RESUMEN
Daily changes in gamma-aminobutyric acid (GABA) turnover rate were studied in the golden hamster retina. This parameter showed significant variations throughout the light-dark cycle, with minimal values during the day. Retinal glutamic acid decarboxylase (GAD) activity was higher at midnight than at noon. Moreover, [3H]GABA binding significantly varied throughout the 24-h cycle, with maximal values during the day. Saturation studies performed at 12:00 and 24:00 h indicated that the maximal concentration of [3H]GABA binding sites (Bmax) was significantly higher at noon, whereas the dissociation constant (Kd) remained unchanged. High K+-induced GABA release was significantly higher at midnight than at midday. Daily variations in retinal GABA turnover rate, GABA release, and in its specific binding persisted in golden hamsters exposed to constant darkness. In summary, these results support the idea of a circadian clock-controlled GABAergic activity in the hamster retina.
Asunto(s)
Ritmo Circadiano/fisiología , Glutamato Descarboxilasa/metabolismo , Mesocricetus/metabolismo , Inhibición Neural/fisiología , Neuronas/metabolismo , Retina/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Radioisótopos de Carbono/farmacocinética , Cricetinae , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacocinética , Masculino , Mesocricetus/anatomía & histología , Neuronas/citología , Neuronas/efectos de los fármacos , Estimulación Luminosa , Ensayo de Unión Radioligante , Retina/citología , Retina/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacocinéticaRESUMEN
PURPOSE: To study the presence of hyaluronidase activity in the rabbit trabecular meshwork and its regulation by brimonidine. METHODS: A spectrophotometric assay that consists of the assessment of N-acetylhexosamine groups released from hyaluronic acid was used to examine hyaluronidase activity. Cyclic adenosine monophosphate (cAMP) levels were assessed by radioimmunoassay. RESULTS: Hyaluronidase activity was detected in the rabbit trabecular meshwork. Its optimal activity was in the acid range of pH 3.8. Brimonidine significantly increased trabecular hyaluronidase-specific activity and decreased cAMP accumulation. Yohimbine significantly inhibited the effect of brimonidine on both hyaluronidase activity and cAMP accumulation. CONCLUSIONS: The finding of endogenous hyaluronidase activity in rabbit trabecular meshwork supports the hypothesis that this tissue can metabolize its own glycosaminoglycan (GAG) products. The present results suggest, however, that the hypotensive effect of brimonidine could be mediated, at least in part, by its ability to increase GAG catabolism, probably through a cAMP-independent mechanism.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Hialuronoglucosaminidasa/metabolismo , Quinoxalinas/farmacología , Malla Trabecular/efectos de los fármacos , Antagonistas Adrenérgicos alfa/farmacología , Animales , Tartrato de Brimonidina , AMP Cíclico/metabolismo , Concentración de Iones de Hidrógeno , Masculino , Quinoxalinas/antagonistas & inhibidores , Conejos , Radioinmunoensayo , Malla Trabecular/enzimología , Yohimbina/farmacologíaRESUMEN
The effect of GABA on melatonin content in vitro was studied in the golden hamster retina. GABA significantly increased melatonin levels in a dose-dependent manner, its effect being reversed by a GABA(A) receptor antagonist, bicuculline, but not by saclofen, a GABA(B) antagonist. Moreover, an equimolar concentration of muscimol, a GABA(A) receptor agonist, significantly increased retinal melatonin content, whereas baclofen, a GABA(B) receptor agonist, was ineffective. The darkness-induced increase in melatonin content in vitro was inhibited by bicuculline, whereas saclofen was ineffective. Retinal GABA turnover rate was significantly higher at midnight than at midday. GABA significantly decreased cyclic AMP and increased cyclic GMP accumulation in the golden hamster retina. The effect of GABA on both nucleotide levels was reversed by bicuculline, but baclofen had no effect. Cyclic GMP analogues (i.e., 8-bromoguanosine 3',5'-cyclic monophosphate and 2'-O-dibutyrylguanosine 3',5'-cyclic monophosphate) significantly increased retinal melatonin content in vitro. Taken together, these results support the hypothesis that GABA may be important for the "dark message" in the hamster retina.
Asunto(s)
Melatonina/metabolismo , Retina/metabolismo , Ácido gamma-Aminobutírico/farmacología , 1-Metil-3-Isobutilxantina/farmacología , Animales , Bicuculina/farmacología , Cricetinae , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Oscuridad , Relación Dosis-Respuesta a Droga , Antagonistas del GABA/farmacología , Masculino , MesocricetusRESUMEN
A decrease in amplitude of wheel running circadian rhythms was found in old (18 month old) Syrian hamsters, as compared with young (3 month old.) animals. In a plus-maze paradigm, amplitude of variation of anxiety-related variables (2400 vs. 1600 h) was significantly impaired in aged hamsters. Cerebral cortex, hypothalamic and pineal gamma-aminobutyric acid (GABA) turnover was higher at night, amplitude of variation being significantly smaller in aged hamsters. The results further support the existence of impaired amplitude of circadian rhythms in aged Syrian hamsters.
Asunto(s)
Envejecimiento/fisiología , Ansiedad/fisiopatología , Ritmo Circadiano/fisiología , Locomoción/fisiología , Ácido gamma-Aminobutírico/metabolismo , Factores de Edad , Envejecimiento/metabolismo , Animales , Cricetinae , MasculinoRESUMEN
Daily variations in cGMP, guanylate cyclase and phosphodiesterase activity in golden hamster retina were studied. Cyclic GMP content exhibited significant variations throughout the 24-hr cycle with maximal values during the dark phase. In order to establish the relative participation of nucleotide synthesis and breakdown during a 24-hr cycle, guanylate cyclase and phosphodiesterase activity were measured in hamsters killed at eight intervals. Guanylate cyclase activity increased at night, peaking at 22.00 hr. Phosphodiesterase activity did not change significantly throughout the light-dark cycle. Light exposure during the night inhibited the nocturnal increase in cGMP content and guanylate cyclase activity, while phosphodiesterase remained unchanged. From these results, it might be presumed that in response to continuous (in a range of hr) light or dark stimuli, the retina would process the photic signal in a different way from that in the short term (in a range of msec).
Asunto(s)
GMP Cíclico/metabolismo , Guanilato Ciclasa/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Retina/metabolismo , Animales , Ritmo Circadiano , Cricetinae , Oscuridad , Luz , Masculino , Mesocricetus , Retina/enzimologíaRESUMEN
Melatonin effect on retinal cyclic GMP accumulation, guanylate cyclase activity, cyclic GMP content and cyclic GMP phospho-diesterase activity was examined in the Syrian hamster retina. Melatonin increased significantly cyclic GMP accumulation at picomolar concentrations and in a time-dependent manner. The kinetic analysis of guanylate cyclase activity revealed a significant increase of both apparent Vmax and K(m), induced by 10 nM melatonin. The effect of melatonin was higher in the absence, than in the presence of the phoshodiesterase inhibitor (IBMX), suggesting an effect on cyclic GMP catabolism. Phosphodiesterase activity was significantly decreased by melatonin. The results show a dual effect of melatonin on cyclic GMP levels, i.e. by increasing the synthesis and inhibiting the degradation, both resulting in an increase of cyclic GMP levels. Taking into account the key role of cyclic GMP in visual mechanisms, the results would suggest the participation of melatonin in retinal physiology.
Asunto(s)
GMP Cíclico/metabolismo , Melatonina/farmacología , Retina/efectos de los fármacos , Animales , Cricetinae , Relación Dosis-Respuesta a Droga , Guanosina Trifosfato/farmacología , Masculino , Radioinmunoensayo , Factores de TiempoRESUMEN
Hypocalcemia is a common finding during stress. The objective of this study was to examine: (a) the changes in circulating calcium, parathyroid hormone (PTH) and calcitonin (CT) concentration in rats stressed by being given a subcutaneous injection of turpentine oil, and (b) the involvement of the sympathetic cervical pathway in stress-induced changes of calcium homeostasis. Four hours after receiving turpentine oil or vehicle, rats were subjected either to hypocalcemia, by being given EDTA intraperitoneally, or to hypercalcemia, by being injected CaCl2 intraperitoneally. Significant changes in serum calcium (10% decrease), serum PTH (28% increase) and CT levels (40% decrease) were observed in stressed rats. EDTA administration brought about a significantly greater hypocalcemia, and a higher PTH secretory response in turpentine oil-stressed rats. During stress, the increase of serum calcium after CaCl2 was significantly smaller, and the rise of CT was greater than in controls. In the case of CT the changes were still observed in rats subjected to superior cervical ganglionectomy (SCGx) 14 days earlier. In the case of PTH, the increase found in stressed rats, but not the augmented response after EDTA, was blunted by SCGx. The potentiation of hypocalcemia brought about by turpentine oil was no longer observed in SCGx rats. In vehicle-treated controls, SCGx delayed PTH response to hypocalcemia, but did not affect the increased response of CT to CaCl2 challenge. The results indicate that a number of changes in calcium homeostasis arise during turpentine oil stress in rats. SCGx was effective to modify the set point for PTH release, but played a minor role in affecting the augmentation of CT release during stress.
Asunto(s)
Calcio/sangre , Homeostasis , Estrés Fisiológico/sangre , Sistema Nervioso Simpático/fisiopatología , Trementina , Animales , Calcitonina/sangre , Cloruro de Calcio/farmacología , Ácido Edético/farmacología , Femenino , Ganglios Simpáticos/fisiopatología , Ganglionectomía , Cinética , Hormona Paratiroidea/sangre , Ratas , Estrés Fisiológico/inducido químicamenteRESUMEN
The effect of lipoxygenase inhibition, leukotriene agonists and antagonists, and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) was examined in the rat pineal gland in organ culture. To study melatonin secretion pineal explants were incubated for 6 h in tissue culture medium 199 with the different drugs. Melatonin concentration in the pineal gland and the medium was measured by RIA. Exposure of explants to norepinephrine (NE) brought about a 2- to 5-fold increase in both parameters, an effect that was reduced but not abolished, by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 10(-5) M). Lilly 171883 (10(-5) M) or FPL 55712 (10(-5) M; both antagonists of leukotrienes) reduced NE-induced melatonin production. Neither NDGA nor Lilly 171883 affected melatonin production in the absence of NE. Leukotrienes C4 and D4 increased melatonin release to the media at all concentrations tested (1-1,000 nM) with a maximum effect at 1 nM (leukotriene C4) and 10 nM (leukotriene D4). Significantly higher tissue melatonin concentrations as compared to controls were observed after exposure of pineal explants to 1 and 100 nM of leukotriene C4, or 100 nM of leukotriene D4. Another 5-lipoxygenase metabolite, 5-HETE, increased pineal melatonin content at concentrations of 1, 10 and 100 nM whereas only 1,000 nM stimulated melatonin release. These results suggest that the 5-lipoxygenase pathway plays a significant role in NE-stimulated melatonin production by the rat pineal gland.
Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Araquidonato Lipooxigenasas/metabolismo , Melatonina/biosíntesis , Norepinefrina/farmacología , Glándula Pineal/metabolismo , Acetofenonas/farmacología , Animales , Cromonas/farmacología , Ácidos Hidroxieicosatetraenoicos/farmacología , Inhibidores de la Lipooxigenasa , Masculino , Masoprocol/farmacología , Técnicas de Cultivo de Órganos , Glándula Pineal/efectos de los fármacos , Glándula Pineal/enzimología , Ratas , Ratas Endogámicas , SRS-A/antagonistas & inhibidores , SRS-A/farmacología , Tetrazoles/farmacologíaRESUMEN
The time course for the decrease in norepinephrine concentration of rat pineal explants in culture indicated a significant fall starting at the 4th hour and completed after 16-24 h of incubation. Significant decreases of serotonin and 5-hydroxyindoleacetic acid (HIAA) levels in tissue, an increase of HIAA/serotonin ratio, and an increase of melatonin production rate in vitro were also observed as a function of the incubation time. Estradiol (10(-7)-10(-5) M) increased rat pineal melatonin content, testosterone (10(-5) M) decreased it and progesterone was devoid of activity when incubated with explants for up to 6 h. The in vitro stimulatory effect of estradiol on rat pineal methoxyindole synthesis was blocked by propranolol but not by phentolamine; propranolol also blocked the increase of nuclear estradiol-receptor complex produced by estrogen exposure of pineal explants. TSH (1-100 ng/ml), growth hormone (10-100 ng/ml) and LH (10 ng/ml) augmented rat pineal melatonin content while 100 ng/ml of FSH decreased it significantly. Prolactin exerted a biphasic effect on rat pineal explants, the lowest concentration augmenting melatonin content while the high concentration depressed it. Deep, intermediate and superficial segments of guinea-pig pineal glands showed an increase in melatonin concentration after a 6-h incubation in the presence of 10(-7)-10(-5) M estradiol.
Asunto(s)
Hormonas Esteroides Gonadales/farmacología , Melatonina/biosíntesis , Glándula Pineal/efectos de los fármacos , Hormonas Adenohipofisarias/farmacología , Animales , Cobayas , Ácido Hidroxiindolacético/metabolismo , Masculino , Norepinefrina/metabolismo , Técnicas de Cultivo de Órganos , Fentolamina/farmacología , Glándula Pineal/metabolismo , Propranolol/farmacología , Ratas , Receptores de Estradiol/metabolismo , Serotonina/metabolismoRESUMEN
In 3 000g-supernatants of rat pineal homogenates a single population of benzodiazepine (BZP) binding sites with dissociation constant= 97-102 nM and maximal number of sites= 6.5-9 pmoles/mg protein was detected by employing 3H-flunitrazepam (FNZP) as a radioligand. The following order of affinity for several BZP was found (Ki, nM): Ro 5-4864 (8), FNZP (99), clonazepam (7,900) Ro 15-1788 (10,000). Two weeks after bilateral superior cervical ganglionectomy (SCGx) a 18-28% reduction of site number without significant changes in affinity of 3H-FNZP binding was detectable in rat pineal glands. In pineal explants priorly incubated with 3H-norepinephrine, exposure to 0.1-10 microM of Ro 5-4864 or diazepam decreased significantly transmitter release elicited by 80 mM K+, whereas clonazepam did not affect it significantly. At 10 microM-concentrations, Ro 5-4864, diazepam or clonazepam increased pineal melatonin content of explants incubated for 6 h with the drug. In pineal explants of rats subjected to SCGx 14 days earlier, only 10 microM of Ro 5-4864 increased melatonin content significantly to about half of the percent increase detected in innervated glands. These results suggest that BZP decrease transmitter release from pineal sympathetic nerves by acting on peripheral BZP binding sites, an effect which is about 2 orders of magnitude greater than the postsynaptic stimulation of pineal melatonin synthesis.
Asunto(s)
Benzodiazepinas/farmacología , Melatonina/metabolismo , Norepinefrina/metabolismo , Glándula Pineal/metabolismo , Animales , Sitios de Unión , Técnicas In Vitro , Masculino , Ratas , Ratas EndogámicasRESUMEN
In 3 000g-supernatants of rat pineal homogenates a single population of benzodiazepine (BZP) binding sites with dissociation constant= 97-102 nM and maximal number of sites= 6.5-9 pmoles/mg protein was detected by employing 3H-flunitrazepam (FNZP) as a radioligand. The following order of affinity for several BZP was found (Ki, nM): Ro 5-4864 (8), FNZP (99), clonazepam (7,900) Ro 15-1788 (10,000). Two weeks after bilateral superior cervical ganglionectomy (SCGx) a 18-28
reduction of site number without significant changes in affinity of 3H-FNZP binding was detectable in rat pineal glands. In pineal explants priorly incubated with 3H-norepinephrine, exposure to 0.1-10 microM of Ro 5-4864 or diazepam decreased significantly transmitter release elicited by 80 mM K+, whereas clonazepam did not affect it significantly. At 10 microM-concentrations, Ro 5-4864, diazepam or clonazepam increased pineal melatonin content of explants incubated for 6 h with the drug. In pineal explants of rats subjected to SCGx 14 days earlier, only 10 microM of Ro 5-4864 increased melatonin content significantly to about half of the percent increase detected in innervated glands. These results suggest that BZP decrease transmitter release from pineal sympathetic nerves by acting on peripheral BZP binding sites, an effect which is about 2 orders of magnitude greater than the postsynaptic stimulation of pineal melatonin synthesis.