CB(1) modulation of hormone secretion, neuronal activation and mRNA expression following extracellular volume expansion

The endocannabinoid system includes important signaling molecules that are involved in several homeostatic and neuroendocrine functions. In the present study, we evaluated the effects of the type 1 cannabinoid (CB(1)) receptor antagonist, rimonabant (10 mg/kg, p.o.), on hormone secretion, neuronal activation and mRNA expression in the hypothalamus following isotonic (I-) or hypertonic (H-) extracellular volume expansion (EVE). The total nitrate content in the PVN and SON was also assessed under the same experimental conditions. Our results showed that OT and AVP plasma concentrations were increased in response to H-EVE, while decreased AVP levels were found following I-EVE. Accordingly, both I- and H-EVE stimulated oxytocinergic neuronal activation, as evidenced by the increased number of c-Fos/OT double labeled neurons in the hypothalamus. The vasopressinergic cells of the PVN and SON, however, were only activated in response to H-EVE. Furthermore, increased amounts of both AVP and OT mRNAs were found in the hypothalamus following EVE. Pretreatment with rimonabant significantly potentiated hormone secretion and also vasopressinergic and oxytocinergic neuronal activation induced by EVE, although decreased AVP and OT mRNA expression was found in the hypothalami of rimonabant pretreated groups. In addition, the nitrate content in the PVN and SON was not altered in response to EVE or rimonabant pretreatment. Taken together, these results suggest that the CB(1) receptor may modulate several events that contribute to the development of appropriate responses to increased fluid volume and osmolality. (C) 2010 Elsevier Inc. All rights reserved.

GABAergic mediation of stress-induced secretion of corticosterone and oxytocin, but not prolactin, by the hypothalamic paraventricular nucleus

The hypothalamus-pituitary-adrenal axis (HPA) participates in mediating the response to stressful stimuli. Within the HPA, neurons in the medial parvocellular region of paraventricular nucleus (PVN) of the hypothalamus integrate excitatory and inhibitory signals triggering secretion of corticotropin-releasing hormone (CRH), the main secretagogue of adrenocorticotropic hormone (ACTH). Stressful situations alter CRH secretion as well as other hormones, including prolactin and oxytocin. Most inputs to the PVN are of local origin, half of which are GABAergic neurons, and both GABA-A and GABA-B receptors are present in the PVN. The objective of the present study was to investigate the role of GABA-A and GABA-B receptors in the PVN`s control of stress-induced corticosterone, oxytocin and prolactin secretion. Rats Were microinjected with saline or different doses (0.5, 5 and 50 pmol) of GABA-A (bicuculine) or GABA-B (phaclofen) antagonists in the PVN. Ten minutes later, they were subjected to a stressor (ether inhalation) and blood samples were collected 30 min before and 10, 30, 60, 90 and 120 min after the stressful stimulus to measure hormone levels by radioimmunoassay. Our results indicate that GABA acts in the PVN to inhibit stress-induced corticosterone secretion via both its receptor subtypes, especially GABA-B. In contrast, GABA in the PVN stimulates oxytocin secretion through GABA-B receptors and does not alter prolactin secretion. (C) 2008 Elsevier Inc. All rights reserved.

Volume-activated chloride channels in mice Leydig cells

Production and secretion of testosterone in Leydig cells are mainly controlled by the luteinizing hormone (LH). Biochemical evidences suggest that the activity of Cl(-) ions can modulate the steroidogenic process, but the specific ion channels involved are not known. Here, we extend the characterization of Cl(-) channels in mice Leydig cells (50-60 days old) by describing volume- activated Cl(-) currents (I(Cl,swell)). The amplitude of I(Cl,swell) is dependent on the osmotic gradient across the cell membrane, with an apparent EC(50) of similar to 75 mOsm. These currents display the typical biophysical signature of volume- activated anion channels (VRAC): dependence on intracellular ATP, outward rectification, inactivation at positive potentials, and selectivity sequence (I(-)>Cl(-)>F(-)). Staurosporine (200 nM) did not block the activation of I(Cl), swell. The block induced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 128 mu M), SITS (200 mu M), ATP (500 mu M), pyridoxalphosphate-6- azophenyl-2`,4`-disulfonate (PPADS; 100 mu M), and Suramin (10 mu M) were described by the permeant blocker model with apparent dissociation constant at 0 mV K(d)(0) and fractional distance of the binding site (delta) of 334 mu M and 47%, 880 mu M and 35%, 2,100 mu M and 49%, 188 mu M and 27%, and 66.5 mu M and 49%, respectively. These numbers were derived from the peak value of the currents. We conclude that ICl, swell in Leydig cells are activated independently of purinergic stimulation, that Suramin and PPADS block these currents by a direct interaction with VRAC and that ATP is able to permeate this channel.

Obesity due to Melanocortin 4 Receptor (MC4R) Deficiency Is Associated with Increased Linear Growth and Final Height, Fasting Hyperinsulinemia, and Incompletely Suppressed Growth Hormone Secretion

Context: Melanocortin receptor 4 (MC4R) deficiency is characterized by increased linear growth greater than expected for the degree of obesity. Objective: The objective of the investigation was to study the somatotroph axis in obese MC4R-deficient patients and equally obese controls. Patients and Methods: We obtained anthropometric measurements and insulin concentrations in 153 MC4R-deficient subjects and 1392 controls matched for age and severity of obesity. We measured fasting IGF-I, IGF-II, IGF binding protein (IGFBP)-1, IGFBP-3, and acid-labile subunit levels in a subset of 33 MC4R-deficient patients and 36 control subjects. We examined pulsatile GH secretion in six adult MC4R-deficient subjects and six obese controls. Results: Height so score was significantly greater in MC4R-deficient children under 5 yr of age compared with controls (mean +/- SEM: 2.3 +/- 0.06 vs. 1.8 +/- 0.04, P < 0.001), an effect that persisted throughout childhood. Final height (cm) was greater in MC4R-deficient men (mean +/- SEM 173 +/- 2.5 vs. 168 +/- 2.1, P < 0.001) and women (mean 165 +/- 2.1 vs. 158 +/- 1.9, P < 0.001). Fasting IGF-I, IGF-II, acid-labile subunit, and IGFBP-3 concentrations were similar in the two groups. GH levels were markedly suppressed in obese controls, but pulsatile GH secretion was retained in MC4R deficiency. The mean maximal GH secretion rate per burst (P < 0.05) and mass per burst (P < 0.05) were increased in MC4R deficiency, consistent with increased pulsatile and total GH secretion. Fasting insulin levels were markedly elevated in MC4R-deficient children. Conclusions: In MC4R deficiency, increased linear growth in childhood leads to increased adult final height, greater than predicted by obesity alone. GH pulsatility is maintained in MC4R deficiency, a finding consistent with animal studies, suggesting a role for MC4R in controlling hypothalamic somatostatinergic tone. Fasting insulin levels are significantly higher in children carrying MC4R mutations. Both of these factors may contribute to the accelerated growth phenotype characteristic of MC4R deficiency. (J Clin Endocrinol Metab 96: E181-E188, 2011)

The role of nutritional profile in the orexigenic neuropeptide secretion in nonalcoholic fatty liver disease obese adolescents

Background Little progress has been made to identify the central neuroendocrine pathway involved in the energy intake control in nonalcoholic fatty liver disease (NAFLD) patients. Objective To assess the influence of orexigenic neuropeptides in the nutritional aspects of NAFLD obese adolescents submitted to a long-term interdisciplinary approach. Methods Fifty adolescents aged 15-19 years, with body mass index at least 95th percentile, consisting of 25 patients without NAFLD and 25 with NAFLD. The NAFLD diagnosis was determined by ultrasonography. Blood samples were collected to analyze glycemia, hepatic transaminases, and lipid profile. Insulin resistance was estimated by Homeostasis Model Assessment Insulin Resistance Index. Neuropeptide Y (NPY) and agouti related protein concentrations were measured by enzyme-linked immunosorbent assay. Analyses of food intake were made by 3 days recordatory inquiry. Results At baseline conditions, the patients with NAFLD had significantly higher values of body mass, body mass index, visceral fat, triglycerides, VLDL-C, and hepatic transaminases. After the long-term intervention, they presented a significant reduction in these parameters. In both the groups, it was observed a significant decrease in energy intake, macronutrients and dietetic cholesterol. Only the patients with NAFLD presented a positive correlation between the saturated fatty acids intake and the orexigenic neuropeptides NPY and agouti related protein, and carbohydrate with NPY. Indeed, it was observed a positive correlation between energy intake, lipid (%) and saturated fatty acids with visceral fat accumulation. Conclusion Our findings showed an important influence of diet composition in the orexigenic system, being essential consider that the excessive saturated fatty acids intake could be a determinant factor to increase nonalcoholic fatty liver disease. Eur J Gastroenterol Hepatol 22:557-563 (C) 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Protective effect of Calendula officinalis extract against UVB-induced oxidative stress in skin: Evaluation of reduced glutathione levels and matrix metalloproteinase secretion

Background and purpose: Calendula officinalis flowers have long been employed time in folk therapy, and more than 35 properties have been attributed to decoctions and tinctures from the flowers. The main uses are as remedies for burns (including sunburns), bruises and cutaneous and internal inflammatory diseases of several origins. The recommended doses are a function both of the type and severity of the condition to be treated and the individual condition of each patient. Therefore, the present study investigated the potential use of Calendula officinalis extract to prevent UV irradiation-induced oxidative stress in skin. Methods: Firstly, the physico-chemical composition of marigold extract(ME) (hydroalcoholic extract)was assessed and the in vitro antioxidant efficacy was determined using different methodologies. Secondly, the cytotoxicity was evaluated in L929 and HepG2 cells with the MTT assay. Finally, the in vivo protective effect of ME against UVB-induced oxidative stress in the skin of hairless mice was evaluated by determining reduced glutathione (GSH) levels and monitoring the secretion/activity of metalloproteinases. Results and conclusions: The polyphenol, flavonoid, rutin and narcissin contents found in ME were 28.6 mg/g, 18.8 mg/g, 1.6 mg/g and 12.2 mg/g, respectively and evaluation of the in vitro antioxidant activity demonstrated a dose-dependent effect of ME against different radicals. Cytoxicity experiments demonstrated that ME was not cytotoxic for L929 and HepG2 cells at concentrations less than or equal to of 15 mg/mL However, concentrations greater than or equal to 30 mg/mL, toxic effects were observed. Finally, oral treatment of hairless mice with 150 and 300 mg/kg of ME maintained GSH levels close to non-irradiated control mice. In addition, this extract affects the activity/secretion of matrix metalloproteinases 2 and 9 (MMP-2 and -9) stimulated by exposure to UVB irradiation. However, additional studies are required to have a complete understanding of the protective effects of ME for skin. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Alteration of Ca(2+)-ATPase activity in the homogenate, plasma membrane and microsomes of the salivary glands of streptozotocin-induced diabetic rats

Diabetes has been implicated in the dryness of the mouth, loss of taste sensation, sialosis, and other disorders of the oral cavity, by impairment of the salivary glands. The aim of the present study was to examine the plasma membrane, microsomal, and homogenate Ca(2+)-ATPase activity in the rat submandibular and parotid salivary glands of streptozotocin-induced diabetes. We have also examined the influence of the acidosis state oil this parameter. Diabetes was induced by an intraperitoneal injection of streptozotocin and acidosis was induced by daily injection of NH(4)Cl. At 15 and 30 days after diabetes induction, the animals were euthanized and the submandibular and parotid salivary glands were removed and analyzed. Ca(2+)-ATPase (total, independent, and dependent) was determined in the homo-enate, microsomal, and plasma membranes of the salivary glands of diabetic and control rats. Calcium concentration was also determined in the glands and showed to be hi-her in the diabetic animals. Ca(2+)-ATPase activity was found to be reduced in all cell fractions studied in the diabetic animals compared with control. Similar results were obtained for the submandibular salivary glands of acidotic animals; however in the parotid salivary glands it was found an increase in the enzyme activity. Copyright (c) 2009 John Wiley & Sons, Ltd.

Activity of Hypothalamic Dopaminergic Neurones During the Day of Oestrus: Involvement in Prolactin Secretion

A secretory surge of prolactin occurs on the afternoon of oestrus in cycling rats. Pituitary prolactin is inhibited by dopamine. We evaluated the activity of the neuroendocrine dopaminergic neurones during oestrus and dioestrus, as determined by dopaminergic activity in the median eminence and neurointermediate lobe of the pituitary, as well as Fos-related antigen expression in tyrosine hydroxylase (TH)-immunoreactive (ir) neurones of the arcuate nucleus (ARC) and periventricular nucleus (Pe). During oestrus, the 4-dihydroxyphenylacetic acid/dopamine ratio in the median eminence decreased at 16.00 h, coinciding with the increase in plasma prolactin levels. Similarly, the expression of Fos-related antigen in TH-ir neurones of Pe and rostral-, dorsomedial- and caudal-ARC also decreased at 16.00 h. On dioestrus, 4-dihydroxyphenylacetic acid/dopamine ratio in the median eminence and Fos-related antigen expression in TH-ir neurones of Pe and rostral-ARC decreased at 18.00 h, whereas prolactin levels were unaltered. No variation in dopaminergic activity was found in the neurointermediate lobe of the pituitary on either oestrus or dioestrus. The number of TH-ir neurones in the ARC and parameters of dopaminergic activity were found to be generally lower on oestrus compared to dioestrus. The transitory decrease in the activity of neuroendocrine dopaminergic neurones temporally associated with the prolactin surge on the afternoon of oestrus suggests a role for dopamine in the generation of the oestrous prolactin surge.

Noradrenaline Involvement in the Negative-Feedback Effects of Ovarian Steroids on Luteinising Hormone Secretion

Noradrenaline has been shown to modulate the ovarian-steroid feedback on luteinising-hormone (LH) release. However, despite the high amount of evidence accumulated over many years, the role of noradrenaline in LH regulation is still not clearly understood. The present study aimed to further investigate the involvement of noradrenaline in the negative-feedback effect of oestradiol and progesterone on basal LH secretion. In experiment 1, ovariectomised (OVX) rats received a single injection of oil, oestradiol, or progesterone at 09.00-10.00 h and were decapitated 30 or 60 min later. Levels of noradrenaline and its metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), were determined in microdissections of the preoptic area (POA) and medial basal hypothalamus-median eminence (MBH-ME) and correlated with LH secretion. Basal LH levels were decreased 30 and 60 min after oestradiol or progesterone injection, and this hormonal response was significantly correlated with a reduction in POA MHPG levels, which reflect noradrenaline release. In addition, noradrenaline levels in the POA were increased, whereas noradrenaline turnover (MHPG/noradrenaline ratio) was decreased 60 min after the injection of both hormones. No effect was found in the MBH-ME. In experiment 2, i.c.v. administration of noradrenaline (60 nmol), performed 15 min before oestradiol or progesterone injection in jugular vein-cannulated OVX rats, completely prevented the ovarian steroid-induced inhibition of LH secretion. The data obtained provide direct evidence that LH secretion in OVX rats is positively regulated by basal noradrenergic activity in the POA, and its reduction appears to play a role in the negative-feedback effect of ovarian steroids on LH secretion in vivo.

The cystic fibrosis transmembrane conductance regulator (CFTR) inhibits ENaC through an increase in the intracellular Cl- concentration

Activation of the CFTR Cl- channel inhibits epithelial Na+ channels (ENaC), according to studies on epithelial cells and overexpressing recombinant cells. Here we demonstrate that ENaC is inhibited during stimulation of the cystic fibrosis trans-membrance conductance regulator (CFTR) in Xenopus oocytes, independent of the experimental set-up and the magnitude of the whole-cell current. Inhibition of ENaC is augmented at higher CFTR Cl- currents. Similar to CFTR, ClC-0 Cl- currents also inhibit ENaC, as well as high extracellular Na+ and Cl- in partially permeabilized oocytes. Thus, inhibition of ENaC is not specific to CFTR and seems to be mediated by Cl-.

The response of molecular isoforms of growth hormone to acute exercise in trained adult males

Circulating GH consists of multiple molecular isoforms, all derived from the one gene in nonpregnant humans. To assess the effect of a potent stimulus to pituitary secretion on GH isoforms, we studied 17 aerobically trained males (age, 26.9 +/- 1.5 yr) in a randomized, repeat measures study of rest vs. exercise. Exercise consisted of continuous cycle ergometry at approximately 80% of predetermined maximal oxygen uptake for 20 min. Serum was assayed for total, pituitary, 22-kDa, recombinant, non-22-kDa, 20-kDa, and immunofunctional GH. All isoforms increased during, peaked at the end, and declined after exercise. At peak exercise, 22-kDa GH was the predominant isoform. After exercise, the ratios of non-22 kDa/total GH and 20-kDa GH/total GH increased and those of recombinant/pituitary GH decreased. The disappearance half-times for pituitary GH and 20-kDa GH were significantly longer than those for all other isoforms. We conclude that 1) all molecular isoforms of GH measured increased with and peaked at the end of acute exercise, with 22-kBa GH constituting the major isoform in serum during exercise; and 2) the proportion of non-22-kDa isoforms increased after exercise due in part to slower disappearance rates of 20-kDa and perhaps other non-22-kDa GH isoforms. It remains to be determined whether the various biological actions of different GH isoforms impact on postexercise homeostasis.

Preliminary characterisation and extraction of anterior adhesive secretion in monogenean (platyhelminth) parasites

Secreted anterior adhesives, used for temporary attachment to epithelial surfaces of fishes (skin and gills) by some monogenean (platyhelminth) parasites have been partially characterised. Adhesive is composed of protein. Amino acid composition has been determined for seven monopisthocotylean monogeneans. Six of these belong to the Monocotylidae and one species, Entobdella soleae (van Beneden et Hesse, 1864) Johnston, 1929, is a member of the Capsalidae. Histochemistry shows that the adhesive does not contain polysaccharides, including acid mucins, or lipids. The adhesive before secretion and in its secreted form contains no dihydroxyphenylalanine (dopa). Secreted adhesive is highly insoluble, but has a soft consistency and is mechanically removable from glass surfaces. Generally there are high levels of glycine and alanine, low levels of tyrosine and methionine, and histidine is often absent. However, amino acid content varies between species, the biggest differences evident when the monocotylid monogeneans were compared with E. soleae. Monogenean adhesive shows similarity in amino acid profile with adhesives from starfish, limpets and barnacles. However, there are some differences in individual amino acids in the temporary adhesive secretions of, on the one hand, the monogeneans and, on the other hand, the starfish and limpets. These differences may reflect the fact that monogeneans, unlike starfish and barnacles, attach to living tissue (tissue adhesion). A method of extracting unsecreted adhesive was investigated for use in further characterisation studies on monogenean glues.

Association of hypertension and hypokalemia with Cushing's syndrome caused by ectopic ACTH secretion: a series of 58 cases.

Cushing's syndrome is associated with hypertension in approximately 80% of cases. Hypertension contributes to the marked increased mortality risk of past or current Cushing's syndrome, largely because of increased cardiovascular risk. Observation of the pathophysiological effect of chronically elevated ACTH and cortisol values in patients with ectopic ACTH secretion complements the available data from acute studies of the effects of ACTH and glucocorticoid infusions in normal volunteers. In a retrospective case review, we identified 58 patients with Cushing's syndrome caused by ectopic ACTH secretion, who were treated at the National Institutes of Health between 1983-1997. The diagnosis of an ectopic ACTH cause was confirmed by inferior petrosal sinus sampling and/or pathologic examination of tumor. The commonest causes were bronchial carcinoid (40%) and thymic carcinoid (10%), but 18 of 58 (31%) patients had an unknown source of ectopic ACTH. Hypertension (systolic blood pressure >140 mmHg and/or diastolic blood pressure >90 mmHg in adults) was noted in 45 of 58 (78%) ectopic Cushing's patients, a prevalence similar to that noted in other endogenous Cushing's syndrome etiologies. Hypertension was severe, deemed to require 3 or more drugs by the treating physicians, in 26 of 58 (45%) patients. Hypokalemia was much more prevalent than in patients with other causes of Cushing's syndrome, affecting 33 of 58 (57%) patients. The range of plasma ACTH (17-1557 pg/mL, normal

Prolactin-releasing peptide in ewe: cDNA cloning, mRNA distribution and effects on prolactin secretion in vitro and in vivo

RT-PCR followed by 5'- and 3'- rapid amplification of cDNA ends was used to clone and sequence ovine prolactin-releasing peptide (PrRP). The cDNA was characterised by short 5'- and 3'-untranslated regions and a GC-rich (71%) coding region. The nucleotide and deduced amino acid sequences for the coding region showed 95.6 and 94.9% identity with bovine PrRP but the amino acid sequence of PrRP31 was conserved between these species. Northern blot analysis and RT-PCR showed that, as in the rat, the peptide was more abundantly expressed in the brainstem than the hypothalamus. However, in the ovine hypothalamus, PrRP mRNA expression was more widespread than in the rat, with expression detected in both rostral and caudal parts of the mediobasal hypothalamus. The effects of synthetic ovine PrRP on prolactin secretion both in vitro and in vivo were also examined. In primary cultures of sheep pituitary cells, PrRP significantly (P