RESUMEN
In chick spinal cord the presence of low affinity (KD = 2.2 microM) receptors for prostaglandin E2 (PGE2) raises the question whether spinal cord possesses a PGE2 biosynthetic capacity able to activate these receptors. The production of PGE2 in spinal cord and meninges was investigated by enzyme immunoassay. Spinal cord exhibited a 30- to 100-fold lower PGE2 biosynthetic capacity compared to meninges, but can generate PGE2 resulting in micromolar concentrations, sufficient to activate the low affinity PGE2 receptors. It is suggested that in physiological conditions, PGE2 synthesized within the spinal cord might locally activate the low affinity PGE2 receptors, whereas in pathological situations, after disruption of the blood-spinal cord barrier, PGE2 produced by the meninges might be accessible to spinal cord PGE2 receptors, and thus largely contribute to their saturation.
Asunto(s)
Dinoprostona/biosíntesis , Meninges/metabolismo , Médula Espinal/metabolismo , Animales , Ácido Araquidónico/metabolismo , Radioisótopos de Carbono , Pollos , Técnicas para Inmunoenzimas , Técnicas In Vitro , Cinética , Receptores de Prostaglandina E/metabolismo , Distribución TisularRESUMEN
Prostaglandin D2 is one of the major prostanoids formed from [14C]arachidonic acid by the central nervous system. The aim of the present study is to specify the prostaglandin D2 biosynthetic capacity in the chick spinal cord and to identify the cell type involved in this synthesis. A highly specific and sensitive enzyme immunoassay allowed us to demonstrate that the amount of newly formed prostaglandin D2 increases proportionally with the concentration of free arachidonic acid of either exogenous or endogenous origin and reaches concentration values ranging from 10(-9) to 10(-6) M. The sites of prostaglandin D2 synthesis were localized in Vibratome sections of spinal cord after incubation with antibodies raised against glutathione-independent prostaglandin D synthase; controls were performed with anti-glutathione-dependent prostaglandin D synthase antibodies and non-immune rabbit or goat serum. After immunoprocessing, electron microscope examination revealed that the specific immunoreactivity was confined to small neurons of laminae II and III in the dorsal horn and to motoneurons in the ventral horn of the spinal cord. The immunodeposits were associated with rough endoplasmic reticulum profiles distributed throughout the dorsal horn neurons or restricted to limited subsurface areas of perikarya and dendrites in motoneurons. Since the immunoreactive neurons in the dorsal horn were closely related to blood capillaries, prostaglandin D2 may be suspected to play a role in the regulation of the microcirculation. The accumulation of prostaglandin D synthase in motoneuron areas facing astrocytic membrane stacks suggests that prostaglandin D2 could interact with astrocytic functions.
Asunto(s)
Oxidorreductasas Intramoleculares , Neuronas Motoras/metabolismo , Neuronas/metabolismo , Prostaglandina D2/biosíntesis , Médula Espinal/metabolismo , Animales , Animales Recién Nacidos , Bioquímica/métodos , Pollos , Glutatión/fisiología , Inmunohistoquímica , Isomerasas/metabolismo , Lipocalinas , Microscopía Electrónica , Neuronas Motoras/ultraestructura , Neuronas/ultraestructura , Conejos , Médula Espinal/citología , Médula Espinal/ultraestructura , Distribución TisularRESUMEN
Prostaglandin (PG) D2 is synthesized by primary sensory neurons grown in vitro. The question can be raised of whether the entire population or only a particular subpopulation of primary sensory neurons synthesizes PGD2 in vivo. To clarify this issue it was necessary to demonstrate that PGD synthase activity persists in fresh dorsal root ganglion (DRG) cryostat slices by characterizing newly formed PGD2 from [14C]-arachidonic acid, and to determine by immunocytochemistry and to identify at the ultrastructural level the neuron subpopulation expressing glutathione (GSH)-independent PGD synthase. Among the various procedures tested, the most intense, selective, and reproducible immunostaining pattern was obtained after periodate-lysine-formaldehyde fixation in phosphate buffer, permeabilization with 0.25% Triton X-100, and incubation with 10 micrograms/ml purified antibodies. Under these conditions, a subpopulation of small Class B ganglion cells was strongly immunoreactive, whereas adjacent control sections treated with absorbed antibodies or with non-immune rabbit or goat serum were unreactive. To identify the subclass of the immunoreactive small Class B neurons, immunostained vibratome slices of DRG were embedded in Epon. Ganglion cell bodies loaded with immunoprecipitates in superficially cut sections were first identified and then ultrastructurally analyzed in thin sections taken from a deeper level to obtain improved preservation of the cell architecture. This procedure enabled us to demonstrate that GSH-independent PGD synthase is accumulated in Subclass B1 primary sensory neurons.
Asunto(s)
Oxidorreductasas Intramoleculares , Isomerasas/metabolismo , Neuronas Aferentes/enzimología , Animales , Pollos , Ganglios Espinales/enzimología , Ganglios Espinales/ultraestructura , Inmunohistoquímica/métodos , Lipocalinas , Microscopía Electrónica , Neuronas Aferentes/ultraestructuraRESUMEN
Homogenates of chick dorsal root ganglia (DRG) and in vitro cultures of DRG neurons are known to synthesize prostaglandin (PG) D2. To specify the PGD synthase isozymes controlling PGD2 synthesis in DRG and to identify the DRG cells responsible for this synthesis, we applied polyclonal antibodies raised against rat brain or rat spleen PGD synthase isozymes to vibratome or cryostat slices of DRG previously fixed with a formaldehyde-lysine-periodate mixture and permeabilized with Triton X-100. The immunoreactivity indicating rat spleen PGD synthase, a glutathione (GSH)-requiring enzyme, was located in satellite cells encompassing particular large neurons of class A and in Schwann cells myelinating and enwrapping their initial axonal segments. In contrast, the immunoreactivity of rat brain PGD synthase, a GSH-independent enzyme, was restricted to particular ganglion cell perikarya: 33% of the DRG neurons were immunostained for rat brain PGD synthase, including 2% of large class A neurons and 40% of small class B neurons. Only 3.3% of rat brain PGD synthase-immunoreactive small B neurons coexpressed substance P, indicating that the immunoreactive neurons belong to the B1 subclass. By electron microscopy, 71 of 72 immunoreactive DRG cells were identified as small B neurons of the B1 subclass, and 71 of 77 B1 neurons were immunoreactive for rat brain PGD synthase. These results demonstrate that PGD2 formation in DRG is regulated by two isozymes: the GSH-requiring isozyme located in satellite and Schwann cells and the GSH-independent isozyme-confined to small B1 neurons.
Asunto(s)
Ganglios Espinales/metabolismo , Oxidorreductasas Intramoleculares , Isoenzimas/metabolismo , Isomerasas/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Animales , Encéfalo/metabolismo , Pollos , Ganglios Espinales/citología , Inmunohistoquímica/métodos , Isoenzimas/inmunología , Isomerasas/inmunología , Lipocalinas , Microscopía Electrónica , Ratas , Bazo/metabolismo , Sustancia P/metabolismo , Distribución TisularAsunto(s)
Dinoprostona/biosíntesis , Ganglios Espinales/metabolismo , Oxidorreductasas Intramoleculares , Prostaglandina D2/biosíntesis , Animales , Células Cultivadas , Embrión de Pollo , Pollos , Fibroblastos/metabolismo , Ganglios Espinales/citología , Inmunohistoquímica , Isoenzimas/metabolismo , Isomerasas/metabolismo , Lipocalinas , Neuroglía/metabolismo , Neuronas/metabolismoRESUMEN
In order to specify the source of locally synthesized prostaglandin (PG) E2 which is able to saturate the large class of low affinity PGE2 receptors in chick spinal cord, bioconversion of [1-14C]arachidonic acid into prostanoids was studied in homogenates of chick spinal cord and meninges first without addition of exogenous glutathione (GSH). Homogenates of spinal cord produced 14C-labeled PGE2, PGD2 and PGF2 alpha. Homogenates of meninges accumulated much larger amounts of [14C]PGE2 than spinal cord and surprisingly a 14C-labeled arachidonate metabolite referred to as compound Y. Compound Y generation, which was inhibited by indomethacin and enhanced by esculetin, was therefore mediated through the cyclooxygenase pathway. The fact that no labeled compound Y was detected in homogenates incubated with [3H]PGD2 or [3H]PGE2 indicated that compound Y was not a degradation product of PGs. Secondly, after addition of exogenous GSH, 14C-labeled compound Y was totally converted into [14C]PGE2. The compound Y which is converted into PGFs after a strong reduction with NaBH4 and into PGE2 after a mild reduction with GSH-hemin system or SnCl2 was therefore assumed to be a 15 hydroperoxy-PGE2 (15 HP-PGE2). These results suggest that PGE2 can be synthesized in meninges either by the classical isomerization of PGH2 or by isomerization of PGG2 followed by a GSH-sensitive reaction.
Asunto(s)
Dinoprostona/análogos & derivados , Dinoprostona/biosíntesis , Meninges/metabolismo , Prostaglandina D2/biosíntesis , Médula Espinal/metabolismo , Animales , Ácido Araquidónico/metabolismo , Borohidruros/farmacología , Pollos , Dinoprost/biosíntesis , Glutatión/farmacología , Técnicas In VitroRESUMEN
Newly formed prostaglandins (PGs), which are assumed to act as modulators of afferent sensory messages, were studied in chick dorsal root ganglia (DRG) during development. [1-14C]Arachidonic acid was converted by DRG homogenates from 1-week-old chickens into two major 14C-PGs: PGE2 and PGD2. The enzymatic conversion of arachidonic acid was characterized as follows: (a) Boiled preparations were inactivated; (b) synthesis of PGs was inhibited by pretreatment with aspirin or indomethacin and enhanced by esculetin, a protector of cyclooxygenase; and (c) [14C]PGE2 and [14C]PGD2 accumulation was a protein dose-dependent process. Further fractionation of crude homogenates indicated that PG endoperoxide synthetase (EC 1.14.99.1) and PGE2 synthetase (EC 5.3.99.3) were membrane-bound enzymes, whereas PGD2 synthetase (EC 5.3.99.2) was recovered in the cytosol. During development, from embryonic day 10 to day 14 after hatching, PGD2 synthetase activity remained constant; in contrast, a sharp rise in [14C]PGE2 synthesis was observed from embryonic day 14 to 18. The time curves of PGD2 and PGE2 synthetase specific activity may be related to changes taking place in the cell population of developing DRG. It is therefore suggested that arachidonic acid would be enzymatically converted early into PGD2 by maturing ganglion cells and then later into PGE2 by proliferating fibroblasts.
Asunto(s)
Dinoprostona/biosíntesis , Desarrollo Embrionario y Fetal , Ganglios Espinales/metabolismo , Prostaglandina D2/biosíntesis , Animales , Animales Recién Nacidos , Embrión de Pollo , Inhibidores de la Ciclooxigenasa , Ganglios Espinales/embriología , Ganglios Espinales/crecimiento & desarrollo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Fracciones Subcelulares/enzimologíaRESUMEN
To determine the type and the relative amount of prostaglandins (PGs) synthesized by various neural tissues, homogenates of meninges, dorsal root ganglia (DRG) capsules, decapsulated DRG, and unsheathed sciatic nerves were incubated with [1-14C]arachidonic acid. Homogenates of cultured cells (meningeal cells, fibroblasts, and nonneuronal or neuronal DRG cells) were used to specify the cells producing particular PGs. The highest synthetic capacity was found in fibroblast-rich tissues (meninges and DRG capsules) and in cultures of meningeal cells or fibroblasts. Two major cyclooxygenase products were formed: [14C]PGE2 and an unusual 14C-labeled compound, Y. The accumulation of compound Y, corresponding probably to 15-hydroperoxy PGE2, was completely impaired by addition of exogenous GSH, which conversely enhanced the synthesis of [14C]PGE2 and promoted the formation of [14C]PGD2. In contrast, decapsulated DRG or unsheathed sciatic nerves displayed a 10-20 times lower capacity to synthesize PGs than fibroblast-rich tissues and produced mainly [14C]PGE2 and [14C]PGD2. In this case, [14C]PGE2 or [14C]PGD2 synthesis was neither enhanced nor promoted by addition of exogenous GSH. Neuron-enriched DRG cell cultures allowed us to specify that [14C]PGD2 is the major prostanoid produced by primary sensory neurons as compared with nonneuronal DRG cells. Because PGD2 synthesis in DRG and more specifically in DRG neurons does not depend on exogenous GSH and differs from PGD2 synthesis in fibroblast-rich tissues, it is concluded that at least two distinct enzymatic processes contribute to PGD2 formation in the nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Dinoprostona/biosíntesis , Fibroblastos/metabolismo , Ganglios Espinales/metabolismo , Neuronas/metabolismo , Prostaglandina D2/biosíntesis , Animales , Animales Recién Nacidos , Células Cultivadas , Embrión de Pollo , Fibroblastos/citología , Ganglios Espinales/citología , Ganglios Espinales/embriología , Meninges/metabolismo , Neuronas/citología , Prostaglandinas/biosíntesis , Nervio Ciático/metabolismoRESUMEN
Prostaglandins (PGs) are neuroactive substances which act in the vicinity of their site of synthesis through receptors coupled to G-proteins. Since large amounts of PGE2 can be synthesized by chicken spinal cord, binding sites for PGE2 were looked for in various cell fractions of spinal cord. In the 17 000 g pellet incubated with 0.3 nM [3H]PGE2, 70% of ligand was specifically bound. Two types of PGE2 binding site were characterized (i) high affinity, low capacity binding sites (KD1 1.34 nM, Bmax1 34.5 fmol/mg prot); (ii) low affinity, high capacity binding sites (KD2 2.23 microM, Bmax2 13.2 pmol/mg prot). The high affinity binding sites fulfil several requirements for being receptors to PGE2: (i) since the KD1 is increased in the presence of the GTP analogue, Gpp(NH)p, these binding sites would be regulated by a G-protein; (ii) a desensitization was obtained by an excess of unlabelled PGE2 and reversed by Gpp(NH)p; (iii) the competition experiments between PGE2 and various prostanoids pointed to PGE2 receptors such as EP2 or EP3. The receptor characteristics of the low-affinity binding sites were not investigated. Hence, our results support the presence of two types of PGE2 binding site in the chicken spinal cord; a high affinity site, which corresponds to a PGE2 receptor responding to nanomolar concentrations and a low affinity site sensitive to micromolar concentrations of PGE2.
RESUMEN
High and low affinity binding sites for PGE2 were localized at the cellular level by means of radioautography. Two types of radioautographic reactions were observed in cryostat sections incubated with 0.6 nM [3H]PGE2: (i) a diffuse reaction scattered over the neuropil; (ii) a stronger reaction over the perikarya of some motoneurons. However, not all the motoneurons were equally labelled; the density of silver grains increased 12 times from poorly labelled to highly labelled cells. After incubation with an excess of unlabelled PGE2 the labelling was drastically reduced over motoneurons and the neuropil, so that the specific binding corresponded to 90 and 85% respectively of the total binding. Two facts indicated that the motoneurons possessed mainly high affinity binding sites which could be saturated by PGE2 concentrations in the nanomolar range: (i) a five-fold increase of [3H]PGE2 concentration (from 0.6 to 3 nM) produced only a 1.5 times increase in the density of silver grains over the perikarya; (ii) incubation in presence of 14 nM unlabelled PGE2 induced a drastic reduction of 0.6 nM [3H]PGE2 binding to the motoneurons. The presence of high affinity binding sites in motoneurons suggests that PGE2 could act as a modulator of spinal reflexes.
Asunto(s)
Ácidos Araquidónicos/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Lipooxigenasa/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Útero/metabolismo , Animales , Ácido Araquidónico , Calcimicina/farmacología , Carbacol/farmacología , Femenino , Cobayas , RatasAsunto(s)
Ácidos Araquidónicos/farmacología , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Ácidos Grasos Insaturados/farmacología , Miometrio/efectos de los fármacos , Prostaglandinas/farmacología , Animales , Ácidos Araquidónicos/metabolismo , Calcimicina/farmacología , Carbacol/farmacología , Inhibidores de la Ciclooxigenasa , Ácidos Grasos Insaturados/metabolismo , Femenino , Guanilato Ciclasa/metabolismo , Cobayas , Isoproterenol/farmacología , Lipooxigenasa/metabolismo , Inhibidores de la Lipooxigenasa , Microtúbulos/efectos de los fármacos , Miometrio/metabolismo , Fosfolipasas A/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Ratas , Ratas EndogámicasAsunto(s)
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Lipooxigenasa/metabolismo , Miometrio/enzimología , Fosfolipasas A/metabolismo , Fosfolipasas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Útero/enzimología , Animales , Calcimicina/farmacología , Carbacol/farmacología , Activación Enzimática , Epoprostenol/biosíntesis , Ácidos Grasos Insaturados/farmacología , Femenino , Cobayas , Miometrio/efectos de los fármacos , Fosfolipasas A2RESUMEN
Accumulation of cyclic GMP in estradiol-treated immature guinea pig myometrium was enhanced by carbachol, ionophore A23187, unsaturated fatty acids and their hydroperoxides. Cyclic AMP content was elevated only by arachidonic acid, A23187 and PGI2. Eicosatetraynoic acid (TYA), but not indomethacin prevented all cyclic GMP responses. The effects of A23187 and arachidonate on cyclic AMP were accompanied by a parallel increase (2-3 fold) in the generation of PGI2 by the myometrium. Both events were similarly reduced by indomethacin, TYA, 15-hydroperoxyarachidonic acid and tranylcypromine, suggesting that PGI2 was involved. Omission of Ca2+ or addition of mepacrine or p-bromophenacylbromide abolished the stimulatory effects of A23187 and carbachol on cyclic GMP as well as the A23187-induced elevations in both PGI2 and cyclic AMP generation. Thus, with both exogenous arachidonate as well as with endogenous fatty acid, released through an apparent phospholipase A2-induced activation process, the lipoxygenase pathway was associated with an activation of the cyclic GMP system and the cyclooxygenase pathway, via PGI2 generation, with an activation of the cyclic AMP system. Carbachol failed to alter both cyclic AMP content and the release of PGI2 suggesting a cholinergic receptor-mediated fatty acid release process, selectively coupled to the lipoxygenase route.
Asunto(s)
Antibacterianos/farmacología , Calcimicina/farmacología , Carbacol/farmacología , Lipooxigenasa/metabolismo , Miometrio/metabolismo , Nucleótidos/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Útero/metabolismo , Animales , Ácido Araquidónico , Ácidos Araquidónicos/farmacología , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Epoprostenol/biosíntesis , Epoprostenol/farmacología , Femenino , Cobayas , Ácidos Linoleicos/farmacología , Fosfolipasas A/metabolismo , Fosfolipasas A2Asunto(s)
AMP Cíclico/metabolismo , Endometrio/metabolismo , Epoprostenol/fisiología , Miometrio/metabolismo , Prostaglandinas/fisiología , Útero/metabolismo , Animales , Ácidos Araquidónicos/farmacología , Biotransformación , Femenino , Técnicas In Vitro , Agregación Plaquetaria/efectos de los fármacos , Prostaglandinas/farmacología , Prostaglandinas F/metabolismo , Ratas , Contracción Uterina/efectos de los fármacosAsunto(s)
GMP Cíclico/fisiología , Miometrio/fisiología , Útero/fisiología , Animales , Calcio/farmacología , Carbacol/farmacología , AMP Cíclico/metabolismo , Activación Enzimática , Femenino , Guanilato Ciclasa/metabolismo , Hidroxilaminas/farmacología , Contracción Muscular/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , RatasAsunto(s)
AMP Cíclico/metabolismo , Endometrio/metabolismo , Ácidos Grasos Insaturados/farmacología , Miometrio/metabolismo , Prostaglandinas E/farmacología , Contracción Uterina/efectos de los fármacos , Útero/metabolismo , Ácido 8,11,14-Eicosatrienoico/farmacología , Animales , Ácidos Araquidónicos/farmacología , Calcio/farmacología , Epinefrina/farmacología , Femenino , Técnicas In Vitro , Ratas , Factores de TiempoRESUMEN
In estrogen-pretreated rat myometrium, the relaxing effect exerted by theophylline or epinephrine has been correlated with their ability to raise cyclic AMP levels (Vesin and Harbon, 1974). The present study demonstrates that such a correlation can be quantitatively extended to the degree of saturation of intracellular cyclic AMP receptors. The rise in cyclic AMP induced by theophylline and/or epinephrine in intact myometrial strips was accompanied by a decrease in the ability of the corresponding extracts to bind exogenous 3H-labeled cyclic AMP. Total intracellular cyclic AMP binding sites were not modified and averaged a value of 0.22 muM. Accurate estimation of intracellular receptor-cyclic AMP complex has been correlated with the corresponding level of cyclic AMP in the tissue, the apparent intracellular Kd for cyclic AMP has been evaluated at 450 nm. Stimulation of myometrial strips with prostaglandin E1 (PGE1) which has been shown previously to induce contractions, although elevating cyclic AMP levels, was accompanied by a parallel increase in the saturation of the endogenous receptor, in an identical manner to that found with epinephrine or theophylline. The postulated hypothesis for a compartmentalization of cyclic AMP, or an interference of PGE1 with the intracellular cyclic AMP binding equilibrium has not been verified. The cyclic AMP system cannot be considered as the exclusive mechanism regulating uterine relaxation.