dinoprost and Coronary-Vasospasm

Certain angina and coronary artery disease forms do not respond to Ca2+ channel blockers, and a role for vasoactive eicosanoids such as PGF2alpha in Ca2+ antagonist-insensitive coronary vasospasm is suggested; however, the signaling mechanisms are unclear. We investigated whether PGF2alpha-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+), PGF2alpha (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 PGF2alpha-induced [Ca2+]i increase but only partially inhibited PGF2alpha-induced cell contraction to 17%. Verapamil-insensitive PGF2alpha contraction was inhibited by PKC inhibitors GF-109203X, calphostin C, and epsilon-PKC V1-2. PGF2alpha caused Ca2+-dependent alpha-PKC and Ca2+-independent epsilon-PKC translocation from cytosolic to particulate fractions that was inhibited by calphostin C. Verapamil abolished PGF2alpha-induced alpha-but not epsilon-PKC translocation. PMA (10-6 M), a direct activator of PKC, caused 21% contraction with no significant [Ca2+]i increase and epsilon-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 alpha- or epsilon-PKC translocation. Thus a significant component of PGF2alpha-induced contraction of coronary smooth muscle is Ca2+ antagonist insensitive, involves Ca2+-independent epsilon-PKC activation and translocation, and may represent a signaling mechanism of Ca2+ antagonist-resistant coronary vasospasm.

Topics: Animals; Calcium; Calcium Channel Blockers; Cells, Cultured; Coronary Vasospasm; Coronary Vessels; Dinoprost; Enzyme Activation; Enzyme Inhibitors; Immunoblotting; Intracellular Fluid; Male; Muscle Contraction; Muscle, Smooth, Vascular; Protein Kinase C; Protein Kinase C-epsilon; Protein Transport; Signal Transduction; Swine; Vasoconstriction; Vasoconstrictor Agents

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

Serotonin is one of the most important vasoactive substances and has been implicated in the pathogenesis of coronary artery spasm and of acute coronary syndrome. We have recently demonstrated that local and long-term treatment with interleukin-1beta(IL-1beta) causes coronary arteriosclerotic changes and hyperconstrictive responses to serotonin in pigs in vivo. However, it remains to be examined which serotonergic (5-HT) receptor subtype mediates coronary spasm and whether alterations in serotonergic receptors are involved in the abnormality. In this study, we examined the inhibitory effect of sarpogrelate, a selective 5-HT2A serotonergic receptor antagonist, on the serotonin-induced coronary spasm as well as the possible alterations of serotonergic receptors in our porcine model. A segment of the porcine coronary artery was carefully dissected and aseptically wrapped with cotton mesh absorbing IL-1beta-bound microbeads from the adventitia. Two weeks after the procedure, angiographic study was performed, followed by binding assay for 5-HT1B and 5-HT2A serotonergic receptors and reverse transcription-polymerase chain reaction (RT-PCR) analysis for mRNA of those receptors. Angiographic study showed coronary vasospastic responses to serotonin at the IL-1beta-treated site. Sarpogrelate dose-dependently inhibited the serotonin-induced coronary spasm, but it did not affect the prostaglandin F2alpha-induced vasoconstriction. Radiolabeled receptor-binding assay showed that receptor affinity or receptor number of the 5-HT1B, or 5-HT2A receptors did not differ significantly between the spastic and the control sites. Furthermore, RT-PCR analysis showed that the expression of neither 5-HT2A nor 5-HT1B receptor mRNA was significantly altered at the spastic site. These results indicate that serotonin-induced coronary spasm is mediated primarily by 5-HT2A receptor in our porcine model, although the 5-HT2A receptor was not up-regulated, suggesting that alteration in the signal-transduction pathway for vascular smooth muscle contraction beyond the 5-HT2A receptor plays a primary role in the pathogenesis of coronary spasm in our porcine model.

Topics: Angiography; Animals; Base Sequence; Coronary Vasospasm; Dinoprost; Disease Models, Animal; Dose-Response Relationship, Drug; In Vitro Techniques; Interleukin-1; Male; Molecular Sequence Data; Muscle Contraction; Muscle, Smooth, Vascular; Protein Binding; Receptors, Serotonin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serotonin; Serotonin Antagonists; Succinates; Swine; Up-Regulation; Vasoconstrictor Agents

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

Although platelet activating factor (PAF) is an important vasoactive substance released from activated leukocytes, platelets and endothelial cells, little is known about its effect at the inflammatory coronary lesions in vivo.. To examine the coronary vasomotor responses to PAF at the inflammatory lesions in our swine model with interleukin-1 beta (IL-1 beta) in vivo.. Under aseptic conditions, the proximal segment of the porcine left coronary artery was dissected and wrapped with cotton mesh absorbing IL-1 beta. Two weeks after the operation, coronary vasomotion in response to intracoronary administration of 0.3 and 1 microgram/kg PAF, 1, 3, and 10 micrograms/kg serotonin, 1, 3 and 10 micrograms/kg histamine, and 5 and 50 micrograms/kg prostaglandin F2 alpha was examined by coronary arteriography.. At the IL-1 beta-treated site, PAF, serotonin and histamine, but not prostaglandin F2 alpha, caused hyperconstriction (n = 8). A synergy of the vasoconstricting effects of PAF and serotonin was also noted (n = 6). Administration of TCV-309, a selective PAF receptor antagonist, abolished the hyperconstrictive responses to PAF but not those to other agonists (n = 6). The PAF-induced coronary hyperconstrictions were significantly inhibited by administrations of the protein kinase C inhibitors staurosporine and sphingosine, but not by administrations of ryanodine, thapsigargin, or indomethacin (n = 4 each).. These results indicate that PAF causes hyperconstriction at the inflammatory coronary lesions in vivo by itself as well as in a synergistic manner with serotonin and that the PAF-induced hyperconstrictions are substantially mediated by a protein kinase C-dependent pathway in vivo.

Topics: Animals; Coronary Vasospasm; Coronary Vessels; Dinoprost; Drug Synergism; Histamine; Interleukin-1; Isoquinolines; Male; Platelet Activating Factor; Platelet Aggregation Inhibitors; Protein Kinase C; Pyridinium Compounds; Serotonin; Swine; Tetrahydroisoquinolines; Vasoconstriction

A 56 year old woman underwent cholecystectomy. Postoperative paralytic ileus was treated with an intravenous infusion of prostaglandin F2 alpha. During infusion she complained of oppressive chest pain. This was accompanied by ST segment depression, and was relieved by sublingual glyceryl trinitrate. Coronary arteriography did not show significant stenosis, but subsequent intravenous infusion of prostaglandin F2 alpha provoked multiple segmental spasm of both the right and left coronary arteries.

Topics: Angina Pectoris; Coronary Angiography; Coronary Vasospasm; Dinoprost; Electrocardiography; Female; Humans; Middle Aged

To determine whether coronary reperfusion enhances the production of endothelium-derived contracting factor, we investigated dogs subjected to global cardiac ischemia (45 minutes) followed by reperfusion (60 minutes). Segments of reperfused and control coronary arteries were suspended in organ chambers to measure isometric force. Perfusate hypoxia caused endothelium-dependent contraction in the control and reperfused arteries. However, reperfused arteries exhibited hypoxic contraction that was significantly greater than control segments. The hypoxic contractions in both the control and reperfused arteries could be inhibited by NG-monomethyl-L-arginine (L-NMMA), the blocker of endothelial cell synthesis of nitric oxide from L-arginine. The action of L-NMMA could be reversed by L-arginine but not D-arginine. Thus, after reperfusion, augmented production of endothelium-derived contracting factor occurs by an L-arginine-dependent pathway. We hypothesize that nitric oxide produced by L-arginine metabolism combines with superoxide anion to produce the peroxynitrite anion (ONOO-), which is metabolized to endothelium-derived contracting factor or induces its synthesis. Augmented production of endothelium-derived contracting factor would favor vasospasm after reperfusion.

Topics: Animals; Biological Factors; Carbon Dioxide; Coronary Vasospasm; Dinoprost; Dogs; Endothelins; Female; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth, Vascular; Myocardial Reperfusion; Nitrogen; Oxygen

Mechanism of coronary spasm was examined regarding endothelium-related relaxation and contraction produced by smooth muscle cells of spastic vessels isolated from Göttingen miniature pigs. In these pigs, coronary artery spasm was documented angiographically in vivo three months after endothelial denudation, and spastic and control segments of the coronary artery were suspended in organ chambers at their optimal length for generating tension. Applications of KCl (118 mM), acetylcholine(10(-9) to 10(-4) M), and PGF2 alpha (10(-8) to 3 X 10(-5) M) produced similar tension, at the respective doses, in both the spastic and control coronary arteries. During increasing concentrations of histamine (10(-8) to 3 X 10(-4) M; n = 14) and serotonin (10(-9) to 10(-5) M; n = 13), the maximum tension of the spastic vessel was 136 +/- 6 and 97 +/- 4%, respectively, of the tension produced by 118 mM KCl. That is significantly larger than seen in the control vessels: 98 +/- 4 and 74 +/- 4%, respectively. The ED50 to histamine and serotonin was also significantly less in the spastic vessels. After mechanical removal of the endothelium, the tension generated during the cumulative administration of histamine (n = 8) but not serotonin (n = 8) was larger in the spastic than the control vessels, thereby suggesting the presence of augmented responses of the smooth muscle to histamine in the spastic vessels. The increase in maximum tension after mechanical denudation was greater in the control than the spastic vessels in cases of histamine and serotonin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcimycin; Coronary Vasospasm; Dinoprost; Disease Models, Animal; Endothelium, Vascular; Histamine; Ketanserin; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Prostaglandins F; Serotonin; Swine; Swine, Miniature

Studies have been conducted on isolated segments of the left circumflex coronary artery of the dog to gain information on the mechanism or mechanisms of vasospasm. Coronary arteries contain both postjunctional alpha 1- and beta 1-adrenoceptors, and both are accessible to norepinephrine released from the sympathetic nerves. However, owing to the dominance of the beta 1-adrenoceptors, sympathetic stimulation causes relaxation of the vascular smooth muscle. In the primary branches of the circumflex artery, only beta 1-adrenoceptors are present. In patients with spasm of the coronary arteries, blockade of the beta 1-adrenoceptors may aggravate the spasm by permitting the unopposed constrictor action of the sympathetic nerves on the alpha 1-adrenoceptors on these vessels. The blood platelets contain substances, including 5-hydroxytryptamine (serotonin) and thromboxane A2, which can cause constriction of vascular smooth muscle. These substances are released whenever platelets aggregate. The normal endothelium, by forming and releasing prostacyclin, inhibits platelet aggregation. In addition, in response to platelet products, the normal endothelium forms one or more inhibitory substances that cause relaxation of the underlying smooth muscle. Also, if any thrombin is formed, this also causes an endothelium-mediated relaxation of the artery. Patients with coronary artery spasm usually have morphologic changes in the artery at the site of the spasm. Thus, platelets can aggregate at the site and the resultant release of serotonin and thromboxane A2, acting directly on the smooth muscle, causes constriction of the artery. Hypoxia of the myocardium follows and this augments the constriction.

Topics: Adenosine Diphosphate; Animals; Coronary Vasospasm; Dinoprost; Dogs; Endothelium; Epoprostenol; Heart; Humans; Muscle, Smooth, Vascular; Platelet Aggregation; Prostaglandins F; Receptors, Adrenergic; Serotonin; Swine; Swine, Miniature; Sympathetic Nervous System; Thromboxane A2

In order to diagnose patients in thrombotic state, it is quite important to detect increased concentration of plasma thromboxane B2 (TXB2), a stable catabolite of TXA2. To determine plasma TXB2 levels with high sensitivity and selectivity, we employed gas chromatography-mass spectrometry (GC/MS). The trimethylsilyl (TMS) ether derivatives conventionally employed in GC/MS analysis of prostanoids are not suitable for quantitation of plasma prostanoids, because the mass spectra are deficient in ions with high intensity in the high mass range and TMS ether derivatives are sensitive to moisture. To solve these problems we employed tert-butyldimethylsilyl (t-BDMS) ether derivatives, based on the observation that t-BDMS ether derivatives afforded abundant ions at [M-57]+ and showed good hydrolytic stability. The reaction conditions of tert-butyldimethylsilylation were also examined to optimize the selected ion monitoring response. The t-BDMS ether derivatives of prostanoids were successfully analyzed with a short capillary column with a relatively large diameter, with maintaining good separation. In conjunction with the use of reversed-phase high performance liquid chromatography as purification procedure, a sensitive and reproducible stable isotope dilution assay of plasma TXB2 was developed. The values obtained by this method correlated well with those obtained by the radioimmunoassay we have developed.

Topics: 6-Ketoprostaglandin F1 alpha; Adult; Aged; Angina Pectoris; Coronary Vasospasm; Dinoprost; Dinoprostone; Female; Gas Chromatography-Mass Spectrometry; Humans; Male; Middle Aged; Myocardial Infarction; Organosilicon Compounds; Prostaglandin D2; Prostaglandins D; Prostaglandins E; Prostaglandins F; Silicon; Thromboxane B2; Thromboxanes

Topics: Aged; Angina Pectoris; Arteriosclerosis; Calcium Channel Blockers; Coronary Vasospasm; Coronary Vessels; Dinoprost; Female; Humans; In Vitro Techniques; Prostaglandins F; Serotonin; Vasoconstriction

This study characterizes the inhibiting effect of diltiazem and nitroglycerin on periodic contractions of isolated human coronary arteries. Isometric force of coronary ring segments from sixty-nine cadavers was recorded in a muscle bath. To quantify the experimental results, we used 3 X 10(-6) M prostaglandin F2 alpha to induce the periodic contractions of a certain force. When diltiazem was added during the periodic contractions, the amplitude of oscillations gradually decreased until eventually oscillations ceased completely. The process prior to the cessation of the oscillations was characterized mainly by the inhibition of the contraction phase. The inhibition rate at the time of the complete cessation of oscillations was 49.3 +/- 6.3% at 5 X 10(-7) M. The time required for complete disappearance of oscillations was dependent on the diltiazem concentration. When nitroglycerin was added during periodic contractions, the oscillations did not disappear. Compared to the contraction phase, the relaxation phase was appreciably inhibited. With only 10(-7) M diltiazem, the rate of inhibition of the contraction phase was 22.0 +/- 7.7%, whereas the preliminary treatment with 5 X 10(-8) M nitroglycerin led to a complete cessation of the oscillations, and suppression of the level of the contractions to a significantly greater extent, viz. 58.7 +/- 5.8% (p less than 0.001). Therefore, it is considered more effective in the treatment and prevention of coronary spasm to use diltiazem and nitroglycerin simultaneously rather than individually.

Topics: Benzazepines; Coronary Vasospasm; Coronary Vessels; Diltiazem; Dinoprost; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; Muscle, Smooth, Vascular; Nitroglycerin; Periodicity; Prostaglandins F; Vasoconstriction