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Effects of cotinine at cholinergic nicotinic receptors of the sympathetic superior cervical ganglion of the mouse
Toxicology 144 (2000) 99 – 105 www.elsevier.com/locate/toxicol
Effects of cotinine at cholinergic nicotinic receptors of the sympathetic superior cervical ganglion of the mouse K.-C. Schroff a,*, P. Lovich a, O. Schmitz b, S. Aschhoff a, E. Richter a, J. Remien a b
a Walther Straub-Institute of Pharmacology and Toxicology, Nußbaumstr. 26, 80336 Munich, Germany DKFZ Heidelberg, Department of Molecular Toxicology, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
Abstract Nicotine, the principal alkaloid in tobacco, is generally accepted to be responsible for most neuropharmacological effects due to tobacco use. Little is known about the action of cotinine, the major metabolite from nicotine, at neuronal structures. To evaluate the mode of action of cotinine at neuronal receptors, its effect on the surface compound potential of the sympathetic superior cervical ganglion (SCG) of the mouse was studied. The modulation of nicotine induced surface potentials by cotinine was tested. It was found that 2-min applications of cotinine (0.1 – 30 mmol/l) induced concentration dependent depolarizations at the SCG (EC50 = 1.7 mmol/l) which were followed by hyperpolarizations and weak afterdepolarizations. The intrinsic activity of cotinine compares to that induced by much lower concentrations of nicotine (EC50 =21 mmol/l). These cotinine effects may be mediated at least in part by nicotine impurities which were found by capillary electrophoresis to be 0.1 and 0.8% in different batches of cotinine. Continuous application of 300 mmol/l cotinine shifted the concentration-response curve of nicotine to the right and reduced (IC50 =302 mmol/l) the effects of submaximal nicotine concentrations (30 mmol/l). This effect could not be mimicked by continuous application of a nicotine concentration (0.3 mmol/l) equivalent to the lower impurity in cotinine. Therefore, the antagonistic action of cotinine at peripheral neuronal nicotinic receptors is at least in part independent of nicotine impurity. The observed antagonistic effect of cotinine at nicotinic receptors likely contributes to the neuropharmacological effects of smoking. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Sympathetic ganglion; Mouse; Cotinine; Nicotinic receptors
1. Introduction Cotinine (1-methyl-5-[3-pyridyl]-2-pyrrolidinone) is the major metabolite of the tobacco * Corresponding author. Tel.: +49-89-5160-7258; fax: + 49-89-5160-7207. E-mail address: [email protected] (K.-C. Schroff)
alkaloid nicotine (Gorrod and Jenner, 1975; Benowitz and Jacob, 1994). In humans 86% of systemically absorbed nicotine is metabolized to cotinine (Benowitz, 1983). Since cotinine is present in much higher concentrations in smokers and with a half-time about tenfold longer than nicotine (Langone et al., 1973), cotinine concentrations in blood and urine are widely used as a
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marker of tobacco consumption and abstinence (Zeidenberg et al., 1977; Wilcox et al., 1979). Some studies have shown pharmacological activity of cotinine: cotinine relaxes vascular smooth muscle, dilates blood vessels in vitro (Kim et al., 1968) and decreases blood pressure (Borzelleca et al., 1982) in anesthetized dogs. Cotinine is able to pass the blood-brain barrier (Gorrod and Wahren, 1993; Benowitz et al., 1994; Crooks et al., 1997) and it has been detected in brain of rat, cat and mouse after peripheral nicotine administration (Applegren et al., 1962; Schmiterlo¨w et al., 1967; Stalhandske, 1970; Petersen et al., 1984; Deutsch et al., 1992; Crooks et al., 1995). In brain cotinine underlies no significant biotransformation (Crooks and Dwoskin, 1997). Also neuropharmacological activities of cotinine have been shown. Cotinine increases serotonin turnover in rat cortex (Fuxe et al., 1979), induces electroencephalographic activation in conscious cats (Yamamoto and Domino, 1965) and has, probably as a result of activation of dopamine pathways in brain, intrinsic reinforcing properties (Fibiger and Phillips, 1987; Corrigal et al., 1992, 1994; Balfour and Benwell, 1993). A recent study has shown dopamine release from rat striatal slices triggered by stimulation of nicotinic receptors (Dwoskin et al., 1999). Acute application of cotinine induces minor cognitive impairment in man, tested with a verbal recall task (Herzig et al., 1998). Nothing is known about cotinine actions at the peripheral sympathetic nerve system. We therefore examined the effect of cotinine at nicotinic receptors of the sympathetic superior cervical ganglion of the mouse in vitro.
2. Materials and methods
2.1. Substances ( − )-Cotinine, (− )-nicotine hydrogene tartrate salt and hexamethonium were purchased from Sigma (Taufkirchen, Germany), atropine sulphate and all other chemicals used for Krebs buffer and capillary electrophoresis were obtained from Merck (Darmstadt, Germany).
2.2. Animals Ganglia were excised from mice of the outbred strain NMRI (27–52 g, Charles River, Sulzfeld, Germany). Mice were kept in macrolene cages, type III, under standard animal laboratory conditions (209 2°C; 50910% relative humidity; 12-h light/dark cycle) with ad libitum access to chow (Alma H 1003, Botzenhard, Kempten) and drinking water.
2.3. Superfusion of isolated superior cer6ical ganglia (SCG) Superfusion of the isolated SCG was adopted from the method described previously (Schlagmann et al., 1990). SCG with their pre- and postganglionic nerve trunks were excised from NMRI mice, which were anesthetized with urethane (1.5 g/kg i.p.). The ganglia were carefully desheathed from outer connective tissue and suspended vertically in a temperature controlled glass apparatus. The apparatus was continually perfused with Krebs solution (containing (mmol/l): NaCl: 118.0, KCl: 4.8, NaHCO3: 25.0, KH2PO4: 1.18, CaCl2: 2.52, MgSO4: 1.19, glucose: 10.0) at a rate of 2 ml/min at 34°C. Drug induced d.c. surface compound potential changes were recorded between the ganglion body and the postganglionic internal carotid nerve. Ag/AgCl electrodes were connected to the tissue by agar-saline bridges. After amplification by a microvoltmeter (Keithley DMM 2000, Keithley Instruments, Cleveland, OH, USA) drug induced responses were continuously monitored on a PC using the data acquisition program Visiprog (VOKUS, Munich). The preparations were always allowed to repolarize between each agonist application. To reduce variations in the absolute response amplitude of different experiments, responses were normalized with respect to a standard concentration of agonists as described in the figures. If possible, antagonist potencies (apparent Ki values) were estimated from parallel shifts of the concentration-response curves of the investigated agonists in the presence of a known concentration of the antagonist, assuming a competitive relationship. To determine the potency of each agonist, ex-
K.-C. Schroff et al. / Toxicology 144 (2000) 99–105
pressed as its EC50 (the molar concentration required to evoke half of the maximum response) the graphs of the cumulated data were iteratively fitted by computer, using Graph Pad Prism 2.0, and the EC50 extracted from the equation of best fit. Possible significant differences of inhibitory effects of cotinine on nicotinic agonistic action were evaluated using Kruskal – Wallis test.
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2.4. Capillary electrophoresis measurements A BioFocus 3000 capillary electrophoresis system (BioRad, Munich, Germany) with a fast scanning UV-detector was used for the analysis. The separation took place at 25°C in a fused-silica capillary (Ltot = 50 cm, Leff = 45.4 cm; ID = 50 mmol/l) from CS-Chromatography-Service (Langerwehe, Germany). The field strength was 300 V/cm. Before each run the capillary was rinsed for 30 s with NaOH (1 mol/l) and afterwards for 120 s with electrolyte. The electrolyte contained 20 mmol/l sodium borate buffer, pH 9.3, 75 mmol/l sodium dodecylsulfate and 20% methanol. The sample was introduced hydrodynamically (10 psi) and the capillary outlet was the cathode in all runs.
3. Results
Fig. 1. Typical surface potential changes of nicotine and cotinine in mouse SCG. Nicotine and cotinine were applied for 2 min. , start of agonist superfusion. Upward change in potential represents depolarization, downward change represents hyperpolarization. Calibration bars apply to both traces.
Fig. 2. Isolated mouse SCG were superfused with cotinine (30–30 000 mmol/l, n =7). All response values are normalized to signal intensity ( = 1) of a standard concentration of cotinine (1000 mmol/l).
Application of cotinine in concentrations higher than 100 mmol/l resulted in a depolarization of the surface compound potential of the ganglia, followed by a hyperpolarization and a second smaller depolarization (Fig. 1). Concentration dependent changes in the potential response were recorded and depolarizing potential changes for 3× 10 − 5 to 3× 10 − 2 mol/l cotinine were plotted (Fig. 2). The EC50 value was 1.7 mol/l. Since the time dependent course of potential changes induced from cotinine is similar to that from nicotine (Fig. 1), we examined a possible nicotine presence in the used cotinine batches with a high resolution capillary electrophoresis apparatus. After measurement with a mixed standard of 500 mmol/l cotinine and nicotine, respectively, we examined cotinine in a concentration of 10 mmol/l (Fig. 3). Our experiments show, that the cotinine batches used in the described experiments contain 0.1 and 0.79% nicotine, respectively. Therefore, nicotine concentration in applied cotinine can rise to 237 mmol/l, which is sufficient to induce strong potential changes at ganglia. It is possible that the intrinsic effect of cotinine results, at least in part, from the found nicotine in the cotinine sample. Further, Fig. 1 shows potential curves induced at the same ganglion from 10 mmol/l cotinine and
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Fig. 3. Analysis of a nicotine impurity in the cotinine sample: electropherogram of cotinine (10 mmol/l). Conditions of measurement were as described in Section 2.
effect of nicotine (Figs. 4 and 5). Depolarizations induced from 30 mmol/l nicotine can be inhibited by cotinine (Fig. 4) in a concentration dependent manner (IC50 = 302 mmol/l). The inhibitory action of cotinine against nicotine induced depolarizations is not a desensitizing effect of the applied nicotine itself, but a real antagonistic effect of cotinine, since similar to results described for the rat SCG (Schroff et al., 1998) repetitive addition of 30 mmol/l nicotine has no significant desensitizing effect on its own intrinsic activity (data not shown). Concentration-response curves of the depolarizing effect of nicotine (3×10 − 7 to 3 ×10 − 5 mol/l) were shifted to the right (Fig. 5) by continuous application of 300 mmol/l cotinine. Similar experiments with continuous application of 3 mmol/l nicotine-equivalent to a nicotine impurity of 1% in 300 mmol/l cotinine has antagonistic action against its own intrinsic action. However, nicotine at a concentration of 0.3 mmol/l, equivalent to the lower nicotine impurity (0.1%) which can be found in 300 mmol/l cotinine, showed no inhibitory action against the intrinsic activity of nicotine. Therefore, the antagonistic potency of cotinine is at least in part an inhibitory effect of cotinine, not only of the found nicotine impurity in the cotinine sample.
4. Discussion
Fig. 4. Inhibition of 2-min applications of nicotine (10 mmol/l, n =3, 9S.E.M) induced by increasing concentrations of cotinine (0.3 – 1000 mmol/l). Ordinates: depolarizing responses of 2-min applications of nicotine. All depolarizations are normalized; a value of 1 was attributed to the potential changes obtained after the first application.
from 10 mmol/l nicotine, the minimal nicotine concentration which can be found in 10 mmol/l cotinine. Both curves show the same amplitude of depolarization, which underlines the possible intrinsic effect of the nicotine impurity in the cotinine sample. Cotinine in concentrations higher than 1 mmol/l has an antagonistic potency against the intrinsic
The major metabolite of the tobacco alkaloid nicotine is cotinine (Gorrod and Jenner, 1975; Benowitz and Jacob, 1994). While nicotine has been of great interest in neuropharmacological research, only poor data are available of the mode of action of cotinine at neuronal structures. In this paper, we describe the effect of cotinine at cholinergic receptors of peripheral sympathetic neurons of the superior cervical ganglion of the mouse. Our experiments show that cotinine has antagonistic activity at neuronal nicotinic receptors, but do not support a direct intrinsic activity of cotinine. In a concentration higher than 100 mmol/l cotinine induced a concentration dependent depolarization, which was followed by hyperpolarization and a second weak depolarization. This intrinsic
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effect of cotinine is similar to that of nicotine. However, our measurements using capillary electrophoresis showed, that the intrinsic activity of cotinine seems to be the result of a non-negligible nicotine impurity of up to 0.79% in the used cotinine batches. Some authors, who have used cotinine from the same manufacturer, described an intrinsic activity of cotinine in concentrations over 300 mmol/l (Vainio et al., 1998). We were able to show that cotinine solutions of this concentration contain 0.3 – 2.4 mmol/l nicotine, a concentration possibly high enough to have weak intrinsic activity. However, Dwoskin et al. (1999) recently described the potency of cotinine to evoke release of 3H-labelled dopamine from rat striatal slices with an EC50 of 30 mmol/l. The EC50
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of nicotine for this release has been reported to be 0.1–4.0 mmol/l (Izenwasser et al., 1991). It seems unlikely that only the possible small nicotine impurity of a 30-mmol/l cotinine concentration is responsible for the dopamine release. Therefore, an intrinsic activity of cotinine itself cannot readily be excluded. In our experiments relatively low concentrations (beginning with 1 mmol/l) of cotinine had an inhibitory action (IC50 = 302 mmol/ l) against the intrinsic activity of nicotine (30 mmol/l). Furthermore, the concentration-response curve of nicotine was shifted to the right by cotinine (300 mmol/l). Experiments with continuous application of nicotine in concentrations (0.3 and 3 mmol/l) equivalent to the nicotine impurity in the used cotinine batches showed inhibitory
Fig. 5. Concentration-response curves of nicotine (1–30 mmol/l, n = 3, 9 S.E.M., solid line), followed by dose-response curve of nicotine in normal Krebs solution, in the presence of cotinine (300 mmol/l, n =3, 9S.E.M.), of nicotine (0.3 mmol/l, n =3, 9 S.E.M.) and of nicotine (3 mmol/l, n = 3, 9 S.E.M.). Ordinate: depolarizing responses of 2-min applications were expressed in relation to the response induced by 30 mmol/l nicotine in the absence of antagonists. Abscissa: log molar nicotine concentration.
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action of nicotine against its own intrinsic activity only of the higher nicotine concentration. Since cotinine from both examined batches has antagonistic action against the depolarizing effect of nicotine, cotinine itself has, at least in part an inhibitory action at neuronal nicotinic receptors independent of the nicotine impurity. This is consistent with some previous findings which have demonstrated the opposing effects of nicotine and cotinine: Hatsukami et al. (1997, 1998) found no effect of cotinine on physiological parameters and self-reported tobacco withdrawal symptoms, but an antagonistic action against the beneficial effect of nicotine containing patches on withdrawal symptoms. Further, cotinine can reverse the pressor actions of nicotine in anesthetized dogs (Wilcox et al., 1979) which could be a result of the antinicotinic action of cotinine. In horse aorta microsomes nicotine inhibits prostacyclin biosynthesis, while cotinine stimulates it and antagonizes the effect of nicotine (Crooks and Dwoskin, 1997). A nerve stimulation-induced noradrenaline release in isolated rabbit heart, which can be potentiated with nicotine, can be decreased with cotinine (Applegren et al., 1962). Since blood cotinine concentration in smokers, dependent on tobacco consumption and metabolism, can reach between 200 and 900 ng/ml (i.e. 1.14 – 5.13 mmol/l; Hill and Marquard, 1980; Benowitz et al., 1983), it seems possible with normal tobacco consumption to reach serum concentrations of cotinine, which could lead to significant activity at nicotinic receptors in vivo. The average serum peak concentrations of nicotine have been found to be between 10 and 30 ng/ml (i.e. 0.06 – 0.18 mmol/ l; Hill and Marquard, 1980; Benowitz et al., 1990). In vitro this concentration of nicotine exerts only small depolarizing actions at the SCG, yet it has strong pharmacological effects in man. One can presume that the effective concentration of cotinine in our in vitro system is tenfold higher than effective concentrations at the target organs in vivo in man. In conclusion, in this work we were able to prove the action of the tobacco alkaloid cotinine as an antagonist at nicotinic receptors of the sympathetic superior cervical ganglion of the mouse. While and shortly after smoking a
cigarette, nicotine will have the major neuropharmacological significance. However, nicotine has only a short half-time of 2–3 h compared to 19 h for cotinine (Benowitz et al., 1983). As a consequence of the longer half-time, the blood levels of cotinine are relatively constant throughout the smoking day and reach concentrations which are sufficient to act at nicotinic receptors. The described action of cotinine could have an impact on the action of the major tobacco alkaloid nicotine, resulting in a lower nicotinic effect and therefore in a need for higher cigarette consumption.
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K.-C. Schroff et al. / Toxicology 144 (2000) 99–105 Crooks, P.A., Dwoskin, L.P., 1997. Contribution of CNS nicotine metabolites to the neuropharmacological effects of nicotine and tobacco smoking. Biochem. Pharmacol. 54, 743 – 753. Crooks, P.A., Li, M., Dwoskin, L.P., 1995. Determination of nicotine metabolites in rat brain after peripheral radiolabeled nicotine administration: detection of nornicotine. Drug Metab. Dispos. 23, 1175–1177. Crooks, P.A., Li, M., Dwoskin, L.P., 1997. Metabolites of nicotine in rat brain after peripheral nicotine administration: cotinine, nornicotine and norcotinine. Drug Metab. Dispos. 25, 47 – 54. Deutsch, J., Hegedus, L., Greig, N.H., Rapoport, S.I., Soncrant, T.T., 1992. Electron-impact and chemical ionization detection of nicotine and cotinine by gas chromatographymass spectrometry in rat plasma and brain. J. Chromatogr. 579, 93 – 98. Dwoskin, L.P., Teng, L., Buxton, S.T., Crooks, P.A., 1999. (S)-(− )-cotinine, the major brain metabolite of nicotine, stimulates nicotinic receptors to evoke [3H]dopamine release from rat striatal slices in a calcium-dependent manner. J. Pharmacol. Exp. Ther. 288 (3), 905–911. Fibiger, H.C., Phillips, A.C., 1987. Role of catecholamine transmitters in brain reward systems: implications for the neurobiology of affect. In: Engel, J., Oreland, L. (Eds.), Brain Reward Systems and Abuse. Raven Press, New York, pp. 61 – 74. Fuxe, K., Everitt, B.J., Hokfelt, T., 1979. On the action of nicotine and cotinine on central 5-hydroxytryptamine neurons. Pharmacol. Biochem. Behav. 10, 671–677. Gorrod, J.W., Jenner, P., 1975. The metabolism of tobacco alkaloids. In: Hayes, J. (Ed.), Essays in Toxicology 6. Academic Press, New York, pp. 35–78. Gorrod, J.W., Wahren, J., 1993. Nicotine and Related Alkaloids: Absorption, Distribution, Metabolism, Excretion. Chapman and Hall, London. Hatsukami, D.K., Grillo, M., Pentel, P.R., Oncken, C., Bliss, R., 1997. Safety of cotinine in humans: physiologic, subjective, and cognitive effects. Pharmacol. Biochem. Behav. 57, 643 – 650. Hatsukami, D.K., Pentel, P.R., Jensen, J., Nelson, D., Allen, S.S., Goldman, A., Rafael, D., 1998. Cotinine: effects with and without nicotine. Psychopharmacology 135, 141–150. Herzig, K.E., Callaway, E., Halliday, R., Naylor, H., Benowitz, N.L., 1998. Effects of cotinine on information processing in non-smokers. Psychopharmacology 135, 127–132.
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Hill, P., Marquard, H., 1980. Plasma and urine changes after smoking different brand of cigarettes. Clin. Pharmacol. Ther. 27 (5), 652 – 658. Izenwasser, S., Jacocks, H.M., Rosenberger, J.G., Cox, B.M., 1991. Nicotine indirectly inhibits [3H]dopamine uptake at concentrations that do not directly promote [3H]dopamine release in rat striatum. J. Neurochem. 56, 603 – 610. Kim, K.S., Borzelleca, J.F., Bowman, E.R., McKennis, H., 1968. Effects of some nicotine metabolites and related compounds on isolated smooth muscle. J. Pharmacol. Exp. Ther. 161, 59 – 69. Langone, J.J., Gijika, H.B., van Vunakis, H.V., 1973. Nicotine and its metabolites. Radioimmunoassays for nicotine and cotinine. Biochemistry 12, 5025 – 5030. Petersen, D.R., Norris, K.J., Thompson, J.A., 1984. A comparative study of the disposition of nicotine and its metabolites in three inbred mice strains. Drug Metab. Dispos. 12, 725 – 731. Schlagmann, C., Ulbrich, H., Remien, J., 1990. Bispyridinium (oxime) compounds antagonize the ‘ganglion blocking’ effect of pyridostigmine in isolated superior cervical ganglia of the rat. Arch. Toxicol. 64, 482 – 489. Schmiterlo¨w, C.G., Hansson, E., Andersson, G., 1967. Distribution of nicotine in central nervous system. Ann. N.Y. Acad. Sci. 142, 2 – 14. Schroff, K.-C., Aschhoff, S., Remien, J., 1998. Inhibitory effects of physostigmine and pyridostigmine at cholinergic receptors of the sympathetic superior cervical ganglion of the rat. Naunyn-Schmiedeberg’s Arch. Pharmacol. 357 (Suppl.), R124. Stalhandske, T., 1970. Effects of increased liver metabolism of nicotine on its uptake, elimination and toxicity in mice. Acta Physiol. Scand. 80, 222 – 234. Vainio, P.J., Viluksela, M., Tuominen, R.K., 1998. Nicotinelike effects of cotinine on protein kinase C activity and noradrenal release in bovine adrenal chromatin cells. J. Autonom. Pharmacol. 18, 245 – 250. Wilcox, R.G., Hughes, J., Roland, J., 1979. Verification of smoking history in patients after infarction using urinary nicotine and cotinine measurements. Br. Med. J. 2, 1026 – 1028. Yamamoto, K.-I., Domino, E.F., 1965. Nicotine-induced EEG and behavioral arousal. Int. J. Neuropharmacol. 4, 359 – 373. Zeidenberg, P., Jaffe, J.H., Kanzler, M., Levitt, M.D., Langone, J.J., van Vunakis, H.V., 1977. Nicotine: cotinine levels in blood during cessation of smoking. Comp. Psychiatry 18, 93 – 101.
Effects of cotinine at cholinergic nicotinic receptors of the sympathetic superior cervical ganglion of the mouse K.-C. Schroff a,*, P. Lovich a, O. Schmitz b, S. Aschhoff a, E. Richter a, J. Remien a b
a Walther Straub-Institute of Pharmacology and Toxicology, Nußbaumstr. 26, 80336 Munich, Germany DKFZ Heidelberg, Department of Molecular Toxicology, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
Abstract Nicotine, the principal alkaloid in tobacco, is generally accepted to be responsible for most neuropharmacological effects due to tobacco use. Little is known about the action of cotinine, the major metabolite from nicotine, at neuronal structures. To evaluate the mode of action of cotinine at neuronal receptors, its effect on the surface compound potential of the sympathetic superior cervical ganglion (SCG) of the mouse was studied. The modulation of nicotine induced surface potentials by cotinine was tested. It was found that 2-min applications of cotinine (0.1 – 30 mmol/l) induced concentration dependent depolarizations at the SCG (EC50 = 1.7 mmol/l) which were followed by hyperpolarizations and weak afterdepolarizations. The intrinsic activity of cotinine compares to that induced by much lower concentrations of nicotine (EC50 =21 mmol/l). These cotinine effects may be mediated at least in part by nicotine impurities which were found by capillary electrophoresis to be 0.1 and 0.8% in different batches of cotinine. Continuous application of 300 mmol/l cotinine shifted the concentration-response curve of nicotine to the right and reduced (IC50 =302 mmol/l) the effects of submaximal nicotine concentrations (30 mmol/l). This effect could not be mimicked by continuous application of a nicotine concentration (0.3 mmol/l) equivalent to the lower impurity in cotinine. Therefore, the antagonistic action of cotinine at peripheral neuronal nicotinic receptors is at least in part independent of nicotine impurity. The observed antagonistic effect of cotinine at nicotinic receptors likely contributes to the neuropharmacological effects of smoking. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Sympathetic ganglion; Mouse; Cotinine; Nicotinic receptors
1. Introduction Cotinine (1-methyl-5-[3-pyridyl]-2-pyrrolidinone) is the major metabolite of the tobacco * Corresponding author. Tel.: +49-89-5160-7258; fax: + 49-89-5160-7207. E-mail address: [email protected] (K.-C. Schroff)
alkaloid nicotine (Gorrod and Jenner, 1975; Benowitz and Jacob, 1994). In humans 86% of systemically absorbed nicotine is metabolized to cotinine (Benowitz, 1983). Since cotinine is present in much higher concentrations in smokers and with a half-time about tenfold longer than nicotine (Langone et al., 1973), cotinine concentrations in blood and urine are widely used as a
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marker of tobacco consumption and abstinence (Zeidenberg et al., 1977; Wilcox et al., 1979). Some studies have shown pharmacological activity of cotinine: cotinine relaxes vascular smooth muscle, dilates blood vessels in vitro (Kim et al., 1968) and decreases blood pressure (Borzelleca et al., 1982) in anesthetized dogs. Cotinine is able to pass the blood-brain barrier (Gorrod and Wahren, 1993; Benowitz et al., 1994; Crooks et al., 1997) and it has been detected in brain of rat, cat and mouse after peripheral nicotine administration (Applegren et al., 1962; Schmiterlo¨w et al., 1967; Stalhandske, 1970; Petersen et al., 1984; Deutsch et al., 1992; Crooks et al., 1995). In brain cotinine underlies no significant biotransformation (Crooks and Dwoskin, 1997). Also neuropharmacological activities of cotinine have been shown. Cotinine increases serotonin turnover in rat cortex (Fuxe et al., 1979), induces electroencephalographic activation in conscious cats (Yamamoto and Domino, 1965) and has, probably as a result of activation of dopamine pathways in brain, intrinsic reinforcing properties (Fibiger and Phillips, 1987; Corrigal et al., 1992, 1994; Balfour and Benwell, 1993). A recent study has shown dopamine release from rat striatal slices triggered by stimulation of nicotinic receptors (Dwoskin et al., 1999). Acute application of cotinine induces minor cognitive impairment in man, tested with a verbal recall task (Herzig et al., 1998). Nothing is known about cotinine actions at the peripheral sympathetic nerve system. We therefore examined the effect of cotinine at nicotinic receptors of the sympathetic superior cervical ganglion of the mouse in vitro.
2. Materials and methods
2.1. Substances ( − )-Cotinine, (− )-nicotine hydrogene tartrate salt and hexamethonium were purchased from Sigma (Taufkirchen, Germany), atropine sulphate and all other chemicals used for Krebs buffer and capillary electrophoresis were obtained from Merck (Darmstadt, Germany).
2.2. Animals Ganglia were excised from mice of the outbred strain NMRI (27–52 g, Charles River, Sulzfeld, Germany). Mice were kept in macrolene cages, type III, under standard animal laboratory conditions (209 2°C; 50910% relative humidity; 12-h light/dark cycle) with ad libitum access to chow (Alma H 1003, Botzenhard, Kempten) and drinking water.
2.3. Superfusion of isolated superior cer6ical ganglia (SCG) Superfusion of the isolated SCG was adopted from the method described previously (Schlagmann et al., 1990). SCG with their pre- and postganglionic nerve trunks were excised from NMRI mice, which were anesthetized with urethane (1.5 g/kg i.p.). The ganglia were carefully desheathed from outer connective tissue and suspended vertically in a temperature controlled glass apparatus. The apparatus was continually perfused with Krebs solution (containing (mmol/l): NaCl: 118.0, KCl: 4.8, NaHCO3: 25.0, KH2PO4: 1.18, CaCl2: 2.52, MgSO4: 1.19, glucose: 10.0) at a rate of 2 ml/min at 34°C. Drug induced d.c. surface compound potential changes were recorded between the ganglion body and the postganglionic internal carotid nerve. Ag/AgCl electrodes were connected to the tissue by agar-saline bridges. After amplification by a microvoltmeter (Keithley DMM 2000, Keithley Instruments, Cleveland, OH, USA) drug induced responses were continuously monitored on a PC using the data acquisition program Visiprog (VOKUS, Munich). The preparations were always allowed to repolarize between each agonist application. To reduce variations in the absolute response amplitude of different experiments, responses were normalized with respect to a standard concentration of agonists as described in the figures. If possible, antagonist potencies (apparent Ki values) were estimated from parallel shifts of the concentration-response curves of the investigated agonists in the presence of a known concentration of the antagonist, assuming a competitive relationship. To determine the potency of each agonist, ex-
K.-C. Schroff et al. / Toxicology 144 (2000) 99–105
pressed as its EC50 (the molar concentration required to evoke half of the maximum response) the graphs of the cumulated data were iteratively fitted by computer, using Graph Pad Prism 2.0, and the EC50 extracted from the equation of best fit. Possible significant differences of inhibitory effects of cotinine on nicotinic agonistic action were evaluated using Kruskal – Wallis test.
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2.4. Capillary electrophoresis measurements A BioFocus 3000 capillary electrophoresis system (BioRad, Munich, Germany) with a fast scanning UV-detector was used for the analysis. The separation took place at 25°C in a fused-silica capillary (Ltot = 50 cm, Leff = 45.4 cm; ID = 50 mmol/l) from CS-Chromatography-Service (Langerwehe, Germany). The field strength was 300 V/cm. Before each run the capillary was rinsed for 30 s with NaOH (1 mol/l) and afterwards for 120 s with electrolyte. The electrolyte contained 20 mmol/l sodium borate buffer, pH 9.3, 75 mmol/l sodium dodecylsulfate and 20% methanol. The sample was introduced hydrodynamically (10 psi) and the capillary outlet was the cathode in all runs.
3. Results
Fig. 1. Typical surface potential changes of nicotine and cotinine in mouse SCG. Nicotine and cotinine were applied for 2 min. , start of agonist superfusion. Upward change in potential represents depolarization, downward change represents hyperpolarization. Calibration bars apply to both traces.
Fig. 2. Isolated mouse SCG were superfused with cotinine (30–30 000 mmol/l, n =7). All response values are normalized to signal intensity ( = 1) of a standard concentration of cotinine (1000 mmol/l).
Application of cotinine in concentrations higher than 100 mmol/l resulted in a depolarization of the surface compound potential of the ganglia, followed by a hyperpolarization and a second smaller depolarization (Fig. 1). Concentration dependent changes in the potential response were recorded and depolarizing potential changes for 3× 10 − 5 to 3× 10 − 2 mol/l cotinine were plotted (Fig. 2). The EC50 value was 1.7 mol/l. Since the time dependent course of potential changes induced from cotinine is similar to that from nicotine (Fig. 1), we examined a possible nicotine presence in the used cotinine batches with a high resolution capillary electrophoresis apparatus. After measurement with a mixed standard of 500 mmol/l cotinine and nicotine, respectively, we examined cotinine in a concentration of 10 mmol/l (Fig. 3). Our experiments show, that the cotinine batches used in the described experiments contain 0.1 and 0.79% nicotine, respectively. Therefore, nicotine concentration in applied cotinine can rise to 237 mmol/l, which is sufficient to induce strong potential changes at ganglia. It is possible that the intrinsic effect of cotinine results, at least in part, from the found nicotine in the cotinine sample. Further, Fig. 1 shows potential curves induced at the same ganglion from 10 mmol/l cotinine and
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Fig. 3. Analysis of a nicotine impurity in the cotinine sample: electropherogram of cotinine (10 mmol/l). Conditions of measurement were as described in Section 2.
effect of nicotine (Figs. 4 and 5). Depolarizations induced from 30 mmol/l nicotine can be inhibited by cotinine (Fig. 4) in a concentration dependent manner (IC50 = 302 mmol/l). The inhibitory action of cotinine against nicotine induced depolarizations is not a desensitizing effect of the applied nicotine itself, but a real antagonistic effect of cotinine, since similar to results described for the rat SCG (Schroff et al., 1998) repetitive addition of 30 mmol/l nicotine has no significant desensitizing effect on its own intrinsic activity (data not shown). Concentration-response curves of the depolarizing effect of nicotine (3×10 − 7 to 3 ×10 − 5 mol/l) were shifted to the right (Fig. 5) by continuous application of 300 mmol/l cotinine. Similar experiments with continuous application of 3 mmol/l nicotine-equivalent to a nicotine impurity of 1% in 300 mmol/l cotinine has antagonistic action against its own intrinsic action. However, nicotine at a concentration of 0.3 mmol/l, equivalent to the lower nicotine impurity (0.1%) which can be found in 300 mmol/l cotinine, showed no inhibitory action against the intrinsic activity of nicotine. Therefore, the antagonistic potency of cotinine is at least in part an inhibitory effect of cotinine, not only of the found nicotine impurity in the cotinine sample.
4. Discussion
Fig. 4. Inhibition of 2-min applications of nicotine (10 mmol/l, n =3, 9S.E.M) induced by increasing concentrations of cotinine (0.3 – 1000 mmol/l). Ordinates: depolarizing responses of 2-min applications of nicotine. All depolarizations are normalized; a value of 1 was attributed to the potential changes obtained after the first application.
from 10 mmol/l nicotine, the minimal nicotine concentration which can be found in 10 mmol/l cotinine. Both curves show the same amplitude of depolarization, which underlines the possible intrinsic effect of the nicotine impurity in the cotinine sample. Cotinine in concentrations higher than 1 mmol/l has an antagonistic potency against the intrinsic
The major metabolite of the tobacco alkaloid nicotine is cotinine (Gorrod and Jenner, 1975; Benowitz and Jacob, 1994). While nicotine has been of great interest in neuropharmacological research, only poor data are available of the mode of action of cotinine at neuronal structures. In this paper, we describe the effect of cotinine at cholinergic receptors of peripheral sympathetic neurons of the superior cervical ganglion of the mouse. Our experiments show that cotinine has antagonistic activity at neuronal nicotinic receptors, but do not support a direct intrinsic activity of cotinine. In a concentration higher than 100 mmol/l cotinine induced a concentration dependent depolarization, which was followed by hyperpolarization and a second weak depolarization. This intrinsic
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effect of cotinine is similar to that of nicotine. However, our measurements using capillary electrophoresis showed, that the intrinsic activity of cotinine seems to be the result of a non-negligible nicotine impurity of up to 0.79% in the used cotinine batches. Some authors, who have used cotinine from the same manufacturer, described an intrinsic activity of cotinine in concentrations over 300 mmol/l (Vainio et al., 1998). We were able to show that cotinine solutions of this concentration contain 0.3 – 2.4 mmol/l nicotine, a concentration possibly high enough to have weak intrinsic activity. However, Dwoskin et al. (1999) recently described the potency of cotinine to evoke release of 3H-labelled dopamine from rat striatal slices with an EC50 of 30 mmol/l. The EC50
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of nicotine for this release has been reported to be 0.1–4.0 mmol/l (Izenwasser et al., 1991). It seems unlikely that only the possible small nicotine impurity of a 30-mmol/l cotinine concentration is responsible for the dopamine release. Therefore, an intrinsic activity of cotinine itself cannot readily be excluded. In our experiments relatively low concentrations (beginning with 1 mmol/l) of cotinine had an inhibitory action (IC50 = 302 mmol/ l) against the intrinsic activity of nicotine (30 mmol/l). Furthermore, the concentration-response curve of nicotine was shifted to the right by cotinine (300 mmol/l). Experiments with continuous application of nicotine in concentrations (0.3 and 3 mmol/l) equivalent to the nicotine impurity in the used cotinine batches showed inhibitory
Fig. 5. Concentration-response curves of nicotine (1–30 mmol/l, n = 3, 9 S.E.M., solid line), followed by dose-response curve of nicotine in normal Krebs solution, in the presence of cotinine (300 mmol/l, n =3, 9S.E.M.), of nicotine (0.3 mmol/l, n =3, 9 S.E.M.) and of nicotine (3 mmol/l, n = 3, 9 S.E.M.). Ordinate: depolarizing responses of 2-min applications were expressed in relation to the response induced by 30 mmol/l nicotine in the absence of antagonists. Abscissa: log molar nicotine concentration.
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action of nicotine against its own intrinsic activity only of the higher nicotine concentration. Since cotinine from both examined batches has antagonistic action against the depolarizing effect of nicotine, cotinine itself has, at least in part an inhibitory action at neuronal nicotinic receptors independent of the nicotine impurity. This is consistent with some previous findings which have demonstrated the opposing effects of nicotine and cotinine: Hatsukami et al. (1997, 1998) found no effect of cotinine on physiological parameters and self-reported tobacco withdrawal symptoms, but an antagonistic action against the beneficial effect of nicotine containing patches on withdrawal symptoms. Further, cotinine can reverse the pressor actions of nicotine in anesthetized dogs (Wilcox et al., 1979) which could be a result of the antinicotinic action of cotinine. In horse aorta microsomes nicotine inhibits prostacyclin biosynthesis, while cotinine stimulates it and antagonizes the effect of nicotine (Crooks and Dwoskin, 1997). A nerve stimulation-induced noradrenaline release in isolated rabbit heart, which can be potentiated with nicotine, can be decreased with cotinine (Applegren et al., 1962). Since blood cotinine concentration in smokers, dependent on tobacco consumption and metabolism, can reach between 200 and 900 ng/ml (i.e. 1.14 – 5.13 mmol/l; Hill and Marquard, 1980; Benowitz et al., 1983), it seems possible with normal tobacco consumption to reach serum concentrations of cotinine, which could lead to significant activity at nicotinic receptors in vivo. The average serum peak concentrations of nicotine have been found to be between 10 and 30 ng/ml (i.e. 0.06 – 0.18 mmol/ l; Hill and Marquard, 1980; Benowitz et al., 1990). In vitro this concentration of nicotine exerts only small depolarizing actions at the SCG, yet it has strong pharmacological effects in man. One can presume that the effective concentration of cotinine in our in vitro system is tenfold higher than effective concentrations at the target organs in vivo in man. In conclusion, in this work we were able to prove the action of the tobacco alkaloid cotinine as an antagonist at nicotinic receptors of the sympathetic superior cervical ganglion of the mouse. While and shortly after smoking a
cigarette, nicotine will have the major neuropharmacological significance. However, nicotine has only a short half-time of 2–3 h compared to 19 h for cotinine (Benowitz et al., 1983). As a consequence of the longer half-time, the blood levels of cotinine are relatively constant throughout the smoking day and reach concentrations which are sufficient to act at nicotinic receptors. The described action of cotinine could have an impact on the action of the major tobacco alkaloid nicotine, resulting in a lower nicotinic effect and therefore in a need for higher cigarette consumption.
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