Guinea Pig Ileum Preparations Research Articles

Expression of spectrin in nonerythroid cells

spleen cells to the T-cell mitogens concanavalin A and phytohemagglutimin. The ,f3-endorphin effect was dose-dependent at concentrations similar to those found in rat plasma. ,&Endorphin had no effect on resting, unstimulated spleen cells. Recently, Schweigerer et al. (Nature 296, 572-574, 1982) reported specific nonopiate binding of &-endorphin to both terminal complexes of human complement: the cytolytic, membrane-derived C5b-9(m) complex, and the cytolytically inactive, serum-derived SC5b-9 complex. These observations suggest that ,L?-endorphin may play an important role in the immune system. For the last few years, more than 60 /I-endorphin analogs have been synthesized by the solid-phase method and characterized. Their biological activities have been assessed by opiate receptor binding, inhibition of electrically stimulated contraction of guinea pig ileum preparation and mouse tail-flick analgesic and radioimmunoreactivity assays. These data, recently reviewed (Li, Hormonal Proteins and Peptides 10, l-34, 19811, suggest the presence of three binding sites in the /I-endorphin molecule, as shown in Figure 2. The first site (15 receptors) is located in the Met-enkephalin segment, and the second site (p receptor) resides in the COOH-terminal segment. The middle segment is the antibody-binding site. Among the synthetic ,&endorphin analogs, /I-endorphin-(6-31) (an analog without the Met-enkephalin segment) exhibits interesting properties. It possesses minimal analgesic potency and ileal opiate activity. As mentioned above, P-endorphin-(6-31) binds to the opiate receptor in the neuroblastoma N18TG2 cells. When P-endorphin-(6-31) is injected together with P-endorphin, it inhibits ,&endorphin-induced analgesia. It may be recalled that a segment of human ACTH [a,,-ACTH-(6-3811 is an inhibitor of ACTH action (Li et al., PNAS 75, 4306-4309, 1978). The concept that a peptide segment inside the amino acid sequence of a hormone is an inhibitor of the hormone itself may turn out to be a general phenomenon in biologically active polypeptides.

Improved assays for the assessment of ϰ- and δ-properties of opioid ligands

The highly selective non-equilibrium μ-antagonist β-funaltrexamine (β-FNA) produced a maximal 20-fold shift in the IC 50 for the μ-agonist morphine on the guinea-pig ileum preparation, whilst producing no significant change in the IC 50 for the ϰ-agonist ethylketazocine. On preparations pretreated with β-FNA, the pA 2 values for the interaction of morphine and ethylketazocine with naloxone were similar. These values were similar to the pA 2 value for the interaction of ethylketazocine and naloxone determined on control tissues, but significantly different from the pA 2 value for morphine-naloxone on control tissues, indicating that the agonist actions of morphine on preparations pretreated with high concentrationsof β-FNA are mediated by ϰ-, rather than μ-receptor interaction. On the mouse was deferens preparation, co-incubation with the highly selective δ-agonist Tyr-D-Ser-Gly-Phe-Leu-Thr (DSLET) and the non-selective non-equilibrium opiate antagonist β-chlornaltrexamine (β-CNA) resulted in marked inhibition of the agonist actions of morphine but had no effect upon the agonist actions of the δ-agonist leucine-enkephalin. The pA 2 values for the interactions of naloxone with leucine-enkephalin and etorphine were unaltered by pretreatment with β-CNA and DSLET. In similarly pretreated tissues, the agonist actions of ethylketazocine were markedly inhibited. It is concluded that manipulation of the guinea-pig ileum and mouse vas deferens preparations in the described manner results in assay systems that possess a largely homogeneous receptor population, and as such are valuable tools with which to evaluate opioid activity.

Effects of the combination of tripelennamine and Pentazocine at the behavioral and molecular levels

The purpose of the present experiments was to determine if the antihistamine tripelennamine potentiates the morphine-like effects of the narcotic-antagonist analgesic pentazocine at the behavioral level or the molecular level or both. At the behavioral level, the effects of pentazocine were determined alone and in combination with tripelennamine in rats trained to discriminate between saline and either morphine or the psychotomimetic narcotic derivative SKF 10,047. The interaction between pentazocine and tripelennamine were also evaluated in the guinea-pig ileum preparation and in the [ 3H]-naloxone opiate receptor binding assay. Tripelennamine significantly enhanced the morphine-like discriminative stimulus effects of pentazocine and markedly reduced the SKF 10,047-like stimulus effects of pentazocine. Naloxone antagonized the morphine-like effects of pentazocine plus tripelennamine. Pentazocine significantly constricted pupils in the rat, an effect which was addictive with the mydriatic effects of tripelennamine. Inhibition of the twitch-height of the electrically stimulated guinea-pig ileum by pentazocine was not affected by tripelennamine. Further, tripelennamine did not modify the K e for naloxone in antagonizing pentazocine. Inhibition of specific [ 3H]-naloxone binding by pentazocine was also not affected by tripelennamine. These results are consistent with the hypothesis that the potentiation of the morphine-like effects of pentazocine by tripelennamine which was observed behaviorally was not due to molecular interactions at the morphine receptor. At least a part of this interaction may be attributable to tripelennamine decreasing the psychotomimetic actions of pentazocine.

Selective antagonists at the opiate delta-receptor

Classical opiate antagonists such as naloxone act predominantly at the opiate mu-receptor and exhibit only weak antagonism at the delta-receptor. Using two in vitro models - the field-stimulated mouse vas deferens and guinea pig ileum preparations - we have identified two novel peptide antagonists which selectively and competitively reverse the effects of opiates at the delta-receptor, with little or no activity at either mu or kappa receptors.

Evaluation of spasmolytic activity of clausmarin-A. A novel coumarin from Clausena pentaphylla (Roxb.) DC.

Crude ethanolic (50%) extracts of five species of Clausena exhibited varying degrees of spasmolytic activity on the isolated guinea-pig ileum preparation. The aerial parts of Clausena pentaphylla had maximum activity. Its active principle clausmarin-A, a novel coumarin terpenoid, was evaluated in detail for spasmolytic activity in various in vivo and in vitro test models. The activity of clausmarin A compared favourably with that of papaverine.

Evaluation of ( d-Pro 2, d-Trp 7,9)-substance P as an antagonist of substance P responses in the rat central nervous system

The ( d-Pro 2, d-Trp 7,9) analogue of substance P has been tested for substance P antagonist activity in the caudal trigeminal nucleus in vivo, and in the isolated spinal cord in vitro. In neither case was the analogue found to be a specific antagonist of substance P, although the analogue did have weak antagonist actions in the isolated guinea-pig ileum preparation. It is concluded that the analogue is not a suitable tool for the identification of putative substance P systems in the spinal cord or the caudal trigeminal nucleus of the rat.

The antiserotonin and antihistamine activities of salivary secretion of Rhodnius prolixus

The antiserotonin and antihistamine activities of Rhodnius prolixus salivary secretion were studied using the rat uterus and the guinea pig ileum preparations. Serotonin antagonism was not competitive and the salivary secretion dose which abolished a half-maximal uterine contraction was 13 ± 2 (S.E.) μg protein/ml ( n = 9). No inhibition was seen for the acetylcholine and bradikinin contractures. On the other hand, the observed antihistamine activity was typically competitive. The mean concentration of Rhodnius salivary secretion of 14 ± 2 (S.E.) μg protein/ml ( n = 9) reduced the effect of histamine to that of a half dose. Specificity was demonstrated when testing salivary secretion in the acetylcholine and BaCl 2-induced guinea pig ileum contractures. In both antagonisms, no preincubation was needed for the full expression of the inhibitory effect, which did not persist after washing the preparation. The presence of enzymes that could be degrading the amines was excluded. Susceptibility to trypsin and heat treatments as well as the behaviour of the principles on gel filtration and preparative agarose electrophoresis experiments, suggest that the activities reside in the same peptide molecule with an apparent molecular weight of 39,000 daltons.

Antagonism of pre and postsynaptic alpha-adrenoreceptors by BE 2254 (HEAT) and prazosin.

1 The alpha-adrenoreceptor blocking effects of BE 2254 and prazosin have been studied in the pithed rat and guinea-pig ileum preparations. 2 BE 2254 was a competitive and potent antagonist of NA, Phenylephrine (PE) or stimulation-induced vasoconstriction in the pithed rat, but only exerted a weak antagonism of postsynaptic alpha 2-adrenoreceptor vasoconstriction induced by N,N-diMe-6,7-diOHATN (TL 99) or azepexole (BHT 933). The potency of BE 2254 against NA-induced vasoconstriction was markedly reduced by pretreatment with prazosin and propranolol. The antagonist effects of BE 2254 against TL 99-induced vasoconstriction was not altered by these pretreatments. Prazosin was without effect at postsynaptic alpha 2-adrenoreceptors. Neither antagonist blocked the pressor responses to angiotensin II (A II) nor the tachycardia-induced by exogenous NA, indicating specificity for alpha 2-adrenoreceptors. 3 BE 2254 was a potent antagonist of TL99-induced cardioinhibition (presynaptic alpha 2-adrenoreceptor effects) in the pithed rat, but may not act as a competitive antagonist in this preparation. Prazosin was without antagonist effect in this preparation. 4 In the transmurally stimulated guinea-pig ileum, BE 2254 was a potent antagonist of TL99, or clonidine-induced inhibition of the twitch response (presynaptic alpha 2-adrenoreceptor effects), but did not antagonize the effects of morphine. Prazosin was again without effect. 5 It was concluded that BE 2254 is a potent antagonist at presynaptic alpha 2-adrenoreceptors in vitro and in vivo, and at postsynaptic alpha 1-adrenoreceptors in vivo, but has only weak activity at vascular postsynaptic alpha 2-adrenoreceptors in the pithed rat. The differences in potency of BE 2254 in the pithed rat at pre- and postsynaptic alpha 2-adrenoreceptors suggests that these alpha-adrenoreceptor sub-types may differ.

Relationships between chemical structure and cholinergic activities of furmethides and potentiation of these activities by physostigmine

Summary Furfuryltrimethylammonium (furmethide; FT), 5-methylfurmethide (5-MFT), 5-hydroxymethylfurmethide (5-HMFT) and 5-chloromethylfurmethide (5-ClMFT) have structural similarity to muscarine. The cholinergic activities of these furmethides were compared on a superfused guinea pig ileum preparation. All four compounds were less potent but equally as active as acetylcholine (ACh). The ED50s of these compounds decrease in the following order: 5-ClMFT > 5-HFT > 5-MFT. The electron densities around the furan oxygen of these compounds decrease in the same order. It has been observed that these furmethides display two separate actions: (1) Direct interaction at the muscarinic receptor to elicit a response; and (2) Release of ACh from the presynaptic nerve terminal. In the presence of physostigmine, dose response curves of these compounds were shifted to the left. There was approximately a two-fold shift in the ED50 values for the furmethide. Like muscarine, these compounds were not hydrolyzed by cholinesterase (ChE). At least part of the furmethides response was due to release of ACh, because the responses were increased by ChE inhibition.

8-Phenyltheophylline: A potent P 1-purinoceptor antagonist

8-Phenyltheophylline was more potent than theophylline in antagonizing the inhibitory effects of adenosine in guinea-pig driven left atrium, rabbit basilar artery and electrically stimulated guinea-pig ileum preparations. In guinea-pig atrium and ileum, the antagonism of adenosine responses by the methylxanthines is of a competitive nature, but in rabbit basilar artery it does not appear to be so. In all three preparations, the effects of theophylline and 8-phenyltheophyllin could be reversed by washing. It is concluded that 8-phenyltheophylline is a more potent P 1-purinoceptor antagonist than theophylline.

Multiple opiate receptors in guinea-pig ileum

Actions of the prototypic μ-, κ-, and σ-opiate receptor agonists, morphine (M) ketocyclazocine (K) and SKF-10,047. (S), respectively, were examined and differentiated using the guinea-pig ileum preparation. S, like M and K, depressed the electrically stimulated ileum. Naloxone antagonized the depressant actions of the prototypic drugs with different potencies. PA 2 values of naloxone for M, K, and S, respectively, were 8.81, 7.58 and 7.74. Relative cross tolerance of each prototypic drug to normorphine, a comparison standard, was also examined in morphine-pretreated ilea and quantitatively estimated as follows: (1) the median effective dose of each drug and of the standard drug normorphine were determined in the nontolerant ileum (IC50 NT) and in ilea with varying degrees of tolerance IC50 T); (2) cross-tolerance ratios (IC50 T/IC50 NT) of each drug and of normorphine were calculated for the varying degrees of tolerance; (3) cross-tolerance ratios of each drug were plotted against those of normorphine, the data were fit by a least squares straight line, and the slope of the line determined as the Relative Cross Tolerance Index (RCTI). RCTI for M was 2.21. K and S, however, had lower RCTI's of 0.44 and 0.64 respectively. In the morphine-pretreated tolerant ilea, slopes of the dose response curves of the prototypic drugs were found to differ: while M and K possessed steep and constant slopes for ilea with different degrees of tolerance, the slopes for S became shallower as ilea became more tolerant to morphine. A maximum ceiling effect of less than 50% depression was obtained for S in the most highly tolerant ilea. The above observations are consistent with possible existence of the three types of hypothesized opiate receptors in the guinea-pig ileum.

Lack of postulated opiate-receptor antagonistic properties of compound 48/80.

The previously suggested opiate-receptor antagonistic properties of compound 48/80 were checked both in vivo and in vitro. In electrically stimulated guinea pig ileum and rat vas deferens preparations compound 48/80 did not reverse the inhibition of the twitches caused either by morphine or Met5-enkephalin. In mice hot-plate test compound 48/80 did not decrease the analgesic activity of morphine. In radioreceptor studies compound 48/80 shows rather low affinity to the 3H-naloxone receptor sites from striatal homogenates of the rat, as compared to non-labelled naloxone and on the other hand it produced moderate, as compared to d-butaclamol, displacement of 3H-spiroperidol from rat striatal homogenates. The variation of composition of various polymers making compound 48/80 is suggested for explanation of obtained results.

Opiate receptor antagonism by l-prolyl-l-leucyl-glycinamide, MIF-I

Chronic administration of l-prolyl-l-leucyl-glycinamide (MIF-I) to mice slightly reduced morphine's antinociceptive activity in the hot-plate test and modified the biphasic motor activity response to morphine. MIF-I antagonized the initial depression of activity and potentiated the increased motor activity phase. Chronic treatment of rats with MIF-I prevented morphine's antinociceptive activity in the tail flick test. MIF-I partly antagonized the inhibition by morphine of the coaxially stimulated guinea-pig ileum preparation. The inhibition of the ileum produced by ethylketocyclazocine was weakly antagonized by MIF-I. In contrast, MIF-I had no effect on the inhibition of the stimulated mouse vas deferens produced by Leu-enkephalin. The findings show that MIF-I weakly and selectively inhibits μ-type opiate receptors which suggests that MIF-I could be an endogenous inhibitor of opiate receptors.

Isolation, characterization and opiate activity of beta-endorphin from the pituitary gland of the ostrich, Struthio camelus.

1. Avian beta-endorphin was purified from adenohypophyseal glands of the ostrich Struthio camelus by a procedure involving acid/acetone extraction, NaCl fractionation, CM-cellulose chromatography, Sephadex G-50 chromatography and paper electrophoresis (pH 6.7). 2. The 31-amino acid peptide behaved as a single substance during polyacrylamide-gel electrophoresis and isoelectric focusing, the isoelectric point being 8.84. 3. Ostrich beta-endorphin exhibited significant opiate activity in the guinea-pig ileum preparation.

A pituitary endorphin with novel properties

We describe the further purification of an opioid peptide from a porcine pituitary concentrate. The peptide has typical naloxone-reversible opioid activity in the guinea pig ileum myenteric-plexus preparation and mouse vas deferens, and it inhibits stereospecific binding at opiate receptors. It is distinguished from β-endorphin and the enkephalins by its apparent molecular weight, its slow reversal with washing in the guinea pig ileum preparation, and the resistance of its biologic activity to cyanogen bromide treatment. In beef pituitary, slow-reversing, cyanogen bromide resistant activity is found principally in neurointermediate lobe.

β-Endorphin: Deletion of a single amino acid residue abolishes immunoreactivity but retains opiate potency

Two synthetic analogs of camel β-endorphin, one with omission of Leu-14 and the other with omission of Asn-20, have been assayed for immunoreactivity by radioimmunoassay, opiate activity in the guinea pig ileum preparation and analgesic potency in mice. It was found that the omission analogs had no immunoreactivity, but retained significant biological activities. As far as we are aware, this is the first instance in which deletion of a single amino acid residue in a biologically active peptide abolished immunoreactivity.

Characteristics of the increased response to electrical stimulation of ileum preparations from morphine pretreated guinea pigs

The relationship between electrical stimulus voltage or pulse duration and the tension generated in the resulting contractions has been studied in control and morphine tolerant guinea pig ileum preparations. A greater maximal tension was observed in the morphine tolerant preparations. Since the response of the preparations to exogenous applied acetylcholine was unchanged, the increase in tension probably reflected an increase in the amount of acetylcholine released by each stimulus. No change in threshold voltage for stimulation was observed. A neuronally mediated, opiate insensitive, component of the response to electrical stimulation has been demonstrated. This component was unchanged in morphine tolerant preparations. Naloxone did not affect the relationship between stimulus pulse duration and response tension in control or morphine tolerant preparations. These results provide further evidence that morphine tolerance is associated with general changes in the properties of opiate sensitive neurons which can be demonstrated in the absence of opiate drugs.

ChemInform Abstract: SYNTHESIS AND PROSTAGLANDIN‐LIKE ACTIVITY OF 2‐(TRANS‐3‐HYDROXY‐1‐OCTENYL)‐3‐INDOLEHEPTANOIC ACID

AbstractAzelainsäurediethylester (I) reagiert mit Oxalsäurediethylester zum Triester (III), der zur Ketodicarbonsäure (IV) verseift und decarboxyliert wird.

Synthesis and prostaglandin-like activity of 2-(trans-3-hydroxy-1-octenyl)-3-indoleheptanoic acid

The synthesis of 2-(trans-3-hydroxy-1-octenyl)-3-indoleheptanoic acid (1) is described. The title compound appeared to show a weak prostaglandin-like activity in two different systems. It contracted rat stomach fundus strips and guinea-pig ileum preparations only at concentrations about 10(3)- and 10(2)-fold higher, respectively, than PGE1. Moreover, it stimulated adenylate cyclase from rat liver plasma membrane, but the relative potency was 4--5 X 10(2)-fold lower than the natural compound. The title compound showed also a certain degree of PGE1 antagonism.

Factors determining the potency of cholinomimetic miotic drugs and their effect upon the light reflex in man.

1. Television pupillometry was used to measure the effect of six topically applied cholinomimetic drugs on the resting diameter and light reflex amplitude of the human pupil. Drug potency was obtained from dose response curves. 2. The tertiary amines arecoline, aceclidine and pilocarpine were considerably more effective miotics than the choline esters carbachol, methacholine and acetylcholine. 3. All the drugs which caused miosis also reduced light reflex amplitude proportionally. 4. The in vitro potency of these drugs was also measured on preparations of rabbit iris sphincter and guinea pig ileum. 5. Dose response relationships for pilocarpine in man and in vitro showed evidence of partial agonist activity on the rabbit iris only. 6. A comparison of the in vivo and in vitro results showed that three factors influenced the potency of topically applied miotics: accessibility to the iris; sensitivity to cholinesterase; and cholinoceptor agonist potency.