Ca2+ antagonist-insensitive coronary smooth muscle contraction involves activation of ϵ-protein kinase C-dependent pathway

2003 ◽  
Vol 285 (6) ◽  
pp. C1454-C1463 ◽  
Author(s):  
Andrea Dallas ◽  
Raouf A. Khalil
Keyword(s):  
Smooth Muscle ◽  
Coronary Artery ◽  
Muscle Contraction ◽  
Smooth Muscle Contraction ◽  
Channel Blockers ◽  
Cell Contraction ◽  
Coronary Smooth Muscle ◽  
Calphostin C ◽  
Gf 109203X ◽  
Dependent Pathway

Certain angina and coronary artery disease forms do not respond to Ca2+ channel blockers, and a role for vasoactive eicosanoids such as PGF2α in Ca2+ antagonist-insensitive coronary vasospasm is suggested; however, the signaling mechanisms are unclear. We investigated whether PGF2α-induced coronary smooth muscle contraction is Ca2+ antagonist insensitive and involves activation of a PKC-dependent pathway. We measured contraction in single porcine coronary artery smooth muscle cells and intracellular free Ca2+ concentration ([Ca2+]i) in fura 2-loaded cells and examined cytosolic and particulate fractions for PKC activity and reactivity with isoform-specific PKC antibodies. In Hanks' solution (1 mM Ca2+), PGF2α (10-5 M) caused transient [Ca2+]i increase followed by maintained [Ca2+]i increase and 34% cell contraction. Ca2+ channel blockers verapamil and diltiazem (10-6 M) abolished maintained PGF2α-induced [Ca2+]i increase but only partially inhibited PGF2α-induced cell contraction to 17%. Verapamil-insensitive PGF2α contraction was inhibited by PKC inhibitors GF-109203X, calphostin C, and ϵ-PKC V1-2. PGF2α caused Ca2+-dependent α-PKC and Ca2+-independent ϵ-PKC translocation from cytosolic to particulate fractions that was inhibited by calphostin C. Verapamil abolished PGF2α-induced α-but not ϵ-PKC translocation. PMA (10-6 M), a direct activator of PKC, caused 21% contraction with no significant [Ca2+]i increase and ϵ-PKC translocation that were inhibited by calphostin C but not verapamil. Membrane depolarization by 51 mM KCl, which stimulates Ca2+ influx, caused 36% cell contraction and [Ca2+]i increase that were inhibited by verapamil but not GF-109203X or calphostin C and did not cause α- or ϵ-PKC translocation. Thus a significant component of PGF2α-induced contraction of coronary smooth muscle is Ca2+ antagonist insensitive, involves Ca2+-independent ϵ-PKC activation and translocation, and may represent a signaling mechanism of Ca2+ antagonist-resistant coronary vasospasm.

Effect of oxidative stress on Rho kinase II and smooth muscle contraction in rat stomach

2015 ◽  
Vol 93 (6) ◽  
pp. 405-411 ◽  
Author(s):  
Othman Al-Shboul ◽  
Ayman Mustafa
Keyword(s):  
Oxidative Stress ◽  
Smooth Muscle ◽  
Muscle Contraction ◽  
Muscle Cell ◽  
Rho Kinase ◽  
Smooth Muscle Contraction ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rat Stomach ◽  
Cell Contraction ◽  
Kinase Pathway

Recent studies have shown that both Rho kinase signaling and oxidative stress are involved in the pathogenesis of a number of human diseases, such as diabetes mellitus, hypertension, and atherosclerosis. However, very little is known about the effect of oxidative stress on the gastrointestinal (GI) smooth muscle Rho kinase pathway. The aim of the current study was to investigate the effect of oxidative stress on Rho kinase II and muscle contraction in rat stomach. The peroxynitrite donor 3-morpholinosydnonimine (SIN-1), hydrogen peroxide (H2O2), and peroxynitrite were used to induce oxidative stress. Rho kinase II expression and ACh-induced activity were measured in control and oxidant-treated cells via specifically designed enzyme-linked immunosorbent assay (ELISA) and activity assay kits, respectively. Single smooth muscle cell contraction was measured via scanning micrometry in the presence or absence of the Rho kinase blocker, Y-27632 dihydrochloride. All oxidant agents significantly increased ACh-induced Rho kinase II activity without affecting its expression level. Most important, oxidative stress induced by all three agents augmented ACh-stimulated muscle cell contraction, which was significantly inhibited by Y-27632. In conclusion, oxidative stress activates Rho kinase II and enhances contraction in rat gastric muscle, suggesting an important role in GI motility disorders associated with oxidative stress.

scholarly journals Mechanism of glucose-6-phosphate dehydrogenase-mediated regulation of coronary artery contractility

2011 ◽  
Vol 300 (6) ◽  
pp. H2054-H2063 ◽  
Author(s):  
Hirotaka Ata ◽  
Dhwajbhadur K. Rawat ◽  
Thomas Lincoln ◽  
Sachin A. Gupte
Keyword(s):  
Smooth Muscle ◽  
Coronary Artery ◽  
Vascular Smooth Muscle ◽  
Muscle Contraction ◽  
Rho Kinase ◽  
Smooth Muscle Contraction ◽  
Atpase Inhibitor ◽  
Vasp Phosphorylation ◽  
Vascular Smooth Muscle Contraction ◽  
Glucose 6 Phosphate Dehydrogenase

We previously identified glucose-6-phosphate dehydrogenase (G6PD) as a regulator of vascular smooth muscle contraction. In this study, we tested our hypothesis that G6PD activated by KCl via a phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-protein kinase C (PKC) pathway increases vascular smooth muscle contraction and that inhibition of G6PD relaxes smooth muscle by decreasing intracellular Ca2+ ([Ca2+]i) and Ca2+ sensitivity to the myofilament. Here we show that G6PD is activated by membrane depolarization via PKC and PTEN pathway and that G6PD inhibition decreases intracellular free calcium ([Ca2+]i) in vascular smooth muscle cells and thus arterial contractility. In bovine coronary artery (CA), KCl (30 mmol/l) increased PKC activity and doubled G6PD Vmax without affecting Km. KCl-induced PKC and G6PD activation was inhibited by bisperoxo(pyridine-2-carboxyl)oxovanadate (Bpv; 10 μmol/l), a PTEN inhibitor, which also inhibited ( P < 0.05) KCl-induced CA contraction. The G6PD blockers 6-aminonicotinamide (6AN; 1 mmol/l) and epiandrosterone (EPI; 100 μmol/l) inhibited KCl-induced increases in G6PD activity, [Ca2+]i, Ca2+-dependent myosin light chain (MLC) phosphorylation, and contraction. Relaxation of precontracted CA by 6AN and EPI was not blocked by calnoxin (10 μmol/l), a plasma membrane Ca2+ ATPase inhibitor or by lowering extracellular Na+, which inhibits the Na+/Ca2+ exchanger (NCX), but cyclopiazonic acid (200 μmol/l), a sarcoplasmic reticulum Ca2+ ATPase inhibitor, reduced ( P < 0.05) 6AN- and EPI-induced relaxation. 6AN also attenuated phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Ser855, a site phosphorylated by Rho kinase, inhibition of which reduced ( P < 0.05) KCl-induced CA contraction and 6AN-induced relaxation. By contrast, 6AN increased ( P < 0.05) vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser239, indicating that inhibition of G6PD increases PKA or PKG activity. Inhibition of PKG by RT-8-Br-PET-cGMPs (100 nmol/l) diminished 6AN-evoked VASP phosphorylation ( P < 0.05), but RT-8-Br-PET-cGMPs increased 6AN-induced relaxation. These findings suggest G6PD inhibition relaxes CA by decreasing Ca2+ influx, increasing Ca2+ sequestration, and inhibiting Rho kinase but not by increasing Ca2+ extrusion or activating PKG.

scholarly journals Effect of 3-substituted 1,4-benzodiazepin-2-ones on maximal normalized rate of bradykinin-induced smooth muscle contraction in the presence of calcium channel blockers

2017 ◽  
Vol 8 (2) ◽  
pp. 224-230 ◽  
Author(s):  
P. A. Virych ◽  
O. V. Shelyuk ◽  
T. A. Kabanova ◽  
O. I. Khalimova ◽  
V. S. Martynyuk ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Calcium Channel ◽  
Muscle Contraction ◽  
Calcium Channel Blockers ◽  
Smooth Muscle Contraction ◽  
Channel Blockers ◽  
B2 Receptors ◽  
The One ◽  
Potential Inhibitors ◽  
Contraction And Relaxation

The development of modern organic chemistry and molecular modeling technologies simplify the search for potential inhibitors of various receptor systems and biological processes. The one of the directions is the development of analgesics of broad spectrum and low toxicity. It is important to search for inhibitors of the kinin-kallikrein system that regulates many functions: inflammation, pain, carcinogenesis, vascular tone, smooth muscle contraction and other. Derivatives of 3-substituted 1,4-benzodiazepine-2-ones have a unique spatial conformation that allows one to simulate β-structures of bioactive peptides. The functional activity of compounds is determined by properties of their peripheral chemical radicals. We analyzed the effect of 3-substituted 1,4-benzodiazepin-2-ones derivatives on the normalized maximal rate of bradykinin-induced smooth muscle contraction and relaxation of the stomach in the presence of calcium channel blockers: verapamil (1 μM), gadolinium (300 μM) and 2-aminoethyl diphenylborinate (0.1 μM). The levels of bradykinin and 3-arylamino-1,2-dihydro-3H-1,4-benzodiazepine-2-ones in incubation solution were 10–6 M. Data processing on dynamics of contraction was performed according to the method of Burdyha and Kosterin. Compounds MX-1775 and MX-1925 reduced maximal normalized rate (Vn) of bradykinin-induced smooth muscle contraction in the presence of Gd3+ by 21.2% and 31.0% respectively. Compound MX-1925 increased Vn of relaxation by 11.6%. A similar effect is typical for MX-2011, where there is an increase by 34.6%. In the presence of verapamil this compound additionally decreased Vn contraction by 20.5%. Substances MX-1775, MX-2004 and MX-1925 restored maximal normalized rate of relaxation to original values of bradykinin-induced contraction. In the presence of 2-aminoethyldiphenylborinate MX-1775 additionally reduced Vn of contractions by 7.5%. 3-substituted 1,4-benzo­diazepine-2-ones did not change the maximal normalized rate of contraction and relaxation of carbachol- and potential-induced smooth muscle contraction. Based on the results and previous investigations, the MX-1775 is a potential blocker of kinin B2-receptors. Effects obtained for other compounds require additional research. 

PKC-mediated redistribution of mitogen-activated protein kinase during smooth muscle cell activation

1993 ◽  
Vol 265 (2) ◽  
pp. C406-C411 ◽  
Author(s):  
R. A. Khalil ◽  
K. G. Morgan
Keyword(s):  
Signal Transduction ◽  
Smooth Muscle ◽  
Protein Kinase ◽  
Muscle Contraction ◽  
Map Kinase ◽  
Surface Membrane ◽  
Smooth Muscle Contraction ◽  
Cell Contraction ◽  
Signal Transduction Cascade ◽  
Mitogen Activated Protein

Protein kinase C (PKC) translocates from the cytosol to the surface membrane at the time it mediates agonist-induced contraction of ferret vascular smooth muscle cells (R. A. Khalil and K. G. Morgan. J. Physiol. Lond. 455: 585-599, 1992). However, no direct communication between membrane-associated PKC and the contractile filaments has been identified. Mitogen-activated protein (MAP) kinase is a substrate for PKC and is also capable of phosphorylating the actin-binding protein caldesmon at sites phosphorylated during smooth muscle contraction in vivo (L. P. Adam, C. J. Gapinski, and D. R. Hathaway. FEBS Lett. 302: 223-226, 1992). In the present study, the hypothesis that PKC and MAP kinase are involved in a signal-transduction cascade leading to smooth muscle contraction was tested. Immunofluorescence and digital-imaging microscopy were used to localize the epsilon-PKC isoform and MAP kinase during phenylephrine-induced Ca(2+)-independent activation of ferret aorta cells. We report that maintained phenylephrine-induced translocation of cytosolic PKC to the surface membrane is associated with transient redistribution of cytosolic MAP kinase to the surface membrane before cell contraction. Coincident with cell contraction, MAP kinase undergoes a second redistribution away from the plasmalemma and toward the vicinity of contractile filaments. Redistribution of MAP kinase is not stimulated by Ca2+ but is completely prevented by PKC inhibitors. The transient Ca(2+)-independent but PKC-dependent redistribution of MAP kinase points to MAP kinase as a missing link in the signal-transduction cascade between membrane-bound PKC and smooth muscle activation.

scholarly journals Effect of 3-arylamino-1,2-dihydro-3H-1,4-benzodiazepine-2-ones on the bradykinin-induced smooth muscle contraction

2017 ◽  
Vol 8 (1) ◽  
pp. 30-35
Author(s):  
P. A. Virych ◽  
O. V. Shelyuk ◽  
T. A. Kabanova ◽  
O. I. Khalimova ◽  
V. S. Martynyuk ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Smooth Muscle ◽  
Muscle Contraction ◽  
Competitive Inhibitor ◽  
Smooth Muscle Contraction ◽  
Signal Transduction Pathways ◽  
Channel Blockers ◽  
Normal Functioning ◽  
Inhibitory Interaction ◽  
Additional Reduction

Damage to tissue, inflammation and disruption of normal functioning of organs are often accompanied by pain. In pain perceptions, the kinin-kallikrein system with bradykinin as mediator is very important. Regulatory activity of the kinin-kallikrein system permits the control of inflammation, pain, vascular tone and other functions. A new group of substances that may used for this purpose are 3-substituted 1,4-benzdiazepinones. We analyzed the effect of 3-aryl amino-1,2-dihydro-3H-1,4-benzodiazepine-2-ones derivatives on the normalized maximal rate of bradykinin-induced smooth muscle contraction of the stomach in the presence of calcium channel blockers verapamil (1 μM) and gadolinium (300 μM). The levels of bradykinin and 3-arylamino-1,2-dihydro-3H-1,4-benzodiazepine-2-ones in the incubation solution were 10–6 M. Data processing on the dynamics of contraction was performed according to the method of T. Burdyha and S. Kosterin. Statistically significant changes were found for MX-1828. This compound reduced the maximal normalized rate of bradykinin-induced smooth muscle contraction in the presence of Gd3+ and verapamil by 19.3% and 32.0%, respectively. Also, MX-1828 demonstrated effects similar to those of the competitive inhibitor bradykinin B2-receptor – des-Arg9-bradykinin-acetate, which is possible evidence of its interaction with the receptor or signal transduction pathways. MX-1828 additionally reduced the maximum normalized rate of relaxation by 6.2% in the presence of Gd3+. This effect was demonstrated for MX-1906 in the presence of verapamil with additional reduction of the maximal normalized rate of relaxation, which was 26.4%. The results suggest the presence of inhibitory interaction between MX-1828 and kinin-kallikrein system receptors or signal transduction pathways. The effects which were found for MX-1906 require further studies to clarify the mechanisms of influence on bradykinin-induced smooth muscle contraction.

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