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Biomedical subjects

J A Ramos

Publications and source records attributed to J A Ramos.

At least 37 records · Page 2Linked to original sources
PubMed2000

Cannabinoid CB(1) receptors colocalize with tyrosine hydroxylase in cultured fetal mesencephalic neurons and their activation increases the levels of this enzyme.

The incubation of cultured fetal mesencephalic neurons with Delta(9)-tetrahydrocannabinol (Delta(9)-THC) increased the activity of tyrosine hydroxylase (TH) and this increase was reversed by SR141716A, a specific antagonist for cannabinoid CB(1) receptors. In the present work, we extended these earlier observations by addressing two objectives. First, we characterized at a molecular level the presence of CB(1) receptors in cultured fetal mesencephalic neurons using two strategies: (i) analyzing the presence of CB(1) receptor gene transcripts by Northern blot, and (ii) measuring [3H]WIN-55,212-2 binding in membrane fractions obtained from these cells, as well as evaluating the potential increase in [35S]-guanylyl-5'-O-(gamma-thio)-triphosphate ([35S]GTPgammaS) binding caused by the activation of these receptors with WIN-55,212-2, a synthetic agonist. Northern blot analyses demonstrated the presence of small, but measurable levels of CB(1) receptor mRNA in cultured fetal mesencephalic neurons. The presence of these transcripts was accompanied by the presence of receptor binding protein, as revealed by a small, but specific, [3H]WIN-55, 212-2 binding in membrane fractions obtained from these cells. These CB(1) receptors are coupled to GTP-binding proteins, as the incubation of membrane fractions obtained from these cells with WIN-55,212-2 slightly, but significantly increased [35S]GTPgammaS binding. This fact indicated the existence, not only of receptor binding, but also of a functional receptor transduction pathway. As a second objective, we examined the potential colocalization of CB(1) receptors and TH in these cells by double-labelling immunocytochemistry. We also determined by Western blotting whether the previously observed Delta(9)-THC-induced increase in TH activity was accompanied by increased TH protein levels. Cultured fetal mesencephalic neurons exhibit diverse cell phenotypes, with CB(1) receptors localized only on TH-containing neurons. In addition, we found that the incubation of fetal mesencephalic neurons with medium containing Delta(9)-THC increased TH protein levels, in concordance with the previously reported increase in TH activity. Collectively, our results support the notion that CB(1) receptors are present in cultured fetal mesencephalic TH-containing neurons, despite their absence in the corresponding neurons in the adult brain. Thus, it is likely that the effects of cannabinoids on TH activity are direct. All this data strengthen the view that cannabinoid receptors are atypically located during brain development and that they might play an important role during this process, in particular on the phenotypical expression of TH-containing neurons.

Animals↗
PubMed2000

Unilateral 6-hydroxydopamine lesions of nigrostriatal dopaminergic neurons increased CB1 receptor mRNA levels in the caudate-putamen.

It has been recently suggested that the effects of cannabinoids on motor behavior might be different in rats with lesions of nigrostriatal dopaminergic neurons than in controls. In the present study, we examined the possible alteration in the status of cannabinoid CB1 receptors in the basal ganglia of rats with unilateral lesions of those neurons caused by 6-hydroxydopamine. We used two different experimental groups depending on the duration of the period of recovery after the lesion, and comparisons were done between the lesioned and nonlesioned sides at the level of the basal ganglia. Both groups of lesioned rats exhibited a similar marked reduction in tyrosine hydroxylase (TH)-mRNA levels, measured by in situ hybridization, in the substantia nigra of the lesioned side. In the same way, lesioned rats exhibited the characteristic rotational behavior after a single injection of apomorphine and the intensity of this rotation was stable at the two times analyzed after the lesion. Also as expected, lesioned rats exhibited an increase in proenkephalin mRNA levels in the caudate-putamen, whereas mRNA levels of substance P decreased, although differences between the two times of recovery analyzed were observed in this case. We did not find any significant changes in CB1 receptor binding, measured by [3H]WIN-55,212,2 autoradiography, or in the activation of signal transduction mechanisms, measured by WIN-55,212,2-stimulated [35S]GTPgammaS binding autoradiography, between the lesioned and nonlesioned sides at the level of the lateral caudate-putamen, globus pallidus and substantia nigra in both groups of lesioned rats. However, we found a significant increase in levels of CB1 receptor-mRNA transcripts, measured by in situ hybridization, in the lesioned side in both the lateral and medial caudate-putamen. This occurred 7-10 weeks after the lesion, but the increase was markedly waned after 17-18 weeks. In summary, the unilateral 6-hydroxydopamine lesion of nigrostriatal dopaminergic neurons originated a marked increase in CB1 receptor-mRNA levels in cell bodies of striatal efferent neurons, although accompanied by no changes in CB1 receptor binding and activation of signal transduction mechanisms. This supports a critical role for dopamine in the control of CB1 receptor gene expression. However, the magnitude of the effect significantly waned as a function of the duration of the period after lesion.

Animals↗
PubMed2000

The endogenous cannabinoid system and brain development.

Cannabinoid receptors and their endogenous ligands constitute a novel modulatory system that is involved in specific brain functions, such as nociception, control of movement, memory and neuroendocrine regulation. Recently, it has also been suggested that this system is involved in brain development. Studies have used a variety of techniques to elucidate the effects of cannabinoids during development, as well as to characterize the presence of elements of the endogenous cannabinoid system (receptors and ligands) in the developing brain. Collectively, they suggest that endocannabinoids participate in brain development through the activation of second-messenger-coupled cannabinoid receptors.

Animals↗
PubMed2000

Decreased cannabinoid CB1 receptor mRNA levels and immunoreactivity in pituitary hyperplasia induced by prolonged exposure to estrogens.

Recent studies have demonstrated that cannabinoid CB1 receptor gene expression in the anterior pituitary gland is under the influence of estrogens. Because these receptors have been recently involved in the development of several types of cancer, it would be interesting to examine the changes produced in these receptors by the development of pituitary hyperplasia after a chronic exposure to estrogens. To this end, we measured mRNA levels and immunoreactivity for the CB1 receptor in the anterior pituitary gland of rats implanted with silastic capsules containing diethylstilbestrod (DES), a synthetic estrogen, or empty capsules. Results were as follows. Induction of pituitary hyperplasia with DES produced the expected body weight loss (-38.4%) and increase in pituitary weight (5-fold) and plasma prolactin (PRL) levels (90-fold). In hyperplastic pituitaries, both CB1 receptor mRNA levels and immunoreactivity decreased significantly (-79.4% and -63.2% respectively). Double immuno-labelling studies demonstrated that CB1 receptors colocalized, in hyperplastic pituitaries, with PRL- or luteinizing hormone-containing cells, as they did in normal pituitaries. In summary, estrogen-induced pituitary hyperplastia was associated with a marked reduction in CB1 receptors, despite the fact that these receptors were located, among others, on lactotroph cells which develop hyperplasia during DES exposure. Whether this decrease is involved in the ethiology of pituitary hyperplasia and whether the pharmacological activation of these receptors might affect this process are presently unknown, but this will be subjected of further research.

Animals↗
PubMed2000

Enhancement of anandamide formation in the limbic forebrain and reduction of endocannabinoid contents in the striatum of delta9-tetrahydrocannabinol-tolerant rats.

Recent studies have shown that the pharmacological tolerance observed after prolonged exposure to synthetic or plant-derived cannabinoids in adult rats is accompanied by down-regulation/desensitization of brain cannabinoid receptors. However, no evidence exists on possible changes in the contents of the endogenous ligands of cannabinoid receptors in the brain of cannabinoid-tolerant rats. The present study was designed to elucidate this possibility by measuring, by means of isotope dilution gas chromatography/mass spectrometry, the contents of both anandamide (arachidonoylethanolamide; AEA) and its biosynthetic precursor, N-arachidonoylphosphatidylethanolamine (NArPE), and 2-arachidonoylglycerol (2-AG) in several brain regions of adult male rats treated daily with delta9-tetrahydrocannabinol (delta9-THC) for a period of 8 days. The areas analyzed included cerebellum, striatum, limbic forebrain, hippocampus, cerebral cortex, and brainstem. The same regions were also analyzed for cannabinoid receptor binding and WIN-55,212-2-stimulated guanylyl-5'-O-(gamma-[35S]thio)-triphosphate ([35S]GTPgammaS) binding to test the development of the well known down-regulation/desensitization phenomenon. Results were as follows: As expected, cannabinoid receptor binding and WIN-55,212-2-stimulated [35S]GTPgammaS binding decreased in most of the brain areas of delta9-THC-tolerant rats. The only region exhibiting no changes in both parameters was the limbic forebrain. This same region exhibited a marked (almost fourfold) increase in the content of AEA after 8 days of delta9-THC treatment. By contrast, the striatum exhibited a decrease in AEA contents, whereas no changes were found in the brainstem, hippocampus, cerebellum, or cerebral cortex. The increase in AEA contents observed in the limbic forebrain was accompanied by a tendency of NArPE levels to decrease, whereas in the striatum, no significant change in NArPE contents was found. The contents of 2-AG were unchanged in brain regions from delta9-THC-tolerant rats, except for the striatum where they dropped significantly. In summary, the present results show that prolonged activation of cannabinoid receptors leads to decreased endocannabinoid contents and signaling in the striatum and to increased AEA formation in the limbic forebrain. The pathophysiological implications of these findings are discussed in view of the proposed roles of endocannabinoids in the control of motor behavior and emotional states.

Animals↗
PubMed2000

Neuronal and astroglial response to pre- and perinatal exposure to delta-9-tetra- hydrocannabinol in the rat substantia nigra.

The responses of neurons and astroglial cells to pre- and perinatal exposure to Delta(9)-tetrahydrocannabinol (Delta(9)-THC) were evaluated in the substantia nigra (SN) of male and female rats, at three postnatal ages (PD21, PD30 and PD70), by immunohistochemical detection of tyrosine hydroxylase (TH) in dopaminergic neurons and of glial fibrillary acidic protein (GFAP) in astrocytes. Our results showed that the effects of pre- and perinatal exposure to Delta(9)-THC on neuronal and astroglial immunoreactivities in the SN (compacta and reticulata) varied with sex, with male rats being more susceptible than females. Prenatal exposure to Delta(9)-THC decreased TH immunoreactivity in the SN of males on PD21 when compared to both their controls and Delta(9)-THC-exposed females of the same age. Furthermore, the TH expression decreased with age in Delta(9)-THC-exposed males in the SNc pars compacta, whereas it increased in controls. On the contrary, TH expression was maintained stable in the SN pars compacta of Delta(9)-THC-exposed females from PD21. These differences in neuronal development caused by prenatal Delta(9)-THC exposure were associated with significant differences in GFAP expression by astroglial cells in both sexes. On PD21, GFAP immunoreactivity decreased in the SN in Delta(9)-THC-exposed male rats. Although GFAP expression increased in Delta(9)-THC-exposed males with age, it did not reach control levels by PD70. On the contrary, significantly increased GFAP expression in the Delta(9)-THC-exposed females on PD21 was observed, compared to their controls and also to Delta(9)-THC-exposed male rats; however, the GFAP expression shown by Delta(9)-THC-exposed females stabilized from PD21. These Delta(9)-THC-induced changes in the glial development could indicate that Delta(9)-THC accelerated the maturation of astrocytes in female rats, whereas Delta(9)-THC delayed astrocytic maturation in Delta(9)-THC-exposed males. These findings suggest that pre- and perinatal exposure to Delta(9)-THC can lead to long-term effects in both neurons and glial cells.

Animals↗
PubMed1999

Analysis of cannabinoid receptor binding and mRNA expression and endogenous cannabinoid contents in the developing rat brain during late gestation and early postnatal period.

Cannabinoid CB(1) receptors emerge early in the rat brain during prenatal development, supporting their potential participation in events related to neural development. In the present investigation, we completed earlier studies, analyzing CB(1) receptor binding and mRNA expression by using autoradiography and in situ hybridization, respectively, in the brain of rat fetuses at gestational day (GD) 21 and of newborns at postnatal days (PND) 1 and 5, in comparison with the adult brain. These analyses were paralleled by quantitation of levels of anandamide and its precursor, N-arachidonoyl-phosphatidylethanolamine (NAPE), and of 2-arachidonoyl-glycerol (2-AG), carried out by using gas chromatography / mass spectrometry of the tri-methyl-sylyl-ether derivatives. As expected, CB(1) receptor binding was detected at GD21 in a variety of brain structures. In most of them, such as the hippocampus, cerebral cortex, cerebellum, basal ganglia, and limbic nuclei, there were no marked differences in the density of CB(1) receptors in animals at GD21 as compared to early newborns (PND1 and 5), although it markedly increased in these regions in adulthood. However, with the exception of the cerebellum and, in part, the caudate-putamen, the pattern observed for binding in these regions was clearly different from that observed for mRNA expression of the CB(1) receptor, which currently exhibited the highest levels at PND1 and the lowest in the adult brain. This was also seen in the basolateral amygdaloid nucleus, ventromedial hypothalamic nucleus, medial habenula, and other structures. In the caudate-putamen and, particularly, in the cerebellum, mRNA expression was higher in the adult brain as compared with other ages. As previously reported, specific binding for CB(1) receptors was also detected at GD21 in white matter areas, such as the corpus callosum, anterior commissure, fornix, fimbria, stria medullaris, stria terminalis, and fasciculus retroflexum. With the exception of the anterior commissure and the fimbria, specific binding progressively decreased at PND1 and PND5 until disappearing in the adult brain. In the fimbria, the highest values of binding were seen at PND1, but binding also completely disappeared in the adult brain, whereas in the anterior commissure, specific binding at PND1 and PND5 was lesser than that observed at GD21 and, particularly, in adulthood. CB(1) receptor mRNA expression was not detected in these white matter areas, thus dismissing the possible presence of these receptors in glial cells rather than in neuronal axons. However, mRNA expression was detected in the brainstem, an area also rich in white matter, and it mostly correlated with receptor binding, exhibiting a progressive decrease from GD21 up to adulthood. CB(1) receptor mRNA expression was also detected at GD21 in atypical areas where binding was not detected. These areas are proliferative regions, such as the subventricular zones of the neocortex, striatum, and nucleus accumbens. This atypical location only persisted at PND1 and PND5 in the striatal subventricular zone, but disappeared in the adult brain. We also found measurable levels of different endogenous cannabinoids in the developing brain. High levels of 2-AG, comparable to those found in the adult brain, were measured at GD21, whereas significantly lower levels were measured for anandamide and NAPE at this fetal age compared with the levels found in the adult brain. Levels of anandamide and NAPE increased during the early postnatal period until reaching the maximum in the adult brain. By contrast, 2-AG levels peaked at PND1, with values approximately twofold higher than those found at the other ages. In summary, all these data demonstrate that the endogenous cannabinoid system, constituted by endogenous ligands and receptor signaling pathways, is present in the developing brain, which suggests a possible specific role of this system in key processes of neural development. (c) 1999 Wiley-Liss, Inc.

Age Factors↗
PubMed1999

Cannabinoid receptor binding and mRNA levels in several brain regions of adult male and female rats perinatally exposed to delta9-tetrahydrocannabinol.

The present study was designed to elucidate whether perinatal delta9-tetrahydrocannabinol (delta9-THC) exposure results in changes in cannabinoid receptor binding and mRNA levels in adulthood. Most of the brain areas studied, including the basal ganglia, the cerebellum, the limbic structures, and most of the hippocampal regions exhibited no changes in cannabinoid receptor binding and mRNA levels in adulthood as a consequence of the perinatal delta9-THC exposure. However, some subtle changes could be appreciated in specific regions, although their physiological relevance seems uncertain. For example, delta9-THC-exposed males exhibited a small decrease in binding in the superficial layer of the cerebral cortex, an effect that was not seen in delta9-THC-exposed females and in mRNA levels for both males and females. In the CA2 layer of the Ammon's horn, there was an increase in mRNA levels of delta9-THC-exposed animals, although this was statistically significant only in males. However, the more marked and probably relevant changes were seen in the arcuate nucleus, where delta9-THC-exposed males exhibited an increase in binding, whereas this tended to decrease in delta9-THC-exposed females. In an additional experiment, we analyzed the motor response of these animals to a challenge with SR141716, a specific antagonist for cannabinoid receptors. The delta9-THC-exposed animals tended to show a higher response to SR141716 challenge, with changes apparently more marked in delta9-THC-exposed females, although they did not reach statistical significance. In summary, perinatal cannabinoid exposure does not appear to significantly alter cannabinoid receptor binding and mRNA expression in the brain of adult rats, as well as the motor response caused by the blockade of these receptors with a specific antagonist. There were some changes in the status of cannabinoid receptors but they were very small and, hence, of debatable physiological relevance. The most significant of these effects was the increase in binding observed in the arcuate nucleus of delta9-THC-exposed males.

Animals↗
PubMed1999

Time-dependent differences of repeated administration with Delta9-tetrahydrocannabinol in proenkephalin and cannabinoid receptor gene expression and G-protein activation by mu-opioid and CB1-cannabinoid receptors in the caudate-putamen.

The purpose of the present study was to examine the time-related effects of repeated administration of Delta9-tetrahydrocannabinol during 1, 3, 7 and 14 days on cannabinoid and mu-opioid receptor agonist-stimulated [35S]GTPgammaS binding, and CB1 cannabinoid receptor and proenkephalin gene expression in the caudate-putamen. Repeated administration with Delta9-tetrahydrocannabinol produced a time-related reduction in cannabinoid receptor synthesis and activation of signal transduction mechanisms in the caudate-putamen. Indeed, WIN-55,212-2-stimulated [35S]GTPgammaS binding decreased 24% on day 1 and then progressively decreased finding a 42% decrease on day 14. Similarly, CB1 cannabinoid receptor mRNA levels decreased (22%) on day 3, reaching 50% reduction on day 7. In contrast, a pronounced increase is detected in DAMGO-stimulated [35S]GTPgammaS binding and proenkephalin mRNA levels in the caudate-putamen. The highest degree of increase was reached on day 7 of the treatment (35% of proenkephalin mRNA levels and 62% of DAMGO-stimulated [35S]GTPgammaS binding) and then values slightly decreased on day 14. Taken together, the results of the present study indicate that, in the caudate-putamen, repeated administration with Delta9-tetrahydrocannabinol produces a time-related increase in proenkephalin gene expression and mu-opioid receptor activation of G-proteins, and a time-related decrease in CB1 cannabinoid receptor gene expression and reduction in CB1 cannabinoid receptor activation of G-proteins. These results also suggest a possible interaction between the cannabinoid and opioid systems in the caudate-putamen which may be potentially relevant in the understanding of the alterations of motor behavior that occur after prolonged exposure to cannabinoids.

Analgesics↗
PubMed1999

Brain regional distribution of endocannabinoids: implications for their biosynthesis and biological function.

The amounts, in nine different rat brain regions, of the two endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoylglycerol (2-AG), and of the putative AEA precursor N-arachidonoyl-phosphatidylethanolamine (NArPE), were determined by isotope-dilution gas chromatography-mass spectrometry and compared to the number of cannabinoid binding sites in each region. The distribution of NArPE, reported here for the first time, exhibited a good correlation with that of AEA, the former metabolite being 3-13 times more abundant than the endocannabinoid in all regions. The highest amounts of both metabolites (up to 358.5 and 87 pmol/g wet weight tissue, respectively) were found in the brainstem and striatum, and the lowest in the diencephalon, cortex, and cerebellum. These data support the hypothesis that, in the brain, AEA is a metabolic product of NArPE and may reach levels compatible with its proposed neuromodulatory function. The brain distribution of 2-AG, also described in this study for the first time, was found to correlate with that of AEA with levels ranging from 2.0 to 14.0 nmol/g (in the diencephalon and brainstem, respectively). The distribution of the endocannabinoids did not match exactly with that of cannabinoid binding sites, suggesting either that these compounds are not necessarily produced near their molecular targets, or that they play functional roles additional to the activation of cannabinoid receptors. Regional differences in the ligand/receptor ratios may also lead to predict corresponding differences in the efficiency of receptor activation, as shown by previous studies.

Aging↗
PubMed1999

Extrapyramidal and neuroendocrine effects of AM404, an inhibitor of the carrier-mediated transport of anandamide.

A selective inhibitor of the carrier-mediated transport of endogenous cannabinoids, N-(4-hydroxyphenyl)-arachidonylethanolamide (AM404), has been recently synthesized and proposed as a useful tool for studying the physiological effects of endogenous cannabinoids and as a potential therapeutic agent in a variety of diseases. In the present study, we have examined the effects of this compound in two important brain processes in which a role for anandamide and other endogenous cannabinoids has been claimed: neuroendocrine regulation and extrapyramidal motor activity. A single and well-characterized dose of AM404, which presumably resulted in a significant elevation of the levels of endogenous cannabinoids, produced a marked decrease in plasma prolactin (PRL) levels, with no changes in luteinizing hormone (LH) levels. This decrease in PRL levels was accompanied by an increase in the activity of tyrosine hydroxylase (TH) in the medial basal hypothalamus. Both decreased PRL secretion and increased hypothalamic TH activity have been reported to occur after the administration of anandamide. Administration of AM404 also produced a marked motor inhibition in the open-field test, as also reported for anandamide, with a decrease in ambulatory and exploratory activities and an increase in the time spent in inactivity. This was accompanied by a decrease in the activity of TH in the substantia nigra, an effect also previously observed for anandamide.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1999

Role of endocannabinoids in brain development.

In addition to those functions that have been extensively addressed in this special issue, such as nociception, motor activity, neuroendocrine regulation, immune function and others, the endogenous cannabinoid system seems to play also a role in neural development. This view is based on a three-fold evidence. A first evidence emerges from neurotoxicological studies that showed that synthetic and plant-derived cannabinoids, when administered to pregnant rats, produced a variety of changes in the maturation of several neurotransmitters and their associated-behaviors in their pups, changes that were evident at different stages of brain development. A second evidence comes from studies that demonstrated the early appearance of elements of the endogenous cannabinoid system (receptors and ligands) during the brain development. The atypical location of these elements during fetal and early postnatal periods favours the notion that this system may play a role in specific molecular events related to neural development. Finally, a third evidence derives from studies using cultures of fetal glial or neuronal cells. Cannabinoid receptors are present in some of these cultured cells and their activation produced a set of cellular effects consistent with a role of this system in the process of neural development. All this likely supports that endocannabinoids, early synthesized in nervous cells, play a role in events related to development, by acting through the activation of second messenger-coupled cannabinoid receptors.

Animals↗
PubMed1999

Pharmacological and biochemical interactions between opioids and cannabinoids.

Opioids and cannabinoids are among the most widely consumed drugs of abuse in humans. A number of studies have shown that both types of drugs share several pharmacological properties, including hypothermia, sedation, hypotension, inhibition of both intestinal motility and locomotor activity and, in particular, antinociception. Moreover, phenomena of cross-tolerance or mutual potentiation of some of these pharmacological effects have been reported. In recent years, these phenomena have supported the possible existence of functional links in the mechanisms of action of both types of drugs. The present review addresses the recent advances in the study of pharmacological interactions between opioids and cannabinoids, focusing on two aspects: antinociception and drug addiction. The potential biochemical mechanisms involved in these pharmacological interactions are also discussed together with possible therapeutic implications of opioid-cannabinoid interactions.

Cannabinoids↗
PubMed1999

Cannabinoid receptor and WIN-55,212-2-stimulated [35S]GTPgammaS binding and cannabinoid receptor mRNA levels in several brain structures of adult male rats chronically exposed to R-methanandamide.

We and others have recently demonstrated that the pharmacological tolerance observed after prolonged exposure to plant and synthetic cannabinoids in adult individuals seems to have a pharmacodynamic basis, based on the observed down-regulation of cannabinoid receptors in the brain of cannabinoid-tolerant rats. However, we were unable to elicit a similar receptor down-regulation after a chronic exposure to anandamide, the first discovered endogenous cannabinoid, possibly because of its rapid metabolic breakdown in arachidonic acid and ethanolamine. The present study was designed to progress in these previous studies, by using R-methanandamide, a more stable analog, instead anandamide. In addition, we examined not only cannabinoid receptor binding, but also WIN-55,212-2-stimulated [35S]-GTPgammaS binding, by autoradiography, and cannabinoid receptor mRNA levels, by in situ hybridization. Results were as follows. The daily administration of R-methanandamide for a period of five days produced decreases in cannabinoid receptor binding in the lateral caudate-putamen, cerebellum, entopeduncular nucleus and substantia nigra. The remaining areas, the medial caudate-putamen, globus pallidus, cerebral cortex (layers I and VI), hippocampus (dentate gyrus and Ammon's horn) and several limbic structures (nucleus accumbens, septum nuclei and basolateral amygdaloid nucleus), exhibited no changes in cannabinoid receptor binding. Similarly, the levels of cannabinoid receptor mRNA expression decreased in the lateral and medial caudate-putamen and in the CA1 and CA2 subfields of the Ammon's horn in the hippocampus after the chronic exposure to R-methanandamide, whereas the remaining areas showed no changes. WIN-55,212-2-stimulated [35S]-GTPgammaS binding did not change in the lateral caudate-putamen, cerebral cortex (layer I), septum nuclei and hippocampal structures (dentate gyrus and Ammon's horn) of animals chronically exposed to R-methanandamide, whereas a certain trend to decrease could be observed in the substantia nigra and deep layer (VI) of the cerebral cortex in these animals. In summary, as reported for other cannabinoid receptor agonists, the prolonged exposure of rats to R-methanandamide, a more stable analog of anandamide, was able to produce cannabinoid receptor-related changes in contrast with the absence of changes observed early with the metabolically labile anandamide. The observed changes exhibited an evident regional pattern with areas, such as basal ganglia, cerebellum and hippocampus, responding to chronic R-methanandamide treatment while regions, such as the cerebral cortex and limbic nuclei, not responding.

Animals↗
PubMed1999

Immunocytochemical demonstration of CB1 cannabinoid receptors in the anterior lobe of the pituitary gland.

Both exogenous and endogenous cannabinoids can influence hormone secretion from the anterior pituitary gland. A large body of information proves that the primary target of these effects is the neuroendocrine hypothalamus. However, recent studies using cannabinoid (CB) receptor autoradiography, messenger RNA in-situ hybridization and in-vitro analysis, indicate direct effects of cannabinoids at the level of the anterior pituitary gland itself. In the present paper, the immunocytochemical distribution of CB in the adult rat anterior pituitary was studied using specific polyclonal antibodies against CB1 (central) and CB2 (peripheral) receptors. Due to its resolution, this method allowed identification of individual anterior pituitary cells possessing cannabinoid receptors. The specific hormone immunoreactive cells with receptor-like immunoreactivity were compared on adjacent sections. CB1-like immunoreactivity (CB1ir) was found in the lactotroph cells as well as in luteinizing hormone (LH) secreting gonadotrophs. The CB1ir positive material present in the cytoplasm of these cells was less homogeneous than the hormone immunoreactive material, and it was also seen at the periphery of the cells, presumably on the cell membrane. No CB1ir was found in growth hormone (GH) secreting cells and it was hardly seen in the corticotrophs. No CB1ir was detected in the posterior pituitary. CB2ir was not observed in any part of the pituitary gland. The results support the view that the site of action of cannabinoids on neuroendocrine regulatory mechanisms may be both at pituitary and hypothalamic levels. We suggest that at least the direct effect of cannabinoids on the regulation of LH and prolactin secretion is mediated via CB1 cannabinoid receptors in the anterior pituitary.

Animals↗
PubMed1999

Identification of endocannabinoids and cannabinoid CB(1) receptor mRNA in the pituitary gland.

Most data on effects of natural and synthetic cannabinoids on anterior pituitary hormone secretion point out to a primary impact on the hypothalamus. There is also some evidence, however, of possible direct actions of these compounds on the anterior pituitary, although the presence of cannabinoid receptors in the pituitary has not been documented as yet. In the present study, we evaluated the presence of cannabinoid CB(1) receptor-mRNA transcripts in the pituitary gland by in situ hybridization. We observed CB(1) receptor-mRNA transcripts in the anterior pituitary and to a lesser extent in the intermediate lobe whereas they were absent in the neural lobe. We then examined whether CB(1) receptor-mRNA levels in both pituitary lobes responded to chronic activation by a specific agonist, as did receptors located in adjacent hypothalamic nuclei and in other brain regions. Daily administration of CP-55,940 for 18 days produced a small, but statistically significant paradoxical increase in CB(1) receptor-mRNA levels in the anterior pituitary, with no changes in the intermediate lobe, in contrast to reduced CB(1) receptor-mRNA levels observed in the ventromedial hypothalamic nucleus (VMN), and to decreased CB(1) receptor binding in the VMN and the arcuate nucleus. The time-course of up-regulation of CB(1) receptor-mRNA transcripts in the anterior lobe was biphasic; daily administration of Delta(9)-tetrahydrocannabinol produced an early and marked decrease in CB(1) receptor-mRNA levels after 1 and 3 days, followed by normalization after 7 days and by a small increase after 14 days. We also checked whether endogenous cannabinoid ligands are present in the anterior pituitary and the hypothalamus. Although anandamide itself was detected only in trace amounts, concentrations of its precursor N-arachidonoyl-phosphatidyl-ethanolamine and of 2-arachidonoyl-glycerol were found in both tissues, suggesting that endocannabinoids may be synthetized in the anterior pituitary. In summary, CB(1) receptors and corresponding ligands seem to be expressed in cells of the anterior and intermediate lobes of the pituitary, but the response of CB(1) receptor-mRNA transcripts in the anterior lobe to chronic agonist activation is different than the desensitization observed in hypothalamic nuclei.

Animals↗
PubMed1998

Effects of cannabinoids on prolactin and gonadotrophin secretion: involvement of changes in hypothalamic gamma-aminobutyric acid (GABA) inputs.

CB1 cannabinoid receptors are located in hypothalamic nuclei and their activation alters several hypothalamic neurotransmitters resulting in, among other things, decreased prolactin (PRL) and luteinizing hormone (LH) secretion from the anterior pituitary gland. In the present study, we addressed two related objectives to further explore this complex regulation. First, we examined whether changes in gamma-aminobutyric acid (GABA) and/or dopamine (DA) inputs in the medial basal hypothalamus might occur in parallel to the effects resulting from the activation of CB1 receptors on PRL and gonadotrophin secretion in male rats. Thus, the acute administration of (-)-delta9-tetrahydrocannnabinol (delta9-THC) produced, as expected, a marked decrease in plasma PRL and LH levels, with no changes in follicle-stimulating hormone (FSH) levels. This was paralleled by an increase in the contents of GABA, but not of DA, in the medial basal hypothalamus and, to a lesser extent, in the anterior pituitary gland. The co-administration of delta9-THC and SR141716, a specific antagonist for CB1 receptors, attenuated both PRL and LH decrease and GABA increase, thus asserting the involvement of the activation of CB1 receptors in these effects. As a second objective, we tested whether the prolonged activation of these receptors might induce tolerance with regard to the decrease in PRL and LH release, and whether this potential tolerance might be related to changes in CB1-receptor binding and/or mRNA expression. The chronic administration of R-methanandamide (AM356), a more stable analog of anandamide, the putative endogenous cannabinoid ligand, produced a marked decrease in plasma PRL and LH levels, with no changes in FSH. The decreases were of similar magnitude to those caused by a single injection of this cannabimimetic ligand, thus suggesting the absence of tolerance. In parallel, the analysis of CB1-receptor binding and mRNA expression in several hypothalamic structures proved that the acute or chronic administration of AM356 did not affect either the binding or the synthesis of these receptors. In summary, the activation of CB1 receptors in hypothalamic nuclei produced the expected decrease in PRL and LH secretion, an effect which might be related to an increase in GABAergic activity in the hypothalamus-anterior pituitary axis. The prolonged activation of these receptors for five days did not elicit tolerance in terms of an attenuation in the magnitude of the decrease in PRL and LH, and, accordingly, did not alter CB1-receptor binding and mRNA levels in the hypothalamic nuclei examined.

Animals↗