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Biomedical subjects

H Akil

Publications and source records attributed to H Akil.

At least 127 records · Page 7Linked to original sources
PubMed1994

Primary structure and functional expression of a guinea pig kappa opioid (dynorphin) receptor.

A full-length cDNA encoding the guinea pig kappa opioid (dynorphin) receptor has been isolated. The deduced protein contains 380 aa and seven hydrophobic alpha-helices characteristic of the G protein-coupled receptors. This receptor is 90% identical to the mouse and rat kappa receptors, with the greatest level of divergence in the N-terminal region. When expressed in COS-7 cells, the receptor displays high affinity and stereospecificity toward dynorphin peptides and other kappa-selective opioid ligands such as U50, 488. It does not bind the mu- and delta-selective opioid ligands. The expressed receptor is functionally coupled to G protein(s) to inhibit adenylyl cyclase and Ca2+ channels. The guinea pig kappa receptor mRNA is expressed in many brain areas, including the cerebellum, a pattern that agrees well with autoradiographic maps of classical guinea pig kappa binding sites. Species differences in the pharmacology and mRNA distribution between the cloned guinea pig and rat kappa receptors may be worthy of further examination.

Amino Acid Sequence↗
PubMed1994

mu-Opioid receptor mRNA expression in the rat CNS: comparison to mu-receptor binding.

The distribution of cells expressing mu-receptor mRNA and mu-receptor binding sites were compared in brain and spinal cord tissue sections using a combination of in situ hybridization and receptor autoradiographic techniques. mu-Receptor mRNA was visualized with a 35S-labeled cRNA probe directed to transmembrane III-VI of the rat mu-receptor, while mu-receptor binding sites were labeled with the mu-selective ligand [3H]DAMGO. A high correspondence between the mu-receptor mRNA and receptor binding distributions was observed in the nucleus of the accessory olfactory bulb, anterior olfactory nuclei, striatal patches of the nucleus accumbens and caudate-putamen, endopiriform nucleus, claustrum, diagonal band of Broca, globus pallidus, ventral pallidum, bed nucleus of stria terminalis, most thalamic nuclei, medial and posteriocortical medial amygdala, lateral, dorsomedial, posterior and mammillary nuclei of the hypothalamus, presubiculum, subiculum, rostral interpeduncular nucleus, median raphe, inferior colliculus, parabrachial nucleus, locus coeruleus, central grey, nucleus ambiguus, nucleus of the solitary tract, nucleus gracilis, nucleus cuneatus, and the dorsal motor nucleus of vagus. Differences in mu-receptor mRNA and receptor binding distributions were observed in several regions, including the olfactory bulb, cortex, hippocampus, superior colliculus, spinal trigeminal nucleus, cochlear nucleus and spinal cord, and may be due to mu-receptor transport to presynaptic terminals.

Animals↗
PubMed1994

Inhibition of opiate tolerance by non-competitive N-methyl-D-aspartate receptor antagonists.

Our laboratory and others have previously reported that the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, interferes with the development of tolerance to the analgesic effects of morphine. The present studies were performed in order to further characterize the role of NMDA receptors in opiate tolerance. The results demonstrate that opiate tolerance is inhibited rapidly, and at low doses, by four different non-competitive NMDA receptor antagonists (MK-801, ketamine, dextrorphan and phencyclidine), suggesting that this inhibition results from blockade of NMDA receptors rather than from the 'side-effect' of a particular drug. The NMDA antagonists were found to inhibit the development but not the expression of opiate tolerance; i.e. they were able to prevent but not reverse tolerance. Finally, the results suggest that NMDA receptor antagonists do not interfere with associative tolerance; instead it appears that these drugs may specifically inhibit non-associative tolerance. It thus appears that NMDA receptors may have a fundamental role in the development of opiate tolerance, and that non-competitive NMDA receptor antagonists may be effective adjuncts to opiates in the treatment of chronic pain.

Adaptation, Psychological↗
PubMed1994

Processing of prodynorphin by the prohormone convertase PC1 results in high molecular weight intermediate forms. Cleavage at a single arginine residue.

Processing of rat prodynorphin (proDyn) by the mouse prohormone convertase PC1 was investigated. Recombinant vaccinia virus vectors were used to coexpress proDyn and PC1 in rat PC12 pheochromocytoma and mouse AtT-20 corticotroph cells. In vitro experiments were also conducted by co-incubating purified proDyn and PC1. The results demonstrate that PC1 cleaves proDyn at pairs of basic residues to yield 10 and 16 kDa high molecular weight (HMW) intermediates. Additionally, PC1 cleaves proDyn at a single arginine residue to yield an 8 kDa product and the C-peptide. This demonstrates that PC1 cleaves proDyn at single and pairs of basic residues.

Amino Acid Sequence↗
PubMed1994

Increased evening activation of the hypothalamic-pituitary-adrenal axis in depressed patients.

OBJECTIVE: To determine whether depressed patients demonstrate hypothalamic-pituitary-adrenal (HPA) axis activation during the late afternoon and evening, a time when the HPA axis is usually quiescent in normal subjects. METHODS: We administered metyrapone, an 11-beta-hydroxylase inhibitor of cortisol synthesis, to normal controls and depressed patients between 4 and 10 PM. Metyrapone blockade of cortisol secretion would amplify any HPA axis secretion. RESULTS: In 10 normal control subjects, administration of metyrapone lowered plasma cortisol levels to a mean of 36 nmol/L. No rebound corticotropin or beta-endorphin secretion was seen in these normal controls between 4 and 10 PM, supporting the existence of a period of minimal endogenous corticotropin releasing factor drive. Compared with a group of placebo-treated depressed patients (n = 10), metyrapone-treated depressed subjects (n = 17) had significantly decreased plasma cortisol concentrations. However, in contrast to normal controls treated with metyrapone, metyrapone-treated depressed patients demonstrated rebound corticotroph secretion, particularly between 7:30 and 10 PM (P = .036 for patients vs normal controls for beta-endorphin secretion from 4:30 to 10 PM). CONCLUSION: These data support the hypothesis of increased corticotropin releasing factor drive in the evening in depressed subjects and are in agreement with the longstanding observation of "early escape" from dexamethasone suppression between 4 and 11 PM in depressed patients.

Adrenocorticotropic Hormone↗
PubMed1994

Kappa 1 receptor mRNA distribution in the rat CNS: comparison to kappa receptor binding and prodynorphin mRNA.

Three opioid receptor types have been identified in the CNS and periphery that are referred to as mu, delta, and kappa. The present study examines the mRNA distribution of the kappa 1 receptor in the rat brain and compares it to the distribution of kappa receptor-binding sites and prodynorphin mRNA using a combination of in situ hybridization and receptor autoradiographic techniques. kappa 1 receptor mRNA was localized with a cRNA probe generated with a BamHI-HindIII cDNA fragment of the rat kappa 1 receptor and corresponds to the last 45 bp of the protein coding region and 728 nucleotides of the 3' untranslated region. Prodynorphin mRNA was localized with a cRNA probe corresponding to a 733-bp BamHI-HincII fragment of prodynorphin. kappa receptor-binding sites were labeled in one of two ways: [3H]U69,593 or [3H]bremazocine in the presence of a 300-fold excess of DAMGO and DPDPE. A high degree of correspondence between the kappa 1 receptor mRNA and kappa receptor binding was observed in several brain regions, including the endopiriform nucleus, claustrum, nucleus accumbens, olfactory tubercle, bed nucleus of the stria terminalis, medial preoptic area, paraventricular, supraoptic, suprachiasmatic, dorsomedial and ventromedial hypothalamic nuclei, basolateral, medial and cortical amygdaloid nuclei, midline thalamic nuclei, periaqueductal grey, parabrachial nucleus, locus coeruleus, and the nucleus of the solitary tract. Differences in the localization of kappa 1 receptor mRNA and binding and the relationship between the distribution of kappa 1 receptor and prodynorphin mRNAs are discussed.

Animals↗
PubMed1994

The role of the hippocampal mineralocorticoid and glucocorticoid receptors in the hypothalamo-pituitary-adrenal axis of the aged Fisher rat.

The aging process has been frequently associated with hippocampal neurodegeneration, loss of corticosteroid receptors, and, at the same time, dysfunction of the hypothalamo-pituitary-adrenal (HPA) axis. We were interested in characterizing simultaneously the activity of the HPA axis and status of both corticosteroid receptors (mineralocorticoid or MR and glucocorticoid or GR) in the hippocampus of aged male Fisher-344 rats. We compared intact, adrenalectomized (ADX), and corticosterone-replaced ADX young (5-6 months) and old (26-27 months) rats, examining all the parameters in the same animals. Aged rats exhibited an unaltered basal rhythm and initial corticosterone response to restraint stress. However, the same old animals showed a delayed turn-off of the stress response and did so at different points of the corticosterone circadian cycle. The aged hippocampus showed a 40-50% lower MR and GR binding under all the conditions studied. This aging effect was not attributable to changes in the kinetics, affinity, or nuclear translocation of MR or GR. Intact aged rats exhibited also a 30-40% reduction of hippocampal MR and GR steady-state mRNA levels. Interestingly, after 36 h ADX only the aged hippocampus showed upregulation of MR and GR mRNA content to levels comparable to those in young rats. However, this increase in MR and GR mRNA content was not accompanied by a proportional increase in the Bmax of these receptors, suggesting age-related translational or post-translational alterations. Moreover, corticosterone replacement was able to reverse the ADX-induced increase of MR and GR Bmax in young and old hippocampi but it only reversed the upregulated mRNA levels of MR (and not GR) in the older group. The fact that corticosterone was able to modulate the biosynthetic rate of MR and GR strongly suggests that the decrease of receptors is functional and not simply due to cell death in the aged hippocampus. We propose that in the aged Fisher rat the loss of hippocampal corticosteroid receptors is previous to any change in the circadian rhythm of circulating corticosterone. Furthermore, the altered turn-off of the corticosterone stress response observed in the same animals may be related to the reduction of functional MR and GR but it is not due to high basal levels of corticosterone.

Adrenalectomy↗
PubMed1994

Cloning and expression of the A2a adenosine receptor from guinea pig brain.

A full-length complementary DNA (cDNA) clone encoding the guinea pig brain A2 adenosine receptor has been isolated by polymerase chain reaction (PCR) and low-stringency-hybridization screening of a guinea pig brain cDNA library. This cDNA contains a long open reading frame encoding a 409-amino acid-residue protein which is highly homologous to the A2 adenosine receptors previously cloned from other species. Hydrophobicity analysis of the deduced protein sequence reveals seven hydrophobic regions, characteristic of a member of the G-protein-coupled receptor superfamily. Radioligand binding assay and functional (GTPase and cAMP) assays of the receptor, transiently expressed in mammalian cells, demonstrate typical characteristics of the A2 type adenosine receptor. The messenger RNA (mRNA) of this A2 receptor is found in the brain, heart, kidney and spleen. Receptor autoradiography with [3H]CGS21680, a specific A2 agonist, and in situ hybridization with A2 cRNA probe in guinea pig brain indicate that the receptor is expressed exclusively in the caudate nucleus. The pharmacological profile and anatomical distribution of this receptor indicate that it is of the A2a subtype. This work represents the first cloning of an A2a receptor in a rodent species, offers a complete pharmacological characterization of the receptor and provides an anatomical comparison between binding profile and gene expression of the receptor.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of a pharmacologically distinct A1 adenosine receptor from guinea pig brain.

Three full-length cDNA clones encoding the guinea pig A1 adenosine receptor have been isolated by polymerase chain reaction (PCR) and low-stringency hybridization screening of a guinea pig brain cDNA library. These three cDNAs, though differing in their 5' untranslated regions, contain the same open reading frame encoding a 326 amino acid residue protein with seven hydrophobic alpha-helices long enough to form the transmembrane domains, suggesting that it belongs to the G protein-coupled receptor superfamily. This protein is highly homologous to the A1 adenosine receptors previously cloned from other species. Pharmacological characterization of this receptor transiently expressed in mammalian cells demonstrates that, despite its high homology to A1 adenosine receptors of other species, the guinea pig A1 adenosine receptor displays a unique pharmacological profile: high affinity for the A1-selective antagonist CPX, yet very low affinity for some A1-selective agonists such as CCPA, CHA and R-PIA. Northern blotting for different guinea pig tissues and in situ hybridization for guinea pig brain sections reveal an abundant and broad distribution of mRNA of this A1 subtype receptor in the brain.

Adenosine↗
PubMed1994

Regulation of hippocampal 5-HT1A receptor gene expression by dexamethasone.

The effect of dexamethasone, a selective GR agonist, on hippocampal, 5-HT1A receptor mRNA expression and 5-HT1A binding was examined using in situ hybridization histochemistry and in vitro receptor autoradiography. One week after adrenalectomy, both 5-HT1A receptor mRNA expression and 5-HT1A binding were increased throughout the hippocampus. Administration of dexamethasone at the time of adrenalectomy significantly attenuated the increases in 5-HT1A mRNA expression in all hippocampal subfields (p < .05, Fisher Test), although 5-HT1A mRNA levels remained significantly higher than sham levels in all subfields with the exception of CA1. However, 5-HT1A binding levels were responsive to dexamethasone administration only within particular hippocampal subfields, CA1, and dentate gyrus. We conclude that GR occupation negatively regulates 5-HT1A receptor mRNA expression within the hippocampus and that 5-HT1A receptor sites are most sensitive to modulation in those hippocampal subfields expressing higher levels of GR receptors.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1993

Cloning and pharmacological characterization of a rat kappa opioid receptor.

A full-length cDNA was isolated from a rat striatal library by using low-stringency screening with two PCR fragments, one spanning transmembrane domains 3-6 of the mouse delta opioid receptor and the other unidentified but homologous to the mouse delta receptor from rat brain. The novel cDNA had a long open reading frame encoding a protein of 380 residues with 59% identity to the mouse delta receptor and topography consistent with a seven-helix guanine nucleotide-binding protein-coupled receptor. COS-1 cells transfected with the coding region of this clone showed high-affinity binding to kappa opioid receptor-selective ligands such as dynorphin A and U-50,488 and also nonselective opioid ligands such as bremazocine, ethylketocyclazocine, and naloxone. Not bound at all (or bound with low affinity) were dynorphin A-(2-13), enantiomers of naloxone and levophanol [i.e., (+)-naloxone and dextrorphan], and selective mu and delta opioid receptor ligands. Activation of the expressed receptor by kappa receptor agonists led to inhibition of cAMP. Finally, in situ hybridization revealed a mRNA distribution in rat brain that corresponded well to the distribution of binding sites labeled with kappa-selective ligands. These observations indicate that we have cloned a cDNA encoding a rat kappa receptor of the kappa 1 subtype.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1993

A bacterially expressed mineralocorticoid receptor is associated in vitro with the 90-kilodalton heat shock protein and shows typical hormone- and DNA-binding characteristics.

A recombinant system was developed for generation of steroid-receptor complexes in vitro. The DNA- and steroid-binding domains of the rat mineralocorticoid receptor were expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The identity of the expressed recombinant protein was confirmed by Western blot analysis. Protein preparations purified by affinity chromatography, avoiding the use of detergents or high ionic strength buffers, exhibited negligible steroid binding. However, after incubation of these preparations with rabbit reticulocyte lysate, known to promote the association of isolated steroid receptors with heat shock proteins, the [3H]aldosterone-binding activity gradually increased. This temperature-dependent effect reached a maximum after 1 h at 30 degrees C and was favored by ATP supplementation (Bmax = 22 +/- 3 pmol/mg of protein). The apparent Kd value for aldosterone (0.6 +/- 0.2 nM) and the steroid-binding specificity of the recombinant protein were in accordance with those reported for the native mineralocorticoid receptor. The sedimentation and DNA-cellulose-binding characteristics of the radioactive complexes were also in agreement with those reported for the native heteromeric receptor. Complexes sedimented at 8.9 +/- 0.2 or 4.2 +/- 0.2 S in sucrose gradients containing 20 mM sodium molybdate or 0.4 M KCl, respectively. Monoclonal antibody 8D3 against the 90-kDa heat shock protein (hsp90) was able to bind to the 8.9S complexes, increasing its sedimentation coefficient. Treatment of the complexes with 100 mM sodium thiocyanate, known to activate the native receptor to a DNA-binding state, caused a 79% increase in DNA-cellulose binding over the control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldosterone↗
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

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

Characterization of pro-opiomelanocortin processing in heterologous neuronal cells that express PC2 mRNA.

We have investigated processing of monkey pro-opiomelanocortin (POMC) following transfection into heterologous neuronal Neuro 2A (N2A) cells. In several separately transfected stable cell lines (termed N2A/POMC2-like; n = 4), POMC was processed to beta E only, by direct cleavage from the precursor. Thus, these cell lines did not produce beta E in the orderly manner observed in the pituitary, that is, via the intermediate peptide beta LPH. Analysis of one representative N2A/POMC2 cell line revealed that the extent of processing to beta E appeared to be negatively correlated with precursor expression level, suggesting that the processing enzyme(s) in these cells was present in limiting amounts. Northern analysis of PC1 and PC2, two recently cloned processing enzymes, showed that N2A/POMC2 cells expressed low levels of PC2 mRNA, but no detectable PC1 mRNA. These data suggest that (1) the order of processing observed in the pituitary is not exclusively determined by tertiary folding of the precursor, but rather by the complement of processing enzymes in a particular cell, and (2) if PC2 is responsible for POMC processing in N2A/POMC2 cells, this enzyme, expressed in limiting amounts, appeared to show selectivity for the beta E amino terminal processing site.

Animals↗
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↗