Binding and biologic activity of neurotensin in guinea pig ileum

Experiments were performed to relate receptor binding to biologic activity for the contractile effect of neurotensin (NT) in guinea pig ileum. The contractile response was examined on pieces of ileum under 1 g tension in a 5 ml bath of oxygenated Tyrode's at 38°C. NT contracted the longitudinal muscle (ED₅₀, 0.3 nM), the 2–3 g response peaking at 1 min and fading rapidly. In the presence of atropine (1 μM), ≥50% of the response was blocked and the residual effect gave an ED₅₀ of 1.4 nM. In the presence of atropine and CP-96,345, a substance P receptor antagonist (0.2 μM), no contraction was observed at 20 nM NT. Thus, there were two components to the response, one involving acetylcholine (ED₅₀, 0.3 nM) and one substance P (ED₅₀, 1.4 nM). Using membrane preparations and ¹²⁵I-labeled NT, specific, high affinty receptors for NT were demonstrated in the muscle and myenteric plexus. Scatchard analyses indicated the presence of two binding sites (K_ds: 0.1 nM and 2 nM). Sodiu ion and GTP analogs inhibited binding. Binding and biologic activity were similar in regard to dependence on specific groups within NT and sensitivity to metal ions. The high potency of Hg⁺⁺ was consistent with an involvement of free sulfhydryl group(s) in the binding reaction; this was supported by work with SH-directed agents. The results suggest that two receptor types or configurations may mediate the two components of the contractile effect of NT on guinea pig ileum.     

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.     

Inhibition of enkephalin degradation in the guinea pig ileum

Guinea pig ileum tissue preparations contain enzymes which degrade both leucine and methionine enkephalin by cleavage of the N-terminal tyrosine residue. Similar enkephalin degrading activity is also found in the fluid bath surrounding ileum tissue preparations and appears to arise from serum and broken cell enzymes. Chelating agents such as 1,10-phenanthroline and 8-OH quinoline are effective inhibitors of enkephalin destruction by these enzymes but in the concentrations necessary to inhibit all enzyme activity, they disturb the contractility of the ileum during $\text{in}$$\text{vitro}$ bioassays. The presence of enkephalin degrading enzymes and the lack of appropriate peptidase inhibitors may hinder the determination and quantification of enkephalin release in this tissue.     

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.     

Contractile activity of Trimeresurus flavoviridis phospholipase A2 on guinea pig ileum and artery.

Trimeresurus flavoviridis phospholipase A2 (PLA2) induced strong contractions of the smooth muscles of guinea pig ileum and artery in a concentration-dependent manner (10(-10)-10(-6) M). When the same dose of PLA2 was administered in repetition to the ileal preparation, the contraction diminished progressively and was no longer recovered even by consecutive washings. The enzymatically inactive derivative of PLA2, in which His-47 was p-bromophenacylated, was unable to elicit contraction. Also, no activity was observed when the Ca(2+)-free medium was used. The contraction induced by PLA2 was inhibited completely by 1.0 x 10(-6) M indomethacin, but not by nordihydroguaiaretic acid. These results imply that the PLA2-induced contraction is due essentially to the hydrolytic action of the enzyme against phospholipid membranes to liberate arachidonic acid that is then converted to pharmacologically active prostaglandins. In guinea pig artery, PLA2 caused both contraction and relaxation.     

Differentiation of multiple neurokinin receptors in the guinea pig ileum

We have studied the selectivity and competitiveness of three neurokinin antagonists and atropine against substance P, neurokinin A, and neurokinin B. DPDTNLE-NB, [D-Pro2, D-Trp6,8, Nle10]-neurokinin B is a competitive antagonist of neurokinin B (pA2 = 5.5), but not substance P or neurokinin A. DPDT-SP ([D-Pro2,Trp7,9]-substance P), competitively blocks substance P (pA2 = 6.9) and neurokinin B (pA2 = 6.8), but not neurokinin A. Spantide ([D-Arg1, D-Trp7,9, Leu11]-substance P) competitively blocks substance P (pA2 = 6.7) and at a log unit higher concentration blocks neurokinin A (pA2 = 5.8), but does not block neurokinin B. Atropine is a competitive antagonist of neurokinin B (pA2 = 9.0) at ten times the concentration needed to block acetylcholine (pA2 = 10.1), but does not inhibit the other neurokinins. These results support the hypothesis of multiple neurokinin receptors in the guinea pig ileum and indicate that the site of neurokinin B, but not substance P or neurokinin A is predominantly on intramural neurons. This indirect stimulation appears to be dependent on the release of acetylcholine. Neurokinin B also has activity on smooth muscle receptors since the contractile response could not be completely antagonized by atropine. There appear to be two smooth muscle neurokinin receptors on the basis of results obtained with DPDT-SP and spantide, one predominantly responsive to substance P and the other to neurokinin A. Only spantide appeared to have any effect on the neurokinin A receptor and that was at a much higher concentration than that needed to block substance P.     

The activity of synthetic leukotriene C-1 on guinea pig trachea and ileum

The effects of synthetic leukotriene C-1 (LTC-1) on isolated guinea pig trachea and ileum have been determined and compared to histamine. LTC-1 produced a slow contraction of the trachea and the ileum with pD2 values of 8.7 +/- 0.1 (n = 14) and 8.5 +/- 0.1 (n = 13), respectively. In comparison, the pD2 values for histamine were 5.6 +/- 0.1 (n = 6) and 6.2 +/- 0.1 (n = 6), indicating LTC-1 was 2-3 orders of magnitude more potent. LTC-1 was antagonised by FPL 55712 with pA2 values of 6.9 +/- 0.1 (n = 5) and 6.4 +/- 0.1 (n = 7) on the trachea and ileum, respectively. Incubation with lipoxidase produced a time and enzyme dependent loss of biological activity and a concurrent shift in U.V. absorption spectrum.     

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.     

HPLC preparation of fish waste hydrolysate fractions. Effect on guinea pig ileum and ACE activity

The effect of RP-HPLC-purified fractions of fish waste hydrolysates issued from three fish industries was tested on guinea pig ileum in order to examine the presence of opioid molecules. The evaluation of anti-hypertensive activities of whole hydrolysates and fractions were also tested, monitoring the ability of the fraction to inhibit the activity of angiotensin I-converting enzyme involved in hypertension regulation. Sardine autolysate and cod head hydrolysate powder (50 microg) were able to inhibit near 30% of ACE activity, whereas 50 microg of shrimp hydrolysate allows the inhibition of 57% of ACE activity. HPLC fractionation of cod head hydrolysate and sardine autolysate was necessary to evidence biological activity, whereas HPLC separation of shrimp hydrolysate exhibited low biological activity fractions. Further studies are necessary to characterise bioactive molecules from cod head alcalase hydrolysate and from sardine autolysate.     

Benzilylcholine mustard and spare receptors in guinea pig ileum

A comparison was made of muscarinic receptor occupancy by the irreversible antagonist benzilylcholine mustard (BCM) as determined from shifts in the dose-response curve to a muscarinic agonist and from 3H-QNB binding to homogenates of BCM-treated tissue. Major discrepancies were found. A low concentration of BCM (3x10-8M/15 min.) produced a parallel dose-response curve shift corresponding to 98-99% receptor occupancy by BCM, whereas 3H-QNB binding revealed only 48% receptor occupancy. Possible origins of this discrepancy are discussed. High concentrations of BCM (5x10-5M, 15 min.) fail to completely alkylate all 3H-QNB binding sites even though response is completely lost. Although significant (64%) recovery of response occurs after prolonged tissue washing (240 min.) this is not accompanied by an increase in 3H-QNB binding. The small fraction (approximately 5%) of sites inaccessible to BCM and with reduced affinity for 3H-QNB may represent a subpopulation of muscarinic receptors.     

Morphine diesters. I. Synthesis and action on guinea pig ileum

3,6-Dipropanoylmorphine (DPM), 3,6-dibutanoylmorphine (DBM), and 3,6-dihexanoylmorphine (DHM) were prepared as prospective long-acting narcotic analgesics. The purity and structure of these compounds were authenticated by high pressure liquid chromatography and direct probe mass spectrometry. In aqueous solution the stability of the HCI salts of these compounds decreased with increasing alkyl chain length such that DHM underwent rapid hydrolysis to 6-monohexanoylmorphine (MHM). A comparison of DPM, DBM, and MHM with morphine (MO) and 3,6-diacetylmorphine (DAM, heroin) in the electrically stimulated guinea pig ileum myenteric plexus longitudinal muscle strip preparation (GUPIL) revealed similar potencies and efficacies for all compounds, but marked differences in the onset of drug action (MO greater than DAM greater than MHM greater than DPM greater than DBM, faster to slower). In a periodically washed GUPIL preparation DBM and MHM were five times longer acting than MO, DAM or DPM.     

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.     

Modulation of autonomic neuroeffector transmission by nitric oxide in guinea pig ileum.

NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide synthesis, markedly enhanced tonic ("hump") responses to transmural stimulation in guinea pig ileum longitudinal muscle. The enhancement of the hump responses was probably due to a prejunctional effect on substance P-like neurotransmission, since the action of L-NMMA was exerted also in the presence of atropine, and since responses to substance P, a mimic of nerve stimulation, were unaffected by L-NMMA as were cholinergic twitch responses and the overflow of [3H]choline. Further in support, the hump responses were blocked by the substance P antagonist Spantide. All effects of L-NMMA were stereospecifically reversed by L-arginine. Endogenous nitric oxide thus selectively modulates peptidergic neurotransmission in the gut.     

Functional role of M2 muscarinic receptors in the guinea pig ileum

Muscarinic agonists elicit contraction in the standard guinea pig ileum bioassay through activation of M3 muscarinic receptors that are also linked to phosphoinositide hydrolysis. Surprisingly, the most abundant muscarinic receptor in the ileum is the M2 which causes a specific inhibition of cyclic AMP accumulation elicited by the beta-adrenergic receptor. After most of the M3 receptors are inactivated, the ileum still retains high sensitivity to muscarinic agonists provided that the contractile responses are measured in the presence of histamine and forskolin, which together, have no effect on contraction. Under these conditions, the potencies of antagonists for blocking the contractile response are consistent with those expected for an M2 response. Moreover, the muscarinic contractile response measured in the presence of histamine and forskolin after inactivation of M3 receptors is pertussis toxin sensitive. In contrast, muscarinic contractions in the standard bioassay are pertussis toxin insensitive. These results demonstrate that the M2 muscarinic receptor can cause an indirect contraction of the guinea pig ileum by preventing the relaxing effect of agents that increase cAMP.     

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.     

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.