J. E. T. Fox

Sites and mechanisms of action of neuropeptides on canine gastric motility differ in vivo and in vitro

Motilin, pentagastrin and substance P (SP), injected intra-arterially into the canine gastric corpus in vivo increased the amplitude of contractions by an action dependent on activation of cholinergic nerves; i.e. atropine or tetrodotoxin (TTX) completely blocked the responses to motilin and pentagastrin and increased the ED50 of SP. TTX and atropine were not equally effective in increasing the ED50 for SP in vivo and the effect of combining them depended on the order of their addition. Both were much more effective than the SP analog D-Pro2, D-Trp7,9 SP (DSP) which appeared to be a weak antagonist of actions dependent on neural activity. In strips from the same region in vitro no receptors dependent on cholinergic nerve activation could be demonstrated for any peptide; i.e., all were atropine- and TTX-insensitive. Motilin, as expected in the absence of such receptors caused no contractile response in vitro. SP, also as predicted, caused contractions suggesting that a smooth muscle receptor, independent of nerve activation was present. However contrary to expectation pentagastrin induced an atropine and TTX-insensitive increase in the amplitude and frequency of contractions. These results show that 1) the most sensitive sites of action of a number of excitatory peptides depend on cholinergic nerve function in vivo; 2) such sites or the nerve activity on which they depend cannot be demonstrated in vitro; 3) SP has an additional site of action on smooth muscle demonstrable in vivo and in vitro, but motilin does not; 4) pentagastrin has only an action dependent on nerve function in vivo, but manifests an action independent of nerve function in vitro. We conclude that sites and mechanisms of action of peptides cannot be assumed to be identical in vivo and in vitro. Actions dependent on nerves are often lost in vitro and not all smooth muscle actions can be demonstrated in vivo.

Galanin: an inhibitory neural peptide of the canine small intestine.

Galanin injected intraarterially during phasic activity of the canine small intestine in vivo produced inhibition. Fifty percent inhibition occurred at 1.5 +/- 0.5 X 10(-10) mols lasting for 0.7 min. The inhibitory response was not decreased by treatment with atropine, hexamethonium, yohimbine or naloxone, suggesting that muscarinic, nicotinic, alpha 2 adrenergic or opiate receptors were not being stimulated. Since tetrodotoxin blockade of nerves did not reduce the response and galanin at 10(-10) mols was able to eliminate the smooth muscle response to intraarterial acetylcholine, we suggest that galanin acts to inhibit smooth muscle directly. Galanin 10(-9) M added to the muscle bath also inhibited phasic activity of the canine ileum circular muscle in vitro in the presence of tetrodotoxin. These results suggest that the neural peptide galanin may be a non-adrenergic, non-cholinergic, non-opioid neurotransmitter in the canine small intestine.

Extrinsic and intrinsic neural control of pyloric sphincter pressure in the dog.

1. In chloralose‐urethane‐anaesthetized dogs a manometric assembly was inserted via a gastrostomy to monitor pyloric pressure with a sleeve sensor. Antral and duodenal contractions were monitored with both manometric side holes and serosal strain gauges. 2. Subserosal silver wire electrodes were placed in the antrum 5 cm orad and the duodenum 3 cm aborad to the pylorus to facilitate field stimulation of intramural nerves. 3. The pylorus exerted spontaneous tone (10.8 +/‐ 4.8 mmHg) with phasic contractions occurring at a rate varying from 1‐5 min‐1 and, at times, with a superimposed higher frequency up to 15 min‐1. Atropine (30 micrograms kg‐1 I.V. and 10 micrograms I.A.) reduced and tetrodotoxin (50‐100 micrograms I.A.) enhanced the phasic activity significantly. 4. Bilateral cervical vagal section had no consistent influence on pyloric motility. 5. Stimulation of the distal ends of the cervical vagal nerves at low frequencies (0.2‐0.5 Hz, 1‐3 ms, 20 V) induced phasic pyloric contractions, which were abolished by atropine or hexamethonium (10 mg kg‐1 I.V. and 1 mg I.A.). Higher frequencies (greater than 0.7 Hz) of stimulation inhibited both phasic and tonic contractions and this inhibition was unaffected by atropine, hexamethonium, phentolamine (1.5 mg kg‐1 I.V. and 100 micrograms I.A.) or propranolol (1 mg kg‐1 I.V. and 100 micrograms I.A.). All neural responses were blocked by tetrodotoxin (50‐100 micrograms I.A.). 6. Duodenal field stimulation (0.2‐5 Hz, 0.5 ms, 40 V) induced strong phasic and tonic contractions in the pylorus. This excitation was blocked by atropine, hexamethonium, tetrodotoxin (50‐100 micrograms I.A.) or duodenal transection orad to the stimulating electrodes. 7. Antral field stimulation (0.5‐1 Hz, 0.5 ms, 40 V) completely abolished phasic activity in the pylorus and reduced tonic activity, regardless of whether the contractile activity was spontaneous or induced by neural stimulation. This inhibitory action was unaffected by atropine, hexamethonium or propranolol but was blocked by tetrodotoxin and antral transection aborad to the stimulating electrodes. Phentolamine attenuated the inhibitory effect of antral field stimulation on pyloric motility. 8. It is concluded that the distal canine pylorus exhibits myogenic tone and phasic activity which is modulated by extrinsic and intrinsic nerve pathways. Vagal nerves contain fibres, activated by different stimulus parameters which can either excite or inhibit pyloric activity. Activation of antral nerves inhibits pyloric activity, with both non‐adrenergic, non‐cholinergic and phentolamine‐sensitive pathways contributing to this inhibitory response.(ABSTRACT TRUNCATED AT 400 WORDS)

The mechanism of motilin excitation of the canine small intestine

Close intraarterial injections of motilin to the small intestine of the anaesthetized dog produce prolonged phasic contractions. Tetrodotoxin infused intraarterially blocked field stimulated contractions and abolished the response to motilin as did treatment with a combination of hexamethonium and atropine. Atropine alone increased the dose of motilin required to induce responses. Hexamethonium alone similarly increased the dose of motilin required in the jejunum, but not for the ileum. These results suggest that motilin acts to contract small intestine by stimulation of intrinsic excitatory nerves, some of which are post-ganglionic cholinergic and some of which are not, but are activated by a pathway with a nicotinic synapse. The ED50 for ileal contractions was greater than that for the jejunum and the time to reach maximum contractions longer suggesting a decreased responsiveness of the lower small intestine to motilin as compared to the upper gastrointestinal tract. These results and the lesser quantity of immunoreactive motilin in the ileum than in the jejunum may explain the lack of relationship of the activity front of the migrating motor complex in the lower small intestine to venous motilin concentrations.

Actions of galanin fragments on rat, guinea-pig, and canine intestinal motility

The 1-20 fragment of synthetic porcine galanin, prepared by tryptic digestion of the intact molecule, was equipotent to synthetic porcine galanin 1-29 in the smooth muscle actions of exciting the rat jejunal longitudinal muscle in vitro and inhibiting circular muscle contractions of the canine small intestine in vitro and in vivo, but was less potent in inhibiting nerve-stimulated contractions of the guinea-pig taenia coli. Fragment 21-29 was effective at high doses only in the canine ileum. Activity of galanin 1-11 was greatly reduced in the dog in vivo. These results may reflect species or cell type differences.

Jejunal Circular Muscle Motility Is Decreased in Nematode-Infected Rat

Jejunal circular muscle motility was studied in vitro in rats 8-10 days after inoculation with the inflammation-inducing nematode Nippostrongylus brasiliensis. The passive properties of the muscle, i.e., the development of passive tension and the optimal amount of stretch for active contractions, were unchanged by infection. Infection decreased the development of active resting tension, spontaneous contractions, muscle contraction to muscarinic receptor activation, and direct electrical stimulation. Relaxation to beta-adrenergic stimulation was also decreased in tissues from infected animals. Response to cholinergic stimulation, spontaneous contractions, and active resting tension were completely dependent on extracellular calcium. The dominant response to electrical stimulation of intrinsic nerves was relaxation in control tissue and contraction in tissue from infected rats. In the presence of atropine, all tissues from control rats but only 33% of the tissues from infected rats relaxed, suggesting a marked difference in functional inhibitory innervation. The inflammation may have either decreased the circular muscle responsiveness to the inhibitory transmitter or decreased the release of this transmitter. Thus, a nematode infection produces decreased responsiveness of the intestinal circular muscle to both contracting and relaxing stimuli and causes a reduction in functional inhibitory innervation in this layer. These changes suggest mechanisms for the reduction of intestinal transit observed after some nematode infections.

Characterization of neurotensin binding to rat gastric smooth muscle receptor sites

The binding of monoiodo 125I-Trp11-neurotensin to purified rat gastric fundus smooth muscle plasma membranes was characterized. Specific binding of ligand in subcellular fractions from rat fundus smooth muscle showed a distribution that paralleled that of several plasma membrane marker enzymes. 125I-Trp11-neurotensin binding to smooth muscle plasma membranes at 25 degrees C was maximal at 30 min, reversible and saturable. Scatchard analysis of equilibrium data indicated the existence of two classes of binding sites with dissociation constants (Kd) of 56 pmol and 1.92 nM, and corresponding binding capacities (Bmax) of 6.6 fmol/mg and 11.4 fmol/mg of membrane protein. Analogues and fragments of neurotensin competed for 125I-Trp11-neurotensin binding with a rank order of potency similar to that previously reported for their contracting effect in rat fundus strips. Na+ decreased in a concentration dependent manner the binding of labelled ligand to the high affinity site. At 100 mM, Na+ induced a 6-fold increase in the IC50 of neurotensin for inhibition of 125I-Trp11-neurotensin binding. At this concentration of Na+, the IC50 for neurotensin was 1 nM, a value close to the Kd of the low affinity site.

Immunoreactive α melanocyte stimulating hormone, its distribution in the gastrointestinal tract of intact and hypophysectomized rats

Abstract The presence of immunoreactive α melanocyte stimulating hormone (IαMSH) was investigated in both mucosal and muscular layers of the various areas of the gastrointestinal tract. IαMSH was present in both layers in all areas of the gastrointestinal tract but the esophageal mucosa and muscularis in saline extracts. The highest concentrations were found in the duodenum. Hypophysectomized males tended to have higher content than intact males. There was no difference between intact estrogen-primed females and hypophysectomized females up to 1 month post hypophysectomy in any area. The tract of 3 month hypophysectomized females showed lower levels than the intact estrogen-primed females in 5 areas; however, in similar groups of 3 month hypophysectomized females which were estrogen primed, 8 of the 10 areas contained more IαMSH than the intact estrogen-primed females. Acid extracts from female rats during the estrous cycle showed no cycle-dependent differences. Comparison of acid and saline extracts showed an absence of IαMSH in gastric tissues and a decrease in the duodenal muscularis in acid extracts but no consistent differences were found in other areas. These results suggest that the IαMSH found in the gastrointestinal tract is not of pituitary origin but may be produced in the gastrointestinal tract. The induction of increased content by estrogen priming in hypophysectomized rats suggests that estrogen priming may induce production. The absence of IαMSH in acid extracts of the stomach suggests that a difference in distribution of pro-opio-cortin products may exist in the gastrointestinal tract.

The actions of neurokinins and substance P in canine pylorus, antrum and duodenum

Analogues highly selective for receptors for substance P [beta-Ala4,Sar9,Met(02)11]-SP(4-11), for neurokinin A, [Nle10]-NKA(4-10), and for neurokinin B, [beta-Asp4,MePhe7]-NKB(4-10), were administered intraarterially before and after atropine or tetrodotoxin, to characterize the locations on nerve and muscle of the different receptor subtypes in the canine antrum, pylorus and duodenum. Circular muscle strips from each region were also studied in vitro. The NK-2 receptors in the antrum and the pylorus were located postsynaptically on smooth muscle. The NK-3 receptors, on the other hand, were located on neuronal sites in the antrum and duodenum. NK-1 receptors were located on neuronal and nonneuronal sites in the antrum, pylorus and duodenum. Only nonneural receptors could be activated in vitro.

Motor effects of gastrin releasing peptide (GRP) and bombesin in the canine stomach and small intestine.

Close intraarterial injections of synthetic porcine gastrin releasing peptide (GRP) or bombesin stimulated contractions in the stomach and inhibited ongoing contractile activity in the small intestine of anaesthetized dogs. Contractile activity of the circular muscle was recorded by serosal strain gauges and phasic activity when desired was elicited by local field stimulation or intraarterial motilin injections. In the stomach (corpus and antrum) following tetrodotoxin blockade of field-stimulated contractions, the contractile response to either peptide was not present, suggesting that stimulation of receptors on nerves initiated contractions in the stomach. Similarly, in the small intestine, the inhibitory response was eliminated by tetrodotoxin suggesting a neural receptor. Pre-treatment with reserpine did not alter the inhibitory response, either in the presence or absence of atropine, therefore, adrenergic inhibitory mechanisms did not appear to be involved. The concentration of bombesin producing 50% inhibition of field stimulation (ED50) was increased following treatment with the putative M1 muscarinic antagonist, pirenzipine suggesting activation of M1 cholinergic inhibitory receptors by bombesin. After blockade by atropine of field-stimulated contractions and the contractile response to intraarterial acetylcholine, the ED50 for bombesin inhibition of motilin contractions was increased. After muscarinic blockade, the residual inhibitory response of GRP/bombesin may involve activation of a neural non-cholinergic non-adrenergic inhibitory mechanism. These results suggest that GRP and bombesin act to alter motility in the dog in vivo by affecting neural activity.