Participation of nitric oxide in the electrical field stimulation-induced nonadrenergic, noncholinergic (NANC) relaxation in various intestinal regions was studied in 2- to 50-week-old Wistar rats. In the jejunum of 2-week-old rats, the extent of the nitric oxide-mediated component of the relaxation of longitudinal muscle was approximately 60-70%, whereas the component was 40-50% in 4-week-old rats and was absent in 8- and 50-week-old rats. Thus, nitric oxide seems to be the most important mediator at young ages but its significance is lost with age. The same tendency as that in the jejunum was also shown in longitudinal muscle of the ileum, proximal and distal colon, and rectum. The tendency was also shown in the circular muscle of the rectum. Sensitivity of the longitudinal muscle of the jejunum and proximal colon to exogenously added nitric oxide was high in younger rats. Immunoreactive structures for nitric oxide synthase were observed in the circular muscle layer of the rectum. The population of the structures was denser in 4-week-old than that in 50-week-old. The results suggest that NANC relaxation in every region of the intestine at 2-week-old is almost solely mediated by nitric oxide, and its significance as an inhibitory mediator gradually or rapidly decreases with age.

ABSTRACT. Participation of nitric oxide, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) in nonadrenergic, noncholinergic (NANC) relaxation of longitudinal muscle of various intestinal regions in Sprague Dawley rats (8-weekold) was studied in vitro. Nitric oxide was suggested to participate in NANC relaxation of every intestinal region studied. But the participation was partial and its extent varied among the regions: significant in the proximal colon and rectum, and moderate in the jejunum, ileum and distal colon. Participation of PACAP in NANC relaxation was suggested only in the distal colon, while that of VIP was not detected in any of regions. Results obtained in the present study indicate that extent of participation of nitric oxide in NANC relaxation in Sprague Dawley rat intestine is more significant than those of other strains, Wistar and Wistar-ST.KEY WORDS: NANC (nonadrenergic noncholinergic) relaxation, nitric oxide, PACAP, rat intestine, VIP.J. Vet. Med. Sci. 62(8): 821-828, 2000 Nitric oxide has been suggested to mediate nonadrenergic, noncholinergic (NANC) relaxation in many regions of the gastrointestinal tract [22,23]. However, accumulated evidence suggests that the role of nitric oxide in NANC relaxation is not uniform throughout the gastrointestine, but quite variable in region to region. Wistar-ST rat intestine is a case in point. That is, an essential role of nitric oxide in the relaxation was suggested in circular [11] and longitudinal [24,26] muscles of the proximal colon, and in circular muscle of the rectum [27], while only minor or no role was suggested in longitudinal muscle of the jejunum [19] or the distal colon [16,24], respectively. In addition to regional difference, we recently found a strain difference in the mediator of NANC relaxation of the distal colon of rats between the Sprague Dawley and Wistar-ST strains: nitric oxide was suggested to partially mediate NANC relaxation of the Sprague Dawley but not Wistar-ST strain [21].In addition to nitric oxide, vasoactive intestinal peptide (VIP) is another candidate for the mediator of NANC relaxation in several gastrointestinal regions: lower oesophageal sphincter of opossums [5] and rabbits [2], stomach of dogs [1], guinea pigs [6, 10] and rats [13], taenia coli of guinea pigs [7,10], and colon of rats [9,24]. Pituitary adenylate cyclase activating peptide (PACAP) was also suggested to mediate NANC relaxation in some regions: lower oesophageal sphincter of cats and humans [20], stomach of guinea pigs [14], and colon of guinea pigs [12] and rats [8,15]. We suggested that VIP and PACAP mediate NANC relaxation of longitudinal muscle of the distal colon of Wistar-ST rats via opening of charybdotoxin-and apamin-sensitive K + channels, respectively [15,25]. However, participation of the peptides as well as nitric oxide in NANC relaxation does not seem to be uniform throughout the gastrointestine. For example, neither role of VIP in longitudinal muscle of the proximal and mid colon [24], a...

1 The mediators of nonadrenergic, noncholinergic (NANC) relaxation in longitudinal muscle of the jejunum and ileum of Wistar rats were examined in vitro. 2 Treatment of the jejunal and ileal segments with a-chymotrypsin resulted in decreases in the NANC relaxations induced by electrical ®eld stimulation (EFS) by about one half. 3 The NANC relaxations were also decreased by about one half after the segments had been desensitized to neurotensin. A neurotensin receptor antagonist, SR48692 (10 mM) inhibited the NANC relaxation by 56 and 34% in the jejunal and ileal segments, respectively. 4 An inhibitor of small conductance Ca 2+ -activated K + channel (SK channel), apamin (100 nM) also inhibited the NANC relaxation by 83 and 63%, respectively. Exogenous neurotensin-induced relaxations of the two segments were abolished by apamin. 5 In the ileal segments, N G -nitro-L-arginine (L-NOARG, 100 mM), inhibited the NANC relaxation by 43%. L-NOARG, but not apamin, further inhibited the relaxation which persisted after the desensitization to neurotensin. Apamin with SR48692 inhibited the relaxation only to the same extent as apamin alone. 6 EFS induced inhibitory junction potentials (i.j.ps) in the longitudinal muscle cells of the ileum. I.j.ps consisted of a rapid and a delayed phase. L-NOARG signi®cantly inhibited only the delayed phase. 7 EFS induced only a rapid i.j.ps in the jejunum. SR48692 and apamin inhibited the i.j.ps. 8 These ®ndings suggest that neurotensin and unknown substance(s) mediate NANC relaxation via SK channels in the jejunum of Wistar rats, and that neurotensin via SK channels and nitric oxide not via SK channels separately mediate the relaxation in the ileum.

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated from ovine hypothalamus and shown to stimulate adenylate cyclase (Miyata et al. 1989). Later, PACAP was also shown to be widely distributed in the gastrointestinal tract (Sundler et al. 1991). Katsoulis & Schmidt (1996) and Schw orer et al. (1992) reported that PACAP relaxed the smooth muscle preparations of guineapig ileum and taenia coli, rat fundus, ileum and colon, pig ileum, and human colon. We have previously suggested that PACAP mediates non-adrenergic, non-cholinergic (NANC) inhibitory responses of longitudinal muscle of rat distal colon via the activation of apamin-sensitive K¤ channels (Kishi et al. 1996). PACAP has also been shown to be released by nerve stimulation and to be responsible for the apaminsensitive component of electrical field stimulation (EFS)-induced relaxation and inhibitory junction potentials (IJPs) of guinea-pig taenia coli (Jin et al. 1994a;McConalogue et al. 1995b). These findings strongly suggest that PACAP is a member of a group of potential neurotransmitters responsible for NANC relaxation, which activates apamin-sensitive K¤ channels in some regions of the gastrointestinal tract. However, the mechanism by which PACAP activates apaminsensitive K¤ channels is still unknown. Increases in cyclic AMP concentrations result in the stimulation of large conductance Ca¥-activated K¤ channels in rat aorta (Sadoshima et al. 1988) and rabbit trachea (Kume 1. It has been suggested that pituitary adenylate cyclase activating peptide (PACAP) may be involved in the non-adrenergic, non-cholinergic (NANC) inhibitory response of longitudinal muscle of rat distal colon. In this study, we have investigated the intracellular mechanism of PACAP-induced relaxation in this muscle. 2. PACAP induced an apamin-sensitive relaxation of the longitudinal muscle. The tyrosine kinase inhibitors genistein at 10 ìÒ and tyrphostin 25 at 30 ìÒ, but not the cyclic AMPdependent protein kinase inhibitor Rp-8-bromoadenosine-3',5'-cyclic monophosphorothioate at 30 ìÒ significantly inhibited the PACAP-induced relaxation to 60% and 25% of control values, respectively. PACAP did not increase the cyclic AMP content of the muscle. 3. Tyrphostin 25 at 10 ìÒ significantly inhibited the relaxation of longitudinal muscle induced by electrical field stimulation (EFS), to 50% of control values. Apamin at 1 ìÒ, an antagonist of small conductance Ca¥-activated K¤ channels, also inhibited the relaxation, to 42% of control values. The inhibitory effects of tyrphostin 25 and apamin were not additive (44% of control values). 4. PACAP induced an apamin-sensitive, slow hyperpolarization of the cell membrane of the muscle. Tyrphostin 25 at 3 ìÒ inhibited this PACAP-induced hyperpolarization. Tyrphostin 25 at 10 ìÒ and genistein at 10 ìÒ inhibited the apamin-sensitive inhibitory junction potentials induced by a single pulse of EFS. 5. The PACAP-induced relaxation of longitudinal muscle occurred with a concomitant decrease in intracellular Ca¥ levels ([Ca¥]é). Tyr...

1 Changes in participation of vasoactive intestinal peptide (VIP) in nonadrenergic noncholinergic (NANC) relaxation of longitudinal muscle of the distal colon with age were studied in 2-to 50-week-old Wistar rats in vitro. 2 The extent of the VIP-mediated component of the relaxation induced by electrical ®eld stimulation (EFS) was determined by the eect of VIP 10 ± 28 , a VIP receptor antagonist. In 2-weekold rats, the extent of the VIP-mediated component of the relaxation was scarce, about 10%, whereas the component gradually increase with age and reached the maximum extent 66% at 50-week-old. 3 Since our previous results suggest that VIP induces NANC relaxation via activation of charybdotoxin (ChTx, a blocker of large conductance Ca 5 Exogenously added VIP induced no appreciable change in the membrane potential of longitudinal muscle cells of 2-week-old, whereas it induced slight slow hyperpolarization of the cell membrane in 4-week-old and magnitude of the hyperpolarization increased with age. On the other hand, relaxant response of the longitudinal muscle to exogenously added VIP was high in younger rats. 6 The present results suggest that the role of VIP in mediating NANC relaxation of longitudinal muscle of the Wistar rat distal colon is very little at neonatal stage, but it increases with age.