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.     

Characterization of melatonin receptors in the ram pars tuberalis: influence of light

The present study was conducted to assess the binding of [125I]melatonin to frozen unfixed sections of pars tuberalis/median eminence tissue from Ile-de-France rams exposed or not exposed to light before slaughter. The specificity of [125I]melatonin binding to the pars tuberalis tissue was revealed by autoradiography and the magnitude of binding as related to the pars tuberalis area was determined after incubation and counting of pars tuberalis/median eminence sections. Subsequent studies with sections incubated with [125I]melatonin indicated that 1. the binding sites were saturable; 2. binding was stable for 24 h at 20 degrees C, but unstable at 28 or 37 degrees C; 3. melatonin and [127I]melatonin had a similar potency to compete with [125I]melatonin for binding sites, whereas other ligands such as serotonin or N-acetylserotonin were devoid of activity, and 4. by Scatchard analysis, the constant affinity Ka was found to be high in the 10(10) l/mol range. Rams exposed to light throughout the night prior to slaughter presented a significant increase in the apparent number of [125I]melatonin binding sites in comparison to animals maintained under darkness (2.25 +/- 0.30 vs 1.01 +/- 0.17 fmol/mm2 pars tuberalis, p less than 0.01), whereas Ka values were similar in both groups. These results indicate the presence of true melatonin receptors in the pars tuberalis of the ram. Furthermore, they suggest that their apparent number is light-dependent.     

Melatonin-binding sites in the rat brain and pituitary mapped by in-vitro autoradiography

Using picomolar concentrations of [125I]iodomelatonin and in-vitro autoradiography, specific melatonin-binding sites have been mapped in the rat brain and pituitary. Using this same technique, high-affinity melatonin receptors had previously been identified in the suprachiasmatic nucleus (SCN) and median eminence regions of the rat hypothalamus. The presence of melatonin binding in the SCN has been confirmed, but the second area of binding has been identified as the pars tuberalis of the pituitary, and a completely novel area of binding is also reported in the area postrema. The existence of lower affinity melatonin receptors in the rat brain was also investigated using in-vitro autoradiography and higher concentrations of [125I]iodomelatonin. No further sites of specific binding were, however, disclosed.     

125I]melatonin binding sites in membrane preparations of quail brain: characteristics and diurnal variations

The binding of [125I]melatonin, a labelled ligand of melatonin, to quail brain membrane preparations was studied. Kinetic studies indicated that the binding of [125I]melatonin to membrane preparations of quail brains collected at mid-light was time-dependent and reversible. The Kd value determined from kinetic analysis was 614 pmol/l, similar to the Kd 705 +/- 81 pmol/l obtained from saturation experiments. The [125I]melatonin binding sites in quail brain membrane had the following order of pharmacological affinities: melatonin greater than 6-chloromelatonin greater than N-acetylserotonin greater than 5-hydroxytryptamine much greater than 5-methoxytryptophol, tryptamine, L-tryptophan, 5-hydroxytryptophan, 5-hydroxyindole-3-acetic acid, 1-acetylindole-3-carboxaldehyde, 3-acetylindole. Compounds known to act on serotonergic, adrenergic or acetylcholinergic receptors were inactive as compared with melatonin. Our results demonstrated the presence of specific melatonin binding sites in the quail brain membrane preparations. In addition, saturation studies demonstrated that [125I]melatonin binding sites in quail brain membrane preparations were 42% higher at mid-light (39.6 +/- 2.2 fmol/mg protein) than at mid-dark (22.6 +/- 2.5 fmol/mg protein), with no significant variation in their binding affinities.     

Demonstration of melatonin-binding sites on the pars tuberalis of the rat

Melatonin-binding sites have previously been identified in the suprachiasmatic nucleus (SCN) and median eminence (ME) of the rat. We have further investigated the localization of melatonin-binding sites in the rat hypothalamus and pituitary using the ligand [125I]iodomelatonin and in-vitro autoradiography. The presence of specific melatonin-binding sites in the SCN is confirmed; however the second area of melatonin binding is identified as the pars tuberalis of the pituitary and not the ME as previously described. No other areas which bound melatonin were found in either the pituitary or the hypothalamus.     

Characterization and mapping of melatonin receptors in the brain of three mammalian species: rabbit, horse and sheep. A comparative in vitro binding study.

Melatonin receptors were characterized in the brains of three mammals (rabbit, horse and sheep) by an in vitro binding technique, using 2-[125I]iodomelatonin as labelled ligand. Although binding sites for melatonin have been described recently in several vertebrate species (including the sheep), the rabbit and the horse have not been the subject of investigation so far. Apart from characterization, the present report describes receptor distribution in a number of brain regions, thus allowing for direct interspecies comparison under the same methodological conditions. 2-[125I]iodomelatonin labelled high-affinity binding sites in crude membrane preparations from these species. A series of kinetic and saturation experiments revealed that the binding was rapid, stable, saturable, reversible, of high affinity (Kd in the low picomolar range) and low capacity (Bmax between 1 and 20 fmol/mg protein). The competition studies showed that the relative order of potency of a variety of indoles for inhibition of 2-[125I]iodomelatonin binding was as follows: 2-iodomelatonin greater than 6-chloromelatonin greater than melatonin much much greater than 5-methoxytryptophol greater than 5-methoxytryptamine, and that it was similar in the different brain regions. Prazosin, which has been reported as an extremely potent melatonin analog in the hamster brain, possessed no potency in all preparations from different regions in the three species under investigation. The regional distribution of the receptor showed insignificant species differences. Highest density was always recorded in the median eminence/pars tuberalis (ME/PT) area. Other regions (SCN, POA and certain cortical areas), showed lower, but significant, receptor content. Saturation and competition studies revealed that these binding sites were also of high affinity, low capacity and high specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Melatonin signal transduction in hamster brain: inhibition of adenylyl cyclase by a pertussis toxin-sensitive G protein

Melatonin signal transduction was examined in median eminence/pars tuberalis (ME/PT) explants from Djungarian hamsters. High affinity melatonin receptors in hamster ME/PT were first quantified by in vitro autoradiography using the potent melatonin agonist 125I-labeled melatonin ([125I]MEL). Scatchard analysis of [125I]MEL binding in ME/PT revealed high affinity receptors [dissociation constant (Kd) = 2.75 X 10(-11) M]. [125I]MEL binding was markedly reduced by guanine nucleotides; treatment with the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) caused a 10-fold decrease in receptor affinity. Melatonin (10 nM) significantly inhibited forskolin-stimulated cAMP accumulation in ME/PT, but not in pituitary or pineal glands. In ME/PT explants, melatonin and 6-chloromelatonin inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner with similar potency (significant inhibition for each at concentrations greater than or equal to 100 pM). Serotonin significantly inhibited forskolin-stimulated cAMP levels only at doses greater than or equal to 100 microM. Inhibition of [125I]MEL binding in ME/PT by these three indolamines paralleled that determined for inhibition of forskolin-stimulated cAMP accumulation. Pertussis toxin treatment (1 microgram/ml) blocked the ability of melatonin (10 nM) to inhibit forskolin-stimulated cAMP accumulation and significantly reduced [125I]MEL binding. Pertussis toxin ADP-ribosylated the alpha-subunits of at least two guanine nucleotide-binding proteins in ME/PT explants with molecular weights of approximately 40 K. Melatonin did not increase phosphodiesterase activity in ME/PT explants. The results strongly suggest that a signal transduction pathway for melatonin in mammals involves inhibition of adenylyl cyclase by a pertussis toxin-sensitive guanine nucleotide-binding protein.     

Identification of binding sites for an insulin-like growth factor (IGF-I) in the median eminence of the rat brain by quantitative autoradiography

The microanatomical location of IGF-I binding in the rat brain was determined by in vitro autoradiography with slide-mounted sections of frozen brain. Sections incubated in 0.1 nM [125I]-iodo-IGF-I produced a dense grain concentration in regions of the autoradiographic image corresponding to the external palisade zone of the median eminence; other hypothalamic regions were not so heavily labeled. This reaction was significantly reduced in the presence of 100 nM IGF-I. Measurement of binding by computer digital image analysis of autoradiographic images showed that specific binding for IGF-I in the median eminence was 41.3 +/- 8 X 10(-3) fmol/mm2 (mean +/- SEM); nonspecific binding was 11.9 +/- 1.8 X 10(-3) fmol/mm2. In contrast, specific binding to other hypothalamic regions was uniformly lower. In a separate experiment, 1000 nM unlabeled insulin was added. Without insulin, specific binding was 23 +/- 0.9 X 10(-3) fmol/mm2; nonspecific binding was 8 +/- 0.5 X 10(-3) fmol/mm2. In the presence of 1000 nM unlabeled insulin, specific binding for [125I]-iodo-IGF-I was 23 +/- 1 X 10(-3) fmol/mm2. The results suggest that a high concentration of receptors for an IGF-I-like molecule is present in the median eminence.     

Melatonin binding sites in the Harderian gland of Syrian hamsters: Sexual differences and effect of castration

Abstract: The presence of specific melatonin binding sites in the Harderian gland of Syrian hamsters was studied using [¹²⁵I]melatonin. Saturation binding experiments conducted with [¹²⁵I] melatonin at 37°C using Harderian glands of both male and female Syrian hamsters revealed a single nanomolar-affinity site. The dissociation constants (Kd) were 6.47 and 6.94 nM for males and females, respectively. The concentration of the binding sites was 7.58 fmol/mg protein for males and 13.50 fmol/mg protein for females. Castration of male hamsters resulted in a significant increase in [¹²⁵I] melatonin binding sites while chronic melatonin administration did not modify the binding properties. The results confirm the presence of melatonin binding sites in the Harderian glands of rodents. The gender-associated differences found together with the effects of castration in male hamsters suggest an androgenic control in [¹²⁵I] melatonin binding sites of the Syrian hamster Harderian gland.     

Opioid receptors are present in the hypothalamus but not detectable in the anterior pituitary of the developing ovine fetus.

Exogenous opioids stimulate adrenocorticotropic hormone (ACTH) release in fetal sheep after day 125 of pregnancy (term 145 days), but not at day 110. To determine if the different response is due to an alteration in opioid-binding sites and to examine sites of opioid action on ACTH release, we established an in vitro binding assay to study changes and characteristics of opioid receptor binding sites in ovine fetal hypothalamus and pituitary during development. [3H]-naloxone was used as the radiolabelled ligand. The binding of [3H]-naloxone to hypothalamic membrane preparations was specific, saturable with respect to [3H]-naloxone concentration, and linear with the hypothalamic membrane protein content. Binding assays were conducted on tissues collected from fetuses at three gestational ages: days 110-115 and 125-130 and term. In all cases, Scatchard analysis revealed a single class of binding sites with high binding affinity (0.9-1.2 nM) which did not differ significantly with gestational ages. The binding capacity increased significantly from 45.4 +/- 2.5 fmol/mg protein at days 110-115 to 76.8 +/- 2.3 fmol/mg at days 125-130, but did not change further in term fetuses. When anterior pituitaries from the three groups of fetuses were processed and analyzed in a similar manner, no detectable binding was found. These results indicate (1) that endogenous opioid peptides may act at the hypothalamus rather than pituitary to regulate hypothalamic-pituitary-adrenal activity in the ovine fetus, and (2) the developmental changes in the binding capacity may contribute, in part, to the altered ACTH response reported in previous in vivo studies.     

Evidence for the presence and characterization of angiotensin II receptors in rat anterior pituitary membranes

Previous work from this laboratory (1) indicates that angiotensin II (AII) can affect release of several anterior pituitary hormones, both in vivo and in vitro. To ascertain whether specific receptors mediate the effects of AII on the anterior pituitary, specific binding as well as the kinetics of [125I] AII binding to rat anterior pituitary membranes were analyzed. Binding of [125I] AII was rapid, reaching equilibrium within 4 min at 37 C. Specific binding was approximately 90%. Increasing concentrations of ligand resulted in saturation of binding, with equilibrium attained at [125I] AII = 2 nM. Scatchard analysis of the data indicated a single class of binding sites, with an equilibrium dissociation constant, Kd = 0.49 nM, and a maximum binding capacity of 40 fmol/mg protein. Specific binding was directly proportional to membrane protein concentration (range 20-240 micrograms protein). Binding was competitively inhibited on an equimolar basis by (Sar1, Ala8) AII (Saralasin), a specific AII receptor antagonist. The decapeptide Angiotensin I was about 10-20-fold less potent in inhibiting specific AII binding. These studies demonstrate and characterize specific receptor sites for AII in the anterior pituitary gland and offer additional evidence for a role of AII in the regulation of anterior pituitary hormone secretion.     

Binding Characteristics, Regional Distribution and Diurnal Variation of [125I]-Iodomelatonin Binding Sites in the Chicken Brain

The [125I]-iodomelatonin binding sites in chicken brain membrane preparations were studied. The binding of [125I]-iodomelatonin to the membrane preparations of chicken brain was rapid, stable, saturable, and reversible. The order of pharmacological affinities of [125I]-iodomelatonin binding sites in the chicken brain membrane preparations was: melatonin greater than 6-chloromelatonin greater than N-acetylserotonin greater than 5-hydroxytryptamine greater than tryptamine greater than 5-methoxytryptophol, much greater than 1-acetylindole-3-carboxaldehyde, 5-hydroxyindole-3-acetic acid, L-tryptophan, 5-hydroxytryptophan, 3-acetylindole. Compounds known to act on the receptor of norepinephrine or acetylcholine were inactive as compared to melatonin. Among the various brain regions studied, melatonin binding had maximal level in the hypothalamus, intermediate levels in the mid-brain, ponsmedulla, and telencephalon, and minimum level in the cerebellum. Subcellular fraction studies indicated that 40% of the binding was located in the mitochondrial fraction, 27% in the nuclear, 26% in the microsomal, and 6% in the cytosol fraction. Scatchard analysis of the membrane preparations revealed a dissociation constant (Kd) of 199.6 +/- 17 pM and a total number of binding sites (Bmax) of 16.6 +/- 0.75 fmol/mg protein at midlight. Thus, our results showed the presence of specific melatonin binding sites in the chicken brain membrane preparations. Saturation studies demonstrated that [125I]-iodomelatonin binding capacity in chicken brain membrane preparations were 40% greater at midlight (16.6 +/- 0.75 fmol/mg protein) than at middark (10.6 +/- 0.56 fmol/mg protein), with no significant variation in their binding affinities.     

Rat pineal alpha 1-adrenoceptors: identification and characterization using [125I]iodo-2-[beta-(4-hydroxyphenyl)-ethylaminomethyl]tetralone

[125I]Iodo-2-[beta-(4-hydroxyphenyl)-ethylaminomethyl]tetralone ([125I]HEAT), a selective, high affinity, high specific activity alpha 1-adrenoceptor ligand, is used to characterize alpha-adrenoceptors in the rat pineal gland. Binding of [125I]HEAT to membranes is rapid [association rate constant (Kon) = 3.1 nM-1 min-1] and readily reversible either by 100-fold dilution or by addition of excess unlabeled HEAT [apparent dissociation rate constant (Koff) = 0.153 min-1). Saturation experiments indicate a single class of noncooperative binding sites with an equilibrium binding constant (KD) of 41 +/- 9 pM and a Bmax of 399 +/- 63 fmol/mg protein. The relative potency of a number of adrenoceptor agonists and antagonists in competing with [125I] HEAT indicates the receptor is an alpha 1-subtype. In addition, inhibition of binding is stereospecific; (-)epinephrine and norepinephrine are more than 100-fold more potent than their (+)isomers. The identification of alpha 1-adrenoceptors in the pineal gland is consistent with evidence indicating a role for these receptors in the regulation of melatonin synthesis and phosphatidylinositol turnover.     

Identification of endothelin receptor subtypes in human renal cortex and medulla using subtype-selective ligands.

High affinity and high density endothelin (ET)-binding sites were identified in membranes prepared from human kidney cortex and medulla. Saturation binding experiments performed in membranes prepared from cortex and medulla using [125I]ET-1 and [125I]ET-3 revealed that the proportion of [125I]ET-3-binding sites was 30-35% less than that of [125I]ET-1-binding sites. The apparent dissociation constants and maximum binding for [125I]ET-1 and [125I]ET-3 to membranes from cortex were 91 +/- 5 pM and 165 +/- 10 fmol/mg protein, and 117 +/- 9 pM and 110 +/- 7 fmol/mg protein, respectively, whereas in medulla they were 139 +/- 10 pM and 360 +/- 11 fmol/mg protein, and 142 +/- 11 pM and 245 +/- 15 fmol/mg protein, respectively. In the presence of 10 nM sarafotoxin-6c, which is selective for ETB receptors, [125I]ET-1 binding was decreased by 65-70%, whereas [125I]ET-3 binding was totally abolished, suggesting that 65-70% of [125I]ET-1 binding and 100% of [125I]ET-3 binding was to ETB receptors. This was further confirmed by the use of a cyclic pentapeptide [cyclo(D-Trp,D-Asp,L-Pro, D-Val,L-Leu)] (BQ123), which is selective for ETA receptors. In the presence of 1 microM BQ123, [125I]ET-1 binding was decreased by 25-30%, whereas [125I]ET-3 binding was unaffected, confirming that 30-35% of ET receptors belong to the ETA subtypes, and that [125I]ET-1 bound to both ETA and ETB receptors with the same high affinity, but [125I]ET-3 bound only to ETB receptors with high affinity. These results suggest that human kidney cortex and medulla contain ETA and ETB receptors in a ratio of 30:70, and that sarafotoxin-6c and BQ123 are valuable tools in identifying the subtype of ET receptors in various tissues.     

Characterization of growth hormone-releasing hormone receptors in pituitary adenomas from patients with acromegaly

GHRH receptors in pituitary adenoma cell membranes from five patients with acromegaly were characterized using [125I] [His1,Nle27]GHRH-(1-32)NH2 ([125I]GHRHa) as a ligand. Specific binding of [125I]GHRHa to adenoma cell membranes was maximal within 20 min at 24 C, remained stable for 60 min, and was reversible in the presence of 500 nmol/L human GHRH-(1-44)NH2 (hGHRH). The specific binding increased linearly with 10-160 micrograms cell membrane protein. This binding was inhibited by 10(-11)-10(-6) mol/L hGHRH in a dose-dependent manner, with an ID50 of 0.20 nmol/L, but not by 10(-7) mol/L vasoactive intestinal peptide, glucagon, somatostatin-14, somatostatin-28, TRH, LHRH, and CRH. The specific binding of [125I]GHRHa to the membranes was saturable, and Scatchard analysis of the data revealed an apparent single class of high affinity GHRH receptors in five adenomas from acromegalic patients; the mean dissociation constant was 0.30 +/- 0.07 (+/- SE) nmol/L, and the mean maximal binding capacity was 26.7 +/- 7.0 (+/- SE) fmol/mg protein. In three nonfunctioning pituitary adenomas, GHRH receptors were not detected. The plasma GH response to hGHRH (100 micrograms) injection was studied in four acromegalic patients before surgery. Plasma GH levels increased variably in response to hGHRH injection in all four patients. However, there was no correlation between the characteristics of the tumor GHRH receptors and plasma GH responsiveness in these patients. We conclude that pituitary GH-secreting adenomas have specific GHRH receptors. Exogenously administered GHRH presumably acts via these receptors, but the variations in plasma GH responsiveness to hGHRH in these patients cannot be directly related to the variations in binding characteristics of the GHRH receptors on the GH-secreting adenoma cells.     

Melatonin binding sites in estrogen receptor-positive cells derived from human endometrial cancer

Our previous work showed that melatonin (N-acetyl-5-methoxytryptamine) inhibits proliferation of the human endometrial cancer cell line, Ishikawa, which is estrogen receptor-positive. The aim of the present study was to determine whether Ishikawa cells possess membrane melatonin receptors. Binding of the radioligand 2-[125I]-iodomelatonin to membrane preparations obtained from Ishikawa cells was detectable, saturable and stable. Scatchard analysis revealed that the dissociation constant (Kd) of the binding sites was 179.0 pm (similar to that of the MT2 [Mel1b] melatonin receptor subtype), and that the concentration (Bmax) of the binding sites was 12.9 fmol/mg protein. Luzindole, a selective MT2 melatonin receptor antagonist, significantly suppressed binding of 2-[125I]-iodomelatonin at all concentrations tested (10(-8) to 10(-4) m). These results suggest that the MT2 melatonin receptor subtype is present in the membranes of Ishikawa cells, and that the antiproliferative effect of melatonin on Ishikawa cells is mediated via the MT2 receptor. This may have implications for the use of melatonin in endometrial cancer therapy.     

Angiotensin II receptors in normal and failing human hearts

To demonstrate the existence and help clarify the function of angiotensin II (Ang II) receptors in the human heart, we characterized the cardiac Ang II receptor and examined the levels and distribution of ventricular Ang II receptors in normal (n = 6) and failing (n = 14) hearts. Ang II receptors were characterized using the Ang II receptor agonist [125I]Ang II. Cardiac [125I]Ang II-binding sites were of high affinity (Kd, approximately 1 nmol/L) and low capacity (Bmax, approximately 3 fmol/mg membrane protein) and were pharmacologically specific [IC50 values for Ang II, [Sar1,Ile8]Ang II, and Ang III were 1.2, 3.0, and 400 nmol/L, respectively; the inactive Ang II metabolite Ang-(1-5), at a concentration of 1 mumol/L, inhibited [125I]Ang II binding by less than 10%]. These characteristics of cardiac [125I]Ang II-binding sites are similar to those of previously characterized mammalian heart Ang II receptors. In normal adult donor hearts (n = 5), Ang II receptor density in the left ventricle [LV, 2.90 +/- 1.40 (+/- SE) fmol/mg] was similar to that in the right ventricle (RV, 3.82 +/- 1.10 fmol/mg). The ventricular Ang II receptor density in adult patients with idiopathic (LV, 1.77 +/- 0.35 fmol/mg; RV, 1.58 +/- 0.29 fmol/mg; n = 8) or dilated cardiomyopathy (LV, 2.00 +/- 0.58 fmol/mg; RV, 2.56 +/- 0.52 fmol/mg n = 5) was similar to that in the normal heart. Ventricular Ang II receptors, localized by autoradiography using the Ang II receptor antagonist [125I]-[Sar1,Ile8]Ang II, were consistently found in the myocardium, cardiac adrenergic nerves, and coronary vessels of normal and failing ventricles. In human ventricles Ang II receptor levels were not correlated with age. Because ventricular Ang II receptor density in a normal neonatal human heart and that in a heart from an adolescent patient with idiopathic cardiomyopathy were more than 10-fold and more than 5-fold higher, respectively, than in normal adult ventricles, we investigated whether postnatal changes occur in ventricular Ang II receptors in rats. In male and female rats ventricular Ang II receptor density was about 2-fold higher in 1-day-old rats compared to that in 10-day-old or peripubertal rats. These data suggest developmental regulation of ventricular Ang II receptors. Our findings suggest that direct and neural angiotensinergic inputs to the myocardium play a role in the regulation of cardiac function in man and that these inputs are preserved in the failing heart.     

Variations of mt1 melatonin receptor density in the rat uterus during decidualization,the estrous cycle and in response to exogenous steroid treatment

The expression of mt1 receptor protein in the rat uterus was investigated using an anti-mt1 polyclonal antibody against the rat mt1 receptor. A melatonin receptor protein of 37 kDa was detectable by Western blotting in the rat uterine membrane preparations. Autoradiography with the melatonin ligand, 2-[125I]iodomelatonin, was used to localize melatonin receptors in the uterus of the estrous rats and to study the changes of melatonin receptors in pregnancy. Melatonin receptors were found to be localized in the estrous rat uterine antimesometrial stroma. As decidualization of the uterine stroma progressed during pregnancy, the melatonin binding sites were progressively reduced and became confined to the antimesometrial non-decidualized outer stroma. 2-[125I]Iodomelatonin binding sites were not seen in the mesometrial stromal cells during pregnancy. The role of ovarian hormones in the regulation of uterine melatonin receptors was examined by studying the binding at various phases of the estrous cycle, after ovariectomy with and without follow-on treatment of estradiol (E2), progesterone (P4) or both. 2-[125I]Iodomelatonin binding in the rat uterus fluctuated during the estrous cycle, being lowest during metestrus. Ovariectomy caused an almost 70% reduction of 2-[125I]iodomelatonin binding compared with the control. Injections of ovariectomized (OVX) rats with E2 or P4 alone or in combination for 11 days induced a partial restoration of 2-[125I]iodomelatonin binding in the OVX rats. The results show that mt1 melatonin receptors in the rat antimesometrial stroma are regulated by ovarian hormones.     

Differential development of insulin-like growth factor I binding in the suprachiasmatic nucleus and median eminence of the rat hypothalamus.

We investigated the in vitro binding of [125I]insulin-like growth factor I ([125I]IGF-I) within the suprachiasmatic nucleus (SCN) and median eminence (ME) of the rat by quantitative autoradiography. Binding of [125I]IGF-I within the adult SCN was saturable, reversible and to a single class of sites with an estimated Kd of 3.01 x 10(-10) M and Bmax of 131 fmol/mg protein. Competition studies revealed [125I]IGF-I binding within the SCN and ME to have the pharmacological specificity characteristic of the brain IGF-I receptor. The most potent competitors were IGF-I and IGF-II, while insulin displaced binding with a much lower potency and nerve growth factor 7S had no effect. Binding within the SCN was evident by embryonic day 18, peaked perinatally, declined to adult levels by day 6 and remained constant through middle age. The potency of IGF-I to displace [125I]IGF-I binding from SCN was similar among all ages. Binding in the ME was not evident until postnatal day 2, peaked near the end of the 1st week, declined to adult levels by day 9 and remained constant thereafter. There was no difference in binding in SCN or ME between day and early night, in animals with acute or long-term eye enucleation, or in animals fed a high sucrose diet. These results demonstrate differential development of [125I]IGF-I binding within the SCN and ME and support a role for IGFs in both these nuclei during development and maturity.     

Effects of Melatonin Agonists and Antagonists on Reproduction and Body Weight in the Siberian Hamster

This study examined whether melatonin-induced inhibition of testicular weight and body weight in vivo could be antagonized by luzindole, a competitive melatonin receptor antagonist, or methysergide, a competitive serotonin receptor antagonist. Adult male Siberian hamsters were exposed to a long photoperiod (16L:8D) and given daily injections of drugs 3 h before lights off for 7 weeks. Hamsters treated with melatonin (0.375 mg/kg) exhibited testicular regression and loss of body weight. These effects were also marked in hamsters treated concomitantly with melatonin (0.375 mg/kg) and luzindole (10 mg/kg). In other studies, chronic injections of luzindole (30 mg/kg) to juvenile hamster failed to antagonize testicular regression induced by either melatonin injections or exposure to a short day photoperiod (12L: 12D). In contrast, concommitant injections of methysergide (6.25 mg/kg) and melatonin attenuated testicular regression and loss of body weight. When administered alone, neither luzindole nor methysergide affected testicular weight or body weight, whereas chronic injections of 5-methyoxyluzindole (10 mg/kg) mimicked the inhibitory effects of melatonin. 5-Methoxyluzindole inhibits 2-[125I]-iodomelatonin binding to median eminence/pars tuberalis membranes with an affinity similar to that of melatonin. Luzindole shows lower affinity for the inhibition of 2-[125I]-iodomelatonin binding than melatonin, which may explain why luzindole is not an effective melatonin receptor antagonist when administered in vivo. Methysergide, which has a very low affinity for inhibition of 2-[125I]-iodomelatonin binding, probably inhibits the effects of melatonin by blocking serotonergic neurotransmission.