RESUMEN
The trimeric derivative of 16,16-dimethyl-15-dehydroprostaglandin B1 (termed tri-Calciphor), which protects tissues against ischemic damage, induced Ca2+ efflux and swelling in mitochondria in the absence of phosphate, Mg2+ and ATP. When glutamate/malate rather than succinate was the substrate, higher tri-Calciphor concentrations were required for the ionophoretic activity. Ca2+ efflux and mitochondrial swelling induced by tri-Calciphor were completely inhibited by ATP, phosphate and Mg2+ added together, and partially inhibited with phosphate plus either ATP or Mg2+. Between 0 and 7 microM added Ca2+ and in the presence of phosphate, ATP and Mg2+, tri-Calciphor stimulated the uptake of Ca2+ by mitochondria and increased the efficiency of buffering of extramitochondrial Ca2+. Thus, depending on the assay conditions, two different effects involving Ca2+ movements and mitochondria are observed with tri-Calciphor.
Asunto(s)
Calcio/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Fosfatos/farmacología , Prostaglandinas B/farmacología , Adenosina Trifosfato/farmacología , Animales , Glutamatos/metabolismo , Ácido Glutámico , Magnesio/farmacología , Masculino , Mitocondrias Hepáticas/metabolismo , Oligomicinas , Ratas , Ratas Wistar , Succinatos/metabolismo , Ácido SuccínicoRESUMEN
Chemically synthesized dimers, trimers and tetramers of 15-dehydroprostaglandin B1 and 16,16'-dimethyl-15-dehydroprostaglandin B1 facilitate the release of Ca2+ from isolated rat liver mitochondria. The parent monomeric prostaglandins had no significant activity. The rate of release was stimulated by exogenous K+ or Na+, suggesting an antiport exchange of monovalent cations for intra-mitochondrial Ca2+. The activity depended upon the presence of ruthenium red, which prevented recycling of Ca2+; comparison of the activity with A23187 and carbonyl cyanide p-trifluoromethoxyphenylhydrazone indicated that the prostaglandin B1 oligomers were functioning as ionophores and the release of Ca2+ was not caused by an uncoupling of oxidative phosphorylation. The oligomers caused a major decrease in the membrane potential but only when the mitochondria were preloaded with exogenous Ca2+, and even then, the Ca2+ efflux was completed before the membrane potential decreased to less than 90 mV. The oligomeric molecules were able to form supramolecular aggregates in the presence of Ca2+ as detected by light scattering. They extracted Ca2+ into an organic phase, and translocated Ca2+ from one aqueous domain to another across an organic barrier; K+ and Na+ modulated these processes. The prostaglandin B1 derivatives also translocated Rb+ from one aqueous phase to another across an organic barrier when Ca2+ was translocated.
Asunto(s)
Calcio/metabolismo , Ionóforos/farmacología , Mitocondrias Hepáticas/metabolismo , Prostaglandinas B/farmacología , Prostaglandinas/farmacología , Animales , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/fisiología , Cinética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Potasio/farmacología , Prostaglandinas B/síntesis química , Ratas , Ratas Endogámicas , Sodio/farmacología , Relación Estructura-ActividadRESUMEN
Our previous studies have demonstrated that platelets possess ATP purinergic receptors in addition to the ADP, P2T, receptor. Occupancy of the P2 receptor by ATP inhibited agonist-induced platelet aggregation. This study demonstrated that the mechanism of inhibition may involve ATP inhibition of agonist-induced mobilization of internal calcium. Within the cardiovascular system, the ATP inhibition of calcium mobilization is unique to platelets. All other cell types in the cardiovascular system, where calcium mobilization is affected by extracellular ATP, responded with an increased mobilization as opposed to inhibition. The platelet inhibitory response to ATP was enhanced by the addition of an ATP generating system, creatine phosphate/phosphocreatine kinase. ATP and ATP analogues were found to inhibit calcium mobilization with a rank order of alpha beta-methylene ATP, beta gamma-methylene ATP approximately ATP > benzoyl ATP > 2 methylthio ATP which is a characteristic of P2x-like receptors. The inhibitory effect of ATP could be abrogated by prolonged treatment of platelets with the P2x desensitizing agent, alpha beta-methylene ATP. Also, UTP and CTP were approximately as effective inhibitors as ATP while GTP was not. ATP competition with ADP for the P2T receptor was excluded in studies with platelets derived from an aspirin-treated individual which were essentially insensitive to ADP. The agonist-induced calcium mobilization and inhibition by ATP occurred with the thromboxane A2 mimetic, U46619, collagen and thrombin; however, the kinetics of mobilization varied somewhat with the different agonists. The responses to extracellular ATP were independent of extracellular Ca2+, where 1 mM calcium or 0.3 mM EGTA was added to the reaction mixture. The inhibition of calcium mobilization coupled to inhibition of platelet aggregation by extracellular ATP may serve an important physiologic role. ATP, released from activated platelets at localized sites of vascular injury, may help to limit the size of the platelet plug-clot that, if left unregulated, could occlude the injured blood vessel.
Asunto(s)
Adenosina Trifosfato/farmacología , Plaquetas/metabolismo , Calcio/metabolismo , Agregación Plaquetaria/fisiología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Adenosina Difosfato/farmacología , Adenosina Trifosfato/análogos & derivados , Plaquetas/efectos de los fármacos , Colágeno/farmacología , Creatina Quinasa/farmacología , Humanos , Cinética , Nucleótidos/farmacología , Fosfocreatina/farmacología , Agregación Plaquetaria/efectos de los fármacos , Endoperóxidos de Prostaglandinas Sintéticos/farmacología , Receptores Purinérgicos P2/fisiología , Trombina/farmacología , Tromboxano A2/análogos & derivados , Tromboxano A2/farmacologíaRESUMEN
Previous studies conducted with intact rats had demonstrated that protein synthesis was reversibly inhibited by cycloheximide. Polysome aggregation occurred during inhibition with a return to normal during recovery. Suggesting that the block of translational activity involved termination and release of polypeptides. This study involving freshly isolated hepatocytes was undertaken to clarify the mechanism of the biphasic response to cycloheximide. Cycloheximide at 1 microM inhibited [3H]leucine incorporation into both cellular and secreted proteins by at least 86%, without having deleterious effects on membrane integrity as indicated by trypan blue uptake and lactate dehydrogenase (LDH) (EC 1.1.1.27) release. After removal of cycloheximide, incorporation of labeled amino acids into cellular protein and protein secreted into the medium returned to control levels. Kinetically, incorporation into secreted protein exhibited a lag of 30-45 min, indicating that a longer recovery period for restoration of proteosynthetic ability is required for membrane-bound polysomes. During the first 100 min of the recovery period, 30% of the cellular protein, which had been prelabeled during cycloheximide inhibition, was secreted into the medium; treated cells, however, secreted prelabeled protein at a lower initial rate. To elucidate the mechanism of action of cycloheximide, the content of the cytoplasmic ribonucleoprotein complexes (RPC), polysome size classes, and the distribution of radioactivity among the various ribosome classes were determined during inhibition and recovery. Larger size class polysomes (7+) were increased by cycloheximide treatment and remained increased during recovery. During inhibition, there was enhanced [3H]leucine labeling with increasing polysome size, implicating termination as the rate-limiting step, whereas during the recovery phase the labeled nascent polypeptides were removed from the ribonucleoprotein complex at a 3- to 4-fold greater rate than control, indicating an accelerated release of completed proteins.
Asunto(s)
Cicloheximida/farmacología , Hígado/metabolismo , Biosíntesis de Proteínas , Animales , Técnicas In Vitro , Leucina/metabolismo , Polirribosomas/metabolismo , Ratas , Factores de TiempoRESUMEN
Molecular sites of perturbation by the hepatocarcinogen aflatoxin B1 (AFB1) in the protein synthesis initiation complex were assessed using isolated hepatocytes and a cell-free activating system containing microsomes and cytoplasmic ribonucleoprotein complexes (cRPC). Ribosomal proteins showed no detectable modification by the toxin in either system. With hepatocytes, initiation factors demonstrated only slight modification by AFB1. RNAs from both hepatocytes and the cell-free system with microsomes and cRPC were modified, with poly(A)-containing RNA exhibiting at least a 5-fold higher modification than poly(A)-lacking RNA. The poly(A)-lacking RNAs were modified in the order 28S rRNA greater than 18S rRNA greater than 5-6S rRNA greater than 4S tRNA. Guanine was the target base of AFB1, but only 10% of the AFB1-GMP adducts were on guanines located in a poly(G) region. These results suggest that guanine modification in RNAs may be responsible for the observed inhibition of translational initiation by AFB1 to a greater extent than modification of either ribosomal intrinsic or associated proteins.
Asunto(s)
Aflatoxinas/farmacología , Hígado/efectos de los fármacos , Biosíntesis de Proteínas , Aflatoxina B1 , Aflatoxinas/metabolismo , Animales , Sitios de Unión , Hidrólisis , Técnicas In Vitro , Hígado/metabolismo , Masculino , Factores de Iniciación de Péptidos/metabolismo , Unión Proteica , ARN Mensajero/efectos de los fármacos , Ratas , Ratas Endogámicas , Ribonucleoproteínas/metabolismo , Proteínas Ribosómicas/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
The ionophoretic activity of PGBx, an oligomeric mixture synthesized from 15-dehydro PGB1, with different cations was measured using arsenazo III-entrapped liposomes. The order of ionophoretic activity was Zn2+ greater than Co2+ greater than Mn2+ greater than Cu2+ greater than Ca2+ greater than Ba2+ greater than Sr2+ greater than Mg2+. The intrinsic fluorescence of PGBx was quenched by the binding of divalent cations as well as by La3+ and H+. Quenching by K+ and Na+ was minimal. The order of quenching strength of divalent cations was Zn2+ greater than Co2+ greater than Cu2+ = Mn2+ greater than Ca2+ greater than Ba2+ greater than Sr2+ greater than Mg2+. Binding affinities of these cations determined by a murexide indicator method were in good agreement with that determined by the fluorescence quenching reaction. The cation binding affinity of PGBx in aqueous solutions correlates with the ionophoretic activity in liposomes. The binding affinity for K+ was estimated from the inhibition by K+ of Ca2+ binding by PGBx. Although PGBx has a lower selectivity for divalent cation binding than the ionophore A23187, the characteristics of the binding affinity of these two compounds for various ions were similar. The pK of PGBx as determined by fluorescence quenching was 6.7. The molecular weight of the divalent cation binding unit was estimated to be about 680, with each PGBx molecule having three such binding sites. The binding of Ca2+ to such a site is one-to-one.
Asunto(s)
Cationes Bivalentes/metabolismo , Ionóforos , Polímeros/farmacología , Prostaglandinas B/farmacología , Prostaglandinas Sintéticas/farmacología , Prostaglandinas/farmacología , Animales , Indicadores y Reactivos , Cinética , Liposomas , Naftalenosulfonatos , Espectrometría de FluorescenciaRESUMEN
Post-ischemic treatment of di-Calciphor (16,16'-dimethyl-15- dehydroprostaglandin B1) significantly improves animal survival and prevents ischemia-induced neurodegeneration of vulnerable forebrain regions assessed with histochemical and biochemical techniques in gerbils. Neuronal degeneration seen by Cresyl violet staining and silver impregnation in the CA1 sector of the hippocampus and the dorso-lateral sector of the striatum was significantly reduced in animals treated with di-Calciphor. In addition, the early onset of selective degradation of calpain I substrates spectrin and microtubule-associated protein (MAP2) in these same vulnerable regions was prevented. The lack of adverse side effects may facilitate the potential therapeutic use of this drug in preventing neuronal damage caused by stroke.
Asunto(s)
Isquemia Encefálica/patología , Prostaglandinas B/farmacología , Animales , Isquemia Encefálica/metabolismo , Cuerpo Estriado/metabolismo , Gerbillinae , Hipocampo/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Degeneración Nerviosa/efectos de los fármacos , Flujo Sanguíneo Regional/efectos de los fármacos , Reperfusión , Espectrina/metabolismoRESUMEN
Following 20 min occlusion of both carotid arteries, female gerbils were subjected to treatment with di- or tri-Calciphor (dimer or trimer of 16,16'-dimethyl prostaglandin B1). Dimer was injected i.p. at 5 and 10 mg/kg at 5 min and again at 24 h, 30 min and 24 h, 60 min and 24 h or 180 min and 24 h postischemia (N = 25/group). Trimer was given i.p. at 5, 10 or 15 mg/kg at 5 min and 24 h postischemia (N = 25/group.) The controls (N = 25) were injected with the vehicle. Neurological status and postischemic survival of the animals were monitored for 14 days postischemia. Survival of the treated gerbils was significantly improved following the treatment with either di- or tri-Calciphor administered at 10 mg/kg at 5 min and 24 h postischemia (36 vs. 68% di- and 64% tri-Calciphor, P less than 0.05), and with di-Calciphor at 5 mg/kg at 180 min and 24 h postischemia (64%). All other treatment regimens with either drug resulted in a numerical, statistically insignificant improvement. In addition, treatment with either drug reduced the intensity of postischemic neurological impairment. Treatment with di-Calciphor injected at 10 mg/kg at 5 min and 24 h post 20 min ischemia substantially reduced the period of postischemic locomotor hyperactivity. The drug had no impact on either body temperature or blood pressure. There is evidence that the effects of Calciphor may be mediated via calcium regulatory mechanisms. The results of the present study are discussed in the light of such possibility.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Temperatura Corporal/efectos de los fármacos , Isquemia Encefálica/tratamiento farmacológico , Actividad Motora/efectos de los fármacos , Prostaglandinas B/uso terapéutico , Análisis de Varianza , Animales , Isquemia Encefálica/fisiopatología , Femenino , Gerbillinae , Prostaglandinas B/farmacologíaRESUMEN
The effects of MK-801 on postischemic recovery, survival and neuronal preservation in the cortex, hippocampus and striatum were studied in Mongolian gerbils. The drug was administered 30 min prior to 20 of min forebrain ischemia induced by bilateral ligation of the carotids. Neurological recovery and survival were monitored for 7 days. At the end of the monitoring period neuronal damage was analyzed in the brains of the survivors in both groups. Treatment with MK-801 did not improve either neurological recovery or end-point survival. However, significant (P < 0.01) neuronal protection was observed in the hippocampi and striata of the drug treated animals while cortical neurons were not significantly protected. These findings demonstrate that protection against ischemic neuronal damage can be observed without concomitant improvement in either postischemic neurological recovery or survival. Protection of selectively vulnerable brain regions, often used as the predictor of the therapeutic potential of an agent, does not appear to correlate well with postischemic survival in this animal model of ischemia.
Asunto(s)
Maleato de Dizocilpina/uso terapéutico , Ataque Isquémico Transitorio/tratamiento farmacológico , Neuronas/efectos de los fármacos , Animales , Presión Sanguínea/efectos de los fármacos , Temperatura Corporal/efectos de los fármacos , Gasto Cardíaco/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Formaldehído , Gerbillinae , Ataque Isquémico Transitorio/mortalidad , Ataque Isquémico Transitorio/patologíaRESUMEN
Chronic treatment of gerbils with 1-aminocyclopropanecarboxylic acid (a high affinity, partial agonist at strychnine-insensitive glycine receptors) resulted in a 3-fold increase in survival, a significant improvement in neurological status, and an extensive protection of vulnerable brain regions following severe forebrain ischaemia. A bolus of 1-aminocyclopropanecarboxylic acid 30 min prior to ischaemia did not further improve outcome compared to gerbils receiving their last injection 24 h prior to ischaemia. These findings are consistent with the hypothesis that chronic treatment with a glycine partial agonist desensitizes the N-methyl-D-aspartate receptor complex. Pharmacological intervention at the strychnine-insensitive glycine receptor may be an effective means of ameliorating the consequences of neuronal degeneration caused by excitotoxic phenomena.
Asunto(s)
Aminoácidos Cíclicos , Aminoácidos/uso terapéutico , Isquemia Encefálica/tratamiento farmacológico , Receptores de Neurotransmisores/efectos de los fármacos , Animales , Femenino , Gerbillinae , Receptores de Glicina , Receptores de N-Metil-D-Aspartato/efectos de los fármacosRESUMEN
BACKGROUND: The mechanisms whereby rat hepatocytes undergo irreversible injury due to a lack of oxygen have not been established. METHODS: Liver cells were used for reperfusion injury, and four compartmentalized pathways were evaluated during hypoxia (N2/CO2, 19:1) for 30 min followed by oxygen (O2/CO2, 19:1) for 30 min. RESULTS: Cell viability decreased during the hypoxic, but not during the reoxygenation, phase. Glycogenolysis, as measured by glucose release, was significantly increased during hypoxia as compared to controls in oxygen (205 +/- 15 vs. 155 +/- 10 nmol glucose/mg protein/h, respectively), and did not return to normal levels by reoxygenation. Gluconeogenesis was importantly decreased during hypoxia (102 +/- 10 vs. 8 +/- 2 nmol glucose/mg protein/h) with partial recovery during reoxygenation. Ureagenesis diminished in hypoxia, but recovered during reoxygenation. Additionally, 3-hydroxybutyrate formation was augmented by hypoxia, with some recovery when oxygen was present. CONCLUSIONS: These results suggest that compartmentalized pathways are protected from hypoxic injury in isolated hepatocytes, and also suggest it as a model to test the idea that enzymes of those pathways are organized into multienzyme complexes in vivo.
Asunto(s)
Hígado/metabolismo , Consumo de Oxígeno , Ácido 3-Hidroxibutírico/metabolismo , Animales , Compartimento Celular , Hipoxia de la Célula , Gluconeogénesis , Glucógeno Hepático/metabolismo , Masculino , Ratas , Ratas Wistar , Urea/metabolismoRESUMEN
The effect of Prostaglandin Bx (PGBx) on the development of gastric stress ulceration and bleeding in rats was investigated. The rats were divided into five groups and each rat was injected intravenously with either Saline (control), 0.5 mg, 1 mg, 5 mg, or 25 mg/kg of PGBx, according to group. The rats were then placed in wire conduit cages and placed in a cold room at 4 degrees C for three hours and sacrificed immediately afterwards. Results showed a significant increase in gastric bleeding and ulceration in the groups that received 1 and 5 mg/kg (PGBx) when compared to the control group (p less than 0.01). There was, however, a significant increase in gastric acid output in the group that received 1 mg/kg PGBx when compared to the control group (p less than 0.01). Our results suggest that PGBx, by a mechanism other than the inhibition of gastric acid secretion, maintains the integrity of the gastric mucosa.
Asunto(s)
Polímeros/uso terapéutico , Prostaglandinas B/uso terapéutico , Prostaglandinas/uso terapéutico , Úlcera Gástrica/prevención & control , Estrés Fisiológico/complicaciones , Animales , Relación Dosis-Respuesta a Droga , Mucosa Gástrica/patología , Masculino , Úlcera Péptica Hemorrágica/prevención & control , Ratas , Ratas Endogámicas , Úlcera Gástrica/patologíaAsunto(s)
Antineoplásicos/farmacología , Aminoácidos/metabolismo , Animales , Técnicas de Cultivo , Fructosa-Bifosfato Aldolasa/antagonistas & inhibidores , Glucosiltransferasas/antagonistas & inhibidores , Glicerolfosfato Deshidrogenasa/antagonistas & inhibidores , Glucólisis/efectos de los fármacos , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Malato Deshidrogenasa/antagonistas & inhibidores , Pentosas , Biosíntesis de Proteínas , TimidinaAsunto(s)
Antineoplásicos/farmacología , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas de Alcohol/antagonistas & inhibidores , Glicerolfosfato Deshidrogenasa/antagonistas & inhibidores , Técnicas In Vitro , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Malato Deshidrogenasa/antagonistas & inhibidoresAsunto(s)
Liposomas , Fosfatidilcolinas , Esteroles , Colestanos , Colestanonas , Colesterol , Difenilhexatrieno , Polarización de Fluorescencia , TemperaturaAsunto(s)
Aspartato Aminotransferasas/metabolismo , Creatina Quinasa/metabolismo , Cicloheximida/farmacología , L-Lactato Deshidrogenasa/metabolismo , Proteínas/metabolismo , Albúminas/metabolismo , Animales , Aspartato Aminotransferasas/sangre , Creatina Quinasa/sangre , Citosol/enzimología , Fibrinógeno/metabolismo , L-Lactato Deshidrogenasa/sangre , Hígado/citología , Masculino , Ratas , Factores de TiempoRESUMEN
Phosphorylase activity of isolated rat liver cells was increased about 2-fold on addition of tri-Calciphor (trimer of 16, 16-dimethyl-15-dehydroprostaglandin B1), epinephrine or the Ca2+ ionophore A23187, in all cases presumably due to an increase in cytosolic Ca2+. Extracellular Ca2+ was required with A23187, but not with either tri-Calciphor or epinephrine. Tri-Calciphor, however, did not stimulate a sustained release of glucose from hepatocytes as compared to the other Ca2+ mobilizing agents, even at concentrations 10-fold higher than that required to stimulate the phosphorylase activity. Tri-Calciphor did not alter the glucose release by epinephrine. It is concluded that tri-Calciphor can alter cytosolic Ca2+, but that its mechanism of action is more complex than that of a simple Ca2+ ionophore.