Oestradiol Potentiates Hormone Secretion and Neuronal Activation in Response to Hypertonic Extracellular Volume Expansion in Ovariectomised Rats

Secretion of vasopressin (VP), oxytocin (OT) and atrial natriuretic peptide (ANP) is an essential mechanism for the maintenance of hydromineral homeostasis. Secretion of these hormones is modulated by several circulating factors, including oestradiol. However, it remains unclear how oestradiol exerts this modulation. In the present study we investigated the participation of oestradiol in the secretion of VP, OT and ANP and in activation of vasopressinergic and oxytocinergic neurones of the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus in response to extracellular volume expansion (EVE). For this purpose, ovariectomised (OVX) rats treated for 7 days with vehicle (corn oil, 0.1 ml/rat, OVX+O group) or oestradiol (oestradiol cypionate, 10 mu g/kg, OVX+E group) were subjected to either isotonic (0.15 m NaCl, 2 ml/100 g b.w., i.v.) or hypertonic (0.30 m NaCl, 2 ml/100 g b.w., i.v.) EVE. Blood samples were collected for plasma VP, OT and ANP determination. Another group of rats was subjected to cerebral perfusion, and brain sections were processed for c-Fos-VP and c-Fos-OT double-labelling immunohistochemistry. In OVX+O rats, we observed that both isotonic and hypertonic EVE increased plasma OT and ANP concentrations, although no changes were observed in VP secretion. Oestradiol replacement did not alter hormonal secretion in response to isotonic EVE, but it increased VP secretion and potentiated plasma OT and ANP concentrations in response to hypertonic EVE. Immunohistochemical data showed that, in the OVX+O group, hypertonic EVE increased the number of c-Fos-OT and c-Fos-VP double-labelled neurones in the PVN and SON. Oestradiol replacement did not alter neuronal activation in response to isotonic EVE, but it potentiated vasopressinergic and oxytocinergic neuronal activation in the medial magnocellular PVN (PaMM) and SON. Taken together, these results suggest that oestradiol increases the responsiveness of vasopressinergic and oxytocinergic magnocellular neurones in the PVN and SON in response to osmotic stimulation.

Carbon monoxide and nitric oxide modulate hyperosmolality-induced oxytocin secretion by the hypothalamus in vitro

OT (oxytocin) is secreted from the posterior pituitary gland, and its secretion has been shown to be modulated by NO (nitric oxide). In rats, OT secretion is also stimulated by hyperosmolarity of the extracellular fluid. Furthermore, NOS (nitric oxide synthase) is located in hypothalamic areas involved in fluid balance control. In the present study, we evaluated the role of the NOS/NO and HO (haem oxygenase)/CO (carbon monoxide) systems in the osmotic regulation of OT release from rat hypothalamus in vitro. We conducted experiments on hypothalamic fragments to determine the following: (i) whether NO donors and NOS inhibitors modulate OT release and (ii) whether the changes in OT response occur concurrently with changes in NOS or HO activity in the hypothalamus. Hyperosmotic stimulation induced a significant increase in OT release that was associated with a reduction in nitrite production. Osmotic stimulation of OT release was inhibited by NO donors. NOS inhibitors did not affect either basal or osmotically stimulated OT release. Blockade of HO inhibited both basal and osmotically stimulated OT release, and induced a marked increase in NOS activity. These results indicate the involvement of CO in the regulation of NOS activity. The present data demonstrate that hypothalamic OT release induced by osmotic stimuli is modulated, at least in part, by interactions between NO and CO.

Ghrelin and PYY(3-36) in gastrectomized and vagotomized patients: relations with appetite, energy intake and resting energy expenditure

Background/Objectives: Reduced food intake, appetite loss and alteration of ghrelin and PYY(3-36) secretion have been suggested to have a function in the loss of body weight commonly observed after gastrectomy. The objective of this study was to investigate the circulating concentrations of ghrelin and PYY(3-36) and their relationships with food intake, appetite and resting energy expenditure (REE) after gastrectomy plus vagotomy. Subjects/Methods: Seven patients with total gastrectomy (TG), 14 with partial gastrectomy (PG) and 10 healthy controls were studied. Habitual food intake and REE was assessed; fasting and postprandial plasma total ghrelin, PYY(3-36) concentrations and appetite ratings were determined after ingestion of a liquid test meal. Results: Differently from PG and controls, fasting ghrelin correlated with REE, and a higher energy intake was observed in the TG group. Fasting plasma ghrelin concentrations were lower in TG compared with controls, and no ghrelin response to the meal was observed in either PG or TG. Fasting plasma PYY(3-36) concentrations were not different among the groups. There was an early and exaggerated postprandial rise in PYY(3-36) levels in both PG and TG groups, but not in controls. No effect of ghrelin or PYY(3-36) concentrations was observed on hunger, prospective consumption or fullness ratings. Conclusions: Total ghrelin and PYY(3-36) do not seem to be involved with appetite or energy intake regulation after gastrectomy plus vagotomy. Ghrelin secreted by sources other than stomach is likely to have a function in the long-term regulation of body weight after TG. European Journal of Clinical Nutrition (2010) 64, 845-852; doi: 10.1038/ejcn.2010.88; published online 19 May 2010

Differential immunoreactivity of glucocorticoid receptors and vasopressin in neurons of the anterior and medial parvocellular subdvisions of the hypothalamic paraventricular nucleus

arginine-vasopressin in the parvocellular neurons of the hypothalamic paraventricular nucleus is known to play an important role in the control of the hypothalamo-pituitary-adrenal axis. In the present study, we verify plasma corticosterone levels, the distribution of glucocorticoid receptor- and arginine-vasopressin-positive neurons, and the co-localization of both glucocorticoid receptors and arginine-vasopressin in neurons in the anterior and medial parvocellular subdivisions of the paraventricular nucleus after manipulations of the hypothalamus-pituitary-adrenal axis. Normal, sham surgery, and adrenalectomized male rats were subjected to intraperitoneal injections of saline or dexamethasone to measure plasma corticosterone levels by a radioimmunoassay. We also examined arginine-vasopressin and glucocorticoid receptor immunofluorescence in sections from the paraventricular nucleus. Our results showed that the immunoreactivity of arginine-vasopressin neurons increased in the anterior parvocellular subdivision and decreased in the medial parvocellular subdivision from adrenalectomized rats treated with dexamethasone. On the other hand, we showed that the immunoreactivity of glucocorticoid receptors increased in the anterior and medial parvocellular subdivisions of these same animals. However, the immunoreactivity of glucocorticoid receptors is higher in the medial parvocellular than anterior parvocellular subdivision. The co-localization of arginine-vasopressin and glucocorticoid receptors was found only in the medial parvocellular subdivision. These findings indicate that glucocorticoids have direct actions on arginine-vasopressin-positive neurons in the medial parvocellular but not anterior parvocellular subdivision. There is a differentiated pattern of arginine-vasopressin-positive neuron expression between the anterior and medial parvocellular subdivisions. (C) 2010 Elsevier Inc. All rights reserved.

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.

Role of homocysteic acid in the guinea pig (Cavia porcellus) anterior cingulate cortex in tonic immobility and the influence of NMDA receptors on the dorsal PAG

Tonic immobility (TI) is an innate defensive behaviour elicited by physical restriction and Postural inversion, and is characterised by a profound and temporary state of akinesis. Our previous studies demonstrated that glutamatergic stimulation of the dorsomedial/dorsolateral Portion of periaqueductal gray matter (dPAG) decreases the duration of TI in guinea pigs (Cavia porcellus). Furthermore, evidence suggests that the anterior cingulate cortex (ACC) constitutes an important Source of glutamate for the dPAG. Hence, in the current study, we investigated the effects of microinjection of the excitatory amino acid (EAA) agonist DL-homocysteic acid (DLH) and the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 into the ACC on the duration of TI in guinea pigs. We also assessed the effect of the NMDA receptor antagonist (MK-801) into the dorsal periaqueductal gray matter (dPAG) prior to DLH microinjection into the ACC on the TI duration in the guinea pig. Our results demonstrated that DLH microinjections into the ACC decreased the duration of TI. This effect was blocked by previous MK-801 microinjections into the ACC or into the dPAG. The MK-801 microinjections alone did not influence TI duration. These results provide the new insight that EAAs in the ACC can decrease the duration of TI. The mechanism seems to be dependent on the NMDA receptors present in the ACC and in the dPAG. (C) 2009 Elsevier B.V. All rights reserved.

HYPOTHALAMIC COCAINE- AND AMPHETAMINE-REGULATED TRANSCRIPT AND CORTICOTROPHIN RELEASING FACTOR NEURONS ARE STIMULATED BY EXTRACELLULAR VOLUME AND OSMOTIC CHANGES

Several studies suggest that hypothalamic cocaine- and amphetamine-regulated transcript (CART) may interact with the hypothalamic-pituitary-adrenal (HPA) axis in the control of neuroendocrine function and may also participate in cardiovascular regulation. Therefore, this study aimed to evaluate, in experimental models of isotonic (I-EVE) and hypertonic (H-EVE) extracellular volume expansion and water deprivation (WD), the activation of CART- and corticotrophin releasing factor (CRF)-immunoreactive neurons, as well as the relative expression of CART and CRF mRNAs in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Both H-EVE (0.30M NaCl, 2mL/100g of body weight, in 1 minute) and 24 hours of WD significantly increased plasma sodium concentrations, producing, respectively, either an increase or a decrease in extracellular volume. I-EVE (0.15M NaCl, 2mL/100g of body weight, in 1 minute) evoked a significant increase in the circulating volume accompanied by unaltered plasma concentrations of sodium. CART-expressing neurons of both magnocellular and parvocellular hypothalamic divisions were activated to produce Fos in response to H-EVE but not in response to I-EVE. Furthermore, increased expression of CART mRNA was found in the PVN of H-EVE but not I-EVE rats. These data show for the first time that EVE not only activates hypothalamic CRF neurons but also increases CRF mRNA expression in the PVN. In contrast, WD increases the number of CART-immunoreactive neurons activated to produce Fos in the PVN and SON but does not change the number of neurons double labeled for Fos and CRF or expression of CRF mRNA in the PVN. These findings provided new insights into the participation of CART in diverse processes within the PVN and SON, including its possible involvement in activation of the HPA axis and cardiovascular regulation in response to changes in extracellular volume and osmolality. (C) 2011 IBRO. Published by Elsevier Ltd. 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.

Nitric oxide modulates the firing rate of the rat supraoptic magnocellular neurons

In vitro, nitric oxide (NO) inhibits the firing rate of magnocellular neurosecretory cells (MNCs) of hypothalamic supraoptic and paraventricular nuclei and this effect has been attributed to GABAergic activation. However, little is known about the direct effects of NO in MNCs. We used the patch-clamp technique to verify the effect Of L-arginine, a precursor for NO synthesis, and N-omega-nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of NOS, on spontaneous electrical activity of MNCs after glutamatergic and GABAergic blockade in Wistar rat brain slices. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 mu M) and DL-2-amino-5-phosphonovaleric acid (DL-AP5) (30 mu M) were used to block postsynaptic glutamatergic currents, and picrotoxin (30 mu M) and saclofen (30 mu M) to block ionotropic and metabotropic postsynaptic GABAergic currents. Under these conditions, 500 mu M L-arginine decreased the firing rate from 3.7 +/- 0.6 Hz to 1.3 +/- 0.3 Hz. Conversely, 100 mu M L-NAME increased the firing rate from 3.0 +/- 0.3 Hz to 5.8 +/- 0.4 Hz. All points histogram analysis showed changes in resting potential from -58.1 +/- 0.8 mV to -62.2 +/- 1.1 mV in the presence of L-arginine and from -59.8 +/- 0.7 mV to -56.9 +/- 0.8 mV by L-NAME. Despite the nitrergic modulator effect on firing rate, some MNCs had no significant changes in their resting potential. In those neurons, hyperpolarizing after-potential (HAP) amplitude increased from 12.4 +/- 1.2 mV to 16.8 +/- 0.7 mV by L-arginine, but without significant changes by L-NAME treatment. To our knowledge, this is the first demonstration that NO can inhibit MNCs independent of GABAergic inputs. Further, our results point to HAP as a potential site for nitrergic modulation. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Modulation of tonic immobility in guinea pig PAG by homocysteic acid, a glutamate agonist

Tonic immobility (TI) is an innate defensive behavior elicited by physical restriction and postural inversion, and is characterized by a profound and temporary state of motor inhibition. The participation of the periaqueductal gray matter (PAG) in TI modulation has previously been described. In addition, the excitatory amino acids (EAA) are important mediators involved in the adjustment of several defensive responses produced by PAG. In the present study, we investigated the effect of microinjection of the EAA agonist DL-homocysteic acid (DLH) and the N-methyl-D-aspartate (NMDA) receptor antagonist (MK-801) into the ventrolateral and dorsal PAG over the duration of TI in guinea pigs. Microinjection of 15 nmol/0.2 mu l of DLH into the ventrolateral PAG (vlPAG) and 30 nmol/0.2 mu l of DLH into the dorsal PAG (dPAG) promoted an increase and decrease in TI duration, respectively. These responses were blocked by prior microinjection of the NMDA receptor antagonist, MK-801 (3.6 nmol/0.2 mu l) at the same site. Microinjection of MK-801 alone into the APAG and dPAG did not alter the duration of TI episodes. These results suggest that NMDA receptors are involved in the modulation of TI in both the vlPAG and dPAG. In addition, PAC excitatory amino acids modulate the TI response via columnar organization of the PAC. In this manner, the vlPAG facilitates TI modulation whereas dPAG has an inhibitory role in TI. (C) 2008 Elsevier Inc. All rights reserved.

Transcription of the Hsp30, Hsp70, and Hsp90 heat shock protein genes is modulated by the PalA protein in response to acid pH-sensing in the fungus Aspergillus nidulans

Heat shock proteins are molecular chaperones linked to a myriad of physiological functions in both prokaryotes and eukaryotes. In this study, we show that the Aspergillus nidulans hsp30 (ANID_03555.1), hsp70 (ANID_05129.1), and hsp90 (ANID_08269.1) genes are preferentially expressed in an acidic milieu, whose expression is dependent on the palA (+) background under optimal temperature for fungal growth. Heat shock induction of these three hsp genes showed different patterns in response to extracellular pH changes in the palA(+) background. However, their accumulation upon heating for 2 h was almost unaffected by ambient pH changes in the palA (-) background. The PalA protein is a member of a conserved signaling cascade that is involved in the pH-mediated regulation of gene expression. Moreover, we identified several genes whose expression at pH 5.0 is also dependent on the palA (+) background. These results reveal novel aspects of the heat- and pH-sensing networks of A. nidulans.

DNA mismatch repair gene methylation in gastric cancer in individuals from northern Brazil

Gastric cancer is one of the most common malignancies. DNA methylation is implicated in DNA mismatch repair genes deficiency. In the present study, we evaluated the methylation status of MLH1, MSH2, MSH6 and PMS2 in 20 diffuse- and 26 intestinal-type gastric cancer samples and 20 normal gastric mucosal of gastric cancer patients from Northern Brazil. We found that none of the nonneoplastic samples showed methylation of any gene promoter and 50% of gastric, cancer samples showed at least one methylated gene promoter. Methylation frequencies of MLH1, MSH2, MSH6 and PMS2 promoter were 21.74%, 17.39%, 0% and 28.26% respectively in gastric cancer samples. MLH1 and PMS2 methylation were associated with neoplastic samples compared to nonneoplastic ones. PMS2:? methylation was associated with diffuse- and intestinal-type cancer compared with normal controls. Intestinal-type cancer showed significant association with MLH1 methylation. Diffuse-type cancer was significantly associated with MSH2 methylation. Our findings show differential gene methylation in tumoral tissue, which allows us to conclude that methylation is associated with gastric carcinogenesis. Methylation of mismatch repair genes was associated with gastric carcinogenesis and may be a helpful tool for diagnosis, prognosis and therapies. However, MSH6 does not seem to be regulated by methylation in our samples.

Identification of genes differentially expressed in a strain of the mold Aspergillus nidulans carrying a loss-of-function mutation in the palA gene

To identify genes differentially expressed in a strain of the mold Aspergillus nidulans carrying a loss-of-function mutation in palA, a gene in the pH-responsive signal transduction pathway, suppression subtractive hybridization was performed between RNA isolated from the biA1 and biA1 palA1 strains grown under limiting inorganic phosphate at pH 5.0. We have identified several genes upregulated in the biA1 palA1 mutant strain that play important roles in mitotic fidelity, stress responses, enzyme secretion, signal transduction mechanisms, development, genome stability, phosphate sensing, and transcriptional regulation among others. The upregulation of eight of these transcripts was also validated by Northern blot. Moreover, we show that a loss of function mutation in the palA gene drastically reduced the neutral sugar content of the acid phosphatase PacA secreted by the fungus A. nidulans grown at pH 5.0 compared with a control strain.

A single amino acid substitution in one of the lipases of Aspergillus nidulans confers resistance to the antimycotic drug undecanoic acid

A plausible approach to evaluate the inhibitory action of antifungals is through the investigation of the fungal resistance to these drugs. We describe here the molecular cloning and initial characterization of the A. nidulans lipA gene, where mutation (lipA1) conferred resistance to undecanoic acid, the most fungitoxic fatty acid in the C(7:0)-C(18:0) series. The lipA gene codes for a putative lipase with the sequence consensus GVSIS and WIFGGG as the catalytic signature. Comparison of the wild-type and LIP1 mutant strain nucleotide sequences showed a G -> A change in lipA1 allele, which results in a Glu(214) -> Lys substitution in LipA protein. This ionic charge change in a conserved LipA region, next to its catalytic site, may have altered the catalytic properties of this enzyme resulting in resistance to undecanoic acid.

In vitro susceptibility to antimycotic drug undecanoic acid, a medium-chain fatty acid, is nutrient-dependent in the dermatophyte Trichophyton rubrum

The antimycotic activity of fatty acids has long been known, and their presence in human skin and sweat appears to protect the host against superficial mycoses. Undecanoic acid is a medium-chain fatty acid that has been used in the treatment of dermatophytoses in humans. In this study, we selected one Trichophyton rubrum undecanoic acid-resistant strain that showed a marked reduction in its capacity to grow on human nail fragments, which correlated with the reduced activity of secreted keratinolytic proteases. Moreover, the susceptibility of T. rubrum to undecanoic acid is also dependent on the carbon source utilized by both control and resistant strains. The growth of the control strain was strongly inhibited by undecanoic acid in Sabouraud medium or in cultures supplemented with low-fat milk, whereas it was ineffective when the cultures were supplemented with Tween 20 or keratin as the carbon source, suggesting that nutrient conditions are crucial in establishing a susceptibility to antifungal drugs, which is helpful for the isolation and characterization of resistant strains, and in the screening for new antifungal drugs.