LES) and internal anal sphincter (IAS) smooth muscles. Studies were performed in the isolated smooth muscles and cells (SMC). ANG II-induced changes in the levels of phosphorylation of different signal transduction and effector proteins were determined before and after selective inhibitors. ANG II-induced contraction of the rat LES and IAS SMC was inhibited by genistein, PD-98059 [a specific inhibitor of MAPK kinases (MEK 1/2)], herbimycin A (a pp60(c-src) inhibitor), and antibodies to pp60(c-src) and p(120) ras GTPase-activating protein (p(120) rasGAP). ANG II-induced contraction of the tonic smooth muscles was accompanied by an increase in tyrosine phosphorylation of p(120) rasGAP. These were attenuated by genistein but not by PD-98059. ANG II-induced increase in phosphorylations of p(44/42) MAPKs and caldesmon was attenuated by both genistein and PD-98059. We conclude that pp60(c-src) and p(44/42) MAPKs play an important role in ANG II-induced contraction of LES and IAS smooth muscles.
Subject(s)
Angiotensin II/pharmacology , Digestive System/drug effects , Mitogen-Activated Protein Kinase 1/physiology , Mitogen-Activated Protein Kinases/physiology , Muscle Contraction/physiology , Muscle, Smooth/drug effects , Proto-Oncogene Proteins pp60(c-src)/physiology , Animals , Antibodies/pharmacology , Benzoquinones , Calmodulin-Binding Proteins/metabolism , Digestive System Physiological Phenomena , Enzyme Inhibitors/pharmacology , Female , Flavonoids/pharmacology , Genistein/pharmacology , In Vitro Techniques , Lactams, Macrocyclic , Male , Mitogen-Activated Protein Kinase 3 , Muscle Contraction/drug effects , Muscle, Smooth/physiology , Phosphorylation , Protein-Tyrosine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins pp60(c-src)/antagonists & inhibitors , Proto-Oncogene Proteins pp60(c-src)/immunology , Quinones/pharmacology , Rats , Rats, Sprague-Dawley , Rifabutin/analogs & derivatives , Tyrosine/metabolism , p120 GTPase Activating Protein/immunology , p120 GTPase Activating Protein/pharmacologyABSTRACT
Studies were performed to compare the actions of Ang II in the internal anal sphincter (IAS) vs. lower esophageal sphincter (LES) smooth muscles in vitro, in opossum and rabbit. Studies also were carried out in isolated smooth muscle cells. In opossum, Ang II produced no discernible effects in the IAS, but did produce a concentration-dependent contraction in the LES. Conversely, in the rabbit, while Ang II caused a modest response in the LES, it caused a significant contraction in the IAS. The contractile responses of Ang II in the opossum LES were mostly resistant to different neurohumoral antagonists but were antagonized by AT1 antagonist losartan. AT2 antagonist PD 123,319, rather than inhibiting, prolonged the contractile action of Ang II. The contractile actions of Ang II in the opossum LES were not modified by the tyrosine kinase inhibitors (genistein and tyrphostin 1 x 10(-6) M) but were partially attenuated by the PKC inhibitor H-7 (1 x 10(-6) M), Ca2+ channel blocker nicardipine (1 x 10(-5) M), Rho kinase inhibitor HA-1077 (1 x 10(-7) M) or p(44/42) MAP kinase inhibitor PD 98059 (5 x 10(-5) M). The combination of HA-1077 and H-7 did not cause an additive attenuation of Ang II responses. Western blot analyses revealed the presence of both AT1 and AT2 receptors. We conclude that Ang lI-induced contraction of sphincteric smooth muscle occurs primarily by the activation of AT1 receptors at the smooth muscle cells and involves multiple pathways, influx of Ca2+, and PKC, Rho kinase and p(44/42) MAP kinase.
Subject(s)
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , Anal Canal/drug effects , Angiotensin II/pharmacology , Esophagus/drug effects , Muscle, Smooth/drug effects , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Anal Canal/physiology , Animals , Dose-Response Relationship, Drug , Drug Combinations , Esophagus/physiology , Female , Flavonoids/pharmacology , Genistein/pharmacology , Imidazoles/pharmacology , Losartan/pharmacology , Male , Muscle Contraction/drug effects , Muscle Contraction/physiology , Muscle, Smooth/physiology , Nicardipine/pharmacology , Opossums , Pyridines/pharmacology , Rabbits , Species Specificity , Tyrphostins/pharmacologyABSTRACT
Effect of ANG II was investigated in in vitro smooth muscle strips and in isolated smooth muscle cells (SMC). Among different species, rat internal and sphincter (IAS) smooth muscle showed significant and reproducible contraction that remained unmodified by different neurohumoral inhibitors. The AT(1) antagonist losartan but not AT(2) antagonist PD-123319 antagonized ANG II-induced contraction of the IAS smooth muscle and SMC. ANG II-induced contraction of rat IAS smooth muscle and SMC was attenuated by tyrosine kinase inhibitors genistein and tyrphostin, protein kinase C (PKC) inhibitor H-7, Ca(2+) channel blocker nicardipine, Rho kinase inhibitor Y-27632 or p(44/42) mitogen-activating protein kinase (MAPK(44/42)) inhibitor PD-98059. Combinations of nicardipine and H-7, Y-27632, and PD-98059 caused further attenuation of the ANG II effects. Western blot analyses revealed the presence of both AT(1) and AT(2) receptors. We conclude that ANG II causes contraction of rat IAS smooth muscle by the activation of AT(1) receptors at the SMC and involves multiple intracellular pathways, influx of Ca(2+), and activation of PKC, Rho kinase, and MAPK(44/42).
