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Effect of acute and short-term administration of cholinomimetic drugs on corticosterone secretion in the rat
European Journal of Pharmacology, 241 (1993) 245-248
245
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53282
Effect of acute and short-term administration of cholinomimetic drugs on corticosterone secretion in the rat L o r e n a C a t t a n e o a, G i a n P i e t r o B o n d i o l o t t i a, E u g e n i o E. M u l l e r a a n d D a n i e l a C o c c h i b a Department of Pharmacology, University of Milan, Via Vanvitelli 32, Milan, Italy, and b Department Farmaco-Biologico, University ofBari, Bari, Italy Received 26 May 1993, revised MS received 25 June 1993, accepted 6 July 1993
Centrally acting cholinomimetic drugs have been proposed for the therapy of cognitive disorders in aged subjects. Among the possible adverse side effects of this class of compounds, of great relevance is the stimulatory action on the adrenocortical axis, in view of the toxicity of glucocorticoids for hippocampal neurons and the immune system. The aim of the present study was to evaluate in conscious male rats the effect of acute and short-term administration of three novel cholinomimetic drugs on the release of corticosterone. The potent agonist of muscarinic receptors RU 35963 strikingly increased corticosterone levels after acute but not after short-term (6 days) administration. Similar results were obtained after administration of the reversible inhibitor of cholinesterase, eptastigmine. In contrast to RU 35963 and eptastigmine, acute administration of a choline precursor, L-a-glycerylphosphorylcholine, only slightly affected plasma corticosterone concentrations after both acute and short-term administration. It is concluded that activation of adrenocortical function by cholinomimetic drugs is a short-lasting event which does not represent an important side effect of these compounds when given on a long-term basis. Cholinergic drugs; Adrenocortical axis; Corticosterone; Tolerance
1. Introduction
The role of the basal forebrain cholinergic system in cognitive processes is well established, particularly in relation to the functional decline that accompanies normal as well as pathological aging (Bartus et al., 1982). Pharmacological therapies have been devised to enhance central cholinergic function, including the scarcely effective choline precursors (Blusztain and Wurtman, 1983), acetylcholinesterase inhibitors and direct cholinergic agonists which consistently induce memory improvement (Heise, 1987). In rodents, acetylcholine itself and direct agonists of acetylcholine receptors activate the hypothalamo-pituitary-adrenal axis (Fisher et al., 1989; Gilad et al., 1987; Springer et al., 1981), via stimulation of corticotropinreleasing hormone release (Calogero et al., 1989). Elevated levels of glucocorticoids in plasma are thought to promote aging of the brain; in particular, the studies of Landfield et al. (1987) and Landfield and
Eldridge (1989) and Sapolsky et al. (1986) have pointed to the hippocampus as the primary site of glucocorticoid effects on the brain. Elevated glucocorticoid titers would be causally related to the death of hippocampal pyramidal neurons (Kerr et al., 1989). Although the relevance of these findings to humans is far from clear, the activation of the hypothalamopituitary-adrenal axis induced by cholinomimetic drugs could be potentially dangerous for neurons involved in the control of memory and learning (Kupfermann, 1991). The aim of this investigation was to study the effect of acute and short-term administration of three novel cholinomimetic drugs on the hypothalamo-pituitaryadrenal axis in young-adult freely moving rats. The results of these studies should be of use for further investigations in old rats.
2. Materials and methods 2.1. Animals and surgery
Correspondence to: D. Cocchi, c / o Department Pharmacology, University of Milan, Via Vanvitelli 32, 20129 Milano, Italy. Tel. 39-270146215, fax 39-2-70106838.
Barrier-derived germ-free Wistar Kyoto inbred male rats (12-14 weeks old) were obtained from Charles River Italia (Calco, Como). The rats were housed indi-
246
vidually under controlled temperature and humidity, on a 14/10 h light/dark cycle, with food and water available ad libitum (temperature 22 + 2°C, humidity 65%, light from 06.00 to 20.00 h). Chronic intracardiac venous cannulas were implanted under ketamine-xilazine (30 and 24 mg/kg, respectively) anesthesia, as described by Tannenbaum and Martin (1976). The experiments were performed 3 days after surgery, and, during the recovery period, frequent handling of the animals for daily checking of cannulas permitted habituation to the blood sampling procedure.
2.2. Experimental procedure Animals were treated once daily at 10.00 h for 6 days with a cholinomimetic drug or isovolumetric amounts of physiologic saline. On the 7th day, 24 h after the last injection, they received an acute injection of the drug or placebo and blood was sampled from the venous cannula, at time 0 min and then different post-injection times (see figures). The experiments were performed in the morning to avoid the interference effected by the spontaneous circadian corticosterone increase occurring around 6 p.m. Blood samples were immediately centrifuged and plasma was separated and stored until radioimmunoassay determination of corticosterone with a commercial kit (ICN Biomedica, Costa Mesa, CA, USA) or determination of cholinesterase activity by a colorimetric method (Ellman et al., 1961; Boehringer-Mannheim, Mannheim, Germany). Intraand inter-assay coefficients of variation of the corticosterone assay were less than 10%.
3. Results
Figure 1 shows the effect of acute and short-term administration of RU 35963 on plasma corticosterone concentrations. Acute administration of the drug induced a marked increase in plasma corticosterone concentrations between 30 and 90 min post-treatment with peak levels occurring at 60 min. Administration of RU 35963 after a 6-day treatment induced only a slight increase of plasma corticosterone concentrations, not significantly different from those present in placebo-injected controls. Acute administration of eptastigmine tartrate induced a clear-cut increase in plasma corticosterone concentrations, although the increase occurred later than that elicited by RU 35963 and the peak response was present at 180 min. The stimulatory effect of eptastigmine tartrate on hypothalamo-pituitary-adrenal axis function was completely abolished by the previous short-term treatment with the drug (fig. 2). Measurement of plasma cholinesterase activity 180 min after drug administration revealed a significant inhibition of enzyme activity by eptastigmine tartrate, either after acute and, more consistently, after shortterm administration (139.5 _+5.5 vs. 102.6 _+3.8 and 77.2_+ 6.2 U / l for controls, acute and short-term eptastigmine tartrate, respectively, P < 0.01; P < 0.01) (data not reported). In contrast to RU 35963 and eptastigmine tartrate, acute administration of L-a-glycerylphosphorylcholine only slightly affected plasma corticosterone concentrations in both acute and short-term conditions (fig. 3). No statistical difference could be detected between
2.3. Drugs The following drugs were used: 1-alkyl-l,2,5,6-tetrahydropyridine-3-carboxaldehyde-O-methyloxime hydrochloride (RU 35963, Roussel-UCLAF, Romainville, France), a direct muscarinic receptor agonist and eptastigmine tartrate (Mediolanum, Milan, Italy), a reversible inhibitor of cholinesterase, were administered s.c. at a dose of 0.1 and 2 mg/kg, respectively. The choline precursor L-a-glycerylphosphorylcholine (Sandoz, Milan, Italy) was administered i.p. at a dose of 300 mg/kg. The doses used were chosen on the basis of the results of preliminary experiments; they represent the highest doses which can be administered to rats without eliciting behavioral side effects.
2.4. Statistical analysis The results are expressed in n g / m l as m e a n s _ S.E.M. The statistical significance of differences among groups was calculated by Dunnett's t-test for multiple comparisons preceded by an ANOVA.
700
E ~00 v
~0 400 (D Q
o 100
0
30
60
90
120
150
180
Time (min) Fig. 1. Effect of acute and short-term administration of R U 35963 (0.1 m g / k g s.c.) on plasma corticosterone levels in freely moving male rats. Each point is the mean +S.E.M. of 7-8 determinations. * P < 0.05 and * * P < 0.01 vs. controls at each time interval ( + controls; ~ short-term RU 35963; • acute R U 35963).
247
700 600
E 50o iv
~t~t
400
C
O 300 O 0 200 0 100 (.)
0
60
90
120
150
180
210
TIME (min) Fig. 2. Effect of acute and short-term administration of eptastigmine (eptastigmine tartrate, 2 m g / k g s.c.) on plasma corticosterone levels in freely moving male rats. Each point is the mean 5: S.E.M. of 7-8 determinations. * P < 0.05 and * * P < 0.01 vs. controls at each time interval ( + controls; ~ short-term eptastigmine; • acute eptastigmine).
corticosterone values in L-a-glycerylphosphorylcholineand placebo-injected rats. None of the drugs, at the doses administered, elicited significant behavioral effects.
4. Discussion
Considerable effort is currently being put into developing new and effective drugs for the therapy of cognitive disorders. RU 35963, as well as other drugs of the family of 1-alkyl-l,2,5,6-tetrahydropyridine-3-carbox-
7°°I __ 600 /
/ ~
200 /
,01/ 0
15
30
45
60
90
120
150
180
TIME (rain) Fig. 3. Effect of acute and short-term administration of L-aglycerylphosphorylcholine (300 m g / k g i.p.) on plasma corticosterone levels in freely moving male rats. Each point is the mean 4- S.E.M. of 7-8 determinations ( • controls; + short-term L-a-glycerylphosphorylcholine; ~ acute L-a-glycerylphosphorylcholine).
aldehyde-o-alkyloximes, has shown stronger antiamnesic properties, higher potency, and longer duration of action than the parcnt molecule arecoline (Toja et al., 1991). Eptastigmine is a cholinesterase inhibitor with a potency similar to that of physostigmine but a more favorable pharmacodynamic profile, i.e. longer half-life, greater bioavailability, attainment of higher brain concentrations (Brufani et al., 1986). Finally, L-a-glycerylphosphorylcholine, a choline precursor, prevents scopolamine-induced amnesia with an effect which is well correlated with its neurochemical properties. The drug, in fact, has an initial short-lasting stimulatory effect on aeetylcholine release paralleled by a significant and long-lasting increase in acetylcholine synthesis (Sigala et al., 1992). Much data in the literature suggests that acetylcholine has a stimulatory role on hypothalamo-pituitary-adrenal axis function, and in vitro studies have clearly shown this effect to be mediated by functional activation of corticotropin-releasing hormone-producing neurons in the rat hypothalamus (Calogero et al., 1989) and involving both muscarinic and nicotinic receptors (Calogero et al., 1988). Consistent with these data, in our study, acute administration of RU 35963 and eptastigmine tartrate induced a striking increase in plasma corticosterone levels whereas acute administration of L-a-glycerylphosphorylcholine was ineffective. In view of the potent stimulatory effect of L-aglycerylphosphorylcholine, administered at the same dose and route of administration, on in vivo and in vitro hippocampal and striatal acetylcholine synthesis and release (Sigala et al., 1992), the last finding is difficult to explain. However, no data are yet available on the ability of this drug to increase cholinergic neurotransmission at the hypothalamic level, where cholinergic nerve terminals synapse with corticotropin-releasing hormone-producing neurons (Muller and Nisticb, 1989). This topic is worthy of further investigation. According to a hypothesis promulgated by Sapolsky et al. (1986), increased circulating corticosterone levels threaten the viability of brain cells, rendering them more vulnerable to noxious stimuli (Aus der Muhlen and Ockenfels, 1969; Sapolsky et al., 1986). In addition, elevated corticosteroid levels would adversely affect the function of the immune system (Berczi, 1986), particularly in aged subjects where this system is already vulnerable (Miller, 1991). Therefore, were the stimulatory effect of RU 35963 and eptastigmine tartrate on hypothalamo-pituitary-adrenal axis function to be persistent, it would represent an undesirable side effect, opposing the beneficial effects of the drugs on cognitive function. The stimulatory role of acetylcholine on hypothalamo-pituitary-adrenal axis function is well established in rodents (see above), but is less clear in humans. According to Ghigo et al. (1990), acute administration
248
of pyridostigmine, a cholinesterase inhibitor, does not affect cortisol secretion in normal young volunteers. However, of interest for our discussion is the finding that physostigmine, while being ineffective in stimulating cortisol secretion in young subjects, elicits a significant increase in hormone levels in aged subjects (Raskind et al., 1990). The current study clearly shows that two cholinomimetic drugs, RU 35963 and eptastigrnine tartrate, lost their ability to stimulate corticosterone secretion after short-term treatment, a phenomenon that is probably related to desensitization of hypothalamic acetylcholine receptors impinging on corticotropin-releasing factor-producing neurons (Siegelbaum and Koester, 1991). It is not likely that desensitization occurred at a site upstream of the receptor; experiments performed with eptastigmine showed that the inhibitory action of the drug on cholinesterase activity was increased at a time when the stimulation of corticosterone secretion was no longer apparent. It would seem, therefore, that activation of hypothalamo-pituitary-adrenal function by these compounds is a short-lasting event which does not represent an adverse side effect on a long-term basis. Extension of these acute and short-term experiments to old rats is warranted.
Acknowledgements We thank Mediolanum Farmaceutici (Milan) and Sandoz Prodotti Farmaceutici S.p.A. (Milan) for providing eptastigmine and L-aglycerylphosphorylcholine and for supporting part of the study. We thank also Roussel Uclaf (Romainville) for providing RU 35963.
References Aus Der Muhlen, K.A. and H. Ockenfels, 1969, Morphologische Ver~inderungen in Diencephalon and Telencefalon: St6rungen des Regelkreises Adenohypophyse-nebennierenrinde, Z. Zellforsch. 93, 126. Bartus, R.T., R.L. Dean, III, B. Beer and A.S. Lippa, 1982, The cholinergic hypothesis of geriatric memory dysfunction, Science 217, 408. Berczi, I., 1986, The influence of pituitary-adrenal axis on the immune system, in: Pituitary Function and Immunity, ed. I. Berczi (CRC Press, Boca Raton, FL) p. 49. Blusztain, J.K. and R.J. Wurtman, 1983, Choline and cholinergic neurons, Science 221, 614. Brufani, M., M. Marta and M. Pomponi, 1986, Anticholinesterase activity of a new carbamate, heptylphysostigmine, in view of its use in patients with Alzheimer-type dementia, Eur. J. Biochem. 157, 115. Calogero, A.E., W.T. Gallucci, R. Bernardini, C. Saoutis, P.W. Gold and G.P. Chrousos, 1988, Effect of cholinergic agonists and
antagonists on rat hypothalamic corticotropin-releasing hormone secretion in vitro, Neuroendocrinology 47, 303. Calogero, A.E., T.C. Kamilaris, M.T. Gomez, E.O. Johnson, M.E. Tartaglia, p.w. Gold and G.P. Chrousos, 1989, The muscarinic cholinergic agonist arecoline stimulates the rat hypothalamicpituitary-adrenal axis through a centrally-mediated corticotropinreleasing hormone-dependent mechanism, Endocrinology 125, 2445. Ellman, G.L., K.D. Courtney, V. Andres, Jr. and R.M. Feather-Stone, 1961, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7, 88. Fischer, W., F.H. Gage and A. Bjorklund, 1989, Degenerative changes in forebrain cholinergic nuclei correlate with cognitive impairments in aged rats, Eur. J. Neurosci. 1, 34. Ghigo, E., E. Arvat, M.R. Valetto, E. Mazza, G. Dantona, G. Rizzi and F. Camanni, 1990, Further evidence against a stimulatory role of the cholinergic system on the hypothalamic pituitary adrenal axis in man, Neuroendocrinol. Lett. 12, No. 2. Gilad, G.M., J.M. Rabey, Y. Tizabey and V.H. Gilad, 1987, Age-dependent loss and compensatory changes of septo-hippocampal cholinergic neurons in two rat strains differing in longevity and response to stress, Brain Res. 436, 311. Heise, G.A., 1987, Facilitation of memory and cognition by drugs, Trends Pharmacol. Sci. 8, 65. Kerr, D.S., L.W. Campbell, S.Y. Hao and P.Y. Landfield, 1989, Corticosteroid modulation of hippocampal potentials: increased effect of aging, Science 245, 1505. Kupfermann, I., 1991, Learning and memory, in: Principles of Neural Sciences, eds. E.R. Kandel, J.I. Schwartz and T.M. Jessell (Elsevier, New York) p. 997. Landfield, P.W., 1987, Modulation of brain aging correlates by long-term alteration of adrenal steroids and neuraUy-active peptides, Prog. Brain Res. 72, 279. Landfield P.W. and J.C. Eldridge, 1989, Increased affinity of type II corticosteroid binding in aged rat hippocampus, Exp. Neurol. 106, 110. Miller, R.A., 1991, Aging and immune function, Int. Rev. Cytol. 124, 187. Muller, E.E. and G. Nisticb, 1989, Brain Messengers and the Pituitary (Academic Press, San Diego, CA). Raskind, M.A.E., R. Peskind, C. Veith, W. Wilkinson, D. Federighi and D. Dorsa, 1990, Differential effects of aging on neuroendocrine responses to physostigmine in normal men, J. Clin. Endocrinol. Metab. 70, 1420. Sapolsky, R.M., L.C. Krey and B.S. McEwen, 1986, The neuroendocrinology of stress and aging: the glucocorticoid cascade hypothesis, Endocr. Rev. 7, 284. Siegelbaum, S. and A.J. Koester, 1991, Ion channels, in: Principles of Neural Sciences, eds. E.R. Kandel, J.I. Schwartz and T.M. Jessell (Elsevier, New York) p. 66. Sigala, S., A. Imperato, P. Rizzonelli, P. Casolini, C. Missale and P.F. Spano, 1992, L-a-Glycerylphosphorylcholine antagonizes scopolamine-induced amnesia and enhances hippocampal cholinergic transmission in the rat, Eur. J. Pharmacol. 211,351. Springer, J.E., M.W. Tayren and R. Loy, 1987, Regional analysis of age-related changes in the cholinergic system of the hippocampal formation and basal forebrain of the rat, Brain Res. 407, 180. Tannenbaum, G.S. and J.B. Martin, 1976, Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat, Endocrinology 98, 562. Toja, E., C. Bonetti, A. Butti, P. Hunt, M. Fortin, F. Barzaghi, M.L. Formento, A. Maggioni, A. Nencioni and G. Galliani, 1991, a-Alkyl- 1,2,5,6-tetrahydropyridine-3-carboxaldehyde-O-alkyloximes: a new class of potent orally active muscarinic agonists related to arecoline, Eur. J. Med. Chem. 26, 853.
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© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53282
Effect of acute and short-term administration of cholinomimetic drugs on corticosterone secretion in the rat L o r e n a C a t t a n e o a, G i a n P i e t r o B o n d i o l o t t i a, E u g e n i o E. M u l l e r a a n d D a n i e l a C o c c h i b a Department of Pharmacology, University of Milan, Via Vanvitelli 32, Milan, Italy, and b Department Farmaco-Biologico, University ofBari, Bari, Italy Received 26 May 1993, revised MS received 25 June 1993, accepted 6 July 1993
Centrally acting cholinomimetic drugs have been proposed for the therapy of cognitive disorders in aged subjects. Among the possible adverse side effects of this class of compounds, of great relevance is the stimulatory action on the adrenocortical axis, in view of the toxicity of glucocorticoids for hippocampal neurons and the immune system. The aim of the present study was to evaluate in conscious male rats the effect of acute and short-term administration of three novel cholinomimetic drugs on the release of corticosterone. The potent agonist of muscarinic receptors RU 35963 strikingly increased corticosterone levels after acute but not after short-term (6 days) administration. Similar results were obtained after administration of the reversible inhibitor of cholinesterase, eptastigmine. In contrast to RU 35963 and eptastigmine, acute administration of a choline precursor, L-a-glycerylphosphorylcholine, only slightly affected plasma corticosterone concentrations after both acute and short-term administration. It is concluded that activation of adrenocortical function by cholinomimetic drugs is a short-lasting event which does not represent an important side effect of these compounds when given on a long-term basis. Cholinergic drugs; Adrenocortical axis; Corticosterone; Tolerance
1. Introduction
The role of the basal forebrain cholinergic system in cognitive processes is well established, particularly in relation to the functional decline that accompanies normal as well as pathological aging (Bartus et al., 1982). Pharmacological therapies have been devised to enhance central cholinergic function, including the scarcely effective choline precursors (Blusztain and Wurtman, 1983), acetylcholinesterase inhibitors and direct cholinergic agonists which consistently induce memory improvement (Heise, 1987). In rodents, acetylcholine itself and direct agonists of acetylcholine receptors activate the hypothalamo-pituitary-adrenal axis (Fisher et al., 1989; Gilad et al., 1987; Springer et al., 1981), via stimulation of corticotropinreleasing hormone release (Calogero et al., 1989). Elevated levels of glucocorticoids in plasma are thought to promote aging of the brain; in particular, the studies of Landfield et al. (1987) and Landfield and
Eldridge (1989) and Sapolsky et al. (1986) have pointed to the hippocampus as the primary site of glucocorticoid effects on the brain. Elevated glucocorticoid titers would be causally related to the death of hippocampal pyramidal neurons (Kerr et al., 1989). Although the relevance of these findings to humans is far from clear, the activation of the hypothalamopituitary-adrenal axis induced by cholinomimetic drugs could be potentially dangerous for neurons involved in the control of memory and learning (Kupfermann, 1991). The aim of this investigation was to study the effect of acute and short-term administration of three novel cholinomimetic drugs on the hypothalamo-pituitaryadrenal axis in young-adult freely moving rats. The results of these studies should be of use for further investigations in old rats.
2. Materials and methods 2.1. Animals and surgery
Correspondence to: D. Cocchi, c / o Department Pharmacology, University of Milan, Via Vanvitelli 32, 20129 Milano, Italy. Tel. 39-270146215, fax 39-2-70106838.
Barrier-derived germ-free Wistar Kyoto inbred male rats (12-14 weeks old) were obtained from Charles River Italia (Calco, Como). The rats were housed indi-
246
vidually under controlled temperature and humidity, on a 14/10 h light/dark cycle, with food and water available ad libitum (temperature 22 + 2°C, humidity 65%, light from 06.00 to 20.00 h). Chronic intracardiac venous cannulas were implanted under ketamine-xilazine (30 and 24 mg/kg, respectively) anesthesia, as described by Tannenbaum and Martin (1976). The experiments were performed 3 days after surgery, and, during the recovery period, frequent handling of the animals for daily checking of cannulas permitted habituation to the blood sampling procedure.
2.2. Experimental procedure Animals were treated once daily at 10.00 h for 6 days with a cholinomimetic drug or isovolumetric amounts of physiologic saline. On the 7th day, 24 h after the last injection, they received an acute injection of the drug or placebo and blood was sampled from the venous cannula, at time 0 min and then different post-injection times (see figures). The experiments were performed in the morning to avoid the interference effected by the spontaneous circadian corticosterone increase occurring around 6 p.m. Blood samples were immediately centrifuged and plasma was separated and stored until radioimmunoassay determination of corticosterone with a commercial kit (ICN Biomedica, Costa Mesa, CA, USA) or determination of cholinesterase activity by a colorimetric method (Ellman et al., 1961; Boehringer-Mannheim, Mannheim, Germany). Intraand inter-assay coefficients of variation of the corticosterone assay were less than 10%.
3. Results
Figure 1 shows the effect of acute and short-term administration of RU 35963 on plasma corticosterone concentrations. Acute administration of the drug induced a marked increase in plasma corticosterone concentrations between 30 and 90 min post-treatment with peak levels occurring at 60 min. Administration of RU 35963 after a 6-day treatment induced only a slight increase of plasma corticosterone concentrations, not significantly different from those present in placebo-injected controls. Acute administration of eptastigmine tartrate induced a clear-cut increase in plasma corticosterone concentrations, although the increase occurred later than that elicited by RU 35963 and the peak response was present at 180 min. The stimulatory effect of eptastigmine tartrate on hypothalamo-pituitary-adrenal axis function was completely abolished by the previous short-term treatment with the drug (fig. 2). Measurement of plasma cholinesterase activity 180 min after drug administration revealed a significant inhibition of enzyme activity by eptastigmine tartrate, either after acute and, more consistently, after shortterm administration (139.5 _+5.5 vs. 102.6 _+3.8 and 77.2_+ 6.2 U / l for controls, acute and short-term eptastigmine tartrate, respectively, P < 0.01; P < 0.01) (data not reported). In contrast to RU 35963 and eptastigmine tartrate, acute administration of L-a-glycerylphosphorylcholine only slightly affected plasma corticosterone concentrations in both acute and short-term conditions (fig. 3). No statistical difference could be detected between
2.3. Drugs The following drugs were used: 1-alkyl-l,2,5,6-tetrahydropyridine-3-carboxaldehyde-O-methyloxime hydrochloride (RU 35963, Roussel-UCLAF, Romainville, France), a direct muscarinic receptor agonist and eptastigmine tartrate (Mediolanum, Milan, Italy), a reversible inhibitor of cholinesterase, were administered s.c. at a dose of 0.1 and 2 mg/kg, respectively. The choline precursor L-a-glycerylphosphorylcholine (Sandoz, Milan, Italy) was administered i.p. at a dose of 300 mg/kg. The doses used were chosen on the basis of the results of preliminary experiments; they represent the highest doses which can be administered to rats without eliciting behavioral side effects.
2.4. Statistical analysis The results are expressed in n g / m l as m e a n s _ S.E.M. The statistical significance of differences among groups was calculated by Dunnett's t-test for multiple comparisons preceded by an ANOVA.
700
E ~00 v
~0 400 (D Q
o 100
0
30
60
90
120
150
180
Time (min) Fig. 1. Effect of acute and short-term administration of R U 35963 (0.1 m g / k g s.c.) on plasma corticosterone levels in freely moving male rats. Each point is the mean +S.E.M. of 7-8 determinations. * P < 0.05 and * * P < 0.01 vs. controls at each time interval ( + controls; ~ short-term RU 35963; • acute R U 35963).
247
700 600
E 50o iv
~t~t
400
C
O 300 O 0 200 0 100 (.)
0
60
90
120
150
180
210
TIME (min) Fig. 2. Effect of acute and short-term administration of eptastigmine (eptastigmine tartrate, 2 m g / k g s.c.) on plasma corticosterone levels in freely moving male rats. Each point is the mean 5: S.E.M. of 7-8 determinations. * P < 0.05 and * * P < 0.01 vs. controls at each time interval ( + controls; ~ short-term eptastigmine; • acute eptastigmine).
corticosterone values in L-a-glycerylphosphorylcholineand placebo-injected rats. None of the drugs, at the doses administered, elicited significant behavioral effects.
4. Discussion
Considerable effort is currently being put into developing new and effective drugs for the therapy of cognitive disorders. RU 35963, as well as other drugs of the family of 1-alkyl-l,2,5,6-tetrahydropyridine-3-carbox-
7°°I __ 600 /
/ ~
200 /
,01/ 0
15
30
45
60
90
120
150
180
TIME (rain) Fig. 3. Effect of acute and short-term administration of L-aglycerylphosphorylcholine (300 m g / k g i.p.) on plasma corticosterone levels in freely moving male rats. Each point is the mean 4- S.E.M. of 7-8 determinations ( • controls; + short-term L-a-glycerylphosphorylcholine; ~ acute L-a-glycerylphosphorylcholine).
aldehyde-o-alkyloximes, has shown stronger antiamnesic properties, higher potency, and longer duration of action than the parcnt molecule arecoline (Toja et al., 1991). Eptastigmine is a cholinesterase inhibitor with a potency similar to that of physostigmine but a more favorable pharmacodynamic profile, i.e. longer half-life, greater bioavailability, attainment of higher brain concentrations (Brufani et al., 1986). Finally, L-a-glycerylphosphorylcholine, a choline precursor, prevents scopolamine-induced amnesia with an effect which is well correlated with its neurochemical properties. The drug, in fact, has an initial short-lasting stimulatory effect on aeetylcholine release paralleled by a significant and long-lasting increase in acetylcholine synthesis (Sigala et al., 1992). Much data in the literature suggests that acetylcholine has a stimulatory role on hypothalamo-pituitary-adrenal axis function, and in vitro studies have clearly shown this effect to be mediated by functional activation of corticotropin-releasing hormone-producing neurons in the rat hypothalamus (Calogero et al., 1989) and involving both muscarinic and nicotinic receptors (Calogero et al., 1988). Consistent with these data, in our study, acute administration of RU 35963 and eptastigmine tartrate induced a striking increase in plasma corticosterone levels whereas acute administration of L-a-glycerylphosphorylcholine was ineffective. In view of the potent stimulatory effect of L-aglycerylphosphorylcholine, administered at the same dose and route of administration, on in vivo and in vitro hippocampal and striatal acetylcholine synthesis and release (Sigala et al., 1992), the last finding is difficult to explain. However, no data are yet available on the ability of this drug to increase cholinergic neurotransmission at the hypothalamic level, where cholinergic nerve terminals synapse with corticotropin-releasing hormone-producing neurons (Muller and Nisticb, 1989). This topic is worthy of further investigation. According to a hypothesis promulgated by Sapolsky et al. (1986), increased circulating corticosterone levels threaten the viability of brain cells, rendering them more vulnerable to noxious stimuli (Aus der Muhlen and Ockenfels, 1969; Sapolsky et al., 1986). In addition, elevated corticosteroid levels would adversely affect the function of the immune system (Berczi, 1986), particularly in aged subjects where this system is already vulnerable (Miller, 1991). Therefore, were the stimulatory effect of RU 35963 and eptastigmine tartrate on hypothalamo-pituitary-adrenal axis function to be persistent, it would represent an undesirable side effect, opposing the beneficial effects of the drugs on cognitive function. The stimulatory role of acetylcholine on hypothalamo-pituitary-adrenal axis function is well established in rodents (see above), but is less clear in humans. According to Ghigo et al. (1990), acute administration
248
of pyridostigmine, a cholinesterase inhibitor, does not affect cortisol secretion in normal young volunteers. However, of interest for our discussion is the finding that physostigmine, while being ineffective in stimulating cortisol secretion in young subjects, elicits a significant increase in hormone levels in aged subjects (Raskind et al., 1990). The current study clearly shows that two cholinomimetic drugs, RU 35963 and eptastigrnine tartrate, lost their ability to stimulate corticosterone secretion after short-term treatment, a phenomenon that is probably related to desensitization of hypothalamic acetylcholine receptors impinging on corticotropin-releasing factor-producing neurons (Siegelbaum and Koester, 1991). It is not likely that desensitization occurred at a site upstream of the receptor; experiments performed with eptastigmine showed that the inhibitory action of the drug on cholinesterase activity was increased at a time when the stimulation of corticosterone secretion was no longer apparent. It would seem, therefore, that activation of hypothalamo-pituitary-adrenal function by these compounds is a short-lasting event which does not represent an adverse side effect on a long-term basis. Extension of these acute and short-term experiments to old rats is warranted.
Acknowledgements We thank Mediolanum Farmaceutici (Milan) and Sandoz Prodotti Farmaceutici S.p.A. (Milan) for providing eptastigmine and L-aglycerylphosphorylcholine and for supporting part of the study. We thank also Roussel Uclaf (Romainville) for providing RU 35963.
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