Geert Jm Smits

Study of NO and VIP as non‐adrenergic non‐cholinergic neurotransmitters in the pig gastric fundus

1 The contribution of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) to non‐adrenergic non‐cholinergic (NANC) relaxations in the pig gastric fundus was investigated. 2 Circular and longitudinal muscle strips were mounted for isotonic registration in the presence of atropine and guanethidine; tone was raised with 5‐hydroxytryptamine. Electrical field stimulation with 10 s trains at 5 min intervals induced short‐lasting, frequency‐dependent relaxations while continuous stimulation with cumulative increase of the stimulation frequency induced sustained frequency‐dependent relaxations in both types of strips; the electrically induced responses were abolished by tetrodotoxin. 3 The short‐lasting as well as the sustained electrically induced NANC relaxations were significantly reduced by NG‐nitro‐L‐arginme methyl ester (l‐NAME). Pretreatment with L‐arginine but not D‐arginine, prevented the inhibitory effect of L‐NAME except for sustained relaxations in the longitudinal muscle strips. 4 Sodium nitroprusside, forskolin, zaprinast and 3‐isobutyl‐l‐methylxanthine induced concentration‐dependent relaxations. Exogenous NO mimicked the short‐lasting electrically induced relaxations, while endogenous VIP evoked sustained relaxations. The responses to exogenous NO and VIP were not influenced by tetrodotoxin and L‐NAME. 5 α‐Chymotrypsin abolished the responses to exogenous VIP but only moderately reduced NANC relaxations induced by continuous electrical stimulation. Zaprinast potentiated the relaxant responses to sodium nitroprusside and increased the duration of the NANC relaxations induced by electrical stimulation with 10 s trains in circular muscle strips but not in longitudinal muscle strips. 6 The cyclic GMP and cyclic AMP response to electrical stimulation, NO and VIP was measured in circular muscle strips. Short‐lasting as well as sustained electrical field stimulation induced an approximately 1.5 fold increase in cyclic GMP content, while NO induced nearly a 40 fold increase. An increase in cyclic AMP content was obtained only with sustained electrical field stimulation. 7 Immunocytochemistry for NO synthase (NOS) revealed immunoreactive neuronal cell bodies in the submucous and myenteric plexuses and nerve fibres in both the circular and longitudinal muscle layer; double‐labelling for NOS and VIP showed that VIP coexists in a major part of the intrinsic neurones. NADPH diaphorase‐histochemistry showed the same pattern of nitrergic neurones and nerves as NOS‐immunocytochemistry. 8 It is concluded that a cyclic GMP‐ and a cyclic AMP‐dependent pathway for relaxation is present in both the circular and longitudinal muscle layer of the pig gastric fundus. NO appears to contribute to short‐lasting as well as sustained NANC relaxations. A peptide, possibly VIP, may be involved during sustained stimulation at lower frequencies of stimulation.

Influence of aging on gastric emptying of liquids, small intestine transit, and fecal output in rats

The gastric emptying of a liquid meal, the small bowel transit, and the number of pellets and fecal output produced during a 24-h period, were studied in young (three months), adult (12 months) and old (24 months) male Wistar rats. The gastric emptying after 20 min of an intragastrically administered liquid meal containing phenol red and methylcellulose was significantly decreased in old rats. The small bowel transit after 15 and 30 min of the front of a charcoal and Arabic gum containing intragastrically administered meal was similar in the three age groups. The number of pellets and the mass of the feces produced during a 24-h period were significantly decreased with age, while the food intake was similar. The water content of the pellets was similar in the three age groups. These results show decreased gastric emptying of liquids and decreased stool mass in old rats, corresponding with the previously reported age-related changes in colonic smooth muscle contractility. Small intestinal transit was well maintained with age.

Influence of vasoactive intestinal polypeptide and NG-nitro-L-arginine methyl ester on cholinergic neurotransmission in the rat gastric fundus

The possible modulating effect of vasoactive intestinal polypeptide (VIP) and nitric oxide (NO), on cholinergic neurotransmission was assessed in longitudinal muscle strips of rat gastric fundus. VIP and NO are the putative co-transmitters of the inhibitory non-adrenergic non-cholinergic (NANC) neurones in this tissue. VIP concentration dependently inhibited cholinergic contractions induced by 2-min transmural stimulation, relaxed tissues, the tone of which was continuously raised by transmural stimulation, and shifted to the right the frequency-response curves for contraction induced by transmural stimulation with a cumulative increase of frequency. The same effect was found when contractions were induced with methacholine, suggesting that only functional antagonism at the postsynaptic smooth muscle cell level is involved. On 30-min incubations, 3 x 10(-4) M NG-nitro-L-arginine methyl ester (L-NAME) potentiated cholinergic responses to 20-s transmural stimulation, while not influencing contractions of similar amplitude evoked by methacholine; the cholinergic responses to 2-min transmural stimulation were also not influenced. The potentiating effect of L-NAME was prevented by L-arginine but not D-arginine. These results suggest that endogenous NO released from the inhibitory NANC neurones during short trains of transmural stimulation interferes with cholinergic neurotransmission either by functional antagonism of acetylcholine at the postsynaptic level or by presynaptic inhibition of acetylcholine release.

ATP and nitric oxide: Inhibitory NANC neurotransmitters in the longitudinal muscle-myenteric plexus preparation of the rat ileum.

1 The nature of neurotransmitter(s) involved in non‐adrenergic non‐cholinergic (NANC) relaxations induced by electrical stimulation (10 s trains, 1–8 Hz) was investigated in the precontracted longitudinal muscle‐myenteric plexus preparation of the rat ileum. 2 Electrical stimulation of the tissue induced complex responses, consisting of a primary contraction, a primary relaxation, an off‐relaxation and a rebound contraction, which were all tetrodotoxin(TTX)‐sensitive. 3 Vasoactive intestinal polypeptide (VIP) and carbon monoxide (CO) did not induce relaxations. α‐Chymotrypsin did not reduce the relaxations induced by electrical stimulation, while zinc protoporphyrin IX had non‐specific effects. 4 Nitric oxide (NO) induced concentration‐dependent relaxations. NG‐nitro‐l‐arginine methylester (L‐NAME) abolished the primary contractions and off‐relaxations, while it partially reduced the primary relaxations. 5 ATP induced relaxations and ATP‐desensitization of the tissues partially reduced the primary relaxations. Suramin and reactive blue 2 did not consistently influence the primary relaxations. 6 The ATP‐induced relaxations were not influenced by L‐NAME or TTX. The inhibitory effect of ATP‐desensitization and L‐NAME did not summate. 7 The cyclic AMP content of the tissue did not increase upon electrical stimulation or after addition of NO or ATP. The cyclic GMP content of the tissue increased upon electrical stimulation and addition of NO, but not after addition of ATP. 8 It is concluded that the relaxation induced by electrical stimulation consists of two types of responses. The off‐relaxation is completely nitrergic, while the primary relaxation is mediated by NO, ATP and an as yet unknown transmitter which is not VIP or CO.

Influence of age on cholinergic and inhibitory nonadrenergic noncholinergic responses in the rat ileum

In view of the previously reported marked reduction in the number of neurons in the rat small intestine, the influence of age on the responses to electrical stimulation of the longitudinal muscle-myenteric plexus preparation of the ileum of young (3-4 months), adult (12-13 months) and old (24-25 months) rats was investigated. The differences in responses between the three age groups were varied. At the basal tone level, electrical train stimulation induced complex responses consisting of a primary contraction, which was partly cholinergic, a secondary contraction, which was completely cholinergic and a rebound contraction, which was partly cholinergic. These responses did not differ between young, adult and old rats. The inhibiting effects of atropine and potentiating effects of physostigmine on these responses did not change with age. The acetylcholine-induced concentration-response curve did not differ between the three age groups. Electrical train stimulation of the precontracted longitudinal muscle-myenteric plexus preparation under nonadrenergic noncholinergic (NANC) conditions induced multiphasic responses consisting of a primary contraction, a primary relaxation, a postrelaxation and a rebound contraction. The primary contraction and the postrelaxation, which were nitrergic in nature, were not influenced by age. The primary relaxation was slightly more pronounced in young rats. In young rats, this relaxation was partly inhibited by NG-nitro-L-arginine methyl ester (L-NAME) and ATP desensitization, while in adult and old rats these primary relaxations were only influenced by ATP desensitization, but not by L-NAME. Nitric oxide-induced relaxations were similar in the three age groups. The cAMP content was not increased by electrical stimulation, while the cGMP content increased with electrical stimulation, but no differences due to age were observed. These results suggest that cholinergic responses in the rat small intestine are well-maintained with age while the nitrergic contribution to NANC relaxation decreases with age.

Influence of age on the signal transduction pathway of non-adrenergic non-cholinergic neurotransmitters in the rat gastric fundus.

1 The influence of aging on the relaxant response and the change in cyclic nucleotide content induced by vasoactive intestinal polypeptide (VIP), nitric oxide (NO), electrical field stimulation of the non‐adrenergic non‐cholinergic neurones and substances acting at different levels of the cyclic AMP and cyclic GMP transduction pathways was studied in longitudinal muscle strips of the rat gastric fundus. 2 The relaxant responses to VIP, sustained electrical stimulation, forskolin and 3‐isobutyl‐1‐methylxanthine were reduced with age, while the responses to dibutyryl cyclic AMP were not. The increase in cyclic AMP content induced by sustained electrical stimulation and forskolin was lower in old rats. 3 The relaxant responses to NO and to short train electrical stimulation were similar in the three age groups. The inhibitory effect of NG‐nitro‐1‐arginine methyl ester on relaxations induced by short train electrical stimulation was more pronounced in old rats. The relaxant responses to sodium nitroprusside (SNP), 8‐bromo‐cyclic GMP and zaprinast were reduced with age. SNP induced a similar elevation of the cyclic GMP content in the three age groups. 4 These results suggest that aging differentially affects the cyclic AMP and cyclic GMP pathway for relaxation by VIP and NO in the rat gastric fundus, as the defect seems to occur at the level of the adenylate cyclase and cyclic GMP‐dependent protein kinase respectively.

Development of cholinergic and inhibitory non-adrenergic non-cholinergic responses in the rat gastric fundus

1 Cholinergic contractions and inhibitory non‐adrenergic non‐cholinergic (NANC) relaxations were studied in longitudinal muscle strips of the gastric fundus of 2, 4 and 8 week old rats. 2 Contractions induced by electrical stimulation of the cholinergic neurones and by administration of acetylcholine decreased during development. The potentiating effect of physostigmine was similar in the 3 age groups. 3 Short train stimulation in NANC conditions induced fast relaxations, which were more pronounced in 4 and 8 week than in 2 week old rats. These relaxations were almost completely inhibited by NG‐nitro‐L‐arginine methyl ester (L‐NAME) in the 3 age groups. The nitric oxide‐induced relaxations did not change during development. 4 Sustained electrical stimulation in NANC conditions induced an initial relaxation, which was almost totally blocked by L‐NAME, followed by an almost complete recovery of tone at lower frequencies of stimulation. At higher frequencies of stimulation, the recovery of tone was incomplete or absent. This sustained relaxation was only partially reduced by L‐NAME and almost abolished by L‐NAME plus α‐chymotrypsin. The initial relaxations increased during development, while the sustained relaxations remained similar during this period. Vasoactive intestinal polypeptide‐induced relaxations were also similar in the 3 age groups. 5 These results show that the sensitivity of the gastric fundus to acetylcholine decreases from 2 weeks to 8 weeks postnatally, while the importance of the nitrergic innervation increases during this period.

Modulation of non‐adrenergic non‐cholinergic inhibitory neurotransmission in rat gastric fundus by the α2‐adrenoceptor agonist, UK‐14,304

1 The influence of the α2‐adrenoceptor agonist, UK‐14,304, on non‐adrenergic non‐cholinergic (NANC) relaxation induced by electrical field stimulation was investigated in longitudinal muscle strips of the gastric fundus of reserpinized rats. 2 In tissues where tone was raised by 3 × 10−7 m prostaglandin F2α (PGF2α), the inhibitory effect of 10−6 m UK‐14,304, on the NANC relaxations induced by short train stimulation (40 V, 1 ms, 20 s) was inversely related to the stimulus frequency (1‐4‐16 Hz). UK‐14,304 (10−6 m) did not influence relaxations induced by administration of exogenous nitric oxide (NO, 2 × 10−6 m−10−4 m). The inhibitory effect of UK‐14,304 on the electrically induced relaxations was antagonized by 10−6 m rauwolscine but not by 10−6 m prazosin. 3 UK‐14,304 (10−6 m) also reduced the amplitude of the sustained NANC relaxation, induced by electrical field stimulation (40 V, 1 ms, 4 Hz) for 5 min. The effect of UK‐14,304 was also antagonized by 10−6 m rauwolscine but not by 10−6 m prazosin. UK‐14,304 (10−6 m) did not reduce the relaxation induced by 3 × 10−9 m. vasoactive intestinal polypeptide (VIP). 4 These results suggest that the release of the inhibitory NANC neurotransmitter during short train stimulation, thought to be NO, and during sustained stimulation, thought to be VIP, is inhibited by stimulation of presynaptic α2‐adrenoceptors in the rat gastric fundus.

Influence of age on responsiveness of rat gastric fundus to agonists and to stimulation of intrinsic nerves

The contractile effect of methacholine, prostaglandin F2 alpha (PGF2 alpha) and electrical stimulation of cholinergic neurones, and the relaxant effect of nitric oxide (NO), vasoactive intestinal polypeptide (VIP) and electrical stimulation of inhibitory non-adrenergic non-cholinergic (NANC) neurones were studied in longitudinal smooth muscle strips of the gastric fundus of young (3 months), adult (12 months) and old (24 months) male Wistar rats. The contractile responses to methacholine and to electrical stimulation of cholinergic neurones were not significantly different between the three age groups. The responses to PGF2 alpha were significantly more pronounced in young than in adult and old rats. The relaxant response to electrical stimulation of NANC neurones with a cumulative increase in frequency showed a decreased response in old rats at the higher stimulation frequencies. This was mimicked by a decreased response to VIP, suggesting that there is a decrease in muscular sensitivity to VIP rather than an impaired capacity to VIP release with increasing age. The relaxant response to electrical stimulation of NANC neurones with short trains was similar in the three age groups, while the sensitivity to exogenous NO increased with age. The latter might be a compensatory mechanism for a decrease in stimulation-induced NO release with age.

Investigation of the influence of pregnancy on the role of nitric oxide in gastric emptying and non-adrenergic non-cholinergic relaxation in the rat

The influence of pregnancy on the role of nitric oxide (NO) in gastric emptying and in non-adrenergic non-cholinergic (NANC) relaxation was studied in rats. The gastric emptying of a non-nutrient liquid solution and of polysterene beads was studied in non-pregnant (NP), 6 to 7 days pregnant (P7) and 18 to 20 days pregnant (P20) rats. Longitudinal muscle strips of the gastric fundus and circular muscle strips of the pylorus were isolated from NP and P20 rats and NANC relaxations were induced by electrical field stimulation. The gastric emptying of the liquid meal was significantly increased in P20 rats as compared to NP and P7 rats. In NP rats, NG-nitro-L-arginine methyl ester (L-NAME) dose-dependently (50–150 mg/kg ip) reduced the gastric liquid emptying; the inhibitory effect of 100 mg/kg L-NAME ip was prevented by 400 mg/kg ip L-arginine and was mimicked by 100 mg/kg NG-monomethyl-L-arginine (L-NMMA). The percentage inhibition of the liquid emptying by L-NAME did not differ between the 3 groups, except for the dose of 150 mg/kg ip where it was significantly lower in P20 rats. The gastric emptying of beads was 54% in NP, 36% in P7 and 69% in P20 rats but these values were not significantly different illustrating the great variability. The inhibitory effect of L-NAME (25 and 100 mg/kg ip) on the emptying of beads did not differ between the 3 groups. As evaluated in NP rats, the inhibitory effect of L-NAME on the gastric emptying of the beads was not prevented by L-arginine nor mimicked by L–NMMA. Electrical field stimulation in NANC conditions induced frequency-dependent relaxations in the fundus strips and relaxations followed by rebound contractions in the pyloric strips. These electrically induced NANC relaxations and their reduction by 3×10–4 M L-NAME were not different between NP and P20 rats. It can be concluded that no evidence for a regulatory role of NO in the gastric emptying of the beads was found, and that the nitrergic contribution to the gastric emptying of liquids and to the fundic and pyloric NANC relaxations was not influenced by pregnancy in rats.