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Role of nicotinic acetylcholine receptor subtypes on nicotine-induced neurogenic contractile response alternation in the rabbit gastric fundus
European Journal of Pharmacology 602 (2009) 395–398
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Pulmonary, Gastrointestinal and Urogenital Pharmacology
Role of nicotinic acetylcholine receptor subtypes on nicotine-induced neurogenic contractile response alternation in the rabbit gastric fundus Ismail Mert Vural a,⁎, Gokce Sevim Ozturk Fincan a, Nihan Burul Bozkurt b, Zeynep Sevim Ercan a, Yusuf Sarioglu a a b
Department of Pharmacology, Medical School, Gazi University, Ankara, Turkey Department of Pharmacology, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 14 July 2008 Received in revised form 30 October 2008 Accepted 17 November 2008 Available online 25 November 2008 Keywords: Nicotine Nicotinic acetylcholine receptors subtypes Electrical field stimulation Rabbit Gastric fundus
a b s t r a c t Nicotine is a nonspecific agonist of nicotinic acetylcholine receptors. We previously demonstrated that nicotine increases the electrical field stimulation (EFS)-evoked contractile responses possibly by facilitating neurotransmitters release from nerve terminals by a mechanism dependent on the influx of Ca2+ from voltage gated Ca2+ channels via activation of nicotinic acetylcholine receptor. The aim of this study is to investigate subtypes of presynaptic nicotinic acetylcholine receptors involved in nicotine induced EFSevoked contractile response alternation in the rabbit gastric fundus. EFS-evoked contractile responses were recorded from gastric fundus strips obtained from rabbits with isometric force displacement transducers. Effects of nicotine on EFS evoked contractions were examined. Then the effect of nicotine on the EFS-evoked contractions was examined in the presence of hexamethonium, dihydro-β-erythroidine, mecamylamine or α-bungarotoxin. In our study, nicotine (10− 4, 3 × 10− 4 M) transiently increased neurogenic contraction induced by EFS in the rabbit isolated gastric fundus. While hexamethonium, dihydro-β-erythroidine and mecamylamine inhibited the neurocontractile response to nicotine on EFS, α-bungarotoxin did not alter these responses. The pA2 values of the antagonists were 4.67 (hexamethonium, n = 8), 5.33 (dihydro-βerythroidine, n = 8) and 5.43 (mecamylamine, n = 8). These findings showed that the α3β4 and α4β2 subunits of nicotinic acetylcholine receptors play a role on the nicotine-induced augmentation in EFS-evoked contractile responses in rabbit gastric fundus. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Nicotine acts as an agonist of nicotinic acetylcholine receptors, which belong to a superfamily of pentameric ligand-gated ion channels. These receptors are located in the central nervous system and peripheral nervous system (Newman et al., 2002; McGehee et al., 1995; Todorov et al., 1991). Nicotinic acetylcholine receptor subunits have been detected in molecular biologic studies. These receptors are composed of α (α2–α10, nine types) and β (β2–β4, three types) subunits (Corriveau and Berg, 1993; Mandelzys et al., 1995; Wonnacott, 1997; Vetter et al., 2007). Nicotine modulates neurotransmitter release via nicotinic acetylcholine receptors. It was demonstrated that nicotine increases the release of various neurotransmitters after nerve stimulation in central and peripheral tissues (Nedergaard and Schrold, 1977; Todorov et al., 1991; Yokotani et al., 2000). In the central nervous system, nicotinic acetylcholine receptor subtypes that have a role in various types of neurotransmitter release were detected in many studies. The role of
⁎ Corresponding author. Tel.: +90 312 2026951. E-mail address: [email protected] (I.M. Vural). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.11.032
α7, α4, β2, and β3 subunits on the release of γ-aminobutyric acid (GABA) or dopamine was demonstrated (Alkondon et al., 1999; Puttfarcken et al., 2000; Salminen et al., 2004). Yokotani et al. demonstrated that nicotinic acetylcholine receptor subtypes were involved in gastric noradrenaline release. Electrical stimulation of the splanchnic nerve induced noradrenaline release mediated partially by α3β4 nicotinic receptors, whereas (−)-nicotine induced noradrenaline release mediated by α3β4 and α4β2 nicotinic receptors (Yokotani et al., 2000, 2001). In another study, it was reported that the predominant nicotinic acetylcholine receptor subtype on gastrointestinal projecting neurons in the dorsal motor vagal nucleus is the α7 subtype. Co-expression of the α4β2 subtype in the neurons projecting to the fundus and antrum was also demonstrated (Sahibzada et al., 2002). We demonstrated that nicotine increased the electrical field stimulation (EFS)-evoked contractile or relaxation response, possibly by facilitating neurotransmitter release from nerve terminals, in various rabbit tissues: gastric fundus, bladder, corpus cavernosum, and myometrium. The effect of nicotine was dependent on the influx of Ca2+ from voltage-gated Ca2+ channels via activation of nicotinic acetylcholine receptors in the isolated gastric fundus (Vural et al., 2006; Bozkurt et al., 2007; Nas et al., 2007; Ilhan et al., 2007, 2008).
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In the present study, we aimed to characterize the nicotinic acetylcholine receptor subtypes involved in nicotine induced EFSevoked contractile response alternation in the rabbit gastric fundus. 2. Materials and methods 2.1. Animals Twenty New Zealand albino rabbits weighing 2.5–3 kg were used for the experiments. All animals were kept under controlled temperature (23.2 °C) and humidity (55.5%). They were fed standard laboratory chow and given tap water. This study was approved by Gazi University Ethics Committee for Animals (G.U.ET-05.074). 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.
2.5. Statistics Nicotine-induced increases were expressed as percentages of the control and the average of seven EFS-evoked contractile responses in the presence of the nicotine. 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. pA2 value of antagonists were calculated from the formula at the below as described previously (Furchgott, 1972). pA2 = logðE2 =E1 −1Þ−log½B E2 E1 [B]
half-maximally concentration (EC50) value of agonist in the presence of antagonist EC50 value of agonist in the absence of antagonist Concentration of antagonist P values of b0.05 were considered to be statistically significant.
2.3. Organ chamber experiments
3. Results
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, KCl 4.7, CaCl2·2H2O 1.3, MgCl2·6H2O 0.5, Na2HPO4·2H2O 0.9, NaHCO3 24.9, glucose monohydrate 11). The pH of the solution was 7.4 after bubbling with a mixture of 95% O2 and 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) connected to an ink writing oscillograph (Grass 79 E) via a preamplifier. Thirty minutes after the EFS-evoked responses reached a steady state, to characterize the EFS-evoked contractile responses the tissues were treated with atropine (10− 6 M), nonspecific muscarinic receptor antagonist, tetrodotoxin (3 × 10− 6 M), a blocker of Na+ channels, or ω-conotoxin GVIA (10− 7 M), a blocker of pre-synaptic Ca+2 channels. To test the effects of nicotine, different concentrations (10− 4 M, 3 × 10 −4 M) of nicotine were administered to the preparations. To avoid any possible habituation effect or tachyphylaxis, EFS was stopped after seven contractions, and the preparations were washed for 1 hour in every 15 min as described previously (Ilhan et al., 2007, 2008). Following washing, EFS was delivered again and the same experimental procedure was performed with the same tissue in the presence of hexamethonium (a nonspecific nicotinic acetylcholine receptor antagonist; 10− 5 M, n = 8), dihydro-β-erythroidine (α4β2 nicotinic acetylcholine receptor antagonist; 10− 5 M, n = 8), mecamylamine (α3β4 nicotinic acetylcholine receptor antagonist; 10− 5 M, n = 8) or α-bungarotoxin (α7 nicotinic acetylcholine receptor antagonist; 3 × 10− 7 M, n = 8). Antagonists were added to the organ baths 30 min before the administration of nicotine.
EFS evoked contractile responses in rabbit stomach. The mean amplitude of the EFS-evoked contractile responses was 2.85 ± 0.42 g at a stimulation frequency of 8 Hz. Either tetrodotoxin (10− 6 M) or ω-conotoxin GVIA (10− 7 M) abolished the EFS-induced contractile responses. EFS-evoked contractile responses were also abolished by atropine (10− 6 M) in rabbit gastric fundus strips.
2.4. Drugs All drugs (atropine sulfate, tetrodotoxin, ω-conotoxin GVIA, nicotine, hexamethonium hydrochloride, dihydro-β-erythroidine, mecamylamine, α-bungarotoxin) were obtained from Sigma (St Louis, MO, USA). Stock solutions of drugs were dissolved in distilled water. Solutions were stored at −20 °C until use. The drugs were diluted in Krebs solution to the required final concentration on the day of use.
3.1. Effects of nicotine on neurogenic contractions of rabbit gastric fundus strips Nicotine increased the EFS-induced contractions (10− 4 M, 169.3 ± 10.3%; 3 × 10− 4 M, 221.5 ± 21.9%; P b 0.05). Nicotine-induced enhancements were reproducible and were not significantly changed during the second period of EFS after washing. No tachyphylaxis was observed. At 10− 4 M concentration, nicotine had no contractile effects on non-stimulated preparations. At 3 × 10− 4 M concentration nicotine
Fig. 1. Effect of different nicotinic acetylcholine receptor antagonists [hexamethonium (hexa., a nonspecific nicotinic acetylcholine receptor antagonist; 10− 5 M), dihydro-βerythroidine (DHβE, α4β2 nicotinic acetylcholine receptor antagonist; 10− 5 M), mecamylamine (mecamyl., α3β4 nicotinic acetylcholine receptor antagonist; 10− 5 M) or α-bungarotoxin (α-bung., α7 nicotinic acetylcholine receptor antagonist; 3 × 10− 7 M)] on nicotine (10− 4 M) induced EFS-evoked contractile response alternation in rabbit gastric fundus. 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 means ± S.E.M. (⁎, P b 0.05).
I.M. Vural et al. / European Journal of Pharmacology 602 (2009) 395–398
Fig. 2. Effect of different nicotinic acetylcholine receptor antagonists [hexamethonium (hexa., a nonspecific nicotinic acetylcholine receptor antagonist; 10− 5 M), dihydro-βerythroidine (DHβE, α4β2 nicotinic acetylcholine receptor antagonist; 10− 5 M), mecamylamine (mecamyl., α3β4 nicotinic acetylcholine receptor antagonist; 10− 5 M) or α-bungarotoxin (α-bung., α7 nicotinic acetylcholine receptor antagonist; 3 × 10− 7 M)] on nicotine (3 × 10− 4 M) induced EFS-evoked contractile response alternation in rabbit gastric fundus. 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 means ± S.E.M. (⁎, P b 0.05).
induced contractile responses before the EFS impulse in some tissues. These tissues were excluded from the study. 3.2. Effects of antagonists on nicotine-induced EFS-evoked transient neurogenic contractions Hexamethonium, dihydro-β-erythroidine and mecamylamine inhibited the nicotine induced EFS-evoked contractile response enhancement. α-bungarotoxin did not alter nicotine induced enhancement significantly (Figs. 1 and 2). The pA2 values of the antagonists were 4.67 ± 0.04 (hexamethonium, n = 8), 5.33 ± 0.02 (dihydro-β-erythroidine, n = 8) and 5.43 ± 0.02 (mecamylamine, n = 8). Hexamethonium, dihydro-β-erythroidine and α-bungarotoxin had no significant effect on EFS-evoked contractile responses. Mecamylamine (10− 5 M) enhanced EFS-evoked contractile responses significantly (22.44 ± 8.81%, n = 8; P b 0.05). 4. Discussion In the present study, tetrodotoxin, ω-conotoxin GVIA, or atropine abolished EFS-evoked responses, suggesting that EFS-evoked responses are induced by cholinergic nerve stimulation in the rabbit gastric fundus, as we previously demonstrated (Ilhan et al., 2007, 2008). We previously showed that nicotine, a nonspecific nicotinic acetylcholine receptor agonist, induced transient neurogenic increases in EFS-evoked cholinergic responses in a dose-dependent manner in the rabbit gastric fundus. Nicotine increased EFS-evoked contractions at 3 × 10− 6 M to 1 × 10− 4 M, but had no effect at 10− 6 M (Ilhan et al., 2007). In this study, we investigated the effects of nicotinic acetylcholine receptor antagonists on nicotine-induced alternation at sub-maximal and maximal concentrations of nicotine. Similar to our previous studies at high concentrations of nicotine, the latter transiently increased EFS-induced contractions. Nicotineinduced enhancements were reproducible and tachyphylaxis was not observed. In our previous studies, hexamethonium and cadmium (Cd2+), which block presynaptic voltage-gated calcium channels involved in EFS-evoked responses, prevented the potentiation caused
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by nicotine. This showed that nicotinic acetylcholine receptors are responsible for the effect of nicotine by a mechanism dependent on the influx of Ca2+ from voltage-gated calcium channels (Ilhan et al., 2007, 2008). A high concentration of H2O2 (10− 4 M) inhibited the increase of nicotine-induced neurogenic contractile response, and catalase (500 U/mL) enhanced the effect of nicotine (Ilhan et al., 2008). Endogenous nitric oxide and prostaglandins do not have a role in the regulation of nicotine-induced responses (Ilhan et al., 2007). We demonstrated nicotine-induced enhancements on EFS-induced responses via nicotinic acetylcholine receptors in various rabbit tissues (bladder, corpus cavernosum, myometrium) (Vural et al., 2006; Bozkurt et al., 2007; Nas et al., 2007). In this study, the nonspecific nicotinic acetylcholine receptor antagonist hexamethonium, the α4β2 nicotinic acetylcholine receptor antagonist dihydro-β-erythroidine, and the α3β4 nicotinic acetylcholine receptor antagonist mecamylamine inhibited nicotine-induced enhancements on EFS-induced cholinergic contraction responses. Subtypes of nicotinic acetylcholine receptor which have a role in neurotransmitter release were previously detected in central and peripheral tissues. It was suggested that α3, α4 and β2 are the dominant nicotinic acetylcholine receptor subunits involved in dopamine release in the cortex and striatum of the rat, but differences were observed between regions (Puttfarcken et al., 2000). It was demonstrated that two classes of striatal presynaptic nicotinic acetylcholine receptors mediate dopamine release in mice. One of the subtypes requires a β3 unit (α6β3β2 or α4α6β3β2) and the other subtype required a α4 unit (α4β2 or α4α5β2) (Salminen et al., 2004). The identity of nicotinic acetylcholine receptor subtypes that modulate GABA release was also demonstrated. It was reported that antagonists of α7 and α4β2 subtypes increase the frequency and amplitude of GABA-mediated postsynaptic currents in rat hippocampal slices (Alkondon et al., 1999). In other studies, it was suggested that identifying the nicotinic acetylcholine receptor subtypes that mediate neurotransmitter release in the central nervous system may require a different approach to the pathophysiology or treatment of various neurologic and psychiatric disorders (Albuquerque et al., 2000; Grady et al., 2007). Previously, research groups characterized nicotinic acetylcholine receptor subtypes in various peripheral tissues. In the rat gastric fundus, the roles of α3β4 and α4β2 nicotinic receptors in noradrenaline release were demonstrated; (−)-nicotine-induced noradrenaline release was abolished by mecamylamine and partially inhibited by dihydro-β-erythroidine (Yokotani et al., 2000). In another study, electrical stimulation of the splanchnic nerve induced noradrenaline release that was partially inhibited by mecamylamine, but dihydro-β-erythroidine did not alter noradrenaline release (Yokotani et al., 2001). In another study, α3β4 but not α4β2 nicotinic receptors were involved in catecholamine release from rat adrenal chromaffin cells (Yokotani et al., 2002). Different nicotinic acetylcholine receptor subtypes had been identified in the enteric nervous system of the guinea-pig. Expression of α3β2, α3β4, α3β2β4 and α7 nicotinic acetylcholine receptor subtypes were shown in myenteric plexuses and submucosa. It was also demonstrated that although nicotinic acetylcholine receptor expression varies from neurone to neurone, all subtypes are present in every ganglion and may play a part in the synaptic responses to stimulation (Obaid et al., 2005). In a different study, the role of nicotinic acetylcholine receptor subunits were investigated in mice with null mutations for β2 and β4 subunits. Simultaneous absence of β2 and β4 subunits altered autonomic control of several peripheral organs and affected survival and growth (Xu et al., 1999). The α7 nicotinic acetylcholine receptor antagonist α-bungarotoxin did not alter nicotine-induced enhancements in EFS-induced contraction responses in this study. It was reported that α7 subtype is the predominant nicotinic acetylcholine receptor subtype on gastrointestinal projecting neurons in the dorsal motor vagal nucleus. Co-expression of the α4β2 subtype in the neurons projecting to the
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stomach, as well as co-expression of the α3β4 subtype in the neurons projecting to the cecum, was demonstrated (Sahibzada et al., 2002). In a different study, it was suggested that the α7 nicotinic acetylcholine receptor subtype in the dorsal motor vagal nucleus has different properties from α7 homomeric nicotinic acetylcholine receptors and is located postsynaptically (Ferreira et al., 2001). It was demonstrated that presynaptic α7 nicotinic acetylcholine receptors mediate nicotine-induced vasodilation via nitric oxide in porcine basilar arteries (Si and Lee, 2001). The role of nicotine via the α7 receptor subtype in inflammation has been demonstrated in different studies (de Jonge and Ulloa, 2007; Park et al., 2007). Existence of the α7 nicotinic acetylcholine receptor subtype in the central nervous system was previously demonstrated. It was shown that α7 subtypes modulate GABA release from CA1 interneurons in rat hippocampal slices (Alkondon et al., 1999). Nicotinic acetylcholine receptors are predominantly located on the cholinergic intramural ganglia and presynaptic receptors on postganglionic nerve terminals in the stomach. The longitudinal preparation of the gastric fundus seems to be containing both nicotinic acetylcholine receptors on these places. In our study we demonstrated that α3β4 and α4β2 subtypes of nicotinic acetylcholine receptors play role on nicotine's effect. But we could not suggest the places on which these nicotinic acetylcholine receptor subtypes are located with our results. Previously either ganglionic or presinaptic nicotinic acetylcholine receptors mediated neurotransmitter release alternation was demonstrated in many different studies. Yokotoni et al. demonstrated that α3β4 nicotinic acetylcholine receptor subtypes which are probably located on gastric sympathetic ganglia, induce noradrenalin release (Yokotani et al., 2000). In a different study α7 and α3β4 nicotinic acetylcholine receptor subtypes determinated on neurons projecting to the stomach in the dorsal motor vagal nucleus (Sahibzada et al., 2002). Previously it was shown that different presynaptic nicotinic acetylcholine receptor subtypes including α7 and β2 subunits modulate neurotransmitter release (McGehee et al., 1995; Guo et al., 1998; Wonnacott et al., 2000; Rousseau et al., 2005). In this study, nicotine transiently increased EFS-induced contractile responses in a dose-dependent manner in the isolated gastric fundus of the rabbit. Hexamethonium, dihydro-β-erythroidine and mecamylamine inhibited the neurocontractile response to nicotine on EFS, but α-bungarotoxin did not alter these responses. These findings demonstrated that the α3β4 and α4β2 subtypes of nicotinic acetylcholine receptors play a part in the nicotine-induced augmentation in EFS-evoked cholinergic contractile responses in the rabbit gastric fundus. Further studies are needed to demonstrate the existence of α3β4 and α4β2 subtypes of nicotinic acetylcholine receptor proteins which play role in cholinergic neurotransmission in the gastric fundus. Acknowledgments This work was supported by Gazi University Unit of Scientific Research Projects (Project number: 01/2006-18) and Turkish Academy of Science. References Albuquerque, E.X., Pereira, E.F.R., Mike, A., Eisenberg, H.M., Maelicke, A., Alkondon, M., 2000. Neuronal nicotinic receptors in synaptic functions in humans and rats: physiological and clinical relevance. Behavioural Brain Research 113, 131–141. Alkondon, M., Pereira, E.F.R., Eisenberg, H.M., Albuquerque, E.X., 1999. Choline and selective antagonists identify two subtypes of nicotinic acetylcholine receptors that modulate GABA release from CA1 interneurons in rat hippocampal slices. Journal of Neuroscience 19, 2693–2705. 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. European Journal of Pharmacology 558, 172–178. Corriveau, R.A., Berg, D.K., 1993. Coexpression of multiple acetylcholine receptor genes in neurons: quantification of transcripts during development. Journal of Neuroscience 13, 2662–2671.
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Pulmonary, Gastrointestinal and Urogenital Pharmacology
Role of nicotinic acetylcholine receptor subtypes on nicotine-induced neurogenic contractile response alternation in the rabbit gastric fundus Ismail Mert Vural a,⁎, Gokce Sevim Ozturk Fincan a, Nihan Burul Bozkurt b, Zeynep Sevim Ercan a, Yusuf Sarioglu a a b
Department of Pharmacology, Medical School, Gazi University, Ankara, Turkey Department of Pharmacology, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 14 July 2008 Received in revised form 30 October 2008 Accepted 17 November 2008 Available online 25 November 2008 Keywords: Nicotine Nicotinic acetylcholine receptors subtypes Electrical field stimulation Rabbit Gastric fundus
a b s t r a c t Nicotine is a nonspecific agonist of nicotinic acetylcholine receptors. We previously demonstrated that nicotine increases the electrical field stimulation (EFS)-evoked contractile responses possibly by facilitating neurotransmitters release from nerve terminals by a mechanism dependent on the influx of Ca2+ from voltage gated Ca2+ channels via activation of nicotinic acetylcholine receptor. The aim of this study is to investigate subtypes of presynaptic nicotinic acetylcholine receptors involved in nicotine induced EFSevoked contractile response alternation in the rabbit gastric fundus. EFS-evoked contractile responses were recorded from gastric fundus strips obtained from rabbits with isometric force displacement transducers. Effects of nicotine on EFS evoked contractions were examined. Then the effect of nicotine on the EFS-evoked contractions was examined in the presence of hexamethonium, dihydro-β-erythroidine, mecamylamine or α-bungarotoxin. In our study, nicotine (10− 4, 3 × 10− 4 M) transiently increased neurogenic contraction induced by EFS in the rabbit isolated gastric fundus. While hexamethonium, dihydro-β-erythroidine and mecamylamine inhibited the neurocontractile response to nicotine on EFS, α-bungarotoxin did not alter these responses. The pA2 values of the antagonists were 4.67 (hexamethonium, n = 8), 5.33 (dihydro-βerythroidine, n = 8) and 5.43 (mecamylamine, n = 8). These findings showed that the α3β4 and α4β2 subunits of nicotinic acetylcholine receptors play a role on the nicotine-induced augmentation in EFS-evoked contractile responses in rabbit gastric fundus. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Nicotine acts as an agonist of nicotinic acetylcholine receptors, which belong to a superfamily of pentameric ligand-gated ion channels. These receptors are located in the central nervous system and peripheral nervous system (Newman et al., 2002; McGehee et al., 1995; Todorov et al., 1991). Nicotinic acetylcholine receptor subunits have been detected in molecular biologic studies. These receptors are composed of α (α2–α10, nine types) and β (β2–β4, three types) subunits (Corriveau and Berg, 1993; Mandelzys et al., 1995; Wonnacott, 1997; Vetter et al., 2007). Nicotine modulates neurotransmitter release via nicotinic acetylcholine receptors. It was demonstrated that nicotine increases the release of various neurotransmitters after nerve stimulation in central and peripheral tissues (Nedergaard and Schrold, 1977; Todorov et al., 1991; Yokotani et al., 2000). In the central nervous system, nicotinic acetylcholine receptor subtypes that have a role in various types of neurotransmitter release were detected in many studies. The role of
⁎ Corresponding author. Tel.: +90 312 2026951. E-mail address: [email protected] (I.M. Vural). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.11.032
α7, α4, β2, and β3 subunits on the release of γ-aminobutyric acid (GABA) or dopamine was demonstrated (Alkondon et al., 1999; Puttfarcken et al., 2000; Salminen et al., 2004). Yokotani et al. demonstrated that nicotinic acetylcholine receptor subtypes were involved in gastric noradrenaline release. Electrical stimulation of the splanchnic nerve induced noradrenaline release mediated partially by α3β4 nicotinic receptors, whereas (−)-nicotine induced noradrenaline release mediated by α3β4 and α4β2 nicotinic receptors (Yokotani et al., 2000, 2001). In another study, it was reported that the predominant nicotinic acetylcholine receptor subtype on gastrointestinal projecting neurons in the dorsal motor vagal nucleus is the α7 subtype. Co-expression of the α4β2 subtype in the neurons projecting to the fundus and antrum was also demonstrated (Sahibzada et al., 2002). We demonstrated that nicotine increased the electrical field stimulation (EFS)-evoked contractile or relaxation response, possibly by facilitating neurotransmitter release from nerve terminals, in various rabbit tissues: gastric fundus, bladder, corpus cavernosum, and myometrium. The effect of nicotine was dependent on the influx of Ca2+ from voltage-gated Ca2+ channels via activation of nicotinic acetylcholine receptors in the isolated gastric fundus (Vural et al., 2006; Bozkurt et al., 2007; Nas et al., 2007; Ilhan et al., 2007, 2008).
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In the present study, we aimed to characterize the nicotinic acetylcholine receptor subtypes involved in nicotine induced EFSevoked contractile response alternation in the rabbit gastric fundus. 2. Materials and methods 2.1. Animals Twenty New Zealand albino rabbits weighing 2.5–3 kg were used for the experiments. All animals were kept under controlled temperature (23.2 °C) and humidity (55.5%). They were fed standard laboratory chow and given tap water. This study was approved by Gazi University Ethics Committee for Animals (G.U.ET-05.074). 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.
2.5. Statistics Nicotine-induced increases were expressed as percentages of the control and the average of seven EFS-evoked contractile responses in the presence of the nicotine. 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. pA2 value of antagonists were calculated from the formula at the below as described previously (Furchgott, 1972). pA2 = logðE2 =E1 −1Þ−log½B E2 E1 [B]
half-maximally concentration (EC50) value of agonist in the presence of antagonist EC50 value of agonist in the absence of antagonist Concentration of antagonist P values of b0.05 were considered to be statistically significant.
2.3. Organ chamber experiments
3. Results
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, KCl 4.7, CaCl2·2H2O 1.3, MgCl2·6H2O 0.5, Na2HPO4·2H2O 0.9, NaHCO3 24.9, glucose monohydrate 11). The pH of the solution was 7.4 after bubbling with a mixture of 95% O2 and 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) connected to an ink writing oscillograph (Grass 79 E) via a preamplifier. Thirty minutes after the EFS-evoked responses reached a steady state, to characterize the EFS-evoked contractile responses the tissues were treated with atropine (10− 6 M), nonspecific muscarinic receptor antagonist, tetrodotoxin (3 × 10− 6 M), a blocker of Na+ channels, or ω-conotoxin GVIA (10− 7 M), a blocker of pre-synaptic Ca+2 channels. To test the effects of nicotine, different concentrations (10− 4 M, 3 × 10 −4 M) of nicotine were administered to the preparations. To avoid any possible habituation effect or tachyphylaxis, EFS was stopped after seven contractions, and the preparations were washed for 1 hour in every 15 min as described previously (Ilhan et al., 2007, 2008). Following washing, EFS was delivered again and the same experimental procedure was performed with the same tissue in the presence of hexamethonium (a nonspecific nicotinic acetylcholine receptor antagonist; 10− 5 M, n = 8), dihydro-β-erythroidine (α4β2 nicotinic acetylcholine receptor antagonist; 10− 5 M, n = 8), mecamylamine (α3β4 nicotinic acetylcholine receptor antagonist; 10− 5 M, n = 8) or α-bungarotoxin (α7 nicotinic acetylcholine receptor antagonist; 3 × 10− 7 M, n = 8). Antagonists were added to the organ baths 30 min before the administration of nicotine.
EFS evoked contractile responses in rabbit stomach. The mean amplitude of the EFS-evoked contractile responses was 2.85 ± 0.42 g at a stimulation frequency of 8 Hz. Either tetrodotoxin (10− 6 M) or ω-conotoxin GVIA (10− 7 M) abolished the EFS-induced contractile responses. EFS-evoked contractile responses were also abolished by atropine (10− 6 M) in rabbit gastric fundus strips.
2.4. Drugs All drugs (atropine sulfate, tetrodotoxin, ω-conotoxin GVIA, nicotine, hexamethonium hydrochloride, dihydro-β-erythroidine, mecamylamine, α-bungarotoxin) were obtained from Sigma (St Louis, MO, USA). Stock solutions of drugs were dissolved in distilled water. Solutions were stored at −20 °C until use. The drugs were diluted in Krebs solution to the required final concentration on the day of use.
3.1. Effects of nicotine on neurogenic contractions of rabbit gastric fundus strips Nicotine increased the EFS-induced contractions (10− 4 M, 169.3 ± 10.3%; 3 × 10− 4 M, 221.5 ± 21.9%; P b 0.05). Nicotine-induced enhancements were reproducible and were not significantly changed during the second period of EFS after washing. No tachyphylaxis was observed. At 10− 4 M concentration, nicotine had no contractile effects on non-stimulated preparations. At 3 × 10− 4 M concentration nicotine
Fig. 1. Effect of different nicotinic acetylcholine receptor antagonists [hexamethonium (hexa., a nonspecific nicotinic acetylcholine receptor antagonist; 10− 5 M), dihydro-βerythroidine (DHβE, α4β2 nicotinic acetylcholine receptor antagonist; 10− 5 M), mecamylamine (mecamyl., α3β4 nicotinic acetylcholine receptor antagonist; 10− 5 M) or α-bungarotoxin (α-bung., α7 nicotinic acetylcholine receptor antagonist; 3 × 10− 7 M)] on nicotine (10− 4 M) induced EFS-evoked contractile response alternation in rabbit gastric fundus. 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 means ± S.E.M. (⁎, P b 0.05).
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Fig. 2. Effect of different nicotinic acetylcholine receptor antagonists [hexamethonium (hexa., a nonspecific nicotinic acetylcholine receptor antagonist; 10− 5 M), dihydro-βerythroidine (DHβE, α4β2 nicotinic acetylcholine receptor antagonist; 10− 5 M), mecamylamine (mecamyl., α3β4 nicotinic acetylcholine receptor antagonist; 10− 5 M) or α-bungarotoxin (α-bung., α7 nicotinic acetylcholine receptor antagonist; 3 × 10− 7 M)] on nicotine (3 × 10− 4 M) induced EFS-evoked contractile response alternation in rabbit gastric fundus. 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 means ± S.E.M. (⁎, P b 0.05).
induced contractile responses before the EFS impulse in some tissues. These tissues were excluded from the study. 3.2. Effects of antagonists on nicotine-induced EFS-evoked transient neurogenic contractions Hexamethonium, dihydro-β-erythroidine and mecamylamine inhibited the nicotine induced EFS-evoked contractile response enhancement. α-bungarotoxin did not alter nicotine induced enhancement significantly (Figs. 1 and 2). The pA2 values of the antagonists were 4.67 ± 0.04 (hexamethonium, n = 8), 5.33 ± 0.02 (dihydro-β-erythroidine, n = 8) and 5.43 ± 0.02 (mecamylamine, n = 8). Hexamethonium, dihydro-β-erythroidine and α-bungarotoxin had no significant effect on EFS-evoked contractile responses. Mecamylamine (10− 5 M) enhanced EFS-evoked contractile responses significantly (22.44 ± 8.81%, n = 8; P b 0.05). 4. Discussion In the present study, tetrodotoxin, ω-conotoxin GVIA, or atropine abolished EFS-evoked responses, suggesting that EFS-evoked responses are induced by cholinergic nerve stimulation in the rabbit gastric fundus, as we previously demonstrated (Ilhan et al., 2007, 2008). We previously showed that nicotine, a nonspecific nicotinic acetylcholine receptor agonist, induced transient neurogenic increases in EFS-evoked cholinergic responses in a dose-dependent manner in the rabbit gastric fundus. Nicotine increased EFS-evoked contractions at 3 × 10− 6 M to 1 × 10− 4 M, but had no effect at 10− 6 M (Ilhan et al., 2007). In this study, we investigated the effects of nicotinic acetylcholine receptor antagonists on nicotine-induced alternation at sub-maximal and maximal concentrations of nicotine. Similar to our previous studies at high concentrations of nicotine, the latter transiently increased EFS-induced contractions. Nicotineinduced enhancements were reproducible and tachyphylaxis was not observed. In our previous studies, hexamethonium and cadmium (Cd2+), which block presynaptic voltage-gated calcium channels involved in EFS-evoked responses, prevented the potentiation caused
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by nicotine. This showed that nicotinic acetylcholine receptors are responsible for the effect of nicotine by a mechanism dependent on the influx of Ca2+ from voltage-gated calcium channels (Ilhan et al., 2007, 2008). A high concentration of H2O2 (10− 4 M) inhibited the increase of nicotine-induced neurogenic contractile response, and catalase (500 U/mL) enhanced the effect of nicotine (Ilhan et al., 2008). Endogenous nitric oxide and prostaglandins do not have a role in the regulation of nicotine-induced responses (Ilhan et al., 2007). We demonstrated nicotine-induced enhancements on EFS-induced responses via nicotinic acetylcholine receptors in various rabbit tissues (bladder, corpus cavernosum, myometrium) (Vural et al., 2006; Bozkurt et al., 2007; Nas et al., 2007). In this study, the nonspecific nicotinic acetylcholine receptor antagonist hexamethonium, the α4β2 nicotinic acetylcholine receptor antagonist dihydro-β-erythroidine, and the α3β4 nicotinic acetylcholine receptor antagonist mecamylamine inhibited nicotine-induced enhancements on EFS-induced cholinergic contraction responses. Subtypes of nicotinic acetylcholine receptor which have a role in neurotransmitter release were previously detected in central and peripheral tissues. It was suggested that α3, α4 and β2 are the dominant nicotinic acetylcholine receptor subunits involved in dopamine release in the cortex and striatum of the rat, but differences were observed between regions (Puttfarcken et al., 2000). It was demonstrated that two classes of striatal presynaptic nicotinic acetylcholine receptors mediate dopamine release in mice. One of the subtypes requires a β3 unit (α6β3β2 or α4α6β3β2) and the other subtype required a α4 unit (α4β2 or α4α5β2) (Salminen et al., 2004). The identity of nicotinic acetylcholine receptor subtypes that modulate GABA release was also demonstrated. It was reported that antagonists of α7 and α4β2 subtypes increase the frequency and amplitude of GABA-mediated postsynaptic currents in rat hippocampal slices (Alkondon et al., 1999). In other studies, it was suggested that identifying the nicotinic acetylcholine receptor subtypes that mediate neurotransmitter release in the central nervous system may require a different approach to the pathophysiology or treatment of various neurologic and psychiatric disorders (Albuquerque et al., 2000; Grady et al., 2007). Previously, research groups characterized nicotinic acetylcholine receptor subtypes in various peripheral tissues. In the rat gastric fundus, the roles of α3β4 and α4β2 nicotinic receptors in noradrenaline release were demonstrated; (−)-nicotine-induced noradrenaline release was abolished by mecamylamine and partially inhibited by dihydro-β-erythroidine (Yokotani et al., 2000). In another study, electrical stimulation of the splanchnic nerve induced noradrenaline release that was partially inhibited by mecamylamine, but dihydro-β-erythroidine did not alter noradrenaline release (Yokotani et al., 2001). In another study, α3β4 but not α4β2 nicotinic receptors were involved in catecholamine release from rat adrenal chromaffin cells (Yokotani et al., 2002). Different nicotinic acetylcholine receptor subtypes had been identified in the enteric nervous system of the guinea-pig. Expression of α3β2, α3β4, α3β2β4 and α7 nicotinic acetylcholine receptor subtypes were shown in myenteric plexuses and submucosa. It was also demonstrated that although nicotinic acetylcholine receptor expression varies from neurone to neurone, all subtypes are present in every ganglion and may play a part in the synaptic responses to stimulation (Obaid et al., 2005). In a different study, the role of nicotinic acetylcholine receptor subunits were investigated in mice with null mutations for β2 and β4 subunits. Simultaneous absence of β2 and β4 subunits altered autonomic control of several peripheral organs and affected survival and growth (Xu et al., 1999). The α7 nicotinic acetylcholine receptor antagonist α-bungarotoxin did not alter nicotine-induced enhancements in EFS-induced contraction responses in this study. It was reported that α7 subtype is the predominant nicotinic acetylcholine receptor subtype on gastrointestinal projecting neurons in the dorsal motor vagal nucleus. Co-expression of the α4β2 subtype in the neurons projecting to the
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stomach, as well as co-expression of the α3β4 subtype in the neurons projecting to the cecum, was demonstrated (Sahibzada et al., 2002). In a different study, it was suggested that the α7 nicotinic acetylcholine receptor subtype in the dorsal motor vagal nucleus has different properties from α7 homomeric nicotinic acetylcholine receptors and is located postsynaptically (Ferreira et al., 2001). It was demonstrated that presynaptic α7 nicotinic acetylcholine receptors mediate nicotine-induced vasodilation via nitric oxide in porcine basilar arteries (Si and Lee, 2001). The role of nicotine via the α7 receptor subtype in inflammation has been demonstrated in different studies (de Jonge and Ulloa, 2007; Park et al., 2007). Existence of the α7 nicotinic acetylcholine receptor subtype in the central nervous system was previously demonstrated. It was shown that α7 subtypes modulate GABA release from CA1 interneurons in rat hippocampal slices (Alkondon et al., 1999). Nicotinic acetylcholine receptors are predominantly located on the cholinergic intramural ganglia and presynaptic receptors on postganglionic nerve terminals in the stomach. The longitudinal preparation of the gastric fundus seems to be containing both nicotinic acetylcholine receptors on these places. In our study we demonstrated that α3β4 and α4β2 subtypes of nicotinic acetylcholine receptors play role on nicotine's effect. But we could not suggest the places on which these nicotinic acetylcholine receptor subtypes are located with our results. Previously either ganglionic or presinaptic nicotinic acetylcholine receptors mediated neurotransmitter release alternation was demonstrated in many different studies. Yokotoni et al. demonstrated that α3β4 nicotinic acetylcholine receptor subtypes which are probably located on gastric sympathetic ganglia, induce noradrenalin release (Yokotani et al., 2000). In a different study α7 and α3β4 nicotinic acetylcholine receptor subtypes determinated on neurons projecting to the stomach in the dorsal motor vagal nucleus (Sahibzada et al., 2002). Previously it was shown that different presynaptic nicotinic acetylcholine receptor subtypes including α7 and β2 subunits modulate neurotransmitter release (McGehee et al., 1995; Guo et al., 1998; Wonnacott et al., 2000; Rousseau et al., 2005). In this study, nicotine transiently increased EFS-induced contractile responses in a dose-dependent manner in the isolated gastric fundus of the rabbit. Hexamethonium, dihydro-β-erythroidine and mecamylamine inhibited the neurocontractile response to nicotine on EFS, but α-bungarotoxin did not alter these responses. These findings demonstrated that the α3β4 and α4β2 subtypes of nicotinic acetylcholine receptors play a part in the nicotine-induced augmentation in EFS-evoked cholinergic contractile responses in the rabbit gastric fundus. Further studies are needed to demonstrate the existence of α3β4 and α4β2 subtypes of nicotinic acetylcholine receptor proteins which play role in cholinergic neurotransmission in the gastric fundus. Acknowledgments This work was supported by Gazi University Unit of Scientific Research Projects (Project number: 01/2006-18) and Turkish Academy of Science. References Albuquerque, E.X., Pereira, E.F.R., Mike, A., Eisenberg, H.M., Maelicke, A., Alkondon, M., 2000. Neuronal nicotinic receptors in synaptic functions in humans and rats: physiological and clinical relevance. Behavioural Brain Research 113, 131–141. Alkondon, M., Pereira, E.F.R., Eisenberg, H.M., Albuquerque, E.X., 1999. Choline and selective antagonists identify two subtypes of nicotinic acetylcholine receptors that modulate GABA release from CA1 interneurons in rat hippocampal slices. Journal of Neuroscience 19, 2693–2705. 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. European Journal of Pharmacology 558, 172–178. Corriveau, R.A., Berg, D.K., 1993. Coexpression of multiple acetylcholine receptor genes in neurons: quantification of transcripts during development. Journal of Neuroscience 13, 2662–2671.
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