Effect of L-NAME, a specific nitric oxide synthase inhibitor, on corticotropin-releasing hormone-elicited ACTH and corticosterone secretion.

This study was designed to determine the role of endogenous nitric oxide (NO) in the corticotropin-releasing hormone (CRH)-induced ACTH and corticosterone secretion, as well as possible involvement of hypothalamic dopamine and noradrenaline in that secretion in conscious rats. CRH given i.p. stimulated dose-dependently the pituitary-adrenocortical activity measured 1 h later. Dexamethasone (0.2 mg/kg i.p.) injected 1 h before CRH (1 microg/kg i.p.) totally abolished the CRH-elicited ACTH and corticosterone secretion, indicating a predominantly pituitary site of CRH-evoked stimulation. L-arginine (120 mg/kg i.p.) and N(omega)-nitro-L-arginine methyl ester (L-NAME 5-10 mg/kg i.p.) did not markedly affect the basal plasma ACTH and corticosterone levels. L-NAME given 15 min before CRH markedly, but not significantly, augmented the CRH-induced ACTH response, and enhanced more potently and significantly the corticosterone response. Pretreatment with L-arginine, a substrate for NOS, slightly diminished the CRH-induced ACTH response and considerably reduced the corticosterone response. L-arginine also significantly reversed the L-NAME-evoked increase in the CRH-induced ACTH and corticosterone secretion. L-NAME did not markedly alter the CRH-induced hypothalamic dopamine and noradrenaline levels, while L-arginine significantly increased noradrenaline level. However, those alterations were not directly correlated with the observed changes in ACTH and corticosterone secretion. These results indicate that in conscious rats NO plays a marked inhibitory role in the CRH-induced ACTH secretion and inhibits more potently corticosterone secretion. Hypothalamic dopamine and noradrenaline do not seem to be directly involved in the observed alterations in ACTH and corticosterone secretion.     

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.     

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.     

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.     

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 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.     

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.     

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.     

Nicotine stimulates secretion of corticosterone via both CRH and AVP receptors

Corticosterone-releasing hormone (CRH) and arginine vasopressin (AVP) are crucial components of the hypothalamic-pituitary-adrenal axis that stimulates the release of adrenocorticotropic hormone from the pituitary and mediate the stress response. CRH binds to two subtypes of CRH receptors (CRH-R1 and CRH-R2) that are present in both central and peripheral tissues. We used the CRH-R1-specific antagonist, antalarmin (ANT), the CRH-R1 and CRH-R2 peptide antagonist, astressin (AST), and the CRH-R2-specific peptide antagonist, astressin2b (AST2b), to determine which CRH receptor is involved in the nicotine-stimulated secretion of corticosterone. Male C57BL/6 mice were administered ANT (20 mg/kg, i.p.), AST (0.3 mg/kg, i.p.), AST2b (0.3 mg/kg, i.p.) or vehicle prior to administration of nicotine (1.0 mg/kg, s.c.), CRH (10 μg/kg, s.c.), AVP (10 μg/kg, s.c.) or saline (s.c.), killed 15 min later and trunk blood collected and assayed for corticosterone plasma levels. We found that CRH enhanced corticosterone release, and this response was blocked by both AST and ANT. Nicotine also increased corticosterone secretion, but this effect persisted in the presence of either CRH antagonist. Furthermore, AST but not ANT or AST2b decreased corticosterone levels associated with stress of handling and injection. We also assessed the role of AVP V(1b) -specific receptor antagonist, SSR149415 alone and in combination with AST and AST2b. Although the AVP antagonist did not alter basal or nicotine-stimulated corticosterone secretion, it attenuated the AVP-induced stimulation of corticosterone and its combination with AST but not AST2b completely abolished nicotine-mediated stimulation of corticosterone secretion. Our results demonstrate that the nicotine-induced stimulation of the hypothalamic-pituitary-adrenal axis is mediated by both the CRH-R and the AVP V(1b) receptor and when the CRH receptor is blocked, nicotine may utilize the AVP V(1b) receptor to mediate secretion of corticosterone. These results argue in favor of the development of specific antagonists that block both AVP and CRH receptors to decrease the pleasurable component of nicotine, which may be mediated by corticosterone.     

Effect of subdiaphragmatic vagotomy and cholinergic agents in the hypothalamic-pituitary-adrenal axis activity.

In the present study, we examined whether the vagus nerve is involved in mediating the stimulation of hypothalamic-pituitary-adrenal (HPA) axis by cholinergic muscarinic and nicotinic agonists, carbachol and nicotine. The site of HPA axis muscarinic stimulation was determined using peripheral (i.p.) and intracerebroventricular (i.c.v.) administration of carbachol, atropine sulphate (AtrS) and atropine hydrobromide (AtrBr). The i.p. carbachol-(0.5 mg/kg)-induced corticosterone response was significantly reduced by i.p. pretreatment with AtrBr (0.1 mg/kg), but was not diminished by i.c.v. AtrS (0.1 mug). The increase in corticosterone secretion induced by i.c.v. carbachol (2 microg) was totally abolished by i.c.v. pretreatment with AtrS (0.1 microg) but was not altered by i.p. AtrBr. Subdiaphragmatic vagotomy performed 2 weeks earlier substantially decreased the i.p. carbachol (0.2 mg/kg)-induced ACTH response and markedly augmented ACTH and corticosterone response to a higher dose of carbachol (0.5 mg/kg) in comparison with the responses in sham operated rats. Vagotomy abolished the stimulatory effect of i.p. nicotine in a low dose (1 mg/kg) on ACTH and corticosterone secretion; the ACTH response to higher dose (2.5 mg/kg) was considerably reduced, while corticosterone response remained unaffected. These results suggest that carbachol given i.c.v. evokes considerable corticosterone response by stimulation of central cholinergic muscarinic receptors. A major part of the i.p. carbachol-induced corticosterone secretion results from peripheral cholinergic muscarinic receptor stimulation. Subdiaphragmatic vagotomy moderately intensified the carbachol-induced ACTH and corticosterone secretion. Vagotomy significantly reduced the nicotine-induced ACTH secretion, possibly by the involvement of vagal afferents. The nicotine-induced corticosterone secretion is not exclusively regulated by circulating ACTH but by various intra-adrenal regulatory components.     

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.     

Involvement of D1 dopamine receptors in the nicotine-induced noradrenaline release from hypothalamic and preoptic noradrenaline nerve terminal systems

Nicotine (4 × 2 mg/kg, i.p.) was given every 30 min for 2 h to male rats. Some rats were pretreated with the D1 dopamine (DA) receptor antagonist SCH 23390 (1 mg/kg, i.p.) or with the D2 DA receptor antagonist raclopride (1 mg/kg, i.p.), 5 min before nicotine treatment. Hypothalamic and preoptic catecholamine levels were measured by quantitative histofluorimetry in discrete DA and noradrenaline nerve terminal systems.Nicotine treatment produced a depletion of catecholamine stores in noradrenaline and DA nerve terminals of the hypothalamus, the preoptic area and the median eminence, an action which was counteracted by SCH 23390 but not by raclopride.The results indicate that hypothalamic D1 DA receptors may regulate the sensitivity of the nicotinic cholinoceptors and increase their ability to release hypothalamic noradrenaline. A possible role of D1 DA receptor antagonists to reduce the ability of nicotine treatment to produce rapid increases in LH, prolactin and corticosterone secretion and tonic arousal is implicated.     

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.     

Atrial natriuretic peptide does not affect corticotropin-releasing factor-, arginine vasopressin- and angiotensin II-induced adrenocorticotropic hormone release in vivo or in vitro

The effect of synthetic atrial natriuretic peptide (ANP) was examined on the in vivo and in vitro release of ACTH. Intravenous ANP (4 micrograms/kg body weight) administration did not affect the corticotropin releasing factor (CRF, 4 micrograms/kg body weight)-, arginine vasopressin (AVP, 2 micrograms/kg body weight)- and angiotensin II (A II, 4 micrograms/kg body weight)-induced ACTH release in unanesthetized freely moving rats. ANP did not inhibit the basal, CRF- and AVP-induced release of ACTH in pituitary cell cultures. ANP did not affect the CRF- and AVP-induced plasma corticosterone elevation, while it attenuated the AVP-induced corticosterone elevation. These results indicate that ANP does not affect the ACTH release at the pituitary level in vivo and in vitro.     

Adrenergic stimulation of adrenocortical secretion in immature fowl

In 5-6-week-old cockerels the circulating corticosterone concentration was significantly increased in birds i.m. injected 30 and 60 min previously with adrenaline (0.33 mg/kg), noradrenaline (0.33 mg/kg) or a beta-adrenergic agonist (isoprotorenol, 1 mg/kg), but was reduced in birds pretreated with an alpha-adrenergic agonist (phenylephrine, 1 mg/kg). The stimulation of corticosterone secretion induced by a 30 min period of forced exercise (0.04 km/hr; 0 degree incline) was potentiated by noradrenaline, isoprotorenol and phentolamine pretreatment. In response to exogenous adrenocorticotrophin (ACTH, 8 i.u./kg), administered i.v., the increase in the plasma corticosterone concentration was elevated above that in the controls in birds pretreated with adrenaline, noradrenaline, isoprotorenol or phentolamine (an alpha antagonist administered at 1 mg/kg). The corticosterone response to ACTH was suppressed by phenylephrine pretreatment. These results demonstrate that both basal and stimulated levels of adrenocortical activity may be subtly regulated by adrenergic mechanisms acting at a site(s) within the hypothalamo-pituitary-adrenal axis.     

Pentadecapeptide BPC 157 Reduces Bleeding and Thrombocytopenia after Amputation in Rats Treated with Heparin,Warfarin, L-NAME and L-Arginine

Background

BPC 157 is a stable gastric pentadecapeptide recently implicated with a role in hemostasis. While NO is largely implicated in hemostatic mechanisms, in tail-amputation-models under heparin- and warfarin-administration, both the NO-synthase (NOS)-blocker, L-NAME (prothrombotic) and the NOS-substrate L-arginine (antithrombotic), were little investigated. Objective. To investigate the effect of L-NAME and L-arginine on hemostatic parameters, and to reveal the effects of BPC 157 on the L-NAME- and L-arginine-induced hemostatic actions under different pathological condition: tail amputation without or with anticoagulants, heparin or warfarin.

Methods

Tail amputation, and/or i.v.-heparin (10 mg/kg), i.g.-warfarin (1.5 mg/kg/day for 3 days) were used in rats. Treatment includes BPC 157, L-NAME, L-arginine, per se and their combination.

Results

After (tail) amputation, with or without i.v.-heparin or i.g.-warfarin, BPC 157 (10 μg/kg, 10 ng/kg, i.p., i.v. (heparin), 10 μg/kg i.g. (warfarin)) always reduced bleeding time and/or haemorrhage and counteracted thrombocytopenia. As for L-NAME and/or L-arginine, we noted: L-arginine (100 mg/kg i.p.)–rats: more bleeding, less/no thrombocytopenia; L-NAME (5 mg/kg i.p.)-rats: less bleeding (amputation only), but present thrombocytopenia; L-NAME+L-arginine-rats also exhibited thrombocytopenia: L-NAME counteracted L-arginine-increased bleeding, L-arginine did not counteract L-NAME-thrombocytopenia. All animals receiving BPC 157 in addition (BPC 157μg+L-NAME; BPC 157μg+L-arginine, BPC 157μg+L-NAME+L-arginine), exhibited decreased haemorrhage and markedly counteracted thrombocytopenia.

Conclusions

L-NAME (thrombocytopenia), L-arginine (increased haemorrhage) counteraction and BPC 157 (decreased haemorrhage, counteracted thrombocytopenia) with rescue against two different anticoagulants, implicate a BPC 157 modulatory and balancing role with rescued NO-hemostatic mechanisms.     

Stimulatory effects of endogenous and exogenous nitric oxide on gastric acid secretion in anesthetized rats

We previously reported the stimulatory effect of endogenous nitric oxide (NO) on gastric acid secretion in the isolated mouse whole stomach and histamine release from gastric histamine-containing cells. In the present study, we investigated the effects of endogenous and exogenous NO on gastric acid secretion in urethane-anesthetized rats. Acid secretion was studied in gastric-cannulated rats stimulated with several secretagogues under urethane anesthesia. The acid secretory response to the muscarinic receptor agonist bethanechol (2 mg/kg, s.c.), the cholecystokinin(2) receptor agonist pentagastrin (20 microg/kg, s.c.) or the centrally acting secretagogue 2-deoxy-D-glucose (200 mg/kg, i.v.) was dose-dependently inhibited by the NO synthase inhibitor N(omega)-nitro-L-arginine (L-NNA, 10 or 50 mg/kg, i.v.). This inhibitory effect of L-NNA was reversed by a substrate of NO synthase, L-arginine (200 mg/kg, i.v.), but not by D-arginine. The histamine H(2) receptor antagonist famotidine (1 mg/kg, i.v.) completely inhibited the acid secretory response to bethanechol, pentagastrin or 2-deoxy-D-glucose, showing that all of these secretagogues induced gastric acid secretion mainly through histamine release from gastric enterochromaffin-like cells (ECL cells). On the other hand, histamine (10 mg/kg, s.c.)-induced gastric acid secretion was not inhibited by pretreatment with L-NNA. The NO donor sodium nitroprusside (0.3-3 mg/kg, i.v.) also dose-dependently induced an increase in acid secretion. The sodium nitroprusside-induced gastric acid secretion was significantly inhibited by famotidine or by the soluble guanylate cyclase inhibitor methylene blue (50 mg/kg, i.v.). These results suggest that NO is involved in the gastric acid secretion mediated by histamine release from gastric ECL cells.     

A single corticosterone pretreatment inhibits the hypothalamic-pituitary-adrenal responses to adrenergic and cholinergic stimulation.

The purpose of the present study was to determine whether an increased plasma corticosterone or dexamethasone levels induced by a single corticosterone or dexamethasone injection to conscious rats affects the hypothalamic-pituitary-adrenocortical (HPA) activity induced by adrenergic and cholinergic agonists. Male Wistar rats were pretreated subcutaneously (s.c.) with a single dose of dexamethasone (5 mg/kg) or corticosterone (25 mg/kg) 24 or 48 h before intraperitoneal (i.p.) administration of adrenergic agonists: phenylephrine, an alpha1-adrenergic receptor agonist, clenbuterol, a beta2-adrenergic agonist and noradrenaline acting predominantly on alpha1-adrenoreceptors, and cholinergic agonists: carbachol, a predominant muscarinic receptor agonist and nicotine, a nicotinic receptor agonist. Dexamethasone profoundly decreased the resting ACTH levels in control rats and given 24 h before each of the stimulatory agonist abolished the adrenergic- and cholinergic agonists-induced ACTH and corticosterone responses. Pretreatment with corticosterone of control rats did not substantially alter the resting plasma ACTH and serum corticosterone levels measured 24 and 48 h later. A single pretreatment with corticosterone abolished or powerfully inhibited, perhaps by a feedback mechanism, the ACTH and corticosterone responses induced 24 and 48 h later by all adrenergic and cholinergic agonists used in this study. These results indicate that prolonged administration of corticosterone is not necessary to induce almost complete suppression of the HPA responsiveness to adrenergic or cholinergic stimulation. Chronic treatment with corticosteroids to achieve glucocorticoid receptors desensitization does not seem to be required.     

Twenty-four-hour variation of L-arginine/nitric oxide/cyclic guanosine monophosphate pathway demonstrated by the mouse visceral pain model

The L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway is known to be involved in central and peripheral nociceptive processes. This study evaluated the rhythmic pattern of the L-arginine/NO/cGMP pathway using the mouse visceral pain model. Experiments were performed at six different times (1, 5, 9, 13, 17, and 21 h after light on) per day in male mice synchronized to a 12 h:12 h light-dark cycle. Animals were injected s.c. with saline, 2 mg/kg L-arginine (a NO precursor), 75 mg/kg L-N(G)-nitroarginine methyl ester (L-NAME, a NOS inhibitor), 40 mg/kg methylene blue (a soluble guanylyl cyclase and/or NOS inhibitor), or 0.1 mg/kg sodium nitroprusside (a nonenzymatic NO donor) 15 min before counting 2.5 mg/kg (i.p.) p-benzoquinone (PBQ)-induced abdominal constrictions for 15 min. Blood samples were collected after the test, and the nitrite concentration was determined in serum samples. L-arginine or L-NAME caused both antinociception and nociception, depending on the circadian time of their injection. The analgesic effect of methylene blue or sodium nitroprusside exhibited significant biological time-dependent differences in PBQ-induced abdominal constrictions. Serum nitrite levels also displayed a significant 24 h variation in mice injected with PBQ, L-NAME, methylene blue, or sodium nitroprusside, but not saline or L-arginine. These results suggest that components of L-arginine/NO/cGMP pathway exhibit biological time-dependent effects on visceral nociceptive process.     

Effect of nitric oxide on the somatostatinergic system in the rat exocrine pancreas

Nitric oxide (NO) and somatostatin (SS) are two important mediators of the exocrine and endocrine pancreas, exerting opposite effects on this organ. There is strong evidence suggesting an interaction between pancreatic NO and SS. The aim of this study was to determine whether L-arginine (L-Arg), the substrate for NO synthase (NOS), and Nomega-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, regulate pancreatic somatostatin-like immunoreactivity (SSLI) content and the SS mechanism of action in pancreatic acinar cell membranes. L-Arg (150 mg/kg, intraperitoneally (i.p.)), L-NAME (50 mg/kg, i.p.) or L-NAME plus L-Arg were injected twice daily at 8 h intervals for 8 days. L-Arg decreased pancreatic SSLI content as well as the number of SS receptors in pancreatic acinar cell membranes whereas L-NAME increased both parameters. The stable SS analogue SMS 201-995 induced a significantly lower inhibition of forskolin-stimulated adenylyl cyclase activity in pancreatic acinar cell membranes from L-Arg-treated rats whereas an increased inhibition was observed in pancreatic acinar membranes from L-NAME-treated rats. These results indicate that the NO system may contribute to the regulation of the pancreatic somatostatinergic system.