Multiple prolactin-releasing activity in the bovine hypothalamic extract.

The prolactin (PRL)-releasing activity (PRA) in the bovine hypothalamic extract (BHE) was compared to that of known substances with PRA and further characterized by gel filtration and reversed-phase high performance liquid chromatography (RP-HPLC). Crude BHE produced marked dose-dependent stimulation of PRL secretion from the cultured rat adenohypophysial cells. Among the synthetic substances examined, vasoactive intestinal peptide (VIP), thyrotropin-releasing hormone (TRH) and beta-endorphin (END) showed significant PRA. However, the flatter dose-response slope for TRH compared with BHE or the small amounts of VIP and END in BHE suggested that these peptides could not account for the major active elements of BHE. Oxytocin and interleukin-1beta were also tested, but they exhibited no PRA in our assay system. Gel filtration of BHE on the Sephadex G-100 column yielded two peaks of PRA distinct from TRH, VIP and END. One eluted in the void and the other in more retarded fractions. The latter fractions were pooled and subjected to the two-step RP-HPLC. The PRA was separated into three peaks designated peaks I, II and III in the first RP-HPLC experiment. Furthermore, the second RP-HPLCs with finer resolution revealed that peak II as well as peak III consisted of three peaks, while peak I eluted as a single peak. Most of these seven PRA peaks exhibited different RP-HPLC profiles from those of the newly characterized PRL-releasing peptides. These findings again provide confirmatory evidence that BHE contained unique factors different from the above known substances.     

Rat hypothalamic extract inhibits vasopressin-stimulated Na+-K+-ATPase activity in the rat renal medulla

Arginine vasopressin stimulates Na⁺-K⁺-ATPase activity located in the rat thick ascending limb of s'Henle loop. Mammalian hypothalamus appears to produce a factor capable of inhibiting Na⁺-K⁺-ATPase activity in a variety of tissues. The effect of a purified rat hypothalamic extract with and without AVP on rat renal Na⁺-K⁺-ATPase activity was evaluated by a cytochemical technique. The hypothalamic extract alone failed to affect basal Na⁺-K⁺-ATPase activity throughout renal segments after 10 min exposure. Na⁺-K⁺-ATPase activity stimulated by AVP (1–10 fmol l^?1) for 10 min was inhibited by rat hypothalamic extract over the concentration range 10^?7–10^?3 U ml^?1 in a dose-dependent manner. Complete inhibition of AVP-stimulated Na⁺-K⁺-ATPase activity occurred at a hypothalamic extract concentration of 10^?3 U ml^?1. Only Na⁺-K⁺-ATPase activity located in the renal medullary thick ascending limb was influenced by the rat hypothalamic extract.     

Glucagon-degrading activity in acid-ethanol extract of rat submandibular gland

To investigate whether immunoreactive glucagon really exists in salivary gland, the integrity of glucagon radioimmunoassay was tested in the acid-ethanol extract of rat submandibular gland. Though immunoreactive glucagon was apparently measured in acid-ethanol extract of rat submandibular gland, the extract contained a significant amount of intact glucagon-degrading activity. The apparent % bound in radioimmunoassay highly correlated with the degradation of [125I] glucagon during incubation. Gel filtration profiles of [125I] glucagon incubated with acid-ethanol extract were the same as those of [125I] glucagon damaged by submandibular acid-saline extract. These data suggest that the immunoreactive glucagon in acid-ethanol extract is, as in the case of acid-saline extract, an artifact due to degradation of [125I] glucagon during radioimmunoassay.     

白藜芦醇抑制大鼠下丘脑脑片室旁核神经元放电

本文旨在研究白藜芦醇(resveratrol)对下丘脑脑片室旁核神经元放电的影响.应用玻璃微电极细胞外记录单位放电技术,在下丘脑脑片上观察白藜芦醇对静息状态下室旁核神经元放电的影响.结果如下:(1)在29张下丘脑脑片室旁核神经元放电单位给予白藜芦醇(O.05,0.5,5.0 μmol/L)2 min,有28张脑片(96.6%)放电频率显著降低,且呈剂量依赖性;(2)预先用0.2mmol/L的L.glutamate灌流8张下丘脑脑片,8张脑片(100%)放电频率显著增加,表现为癫痫样放电,该放电可被白藜芦醇(5.0 μmol/L)灌流2 min抑制:(3)预先用L型钙通道开放剂Bay K8644(0.1μmol/L)灌流8张下丘脑脑片,8张脑片(100%)放电频率显著增加,该放电可被白藜芦醇(5.0 μmol/L)灌流2 min抑制;(4)用一氧化氮合酶抑制剂Nω-nitro.L-arginine methyl ester(L-NAME)50μmol/L灌流8张下丘脑脑片,7张脑片(87.5%)放电频率显著增加,该放电可被白藜芦醇(5.0 μmol/L)灌流2 min抑制.以上结果提示,白藜芦醇抑制下丘脑室旁核神经元自发放电,可能通过降低心血管中枢的活动性而产生中枢保护作用.这种抑制作用可能与白藜芦醇抑制L型钙通道、减少钙内流有关,与NO释放无关.     

Gonadotropin-releasing hormone rapidly alters polyphosphoinositide metabolism in rat granulosa cells

This report describes the rapid effects of GnRH and an agonist [D-Ala6, des-Gly10] GnRH ethylamide (GnRHa) on polyphosphoinositide metabolism in rat granulosa cells. As indicated by the depletion of cellular levels of 32P-prelabeled triphosphoinositide (TPI) and diphosphoinositide (DPI), GnRHa rapidly stimulated the hydrolysis of TPI and DPI. The effect of GnRHa was maximal at the earliest time point examined (30 sec) and preceded GnRHa-induced increases in labeling of phosphatidylinositol. A specific GnRH antagonist had no effect on TPI or DPI levels, but prevented the polyphosphoinositide depletion induced by GnRH. LH did not stimulate depletion of 32P-polyphosphoinositides. The rapid and specific effects of GnRH on polyphosphoinositide depletion may represent an early and possibly initiating event in the action of GnRH.     

Potentiating activity of rat serum extract on ACTH-induced corticosteroidogenesis in isolated rat adrenal cells

ACTH potentiating activity was found in rat serum. The extract, obtained from ACTH-free rat serum by the QUSO G 32 adsorption method, potentiated ACTH1-24-induced corticosterone production in isolated rat adrenal cells. In our assay system, the maximal potentiation was observed with the extract of 0.5 ml of rat serum. With the extract, the log dose response curve for ACTH1-24 shifted to the side of lower doses of ACTH1-24. The potentiating substance was stable in the serum: the activity was hardly decreased even after leaving the serum stand for six days at room temperature. On Sephadex G-100 gel filtration of the extract, the most of activity was found between about 40,000 to 9,000 in molecular weight and a small portion of the activity was in the range of lower molecular weight. After hypophysectomy, the potentiating activity found in the fractions was markedly decreased, but a part of the activity still remained 30 days after the operation. This result suggests that the potentiating substance is produced mainly by the pituitary, but also produced by the other organ(s). SDS polyacrylamide gel electrophoresis of the active fractions revealed five peptides which were decreased quantitatively by hypophysectomy.     

Dynamics of spontaneous activity of hypothalamic neurons in the rat during emotional stress

Statistic parametres of discharges of separate cells in various hypothalamic nuclei were studied on Wistar rats in acute experiment under aethernembutal anesthesia. By logarithmic transformation of activity parameters a difference was established and some similar features of background firing of neurones in the frontal and posterior zones of the hypothalamus. Changes were shown in quantitative characteristics of impulse activity of hypothalamic neurones during development of emotional stress in conditions of long-term sexual conflict situations.     

Effect of oxytocin on the activity of posterior hypothalamic neurons in the rat

Changes of unit activity in different nuclei of the posterior hypothalamus induced by intravenous oxytocin injections were studied in acute experiments on Wistar rats anaesthetized with nembutal. Influence of nonapeptide was dose-dependent. Latencies of various neuronal reactions were different. Mechanisms of oxytocin regulation of neuronal activity in the posterior hypothalamus are discussed.     

Influence of naloxone on hypothalamic L-aminopeptidase activity in the normal and ovariectomized rat

The effect of naloxone on the L-leucinaminopeptidase (LAP) activity has been determined in the hypothalamus of normal female rats or after different periods of time from ovariectomy (15th or 30th day). Castration at 15th and 30th days produced a not very important fall of LAP activity. The naloxone injections (2.5 or 5 mg/kg vía i.p.) determined a significant decrease in LAP activity in the intact and ovariectomized rats, greater for 5 mg/kg. A significant LAP activity decrease was found only after a 30 day postcastration period when naloxone treated intact animals were compared with the castrated rats. These data are discussed in relation to the physiological significance of brain peptidases and the pharmacological effect of naloxone on the function of the hypothalamic-pituitary-gonadal axis.     

Gonadotropin-releasing hormone analog buserelin causes neuronal loss in rat gastrointestinal tract

Gonadotropin-releasing hormone (GnRH) analogs are given to women undergoing in vitro fertilization. Case reports describing the development of chronic intestinal pseudo-obstruction and auto-antibodies against GnRH after such treatment suggest a strong association between intestinal dysfunction and GnRH analogs. No experimental model for studying such a relationship is currently at hand. Our main goal was to investigate possible enteric neurodegeneration and titers of GnRH antibodies in response to repeated administration of the GnRH analog buserelin in rat. Rats were treated for 1–4 sessions with daily subcutaneous injections of buserelin or saline for 5 days, followed by 3 weeks of recovery. Buserelin treatment caused significant loss of submucous and myenteric neurons in the fundus, ileum, and colon. The loss of enteric neurons can, at least partly, be explained by increased apoptosis. No GnRH- or GnRH-receptor-immunoreactive (IR) enteric neurons but numerous luteinizing hormone (LH)-receptor-IR neurons were detected. After buserelin treatment, the relative number of enteric LH-receptor-IR neurons decreased, whereas that of nitric-oxide-synthase-IR neurons increased. No intestinal inflammation or increased levels of circulating interleukins/cytokines were noted in response to buserelin treatment. Serum GnRH antibody titers were undetectable or extremely low in all rats. Thus, repeated administrations of buserelin induce neurodegeneration in rat gastrointestinal tract, possibly by way of LH-receptor hyperactivation. The present findings suggest that enteric neurodegenerative effects of GnRH analog treatment in man can be mimicked in rat. However, in contrast to man, no production of GnRH auto-antibodies has been noted in rat.     

Modifications of neuronal activity in the rat hypothalamic supraoptic nucleus after long-lasting vibrational stimulation

We analyzed the background impulse activity (BIA) generated by neurons of the rat hypothalamic supraoptic nucleus in the norm and under conditions of long-lasting vibrational stimulation (exposure 5, 10, or 15 days). Distributions of neurons by the level of regularity, dynamics of discharge trains, form of histograms of interspike intervals (ISIs), as well as distributions of neurons by the BIA frequency ranges, were studied. We also calculated the mean frequency of impulsation of the neurons under study and the coefficient of variation of ISIs. After vibrational influences, we found modifications of both the internal structure of the recorded spike trains and the mean frequency of impulsation within the entire studied group and different frequency subgroups. Neirofiziologiya/Neurophysiology, Vol. 38, No. 3, pp. 224–230, May–June, 2006.     

Gonadotropin-releasing hormone inhibits cyclic nucleotide accumulation in cultured rat granulosa cells

Ovarian granulosa cells obtained from hypophysectomized, diethylstilbestrol-treated rats were cultured in the presence of ovine follicle-stimulating hormone (FSH) and gonadotropin-releasing hormone (GnRH). FSH stimulated the production and accumulation of both cAMP and cGMP, as well as progesterone, during a 48-h incubation period. Addition of GnRH or an agonist analog, [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa), did not influence the cyclic nucleotide response to FSH in the first 6 h of incubation, but caused dose-dependent inhibition of the FSH-induced rise in cyclic nucleotide production from 24 to 48 h of incubation. Cellular production of both cyclic nucleotides and progesterone was decreased by GnRHa concentrations as low as 10(-12) M, with maximum inhibition at 10(-9) M GnRHa. These results suggest that the in vitro antigonadal actions of GnRH and related peptides are expressed through inhibition of cyclic nucleotide production.     

Plasma leptin-binding activity and hypothalamic leptin receptor expression during pregnancy and lactation in the rat

Leptin, the 16-kDa peptide hormone product of the ob gene, regulates body weight via the hypothalamus but also influences several aspects of reproductive function. Results of previous studies have suggested that pregnancy is a state of leptin resistance, because food consumption remains stable or increases despite a progressive rise in plasma leptin across most of gestation. In the present study, we assessed whether this apparent leptin resistance during rat pregnancy was due to either increased plasma leptin-binding activity and/or reduced expression of hypothalamic leptin receptor. Plasma leptin increased from 2.2 +/- 0.4 ng/ml before pregnancy to a maximum at midgestation (4.2 +/- 0.8 ng/ml on Day 12) and then fell by Day 22 and remained low throughout lactation. Despite the higher plasma leptin levels in pregnancy, food consumption increased from a minimum of 13.6 +/- 0.5 g/day before pregnancy to a peak of 21.9 +/- 0.6 g/day on Day 19, then fell before parturition (11.9 +/- 0.4 g/day on Day 22). At least part of the increase in plasma leptin during pregnancy was attributable to a marked increase (P < 0.001) in plasma leptin-binding activity between diestrus and late pregnancy, which then fell after birth but remained at midpregnancy levels to at least Day 12 of lactation. Hypothalamic expression of mRNA encoding the long form of the leptin receptor (Ob-Rb) was elevated in early pregnancy (Day 7) but returned to prepregnancy levels by midgestation and remained stable thereafter. The results of this study confirm that pregnancy in the rat is a state of relative leptin resistance, which is due primarily to increased plasma leptin-binding activity rather than to changes in hypothalamic Ob-Rb expression.     

Gonadotropin-releasing hormone (GnRH) neurons and pathways in the rat brain

Summary Gonadotropin-releasing hormone (GnRH) neurons and their pathways in the rat brain were localized by immunocytochemistry in 6-to 18-day-old female animals, by use of thick frozen or vibratome sections, and silver-gold intensification of the diaminobenzidine reaction product. GnRH-immunoreactive perikarya were observed in the following regions: olfactory bulb and tubercle, vertical and horizontal limbs of the diagonal band of Broca, medial septum, medial preoptic and suprachiasmatic areas, anterior and lateral hypothalamus, and different regions of the hippocampus (indusium griseum, Ammon's horn). In addition to the known GnRH-pathways (preoptico-terminal, preoptico-infundibular, periventricular), we also observed GnRH-immunopositive processes in several major tracts and areas of the brain, including the medial and cortical amygdaloid complex, stria terminalis, stria medullaris thalami, fasciculus retroflexus, medial forebrain bundle, indusium griseum, stria longitudinalis medialis and lateralis, hippocampus, periaqueductal gray of the mesencephalon, and extracerebral regions, such as the lamina cribrosa, nervus terminalis and its associated ganglia. By use of the silver-gold intensification method we present Golgi-like images of GnRH perikarya and their pathways. The possible distribution of efferents from each GnRH cell group is discussed.     

Identification of calcitonin gene related peptide in ovine hypothalamic extract.

Calcitonin gene-related polypeptide (CGRP) was purified from ovine hypothalamic extracts. Its amino acid sequence was determined as: Ser-(Cys)-Asn-Thr-Ala-Thr-(Cys)-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-Ser- Arg-Ser - Gly-Gly-Val-Val-Lys-Ser-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-Ser-Gln-Ala-Phe- NH2. This sequence differs from rat CGRP by two amino acid substitutions (Ser for Asp25 and Gln for Glu35). Adenylate cyclase stimulating activity in rat pituitary cell cultures was monitored during the isolation. CGRP had adenylate cyclase stimulating activity comparable to corticotropin-releasing hormone, suggesting a hypophysiotropic role for CGRP. This is the first chemical characterization of CGRP in the brain (hypothalamus).