Several experimental evidences suggest that acetylcholine (ACh) is excitatory to the hypothalamic-pituitary-adrenal axis. Since acetylcholine does not affect pituitary ACTH secretion in vitro it may stimulate the HPA axis by causing corticotropin-releasing hormone secretion. In single rat hypothalami in vitro acetylcholine stimulated IR-CRH secretion which was antagonized by simultaneous presence of atropine and hexamethonium, a muscarinic and nicotinic receptor antagonist (1).

Nicotinic receptors are widespread in the mammalian central nervous system (CNS) (2, 3) and have been implicated in various physiological and pathological conditions. A large body of evidence indicates that there is a direct participation of nicotinic acetylcholine receptors (nAChRs) in the control of neuronal function in the CNS including the regulation of HPA function (4, 5). In the median eminence corticotropin-releasing hormone axon terminals coexist with nicotinic cholinergic receptors (6), which stimulate the release of CRH and ACTH.

Nicotine can penetrate into the brain easily and it acts via a central mechanism. Peripheral (i.v.) administration of nicotinic receptor agonist which does not cross the blood-brain barrier (BBB) was ineffective in stimulating ACTH release in vivo. Similary, the antagonist hexamethonium (i.v.) a quarternary amine that does not penetrate the BBB, could not block nicotine-stimulated ACTH release, wheras the centrally active antagonist, mecamylamine inhibited the HPA response tonicotine (7, 8). Moreover, nicotine alone has no effect on cultured pituitary corticotropes. These findings demonstrate that systemic nicotine must act within the brain to stimulate the release of ACTH from the pituitary (4).

Nicotine increases the secretion of adrenaline and noradrenaline in humans and animals from adrenal chromaffin cells by direct stimulation of nACh receptors present on these cells. Nicotine given repeatedly is known to activate thyrosine hydroxylase (TH), the enzyme that catalyses the rate-limiting step in catecholamine biosynthesis, and TH gene transcription rate in rat adrenal glands (9). Stimulation by nicotine directly postganglionic sympathetic nerve endings (10) to increase catecholamine release is not a sole mechanism in inducing ACTH and corticosterone secretion. Rather action of nicotine on cholinergic receptors in the brainstem catecholaminergic neurons and the resultant release of noradrenaline in the PVN, which would lead to the release of ACTH play a significant role in the central effect of nicotine on HPA activation (11-13).

Prostaglandins are derived from arachidonic acid by phospholipase A2 and the two cyclooxygenase isoforms. Under basal conditions prostaglandins are synthesized by constitutive cyclooxygenase (COX-1) which is distributed in most cell types (14). Presence of inducible cyclooxygenase (COX-2) was found in various tissues under physiological conditions (15). In the CNS prostaglandins are involved in regulation of the HPA activity by neurotransmitters and neuropeptides (16). Prostaglandins are also involved in the nicotine-induced ACTH secretion (8).

Indomethacin significantly inhibits ACTH response to alpha1- and alpha2-adrenergic receptors stimulation by phenylephrine and clonidine, but does not substantially affect the response to ß- or ß2-adrenergic agonists, isoprenaline or clenbuterol (17). We have shown that, under basal conditions, prostaglandins synthesized by constitutive cyclooxygenase mainly mediate the stimulatory effect of alpha1- and alpha2-adrenergic agonists on the HPA activity. Prostaglandins synthesized by inducible cyclooxygenase are of lesser importance in this stimulation (18).

Since in the stimulatory effect of nicotine on HPA activity central adrenergic system and alpha-adrenergic receptors are significantly involved we investigated the involvement of adrenergic receptors and prostaglandins synthesized by COX-1 and COX-2 isoenzymes in the stimulation by nicotine of ACTH and corticosterone secretion in intact rats under basal conditions.


MATERIALS AND METHODS
Male Wistar rats weighing 190-220g were used in these studies. The animals were housed 6 per cage and were provided with unlimited access to commercial food and water. The animal room was maintained on a 12-hour light-dark cycle with lights on at 7.00 a.m. All animals were given a one-week acclimation period before the onset of experimentation. The experiments were approved by the local Ethical Committee.

Treatment

Experiments were carried out in two separate series. First series of experiments was performed to determine the effects of adrenergic receptor blockers on the nicotine-induced stimulation of the pituitary-adrenocortical axis. The following groups of animals were used in this series of the experiment: 1) control rats injected i.p. with saline (0.9% NaCl), 2) rats injected i.p. with nicotine (5 mg/kg), 3) rats injected i.p. with prazosin (0.01-0.1mg /kg), an alpha1-adrenergic receptor antagonist, 15 min before nicotine, 4) rats treated i.p. with yohimbine, (0.1-1.0 mg /kg), an alpha2-adrenergic antagonist, 15 min before nicotine and animals injected i.p. with propranolol (0.1; 2.0; 10 mg/kg, a ß-adrenergic antagonist, 15 min prior to nicotine. One hour after the last injection the rats were decapitated and their trunk blood was collected. Second series of experiments was performed to determine the effects of a constitutive cyclooxygenase (COX-1) and inducible cyclooxygenase (COX-2) blockers on the nicotine-induced ACTH and corticosterone secretion. The following groups of animals were used in this series of experiment:1) control rats injected i.p. with saline or solvent, 2) rats injected i.p. with nicotine (2.5-5 mg/kg i.p.), 3) rats injected i.p. with indomethacin (2.0 mg/kg i.p.), a non -selective COX blocker, 15 min prior to nicotine, 4) rats injected i.p. with piroxicam (0.2; 2.0; 5.0 mg/kg i.p.), a COX-1 blocker, 15 min prior to nicotine and 5) animals injected i.p. with compound NS-398 (0.2; 2.0; 5.0 mg/kg i.p.), a selective COX-2 blocker, 15 min before nicotine.

Preparation of drugs

Drugs used in this study were: prazosin hydrochloride (Pfizer), yohimbine hydrochloride, DL-propranolol hydrochloride, nicotine, piroxicam (Sigma) and NS-398 (Cayman Chemical Co). Piroxicam was prepared for injection by sonication in 1% Tween solution, NS-398 was dissolved in ethanol and remaining drugs were dissolved in saline. Solutions were prepared immediately before use. The required doses of drugs or solvents were injected i.p. in a volume of 2 ml/kg.

ACTH and corticosterone determinations

One hour after the last injection the rats were decapitated immediately after their removal from the cage and their trunk blood samples were collected on ice in plastic conical tubes containing 200 ml of a solution of 5 mg/ml EDTA and 500 TIU of aprotinin (Sigma). Control rats were decapitated concurrently with the experimantal group. Plasma was separated by centrifugation in a refrigerated centrifuge within 30 min and frozen at -20°C until the time of assay. Plasma ACTH concentrations were measured using the double antibody 125I radioimmunoassay obtained from CIS Bio International and calculated as pg/ml of plasma. The concentration of serum corticosterone was measured fluorometrically and expressed as µg per 100 ml. To avoid circadian variability, all experiments were performed between 10-11 a.m. and all decapitations between 11-12 a.m., when plasma hormones are at a relatively low levels.

Statistics

The results were calculated as a group mean ± standard error of the mean. Statistical evaluation was performed by an analysis of variance, followed by individual comparisons with Duncan`s test. The results were considered to be significantly different when p<0.05.


RESULTS
In control rats i.p. injection of saline or 1% Tween solution or diluted ethanol in a volume of 2 ml/kg did not alter the resting plasma ACTH and corticosterone levels 1 hr after administration.

Effect of adrenergic antagonists on the nicotine-induced ACTH and corticosterone secretion

The doses of adrenergic receptor antagonists used in the present experiment did not affect the basal plasma ACTH and corticosterone levels 1 hr after i.p. administration.

Pretreatment of rats with alpha1-adrenergic antagonist, prazosin (0.01 and 0.1 mg/kg i.p.) 15 min prior to nicotine (5.0 mg/kg i.p.) considerably diminished the nicotine-induced increase in ACTH and corticosterone secretion. A most potent inhibition by 48.4% of ACTH response was induced by a lower dose of prazosin (0.01 mg/kg i.p.), while similar decrease in corticosterone secretion (by 32%) was evoked by a larger dose of prazosin (0.1 mg/kg i.p.) (Fig. 1).

Fig. 1. Effect of prazosin on the nicotine-induced ACTH and corticosterone secretion. Prazosin was injected i.p. 15 min before i.p. nicotine. One hour after the last injection the rats were decapitated. ++p<0.01 vs. saline control; **p<0.01 vs. nicotinetreated group. In Fig. 1-6 values represent the mean ± SEM of 6 rats.

Pretreatment with yohimbine (0.1 and 1 mg/kg i.p.), an alpha2-adrenergic antagonist, did not markedly affect the nicotine-induced hormone secretion. This blocker slightly diminished the nicotine-induced increase in ACTH secretion (by 12%) and increased the nicotine-elicited corticosterone secretion (by 12%) (Fig. 2).

Propranolol (0.1-10 mg/kg i.p.) a ß-adrenergic receptor antagonist, given 15 min prior to nicotine did not significantly affect the nicotine-induced ACTH and corticosterone secretion. This blocker diminished the nicotine-induced ACTH response (by 3.5-20%) and moderately altered corticosterone response (by -17 to +24%) (Fig. 3).

Effect of indomethacin on nicotine-induced ACTH and corticosterone secretion

A non-selective cyclooxygenase inhibitor indomethacin (2 mg/kg i.p.) significantly decreased (by 33.1%) the secretion of ACTH elicited by nicotine (2.5 mg/kg) given 15 min later. Pretreatment with indomethacin diminished to a lesser extent (by 13.6%) the nicotine-induced corticosterone secretion (Fig. 4).

Fig. 2. Effect of yohimbine on the nicotine induced ACTH and corticosterone secretion. Yohimbine was injected i.p. 15 min before i.p. nicotine. One hour after the last injection the rats were decapitated. ++p<0.01 vs. saline-treated group.

Effect of COX-1 and COX-2 antagonists on nicotine-induced ACTH and corticosterone secretion

Piroxicam (0.2, 2 and 5 mg/kg i.p.), a COX-1 antagonist, significantly and dose-dependently diminished the ACTH and corticosterone response to nicotine (2.5 mg/kg i.p.) given 15 min later. Piroxicam gradualy decreased the nicotine induced ACTH secretion, by 25, 45 and 71%, and corticosterone secretion by 33, 58 and 63% in comparison with nicotine-induced secretion (Fig. 5). These data indicate a very potent involvement of PGs generated by COX-1 in the nicotine-induced activation of ACTH and corticosterone secretion. Under basal conditions a selective COX-2 inhibitor, compound NS-398 (0.2-5 mg/kg), injected i.p. did not significantly alter the stimulatory effect of nicotine (2.5 mg/kg i.p.), given 15 min later on ACTH and corticosterone secretion. The observed fluctuations in plasma hormone levels were neither dose-dependent nor statistically significant (Fig. 6). This finding indicates that, under basal conditions prostaglandins synthesized by COX-2 isoenzyme do not markedly participate in the nicotine-evoked ACTH and corticosterone secretion.

Fig. 3. Effect of propranolol on the nicotine-induced ACTH and corticosterone secretion. Propranolol was injected i.p. 15 min before i.p. nicotine. One hour after the last injection the rats were decapitated. ++p<0.01 vs. saline-treated group.

DISCUSSION
In the present experiment nicotine (2.5-5 mg/kg) given i.p. significantly increased plasma ACTH and corticosterone levels 1 hr after administration. It is established that both carbachol, a muscarinic receptor agonist, and nicotine, a nicotinic receptor agonist, stimulate IR-rCRH secretion from rat hypothalami in vitro, by activation of muscarinic and nicotinic receptors (1). After systemic administration nicotine easily reaches the anterior hypophysis and the median eminence which are devoid of the blood-brain barrier (BBB). Nicotine can easily penetrate the hypothalamic paraventricular nucleus and stimulate the secretion of CRH via activation of nACh receptors on CRH containing cell bodies. Also in the median eminence of the rat axon terminals CRH coexists with nicotinic receptors. It is not clear to what extent nicotine can directly stimulate CRH release via activation of nACh receptors. Nicotine is known to stimulate sympathetic neurotransmission which considerably mediates the nicotine-induced activation of brain structures involved in the regulation of HPA axis. Nicotine given i.p. in the present experiment probably produces comparable increases in plasma concentration of both adrenaline and noradrenaline (19) since systemically administered nicotine acts directly on both the adrenal medulla and postganglionic sympathetic neurons by acting on adrenergic receptors.

Fig. 4. Effect of indomethacin on the nicotine-induced ACTH and corticosterone secretion. Indomethacin was injected i.p. 15 min before i.p. nicotine. One hour after the last injection the rats were decapitated. ++p<0.01 vs. saline-treated group; **p<0.01 vs. nicotine-treated group.

Fig. 5. Effect of piroxicam on the nicotine-induced ACTH and corticosterone secretion. Piroxicam was injected i.p. 15 min before i.p. nicotine. One hour after the last injection the rats were decapitated. ++p<0.01 vs. saline-treated group; *p<0.05 and **p<0.01 vs. nicotine-treated group.

In our experiment pretreatment with prazosin (0.01 and 0.1 mg/kg i.p.), an alpha1-adrenergic receptor antagonist, considerably inhibited the nicotine-induced ACTH and corticosterone secretion, by 48.4 and 32%, respectively. Yohimbine (0.1 and 1 mg/kg i.p.), an alpha2-adrenergic receptor antagonist, slightly diminished the nicotine-evoked ACTH response (by 12%) and in the same manner it augmented corticosterone response. This finding indicates that postsynaptic alpha1-adrenergic receptors considerably mediate the nicotine-induced ACTH and corticosterone response. Propranolol (0.1-10 mg/kg i.p.), a non-selective ß-adrenergic receptor antagonist, did not markedly affect the nicotine-induced ACTH and corticosterone response in the present experiment. This adrenergic antagonist administered into the third cerebral ventricle was similarly ineffective in inducing any marked alterations in the ACTH response to nicotine (1 mg) given into the fourth cerebral ventricle while prazosin significantly reduced this response (20). Nicotine is known to facilitate noradrenaline release in different brain structures including these involved in the regulation of HPA axis. Our present results suggest that noradrenaline, which affects predominantly alpha1-adrenergic receptors, seems to be a main mediator in the nicotine-evoked ACTH and corticosterone secretion observed in our experiment. A lack of involvement of ß-adrenergic receptors, stimulated mainly by adrenaline, in the nicotine-induced activation of HPA axis suggests a minor role of adrenaline in this activation.

Fig. 6. Effect of NS-398 on the nicotine-induced ACTH and corticosterone secretion. NS-398 was injected i.p. 15 min before i.p. nicotine. One hour after the last injection the rats were decapitated. ++p<0.01 vs. saline-treated group.

Nicotine may also stimulate nicotinic acetylcholine receptors on vasopressin (AVP)- producing cell bodies and on presynaptic nerve terminals in the supraoptic nucleus and in the hypothalamic AVP containing CRH neurons (21, 22). In our present experiment both anti-CRH antibody and anti-AVP antibody (1µg/kg i.p.) considerably impaired the nicotine-induced ACTH and corticosterone secretion (data not shown).

The present results show that endogenous prostaglandins are involved in the stimulation of HPA axis by nicotine. Indomethacin (2 mg/kg) a non-selective cyclooxygenase inhibitor, given i.p. in a dose that effectively blocked the HPA response to adrenergic (23) and vasopressin stimulation (24) diminished significantly the ACTH and, to a lesser extent, corticosterone secretion induced by nicotine (2.5 mg/kg) administered by the same route 15 min later. Because of limited penetration of the blood-brain-barrier from peripheral circulation, indomethacin acts mainly directly on median eminence and anterior pituitary to inhibit PGs synthesis that mediate CRH and ACTH release. However, via fenestrated capillaries of the circumventricular organs, indomethacin may also penetrate hypothalamic PVN and inhibit nicotine-induced PGs synthesis which is known to mediate CRH release. Since in our earlier experiment i.c.v. indomethacin evoked considerable inhibition of the nicotine-induced ACTH secretion, hypothalamic site of PGs interaction with the nicotine-induced stimulation is strongly suggested (8).

The present experiments show that constitutive cyclooxygenase, that is present in different tissues under basal conditions, has a major role in PGs synthesis and mediation of the nicotine-induced ACTH secretion. Piroxicam (0.2 and 2 mg/kg i.p.), a COX-1 isoenzyme blocker, considerably impaired the nicotine-elicited ACTH and corticosterone secretion, by 48 and 32%, respectively. By contrast, compound NS-398, a selective COX-2 blocker, did not markedly alter the nicotine-induced ACTH and corticosterone secretion. This finding indicates that COX-2 isoenzyme is either absent or not active in brain structures involved in the regulation of HPA axis under basal conditions.

The results of the present study indicate that systemic nicotine stimulates the HPA axis indirectly via postsynaptic alpha1-adrenergic receptors activation by released noradrenaline and by releasing prostaglandins generated by constitutive cyclooxygenase isoenzyme.


REFERENCES

1. Calogero AE, Gallucci WT, Bernardini R, Saoutis C, Gold PW, Chrousos GP. Effect of cholinergic agonists and antagonists on rat hypothalamic corticotropin-releasing hormone secretion in vitro. Neuroendocrinology 1988; 47: 303-308.
2. Galzi J-L, Changeux J-P. Neurotransmitter receptors. VI. Neuronal nicotinic receptors: molecular organization and regulations. Neuropharmacology 1995; 34: 563-582.
3. Gotti C, Fornasari D, Clementi F. Human neuronal nicotinic receptors. Prog Neurobiol 1997; 53: 199-237.
4. Matta SG, Fu Y, Valentine JD, Sharp BM. Response of the hypothalamo-pituitary-adrenal axis to nicotine. Psychoneuroendocrinology 1998; 23: 103-113.
5. Rosecrans JA, Karin LD. Effects of nicotine on the hypothalamic-pituitary-axis (HPA) and immune function: introduction to the Sixth Nicotine Round Table Satellite, American Society of Addiction Medicine Nicotine Dependence Meeting, November15, 1997. Psychoneuroendocrinology 1998; 23: 95-102.
6. Okuda H, Shioda S, Nakai Y, Nakayama H, Okamoto M, Nakashima T. The presence of corticotropin-releasing factor-like immunoreactive synaptic vesicles in axon terminals with nicotinic acetylcholine receptor-like immunoreactivity in the median eminence of the rat. Neurosci Lett 1993; 161: 183-186.
7. Matta SG, Beyer HS, McAllen KM, Sharp BM. Nicotine elevates rat plasma ACTH by a central mechanism. J Pharmacol Exp Ther 1987; 243: 217-226.
8. Bugajski J, G¹dek-Michalska A, Borycz J, G³ód R. Effect of indomethacin on nicotine-induced ACTH and corticosterone response. J Physiol Pharmacol 1998; 49: 165-173.
9. Sterling CR, Tank AW. Adrenal tyrosine hydroxylase activity and gene expression are increased by intraventricular administration of nicotine. J Pharmacol Exp Ther 2001; 296: 15-21.
10. Haass M, Kubler W. Nicotine and sympathetic neurotransmission. Cardiovasc Drugs Ther 1996; 10: 657-665.
11. Matta SG, Foster CA, Sharp BM. Selective administration of nicotine into catecholaminergic regions of rat brainstem stimulates adrenocorticotropin secretion. Endocrinology 1993; 133: 2935-2942.
12. Matta SG, McCoy JG, Foster CA, Sharp BM. Nicotinic agonists administered into the fourth ventricle stimulate norepinephrine secretion in the hypothalamic paraventricular nucleus: an in vivo microdialysis study. Neuroendocrinology 1995; 61: 383-392.
13. Fu Y, Matta SG, Valentine JD, Sharp BM. Adrenocorticotropin response and nicotine-induced norepinephrine secretion in the rat paraventricular nucleus are mediated through brainstem receptors. Endocrinology 1997; 138: 1935-1943.
14. Vane JR, Bakhle YS, Botting RM. Cyclooxygenase 1 and 2. Annu Rev Pharmacol Toxicol 1998; 38: 97-120.
15. Katori M, Majima M. Cyclooxygenase-2: its rich diversity of roles and possible application of its selective inhibitors. Inflamm Res 2000; 49: 367-392.
16. Bugajski J. Role of prostaglandins in the stimulatory action of the hypothalamic-pituitary-adrenal axis by adrenergic and neurohormone system. J Physiol Pharmacol 1996; 47: 559-575.
17. Glod R, Gadek-Michalska A, Bugajski J. The influence of indomethacin on the ACTH secretion induced by central stimulation of adrenergic receptors. J Physiol Pharmacol 2000; 51: 347-357.
18. Bugajski J, Glod R, Gadek-Michalska A, Bugajski AJ. Involvement of constitutive (COX-1) and inducible cyclooxygenase (COX-2) in the adrenergic-induced ACTH and corticosterone secretion. J Physiol Pharmacol 2001; 52: 795-809.
19. Kiritsy JA, Mousa SA, Appel NM, VanLoon GR. Tolerance to nicotine-induced sympathoadrenal stimulation and cross-tolerance to stress: diferential central and peripheral mechanisms in rats. Neuropharmacology 1990; 29: 579-589.
20. Matta SG, Singh J, Sharp BM. Catecholamines mediate nicotine-induced adrenocorticotropin secretion via alpha-adrenergic receptors. Endocrinology 1990; 127: 1646-1655.
21. Liu X, Onaka T, Yagi K. Nicotine facilitates noradrenaline release in the rat supraoptic nucleus. Neuroreport 2001; 12: 641-643.
22. Stalke J, Hader O, Bahr V, Hensen J, Scherer G, Oelkers W. The role of vasopressin in the nicotine-induced stimulation of ACTH and cortisol in men. Clin Invest 1992; 70: 218-223.
23. Bugajski J, Gadek-Michalska A, Borycz J, Glod R, Bugajski AJ. Effect of indomethacin on the pituitary-adrenocortical response to adrenergic stimulation. Life Sci 1996; 59: 1157-1164.
24. Bugajski J, Olowska A, Gadek-Michalska, Borycz J, Glod R, Bugajski A. Effect of indomethacin on the CRH- and VP-induced pituitary-adrenocortical response during social stress. Life Sci 1996; 58: 67-72.