Estrogen induction of progestin receptors in microdissected hypothalamic and limbic nuclei of female guinea pigs

Estrogen induction of cytosolic progestin receptors (CPRs) in the hypothalamus-preoptic area of the female guinea pig is correlated with facilitation of female sexual behavior and gonadotropin secretion by progesterone. The present study validated a CPR microassay and determined where, within microdissected areas of the hypothalamus-preoptic area of the female guinea pig, induction of CPRs by estradiol occurs. Ovariectomized adult guinea pigs were given 20 micrograms estradiol benzoate (EB) or oil vehicle for 3 successive days. CPRs were measured using the synthetic progestin [3H]-R5020. The highest basal (no estrogen treatment) level of CPRs was seen in the arcuate-median eminence (34.1 +/- 3.7 fmol/mg). With EB treatment, the highest level of CPRs was again in the arcuate-median eminence (178.0 +/- 12.0 fmol/mg). EB-treated females also had high CPR levels in the periventricular area (88.5 +/- 10.8 fmol/mg) and the medial preoptic area (86.3 +/- 9.3 fmol/mg). Moderate levels were seen in the ventromedial nucleus of the hypothalamus (32.7 +/- 3.0 fmol/mg) and in the anterior hypothalamic nucleus (13.0 +/- 2.1 fmol/mg), but these were not significantly different from the low levels in the medial amygdala (4.5 +/- 1.2 fmol/mg) and in the dorsomedial nucleus of the hypothalamus (5.4 +/- 1.1 fmol/mg) of EB-treated females. However, EB caused a significant induction over baseline levels not only in the arcuate-median eminence, periventricular area, and medial preoptic area, but also in the ventromedial nucleus of the hypothalamus and the anterior hypothalamic nucleus. EB did not increase CPRs in the medial amygdala or the dorsomedial nucleus of the hypothalamus.     

Quantitative distribution of nuclear androgen receptors in microdissected areas of the rat brain

The binding of androgens to specific high-affinity receptor sites in brain tissue is postulated as an initial event in the mechanism of central androgenic action. In an effort to assess the functional capacity of the androgen receptor system in the central nervous system, we measured the concentration of nuclear (ARn) as well as cytosolic androgen receptors (ARc) in 13 microdissected brain samples from intact male and female Sprague-Dawley rats. Tissues from 6 rats were combined for each determination and androgen receptor contents were measured with single-point in vitro assays that used saturating concentrations of high specific activity 3[H]dihydrotestosterone. We found that ARc levels tended to be higher in females than males although the general patterns of distribution were very similar. As expected, ARn concentrations were significantly higher in males than females. The highest concentrations of ARn (greater than 100 fmol/mg DNA) in males were measured in the ventromedial nucleus of the hypothalamus and medial amygdala; intermediate levels (50-100 fmol/mg DNA) were found in arcuate nucleus-median eminence, medial preoptic nucleus, periventricular preoptic area, bed nucleus of the stria terminalis, anterior hypothalamus, periventricular anterior hypothalamus, lateral septum, and parietal cortex, and low levels (less than 50 fmol/mg DNA) were measured in lateral preoptic nucleus and cortical amygdala. With the exception of the periventricular preoptic area (74 +/- 33 fmol/mg DNA), only very low concentrations of ARn were measured in females. These data provide the first quantitative profile of ARn in discrete brain nuclei and subregions of the rat.     

The quantitative distribution of cytosolic androgen receptors in microdissected areas of the male rat brain: effects of estrogen treatment

Estrogen and androgen synergize in the regulation of various neuroendocrine functions. To determine a potential cellular basis of this synergism, we measured androgen receptor (AR) in the cytosol of 16 hypothalamic and limbic nuclei and subregions in castrated male rats and castrated rats treated with estradiol. Androgen receptor was measured by a previously validated in vitro binding assay using the synthetic androgen methyltrienolone [( 3H]R1881). Male Sprague-Dawley rats (250-350 g) were castrated 2 weeks before the implantation of a 2.5-cm Silastic capsule filled with crystalline 17 beta-estradiol. Control rats were sham implanted. Estrogen treatment lasted for 1 week, after which time the animals were killed, their brains were frozen and sectioned, and individual nuclei and subregions were removed by a tissue punch technique. Tissue from six rats were combined for each determination. The highest levels of AR were found in the ventromedial nucleus (16.5 +/- 1.4 fmol/mg protein), medial preoptic area (12.1 +/- 1.4 fmol/mg protein), bed nucleus of the stria terminalis (11.6 +/- 1.4 fmol/mg protein), lateral septum (11.4 +/- 1.4 fmol/mg protein), arcuate nucleus-median eminence (10.9 +/- 2.1 fmol/mg protein), and medial amygdala (10.3 +/- 0.9 fmol/mg protein). Estrogen treatment resulted in significant increases in AR in medial preoptic area (14.8 +/- 0.6 fmol/mg protein; P less than 0.05) and medial amygdala (14.6 +/- 1.2 fmol/mg protein; P less than 0.02). Subsequent studies using block-dissected hypothalamus-preoptic area, anterior pituitary, and prostate revealed significant estrogen-mediated elevations in AR in anterior pituitary cytosol [42.2 +/- 3.0 vs. 26.4 +/- 1.6 fmol/mg protein (control); P less than 0.01], but not in hypothalamus-preoptic area or prostate cytosols. Estrogen treatment had no effect on AR affinity. The binding of [3H]R1881 was specific for AR and was not affected by the addition of radioinert progesterone to the incubation tube. Estimates of AR concentration were similar regardless of whether [3H]R1881 or [3H]dihydrotestosterone was used as the ligand. In this study, we describe the distribution of AR throughout the hypothalamus and limbic areas using biochemical techniques. In addition, we have identified some cellular events that may mediate the synergistic actions of estrogen and androgen on the neuroendocrine system.     

Pertussis toxin treatment of whole blood. A novel approach to assess G protein function in congestive heart failure

This study was designed to assess G protein function in mononuclear leukocytes (MNL) of patients with congestive heart failure (CHF). MNL membranes were ADP-ribosylated in vitro in the presence of pertussis or cholera toxin. The amount of pertussis toxin substrates did not differ significantly between CHF patients (6,100 +/- 224 fmol/mg, n = 23) and age-matched healthy control subjects (5,812 +/- 972 fmol/mg protein, n = 19). Among the CHF patients, no differences were observed between those with idiopathic and ischemic CHF. The amount of cholera toxin substrates also did not differ significantly between CHF patients (7,522 +/- 1,405 fmol/mg protein, n = 11) and control subjects (5,654 +/- 707 fmol/mg protein, n = 14). Moreover, basal and isoproterenol- and prostaglandin E1-stimulated cyclic AMP (cAMP) accumulation in MNL was similar in control subjects and patients. To detect more subtle alterations of the cAMP-generating system, we incubated anticoagulated blood with 250-400 ng/ml pertussis toxin for 4 hours at 37 degrees C. This treatment completely ADP-ribosylated the MNL pertussis toxin substrates. Incubation with pertussis toxin did not change basal or prostaglandin E1-stimulated cAMP generation in MNL of control subjects, but it significantly enhanced stimulated generation (443 +/- 44 vs. 643 +/- 93 pmol/10(7) cells, p less than 0.025) in MNL of CHF patients. This enhancement was most pronounced in the most severely ill patients (New York Heart Association class IV) and correlated with plasma norepinephrine levels, another marker of CHF severity (r = 0.798, n = 11, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcitonin gene-related peptide and its binding sites in the human central nervous system and pituitary.

Binding sites for synthetic human 125I-labeled calcitonin gene-related peptide (125I-CGRP) have been demonstrated in membranes of the human nervous system. Binding was high in the cerebellar cortex (1.35 +/- 0.27 fmol/mg of tissue; mean +/- SEM), spinal cord (1.06 +/- 0.27 to 1.27 +/- 0.23 fmol/mg), and nucleus dentatus (1.02 +/- 0.15 fmol/mg), intermediate in the inferior colliculus (0.80 +/- 0.14 fmol/mg) and substantia nigra (0.75 +/- 0.14 fmol/mg), low in the neocortex, globus pallidus, nucleus caudatus, hippocampus, amygdala, superior colliculus, thalamus, and hypothalamus (0.15-0.32 fmol/mg), and negligible in spinal and sympathetic ganglia and pituitary (less than 0.04 fmol/mg). Autoradiography showed distinct 125I-CGRP binding over the molecular and Purkinje cell layers of the cerebellar cortex and over the substantia gelatinosa posterior of the spinal cord. The highest levels of CGRP-like components were recognized in the dorsal part of the spinal cord and the pituitary gland. In the ventral part of the spinal cord as well as in the pituitary and thyroid glands, CGRP values were higher when measured by radioreceptorassay as compared to RIA, indicating that at least two CGRP-like components are present. The predominant CGRP-like peak on HPLC had the retention time of synthetic human CGRP. Immunohistochemistry revealed the presence of a dense plexus of CGRP immunoreactive nerve fibers in the dorsal horn of the spinal cord.     

Endothelin in human brain and pituitary gland: presence of immunoreactive endothelin, endothelin messenger ribonucleic acid, and endothelin receptors.

The presence of immunoreactive (IR) endothelin, endothelin mRNA, and endothelin receptors in human brain and pituitary gland has been studied by RIA, Northern blot hybridization, and receptor assay. IR endothelin was detected in all five brain regions examined (cerebral cortex, cerebellum, brain stem, basal ganglia, and hypothalamus) (6-10 fmol/g wet wt) and spinal cord (22 +/- 6 fmol/g wet wt, n = 7, mean +/- SEM). Higher concentrations of IR endothelin were found in the pituitary gland (147 +/- 30 fmol/g wet wt). Fast protein liquid chromatographic analysis of the IR endothelin in pituitary gland showed a large IR peak in the position of endothelin-3 and a smaller peak in the position of endothelin-1, whereas IR endothelin in the hypothalamus and brain stem was mainly endothelin-1. Endothelin messenger RNA was detected by Northern blot hybridization in the pituitary but not in hypothalamus. The receptor assay showed that 125I-endothelin-1 binding sites were present in large numbers in all five brain regions but were much less abundant in the pituitary gland. Binding capacity and dissociation constant were 5052 +/- 740 fmol/mg protein and 0.045 +/- 0.007 nM in brain stem and 963 +/- 181 fmol/mg protein and 0.034 +/- 0.009 nM in hypothalamus. In the pituitary gland, there were two classes of binding sites for endothelin with dissociation constants of 0.059 +/- 0.002 nM (binding capacity = 418 +/- 63 fmol/mg protein) and 0.652 +/- 0.103 nM (binding capacity = 1717 +/- 200 fmol/mg protein). Endothelin-1, -2 and -3 were almost equipotent in displacing the binding (IC50 approximately 0.04 nM). These findings are in accord with the possibility that endothelin acts as a neurotransmitter, neuromodulator or neurohormone in man.     

Localization of growth hormone-releasing hormone in the human hypothalamus and pituitary stalk

The localization of the recently identified GH-releasing hormone (GHRH) in the human hypothalamus and pituitary stalk was determined by microdissection techniques and a specific RIA for GHRH. The highest concentrations of GHRH immunoreactivity (IR-GHRH) in the hypothalamus were found in the area of the infundibular nucleus (83 +/- 4 ng/mg protein; average +/- range). Lower quantities were found in other hypothalamic regions. Very high concentrations of IR-GHRH were present in the upper portion of the pituitary stalk (1454 +/- 48 ng/mg protein), and they decreased gradually toward the distal end of the stalk (21 +/- 3 ng/mg). This concentration gradient suggests that the peptide reaches the anterior pituitary mainly by way of the long portal vessels. Somatostatin, the second neuropeptide involved in the regulation of GH secretion from the anterior pituitary, had a pattern of distribution along the pituitary stalk very similar to that of IR-GHRH.     

Posterior hypothalamic lesions advance the onset of puberty in the female rhesus monkey

The effects of experimental lesions in the posterior hypothalamus and the anterior hypothalamus on menarche and first ovulation were examined in nonhuman primates. With the aid of x-ray ventriculography, bilateral lesions were made by passing a radiofrequency current through a thermister electrode in the posterior hypothalamus (n = 7) or the anterior hypothalamus (n = 6) of female rhesus monkeys at 18 months of age. Four animals that received sham lesions as well as four normal females of a similar age served as controls. All animals were caged individually and examined daily for vaginal bleeding and sex skin color change. Developmental changes in gonadotropins, ovarian steroids, body weight, and nipple size were monitored throughout the experiments. The time of first ovulation was determined by laparoscopic observation of the newly formed corpus luteum and by the level of circulating progesterone. Histological examination confirmed that the bilateral lesions in the hypothalamus were approximately 2-3 mm in diameter and overlapped midline. Primary sites of posterior hypothalamic lesions included the premamillary area and the posterior nucleus, while the infundibular nucleus and the median eminence were entirely spared. The posterior lesions encroached upon the mamillary nuclei caudally in most cases and upon the ventromedial nucleus rostrally in some cases. Primary sites of anterior hypothalamic lesions included the medial preoptic area, the periventricular preoptic nucleus, and the anterior hypothalamic nucleus. Partial lesions of the diagonal bundle of Broca, the medial preoptic nucleus, and the paraventricular nucleus were also detected. Posterior hypothalamic lesions advanced the ages at menarche (22.2 +/- 1.3 months; P less than 0.001) and first ovulation (40.7 +/- 2.7 months; P less than 0.05) compared to those of control animals (menarche, 30.3 +/- 3.1; first ovulation, 51.2 +/- 3.3 months). The body weight at menarche of these lesioned animals (2.62 +/- 0.11 kg) was smaller (P less than 0.05) than that of controls (3.14 +/- 0.20 kg), but the body weight at first ovulation of lesioned animals (4.36 +/- 0.28 kg) was not different from that of controls (4.57 +/- 0.13 kg). Hormonal and physical changes during maturation, i.e. an increase in circulating estradiol and growth in nipple size before menarche and first ovulation, occurred earlier in the lesioned animals and the growth spurt before first ovulation not only began earlier but also attained mature levels several months earlier than that in control animals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regional distribution of human growth hormone-releasing hormone in the human hypothalamus by radioimmunoassay

The regional distribution of human GH-releasing hormone (hGHRH) in the hypothalamus was determined by RIA using a standardized microdissection technique. The antiserum used in the RIA is highly specific for human GHRH-44, and cross-reactivity to hGHRH-40 is minimal (less than 0.06%). The concentration and content of immunoreactive hGHRH-44 (IR-GHRH) were measured in extracts of 12 nuclei and areas microdissected from hypothalami from 5 autopsy subjects. Extracts of proximal and distal pituitary stalk were similarly analyzed for IR-GHRH. The highest concentration of IR-GHRH in the hypothalamus was in the infundibular nucleus, with lesser concentrations in the periventricular area and paraventricular and supraoptic nuclei. All other regions had measurable concentrations, except the mammillary nuclei. There was 56.1 +/- 12.4 (+/- SD) ng (11.1 +/- 1.2 pmol) IR-GHRH in the human hypothalamus and pituitary stalk, with 62% in the pituitary stalk. IR-GHRH in tissue extracts produced the same dose-response curve as did hGHRH in the RIA and coeluted with synthetic hGHRH-44 on gel filtration. Our results indicate the following. 1) The regional distribution of IR-GHRH in the human hypothalamus differs from the regional distributions of other neuropeptides. 2) The infundibular nucleus contains the greatest IR-GHRH regional concentration, in accord with immunohistochemical studies. 3) Appreciable concentrations of IR-GHRH are found in regions not previously identified by immunohistochemical techniques. 4) The IR-GHRH molar content in the hypothalamus-pituitary stalk is lower than that reported for other hypothalamic releasing hormones.     

Distribution of neurotensin-like immunoreactivity (NTLI) and neurotensin receptor in the rat brain and effects of various drugs on NTLI concentration and NT receptor in the rat cerebral cortex

A radioimmunoassay for neurotensin(NT) and binding assay for NT receptor have been developed to determine neurotensin-like immunoreactivity (NTLI) concentration and to characterize NT receptor in the rat brain. The effect of various drugs affecting the dopaminergic system in the brain were also investigated to clarify the relationship between NT and dopamine. NTLI was widely distributed in various regions of the rat brain: the highest level was 53.1 +/- 14.1 ng/g wet wt in the hypothalamus, 11.9 +/- 6.0 ng/g wet wt in the thalamus, 6.9 +/- 0.7 ng/g wet wt in the cerebral cortex and 0.7 +/- 0.2 ng/g wet wt in the cerebellar cortex. The Bmax of NT receptor was 20.2 +/- 4.6 fmol/mg protein in the cerebral cortex, 16.0 +/- 4.9 fmol/mg protein in the hypothalamus, 13.3 +/- 3.7 fmol/mg protein in the thalamus and no detectable level in the cerebellar cortex. The Kd of NT receptor was 2.7 +/- 0.7 nM in the cerebral cortex, 1.9 +/- 0.8 nM in the hypothalamus, 1.1 +/- 0.2 nM in the thalamus. Intraperitoneal(ip) bolus administration of L-DOPA caused the reduction in NTLI concentration from 9.2 +/- 1.0 ng/g wet wt to 5.3 +/- 2.1 ng/g wet wt and Bmax of NT receptor from 19.7 +/- 3.1 fmol/mg protein to 13.9 +/- 2.6 fmol/mg protein with no change in Kd. Intracerebroventricular administration of dopamine also induced a significant reduction of NTLI concentration and Bmax of NT receptor with no change in Kd. Inversely, ip bolus administration of alpha-methyldopa evoked an increase in NTLI concentration from 6.9 +/- 0.9 ng/g wet wt to 12.0 +/- 3.0 ng/g wet wt and that in Bmax of NT receptor from 15.9 +/- 2.9 fmol/mg protein to 24.2 +/- 4.6 fmol/mg protein. Serial administration of haloperidol evoked an increase in Bmax of NT receptor from 16.9 +/- 1.5 fmol/mg protein to 20.5 +/- 1.9 fmol/mg protein with no changes in Kd and NTLI concentration. These results suggest that dopamine may modulate the action of NT in the rat cerebral cortex.     

Differential distribution of muscarinic cholinergic and putative nicotinic cholinergic receptors within the hypothalamo-neurohypophysial system of the rat

Binding of the muscarinic cholinergic receptor probe [3H]quinuclidinylbenzilate ([3H]QNB) and the putative nicotinic receptor probe [125I]alpha-bungarotoxin ([125I]alpha BTX) to vasopressin (VP) and oxytocin (OT) neuroendocrine cells was investigated with a combination of quantitative receptor binding, autoradiography and immunocytochemistry. A single high-affinity site was labelled by [3H]QNB in the hypothalamus and pituitary (KD = 0.76-1.44 X 10(-10) M) with a mean hypothalamic density of 213 fmol/mg protein compared with only 56 fmol/mg protein in the pituitary. Analysis of autoradiographic silver grains from [3H]QNB binding revealed a relative absence of binding associated with magnocellular VP and OT cell groups in the hypothalamus. The median eminence and neural lobe of the pituitary contained low levels of [3H] QNB binding, which, however, were the highest within the hypothalamo-neurohypophysial system. The ligand [125I]alpha BTX binds with both a high and low affinity to sites within the hypothalamus and pituitary (high-affinity KD = 0.77-1.03 X 10(-10) M). In the hypothalamus the density of high-affinity binding sites (25 fmol/mg protein) is approximately 2.5 times greater than in the pituitary. In contrast to [3H]QNB, high-affinity binding of [125I]alpha BTX was found to be highly concentrated within the supraoptic nucleus, nucleus circularis, and the magnocellular areas of the paraventricular nucleus. Autoradiographic silver grains were distributed over both VP and OT immunoreactive neurons and processes. Binding within the neural lobe was very low. These data suggest that the cholinergic regulation of VP and OT release may occur via nicotinic cholinergic receptors at the level of the magnocellular cell bodies and predominantly via muscarinic cholinergic receptors within the neural lobe.     

Effects of thyroxine on oestrogen receptor concentrations in anterior pituitary and hypothalamus of hypothyroid rats

The effects of thyroxine (T4) were studied on the concentration of oestrogen receptors in the anterior pituitary gland and hypothalamus of ovariectomized euthyroid and hypothyroid rats. A group of rats was made hypothyroid by the administration of 131I. Seven days after ovariectomy, animals were separated into five groups: I, euthyroid controls; II, hypothyroid controls; III, hypothyroid and injected with oestradiol benzoate (10 micrograms/day for 10 days); IV, hypothyroid and injected with T4 (4 micrograms/day for 10 days) and V, hypothyroid and injected with both oestradiol and T4 as described above. In group I, oestrogen receptor levels in pituitary cytosol were 44.4 +/- 3.4 (S.D.) fmol/mg protein and in the nucleus 47.7 +/- 4.0 fmol/mg DNA. In group II the respective values were 12.8 +/- 1.7 fmol/mg protein (P less than 0.01) and 12.7 +/- 1.7 fmol/mg DNA (P less than 0.01 compared with group I). In group III, cytosolic receptor concentrations decreased when compared with those in group II (P less than 0.05), whereas nuclear receptor concentrations rose significantly (P less than 0.01). Group IV had both pituitary cytosolic and nuclear receptors increased (P less than 0.01 compared with group II). In group V there were no changes in cytosolic receptor concentrations but a significant (P less than 0.01) rise in nuclear receptors as compared with group II. Hypothalamic oestrogen receptors in untreated hypothyroid rats (group II) were unchanged in the cytosol and diminished (P less than 0.01) in the nucleus in relation to euthyroid controls (group I). Thyroxine, but not oestrogen, was effective in increasing the concentration of cytosolic receptors (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and regulation of aromatase activity in the rat hypothalamus and limbic system

Conversion of androgen to estrogen in the rat brain is catalyzed by aromatase enzymes. The maximum concentrations of these enzymes are found within the hypothalamus and amygdala, where they appear to play an important role in the process by which androgens affect both behavior and neuroendocrine function. In the present study, we measured the levels of aromatase activity (AA) in 20 nuclei and brain regions of the adult rat brain. Individual nuclei were microdissected from 600-micron frozen sections. Tissues from 3 animals were pooled, and AA was measured by an in vitro radiometric assay that quantifies the stereospecific production of 3H2O from [1 beta-3H]androstenedione as an index of estrogen formation. We report that AA is heterogeneously distributed within the rat brain. The greatest amounts of activity were found in the bed nucleus (n.) of the stria terminalis (700 protein fmol/h . mg) and in the medial (MA) and cortical amygdala (400-600 fmol/h . mg protein) of the male. There was an evident rostral-caudal and medial-lateral gradient in AA throughout the diencephalon. Activity was high in the periventricular preoptic n. and medial preoptic n.; intermediate in the suprachiasmatic preoptic n., anterior hypothalamus, periventricular anterior hypothalamus, and ventromedial n.; and low in the arcuate n.-median eminence, lateral preoptic n., supraoptic n., dorsomedial n., and lateral hypothalamus. Regions devoid of measurable AA included the medial and lateral septum, caudate-putamen, hippocampus, and parietal cortex. In the female, AA was greatest in the MA and cortical amygdala. We found that AA in the MA, stria terminalis n., suprachiasmatic preoptic n., periventricular preoptic in., medial preoptic n., anterior hypothalamus, and ventromedial n. was significantly greater (P less than 0.05) in males than in females. Orchidectomy reduced AA to levels seen in females, and administration of testosterone to castrated males restored AA in these areas. No significant sex differences were observed in any other hypothalamic or amygdaloid nuclei, although AA was increased by testosterone treatment in the periventricular anterior hypothalamus, arcuate n.-median eminence, and lateral hypothalamus. Our results provide a quantitative profile of AA in specific hypothalamic and limbic nuclei of the rat brain as well as information on the control of AA within these discrete regions.     

Human corpus luteum: luteinizing hormone and chorionic gonadotropin receptors during the menstrual cycle

To characterize and determine the concentration of LH/hCG receptors in human corpora lutea of the menstrual cycle, we measured occupied and unoccupied receptors and determined the association (Ka) and dissociation (Kd) constants individually in 23 corpora lutea (CL) and 4 corpora albicantia obtained at the time of tubal ligation from 25 normal cycling women. We found no [125I]hCG binding in any of the corpora albicantia. Scatchard plot analysis for each CL revealed a linear binding plot indicative of a single set of LH/hCG receptors. The mean concentration of unoccupied receptors was 36 +/- 10 (+/- SE) fmol/mg protein in the early luteal phase (days 15-19; n = 5), 64 +/- 11 fmol/mg protein in the midluteal phase (days 20-25; n = 13), and 42 +/- 19 fmol/mg protein in the late luteal phase (days 26-30; n = 5). The concentrations of occupied receptors were 56 +/- 8, 46 +/- 6, and 54 +/- 12 fmol/mg protein in the early, mid-, and late luteal phases, respectively. Total (occupied plus unoccupied) receptor concentrations reached maximum levels of 110 +/- 11 fmol/mg protein in the midluteal phase. Ka increased progressively from 12 +/- 4 X 10(9) mol/L-1 in the early luteal phase to 19 +/- 7 X 10(9) and 21 +/- 8 X 10(9) mol/L-1 in the mid- and late luteal phases. We conclude that in normal CL, 1) total and unoccupied LH/hCG receptor levels parallel progesterone secretion; 2) changes in the binding affinity may be important in sustaining and/or rescuing the CL; and 3) loss of LH/hCG receptors is probably related to luteolysis.     

In vivo heterologous regulation of rat glomerular and vascular atrial natriuretic factor receptors by angiotensin II

Since glomerular and vascular atrial natriuretic factor (ANF) receptor density may vary inversely with circulating ANF concentration, and the latter may respond to changes in blood pressure, we investigated whether ANF receptors are modified by a pressor agent, angiotensin II. Rats were infused intraperitoneally for 6 days with either a non-pressor (200 ng/kg per min) or pressor dose (800 ng/kg per min) of angiotensin II. Blood pressure was higher and plasma renin activity lower in the pressor than in the non-pressor or sham-infused groups. Plasma ANF was elevated only in animals infused with a pressor dose of angiotensin II. Glomerular ANF receptor density was higher in the non-pressor (Bmax = 1308 +/- 360 fmol/mg protein) and lower in the pressor (Bmax = 459 +/- 13 fmol/mg protein) than in the sham-infused group (Bmax = 755 +/- 303 fmol/mg protein). In vitro, angiotensin II-precontracted isolated glomeruli from animals infused with a pressor dose of angiotensin II were less sensitive to the relaxant effect of low ANF concentrations (10(-12) and 10(-11) mol/l) than those receiving a non-pressor dose of angiotensin II or sham-infused. Vascular ANF receptor density was similar in the control and non-pressor groups (Bmax = 64 +/- 12 and 62 +/- 20 fmol/mg protein, respectively) and decreased in the pressor group (Bmax = 30 +/- 4 fmol/mg protein). Norepinephrine-precontracted aorta strips from rats receiving a non-pressor dose of angiotensin II were more sensitive to the relaxant effect of ANF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased neuropeptide Y content in individual hypothalamic nuclei, but not neuropeptide Y mRNA, in diet-induced obesity in rats.

Neuropeptide Y (NPY) is the most powerful appetite stimulant known, and chronic administration leads to obesity. The hypothalamic content of NPY varies with nutritional status, suggesting that it is of physiological importance. We measured NPY in specific hypothalamic nuclei and NPY mRNA in the hypothalamus by Northern blotting in rats made obese by feeding a highly palatable diet compared with controls fed standard chow. In animals fed the palatable diet, NPY concentrations were increased in the paraventricular nucleus (mean +/- S.E.M.; 19.5 +/- 2.3 vs 11.1 +/- 1.1 fmol/micrograms protein, P less than 0.02), the arcuate nucleus (20.4 +/- 3.3 vs 9.3 +/- 0.6 fmol/micrograms protein, P less than 0.01), the medial preoptic area (9.1 +/- 0.9 vs 5.9 +/- 0.7 fmol/micrograms protein, P less than 0.02) and the anterior hypothalamus (2.7 +/- 0.2 vs 2.0 +/- 0.1 fmol/micrograms, P less than 0.02). Hypothalamic NPY mRNA measured by Northern blot analysis was, however, unchanged. These results suggest that the increase in NPY was due to decreased release rather than increased NPYergic activity. The findings are in accord with the neuroendocrine disturbance and increased thermogenesis observed in this model of obesity.     

Glucocorticoid receptors in leukocytes and gill of juvenile coho salmon (Oncorhynchus kisutch)

We demonstrated that cytosol from the gill of coho salmon (Oncorhynchus kisutch) had saturable, high affinity, low capacity binding to radiolabeled [3H]cortisol (Kd = 2.24 +/- 0.28 nM, mean +/- 1 SE; Nmax = 41.4 +/- 7.4 fmol/mg protein) and radiolabeled [3H]triamcinolone acetonide (TA; Kd = 0.38 +/- 0.03 nM, Nmax = 37.8 +/- 4.9 fmol/mg protein). Similarly, TA bound to cytosolic fractions of leukocytes harvested from spleen (Kd = 0.32 +/- 0.03 nM, Nmax = 8.3 +/- 2.0 fmol/mg protein) and anterior kidney (Kd = 0.37 +/- 0.03 nM, Nmax = 30.2 +/- 5.2 fmol/mg protein) and to whole leukocytes from spleen (Kd = 0.30 +/- 0.04 nM, Nmax = 445 +/- 57 sites/cell) and anterior kidney (Kd = 0.40 +/- 0.04 nM, Nmax = 1198 +/- 180 sites/cell). The competition hierarchies of steroid competitors were the same for both ligands and all tissues (TA greater than cortisol greater than 17 alpha-hydroxyprogesterone greater than cortisone greater than aldosterone greater than testosterone). The differences in ligand binding in leukocytes from spleen and anterior kidney are consistent with previously reported organ-dependent sensitivity of leukocytes to cortisol.     

Lung beta-adrenoceptors in pulmonary hypertension. A study of biopsy specimens in children with congenital heart disease.

Characteristics of beta-adrenoceptors were analyzed using radioligand-binding techniques with 3H-dihydroalprenolol in lung specimens from 11 children with pulmonary hypertension (median age, three years) undergoing surgical repair of congenital heart defects and four pediatric control subjects (median age, five years) undergoing thoracotomy for removal of neoplasms or cysts. Scatchard analysis of 3H-DHA binding to lung membranes showed similar values of the dissociation constant in both groups (Kd = 0.72 +/- 0.22 nM in patients vs 1.22 +/- 0.22 nM in controls; p = NS). The receptor density was significantly increased in patients in comparison with controls, with respective values of 164 +/- 19 and 95 +/- 13 fmol/mg of protein (p less than 0.025), and correlated directly with mean pulmonary arterial pressure (r = 0.82; p less than 0.0005). No significant relationship was observed between receptor number and pulmonary arterial medial thickness. Thus, the increase in receptor density in these patients may be related to adaptative changes in cells other than vascular smooth muscle.