RESUMEN
OBJECTIVE: To determine the roles of the eicosanoids thromboxane and prostacyclin, and their compartmentalization, in the regulation of placental blood flow. METHODS: First, the sites of production of thromboxane and prostacyclin were determined within the placental villus using immunohistochemical staining for thromboxane and prostacyclin synthetase. Second, the production of both eicosanoids was studied in cultured trophoblasts and compared with that in the villous core by measuring the metabolites thromboxane B2 and 6-keto-prostaglandin F 1 alpha. Finally, eicosanoid production was assessed in intact villi after stimulation by an acute change in oxygen content, 5% to 95%. RESULTS: Immunohistochemical staining showed that thromboxane production was primarily within the trophoblasts, whereas prostacyclin production was localized to the endothelial cells within the villi. In culture, we found preferential production of prostacyclin by the villous core cells and increased production of thromboxane by trophoblasts. Perifusion of intact villi demonstrated increased production of thromboxane by trophoblasts in response to an increase in oxygen content. Prostacyclin levels were too low to be detected. CONCLUSIONS: Placental blood flow appears to be regulated by compartmentalized eicosanoids, with thromboxane affecting primarily the maternal side of the placental circulation and prostacyclin affecting primarily the fetal side.
Asunto(s)
6-Cetoprostaglandina F1 alfa/biosíntesis , Vellosidades Coriónicas/metabolismo , Oxidorreductasas Intramoleculares , Placenta/irrigación sanguínea , Tromboxano B2/biosíntesis , Trofoblastos/metabolismo , 6-Cetoprostaglandina F1 alfa/fisiología , Células Cultivadas , Vellosidades Coriónicas/enzimología , Sistema Enzimático del Citocromo P-450/análisis , Sistema Enzimático del Citocromo P-450/inmunología , Eicosanoides/biosíntesis , Eicosanoides/metabolismo , Femenino , Humanos , Inmunohistoquímica , Isomerasas/análisis , Isomerasas/inmunología , L-Lactato Deshidrogenasa/metabolismo , Intercambio Materno-Fetal/fisiología , Oxígeno/metabolismo , Placenta/citología , Placenta/enzimología , Placenta/ultraestructura , Embarazo , Tromboxano B2/fisiología , Tromboxano-A Sintasa/análisis , Tromboxano-A Sintasa/inmunología , Factores de Tiempo , Trofoblastos/citología , Trofoblastos/enzimologíaRESUMEN
We have previously shown that a metabolite of NAD+ generated by an enzyme present in sea urchin eggs and mammalian tissues can mobilize intracellular Ca2+ in the eggs. Structural determination established it to be a cyclized ADP-ribose, and the name cyclic ADP-ribose (cADPR) has been proposed. In this study, Ca2+ mobilizations induced by cADPR and inositol trisphosphate (IP3) in sea urchin egg homogenates were monitored with Ca2+ indicators and Ca2(+)-specific electrodes. Both methods showed that cADPR can release Ca2+ from egg homogenates. Evidence indicated that it did not act as a nonspecific Ca2(+)-ionophore or as a blocker of the microsomal Ca2(+)-transport; instead, it was likely to be operating through a specific receptor system. This was supported by its half-maximal effective concentration of 18 nM, which was 7 times lower than that of IP3. The receptor for cADPR appeared to be different from that of IP3 because heparin, an inhibitor of IP3 binding, had no effect on the cADPR action. The Ca2+ releases induced by cADPR and IP3 were not additive and had an inverse relationship, indicating overlapping stores were mobilized. Microinjection of cADPR into intact eggs induced transient intracellular Ca2+ changes and activated the cortical reaction. The in vivo effectiveness of cADPR was directly comparable with IP3 and neither required external Ca2+. In addition, both were effective in activating the eggs to undergo multiple nuclear cycles and DNA synthesis. These results suggest that cADPR could function as a second messenger in sea urchin eggs.
Asunto(s)
Adenosina Difosfato Ribosa/farmacología , Calcio/metabolismo , Inositol 1,4,5-Trifosfato/farmacología , Sistemas de Mensajero Secundario , Adenosina Difosfato Ribosa/fisiología , Animales , Compartimento Celular , Ciclo Celular/efectos de los fármacos , ADP-Ribosa Cíclica , Replicación del ADN/efectos de los fármacos , Heparina/farmacología , Inositol 1,4,5-Trifosfato/fisiología , Ionomicina/farmacología , Microinyecciones , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Óvulo/efectos de los fármacos , Óvulo/metabolismo , Partenogénesis/efectos de los fármacos , Erizos de MarRESUMEN
Inositol trisphosphate (IP3) was previously shown to release Ca2+ from a nonmitochondrial store in sea urchin eggs. In this study, egg homogenates and purified microsomes were monitored with either fura 2 or Ca2+-sensitive minielectrodes to determine whether other stimuli would induce Ca2+ release. Pyridine nucleotides (whose concentrations are known to change at fertilization) were found to release nearly as much Ca2+ as did IP3. Average releases/ml of homogenate were: 0.6 microM IP3, 10.9 nmol of Ca2+; 50 microM NADP, 7.3 nmol of Ca2+; and 100 microM NAD, 6.5 nmol of Ca2+ (n = 6). Specificity was demonstrated by screening a series of other phosphorylated metabolites, and none was found to reproducibly release Ca2+. Calcium release induced by IP3 or NADP was immediate, whereas a lag of 1-4 min occurred with NAD. This lag before NAD-induced Ca2+ release led to the discovery that a soluble egg factor (Mr greater than 100,000) converts NAD into a highly active metabolite that releases Ca2+ without a lag. The NAD metabolite (E-NAD) was purified to homogeneity by high pressure liquid chromatography and produced half-maximal Ca2+ release at about 40 nM. Injection of E-NAD into intact eggs produced both an increase in intracellular Ca2+ (as assayed with indo-1) and a cortical reaction. Following Ca2+ release by each of the active agents (IP3, NAD, and NADP), the homogenates resequestered the released Ca2+ but were desensitized to further addition of the same agent. A series of desensitization experiments showed that homogenates desensitized to any two of these agents still responded to the third, indicating the presence of three independent Ca2+ release mechanisms. This is further supported by experiments using Percoll density gradient centrifugation in which NADP-sensitive microsomes were partially separated from those sensitive to IP3 and NAD.