Pedro Abreu

Evidence of melatonin synthesis by human lymphocytes and its physiological significance: possible role as intracrine, autocrine, and/or paracrine substance

It has been historically assumed that the pineal gland is the major source of melatonin (N‐acetyl‐ 5‐methoxytryptamine) in vertebrates. Melatonin plays a central role in fine‐tuning circadian rhythms in vertebrate physiology. In addition, melatonin shows a remarkable functional versatility exhibiting antioxidant, oncostatic, antiaging, and immunomodulatory properties. Melatonin has been identified in a wide range of organisms from bacteria to human beings. Its biosynthesis from tryptophan involves four well‐defined intracellular steps catalyzed by tryptophan hydroxylase, aromatic amino acid decarboxylase, serotonin‐N‐acetyltransferase, and hydroxyndole‐O‐methyltransferase. Here, for the first time, we document that both resting and phytohemagglutinin‐stimulated human lymphocytes synthesize and release large amounts of melatonin, with the melatonin concentration in the medium increasing up to five times the nocturnal physiological levels in human serum. Moreover, we show that the necessary machinery to synthesize melatonin is present in human lymphocytes. Furthermore, melatonin released to the culture medium is synthesized in the cells, because blocking the enzymes required for its biosynthesis or inhibiting protein synthesis in general produced a significant reduction in melatonin release. Moreover, this inhibition caused a decrease in IL‐2 production, which was restored by adding exogenous melatonin. These findings indicate that in addition to pineal gland, human lymphoid cells are an important physiological source of melatonin and that this melatonin could be involved in the regulation of the human immune system, possibly by acting as an intracrine, autocrine, and/or paracrine substance

Increased or decreased locomotor response in rats following repeated administration of apomorphine depends on dosage interval

Administration of drugs that reduce the influence of dopamine at its receptor site can lead to postsynaptic supersensitivity, whereas treatment with dopamine (DA) agonists can cause postsynaptic subsensitivity. Both unaltered and enhanced postsynaptic responses to DA have been shown after pretreatment with DA agonists. In the present manuscript pretreatment with apomorphine, a dopaminergic agonist, is shown to induce either increased or reduced locomotor activity. When a drug-free period between successive injections was allowed, apomorphine induced an enhanced locomotor response, whereas a reduced response occurred when each dose was injected before the previous apomorphine dose had been completely metabolized. Pretreatment with both high (1 and 3 mg/kg) and low (0.05 mg/kg) apomorphine doses enhanced the response. Apomorphine treatment that caused enhanced locomotor responses did not modify the stereotypy response to the drug. Similar enhanced or reduced response were found in rats with partial lesions of the nigrostriatal system. These altered responses to DA agonists may have important clinical consequences. The present data also suggest the existence of a different DA systems for locomotor and stereotypy actions of dopaminergic agonists.

Changes in Mating Behavior, Erectile Function, and Nitric Oxide Levels in Penile Corpora Cavernosa in Streptozotocin-Diabetic Rats

Abstract This study assessed whether the in vivo production of nitric oxide (NO) in the penis is impaired in experimental diabetes and whether this phenomenon can be explained by abnormal levels of NO synthase isoenzymes and/or plasma androgens. Adult male Sprague-Dawley rats were injected with streptozotocin (STZ) (40 mg/kg, i.p.) or vehicle. One half of the STZ-treated animals received daily insulin replacement. Twelve weeks later, the animals were tested for mating behavior and erectile reflexes. They were then anesthetized with urethane (1 g/kg), and the NO levels in their corpora cavernosa were monitored electrochemically with porphyrin microsensors before and after electrostimulation of the cavernous nerve. The intracavernous pressure (ICP) was measured simultaneously. The diabetic animals had substantial impairment in the mating and erectile reflexes tests, decreased basal and stimulated NO levels in the corpora, and a reduced ICP response to cavernous nerve stimulation. Insulin replacement fully reversed the effects of diabetes on the mating reflexes, the basal NO signals, and the ICP responses to electrical field stimulation and partially restored the stimulated NO release. Neither diabetes nor diabetes with insulin treatment had significant effects on serum testosterone levels or NOS isoform (nNOS, eNOS, and iNOS) protein content in penile homogenates, indicating that the changes found in erectile function were independent of such variables. These results also suggest that the diabetes-induced reduction in corporeal NO levels could be mainly due to the lack of some essential cofactors for NOS activity rather than to changes in the amount of enzyme proteins.

Gonadal steroid modulation of neuroendocrine transduction: A transynaptic view

Summary1. Steroid hormones act on neuronal communication through different mechanisms, ranging from transynaptic modulation of neurotransmitter synthesis and release to development and remodeling of synaptic circuitry. Due the wide distribution of putative brain targets for steroid hormones, acute or sustained elevations of their circulating levels may affect, simultaneously, a variety of neuronal elements. In an elementary mode of interaction, steroids are able to modulate both the synthesis and release of a neurotransmitter at a particular synapse, and the response of its target postsynaptic cells. Using two neuroendocrine transducing systems—the rat pineal gland and the GT1–7 cell line—we have examined these interactions and the following findings are discussed in this article.2. In the rat, pineal melatonin production is partially controlled by gonadal hormones. In females, melatonin synthesis and secretion is reduced during the night of proestrus, apparently as a consequence of elevated estradiol and progesterone levels. In males, circulating testosterone seems to be necessary to maintain the amplitude of the nocturnal melatonin peak.3. Some gonadal effects on pineal activity are exerted on its noradrenergic input, since changes in circulating steroid hormone levels are able to induce acute modifications of tyrosine hydroxylase activity in pineal sympathetic nerve terminals.4. Gonadal steroids are also able to regulate the response of pineal cells to adrenergic stimulation, since in vivo treatment of both male and female rats with steroid hormone blockers induces profound modifications in adrenergically-induced accumulation of cyclic AMP (cAMP) in dispersed pinealocytes.5. Direct exposure of pineal cells from gonadectomized female and male rats to estradiol (E2) or testosterone (T), respectively, potentiates pinealocyte response to adrenergic activation. In addition, short-term (15 min) exposure to either progesterone (Pg) or progesterone coupled to bovine serum albumin (P-3-BSA) suppresses the E2-dependent potentiation of adrenergic response in female rat pinealocytes.6. Exposure of GT1–7 cells to E2 completely blocked the norepinephrine (NE)-induced elevation of cAMP content. In E2-treated GT1–7 cells, additional exposure (15 min) to either Pg or P-3-BSA abolished E2-dependent inhibition of NE responsiveness. In addition, P-3-BSA alone increased basal cAMP levels in GT1–7 cells, regardless previous exposure to E2.7. In conclusion, there are evidences, both from the current literature and from the present results, supporting the view that in some neuroendocrine systems gonadal hormones modulate neurotransmission by acting, simultaneously, at pre- and postsynaptic sites. The models presented here constitute appropriate examples of this transynaptic mode of steroid action and, therefore, may offer a useful approach to investigate steroid hormone actions on the brain.

Dopamine transporter glycosylation correlates with the vulnerability of midbrain dopaminergic cells in Parkinson's disease

The dopamine transporter (DAT) is a membrane glycoprotein responsible for dopamine (DA) uptake, which has been involved in the degeneration of DA cells in Parkinsons disease (PD). Given that DAT activity depends on its glycosylation status and membrane expression, and that not all midbrain DA cells show the same susceptibility to degeneration in PD, we have investigated a possible relationship between DAT glycosylation and function and the differential vulnerability of DA cells. Glycosylated DAT expression, DA uptake, and DAT V(max) were significantly higher in terminals of nigrostriatal neurons than in those of mesolimbic neurons. No differences were found in non-glycosylated DAT expression and DAT K(m), and DA uptake differences disappeared after deglycosylation of nigrostriatal synaptosomes. The expression pattern of glycosylated DAT in the human midbrain and striatum showed a close anatomical relationship with DA degeneration in parkinsonian patients. This relationship was confirmed in rodent and monkey models of PD, and in HEK cells expressing the wild-type and a partially deglycosylated DAT form. These results strongly suggest that DAT glycosylation is involved in the differential vulnerability of midbrain DA cells in PD.

Magnesium sulphate injected subcutaneously suppresses autotomy in peripherally deafferented rats

&NA; In rats, recent evidence suggests that injury discharge caused by peripheral nerve section releases excitatory amino acids into the spinal cord which in turn influences decisively the development of autotomy, a self‐mutilation behaviour directed towards the denervated areas. Autotomy has been proposed as a behavioural correlate of the neuropathic pain which occurs in humans after complete nerve lesions. Mg2+ ions have been shown to offer protection from neurological and degenerative disorders in which excitatory amino acids are putatively involved. To ascertain the preventive value of Mg2+ administration on autotomy, male rats underwent unilateral ligation and transection of the sciatic and saphenous nerves 30 min after being injected subcutaneously (s.c.) with 300 or 600 mg/kg MgSO4 or saline. Thereafter, autotomy was monitored for 8 weeks. Serum, lumbosacral (L1–S1) and brain magnesium levels were analyzed 0, 30, 60, 120, 180, 240, 360 min and 24 h after the s.c. injection of 600 mg/kg MgSO4. Serum magnesium levels increased quickly from 1.02 mM (0 time) to 4.52 mM (at 60 min) and dropped afterwards to reach physiological levels at 6 h. Peak increments in L1–S1 and brain Mg2+ levels were smaller (32% and 30%, respectively) although maintained for at least 6 h. Magnesium pretreatment in a significant and dose‐dependent manner (1) largely suppressed autotomy, (2) decreased final autotomy scores, (3) delayed autotomy onset, and (4) decreased the percentage of animals engaged in high autotomy behaviors. The data support a role for excitatory amino acids in determining susceptibility to autotomy and suggest a hopeful way to prevent neuropathic pain in humans after peripheral deafferentation.

Aging effects on the dopamine transporter expression and compensatory mechanisms

Several studies report that the striatal dopamine (DA) uptake declines with age, but the underlying mechanisms are still unclear. The use of molecular, biochemical and morphological techniques, and antibodies which detect the glycosylated (80 kDa) and non-glycosylated (50 kDa) DA transporter (DAT) forms in the rat mesostriatal system, reveals that DAT is pre- and post-translationally damaged during aging. In middle age (18 months), the glycosylated DAT form decreases in the plasma membrane of striatal terminals, and the non-glycosylated form is accumulated in the endoplasmic reticulum-Golgi complex. Thereafter, in aged rats (24 months), DAT synthesis is also affected as the decrease in both DATmRNA and total DAT protein levels suggests. However, the evidence of a decrease in both DAT expression in the endosomal (vesicle-enriched) compartment and the phosphorylated DAT fraction from middle age, as well as its compartmental redistribution towards the terminal plasma membrane, with an increase in the membrane DAT/total DAT ratio in striatal synapotosomes, in aged rats, indicate that DA-cells activate compensatory mechanisms directed at maintaining DAT function during normal aging.

Aging of the rat mesostriatal system: Differences between the nigrostriatal and the mesolimbic compartments

The impairment of the mesostriatal dopaminergic system has been considered responsible for motor and affective disturbances associated with aging and a risk factor for Parkinsons disease. However, the basic mechanisms underlying this phenomenon are still unknown. Here we used biochemical, molecular and morphological techniques directed at detecting flaws in the dopamine synthesis route and signs of dopaminergic degeneration in the rat mesostriatal system during normal aging. We found two different age-related processes. One is characterized by a dopa decarboxylase decrease, and involves both the nigrostriatal and mesolimbic compartments, and is responsible for a moderate dopamine loss in the dorsal striatum, where other parameters of dopamine synthesis are not affected. The other is characterized by axonal degeneration with aggregation of phosphorylated forms of tyrosine hydroxylase (TH) and amyloid precursor protein in degenerate terminals, and alpha-synuclein in their original somata. This process is restricted to mesolimbic regions and is responsible for the decline of TH activity and l-dopa levels and the greater decrease in dopamine levels in this compartment. These findings suggest that both the nigrostriatal and the mesolimbic systems are vulnerable to aging, but in contrast to what occurs in Parkinsons disease, the mesolimbic system is more vulnerable to aging than the nigrostriatal one.

Interleukin-6 and Nitric Oxide Synthase Expression in the Vasopressin and Corticotrophin-releasing Factor Systems of the Rat Hypothalamus

Nitric oxide synthase (NOS) and interleukin-6 (IL-6) are constitutively expressed in hypothalamic cells. However, phenotypic and functional aspects of these cells remain unknown. We have studied the expression pattern of these two molecules in hypothalamic cells expressing corticotropin-releasing factor (CRF) and arginin-vasopressin (AVP), two major regulatory peptides in the hypothalamus-pituitary system, using immunofluorescence, intracerebroventricular injection of colchicine, and the study in parallel of the labeling pattern of axons in the median eminence. Within AVP cells, we distinguished two different populations: large, intensely stained AVP cells coexpressing IL-6; and large, intensely stained AVP cells coexpressing IL-6 and NOS. Within the CRF cells, we distinguished three different populations: large, intensely stained CRF cells immunonegative for AVP, NOS, and IL-6; large cells weakly stained for CRF and AVP, immunopositive for NOS and immunonegative for IL-6; and small cells intensely stained for CRF and AVP and immunonegative for IL-6 and NOS. In addition, we also found AVP cells containing IL-6 in the suprachiasmatic nucleus. These results suggest that neuronal NOS and IL-6 may be involved in different modulatory processes in hypophysiotropic and non-hypophysiotropic cells.

Ovarian steroids block the isoproterenol-induced elevation of pineal melatonin production in the female rat

Changes in pineal indole metabolism during the estrous cycle, as well as in response to estrogen administration, were studied in female rats. Tyrosine hydroxylase (TH) activity, norepinephrine (NE) and indoleamine levels were determined by high-performance liquid chromatography (HPLC). Pineal melatonin was measured by radioimmunoassay (RIA). Both 5-hydroxytryptamine (5-HT) and melatonin levels, but not TH activity or NE content, were reduced during proestrus. Ovarian hormones blocked the isoproterenol-induced elevation of pineal melatonin, and reduced 5-HT levels in ovariectomized rats. These results suggest that an estrogen-induced inhibition of the pineal response to adrenergic stimulation, could be responsible for the reduction in pineal melatonin production occurring during the proestrus stage of the rat estrous cycle.