The involvement of nitric oxide and prostaglandins in the cholinergic stimulation of hypothalamie-pituitary-adrenal response during crowding stress.

The aim of the present study was to determine the effect of social crowding stress and significance of nitric oxide (NO) and prostaglandins (PG) generated by constitutive and inducible nitric oxide synthase (NOS) and cyclooxygenase (COX) in the stimulation of hypothalamic-pituitary-adrenal (HPA) axis by cholinergic muscarinic receptor agonist carbachol. Inhibitors of neuronal NOS (nNOS) L-NNA, general NOS L-NAME and inducible NOS (iNOS) aminoguanidine, as well as inhibitors of COX-1, piroxicam, and COX-2, compound NS-398 were administered 15 min prior to carbachol to control or crowded rats (24 rats in cage for 7, during 3 and 7 days). In stressed rats L-NAME, L-NNA and aminoguanidine significantly intensified the carbachol-induced ACTH and corticosterone secretion, like in control rats. Piroxicam, markedly decreased the carbachol-induced ACTH and corticosterone response under either basal or stress conditions. Compound NS-398 did not markedly alter the carbachol-induced HPA response in control and stressed rats. Crowding stress (3 days) significantly impaired the i.c.v. prostaglandin E(2)-induced ACTH response. Corticotropin releasing hormone (CRH) receptor antagonists, alpha-helical CRH [9-14], given i.c.v. did not alter the PGE(2)-evoked corticosterone response in either control or stressed rats, indicating that hypothalamic CRH is not involved in the PGE(2)-induced central stimulation of HPA axis. In control rats L-NAME considerably enhanced, while L-arginine, a physiological NOS substrate, abolished the PGE(2)-induced ACTH and corticosterone response. In stressed rats this NOS blocker significantly increased and L-Arg reduced the stimulatory effect of PGE(2) on ACTH and corticosterone secretion. The carbachol-induced corticosterone response was significantly increased by pretreatment with nNOS inhibitor L-NNA and was considerably reduced by indomethacin, a general COX inhibitor. Pretreatment with both antagonists left the carbachol-induced corticosterone level unchanged, suggesting an independent and reciprocal effect of NO and PG in the cholinergic stimulation of pituitary-adrenocortical response. These results indicate that in the stimulatory action of muscarinic agonist, carbachol, NO is an inhibitory transmitter under basal and crowding stress conditions. This psychosocial stress does not functionally affect the NOS/NO systems. Prostaglandins are involved in the cholinergic muscarinic-induced stimulation of HPA response to a significant extent in non-stressed rats. PGE(2) may be involved in the carbachol-elicited HPA response under basal and stress conditions. Prostaglandins released in response to muscarinic stimulation did not evoke the hypothalamic CRH mediation. NO significantly impairs and PG stimulates the carbachol-induced HPA response in rats under basal and social stress conditions.     

Nitric oxide mediates the interleukin-1beta- and nicotine-induced hypothalamic-pituitary-adrenocortical response during social stress.

We investigated the role of nitric oxide (NO) in the interleukin 1beta (IL-1beta) and nicotine induced hypothalamic-pituitary-adrenal axis (HPA) responses, and a possible significance of CRH and vasopressin in these responses under basal and social stress conditions. Male Wistar rats were crowded in cages for 7 days prior to treatment. All compounds were injected i.p., nitric oxide synthase (NOS) inhibitors, alpha-helical CRH antagonist and vasopressin receptor antagonist 15 min before IL-1beta or nicotine. Identical treatment received control non-stressed rats. Plasma ACTH and serum corticosterone levels were measured 1 h after IL-1beta or nicotine injection. L-NAME (2 mg/kg), a general nitric oxide synthase (NOS) inhibitor, considerably reduced the ACTH and corticosterone response to IL-1beta (0.5 microg/rat) the same extent in control and crowded rats. CRH antagonist almost abolished the nicotine-induced hormone responses and vasopressin antagonist reduced ACTH secretion. Constitutive endothelial eNOS and neuronal nNOS inhibitors substantially enhanced the nicotine-elicited ACTH and corticosterone response and inducible iNOS inhibitor, aminoguanidine, did not affect these responses in non-stressed rats. Social stress significantly attenuated the nicotine-induced ACTH and corticosterone response. In crowded rats L-NAME significantly deepened the stress-induced decrease in the nicotine-evoked ACTH and corticosterone response. In stressed rats neuronal NOS antagonist did not alter the nicotine-evoked hormone responses and inducible NOS inhibitor partly reversed the stress-induced decrease in ACTH response to nicotine. These results indicate that NO plays crucial role in the IL-1beta-induced HPA axis stimulation under basal and social stress conditions. CRH and vasopressin of the hypothalamic paraventricular nucleus may be involved in the nicotine induced alterations of HPA axis activity. NO generated by eNOS, but not nNOS, is involved in the stress-induced alterations of HPA axis activity by nicotine.     

Nitric oxide and prostaglandin systems in the stimulation of hypothalamic-pituitary-adrenal axis by neurotransmitters and neurohormones.

The review presents our results on the regulatory role of prostaglandins (PG) and nitric oxide (NO) in the activation of hypothalamic-pituitary-adrenal (HPA) axis by cholinergic, adrenergic and histaminergic systems and by neurohormones: corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) under basal conditions. The synthesis of endogenous PG or NO was inhibited by non-selective and selective cyclooxygenase (COX) antagonists and nitric oxide synthase (NOS) blockers given 15 min before the respective receptor agonist and HPA axis activity was assessed 1 h later by measuring plasma ACTH and serum corticosterone levels. The muscarinic agent - carbachol-induced HPA response was considerably supressed by piroxicam, a predominantly constitutive cyclooxygenase (COX-1) inhibitor and significantly diminished by indomethacin, a non-selective COX blocker, but was unaffected by compound NS-398, an inducible cyclooxygenase (COX-2) antagonist. A non-selective NOS antagonist L-NAME and neuronal NOS blocker L-NNA significantly intensified the carbachol-induced corticosterone secretion. The nicotine-induced increase in ACTH and corticosterone response was significantly supressed by piroxicam, and diminished by indomethacin, but was significantly augmented by L-NAME and L-NNA. The inhibition of PG synthesis by indomethacin totally abolished or reversed the increase of nicotine-induced hormone responses to both NOS blockers. The i.c.v. phenylephrine, an alpha(1)-adrenergic receptor agonist - evoked HPA response was significantly impaired by piroxicam and compound NS-398 and more potently reduced by L-NAME. The i.c.v. clonidine, an alpha(2)-adrenergic agonist - elicited HPA response was also considerably decreased by piroxicam, compound NS-398 and L-NAME. By contrast, the stimulatory effect of i.c.v. isoprenaline, a non-selective beta-adrenergic agonist, was not altered by either COX or NOS inhibitors. The i.c.v. histamine- and HTMT, a histamine H(1)-agonist-induced ACTH and corticosterone response were significantly diminished by piroxicam and indomethacin, respectively. Compound NS-398, did not markedly alter the HPA response to HTMT or amthamine, a histamine H(2) receptor agonist. Inhibition of endogenous NO synthesis by a neuronal NOS inhibitor 7-nitroindazole markedly enhanced the histamine-induced hormone secretion, abolished the HTMT-induced response and did not substantially alter the amthamine-evoked ACTH and corticosterone secretion. COX blockers did not significantly affect the CRH-induced HPA response and the inhibition of NO synthesis by L-NNA markedly intensified ACTH response. The vasopressin-stimulated increase in HPA response, was considerably reduced by the inhibition of PG synthesis by both COX antagonists while inhibition of NO synthesis by NOS blockers greatly enhanced this response. The involvement of PG and NO in the neurohormonal regulation of HPA activity depends mainly on greatly complex and tightly regulated mechanisms at the level of second messengers IP(3) and adenylyl cyclase systems.     

Role of prostaglandins and nitric oxide in the lipopolysaccharide-induced ACTH and corticosterone response.

This study was designed to determine the role of endogenous prostaglandins (PG) and nitric oxide (NO) in the lipopolysaccharide (LPS)-induced ACTH and corticosterone secretion in conscious rats. LPS (0.5 and 1 mg/kg) given i.p. stimulated the hypothalamic-pituitary-adrenocortical (HPA) activity measured 2 h later. A non-selective cyclooxygenase inhibitor indomethacin (10 mg/kg i.p.), piroxicam (2 mg/kg i.p.), a more potent antagonist of constitutive cyclooxygenase (COX-1) and compound NS-398 (2 mg/kg i.p.), a selective inhibitor of inducible cyclooxygenase (COX-2) given 30 min before LPS (1 mg/kg i.p.) significantly diminished both the LPS-induced ACTH and corticosterone secretion. COX-2 blocker was the most potent inhibitor of ACTH secretion (72.3%). Nomega-nitro-L-arginine methyl ester (L-NAME 2 and 10 mg/kg i.p.), a non-selective nitric oxide synthase (NOS) blocker given 15 min before LPS did not substantially alter plasma ACTH and corticosterone levels 2 h later. Aminoguanidine (AG 100 mg/kg i.p.), a selective inducible nitric oxide synthase (iNOS) inhibitor, considerably enhanced ACTH and corticosterone secretion induced by a lower dose (0.5 mg/kg) of LPS and did not significantly alter this secretion after a larger dose (1 mg/kg) of LPS. L-NAME did not markedly affect the indomethacin-induced inhibition of ACTH and corticosterone response. By contrast, aminoguanidine abolished the indomethacin-induced reduction of ACTH and corticosterone secretion after LPS. These results indicate an opposite action of PG generated by cyclooxygenase and NO synthesized by iNOS in the LPS-induced HPA-response.     

Role of nitric oxide in the nicotine-induced pituitary-adrenocortical response.

Nitric oxide (NO) is a major signaling molecule and biological mediator of the hypothalamic-pituitary-adrenal (HPA) axis. We investigated the role of NO formed by endothelial (e), neuronal (n) and inducible (i) nitric oxide synthase (NOS) in the stimulatory effect of nicotine on the HPA axis in rats under basal conditions. Also possible interaction of NOS systems with endogenous prostaglandins (PG) in that stimulation was assessed. NOS and cyclooxygenase inhibitors were administered i.p. 15 min prior to nicotine (2, 5 mg/kg i.p.). Plasma ACTH and serum corticosterone levels were measured 1 h after nicotine injection. NOS blockers given alone did not markedly affect the resting ACTH and corticosterone levels. L-NAME (2-10 mg/kg), a broad spectrum NOS inhibitor considerably and dose dependently enhanced the nicotine-induced ACTH and corticosterone secretion. L-NNA (2 mg/kg) and 7-nitroindazole (7-NI 20 mg/kg), neuronal NOS inhibitors in vivo also significantly augmented the nicotine-induced ACTH and corticosterone levels. L-arginine greatly impaired the nicotine-induced hormone responses and reversed the L-NNA elicited enhancement of the nicotine-evoked ACTH and corticosterone response. In contrast to the constitutive eNOS and nNOS antagonists, an inducible NOS antagonist guanethidine (50-100 mg/kg i.p.) did not substantially affect the nicotine-elicited pituitary-adrenocortical responses. Indomethacin (2 mg/kg i.p.), a non-selective cyclooxygenase blocker abolished the L-NAME and L-NNA-induced enhancement of the nicotine-evoked ACTH and corticosterone response. These results indicate that NO is an inhibitory mediator in the HPA axis activity. Inhibition of its generation by eNOS and nNOS significantly enhances the nicotine-induced HPA response. Under basal conditions iNOS is not involved in the nicotine-induced ACTH and corticosterone secretion. Prostaglandins play an obligatory role in the response of HPA axis to systemic nicotine administration.     

Effect of cyclooxygenase inhibitors on the vasopressin induced ACTH and corticosterone response during crowding stress.

The aim of the present study was to compare the effect of social stress on the corticotropin releasing hormone (CRH) and arginine vasopressin (AVP)-induced pituitary-adrenocortical activity. Also the significance of prostaglandins (PG) generated by constitutive and inducible cyclooxygenase (COX-1 and COX-2) in the stimulation of hypothalamic-pituitary-adrenal (HPA) axis by AVP under basal and crowding stress conditions was investigated. The control rats were housed 7 in a standard cage and stressed rats were crowded 24 in a cage of the same size during 7 days. The activity of HPA axis was determined by measuring plasma ACTH and serum corticosterone levels 1 h after i.p. AVP administration. Indomethacin (2.0 mg/kg i.p.), a non-selective COX inhibitor, piroxicam (0.2, 2.0, and 5.0 mg/kg), a more potent COX-1 than COX-2 inhibitor, and compound NS-398 (0.2 and 2.0 mg/kg) a selective COX-2 inhibitor, were administered i.p. 15 min prior to AVP (5.0 microg/kg i.p.) to control or crowded rats. The obtained results indicate that social stress for 7 days considerably inhibits the stimulatory action of AVP on ACTH secretion, while it intensifies the CRH-induced ACTH secretion. Indomethacin, piroxicam and NS-398 significantly diminished the AVP-elicited ACTH and corticosterone secretion in non-stressed rats. None of these COX antagonist induced any significant inhibition of the AVP-induced ACTH and corticosterone secretion in stressed rats. Therefore, PG generated by COX-1 or COX-2 do not participate to a significant extent in the HPA stimulation by AVP during crowding stress. These results suggest that social crowding stress desensitizes the PG stimulatory mechanism which considerably mediates the AVP-induced HPA stimulation under basal conditions. The results contrast with a lack of any involvement of PG in the CRH-induced stimulation of HPA response under basal or crowding stress conditions.     

Repeated handling, restraint, or chronic crowding impair the hypothalamic-pituitary-adrenocortical response to acute restraint stress.

The purpose of the present study was to assess whether, and to what extent prior handling, restraint or social crowding stress during 3-10 days affects the hypothalamic-pituitary-adrenocortical (HPA) response to an acute short-lasting restraint stress. Also the effect of a feedback inhibitory mechanism of corticosterone in the impairment of HPA axis by these stressors was investigated. Male Wistar rats were pretreated with handling 1 min/day for 3-10 days, restraint 2 times daily for 3-7 days and crowding stress for 7 days before exposure to acute restraint stress in metal tubes for 10 min. Some group of rats received exogenous s.c. corticosterone either once 25 mg/kg or 2 times daily 10 mg/kg for 3-10 days before restraint stress. After the last restraint the rats were decapitated and their trunk blood was collected for the measurement of plasma ACTH and serum corticosterone levels. Handling for 3-7 days, restraint for 3-7 days, and crowding for 7 days and a single pretreatment with corticosterone--all significantly and to a similar extent inhibited the restraint stress-induced increase in ACTH and corticosterone secretion. Chronic pretreatment with corticosterone blunted the restraint stress-induced increase in HPA axis activity. These results indicate that repeated short-lasting stress induced by handling, restraint, or crowding potently attenuates the acute restraint stress-induced stimulatory action of the HPA axis. They also indicate adaptive action of moderate stress on the HPA axis response to acute stress. The results also suggest that a short-lasting hypersecretion of corticosterone during psychological stress may induce a prolonged feedback inhibition of the HPA axis activity. The attenuation of HPA axis response by prior handling has also obvious methodological implications.     

Effect of cyclooxygenase inhibitors on the CRH-induced pituitary-adrenocortical activity during crowding stress.

The aim of the present study was to determine the effect of social stress and significance of prostaglandins (PG) generated by constitutive and inducible cyclooxygenase (COX-1 and COX-2) in the stimulation of hypothalamic-pituitary-adrenal (HPA) axis by corticotropin releasing hormone (CRH) under basal and social crowding stress conditions. The stressed rats were crowded in groups of 24 to a cage for 3 or 7 days, whereas the control animals were haused in groups of 7 to a cage of the same size. The activity of HPA axis was determined by measuring plasma ACTH and serum corticosterone levels 1 h after i.p. CRH administration. Inhibitors of COX-1, piroxicam (0.2, 2.0, and 5.0 mg/kg), and COX-2, compound NS-398 (0.2 and 2.0 mg/kg), were administered i.p. 15 min prior to CRH (0.1 microg/kg i.p.) to control or crowded rats. The obtained results indicate that social stress for 3 and 7 days markedly intensifies the stimulatory action of CRH on ACTH secretion. Neither piroxicam nor NS-398 induce any significant effect on the CRH-elicited ACTH and corticosterone secretion in non-stressed or crowded rats. Therefore, PG generated by COX-1 or COX-2 do not participate to a significant extent in the stimulation of HPA axis by CRH under either basal conditions or during crowding stress. These results also indicate that the stimulatory action of CRH on ACTH secretion is not only completely resistant to desensitization but is sensitized during social crowding stress. The results contrast with a significant involvement of PG in the vasopressin-induced stimulation of HPA response during crowding stress.     

Social stress adapts signaling pathways involved in stimulation of the hypothalamic-pituitary-adrenal axis.

In socially organized mammals the predominating stressors are not physical events but arise from the immediate social environment of the animal. Crowding typically evokes social stress reactions with prominent psychosocial components mimicking emotional state alterations. Depending on the nature, intensity and duration of the initial stimuli, they can either reduce or increase the response of the hypothalamic-pituitary adrenal (HPA) axis. In homologous desensitization only stimulation by desensitizing hormone is attenuated, in heterologous desensitization diminished responsiveness to additional activators occurs. Social stress of crowding (21 rats in a cage for 7) for 3, 7, 14 and 21 days considerably reduced the corticosterone response to intracerebroventricular (icv) administration of carbachol, a cholinergic muscarinic receptor agonist due to a homologous desensitization and down-regulation of central muscarinic receptors by an increased secretion of acetylcholine. Crowding stress significantly reduced the HPA response to icv isoprenaline, a beta-adrenergic agonist and clonidine, an alpha2-adrenergic agonist and only moderately diminished the response to phenylephrine -- an alpha1-adrenergic agonist. The stimulatory effect of dimaprit, a nonselective histamine H2-receptor agonist on HPA axis was considerably impaired in crowded rats while the response to 2-pyridylethylamine, a H1-receptor agonist was moderately affected. Social crowding stress did not substantially alter the CRH-induced ACTH and corticosterone response while it suppressed the vasopressin-induced responses. Indomethacin did not change basal plasma ACTH and corticosterone levels, indicating that prostaglandins are not involved in basal regulation of the HPA activity. Inhibition of prostaglandins synthesis by indomethacin significantly diminished the vasopressin-induced HPA response under both basal and social stress conditions, whereas it did affect the CRH-elicited HPA stimulation under both these circumstances. Social stress inhibits the nitric oxide effect on the CRH-induced ACTH response but it does not alter the AVP-induced responses. These results indicate a specific and distinct influences of social crowding stress on the neurotransmitters- neurohormones- prostaglandins- and nitric oxide-induced HPA responses.     

Vascular effects of red wine polyphenols in chronic stress-exposed Wistar-Kyoto rats

Present study investigated the effect of red wine polyphenolic compounds (Provinols) on blood pressure (BP), nitric oxide synthase (NOS) activity and vascular function in Wistar-Kyoto (WKY) rats exposed to chronic social stress produced by crowding. Adult male rats were divided into four groups: control (480 cm(2)/rat), Provinols-treated (20 mg/kg/day, 480 cm(2)/rat), crowded (200 cm(2)/rat) and crowded treated with Provinols (20 mg/kg/day, 200 cm(2)/rat) for 8 weeks. No differences in BP were observed among the groups at the end of experiment, however, reduced BP was observed in Provinols-treated rats after 3 weeks of treatment. NOS activity in the aorta was significantly elevated in crowded rats, while Provinols alone had no effect on nitric oxide (NO) production. Acetylcholine-induced relaxation of the femoral artery was significantly improved in stressed and Provinols-treated rats vs. control, without significant changes in their noradrenaline-induced vasoconstriction. Interestingly, Provinols blunted the elevation of NO production and vasorelaxation during crowding. Increased endothelium-dependent vasorelaxation and NO synthesis in crowded rats may represent the adaptation mechanisms, resulting in unaltered blood pressure in stress-exposed normotensive rats. This study further demonstrated that elevated release of NO during chronic stress may be prevented by Provinols. Thus, Provinols might maintain equilibrium between endothelium-derived vasoconstrictor and vasodilator factors in stress.     

Intracerebroventricular injection of neuronal and inducible nitric oxide synthase inhibitors attenuates fever due to LPS in rats.

Nitric oxide (NO) has been shown to be an important mediator of febrile response to lipopolisaccharide (LPS). To clarify the role of different isoforms of NO synthase (NOS) in febrile response to immune challenge, effects of selective iNOS and nNOS inhibitors on fever to LPS were examined in freely moving biotelemetered rats. Vinyl-L-NIO (N(5) - (1-Imino-3-butenyl) - ornithine (vL-NIO), a neuronal nitric oxide synthase (nNOS) inhibitor, and aminoguanidine hydrochloride, an inducible nitric oxide synthase (iNOS) inhibitor, were injected intracerebroventricularly at a dose of 10 microg/rat just before intraperitoneal injection of LPS at a dose of 50 microg/kg. Both inhibitors injected at a selected doses had no effect on normal day-time body temperature (T(b)) and normal night-time T(b). vinyl-L-NIO and aminoguanidine injected intracerebroventricularly at a dose of 10 microg/animal suppressed the LPS-induced fever in rats. The fever index calculated for rats pretreated with v-LNIO or with aminoguanidine and injected with LPS was reduced by 43% and 72%, respectively, compared to that calculated for water-pretreated and LPS-injected rats. Whereas vL-NIO partly attenuated both phases of febrile rise in T(b), administration of aminoguanidine into the brain completely prevented fever induced by LPS. These data indicate that activation of iNOS inside the brain is not only responsible for triggering but also for maintaining of LPS-induced fever in rats. It is, therefore, reasonable to hypothesize that, activation of iNOS inside the brain is more important in fever development than activation of nNOS.     

Effect of adrenergic antagonists and cyclooxygenase inhibitors on the nicotine-induced hypothalamic-pituitary-adrenocortical activity.

Nicotine is a potent stimulus for the hypothalamic-pituitary-adrenal (HPA) axis. Systemic nicotine acts via central mechanisms to stimulate by multiple pathways the release of ACTH from the anterior pituitary corticotrops and corticosterone from the adrenal cortex. Nicotine may stimulate indirectly the hypothalamic paraventricular nucleus, the site of the corticotropin-releasing hormone (CRH) neurons which activates ACTH release. In the present studies an involvement of adrenergic system and prostaglandins synthesized by constitutive cyclooxygenase (COX-1) and inducible cyclooxygenase (COX-2) in the nicotine-induced HPA response in rats was investigated. Nicotine (2.5-5 mg/kg i.p.) significantly increased plasma ACTH and corticosterone levels measured 1 hr after administration. Adrenergic receptor antagonists or COX inhibitors were injected i.p. 15 min prior to nicotine and the rats were decapitated 1 hr after the last injection. Prazosin (0.01-0.1 mg/kg), an alpha1-adrenergic antagonist, significantly decreased the nicotine-evoked ACTH and corticosterone secretion. Yohimbine (0.1-1.0 mg/kg), an alpha2-adrenergic antagonist, moderately diminished ACTH response, and propranolol (0.1-10 mg/kg), a beta-adrenergic antagonist, did not significantly alter the nicotine-induced hormones secretion. Pretreatment with piroxicam (0.2-2.0 mg/kg), a COX-1 inhibitor, considerably impaired the nicotine-induced ACTH and corticosterone secretion. Compound NS-398 (0.2-5.0 mg/kg), a selective COX-2 blocker did not markedly alter these hormones secretion, and indomethacin (2 mg/kg), a non-selective COX inhibitor significantly diminished ACTH response. These results indicate that systemic nicotine stimulates the HPA axis indirectly, and both adrenergic system and prostaglandins are significantly involved in this stimulation. Noradrenaline, stimulating postsynaptic alpha1-adrenergic receptors, and prostaglandins, synthesized by COX-1 isoenzyme, are of crucial significance in the nicotine-induced ACTH and corticosterone secretion.     

Blockade of nitric oxide formation impairs adrenergic-induced ACTH and corticosterone secretion.

It has been suggested that adrenergic agents might modulate the L-arginine-NO pathway. Sympathomimetic agonists enhance the basal release of NO, and noradrenaline increases the synthesis of nitric oxide synthase (NOS) in the medial basal hypothalamus in vitro. In the present study possible involvement of NO in central stimulation of the hypothalamic-pituitary-adrenal (HPA) axis by adrenergic agents was investigated in conscious rats. The nitric oxide synthase blocker N(omega)-nitro-L-arginine methyl ester (L-NAME 2 and 10 microg) was administered intracerebroventricularly (i.c.v.) 15 min before the adrenergic agonist given by the same route; 1 h later the rats were decapitated. Plasma levels of ACTH and corticosterone were measured. L-NAME significantly diminished the ACTH and corticosterone response to phenylephrine (30 microg), an alpha1-adrenergic receptor agonist. These hormone responses to clonidine (10 microg), an alpha2-receptor agonist, were dose-dependently suppressed or totally abolished by L-NAME. A significant rise in the ACTH and corticosterone secretion induced by isoprenaline (10 microg), a beta-adrenergic receptor agonist, was only moderately diminished by pretreatment with L-NAME. These results indicate that NOS is considerably involved in central stimulation of the HPA axis by alpha1- and alpha2-adrenergic receptor agonists, and that NO mediates the stimulatory action of these agonists on ACTH and corticosterone secretion. The stimulation induced by beta-adrenergic receptors is only moderately affected by endogenous NO.     

Lipopolysaccharide-induced increase of prostaglandin E(2) is mediated by inducible nitric oxide synthase activation of the constitutive cyclooxygenase and induction of membrane-associated prostaglandin E synthase

NO produced by the inducible NO synthase (NOS2) and prostanoids generated by the cyclooxygenase (COX) isoforms and terminal prostanoid synthases are major components of the host innate immune and inflammatory response. Evidence exists that pharmacological manipulation of one pathway could result in cross-modulation of the other, but the sense, amplitude, and relevance of these interactions are controversial, especially in vivo. Administration of 6 mg/kg LPS to rats i.p. resulted 6 h later in induction of NOS2 and the membrane-associated PGE synthase (mPGES) expression, and decreased constitutive COX (COX-1) expression. Low level inducible COX (COX-2) mRNA with absent COX-2 protein expression was observed. The NOS2 inhibitor aminoguanidine (50 and 100 mg/kg i.p.) dose dependently decreased both NO and prostanoid production. The LPS-induced increase in PGE(2) concentration was mediated by NOS2-derived NO-dependent activation of COX-1 pathway and by induction of mPGES. Despite absent COX-2 protein, SC-236, a putative COX-2-specific inhibitor, decreased mPGES RNA expression and PGE(2) concentration. Ketoprofen, a nonspecific COX inhibitor, and SC-236 had no effect on the NOS2 pathway. Our results suggest that in a model of systemic inflammation characterized by the absence of COX-2 protein expression, NOS2-derived NO activates COX-1 pathway, and inhibitors of COX isoforms have no effect on NOS2 or NOS3 (endothelial NOS) pathways. These results could explain, at least in part, the deleterious effects of NOS2 inhibitors in some experimental and clinical settings, and could imply that there is a major conceptual limitation to the use of NOS2 inhibitors during systemic inflammation.     

Effect of constitutive- and inducible-cyclooxygenase in the carbachol-induced pituitary-adrenocortical response during social stress.

Acetylcholine potently stimulates the hypothalamic-pituitary-adrenal (HPA) axis. Cholinergic receptor agonist carbachol, given intraperitoneally (i.p.) or into the lateral cerebral ventricle (i.c.v.) to non-anesthetized rats acts via multiple pathways to stimulate the HPA axis. The present study sought to determine 1) the functional selectivity of carbachol for cholinergic muscarinic and/or nicotinic receptors involved in the stimulation of HPA axis; 2) the involvement of prostaglandins (PGs) generated by constitutive and inducible cyclooxygenase (COX-1 and COX-2) in the carbachol-induced ACTH and corticosterone secretion in non-stressed rats and animals exposed to social crowding stress for 7 days (24 per a cage for 6). Carbachol was given i.c.v. or i.p. and cholinergic receptor antagonists or cyclooxygenase isoenzyme antagonists were given by the same routes 15 min earlier. One hour after the last injection trunk blood was taken for ACTH and corticosterone determinations. Atropine (0.1 microg i.c.v.), a cholinergic receptor antagonist, totally abolished the carbachol (2 microg i.c.v.)-induced ACTH and corticosterone secretion and mecamylamine (20 microg i.c.v.), a selective nicotinic receptor antagonist, did not affect this secretion. This finding indicates that carbachol functions as a selective central cholinergic muscarinic receptor agonist for the HPA axis stimulation. Crowding stress significantly diminished the carbachol (0.2 mg/kg i.p.)-induced plasma ACTH and corticosterone levels measured 1 hr after administration. Pretreatment with indomethacin (2 mg/kg i.p.), a non-selective cyclooxygenase inhibitor, significantly diminished the ACTH and corticosterone responses to carbachol (0.2 mg/kg i.p.) in control rats and moderately decreased these responses in stressed rats. Piroxicam (0.2 and 2.0 mg/kg i.p.), a COX-1 inhibitor, considerably impaired the carbachol-induced ACTH and corticosterone responses in control rats and markedly diminished these responses in stressed rats. A selective COX-2 blocker, compound NS-398 (0.2 and 2.0 mg/kg i.p.), substantially decreased the carbachol-induced hormones secretion in control rats but did not markedly alter this secretion in stressed rats. These results indicate that in the carbachol-induced HPA axis activation PGs generated by COX-1 are considerably and to a much greater extent involved than PGs generated by COX-2. Social stress markedly diminishes the mediation of PGs generated by COX-1 but PGs synthesized by COX-2 do not substantially participate in the carbachol-induced HPA response.     

Roles of nitric oxide and prostaglandins in pathogenesis of delayed colonic transit after burn injury in rats

Burn injury has been shown to impair gut transit, but the exact mechanism remains unknown. The present study investigated whether nitric oxide synthase (NOS) and cyclooxygenase (COX) mediated changes in burn-induced colonic transit. After rats underwent 30% total body surface area burn injury, they were injected with S-methylisothiourea (SMT, selective inducible NOS inhibitor), 7-nitronidazole (7-NI, selective neuronal NOS inhibitor), and nimesulide (NIM, selective COX-2 inhibitor), respectively. The protein and mRNA of NOS and COX-2 were measured by Western blot analysis and real-time RT-RCR, and localization of NOS and COX-2 protein was determined by immunohistochemistry. Our results showed that colonic transit assessed by the geometric center was delayed from 3.47+/-0.28 in controls to 2.21+/-0.18 after burn (P<0.009). SMT and NIM significantly improved colonic transit in burned rats but had no effect in sham-operated rats. 7-NI failed to modify delayed transit in burned rats but significantly delayed colonic transit in sham-operated rats. Both protein and mRNA of inducible NOS and COX-2 increased significantly but not neuronal NOS in burned rats. Inducible NOS protein expression was noted not only in epithelial cells but also in neurons of the myenteric ganglia in burned rats. These findings suggest that nitric oxide (NO) produced by neuronal NOS plays an important role in mediating colonic transit under the physiological condition. NO produced by inducible NOS and prostaglandins synthesized by COX-2 are both involved in the pathogenesis of delayed colonic transit after burn injury. Inducible NOS expression in neurons of the myenteric ganglia may contribute to dysmotility with burn injury.     

Influence of nitric oxide synthase inhibitors on the vasopressin-induced pituitary-adrenocortical activity and hypothalamic catecholamine levels.

In the present study the role of endogenous nitric oxide (NO) in the vasopressin-induced ACTH and corticosterone secretion was investigated in conscious rats. Vasopressin (AVP 5 microg/kg i.p.) considerably augmented ACTH and corticosterone secretion. L-arginine (120 and 300 mg/kg i.p.) did not significantly alter the AVP-induced secretion of those hormones. Nitric oxide synthase (NOS) blockers N(omega)-nitro-L-arginine (L-NNA) and its methyl ester (L-NAME) given i.p. 15 min before AVP markedly increased the AVP-induced ACTH secretion. L-NNA (2 mg/kg) more potently and significantly increased the AVP-induced ACTH secretion, whereas L-NAME elicited a weaker and not significant effect. Both those NOS antagonists intensified significantly and to a similar extent the AVP-induced corticosterone secretion. L-arginine (120 mg/kg i.p.) reversed the L-NNA-induced rise in the AVP-stimulated ACTH secretion and substantially diminished the accompanying corticosterone secretion. Neither vasopressin alone nor in combination with L-arginine and L-NAME evoked any significant alterations in the hypothalamic noradrenaline and dopamine levels. L-NNA (2 and 10 mg/kg i.p.) elicited a dose dependent and significant decrease in the hypothalamic noradrenaline level. The hypothalamic dopamine level was not significantly altered by any treatment. These results indicate that in conscious rats endogenous NO has an inhibitory influence on the AVP-induced increase in ACTH and corticosterone secretion. L-NNA is significantly more potent than L-NAME in increasing the AVP-induced ACTH secretion. This may be connected with a considerable increase by L-NNA of hypothalamic noradrenergic system activation which stimulates the pituitary-adrenal axis in addition to specific inhibition of NOS.     

NO mediates effects of estrogen on central regulation of blood pressure in restrained,ovariectomized rats

We tested the hypotheses that estrogen replacement in ovariectomized (OVX) rats attenuates cardiovascular responses to psychological stress and that nitric oxide (NO) in the brain mediates these effects. Female rats were OVX; one group received 17beta-estradiol (OVX-E) for 11-12 days and the other received vehicle (OVX-V). Seven days after OVX, OVX-E and OVX-V rats were chronically instrumented for arterial pressure measurements and intracerebroventricular injections. Later (4-5 days), OVX-E and OVX-V rats received intracerebroventricular injections of NG-nitro-l-arginine (88 microg/kg), an inhibitor of constitutive NO production, or vehicle. Mean arterial pressure (MAP) and heart rate responses were then measured in conscious rats exposed to two cycles of 1-h restraint/1-h rest. We show that MAP responses in restrained OVX-E rats were attenuated both during restraint and during rest. Although inhibition of NO production in the brain had no effect on MAP responses to restraint in OVX-V rats, it augmented responses in restrained OVX-E rats, especially during periods of rest, so that MAPs in restrained OVX-E and OVX-V rats were indistinguishable. Finally, NO levels in hypothalami and brain stems were elevated in restrained OVX-E, but not OVX-V, rats compared with their respective unrestrained controls. These results show that estrogen replacement in OVX rats reduces arterial pressure responses to psychological stress and that these effects are mediated, at least in part, by NO.     

Contribution of capsaicin-sensitive sensory neurons to stress-induced increases in gastric tissue levels of prostaglandins in rats

We examined whether capsaicin-sensitive sensory neurons might be involved in the increase in the gastric tissue level of prostaglandins, thereby contributing to the reduction of water immersion restraint stress (WIR)-induced gastric mucosal injury in rats. Gastric tissue levels of calcitonin gene-related peptide (CGRP), 6-keto-PGF1alpha, and PGE2 were transiently increased 30 min after WIR. These increases were significantly inhibited by subcutaneous injection of capsazepine (CPZ), a vanilloid receptor antagonist, and by functional denervation of capsaicin-sensitive sensory neurons induced by the administration of high-dose capsaicin. The administration of capsaicin (orally) and CGRP (intravenously) significantly enhanced the WIR-induced increases in the gastric tissue level of prostaglandins 30 min after WIR, whereas CGRP-(8-37), a CGRP receptor antagonist, significantly inhibited them. Pretreatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nonselective inhibitor of nitric oxide (NO) synthase (NOS), and that with indomethacin inhibited the WIR-induced increases in gastric tissue levels of prostaglandins, whereas either pretreatment with aminoguanidine (AG), a selective inhibitor of the inducible form of NOS, or that with NS-398, a selective inhibitor of cyclooxygenase (COX)-2, did not affect them. CPZ, the functional denervation of capsaicin-sensitive sensory neurons, and CGRP-(8-37) significantly increased gastric MPO activity and exacerbated the WIR-induced gastric mucosal injury in rats subjected to 4-h WIR. The administration of capsaicin and CGRP significantly increased the gastric tissue levels of prostaglandins and inhibited both the WIR-induced increases in gastric MPO activity and gastric mucosal injury 8 h after WIR. These effects induced by capsaicin and CGRP were inhibited by pretreatment with L-NAME and indomethacin but not by pretreatment with AG and NS-398. These observations strongly suggest that capsaicin-sensitive sensory neurons might release CGRP, thereby increasing the gastric tissue levels of PGI2 and PGE2 by activating COX-1 through activation of the constitutive form of NOS in rats subjected to WIR. Such activation of capsaicin-sensitive sensory neurons might contribute to the reduction of WIR-induced gastric mucosal injury mainly by inhibiting neutrophil activation.