Browsing by Author "BELMAR, J"

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    BIOCHEMICAL-EVIDENCE FOR THE PRESENCE OF SMALL AND LARGE NORADRENERGIC STORAGE-VESICLES ISOLATED FROM CAT OVARY IN ISOOSMOTIC CONDITIONS - DISTRIBUTION OF DOPAMINE-BETA-HYDROXYLASE ACTIVITY
    (1989) LARA, H; BELMAR, J
    The cat ovary presents unusually high levels of noradrenaline that change according to the endocrine status of the animal. Their functional meaning remains unknown. The cat ovary innervation, unlike that of other organs receiving noradrenergic innervation, has been poorly characterized on biochemical grounds. We present here a biochemical characterization of the neurotransmitter storage. By using hyperosmotic and isoosmotic gradients evidence is presented that noradrenaline is associated to two different populations of vesicles. In hyperosmomolarity conditions (sucrose gradients) "light" vesicles (density 1.12 g/ml) and "heavy" vesicles (density 1.17 g/ml) appeared. In both vesicles, noradrenaline and dopamine-.beta.-hydroxylase were found. In isoosmotic Percoll gradients distribution of the markers also suggested the presence of two vesicle populations. Light vesicles (density 1.033 g/ml) with high dopamine-.beta.-hydroxylase activity but very low levels of noradrenaline and adenosine triphosphate; [3H]noradrenaline, used as a specific exogenous vesicle marker, was feebly incorporated in this fraction. Heavy vesicles (density 1.041 g/ml) containing high levels of noradrenaline, adenosine triphosphate, low levels of dopamine-.beta.-hydroxylase activity are able to incorporate high amounts of [3H]noradrenaline. In these gradients, Mg2+ activated ATPase activity was present in both vesicle fractions. Sedimentation analysis by analytical differential centrifugation also disclosed two types of vesicles; large vesicles with a sedimentation coefficient between 348 and 308 .hivin.S and small vesicles with a sedimentation coefficient of 96 .hivin.S. Large vesicles were associated with noradrenaline-.beta.-hydroxylase activity, while small vesicles were associated only with noradrenaline. In isoosmotic conditions the use of other microsomal markers allowed us to define the degree of contamination of the vesicle fractions. It was found that the noradrenergic heavy vesicles fraction presented under 11% of 5-nucleotidase activity of the total activity present in the gradient and less than 5% of acid phosphatase, NADH-cytochrome c reductase and monoaminooxidase of the total activities in the gradients. In isoomotic conditions the physical properties of presumed vesicles were apparently undisturbed supporting the current morphometric observations. Our results then suggest prevailing roles for each type of vesicle: synthesis for light vesicles, and storage and/or release for heavy ones.
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    BRADYKININ MODULATES THE RELEASE OF NORADRENALINE FROM VAS-DEFERENS NERVE-TERMINALS
    (1991) LLONA, I; GALLEGUILLOS, X; BELMAR, J; HUIDOBROTORO, JP
    To assess whether bradykinin influences the release of noradrenaline from the adrenergic varicosities of the vas deferens, tissues were loaded with 3H-noradrenaline. Upon electrical depolarization bradykinin increased in a concentration-dependent fashion, the overflow of tritium from the mouse or rat vas deferens. The 3H-overflow is dependent on the external Ca2+ concentration suggesting neuronal release of 3H-noradrenaline. The present results add evidence to the hypothesis that bradykinin modulates the release of noradrenaline from peripheral sympathetic nerve terminals via the activation of a presynaptic mechanism.
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    CHANGES IN NORADRENERGIC VESICLE MARKERS OF RABBIT OVIDUCTS DURING PROGESTERONE TREATMENT
    (1983) BELMAR, J; LARA, H; GALLEGUILLOS, X
    The effect of progesterone (P) on norepinephrine (NE), [3H]norepinephrine ([3H]NE) and dopamine .beta.-hydroxylase (DBH) in noradrenergic vesicles from rabbit oviducts was studied after daily injections of the hormone during different periods (4, 7 and 15 days). Progesterone induced a concomitant increase in NE and DBH activity and [3H]NE uptake. To study the mechanism involved in such effects, 4 tissue fractions were obtained by differential centrifugation of the oviducts of which the vesicular fraction was applied over continuous sucrose gradients (0.3-2 M). The changes induced by P in markers of tissue and gradient fractions showed an increase of the NE storage capacity which could be ascribed to an increase in the number of storage vesicles, and/or to a higher extravesicular storage capacity. The occurrence of these mechanisms during pregnancy or after P treatment could account for the (long-lasting) high levels of NE observed in such instances.
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    CHANGES OF NOREPINEPHRINE LEVELS, TYROSINE-HYDROXYLASE AND DOPAMINE-BETA-HYDROXYLASE ACTIVITIES AFTER CASTRATION AND TESTOSTERONE TREATMENT IN VAS-DEFERENS OF ADULT-RATS
    (1989) BUSTAMANTE, D; LARA, H; BELMAR, J
    Norepinephrine levels and tyrosine hydroxylase and dopamine-.beta.-hydroxylase activities have been used to evaluate the effect of castration and testosterone treatment on the sympathetic innervation of the adult was deferens. Castration was followed by a decrease in both norepinephrine content and tyrosine hydroxylase activity, even though the changes were not concomitant. Treatment of castrated animals with testosterone reversed the effect of castration on organ weight and norepinephrine content, but only a short-lasting increase in tyrosine hydroxylase activity occurred at the beginning of testosterone treatments. In contrast, the testosterone-induced recovery of norepinephrine content observed at this time was accompanied by a marked increase in dopamine-.beta.-hydroxylase activity. The results suggest that in rat was deferens, norepinephrine levels are under androgenic control and that this regulation mainly involves changes in dopamine-.beta.-hydroxylase activity rather than a modulation of tyrosine hydroxylase.
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    EARLY MALNUTRITION AND CHANGES IN THE INDUCED RELEASE OF NORADRENALINE IN THE PREFRONTAL CORTEX OF ADULT-RATS
    (1987) SOTOMOYANO, R; HERNANDEZ, A; PEREZ, H; RUIZ, S; DIAZVELIZ, G; BELMAR, J
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    EFFECT OF CASTRATION AND TESTOSTERONE ON NOREPINEPHRINE STORAGE AND ON THE RELEASE OF [H-3]NOREPINEPHRINE FROM RAT VAS-DEFERENS
    (1985) LARA, H; GALLEGUILLOS, X; ARRAU, J; BELMAR, J
    Norepinephrine and dopamine-.beta.-hydroxylase, used as noradrenergic vesicle markers, were decreased in the rat vas deferens 10 days after castration. Five days of testosterone administration to castrated animals increased the enzyme activity over that of controls but did not modify norepinephrine content. In tissue fractions obtained by differential centrifugation, the highest activities of the noradrenergic markers appeared in the vesicular fraction of controls and in the soluble fraction of castrated animals. Testosterone reversed the effect of castration: it increased dopamine-.beta.-hydroxylase activity in the vesicular and soluble fractions, while norepinephrine increased only in the vesicular fraction. Results obtained after continuous sucrose gradient centrifugation of vesicular fractions suggested that these changes principally affected the number of light noradrenergic vesicles while testosterone increased the number of vesicles reduced by castration. Hormonal manipulations also modified some functional properties of nerve endings: norepinephrine depletion after transmural stimulation in the presence of tetraethylammonium, as well as the release of the neurotransmitter, were decreased after castration. These effects were reversed by testosterone. A modulatory effect of testosterone on the norepinephrine storage system and on the functional properties of the adrenergic innervation of vas deferns is suggested.
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    EFFECT OF CLONIDINE EARLY IN LIFE ON BRAIN MORPHOFUNCTIONAL DEFICITS INDUCED BY NEONATAL MALNUTRITION IN THE RAT
    (1989) SOTOMOYANO, R; HERNANDEZ, A; PEREZ, H; RUIZ, S; GALLEGUILLOS, X; BELMAR, J
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    IN-VITRO PROGESTERONE EFFECTS ON H-3 DOPAMINE RELEASE FROM RAT CORPUS STRIATUM SLICES OBTAINED UNDER DIFFERENT ENDOCRINE CONDITIONS
    (1993) CABRERA, R; DIAZ, A; PINTER, A; BELMAR, J
    The release of dopamine (DA) from corpus striatum is affected by the endocrine state of the animal being progesterone suggested as a potential hormonal modulatory signal. Most of its actions have been described on endogenous DA release induced by amphetamine. However the release of DA and the mechanism of the drug effect have been shown to be highly complexes. Considering that DA recently incorporated and/or synthetized is preferentialy used we have characterized the effect of progesterone in vitro on the K+-induced release of H-3-dopamine)H-3-DA) from rat corpus striatum slices. These were obtained during the estrous cycle or under conditions of high or low levels of endogenous progesterone (pregnant and ovariectomized rats). The release of H-3-DA was independent of the cycle. However, progesterone in vitro modified the induced release in a cyle-dependent way. Low concentrations of the hormone (100-200 mM) reduced the K+ (30 mM) effect while higher doses (300-500 mM) were facilitatories. After 7 days of ovariectomy, the induced release of H-3-DA was unchanged while in pregnant rats it was found decreased. In both cases the inhibitory effect of the hormone dissapeared. Both progesterone (200 nM) and omission of Ca++ from the superfusion medium did not modified tyramine(20 muM) or K+ induced release, respectively. Data suggest that the pool of DA, related to exocytotic mechanism of release, could be specifically affected by progesterone, in a bimodal way, probably through independent genomic and non-genomic influences.
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    IN-VITRO PROGESTERONE EFFECTS ON H-3 NOREPINEPHRINE RELEASE FROM RAT CEREBRAL-CORTEX SLICES
    (1993) PINTER, A; BELMAR, J
    THE effect of progesterone in vitro (0.05-5 muM) on K+-induced, Ca2+-dependent release of H-3-NE from brain slices obtained from the occipital region of rats, during the oestrous cycle or 7 days after ovariectomy or on the fifth day of pregnancy was studied. The hormone reduced the K+ (20 mM) effect in all the stages of the cycle, but did not modify the reduced release found- after ovariectomy or pregnancy or the release induced from male slices. Yohimbine blocked the effect of progesterone. The results suggest the releasing process of norepinephrine in the cerebral cortex as a target for progesterone modulatory influences on brain function.
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    NONNEURONAL ENDOGENOUS GABA EFFLUX FROM THE RAT OVIDUCT
    (1993) FORRAY, MI; HIDALGO, P; DIAZ, F; BELMAR, J
    The subcellular distribution of Gamma-aminobutyric acid (GABA) was studied in the rat oviduct. The highest content of GABA was found in the soluble fraction. The effect of chemical stimulation of the endogenous GABA efflux from the rat oviduct was examined. High K+ concentrations could not induce elevation of the GABA efflux. Instead, a continuous spontaneous GABA efflux without change for long periods of time was observed. The total GABA content and GABA concentration were determined in the rat oviduct on days 1, 5, 10, 15, 30, 35 and 40 of the postnatal period and also during the estrous cycle. During postnatal development the GABA levels increase gradually with time reaching at prepuberal age a concentration similar to that found in diestrous rats. In the estrous cycle both GABA content and GABA concentration reached the highest value in the proestrous and the lowest value in the estrous phase. These findings support the hypothesis that GABA efflux may be modulated by the changes in oviductal fluid volume during the estrous cycle.
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    RELEASE OF H-3 NORADRENALINE FROM THE RAT OVIDUCT - CHANGES DURING ESTROUS-CYCLE AND BY PROGESTERONE INVITRO
    (1990) CHIAPPE, P; GALLEGUILLOS, X; LARA, H; FUENTEALBA, B; FORRAY, I; BELMAR, J
    Rat oviduct noradrenergic innervation seems to be under the influence of hormonal modulation. Noradrenaline level is specifically affected, but other processes like the release of the neurotransmitter have not yet been studied. In this work the release of 3H-noradrenaline (3H-NA) during the estrous cycle and its modification by progesterone "in vitro", were studied. The basal 3H-NA outflow was unchanged during the estrous cycle. However, the induced release (K+, 80 mM) was maximal during estrous. After progesterone, induced 3H-NA release was inhibited at low concentrations (5 .mu.M); under higher concentrations (50 .mu.M) the effect persisted but basal outflow of radioactivity was increased. The inhibitory effect was potentiated by RU-486 (Progesterone blocker). Results suggest a modulatory role for the hormone and it could be related to interactions with nerve-ending membranes and not with classical nuclear receptors.
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    RELEASE OF NOREPINEPHRINE FROM THE CAT OVARY - CHANGES AFTER OVULATION
    (1991) LARA, HE; BELMAR, J
    The distribution of intraneuronal constituents involved in norepinephrine (NE) storage, uptake, and release were used to estimate changes in NE secretion from the cat ovary after ovulation induced with eCG plus hCG. The content of NE and ATP, which are principally stored in small noradrenergic vesicles (isolated at a density of 1.041 g/ml in Percoll gradient), decreased after ovulation. However, the activity of dopamine beta-hydroxylase which is principally associated with large noradrenergic vesicles (isolated at a density of 1.033 g/ml in Percoll gradient), was only slightly decreased. Mg2+-dependent ATPase, located in both large and small storage vesicles, decreased only in the small storage vesicles, suggesting that preferential secretion from small noradrenergic vesicles occurred. The hormonal treatment also affected the functional capacity of the vesicles, as evidenced by the decrease in uptake and storage capacity as well as the decrease in the stimulated release of H-3-NE observed after ovulation. The aforementioned changes are characteristically seen after a sympathetic discharge; thus they strongly support the notion that ovarian sympathetic activity increases during the ovulatory process, resulting in the postovulatory decrease in both the size and functional capacity of the intraneuronal compartment where NE is stored.
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    YOHIMBINE EARLY IN LIFE ALTERS FUNCTIONAL-PROPERTIES OF INTERHEMISPHERIC CONNECTIONS OF RAT VISUAL-CORTEX
    (1991) SOTOMOYANO, R; HERNANDEZ, A; PEREZ, H; RUIZ, S; GALLEGUILLOS, X; BELMAR, J
    It has been shown that noradrenaline (NA) is an important regulator of normal regressive processes occurring during synaptogenesis such as cell death, axonal pruning and synaptic elimination. The present study was designed to investigate whether enhanced NA release induced by chronic yohimbine administration early in life may alter in the rat the normal pattern of functional interhemispheric connections of the visual cortex. Yohimbine administration to rats between days 5 and 16 of postnatal life (2.5 mg/kg, IP, daily) resulted in changes in the pattern of transcallosal responses evoked in the visual cortex, characterized by a reduction in the peak-to-peak amplitude as well as a reduction of the extent of projecting fields of maximal activity, when examined at 30-35 days following termination of the drug treatment regimen. The results indicate that yohimbine treatment early in life induces functional alterations in the interhemispheric connectivity of the visual areas, probably by disrupting the normal trophic role of NA during synaptogenesis.