Extrinsic control of the release of galanin and VIP from intrinsic nerves of isolated, perfused, porcine ileum.

By immunohistochemistry galanin-like immunoreactivity and vasoactive intestinal polypeptide (VIP)-like immunoreactivity were found in nerve cell bodies mostly in the submucous plexus and in nerve fibres in the mucosa, submucosa and muscularis including the myenteric plexus of the porcine ileum and were found to co-exist in most of these structures. Using isolated, perfused porcine ileum we studied the release of galanin and VIP in response to electrical stimulation of the mixed periarterial nerves or to intraarterial infusions of different neuroactive agents. Nerve stimulation (4-10 Hz) inhibited the basal release of galanin and VIP from the ileum (to 69 +/- 6 and 62 +/- 6% of basal release). After infusion of the alpha-adrenergic blocker, phentolamine, (10(-6) M) electrical stimulation increased the release of both galanin and VIP (to 140 +/- 12 and 133 +/- 13% of basal output). This increase was abolished by atropine (10(-6) M) and by hexamethonium (3.10(-5) M). Infusion of norepinephrine (10(-6) M) inhibited, whereas acetylcholine (10(-6) M) stimulated the release of both peptides. The effect of the latter was abolished by atropine. The inhibitory effect of nerve stimulation was not influenced by atropine. Our results suggest that the galanin- and VIP-producing intrinsic neurons receive inhibitory signals by noradrenergic nerve fibers and stimulatory signals mediated by cholinergic nerves, possibly via a cholinergic interneuron.     

Galanin induces contraction of isolated cells from circular muscle layer of pig ileum.

The effects of galanin and its interaction with cholecystokinin and acetylcholine on smooth muscle cells were studied in vitro on isolated cells obtained from pig ileum circular muscle layer. Galanin induced a concentration-dependent cell contraction with a maximal contraction (24.5% decrease in cell length from control) obtained at 1 nM. The concentration of galanin inducing a half-maximal contraction was 3 pM. Tetrodotoxin (10 microM) failed to inhibit cell contraction induced by galanin (1 nM), pentagastrin (10 nM) and acetylcholine (1 microM). Atropine abolished the contraction induced by acetylcholine (1 microM), but had no effect on galanin- and pentagastrin-induced contraction. L 364,718 inhibited the contraction induced by CCK8 but not the galanin-induced contraction. At the uneffective concentration of 10 fM, galanin had a synergistic effect with an uneffective concentration of CCK8 (1 pM). These results suggest that (i) galanin contracts smooth muscle cells from pig ileum by acting on a specific receptor; (ii) galanin and either CCK or acetylcholine may act in a synergistic way to induce cell contraction.     

The metabolism of the isolated perfused canine liver

An isolated liver perfusion was used for metabolic interrelation studies in our laboratory. The liver slices, after a 2-hr perfusion period in various pretreated groups, were also studied for carbohydrate metabolism. It was found that aerobic and anaerobic metabolism of liver slices treated with deoxycorticosterone acetate, testosterone, and partial ligation of the thoracic inferior vena cava, were the same as in normal livers. We also observed depression of the glycolytic pathway for utilizing exogenous fructose in the group pretreated with carbon tetrachloride and common bile duct ligation. An increase in oxygen ocnsumption in common bile duct-pretreated animals was also observed. Such studies suggest that hepatic metabolic performance in vitro or after perfusion cannot, therefore, provide infallible information on the prior presence of important host drug treatments in hepatic disease states. Such features may complicate donor considerations in hepatic transplantation patients.     

The effect of vasoactive intestinal polypeptide (VIP) on the canine gallbladder motility.

1. VIP at doses of 10(-9) to 10(-8) M was ineffective and at doses of 5 x 10(-8) to 10(-7) M exerted a slight inhibitory effect on the tone of the canine gallbladder muscle strip. However, VIP (0.1-1 micrograms/kg) injected intravenously (i.v.) in conscious dogs dose-dependently decreased the gallbladder pressure. 2. VIP did not influence significantly the acetylcholine (ACh)- or carbachol- induced contractions of canine gallbladder under in vitro or in vivo conditions, but it decreased the electrically-induced, atropine-sensitive contractions of gallbladder muscle strips. 3. VIP (5 x 10(-9) to 5 x 10(-8) M) did not influence significantly the dose-response curve for cholecystokinin octapeptide (CCK OP) of canine and guinea-pig gallbladder muscle strips. VIP injected i.v. (0.1-0.5 micrograms/kg) in conscious dogs greatly decreased the CCK OP-induced gallbladder pressure.     

Factors affecting amylase output by the isolated perfused liver

During the perfusion of isolated rat livers with either diluted whole rat blood or washed red blood cells in a reconstituted medium, the amylase content of the plasma increased two- to threefold, and it was determined that there was a net gain in the amylase of the total system. Amylase was found in the bile excreted from these livers during the perfusion. Administration of N-nitrosodimethylamine to rats produced damage to their livers. Amylase output and bile formation by these damaged livers were below normal as were the amylase contents of the livers themselves. Lack of oxygenation of the perfusing blood resulted in lowered amylase output and bile formation by the perfused livers. No significant effects of the hormones, glucagon and insulin, on amylase production were noted. Epinephrine in large amounts inhibited amylase output probably because of its effect on blood flow through the liver. Amylase was found to be uniformly distributed throughout all lobes of the rat liver.     

Characteristics of spontaneous and evoked motility in the isolated perfused porcine duodenum.

The aims of the study were to evaluate characteristics of spontaneous motility and of the ascending excitatory peristaltic reflex (AEPR) and intraluminal cross-sectional area in the isolated perfused porcine duodenum. The parameters were measured by an intraluminal catheter by use of the perfused side-hole technique and impedance planimetry. Respiratory parameters such as pH and oxygen consumption and the arterial perfusion pressure were monitored and did not vary significantly throughout the study time. Spontaneous motility was intense at the beginning but declined and disappeared within 45-90 min. It was abolished by atropine, epinephrine, and UK-14,304 (an alpha 2-adrenoceptor agonist). Secondary motility was evoked by intraluminal balloon distensions by raising the balloon pressure to 1.5 kPa for 1-min periods. Reproducible results regarding the AEPR, external balloon diameters to elicit the AEPR, and intraluminal cross-sectional area were obtained. The order of potency (pD2 values) for inhibition of the AEPR was the selective M3-receptor antagonist 4-DAMP greater than atropine greater than the selective M2-receptor antagonist AFDX-116 greater than the selective M1-receptor antagonist pirenzepine greater than hexamethonium. 4-DAMP was 16 and 29 times more potent than AFDX-116 (P less than 0.02) and pirenzepine (P less than 0.02). None of the drugs altered the intraluminal cross-sectional area during the balloon distensions. The model provides the opportunity for physiological and pharmacological studies of duodenal motility and duodenal cross-sectional area devoid of extrinsic neural and endocrine effects. The abolishment of the AEPR by atropine is caused by blockade of the M3-receptor in the porcine duodenum.     

Biliary protein output by isolated perfused rat livers. Effects of bile salts.

The output of proteins into bile was studied by using isolated perfused rat livers. Replacement of rat blood with defined perfusion media deprived the liver of rat serum proteins (albumin, immunoglobulin A) and resulted in a rapid decline in the amounts of these proteins in bile. When bovine serum albumin was incorporated into the perfusion medium it appeared in bile within 20 min and the amount in the bile was determined by the concentration of the protein in the perfusion medium. The use of a defined perfusion medium also deprived the livers of bile salts and the amounts of these, and of plasma-membrane enzymes [5'-nucleotidase (EC 3.1.3.5) and phosphodiesterase I], in bile declined rapidly. Introduction of micelle-forming bile salts (taurocholate or glycodeoxycholate) to the perfusion medium 80 min after liver isolation markedly increased the output of plasma-membrane enzymes but had no effect on the other proteins. The magnitude of this response was dependent on the bile salt used and its concentration in bile; there was little effect on plasma-membrane enzyme output until the critical micellar concentration of the bile salt had been exceeded in the bile. A bile salt analogue, taurodehydrocholate, which does not form micelles, did not produce the enhanced output of plasma-membrane enzymes. This work supports the view that the output of plasma-membrane enzymes in bile is a consequence of bile salt output and also provides evidence for mechanisms by which serum proteins enter the bile.     

Jejunal factor stimulating insulin release in the isolated perfused canine pancreas and jejunum.

In the present study using an isolated perfused preparation of canine jejunum and pancreas, an insulin-releasing factor was found in the venous effluent of the jejunum. Insulin secretion by the pancreas rose twofold after 10% glucose was infused in the lumen of the jejunum and remained at a high level even after the stimulus was discontinued. No modification of the exocrine pancreatic secretion occurred during the insulin release, and therefore it seems unlikely that gastrin, secretin or cholecystokinin-pancreozymin were released by the jejunal mucosa. In control experiments the values of hyperglycaemia observed previously and intraluminal hyperosmolarity were tested: at these levels, they did not affect insulin secretion. The nature of this intestinal insulin-releasing factor remains unknown however, but may be identifiable when intestinal hormones in blood can be assayed reliably.     

Calcitonin gene-related peptide and capsaicin inhibit the circular muscle of the guinea-pig ileum

Calcitonin gene-related peptide (CGRP) is known to excite, through the release of acetylcholine, the circular muscle (CM) of the guinea-pig ileum in vitro. In the present experiments, the effect of rat CGRP was investigated on the CM of tetrodotoxin-treated, spontaneously active ileum preparations. CGRP (1-10 nM) caused concentration-dependent inhibition of both the amplitude and frequency of spontaneous CM contractions. Capsaicin (a sensory stimulant known to release CGRP from primary afferents) also inhibited CM activity. The effect of 1 microM capsaicin underwent rapid desensitization, indicating specific action on afferent structures, whereas a high concentration of the drug (33 microM) inhibited CM activity most probably on the smooth muscle itself.     

Mechanism of noncholinergic excitation of canine ileal circular muscle by motilin.

In the isolated perfused canine ileal segment, exogenous motilin infused for 9 min, at concentrations from 10(-10) M and 10(-8) M, increased circular muscle motility concomitant with inhibiting tonic VIP release, maximum at 10(-8) M. Both effects increased with increasing motilin concentrations. Atropine 10(-7) M pretreatment did not alter these responses. Naloxone 10(-7) M pretreatment eliminated both the increase in motor activity and the inhibition of VIP levels. Thus the nonmuscarinic neural pathway responsible for motor activation by motilin probably involves the stimulation of release of opiates, which in turn inhibit the release of VIP. Reduction of tonic inhibition of the muscle by continuous VIP release may in part account for increases in motor activity induced by motilin.     

Interactions of neurogenic responses of longitudinal and circular muscle in the guinea-pig ileum

The relationship between neurogenic responses of longitudinal and circular muscle was studied by measuring contractions and EMG or nonadrenergic, non-cholinergic (NANC) relaxations and NANC inhibitory junction potentials in different preparations of the guinea-pig ileum. NANC relaxation of longitudinal muscle was observed also without any preceding or concomitant circular muscle contraction ruling out the possibility that the latter might be the cause of the NANC relaxation. Circular muscle twitches or powerful contractions were absent if there was no preceding neurogenic or myogenic excitation of longitudinal muscle; in preparations with myenteric plexus-longitudinal muscle layers removed only small residual responses were seen although still under neurogenic influences. Thus excitation of longitudinal muscle seemed a prerequisite for synchronized and powerful contractions of circular muscle to occur. Cholinergic contraction and NANC relaxation of longitudinal muscle evoked by field stimulation were partly inhibited if the submucous plexus was also present suggesting the involvement of a more complex neuronal circuitry in these responses.     

Effects of noradrenaline and galanin on duodenal motility in the isolated perfused porcine pancreatico-duodenal block.

The influence of neurotransmitters on gastrointestinal motility is different in different segments of the gastrointestinal tract. To clarify the regulation of duodenal motility, the aim of the present study was to investigate the effects of alpha-adrenoceptor agonism and blockade and of galanin on duodenal motility. The study was undertaken in the isolated perfused porcine pancreatico-duodenal block. The agents under investigation were administered arterially. Duodenal motility was measured by means of a low-compliance perfusion system using an intraluminal catheter. In addition the concentration of galanin was measured in the portal effluent. We found that spontaneous motility was abolished by noradrenaline by an effect that was counteracted by the alpha 2-adrenoceptor antagonist idazoxan. In contrast, the selective alpha 1-adrenoceptor antagonist prazosin did not influence the effect of noradrenaline. Galanin, like noradrenaline, abolished duodenal motility. Furthermore, the concentration of galanin in the portal effluent was decreased by noradrenaline by an alpha 2-adrenoceptor mediated mechanism. We conclude that alpha 2-adrenoceptor activation and galanin inhibit duodenal motility and that the release of galanin from the pancreatico-duodenal preparation is reduced by alpha 2-adrenoceptors.     

Characteristics of ATPase activity in the sarcolemma of longitudinal and circular muscles of the canine ileum

The subcellular fraction enriched in sarcolemmal vesicles was isolated from the longitudinal muscle (LM) and the circular muscle (CM) of the canine ileum by sucrose density gradient centrifugation. Treatment of the LM and CM membranes with sodium dodecylsulfate (0.2 mg/kg protein) led to a 3-fold increase in Na,K-ATPase activity (up to 24 and 39 mumol Pi/mg protein/h, respectively) and to a 90-95% inactivation of Mg-ATPase which was 2 and 8 times (for the CM and the LM, respectively) more active than Na,K-ATPase in the untreated sarcolemma. A specific inhibition of Na,K-ATPase activity by acetylcholine (Ach) and serotonin (ST) was observed which could de blocked in the presence of muscarinic and serotonin receptor antagonists. Sensitivity of the enzyme to ST was more than one order of magnitude higher than to Ach (IC50 = 10(-8) vs 1.2 x 10(-7) M). The inhibition of Na,K-ATPase activity by the neurotransmitters was more pronounced in the LM membranes (30-40%) than in the CM ones (10-20%). These data indicate that cell membranes of the LM and CM differ both in specific ATPase activities and the responsiveness of Na,K-ATPase to the receptor-mediated effects of Ach and ST.     

Alpha 2-adrenergic receptors on the circular smooth muscle of canine small intestine.

We have studied the distribution and properties of alpha 2-adrenergic receptors in the circular muscle layer (containing deep muscular plexus) of canine small intestine. Using radioactivity labelled rauwolscine, we located the binding sites to the neuronal membranes supporting the prejunctional action of alpha 2-adrenergic agents in the gut. Moreover, although the functional data to suggest the existence of postjunctional alpha 2-adrenergic receptors coupled to contraction are not available so far, we measured a substantial number of rauwolscine binding sites on the smooth muscle plasma membranes. Scatchard and Hill analyses of the saturation data were indicative of the presence of a single high affinity site (Hill coefficient 0.996) with a KD value of 8.8 nM and the maximum number of binding sites (Bmax) of 313 fmol/mg of protein. Competition studies suggested the presence of multiple subtypes of alpha 2-adrenoceptors.     

Functional characterisation of tachykinin receptors in the circular muscle layer of the mouse ileum

OBJECTIVES: Tachykinins are important mediators in neuromuscular signalling but have not been thoroughly characterised in the mouse gut. We investigated the participation of tachykinin receptors in contractility of circular muscle strips of the mouse ileum. RESULTS: Electrical field stimulation (EFS) of excitatory nonadrenergic noncholinergic (NANC) nerves induced frequency-dependent contractions which were mimicked by substance P (SP). Desensitisation of SP and NK(1), NK(2) or NK(3) receptors significantly reduced contractions to EFS. The NK(1) receptor blocker RP67580 significantly inhibited NANC contractions to EFS. The NK(2) and NK(3) receptor blockers nepadutant and SR142801 did not affect NANC contractions per se but increased the RP67580-induced inhibition of NANC contractions to EFS. Contractions to SP were significantly reduced by RP67580 but not affected by nepadutant or SR142801. The NK(1) and NK(2) receptor agonists, septide and [beta-ala(8)]-NKA 4-10 (beta-A-NKA), respectively, but not the NK(3) receptor agonist senktide-induced dose-dependent contractions. Atropine inhibited and l-NNA augmented contractions to septide. Contractions to beta-A-NKA were insensitive to atropine but augmented by l-NNA. CONCLUSIONS: Tachykinins mediate NANC contractions to EFS in the mouse small intestine. Endogenously released tachykinins activate mainly NK(1) receptors, located on cholinergic nerves and smooth muscle cells and, to a lesser degree, NK(2) and NK(3) receptors, most likely located presynaptically.     

High degree of conversion of angiotensin I to angiotensin II in the mesenteric circulation of the isolated perfused terminal ileum of the cat.

Conversion of AI to AII has been studied in the mesenteric circulation of the isolated perfused cat terminal ileum. Infusion of AI through the mesenteric circulation induced a significantly potentiated response when the venous return was superfused over the rat colon and the rabbit aortic strip. Addition of converting-enzyme inhibitor, SQ 20881 to the perfusion medium competitively prevented the potentiation of AI on the assay organs without altering its direct effects. The percent conversion of AI to AII was found to be 68 in the mesenteric circulation. In contrast, infusion of AII through the mesenteric circulation has lost about 40% of its biological activity as measured on the same assay organs. SQ 20881 abolished the inactivation of AII in the mesenteric circulation. It is concluded that the mesenteric circulation of the isolated perfused cat terminal ileum is one of the major conversion areas of AI to AII. SQ 20881 prevented the conversion of AI to AII as well as abolishing the inhibition of AII passing through the mesenteric circulation.     

Multiple responses to electrical field stimulation in circular muscle of canine gastric corpus

Innervation of circular muscle of the canine stomach studied in vitro was investigated by subjecting muscle strips to electrical field stimulation. Strips were cut from the lesser curvature of the gastric corpus and stimulated with 10-s trains of 0.5-ms pulses at 0.5-20 Hz, 40 V. Most responses were classified into one of three types. In general, field stimulation tended to elicit sequences of varying magnitudes of transient on-contraction, on-relaxation, off-relaxation, off-contraction. Responses were abolished by tetrodotoxin. On-contraction was almost abolished by atropine plus desensitization by 5-hydroxytryptamine (5-HT) or substance P. On-relaxation and off-relaxation were not affected by adrenergic blockade, methysergide, apamin, or 4-aminopyridine. ATP usually caused contraction and slightly diminished relaxation to field stimulation. Vasoactive intestinal polypeptide (VIP) had little effect on tone and response to field stimulation. Relaxation disappeared after scorpion venom treatment. This probably resulted from depletion of the transmitter which mediates relaxation. Off-contraction was reduced by atropine, desensitization by 5-HT or substance P, cromoglycate, indomethacin or ATP, but was not affected by adrenergic blockade, hexamethonium, methysergide, mepyramine, or VIP. The findings suggest that innervation of gastric corpus circular muscle included excitatory cholinergic and both excitatory and inhibitory noncholinergic, nonadrenergic innervation. However, the responses of circular muscle to field stimulation in vitro were drastically different from those obtained previously in vivo, suggesting damage or altered inputs to circular muscle when strips of circular muscle are studied.