RESUMEN
We have performed chromosomal monitoring on 18 MRC-5 cell banks using the G-banding technique. A higher frequency of structural abnormalities and hyperdiploidy was detected with respect to the numerical values that were established with conventional staining techniques and reported in the 1979 Ad Hoc Committee guideline on karyology controls of human cell substrates. These numerical criteria have been adopted by international regulatory agencies to release human diploid cell banks (WI-38 and MRC-5) for vaccine manufacture. In the process of characterizing these 18 cell banks, a 7;12 translocation clone was detected in 7 cell banks at a frequency ranging from 0.2 to 5.6%. The presence of the t(7;12) appears to be biphasic. At low population doubling levels (PDLs) (< 30), t(7;12) is rarely observed. However, the incidence of t(7;12) increases and plateaus between PDL 40-50. A decrease in frequency is observed at higher PDLs. Before senescence of the cell bank, t(7;12) is not observed. Investigation of the growth characteristics of MRC-5 cells revealed that cell banks containing the translocation senesced at similar PDLs compared to MRC-5 cells with no detectable 7;12 translocation. In addition, MRC-5 cell banks containing the t(7;12) have successfully completed tumorigenicity testing in a nude mouse model. We conclude that MRC-5 cells obtained from either National Institute for Biological Standards and Control (NIBSC) or American Type Culture Collection (ATCC) contain a 7;12 translocation at a low frequency. This abnormality does not provide MRC-5 cell bank mass cultures with a growth advantage nor is it tumorigenic in nude mice. Furthermore, the presence of this clone and employment of the G-banding technique may be responsible for the increased observation of structural abnormalities in our laboratories. In view of this information, the cytogenetic criteria that were established in 1979 with conventional staining techniques are not appropriate for human diploid cell banks that are examined with more sensitive methodology. Since it has been recognized that MRC-5 and WI-38 cells are safe biological substrates, we recommend that MRC-5 and WI-38 cell banks should only be identified by using an appropriate identity test and should not require any chromosomal analysis before being used as a cell substrate for the manufacture of live virus vaccines.
Asunto(s)
Bandeo Cromosómico/métodos , Bancos de Tejidos , Animales , Línea Celular , Cromosomas Humanos Par 12 , Cromosomas Humanos Par 7 , Humanos , Cariotipificación , Ratones , Translocación Genética , Células Tumorales Cultivadas , Cultivo de VirusRESUMEN
Bovine coronary arteries relax in response to bradykinin, methacholine, sodium nitroprusside, isoproterenol, and arachidonic acid in a concentration-dependent manner. The relaxations to methacholine, bradykinin, and arachidonic acid are lost when endothelium is removed. Indomethacin, a cyclooxygenase inhibitor, attenuated the relaxations to methacholine, bradykinin, and arachidonic acid and shifted the EC50 (control versus indomethacin) to each (1 x 10(-7) versus 3 x 10(-7) mo1/L, 3 x 10(-10) versus 2 x 10(-9) mo1/L, and 3 x 10(-7) versus 2 x 10(-6) mo1/L, respectively). Nitro-L-arginine, a nitric oxide synthase inhibitor, also attenuated the relaxations to methacholine, bradykinin, and arachidonic acid and shifted the EC50 (control versus nitro-L-arginine) to each (1 x 10(-7) versus 3 x 10(-7) mo1/L, 3 x 10(-10) versus > 10(-9) mo1/L, and 3 x 10(-7) versus > 10(-6) mo1/L, respectively). The combination of indomethacin and nitro-L-arginine blunted the relaxations to these agents and also shifted the EC50 values (control versus indomethacin plus nitro-L-arginine) to each (1 x 10(-7) versus 5 x 10(-7) mo1/L, 3 x 10(-10) versus > 10(-9) mo1/L, and 3 x 10(-7) versus > 10(-6) mo1/L, respectively). Methacholine, bradykinin, and arachidonic acid stimulated the release of prostaglandin I2, measured as 6-keto-PGF1 alpha. Indomethacin, but not nitro-L-arginine, inhibited arachidonic acid-induced release of 6-keto-PGF1 alpha. Vascular cGMP content was unchanged by arachidonic acid but was significantly elevated by bradykinin. Relaxations to prostaglandin I2 and sodium nitroprusside, but not 8,9-epoxyeicosatrienoic acid or isoproterenol, were inhibited by nitro-L-arginine. We conclude that the endothelium-dependent relaxations to methacholine, bradykinin, and arachidonic acid are partly due to prostaglandin I2 release. The remainder of the responses to these agents is due to the release of other relaxing factor or factors. Since bradykinin increased cGMP and nitro-L-arginine partially inhibited its relaxant effects, nitric oxide also appears to participate in the bradykinin-induced effect. Since the combination of indomethacin and nitro-L-arginine failed to completely block the relaxations to methacholine, bradykinin, and arachidonic acid, another endothelial factor must contribute to their vascular effects. Surprisingly, nitro-L-arginine attenuated the relaxations to arachidonic acid; however, L-arginine failed to reverse the effects of nitro-L-arginine on arachidonic acid-induced relaxations. In addition, arachidonic acid failed to increase cGMP. Nitro-L-arginine also reduced the responses to prostaglandin I2 and sodium nitroprusside. These data indicate that these arginine analogues may have effects other than competitive inhibition of nitric oxide synthase.
Asunto(s)
Ácido Araquidónico/fisiología , Vasos Coronarios/fisiología , Epoprostenol/fisiología , Contracción Muscular , Músculo Liso Vascular/fisiología , Óxido Nítrico/fisiología , Análisis de Varianza , Animales , Ácido Araquidónico/metabolismo , Ácido Araquidónico/farmacología , Bradiquinina/farmacología , Bovinos , Cromatografía Líquida de Alta Presión , Vasos Coronarios/efectos de los fármacos , GMP Cíclico/análisis , Epoprostenol/análisis , Epoprostenol/biosíntesis , Técnicas In Vitro , Cloruro de Metacolina/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , RadioinmunoensayoRESUMEN
Both polymorphonuclear (PMN) leukocytes and metabolites of arachidonic acid, especially lipoxygenase products, have been reported to contribute to myocardial damage after coronary artery occlusion and reperfusion. While canine models of myocardial ischemia were used in many of these studies, very little is known about arachidonic acid metabolism by canine PMNs. Moreover, it is unclear whether arachidonic acid metabolites released by canine PMNs affect vascular tone. Therefore, we characterized arachidonic acid metabolism by canine PMNs and determined the effect of these metabolites on vascular tone of isolated canine coronary arteries. Suspensions of canine PMNs were incubated with [14C]arachidonic acid and the calcium ionophore A23187. The incubation media was extracted, and the metabolites resolved by HPLC. 20-Hydroxy-leukotriene B4 (LTB4), 12,20-dihydroxyeicosatetraenoic acid (diHETE), LTB4, 12-hydroxyheptadeclatrienoic acid (HHT), and 12-(S)-hydroxyeicosatetraenoic acid (HETE) were isolated, and their structures confirmed by gas chromatography/mass spectrometry. There was also evidence for the formation of 20-HETE, thromboxane B2 (TXB2), 5-HETE, and several isomers of LTB4. None of the arachidonic acid metabolites that were isolated from incubates of canine PMNs augmented vascular tone, but material migrating with 12,20-diHETE relaxed canine coronary arteries. Authentic 12(S),20-diHETE also produced a concentration-related relaxation of canine coronary artery. 12(R), 20-diHETE was inactive. 20-HETE inhibited A23187-induced PMN aggregation. Thus, arachidonic acid is metabolized in canine PMNs through the cyclooxygenase, lipoxygenases and cytochrome P-450 pathways. Whether these metabolites contribute to myocardial injury remains to be determined.
Asunto(s)
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/análogos & derivados , Ácido Araquidónico/metabolismo , Vasos Coronarios/efectos de los fármacos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacología , Neutrófilos/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Animales , Arterias/efectos de los fármacos , Arterias/fisiología , Agregación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Vasos Coronarios/fisiología , Perros , Ácidos Grasos Insaturados/metabolismo , Ácidos Grasos Insaturados/farmacología , Ácidos Hidroxieicosatetraenoicos/análisis , Técnicas In Vitro , Leucotrieno B4/análogos & derivados , Leucotrieno B4/metabolismo , Leucotrieno B4/farmacología , Espectrometría de Masas , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Endoperóxidos de Prostaglandinas Sintéticos/farmacología , Tromboxano A2/análogos & derivados , Tromboxano A2/farmacología , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Vasodilatación/efectos de los fármacosRESUMEN
Endothelial cells release several factors which influence vascular tone, leukocyte function and platelet aggregation. Some of these factors are metabolites of arachidonic acid, most notably prostacyclin. However, many of the endothelial metabolites of arachidonic acid have not been positively identified. The purpose of these studies is to identify the arachidonic acid metabolites synthesized by bovine coronary endothelial cells. Cultured bovine coronary artery endothelial cells were incubated with [14C]arachidonic acid. The incubation media was extracted and the radioactive metabolites resolved by a combination of reverse phase- and normal phase-high pressure liquid chromatography (HPLC). The cells synthesized 6-keto prostaglandin (PG)F1 alpha, PGE2, 12-hydroxyheptadecatrienoic acid (HHT), 12-, 15-, and 11-hydroxyeicosatetraenoic acids (HETE), and 14,15-, 11,12-, 8,9-, and 5,6-epoxyeicosatrienoic acids (EET). Several of the HETEs were further analyzed by chiral-phase HPLC. The cells synthesized predominately 12(S)-, 15(S)-, and 11(R)-HETE. The synthesis of the S optical isomers of 12- and 15-HETE suggested that the 12- and 15-lipoxygenases were present in these cells. 11(R)-HETE is probably derived from cyclooxygenase. They also synthesized smaller amounts of 9-, 8- and 5-HETEs. The structures of the HETEs and EETs were confirmed by mass spectrometry. The release of 6-keto PGF1 alpha and 15-HETE was measured by specific radioimmunoassays. Melittin, thrombin, arachidonic acid and A23187 stimulated the release of both eicosanoids in a concentration-related matter. Under all conditions, the release of 6-keto PGF1 alpha exceed the release of 15-HETE. Therefore, cultured bovine coronary artery endothelial cells synthesize cyclooxygenase, lipoxygenase and cytochrome P-450 metabolites of arachidonic acid.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/metabolismo , Endotelio Vascular/metabolismo , Compuestos Epoxi/metabolismo , Ácidos Hidroxieicosatetraenoicos/biosíntesis , 6-Cetoprostaglandina F1 alfa/biosíntesis , Ácido 8,11,14-Eicosatrienoico/química , Animales , Ácido Araquidónico/metabolismo , Ácido Araquidónico/farmacología , Radioisótopos de Carbono , Bovinos , Células Cultivadas/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Vasos Coronarios , Sistema Enzimático del Citocromo P-450/metabolismo , Endotelio Vascular/enzimología , Compuestos Epoxi/química , Ácidos Hidroxieicosatetraenoicos/química , Ácidos Hidroxieicosatetraenoicos/aislamiento & purificación , Lipooxigenasa/metabolismo , Espectrometría de Masas , Prostaglandina-Endoperóxido Sintasas/metabolismo , EstereoisomerismoRESUMEN
Papillomaviruses infect epithelia of the skin and mucous membranes and cause benign or malignant tumours in animals and in humans. The viruses are highly species-specific, and cell culture systems for propagating human papillomaviruses (HPVs) do not exist. However, there are several animal papillomavirus models. In the cottontail rabbit papillomavirus (CRPV) system, we demonstrated that recombinant CRPV virus-like particles (VLPs) consisting of the capsid proteins L1 or L1+L2 can be produced in the yeast Saccharomyces cerevisiae. Three immunizations with L1 VLPs formulated on aluminum adjuvant at 1-100 micrograms dose-1 efficiently protected rabbits from challenge with CRPV. Sera of immunized rabbits were shown to contain high-titered serum antibodies to CRPV L1 VLPs and to neutralize CRPV in vitro. Our results suggest that recombinant yeast-derived VLPs could be the basis for a candidate HPV vaccine.
Asunto(s)
Papillomavirus del Conejo de Rabo Blanco/inmunología , Papiloma/prevención & control , Papiloma/virología , Infecciones por Papillomavirus/prevención & control , Saccharomyces cerevisiae/metabolismo , Infecciones Tumorales por Virus/prevención & control , Vacunas Virales/farmacología , Animales , Anticuerpos Antivirales/biosíntesis , Secuencia de Bases , Cápside/biosíntesis , Cápside/inmunología , Femenino , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/inmunología , Conejos , Infecciones Tumorales por Virus/complicaciones , Infecciones Tumorales por Virus/inmunología , Vacunas Virales/inmunología , Virión/inmunología , Virión/metabolismoRESUMEN
Human papillomavirus 6a (HPV6a), the most abundant HPV6 subtype, was detected in a vulvar condyloma acuminatum. The complete genome of HPV6a was cloned, and its DNA sequence was shown to be over 97% identical to the HPV6b sequence. Of the eight open reading frames (ORFs) of HPV6a, only the imputed amino acid sequence of the major capsid protein L1 was identical to the corresponding HPV6b sequence; all other HPV6a ORFs showed amino acid changes compared to the HPV6b ORFs. The HPV6a L1 or the L1 + L2 ORFs were expressed in the yeast Saccharomyces cerevisiae. Self-assembly of the L1 capsid protein into virus-like particles (VLPs) was demonstrated both in the L1 as well as L1 + L2 coexpressing yeast strains. Copurification of the L1 and L2 proteins showed complex formation of the L1 and L2 proteins in the yeast-derived VLPs of coexpressing strains.
Asunto(s)
Genoma Viral , Papillomaviridae/genética , Saccharomyces cerevisiae , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Codón/genética , Condiloma Acuminado/virología , Cartilla de ADN , ADN Viral/genética , ADN Viral/aislamiento & purificación , Femenino , Variación Genética , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Papillomaviridae/clasificación , Papillomaviridae/aislamiento & purificación , Mutación Puntual , Reacción en Cadena de la Polimerasa , Trastornos Puerperales/virología , Mapeo Restrictivo , Enfermedades de la Vulva/virologíaRESUMEN
Reactive oxygen metabolites have been reported to affect platelet aggregation. However, this phenomenon is still poorly understood. In the present study we investigated the effects of superoxide radical and hydrogen peroxide (H2O2) on platelet function in vitro and correlated those effects to possible changes of platelet concentrations of cyclic nucleotides and thromboxane, since these systems play a key role in the response of platelets to activating stimuli. Human platelets were exposed to xanthine-xanthine oxidase (X-XO), a system that generates both superoxide radicals and H2O2. Sixty seconds of incubation with X-XO impaired aggregation in response to ADP (by 48%), collagen (by 71%), or the thromboxane mimetic U-46619 (by 50%). This effect was reversible and occurred in the absence of cell damage. Impairment of aggregation in platelets exposed to X-XO was due to H2O2 formation, since it was prevented by catalase but not by superoxide dismutase. Similarly, incubation with the pure H2O2 generator glucose-glucose oxidase also markedly inhibited ADP-induced platelet aggregation in a dose-dependent fashion. Impaired aggregation by H2O2 was accompanied by a > 10-fold increase in platelet concentrations of guanosine 3',5'-cyclic monophosphate (cGMP), whereas adenosine 3',5'-cyclic monophosphate levels remained unchanged. The inhibitory role of increased cGMP formation was confirmed by the finding that H2O2-induced impairment of platelet aggregation was largely abolished when guanylate cyclase activation was prevented by incubating platelets with the guanylate cyclase inhibitor, LY-83583. Different effects were observed when arachidonic acid was used to stimulate platelets. Exposure to a source of H2O2 did not affect aggregation to arachidonate. Furthermore, in the absence of exogenous H2O2, incubation with catalase, which had no effects on platelet response to ADP, collagen, or U-46619, virtually abolished platelet aggregation and markedly reduced thromboxane B2 production (to 44% of control) when arachidonic acid was used as a stimulus. In conclusion, our data demonstrate that H2O2 may exert complex effects on platelet function in vitro. Low levels of endogenous H2O2 seem to be required to promote thromboxane synthesis and aggregation in response to arachidonic acid. In contrast, exposure to larger (but not toxic) concentrations of exogenous H2O2 may inhibit aggregation to several agonists via stimulation of guanylate cyclase and increased cGMP formation.
Asunto(s)
Plaquetas/efectos de los fármacos , Especies Reactivas de Oxígeno/farmacología , Nucleótidos de Adenina/metabolismo , Plaquetas/metabolismo , Plaquetas/fisiología , Catalasa/farmacología , Humanos , Peróxido de Hidrógeno/farmacología , Nucleótidos Cíclicos/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Superóxido Dismutasa/farmacología , Superóxidos/farmacología , Tromboxanos/biosíntesisRESUMEN
Metabolites of arachidonic acid regulate several physiological processes, including vascular tone. The purpose of this study was to determine which metabolites of arachidonic acid are produced by bovine coronary arteries and which may regulate coronary vascular tone. Arachidonic acid induced a concentration-related, endothelium-dependent relaxation [one-half maximum effective concentration (EC50) of 2 x 10(-7) M and a maximal relaxation of 91 +/- 2% at 10(-5) M] of bovine coronary arteries that were contracted with U-46619, a thromboxane mimetic. The concentration of 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), a metabolite of prostaglandin I2 (PGI2), increased from 82 +/- 6 to 328 +/- 24 pg/ml with arachidonic acid (10(-5) M). Treatment with the cyclooxygenase inhibitor indomethacin attenuated arachidonic acid-induced relaxations by approximately 50% and blocked the synthesis of 6-keto-PGF1 alpha. PGI2 caused a concentration-related relaxation (EC50 of 10(-8) M and a maximal relaxation of 125 +/- 11% at 10(-7) M). BW755C, a cyclooxygenase and lipoxygenase inhibitor, inhibited arachidonic acid-induced relaxation to the same extent as indomethacin. When vessels were treated with both indomethacin and BW755C, the inhibition of relaxation was the same as either inhibitor alone. SKF 525a, a cytochrome P-450 inhibitor, reduced arachidonic acid-induced relaxation by approximately 50%. When SKF 525a was given in combination with indomethacin, the relaxation by arachidonic acid was almost completely inhibited. SKF 525a inhibited the synthesis of epoxyeicosatrienoic acids (EETs).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido Araquidónico/farmacología , Vasos Coronarios/efectos de los fármacos , Epoprostenol/fisiología , Vasodilatación , Ácido 8,11,14-Eicosatrienoico/farmacología , Animales , Arterias/efectos de los fármacos , BovinosRESUMEN
The goal of this study was to test the hypotheses that endogenous ADP plays an important role in vivo in mediating platelet aggregation and cyclic coronary artery blood flow variations (CFVs) in stenosed and endothelium-injured coronary arteries in an experimental canine model. Anesthetized animals were studied and coronary blood flow velocities monitored by a pulsed Doppler flow probe positioned around the left anterior descending coronary artery. CFVs were established by an external constrictor positioned at sites with injured endothelium. Apyrase, an ADP-removing enzyme, was infused into the left anterior descending coronary artery (0.3-1.8 units/min) 30 minutes or 2 hours after the establishment of CFVs. Complete abolition of CFVs was achieved in 81% (13/16) of dogs with 30-minute CFVs and in 83% (five of six) of dogs with 2-hour CFVs. In other dogs, a potent inhibitor of ADP-induced platelet aggregation, clopidogrel, was administered as a 10 mg/kg i.v. bolus and a 2.5 mg/kg/hr infusion 30 minutes and 3 hours after the establishment of CFVs. This treatment resulted in complete abolition of CFVs in 14 dogs (100%) with either 30-minute or 3-hour CFVs. Epinephrine was infused into some dogs after CFVs had ceased as a result of either apyrase or clopidogrel administration and into some dogs in whom SQ29548, a thromboxane A2 receptor antagonist, had been given when apyrase failed to abolish CFVs. Epinephrine restored CFVs in all dogs treated with apyrase alone, 67% (four of six) of dogs treated with the combination of apyrase and SQ29548, and 29% (two of seven) of dogs treated with clopidogrel. The plasma epinephrine levels required for CFV restoration were 20 times higher than baseline values in dogs receiving apyrase alone, 100 times higher when a combination of apyrase and SQ29548 had been given, and more than 5,000 times higher in dogs receiving clopidogrel. In vitro studies showed that apyrase only inhibited ADP-induced platelet aggregation, whereas clopidogrel not only inhibited ADP-induced platelet aggregation, but also reduced platelet aggregation induced by the thromboxane mimetic U46619 and serotonin. These data suggest that 1) ADP is an important mediator of platelet aggregation and CFVs in vivo and 2) combined inhibition of thromboxane A2 and ADP's effects provides marked protection against CFVs in experimentally stenosed and endothelium-injured canine coronary arteries. These data and our previous observations are consistent with the possibility that specific antagonists of thromboxane A2, serotonin, and ADP, alone and together, may provide substantial protection against platelet aggregation leading to CFVs at sites of endothelial injury and coronary artery stenosis.
Asunto(s)
Adenosina Difosfato/fisiología , Circulación Coronaria , Enfermedad Coronaria/fisiopatología , Vasos Coronarios/fisiopatología , Endotelio Vascular/lesiones , Agregación Plaquetaria , Animales , Apirasa/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes , Clopidogrel , Circulación Coronaria/efectos de los fármacos , Perros , Ácidos Grasos Insaturados , Femenino , Hidrazinas/farmacología , Masculino , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Serotonina/farmacología , Tromboxano A2/antagonistas & inhibidores , Ticlopidina/análogos & derivados , Ticlopidina/farmacologíaRESUMEN
We have reported that thromboxane A2 and serotonin are two important mediators of coronary cyclic flow variations (CFVs) caused by recurrent platelet aggregation and dislodgement on a stenosed coronary arterial wall with endothelial injury. To test the hypothesis that blocking the synthesis of thromboxane A2 would not prevent serotonin release, 1.1, 4.6, and 9.2 mg/kg of aspirin were administered through the left atrium to 27 dogs with CFVs. The CFV elimination rate was 70% in the aspirin-treated dogs. Thromboxane B2 and serotonin concentrations were measured in different coronary arterial segments. There were significantly lower thromboxane B2 and 6-keto-PFG1a levels in the stenosed left arterior descending (LAD) segments with increasing dosage of aspirin-208 +/- 36, 24 +/- 31, 50 +/- 6 ng/g (P less than 0.0001) and 125 +/- 27, 58 +/- 38, 25 +/- 5 ng/g (P less than 0.0001), respectively. Serotonin levels were significantly higher in stenosed LAD (265.7 +/- 131.2 ng/g) than in LAD segments proximal or distal to the stenosis and in corresponding circumflex coronary artery segments, 17.1 +/- 3.7, 18.6 +/- 3.7, and 19.2 +/- 5.1 ng/g, respectively (P less than 0.05) following the highest dose of aspirin. In 41 additional dogs, electrical injury was used to initiate thrombosis in the circumflex artery and in those receiving aspirin (15 mg/kg) (n = 5), occlusive thrombus formation was inhibited. However, the local accumulation of serotonin was not significantly different between the control (194 +/- 27 ng/g) (n = 36) and the aspirin-treated group (167 +/- 19 ng/g) (n = 5). In vitro platelet aggregation induced by arachidonic acid was inhibited by the in vivo administration of 1.1 mg/kg of aspirin and abolished by 4.6 + 1.1 and 9.2 + 4.6 + 1.1 mg/kg of aspirin. However, serotonin-induced platelet aggregation was not affected following all doses of aspirin. Thus, aspirin eliminates CFVs in 70% of dogs, and markedly diminishes thromboxane A2 and prostacyclin concentrations in stenosed canine coronary arteries, but it does not prevent local serotonin accumulation. Similarly, aspirin prevents occlusive coronary thrombosis in dogs with electrically-induced endothelial injury, but it did not prevent local assumulation of serotonin. These experimental findings suggest that cyclo-oxygenese inhibition does not prevent serotonin accumulation at sites of coronary artery endothelial injury, and they thereby help provide a potential explanation of the lack of complete protection provided by aspirin in eliminating CFVs in this experimental model.
Asunto(s)
Aspirina/farmacología , Circulación Coronaria/efectos de los fármacos , Enfermedad Coronaria/metabolismo , Serotonina/metabolismo , Tromboxano B2/biosíntesis , 6-Cetoprostaglandina F1 alfa/biosíntesis , Animales , Trombosis Coronaria/metabolismo , Perros , Electricidad , Femenino , Masculino , Agregación PlaquetariaRESUMEN
To assess endothelium-dependent responses of blood vessels in vitro, endothelial cells are removed by a variety of mechanical means. We sought to determine if the method of removal of the endothelium affected arachidonic acid metabolism and vascular reactivity of isolated strips of rabbit aorta. Thoracic aorta of New Zealand White rabbits were excised and sectioned into strips with a sharp razor blade. The luminal surface of the vessel was then gently stroked (denuded-1) or forcefully rubbed (denuded-2) with a moist cotton swab. Vessels were then either fixed in 3% glutaraldehyde and processed for electron microscopy, incubated with [14C]arachidonic acid and 20 microM A23187 for determination of arachidonic acid metabolism, incubated with 20 microM A23187 for measurement of endogenous release of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and 12-hydroxyeicosatetraenoic acid (12-HETE) by specific radioimmunoassays, or suspended in an organ chamber filled with Krebs bicarbonate solution for vascular reactivity experiments. Electron micrographs showed that denuded-1 vessels lacked an endothelial cell layer and had slight degeneration of the smooth muscle cells. Additionally, these vessels had a diminished capacity to produce 6-keto-PGF1 alpha as compared to control vessels (214 +/- 25 vs. 360 +/- 36 pg/mg of tissue, P less than 0.05). Denuded-2 vessels contained severe degeneration and rupture of smooth muscle cells in addition to the loss of the endothelial cell layer. While the 6-keto-PGF1 alpha concentration (168 +/- 23 pg/mg) was less in denuded-2 vessels, HPLC indicated that the production of [14C]12-HETE was markedly increased in these vessels as compared to control or denuded-1 vessels.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Aorta Torácica/metabolismo , Ácidos Araquidónicos/metabolismo , Endotelio Vascular/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , 6-Cetoprostaglandina F1 alfa/biosíntesis , Animales , Aorta Torácica/ultraestructura , Ácido Araquidónico , Endotelio Vascular/ultraestructura , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Microscopía Electrónica de Rastreo , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Conejos , RadioinmunoensayoAsunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Vasos Coronarios/efectos de los fármacos , Endotelio Vascular/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido 8,11,14-Eicosatrienoico/farmacología , Animales , Bovinos , Células Cultivadas , Vasos Coronarios/citología , Perros , Epoprostenol/farmacología , Ácidos Hidroxieicosatetraenoicos/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Endoperóxidos de Prostaglandinas Sintéticos/farmacología , Vasodilatación/efectos de los fármacosRESUMEN
We tested the hypothesis that simultaneous inhibition of TxA2 synthase and blockade of TxA2/PHG2 receptors is more effective in enhancing thrombolysis and preventing reocclusion after discontinuation of tissue plasminogen activator (t-PA) than either intervention alone. Coronary thrombosis was induced in 35 dogs by placing a copper coil into the left anterior descending coronary artery. Coronary flow was measured with a Doppler flow probe. 30 min after thrombus formation, the animals received saline (controls, n = 10); SQ 29548 (0.4 mg/kg bolus + 0.4 mg/kg per h infusion), a TxA2/PGH2 receptor antagonist (n = 8); dazoxiben (5 mg/kg bolus + 5 mg/kg per h infusion), a TxA2 synthase inhibitor (n = 9); or R 68070 (5 mg/kg bolus + 5 mg/kg per h infusion), a drug that blocks TxA2/PGH2 receptors and inhibits TxA2 synthase (n = 8). Then, all dogs received heparin (200 U/kg) and a bolus of t-PA (80 micrograms/kg) followed by a continuous infusion (8 micrograms/kg per min) for up to 90 min or until reperfusion was achieved. The time to thrombolysis did not change significantly in SQ 29548-treated dogs as compared with controls (42 +/- 5 vs. 56 +/- 7 min, respectively, P = NS), but it was significantly shortened by R 68070 and dazoxiben (11 +/- 2 and 25 +/- 6 min, respectively, P less than 0.001 vs. controls and SQ 29548-treated dogs). R 68070 administration resulted in a lysis time significantly shorter than that observed in the dazoxiben-treated group (P less than 0.01). Reocclusion was observed in eight of eight control dogs, five of seven SQ 29548-treated dogs, seven of nine dazoxiben-treated dogs, and zero of eight R 68070-treated animals (P less than 0.001). TxB2 and 6-keto-PGF1 alpha, measured in blood samples obtained from the coronary artery distal to the thrombus, were significantly increased at reperfusion and at reocclusion in control animals and in dogs receiving SQ 29548. R 68070 and dazoxiben prevented the increase in plasma TxB2 levels, whereas 6-keto-PGF1 alpha levels were significantly increased with respect to control and SQ 29548-treated dogs. Thus, simultaneous inhibition of TxA2 synthase and blockade of TxA2/PGH2 receptors is more effective than either intervention alone in this experimental model in enhancing thrombolysis and preventing reocclusion after t-PA administration.
Asunto(s)
Trombosis Coronaria/tratamiento farmacológico , Hidrazinas/uso terapéutico , Imidazoles/uso terapéutico , Endoperóxidos de Prostaglandina/fisiología , Receptores de Prostaglandina/efectos de los fármacos , Tromboxano A2/antagonistas & inhibidores , Tromboxano-A Sintasa/antagonistas & inhibidores , Activador de Tejido Plasminógeno/uso terapéutico , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes , Trombosis Coronaria/etiología , Cricetinae , Perros , Ácidos Grasos Insaturados , Fibrinólisis/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Prostaglandinas/biosíntesis , Receptores de Tromboxanos , Receptores de Tromboxano A2 y Prostaglandina H2RESUMEN
The purpose of this study was to test the hypothesis that combined thromboxane A2 synthetase inhibition and receptor blockade is superior to either action alone in preventing cyclic flow variations in stenosed and endothelially injured canine coronary arteries. Forty-five dogs developed coronary cyclic flow variations after a plastic constrictor was placed around the left anterior descending coronary artery at the site where the endothelium was injured and received different interventions. In Group I, 17 dogs were treated with SQ 29,548, a thromboxane A2-prostaglandin H2 receptor antagonist. In Group II, 11 dogs received dazoxiben, a thromboxane A2 synthetase inhibitor. In Group III, R 68,070, a dual thromboxane A2 synthetase inhibitor and thromboxane A2-prostaglandin H2 receptor antagonist, was administered to 11 dogs. Group IV comprised six dogs that received aspirin before receiving R 68,070. Complete abolition of cyclic flow variations was achieved in 71% of dogs in Group I, 82% in Group II, 100% in Group III (p = 0.06 compared with Group I) and 50% in Group IV (p = 0.03 compared with Group III). Epinephrine was infused into dogs with abolished cyclic flow variations: all dogs in Group I had cyclic flow variations restored, 44% in Group II (p = 0.01 compared with Group I) and 64% in Group III (p = 0.04 compared with Group I). The plasma epinephrine levels required to restore cyclic flow variations were 2.2 +/- 0.5 ng/ml (control 0.04 +/- 0.01) in Group I, 8.7 +/- 4.5 ng/ml (control 0.05 +/- 0.02) in Group II and 7.4 +/- 2.6 ng/ml (control 0.07 +/- 0.02) in Group III.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Circulación Coronaria/efectos de los fármacos , Enfermedad Coronaria/tratamiento farmacológico , Receptores de Prostaglandina/efectos de los fármacos , Tromboxano A2/antagonistas & inhibidores , Tromboxano-A Sintasa/antagonistas & inhibidores , Animales , Aspirina/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes , Perros , Epinefrina/farmacología , Epoprostenol/fisiología , Ergolinas/uso terapéutico , Ácidos Grasos Insaturados , Femenino , Hidrazinas/uso terapéutico , Imidazoles/uso terapéutico , Masculino , Ácidos Pentanoicos/uso terapéutico , Piridinas/uso terapéutico , Receptores de Tromboxanos , Antagonistas de la Serotonina/uso terapéuticoRESUMEN
Coronary vascular injury promotes blood cell-vessel wall interactions that influence arachidonic acid metabolism and coronary blood flow patterns. Since lipoxygenase and cytochrome P-450 epoxygenase metabolites of arachidonic acid are synthesized by vascular and inflammatory cells and have a variety of important biological actions, we investigated the metabolism of arachidonic acid by these pathways in normal and stenosed, endothelially injured canine coronary arteries. We found and confirmed by gas chromatography/mass spectrometry that primarily 12- and 15-hydroxyeicosatetraenoic acids (HETEs) are synthesized by both coronary artery segments. Lesser amounts of 11-, 9-, 8-, and 5-HETEs are also produced. 15-Ketoeicosatetraenoic acid is also synthesized. The synthesis of 14C-HETEs is fivefold to 10-fold greater by the stenosed than the normal coronary artery. Specific radioimmunoassays indicated that the stenosed coronary artery synthesized 93 +/- 14 and 1,102 +/- 154 ng/g of tissue of 15- and 12-HETE, respectively, while the normal coronary artery produced 17 +/- 3 and 162 +/- 68 ng/g of tissue of 15- and 12-HETE, respectively. Products comigrating with 14,15-; 11,12-; 8,9-; and 5,6-epoxyeicosatrienoic acids (EETs) and the corresponding dihydroxyeicosatrienoic acids (DHETs) were detected predominantly in stenosed coronary arteries by high-pressure liquid chromatography. The structures of the EETs were confirmed by GC/MS. The EETs and prostaglandin I2 produced endothelium-independent, concentration-related relaxations of dog coronary artery rings. These data indicate that normal and stenotic coronary arteries metabolize arachidonic acid to HETEs, DHETs, and EETs along with prostaglandins; however, the synthesis of these metabolites is greater in the stenosed, endothelially injured vessel. The EETs may be synthesized during the development of cyclic flow variations and counteract the vasoconstrictor effects of thromboxane A2.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Enfermedad Coronaria/metabolismo , Vasos Coronarios/metabolismo , Sistema Enzimático del Citocromo P-450 , Lipooxigenasa/biosíntesis , Oxigenasas/biosíntesis , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Ácido Araquidónico , Ácidos Araquidónicos/biosíntesis , Constricción Patológica , Citocromo P-450 CYP2J2 , Perros , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Prostaglandinas/biosíntesis , Valores de ReferenciaRESUMEN
In Aplysia mechanosensory neurons, the neuropeptide FMRFamide increases the opening of the background S-K+ channel. This action is mediated by activation of arachidonic acid metabolism. Arachidonic acid in Aplysia nervous tissue is transformed through the 12-lipoxygenase pathway to 12-HPETE, which undergoes further metabolism. In intact sensory cells, 12-HPETE simulates the FMRFamide response, raising the question of whether 12-HPETE is the messenger molecule ultimately acting on the S-K+ channel. Here we show that in cell-free (inside-out) patches from sensory cells, 12-HPETE fails to modulate the S-K+ channel, but in the presence of hematin (which catalyzes 12-HPETE metabolism), it triggers sharp increases in the channel opening probability. We also found that SKF-525A, an inhibitor of the cytochrome P450, reduces the response to FMRFamide, arachidonic acid, and 12-HPETE in intact cells. We conclude that a heme-catalyzed transformation of 12-HPETE is necessary and sufficient to promote the opening of the S-K+ channel and a heme-containing enzyme such as cytochrome P450 might play this key role.
Asunto(s)
Ácidos Araquidónicos/farmacología , Hemo/farmacología , Leucotrienos/metabolismo , Canales de Potasio/efectos de los fármacos , Animales , Aplysia , Sistema Libre de Células , FMRFamida , Hemina/farmacología , Leucotrienos/farmacología , Neuropéptidos/farmacología , Piridinas/farmacologíaRESUMEN
Feeding a diet rich in eicosapentaenoic acid (EPA) to Watanabe heritable hyperlipidemic (WHHL) rabbits resulted in an attenuated aortic contractile response to the vasoconstrictor agent serotonin when compared to responses from WHHL rabbits fed normal rabbit chow. In contrast, only the maximal contractile response to norepinephrine was reduced in EPA-fed rabbit aortas. Additionally, methacholine-induced relaxations were potentiated in aortas obtained from the EPA-fed rabbits. When platelets obtained from EPA-fed rabbits were incubated with arachidonic acid, there was a reduced ability of the platelets to adhere to albumin-coated discs in comparison to control rabbit platelets. These data indicate a potentially beneficial effect of EPA in atherosclerotic WHHL rabbits.
Asunto(s)
Ácido Eicosapentaenoico/farmacología , Hemodinámica/efectos de los fármacos , Hiperlipidemias/sangre , Adhesividad Plaquetaria/efectos de los fármacos , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Ácido Araquidónico , Ácidos Araquidónicos/farmacología , Dieta , Ácidos Grasos/metabolismo , Femenino , Hiperlipidemias/genética , Técnicas In Vitro , Masculino , Cloruro de Potasio/farmacología , ConejosRESUMEN
The goal of the present study was to demonstrate that intracoronary platelet deposition may trigger intense vasoconstriction of large epicardial coronary arteries in vivo and that this is largely mediated by thromboxane A2 and serotonin released by activated platelets. Cyclic flow variations (progressive declines in blood flow followed by sudden restorations of flow) due to recurrent intracoronary platelet activation and thrombus formation were induced by damaging the endothelium and placing a cylindrical constrictor on the left anterior descending coronary artery (LAD) in open-chest, anesthetized dogs. Coronary diameters were measured in vivo by means of ultrasonic crystals sutured on the LAD immediately distal to the constrictor (LAD1) and 1 cm below (LAD2) and on the circumflex coronary artery (Cx). Coronary artery diastolic diameters were measured continuously before and during cyclic flow variations and after they were abolished by administration of LY53857, a serotonin-receptor antagonist (group 1, n = 7), or SQ29548, a thromboxane-receptor antagonist (group 2, n = 7). During cyclic flow variations, at the nadir of coronary flow, LAD1 (a site of maximal platelet accumulation) cross-sectional area decreased by 52 +/- 10% and 38 +/- 6% in group 1 and 2 animals, respectively (p less than 0.001 compared with values recorded during a brief LAD occlusion obtained by a suture snare), whereas LAD2 (a site of minimal or no platelet accumulation) cross-sectional area did not differ from that recorded during the brief LAD occlusion. SQ29548 abolished cyclic flow variations in seven of seven dogs and LY53857 in six of seven, but they affected the increased coronary vasoconstriction differently: LAD1 cross-sectional area increased by 32 +/- 6% of the control value in SQ29548-treated animals, whereas it returned to baseline dimension values in the LY53857-treated group as these interventions also abolished the cyclic flow variations. We conclude that a marked coronary vasoconstriction may be triggered by local platelet deposition and that thromboxane A2 and serotonin are mediators of this vasoconstriction.
Asunto(s)
Plaquetas/fisiología , Vasos Coronarios/fisiología , Serotonina/fisiología , Tromboxano A2/fisiología , Vasoconstricción , Animales , Supervivencia Celular , Vasos Coronarios/patología , Perros , Femenino , Hemodinámica , Masculino , Pericardio , Agregación PlaquetariaRESUMEN
The aim of this study was to determine the effect of blood coagulation and platelet aggregation on the perfusability of arterioles (19-50 micron) and capillaries in subepicardial and subendocardial ischemic and nonischemic myocardium of anesthetized open-chest rabbits. Fluorescein isothiocynate-dextran (MW 150,000) was injected intravenously to label perfusable myocardial microvessels of rabbits that were subjected to 60 min of coronary artery occlusion. Fluorescent microscopy was used to identify the perfusable vessels and an alkaline phosphatase stain was employed to locate the total microvasculature of the heart. Stereological principles were utilized to determine various morphometric parameters. About 25% of the capillaries were incapable of being perfused but virtually all arterioles were perfusable in occluded myocardium of the control group. Essentially all capillaries and arterioles were perfusable in nonoccluded myocardium. Collagen infusion produced a perfusion defect in 14% of the capillaries and arterioles in nonoccluded myocardium and in 33% of the capillaries and arterioles in occluded myocardium. Heparin, prostaglandin E1 (PGE1), or PGE1 + heparin did not prevent the perfusion defect in capillaries of occluded myocardium. It is concluded that while promotion of blood coagulation and platelet aggregation was able to produce microvessel obstruction, these hemostatic mechanisms were not primarily responsible for the capillary obstruction observed during myocardial ischemia in the rabbit heart.
Asunto(s)
Arterias/fisiopatología , Arteriolas/fisiopatología , Coagulación Sanguínea , Capilares/fisiopatología , Enfermedad Coronaria/fisiopatología , Agregación Plaquetaria , Alprostadil/farmacología , Animales , Capilares/efectos de los fármacos , Colágeno/farmacología , Circulación Coronaria , Enfermedad Coronaria/sangre , Hemodinámica , Heparina/farmacología , Microcirculación , Microscopía Fluorescente , Perfusión , ConejosRESUMEN
The aim of this study was to identify the percentage of the capillary and arteriolar beds that was perfused at a given time or that was capable of being perfused within subepicardium and subendocardium of ischemic and nonischemic myocardium of anesthetized, open-chest rabbits. Fluorescein isothiocyanate-dextran (150,000 MW) was injected intravenously to label perfused microvessels of rabbits that were subjected to 60 min of coronary artery occlusion. Fluorescent microscopy was used to identify the perfused vessels and an alkaline phosphatase stain was employed to locate the total microvasculature. Stereological principles were utilized to determine various morphometric parameters. After 14 sec of dextran injection, approximately 66 +/- 2% (mean +/- SEM) of the capillaries and 65 +/- 5% of the arterioles (19-50 microns) were perfused within ischemic myocardium. Two minutes after dextran injection, 72 +/- 3% of the capillaries and 94 +/- 4% of the arterioles were perfused within the ischemic myocardium. In the nonischemic myocardium, 64 +/- 2% of the capillaries and 59 +/- 5% of the arterioles were perfused 14 sec after dextran injection, while 94 +/- 1% and 96 +/- 2% of the capillaries and arterioles, respectively, were perfused 2 min after dextran injection. It is concluded that a significant unperfused reserve of arterioles exists, but a significant portion of the capillary bed is incapable of being perfused within ischemic rabbit myocardium.