Biomedical subjects

H Akil

Publications and source records attributed to H Akil.

At least 73 records · Page 4Linked to original sources

PubMed1998

Controlling signaling with a specifically designed Gi-coupled receptor.

We are developing a system to control G protein signaling in vivo to regulate a broad range of physiologic responses. Our system utilizes G protein-coupled peptide receptors engineered to respond exclusively to synthetic small molecule ligands and not to their natural ligand(s). These engineered receptors are designated RASSLs (receptor activated solely by a synthetic ligand). We have made two prototype RASSLs that are based on the human kappa opioid receptor. Small molecule drugs that activate the kappa receptor are nonaddictive and safe to administer in vivo. Binding and signaling assays reveal 200-2000-fold reductions in the ability of our RASSLs to bind or be activated by dynorphin, an endogenous peptide ligand of the kappa opioid receptor. In a high-throughput signaling assay, these prototype RASSLs expressed in Chinese hamster ovary K1 cells showed little or no response to a panel of 21 opioid peptides but still signaled normally in response to small molecule drugs such as spiradoline. Activation of a RASSL by spiradoline also caused proliferation of rat-1a tissue culture cells. These data provide evidence that G protein-coupled receptors can be made into RASSLs. The potential in vivo applications for RASSLs include the positive enrichment of transfected cells and the development of new animal models of disease.

Adenylyl Cyclases↗

PubMed1998

Creating a functional opioid alkaloid binding site in the orphanin FQ receptor through site-directed mutagenesis.

Although much has been learned about the mechanisms of ligand selectivity between different opioid receptor subtypes, little is known about the common opioid binding pocket shared by all opioid receptors. The recently discovered orphanin system offers a good opportunity to study the mechanisms involved in the binding of opioid versus nonopioid ligands. In the current study, we adopt a "gain of function" approach aimed at shifting the binding profile of the orphanin FQ receptor toward that of the opioid receptors. After two rounds of mutagenesis, several orphanin FQ receptor mutants can be labeled with the opiate alkaloid [3H]naltrindole and show greatly increased affinities toward the opiate antagonists naltrexone, nor-binaltrophine HCl, and (-)-bremazocine. These orphanin FQ receptor mutants also display stereospecificity similar to that of opioid receptors. Furthermore, the orphanin FQ receptor mutant that has the best affinities toward the opioid alkaloids shows, in the presence of GTP and high salt concentration, an affinity-shift profile similar to that of the delta receptor. Most strikingly, the same mutant exhibits naltrindole-sensitive etorphine-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding, whereas the effect of etorphine on GTP binding cannot be inhibited by naltrindole in the wild-type receptor. Our results indicate that 1) several residues in the orphanin FQ receptor are critical to its selectivity against the opiate alkaloids, particularly antagonists; and 2) mutating these residues to those of the opioid receptor at the corresponding position preserves the agonist/antagonist nature of opiate alkaloids as they interact with the mutant receptor. It is reasonable to hypothesize that the corresponding residues in the opioid receptors may form a functional common binding pocket for opiate alkaloids. These findings may be helpful to medicinal chemists in designing ligands for the orphanin FQ receptor based on the structure of the opiate alkaloids.

Amino Acid Substitution↗

PubMed1998

Alpha, beta, and gamma mineralocorticoid receptor messenger ribonucleic acid splice variants: differential expression and rapid regulation in the developing hippocampus.

Two different types of corticoid receptor molecules bind circulating corticosterone in brain: mineralocorticoid receptors (MR) and glucocorticoid receptors. MR exhibit the highest affinity for the endogenous glucocorticoid in the rat, corticosterone. During development, low corticosterone levels influence neurogenesis, and these effects are probably MR mediated. Three MR complementary DNA clones, alpha, beta, and gamma, have been identified in the rodent. All of these MR complementary DNA clones have identical coding regions, but differ significantly at the 5'-untranslated end. Although the functional significance of these three messenger RNA (mRNA) species remains unknown, one hypothesis is that they reflect the ability of the brain to regulate the expression of MR, allowing multiple factors to differentially control transcription in a tissue- and time-specific manner. To investigate this possibility, we examined the presence of these distinct mRNA forms in the developing rat hippocampus (HC). In situ hybridization with specific alpha, beta, and gamma complementary RNA probes was performed in the HC of 3-, 5-, 7-, 12-, 14-, 28-, 35-, and 65-day-old animals. We found that there is differential expression of these forms in each of the HC subfields from infancy to adulthood. y expression appears to be associated with periods of cell birth and increased axonal sprouting. beta expression, on the other hand, may be best linked to periods of synaptogenesis, growth of commissural and associative terminal fields, and possibly active pruning. To explore the possibility that the differential gene expression may be related to corticosterone environment, adrenalectomy was performed. A rapid modulation of the MR mRNA variants (14 h) in an age- and site-specific fashion was seen. These findings suggest that the variation in expression and regulation during development of the multiple MR transcripts could reflect a complex pattern of developmental regulation that may involve a multitude of factors unique to each postnatal age and to the different neuronal populations within the hippocampal formation.

Adrenalectomy↗

PubMed1998

The role of mineralocorticoid receptors in hypothalamic-pituitary-adrenal axis regulation in humans.

In rodents, two types of glucocorticoid receptors, the mineralocorticoid (MR; type I) and the glucocorticoid (type II) receptors, have been demonstrated to play a role in hypothalamic-pituitary-adrenal (HPA) axis regulation. Because MR shows a very high affinity for cortisol, it has been suggested that MR plays an important role in restraint of CRH and ACTH secretion during the nadir of the circadian rhythm. Although a number of studies have established the importance of MR in rodents, the functional role of MR in humans has not been determined. These studies evaluated whether spironolactone, an MR antagonist, had a detectable effect on HPA axis regulation in humans, and whether the effect was greatest during the evening, when plasma cortisol concentrations are in the MR range. Compared to the placebo day, after a single dose of spironolactone at either 0800 or 1600 h, there is a significant increase in plasma cortisol, which is preceded by a rise in ACTH and beta-endorphin. A significant effect of spironolactone on cortisol secretion was demonstrated with no differences between the morning and evening. Because the effect of spironolactone on cortisol was short lived, a second experiment was conducted using two doses of spironolactone, again sampling in the morning and evening. After two doses of spironolactone, plasma cortisol levels showed a significant and sustained spironolactone-induced elevation for the entire sampling period. However, neither plasma beta-endorphin nor ACTH was increased compared to levels on the placebo day. These data suggest that MR appear to play a clear role in HPA axis regulation during the time of the circadian peak as well as the trough. Furthermore, MR blockade may affect the sensitivity of the adrenal to ACTH.

Adrenal Glands↗

PubMed1998

Nitric oxide in the stress axis.

In recent years nitric oxide (NO) has emerged as a unique biological messenger. NO is a highly diffusible gas, synthesized from L-arginine by the enzyme nitric oxide synthase (NOS). Three unique subtypes of NOS have been described, each with a specific distribution profile in the brain and periphery. NOS subtype I is present, among other areas, in the hippocampus, hypothalamus, pituitary and adrenal gland. Together these structures form the limbic-hypothalamic-pituitary-adrenal (LHPA) or stress axis, activation of which is one of the defining features of a stress response. Evidence suggests that NO may modulate the release of the stress hormones ACTH and corticosterone, and NOS activity and transcription is increased in the LHPA axis following various stressful stimuli. Furthermore, following activation of the stress axis, glucocorticoids are thought to down-regulate the transcription and activity of NOS via a feedback mechanism. Taken together, current data indicate a role for NO in the regulation of the LHPA axis, although at present this role is not well defined. It has been suggested that NO may act as a cellular communicator in plasticity and development, to facilitate the activation or the release of other neurotransmitters, to mediate immune responses, and/or as a vasodilator in the regulation of blood flow. In the following review we summarize some of the latest insights into the function of NO, with special attention to its relationship with the LHPA axis.

Animals↗

PubMed1998

Stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate binding by endogenous opioids acting at a cloned mu receptor.

The ability of endogenous opioids to activate G proteins was measured in membranes from C6 rat glioma cells stably expressing a cloned rat mu receptor. Peptides representing each of the three known families of endogenous opioids (enkephalins, endorphins and dynorphins) were studied, as well as two recently discovered endogenous opioids, endomorphin-1 and -2, which are thought to represent a fourth family of endogenous opioid peptides. Stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding to membranes was used as a measure of G protein activation. It was possible to differentiate high efficacy compounds such as Tyr-D-Ala-Gly-(Me)Phe-Gly-ol from lower-efficacy agonists such as morphine or meperidine. Met- and leu-enkephalin, beta endorphin and dynorphin A were all found to have high efficacy at the mu receptor, as were the peptide fragments beta endorphin-1(1-27) and dynorphin A-(1-13). Endomorphin-1 and -2 were found to be partial agonists, capable of both stimulating [35S]GTP gamma S binding and antagonizing the stimulation produced by the higher-efficacy agonist Tyr-D-Ala-Gly-(Me)Phe-Gly-ol. Binding affinities for the opioid agonists at the cloned mu receptor were measured by the displacement of radiolabeled antagonist. It was found that the Ki values closely matched the EC50 values for [35S]GTP gamma S binding stimulation, indicating that a large receptor reserve does not exist for the complete activation of G proteins in this system.

Animals↗

PubMed1998

Pattern of c-fos mRNA induction in rat brain by acute morphine.

Initially, opioid signaling had been thought to be mainly inhibitory in nature. However, it has been shown that opioids can activate specific signaling pathways and induce immediate early gene (IEG) transcription in brain. IEGs can then regulate the transcription of other genes, leading to changes in neuronal function in response to extracellular stimuli. This study was designed to identify brain regions that demonstrate specific induction of the IEG c-fos, a component of the AP-1 transcription factor, in response to acute morphine, and to contrast this induction with the stressful effects of the injection itself. Rats received either 10 mg/kg morphine or an equivalent volume of saline injected subcutaneously. Animals were then sacrificed 15, 30, or 60 min after injection. Specific induction of c-fos mRNA by morphine was seen in dorsomedial caudate-putamen, paraventricular nucleus of the thalamus, central and intralaminar thalamic nuclei, dorsal central grey, superior colliculus, lateral parabrachial nucleus, inferior olivary complex, and caudal nucleus tractus solitarius. These findings represent the first complete anatomical mapping of c-fos induction in rat brain, and show that acute morphine administration alters gene expression in several areas related to known functional properties of opioids. However, regions showing c-fos induction are not all classically associated with opioid receptors and opioid-mediated effects. These findings are considered in the context of the effects of opioids on neural circuitry as well as direct, receptor-mediated effects of morphine on neural cells.

Animals↗

PubMed1997

Synthesis and characterization of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-labeled fluorescent ligands for the mu opioid receptor.

A series of opioid ligands utilizing the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3-propionic acid or 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza- s-indacene-3-propionic acid were synthesized and characterized for their ability to act as a suitable fluorescent label for the mu opioid receptor. All compounds displaced the mu opioid receptor binding of [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol in monkey brain membranes with high affinity. The binding of fluorescent ligands to delta and kappa receptors was highly variable. 5,7-Dimethyl-BODIPY naltrexamine, "6-BNX," displayed subnanomolar affinities for the mu and kappa opioid receptors (Ki 0.07 and 0.43 nM, respectively) and nanomolar affinity at the delta (Ki 1.4 nM) receptor. Using fluorescence spectroscopy, the binding of 6-BNX in membranes from C6 glioma cells transfected with the cloned mu opioid receptor was investigated. In these membranes containing a high receptor density (10-80 pmol/mg protein), 6-BNX labeling was saturable, mu opioid specific, stereoselective (as determined with the isomers dextrorphan and levorphanol), and more than 90% specific. The results describe a series of newly developed fluorescent ligands for the mu opioid receptor and the use of one of these ligands as a label for the cloned mu receptor. These ligands provide a new approach for studying the structural and biophysical nature of opioid receptors.

Animals↗

PubMed1997

Social stress in hamsters: defeat activates specific neurocircuits within the brain.

During an agonistic encounter, subordinate male hamsters display defensive and submissive postures and show increased secretion of glucocorticoids, whereas dominant males do not. To determine whether specific neuronal pathways are activated during the behavioral and neuroendocrine responses of subordinate males, expression of c-fos mRNA within the brains of subordinate males was compared with the pattern in dominant males after fighting. After 1 week of handling, pairs of hamsters were either swapped between cages (handled control males), or were allowed to interact for 30 min [dominant (DOM) males and subordinate (SUB) males]. A second group of control animals that received no handling or social stimulation (unhandled control males) were also included. After testing, all animals were killed by decapitation, their brains were removed for c-fos in situ hybridization, and trunk blood was collected for analysis of plasma cortisol and corticosterone levels. Exposure of males to their partner's cage for 30 min resulted in increased expression of c-fos mRNA in multiple brain regions. In addition, fighting increased c-fos expression in the medial amygdaloid nucleus of both DOM and SUB males as well as having more selective effects. In DOM males, c-fos expression was elevated within the supraoptic nucleus of the hypothalamus. In SUB males, c-fos expression increased within a multitude of brain areas, including cingulate cortex, lateral septum, bed nucleus of the stria terminalis, medial preoptic area, several hypothalamic nuclei, central amygdaloid nucleus, amygdalohippocampal area, dorsal periaqueductal gray, dorsal raphe, cuneiform nucleus, and locus coeruleus. These findings are discussed in relation to neurocircuits associated with behavioral arousal and stress.

Aggression↗

PubMed1997

Lesions of the medial geniculate nuclei specifically block corticosterone release and induction of c-fos mRNA in the forebrain associated with audiogenic stress in rats.

Audiogenic stress is known to activate the hypothalamo-pituitary-adrenocortical (HPA) axis in rats. The goal of the present study was to determine whether the medial geniculate nuclei (including all auditory nuclei of the thalamus), which are obligatory relays in the transmission of auditory information to the forebrain, are critically involved in HPA activation by audiogenic stress. To this end, corticosterone levels and regional brain activity indexed by c-fos mRNA induction, elicited by 30 min of 105 dB white noise, were measured. Compared with unoperated and sham-operated rats, complete medial geniculate nuclei lesions blocked corticosterone release normally induced by loud noise. The effects of the lesions were specific to loud noise insofar as corticosterone release in response to restraint or ether stress was not reduced in lesioned rats. We have determined previously that audiogenic stress is associated with a specific regional pattern of c-fos mRNA induction. Rats sustaining complete medial geniculate lesions demonstrated a blockade of c-fos mRNA induction in several audiogenic stress responsive regions, also known to directly innervate medial parvocellular neurons of the paraventricular hypothalamic nucleus. Thus, in addition to blockade in the paraventricular hypothalamic nucleus, c-fos mRNA induction in the lesioned animals was abolished in the bed nucleus of the stria terminalis, especially its anterior medial and ventral aspects, the septohypothalamic nucleus, and the anteroventral preoptic area, compared with unoperated and sham-operated rats. Several additional regions in the lesioned rats failed to show reliable c-fos mRNA induction compared with naive rat controls. Nearly all other regions that showed reliable c-fos mRNA induction in the unoperated and sham-operated rats displayed either similar or slightly reduced levels in complete medial geniculate-lesioned rats, suggesting that these regions are not part of a critical HPA activational circuit in response to audiogenic stress. On the basis of these results, putative circuits from the medial geniculate nuclei to the paraventricular nucleus of the hypothalamus involved in activation of the HPA axis by audiogenic stress are discussed.

Animals↗

PubMed1997

Normal pituitary response to metyrapone in the morning in depressed patients: implications for circadian regulation of corticotropin-releasing hormone secretion.

Excess secretion of cortisol in depressed patients has been documented by a number of investigators, which is presumed secondary to increased corticotropin (ACTH) and ACTH-releasing hormone (CRH) secretion. To unmask the proposed increased central (CRH) drive, we administered metyrapone in the AM to 13 depressed and 13 age- and sex-matched normal control subjects. Metyrapone administration resulted in a prompt decrease in plasma cortisol and in an increase in 11-deoxycortisol, the inactive precursor, in all subjects. Both depressed patients and normal control subjects demonstrated clear increases in ACTH and beta-lipotropin/beta-endorphin production. There were no significant differences between patients and controls in any hormonal measures following metyrapone administration. These data suggest that: 1) in the absence of negative feedback (cortisol blockade), mildly to moderately depressed outpatients do not manifest increased central drive in the morning; and 2) the secretory capacity of the corticotropes do not differ between such depressed patients and controls.

Adrenocorticotropic Hormone↗

PubMed1997

Opioid receptor expression in the rat gastrointestinal tract: a quantitative study with comparison to the brain.

The present study was undertaken to analyze the expression of two opioid receptor genes (mu and kappa) in different gastrointestinal regions of the rat. A combination of mRNA quantification and immunohistochemical visualization was used to characterize their expression. Using naive animals, RNA was extracted from tissues and used in RNase protection assays: both receptor mRNAs were expressed in all investigated areas but displayed different expression profiles across the various regions of the digestive tract. Stomach and proximal colon appeared to have the highest expression levels of both receptors, whereas the lowest expression levels were found in the duodenum. Expression levels for both receptors were always lower in the gastrointestinal tract compared to the brain. However, the kappa-receptor expression in the proximal colon represented 40% of the amount found in the brain, which is almost 4 times as high as the respective mu-receptor expression. In contrast to smooth muscle cells, myenteric plexus perikarya of the rat stomach and colon were immunoreactive with antibodies raised against the C-termini of both kappa- and mu-opioid receptors. Numerous nerve fibers were also immunoreactive for both mu- and kappa-receptors and distributed in the longitudinal and circular muscle layers. Small perikarya immunoreactive for mu-receptor were localized around the myenteric plexus and at the submucosal border of the circular muscle, whereas only few perikarya were immunoreactive for the kappa-receptor. We conclude that at least in rat stomach and colon, mu- and kappa-opioid receptors may directly control neuronal communication but seem to have no direct influence on smooth muscle cells.

Animals↗

PubMed1997

The interleukin-1beta-mediated regulation of proenkephalin and opioid receptor messenger RNA in primary astrocyte-enriched cultures.

Opioids have been found to modulate the function of the immune system by regulating the biochemical and proliferative properties of its cellular components. The interaction of opioid and immune systems, however, is not unidirectional, but rather, bidirectional in nature. In the CNS, one cellular target of immune system activation is the astrocytes, glial cells known to synthesize proenkephalin. We have recently shown that these cells also express the messenger RNA transcripts for the opioid receptors mu, delta and kappa, raising the question of the functional significance of this opioid peptide and the related receptors in the astrocytes. That is, why do astrocytes express proenkephalin and opioid receptors, and are these molecules responsive to a factor to which the astrocytes could be exposed in vivo? Furthermore, do these molecules respond to this factor in a region-specific fashion? In the present study, in order to characterize the astrocytic opioid response to an immune factor, we examined the concomitant regulation of mu, delta, kappa and proenkephalin messenger RNAs by interleukin-1beta (1 ng/ml=60 pM, 24 h) in primary astrocyte-enriched cultures derived from the rat (post-natal day 1-2) cortex, striatum, cerebellum, hippocampus and hypothalamus. Interleukin-1beta treatment was found to increase by 55-75% the level of mu receptor messenger RNA in striatal, cerebellar and hippocampal cultures, but not in cultures derived from the cortex or hypothalamus. However, the cytokine had no effect on the level of delta receptor messenger RNA in any of the five cultures examined. In marked contrast to its stimulatory effects on mu receptor messenger RNA levels and its lack of an effect on 6 receptor messenger RNA expression, interleukin-1beta reduced to 10-30% of control levels the kappa receptor messenger RNA levels in all cultures. Interleukin-1beta had no effect on the level of proenkephalin messenger RNA in cortical, striatal, cerebellar and hypothalamic cultures, but did significantly decrease the expression of proenkephalin messenger RNA in hippocampal cultures to 40% of the control level. Therefore, interleukin-1beta differentially regulated opioid receptor messenger RNA in astrocyte-enriched cultures in a manner dependent upon both the receptor type and the brain region from which the culture was derived. The cytokine also differentially regulated proenkephalin messenger RNA in a region-dependent fashion. These findings suggest a capacity for astrocytes to differentially regulate opioid peptide and receptor messenger RNAs in response to an immune factor, supporting the potential existence of a novel immune-opioid system interaction in the CNS.

Animals↗

PubMed1997

Cellular localization and distribution of the cloned mu and kappa opioid receptors in rat gastrointestinal tract.

Several pharmacological and electrophysiological studies have shown that the opioid receptors are widely distributed in the gastrointestinal tract. Despite such consensus, there are conflicting findings regarding their effects in intestinal function, and their precise site of action remained unclear. The aim of the present study was therefore to delineate the cellular localization of mu and kappa opioid receptors in rat gastrointestinal tract using polyclonal antibodies generated to C-terminal end of the cloned mu (63 amino acids) and kappa (41 amino acids) receptors. The distribution of mu differs from that of kappa receptors within the gastrointestinal wall, with a greater abundance of mu receptor-like immunoreactive fibres in all intestinal layers. Numerous neurons expressing mu receptor-like proteins were found in the submucosal plexus with comparatively few in the myenteric plexus. In contrast, a higher number of neurons expressing kappa receptor-like immunoreactivity were visualized in the myenteric plexus with a small number in the submucosal plexus. A high number of immunopositive neurons were found in the myenteric plexus of the stomach and the proximal colon with both antibodies. In the submucosal and mucosal layers. mu receptor-immunoreactive fibres were more abundant and distributed around the crypts, blood vessels and lymphatic nodes. Interestingly, numerous mu and fewer kappa receptor-immunoreactive interstitial cells are localized in the region of myenteric plexus and at the internal border of the circular muscle. Finally, smooth muscle cells did not demonstrate any mu- nor kappa-receptor immunoreactivity. These findings suggest that in the rat gastrointestinal tract, mu and kappa opioid receptors may directly influence neuronal and interstitial cell activity. This appears not to be the case for the smooth muscle cells. In the muscular layers, the anatomical data point to mu receptor actions being mediated by nerve terminals, whereas kappa receptor effects may be mediated by both nerve terminals and somatodendritic synaptic mechanisms. In contrast, in the submucosal and mucosal layers, mu receptors predominate and are localized on both nerve terminals and somatodendritic synaptic elements.

Animals↗

PubMed1997

Distribution of alpha 1a-, alpha 1b- and alpha 1d-adrenergic receptor mRNA in the rat brain and spinal cord.

The technique of in situ hybridization with specific ribonucleotide probes was used to determine the distribution patterns of mRNA encoding the alpha 1a-, alpha 1b- and alpha 1d-adrenoceptor (AR) subtypes in rat brain and spinal cord. The expression pattern of alpha 1a-AR mRNA has not been reported previously, and was found to be widespread throughout the rat central nervous system. High levels were found in regions of the olfactory system, several hypothalamic nuclei, and regions of the brainstem and spinal cord, particularly in areas related to motor function. Regions expressing moderate levels of mRNA for this receptor were the septum, bed nucleus of the stria terminalis, cerebral cortex, amygdala, cerebellum and pineal gland. Low expression levels were detected in the hippocampal formation. Most nuclei in the basal ganglia and thalamus expressed extremely low or undetectable levels of alpha 1a-AR mRNA. The expression patterns of the alpha 1b- and alpha 1d-AR mRNAs were similar to those described using oligonucleotide probes in earlier studies. High expression of alpha 1b-AR mRNA was noted in the pineal gland, most thalamic nuclei, lateral nucleus of the amygdala and dorsal and median raphe nuclei. Moderate expression levels were noted throughout the cerebral cortex, and in some olfactory, septal, and brainstem regions. The distribution of alpha 1d-AR mRNA was the most discrete of the three receptors examined. Expression was strong in the olfactory bulb, cerebral cortex, hippocampus, reticular thalamic nucleus, regions of the amygdala, motor nuclei of the brainstem, inferior olivary complex and spinal cord. Comparison of the distributions of the alpha 1a-, alpha 1b- and alpha 1d-AR mRNA suggests unique functional roles for each of these receptors.

Animals↗

PubMed1997

Motivational properties of oxytocin in the conditioned place preference paradigm.

We hypothesized that oxytocin might have intrinsic reinforcing properties and studied it using a conditioned place preference. Three studies examining motivational properties of oxytocin in nonpreferred, preferred, and balance designs were performed utilizing two compartment apparatus. On alternate days, compartments were paired with subcutaneously injected oxytocin (6 mg/kg) or saline, and animal pre- and post-conditioning place preference was compared. Whereas in animals paired with saline there was a shift to a lack of preference, oxytocin-treated animals reversed their preference, spending more time in a previously unpreferred, compartment. In preferred compartment design, oxytocin-treated animals further increased their preference, whereas saline-treated animals decreased their preference toward a nonpreference for either compartment. Our results demonstrate that oxytocin produces a reliable and robust preference for the environment with which it is repeatedly associated, and has rewarding or potentially anti-aversive properties. Future studies are needed to distinguish among these possibilities.

Animals↗

PubMed1997

Kinetics of radiolabeled adrenocorticotropin hormone in infant and weanling rats.

Unlike the adult animal, the developing rat has a diminished ability to activate and inhibit the hypothalamic pituitary adrenal axis. In general, a gradual ACTH and corticosterone response to stressors appear after postnatal day 10 and is well established to adult level by weaning age. Although at this age the peak ACTH level is comparable to that of the adult, ACTH levels remain elevated for a longer period of time. The purpose of this study was to investigate the possibility that ACTH metabolism can, in part, explain this prolonged ACTH elevation after a challenge. The plasma half life of disappearance (t1/2, the apparent volume of distribution and metabolic clearance rate (MCR) were determined after injection of a tracer dose of 3-I125-Iodotyrosyl23 ACTH1-39 in rats at 14 and 25 days of age. An adult animal group (65 days old) was used for comparison. The t1/2 for ACTH decreases with age (14 day old = 7.47 +/- 0.9 min; 25 day old = 6.48 +/- 0.4 min; adult = 4.46 +/- 0.2 min) while the volume of distribution remains constant. The MCR is also decreased in the young animals (14 day old = 1.5 +/- 0.19 min; 25 day old = 1.6 +/- 0.18 min; adult = 3.0 +/- 0.56 min). For the first time, it is established that the young animals require longer to clear ACTH from an equivalent volume of blood when compared to the adult. Thus, the kinetic properties of ACTH are different in the developing animal and this partly explains the prolonged ACTH elevation observed after stress challenges.

Adrenocorticotropic Hormone↗

PubMed1997

Key residues defining the mu-opioid receptor binding pocket: a site-directed mutagenesis study.

Structural elements of the rat mu-opioid receptor important in ligand receptor binding and selectivity were examined using a site-directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the mu, delta, and kappa receptors (Asn150 to Ala, His297 to Ala, and Tyr326 to Phe) and two designed to test for mu/delta selectivity (Ile196 to Val and Val202 to Ile). Mutation of His297 in transmembrane domain 6 (TM6) resulted in no detectable binding with [3H]DAMGO (3H-labeled D-Ala2, N-Me-Phe4, Gly-ol5-enkephalin), [3H]bremazocine, or [3H]ethylketocyclazocine. Mutation of Asn150 in TM3 produces a three- to 20-fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, beta-endorphin1-31, JOM-13, deltorphin II, dynorphin1-13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor-binaltorphamine. In contrast, the Tyr326 mutation in TM7 resulted in a decreased affinity for a wide spectrum of mu, delta, and kappa agonists and antagonists. Altering Val202 to Ile in TM4 produced no change on ligand affinity, but Ile196 to Val resulted in a four- to fivefold decreased affinity for the mu agonists morphine and DAMGO, with no change in the binding affinities of kappa and delta ligands.

Animals↗