endothelin-1 and Coronary-Vasospasm

Smooth muscle contraction is activated primarily by phosphorylation at Ser19 of the regulatory light chain subunits (LC20) of myosin II, catalysed by Ca(2+)/calmodulin-dependent myosin light chain kinase. Ca(2+)-independent contraction can be induced by inhibition of myosin light chain phosphatase, which correlates with diphosphorylation of LC20 at Ser19 and Thr18, catalysed by integrin-linked kinase (ILK) and zipper-interacting protein kinase (ZIPK). LC20 diphosphorylation at Ser19 and Thr18 has been detected in mammalian vascular smooth muscle tissues in response to specific contractile stimuli (e.g. endothelin-1 stimulation of rat renal afferent arterioles) and in pathophysiological situations associated with hypercontractility (e.g. cerebral vasospasm following subarachnoid hemorrhage). Comparison of the effects of LC 20 monophosphorylation at Ser19 and diphosphorylation at Ser19 and Thr18 on contraction and relaxation of Triton-skinned rat caudal arterial smooth muscle revealed that phosphorylation at Thr18 has no effect on steady-state force induced by Ser19 phosphorylation. On the other hand, the rates of dephosphorylation and relaxation are significantly slower following diphosphorylation at Thr18 and Ser19 compared to monophosphorylation at Ser19. We propose that this diphosphorylation mechanism underlies the prolonged contractile response of particular vascular smooth muscle tissues to specific stimuli, e.g. endothelin-1 stimulation of renal afferent arterioles, and the vasospastic behavior observed in pathological conditions such as cerebral vasospasm following subarachnoid hemorrhage and coronary arterial vasospasm. ILK and ZIPK may, therefore, be useful therapeutic targets for the treatment of such conditions.

Topics: Acute Kidney Injury; Animals; Catalysis; Coronary Vasospasm; Death-Associated Protein Kinases; Endothelin-1; Humans; Hypertension; Microcirculation; Molecular Targeted Therapy; Muscle, Smooth, Vascular; Myosin Type II; Myosin-Light-Chain Kinase; Myosin-Light-Chain Phosphatase; Phosphorylation; Protein Serine-Threonine Kinases; Rats; Renal Circulation; Vasoconstriction; Vasospasm, Intracranial

One year after the revelation by Dr. Furchgott in 1980 that the endothelium was obligatory for acetylcholine to relax isolated arteries, it was clearly shown that the endothelium could also promote contraction. In 1988, Dr. Yanagisawa's group identified endothelin-1 (ET-1) as the first endothelium-derived contracting factor. The circulating levels of this short (21-amino acid) peptide were quickly determined in humans, and it was reported that, in most cardiovascular diseases, circulating levels of ET-1 were increased, and ET-1 was then tagged as "a bad guy." The discovery of two receptor subtypes in 1990, ET(A) and ET(B), permitted optimization of the first dual ET-1 receptor antagonist in 1993 by Dr. Clozel's team, who entered clinical development with bosentan, which was offered to patients with pulmonary arterial hypertension in 2001. The revelation of Dr. Furchgott opened a Pandora's box with ET-1 as one of the actors. In this brief review, we will discuss the physiological and pathophysiological role of endothelium-derived ET-1 focusing on the regulation of the vascular tone, and as much as possible in humans. The coronary bed will be used as a running example in this review because it is the most susceptible to endothelial dysfunction, but references to the cerebral and renal circulation will also be made. Many of the cardiovascular complications associated with aging and cardiovascular risk factors are initially attributable, at least in part, to endothelial dysfunction, particularly dysregulation of the vascular function associated with an imbalance in the close interdependence of nitric oxide and ET-1.

Topics: Aging; Animals; Atherosclerosis; Coronary Vasospasm; Coronary Vessels; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Humans; Peptides, Cyclic; Receptor, Endothelin A; Receptor, Endothelin B; Vasoconstriction; Vasodilation

Topics: Acetylation; Animals; Cardiac Myosins; Cell Proliferation; Cell Shape; Cells, Cultured; Coronary Vasospasm; Coronary Vessels; Disease Models, Animal; Endothelin-1; Male; Muscle, Smooth, Vascular; Myosin Light Chains; Myosin-Light-Chain Kinase; NF-kappa B; Rats, Nude; Rats, Sprague-Dawley; Signal Transduction; Sirtuin 1; Vasoconstriction

We investigated the role of endothelin-1 (ET-1) in coronary microvascular spasm in a porcine model.. A flowmeter was implanted around the left anterior descending coronary artery (LAD), and epicardial coronary artery endothelial denudation (ED) was performed just distal to the flowmeter every 2 weeks (W) until 6 W in 10 pigs (ED group). Ten pigs were chronically treated with endothelin type A receptor (ET(A)) antagonist (TA-0201, 0.1 mg/kg/day, ED+ET(A) group), while neither ED nor administration of ET(A) antagonist was performed in 12 pigs (Control group). We assessed changes in LAD blood flow and the denuded site diameter induced by acetylcholine (ACh, 0.05 microg/kg i.c.).. In the ED group, administration of ACh to LAD induced zero flow without LAD diameter reduction at 8 W. In the ED+ET(A) group, the decrease in LAD blood flow response to ACh was suppressed. Chronic administration of TA-0201 suppressed ROS generation in the myocardium. Decreases of eNOS and intimal thickening were smaller in the TA-0201 administration group than the non-TA-0201 administration group.. Chronic administration of ET(A) receptor antagonist is effective to prevent coronary microvascular spasm.

Topics: Acetylcholine; Animals; Body Weight; Coronary Circulation; Coronary Sinus; Coronary Vasospasm; Coronary Vessels; Disease Models, Animal; Endothelin A Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Microcirculation; Nitric Oxide Synthase Type III; Pyrimidines; Reactive Oxygen Species; Receptor, Endothelin A; Sulfonamides; Sus scrofa; Vascular Resistance; Vasodilator Agents

A relationship between coronary artery spasm (CAS) and myocardial bridge (MB) has been noticed. This study was designed to investigate the differences of stress tests and symptoms between CAS patients with or without MB.. Two hundred and sixteen patients with atypical chest pain who underwent coronary angiography and acetylcholine provocation test were divided into MB group (n=68) and nonmyocardial bridge group (NMB, n=148). The results of acetylcholine test, treadmill exercise electrocardiography, myocardial scintigraphy, and levels of plasma endothelin-1 and nitric oxide were compared between the two groups.. Among these atypical chest pain patients, CAS was induced by acetylcholine in 85% MB patients and 53% NMB patients (P<0.001). A positive exercise electrocardiogram was identified in 71% MB patients and 8% NMB patients (P<0.001). Myocardial scintigraphy revealed ischemic changes in 67% MB patients and 9% NMB patients (P<0.001) and reverse redistribution in 87 and 69% (P<0.01), respectively. MB patients experienced exertional chest pain as well as at rest more frequently than NMB patients. Endothelin-1 levels were elevated in MB group complicated with CAS (P<0.01), whereas nitric oxide levels were reduced in the same cohort (P<0.05) compared with NMB group.. MB might predispose to CAS in which endothelial dysfunction may play a part. CAS patients with MB usually present mixed chest pain and positive stress tests as well as reversal redistribution on myocardial scintigraphy whereas CAS patients without MB displayed chest pain at rest, negative stress test and reversal redistribution.

Topics: Acetylcholine; Adult; Aged; Biomarkers; Case-Control Studies; Chest Pain; Coronary Angiography; Coronary Vasospasm; Electrocardiography; Endothelin-1; Exercise Test; Female; Humans; Male; Middle Aged; Myocardial Bridging; Myocardial Perfusion Imaging; Nitric Oxide; Predictive Value of Tests; Retrospective Studies

The role of endothelial dysfunction in coronary vasospasm is controversial. We hypothesized that reactive oxygen species (ROS) and endothelin-1 (ET-1) are plausible candidates as the mediator of vasospasm is linked to endothelial dysfunction. In a pig model with repetitive endothelial injury in coronary arteries, intracoronary administration of serotonin induced a vasospasm at the endothelial injury site. The level of endothelin-converting enzyme was upregulated at that site where, upon exposure to serotonin, there were also increases in p47(phox), ROS, and ET-1 fluorescence intensities, and myosin light chain phosphorylation and RhoA activation were detected. The nicotinamide adenine dinucleotide phosphate oxidase inhibitor, apocynin, had the effect of extinguishing not only ROS but also the appearance of ET-1. The chronic blockade of endothelin type-A receptor prevented a serotonin-triggered vasospasm along with the inhibition of ROS generation and myosin light chain phosphorylation. Under the coronary artery endothelial dysfunction, ET-1 is essential for an ROS-dependent coronary vasospasm. Our findings suggest that endothelial dysfunction plays a critical role in clinically defined human Prinzmetal angina.

Topics: Acetophenones; Angina Pectoris, Variant; Animals; Aspartic Acid Endopeptidases; Coronary Angiography; Coronary Vasospasm; Disease Models, Animal; Endothelin A Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Endothelium, Vascular; Enzyme Inhibitors; Metalloendopeptidases; Myosin Light Chains; NADPH Oxidases; Phosphorylation; Pyrimidines; Reactive Oxygen Species; Receptor, Endothelin A; rhoA GTP-Binding Protein; Serotonin; Signal Transduction; Sulfonamides; Swine; Time Factors; Vasoconstriction

In this study, basal and thrombin-stimulated release of nitric oxide and endothelin-1 in the internal mammary artery and the radial artery were measured, together with superoxide radicals generated after anoxia and reoxygenation. Arterial segments were obtained from patients undergoing coronary bypass operations. Quantification of nitric oxide was performed by measuring the stable oxidation products of nitric oxide. Endothelin levels were measured by an enzyme immunoassay kit, and the superoxides were measured by lucigenin-enhanced chemiluminescence. Basal and stimulated release of nitric oxide from the internal mammary artery is significantly higher than that in the radial artery. On the other hand, basal release of endothelin-1 is less in the internal mammary artery than in the radial artery, but similar after stimulation. In our study, the quantity of superoxide radicals produced by the internal mammary artery was greater than that produced by the radial artery. Our results show that there are differences between these 2 arteries in regard to production of nitric oxide, endothelin-1, and superoxide radicals. These differences may have a role in the process of atherogenesis and may contribute to long-term patency of arterial bypass grafts. These results may also explain the mechanism of radial artery graft spasm in coronary artery surgery and may constitute a basis for future pharmacological and clinical improvements for successful surgical application.

Topics: Atherosclerosis; Coronary Artery Bypass; Coronary Vasospasm; Endothelin-1; Humans; Luminescence; Mammary Arteries; Nitric Oxide; Radial Artery; Superoxides; Vascular Patency

Endothelin is implicated in graft spasm after coronary artery bypass grafting. We assessed reversal by the endothelium-derived vasodilator C-type natriuretic peptide of prior contraction of radial artery and other vessels commonly used for coronary artery bypass surgery.. Segments of human radial artery, saphenous vein, and internal mammary artery were mounted in organ baths after removal from patients undergoing cardiac surgery (n = 34; 64 +/- 2 years). Effects of increasing concentrations of C-type natriuretic peptide (with or without aprotinin, 1,000 U/mL) on endothelin-induced contraction were compared with acetylcholine, sodium nitroprusside, and papaverine.. C-type natriuretic peptide relaxed endothelin precontraction in all vessels (F = 17.8, 36.3, and 48.4, respectively; p < 0.001), with maximum relaxations of 44%, 54%, and 66% in saphenous vein, internal mammary artery, and radial artery, respectively. Aprotinin did not affect relaxation to C-type natriuretic peptide. Acetylcholine relaxed the saphenous vein weakly, with maximal relaxation of 9% at 10(-6) M. However, the radial artery and internal mammary artery relaxed strongly to acetylcholine. The highest concentration of papaverine completely relaxed all vessels, but responses were less sensitive than to sodium nitroprusside or acetylcholine.. C-type natriuretic peptide reverses endothelin-induced constriction in arterial and venous conduits used for coronary artery bypass, particularly the radial artery. Proteolytic breakdown of C-type natriuretic peptide by local vascular enzymes appears of little importance in vitro. This signals the therapeutic potential of using C-type natriuretic peptide as an antagonist of graft vasospasm after coronary artery bypass surgery.

Topics: Acetylcholine; Coronary Artery Bypass; Coronary Vasospasm; Dose-Response Relationship, Drug; Endothelin-1; Humans; In Vitro Techniques; Mammary Arteries; Middle Aged; Natriuretic Agents; Natriuretic Peptide, C-Type; Papaverine; Radial Artery; Saphenous Vein; Transplants; Treatment Outcome; Vasoconstriction; Vasodilator Agents

Topics: Coronary Artery Bypass; Coronary Vasospasm; Dose-Response Relationship, Drug; Endothelin-1; Humans; In Vitro Techniques; Natriuretic Agents; Natriuretic Peptide, C-Type; Transplants; Vasoconstriction; Vasodilator Agents

Certain forms of coronary artery disease do not respond to treatment with Ca2+ channel blockers, and a role for endothelin-1 (ET-1) in Ca2+ antagonist-insensitive forms of coronary vasospasm has been suggested; however, the signaling mechanisms involved are unclear. We tested the hypothesis that a component of ET-1-induced coronary smooth muscle contraction is Ca2+ antagonist-insensitive and involves activation of protein kinase C (PKC). Cell contraction was measured in smooth muscle cells isolated from porcine coronary artery, [Ca2+]i was measured in fura-2 loaded cells, and the cytosolic and particulate fractions were examined for PKC activity and reactivity with isoform-specific PKC antibodies using Western blot analysis. In Hank's solution (1 mmol/L Ca2+), ET-1 (10(-7) mol/L) caused a transient increase in [Ca2+]i (236+/-14 nmol/L) followed by a maintained increase in [Ca2+]i (184+/-8 nmol/L) and 35% cell contraction. The Ca2+ channel blockers verapamil and diltiazem (10(-6) mol/L) abolished the maintained ET-1-induced [Ca2+]i, but only partially inhibited ET-1-induced cell contraction to 18%. The verapamil-insensitive component of ET-1 contraction was inhibited by the PKC inhibitors calphostin C and epsilon-PKCV1-2. ET-1 caused translocation of Ca2+-dependent alpha-PKC and Ca2+-independent epsilon-PKC from the cytosolic to the particulate fraction that was inhibited by calphostin C. Verapamil abolished ET-1-induced translocation of alpha-PKC, but not that of epsilon-PKC. Phorbol 12-myristate 13-acetate (10(-6) mol/L), a direct activator of PKC, caused 22% cell contraction, with no increase in [Ca2+]i, and translocation of epsilon-PKC that was inhibited by calphostin C, but not by verapamil. KCl (51 mmol/L), which stimulates Ca2+ influx, caused 35% cell contraction and increase in [Ca2+]i (291+/-11 nmol/L) that were inhibited by verapamil, but not by calphostin C, and did not cause translocation of alpha- or epsilon-PKC. In Ca2+-free (2 mmol/L EGTA) Hank's solution, ET-1 caused 15% cell contraction, with no increase in [Ca2+]i, and translocation of epsilon-PKC that were inhibited by epsilon-PKC V1-2 inhibitory peptide. Thus, a significant component of ET-1-induced contraction of coronary smooth muscle is Ca2+ antagonist-insensitive and involves activation and translocation of Ca2+-independent epsilon-PKC, and may represent a signaling mechanism of Ca2+ antagonist-resistant forms of coronary vasospasm.

Topics: Animals; Calcium; Calcium Channel Blockers; Coronary Vasospasm; Coronary Vessels; Diltiazem; Disease Models, Animal; Drug Resistance; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Male; Muscle, Smooth, Vascular; Naphthalenes; Peptide Fragments; Protein Kinase C; Protein Kinase C-epsilon; Protein Transport; Signal Transduction; Swine; Tetradecanoylphorbol Acetate; Vasoconstriction; Verapamil

Although beta-blockers can not be used for the treatment of vasospastic angina, the effect of beta-blockers with vasorelaxant property on coronary vasospasm remains uncertain. In this study, we evaluated the effect of betaxolol, a new beta-blocker with calcium antagonistic property, as an additional therapy on vasospastic angina (VSA) with anginal attacks on effort. We enrolled 12 patients with VSA and anginal attacks with ST segment depression during exercise stress test. All patients received 1.25-5 mg of betaxolol for 3 months. Treadmill exercise stress test and adenosine triphosphate stress thallium-201 myocardial scintigraphy were performed before and 3 months after the onset of the betaxolol treatment. The other drugs including calcium antagonists, nitrates and nicorandil were continued. No patients experienced the exacerbation of angina during the betaxolol treatment. Exercise time to chest pain (317.5+/-72.1-454.2+/-75.5 s, P<0.01) and maximal ST segment depression (1.67+/-0.67-1.16+/-0.46 mm, P<0.01) obtained by exercise stress test, the defect score (8.6+/-2.7-5.3+/-2.1, P<0.01), the extent score (14.8+/-5.8-8.8+/-4.6%, P<0.01), the severity score (17.5+/-7.3-11.3+/-5.2, P<0.01) and washout rate (31.4+/-5.6-37.6+/-5.0%, P<0.01) obtained by the scintigraphy were improved by betaxolol. Our results suggest that betaxolol increases regional myocardial blood flow and improves exercise capacity in patients with VSA. Betaxolol may become a drug for a new potential therapy for VSA.

Topics: Adrenergic beta-1 Receptor Antagonists; Adrenergic beta-Antagonists; Aged; Betaxolol; Biomarkers; Blood Pressure; Calcium Channel Blockers; Coronary Circulation; Coronary Vasospasm; Drug Evaluation; Electrocardiography; Endothelin-1; Endothelium, Vascular; Exercise; Exercise Test; Exercise Tolerance; Female; Heart Conduction System; Heart Rate; Humans; Male; Middle Aged; Motor Activity; Myocardial Ischemia; Nitric Oxide; Oxygen Consumption; Severity of Illness Index; Treatment Outcome; Vasoconstriction; Vasodilation

To quantify the gene expression levels of the ABC-proteins MDR1 (P-glycoprotein) and MRP (multidrug resistance-associated protein) isoforms in isolated mononuclear cells of vasospastic persons with increased Endothelin-1 plasma levels.. Quantitative real-time RT-PCR was performed to determine the expression levels of the MDR1 (P-glycoprotein) gene and MRP1 to MRP5 genes as well as the expression of the ETA and ETB receptor in mononuclear cells derived from 11 vasospastic subjects compared to 10 healthy controls.. Mononuclear cells of vasospastic subjects showed a significant decrease in the expression of MDR1 (P-glycoprotein) gene (p=0.029), MRP2 gene (p=0.003), and MRP5 gene (p=0.013) when compared to healthy controls. These effects were poorly correlated with ET-1 plasma levels. No significant ETA and ETB receptor expression was observed in both groups.. Vasospastic persons differ in their expression pattern of MDR proteins from healthy controls. This might be an indirect effect of elevated ET-1 levels.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Coronary Vasospasm; Endothelin-1; Female; Gene Expression Regulation; Humans; Male; Multidrug Resistance-Associated Proteins; Protein Isoforms; Receptor, Endothelin A; Receptor, Endothelin B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vasoconstriction

It has been shown that the adenosine concentration in the pericardial fluid of the normal heart is higher by one order of magnitude than that of the venous plasma. A further increase in the pericardial adenosine concentration was also demonstrated in myocardial ischaemia or hypoxia. It was proposed that pericardial nucleoside levels may represent the interstitial concentrations of the adenine nucleosides. An experimental model was designed to determine the intrapericardial concentrations of adenosine, inosine and hypoxanthine during coronary spasm provoked by intracoronary administration of endothelin-1 (ET-1; 0.08+/-0.02 nmol/g of myocardial tissue). In the in situ dog heart (n=10), adenosine, inosine and hypoxanthine concentrations were determined by HPLC in fluid samples collected from the closed pericardial sac before and after ET-1 administration, and from the systemic arterial blood. Systemic blood pressure, heart rate and standard ECG were registered continuously. We found that the nucleoside concentrations in the infusate samples increased significantly during coronary spasm [adenosine, 1.49+/-0.44 compared with 0.37+/-0.07 microM (P<0.05); inosine, 27.43+/-11.51 compared with 0.47+/-0.11 microM (P<0.05); hypoxanthine, 21.17+/-6.49 compared with 4.91+/-1.24 microM (P<0.05)], while a significant decrease in blood pressure and an elevation in ECG ST segments were observed. The levels of the purine metabolites did not change in the systemic blood. The data indicate that changes in adenine nucleoside levels measured in pericardial infusate samples reflect activation of coronary metabolic adaptation in this model of spastic ischaemia, and that pericardial nucleoside levels may characterize alterations in interstitial adenine nucleoside concentrations.

Topics: Adenosine; Animals; Coronary Vasospasm; Dogs; Endothelin-1; Hypoxanthine; Inosine; Models, Animal; Myocardial Ischemia; Pericardium; Vasoconstrictor Agents

Endothelin-1 (ET-1) has been implicated in coronary vasospasm by enhancing coronary vasoconstriction to vasoactive eicosanoids, and a role for protein kinase C (PKC) activation has been suggested. However, the cellular mechanisms downstream from PKC activation are unclear. We investigated whether physiological concentrations of ET-1 enhance coronary smooth muscle contraction by activating a PKC-mediated signaling pathway involving tyrosine phosphorylation and activation of mitogen-activated protein kinase (MAPK). Cell contraction was measured in smooth muscle cells isolated from porcine coronary artery, [Ca(2+)](i) was measured in fura-2 loaded cells, and tissue fractions were examined for reactivity with anti-phosphotyrosine (P-Tyr) and anti-MAPK antibodies using immunoprecipitation and immunoblot analysis. In Hanks' solution (1 mmol/L Ca(2+)), ET-1 (10 pmol/L) did not increase basal [Ca(2+)](i) (81 +/- 2 nmol/L) but caused cell contraction (10%) that was inhibited by calphostin C (10(-6) mol/L), inhibitor of PKC, tyrphostin (10(-6) mol/L), inhibitor of tyrosine kinase, and PD098059 (10(-6) mol/L), inhibitor of MAPK kinase. The vasoactive eicosanoid prostaglandin F(2alpha) (PGF(2alpha); 10(-7) mol/L) caused increases in cell contraction (11%) and [Ca(2+)](i) (122 +/- 9 nmol/L) that were inhibited by the Ca(2+) channel blocker verapamil (10(-6) mol/L) but not by calphostin C, tyrphostin, or PD098059. Pretreatment with ET-1 for 10 minutes enhanced cell contraction to PGF(2alpha) (33%) with no additional increase in [Ca(2+)](i) (124 +/- 10 nmol/L). Activation of PKC by phorbol 12-myristate 13-acetate (PMA; 10(-7) mol/L) caused cell contraction and enhanced PGF(2alpha) contraction (32%) with no additional increase in [Ca(2+)](i) (126 +/- 9 nmol/L). The ET-1-- and PMA-induced enhancement of PGF(2alpha) contraction was abolished by verapamil or calphostin C but not by tyrphostin or PD098059. ET-1 and PMA caused significant increases in tyrosine phosphorylation of MAPK that were inhibited by calphostin C, tyrphostin, and PD098059. PGF(2alpha) did not cause any additional increases in tyrosine phosphorylation of MAPK in tissues untreated or pretreated with ET-1 or PMA. Thus, physiological concentrations of ET-1 activate a Ca(2+)-independent PKC-mediated signaling pathway that involves tyrosine phosphorylation and activation of MAPK. The enhancement of PGF(2alpha)-induced coronary smooth muscle contraction by ET-1 involves additional activation of a Ca(2+)-sensit

Topics: Animals; Calcium; Cells, Cultured; Coronary Vasospasm; Eicosanoids; Endothelin-1; Male; Mitogen-Activated Protein Kinases; Muscle Contraction; Muscle, Smooth, Vascular; Protein Kinase C; Signal Transduction; Swine; Vasoconstriction

Endothelin-1 (ET-1), a potent vasoconstrictor, has been implicated in the pathogenesis of coronary vasospasm by enhancing coronary vasoconstriction to vasoactive eicosanoids; however, the cellular mechanisms involved are unclear. We investigated whether physiological concentrations of ET-1 enhance coronary smooth muscle contraction to vasoactive eicosanoids by activating specific protein kinase C (PKC) isoforms. Cell contraction was measured in single smooth muscle cells isolated from porcine coronary arteries, intracellular free Ca(2+) ([Ca(2+)](i)) was measured in fura-2-loaded cells, and the cytosolic and particulate fractions were examined for PKC activity and reactivity with isoform-specific anti-PKC antibodies using Western blots. In Hanks' solution (1 mmol/L Ca(2+)), ET-1 (10 pmol/L) did not increase basal [Ca(2+)](i) (81+/-2 nmol/L), but it did cause cell contraction (9%) that was inhibited by GF109203X (10(-6) mol/L), an inhibitor of Ca(2+)-dependent and Ca(2+)-indpendent PKC isoforms. The vasoactive eicosanoid prostaglandin F(2alpha) (PGF(2alpha), 10(-7) mol/L) caused increases in cell contraction (11%) and [Ca(2+)](i) (108+/-7 nmol/L) that were inhibited by the Ca(2+) channel blocker diltiazem (10(-6) mol/L). Pretreatment with ET-1 (10 pmol/L) for 10 minutes enhanced cell contraction to PGF(2alpha) (35%) with no additional increase in [Ca(2+)](i) (112+/-8 nmol/L). Direct activation of PKC by phorbol 12,13-dibutyrate (PDBu, 10(-7) mol/L) caused cell contraction (10%) and enhanced PGF(2alpha) contraction (33%) with no additional increase in [Ca(2+)](i) (115+/-7 nmol/L). The ET-1-induced enhancement of PGF(2alpha) contraction was inhibited by Gö6976 (10(-6) mol/L), an inhibitor of Ca(2+)-dependent PKC isoforms. Both ET-1 and PDBu caused an increase in PKC activity in the particulate fraction and a decrease in the cytosolic fraction and increased the particulate/cytosolic PKC activity ratio. Western blots revealed the Ca(2+)-dependent alpha-PKC and the Ca(2+)-independent delta-, epsilon-, and zeta-PKC isoforms. In resting tissues, alpha- and epsilon-PKC were mainly cytosolic, delta-PKC was mainly in the particulate fraction, and zeta-PKC was equally distributed in the cytosolic and particulate fraction. ET-1 (10 pmol/L) alone or PDBu (10(-7) mol/L) alone caused translocation of epsilon-PKC from the cytosolic to the particulate fraction, localized delta-PKC more in the particulate fraction, but did not change the distribution of zeta-PKC. PGF(2alpha

Topics: Animals; Blotting, Western; Calcium; Calcium Channel Blockers; Cell Separation; Coronary Vasospasm; Coronary Vessels; Diltiazem; Dinoprost; Dose-Response Relationship, Drug; Endothelin-1; Enzyme Activation; Fura-2; In Vitro Techniques; Isoenzymes; Male; Muscle Contraction; Muscle, Smooth, Vascular; Phorbol Esters; Protein Kinase C; Subcellular Fractions; Swine

The cellular mechanisms of coronary vasospasm are unclear, and a role for protein kinase C (PKC) activation by the endogenous vasoconstrictors endothelin-1 (ET-1) and prostaglandin F2alpha (PGF2alpha) has been suggested. In this study, we developed a phorbol ester-induced PKC downregulation protocol to investigate the relation between the amount and activity of specific PKC isoforms in coronary arterial smooth muscle and coronary vasoconstriction by ET-1 and PGF2alpha. Isometric tension was measured in deendothelialized porcine coronary artery strips, [Ca2+]i was monitored in single coronary smooth muscle cells loaded with fura-2, and the whole tissue, cytosolic, and particulate fractions were examined for PKC activity and reactivity with isoform-specific anti-PKC antibodies using Western blot analysis. In Ca(2+)-free (2 mM EGTA) Krebs solution, ET-1 (10(-7) M), PGF2alpha (10(-5) M) and PKC activator phorbol 12,13-dibutyrate (PDBu) (10(-6) M) caused significant contractions that were completely inhibited by the PKC inhibitors staurosporine and calphostin C, no significant change in [Ca2+]i, and significant activation and translocation of the Ca(2+)-independent epsilon-PKC but not the Ca(2+)-dependent alpha-PKC. In Ca(2+)-free Krebs, a single application of PDBu produced maximal contraction and PKC activity after 30 min, which declined to basal levels in 3 h and remained steady for 24 h, but did not prevent subsequent increases in contraction and PKC activity with a new addition of PDBu and did not significantly decrease the amount of alpha- or epsilon-PKC. Repeated (five to eight) applications of PDBu in Ca(2+)-free Krebs at 3-h intervals completely inhibited subsequent increases in contraction and PKC activity to PDBu, ET-1, or PGF2alpha, and significantly decreased the amount of epsilon-PKC but not that of alpha-PKC. These results provide evidence that a Ca(2+)-independent coronary vasoconstriction induced by ET-1 and PGF2alpha is associated with activation of the epsilon-PKC isoform. The results suggest that, in coronary artery smooth muscle, downregulation of PKC is isoform specific and is more dependent on the frequency rather than the duration of PKC activation. The results also suggest that repeated downregulation of epsilon-PKC might play a role in preconditioning of the coronary artery against vasoconstriction by ET-1 and PGF2alpha.

Topics: Animals; Carcinogens; Coronary Vasospasm; Coronary Vessels; Dinoprost; Down-Regulation; Endothelin-1; Enzyme Activation; Male; Muscle, Smooth, Vascular; Oxytocics; Phorbol 12,13-Dibutyrate; Protein Kinase C; Swine; Vasoconstriction

Vasospastic angina as a result of alcohol ingestion has been reported, but the mechanism of alcohol-induced coronary artery spasm is presently unknown. This report presents 2 cases of alcohol-induced variant angina (VA) with elevated levels of plasma endothelin-1 after alcohol ingestion. In case 1, the plasma endothelin-1 concentration was 3.15 pg/ml before drinking (normal <2.30 pg/ml) and increased to 4.09 pg/ml when measured 5 h after alcohol ingestion. After 2 months of abstinence, the plasma endothelin-1 concentration was 2.88 pg/ml and 6 months after abstinence, it decreased to 2.03 pg/ml (normal range). In case 2, the plasma endothelin-1 concentration was 2.44 pg/ml before drinking and increased to 4.36 pg/ml when measured 5 h after alcohol ingestion. After 2 months of abstinence, the plasma endothelin-1 concentration was 3.04 pg/ml and 6 months after abstinence, it decreased to 2.09 pg/ml (normal range). These 2 cases suggest that a relationship may exist between alcohol-induced VA and elevation in the plasma endothelin-1 concentration after alcohol ingestion.

Topics: Aged; Alcoholic Beverages; Angina Pectoris, Variant; Chest Pain; Coronary Angiography; Coronary Vasospasm; Electrocardiography; Endothelin-1; Ethanol; Humans; Male; Temperance; Time Factors

Cardiotoxicity is an uncommon side-effect of 5-FU-based chemotherapy. Coronary artery vasospasms have been postulated to be involved in the pathogenesis of this rare but serious problem. We found high plasma levels of ET-1, a potent natural vasoconstrictor, in two patients who experienced two of the commonest clinical manifestations of 5-FU-induced cardiac toxicity--i.e., angina pectoris and chronic heart failure. We, therefore, propose ET-1 as the ultimate mediator of this toxicity, even though the mechanism of ET-1 increase in peripheral venous blood is still unknown. Finally, another important question still remains unresolved: is the release of ET-1 from normal coronary endothelial cells the prime cause or simply the consequence of 5-FU-related cardiotoxicity?

Topics: Aged; Antimetabolites, Antineoplastic; Coronary Vasospasm; Endothelin-1; Female; Fluorouracil; Heart Diseases; Humans; Male; Middle Aged

Endothelin participates in regulating the vascular tone, and it is also involved in the pathogenesis of vasospasm following subarachnoid hemorrhage (SAH). Endothelin-1 (ET-1) induced cerebral vasospasm is inhibited by ETA receptors specific antagonist-BQ-123; this protects the neurons from ischemic damage. The present study evaluates the dynamics of ET-1 concentration changes in the plasma of rats in the acute phase of vasospasm after SAH, which was induced by administering 100 microliters non-heparinized fresh autologous arterial blood into the brain cisterna magna (CM). The study also assesses the effect of blocking ETA receptors on the changes in ET-1 level. BQ-123, the specific ETA receptors antagonist, was administered to cerebrospinal fluid (CSF) through a cannula inserted into CM; the antagonist--40 nmol in 50 microliters CSF--was given 20 minutes prior to SAH. In the control group, sham SAH was induced by administering 100 microliters artificial CSF (aCSF) to CM. ET-1 concentration in the plasma of rats in the acute phase of vasospasm was assessed by radioimmunoassay 30 and 60 minutes after SAH or sham SAH. It has been showed that both SAH and sham SAH cause significant increase in the ET-1 concentration (p < 0.05) in the rat plasma after 30 minutes; the concentration returns to an initial value after following 30 minutes, which may suggest that ET-1 released binds to its receptors in the acute phase of the vasospasm. On the other hand, in the two groups of rats with blocked ETA receptors there was a significant rise in ET-1 concentration 30 minutes after SAH or sham SAH, and a still further rise was observed 60 minutes after the procedure. The rise was significantly higher in animals with SAH (p < 0.05). The dynamics of the ET-1 concentration changes observed in rats with blocked ETA receptor suggests that SAH is an ET-1 production stimulator significantly more potent than other factors assessed in the study, such as a rise in the intracranial pressure resulting from administering aCSF to CM. Blocking ETA receptors makes it impossible for the ET-1 released to bind to the receptors, which may be a factor preventing the occurrence of cerebral vasospasm following SAH.

Topics: Animals; Coronary Vasospasm; Endothelin Receptor Antagonists; Endothelin-1; Intracranial Pressure; Male; Peptides, Cyclic; Rats; Rats, Wistar; Receptor, Endothelin A; Subarachnoid Hemorrhage