Three dimensional culture of pineal cell aggregates: a model of cell-cell co-operation.

Three dimensional (3-D) cultures of pineal cell aggregates were obtained by constant gyratory shaking the heterogenous cell populations, obtained from the rat pineals, in the DMEM (Dulbecco's modified Eagle's medium). Within 4 days, the pineal cells became organized into a tissue like configuration appearing as a compact ball, evidenced by the scanning electron microscopy. The 3-D aggregates seemed to be mainly composed of pinealocytes (round-oval cells), glial (elongated cells) and other unknown cells. The heterogenous cells were separated by intercellular spaces. The ultrastructural characteristics revealed by transmission electron microscopy exhibited the presence of granular lysosomes, typical of pinealocytes actively involved in the secretion. These pineal cell aggregates secreted melatonin and other indole amines i.e. 5-methoxytryptamine (5-MT), indole acetic acid (IAA), 5-methoxy-3-indole acetic acid (5-MIAA), tryptophol (TOL) and 5-methoxytryptophol (5-MTL) in the culture medium, indicating the functional aspect of pinealocytes. The 3-D aggregates cultures had advantages over the pineal monolayer cultures as, after 4 days of culture, the amounts of indole amines secreted by 3-D aggregates were higher than those secreted by monolayer cultures. Besides, the 3-D aggregates remained functional till 24 days in the gyratory culture conditions. In the continuous perifusion system, the 3-D aggregates secreted melatonin while challanged with isoproterenol. This 3-D model of pineal cell aggregates might be useful, in future, to perform other kinetic studies of the release of indole amines in perifusion experiments as this system allows the maintenance of pineal cells for a long period of time.

Lack of uptake, release and action of UTP at sympathetic perivascular nerve terminals in rabbit ear artery.

A possible role of uridine 5'-triphosphate (UTP) and uridine at sympathetic nerve terminals was studied in the rabbit ear artery after incubation of isolated vessels with [3H]uridine or [3H]noradrenaline. It was found that [3H]uridine was taken up by rabbit ear artery. This uptake was largely suppressed after the removal of endothelium and was inhibited by ethidium bromide and dipyridamole. Chemical denervation of the vessels with 6-hydroxydopamine did not reduce the uptake. Following pre-incubation of the isolated vessels with [3H]uridine, there was a release of radioactivity from the superfused rabbit ear artery. UTP, UDP, UMP and uridine were detected by thin layer chromatography both in the superfusate and inside the vessels. Transmural electric stimulation (30 V, 5 Hz) induced a contraction of the vessels but did not increase the release of uridine nucleotides into the superfusate. [3H]Noradrenaline was released during electric stimulation and the addition of UTP (100 microM) had no effects on this release. To conclude, this study shows that in contrast to endothelial cells, the sympathetic nerve terminals of the rabbit ear artery do not take up uridine and do not release uridine-derived nucleotides. UTP at 100 microM is also unable to modulate the evoked release of noradrenaline. These results mainly confine the role of UTP in endothelium-derived vasodilatation via P2Y2 and/or P2Y4 receptors.

Free radical involvement in endothelium-dependent responses of the rat thoracic aorta in moderate hypoxic conditions.

This study investigates the effects of agents which act on the production or efficacy of free radicals on the hypoxic responses of rat aorta rings. Under moderate hypoxic conditions, the resting tension of the rings was not changed but in rings precontracted with 5-hydroxytryptamine, there was a relaxation followed by a contraction. Removal of the endothelium with saponin suppressed relaxation to acetylcholine and abolished the contractions produced by hypoxia. In rings with a functional endothelium, hypoxic vasoconstriction was strongly inhibited by mannitol and exifone, but was not reduced by N(G)-nitro-L-arginine methyl ester, superoxide dismutase + catalase, or deferoxamine. Hypoxic vasodilatation was only partially inhibited by mannitol. To conclude, hypoxic constriction of the rat thoracic aorta is largely endothelium-dependent and involves free radicals whereas hypoxic dilatation is partially endothelium-dependent and partially involves free radicals. There is also indirect evidence for lack of direct involvement of nitric oxide/endothelium-derived relaxing factor (NO*/EDRF), hydroxyl radical (OH*) and superoxide anion in the hypoxic constriction and relaxation of the rat aorta.

Rat pineal cell aggregates: ultrastructural and functional characteristics.

The aggregates were obtained by constant gyratory shaking of suspension cells freshly isolated from adult rat pineal glands. Their sizes ranged from 60 to 120 microns. Within 4-5 days, the aggregates formed by pinealocytes, astrocytes, and other unidentified cells became organized in a tissue-like configuration. There was no proliferation of the fibroblast cells. Ultrastructural characteristics of the aggregates were revealed by the presence of granular lysosomes, which are typical of pinealocytes, and are actively involved in the secretion. Functional characteristics were studied in static incubation. The aggregates secreted melatonin and other indole amines in culture medium. Basal melatonin release was detected until Day 24 of culture. This secretion was stimulated 230% with Isoproterenol (beta-adrenergic agonist), 725% with Epinephrine (alpha- and beta-adrenergic agonists), and 140% with Vasoactive Intestinal Peptide after 5 days in culture, then > 1200% with Forskolin 9 days later (14-day-old aggregates). The results indicate that three-dimensional aggregates obtained from isolated pineal gland cells were the functional multicellular structures with in vivo characteristics.

Effect of naloxone and beta-casomorphin on the hypothalamic-pituitary-luteinizing hormone axis in vitro.

The effect of naloxone and beta-casomorphin on luteinizing hormone (LH) release from pituitary cell aggregates, obtained by three-dimensional culture, with or without mediobasal hypothalamic fragments was studied in vitro. Short-term naloxone perifusion at a concentration of 10(-5)M did not modify either basal or LHRH-stimulated LH release from the pituitary cell aggregates. In contrast, a 12-min naloxone perifusion at the same concentration caused an increase in LH release in the mediobasal hypothalamic-pituitary cell aggregate axis. This increase was rapid (12-16 min after time pulse), marked [up to 10 times (p less than 0.004) the initial base line], short (return to the base line secretion 32-40 min after the beginning of the time pulse) and dose-dependent, with a rise greater than 1000% at a concentration of 10(-4) (p less than 0.006). The same effect was observed when a second pulse was applied 48 min after the first one. LH release induced by naloxone was antagonized 56 +/- 2% (p less than 0.03) by beta-casomorphin (an exogenous opiate) at a concentration of 10(-5) M. beta-casomorphin alone did not modify LH basal secretion, but inhibited 25.1 +/- 2.4% (p less than 0.008) LH release enhanced by LHRH. These results indicate that naloxone, an opiate antagonist, markedly increases LH release via a mu-type opioid receptor mechanism at the hypothalamic level only, during short-term exposure.

Melatonin modifies prolactin release induced by opiate antagonists in male rats.

The effect of melatonin on the prolactin (PRL) release induced by treatment with naloxone, naloxone methyliodide, naltrexone and nalmefene were studied in adult male rats. Subcutaneous melatonin injection (1.4 mg/Kg) had no significant effect on serum PRL levels, but decreased by 29% and 26% respectively the inhibitory effect potency of naltrexone (2.5 mg/Kg) and nalmefene (2 mg/Kg) on PRL secretion after simultaneous injections. The inhibitory effect potency of naloxone on PRL release increased (16%) when it was administered with melatonin. Simultaneous injection of melatonin with naloxone methyliodide (2.8 mg/Kg) inhibited PRL release (77.5%) while naloxone methyliodide alone did not modify this secretion. The results obtained with a quaternary opioid antagonist indicate that the opioid receptor type which mediates PRL response is located inside the blood-brain barrier. Our findings show that opiate antagonists and their quaternary ammonium salts affect secretion of PRL through mechanism susceptible to the influence of melatonin.

Stimulatory effect of perhexiline maleate on the basal and LHRH-stimulated luteinizing hormone release from rat pituitary cell aggregates in vitro.

Perhexiline maleate (PM) is an anti-anginal agent of amphiphilic character involved in lipidosis disorders. Experiments were carried out to study PM action on LH release from rat anterior pituitary cell aggregates. PM caused significant and dose-dependent increases of basal LH release when the aggregates had been previously treated with PM for 20 min, then incubated for 30 min with the same concentration of drug after renewal of the culture medium. The increases of basal LH secretion induced by PM were 69% (p < 0.001) at a concentration of 10(-7)M, 130% (p < 0.001) at a concentration of 10(-5)M and 250% (p < 0.001) at a concentration of 10(-4)M. PM also increased the LHRH-stimulated LH release by 25.5% (p < 0.05) at a concentration of 10(-5)M and by 74.5% (p < 0.01) at the concentration of 10(-4)M. The results showed that PM was more potent on basal LH release than on LH stimulated by LHRH. The mechanism of this action remains unclear. It may be due to the lipidosis property of the drug.

Alpha-L-fucosidase in rat testis during sexual maturity.

alpha-L-fucosidase (EC 3.2.1.51) activity, concentration of testosterone (T), and its metabolite dihydrotestosterone (DHT) were tested in rat testis during the onset of puberty. This study was also carried out in testes of rats treated with 17-beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5-alpha-androstane-3-one (DMAA), an inhibitor of the 5-alpha-reductase-mediated conversion of T into DHT. alpha-L-fucosidase activity in seminiferous tubules of control and DMAA-treated rats was found to be relatively high on the 25th day after birth (approximately 24 units) but decreased and remained relatively constant the following days (approximately 10 units). In contrast, alpha-L-fucosidase activity was nearly undetectable in the immature interstitium of the control rats but sharply increased the following days. A maximum was reached at the 55th day, followed by a rapid decrease. alpha-L-fucosidase activity evolved in parallel with an increase and decrease of DHT concentration. In DMAA-treated rats with DHT levels and an alpha-L-fucosidase activity significantly lower than in the control rats between the 55th and the 65th days, this parallelism existed as well.

Newly evidenced pyrimidinoceptors and the P2x purinoceptors are present on the vascular smooth muscle and respectively mediate the UTP- and ATP-induced contractions of the dog maxillary internal vein.

P2 purinergic receptors and pyrimidinoceptors were studied by comparing the contractile responses to UTP with those to ATP, in the dog internal maxillary vein (IMV) after endothelium removal. The contraction curves were very different: rapid subsidence with ATP and sustained contraction with UTP. alpha beta methylene ATP induced desensitization to ATP, whereas it did not antagonize the UTP-induced contractions. Reactive blue 2 (RB2) was incapable of antagonizing contractions to ATP and UTP in this vessel. We showed that Nicardipine, a calcium antagonist, was more potent on the UTP-induced than on the ATP-induced contractions. The fact that RB2 did not potentialize the UTP-induced contractions, unlike what has been observed in the dog saphenous vein, suggests that a new category of pyrimidinoceptors have been evidenced on the IMV. Our results also indicate that ATP induces contraction via P2x rather than P2y purinoceptors. The receptors described here are localized on the vascular smooth muscle.

Effects of simultaneous active immunization against 17 beta-estradiol and testosterone on pituitary and ovarian activity in the rat.

Female rats were immunized with mixed 17 beta-estradiol-6-carboxymethyloxime-bovine serum albumin and testosterone-3-carboxymethyloxime-bovine serum albumin. All the animals produced antibodies against the 2 haptens and were better immunized against 17 beta-estradiol than against testosterone. Nevertheless the disappearance of cyclic ovarian function was only obtained in animals highly immunized against the two antigens. The 17 beta-estradiol serum concentration increased with the level of immunization whereas the progesterone concentration fell. Persistent vaginal cornification was observed and ovarian lesions (large fluid filled cysts, hemorrhagic follicles and absence of corpora lutea) coincided with increased serum levels of luteinizing hormone and prolactin.

Effects of melatonin in vivo upon luteinizing hormone and prolactin releases induced by opiate receptor antagonists in adult male rats.

The effects of melatonin on LH and PRL releases induced by treatment with naloxone, naloxone methyliodide and nalmefene were studied in adult male rats. Subcutaneous melatonin injection (1.4 mg/Kg) had no effect on LH secretion, but caused an inhibition effect (84%) on LH release induced by naloxone (2.4 mg/Kg). Melatonin too totally inhibited LH secretion induced by naloxone methyliodide (2.8 mg/Kg) and nalmefene (2 mg/Kg) when it was simultaneously administered with each opioid receptor antagonist. Melatonin alone had no significant effect on serum PRL levels, but decreased by 25.5% the inhibitory effect potency of nalmefene on PRL secretion after simultaneous injections. The inhibitory effect potency of naloxone on PRL release increased (16%) when it was administered with melatonin. Simultaneous injection of melatonin with naloxone methyliodide inhibited PRL release (78%) while naloxone methyliodide alone did not modify this secretion. The results obtained with a quaternary opioid antagonist indicate that the opioid receptor type which mediates LH and PRL responses is located respectively outside and inside the blood-brain barrier. Our findings show that opiate antagonists and their quaternary ammonium salts affect secretion of LH and PRL through different mechanisms susceptible to the influence of melatonin.

Inhibition of (Na+,K+)-ATPase and Mg++-ATPase by a lysosomotropic drug: perhexiline maleate.

Human clinical observations and in vivo studies have shown that the amphiphilic drug perhexiline maleate is responsible for lipidosis storage disorders. When the drug was incubated in vivo with rat brain homogenates, the ouabain-sensitive (Na+,K+)-ATPase and the Mg++-ATPase activities were inhibited. 50% inhibition occurred at the drug concentrations 5.10(-5) M for (Na+,K+)-ATPase and at 10(-4) M for Mg++-ATPase, respectively. Kinetic studies performed on rat brain homogenates showed a mixed type inhibition of these enzymes by perhexiline maleate. The effect of other lysosomotropic drugs (imipramine, chlorpromazine, thioridazine and tamoxifen) on (Na+,K+)-ATPase and on Mg++-ATPase activities was found to be similar to that induced by perhexiline maleate. These results indicate that the inhibitory effect of perhexiline maleate on (Na+,K+)-ATPase and Mg++-ATPase may be a common feature shared by the lysosomotropic drugs.

Effects of perhexiline maleate on asialo-orosomucoid receptor endocytosis and recycling in HTC cells.

Perhexiline is a lysosomotropic agent which has proved to be very valuable to certain patients suffering from angina pectoris. However long-term administration of the drug may induce hepato- and neuro-toxicity. Using HTC cells (a rat hepatoma-derived cell line) whose plasma membranes were labeled with NaB[3H]4 after oxidation by NaIO4, endocytosis and recycling of labeled asialo-orosomucoid (ASOR) receptors were investigated in the presence of 50 mumols/l perhexiline maleate. The results demonstrate that the drug induces a significant decrease of the rate of both the internalization and the recycling of ASOR receptors. The mechanisms responsible for these effects have not yet been elucidated. However, the current findings may be related to the previously observed inhibitory effect of perhexiline on cellular (Na+, K+)-ATPase and Mg++-ATPase activities. Our findings would then reflect insufficient cellular energy production, resulting from depressed ATP hydrolysis in the presence of perhexiline.