Hydrogen peroxide and antioxidizing enzymes involved in modulation of transient facilitatory effects of nicotine on neurogenic contractile responses in rat gastric fundus

(1)

Hydrogen peroxide and antioxidizing enzymes involved in modulation of

transient facilitatory effects of nicotine on neurogenic

contractile responses in rat gastric fundus

Sevil Özger

İlhan

a

, Yusuf Sarioglu

b

,

İsmail Mert Vural

b,

⁎

, Ergin Dileköz

c

,

Gökçe Sevim Öztürk

b

, Zeynep Sevim Ercan

b

a_{Department of Pharmacology, Medical School, UfukUniversity, Ankara, Turkey} b_{Department of Pharmacology, Medical School, Gazi University, 06510 Be}_{şevler, Ankara, Turkey} c

Massachusetts General Hospital, Harvard Medical School Department of Radiology Charlestown, MA 02129 USA Received 19 June 2007; received in revised form 3 March 2008; accepted 14 March 2008

Available online 1 April 2008

Abstract

Nicotine acts as an agonist of nicotinic acetylcholine receptors. Nicotinic acetylcholine receptors play a role in the modulation of neurotransmitter release in both the central and the peripheral nervous system. Moderate reactive oxygen species levels modulate the regulation of physiological functions e.g. neurotransmitter release. Previously in rabbit gastric fundus we demonstrated that nicotine transiently increased neurogenic contraction induced by electrical field stimulation (EFS). In this study we aimed to investigate the effects of hydrogen peroxide (H2O2), antioxidizing enzymes

catalase and superoxide dismutase (SOD) on nicotine induced increases at cholinergic neurotransmission in rabbit gastric fundus. Although H2O2did

not alter nicotine induced transient neurogenic contractions at concentrations of 10− 6and 10− 5M, at high concentration (10− 4M) H2O2inhibited

nicotine induced increases. Catalase (500 units/ml), enhanced the effect of nicotine but did not alter nicotine induced transient neurogenic contractions at the concentrations of 100 and 250 units/ml. SOD (75,150 and 225 units/ml) did not alter nicotine induced transient neurogenic contractions. In conclusion, at high concentration H2O2(10− 4M) inhibited nicotine's transient ability to augment neurogenic contractions and

Keywords: Nicotine; Rabbit gastric fundus; Cholinergic neurotransmission; Nicotinic acetylcholine receptor; Hydrogen peroxide (H2O2); Catalase; Superoxide

dismutase (SOD)

1. Introduction

Nicotine, an alkaloid isolated from the leaves of tobacco plant, is the nonspecific agonist of nicotinic acetylcholine receptors. Nicotinic acetylcholine receptors belong to a super-family of pentameric ligand-gated ion channels and have seventeen subunits identified as of the present time. These receptors are located in both the central and peripheral nervous

system (Newman et al., 2002; McGehee et al., 1995; Todorov

et al., 1991). Previously, different research groups demonstrated that, noradrenalin release is increased by nicotine via peripheral nicotinic acetylcholine receptors in various tissues such as guinea pig vas deferens, rat stomach, adrenal gland and

anococcygeus muscle (Todorov et al., 1991; Nedergaard and

Schrold, 1977; Yokotani et al., 2002, 2000; Rand and Li, 1992). Similarly, nicotine modulates acetylcholine release via nicotinic acetylcholine receptors in aganglionic vas deferens and guinea

pig ileum myenteric plexus (Cuprian et al., 2005; Briggs and

Cooper, 1982). Nicotine triggers influx of Ca+2through

ligand-gated channels and/or voltage-ligand-gated Ca2+channels (VGCC) via

activation of nicotinic acetylcholine receptors. Then the release

European Journal of Pharmacology 587 (2008) 267–272

www.elsevier.com/locate/ejphar

⁎ Corresponding author. Gazi University, Faculty of Medicine, Department of Pharmacology Besevler 06510 Ankara, Turkey. Tel.: +90 312 2026951, +90 312 2024622; fax: +90 312 212 46 47.

E-mail address:imvural@yahoo.com(İ.M. Vural).

(2)

of Ca2+ from intracellular calcium stores is triggered. Ca+2 activation plays an important role in the action of nicotine via activation of the presynaptic nicotinic acetylcholine receptors and as a result neurotransmitter release increases (Brain et al., 2001; Dolezal et al., 1998; Shoop et al., 2001; Sharma and Vijayaraghavan, 2001).

There are numerous systems that produce reactive oxygen

species (hydrogen peroxide, H2O2; superoxideradical, O2-;

hydroxyl radical, OH.) in the organism. Reactive oxygen species are responsible for several biological phenomena including carcinogenesis, ischemia-reperfusion injury,

radia-tion damage and neurodegenerative diseases (Toyokuni,1999;

Richter et al., 1995). But it was shown that moderate levels of reactive oxygen species modulate the regulation of critical physiological functions, e.g. signaling cascades, control of

gene expression (Nose, 2000). It has been demonstrated that

H2O2is involved in the regulation of vascular tone. In the rat

aorta, H2O2 induces contractile responses by increasing

intracellular Ca2+levels and this effect is completely reduced by catalase (Yang et al., 1998). Also in smooth muscles it was

shown that voltage-dependent Ca2+ entry is potentiated by

H2O2(Oba et al., 1998). As a result in several studies it was

suggested that reactive oxygen species may modulate

intracellular Ca2+concentration. Although effects of reactive

oxygen species on synaptic neurotransmission was investi-gated by different research groups previously, this

relation-ship remains poorly understood (Pellmar, 1987; Chen et al.,

2001; Vural et al., 2006). Previously in rabbit bladder we demonstrated that reactive oxygen species do not play a physiological role on the regulation of nicotinic acetylcholine

receptors dependant neurotransmitter releases (Vural et al.,

2006).

In our previous studies, we demonstrated that nicotine increases the electrical field stimulation (EFS)-evoked contractile response, possibly by facilitating neurotransmitter release from

nerve terminals by a mechanism dependant on the influx of Ca2+

from VGCCs via activation of nicotinic acetylcholine receptors in isolated rabbit bladder, corpus cavernosum and gastric fundus. NO and prostaglandins do not have a physiological role on the regulation of neurotransmitter release in both rabbit gastric fundus and bladder (Bozkurt et al., 2007; Vural et al., 2007; Ilhan et al.,

2007). In this study we aimed to investigate the effects of

hydrogen peroxide (H2O2) and the antioxidizing enzymes

catalase and superoxide dismutase on nicotine induced increases in nerve evoked contraction in rabbit gastric fundus.

2. Materıals and methods

2.1. Animals

Twenty New Zealand albino rabbits weighing 2.5−3.0 kg were

used for the experiments. All animals were kept under controlled temperature (23.2 °C) and humidity (55.5%) with a 14-h light and 10-h dark cycle. They were fed standard laboratory chow and given tap water. All experiments were performed in accordance with the ethical regulations of the Helsinki Declaration. This study was approved by the Gazi University Ethics Committee for Animals.

2.2. Tissues

Animals were sacrificed by exsanguination and their stomachs were rapidly excised, opened lengthwise, and emptied. Adherent fat, gross connective tissues, and gastric mucosa were removed, and uniform longitudinal strips (15 mm × 3 mm) were prepared from the smooth muscle of the gastric fundus.

2.3. Organ chamber experiments

Each strip was mounted under 1 g isometric resting tension in an organ bath containing 15 mL Krebs−Henseleit solution

(composition in mmol/L: NaCl 118.0, KCl 4.7, CaCl2.2H2O

1.3, MgCl2.6H2O 0.5, Na2HPO4.2 H2O 0.9, NaHCO3 24.9,

glucose monohydrate 11.0). The pH of the solution was 7.4 after bubbling with a mixture of 95% O2and 5% CO2, and the solution

was maintained at 37 °C. The tissues were allowed to equilibrate for at least 1 h before experimental procedures. Isometric contractions were evoked by EFS through a pair of platinum electrodes with an 8 Hz stimulation frequency by 10 s trains of impulses delivered every 2 min. A stimulator (S48; Grass Instruments, Quincy, MA, USA) delivered 60 V pulses of 1 ms duration. EFS-evoked responses were recorded via Grass isometric force displacement transducers (Grass FT 03) con-nected to an ink writing oscillograph (Grass 79 E) via a preamplifier. Thirty minutes after the EFS-evoked responses reached a steady state, to test the contribution of the cholinergic component, the tissues were treated with atropine (10− 6M), non-selective competitive antagonist of muscarinic receptor, or neostigmine (10− 5M), a reversible anticholinesterase drug. The effects of tetrodotoxin (3 × 10− 6 M), H2O2 (10− 6–10− 4 M),

catalase (100 units/ml, 250 units/ml and 500 units/ml) or superoxide dismutase (SOD) (75,150 and 225 units/ml) on the EFS-evoked responses were tested similarly in separate strips. The tissues were exposed with drugs at least 30 min. To test the effects of nicotine, different concentrations (3 × 10− 5M, 10− 4M) of nicotine were administered to the separate preparations. To avoid any possible habituation effect or tachyphylaxis, EFS was stopped after seven contractions and the preparations were washed four times every 15 min for 1 h as in our previous study (Ilhan et al., 2007). To investigate the effects of reactive oxygen species on the nicotine induced EFS-evoked contractile response alternations, same experimental procedure was repeated in the

presence of H2O2 (10− 6–10− 4 M), catalase (100 units/ml,

250 units/ml and 500 units/ml) or superoxide dismutase (SOD) (75,150 and 225 units/ml). H2O2and enzymes were added to the

organ baths 30 min before the administration of nicotine. 2.4. Drugs

All of the following drugs were obtained from Sigma (St. Louis, MO, USA): nicotine, atropine sulfate, superoxide dismutase (EC 1.15.1.1) from bovine liver, catalase (EC 1.11.1.6) from bovine liver, tetrodotoxin except hydrogen peroxide which was obtained from Merck Schuchardt. Stock solutions of drugs were dissolved in distilled water. Solutions were stored at−20 °C.

(3)

The drugs were diluted in Krebs to the required final concentra-tion on the day of use.

2.5. Statistics

Nicotine-induced increases were expressed as percentage variation of the average of seven EFS-evoked contractile responses in the presence of the drug with respect to control values. The value of the last contraction before the application of nicotine was taken as the control value.

Experimental values were expressed as the mean ± S.E.M. Groups were compared statistically using general linear models of analysis of variance (ANOVA) followed by post hoc analysis with the Bonferroni test.

P values of b0.05 were considered to be statistically

significant. 3. Results

EFS evoked contractile responses in rabbit gastric fundus. Mean amplitude of the EFS-evoked contractile responses was 2.85 ± 0.42 g at 8 Hz of stimulation frequency.

Tetrodotoxin (3 × 10−6M), a blocker of Na+channels, abolished the EFS-evoked contractile responses in rabbit gastric fundus strips.

EFS-evoked contractile responses were abolished by atro-pine at the 10− 6M concentration in rabbit gastric fundus strips. Neostigmine led to increase in the amplitudes of the

EFS-evoked contractile responses (10− 6 M, 11.1%; 3 × 10− 6 M,

100%; 10− 5M, 722.2%; Pb0.05).

3.1. Effects of nicotine on EFS-induced contractile responses Nicotine increased the EFS-induced contractions transiently

(3 × 10− 5 M–113.21±20.09%; 10− 4 M–151.66±16.02%) in

this study. Nicotine had no contractile effects on non-stimulated preparations at the concentrations listed above (3 × 10− 5M to 10− 4M).

3.2. Effects of H2O2 and antioxidizing enzymes on nicotine

induced transient neurogenic contractions

H2O2 was used at various concentrations (10− 6–10− 4 M).

H2O2increased the basal tonus of the tissue at 10−5 and 10−4 M

concentrations but not alter the EFS-evoked contractile

responses. Although H2O2 did not alter nicotine induced

transient neurogenic contractions at concentrations of 10− 6

and 10− 5 M, at high concentration (10− 4 M, 55.95 ± 5.09%,

pb0.05) H2O2inhibited nicotine induced increases (Fig. 1). In

all the experiments nicotine's concentration was 10− 4M. Catalase did not alter nicotine induced transient neurogenic contractions at the concentration of 100 and 250 units/ml. At 500 units per ml concentration catalase augmented the transient neurogenic contractions induced by nicotine. In all the experiments nicotine's concentration was 3 × 10− 5M (Fig. 2).

SOD (75, 150 and 225 units/ml) did not alter nicotine induced transient neurogenic contractions. Nicotine was used at 3 × 10− 5M concentrations (Fig. 3).

Neither catalase nor SOD altered EFS-evoked contractile responses and basal tonus of the tissue.

Fig. 1. Effects of the variant concentrations of H2O2[10− 6M (n = 8), 10− 5M (n = 8)

and 10− 4M (n = 8)] on nicotine (nic.) (10− 4M)-induced increases at EFS-evoked contractile responses. Each point is expressed as a percentage of the control and the average of seven EFS-evoked contractile responses. All points are given as the mean ± S.E.M. (⁎, Pb0.05).

Fig. 2. Effects of the variant concentrations of catalase [100 units/ml (n = 8), 250 units/ml (n = 8), 500 units/ml (n = 8) and 1000 unit/ml (n = 8)] on nicotine (nic., 3 × 10− 5M)-induced increase at EFS-evoked contractile responses. Each point is expressed as a percentage of the control and the average of seven EFS-evoked contractile responses. All points are given as the mean ± S.E.M. (⁎, Pb0.05).

Fig. 3. Effects of variant concentrations of superoxide dismutase [SOD; 75 units/ml (n = 8), 150 units/ml (n = 8), 225 units/ml (n = 8)] on the nicotine (nic., 3 × 10− 5 M)-induced increase at EFS-evoked contractile responses. Each point is expressed as a percentage of the control and the average of seven EFS-evoked contractile responses. All points are given as the mean ± S.E.M.

(4)

4. Discussion

The gastric fundus is supplied with cholinergic nerves that produce contractions via muscarinic receptors and these cholinergic nerves play an important role in the regulation of

gastrointestinal motility (Hammer, 1980; Heitkemper and

Marotta, 1983; Leclere and Lefebvre, 2002a,b). In the present

experiments, tetrodotoxin, a blocker of Na+ channels,

abolished EFS-evoked responses, suggesting that EFS-evoked responses are induced by nerve stimulation. In this study, we used atropine and neostigmine to evaluate the involvement of cholinergic activation in EFS-evoked contractions. Atropine abolished and neostigmine enhanced EFS-evoked contractile responses, indicating presence of the cholinergic system in rabbit gastric fundus, which was shown in previously (Hammer, 1980; Heitkemper and Marotta, 1983; Leclere and Lefebvre, 2002a,b).

Previously in rabbit gastric fundus we demonstrated that nicotine which is a nonspecific nicotinic acetylcholine receptor agonist, induced a transient neurogenic increase in EFS-evoked cholinergic responses in a concentration depen-dent manner. Hexamethonium, a nonspecific nicotinic

acetylcholine receptor antagonist, and cadmium (Cd2+)

which blocks presynaptic voltage-gated calcium channels involved in the EFS-evoked responses prevented the potentiation caused by nicotine, showing that nicotinic acetylcholine receptors are responsible for the effect of

nicotine by a mechanism dependent on the influx of Ca2+

from voltage-gated Ca2+channels (VGCC). NeitherL-NAME

nor indomethacin altered nicotine induced transient neuro-genic increases indicating that NO and prostaglandins do not

play a physiological role on this mechanism (Ilhan et al.,

2007). Although nicotine has been shown to have a

facili-tatory effect on neurotransmitter release through acting on nicotinic acetylcholine receptors, little is known about the

exact mechanisms of this action (Brain et al., 2001; Wang

et al., 2000). Similarly, in our previous studies, nicotine induced the transient neurogenic contractions via nicotinic

acetylcholine receptors in rabbit myometrium (Nas et al.,

2007) and bladder (Vural et al., 2006, 2007). In our different study nicotine evoked the EFS-induced smooth muscle

relaxation in rabbit corpus cavernosum (Bozkurt et al.,

2007). Nicotine was demonstrated to reduce the EFS-induced

contractions in human circular muscle. This reduction was

blocked by L-NAME (McKirdy et al., 2004). Nicotinic

acetylcholine receptors have been reported to be involved in the nicotine-induced enhancement of neurotransmitter release

in both central and peripheral neurons (McGehee et al., 1995;

Rose et al., 1999). Nicotine was shown to induce noradrena-line release in rat stomach, due to activation of ganglionic nicotinic acetylcholine receptors localized at the celiac ganglia. This effect of nicotine was sensitive to

hexametho-nium and was inhibited by tetrodotoxin (Yokotani et al.,

2000). In guinea pig ileum myenteric plexus nicotinic

acetylcholine receptors activation stimulated the release of

neurotransmitters including acetylcholine (Galligan, 1999).

Similarly in mouse isolated vas deferens acetylcholine

release from cholinergic nerve terminals enhanced by

activation of nicotinic acetylcholine receptors (Cuprian

et al., 2005). It has been shown in various studies that influx

of Ca2+ through ligand-gated and/or voltage-gated Ca2+

channels, via activation of nicotinic acetylcholine receptors

by nicotine, triggers the release of Ca2+ from intracellular

calcium stores. This Ca2+ activation plays an important role

in the effect of nicotine on neurotransmitter release (Brain

et al., 2001; Dolezal et al., 1998; Shoop et al., 2001; Sharma and Vijayaraghavan, 2001).

Reactive oxygen species are formed and degraded by all aerobic organisms. Although reactive oxygen species are known mediators of intracellular signaling cascades, they are also responsible for several biological phenomena including carcinogenesis, ischemia-reperfusion injury, radiation damage

and neurodegenerative diseases (Toyokuni, 1999; Nose, 2000;

Nordberg and Arner, 2001). In our experiments at high

concentration (10− 4 M) H2O2 inhibited nicotine induced

transient neurogenic contractions. Although SOD did not alter nicotine induced transient neurogenic contractions, at 500 units/ ml concentration catalase augmented the transient neurogenic contractions induced by nicotine. In rabbit gastric fundus in both our previous study (Ilhan et al., 2007) and this study we investigated the effects of different drugs on nicotine-induced transient neurogenic contractions at nicotine's 10–4 M con-centration. But in this study in preliminary experiments catalase enhanced nicotine-induced transient neurogenic contractions. After than we decided to investigate the enhancement effect of

catalase on nicotine's lower concentration (3 × 10−5 M) for

observing this effect more clearly. Previously it was shown that moderate levels of reactive oxygen species modulates the regulation of critical physiological functions, e.g. signaling cascades, control of gene expression (Nose, 2000). But in this

study both H2O2 and catalase altered nicotine induced

responses at high concentrations. It has been demonstrated that H2O2might be involved in the regulation of vascular tone.

Also in this study H2O2increased the basal tonus of the tissue

at 10− 5 and 10 − 4 M concentrations. Similarly in rat aorta at pathophysiological concentrations H2O2induced contractile

responses with a Ca+ 2dependent mechanism. This contractile

effect of H2O2is completely inhibited by catalase (Yang et al.,

1998). Also in smooth muscles it was shown that

voltage-dependant Ca2+entry is potentiated by H2O2(Oba et al., 1998).

As a result in several studies it was suggested that reactive

oxygen species may modulate intracellular Ca2+concentration.

In different studies an inhibitory effect of reactive oxygen species on synaptic neurotransmission was demonstrated (Pellmar, 1987; Chen et al., 2001; Zoccarato et al., 1995). It was shown that hydrogen peroxide inhibits neurotransmitter release possibly by decreasing Ca2+entry via presynaptic Ca2+

channels (Pellmar, 1987). Also it was demonstrated that

endogenous Ca2+dependent H2O2production inhibits synaptic

dopamine release in guinea pig striatal slices (Chen et al.,

2001). Similarly H2O2 inhibited the glutamate release in

cerebrocortical synaptosomes from guinea pig (Zoccarato

et al., 1995). But previously in the rabbit bladder we demon-strated that reactive oxygen species do not play a physiological

(5)

role on the regulation of nicotinic acetylcholine receptors dependant neurotransmitter releases (Vural et al., 2006). In this study although SOD did not alter nicotine induced transient neurogenic contractions, at high concentration catalase aug-mented the transient neurogenic contractions induced by nicotine. In the rat aorta H2O2effects were blocked by catalase

but not by SOD suggesting that the formation of hydroxyl radicals is not implicated in H2O2′s effects (Gil-Longo and

Gonzalez-Vazquez, 2005). In the guide of our results we can

suggest that H2O2 may assist in the mechanism of nicotine

induced EFS enhancement via nicotinic acetylcholine receptors.

In conclusion nicotine augmented EFS-induced contractile responses by increasing acetylcholine release or additional

mechanisms. At high concentration H2O2 (10−4 M) inhibited

nicotine's transient ability to augment EFS-induced neurogenic contractile responses and catalase (500 U/ml) enhanced the effect of nicotine. But SOD did not alter nicotine induced increases. Nicotinic acetylcholine receptor subtypes involved in nicotine-induce enhancement and modulation of this mechanism needs further investigation.

Acknowledgments

This work was supported partially by the Gazi University Unit of Scientific Research Projects (Project number: 11/2003-09) and the Turkish Academy of Science.

References

Brain, K.L., Trout, S.J., Jackson, V.M., Dass, N., Cunnane, T.C., 2001. Nicotine induces calcium spikes in single nerve terminal varicosities: a role for intracellular calcium stores. Neuroscience 106, 395–403.

Briggs, C.A., Cooper, J.R., 1982. Cholinergic modulation of the release of [3H] acetylcholine from synaptosomes of the myenteric plexus. J. Neurochem. 38, 501–508.

Bozkurt, N.B., Vural, I.M., Sarioglu, Y., Pekiner, C., 2007. Nicotine potentiates the nitrergic relaxation responses of rabbit corpus cavernosum tissue via nicotinic acetylcholine receptors. Eur. J. Pharmacol. 558, 172–178. Chen, B.T., Avshalumov, M.V., Rice, M.E., 2001. H(2)O(2) is a novel,

endogenous modulator of synaptic dopamine release. J. Neurophysiol. 85, 2468–2476.

Cuprian, A.M., Solanki, P., Jackson, M.V., Cunnane, T.C., 2005. Cholinergic innervation of the mouse isolated vas deferens. Br. J. Pharmacol. 146, 927–934.

Dolezal, V., Schobert, A., Hertting, G., 1998. Presynaptic nicotinic receptors stimulate increases in intraterminal calcium of chick sympathetic neurons in culture. J. Neurochem. 65, 1874–1879.

Galligan, J.J., 1999. Nerve terminal nicotinic cholinergic receptors on excitatory motoneurons in the myenteric plexus of guinea pig intestine. J. Pharmacol. Exp. Ther. 291, 92–98.

Gil-Longo, J., Gonzalez-Vazquez, C., 2005. Characterization of four different effects elicited by H2O2in rat aorta. Vasc. Pharmacol. 43, 128–138.

Hammer, R., 1980. Muscarinic receptors in the stomach. Scand. J. Gastroenterol. Suppl. 66, 5–11.

Heitkemper, M.M., Marotta, S.F., 1983. Development of neurotransmitter enzyme activity in the rat gastrointestinal tract. Am. J. Physiol. 244, G58–G64.

Ilhan, S.O., Vural, I.M., Dilekoz, E., Ozturk, G.S., 2007. Enhancement effects of nicotine on neurogenic contractile responses in rabbit gastric fundus. Eur. J. Pharmacol. 561, 182–188.

Leclere, P.G., Lefebvre, R.A., 2002a. Characterization of pre- and postsynaptic muscarinic receptors in circular muscle of pig gastric fundus. Br. J. Pharmacol. 135, 1245–1254.

Leclere, P.G., Lefebvre, R.A., 2002b. Presynaptic modulation of cholinergic neurotransmission in the human proximal stomach. Br. J. Pharmacol. 135, 135–142.

McGehee, D.S., Heath, M.J., Gelber, S., Devay, P., Role, L.W., 1995. Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors. Science 269, 1692–1696.

McKirdy, H.C., Richardson, C.E., Green, J.T., Rhodes, J., Williams, G.T., Marshall, R.W., 2004. Differential effect of nitric oxide synthase inhibition on sigmoid colon longitudinal and circular muscle responses to nicotine and nerve stimulation in vitro. Br. J. Surg. 91, 229–234.

Nas, T., Barun, S., Ozturk, G.S., Vural, I.M., Ercan, Z.S., Sarioglu, Y., 2007. Nicotine potentiates the electrical field stimulation-evoked contraction of non-pregnant rabbit myometrium. Tohoku J. Exp. Med. 211, 187–193.

Nedergaard, O.A., Schrold, J., 1977. The mechanism of action of nicotine on vascular adrenergic neuroeffector transmission. Eur. J. Pharmacol. 42, 315–329.

Newman, M.B., Arendash, G.W., Shytle, R.D., Bickford, P.C., Tighe, T., Sanberg, P.R., 2002. Nicotine's oxidative and antioxidant properties in CNS. Life Sci. 71, 2807–2820.

Nordberg, J., Arner, E.S., 2001. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic. Biol. Med. 31, 1287–1312.

Nose, K., 2000. Role of reactive oxygen species in the regulation of physiological functions. Biol. Pharm. Bull. 23, 897–903.

Oba, T., Ishikawa, T., Yamaguchi, M., 1998. Sulfhydryl associated with H2O2

-induced cannel activation are on luminal side of ryanodine receptors. Am. J. Physiol. 274, C914–C921.

Pellmar, T.C., 1987. Peroxide alters neuronal excitability in the CA1 region of guinea-pig hippocampus in vitro. Neuroscience 23, 447–456.

Rand, M.J., Li, C.G., 1992. Activation of noradrenergic and nitrergic mechanisms in the rat anococcygeus muscle by nicotine. Clin. Exp. Pharmacol. Physiol. 19, 103–111.

Richter, C., Gogvadze, V., Laffranchi, R., Schlapbach, R., Schweizer, M., Suter, M., Walter, P., Yaffee, M., 1995. Oxidants in mitochondria: from physiology to diseases. Biochim. Biophys. Acta 24, 67–74.

Rose, J.E., Westman, E.C., Behm, F.M., Jonhson, M.P., Goldberg, J.S., 1999. Blockade of smoking satisfaction using the peripheral nicotinic antagonist trimethaphan. Pharmacol. Biochem. Behav. 62, 165–172.

Sharma, G., Vijayaraghavan, S., 2001. Nicotinic cholinergic signaling in hippocampal astrocytes involves calcium-induced calcium release from intracellular stores. Proc. Nati. Acad. Sci. USA 98, 3631–3632.

Shoop, R.D., Chang, K.T., Ellisman, M.H., Berg, D.K., 2001. Synaptically driven calcium transients via nicotinic receptors on somatic spines. J. Neurosci. 21, 771–781.

Todorov, L., Windisch, K., Shersen, H., Lajtha, A., Papasova, M., Vizi, E.S., 1991. Prejunctional nicotinic receptors involved in facilitation of stimula-tion-evoked noradrenaline release from the vas deferens of the guinea-pig. Br. J. Pharmacol. 102, 186–190.

Toyokuni, S., 1999. Reactive oxygen species-induced molecular damage and its application in pathology. Pathol. Int. 49, 91–102.

Vural, I.M., Ozturk, G.S., Ercan, Z.S., Sarioglu, Y., 2006. Effects of the nicotine on the neurogenic contractile responses via nicotinic acetylcholine receptors in rabbit bladder tissue: reactive oxygen species do not plays a role in this mechanism. Pharmacology Online 2, 54–62.

Vural, I.M., Ozturk, G.S., Ercan, Z.S., Sarioglu, Y., 2007. Nicotine potentiates the neurogenic contractile response of rabbit bladder tissue via nicotinic acetylcholine receptors: nitric oxide and prostaglandins have no role in this process. Life Sci. 80, 1123–1127.

Wang, M., Okada, S., Murakami, Y., Yokotani, K., 2000. Nicotine-induced noradrenaline release from the isolated rat stomach by activation of L- and N-type calcium channels. Jpn. J. Pharmacol. 83, 102–106.

Yang, Z.W., Zheng, T., Zang, A., Altura, B.T., Altura, B.M., 1998. Mechanism of hydrogen peroide-induced contraction of rat aorta. Eur. J. Pharmacol. 344, 169–181.

(6)

Yokotani, K., Okada, S., Nakamura, K., 2000. Characterization of functional nicotinic acetylcholine receptors involved in catecholamine release from the isolated rat adrenal gland. Eur. J. Pharmacol. 446, 83–87.

Yokotani, K., Wang, M., Okada, S., Murakami, Y., Hirata, M., 2002. Characterization of nicotinic receptor-mediated noradrenaline release from the isolated rat stomach. Eur. J. Pharmacol. 402, 223–229.

Zoccarato, F., Valente, M., Alexandre, A., 1995. Hydrogen peroxide induces a long-lasting inhibition of the Ca(2+)-dependent glutamate release in cerebrocortical synaptosomes without interfering with cytosolic Ca2+. J. Neurochem. 64, 2552–2558.