No abstract

1 The interaction between nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) was investigated in isolated circular smooth muscle cells and strips of the guinea-pig gastric fundus. 2 VIP induced a concentration-dependent inhibition of carbachol-induced contraction in smooth muscle cells with a maximum at 10 76 M. The relaxation by 10 76 M VIP was inhibited for 79.1+5.8% (mean+s.e.mean) by the NO-synthase (NOS) inhibitor L-N G -nitroarginine (L-NOARG; 10 74 M) in a L-arginine reversible way. Also the inducible NOS (iNOS) selective inhibitor N-(3-(acetaminomethyl)-benzyl)acetamide (1400 W; 10 76 M) inhibited the VIP-induced relaxation, but its inhibitory e ect was not reversed by L-arginine. 3 When cells were incubated with the guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ, 10 76 M), the protein kinase A-inhibitor (R)-p-cyclic adenosine-3',5'-monophosphothioate ((R)-p-cAMPS, 10 76 M) and the glucocorticoid dexamethasone (10 75 M), the relaxant e ect of VIP was decreased by respectively 80.9+7.6, 77.0+11.6 and 87.1+4.5%. 4 In circular smooth muscle strips of the guinea-pig gastric fundus, the VIP (10 79 ± 10 77 M)-induced relaxations were not signi®cantly in¯uenced by 10 74 M L-NOARG, 10 76 M 1400 W, 10 76 M ODQ and 10 75 M dexamethasone. 5 These results suggest that iNOS, possibly induced by the procedure to prepare the smooth muscle cells, is involved in the relaxant e ect of VIP in isolated smooth muscle cells but not in smooth muscle strips of the guinea-pig gastric fundus. This study illustrates the importance of the experimental method when studying the in¯uence of NOS inhibitors on the relaxation induced by VIP in gastrointestinal smooth muscle preparations.

It is widely accepted that nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) both play an important role as inhibitory neurotransmitters in non-adrenergic noncholinergic (NANC) smooth muscle responses throughout the gastrointestinal tract (Sanders & Ward, 1992; Brookes, 1993; Shuttleworth & Keef, 1995; Rand & Li, 1995).NO is produced from -arginine by one of three nitric oxide synthase (NOS) enzymes, namely neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) (Förstermann et al. 1995). The source of NO as an inhibitory regulator of gastrointestinal motility is usually thought to be nNOS in so-called nitrergic nerves; by immunochemistry, nNOS has indeed been localized to neurones and nerve endings throughout the gastrointestinal tract (see e.g. Ekblad et al. 1994a). Investigations into whether myenteric nitrergic neuron cell bodies and nerve endings also contain VIP have yielded variable results (Barbiers et al. 1993; Ekblad et al. 1994b). VIP and NO, being released from the same or separate neurones, are in general considered to contribute in parallel to a given inhibitory motor response. At the level of the gastric fundus, NO and VIP initiate and sustain NANC relaxation, respectively, in some species such as the rat and the ferret (Li & Rand, 1990; Boeckxstaens et al. 1992; D'Amato et al. 1992; Grundy et al. 1993), while NO seems to be the major neurotransmitter, to both initiate and sustain NANC relaxation, in the gastric fundus of other species such as the guinea-pig, the cat and the pig (Barbier & Lefebvre, 1993; Desai et al. 1994; Lefebvre & Vandekerkhove, 1998). The stimulation frequency of the myenteric neurones is important, as recently illustrated in the human gastric fundus; low frequency stimulation caused only NO release whereas high frequency stimulation induced both NO and VIP release (Tonini et al. 2000). It has also been proposed that NO is released from gastrointestinal smooth muscle cells in response to VIP. In isolated smooth muscle cells and smooth muscle strips of the guinea-pig gastric fundus and rat colon, the relaxant effect of VIP was indeed inhibited by NOS inhibitors (Grider et al. 1992; Grider, 1993; Jin et al. 1993). Although Western and immunoblot analysis of rabbit gastric smooth muscle did not reveal the presence of NOS, eNOS mRNA was localized in these cells by RT-PCR (Teng et al. 1998). Additionally, nNOS mRNA has been detected in gastrointestinal smooth muscle cells (Chakder et al. 1997). All these results correlate with the possibility that NO is a mediator synthesized in smooth muscle cells by VIP. In many gastrointestinal tissues nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) both play a role as inhibitory non-adrenergic non-cholinergic neurotransmitters. As the mode of interaction between NO and VIP remains controversial, the aim of this study was to investigate the interplay between NO and VIP in the mouse gastric fundus and to evaluate the nitric oxide synthase (NOS) isoform involved in VIP-induced relaxation by using induci...

No abstract

The inhibitory potency of different classes of nitric oxide synthase (NOS) inhibitors (amino acid-based substances, guanidines, isothioureas, imidazoles and indazoles) versus peripheral neuronal NOS in the pig gastric fundus was investigated by studying their influence on electrically induced relaxations in non-adrenergic noncholinergic conditions. Circular muscle strips were mounted for isotonic registration in the presence of atropine and guanethidine, and tone was raised with 5-hydroxytryptamine. Electrical field stimulation (40 V, 0.1 ms, 4 Hz, 10 s) induced short-lasting relaxations. The inhibitory effect of 1-phenylimidazole could not be evaluated because it nearly abolished the 5-hydroxytryptamine-induced tone of the tissues. 7-Nitroindazole, imidazole, 2-iminobiotin and aminoguanidine did not inhibit the electrically induced relaxations, while the other 9 substances tested were able to do so. The influence of the incubation period was tested by studying the inhibitory effect after incubation for 10 up to 60 min. For N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-nitro-L-arginine (L-NNA), L-N5-(1-iminoethyl)-ornithine (L-NIO), L-N6-(1-iminoethyl)-lysine (L-NIL), S-methyl-L-thiocitrulline and S-isopropyl isothiourea there was a moderate increase in the inhibitory effect up to 30 min of incubation so that they were incubated for 30 min to study their inhibitory potency. For L-thiocitrulline, S-methyl isothiourea and S-ethyl isothiourea, an incubation period of 60 min was used. The 9 substances concentration-dependently inhibited the electrically induced relaxations with a maximal inhibitory effect of approximately 80% except for S-methyl isothiourea (Emax of 53%). The overall order of potency was: S-isopropyl isothiourea> S-ethyl isothiourea > or = S-methylL-thiocitrulline > or = L-NNA > L-NIO > L-NAME > S-methyl isothiourea > L-thiocitrulline > L-NIL. While the potency for S-isopropyl isothiourea (EC50: 3.1 x 10(-5) M, n = 6) to S-methyl isothiourea (EC50: 11.5 x 10(-5) M, n = 5) was in the same range, the potency of L-thiocitrulline and L-NIL was clearly lower. This study showed several compounds to be potent inhibitors of peripheral neuronal NOS in the pig gastric fundus while some compounds, that were reported to inhibit brain neuronal NOS were not effective. The EC50 values found for the effective substrates in this functional study may be a guideline for the concentrations required to evaluate the role of NO in NANC neurotransmission in gastrointestinal smooth muscle preparations.