Expression and localization of cannabinoid receptors in human immature oocytes and unfertilized metaphase-II oocytes.

Endocannabinoid anandamide and cannabinoid receptors have been described in some organs of the female reproductive system, but little is known about the expression of these receptors in human oocytes. The aim of the study was to describe the expression of cannabinoid receptors in human oocytes and to investigate their differential distribution at various stages of meiotic resumption in human oocytes. A total of 750 human oocytes from 214 patients were analysed by Western blot, immunocytochemistry and PCR. For this study, oocytes that were not suitable for intracytoplasmic sperm injection (ICSI) (germinal-vesicle and metaphase-I stages), as well as metaphase-II oocytes that had not developed into an embryo after ICSI were used. Western blot analysis revealed the presence of CB1 and CB2 receptor proteins in human oocytes. CB1 and CB2 receptor immunostaining patterns changed during the various stages of meiotic resumption. Localization of CB1 receptor was peripheral at germinal-vesicle stage, homogeneous over the entire oocyte at metaphase I and peripheral at mature metaphase II. CB2 receptor localization was peripheral at germinal-vesicle and metaphase-I stages but homogeneous over the entire cell at metaphase II. This finding suggests a possible role for endocannabinoids, acting via receptors, in the maturation of female gametes and fertilization. The number of couples with sterility problems attending fertility programmes is rising but treatment is not always successful. Important problems associated with failure to conceive remain unresolved because many physiological aspects of human reproduction are still unknown. Endocannabinoids are endogenous chemical compounds that mimic the action of the main psychoactive component of marijuana, delta-9-tetrahydrocannabinol. An endogenous cannabinoid named anandamide has been found in human follicular fluid. Thus, in order to develop knowledge in this field, in the present study we have described the presence of the cannabinoid receptors CB1 and CB2 (the proteins required to mediate the action of the cannabinoids) in the early stages of meiotic resumption of oocytes (the stages before ovulation) and we could postulate that the endocannabinoids could act in the regulation of maturation of oocytes. Our study, together with other studies, indicates that the endocannabinoid system may play a role in human reproduction.

Immunohistochemical localization of CB1 cannabinoid receptors in frontal cortex and related limbic areas in obese Zucker rats: effects of chronic fluoxetine treatment.

In the present study, we report on the application of two specific polyclonal antibodies to different intracellular domains of the CB1 cannabinoid receptor to define the expression of the neural CB1 cannabinoid receptor at the histochemical level in frontal cortex and related limbic areas of the obese Zucker rats. Higher levels of CB1 receptor expression in frontal, cingulated and piriform cortex, without differences in temporal, parietal and occipital cortex, were observed in obese Zucker rats, with respect to their lean littermates. CB1 phosphorylated receptor (CB1-P) levels were also higher in frontal, temporal, parietal and occipital cortex in obese rats with respect to lean controls. Potential involvement of brain cortical CB1 cannabinoid receptors in the long-term effects of fluoxetine was studied. Experimental animals were administered with fluoxetine (10 mg/kg, i.p.) daily for 3 weeks, whereas the control group was given 0.9% NaCl solution. In obese Zucker rats, a significant decrease in CB1 receptor levels, measured by western blot, was observed in brain cortex after fluoxetine treatment. Immunostaining for CB1 receptor expression was also carried out, showing a significant decrease in the density of neural cells positive for CB1 receptor in frontal, cingulate and piriform cortex, without changes in parietal, temporal and occipital regions. Regional prosencephalic immunostaining for CB1-P receptor level showed a significant decrease in the density of stained neural cells in frontal, temporal and parietal cortex, without changes in cingulated, piriform and occipital cortex. These results suggest the involvement of endocannabinoid system in the chronic effects of fluoxetine, especially in the frontal cortex.

Type 1 diabetes alters brain cannabinoid receptor expression and phosphorylation status in rats.

One of the most common symptoms of diabetes is extreme hunger, but the brain mechanism underlying this hyperphagia is unknown. The endocannabinoid system has emerged as one of the main food intake regulators in the brain. However, the effects of type 1 diabetes on the endocannabinoid system are not completely known. Thus, the aim of the present work is to establish the possible alterations induced by type 1 diabetes on the brain endocannabinoid system in rats. Western blot and immunocytochemistry were used to measure CB1 and phosphorylated CB1 receptor expression in several prosencephalic regions in streptozotocin-induced type 1 diabetic rats. Serum leptin levels were measured by ELISA. CB1 receptor expression was increased in striatum and hypothalamus of diabetic animals, with no changes in other brain areas studied. CB1 receptor phosphorylation was also increased in the same brain areas. Type 1 diabetes induced significant weight loss, and serum leptin levels were severely decreased. These results reinforce the possible role of the CB1 receptor as a pharmacological target for the clinical management of appetite in diabetic patients.

Effects of fluoxetine administration on hypothalamic melanocortin system in obese Zucker rats.

The aim of the present work was to study the potential involvement of melanocortin system in the anorectic mechanism of fluoxetine, a selective serotonin reuptake inhibitors, in obese Zucker rats. Male obese Zucker (fa/fa) rats were administered fluoxetine (10 mg/kg; i.p.) daily for two weeks. The control group was given 0.9% NaCl solution. RT-PCR for pro-opiomelanocortin (POMC), Agouti gene related peptide (AgRP) and melanocortin receptor 4 (MC4-R) in the hypothalamus, as well as regional immunostaining for alpha-melanocyte stimulating hormone (alpha-MSH) and MC4-R were carried out. Fluoxetine administration increased POMC expression and reduced MC4-R expression in the hypothalamus, without changes in AgRP mRNA levels. Moreover, an increase in the numbers of alpha-MSH positively immunostained neural cells in the hypothalamic arcuate nucleus (ARC), as well as a significant decrease in the numbers of neural cells positively immunostained for MC4-R in the paraventricular nucleus (PVN), without changes in lateral hypothalamic area (LHA), were observed. These results suggest the involvement of alpha-MSH in central fluoxetine anorectic action.

Peptide metabolism and the control of body fluid homeostasis.

In this paper, we review our current understanding of the medicinal chemistry of the major peptide systems, which influence body fluid homeostasis. Electrolytes play pivotal roles in intra- and intercellular communication, acid-base equilibrium and, when bound to several macromolecules, they regulate a myriad of enzymatic proteins, receptors and transcription factors. Cell turgor influences the plasma membrane, which activates mechanically-gated ion channels or mechanoreceptors, and the expression of a number of genes which underlie long-term metabolic responses to hormones, substrates and reactive oxygen intermediates. The altered kinetics and enzymatic cleavage of peptides during water-electrolyte imbalance can contribute to cardiac and renal damage associated with elevated blood pressure. Identification of the enzymes which are responsible for cleavage, together with emerging information about the mechanisms of action and structures of regulatory and effector peptides, has laid a foundation for the discovery of novel drugs, some of which are in use or are now undergoing evaluation in experimental trials. The development of models of hydrosaline challenge with relative efficiency to induce selective water-electrolyte imbalance has permitted the identification of kallikrein-kinin, renin-angiotensin-aldosterone, vasopressin-oxytocin, thyrotropin-releasing hormone and luteinizing hormone-releasing hormone as susceptible substrates. At present, the angiotensin-I converting enzyme inhibitors are well-known efficacious, orally active, blood pressure-lowering agents which have been used in hypertensive patients. In addition to several new analogues of this class of drug, some selective dual inhibitors of angiotensin-I converting enzyme and neutral endopeptidase and inhibitors of aminopeptidases are now also being rationally assayed and their beneficial effects on hypertension and hydromineral balance indicate that this type of drug may have powerful therapeutic effects for disorders of body fluid homeostasis.

Sibutramine decreases body weight gain and increases energy expenditure in obese Zucker rats without changes in NPY and orexins.

The aim of the present work was to describe the effects of sibutramine on body weight and adiposity and to establish the potential involvement of neuropeptide Y (NPY) and orexins in the anorectic action of this drug. Male obese Zucker rats were daily administered with sibutramine (10 mg/kg, intraperitoneal) for two weeks. Carcass composition was assessed using the official methods of the Association of Official Analytical Chemists. Total body oxygen consumption was measured daily for 60 min before sibutramine or saline injection and for 30 min (from 60 to 90 min) after drug or saline injection. Hypothalamic arcuate and paraventricular nuclei, and the lateral hypothalamic area were immunostained for NPY, orexin A and orexin B. Commercial kits were used for serum determinations. Reductions in body weight and adipose tissue weights were observed after sibutramine treatment in obese Zucker rats. No changes in NPY immunostaining in the arcuate and paraventricular nuclei were found. Orexin A and orexin B immunostaining was not modified in the lateral hypothalamic area in treated rats. The reduction in body weight and adiposity induced by sibutramine was achieved by both a reduction in food intake and an increase in energy expenditure. NPY and orexins do not seem to be involved in the anorectic effect of sibutramine.

Effect of the disruption of body fluid balance on pyroglutamyl aminopeptidase (Type-1) in rat brain structures.

The activity of soluble and membrane-bound pyroglutamyl aminopeptidase Type-1 (PAP I) was evaluated in the hypothalamus, hippocampus, thalamus, brain cortex, and pituitary gland of rats after applying certain hydromineral challenges. Compared to euhydrated rats, decreased enzyme activity was found in the hypophysis of rats deprived of water for 48 h, or rats drinking ad libitum hypertonic sodium chloride solution (2%) for 6 days or distilled water for 6 days and then submitted to acute water overload. PAP I cleaves the pGlu-amino acid bond of neuropeptides such as thyroliberin, luliberin, neurotensin, and bombesin. The decay of particulate PAP I activity may cause an increase of these pyroglutamate peptides in the whole pituitary. Although the deleterious or pro-homeostatic influence of this decay remains to be elucidated, the present data provide evidence for the involvement of this enzyme activity at this anatomical location in the water-electrolyte imbalance.

Nefazodone alters NPY immunostaining in rat arcuate-paraventricular projection without changes in food intake and body weight.

Nefazodone is an antidepressant drug that inhibits serotonin and noradrenaline reuptake. The aim of the present work was to study the effects of nefazodone on food intake, body weight, adiposity and hypothalamic NPY immunostaining in rats. For this purpose, male Sprague-Dawley rats (3-month-old) were administered with nefazodone (20 mg/kg; i.p) daily for two weeks. The control group was given 0.9% NaCl solution. Hypothalamic arcuate, paraventricular, periventricular, supraoptic and suprachiasmatic nuclei and the lateral hypothalamic area were immunostained for NPY. Chronic nefazodone administration in rats did not modify food intake, body weight and adipose depot size (subcutaneous, perirenal and epididymal white adipose tissues, and interscapular brown adipose tissue). However, a significant decrease in paraventricular NPY immunostaining was found in the nefazodone group compared with the control group. No changes in other hypothalamic regions such as arcuate, periventricular, supraoptic and suprachiasmatic nuclei, and lateral and medial preoptic areas were observed. Because nefazodone is an alpha1-adrenoceptor antagonist, it can be proposed that the expected decrease in food intake after nefazodone administration, due to its effects on NPY arcuate-paraventricular projection, could be masked by the opposite orexigenic effect of alpha1-adrenoceptor blockade.

Effects of hydrosaline treatments on prolyl endopeptidase activity in rat tissues.

Enzymatic cleavage of some peptide hormones, neurotransmitters and neuromodulators could be implicated in the regulation of extra- and intracellular fluid volume and osmolality. Prolyl endopeptidase is known to hydrolyze several peptides, which act on hydromineral balance, such as angiotensins, bradykinin, vasopressin, oxytocin, thyrotropin-releasing hormone, neurotensin and opioids. In this work, we analyzed the effects of certain volume and/or osmotic changes in the activity of the soluble and membrane-bound prolyl endopeptidase in several brain areas, heart, lungs, kidney and adrenal and pituitary glands of the rat. Soluble prolyl endopeptidase activity was higher in the renal cortex of the chronic salt-loaded rats than in the control rats. In the water-deprived and polyethylene glycol-treated rats, heart particulate prolyl endopeptidase was lower than in the control rats. Particulate prolyl endopeptidase was also lower in the adrenal gland of the acute salt-loaded rats and in the brain cortex of the water-loaded rats than in the control rats. Data suggest that tissue-dependent peptide hydrolysis evoked by prolyl endopeptidase activity is involved in the water-electrolyte homeostasis.

Aminopeptidase activity in human saliva.

In this work, different aminopeptidase activity levels in whole human saliva are described. Aminopeptidase activities were studied by measuring the rate of hydrolysis of the artificial substrates Ala-, pGlu-, Pro-, Arg-, Asp- y Cis-2-naphthylamides (fluorimetrically detected at 412 rim with excitation at 345 nm). The presence of these enzyme activities in the saliva suggests that the active levels of saliva peptides can be controlled by homeostatic mechanism similar to those that have been described in other tissues, such as plasma, the central nervous system, and immunocompetent cells.

Regional and subcellular distribution of soluble aminopeptidase in the human and the rat brain: a comparative study.

In an attempt to elucidate the cellular function of the soluble aminopeptidases, we have analysed their activity in several subcellular fractions (synaptosomal, mitochondrial, microsomal, nuclear and cytosolic fraction) and in different areas (amygdala, hypothalamus, hippocampus, striatum, frontal cortex, occipital cortex and parietal cortex) of the human and the rat brain. The enzymes assayed in this study were five cytosolic aminopeptidases identified inmammalian brain tissues: alanyl-aminopeptidase, arginyl-aminopeptidase, leucyl-aminopeptidase, pyroglutamyl-peptidase I and aspartyl-aminopeptidase. The regional comparative study revealed significantly higher activities of alanyl-aminopeptidase activity in the human brain, with arginyl-aminopeptidase activities being higher in the rat brain. In the subcellular study, while the alanyl- and arginyl-aminopeptidase activities were quite homogeneous in all the subcellular fractions, the leucyl-aminopeptidase, pyroglutamyl-peptidase I and aspartyl-aminopeptidase activities were significantly higher in the synaptosomal fraction. The differential distribution of these enzymes could suggest that these activities have different functions in the distinct subcellular structures of the human and the rat brain.

Interactions among challenges of hydromineral balance, angiotensin-converting enzyme, and cystine aminopeptidase.

Enzymatic cleavage of some peptides could be included among the mechanisms of water-electrolyte homeostasis. To test this hypothesis, the angiotensin-converting activity (ACE) of plasma and the L-cystine-di-beta-naphthylamidase activity (CAP) of plasma and of soluble and particulate fractions from different areas of the central nervous system (CNS) were investigated in rats submitted to treatments eliciting hydromineral imbalance. CAP in the CNS was unchanged by hydromineral challenges. The correlations observed between plasma osmolality and CAP, and plasma CAP and ACE suggested a contribution of these activities to the restoration of basal water-electrolyte and blood pressure conditions through the hydrolysis of vasopressin, oxytocin, angiotensin I and bradykinin.

Comparación de los efectos de la administración de fluoxetinay sertralina sobre el inmunomarcajedel receptor opioide mu en el cerebro de la rata / Comparison of the effects of the administration of fluoxetine and sertraline on the immunomarking of the mu opioid receptor in rat brain

Introducción. La administración crónica de fluoxetina en ratas origina un incremento en el número de células nerviosas que expresan el receptor opioide mu en diversas regiones prosencefálicas, siguiendo un patrón muy parecido al desencadenado por la administración de imipramina. Objetivo. Este trabajo pretende describir los efectos de la administración crónica de sertralina sobre el inmunomarcaje de receptores opioides mu en diversas regiones prosencefálicas de la rata y comparar su acción con la ejercida por la fluoxetina. Material y métodos. Se emplearon ratas tratadas por vía intraperitoneal con el fármaco, utilizándose posteriormente un método inmunocitoquímico y un equipo de análisis de imagen para determinar el número de células nerviosas por unidad de superficie que expresaban el receptor opioide mu en las regiones prosencefálicas estudiadas. Resultados. Aunque la administración crónica de sertralina en ratas genera un incremento estadísticamente significativo en el número de células nerviosas que expresan el receptor opioide mu en el caudado putamen, el giro dentado, el septo lateral y las cortezas frontal, parietal y piriforme, existen algunas ligeras diferencias regionales en su acción con respecto a lo descrito anteriormente para el caso de la fluoxetina, tales como un mayor efecto de la sertralina sobre la corteza parietal y el septo y una acción menos intensa sobre la corteza frontal. Conclusión. Las diferencias regionales observadas en la acción de la sertralina (con respecto a fluoxetina) sobre la expresión del receptor opioide mu en el prosencéfalo de la rata podrían relacionarse con una menor incidencia de efectos adversos de tipo psicomotor (AU)

Subcellular distribution of pyroglutamyl-peptidase I activity in the developing rat cerebellum.

In this work we analyzed the activity of pyroglutamyl (pGlu)-peptidase I in several subcellular fractions of the rat cerebellum during its development. The aim of this study is to determine if there is a developmental redistribution of this enzyme activity and if this fact could indicate changes in the role of the enzyme as age progresses. Results show that pGlu-peptidase I is widely distributed, but not homogeneously, in all the subcellular fractions that were studied, in both soluble and particulate forms. Significantly the distribution of the enzyme changes with development. Thus, in the soluble synaptosomal fraction, the pGlu-peptidase I activity is low until PD9 and the activity increases significantly from PD9 to PD15, when it reaches adult levels. In contrast, in the cytosolic fraction, the pGlu-peptidase I activity is high from fetal day 22 to postnatal day 6, and then decreases significantly until postnatal day 90.

Subcellular ontogeny of brain pyroglutamyl peptidase I.

The present work studies pyroglutamyl-peptidase I activity in several subcellular fractions of the developing brain. In the synaptosomal fraction, soluble pGlu-peptidase I activity is low until postnatal Day 9 (with a peak at postnatal Day 2) and the activity increases at postnatal Day 15. In later stages there are not significant changes of synaptosomal pGlu-peptidase I activity. However, in the cytosolic fraction the activity is high from ED22 until postnatal Day 9, and afterwards decreases. The changes in the particulate fractions are generally less drastic.

Lithium alters mu-opioid receptor expression in the rat brain.

Lithium can potentiate the effects of antidepressant drugs and alters morphine analgesia and phosphoinositide turnover. Analysis of mu-opioid receptor immunostaining after chronic lithium administration in rats revealed an increase in the density of cells expressing mu-opioid receptors in the caudatus-putamen, the dentate gyrus, the lateral septum and the frontal, parietal and piriform cortices. These data suggest that mu-opioid receptor expression in the rat forebrain is altered by in vivo chronic lithium treatment. This could be a compensatory mechanism, induced in part by the effects of lithium on mu-opioid receptor transduction mechanism.