Isolation of a novel tetrapeptide with opiate and antiopiate activity from human brain cortex: Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1).

A novel tetrapeptide, Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1), was purified from extracts of frontal cortex of human brain tissue by several consecutive reversed-phase high performance liquid chromatographic steps followed by a radioimmunoassay originally developed for Tyr-Pro-Leu-Gly-NH2 (Tyr-MIF-1). Sequencing, mass spectrometric analysis, and comparison of its chromatographic behavior with that of the synthetic peptide confirmed the structure. Like Tyr-MIF-1, which was previously isolated from human brain tissue, Tyr-W-MIF-1 can inhibit the binding of 3H-DAMGO (selective for mu opiate receptors) to rat brain and can act as an opiate agonist as well as antagonist. Tyr-W-MIF-1 was a more potent opiate agonist than Tyr-MIF-1, the free acid of Tyr-W-MIF-1, and the structurally related hemoglobin-derived opiate peptide hemorphin-4 (Tyr-Pro-Trp-Thr) in the guinea pig ileum. Each of these peptides acted as opiate antagonists on the ileum from morphine-tolerant guinea pigs; the free acid of Tyr-W-MIF-1 was the most potent antagonist in inhibiting the activity of DAMGO. The results demonstrate the presence in human brain of a new member of the Tyr-MIF-1 family of biologically active peptides.     

Hemorphins, cytochrophins, and human beta-casomorphins bind to antiopiate (TYR-MIE-1) as well as opiate binding sites in rat brain

Novel peptides with opiate activity, derived from endogenous sources (human and bovine casomorphins from milk, hemorphins from hemoglobin, and cytochrophins from mitochondrial cytochrome b), were tested for their ability to inhibit binding of the brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) to its high affinity sites in rat brain. The order of potency in inhibiting binding of 125I-Tyr-MIF-1 was: hemorphin and bovine casomorphins greater than Tyr-MIF-1 greater than cytochrophins greater than human casomorphins. Naloxone and DAMGO were ineffective at inhibiting Tyr-MIF-1 binding. The results provide evidence that, in addition to their ability to bind to mu opiate receptors, these novel endogenous peptides with opiate activity and a peptide (Tyr-MIF-1) with antiopiate properties also bind to a non-opiate site labeled by Tyr-MIF-1. These sites could be involved in a balance between opiate and antiopiate peptides.     

Prejunctional alpha 2 adrenoceptors inhibit contraction of tracheal smooth muscle by inhibiting cholinergic neurotransmission

Ring preparations obtained from the guinea pig trachea contracted on short trains of electrical field stimulation. These contractions were mediated by activation of cholinergic nerves since they were abolished by atropine or tetrodotoxin. In the presence of beta blocking drugs noradrenaline and adrenaline dose-dependently inhibited contractions induced by field stimulation. By contrast, contractions on exogenous acetylcholine were left completely unaffected. It is concluded that the adrenergic agonists inhibited cholinergic neurotransmission by a prejunctional action. In order to characterize the noradrenaline receptor the effects of alpha₁ and alpha₂ blockers were evaluated using the Schild plot. For comparison experiments were also conducted on the guinea pig aorta and electrically stimulated guinea pig ileum. The results indicate that in guinea pig trachea and ileum noradrenaline inhibits cholinergic neurotransmission by acting on prejunctional alpha₂ receptors whereas in guinea pig aorta it induces contraction by stimulating alpha₁ receptors.     

Sodium-induced alterations of opiate effects on the binding of 3H-dihydromorphine to mouse brain homogenates.

The inhibitory potency of opiate agonists on the stereo-specific binding of 3H-dihydromorphine in mouse brain homogenates was not affected by the presence of sodium ions. That of pure antagonists was greatly enhanced by NaCl whereas the inhibitory effects of mixed agonist-antagonists were reduced by NaCl, indicating that sodium ions might affect the agonist component more than the antagonist component of narcotic-antagonist analgesics. The inhibitory potency of the opiates tested in our system agrees with their potency in reducing the stereospecific binding of 3H-naloxone to rat membranes, the contractions of co-axially stimulated guinea pig ileum and their analgesic potency in animals and humans.     

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.     

MIF-1 and Tyr-MIF-1 augment GABA-stimulated benzodiazepine receptor binding

Behavioral evidence in laboratory animals and human beings indicates possible links between the endogenous opiate and gamma-aminobutyric acid (GABA)-benzodiazepine receptor systems, especially with regard to antagonistic properties. To assess possible interactions between endogenous opiate antagonists and benzodiazepine receptor binding, we evaluated the effects of the peptides MIF-1 and Tyr-MIF-1 on benzodiazepine receptor binding in mouse brain membranes. Neither peptide affected receptor binding in cortex over a broad dose range, but both peptides significantly augmented GABA-stimulated benzodiazepine receptor binding at GABA concentrations of 10(-8) and 10(-7) M. Rosenthal-Scatchard analysis indicated that the increase in binding was largely due to increased apparent affinity. Both peptides augmented GABA-enhanced binding at low doses (MIF-1 10(-11) M, Tyr-MIF-1 10(-13) M) with decreased effects at higher doses. In cerebellum and brainstem, MIF-1 tended to enhance GABA-stimulated binding but Tyr-MIF-1 was inactive. These results indicate benzodiazepine-opiate and benzodiazepine-peptide interactions.     

Inhibitory influences of MIF-1 (PLG) and Tyr-MIF-1 (YPLG) on aggression and defeat-induced analgesia in mice

The effects of prolyl-leucyl-glycinamide (MIF-1, PLG), tyrosine-prolyl-leucyl-glycinamide (Tyr-MIF-1, YPLG) and the exogenous opiate antagonist, naloxone, on aggressive interactions and defeat-induced analgesia were examined in male mice. All three substances reduced the number of bites required to obtain defeat in subordinate mice during aggressive encounters, as well as blocking subsequent defeat-induced analgesia. Tyr-MIF-1 had significantly greater inhibitory effects than MIF. These results suggest that both MIF and Tyr-MIF-1 may function as endogenous opioid antagonists and have inhibitory influences on aggression, with the antagonistic effects of Tyr-MIF-1 being more potent than those of MIF-1.     

Lithium does not synergize the peripheral action of cholinomimetics as seen in the central nervous system

Lithium is known to synergize the action of cholinomimetics in the CNS such that pilocarpine induces seizures in low concentration (1/13th of per se dose) in rats. The present study was undertaken to see if lithium priming also enhances the peripheral effects of acetylcholine and pilocarpine i.e. change in blood pressure in rats and contractions of the isolated guinea pig ileum. In anaesthetized rats the blood pressure was recorded from cannulated carotid artery connected through the pressure transducer to Coulbourn polygraph. The blood pressure response of pilocarpine was not different either in magnitude or in duration when administered 1, 2 and 4 h after lithium chloride (3 meq/kg) pretreatment as compared to the control. Similarly acetylcholine effect remained unchanged after lithium chloride priming. In the isolated guinea pig ileum experiments, ileum was incubated for 1 h in different concentrations of lithium chloride and effect on acetylcholine induced contractions were observed. Lithium in concentration of 2.8 x 10(-3) M had no effect on acetylcholine induced contractions while incubation with higher concentrations of 1.4 x 10(-2) M and 2.8 x 10(-2) M significant inhibition of acetylcholine contractions were observed. At this concentration, histamine induced contractions were also inhibited. The results indicate that lithium does not synergize the action of cholinomimetics in the periphery as that seen in the CNS. The inhibition of acetylcholine and histamine induced contractions in guinea pig ileum at high concentration of lithium seems to be non-specific effect.     

The effect of calcium antagonists on contractions of the sensitized and normal guinea pig ileum

The purpose of this study was to learn wether a number of Ca²⁺ antagonists were effective in reducing contractile response of the isolated ileum of the sensitized and normal guinea pig. Contractions of the normal ileum in response to LTD₄, acetylcholine, histamine, and potassium chloride were obtained before and after verapamil, diltiazen and papaverine. Ovalbumin-induced contractions of the ovalbumin-sensitized ileum were obtained in the presence of the three Ca²⁺ antagonists. In the normal ileum, all the Ca²⁺ antagonists were highly effective in diminishing the contractile responses to LTD₄, acetylcholine, histamine and potassium chloride. In the sensitized ileum, ovalbumin-evoked contractions, with subsequent release of a potent contractile mediator (presumably SRS-A), were Ca²⁺-dependent since verapamil, diltiazem and papaverine caused a concentration-related reduction of contractions. Thus, the influx of extracellular Ca²⁺ plays a key role in the contractile responses of the normal and sensitized guinea pig ileum when stimulated by various potent agonists acting on specific receptors or on the cell membrane.     

Isolation of a heptapeptide Val-Val-Tyr-Pro-Trp-Thr-Gln (valorphin) with some opiate activity.

Bovine hypothalamic tissue was extracted and purified by solid phase extraction and several reversed-phase HPLC steps. The amino acid sequence of the purified peptide was determined by Edman degradation to be Val-Val-Tyr-Pro-Trp-Thr-Gln. This was confirmed by comparison of its chromatographic behavior with that of the synthetic peptide, and mass spectrometric analysis resulted in a mass identical to the calculated mass for this peptide. This heptapeptide shows homology with residues 32-38 of the beta-chain of bovine hemoglobin. The peptide inhibited the electrically induced contractions of the guinea pig ileum muscle preparation; this inhibition was reversible by naloxone. It also inhibited the binding of 125I-DAMGO (selective for mu receptors) to rat brain with an IC50 of 10 microM and the binding of 3H-DPDPE (selective for sigma receptors) with an IC50 of 185 microM. With two valines at the N-terminus and some opiate activity, valorphin seems a suitable name for this newly isolated peptide.     

Synthesis and opioid activity of new fentanyl analogs

Three new fentanyl analogs (compounds 3-4-5) have been synthesized and evaluated for antinociceptive properties using the writhing test. The analgesic property of the active compound, N-[1-phenylpyrazol-3-yl]-N-[1-(2-phenethyl)-4-piperidyl)] propenamide (compound 4), was tested using the hot plate test in mice. Its opioid agonistic activity was characterized using three isolated tissues: guinea pig ileum, mouse vas deferens, and rabbit vas deferens. Compound 4 was as effective as fentanyl or morphine and it showed less antinociceptive potency than fentanyl but it was more potent than morphine. The duration of the antinociception was similar to that of fentanyl. This compound inhibited the electrically evoked contractions of myenteric plexus-longitudinal muscle strips of guinea pig ileum and of mouse vas deferens but not those of rabbit vas deferens. These effects could be reversed by micro selective antagonists (naloxone and/or CTOP) but not by the delta selective antagonist naltrindole, thus indicating that the compound acted as a micro opioid agonist. Finally, the binding data confirmed that compound 4 had high affinity and selectivity for the micro-receptor.     

A peptide-like substance from pituitary that acts like morphine. I. Isolation.

A method is described for extraction from bovine pituitary glands of a substance that shows several properties characteristic of opiates. The material inhibits the twitch tension of the electrically stimulated guinea pig longitudinal muscle-myenteric plexus preparation and of the electrically stimulated mouse vas deferens. This inhibition is reversed and blocked by the opiate antagonist naloxone. The extract also inhibits binding of the opiate agonist etorphine and the opiate antagonist naloxone to stereospecific binding sites in synaptic membranes of guinea pig brain. The inhibition of naloxone binding is decreased by Na⁺ in the manner characteristic of opiate agonists. The physiologic role of the pituitary opioid remains to be investigated.     

Beta-endorphin. Biological activity of analogs containing dermorphin and dynorphin sequences: ileum and vas deferens assays

Biological activity of synthetic beta-endorphin (beta-EP) analogs containing dermorphin or dynorphin-A-(1-13) structure has been investigated using the guinea pig ileum and the vas deferens of the mouse, rat and rabbit. Replacement of NH2-terminal 1-7 segment of camel beta-EP [beta c-EP-(1-7)] with dermorphin caused a great increase in opiate potency of the analog. [Dermorphin (1-7)]-beta c-EP was 120 times more potent than beta c-EP in the guinea pig ileum assay, 49 times more potent in the mouse vas deferens assay; and only 4 times more potent in the rat vas deferens assay. Replacement of NH2-terminal 1-13 segment of human beta-EP [beta h-EP-(1-13)] with dynorphin-A-(1-13) caused an increase in opiate potency in both the guinea pig ileum and rabbit vas deferens assays, a complete loss of potency in the rat vas deferens assay, and no change in the mouse vas deferens assay. In comparison with dynorphin-A-(1-13), the hybrid peptide was less potent in the guinea pig ileum assay as well as in mouse and rabbit vas deferens assay. It is suggested that beta c-EP-(8-31) facilitates the dermorphin moiety to act on opiate mu and delta receptors but not on the epsilon receptor, while beta h-(14-31) reduces the action of dynorphin on mu, delta and kappa receptors.     

Humoral endorphin: A new endogenous factor with opiate-like activity

Humoral (H) endorphin, a novel endogenous opioid ligand detected in brain, blood and cerebrospinal fluid was tested in a series of opiate sensitive assays. H-endorphin displaced radiolabeled enkephalin from its specific bindings sites and inhibited the electrically evoked contraction of the guinea pig ileum and mouse vas deferens. When injected to unanesthesized animals, humoral endorphin induced analgesia in rats and mydriasis in mice. The activity of H-endorphin both $\text{in}\text{vitro}$ and $\text{in}\text{vivo}$ attests to its opioid nature. However, while its antinociceptive effect was blocked by naloxone, mydriasis induced by H-endorphin was resistant to the effect of the opiate antagonist. Similarly, intermediate concentrations of naloxone inhibited the effect of H-endorphin on the guinea pig ileum while its effect on the mouse vas deferens was completely refractory to naloxone. The physiological function of humoral endorphin in various naturally occuring states that show similar paradoxical interactions with naloxone is discussed.     

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.     

Mechanisms of the contractile effect of the alcoholic extract of Aegle marmelos Corr. on isolated guinea pig ileum and tracheal chain

In the present work, the effect of the alcoholic extract of the leaves of Aegle marmelos Corr. on guinea pig isolated ileum and tracheal chain was investigated, as this plant is used traditionally to treat asthma and related afflictions. These effects were investigated using the isolated organ bath method. 1 mg/ml and 2 mg/ml doses of the alcoholic extract of this plant produced a positive relaxant effect in isolated guinea pig ileum and tracheal chain, respectively. In addition, they antagonized the contractions, which are produced by histamine. Because the alcoholic extracts elicited the antagonistic effect against histamine and also relaxed the histamine-induced contractions, it can be concluded that relaxations induced by A. marmelos in both guinea pig ileum and tracheal chain were due to the depression of H₁-receptors. Since we observed a complete relaxation of the guinea pig ileum and tracheal chain produced by the extract, we investigated its antagonistic effect against histamine. These results were due to the presence of one or more anti-histaminic constituents present in the alcoholic extract of this plant, therefore supporting to the traditional use of A. marmelos in asthmatic complaints.     

Comparative effects of somatostatin and enkephalins on the guinea pig ileum and the rat vas deferens

The action of somatostatin (SRIF (somatotrophin release inhibiting factor)) was compared with that of Met-enkephalin (Tyr-Gly-Gly-Phe-Met) in the electrically stimulated guinea pig ileum myenteric plexus longitudinal muscle and with that of an enkephalin analogue (FK 33-824 (Tyr-D-Ala-Gly-MePhe-Met-(O)-ol)) in the rat vas deferens. In both tissues SRIF produced a twitch inhibition which was not antagonized by naloxone and which showed a long-lasting tachyphylaxis. The enkephalins tested produced a naloxone-antagonizable inhibition of twitch in both tissues but no tachyphylaxis. Therefore we conclude that SRIF is not acting at opiate receptors in these tissues.     

Reduction in opiate receptor reserve in morphine tolerant guinea pig ilea

Spare opiate receptors in the guinea pig ileum have been detected by the use of the opiate receptor alkylating agent beta-chlornaltrexamine (CNA). Treatment of the guinea pig ileum longitudinal muscle in vitro with low concentrations (less than 10nM) of CNA resulted in an irreversible parallel shift to the right of the normorphine log concentration response curve. With increasing concentration of the reagent, the agonist EC50 becomes progressively greater. Finally a point is reached at which the maximal agonist effect decreases, so that parallelism is no longer seen. The maximal parallel shift provides a measure from which one can estimate the spare receptor fraction that is present in untreated tissue. In ilea from normal guinea pigs, roughly 80-90% of the opiate receptors for normorphine were found to be spare. Even after the largest parallel shifts that could be achieved, the naloxone Ke value for antagonism was unchanged, indicating that normorphine acts through spare mu receptors. Ilea from guinea pigs made tolerant by chronic morphine pellet implantation were found to be more sensitive to the effects of CNA treatment; there was a reduction in the number of spare receptors for normorphine. It is suggested that the opiate spare receptor fraction is physiologically modulated to control neuronal sensitivity to opioid effect.     

A non-peptide morphine-like compound from brain

A non-peptide morphine-like compound was extracted from calf brain and partially purified by immunoabsorption to antimorphine antibodies. The material was able to depress electrically stimulated contractions of the myenteric plexus-longitudinal muscle of the isolated guinea pig ileum. This effect could be prevented and reversed by naloxone and by antimorphine antibodies. Biological activity and morphine-like immunoreactivity were not affected by treatment with proteolytic enzymes. These results represent the first independent confirmation of the non-peptide morphine-like compound reported by Gintzler $\text{et al}$.     

Inhibition of intestinal smooth muscle function by tamoxifen and clomiphene

The acute in vitro effects of the oestrogen antagonists clomiphene (a racemic mixture) and tamoxifen were examined upon spasmogen induced contractions of the guinea pig ileum. Both oestrogen antagonists inhibited the contractions produced by acetylcholine, the muscarinic agonist beta-methylcholine, histamine and bradykinin in a manner which suggests non competitive antagonism. Concentration effect curves to each of the spasmogens were unaffected using 0.1 mumoles/litre of either of the oestrogen antagonists. Clomiphene at 1 mumole/litre reduced the maximum response to the spasmogens by 20 to 50%. Tamoxifen at the same concentration also inhibited the contractions but to a much smaller extent. Clomiphene at the highest concentration tested 10 mumoles/litre completely suppressed contractions to all the spasmogens. Tamoxifen at 10 mumoles/litre completely suppressed contractions to beta-methylcholine and bradykinin. A residue of a response to acetylcholine and histamine remained and this was abolished by raising the concentration to 100 mumoles/litre. The data indicated that clomiphene was more potent than tamoxifen upon the inhibition of the contractions of the ileum. The results demonstrate a non specific effect of the oestrogen antagonists upon smooth muscle function and do not substantiate early reports of specific anticholinergic activity. The possibility of these effects contributing to both the side effects and the antitumor activity of the oestrogen antagonists is discussed.