Distribution of c-fos mRNA in the brain following intracerebroventricular injection of nitric oxide (NO)-releasing compounds: possible role of NO in central cardiovascular regulation.

We injected nitric oxide (NO)-releasing compounds and NO synthase (NOS) inhibitors into the brains of conscious, freely moving rats and measured the effects on mean arterial blood pressure (MAP) and heart rate, as well as on the expression of c-fos mRNA, neuronal NOS (nNOS) mRNA and NADPH-diaphorase, an indicator of NOS activity. When administered i.c.v., the NO donor, NOC-18, caused a significant fall in MAP and heart rate, whereas the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), induced a significant rise in MAP. The same dose of NOC-18 or L-NAME when administered i.v. did not affect MAP and heart rate. Centrally administered NOC-18 induced c-fos mRNA expression in several regions of the brain involved in the baroreceptor response, including the nucleus of the solitary tract, the area postrema and the rostral ventrolateral medulla, as well as areas involved in the integration of autonomic, neuroendocrine and behavioural responses, including the medial preoptic area, the organum vasculosum lamina terminalis, the bed nucleus of stria terminalis, the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the central nucleus of amygdala (CeA) and the locus coeruleus. Most of the areas that expressed c-fos also contained nNOS mRNA and/or NADPH-d-positive neurones and fibres. i.c.v. injection of L-NAME induced c-fos mRNA expression in PVN, SON, locus coeruleus and NTS, suggesting a tonic inhibition of neuronal activity by NO or stimulation of neuronal activity by endogenous NO. i.v. injection of NOC-18 or L-NAME did not induce any significant c-fos mRNA expression in rat brain. These results demonstrate that NO acts directly in the brain to reduce the systemic blood pressure, and that the endogenous NO pathway may play a role in cardiovascular and autonomic regulation by modulating neuronal activities in discrete regions of the brain.

A defect of lipopolysaccharide-induced nitric oxide synthase gene expression in the paraventricular nucleus of Lewis rats.

The hypothalamo-pituitary-adrenal (HPA) axis activity of Lewis rats has been reported to be hyporesponsive to immune challenge, and nitric oxide (NO) has been demonstrated to be involved in the regulation of the HPA axis during the response to immune challenge. The present study investigates the effect of systemic injection of lipopolysaccharide (LPS) on NO production within the paraventricular nucleus (PVN) of immature female Fisher-344 (F344) and Lew/N rats (Lew/N), to clarify the pathophysiological role of NO in the dysregulation of the HPA axis in Lewis/N. Intraperitoneal injection of 25 mg/kg LPS significantly increased neuronal NO synthase (nNOS) mRNA expression in the PVN of F344 rats, but did not change nNOS mRNA levels in the PVN of Lew/N rats. CRF mRNA levels in the PVN significantly increased in response to LPS injection only in F344 rats. In contrast, inducible NOS (iNOS) mRNA increased similarly in both strains. These results demonstrated a defect of up-regulation of nNOS gene expression in the PVN of Lew/N rats following immune challenge. The defect appears to be associated with the dysfunction of the HPA axis in this strain. An increase in iNOS mRNA may partially restore NO production in the PVN.

Urocortin mRNA is expressed in the enteric nervous system of the rat.

The expression of the urocortin gene in the gastrointestinal tract was investigated using reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization histochemistry. PCR demonstrated the presence of urocortin mRNA in the rat brain, duodenum, small intestine, and colon. By in situ hybridization, urocortin-containing cells were exclusively localized to the submucosal plexus and myenteric plexus in the duodenum, small intestine and colon. These results suggest that urocortin may play an important role in the regulation of gastrointestinal motor function throughout the enteric nervous system.

Distinct distribution and time-course changes in neuronal nitric oxide synthase and inducible NOS in the paraventricular nucleus following lipopolysaccharide injection.

Nitric oxide (NO) is known to be involved in the modulation of neuroendocrine function. To clarify the role of different isoforms of NO synthase (NOS) in the neuroendocrine response to immune challenge, the expressions of neuronal NOS (nNOS) and inducible NOS (iNOS) genes in the hypothalamus following lipopolysaccharide (LPS) injection were examined using in situ hybridization. NOS activity was also determined by NADPH-diaphorase (NADPH-d) histochemistry. LPS (25 mg/kg) or sterile saline was injected intraperitoneally to male Wistar rats and the rats sacrificed 30 min, or 1, 2, 3, 5, 12 or 24 h after injection. nNOS mRNA expression in the paraventricular nucleus (PVN) was significantly increased 2 h after LPS injection. iNOS mRNA, which was not detected until 2 h after LPS injection, was significantly increased in the PVN 3 h after LPS injection. Both RNA expressions had returned to basal levels by 12 h after LPS injection. The number of NADPH-d positive cells was significantly increased 5 h after LPS injection. iNOS expression was more robust in parvocellular PVN, while nNOS was distributed mainly in the magnocellular PVN. Double in situ hybridization histochemistry revealed that some of the iNOS- (48.4%) or nNOS-positive cells (34. 3%) in the parvocellular PVN expressed CRF mRNA. The results demonstrate that LPS-induced sepsis causes significant increases in nNOS and iNOS gene expression with different time-courses and distributions, and that iNOS mRNA was more frequently co-localized with CRF-producing parvocellular neurons in the PVN. Thus, NO produced by iNOS and nNOS may play an important role in the neuroendocrine response to an immune challenge. Distinct differences in the distribution and time-course changes of iNOS and nNOS suggest different roles for the hypothalamic-pituitary-adrenal axis and/or neurohypophyseal system.

Stress-induced changes of gene expression in the paraventricular nucleus are enhanced in spontaneously hypertensive rats.

Heightened hypothalamic-pituitary-adrenal (HPA) axis responses have been implicated in hypertension in the spontaneously hypertensive rat (SHR), but the exact mechanisms involved are poorly understood. To determine changes in gene expression in SHR in the paraventricular nucleus (PVN), stress-induced accumulation of CRF, CRF type 1 receptor (CRFR-1) genes, and immediate-early genes were examined using in situ hybridization in young (5 weeks old) and adult (12 weeks old) stroke-prone SHR (SHRSP), compared with normotensive Wistar Kyoto (WKY) rats. Restraint stress-induced accumulation of c-fos, jun B, and NGFI-B mRNA, and CRF hnRNA in the PVN was significantly higher in young and adult SHRSP than in WKY rats at 30 min, except for c-fos in young rats. CRFR-1 mRNA expression in the PVN was also significantly higher in adult SHRSP than in WKY rats at 120 min after stress onset. CRF mRNA was increased in response to stress in young SHRSP. The basal CRF mRNA level in the PVN was significantly lower in adult SHRSP than in WKY rats. Young SHRSP exhibit greater ACTH responses to stress without significant changes in plasma corticosterone concentrations. The adult SHRSP exhibited lower baseline concentrations of corticosterone and similar corticosterone response to stress with enhanced secretion of ACTH. Overall, these results demonstrated that stress-induced activation of immediate early genes and CRF gene transcription in the PVN, and ACTH secretion is enhanced in early hypertensive, young, and adult SHRSP, suggesting that they are probably not the result of chronic alterations in blood pressure. The abnormal hypothalamic-pituitary response to stress thus appears to be related to the development of hypertension.

Different expression of immediate-early genes in the rat paraventricular nucleus induced by stress: relation to corticotropin-releasing factor gene transcription.

Stressful stimuli induce the transcriptional activation of the corticotropin-releasing factor (CRF) gene as well as that of various immediate-early genes (IEGs). Among them, members of the fos/jun families play an important role in the regulation of gene expression. The possible relation between stress-induced IEG expression and CRF gene transcription was investigated by analysis of stress-induced changes in the abundance of c-fos, c-jun, jun-B, jun-D, and NGFI-B mRNA as well as CRF heteronuclear RNA (hnRNA), which reflects gene transcription, in the rat paraventricular nucleus (PVN) by in situ hybridization. Restraint stress induced rapid (within 5 min) and transient increases in the level of c-fos mRNA and CRF hnRNA in the PVN, with peak expression apparent 30 min after the onset of stress. The induction of jun-B and NGFI-B gene expression was also rapid and transient, but was delayed relative to that of c-fos mRNA and the CRF hnRNA. In contrast, both jun-D and c-jun were constitutively expressed in the PVN, and the amount of the corresponding transcripts increased only slightly in response to stress. These results reveal differential patterns of expression of IEGs in the PVN in response to stress. The observation that the level of mRNAs corresponding to these IEGs did not increase before that of CRF hnRNA may suggest that the products of these genes do not seem to directly mediate the stress-induced increase in CRF gene transcription in the PVN.

Corticotropin-releasing factor up-regulates its own receptor mRNA in the paraventricular nucleus of the hypothalamus.

We investigated the role of CRF in regulating receptor expression in the paraventricular nucleus (PVN). First, to clarify the effect of exogenously administered CRF, 1 microgram of ovine CRF was injected into rat lateral ventricle and changes in concentration of the CRF type 1 receptor (CRF1-R) and CRF mRNA in the PVN were semiquantified after in situ hybridization. Second, we determined the effect of stress, as a stimulant of endogenous CRF secretion, on mRNA accumulation. While CRF1-R mRNA expression was low to be undetectable in the PVN of controls, both intracerebroventricular administration of CRF and restraint significantly increased CRF1-R and CRF signals in the parvocellular PVN. Thus CRF may modulate CRF production and release from the PVN, by regulating CRF1-R expression.

Chlordiazepoxide attenuates stress-induced activation of neurons, corticotropin-releasing factor (CRF) gene transcription and CRF biosynthesis in the paraventricular nucleus (PVN).

Corticotropin-releasing factor (CRF) plays a role in coordinating endocrine, autonomic, and behavioral responses to stressful stimuli. Benzodiazepines exert many effects which oppose those of CRF, including anxiolysis and suppression of the pituitary-adrenal axis. In the present study, we employed in situ analysis of CRF heteronucleous RNA (hnRNA) and c-fos mRNA to assess stimulus-induced CRF gene transcription rate following stress and its modulation by chlordiazepoxide (CDP). Male albino rats were exposed to restraint stress for 30 min and sacrificed 30 and 120 min after the onset of stress. Either CDP or vehicle was given intraperitoneally 60 min before stress. To determine plasma ACTH levels by immunoradiometric assay, another group of rats was decapitated 10 min after the onset of restraint stress. Restraint stress induced rapidly and significantly c-fos mRNA and CRF hnRNA expression in the PVN at the 30 min time point. Increases in both RNA copies were significantly inhibited by administration of CDP at doses of 5 and 10 mg/kg. CRF mRNA concentrations were increased significantly in the PVN 120 min after stress and again, CDP attenuated significantly these increases in the PVN. The plasma ACTH increase in response to stress was inhibited significantly by CDP administration at every dose tested. CDP did not change CRF mRNA levels in the non-stressed animal.(ABSTRACT TRUNCATED AT 250 WORDS)

Stress-induced activation of neuronal activity and corticotropin-releasing factor gene expression in the paraventricular nucleus is modulated by glucocorticoids in rats.

Intronic in situ hybridization methodology provides a means of determining the rate of gene transcription under basal and stimulated conditions. In the present study, we have used intronic in situ hybridization to the corticotropin-releasing factor (CRF) gene to measure hypothalamic CRF gene transcription after stress as well as its modulation by glucocorticoids. Using this and conventional exonic in situ hybridization we examined the time course of changes in c-fos mRNA, and CRF heteronuclear RNA (hnRNA) and mRNA concentrations in the paraventricular nucleus (PVN) of male Wistar rats after restraint stress. In addition, we determined the effects of adrenalectomy and dexamethasone administration on c-fos and CRF gene expression in the PVN. Restraint stress induced a rapid induction (within 5 min) of c-fos mRNA and CRF hnRNA expression in the PVN. Both RNA concentrations peaked at 30 min then decreased and were undetectable 2 h after stress onset. In contrast, the concentration of CRF mRNA increased gradually and a significant elevation was first detected 60 min after the beginning of stress. Adrenalectomy augmented and dexamethasone pretreatment inhibited c-fos mRNA, CRF hnRNA, and mRNA induction after stress. The data suggest that stress-induced activation of neurons, CRF gene transcription, and CRF synthesis in the PVN are modulated by glucocorticoids.

[Effect of aging and physical inactivity on glucose tolerance and insulin sensitivity].

It has been well documented that glucose intolerance is associated with aging, but it is not yet clear whether this phenomenon is due to the aging process itself or is secondary to the appearance of other age-related conditions among which physical inactivity is one of most important variables. To evaluate the effect of aging process and/or physical inactivity on insulin action, this study was undertaken using the euglycemic insulin clamp technique and the oral glucose tolerance test (OGTT). Subjects without diabetes mellitus and other serious diseases consisted of 14 non-obese aged individuals and 10 young controls (YC group), ranging in age from 63 to 85 yrs, and from 19 to 21 yrs, respectively. The aged individuals were further divided into two groups (one was termed as the AS group, in which 7 aged subjects had been confined to bed for at least 3 months and the other was termed as the AC group in which 7 aged controls kept their daily physical activity such as walking). The results of OGTT did not show any remarkable differences between AC and YC groups. In the AS group, however, glucose intolerance and low insulin response during OGTT were observed. In view of the tissue insulin action, MCR, which is thought as a reliable marker for tissue insulin action, evaluated by euglycemic insulin clamp was 5.31 +/- 0.68, 8.57 +/- 1.20, 9.60 +/- 0.35 ml/kg/min in the AS, AC and YC groups, respectively (AS less than AC, p less than 0.05, AS less than YC, p less than 0.01, AC less than YCM, N.S.).(ABSTRACT TRUNCATED AT 250 WORDS)