Effect of inhibitors of enkephalin degradation in the isolated guinea-pig ileum

Bestatin and high concentration of puromycin increase the depressing effect of [Met] enkephalin on the twitch response of the electrically stimulated guinea-pig ileum. Thiorphan (enkephalinase A inhibitor) is hardly effective, but phelorphan (mercapto-acetyl-Phe-Phe) a newly synthesized enzyme-inhibitor which effectively inhibits the enkephalinase A, enkephalinase B and soluble aminopeptidase activity, potentiates the effect of enkephalin dose-dependently and in low concentrations (0.01-1 microM). Enkephalinase A, though present in these tissues, is not functional under the conditions of the test, because it is inhibited by the physiological buffer itself. These results demonstrate that enkephalinase B and the membrane bound aminopeptidase, but not the soluble aminopeptidase or enkephalinase A hydrolyse enkephalins in the isolated guinea-pig ileum.     

FMRFamide enhances acetylcholine-induced contractions of guinea pig ileum

Isolated guinea pig ilea were contracted with acetylcholine (ACh) in the absence and presence of the neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2). FMRFamide (0.17-17 microM) enhanced ACh-induced contractions (observed as a leftward shift of the dose-response curve and increase in Emax) with maximal effect at 1.7 microM. FMRFamide had no effect when administered alone. These results extend the demonstration of a FMRFamide/ACh interaction to mammalian tissue and support the concept that FMRFamide, or mammalian equivalents, could play a modulatory role in mammals.     

Opioid activity of delta O-endorphin in the guinea pig ileum.

The oligopeptides beta- and delta O-endorphin were isolated from porcine and bovine pituitary respectively. Their opiate activity was determined in the guinea pig ileum and compared to that of the pentapeptide methionine-enkephalin and morphine. The rank order of opioid activity was found to be: morphine greater than beta-endorphin = Met-enkephalin greater than delta O-Endorphin which lacks the four C-terminal amino acids of beta-endorphin displayed 60% of the activity of beta-endorphin. These results indicate, that C-terminal amino acids contribute little to the affinity of beta-endorphin for opiate receptors in the guinea pig ileum.     

Myenteric neurons activate submucosal vasodilator neurons in guinea pig ileum

This study examined whether myenteric neurons activate submucosal vasodilator pathways in in vitro combined submucosal-myenteric plexus preparations from guinea pig ileum. Exposed myenteric ganglia were electrically stimulated, and changes in the outside diameter of submucosal arterioles were monitored in adjoining tissue by videomicroscopy. Stimulation up to 18 mm from the recording site evoked large TTX-sensitive vasodilations in both orad and aborad directions. In double-chamber baths, which isolated the stimulating myenteric chamber from the recording submucosal chamber, hexamethonium or the muscarinic antagonist 4-diphenylacetoxy-N-(2-chloroethyl)-piperdine hydrochloride (4-DAMP) almost completely blocked dilations when superfused in the submucosal chamber. When hexamethonium was placed in the myenteric chamber approximately 50% of responses were hexamethonium sensitive in both orad and aboard orientations. The addition of 4-DAMP or substitution of Ca(2+)-free, 12 mM Mg(2+) solution did not cause further inhibition. These results demonstrate that polysynaptic pathways in the myenteric plexus projecting orad and aborad can activate submucosal vasodilator neurons. These pathways could coordinate intestinal blood flow and motility.     

Opiate-like activity of salsolinol on the electrically stimulated guinea pig ileum

Contractions elicited by electrical stimulation of the guinea pig ileum are reduced partially by the tetrahydroisoquinoline compound, salsolinol. This action is antagonized by pretreatment with the narcotic antagonist, naloxone, but not reversed by this agent once the effect is initiated. In addition, salsolinol can reduce the inhibitory activity of morphine. These results suggest that salsolinol may be a partial agonist on this opiate-sensitive preparation.     

GABA evoked ACh release from isolated guinea pig ileum

To identify the target cells of GABAergic neurons located in the myenteric plexus, the action of γ-aminobutyric acid (GABA) on the release of acetylcholine (ACh) and on the contractions was studied using the isolated guinea pig ileum. GABA evoked a release of ³H-ACh from the contracting ileum, under conditions of loading with ³H-choline. As both the GABA-evoked release of ³H-ACh and the contractions were inhibited by bicuculline, tetrodotoxin and furosemide, but not by hexamethonium, this release seems to be evoked through GABA receptors which are bicuculline sensitive and associated with the Cl- ion channel.     

Unexpected reversal effects of naloxone on the guinea pig ileum.

Distension of the guinea pig ileum segment elicits peristaltic activity. If the distension is maintained the peristaltic activity disappears gradually; naloxone restores normal activity in such “fatigued” preparations. The bath solution surrounding a fatigued preparation inhibits peristaltic reflex activity in non-fatigued segments; this inhibitory effect is reversed by naloxone. The latter also antagonizes the inhibitory effects of adenine-nucleotides. These results indicate that during fatigue a substance is liberated which blocks peristalsis. They further suggest that naloxone-induced reversal of inhibition in the guinea pig ileum does not necessarily demonstrate that the inhibition is caused by a direct action on morphine receptors.     

Cholinesterases and choline acetyltransferase in the longitudinal muscle of the guinea pig ileum

In order to gain insight into the possible role of the ACh-system in the smooth muscle cell, the presence of choline acetyltransferase, acetylcholinesterase and butyrylcholinesterase was studied in the longitudinal muscle of the guinea-pig ileum after the mechanical removal of Auerbach's plexus. Such treatment completely removes all nerve elements as confirmed by histochemistry and electron-microscopic examination. It was found that in the longitudinal muscle devoid of all nervous elements a substantial percentage of the activity of all three enzymes still remained. Ultrastructural localization of acetylcholinesterase and butyrylcholinesterase was observed on the sarcolemma, sarcoplastic reticulum, nuclear membrane and invaginations of the sarcolemma. The localization of cholinesterases coincides with sites which are presumably involved in calcium movements during contraction and relaxation. It is well known that the depolarized smooth muscle responds to exogenous ACh with a reversible, calcium dependent contraction and it was suggested that ACh may act by increasing the influx of calcium through the cell membrane or by liberating calcium from its bound form. The presence of choline acetyltransferase and cholinesterase activities in the muscle cell proper, as well as the localization of cholinesterases on structures connected with calcium movements, support the coexistence of an intrinsic cholinergic mechanism in the smooth muscle.     

Cholinesterases and choline acetyltransferase in the longitudinal muscle of the guinea pig ileum

Summary In order to gain insight into the possible role of the ACh-system in the smooth muscle cell, the presence of choline acetyltransferase, acetylcholinesterase and butyrylcholinesterase was studied in the longitudinal muscle of the guinea-pig ileum after the mechanical removal of Auerbach's plexus. Such treatment completely removes all nerve elements as confirmed by histochemistry and electron-microscopic examination.It was found that in the longitudinal muscle devoid of all nervous elements a substantial percentage of the activity of all three enzymes still remained. Ultrastructural localization of acetylcholinesterase and butyrylcholinesterase was observed on the sarcolemma, sarcoplasmic reticulum, nuclear membrane and invaginations of the sarcolemma. The localization of cholinesterases coincides with sites which are presumably involved in calcium movements during contraction and relaxation. It is well known that the depolarized smooth muscle responds to exogenous ACh with a reversible, calcium dependent contraction and it was suggested that ACh may act by increasing the influx of calcium through the cell membrane or by liberating calcium from its bound form.The presence of choline acetyltransferase and cholinesterase activities in the muscle cell proper, as well as the localization of cholinesterases on structures connected with calcium movements, support the coexistence of an intrinsic cholinergic mechanism in the smooth muscle.Part of this work was presented at the 6^th International Meeting of the International Society for Neurochemistry, Copenhagen, 1977     

A method for the rapid development of opiate tolerance in the guinea pig ileum

The rate and degree of in vitro tolerance development to morphine, normorphine and d,1-methadone were assessed on the excised guinea pig ileum. Agonists in fixed concentrations at 1/4, 1/2, 1 and 2 x IC50 were incubated with the tissue for 1, 2 or 4 hours. The degree of tolerance development was expressed as a ratio of the IC50 after and before incubation. A high degree of tolerance developed to all three agonists and the effect could be prevented by co-incubation with naloxone. Tolerance development was stereo-specific; levorphanol and 1-methadone developed much higher degrees of tolerance than their respective d-isomers. Furthermore, under the same conditions, subsensitivity to acetylcholine, norepinephrine, and adenosine monophosphate did not develop. The in vitro tolerance was accompanied by physical dependence development as evidenced by the fact that naloxone elicited muscular contracture in the tolerant ileum. cAMP enhanced the development of tolerance to normorphine and cycloheximide could reduce this phenomenon. It is concluded that the procedure may facilitate studies on the mechanisms involved in the development of opiate tolerance and physical dependence.     

Possible existence of a new tachykinin receptor subtype in the guinea pig ileum.

The guinea pig ileum possesses NK-1 and NK-3 tachykinin receptors. As expected, [Pro9]SP and senktide, which are selective agonists of NK-1 and NK-3 receptors, respectively, were found to be highly potent in contracting the guinea pig ileum. Surprisingly, similar observations were made with septide, SP-O-CH3, [Apa9-10]SP, or [Pro9,10]SP although, in contrast to [Pro9]SP, these four peptides showed a low affinity for 3H-[Pro9]SP-specific NK-1 binding sites on membranes from the guinea pig ileum. They were also devoid of affinity for NK-2 and NK-3 binding sites. GR 71251, a compound which has been described as a NK-1 antagonist, was more potent in inhibiting the septide- than the [Pro9]SP-evoked contracting response. Altogether, these results suggest that septide, [Apa9-10]SP, and [Pro9,10]SP exert their high contracting activity in the guinea pig ileum by acting on a new subtype of tachykinin receptors.     

Potentiation of the effects of bradykinin on its receptor in the isolated guinea pig ileum

Angiotensin I-converting enzyme (ACE/kininase II) inhibitors potentiated guinea pig ileum's isotonic contractions to bradykinin (BK) and its analogues, shifting the BK dose-response curve to the left. ACE inhibitors added at the peak of the contraction immediately enhanced it further (343 +/- 40%), although the ileum inactivated BK slowly (t(1/2) = 12-16 min). Chymotrypsin and cathepsin G also augmented the activity of BK up to three- or four-fold, but in a manner slower than that of ACE inhibitors. The BK B(2) receptor blocker HOE 140 inhibited all effects. Histamine and angiotensin II were not potentiated. ACE inhibitors potentiate BK independent of blocking its inactivation by inducing crosstalk between ACE and the BK B(2) receptor; proteases activate the receptor by different mechanism.