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. 2003 Jun 6;550(Pt 3):863–871. doi: 10.1113/jphysiol.2003.043737

Interplay between Nitric Oxide and Vasoactive Intestinal Polypeptide in Inducing Fluid Secretion in Rat Jejunum

F H Mourad *,†, K A Barada *,†, N Abdel-Malak †, N A Bou Rached †, C I Khoury †, N E Saadé †, C F Nassar †
PMCID: PMC2343080  PMID: 12794180

Abstract

Nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) interact in the regulation of neuromuscular function in the gut. They are also potent intestinal secretogogues that coexist in the enteric nervous system. The aims of this study were: (1) to investigate the interaction between NO and VIP in inducing fluid secretion in the rat jejunum, and (2) to determine whether the NO effect on intestinal fluid movement is neurally mediated. The single pass perfusion technique was used to study fluid movement in a 25 cm segment of rat jejunum in vivo. A solution containing 20 mml-arginine, a NO precursor, was perfused into the segment. The effect of the NO synthase inhibitors (l-NAME and l-nitroindazole (l-NI)) and the VIP antagonist ([4Cl-D-Phe6,Leu17]VIP (VIPa)) on l-arginine-induced changes in fluid movement, expressed as μl min−1 (g dry intestinal weight)−1, was determined. In addition, the effect of neuronal blockade by tetrodotoxin (TTX) and ablation of the myenteric plexus by benzalkonium chloride (BAC) was studied. In parallel groups of rats, the effect of l-NAME and l-NI on VIP-induced intestinal fluid secretion was also examined. Basal fluid absorption in control rats was (median (interquartile range)) 65 (45–78). l-Arginine induced a significant fluid secretion (−14 (−20 to −5); P < 0.01). This effect was reversed completely by l-NAME (60 (36–65); P < 0.01) and l-NI (46 (39–75); P < 0.01) and partially by VIPa (37 (14–47); P < 0.01). TTX and BAC partially inhibited the effect of l-arginine (22 (15–32) and 15 (10–26), respectively; P < 0.05). The effect of VIP on fluid movement (−23 (−26 to −14)) was partially reversed by l-NAME (24 (8.4–35.5); P < 0.01) and l-NI (29 (4–44); P < 0.01). The inhibition of VIP or NO synthase prevented l-arginine- and VIP-induced intestinal fluid secretion through a neural mechanism. The data suggest that NO enhances the release of VIP from nerve terminals and vice versa. Subsequently, each potentiates the other's effect in inducing intestinal fluid secretion.

Since Palmer et al. (1987b) and Ignarro et al. (1987) showed that vascular endothelial cells could synthesise nitric oxide (NO), this soluble gas has emerged as an important mediator, messenger and regulator of cell function in a number of physiological systems and pathophysiological states (Moncada & Higgs, 1991; Moncada et al. 1991). In the gastrointestinal tract, there is enough evidence to indicate that NO mediates relaxation of the muscularis externa and plays an important role in mucosal blood flow, mucosal protection, the haemodynamic response to liver disease, regulation of hepatocyte function and hepatotoxicity (Stark & Szurszewski, 1992). The effects of NO on the intestinal epithelium, the local microcirculation, the enteric nervous system and inflammatory cascades are suggestive of a role for NO as a potential mediator of intestinal fluid and electrolyte transport (Salzman, 1995). NO is formed from l-arginine by the action of a stereospecific group of enzymes called nitric oxide synthases (NOS) which exist as the constitutive (cNOS) and the inducible (iNOS) isoforms (Lowenstein et al. 1994). cNOS may be further subdivided into endothelial NOS (eNOS) and neuronal NOS (nNOS), and also may be present in epithelial cells. In the gut, nNOS has been found to be localised mainly in the myenteric neurons of most animal species studied (Bredt et al. 1990; Furness et al. 1992; Llewellyn-Smith et al. 1992; Costa et al. 1992; Kostka et al. 1993; Li et al. 1995; Toole et al. 1998) and almost always coexists with vasoactive intestinal polypeptide (VIP) in the rat and guinea-pig (Furness et al. 1992; Costa et al. 1992; Li et al. 1995), but not in the golden hamster (Toole et al. 1998). It has also been recently shown that VIP and nNOS coexist in the submucosal plexus of the rat intestine (Chino et al. 2002), providing evidence that the distribution of nNOS in the enteric nervous system is species dependent.

NO has been studied as a regulator of the basal intestinal fluid transport, as an effector substance in many laxatives and as a mediator of pathological conditions where disturbance in fluid transport plays an important role. In vitro studies demonstrated an increase in short circuit current (Isc) in guinea-pig small intestine after serosal addition of the NO donors sodium nitroprusside (SNP) and isosorbide dinitrate (ISDN) (MacNaughton, 1993) and in rat ileum after addition of SNP (Rolfe & Levin, 1994). The same results were obtained in rat and human colon in vitro, after adding different NO donors such as SNP, S-nitroso acetyl penicillamine (SNAP) or saturated NO solutions (MacNaughton, 1993; Wilson et al. 1993; Tamai & Gaginella, 1993; Stack et al. 1996) suggesting that NO, at high doses, has a secretory effect. Other studies, however, have demonstrated that NO could have a basal proabsorptive tone in the intestine (Shirgi-Degen & Beubler, 1998) or even both proabsortive and prosecretory roles in cholera toxin-induced secretion (Turvill et al. 1999), thus proving that the effect of NO is multifaceted.

Even before the discovery of NO, Hellier et al. (1973) and Hegarty et al. (1981) demonstrated that l-arginine, unlike other amino acids, induced fluid secretion when perfused in human jejunum. Similarly, we found that intraluminal infusion of l-arginine (20 mm) in rat jejunum induced fluid and electrolyte secretion which could be inhibited by a low concentration (0.1 mm) of l-NAME (Mourad et al. 1996), implying that this effect is mediated by NO. The question of how NO induces fluid secretion remains unanswered. Whether NO, produced by nNOS or eNOS, acts directly on enterocytes or indirectly through neuronal reflexes is not known. A few in vitro studies have shown that the enteric nervous system may play a role in NO-induced secretion (Tamai & Gaginella, 1993; Wilson et al. 1993; Rolfe & Levin, 1994; Stack et al. 1996) and that the NO effect depends on an intact myenteric plexus (Rolfe & Levin, 1994). This effect has not been studied in vivo. It has also been shown that NO coexists with VIP (Bredt et al. 1990; Furness et al. 1992; Costa et al. 1992; Li et al. 1995; Toole et al. 1998; Chino et al. 2002) and that both interact to cause relaxation in smooth muscles with NO amplifying the effect of VIP and vice versa (Grider, 1993; Daniel et al. 1994; Keef et al. 1994; Murthy et al. 1996). Whether a similar interaction occurs in the control of intestinal fluid movement has not been previously investigated. The aims of this study were therefore to: (1) investigate the mechanism of l-arginine-induced fluid secretion by studying how it is affected by neuronal blockade and nNOS inhibition, and (2) explore the interplay between VIP and NO in the regulation of fluid transport in the rat small intestine in vivo.

METHODS

All animal experiments were approved by the Institutional Review Board-Animal Care Committee (IRB-ACC) and the University Research Board of the American University of Beirut.

Intestinal perfusion

Adult Sprague-Dawley rats (180–220 g body weight) (n = 5–11 in each group), fasted for 18 h, were anaesthetised with an intraperitoneal (I.P.) injection of sodium pentobarbitone (45 mg kg−1). Anaesthesia was maintained throughout the experiments by intermittent I.P. injections (15–30 mg kg−1) as necessary. The abdomen was opened through a midline incision and cannulae were inserted into the jejunum proximally (5 cm distal to the duodenojejunal junction) and 25 cm distally and fixed by ligation as described previously (Rolston et al. 1987). The intestine was returned to the abdominal cavity and the abdomen was closed. The femoral vein was cannulated for intravenous (I.V.) administration of saline or drugs. The isolated intestinal segment was perfused in situ at a rate of 0.5 ml min−1 with a plasma electrolyte solution (PES) (mequiv l−1: Na+ 140, K+ 4, Cl− 104, CO2 40) containing 4 μCi l−1 [14C]polyethylene glycol ([14C]PEG) as a non-absorbable volume marker (control) or with the same solution to which 20 mml-arginine (PES + l-arginine) or d-arginine was added. Thirty minutes were allowed to elapse to ensure establishment of a steady state, following which consecutive 10 min collections of the effluent were obtained from the distal cannula for 1 h. At the end of the experiments, the animals were killed by an overdose of pentobarbitone and the perfused intestinal segment was removed, rinsed, blotted and desiccated in an oven at 100 °C to obtain the dry weight. The samples of effluent were analysed immediately or kept frozen at −20 °C until analysed within 2 weeks. Short segments of the perfused intestine were taken and stored in 10 % formalin for histological investigations when needed.

Nitric oxide synthase inhibition and VIP antagonism

The NOS inhibitor, nitro-l-arginine methyl ester (l-NAME) at concentrations ranging from 0.1 to 20 mm was added to PES or PES + l-arginine and perfused in the intestine as described above. The same experiments were repeated after administration of the nNOS inhibitor l-nitroindazole (l-NI) (50 mg kg−1 in 10 mg kg−1 peanut oil I.P.) instead of l-NAME.

In further groups of rats, an infusion of the VIP antagonist [4Cl-D-Phe6,Leu17]VIP at a dose of 2 μg kg−1min−1 was started, and at the same time the intestine was perfused with either PES alone or PES + l-arginine.

Neuronal blockade or ablation by TTX, BAC or capsaicin

To determine whether the effects of l-arginine on fluid movement were neurally mediated, different protocols for neuronal blockade or ablation were used.

(i) TTX at a concentration of 0.2 μm was added to the intestinal perfusate of PES or PES + l-arginine.

(ii) The myenteric plexus was ablated with benzylalkonium chloride (BAC) (Sakata et al. 1979; Holle & Forth, 1990; Ramalho et al. 1993). Rats were anaesthetised with pentobarbitone sodium (45 mg kg−1) and the abdominal cavity was opened by a midline incision. A 25 cm segment of the proximal jejunum, 5 cm distal to the ligament of Treitz, was exteriorised and soaked for 30 min in a sterile Petri dish containing 3 mm BAC solution. The segment was then thoroughly washed with saline and returned to the abdominal cavity. The abdominal wall was closed, and the animal was left to recover for a period of 2–3 weeks. This procedure results in ablation of the myenteric plexus of the treated segment (Sakata et al. 1979; Holle & Forth, 1990; Ramalho et al. 1993). Sham rats underwent the same surgical procedure, and the jejunal segment was soaked with saline for an equal length of time. Animals had access to only water for the first 24 h after treatment. Following this initial period, they were provided with food and water. The antibiotic penicillin G (250 000 u kg−1) was given intramuscularly daily for 2 days after the surgery. The effect of intestinal perfusion of l-arginine on fluid transport was then examined as described above.

(iii) Capsaicin-sensitive primary afferents (CSPA) were chemically ablated. Under ether anaesthesia, rats were injected subcutaneously (S.C.) at time zero with 25 mg kg−1 capsaicin (in 10 % Tween 80, 10 % olive oil, 80 % distilled water) followed by two injections (under ether anaesthesia) of 50 mg kg−1 capsaicin at 8 and 32 h after the first injection (McCafferty et al. 1997). Fifteen days later, rats were checked for successful ablation using the eye wipe test (Hammond & Ruda, 1991) and were entered in the experimental protocol.

Antagonism of VIP-induced intestinal fluid secretion

VIP was started at a dose of 0.6 μg kg−1 min−1 I.V. and the intestine was perfused with PES to which different concentrations of l-NAME were added (0.1–20 mm). In order to determine whether the effect of l-NAME on VIP-induced secretion was specifically NO-mediated, another set of experiments was conducted in which l-arginine was given S.C. (100 mg kg−1) before starting the perfusion with l-NAME to counteract its effect. Further groups of rats were given either l-NI (50 mg kg−1 I.P.) or the VIP antagonist (2 μg kg−1 min−1 I.V.) and the intestine was perfused with PES.

Analytical methods and data analysis

[14C]PEG concentrations were measured in duplicate in the effluent and perfusate by liquid scintillation spectroscopy and fluid movement calculated according to the equation:

graphic file with name tjp0550-0863-mu1.jpg

where PEGp and PEGe are the measured counts per minute (c.p.m.) of the radioactive carbon in the perfusate and effluent samples, respectively.

Fluid movement is expressed as μl min−1 (g dry intestinal weight)−1. Positive values denote net absorption and negative values denote secretion. Steady-state conditions were confirmed by less than 5 % variation in fluid movement between consecutive 10 min collections. Values were accepted only if [14C]PEG recovery fell between 95 and 105 % (Rolston et al. 1987).

Results are expressed as medians and interquartile ranges in each group of animals studied. Multiple comparisons between groups were made using the Kruskall-Wallis test and differences between pairs were tested using the Wilcoxon rank sum test as appropriate.

Materials

VIP, VIP antagonist, l- and d-arginine, l-NAME, l-NI, capsaicin, BAC and TTX were obtained from Sigma Chemical Company (St Louis, Missouri, USA). Radiolabelled polyethylene glycol ([14C]PEG 4000) was obtained from Amersham International (Buckinghamshire, UK). All other reagents were supplied by BDH Chemicals (Poole, UK).

RESULTS

Effect of arginine, NOS inhibition and VIP antagonism on intestinal fluid transport

Basal fluid absorption in control rats was 65 (45–78) μl min−1 g−1 (n = 11). d-Arginine did not produce a significant alteration of basal fluid transport (70 (42–75), n = 6). l-Arginine, however, induced a significant intestinal fluid secretion (−14 (−5 to −20), n = 11; P < 0.01), which was dose dependently reversed by the NOS inhibitor l-NAME (Fig. 1). At a concentration of 1 mm, l-NAME completely abolished the effect of l-arginine on intestinal fluid transport (60 (35–65), n = 5). At higher concentrations, 10 and 20 mm, l-NAME resulted in a drop in fluid absorption (Fig. 1). When l-NAME was perfused in the intestine in the absence of l-arginine, it caused a drop in basal fluid absorption (Fig. 2). Similarly, l-NI administration resulted in a complete inhibition of l-arginine-induced secretion (46 (39–75), n = 5) but l-NI had no effect on basal intestinal fluid absorption (Fig. 3).

Figure 1. Effect of different concentrations of l-NAME (n = 5 in each group) on rat jejunal fluid secretion induced by 20 mml-arginine (l-Arg; n = 10).

Figure 1

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All solutions were perfused in the rat jejunum. Results are expressed as medians (horizontal lines) and interquartile ranges (boxes); positive values denote absorption and negative values denote secretion. P < 0.005, Kruskall-Wallis test; * P < 0.01 compared to control; † P < 0.01 compared to l-arginine and to control; ‡ P < 0.01 compared to l-arginine and P > 0.05 compared to control.

Figure 2. Effect of different concentrations of l-NAME (n = 5 in each group) on rat jejunal basal fluid movement (control; n = 11).

Figure 2

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Solutions were perfused in the rat jejunum. Results are expressed as medians and interquartile ranges; positive values denote absorption and negative values denote secretion. P < 0.005, Kruskall-Wallis test; * P < 0.01 compared to control.

Figure 3. Effect of neuronal NOS inhibition and VIP antagonism on rat jejunal basal fluid movement and l-arginine-induced secretion.

Figure 3

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l-Nitroindazole (NI; 50 mg kg−1) was given I.P. and the VIP antagonist [4Cl-D-Phe6,Leu17]VIP (VIPa; 2 μg kg−1 min−1) was given I.V. The intestine was then perfused with a plasma electrolyte solution with or without 20 mml-arginine (l-Arg) (n = 5 in each group). Results are expressed as medians and interquartile ranges; positive values denote absorption and negative values denote secretion. * P < 0.01 compared to l-Arg and P > 0.05 compared to control + l-NI. † P < 0.01 compared to l-Arg and P < 0.05 compared to control + VIPa.

The VIP antagonist [4Cl-D-Phe6,Leu17]VIP reversed the effect of l-arginine on intestinal fluid movement, from secretion to absorption (37 (14–47), n = 5; P < 0.01), but the absorption rate did not return to normal values (P < 0.05 compared with normal controls) (Fig. 3). The VIP antagonist had no effect on basal fluid transport but completely reversed VIP-induced secretion to basal values (Table 1).

Table 1.

Effect of VIP antagonism on VIP-induced jejunal fluid secretion

n Fluid movement (μl min−1−1)
Control 11 65 (45–78)
Control + VIPa (0.2 μg kg−1 min−1) 5 59 (50–79)
Control + VIPa (2 μg kg−1 min−1) 5 67 (48–82)
VIP 7 23 (−25 to −23)*
VIP + VIPa (0.2 μg kg−1 min−1) 5 22 (13–28)†
VIP + VIPa (2 μg kg−1 min−1) 5 53(40–61)‡
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VIP, vasoactive intestinal polypeptide. VIP given at a dose of 0.6 μg kg−1 min−1 I.V. VIPa, VIP antagonist [4Cl-D-Phe6, Leu17]-VIP given I.V. Data are medians and interquartile ranges.

*

P < 0.01 compared to control

†

P < 0.01 compared to VIP and P < 0.01 compared to control + VIPa (0.2 μg kg−1 min−1)

‡

P < 0.01 compared to VIP and P > 0.05 compared to control + VIPa (2 μg kg−1 min−1).

Effect of TTX, BAC and capsaicin treatments on fluid movement

Intraluminal perfusion of TTX at a concentration of 0.2 μm did not affect basal fluid absorption (62 (49–71), n = 5) but partially reversed the effect of l-arginine (22 (15–32), n = 5; P < 0.05 compared with either the TTX control or l-arginine) (Fig. 4).

Figure 4. Effect of tetrodotoxin (TTX) and benzylalkonium chloride (BAC) treatment on rat jejunal basal fluid movement and on l-arginine-induced secretion.

Figure 4

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TTX (0.2 μm) was added to the intestinal perfusate. BAC treatment of the jejunal segment was performed 2–3 weeks prior to the intestinal perfusion. The intestine was perfused with a plasma electrolyte solution with or without 20 mml-arginine (l-Arg; n = 5 in each group). Results are expressed as medians and interquartile ranges; positive values denote absorption and negative values denote secretion. * P < 0.05 compared to TTX control and P < 0.05 compared to l-Arg. † P < 0.05 compared to BAC control and P < 0.01 compared to sham + l-Arg.

BAC treatment did not induce a significant alteration in the basal fluid transport (51 (40–56), n = 5), but produced an incomplete reversal of the effect of l-arginine (15 (10–26), n = 5; P < 0.05 compared with BAC and P < 0.01 compared with l-arginine) (Fig. 4). On histological examination, jejunal segments treated with BAC showed an noticeable increase in the thickness of the muscle layer and hypertrophy of the crypts and villi. These changes are similar to those described by others (Sakata et al. 1979; Holle & Forth, 1990; Ramalho et al. 1993).

Capsaicin treatment resulted in a significant drop in basal fluid absorption (36 (10–43), n = 5; P < 0.05 compared with control); however, it did not affect l-arginine-induced secretion (−19 (−23 to −13), n = 5).

VIP-induced secretion: effect of l-NAME and l-NI

VIP induced significant intestinal fluid secretion (−23 (−16 to −25), n = 6), which was ameliorated or partially reversed by different concentrations of l-NAME, the optimal concentration being 0.5 mm (24 (8.4–35.5), n = 5; P < 0.01). However, at high concentrations (10 and 20 mm), l-NAME had no effect on VIP-induced secretion (Fig. 5). The effect of 0.5 mml-NAME on VIP-induced secretion, namely changing secretion to absorption, was completely abolished by pretreatment with 100 μg l-arginine given subcutaneously (−13 (−17 to −2), n = 5; P < 0.05 compared with VIP + 0.5 mml-NAME, and P > 0.05 compared with VIP). l-NI also reversed the secretory effect of VIP to absorption (29 (4–44), n = 5; P < 0.01) but did not produce a complete recovery (Fig. 6).

Figure 5. Effect of l-NAME on rat jejunal fluid secretion induced by VIP.

Figure 5

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VIP (0.6 μg kg−1 min−1) was given I.V. The jejunal segment was perfused with either a plasma electrolyte solution alone (n = 7) or after addition of different concentrations of l-NAME (n = 5–6 in each group). Results are expressed as medians and interquartile ranges; positive values denote absorption and negative values denote secretion. P < 0.005, Kruskall-Wallis test. * P < 0.01 compared to control; † P < 0.01 compared to VIP and P < 0.01 compared to control; ‡ P < 0.05 compared to VIP and P < 0.01 compared to control; § P > 0.05 compared to VIP and P < 0.01 compared to control.

Figure 6. Effect of neuronal NOS inhibition on VIP-induced fluid secretion in rat jejunum.

Figure 6

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l-Nitroindazole (l-NI) (50 mg kg−1) was given I.P. (n = 5) and VIP was administered I.V. (0.6 μg kg−1 min−1). The jejunal segment was perfused with a plasma electrolyte solution. Results are expressed as medians and interquartile ranges; positive values denote absorption and negative values denote secretion. * P < 0.01 compared to control; † P < 0.01 compared to VIP and P < 0.01 compared to control.

DISCUSSION

The present experiments suggest that l-arginine-induced intestinal fluid secretion is neurally mediated and depends on the production of NO using the neuronal NOS and on the release of VIP. In addition, VIP induces intestinal fluid secretion either directly through its action on enterocytes and secretomotor neurons, or indirectly through the release of NO.

The effect of NO on intestinal fluid transport has been controversial, with many studies showing a proabsorptive effect and other studies proving a prosecretory effect (Izzo et al. 1998; Mourad et al. 1999). We have previously demonstrated that NO acts as an absorbagogue under basal physiological conditions, and as a secretagogue when given or produced in high concentrations (Mourad et al. 1996). The present experiments consolidate some of our previous findings. The fact that the NOS inhibitor l-NAME at low concentrations reverses l-arginine-induced fluid secretion to control values suggests that the l-arginine effect occurs through formation of NO. Interestingly, when high concentrations of 10 and 20 mml-NAME were added to the perfusate, there was again a drop in fluid absorption to a level similar to that seen with the perfusion of l-NAME alone, suggesting that, at these concentrations, l-NAME totally abolishes the effect of the perfused and basal l-arginine production and that NO has a basal proabsorptive tone. This is in accordance with the findings of Shirgi-Degen & Beubler (1998), who demonstrated that l-NAME, unlike d-NAME, induced fluid secretion in rat jejunum in vivo. Although l-NAME has been found to possess an antimuscarinic effect at high concentrations (Buxton et al. 1993), this is unlikely to play an important role in the present experiments since its muscarinic effect would be expected to increase fluid absorption rather than increase secretion. It seems that there is a certain specific threshold concentration of NO that determines its dual effect on intestinal fluid transport. Our results correlate well with the observations made by Wapnir et al. (1997) showing that a low concentration of l-arginine added to the oral rehydration solution enhanced fluid absorption whereas higher concentrations reversed electrolyte transport.

The fact that the nNOS inhibitor l-NI did not affect basal fluid absorption suggests that the basal proabsorptive tone of NO, which is abolished by l-NAME, but not by l-NI, is produced through endothelial NOS activity rather than neuronal NOS activity. This is further substantiated by the observation that treatment with BAC, which leads to the destruction of the myenteric plexus, did not result in a significant alteration in fluid absorption. Whether this effect is mediated through maintaining blood perfusion to the intestine, through a direct action on enterocytes, or indirectly through extrinsic neuronal mechanisms cannot be answered from our experiments.

The exact mechanism of action of NO in inducing fluid secretion is not fully understood. NO could modulate intestinal fluid transport: (i) by acting directly on the epithelium, (ii) by indirectly stimulating neuronal reflexes, (iii) by stimulating the release of other agents from the epithelium or the intestinal innervation that can modify fluid transport or (iv) by affecting mucosal blood flow (Mourad et al. 1999). Some investigators have alluded to the fact that fluid secretion induced by NO donors such as SNP and SNAP, and NO precursors such as l-arginine could be neurally mediated since TTX (1–1.25 μm) inhibited the changes in short circuit current induced by these agents in guinea-pig ileum, and in human and rat colon mounted in Ussing chambers (Tamai & Gaginella, 1993; Wilson et al. 1993; Rolfe & Levin, 1994; Stack et al. 1996). This effect has not been studied in vivo. In the present report, we provide evidence that NO-induced fluid secretion in vivo is neurally mediated. First, l-arginine-induced secretion was completely blocked by the nNOS inhibitor l-NI which reversed fluid secretion to absorption at a level similar to that seen with controls, implying that l-arginine induces secretion through formation of NO through nNOS. Second, TTX partially reversed the effect of l-arginine but had no effect on basal fluid transport, suggesting that the effects of NO require an intact transmission of neuronal messages. It has previously been shown that the concentration of TTX used (0.2 μm) does not alter the ionic movements in rat colon and small intestine (Zimmerman & Binder, 1983; Jodal et al. 1993) but interfers with synaptic mechanisms (nicotinic) activated by high doses of carbachol (Zimmerman & Binder, 1983) or cholera toxin (Jodal et al. 1993). Third, destruction of myenteric neurons by BAC markedly decreased the effect of l-arginine without interfering with the basal level of fluid absorption. Hence, an intact myenteric plexus is necessary for the full effect of NO. In general, BAC-treated rats have an altered mucosal area due to hypertrophy of both villi and crypts (Holle, 1991; Hadzijahic et al. 1993), but this does not result in a significant decrease in basal fluid absorption. These findings are in line with previous observations that BAC-treated rats do not show malabsorption and have a normal growth pattern (Holle & Forth, 1990). Although ablation of CPSA fibres decreased basal fluid absorption, CSPA fibres do not seem to play a role in l-arginine-induced intestinal fluid secretion. Thus, it seems likely that CSPA fibres are involved in extrinsic reflex mechanisms contributing to the proabsorptive role of NO. However, the lack of an effect of CPSA fibres on l-arginine-induced fluid secretion suggests that NO may act on local intrinsic mechanisms involving mainly the enteric neurons in the myenteric and submucosal plexuses. This hypothesis may open the issue of the involvement of VIP and NO in the intestinal synaptic circuitry.

VIP is present in enteric neurons (Bryant et al. 1976; Costa et al. 1980) and has been proposed as a non-adrenergic non-cholinergic (NANC) inhibitory transmitter in motor neurons, especially in the myenteric plexus (Fujimiya & Inui, 2000) and as a stimulatory transmitter of secretory processes in the submucous plexus and the mucosa (Cooke, 2000). It has been demonstrated that VIP release from rat enteric synaptosomes could be stimulated by the NO donors SNP, 3-(morpholino) sydnonimine (SIN-1), as well as by l-arginine (Allescher et al. 1996). Similarly, in isolated perfused canine ileum, VIP release was reduced by the NOS inhibitor NG-nitro-l-arginine (l-NNA) and increased by NO donors (Daniel et al. 1994); hence NO could constitute a key player in the secretory mechanisms by releasing VIP from nerve terminals.

Our experiments demonstrate that VIP plays an important role in l-arginine-induced fluid secretion. The VIP antagonist [4Cl-D-Phe6,Leu17]VIP reversed l-arginine-induced intestinal secretion to absorption but did not return it to control values, which implies that other neuronal mediators may be also involved or that NO may exert a direct secretory effect on enterocytes. We have previously shown that the VIP antagonist used is a specific VIP inhibitor and can completely reverse VIP-induced changes in fluid absorption in vivo (Mourad & Nassar, 2000) (Table 1). It has been well established that VIP and nNOS are colocalised mainly in the myenteric neurons (Furness et al. 1992; Costa et al. 1992; Li et al. 1995) although some recent data have demonstrated their co-existence in rat submucosal neurons, the axons of which can secrete either VIP or NO (Chino et al. 2002). On the other hand, VIP/NOS terminals in the myenteric plexus synapse on VIP and non-VIP secretomotor neurons that do not contain NOS (Li et al. 1995). This may imply that NO produced in the myenteric plexus, and to a lesser extent in the submucosal plexus, could induce VIP release from either plexus to induce secretion. In addition, sodium nitroprusside, a NO donor, induces the production of cGMP in both the epithelial and subepithelial layers of the rat colon (Wilson et al. 1996) and in VIP secretomotor neurons in the guinea-pig ileum (Young et al. 1993), providing further proof that NO could act at several sites in the intestine through the stimulation of guanylyl cyclase and in particular it could influence the firing of VIP-ergic neuronal fibres. Thus, NO might affect neuronal input to VIP-containing neurons. Furthermore, the observations that exogenous NO can modulate the slow excitatory potential in the myenteric neurons (Tamura et al. 1993) and that neuronal NO can activate local inhibitory enteric neurons and can act as a retrograde neurotransmitter (Yuan et al. 1995) add to the complexity of intestinal mechanisms triggered by NO.

Many studies have shown that VIP releases NO from smooth muscles (Murthy et al. 1993) and from myenteric neurons (Chakder & Rattan, 1996). We therefore investigated whether the VIP secretory effect involves NO. Our data show that VIP, a known intestinal secretagogue which acts by inducing cAMP, is partially inhibited by l-NAME and by the nNOS inhibitor l-NI, proving that this effect is neurally mediated and not dependent on a change in mucosal blood flow that could be induced by l-NAME. The fact that a subcutaneous injection of l-arginine reversed the effect of l-NAME on VIP-induced secretion proves that this effect is specific and mediated by inhibition of NO formation. VIP seems to induce the formation or the release of NO, which may directly enhance fluid secretion or may further increase VIP release from neurons. This suggests the scenario that these two secretagogues could act synergistically, with NO potentially amplifying the biological effects of VIP. The source of NO cannot be determined from the present experiments. The ability of VIP to induce the formation or release of NO seems to vary depending on the tissues, and maybe the animal species, studied. Whereas it has been previously shown that VIP does not induce formation of NO from nerve terminals but may induce NO from smooth muscle of gastric tissues (Murthy et al. 1993, 1996), Chakder et al. (1996) demonstrated that the increase in NO production in response to VIP in opossum internal sphincter occurs mainly from the myenteric neurons, with some contribution from the smooth muscle. This interaction at the neuromuscular junction in rat jejunum has not been previously studied. Whether NO formed from smooth muscles or myenteric neurons will further enhance VIP formation and release needs to be investigated. Furthermore, VIP depolarises many enteric neurons including the secretomotor neurons and the intrinsic sensory neurons (Mihara et al. 1985; Palmer et al. 1987a), which may further enhance fluid secretion independently of the direct action of VIP on enterocytes. Therefore, exogenous VIP could induce secretion by stimulating such secretomotor fibres including NO-containing fibres.

In conclusion, NO produced from l-arginine through nNOS activity stimulates the enteric nervous system with the release of VIP from nerve terminals leading to intestinal fluid secretion. In addition, VIP induces NO production or release which may further enhance its secretory effect.

Acknowledgments

This research was supported by a grant from the University Research Board (URB) and the Medical Practice Plan (MPP) at the American University of Beirut, Lebanon.

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