Introduction

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Acetylcholine (ACh) acting at nicotinic cholinergic receptors (nAChRs) is the principal excitatory neurotransmitter in the myenteric plexus. lntracellular electrophysiological studies have shown that fast excitatory postsynaptic potentials recorded from myenteric neurons are at least partly inhibited by nAChR antagonists such as hexamethonium (Nishi and North, 1973; Hirst et al., 1974; Galligan and Bertrand, 1994). Excitatory and inhibitory intestinal motor reflexes caused by distention of the gut wall or mucosal stimulation are also inhibited by hexamethonium or other nAChR antagonists (Costa and Furness, 1976; Bartho et al., 1987; Holzer, 1989; Johnson et al., 1996). Finally, studies in vivo have shown gastrointestinal motility is inhibited by nAChR antagonists (Sarna et al., 1981; Al-Saffar, 1984; Galligan et al., 1986). These observations highlight the central role of nAChRs in the control of gastrointestinal motor function.

Data from molecular biological and functional studies indicate that there are multiple subtypes of neuronal nAChRs (Sargent, 1993; Boyd, 1997). These receptor subtypes are composed of different combinations of  and  subunits, and specific subunit composition gives these receptors unique pharmacological and functional properties (Colquhoun and Patrick, 1997). However, little is known about the molecular composition, pharmacological properties, or subcellular distribution of enteric nAChRs. Recent, immunohistochemical studies using antibodies that recognize 3, 5, and 4 subunits (MAb-35) or the 7 subunit have localized nAChR immunoreactivity to nerve cell bodies, dendrites and nerve terminals in enteric ganglia (Kirchgessner and Liu, 1998). The traditional model of enteric ganglionic neurotransmission has the nAChR localized to the somatodendritic region of the neuron where it is in a position to mediate fast excitatory neurotransmission (Töröcsik et al., 1991). The localization of nAChRs on the somatodendritic region of enteric neurons is consistent with this model (Kirchgessner and Liu, 1998). However, it has also been established that nAChRs can be localized on nerve endings where these receptors function to cause release of neurotransmitter (McGehee et al., 1995; Gray et al., 1996; Wonnacott, 1997). Agonists acting at these receptors cause transmitter release using a calcium-dependent but action potential-independent mechanism as agonist induced transmitter release from preparations containing intact nerves is resistant to tetrodotoxin (TTX) or nAChR agonists can release transmitter from synaptosomal preparations (Wonnacott, 1997). These data may be important for understanding the actions of nAChR agonists on the gastrointestinal tract as some contractile responses caused by these drugs are partly resistant to TTX (Romano, 1981; Maggi et al., 1985; Börjesson et al., 1997) and nicotine can release transmitter from myenteric synaptosomes (White, 1982).

The purpose of the present study was to examine the actions of nAChR agonists on longitudinal muscle contractions of guinea pig ileum. These experiments were designed to determine whether nAChRs were localized exclusively to the somatodendritic region or to nerve terminals as well.

	Materials and Methods

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Tissue Preparation. All animal use protocols were approved by the All University Committee on Animal Use and Care at Michigan State University. Male guinea pigs (300-450 g) were anesthetized via halothane inhalation and then were stunned and the carotid arteries were severed. A segment of distal ileum was removed from the animal and placed in Krebs' solution of the following composition: NaCl, 117 mM; KCI, 4.7 mM; CaCl₂, 2.5 mM; MgCl₂, 1.2 mM; NaH₂PO₄, 1.2 mM; NaHCO₃, 25 mM; and glucose, 11 mM. The Krebs' solution was oxygenated continuously with 95% O₂, 5% CO₂. Whole segments of ileum cut to 3 cm in length were mounted in Plexiglas tissue holders and the tissue and holder were placed in jacketed baths (20-ml volume). One end of the segment was attached to a screw at the base of the holder using 4-0 silk suture whereas the other end was attached to a force transducer (FT03C; Grass Instruments, Quincy, MA) also using 4-0 silk suture. Tissues were placed under 10 mN of tension and were allowed to equilibrate for 30 min before starting experimental protocols.

Agonist-Concentration Response Curves. The nAChR agonists, cytisine, nicotine, and dimethylphenylpiperazinium iodide (DMPP) were added to the baths in volumes of 20 to 60 µl. Concentration-response curves were obtained by measuring the peak contraction caused by each concentration of agonist. Successive agonist concentrations were applied at 20-min intervals and each concentration was applied for 1 min. The order in which concentrations of agonist were applied was randomized among tissues.

Antagonist Studies. In all tissues, a control concentration-response curve for an agonist was obtained before testing the effects of an antagonist. Increasing concentrations (10, 30, and 100 µM) of the competitive nAChR antagonist, dihydro--erythroidine (DHE), was incubated with the tissues for 20 to 30 min before obtaining a subsequent agonist concentration-response curve. A control curve and curves in the presence of increasing concentrations of antagonist were obtained in each tissue. The DHE K_B value was determined using the method of Arunlakshana and Schild (1959). Studies of noncholinergic contractions caused by nAChR agonists were accomplished by adding 1 µM scopolamine to the Krebs' solution. The neurokinin-1 (NK-1) receptor antagonist CP 96,345-1 (Snider et al., 1991) was used to demonstrate that the noncholinergic contractions were mediated by a tachykinin peptide. Concentration-response curves for nAChR agonists were obtained first in the presence of scopolamine and a second agonist concentration-response curve was obtained in the presence of scopolamine and 0.3 µM CP 96345-1. CP 96345-1 was incubated with the tissues for 20 min before agonist addition.

Statistics. Agonist concentration-response curves from individual preparations were fit using the logistic function:
where "Y_min" and "Y_max" are minimum and maximum responses, respectively, "EC₅₀" is the half-maximal effective concentration, and "m" is the slope factor. Agonist mean EC₅₀ values and 95% confidence intervals were determined from individual fits. In studies in which complete concentration-response curves were not obtained, agonist response amplitudes in the absence and presence of an antagonist were compared using the univariate procedure from SAS/STAT version 6.12 (SAS Institute Inc., Cary, NC) to test for normality and for subsequent comparisons using the sign test. Antagonist K_B values were determined from Schild plots. Schild regressions were constructed from plots of the ratio of individual agonist EC₅₀ values obtained in the absence and presence of antagonist. When the slope of the regression did not differ from unity, it was constrained to a value of 1 and the K_B was determined from the x-intercept (pA₂ value). Data are mean values ± S.E. "N" values refer to the number of tissues from which data were obtained.

	Results

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Agonist Concentration-Response Curves. Nicotine, DMPP, and cytisine caused concentration-dependent contractions of whole ileal preparations. The contractions were biphasic with an initial, rapidly developing peak contraction followed by a slower developing but longer lasting secondary contraction. Nicotine caused the most prominent secondary contraction (Fig. 1). When the peak contraction was measured, the agonist rank order potency was DMPP > cytisine = nicotine (Fig. 2, Table 1).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Representative traces of longitudinal muscle contractions caused by increasing concentrations of nAChR agonists. Each agonist elicited a biphasic response. A, contractions caused by DMPP at the indicated µM concentrations. B, contractions caused by nicotine at the indicated µM concentrations. C, contractions caused by cytisine at the indicated µM concentration. Scale bars are approximately 10 mN and 1 min.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Concentration response curves for the peak contraction caused by DMPP (; n = 22 tissues from 5 animals), nicotine (; n = 19 tissues from 5 animals), and cytisine (; n = 19 tissues from 5 animals). Curves are fits using a logistic function as described in Materials and Methods.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Longitudinal muscle contractions caused by DMPP and nicotine are blocked by TTX. A, peak contraction caused by the indicated concentrations of DMPP are inhibited by TTX. B, peak contraction caused by the indicated concentrations of nicotine are inhibited by TTX. For both figures, data are the mean + S.E. obtained from eight preparations from four animals. *, significantly different from control responses (sign test, P < .05).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   DHE competitively inhibits contractions caused by DMPP. A, DMPP concentration-response curves in the absence and presence of the indicated concentrations of DHE (, control; , 10 µM; , 30 µM; , 100 µM). Increasing concentrations of DHE cause successive rightward shifts in the DMPP concentration-response curves. Data are expressed as mean ± S.E. and were obtained from six preparations from two animals. B, Schild regression of the rightward shifts in the DMPP concentration-response curves caused by DHE shown in A. Points are the individual log dose ratio-1 values from each preparation. Solid line is the best fit (slope constrained to 1) and the dotted lines are the 95% confidence limits of the fit. The pA2 value was 5.4.

Noncholinergic Contractions. To study excitation of noncholinergic excitatory motoneurons by nAChR agonists, 1 µM scopolamine was added to the Krebs' solution for all of these studies. DMPP, nicotine, and cytisine caused concentration-dependent contractions of the longitudinal muscle (Fig. 5). The maximum contractions caused by each agonist in the presence of scopolamine were less than half the amplitude caused by the same agonist concentrations in normal Krebs' solution (Fig. 6; compare with Fig. 2). DMPP and cytisine were approximately 8- and 2.5-fold less potent in causing noncholinergic contractions compared with contractions studied in normal Krebs' solution (Table 1). Nicotine was equipotent in eliciting contractions in normal Krebs' solution and in scopolamine-containing Krebs' solution (Table 1). The agonist rank order potency for noncholinergic contractions was DMPP = nicotine > cytisine (Fig. 6, Table 1). Because the peak amplitude of the contractions caused by cytisine was relatively small (see Fig. 6), characterization of responses caused by cytisine was not attempted.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Representative traces of noncholinergic contractions of longitudinal muscle caused by DMPP, nicotine, and cytisine. All responses were obtained in the presence of 1 µM scopolamine. A, contractions caused by the indicated µM concentrations of DMPP. B, contractions caused by the indicated µM concentrations of nicotine. C, contractions caused by the indicated µM concentrations of cytisine. Scale bar is 10 mN and 1 min.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Concentration-response curves for noncholinergic contractions caused by nicotine (; n = 18 tissues from 5 animals), DMPP (; n = 16 tissues from 5 animals), and cytisine (; n = 12 tissues from 4 animals). Data are expressed as grams tension and are the mean ± S.E. Scopolamine (1 µM) was present throughout these experiments. Curves are fits using a logistic function as described in Materials and Methods.

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Noncholinergic contractions caused by DMPP and nicotine are only partly blocked by TTX. A, peak contraction caused by the indicated concentrations of DMPP are inhibited by 60 to 100% by TTX. B, peak contraction caused by the indicated concentrations of nicotine are inhibited 40 to 100% by TTX. For both figures, data are mean + S.E. of 8 to 18 preparations from two to five animals per bar. *, significantly different from control responses (P < .05, sign test).

View larger version (23K): [in this window] [in a new window]   Fig. 8.   DHE competitively inhibits noncholinergic contractions caused by DMPP. A, DMPP concentration-response curves in the absence () and presence of the indicated concentrations (, 10 µM; , 30 µM; , 100 µM) of DHE. Increasing concentrations of DHE cause successive rightward shifts in the DMPP concentration response curves. Data are mean ± S.E. and were obtained from six preparations from two animals; 1 µM scopolamine was present throughout. B, Schild regression of the rightward shifts in the DMPP concentration-response curves caused by DHE shown in A. Points are individual log dose ratio-1 values from each preparation. Solid line is the best fit (slope constrained to 1) and the dotted lines are the 95% confidence limits of the fit. The pA2 value was 5.4.

View larger version (17K): [in this window] [in a new window]   Fig. 9.   The NK-1 receptor antagonist, CP 96,345-1, inhibits noncholinergic contractions caused by DMPP and nicotine. A, concentration-response curves for DMPP-induced contractions in the absence () and presence () of 0.3 µM CP 96,345-1. B, concentration-response curves for nicotine-induced contractions in the absence () and presence () of 0.3 µM CP 96,345-1. In both figures data are the mean ± S.E. of eight preparations obtained from two animals; *, indicates points significantly different from control responses (sign test, P < .05). Scopolamine (1 µM) was present throughout these experiments.

	Discussion

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Synaptic interactions between myenteric nerves that result in normal contractions and relaxations of gastrointestinal smooth muscle are complex and involve multiple transmitters and synaptic mechanisms (Kunze and Furness, 1999). Most studies of these synaptic events have focused on postsynaptic mechanisms. However, data from the present study indicate that presynaptic receptors may be an additional site at which neurotransmitters can modulate the activity of other neurons. The data presented here indicate that there are presynaptic nAChRs on nerve terminals that release tachykinins to cause contractions of longitudinal smooth muscle.

Presynaptic nAChRs. In normal Krebs' solution, nicotine, DMPP, and cytisine caused longitudinal muscle contractions that were nerve-mediated as they were blocked by TTX. These data are consistent with established enteric circuitry in which motoneurons in the gastrointestinal tract express somal nAChRs (Bornstein et al., 1994). The contraction caused by each nAChR agonist was also due largely to excitation of cholinergic motoneurons as agonist-induced contractions were inhibited by the muscarinic cholinergic receptor antagonist scopolamine. However, each nAChR agonist induced a residual contraction that persisted in the presence of scopolamine. The noncholinergic contractions are due to release of the tachykinin peptides, SP and NKA (Grider, 1989; Holzer, 1989; Yunker and Galligan, 1996). This observation was confirmed in the present study when it was shown that the noncholinergic contractions caused by nicotine and DMPP were blocked by the NK-1 receptor antagonist CP 96,345-1. However, it was also shown that contractions caused by nicotine and DMPP were incompletely inhibited by TTX. It is unlikely that the concentration of TTX used was insufficient to block nerve-mediated responses as electrically-evoked contractions of the nerve muscle preparations of guinea pig ileum are completely blocked by the concentration of TTX (0.3 µM) used here (see Yunker and Galligan, 1996 for example). Therefore, DMPP and nicotine were acting via a TTX-resistant mechanism to cause noncholinergic contractions.

Pharmacological Properties of Myenteric Nicotinic Receptors. The subunit composition of neuronal nAChRs determines the functional and pharmacological properties of the receptor (Colquhoun and Patrick, 1997). In normal Krebs' solution or in scopolamine-containing solutions, cytisine was either equipotent with or less potent than nicotine or DMPP in causing longitudinal muscle contractions. For receptors containing 4 subunits, cytisine is the most potent agonist (Luetje and Patrick, 1994; Papke and Heinemann, 1994) so it is unlikely that the nAChR(s) involved in the responses studied here contained 4 subunits. lmmunohistochemical studies of myenteric neurons using an antibody that recognizes the 3, 5, and 4 subunits (MAb-35; Vernallis et al., 1993) showed immunoreactivity in neurons that also contain calretinin, a marker for excitatory longitudinal muscle motoneurons (Brookes et al., 1992; Kirchgessner and Liu, 1998). Data from the present pharmacological studies suggest that nAChRs containing 4 subunits are not expressed by excitatory longitudinal muscle motoneurons and that MAb-35 immunoreactivity indicates the expression of either (3, 5, or both of these subunits in excitatory longitudinal muscle motoneurons.

Conclusions. In guinea pig ileum myenteric plexus, excitatory longitudinal muscle motoneurons express nAChRs on the somatodendritic region and on nerve terminals. Nerve terminal nAChRs function to stimulate release of SP/NKA as mediators of noncholinergic contractions of the longitudinal muscle layer (Fig. 10). The nerve terminal nAChRs may act as facilitatory autoreceptors responding to ACh coreleased from the same nerves releasing SP/NKA.

View larger version (24K): [in this window] [in a new window]   Fig. 10.   nAChRs localized to the somatodendritic and nerve terminal regions of excitatory motoneurons mediate noncholinergic contractions of the longitudinal muscle layer. A, DMPP, nicotine, and cytisine act at somatodendritic and nerve terminal nAChRs to cause release of ACh and SP/NKA. As scopolamine was present, ACh can not activate muscarinic receptors (M) but SP/NKA can activate NK-1 receptors localized to the longitudinal smooth muscle. B, in the presence of TTX, DMPP, nicotine, and cytisine can activate somatodendritic nAChRs, but action potentials initiated in the somatodendritic region can not propagate to the nerve terminal. DMPP and nicotine cause longitudinal muscle contractions by activating nerve terminal nAChRs to cause release of ACh and SP/NKA. As ACh and SP/NKA are colocalized in longitudinal muscle motoneurons, nerve terminal nAChRs may function as autoreceptors for ACh released from these nerve endings (?).