Milagros Méndez

Role of mu and delta opioid receptors in alcohol drinking behaviour

The dopaminergic mesolimbic system plays a key role in the mechanisms of reinforcement elicited by alcohol (ethanol) and other drugs of abuse. Numerous lines of evidence indicate that ethanol reinforcement mechanisms involve, at least partially, the ethanol-induced activation of the endogenous opioid system. Ethanol may alter opioidergic transmission at different levels, including the biosynthesis, release, and degradation of opioid peptides, as well as binding of endogenous ligands to opioid receptors. Several studies suggest that mu and delta opioid receptors play a major role in ethanol reinforcement and dependence. These studies implicate enkephalins and beta-endorphin as physiological mediators of ethanols actions in the brain. In this review we describe the pharmacological characteristics of opioid receptors and their distribution in brain, as well as the major functions of their endogenous ligands. Thereafter, we present evidence supporting the participation of mu and delta opioid receptors in ethanol reinforcement mechanisms and high alcohol drinking behaviour. The use of opioid receptor agonists and antagonists, as well as ethanol-preferring selected rodents and knockout mice, has contributed to understand the role of mu and delta receptors in these processes. The effects of ethanol on binding of selective ligands to opioid receptors in different experimental models are also reviewed. The relevance of opioid receptors in human alcoholism is further evidenced by the association of mu receptor polymorphisms with ethanol dependence. The clinical implication of these findings is discussed regarding the differential responses observed in some alcoholic patients to treatment with opioid receptor antagonists such as naltrexone.

Regional distribution of the membrane-bound pyroglutamate amino peptidase-degrading thyrotropin-releasing hormone in rat brain☆

The brain regional distribution of membrane-bound pyroglutamate aminopeptidase-degrading thyrotropin-releasing hormone (TRH) in rat was studied using a specific radiometric assay. The distribution was not homogeneous: a 10-fold difference was observed between regions. The highest activity was detected in olfactory bulb while the lowest was in the cervical part of spinal cord. There was no correlation with the regional distribution of enzyme activity vs TRH levels, previously reported TRH receptors or in vitro TRH release. The differential distribution of this enzyme is consistent with the hypothesis that it is responsible for extracellular degradation of neuroactive peptides.

Acute ethanol administration differentially modulates μ opioid receptors in the rat meso-accumbens and mesocortical pathways

Biochemical and pharmacological evidence suggest that the dopaminergic mesolimbic system plays a key role in mediating the reinforcing properties of alcohol and other drugs of abuse. Alcohol reinforcement and high alcohol drinking behavior have been postulated to be partially mediated by a neurobiological mechanism involving the alcohol-induced activation of the endogenous opioid system. The aim of this work was to study the effect of the in vivo acute administration of ethanol on mu (mu) opioid receptors in the rat dopaminergic meso-accumbens and mesocortical pathways by quantitative receptor autoradiography. [(3)H]DAMGO binding was significantly decreased in the ventral tegmental area (VTA) 30 min after ethanol administration. A small ethanol-induced reduction was observed in the shell region of the nucleus accumbens 1 h after exposure. In contrast, 2 h after ethanol administration, [(3)H]DAMGO binding was significantly increased in the frontal and prefrontal cortices. The observed changes correlated well with high ethanol plasma levels. Our results suggest that the reinforcing properties of ethanol may be partially mediated by mechanisms involving the ethanol-induced down- and up-regulation of mu receptors in the dopaminergic mesolimbic system. Mu receptors in the VTA and the frontal and prefrontal cortices may be involved in the in vivo acute responses to ethanol and could play a key role in modulating the dopaminergic activity of the mesocortical pathway in response to the drug. In contrast, the contribution of both mu and delta receptors in the nucleus accumbens might be relevant in these processes.

Pyroglutamyl peptidase II inhibition specifically increases recovery of TRH released from rat brain slices

Pyroglutamyl peptidase II (EC 3.4.19-) is a highly specific membrane-bound thyrotropin releasing hormone (TRH) degrading enzyme. To study the functional significance of pyroglutamyl peptidase II in TRH degradation, we synthesized the reversible inhibitor N-1-carboxy-2-phenylethyl (Nimbenzyl)-histidyl-beta-naphthylamide (CPHNA). CPHNA inhibited the enzyme with a Ki of 8 microM, but had no effect no TRH receptors or no prolyl endopeptidase (EC 3.4.21.26). It weakly inhibited cytosolic pyroglutamyl peptidase I (EC 3.4.19.3). CPHNA at a concentration of 10(-4) M increased both the basal and potassium stimulated recovery of TRH released from hypothalamic slices by approximately two-fold. An even higher recovery was observed in slices from brain regions with relatively high levels of pyroglutamyl peptidase II. CPHNA had no effect on the basal recovery of gamma-aminobutyric acid or Met-enkephalin released from brain slices but decreased the potassium stimulated recovery of both Metenkephalin and gamma-aminobutyric acid. These data further support the involvement of pyroglutamyl peptidase II in the extracellular inactivation of brain TRH.

Acute ethanol administration induces changes in TRH and proenkephalin expression in hypothalamic and limbic regions of rat brain

Thyrotropin releasing hormone (TRH) present in several brain areas has been proposed as a neuromodulator. Its administration produces opposite effects to those observed with acute ethanol consumption. Opioid peptides, in contrast, have been proposed to mediate some of the effects of alcohol intoxication. We measured TRH content and the levels of its mRNA in hypothalamic and limbic zones 1-24 h after acute ethanol injection. We report here fast and transient changes in the content of TRH and its mRNA in these areas. The levels of proenkephalin mRNA varied differently from those of proTRH mRNA, depending on the time and region studied. Wistar rats were administered one dose of ethanol (intraperitoneal, 3 g/kg body weight) and brains dissected in hypothalamus, hippocampus, amygdala, n. accumbens and frontal cortex, for TRH quantification by radioimmunoassay or for proTRH mRNA measurement by RT-PCR. After 1 h injection, TRH levels were increased in hippocampus and decreased in n. accumbens; after 4 h, it decreased in the hypothalamus, frontal cortex and amygdala, recovering to control values in all regions at 24 h. ProTRH mRNA levels increased at 1 h post-injection in total hypothalamus and hippocampus, while they decreased in the frontal cortex. The effect of ethanol was also studied in primary culture of hypothalamic cells; a fast and transient increase in proTRH mRNA was observed at 1 h of incubation (0.001% final ethanol concentration). Changes in the mRNA levels of proTRH and proenkephalin were quantified by in situ hybridization in rats administered ethanol intragastrically (2.5 g/kg). Opposite alterations were observed for these two mRNAs in hippocampus and frontal cortex, while in n. accumbens and the paraventricular nucleus of the hypothalamus, both mRNA levels were increased but with different kinetics. These results give support for TRH and enkephalin neurons as targets of ethanol and, as possible mediators of some of its observed behavioral effects.

Comparison between Low Frequency Magnetic Field Stimulation and Nerve Growth Factor Treatment of Cultured Chromaffin Cells, on Neurite Growth, Noradrenaline Release, Excitable Properties, and Grafting in Nigrostriatal Lesioned Rats

Adrenal chromaffin cells in vitro respond to nerve growth factor (NGF) by expressing neuronal traits. Low frequency magnetic (LFM) field stimulation, while inducing a variety of effects on several cell types, has never been studies as to its effects on chromaffin cell cultures. The purpose of this study was to compare the effects of LFM field stimulation with that of NGF on the morphological phenotype, on noradrenaline (NA) release, and on membrane excitability of cultured chromaffin cells. We also tested the effects of grafting LFM and NGF-treated chromaffin cells into the caudate nucleus of rats with 6-hydroxydopamine lesions of the nigrostriatal pathway. The results of this study showed that LFM field stimulation produced neurite growth of cultured chromaffin cells in a manner similar to that of NGF exposure. The combination of the two procedures did not induce changes above those observed by NGF alone. Both NGF- and LFM-treated chromaffin cells released [3H]NA equally in response to a depolarizing concentration of KCl. On the other, Na+ current density of LFM field stimulation increased, but to a lesser extent than that seen in NGF-treated cells. In addition both types of cells when transplanted into nigrostriatal-lesioned animals induced a similar decrease in the motor asymmetries produced by the lesion. When NGF- or LFM-treated chromaffin cells where compared to untreated control cells, no significant differences were observed in [3H]NA release, on Na+ current densities, or on postgraft motor asymmetries. The results are discussed in terms of the fact that LFM-stimulated cells can be differentiated in a manner similar to NGF-treated cells, by acquiring sympathetic like traits which in turn can diminish motor asymmetries when grafted into nigrostriatal-lesioned rats.

Regional distribution of in vitro release of thyrotropin releasing hormone in rat brain

To increase our knowledge of the TRH functions in brain and the processes of TRH compartmentalization and release, we studied the in vitro release of endogenous TRH in different brain areas. We also determined the correlation between TRH levels and release under both basal and stimulated conditions. TRH concentration was measured in tissues and media by specific radioimmunoassay. TRH-like material detected in olfactory bulb and hypothalamic incubates (basal or K+ stimulated) were shown to be chromatographically identical to synthetic TRH. Different brain regions showed high variability in the basal release of TRH (1-20% of tissue content). This suggests the existence of different pools. The response to depolarizing stimulus (56 mM K+) was significant only in the following regions: median eminence, total hypothalamus, preoptic area, nucleus accumbens-lateral septum, amygdala, mesencephalon, medulla oblongata and the cervical region of the spinal cord. These regions have been shown to contain a high number of receptors, a high concentration of TRH nerve endings and are susceptible to TRH effects. These results support the hypothesis that TRH functions as neuromodulator in these areas.

Neuronal TRH synthesis: developmental and circadian TRH mRNA levels

Peptide biosynthesis within a neuron involves several steps occurring at the soma and during its travel to the nerve terminal, where it accumulates to be released under stimulatory conditions. We have measured hypothalamic TRH and TRH mRNA during ontogeny and circadian cycle and observed that TRH mRNA variations are more prominent than TRH ones. On the basis of these results and in vitro release experiments, we propose a compensatory mechanism working at the nerve terminal which is activated after release.

Evaluation of the role of prolyl endopeptidase and pyroglutamyl peptidase I in the metabolism of LHRH and TRH in brain

Intraneuronal peptide regulatory mechanisms are still poorly understood. The cytosolic enzymes prolyl endopeptidase (EC 3.4.21.26) and pyroglutamyl peptidase I (E.C.3.4.19.3) degrade both TRH and LHRH. Previous studies from this laboratory have not supported a role for these enzymes in the control of TRH levels. These studies have now been extended to cell and organ cultures and examine the effects of enzyme inhibition on LHRH. Exposure of dispersed hypothalamic cells or median eminences in culture to Z-Pro-Prolinal and pyroglutamyl diazomethyl ketone, specific inhibitors of prolyl endopeptidase and pyroglutamyl peptidase I respectively, did not change TRH content or recovery of released TRH. In vivo and in vitro treatment with these inhibitors did not modify the content of LHRH or recovery of this peptide upon release from several brain regions except in the olfactory bulb where an unexpected decrease in levels was observed. Olfactory bulb levels of TRH also decreased but only after prolonged in vivo inhibitor treatment. The decrease in olfactory bulb LHRH and TRH could not be accounted for by enzyme induction and is likely due to a non-specific or indirect effect of the inhibitors on the processing of these peptides. These studies demonstrate that levels of LHRH and TRH in brain are not controlled by cytosolic peptidases.

Specific inhibitors of pyroglutamyl peptidase I and prolyl endopeptidase do not change the in vitro release of TRH or its content in rodent brain

Pyroglutamyl diazomethyl ketone and N-benzyloxycarbonyl prolyl prolinal, specific inhibitors of pyroglutamyl peptidase I and prolyl endopeptidase respectively, were used to study the possible role of these enzymes in the regulation of thyrotropin releasing hormone turnover. In vitro thyrotropin releasing hormone release by male rat hypothalamic slices was studied. Combined in vitro treatment with 10(-5)M of both inhibitors totally inhibited both enzymatic activities. The treatment did not affect basal or 56 mM K+ induced thyrotropin releasing hormone release or thyrotropin releasing hormone levels in slices. Repeated combined intraperitoneal injections of the two inhibitors for up to 12 hours produced a 70%-95% reduction in mouse brain pyroglutamyl peptidase I specific activity and a 65%-85% reduction in prolyl endopeptidase specific activity. Thyrotropin releasing hormone levels were unaffected by this treatment in all regions tested. The data suggest that these two enzymes are not involved in the intra- or extracellular control of thyrotropin releasing hormone levels in brain or hypophysis.