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S J Watson

Publications and source records attributed to S J Watson.

At least 163 records · Page 9Linked to original sources
PubMed1993

Molecular cloning, expression, and gene localization of a fourth melanocortin receptor.

The recent cloning of three melanocortin receptors suggests an unexpected diversity in this family of seven transmembrane G-protein linked receptors. Herein, we report the cloning, expression, and gene localization of a fourth human melanocortin receptor, the melanocortin-4 receptor. By Northern blot analysis and in situ hybridization, this receptor is expressed primarily in the brain, but its expression is notably absent in the adrenal cortex, melanocytes, and placenta. Agonist stimulation of COS-1 cells transiently transfected and L-cells permanently transfected with the coding region of the cloned melanocortin-4 receptor leads to increases in intracellular cyclic 3',5'-adenosine monophosphate. The profile of the responses of the melanocortin-4 receptor to different melanocortins distinguishes it from melanocortin receptors previously described. Using the technique of fluorescent in situ hybridization, the gene encoding the melanocortin-4 receptor was localized to chromosome 18 (q21.3).

Adrenocorticotropic Hormone↗
PubMed1993

Ventral subicular interaction with the hypothalamic paraventricular nucleus: evidence for a relay in the bed nucleus of the stria terminalis.

The axonal projections of the ventral subiculum to the bed nucleus of the stria terminalis (BST) were examined in the rat with the anterograde neuronal tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). Axons originating in the ventral subiculum coursed to the BST through either the fimbria-fornix, or a pathway involving the stria terminalis via the amygdala. Ventral subicular axons gave rise to dense terminal networks that were preferentially distributed in medial and ventral subregions of the BST. The distribution of subicular fibers and terminals was examined in relation to BST neurons that project to the hypothalamic paraventricular nucleus (PVN). In these cases, discrete iontophoretic injections of the retrograde tracer Fluoro-gold were made in the PVN, with PHA-L delivered to the ipsilateral ventral subiculum. An immunocytochemical double-labeling protocol was then employed for the simultaneous detection of PHA-L and Fluoro-gold, and provided light microscopic evidence for subicular input to PVN-projecting cells located within the BST. In a second series of experiments, the gamma-amino butyric acid (GABA)ergic nature of the BST was examined by in situ hybridization histochemistry for detection of transcripts encoding GAD67 mRNA. The studies revealed that a high proportion of BST neurons express GAD67 transcripts. Also, experiments combining Fluoro-gold tracing with GAD67 in situ hybridization suggested that a proportion of PVN-projecting neurons in the BST are GABAergic. Taken together, the results of these sets of studies suggest that the inhibitory influences of the hippocampus on the PVN might be relayed through specific portions of the BST. These findings may have important implications for our understanding of the neural regulation of the hypothalamic-pituitary-adrenal axis.

Amygdala↗
PubMed1993

Molecular cloning of a novel melanocortin receptor.

Using the technique of the polymerase chain reaction primed with oligonucleotides based on the homologous transmembrane regions of seven transmembrane G protein-linked receptors, we isolated three full-length human genes that encode a novel subgroup of this receptor family. Recently, two of these receptors were identified as specific for alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropic hormone. We report the molecular cloning and pharmacologic characterization of a third member of this subgroup. The gene for this receptor encodes a protein of 361 amino acids in length. Its pharmacology characterizes it as an MSH receptor specific to the heptapeptide core common to adrenocorticotropic hormone and alpha-, beta-, and gamma-MSH. By Northern blot hybridization and polymerase chain reaction, it is expressed in brain, placental, and gut tissues but not in melanoma cells or in the adrenal gland. These findings may yield insight into the physiology of peptides derived from pro-opiomelanocortin post-translational processing.

Amino Acid Sequence↗
PubMed1993

Multiple HPA profiles in endogenous depression: effect of age and sex on cortisol and beta-endorphin.

We have previously shown that a number of depressed patients demonstrated a failure to suppress corticotrophic secretion, as measured by beta-Endorphin/beta-Lipotropin (beta-End/beta-LPH levels), following dexamethasone challenge. The current study is an extension and replication of these findings, as well as an analysis of some of the biological variables which may contribute to the variance in beta-End/beta-LPH nonsuppression. We continue to observe a high rate of beta-End/beta-LPH nonsuppression in depressed patients following dexamethasone; this escape at the pituitary level is even observed in a number of patients who demonstrate normal cortisol suppression. Advancing age, particularly in women, led to higher baseline cortisol, lower baseline beta-End/beta-LPH, and a greater likelihood of being a nonsuppressor on one or both measures.

Adrenocorticotropic Hormone↗
PubMed1993

Region specific expression of furin mRNA in the rat brain.

The distribution of furin mRNA was examined in the rat central nervous system. Northern blot analysis reveals the presence of a 4.4 kb band in all brain tissues examined. In situ hybridization analysis of frozen rat brain sections using a radioactively labeled antisense cRNA probe to rat furin demonstrated moderate to low levels of expression in both neuronal and non-neuronal tissue in all areas examined. Interestingly, higher levels of furin were expressed in selective regions which include the ventricles (the choroid plexus and ependymal cells), the islands of Calleja, the hippocampus and the pineal gland. the ubiquitous localization of furin in the brain is consistent with its postulated role as a vital convertase important in the processing of proproteins negotiating the constitutive pathway of secretion. However, the higher expression of furin mRNA in distinct brain areas suggests a more active role in the processing of proproteins synthesized in these tissues.

Animals↗
PubMed1993

Dissociation between pituitary and adrenal suppression to dexamethasone in depression.

OBJECTIVE: To determine if corticotroph nonsuppression, as reflected by beta-endorphin nonsuppression, occurs before cortisol nonsuppression (defined as a cortisol level of > 140 nmol/L) when examining multiple time points in a day. SETTING: The General Medical Clinical Research Center and Inpatient Depression Research Unit, Ann Arbor, Mich. DESIGN: Multiple blood samples were obtained through an intravenous catheter around the time points of 8 AM, noon, and 4 PM and assayed for beta-endorphin and cortisol. PATIENTS: Patients meeting Research Diagnostic Criteria for the diagnosis of major depressive disorder, primary and simple. A total of 73 subjects, both inpatients and outpatients, were studied. INTERVENTION: Samples were obtained both at baseline and 1 day after administration of 1 mg of dexamethasone at 11:30 PM. MEASUREMENTS AND RESULTS: Overall 39 patients (53%) demonstrated beta-endorphin nonsuppression after administration of dexamethasone at any of the three time points, while only eight patients (11%) demonstrated cortisol nonsuppression at any of these time points. Cortisol nonsuppression, but not beta-endorphin nonsuppression, was associated with lower concentrations of dexamethasone in plasma. Baseline cortisol and menopausal status were significantly associated with beta-endorphin nonsuppression in women.

Adult↗
PubMed1993

Cloning and pharmacological characterization of a rat mu opioid receptor.

We have isolated a rat cDNA clone that displays 75% amino acid homology with the mouse delta and rat kappa opioid receptors. The cDNA (designated pRMuR-12) encodes a protein of 398 amino acids comprising, in part, seven hydrophobic domains similar to those described for other G protein-linked receptors. Data from binding assays conducted with COS-1 cells transiently transfected with a CMV mammalian expression vector containing the full coding region of pRMuR-12 demonstrated mu receptor selectivity. In situ hybridization mRNA analysis revealed an mRNA distribution in rat brain that corresponds well to the distribution of binding sites labeled with mu-selective ligands. Based upon these observations, we conclude that pRMuR-12 encodes a mu opioid receptor.

Amino Acid Sequence↗
PubMed1993

Altered ratios of beta-endorphin: beta-lipotropin released from anterior lobe corticotropes with increased secretory drive. I. Effects of diminished glucocorticoid secretion.

Previous studies have demonstrated that acute stress or ovine corticotropin-releasing hormone (oCRH) in vivo, or oCRH in vitro, stimulates release of beta-endorphin over beta-lipotropin from anterior pituitary corticotropes. This occurs despite the predominance of beta-lipotropin in corticotrope peptide stores. In vitro studies with primary anterior pituitary cultures suggested that chronic exposure to oCRH results in a shift towards more beta-lipotropin secretion into the media than with short-term exposure. The current studies explored whether increased secretory drive in vivo results in a similar shift towards more beta-lipotropin. We used removal of glucocorticoids by adrenalectomy or metyrapone blockade of corticosterone synthesis, to stimulate endogenous secretion of CRH and vasopressin. Both treatments resulted in shifts of the ratio of beta-endorphin: beta-lipotropin in plasma of experimental animals in comparison to the sham-treated control rats. In vitro testing with oCRH of anterior lobe cultures from adrenalectomized or metyrapone-treated rats demonstrated similar effects of these treatments on the ratio of beta-endorphin:beta-lipotropin. These changes occurred despite similar ratios of beta-endorphin:beta-lipotropin in anterior pituitary peptide stores.

Adrenalectomy↗
PubMed1993

Altered ratios of beta-endorphin:beta-lipotropin released from anterior lobe corticotropes with increased secretory drive. II. Repeated stress.

A number of stimuli including acute footshock and electrically-induced seizures lead to release of beta-endorphin immunoreactivity from the anterior pituitary corticotropes. Gel filtration of this beta-endorphin immunoreactivity indicates that approximately 3-fold more beta-endorphin than beta-lipotropin is released into plasma following these acute stressors. A similar preponderance of beta-endorphin over beta-lipotropin is seen in the media of short-term anterior lobe cell suspensions stimulated with ovine corticotropin-releasing hormone. Previous studies indicated that footshock stress, when administered repeatedly, can increase the biosynthesis of anterior lobe proopiomelanocortin (POMC) as indicated by increased steady state adrenocorticotropin/beta-endorphin content as well as increased POMC mRNA levels and increased POMC biosynthesis and rate of processing as measured by pulse-labeling and pulse-chase studies. The goal of the present studies was to determine whether this increased biosynthetic drive results in an alteration in the end products secreted with repeated stress. Acute footshock in a rat which has received 14 days of chronic footshock releases proportionately more beta-lipotropin than is released in a naive rat. Chronic electrically-induced seizures, which also increase anterior lobe POMC derived peptide stores, lead to a similar shift in the ratio of beta-lipotropin:beta-endorphin released following stress. These data suggest that chronic drive and the subsequent changes in POMC peptide stores may lead to a decrease in the proportion of beta-endorphin size immunoreactivity in the releasable pool of the anterior lobe corticotrope, thus altering the hormonal signal from the anterior lobe corticotrope.

Adrenocorticotropic Hormone↗
PubMed1993

Diurnal CRH mRNA rhythm in the hypothalamus: decreased expression in the evening is not dependent on endogenous glucocorticoids.

Levels of hypothalamic corticotropin-releasing hormone (CRH) mRNA and plasma glucocorticoids vary diurnally as a result of circadian influences on the hypothalamopituitary-adrenal axis. CRH mRNA expression increases from morning to afternoon in rats but decreases rapidly near the onset of dark as glucocorticoids reach peak concentrations in plasma. Since glucocorticoids are normally inhibitory on hypothalamic CRN mRNA expression, we determined whether the glucocorticoid secretion at the diurnal peak reduced CRH mRNA concentration in the evening. We found that adrenalectomy did not prevent the decrease in CRH mRNA levels near the onset of dark. It appears that the drop in CRH mRNA expression occurs via a steroid-independent mechanism. While the mean CRH mRNA level increased after adrenalectomy, the shape of the CRH mRNA rhythm remained unchanged except in the morning. Interestingly, adrenalectomy increased CRH mRNA levels disproportionately in the morning, producing a sharp rise followed by a plateau during the light phase instead of the gradual rise observed in intact animals. We subsequently treated adrenalectomized animals with corticosterone pellets to determine whether a constant steroid signal was sufficient in restoring the normal shape of the mRNA rhythm during the light phase. Results indicate that the endogenous steroid rhythm is not necessary for generating the normal CRH mRNA rhythm during the light phase. Instead, a constant exposure to corticosterone at approximately 50% of the daily mean (2.4-3 micrograms/dl) appears to be sufficient for regulation of the mRNA rhythm.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗
PubMed1993

5'-Heterogeneity of the mineralocorticoid receptor messenger ribonucleic acid: differential expression and regulation of splice variants within the rat hippocampus.

The mineralocorticoid receptor (MR) cDNA we previously isolated from the rat hippocampus differs from the clone isolated from the kidney at the 5'-untranslated (5'UT) region. The kidney clone (alpha MR mRNA) and the hippocampal clone (beta MR mRNA) possess unique 5'UT sequences of 220 and 300 nucleotides, respectively, but share an invariant peptide-coding domain and appear to encode an identical MR protein. The two mRNA variants may represent tissue-specific forms of the MR or may be coexpressed in the rat hippocampus along with other 5'UT variants. Here, we report that three mRNA subtypes were found in the hippocampus; their relative abundance was as follows: alpha = beta >> gamma. The three mRNA variants were differentially distributed within the hippocampal subfields, with the alpha form being highly enriched in CA2, dentate, the fasciculum cinereum, and the indusium griseum, whereas beta and gamma forms were evenly distributed through CA1-4. Adrenalectomy selectively increased alpha MR mRNA content, but the changes were restricted to CA1, CA2, and CA3 regions. We conclude that multiple MR mRNAs are differentially expressed in the rat hippocampus. The expression of alpha MR mRNA is specifically increased during adrenalectomy, suggesting that the increase in total MR mRNA content documented previously arises from a substantial increase in a single MR variant that elevates the total MR mRNA content, with the apparent elevation reflecting the average of regulated and unregulated transcripts. It is suggested from our data that a complex mechanism involving transcription and translation regulates MR expression in the rat hippocampus.

Adrenalectomy↗
PubMed1993

Gene expression of prohormone and proprotein convertases in the rat CNS: a comparative in situ hybridization analysis.

Posttranslational processing of proproteins and prohormones is an essential step in the formation of bioactive peptides, which is of particular importance in the nervous system. Following a long search for the enzymes responsible for protein precursor cleavage, a family of Kexin/subtilisin-like convertases known as PC1, PC2, and furin have recently been characterized in mammalian species. Their presence in endocrine and neuroendocrine tissues has been demonstrated. This study examines the mRNA distribution of these convertases in the rat CNS and compares their expression with the previously characterized processing enzymes carboxypeptidase E (CPE) and peptidylglycine alpha-amidating monooxygenase (PAM) using in situ hybridization histochemistry. Furin mRNA was ubiquitously distributed and detected both in neurons and non-neuronal tissue throughout the brain with a higher abundance in ependyma, the circumventricular organs, the islands of Calleja, hippocampus, and allocortex. The cellular localization of PC1 and PC2 was exclusively neuronal with highest concentrations in known neuropeptide-rich brain regions. In general, PC2 was more widely expressed than PC1 in the CNS, although many regional variations were detected. The identification of specific combinations of convertase expression together with CPE and PAM expression in neuropeptide-rich brain regions suggests that specific enzymatic pathways are involved in neuropeptide precursor processing, and that these specific combinations are responsible for region-specific differences of posttranslational processing.

Animals↗
PubMed1993

Corticosteroids regulate brain hippocampal 5-HT1A receptor mRNA expression.

Using in situ hybridization techniques, the expression of 5-HT1A receptor mRNA was measured within the hippocampal formation after bilateral adrenalectomy (ADX). After 24 hr ADX, 5-HT1A receptor mRNA expression was significantly increased in all hippocampal subfields in ADX animals relative to sham-operated controls (SHAM). The magnitude of the increase was most pronounced within CA2 (127%) and CA3/4 (94%)-subfields of dorsal hippocampus, intermediate in the dentate gyrus (73%), and least within CA1 (60%). Administration of exogenous corticosterone (CORT) at the time of ADX maintained the level of 5-HT1A receptor mRNA expression within the range of SHAM animals. In vitro receptor autoradiographic analysis of 5-HT1A receptors in adjacent sections from the same animals indicated a simultaneous increase in 5-HT1A binding throughout the hippocampus in response to ADX. 5-HT1A binding increased to a similar extent (approximately 30%) in CA subfields and dentate gyrus but remained within SHAM levels in CORT-replaced animals. 5-HT1A receptor mRNA levels were also increased in hippocampal subregions of 1 week ADX animals relative to SHAM animals. Within both CA1 and CA2 subfields, the increments were approximately double those observed after 1 d ADX. 5-HT1A receptor binding was increased in every hippocampal subfield to a similar extent as that observed after 1 d ADX. Increases in both 5-HT1A receptor mRNA expression and 5-HT1A receptor binding were preventable by administration of exogenous CORT at the time of ADX. Hippocampal 5-HT1C receptor mRNA and D1 receptor mRNA expression were not significantly altered by either acute or chronic ADX treatment. These data indicate that adrenal steroids may selectively regulate hippocampal 5-HT1A receptors at the level of 5-HT1A receptor mRNA expression.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1992

Distribution of D5 dopamine receptor mRNA in rat brain.

The distribution of the messenger RNA encoding the dopamine D5 receptor was determined in the rat brain by in situ hybridization. Using [35S]-labelled riboprobes to either the rat or human D5 receptor, this mRNA was localized to the hippocampus and the parafascicular nucleus of the thalamus. This mRNA could not be visualized in the more traditional brain regions associated with dopaminergic cell bodies or projection fields. This unusual distribution suggests a novel function in the brain for this subtype of the dopamine receptor.

Animals↗
PubMed1992

Selective forebrain fiber tract lesions implicate ventral hippocampal structures in tonic regulation of paraventricular nucleus corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) mRNA expression.

The hippocampus appears to be involved in tonic regulation of the hypothalamo-pituitary-adrenocortical axis via interactions with corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP)-containing neurons of the hypothalamic paraventricular nucleus (PVN). To further investigate the anatomical basis of such interactions, lesions were made to forebrain fiber tracts in position to communicate inhibitory information from the hippocampus to the PVN. Total fimbria-fornix transections (TFF) and lateral fimbria-fornix lesions (LFF) both significantly increased CRH mRNA levels in the medial parvocellular PVN, as assayed by semi-quantitative in situ hybridization histochemistry. Medial fimbria-fornix lesions or section of the medial corticohypothalamic tracts (MCHT) did not influence CRH mRNA levels. The LFF group showed increases in both AVP mRNA and ACTH secretion, whereas no other lesion was effective in this regard. The results suggest: (1) hippocampal efferents conferring tonic inhibition of the HPA axis probably originate in regions contributing to the lateral extent of the fornix, representing structures in the ventral subiculum and ventral extent of CA1; (2) projections from the hippocampus to the medial basal hypothalamus (travelling in the MCHT) are unlikely to affect HPA function; (3) hippocampus may influence the PVN CRH/AVP neuron at multiple levels, in that LFF and TFF lesions have differential effects on PVN AVP mRNA levels and ACTH secretion.

Adrenocorticotropic Hormone↗
PubMed1992

Evidence that beta-endorphin is synthesized in cells in the nucleus tractus solitarius: detection of POMC mRNA.

Evidence from a number of sources indicates that the major site of pro-opiomelanocortin (POMC)-producing cells in the CNS is the arcuate nucleus of the hypothalamus. Using immunocytochemical techniques, a second, smaller group of POMC cells has been detected in the nucleus tractus solitarius (NTS) area of the caudal medulla. However, POMC mRNA has never been reported in the NTS even though it has been found in other extrahypothalamic brain regions. Thus, there is some uncertainty as to whether POMC peptides are actually synthesized de novo in the NTS. In the present study, we used biochemical and anatomical techniques to examine whether POMC mRNA is localized in the NTS. Using in situ hybridization, cells containing POMC mRNA were found in the caudal portion of the NTS. The nucleic acid distribution correlated well with the anatomical distribution of 16k POMC peptide immunoreactivity as determined by immunocytochemistry. Northern analysis revealed that the apparent size of POMC mRNA in the NTS was similar to that found in the arcuate nucleus or the pituitary gland. Results of RNase protection assays using a POMC riboprobe complementary to the 5' end of exon 3 suggested that POMC mRNA in the NTS and arcuate nucleus are identical in this region of the message at least. We also calculated POMC peptide product to mRNA ratios in different tissues and found that NTS cells appear to produce less peptide per mRNA molecule than those in the arcuate nucleus or pituitary gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

A comparison of D1 receptor binding and mRNA in rat brain using receptor autoradiographic and in situ hybridization techniques.

D1, a subtype of the dopamine receptors, is widely distributed in the nervous system and has been shown to be positively coupled to adenylate cyclase. Using a combination of in vitro receptor autoradiographic and in situ hybridization techniques, the present study examines the co-distribution of D1 receptor binding sites and D1 receptor mRNA in adjacent rat brain sections. D1 receptor binding sites were labeled using the selective antagonist [3H](R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzaz epin- 7-ol (SCH23390) (4.6 nM), in the presence of 1 microM ketanserin, while the D1 receptor mRNA was visualized with a 35S-labeled riboprobe corresponding to a region between transmembrane domains III and VI of the rat D1 receptor (base pairs 383-843). Analysis of serial sections suggested a good agreement between D1 receptor binding and mRNA in several brain regions, including the paleocortex, caudate-putamen, nucleus accumbens, amygdala, and suprachiasmatic nucleus. Marked discrepancies between D1 receptor binding and mRNA were observed in other brain regions including the entopeduncular and subthalamic nuclei, substantia nigra (pars reticulata), hippocampus, and cerebellum. While technical considerations may contribute to these results, much of the discordance between the distributions is probably due to the differential localization of D1 receptor mRNA in cell bodies and receptor binding sites on fibers and may provide insights into receptor synthesis, transport, and membrane insertion. In the basal ganglia, for instance, D1 receptors are synthesized in the striatum and are either transported to efferent projections in areas such as the substantia nigra, or remain localized in striatal cells bodies. Ibotenic acid lesions in the striatum are consistent with these conclusions and demonstrate a coordinate loss of D1 receptor binding and mRNA in the caudate-putamen that is accompanied by a degeneration of fibers projecting to substantia nigra and a loss of D1 binding in the pars reticulata. Neurons in the dentate gyrus and in the granular layer of the cerebellum, on the other hand, synthesize D1 receptors and transport them entirely to either their dendritic or axonal fields, respectively, in the molecular layer. This analysis provides a better understanding of dopaminergic receptor systems in the CNS and their anatomical organization.

Animals↗