Modulation of cholinergic neuromuscular transmission by nitric oxide in canine colonic circular smooth muscle

M. G. Rae, M. A. Khoyi, and K. D. Keef

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

This study examines the effect of nitric oxide (NO) on cholinergic transmission in strips of canine colonic circular muscle in which neural plexus-pacemaker regions had been removed. Electrical field stimulation gave rise to atropine- and TTX-sensitive excitatory junction potentials (EJPs), the amplitude of which were frequency dependent. In 47% of control muscles, the EJP was followed by an inhibitory junction potential (IJP), whereas in the presence of atropine all preparations exhibited only IJPs. The NO synthase inhibitor Nomega -nitro-L-arginine (L-NNA), the guanylyl cyclase inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxaline-1-one (ODQ), and the protein kinase G (PKG) antagonist Rp-8-bromo-PET-cGMPS all significantly increased EJP amplitude and reduced or abolished IJPs. The potentiation of EJPs by L-NNA was reversed by the NO donors sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine in a manner blocked by ODQ. [14C]ACh overflow was also measured to evaluate the possible prejunctional effects of NO. Both norepinephrine and TTX significantly decreased [14C]ACh overflow; however, L-NNA, ODQ, and SNP were without effect. These data suggest that both cholinergic and nitrergic motoneurons functionally innervate the interior of the circular muscle layer. The inhibitory actions of NO on cholinergic transmission appear to be post- rather than prejunctional and to involve guanylyl cyclase as well as possibly PKG.

smooth muscle electrophysiology; enteric nerves; acetylcholine release; gastrointestinal tract

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

ENTERIC MOTONEURONS IN the canine proximal colon contain a number of excitatory and inhibitory neurotransmitter substances. Of these, two of the most functionally prominent are ACh and nitric oxide (NO). The role of ACh as an excitatory transmitter in gastrointestinal (GI) smooth muscle has been established for more than half a century and is well described in the canine proximal colon (11, 19, 23). NO has been more recently identified as an inhibitory neurotransmitter substance in the GI tract. Nonetheless, a substantial amount of literature now exists that documents the importance of this substance as a mediator of inhibitory neural responses in the GI tract (27, 30). Electrophysiological studies of the canine colon have suggested that inhibitory neural responses in this muscle can largely be accounted for by neuronally released NO (18, 36). Indeed, histological studies of this tissue have demonstrated that varicose nerve fibers containing NO synthase (NOS) are located in neurons throughout the muscularis externa (37).

Because the muscularis externa is innervated by both inhibitory and excitatory motoneurons, the transmitters that are released may have prejunctional neuromodulatory effects and they may modulate the activity of the effector smooth muscle. For example, in a number of different GI preparations, NO suppresses excitatory neuromuscular transmission (2, 24, 26, 33, 38). Consensus, however, remains divided over the relative importance of pre- vs. postjunctional inhibition.

In the present study, the electrical events underlying neuromuscular transmission from enteric excitatory and inhibitory motor nerves were investigated in the canine colonic circular muscle layer. To accomplish this, muscle strips that had neural plexus-pacemaker regions removed were used so that junctional transmission in the interior of the muscle layer could be characterized in the absence of ganglionic events and pacemaker potentials. The neuromodulatory effects of NO on cholinergic transmission were also investigated by examining the actions of selected modulators of the NO pathway on junction potentials recorded with microelectrodes and on the overflow of [14C]ACh.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Mongrel dogs of either sex were killed with an overdose of pentobarbital sodium (45 mg/kg). The abdomen was opened, and a segment of proximal colon, 6-14 cm from the ileocecal sphincter, was removed. The colon was opened along the mesenteric border, cleared of remaining fecal matter, and pinned out in a dissecting dish containing oxygenated (95% O2-5% CO2) Krebs-Ringer bicarbonate solution (KRB) of the following composition (in mM): 142.3 Na+, 5.9 K+, 2.5 Ca2+, 1.2 Mg2+, 130.6 Cl-, 23.8 HCO-3, 1.2 H2PO-4, and 11.0 dextrose, pH 7.4.

Intracellular recording. Strips of the entire muscularis (10 mm long) were cut parallel to the long axis of the circular muscle fibers with a knife consisting of a pair of parallel scalpel blades set 2 mm apart. Experiments were carried out on dissected strips of circular muscle that had both the myenteric and submucosal edges removed. These strips contained approximately one-half of the circular muscle layer and are referred to as interior circular muscle (ICM; Fig. 1). ICM strips were pinned to the floor of the recording chamber, myenteric side face up, with one unpinned end attached to an isometric force displacement tension transducer (FT03; Grass Instruments). Strips were constantly perfused with oxygenated KRB at 37 ± 0.5°C and allowed to equilibrate for at least 1 h before experimentation. Electrical field stimulation (EFS) was provided by a stimulator (S88; Grass Instruments) via a stimulus isolation unit (SIU-5; Grass Instruments).


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Fig. 1.   Diagram of the procedure used to produce interior circular muscle (ICM) from the muscularis externus. Submucosal and myenteric borders of the circular muscle layer were removed by sharp dissection to form a strip of ICM. ICM strips were devoid of both the myenteric and submucosal neuronal plexus-pacemaker regions.

Intracellular electrical recordings were made using conventional capillary glass microelectrodes (1.2 mm OD, 0.6 mm ID; FHC) filled with 3 M KCl and having resistances ranging from 30 to 70 MOmega . Membrane potential was measured with a high-input impedance electrometer (World Precision Instruments Duo 773, Sarasota, FL), and outputs were displayed on an oscilloscope (Nicolet 3091, Madison, WI). Analog electrical and mechanical signals were reproduced on chart paper (Gould 2200) and were digitized and recorded on a videocassette recorder (Panasonic Hi-Tech 4). Data were also stored and analyzed by a computer (Micron Millenia Pro2) using a data acquisition program (AcqKnowledge III; Biopac Systems, Santa Barbara, CA).

[14C]ACh labeling and overflow. In each experiment, four 5-cm lengths of ICM were secured between ring electrodes. The tissues were incubated at 37°C in a water bath in KRB containing [14C]choline chloride (1 µCi/ml) for 30 min in which tissues were continuously stimulated with electrical square-wave pulses (1 ms, 1 Hz). The tissues were then cut into ~5-mm strips, placed into 300-µl perfusion chambers, and superfused with KRB, at 37°C, containing hemicholinium-3 (10 µM) for 90 min (2 ml/min). After 65 min of washing, the tissues were electrically stimulated (5 Hz, 0.05 ms) for 1 min (S1), as preliminary experiments had demonstrated that the first stimulus evoked a very high overflow of [14C]ACh compared with subsequent stimuli. After this washing period, the superfusate was collected for 50 min at 1-min intervals in 7-ml scintillation vials. The tissues were then stimulated at 30-min intervals (S2, 95th min; S3, 125th min) for 1 min (5 Hz, 0.05-ms duration pulses). In experiments in which the effects of Nomega -nitro-L-arginine (L-NNA), 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxaline-1-one (ODQ), sodium nitroprusside (SNP), and TTX were examined, the drugs were allowed to equilibrate in the bathing medium for 20 min before S3. In experiments in which the effect of norepinephrine (NE) was examined, a more acute exposure was desirable, and only 4-min prestimulus incubation was used. The 2-ml samples of KRB were made up to 7 ml with Ecolume scintillant (ICN Biomedical) before being counted twice in a Beckman LS60001C scintillation counter for 3 min; results were then averaged. The tissues were transferred to scintillation vials, solubilized overnight in 1 ml of 10% NaOH, neutralized with HCl, and buffered with HEPES before counting. Overflow of 14C was calculated as a fractional release from the tissue. The average of five 1-min prestimulation counts was deducted from the counts at 1, 2, and 7 min poststimulation (S2 and S3), and the sum of increased overflow was designated as the release due to EFS. The ratio S3/S2 was calculated for control and drug treatments. This technique has been employed to study the release of ACh from a variety of neuroeffector preparations such as guinea pig GI tissues (1). Accordingly, we refer to the release of 14C as release of [14C]ACh.

Analysis of data. For experiments that measured drug effects, each experimental measurement was matched with a control measurement in the same tissue. Results were expressed as means ± SE of n (no. of tissues). Statistical analysis was performed by means of Student's t-test for paired or unpaired data with a value of P < 0.05 considered to be significantly different.

Drugs. The following were used: ACh, atropine sulfate, L-NNA, hemicholinium-3, hexamethonium bromide, norepinephrine hydrochloride (NE), SNP, TTX (all from Sigma Chemical, St. Louis, MO), [14C]choline chloride (DuPont NEN, Boston, MA), S-nitroso-N-acetylpenicillamine (SNAP; from Research Biochemicals International, Natick, MA), ODQ (Tockris Cookson, St. Louis, MO), Rp-beta -phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate (Rp-8-Br-PET-cGMPS; a generous gift from Dr. Hans G. Genieser, Biolog Life Science Institute, distributed by Ruth Langhurst International Marketing, La Jolla, CA).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Responses to EFS in ICM. The mean resting membrane potential (RMP) recorded from cells in strips of ICM was -70 ± 0.6 mV (n = 44 tissues). In contrast, RMP of cells at this position (~75%) in whole circular muscle strips is less negative (i.e., -50 mV; Ref. 15). The more negative RMP in ICM strips may be due to the absence of myenteric pacemaker cells (25). There were no spontaneous oscillations of membrane potential in ICM cells, providing functional evidence that pacemaker cells had been effectively removed. Under control conditions, a single stimulus or trains of five stimuli at 5 Hz (0.3-ms duration pulses, 15 V) evoked frequency-dependent TTX (1 µM; n = 3)-sensitive excitatory junction potentials (EJPs). EJPs elicited with single stimuli averaged 3.3 ± 0.4 mV (n = 58) in amplitude, whereas significantly (P < 0.01) larger EJPs were obtained with multiple stimuli (5.8 ± 0.6 mV, 5 pulses at 5 Hz; n = 58). The time to peak of the EJP (single stimulus) was 0.5 ± 0.04 s (n = 14), and the decay time constant (tau EJP) was 0.4 ± 0.1 s (n = 10; Table 1 and Fig. 2A).

                              
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Table 1.   Effect of selected modulators of nitric oxide activity on EJP amplitude and EJP decay time constant


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Fig. 2.   Effect of Nomega -nitro-L-arginine (L-NNA) and atropine on junction potentials elicited with single stimuli (ss) and multiple stimuli. A: electrical field stimulation (EFS; 0.3 ms duration, 15 V) of nerves gave rise to excitatory junction potentials (EJPs) in ICM cells. Initial deflection seen in left (A) is a stimulus artifact. This was followed by a brief delay before the depolarization phase of the EJP began. When multiple stimuli were applied, summation occurred, leading generally to greater depolarization (see Table 1 for comparison of single stimuli to 5 Hz control EJPs in 28 cells), although not in this particular example. Middle: responses in the presence of L-NNA (300 µM). L-NNA increased the amplitude of EJPs elicited with both single and multiple stimuli. Right: subsequent addition of atropine (1 µM) in the continued presence of L-NNA entirely abolished the EJPs. B: examples of the electrical response to longer trains of stimuli. Left: repetitive stimulation at 10 Hz for 10 s resulted in an initial EJP followed by a more sustained inhibitory junction potential (IJP). Middle: 30-min exposure to L-NNA entirely abolished the IJP and increased the amplitude and duration of nerve-evoked depolarization. Membrane potential fluctuations were often superimposed on the depolarization (as shown in middle). Right: addition of atropine (1 µM) in the continued presence of L-NNA abolished all junction potentials.

In approximately one-half of the recordings (i.e., 47% of cells, n = 58), biphasic responses were observed with multiple stimuli (5 stimuli at 5 Hz). These consisted of an EJP followed by an inhibitory junction potential (IJP). IJPs recorded under control conditions with multiple stimuli averaged 4.5 ± 0.5 mV in amplitude (5 stimuli at 5 Hz; n = 27). In contrast, biphasic responses were observed in only 3 of 58 tissues with single stimuli. When tissues were stimulated for a longer period of time (i.e., 100 stimuli at 10 Hz), IJPs were always observed and these had a mean amplitude of 10.4 ± 4 mV (n = 4; Fig. 2B).

Effect of L-NNA on IJPs and EJPs. There is substantial evidence suggesting that the predominant inhibitory neurotransmitter in the canine colonic circular muscle layer is NO (e.g., Ref. 36). To investigate the contribution of NO to the electrical response observed under control conditions in ICM strips, the NOS inhibitor, L-NNA, was tested. RMP in the presence of L-NNA (300 µM) was not significantly different from control (i.e., -73 ± 1.6 to -72 ± 1.5 mV, n = 21, P > 0.05). However, addition of L-NNA led to a significant increase in the amplitude of EJPs evoked with either single or multiple stimuli (see Fig. 2 and Table 1). L-NNA also significantly increased tau EJP (Table 1) and abolished the IJPs observed with multiple stimuli (Fig. 2B). Interestingly, prolonged stimulation (100 stimuli at 10 Hz) in the presence of L-NNA produced oscillating electrical activity with a peak depolarization of 34 ± 3 mV (n = 4; Fig. 2B).

Effect of cholinergic antagonists on EJPs and IJPs. Previous studies have suggested that cholinergic responses in canine colon are mediated via activation of muscarinic receptors (11, 19). To determine whether the EJPs recorded in segments of ICM were also due to stimulation of muscarinic receptors, experiments were undertaken with the muscarinic antagonist atropine (1 µM). Atropine abolished EJPs evoked with either single or multiple stimuli (n = 17) in the presence of L-NNA (Fig. 2). In the absence of L-NNA, addition of atropine revealed IJPs or significantly (P < 0.05) enhanced the amplitude of existing IJPs (from 3.2 ± 0.7 to 4.8 ± 0.7 mV, n = 16, P < 0.05 for 5 stimuli at 5 Hz, see Fig. 3). All junction potentials were abolished with combined atropine (1 µM) and L-NNA (n = 5; Fig. 2). In contrast to atropine, hexamethonium (100 µM) had no effect on EJPs (n = 4; data not shown), suggesting that nicotinic receptors are not involved in generation of EJPs. Because nicotinic receptors participate in ganglionic transmission, these results also provide further functional evidence that neuronal cell bodies were absent in ICM preparations.


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Fig. 3.   Inhibition of muscarinic receptors enhances IJPs. Sample traces of junction potentials in a single cell before addition of atropine (left), with atropine alone (1 µM; middle), or with atropine plus L-NNA (300 µM; right) in response to EFS (0.3 ms duration, 15 V). Note that under control conditions multiple stimuli (5 pulses at 5 Hz) produced both an EJP and IJP. Atropine (1 µM) abolished the EJPs and revealed or enhanced the amplitude of IJPs (middle). Addition of L-NNA (300 µM), in the continued presence of atropine, abolished all junction potentials (right).

Effect of NO donors on EJPs. The potentiation of EJPs by L-NNA suggested that neuronally released NO inhibits cholinergic activity. To further examine the actions of NO on cholinergic transmission, additional experiments were carried out using two different NO donors, i.e., SNP and SNAP. With L-NNA (300 µM) present throughout, SNP reduced EJP amplitude in a concentration-dependent manner (see Fig. 4 and Table 1). SNP also gave rise to a small but significant membrane hyperpolarization, which was maximal at 1 µM (from -69 ± 2 to -72 ± 2 mV; n = 6, P < 0.05). In most cases, the reduction in EJP amplitude was not associated with a significant change in the time course of the EJP (e.g., 100 µM SNP reduced the EJP amplitude elicited with a single stimulus by 56%, whereas it had no effect on tau EJP; see Table 1). SNAP (1-100 µM) also significantly decreased EJP amplitude (Fig. 4C) and hyperpolarized the membrane (from -72 ± 1 to -76 ± 2 mV, n = 4; P < 0.05).


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Fig. 4.   Effect of nitric oxide (NO) donors on EJPs. A, left: example trace of the response to EFS (0.3 ms duration, 15 V) in the presence of L-NNA (300 µM). EJPs elicited with single stimuli (ss) and multiple stimuli (5 pulses at 5 Hz) were reduced following addition of 1 µM sodium nitroprusside (SNP; right). B: application of higher concentrations of SNP led to a further reduction in EJP amplitude recorded from the same cell. C: summary graph of the effects of increasing concentrations of SNP and S-nitroso-N-acetylpenicillamine (SNAP; both 1-100 µM) on EJP amplitude. Amplitudes were normalized to the response obtained in the presence of L-NNA alone. Mean values ± SE from at least 4 tissues are shown. *, ** Values obtained with NO donor plus L-NNA that were significantly different (P < 0.05 and P < 0.01, respectively) from the response obtained in L-NNA alone.

Effect of NO donors on exogenous ACh. The results obtained with muscarinic antagonists, NOS inhibitors, and NO donors all suggest that ACh release gives rise to a depolarization that is suppressed by neuronally released NO. To provide additional evidence for this hypothesis, we examined the effect of NO donors on exogenously applied ACh. ACh (1 µM) gave rise to a mean depolarization of 20.3 ± 0.9 mV (n = 51) as shown in Fig. 5. In some cases, addition of ACh resulted in rhythmic fluctuations in membrane potential, which were superimposed on the depolarization as previously reported (19). Both SNP (100 µM; n = 5) and SNAP (100 µM; n = 5) evoked a large hyperpolarization in the presence of ACh (i.e., 14.9 ± 2.7 and 13.3 ± 2.9 mV changes in membrane potential, respectively).


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Fig. 5.   Effect of SNAP on ACh-induced depolarization. Sample trace of the effect of ACh (1 µM) and SNAP (100 µM) on membrane potential is shown. Addition of ACh depolarized the tissue by ~20 mV. EFS (0.3 ms, 15 V, 5 stimuli at 10 Hz) in the presence of ACh evoked an IJP followed by a rebound EJP. In the continued presence of ACh, SNAP (100 µM) produced a 26 mV hyperpolarization.

Effect of guanylyl cyclase inhibition on EJPs. One of the principal downstream targets of NO in smooth muscle is soluble guanylyl cyclase (27, 30). To determine whether NO-induced inhibition of EJPs is related to activation of guanylyl cyclase, we investigated the effects of the novel guanylyl cyclase inhibitor ODQ (10). ODQ (10 µM) significantly increased EJP amplitude and tau EJP (Table 1 and Fig. 6A) in the absence of a change in membrane potential (i.e., -72 ± 1.9 vs. -72 ± 1.2 mV, n = 12, P > 0.05). ODQ also abolished IJPs, as previously reported in canine colonic whole circular muscle (8). L-NNA had no significant effect on EJP amplitude when applied in the presence of ODQ (n = 4; data not shown). These data suggest that neuronally released NO inhibits cholinergic activity via activation of guanylyl cyclase. The actions of SNP and SNAP were also tested in the presence of ODQ. After addition of ODQ, both NO donors ceased to inhibit EJPs. Interestingly, a small but significant increase in EJP amplitude was observed instead (Fig. 6, B and C). NO has been reported to have cGMP-independent excitatory effects on some cellular constituents, including cyclooxygenase (28) and some ion channels (e.g., Ref. 22). The excitatory effect of NO on EJPs was not further investigated in this study.


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Fig. 6.   Effects of the guanylyl cyclase inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxaline-1-one (ODQ) and NO donors on EJPs. A: sample traces of junction potentials obtained in a single cell before (left) and after (right) addition of ODQ (10 µM) in response to EFS (0.3 ms duration, 15 V). Note that in this tissue multiple stimuli (5 pulses at 5 Hz) produced both an EJP and an IJP. ODQ abolished the IJP and increased the amplitude of the EJPs (right). B: recording obtained from the same cell as in A following addition of SNP (10 µM) to the superfusate. SNP did not reduce EJP amplitude in the presence of ODQ but rather resulted in a small increase in EJP amplitude. C: summary graph of the effect of ODQ alone or in combination with increasing concentrations of SNP and SNAP (both 1-100 µM) on EJP amplitude. Amplitudes were normalized to the response obtained in the presence of ODQ alone. Mean values ± SE from at least 4 tissues are shown. *, ** Values significantly different (P < 0.05 and P < 0.01, respectively) from the response obtained in ODQ alone.

Effect of a cGMP-dependent protein kinase antagonist on EJPs. cGMP-dependent protein kinase (PKG) is widely recognized as an important downstream mediator of cGMP effects (6). Therefore, the effect of the PKG antagonist Rp-8-Br-PET-cGMPS (5) on EJPs was tested. Rp-8-Br-PET-cGMPS (100 nM) significantly increased the amplitude of EJPs in response to both single stimuli and multiple stimuli (see Table 1 and Fig. 7). The potentiation of EJPs produced with Rp-8-Br-PET-cGMPS was significantly less than that observed with either L-NNA or ODQ (P < 0.01 at 5 and 10 Hz; see Table 1 and Fig. 7). When L-NNA (300 µM) was applied in the continued presence of Rp-8-Br-PET-cGMPS, there was an additional significant increase in EJP amplitude (118.6 ± 45.9%, n = 5, P < 0.05 to single stimuli; Fig. 7).


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Fig. 7.   Effect of the cGMP-dependent protein kinase inhibitor Rp-8-PET-bromoguanosine-3',5'-cyclic monophosphorothioate (Rp-8-Br-PET-cGMPS) on EJPs. A: sample traces of EJPs obtained during a single recording under control conditions (left), after addition of Rp-8-Br-PET-cGMPS (100 nM; middle), and in the presence of both Rp-8-Br-PET-cGMPS and L-NNA (300 µM; right) in response to EFS (0.3 ms, 15 V). Rp-8-Br-PET-cGMPS increased the amplitude of EJPs to both single stimuli (ss) and multiple stimuli (5 stimuli at 5 Hz). Addition of L-NNA, in the continued presence of Rp-8-Br-PET-cGMPS, further enhanced EJP amplitude. B: summary graph of the effect of Rp-8-Br-PET-cGMPS (100 nM) on EJP amplitude (plotted in mV). Rp-8-Br-PET-cGMPS produced a significant increase in EJP amplitude. An additional significant increase in EJP amplitude occurred when Rp-8-Br-PET-cGMPS was combined with L-NNA (300 µM) for both single stimuli and multiple stimuli. Mean values ± SE from at least 4 tissues are shown. *, ** Values significantly different (P < 0.05 and P < 0.01, respectively) from the response obtained in the absence of Rp-8-Br-PET-cGMPS. # Values significantly different (P < 0.05) from the response obtained with Rp-8-Br-PET-cGMPS alone.

[14C]ACh overflow measurements. The experiments with L-NNA, ODQ, and Rp-8-Br-PET-cGMPS suggest that NO modulates cholinergic activity largely via the guanylyl cyclase-cGMP-PKG pathway. However, the site(s) of action of NO (i.e., pre- vs. postjunctional) cannot be determined with any certainty from intracellular measurements. To directly investigate the possible prejunctional actions of NO, additional studies were undertaken that measured [14C]ACh overflow in strips of ICM following EFS (5 Hz, 1 min). Addition of L-NNA (100 µM), either alone or in combination with SNP (3-100 µM) or ODQ (10 µM), did not significantly affect [14C]ACh overflow, suggesting that the action of NO is postjunctional rather than prejunctional. In contrast, [14C]ACh overflow was significantly reduced by NE (10 µM; 52 ± 3%, n = 7) and TTX (1 µM; 82 ± 6%, n = 11). NE also reduced the amplitude of single stimulus EJPs and EJPs evoked with 5 Hz EFS by >90% (n = 3; data not shown). Cumulative release data are shown in Fig. 8. The inhibitory effects of SNP and NE on electrical events were also associated with changes in the contractile response to nerve stimulation, i.e., both SNP and NE reduced the response to 5 Hz nerve stimulation (0.3-ms duration, 15 V, 1 min) by >80% (n = 4; data not shown).


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Fig. 8.   Effect of various drugs on electrically evoked [14C]ACh overflow. Summary graph of the effects of L-NNA (300 µM), SNP (3 and 100 µM) plus L-NNA, ODQ (10 µM), norepinephrine (NE; 10 µM), and TTX (1 µM) on [14C]ACh overflow in response to EFS (5 Hz, 1 min) in strips of canine colonic ICM. Only TTX and NE produced a significant (P < 0.05) reduction in [14C]ACh release. Each bar represents mean ± SE of at least 4 tissues. *** Values significantly (P < 0.001) different from control. S2, 95th min; S3, 125th min.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Both NO and ACh play key roles as enteric inhibitory and excitatory neurotransmitters in the canine colonic circular muscle layer (11, 19, 23, 27, 30). However, due to the complex nature of electrical activity in this muscle, it has been difficult to separate the junctional events underlying neuromuscular transmission from the changes that these nerves produce in pacemaker potential activity. In the present study, we used a simplified preparation of ICM, which lacked both pacemaker regions, to specifically characterize junctional transmission. Our results indicate that the interior nonpacing region is functionally innervated by both excitatory cholinergic and inhibitory nitrergic motoneurons. Other neurotransmitters are known to be colocalized with ACh and NO in motoneurons (9). Our results do not preclude a significant role for these other cotransmitters, but they do suggest that the electrical events observed under the conditions of our experiments are largely mediated by ACh and NO. A number of experiments were undertaken to explore how nitrergic nerves and NO modulate the cholinergic neuronal response at the level of terminal nerve varicosities. These data suggest that neuronal release of NO suppresses cholinergic responses via postjunctional mechanisms, which require activation of guanylyl cyclase.

Single stimuli elicited in the presence of L-NNA gave rise to atropine-sensitive EJPs that averaged 10 mV in amplitude, indicating strong functional innervation of ICM by cholinergic nerves. Cholinergic EJPs have been recorded from various other GI preparations (15) but not from the canine colon. Repetitive EFS led to summation of EJPs, and, in the presence of L-NNA, prolonged stimulation (i.e., 100 stimuli at 10 Hz) gave rise to rhythmic fluctuations in membrane potential. Rhythmic oscillations in membrane potential were also observed when ACh was applied exogenously to ICM strips (Ref. 19 and present study). These oscillations are likely to be involved in interactions between several voltage-gated and receptor-operated conductances. Further studies are required to clarify this interaction.

Although EJPs were recorded under control conditions, their amplitude was dramatically increased when NOS was inhibited with L-NNA, suggesting that NO inhibits cholinergic responses. This conclusion was further supported by our observation that NO donors led to concentration-dependent inhibition of EJP amplitude in the presence of L-NNA. Activation of guanylyl cyclase appears to play a central role in this process, since the guanylyl cyclase inhibitor ODQ enhanced EJP amplitude to the same extent as L-NNA. Perhaps even more compelling was the observation that the effects of L-NNA and ODQ were nonadditive, suggesting that these inhibitors block two different steps in the same pathway. Guanylyl cyclase activity has also been shown to be essential for generation of IJPs in this tissue (8).

EJP amplitude was also increased when the PKG blocker Rp-8-Br-PET-cGMPS was applied. This observation suggests that EJP amplitude is suppressed under control conditions by activated PKG. Although PKG is an important downstream mediator of the effects of cGMP (6), we found that blocking PKG with Rp-8-Br-PET-cGMPS was less effective than blocking either NOS with L-NNA or guanylyl cyclase with ODQ. Furthermore, some additional enhancement of EJP amplitude could still be obtained when L-NNA was added in the presence of Rp-8-Br-PET-cGMPS. The concentration of Rp-8-Br-PET-cGMPS used for this study (i.e., 100 nM) was significantly greater than the inhibition constant reported for biochemical studies (5). However, it is possible that the concentration of Rp-8-Br-PET-cGMPS reached intracellularly in the intact tissue was <100 nM, which led to incomplete inhibition of PKG. Alternatively, another cGMP-dependent, PKG-independent pathway may be involved, as in the perfused rat lung, where NO-induced modulation of basal tone involves a cGMP-dependent, PKG-independent pathway (7).

Although our results suggest that cholinergic responses in ICM are suppressed by the concomitant release of NO, the site(s) of action of NO (i.e., pre- vs. postjunctional) cannot be determined with any certainty from intracellular measurements alone. Likewise, although inhibition appears to involve guanylyl cyclase, the location of this target enzyme cannot be confidently assigned to either a pre- or postjunctional site from intracellular measurements. To directly address the possible prejunctional actions of NO, we therefore measured [14C]ACh overflow in ICM strips. Blockade of NOS with L-NNA or guanylyl cyclase with ODQ did not significantly affect [14C]ACh overflow, suggesting that potentiation of EJPs by these drugs was due entirely to postjunctional mechanisms. Likewise, all concentrations of SNP tested (3-100 µM) were without effect on [14C]ACh overflow. In contrast, [14C]ACh overflow was significantly decreased by TTX and NE, providing evidence that ACh release was neural in origin and that it could be modified under the conditions of our experiments.

Several other groups have also investigated the actions of NO on ACh release in GI and tracheal preparations. In some of these studies inhibition of ACh release by NO donors (20, 31) and enhancement of release by blockade of NOS (16, 20, 21, 31) were reported. In contrast, others (3, 13, 34, 35) have suggested that NO donors and blockade of NOS do not modify ACh release in either tracheal or intestinal preparations. Finally, Wiklund et al. (38) reported that NO donors inhibited ACh release in guinea pig ileum, whereas NOS inhibitors were without effect. An important difference between the present study and most of the previous studies listed above is that we measured ACh release in the absence of ganglia. Hence, the potential modulatory role of NO at the level of the varicosity was separated from its action at the ganglia. One exception to this is the study of Hryhorenko et al. (16) in which NG-nitro-L-arginine methyl ester (and to a lesser extent L-NNA) significantly increased ACh release in ICM strips of the canine small intestine. Whether these conflicting data reflect differences in experimental conditions or reflect differences between the large and small intestine of the dog remains to be determined.

Because the inhibitory effects of NO were guanylyl cyclase dependent, it follows that the target cell regulated by NO must exhibit significant guanylyl cyclase activity. Immunohistochemical studies of the canine colon indicate that both exogenous and neuronally released NO raise cGMP levels in smooth muscle cells, in interstitial cells of Cajal (ICC), and in ~10% of the myenteric and submucosal ganglionic neurons (32). Few studies of ACh release have directly addressed the possible role of guanylyl cyclase as a mediator of NO-induced effects. One exception is a recent study of ACh release in the guinea pig ileum, which reported that blockade of guanylyl cyclase with ODQ enhanced nerve-evoked ACh release, whereas the guanylyl cyclase activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole inhibited ACh release (14). In the present study, ODQ did not affect ACh release. The differing results of these studies may reflect the use of ganglion-intact muscle strips (14) vs. ganglion-free muscle strips (present study).

A variety of postjunctional mechanisms could account for the inhibition of EJPs by NO. In isolated cells from this tissue, the NO-guanylyl cyclase-cGMP pathway can activate at least three different K+ channels (22). It is therefore possible that the inward current associated with the EJP is simply short circuited by the outward current generated via the opening of K+ channels. Additional support for this possibility is the observation that NO donors led to a hyperpolarization of RMP and a significantly greater hyperpolarization when membrane potential was depolarized away from the K+ equilibrium potential with ACh. Another possible mechanism that may contribute to the inhibition of EJPs is modification by NO of some step in the pathway leading to inward current generation. ACh activates a Ca+-sensitive nonselective cation channel current in canine colonic smooth muscle cells (23) so it is possible that NO inhibits these channels by decreasing intracellular Ca2+ concentration (12).

There is substantial evidence that pacemaker potentials in the colon are generated by ICC located at both the myenteric and submucosal edges of the circular muscle layer (29). By recording from strips of ICM, we have effectively isolated neuromuscular junction events from the effects of nerves on these pacemaker cells. However, other morphologically distinct types of ICC are present in the interior of the circular muscle layer, which do not generate pacemaker potentials (39). This nonpacing type of ICC is probably involved in transmission of signals from nitrergic nerves to the effector smooth muscle (4). Hence, although the responses we observed were independent of pacemaker cells, they may still have involved ICC.

In conclusion, our results suggest that the interior of the circular muscle layer is functionally innervated by neurons that release ACh and neurons that release NO. These two transmitters appear to account for all of the electrical changes that are observed with brief trains of stimuli. The ability of NO to suppress cholinergic activity is predominantly postjunctional in nature and involves activation of guanylyl cyclase and possibly PKG. Because there is basal release of NO from nerves in the canine colon in the absence of extrinsic stimuli (17), cholinergic motor responses may be subject to some degree of continuous postjunctional modulation by neuronally released NO in vitro as well as in vivo.

    ACKNOWLEDGEMENTS

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-45376 (to K. D. Keef) and postdoctoral fellowship money from the National Science Foundation, Nevada Experimental Program to Stimulate Competitive Research Award EPS 9353227, Women in Science and Engineering component.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: K. D. Keef, Dept. of Physiology and Cell Biology/352, Univ. of Nevada, Reno, NV 89557.

Received 30 April 1998; accepted in final form 28 August 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(6):G1324-G1332
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