Discovery of the first non-peptide antagonist of the motilin receptor.

A first-in-class non-peptide antagonist of the motilin receptor was identified through electronic screening of our corporate database against a 3D pharmacophore. The pharmacophore was developed from the motilin 22 residue endogenous peptide using NMR structural data, principles of peptide folding, and peptide structure activity relationships. The NMR data supported helical content within the peptide, and both the hydrophobic staple and N-capping box motifs were identified in the motilin sequence. The conformational features of these motifs were imposed on the peptide structure, providing a constrained conformer as a starting point for database searching. A trisubstituted cyclopentene lead was identified directly from the electronic search. Compounds in this series inhibit the binding of 125I-motilin to human antral smooth muscle membrane and antagonize motilin-induced intracellular calcium mobilization in cells expressing the human motilin receptor. A potent compound developed through optimization, RWJ 68023, is active in binding and cell-based functional assays and is also effective in inhibiting motilin-induced contractility in segments of rabbit duodenum. This orally active compound is currently undergoing clinical evaluation for the treatment of gastrointestinal disorders associated with altered motility.

A branched DNA signal amplification assay to quantitate messenger RNA of human uncoupling proteins 1, 2, and 3.

Uncoupling proteins (UCP) are inner mitochondrial membrane transporters which dissipate the proton gradient, releasing stored energy as heat. Three subtypes of UCP have been identified so far. The regulation of UCP expression is mainly controlled at the transcriptional level, thus making the measurement of UCP mRNA beneficial for both diagnosis and research of weight disorders and diabetes. We have developed an assay using the branched DNA signal amplification assay (bDNA assay) to quantitatively measure the mRNA levels for human UCP1, 2, and 3. UCP-subtype-specific primers were designed for the assay. RNA transcripts of each UCP generated by in vitro transcription were used to validate the specificity and sensitivity of the assay. The quantitative measurement of UCP mRNA was further demonstrated with cultured cells and human tissue. A comprehensive survey of UCP expression from 17 human tissues measured by the newly developed assay is provided. The method described here offers a rapid, sensitive, specific, and quantitative assay for measurement of human UCP mRNA.

Characterization of the progestin receptors in the human TE85 and murine MC3T3-E1 osteoblast-like cell lines.

Progestins are believed to exert positive effects on bone density through receptors located in osteoblasts. In the present studies, the binding characteristics and regulation of the progestin receptors in two osteoblast-like cell lines were compared with those in human breast lines. Human TE85 and murine MC3T3-E1 osteoblast-like cells contain a single, high-affinity progestin binding site whose affinity and concentration are lower than in human breast cells. The osteoblastic progestin binding sites showed the expected steroid specificity and associated with the cell nuclei when occupied by ligand. The progestin receptors in osteoblastic cells also had sedimentation coefficients similar to those receptors in breast cells. The regulation of the progestin receptor in the osteoblast-like cells was explored by treating them with estradiol. In contrast to the large, rapid change seen in the breast cells, the progestin receptor levels in the MC3T3-E1 cells showed only a small, delayed up-regulation with estradiol treatment. The progestin receptor number in the TE85 cells was unaffected by estradiol. Down-regulation of the progestin receptors was explored by treating the cells with the progestin, norethindrone (NET). NET administration produced a rapid drop in progestin binding sites in the breast cells and a smaller, more gradual decline in MC3T3-E1 progestin binding. While the maximal decrease in receptor number occurred within 24 h in the breast cells, the receptor number was still continuing to fall after 72 h in the MC3T3-E1 cells. The data presented here demonstrate that both human and murine osteoblast-like cells contain a functional progestin receptor whose binding characteristics and regulation are similar, but not identical, to those receptors in other progestin target tissues such as the breast.

Nonsteroidal progesterone receptor ligands. 2. High-affinity ligands with selectivity for bone cell progesterone receptors.

A novel series of nonsteroidal heterocycles was discovered which display cell-type selective, high-affinity (nanomolar) binding to the progesterone receptors from TE85 osteosarcoma cells but > 1 microM binding affinity to the progesterone receptors from T47D and ZR75 human breast carcinoma cells. Structure-activity relationships were developed for a set of these compounds, and a representative analog 1-(3,4-dichlorobenzoyl)-3-phenyl-1,4,5,6-tetrahydropyridazine++ + (1i, RWJ 25333) was chosen for further evaluation. RWJ 25333 stimulated the in vitro proliferation of human osteoblast-like cells but not human breast cells.

Development of a heterologous radioimmunoassay for canine osteocalcin.

The aim of this study was to develop a routine and reliable radioimmunoassay (RIA) for dog osteocalcin. Two peaks of dog osteocalcin were purified to apparent homogeneity according to N-terminal sequence analysis. Amino acid composition analysis suggested that the second peak was intact dog osteocalcin whereas the first peak could be a truncated molecule. High titer (> 1:5,000) anti-dog osteocalcin antisera were produced in rabbits. The antiserum recognized dog and rat osteocalcins but not that in serum of human, bovine, rabbit, mouse, guinea pig, or goat. A homologous RIA using anti-dog osteocalcin as the antibody and dog osteocalcin as the tracer and standard was developed. Taking advantages of the facts that (1) anti-dog osteocalcin cross-reacted in parallel with rat osteocalcin and (2) purified rat osteocalcin is commercially available, we devised an approach that used rat osteocalcin as the tracer and standard, and anti-dog osteocalcin as the antibody to develop a heterologous RIA. This assay recognized dog serum osteocalcin and diluted in parallel with rat and dog osteocalcins. Quantitation was done using rat osteocalcin to construct standard curves, and results were expressed in ng/ml of rat osteocalcin-equivalent. The detection limit of the assay was 5 ng/ml rat osteocalcin-equivalent, and half-maximal displacement was seen at 30-40 ng/ml rat osteocalcin-equivalent. The inter- and intraassay variations were 16.1% and 8.5%, respectively. The assay accurately determined the amount of exogenously added dog osteocalcin in serum. The results quantitated with this RIA correlated well (r = 0.975, n = 86) with those obtained with the homologous RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of a sustained release formulation of the gonadotrophin-releasing hormone agonist histrelin on serum concentrations of gonadotrophins and oestradiol, and ovarian LH/human chorionic gonadotrophin receptor content in the rat.

Pituitary and ovarian function were studied during the loss and recovery of oestrous cyclical activity in rats following treatment with a sustained release formulation of the gonadotrophin-releasing hormone (GnRH) agonist [imidazole benzyl-D-His6,Pro9-ethylamide]-GnRH (histrelin). A single s.c. injection of microencapsulated histrelin (10-300 micrograms peptide/kg) induced a dose-dependent disruption of normal oestrous cyclical activity with a persistent dioestrous-like vaginal cytology. In preliminary studies, pituitary gland stimulation and desensitization were demonstrated when serum LH and FSH levels were greater 1 week after administration of 10 micrograms microencapsulated histrelin/kg compared with 300 micrograms microencapsulated histrelin/kg. Changes in pituitary and ovarian function were assessed over time following injection of microencapsulated histrelin (100 micrograms peptide/kg). LH secretion was maximal within 8 h and then gradually declined, remaining at dioestrous levels from days 7 to 28. Serum oestradiol concentrations remained low and rose above dioestrous levels only on day 28. In contrast, ovarian LH/human chorionic gonadotrophin (LH/hCG) receptor content fell within 8 h and, after a nadir on day 7, slowly returned to dioestrous levels by day 28. The increase in ovarian LH/hCG receptor content preceded any significant change in pituitary gonadotrophin secretion, indicating a differential pattern of recovery for pituitary and ovarian function. Subsequent studies tested the possibility that these temporal differences in pituitary and ovarian function may result from histrelin acting directly on these tissues. Treatment with histrelin microcapsules (300 micrograms peptide/kg) prevented any increase in LH secretion in response to a GnRH challenge 3 days later, indicating a direct action of histrelin on the pituitary gland.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Methoxy-4-hydroxyphenylethyleneglycol concentrations in discrete hypothalamic nuclei reflect the activity of noradrenergic neurons.

An analytical technique is described which permits the quantitation of picogram concentrations of 3-methoxy-4-hydroxyphenylethylene-glycol (MHPG) in acid hydrolyzed extracts of microdissected regions of the rat brain, and this procedure is used to determine if alterations in the activity of noradrenergic neurons are reflected by changes in the concentrations of MHPG in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the rat hypothalamus. MHPG was not detected in non-hydrolyzed samples of either the PVN or SON, but following acid hydrolysis (heating of samples at 94 degrees C for 5 min in 0.16 M perchloric acid) MHPG was detected in both of these regions. These results indicate that MHPG exists primarily as a conjugate in the PVN and SON. Neurotoxin-induced lesions of the ventral noradrenergic bundle decreased norepinephrine (NE) and MHPG concentrations in the PVN and SON, demonstrating that tissue levels of MHPG in these brain regions are dependent upon the presence of noradrenergic neurons. Electrical stimulation of the locus coeruleus increased MHPG concentrations in the PVN, but not in the SON, whereas electrical stimulation of the medial forebrain bundle increased MHPG concentrations in both of these regions. The alpha 2-adrenergic receptor antagonist idazoxan increased, while the alpha 2-adrenergic receptor agonist clonidine decreased MHPG concentrations in both the PVN and SON, but neither idazoxan nor clonidine altered NE concentrations in these regions. Immobilization of rats in the supine position increased MHPG concentrations in the PVN and SON, and this was accompanied by a decrease in NE concentrations in the SON.(ABSTRACT TRUNCATED AT 250 WORDS)

Stress-induced secretion of alpha-melanocyte-stimulating hormone is accompanied by a decrease in the activity of tuberohypophysial dopaminergic neurons.

The purpose of the present study was to examine the acute effects of stress on the secretion of alpha-melanocyte-stimulating hormone (alpha MSH) and the activity of tuberohypophysial dopamine (DA) neurons in female and male rats. The activity of tuberohypophysial DA neurons was estimated by measuring the accumulation of 3,4-dihydroxyphenylalanine (DOPA) following administration of the decarboxylase inhibitor NSD 1015, and the concentrations of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the intermediate and neural lobes of the posterior pituitary. The combination of brief (2 min) ether exposure followed by 30 min of supine restraint (immobilization in the supine position) decreased the rate of DOPA accumulation in the intermediate, but not in the neural lobe of both female and male rats. Similarly, brief ether exposure followed by 10, 20 or 30 min of supine restraint increased plasma alpha MSH concentrations and decreased DOPAC concentrations in the intermediate lobe of female and male rats. In the absence of ether, tube restraint (confinement in a cylindrical acrylic tube) increased alpha MSH secretion and decreased intermediate lobe DOPAC concentrations, whereas ether in the absence of physical restraint had no effect. These results suggest that the stress-induced activation of alpha MSH secretion in both female and male rats may be due, in part, to a decrease in the activity of tuberohypophysial DA neurons in the intermediate lobe of the posterior pituitary.

Comparison of the effects of ether and restraint stress on the activity of tuberoinfundibular dopaminergic neurons in female and male rats.

The purpose of the present study was to characterize the acute inhibitory effects of restraint stress on the activity of tuberoinfundibular dopamine (DA) neurons as estimated by measuring concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence. The time course of the effects of two types of physical restraint (immobilization in the supine position or confinement in an acrylic cylindrical tube) was determined in unanesthetized and diethylether (ether)-exposed female and male rats. The combination of brief (2 min) exposure to ether followed by 10 and 20 min of supine restraint increased concentrations of prolactin in plasma and decreased DOPAC concentrations in median eminence of both female and male rats. Thirty minutes of supine restraint decreased DOPAC concentrations in the median eminence of female rats that were not exposed to ether, and brief exposure to ether enhanced this effect. By contrast, 30 min of supine restraint failed to alter DOPAC concentrations in the median eminence in either unanesthetized or ether-exposed male rats. Tube restraint in the absence of ether failed to alter DOPAC concentrations in the median eminence of either female or male rats; but in female rats preexposed to ether, 30 min of tube restraint decreased DOPAC concentrations in the median eminence. On the other hand, in the absence of physical restraint, 2 min ether exposure caused a transient increase in prolactin secretion and a concurrent decrease of DOPAC concentrations in median eminence of both female and male rats.(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dihydroxyphenylacetic acid concentrations in the intermediate lobe and neural lobe of the posterior pituitary gland as an index of tuberohypophysial dopaminergic neuronal activity.

Tuberohypophysial dopamine (DA) neurons terminate in the intermediate and neural lobes of the posterior pituitary. The objective of this study was to determine if concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC), a major metabolite of DA in these regions, reflect the activity of tuberohypophysial DA neurons. The concentrations of DOPAC and DA in the intermediate lobe were approximately twice those in the neural lobe, so that the ratios of DOPAC/DA were similar between lobes. The administration of a monoamine oxidase inhibitor pargyline produced a rapid decline (by 5 min) of DOPAC concentrations in both the intermediate and neural lobes. The administration of nomifensine, an inhibitor of DA uptake at the nerve terminal, produced a modest 33% decline in DOPAC concentrations in the intermediate lobe, but was without effect in the neural lobe. Activation of tuberohypophysial DA neurons by electrical stimulation of the pituitary stalk increased both the rate of DA synthesis (accumulation of dihydroxyphenylalanine (DOPA) after administration of the decarboxylase inhibitor NSD 1015) and the concentrations of DOPAC in the intermediate and neural lobes. Administration of the DA antagonist haloperidol increased, and the DA agonist apomorphine decreased both the rate of DOPA accumulation and DOPAC concentrations in the intermediate lobe but not the neural lobe. The results of the present study demonstrate that: (1) elimination of DOPAC from the intermediate lobe and neural lobe is rapid and alterations in DOPAC concentrations reflect dynamic changes in metabolism of DA; (2) DA which is released and recaptured is a minor contributor to DOPAC concentrations; and (3) alterations in the activity of tuberohypophysial DA neurons are accompanied by corresponding changes in DOPAC concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of alterations in the activity of tuberohypophysial dopaminergic neurons on the secretion of alpha-melanocyte stimulating hormone.

Administration of gamma-butyrolactone (GBL), an anesthetic which reduces dopaminergic neuronal activity, decreased the concentration of the dopamine (DA) metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the intermediate lobe of the pituitary gland, and increased alpha-melanocyte stimulating hormone (alpha MSH) concentrations in the serum of male rats. Bilateral electrical stimulation of the rostral arcuate nucleus, which contains perikarya of tuberohypophysial DA neurons, increased DOPAC concentrations in the intermediate lobe and decreased alpha MSH concentrations in the serum of GBL-anesthetized rats. Administration of the DA antagonist haloperidol prevented the decline in serum alpha MSH levels following arcuate nucleus stimulation, but had no effect on serum alpha MSH concentrations in sham-stimulated GBL-treated rats. These results indicate that GBL-induced decreases or stimulation-induced increases in the activity of tuberohypophysial DA neurons are accompanied by corresponding changes in the metabolism of DA in the intermediate lobe of the rat pituitary gland, and by reciprocal changes in the secretion of alpha MSH.

Effect of electrical stimulation of the ventromedial nucleus and the dorsomedial nucleus on the activity of tuberoinfundibular dopaminergic neurons.

Perikarya and terminals of tuberoinfundibular dopaminergic (TIDA) neurons are located in the arcuate nucleus (ARN) and in the median eminence (ME), respectively. Dopamine (DA) released from TIDA terminals in the ME inhibits prolactin secretion from the anterior pituitary. Anatomical studies have described the sources of afferents to ARN and ME, but not to TIDA neurons per se. The ventromedial nucleus (VMN) and the dorsomedial nucleus (DMN) of the hypothalamus project to ARN and ME and have a role in prolactin regulation. In the present study, VMN and DMN were investigated as possible sources of TIDA afferents. Alterations in the activity of TIDA neurons were estimated by measuring plasma concentrations of prolactin and the rates of DA synthesis (3,4-dihydroxyphenylalanine - DOPA - accumulation after administration of the decarboxylase inhibitor NSD 1015) and metabolism (concentrations of the DA metabolite 3,4-dihydroxyphenylacetic acid - DOPAC) in the ME following electrical stimulation of ARN, VMN, and DMN in ovariectomized female rats. Thirty minutes of bilateral stimulation of ARN or DMN increased DOPA accumulation in the ME; stimulation of the VMN had no effect. 5-Hydroxytryptamine synthesis in the ME was unaffected by stimulation of any region. Plasma prolactin levels declined during DMN stimulation, varying with the frequency and duration of the electrical stimulus. DA metabolism within TIDA neurons increased with DMN stimulation, as evidenced by increased DOPAC concentrations in the ME. In females whose basal TIDA activity has been increased by haloperidol treatment or decreased by bromocriptine treatment, DMN stimulation was still able to increase DOPA accumulation in the ME. The present data suggest the presence of stimulatory TIDA afferents originating from or passing through the DMN.

Neuroleptics and neuroendocrine function.

Neuroleptics have been developed primarily to treat psychoses, but they have become invaluable research tools. Because of their selective action on DA receptors, neuroleptics are commonly employed to study the function and regulation of DA neurotransmission. The relationship between the antipsychotic efficacy and the DA receptor affinity of the various neuroleptic drugs has lead to the development of new DA antagonists in hopes of discovering novel antipsychotic agents. This approach has produced interesting new compounds selective for the DA receptor subtypes. The use of DA receptor antagonism as a measure of the potential antipsychotic efficacy of a compound will undoubtedly change as the mechanisms behind the antipsychotic actions of neuroleptics become better understood. Although endocrine side effects of neuroleptic administration are undesired in the clinic, they have provided insight into the neuroendocrine regulation of pituitary hormones. Through the use of neuroleptics, DA neurons in the hypothalamus have been shown to play a role in the regulation of prolactin, GH, and TSH secretion. The ability of DA to act at the pituitary and thereby inhibit the secretion of these three hormones suggests that other regulatory factors must provide the specificity needed for the differential secretion of the individual hormones during varying physiological states. Future research will certainly explore the interactions of DA and these regulatory factors at the pituitary. The role of DA in neuroendocrine regulation is not limited to the pituitary. The presence of DA neurons within the hypothalamus offers the possibility of DA regulation of hypothalamic neurosecretory activity.

Electrical stimulation of the arcuate nucleus increases the metabolism of dopamine in terminals of tuberoinfundibular neurons in the median eminence.

The purpose of the present study was to examine the effects of procedures that alter impulse flow in tuberoinfundibular dopamine (DA) neurons on the metabolism of DA in the median eminence and on the secretion of prolactin from the anterior pituitary. Twenty min following the administration of gamma-butyrolactone (GBL, 1000 mg/kg, i.p.) there was a marked decrease in 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations in the median eminence and an increase in prolactin concentrations in the serum, indicating that a decrease in activity of tuberoinfundibular DA neurons is accompanied by a decrease in DA metabolism in the median eminence and a loss of tonic inhibition of pituitary prolactin secretion. Activation of tuberoinfundibular DA neurons by bilateral stimulation of the arcuate nucleus in GBL-treated rats produced a rapid increase in median eminence DOPAC concentrations and a time-dependent decrease in serum prolactin concentrations. Nomifensine (25 mg/kg, i.p., 30 min), a DA uptake inhibitor, had no effect on median eminence DOPAC concentrations in sham- or arcuate nucleus-stimulated rats, indicating that regardless of the level of activity of tuberoinfundibular neurons, very little DA is recaptured and metabolized in terminals of these neurons in the median eminence. Taken together these results reveal that alterations in impulse flow in tuberoinfundibular DA neurons are accompanied by corresponding changes in the metabolism of DA in the median eminence.

Effect of electrical stimulation of the arcuate nucleus on neurochemical estimates of tuberoinfundibular and tuberohypophysial dopaminergic neuronal activities.

The activity of nigrostriatal dopaminergic neurons has been estimated biochemically by measuring the rates of dopamine (DA) synthesis (accumulation of dihydroxyphenylalanine (DOPA) after NSD 1015) and turnover (decline of DA concentrations after alpha-methyltyrosine) in the striatum. It has been assumed that the activities of tuberoinfundibular dopaminergic (TIDA) and tuberohypophysial dopaminergic (THDA) neurons can also be estimated by making the same measurements in the terminals of these neurons in the median eminence and the posterior pituitary, respectively. In the present study, this assumption was tested directly by measuring the rates of DA synthesis and turnover in the median eminence and posterior pituitary following electrical stimulation of TIDS and THDA cell bodies in the arcuate nucleus. Electrical stimulation of the arcuate nucleus increased the rate of DOPA accumulation and the alpha-methyltyrosine-induced decline of DA concentrations in the median eminence and in the neural and intermediate lobes of the posterior pituitary. gamma-Butyrolactone (GBL), an anesthetic that selectively inhibits DA impulse flow, reduced the rates of DA synthesis and turnover in the median eminence. GBL also increased prolactin secretion which is tonically inhibited by DA released from TIDA neurons. Serum prolactin levels were significantly decreased by arcuate nucleus stimulation in GBL-anesthetized rats. These results indicate that the rates of DA synthesis and turnover within the median eminence and posterior pituitary reflect the activities of TIDA and THDA neurons, respectively.

Effects of gonadal steroids on tuberoinfundibular and tuberohypophysial dopaminergic neuronal activity in male and female rats.

The activities of tuberoinfundibular and tuberohypophysial dopamine (DA) neurons were estimated by measuring the turnover of DA in terminals of these neurons in the median eminence and in the neural and intermediate lobes of the pituitary, respectively. The rate of DA turnover (alpha-methyltyrosine-induced decline of DA) in the median eminence was two to three times faster in females than in males, but no sexual differences in DA turnover rates were noted in the neural and intermediate lobes. Two weeks following gonadectomy the rate of DA turnover in the median eminence was increased in the male but decreased in the female. These effects were reversed by testosterone and estrogen replacement in gonadectomized males and females, respectively. Neither gonadectomy nor steroid replacement altered DA turnover in the neural or intermediate lobes of either males or females. These results indicate that estrogen stimulates and testosterone inhibits tuberoinfundibular DA neuronal activity while neither steroid affects tuberohypophysial DA neuronal activity.

A comparison of biochemical indices of 5-hydroxytryptaminergic neuronal activity following electrical stimulation of the dorsal raphe nucleus.

The activity of 5-hydroxytryptaminergic neurons has been estimated from measurements of: concentrations of 5-hydroxyindoleacetic acid; the ratio of the concentrations of 5-hydroxyindoleacetic acid to 5-hydroxytryptamine; the rate of accumulation of 5-hydroxytryptophan following the administration of an aromatic L-amino acid decarboxylase inhibitor (e.g., NSD 1015); the rate of accumulation of 5-hydroxytryptamine, and the rate of decline of 5-hydroxyindoleacetic acid following the administration of a monoamine oxidase inhibitor (e.g., pargyline). The purpose of the present study was to compare these different methods under conditions of changing neuronal impulse traffic produced by electrical stimulation of 5-hydroxytryptaminergic neurons. Male rats anesthetized with chloral hydrate were killed following 0, 15, or 30 min of electrical stimulation of the dorsal raphe nucleus at a frequency of 0, 5, or 10 Hz. The concentrations of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophan in nucleus accumbens, amygdala, suprachiasmatic nucleus, and dorsomedial nucleus were measured using HPLC coupled to an electrochemical detector. In each brain region, stimulation elicited an increase in the concentration of 5-hydroxyindoleacetic acid and the 5-hydroxyindoleacetic acid/5-hydroxytryptamine concentration ratio in saline-treated animals and an increase in 5-hydroxytryptophan accumulation in NSD 1015-treated animals, but did not alter the concentration of 5-hydroxytryptamine or 5-hydroxyindoleacetic acid in pargyline-treated rats. The results o f this study indicate that although the first three methods serve as valid indices of 5-hydroxytryptaminergic neuronal activity, the pargyline-dependent techniques are not responsive to changes in the rate of 5-hydroxytryptamine nerve firing.

Comparison of the effects of castration and steroid replacement on incertohypothalamic dopaminergic neurons in male and female rats.

The activities of incertohypothalamic (IH) and tuberoinfundibular (TI) dopamine (DA) neurons were compared in selected brain regions of male and female rats by measuring the rate of DA turnover (alpha-methyltyrosine-induced decline in brain DA concentrations). The rates of DA turnover in regions containing TIDA (median eminence) and rostral IHDA (rostral periventricular and medial preoptic nuclei) neurons were greater in diestrous females than in intact males. In contrast, the rate of DA turnover in the caudal IHDA neurons (medial zona incerta), was greater in intact males than diestrous females. These results indicate that the activities of IHDA neurons, like those of TIDA neurons, differ between the sexes but that the sexual differentiation of IHDA neurons is not homogeneous. Two weeks following orchidectomy, the rates of DA turnover were increased in the median eminence and decreased in the medial preoptic nucleus. Testosterone replacement in orchidectomized males produced opposite effects, causing a decrease in DA turnover in the median eminence and an increase in the medial preoptic nucleus. In female rats, the rates of DA turnover were decreased in the median eminence and medial zona incerta and increased in the medial preoptic nucleus 2 weeks following ovariectomy. Only in the median eminence did 2 days of estrogen replacement in ovariectomized rats produce effects opposite those seen after ovariectomy alone. These data show that the activities of IHDA neurons, as estimated from measurements of DA turnover, can be altered by the removal and replacement of the gonadal steroids.

The interaction of the medial preoptic area and the dorsomedial-ventromedial nuclei of the hypothalamus in the regulation of the mating-induced release of prolactin.

The medial preoptic area (MPOA) and the dorsomedial-ventromedial nuclei (DMN-VMN) of the hypothalamus regulate the mating-induced nocturnal and diurnal surges of prolactin in rats. The neural mechanisms governing the release of the two surges differ. For the nocturnal surge the MPOA serves an inhibitory role while the DMN-VMN serves a stimulatory role. The diurnal surge is controlled by both areas functioning as stimulatory centers. The goal of the present study was to explore the possibility of functional interactions between the MPOA and the DMN-VMN in control of mating-induced prolactin secretion. Stimulation of the MPOA in conscious, cervically stimulated (CS) females suppresses the nocturnal surge of prolactin. To determine if the inhibitory effects of the MPOA operate through the DMN-VMN, electrical stimulation was applied to the MPOA of conscious ovariectomized female rats bearing bilateral electrolytic lesions of the DMN-VMN. In the first experiment, control (sham-stimulated, sham-lesioned) CS females exhibited normal nocturnal surges which peaked at 03.00 h. MPOA stimulation (01.00-05.00 h) of both sham-lesioned and DMN-VMN lesioned CS females inhibited the release of their nocturnal surges. This suggests that the inhibitory function of the MPOA is independent of the DMN-VMN. MPOA stimulation can induce the release of a diurnal surge if the females are anesthetized with pentobarbital. In the second experiment, MPOA stimulation (15.00-19.00 h) of sham-lesioned anesthetized females produced elevated prolactin levels with significant peaks at 15.30 and 19.00 h. Anesthetized females with DMN-VMN lesions did not respond to MPOA stimulation with any change in prolactin secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypothalamic regulation of mating-induced prolactin release. Effect of electrical stimulation of the medial preoptic area in conscious female rats.

Lesions of the medial preoptic area (MPOA) induce nocturnal prolactin surges similar to those initiated by cervical stimulation (CS). These same lesions can abolish diurnal prolactin surges previously initiated by CS. Based on these results the MPOA has been suggested to contain two functionally dissimilar sets of neurons, one inhibitory for the nocturnal surge and the other stimulatory for the diurnal surge. The present study sought to demonstrate the existence of these neural elements by electrically stimulating the MPOA of conscious ovariectomized female rats during those times of day when these neurons would be most active. Serial blood samples were collected via cannula before, during and after the stimulation. Stimulation of the MPOA (01.00-05.00 h) on day 2 after CS inhibited the nocturnal surge of prolactin while sham MPOA stimulation of CS females did not disturb the nocturnal surge. MPOA stimulation in non-CS females had no effect upon prolactin secretion. Application of MPOA stimulation (15.00-19.00 h) to CS females also suppressed the diurnal surge of prolactin. Sham-stimulated CS females, however, secreted a diurnal surge peaking at 17.00 h. Basal prolactin levels were unaffected by MPOA stimulation (15.00-19.00 h) in non-CS females. The results from these experiments suggest that the MPOA contains neurons inhibitory for both the nocturnal and diurnal prolactin surges. In a further attempt to show a stimulatory role for the MPOA in prolactin regulation, MPOA stimulation was applied (15.00-19.00 h) to pentobarbital anesthetized non-CS females. Pentobarbital treatment allowed the MPOA stimulation to trigger two prolactin peaks, one at 16.00 h and the other at 19.00 h.(ABSTRACT TRUNCATED AT 250 WORDS)