Tonic inhibition of small intestinal motility by nitric oxide

Journal of the Autonomic Nervous System, 44 (1993) 179 - 187

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© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1838/93/$06.00 JANS 01419

Tonic inhibition of small intestinal motility by nitric oxide Bengt I. Gustafsson and Dick S. Delbro Departments of Physiology and Surgery, University of G6teborg, Sweden (Received 17 November 1992) (Revision received 2 February 1993) (Accepted 26 February 1993)

Key words: Enteric nervous system; Nitric oxide; Small intestine; Vagal nerve Abstract The effects of blocking nitric oxide synthase with the arginine analog N'°-nitro-L-arginine (L-NNA) were investigated in anaesthetized cats, vagotomized and pretreated with guanethidine and atropine. Spontaneous NANC jejunal motility (recorded as the volume changes of an intraluminal balloon) was markedly increased in a dose-dependent and stereospecific manner. The effect of L-NNA was partly reversed by L-arginine, the substrate for nitric oxide (NO) synthesis. Thus, this study presents evidence for a tonic inhibitory influence, via the release of NO, on small intestinal motility in vivo. Furthermore, relaxations upon the L-NNA-induced hypermotility could be elicited by vagal nerve stimulation, which may suggest the existence of another NANC inhibitory transmitter. Hexamethonium abolished such relaxations but did not affect the tone or phasic activity after L-NNA.

Introduction

In a pioneering paper in 1972 [40], Wood suggested that the circular muscle layer of the feline small intestine is subjected to tonic suppression, due to the release of an inhibitory transmitter from myenteric motor neurons. The main evidence for this hypothesis was the finding, in vitro, that the muscle was markedly excited, both electrically and mechanically, when exposed to the nerve blocking agent, tetrodotoxin (TTX). According to Wood, the motility of the gut is regulated not primarily by excitatory motor neurons but rather by the degree of inhibition of the muscle, as determined by the activity in these

Correspondence to: B.I. Gustafsson, Department of Physiology, Medicinaregatan 11, S-413 90 G6teborg, Sweden.

hypothetical, inhibitory motor fibres [40,42]. Several authors have confirmed the original observations, in different species and experimental conditions, in vivo [5,14,39]. The effect of TTX on the feline small intestine, in vivo, can be mimicked by the p.-opioid receptor agonist, morphine [12,21]. Electrophysiological studies indicate that this response could, indeed, be due to interference with enteric, inhibitory nerves [41]. This view was supported by the finding that some opioid peptides, acting at prejunctional # and ~ receptors, could suppress inhibitory motor nerves to the circular muscle [2,31. A physiological correlate to these observations was recently suggested. Thus, vagal nerve stimulation in the anaesthetized cat, after pharmacological blockade of the noradrenergic and muscarinic cholinergic neurotransmission, elicited a

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contractile response in the small intestine that was sensitive to the opioid receptor blocker, naloxone [18,19]. We suggested that this effect could possibly result from a temporary blockade of a prevailing inhibition due to the release of endogenous opioid peptides (presumably enkephalins) from enteric nerves under vagal control [19,21]. In addition, vagal nerve stimulation, when applied after the jejunal tone had been raised by various stimuli, resulted in a rapid relaxation, demonstrating another type of vagally induced NANC response [12,20,21]. The identity of the final neurotransmitter(s) involved in the various NANC inhibitory motor effects is unresolved to date. In the small intestine, NANC inhibitory effects have been described for both the longitudinal and the circular muscle layers in several mammals, including cat [11,40]. Among the substances proposed, adenosine triphosphate (ATP) and vasoactive intestinal peptide (VIP) have hitherto been regarded as the strongest transmitter candidates [24]. In addition, there is growing evidence suggesting that nitric oxide (NO), or a closely related nitroso compound, serves as an important inhibitory transmitter in several parts of the gastrointestinal (GI) tract [35]. The existence of such 'nitroxergic' inhibition of the gut is corroborated by the demonstration of immunoreactivity for nitric oxide synthase (NOS) in enteric neurons [8]. Additionally, diaphorase staining of presumably NO-producing neurons in the gut has been reported [4,43]. NOS catalyses the conversion of e-arginine (L-arg) and oxygen to NO and citrulline [30]. NO causes muscle relaxation by activating guanylate cyclase, which, in turn, increases cyclic guanosine monophosphate [1,25]. NOS can be inhibited stereospecifically by some arginine analogs with a substituted N-guanidino group, where N'-nitro-Larginine (L-NNA) seems to be one of the most potent, while its D-enantiomer (D-NNA) is biologically inactive [31,38]. The aim of the present study was to investigate a possible role for NO in the different inhibitory motor mechanisms, as reported for the feline jejunum, in vivo. This was addressed by analysing the effects on the gut of blockade of NOS by L-NNA.

Materials and Methods

General Experiments were conducted using cats of either sex (3.1-4.7 kg body weight; n = 10). The animals were deprived of food for 24 h prior to the experiments but had free access to water. Anaesthesia was induced with pentobarbital (0.16 mmol/kg, i.p., n = 7) or diethyl ether (n = 3), whereafter a tracheal cannula was inserted. A femoral vein and artery were catheterized and anaesthesia was maintained with chloralose (0.16 mmol/kg, i.v., given as a single dose; a supplementary injection of 0.08 mmol/kg, i.v., was given at least once in the course of each experiment). An infusion (0.1 m l / m i n ) of 0.28 M glucose and 0.1 M sodium bicarbonate in distilled water was administered via the femoral artery to counteract the acidosis caused by the surgical trauma [23]. In two experiments also the left carotid artery was cannulated in the retrograde direction. Ventilation was assisted by means of a respiration pump. Guanethidine sulphate (6 /xmol/kg, i.v.) was injected to prevent noradrenergic inhibition of gastrointestinal motility [15]. After midline laparotomy, the greater and lesser splanchnic nerves were sectioned. In addition, the adrenals were ligated to prevent possible reflex release of catecholamines. Corticosteroid substitution (hydrocortisone sodium succinate, 9 - 3 0 / z m o l / k g , i.m.) was administered to compensate for the elimination of adrenocortical secretion.

Nerve stimulation The cervical vagal nerves were dissected free and cut. The left or right vagus was arranged for bipolar electrical stimulation in the peripheral direction. Square-wave pulses were delivered with a Grass $5 stimulator.

Recording of effector responses Jejunal motility was recorded by means of a thin rubber balloon (length 5 cm) tied onto a tubing (i.d. 3.5 mm) and introduced into the jejunum via an antimesenteric incision about 20 cm anally to the ligament of Treitz. The tip of the balloon was directed 10 cm orally. A drainage tube for bile and pancreatic juice was inserted via

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a duodenal incision. The incisions were closed with purse-string sutures, without interrupting the continuity of the gut. The balloon was filled with body-warm water and was connected to a container (diameter: 3 cm) placed on a force-displacement transducer (Grass FT 03C). The level of intraluminal pressure was set by the height of the container above the abdominal cavity and was kept constant in each experiment (5-12 cm H20). This method allowed intestinal motility to be monitored as volume changes of the balloon at a fairly constant transmural pressure [26]. Blood pressure was recorded by a transducer (Statham P23 AC) connected to the catheter in the femoral artery. Care was taken during surgery to keep the abdominal organs covered with swabs moistened with body-warm, isotonic saline. The abdominal wound was firmly closed with a running catgut suture and clips. Body temperature was maintained at 38°C by radiant heat. All recordings were made on a Grass 7D polygraph.

Experimental protocol and evaluation of results Drug effects on jejunal motility were quantified with respect to changes of both basal volume (i.e. tone, ml) and superimposed phasic contractile activity. Since there is an inverse relationship between tone and volume, an increased muscle tone is reflected by a decrease in jejunal volume, and vice versa. For the quantification of phasic activity, a computerized planimetric method was used where the area under the response graph was estimated and expressed as motility index (arbitrary area units). Gut motility and also mean arterial blood pressure (MABP, mmHg) were evaluated in 5-min periods, 5 min prior to (Control), 25 and 55 min after the administration of D-NNA (10 Izmol/kg, i.v.). L-NNA was then given at two doses (10 and 30 ~zmol/kg, i.v.) at 30-rain intervals, and evaluations were made in the 5-min periods 25 min after each injection (n =7). Thereafter, vagal nerve stimulations were performed (0.5, 1, 2, 4, 8, 16 and 32 Hz; 5 ms, 15-30 V, for 15-30 s at 2 min intervals; n = 7). Sodium nitroprusside (SNP), which releases NO in a non-enzymatic reaction [13], was then injected (10 nmol/kg, i.v., n = 5). After recovery from the effect of SNP, D-arginine (D-arg; 0.1 mmol/kg,

i.v.) was given, followed after 20 min by L-arg (0.1 mmol/kg, i.v.). Evaluations were made in the 5-min periods, immediately before the injection of D-arg (Control), 5 and 15 rain after the administration of either drug (n = 6). In three additional experiments, jejunal motility was increased by morphine (1 ~mol/kg, i.v.) before investigating the effect of L-NNA. In these cats, motility was only qualitatively estimated. The results are presented as means _+ S.E.M. Statistical analyses were performed by a one-way analysis of variance (ANOVA), followed by Fisher's LSD test for repeated measurements [28]. A P value < 0.05 was considered significant.

Drugs The following drugs were used: D- and Larginine (Sigma Chemical Co., St. Louis, MO), N'°-nitro-D-arginine (Serva Feinbiochemica GmbH, Heidelberg, FRG), N'-nitro-L-arginine (Sigma), atropine sulphate (Sigma), a-D(+)gluco-chloralose (E. Merck AG, Darmstadt, FRG), guanethidine sulphate (Ismelin ®, CibaOeigy AG, Basel, Switzerland), hexamethonium chloride (Fluka AG, Buchs, Switzerland), hydrocortisone sodium succinate (Solu-Cortef ®, Upjohn S.A., Puurs, Belgium), morphine hydrochloride (Morfin ®, KabiVitrum AB, Stockholm, Sweden), naloxone hydrochloride (Narcanti ®, Du Pont Pharmaceuticals, Wilmington, DE; Sigma), pentobarbital sodium (Mebumal Vet ®, Nordvacc, Sk~rholmen, Sweden), and sodium nitroprusside (Nitroprussid-natrium-dihydrat, Merck). The solid drugs were dissolved in isotonic saline or distilled water, except for chloralose (0.13 M borax). Ultrasonic disruption of the powder was required to dissolve the arginine analogs.

Results

General At the completion of surgery, the animals were left undisturbed for at least 1 h to alleviate perturbations of the GI motility due to the surgical trauma. Thereafter, the responsiveness of the gut to excitatory stimuli was verified by vagal nerve stimulation (16-32 Hz, 5 ms, 10-30 V). When

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contractions could be elicited reproducibly, atropine (1.4 ~ m o l / k g , followed by an infusion of 0.7 / z m o l / k g / h , i.v.) was administered. Subsequently, the spontaneous motor activity was either sparse or absent. Renewed stimulation produced a contractile response, consisting of a delayed increase in tone and phasic activity, as previously described [18,19]. The possible involvement of NO in NANC jejunal motility was then investigated.

Effect of blockade of nitric oxide synthase

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Fig. 1. Effects of the enantiomers of N'°-nitro-arginine, DN N A and L-NNA, on N A N C jejunal motility (recorded by a volumetric method) and blood pressure, as investigated in a n a e s t h e t i z e d cats, v a g o t o m i z e d and p r e t r e a t e d with guanethidine and atropine. In each animal, evaluations were made in the 5-min periods, immediately prior to (Control), 25 and 55 min after the administration of D-NNA ( 1 0 / x m o l / k g , i.v.) and also 25 min after each injection of L-NNA (10 and 30 p.mol/kg, i.v.) (n = 7). Motility is presented as phasic activity (i.e. motility index; A) and basal level of tone (i.e. jejunal volume; B). Mean arterial blood pressure is expressed in m m Hg (C). Data are given as m e a n s + S . E . M . The comparisons are m a d e with respect to Control. Note that a decrease in volume signifies an increased jejunal tone. *, P < 0.05; **, P < 0.01; * * * , P < 0.001; n.s., not significant.

The effects of D- and L-NNA on jejunal motility and blood pressure are presented in Fig. 1. D-NNA did not influence either parameter investigated, while L-NNA caused a dose-dependent increase in jejunal tone and phasic activity as well as in MABP. The effect of L-NNA on jejunal motility had a rapid onset and appeared to peak within 5-10 min (Fig. 2). Tone and phasic activity then stabilized and were more or less maintained during the observation period. On this augmented jejunal motility in response to NOS blockade, vagal stimulation at increasing frequencies resulted in frequency-dependent, usually biphasic responses. The first component consisted of an inhibition of phasic activity in all animals and, simultaneously, also a decrease in tone in 6 out of 7 cats (in one cat, there was

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phasic activity but no augmented tone). The threshold frequency was 1-2 Hz and near-maximal effect was obtained at 4-8 Hz. Peak relaxation amounted to 0.25-1.2 ml, corresponding to 89 _+7% of the increased level in tone caused by L-NNA. The relaxations were similar to those obtained by vagal stimulations (same parameters) when (NANC) tone instead had been raised by morphine. In the latter situation, peak relaxation was 88 _+2% of the increased level in tone. (These latter data are compiled from four experiments, constituting a part of a recently published study [21]). The second component was evident upon termination of the stimulation. Then, a 'rebound' contractile effect (maximal amplitude: 1.13 + 0.15 ml; duration: 1-2 min) could always be observed at frequencies > 4-8 Hz (Fig. 3). The jejunal hypermotility was sustained until L-arg (see below) was given, i.e. 90-100 rain after the second injection of L-NNA.

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Drug effects on L-NNA induced hypermotility SNP caused a transient relaxatory response in the gut (duration: 3-4 min; amplitude: 79 _+ 6% of L-NNA-induced tone) and a decrease in blood pressure (Fig. 3). This demonstrates that the gut was responsive to exogenous NO despite the pharmacological interference with NOS.

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Fig. 4. Continuation from Fig. 1. Effects of D- and L-arginine (D-arg; L-arg), when administered after L-NNA. D-arg was followed after 20 min by L-arg (either drug at 0.1 m m o l / k g , i.v.; n = 6). Jejunal motility and M A B P were evaluated in the 5-rain periods, immediately prior to D-arg (Control) as well as 5 and 15 rain after each drug injection. Recording parameters as in Fig. 1. L-arg induced a temporary inhibition of phasic activity (A), tone (B) and also a depressor response (C). The comparisons are made with respect to Control. Data are given as m e a n s + S.E.M. Note that an increase in volume signifies a decreased jejunal tone. **, P < 0 . 0 1 ; * * * , P < 0 . 0 0 1 ; n.s., not significant.

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While D-arg did not affect jejunal motor activity or MABP, L-arg caused, within a few min, an inhibition of tone and phasic activity and a concomitant depressor response. The effect on the phasic activity was transient (Fig. 4). Twenty minutes after the administration of L-arg, either morphine (1 /xmol/kg, i.v., n = 3) or a renewed dose of L-NNA (30-60 /~mol/kg, i.v., n = 3) was given. Morphine caused a powerful increase in tone (3.1 + 0.4 ml), which virtually emptied the balloon. L-NNA fully reversed the effect of L-arg within 5-10 min. Renewed vagal stimulations elicited relaxatory responses as above. The hypermotility caused by L-NNA was unaltered by naloxone (2.5 p.mol/kg, i.v., n = 2) or by the nicotinic ganglionic receptor blocker, hexamethonium (0.08 m m o l / k g , i.v., n = 3). However, the relaxations in response to vagal stimulation were abolished by the latter drug.

Effect of NOS blockade after morphine In 3 cats, morphine (1 /zmol/kg, i.v.) was administered prior to L-NNA. In concert with a recently published report [21], this compound caused an immediate increase in tone and phasic activity, upon which frequent spontaneous relaxations were observed. Vagal stimulations (parameters as above) elicited frequency-dependent relaxations (sometimes followed by a rebound contraction) where near-maximal effect was already obtained at 4 Hz. These relaxatory responses were indistinguishable from the spontaneously occurring ones, and were also very similar in character to those elicited upon L-NNA-induced tone (see above). After the administration of LNNA (40-100 /.~mol/kg, i.a. or i.v.), the spontaneous relaxations were absent for 15-40 min, while the morphine-induced jejunal hypermotility was otherwise unaffected. Also the relaxations to vagal stimulation (4 or 16 Hz) were abolished, but reappeared within 5-15 min upon renewed stimulations. The magnitude of these relaxatory effects was equal to those before L-NNA.

Discussion

The identity of the neurotransmitter(s) involved in NANC inhibition of G1 motility has for

long been a matter of intense investigation, much controversy and speculation. Recently, evidence has accumulated indicating that NO could be such a mediator (see Introduction for reference). In some parts of the GI tract, NO seems to be tonically released to maintain a certain degree of suppression of circular muscle activity, since blockade of NOS results in an augmented tone. This effect has been reported for, e.g., the small intestine in vitro [6,7,9,27]. It is not known whether such tonic suppression occurs spontaneously or is reflexogenic. In the longitudinal layer, NOS antagonism does not seem to affect basal motor activity [22,32,36]. The main finding of the present study, performed in vivo, was that the blockade of NOS by L-NNA, in a dose-dependent and stereospecific manner, caused a distinct and long-lasting augmentation of both jejunal tone and phasic activity. This effect was partly reversed by L-arg but not by D-arg. These data thus demonstrate that, under the prevailing experimental conditions, NO exerts tonic inhibition of small intestinal motility. Since hexamethonium did not attenuate the LNNA-induced hypermotility, the release of NO seems independent of a 'conventional' synaptic input. This could suggest that NO is released from spontaneously active myenteric neurons [40,41,42]. However, the presence of the intraluminal balloon can constitute a prevailing stimulus. Therefore, a reflexogenic activation of 'nitroxergic' neurons, via hexamethonium-resistant pathways, cannot be excluded (cf. [34]). The effect of L-NNA, as observed in the present study, is very similar to the motor response to morphine or T T X (see Introduction). Hypothetically, these compounds could elicit gut hypermotility by suppressing a tonic, either spontaneous or reflexogenic, release of NO to the circular muscle layer of the small intestine. Accompanying the intestinal response to NOS blockade was an increase in blood pressure, presumably due to the antagonism of a prevailing vasodilation caused by endothelially or neurally derived NO [29,37]. The onset of the action of L-NNA on the gut was comparable to that reported for the blockade of neurally induced relaxations in the lower o e -

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sophageal sphincter (LOS) and the internal anal sphincter (IAS) of the anaesthetized opossum [33,38]. The effect of L-arg was not regularly sustained. A partial resistance to L-arg reversal of the LNNA-induced antagonism of NANC relaxation was also reported for the guinea pig ileum, in vitro [32] and two possible explanations were offered. L-NNA may dissociate slowly from the isoform of NOS present in enteric neurons, or, alternatively, L-arg is not the preferred substrate for this particular isoenzyme. A nonspecific action of L-NNA was ruled out. During the L-NNA-induced hypermotility, relaxations could readily be elicited by vagal nerve stimulation. These were abolished by hexamethonium, indicating a dependence on a ganglionic nicotinic input, while the motility was otherwise unaltered (cf. [21]). The question arises whether the relaxatory effect is a result of NO release or due to the liberation of another inhibitory substance. When jejunal tone was first raised by morphine, L-NNA caused a short-lasting blockade of spontaneous as well as vagally induced relaxations. Interestingly, the neurally elicited NANC relaxation of the LOS and the IAS in the anaesthetized opossum was abolished for at least 30 min [38] or several hours [33] by L-NNA at doses around 1 0 / z m o l / k g . This should be compared to the total dose of 40-100 /xmol/kg in our study. The discrepancy could hypothetically be explained by an incomplete blockade of NOS in the jejunum. However, the long duration of the LNNA-induced hypermotility in this study indicates an adequate NOS blockade. Thus, our data do not readily suggest NO being of major importance for the spontaneous or vagally induced relaxations upon the raised tone of the feline jejunum. In our view, a more likely mechanism would be the existence of another NANC inhibitory transmitter. Such a transmitter could possibly be co-released [10,16]. Alternatively, there is a more complex mechanism for the release of NO [17]. The identity of the transmitter(s) responsible clearly demands further investigations in order to be elucidated. At moderate or high stimulation frequencies, a

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Fig. 5. Tentative neuronal a r r a n g e m e n t with respect to the N A N C control mechanisms of the circular muscle layer of the small intestine. Muscle excitation is achieved primarily as a consequence of diminished discharge of an inhibitory myenteric neuron (IMN). This nerve, utilizing nitric oxide (NO) as transmitter, can be controlled by morphine (Mo), either at the soma or prejunctionally. Furthermore, the IMN can be excited by preganglionically released acetylcholine (ACh) or inhibited by enkephalins (Enk). The myogenic hyperactivity resulting from diminished discharge of NO may be attenuated by a different, inhibitory transmitter (X). The IMN could hypothetically either be tonically active or receive non-nicotinic (NN) ganglionic input from another enteric, possibly sensory, neuron (?). ( + ) and ( - ) signifies excitatory and inhibitory effects, respectively.

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contractile effect generally occurred at the termination of vagal stimulation. It is likely that such 'rebounds', at least partly, correspond to the previously observed atropine-resistant, naloxone-sensitive contractions [18,19]. However, since naloxone did not affect the e-NNA-induced hypermotility, there seems to be no involvement of endogenous opioids in the effects of NOS blockade. A tentative neuronal arrangement for the motor events observed in this study is presented in Fig. 5. The muscle layer(s) responsible for the vagally induced relaxation cannot be inferred from the present data. For simplicity, it is assumed that the various motor effects discussed are exerted on the circular muscle. To conclude, this study clearly shows that blockade of NOS by L-NNA causes an augmentation of spontaneous NANC motility in the feline jejunum. Thus, evidence is presented for a tonic suppression of the circular muscle, via the release of NO, in the small intestine, in vivo. Furthermore, the relaxations elicited by vagal stimulation upon L-NNA-induced increase in tone can possibly also involve another, as yet unidentified, transmitter.

Acknowledgements The present study was supported by The Swedish Medical Research Council (08286), The G6teborg Medical Society, The Medical Faculty, University of G6teborg, The Swedish Society for Medical Research and The Swedish Society of Medicine. Ismelin ® was generously supplied by Ciba-Geigy AB, V. Fr61unda, Sweden.

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