Carlos F. Reyes-Toso

Vascular reactivity in diabetic rats: effect of melatonin.

The aim of the present study was to evaluate the in vitro contractile response of rat aorta in mild and severe type I diabetes and the effect of melatonin on it. Aortic rings were obtained from male Wistar rats injected with streptozotocin 8–12 wks earlier. Rats were divided into three groups: non‐diabetic rats (NDR), mildly diabetic rats (MDR) and severely diabetic rats (SDR). Dose–response curves for acetylcholine‐induced, endothelium‐related relaxation of aortic rings (after previous exposure to phenylephrine) and for serotonin‐induced vasoconstriction were conducted in the presence or absence of 10–5 mol/L melatonin. This protocol was repeated with rings preincubated in a high glucose solution (44 mmol/L). The contractile response to phenylephrine decreased in SDR, an effect counteracted by preincubation with high glucose. Melatonin decreased phenylephrine‐induced vasoconstriction in MDR and counteracted the effect of high glucose in SDR. Acetylcholine‐evoked relaxation decreased significantly after exposure to a high glucose in SDR, this effect being counteracted by melatonin. Serotonin‐induced vasoconstriction decreased in SDR and augmented in MDR, but only after exposure to high glucose. Melatonin reduced the maximal tension of aortic contraction after serotonin in MDR, both under basal conditions and after preincubation in a high glucose solution. The results support the existence of differences in vasomotor responses as a function of the diabetes state and of an improvement of contractile performance in diabetic rats after exposure to melatonin at a pharmacological concentration (in terms of circulating melatonin levels but not necessarily for some other fluids or tissues).

Effect of melatonin treatment on oxygen consumption by rat liver mitochondria

Summary.The objective of this study was to examine the in vivo effect of melatonin on rat mitochondrial liver respiration. Two experiments were performed: For experiment 1, adult male rats received melatonin in the drinking water (16 or 50 µg/ml) or vehicle during 45 days. For experiment 2, rats received melatonin in the drinking water (50 µg/ml) for 45 days, or the same amount for 30 days followed by a 15 day-withdrawal period. At sacrifice, a liver mitochondrial fraction was prepared and oxygen consumption was measured polarographically in the presence of excess concentration of DL-3-β-hydroxybutyrate or L-succinate. Melatonin treatment decreased Krebs’ cycle substrate-induced respiration significantly at both examined doses. The stimulation of mitochondrial respiration caused by excess concentration of substrate recovered after melatonin withdrawal. Basal state 4 respiration was not modified by melatonin. Melatonin, by curtailing overstimulation of cellular respiration caused by excess Krebs’ cycle substrates, can protect the mitochondria from oxidative damage.

Melatonin restores endothelium-dependent relaxation in aortic rings of pancreatectomized rats.

Abstract:  In rats turned hyperglycemic by a subtotal pancreatectomy, a decreased relaxation response of aortic rings to acetylcholine (ACh) was found; this effect was amplified by preincubation in a high glucose medium (44 mmol/L). The relaxation response to ACh did not occur in endothelium‐denuded rings or after the aortic rings were exposed to l‐nitro‐arginine methyl ester [l‐NAME, a nitric oxide (NO) synthase inhibitor]. Incubation with the NO donor sodium nitroprusside (SNP) restored the impaired relaxation response seen in endothelium‐denuded or l‐NAME‐treated aortic rings. Pancreatectomy decreased the vasorelaxation of aortic rings caused by SNP. Only in pancreatectomized rats, incubation in a high glucose medium impaired the relaxation effect of SNP. To assess whether melatonin preincubation reversed the impaired relaxation response to ACh (intact endothelium aortic rings) or to SNP (endothelium‐denuded or l‐NAME‐treated rings) in hyperglycemic rats, cumulative dose–response curves were performed in the presence of 10−5 mol/L melatonin. Melatonin preincubation did not modify ACh‐induced relaxation of aortic rings in a normal glucose concentration but was highly effective in preventing the impairment of relaxation caused by a high glucose solution. Melatonin was also effective in restoring the impaired SNP‐induced vasorelaxation seen in endothelium‐denuded or l‐NAME‐treated aortic rings from hyperglycemic rats. The results further support the improvement by melatonin of the endothelial‐mediated relaxation in blood vessels of diabetic rats.

Effect of melatonin on vascular responses in aortic rings of aging rats

In old animals a marked reduction in endothelium-dependent relaxation occurs. Since there is evidence that the endothelial dysfunction associated with aging may be partly related to the local formation of reactive oxygen species, the purpose of this study was to examine the effect of the natural antioxidant melatonin (10(-5)mol/l) on in vitro contractility of aged aortic rings under conditions of increased oxidative stress (40 m mol/l glucose concentration in medium). Experiments were carried out in 18-20 months old, Wistar male rats, using adult (6-7 months old) animals as controls. A higher plasma lipid peroxidation was found in aged rats as compared to the younger ones. In a first experiment, dose-response curves for acetylcholine-induced relaxation of aortic rings were conducted. Analyzed as a main factor in a factorial ANOVA, age decreased and melatonin augmented the relaxing response to acetylcholine. melatonins restoring effect on aortic ring relaxation was found in aged aortic rings only and was more pronounced in the presence of a high glucose medium. In a second experiment, the effect of melatonin on the contractility response to phenylephrine of intact or endothelium-denuded aortic rings obtained from aged or control rats was examined in normal or high glucose medium. A main factor analysis in the factorial ANOVA indicated that age and operation augmented, and melatonin decreased, aortic ring contractility response to phenylephrine. Melatonins restoring effect on aortic contractility was seen in aged aortic rings. The effect of age or a high glucose medium on phenylephrine-induced contractility was more pronounced in the absence of an intact endothelium. Aging did not affect the relaxant response of intact or endothelium-denuded rings to sodium nitroprusside. The results support the improvement by melatonin of vascular response in aging rats, presumably via its antioxidant activity.

Effect of melatonin on 24h changes in plasma protein levels during the preclinical phase of Freund's adjuvant arthritis in rats

The 24h rhythms in plasma protein concentration were examined in rats on the third day after injection of Freunds complete adjuvant or adjuvants vehicle, performed 3h after light on. In rats treated with adjuvants vehicle, peak values of albumin and gamma globulin occurred during the nocturnal activity span (P <.02 and P <. 0001, respectively), while those of alpha-1, alpha-2, and beta globulins were found late during the rest span (P <. 002, P <. 0001, and P <. 0004, respectively). Freunds adjuvant administration abolished temporal changes in plasma albumin and beta globulin levels. It also decreased the amplitude of daily changes in alpha-1 and alpha-2 globulin (P <. 05) and diminished mean values of alpha-2 globulin (P <. 01). Pretreatment of rats with melatonin (30 μg daily) for 11 days, 11h after light on, counteracted mycobacterial adjuvant-induced suppression of the 24h rhythms in albumin and alpha-1, alpha-2, and beta globulins. The results further support the existence of preventive properties of a pharmacological dose of melatonin in situations in which a lost of circadian rhythmicity is expected. (Chronobiology International, 18(3), 435–446, 2001)

Effect of ethanol on 24-h hormonal changes in prolactin release mechanisms in growing male rats

This study analyzes the effect of chronic ethanol feeding on 24-h variation of hypothalamic-pituitary mechanisms involved in prolactin regulation in growing male Wistar rats. Animals were maintained under a 12:12 h light/dark photoperiod (lights off at 2000 h), and they received a liquid diet for 4 wk, starting on d 35 of life. The ethanol-fed group received a similar diet to controls except that maltose was isocalorically replaced by ethanol. Ethanol replacement provided 36% of the total caloric content of the diet. Rats were killed at six time intervals every 4 h, beginning at 0900 h. Mean concentration of serum prolactin in ethanol-fed rats was 58.7% higher than in controls. Peak circulating prolactin levels occurred at the early phase of the activity span in both groups of rats, whereas a second peak was found late in the resting phase in ethanol-fed rats only. In control rats, median eminence dopamine (DA), serotonin (5-HT), γ-aminobutyric acid (GABA), and taurine levels exhibited two maxima, the major one preceding prolactin release and a second one during the first part of the resting phase. Median eminence DA and 5-HT turnover (as measured by 3,4-dihydroxyphenylacetic acid, DOPAC/DA, and 5-hydroxyindoleacetic acid, 5-HIAA/5-HT ratio) showed a single maximum preceding prolactin, at 0100 h. Ethanol treatment did not affect median eminence DA or 5-HT levels but it decreased significantly their turnover rate. The midday peak in DA and 5-HT levels (at 1300 h) was abolished and the night peak (at 0100 h) became spread and blunted in the ethanol-fed rats. This was accompanied with the disappearance of the 0100 h peak in DA and 5-HT turnover and the occurrence of a peak in 5-HT turnover at 1700 h. Ethanol intake suppressed the night peak in median eminence GABA and taurine (at 0100 h) as well as the midday peak of GABA. Ethanol augmented pituitary levels of DOPAC and 5-HIAA. The results indicate that chronic ethanol administration affects the mechanisms that modulate the circadian variation of prolactin release in growing male rats.

Comparative effects of melatonin and vitamin E in restoring aortic relaxation in pancreatectomized rats

In a previous study we reported the efficacy of melatonin to restore the decreased relaxation response to acetylcholine (ACh) or to sodium nitroprusside (SNP) in aortic rings of rats turned hyperglycemic by subtotal pancreatectomy. The effect was amplified by pre-incubation in a high (44 mmol/l) glucose solution, a situation that resulted in oxidative stress. We hereby compare the effect of another antioxidant, vitamin E, with that of melatonin on ACh response in intact aortic rings or on SNP response in endothelium-denuded aortic rings obtained from pancreatectomized or sham-operated rats. Dose-response curves to ACh or SNP were performed in the presence or absence of melatonin or vitamin E (10-5 mol/l) in 10 or 44 mmol/l glucose medium. Melatonin was more effective than vitamin E in restoring Ach- or SNP-induced relaxation of aortic rings in a high glucose medium. The differences between the two antioxidants may rely on the ability of melatonin to diffuse readily into intracellular compartments.ResumenEn un estudio previo se describe la eficacia de la melatonina para restablecer la respuesta disminuida a la acetilcolina (ACh) o al nitroprusiato de sodio (SNP) de anillos aórticos de rata con pancreatectomía subtotal. Este efecto fue mayor en el grupo de anillos preincubados en solución de Krebs con elevada concentración de glucosa (44 mmol/l), lo que favorece la producción de estrés oxidativo. En el presente trabajo se comparan los efectos de la vitamina E y la melatonina sobre la respuesta a la ACh y al SNP de anillos aórticos con endotelio intacto o denudado, obtenidos a partir de ratas con pancreatectomía subtotal o con operación simulada (controles). Se realizaron curvas dosis-respuesta a la ACh o al SNP en medios de incubación con glucosa normal o alta con melatonina o vitamina E (10−5 mol/l). La melatonina fue más efectiva que la vitamina E para restablecer la relajación provocada por ACh o SNP en anillos aórticos expuestos a un medio con elevada concentración de glucosa. La diferencia entre el efecto de ambas sustancias antioxidantes podría deberse a la capacidad de la melatonina para difundir hacia el compartimiento intracelular.