Acute Osmotic Stimuli Research Articles

Nitric oxide acutely modulates hypothalamic and neurohypophyseal carbon monoxide and hydrogen sulphide production to control vasopressin, oxytocin and atrial natriuretic peptide release in rats.

Nitric oxide (NO) negatively modulates the secretion of vasopressin (AVP), oxytocin (OT) and atrial natriuretic peptide (ANP) induced by the increase in extracellular osmolality, whereas carbon monoxide (CO) and hydrogen sulphide (H2 S) act to potentiate it; however, little information is available for the osmotic challenge model about whether and how such gaseous systems modulate each other. Therefore, using an acute ex vivo model of hypothalamic and neurohypophyseal explants (obtained from male 6/7-week-old Wistar rats) under conditions of extracellular iso- and hypertonicity, we determined the effects of NO (600μmolL-1 sodium nitroprusside), CO (100μmolL-1 tricarbonylchloro[glycinato]ruthenium [II]) and H2 S (10mmolL-1 sodium sulphide) donors and nitric oxide synthase (NOS) (300μmolL-1 Nω -methyl-l-arginine [LNMMA]), haeme oxygenase (HO) (200μmolL-1 Zn(II) deuteroporphyrin IX 2,4-bis-ethylene glycol [ZnDPBG]) and cystathionine β-synthase (CBS) (100μmolL-1 aminooxyacetate [AOA]) inhibitors on the release of hypothalamic ANP and hypothalamic and neurohypophyseal AVP and OT, as well as on the activities of NOS, HO and CBS. LNMMA reversed hyperosmolality-induced NOS activity, and enhanced hormonal release by the hypothalamus and neurohypophysis, in addition to increasing CBS and hypothalamic HO activity. AOA decreased hypothalamic and neurohypophyseal CBS activity and hormonal release, whereas ZnDPBG inhibited HO activity and hypothalamic hormone release; however, in both cases, AOA did not modulate NOS and HO activity and ZnDPBG did not affect NOS and CBS activity. Thus, our data indicate that, although endogenous CO and H2 S positively modulate AVP, OT and ANP release, only NO plays a concomitant role of modulator of hormonal release and CBS activity in the hypothalamus and neurohypophysis and that of HO activity in the hypothalamus during an acute osmotic stimulus, which suggests that NO is a key gaseous controller of the neuroendocrine system.

Alterations in hypothalamic synaptophysin and death markers may be associated with vasopressin impairment in sepsis survivor rats.

The impairment in arginine vasopressin (AVP) secretion during sepsis is described in clinical and experimental studies and has been associated with oxidative stress, apoptosis, and diminished activation of hypothalamic neurons. Few studies have, however, assessed these abnormalities in sepsis survivors. Here we performed two sets of experiments on Wistar rats that had been subjected to sepsis by cecal ligation and puncture (CLP) or nonmanipulated (naive) as control. In the first set, tissues and blood were collected from survivor rats 10 days after CLP to quantify hypothalamic Bcl-2, cleaved caspase- 3 and synaptophysin content, and bacterial load. In the second set, survivor rats were submitted to an acute osmotic stimulus (hypertonic saline), and after 30 minutes the water intake and AVP secretion were analyzed. The sepsis-surviving rats did not show bacterial load in tissues, but their hypothalamic synaptophysin and Bcl-2 levels were decreased, and the cleaved caspase- 3 level was increased when compared with the control group. However, AVP secretion was significantly attenuated in the CLP survivor animals submitted to an acute osmotic stimulus. These results suggest that the persistent AVP impairment in sepsis survivor animals may be due to a hypothalamic dysfunction associated with a synaptic deficit and decreased anti-apoptotic protein expression. This article is protected by copyright. All rights reserved.

Fos proteins are not prerequisite for osmotic induction of vasopressin transcription in supraoptic nucleus of rats

While it is well known that osmotic stimulation induces the expression of Fos family members in the supraoptic nucleus (SON), it is unclear whether the induced protein products are involved in the regulation of the gene transcription of arginine vasopressin (AVP). In the present study, we examined the in vivo correlation between changes in AVP gene transcription and expression of the various Fos family members in the SON after acute osmotic stimuli. The data demonstrated that the peak of AVP transcription (measured by intronic in situ hybridization) observed 15 min after an injection of hypertonic saline preceded the expression of Fos proteins, which became detectable at 30 min and peaked at 120 min. Electrophoretic mobility shift assay showed that the expressed Fos proteins bound to the composite AP-1/CRE-like site in the AVP promoter. These data suggest that Fos proteins in the SON induced by acute osmotic stimuli could affect AVP gene transcription by binding to the AVP promoter, but they are not prerequisite for the induction of AVP gene transcription.

A ROLE FOR BENZAMIL‐SENSITIVE PROTEINS OF THE CENTRAL NERVOUS SYSTEM IN THE PATHOGENESIS OF SALT‐DEPENDENT HYPERTENSION

1. Although increasing evidence suggests that salt-sensitive hypertension is a disorder of the central nervous system (CNS), little is known about the critical proteins (e.g. ion channels or exchangers) that play a role in the pathogenesis of the disease. 2. Central pathways involved in the regulation of arterial pressure have been investigated. In addition, systems such as the renin-angiotensin-aldosterone axis, initially characterized in the periphery, are present in the CNS and seem to play a role in the regulation of arterial pressure. 3. Central administration of amiloride, or its analogue benzamil hydrochloride, has been shown to attenuate several forms of salt-sensitive hypertension. In addition, intracerebroventricular (i.c.v.) benzamil effectively blocks pressor responses to acute osmotic stimuli, such as i.c.v. hypertonic saline. Amiloride or its analogues have been shown to interact with the brain renin-angiotensin-aldosterone system (RAAS) and to effect the expression of endogenous ouabain-like compounds. Alterations of brain RAAS function and/or endobain expression could play a role in the interaction between amiloride compounds and arterial pressure. Peripheral treatments with benzamil, even at higher doses than those given centrally, have little or no effect on arterial pressure. These data provide strong evidence that benzamil-sensitive proteins (BSPs) of the CNS play a role in cardiovascular responsiveness to sodium. 4. Mineralocorticoids have been linked to human hypertension; many patients with essential hypertension respond well to pharmacological agents antagonizing the mineralocorticoid receptor and certain genetic forms of hypertension are caused by chronically elevated levels of aldosterone. The deoxycorticosterone acetate (DOCA)-salt model of hypertension is a benzamil-sensitive model that incorporates several factors implicated in the aetiology of human disease, including mineralocorticoid action and increased dietary sodium. The DOCA-salt model is ideal for investigating the role of BSPs in the pathogenesis of hypertension, because mineralocorticoid action has been shown to modulate the activity of at least one benzamil-sensitive protein, namely the epithelial sodium channel. 5. Characterizing the BSPs involved in the pathogenesis of hypertension may provide a novel clinical target. Further studies are necessary to determine which BSPs are involved and where, in the nervous system, they are located.

Induction of the galanin-like peptide gene expression in the posterior pituitary gland after acute osmotic stimulus in rats

Galanin-like peptide (GALP) is a 60 amino-acid peptide, and the GALP mRNA is restricted to pituicytes in the posterior pituitary gland (PP) and neurons in the hypothalamic arcuate nucleus (Arc). We examined whether the GALP gene expression in the PP and Arc would be induced after intraperitoneal (i.p.) administration of hypertonic saline, that is, acute osmotic stimulus, in rats. The dose–response (2.8, 4.5, 6.0 and 9.0% NaCl) and time-course (6.0% NaCl, 1, 3, 6, 12 and 24 h) effects of acute osmotic stimulus on GALP mRNA levels in the PP and Arc were examined in rats by using in situ hybridization histochemistry. Plasma osmolality and plasma sodium concentration increased significantly at 1 h, and returned to control level at 6 h after i.p. administration of hypertonic saline (6.0% NaCl). The GALP mRNA level in the PP increased significantly 3 and 6 h after i.p. administration of hypertonic saline (6.0% NaCl), but the level in the Arc did not change. These results showed that acute osmotic stimulus-induced GALP gene expression in the pituicyte of the PP, but not in the neurons in the Arc, and the gene expression in the pituicyte might be regulated by plasma osmolality and/or plasma sodium concentration.

Vasopressin gene transcription increases in response to decreases in plasma volume, but not to increases in plasma osmolality, in chronically dehydrated rats

The synthesis of arginine vasopressin (AVP) in the magnocellular neurons of the supraoptic (SON) and paraventricular nuclei (PVN) is physiologically regulated by plasma osmolality and volume. To clarify how the regulation of AVP gene transcription is affected by chronic dehydration, we examined changes in the transcriptional activities of AVP gene by plasma osmolality and volume in both euhydrated and dehydrated conditions. Euhydrated rats had free access to water, whereas dehydrated rats had been deprived of water for 3 days before experiments. Rats in both conditions were subjected to acute hypertonic stimuli or hypovolemia, and changes in AVP heteronuclear (hn)RNA levels, an indicator of gene transcription, in the SON and PVN were examined with in situ hybridization. The intraperitoneal (i.p.) injection (2% body wt) of hypertonic (1.5 M) saline increased plasma Na levels by approximately 40 meq/l in both euhydrated and dehydrated conditions. However, expression levels of AVP hnRNA in the SON and PVN were increased only in euhydrated, not dehydrated, rats. On the other hand, i.p. injection of polyethylene glycol decreased the plasma volume by approximately 16-20%, and AVP hnRNA levels in the SON and PVN were significantly increased in both conditions. Thus it is demonstrated that signaling pathways regulating AVP gene transcription in the magnocellular neurons were completely refractory to acute osmotic stimuli under the chronic dehydration and that AVP gene transcription could probably respond to acute hypovolemia through different intracellular signal transduction pathways from those for osmoregulation.

Expression of Immediate Early Genes and Vasopressin Heteronuclear RNA in the Paraventricular and Supraoptic Nuclei of Rats After Acute Osmotic Stimulus

Monitoring the expression of immediate early genes (IEGs) is useful for following stress-induced cellular responses in the neuroendocrine system. We have examined the transcriptional activities of four IEGs (c-fos, junB, NGFI-A and NGFI-B) and of the arginine vasopressin (AVP) gene in the hypothalamic paraventicular (PVN) and supraoptic nuclei (SON) of rats after acute osmotic stimuli, using in situ hybridization histochemistry. After intraperitoneal (i.p.) administration of hypertonic saline (2% body weight, 900 mOsm/kg), the expression levels of all IEG mRNAs were increased significantly both in the PVN and SON at as early as 10 min, peaked at 30 min and remained elevated until 60 min. The expression of AVP heteronuclear (hn)RNA also peaked at 30 min, and remained elevated until 180 min. Thirty min after i.p. administration of hypertonic saline (600 mOsm/kg), the expression levels of all IEG mRNAs in the PVN and SON were significantly increased in comparison with those after i.p. administration of isotonic saline (290 mOsm/kg). Regression analysis revealed that expression levels of the IEG mRNAs and AVP hnRNA were positively correlated with the plasma concentration of sodium, and the rates of increase of the expression levels of all IEG mRNAs were similar. The expression levels of all IEG mRNAs examined are useful markers for following the changes of the AVP gene transcription in the PVN and SON after acute osmotic stimuli in rats.

Regulation of neuronal nitric oxide synthase mRNA expression in the rat magnocellular neurosecretory system

We examined the activation of nNOS mRNA expression within the supraoptic and paraventricular nuclei (SON and PVN) of the hypothalamus. In salt-loaded rats nNOS mRNA expression was significantly increased in both nuclei. In rats given i.p. injections of 1.5 M NaCl (4 ml/kg), a small but significant increase in nNOS mRNA expression in the SON and PVN was found 6 h after injection; no change was detected 2 or 4 h after injection. In rats in which hyponatraemia had been induced experimentally, nNOS mRNA was downregulated in the SON, and expression levels were not increased within 4 h after intense acute osmotic stimuli. Finally, neurons of the SON were antidromically-activated by neural stalk stimulation for 2 h. No increase of nNOS mRNA expression in the SON was observed 2 h after stimulation. Thus, increased electrical activity is not directly coupled to rapidly increased expression of nNOS mRNA, and hence acute increases in nNOS mRNA expression are unlikely to play a role in short-term adaptation of the magnocellular system to osmotic stimulation.

Salt loading abolishes osmotically stimulated vasopressin release within the supraoptic nucleus

Central and systemic osmotic stimulation increase vasopressin (VP) release within the supraoptic nucleus (SON) and into the general circulation. We examined whether changes in water/electrolyte balance affect the neurosecretory responses to these stimuli. Urethane-anesthetized control, salt-loaded (2% NaCl for 2 days) or water-deprived (for 2 days) male rats were implanted with an arterial catheter and bilateral microdialysis probes into the SON. Plasma and SON VP levels were measured before and after acute osmotic stimuli were administered intraperitoneally (i.p.) and then directly into the SON. Water deprivation resulted in elevated basal intranuclear and plasma VP levels. Intraperitoneal hypertonic saline (HS) and direct osmotic stimulation of the SON increased VP release into the SON in both the control and water-deprived groups. Salt loading abolished the intranuclear VP response to both stimuli. Osmotically induced release of VP into plasma was not different between the three groups. These data demonstrate that salt loading, but not water deprivation, alters the central neurosecretory VP response to acute osmotic stimulation.

Dehydration attenuates plasma prolactin response to suckling through a dopaminergic mechanism.

The tuberoinfundibular dopaminergic neurons projecting to the median eminence are well accepted as a major physiological regulator of adenohypophyseal PRL secretion. However, recent evidence has shown that dopamine (DA) in the neurointermediate lobe also has an inhibitory effect on PRL secretion by anterior pituitary. Since the neurointermediate is innervated by the tuberohypophyseal dopaminergic (THDA) neurons, which is known to be selectively activated by dehydration of the animal, the aim of this study was to investigate the physiological role of the THDA system in PRL release during lactation. On the day of the experiments, the litters were separated from the mothers for 4 h before initiation of the suckling stimulus. The suckling-induced PRL surge was detected on three consecutive days. On the first day the normal response was tested; then immediately after taking the last blood samples, drinking solutions were changed to the high salt (2.5% saline) containing bottles or were taken away. Suckling-induced PRL response was significantly decreased after 24 h and almost completely blocked 48 h later in dehydrated mothers. This effect could be prevented by haloperidol (a DA receptor antagonist) pretreatment (0.1 mg/kg BW sc), and it was only transient because rehydration of the mothers reestablished basal as well as suckling-induced PRL response. In addition, the effect of an acute osmotic stimulus on the plasma PRL levels (injecting 0.5 ml 10% saline solution iv) was also tested. There was a marked and immediate decrease in PRL concentration within 15 min of injection. Domperidone, another DA receptor blocker (20 micrograms/rat iv) completely abolished the depletion of plasma PRL in response to 10% saline injection. These results support our assumption that the dopaminergic regulation of PRL secretion during lactation involves the THDA system. Furthermore, these data underline the importance of an interaction between regulation of PRL secretion and water and sodium homeostasis.

Vasopressin Gene Expression in the Rodent Hypothalamus: Transcriptional and Posttranscriptional Responses to Physiological Stimulation

The neuropeptide vasopressin (VP) is expressed in the supraoptic nucleus, a discrete group of neurons in the hypothalamus that respond to osmotic stimuli. In the rat the pattern of expression of VP mRNA changes in two ways as a consequence of the physiological stimulation of these neurons. Firstly, there is an accumulation of VP mRNA, and secondly, the poly(A) tail of the VP mRNA increases in length. We asked whether the increase in VP mRNA level is a consequence of transcriptional or posttranscriptional mechanisms. We present evidence from nuclear run-on assays that increases in the transcription of the rat VP gene are sufficient to account for the accumulation of VP mRNA observed in chronically stimulated animals. However, we note that in acutely stimulated animals there are rapid and relatively large increases in VP gene transcription that do not correlate with increases in the VP mRNA level, but coincide with the appearance of a homogeneous class of VP mRNAs with elongated poly(A) tails. We suggest that immediately after the onset of an acute osmotic stimulus, there is a rapid destruction of preexisting VP mRNAs and their replacement with new transcripts bearing longer poly(A) tails. We have also addressed the question of the function of the elongated VP mRNA poly(A) tail. It is unlikely that the poly(A) tail extension is involved in RNA stability; the transcriptional changes observed are sufficient to account for the increase in VP mRNA level, and we show that in the mouse similar increases in VP mRNA level are observed without concomitant changes in poly(A) tail length. We did not observe a change in the polysome distribution of the VP mRNA after osmotic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)