Angela A. Waterfield

EFFECTS OF CHANGES IN THE STRUCTURE OF ENKEPHALINS AND OF NARCOTIC ANALGESIC DRUGS ON THEIR INTERACTIONS WITH μ- AND δ-RECEPTORS

1 The activity pattern of analogues of the enkephalins was determined in four parallel assays, the inhibition of the electrically evoked contraction of the guinea‐pig ileum and mouse vas deferens at 36°C and the inhibition of [3H]‐naltrexone and [3H]‐leucine‐enkephalin binding at 0 to 4°C in homogenates of guinea‐pig brain. 2 The activity pattern was best characterized by the ratio of the potency in the guinea‐pig ileum to that in the mouse vas deferens (G.p.i./M.v.d.) and the ratio of the potency in inhibiting [3H]‐naltrexone binding to that in inhibiting [3H]‐leucine‐enkephalin binding (Nal/Leu). 3 The enkephalins had low G.p.i./M.v.d. (0.02 to 0.09) and low Nal/Leu (0.05 to 0.18) ratios whereas the corresponding values for morphine were 7.0 and 7.5. 4 Analogues obtained by substituting d‐Ala for Gly2and d‐Met or d‐Leu for l‐Met5or l‐Leu5showed only minor changes in G.p.i./M.v.d. (0.01 to 0.11) and in Nal/Leu (0.06 to 0.13) ratios. 5 Analogues in which resistance to enzymatic degradation was brought about by amidation of the C‐terminal carboxylic group or methylation of the amino group of tyrosine or both modifications, had G.p.i./M.v.d. ratios of 1.2 to 5.5 and Nal/Leu ratios of 0.5 to 21. High values (2.1 and 3.4) were found for the potent antinociceptive analogue of Sandoz, Tyr‐d‐Ala‐Gly‐NCH3Phe‐Met(O)‐ol. 6 In the mouse vas deferens, some of the analogues with high G.p.i./M.v.d. and Nal/Leu ratios were tested for antagonism by naloxone and found to require less than the high concentration needed for the natural enkephalins. C57/BL mice, which have a lowered sensitivity to morphine but a normal response to peptides with low G.p.i./M.v.d. and Nal/Leu ratios, had a lowered sensitivity to analogues with high ratios. 7 In the alkaloid‐like series of narcotic analgesic drugs, ketobemidone, levorphanol, methadone, etorphine and the antagonist Mr 2266 had lower Nal/Leu ratios (1.0 to 2.8) than morphine, normorphine, naloxone and naltrexone (8 to 12). 8 It would appear that compounds with low G.p.i./M.v.d. and Nal/Leu ratios interact mainly with δ‐receptors in the brain and peripheral nervous system while compounds with high ratios interact mainly with μ‐receptors. For antinociceptive action μ‐receptors may be more important than δ‐receptors.

In vitro pharmacology of the opioid peptides, enkephalins and endorphins

In the guinea-pig ileum methionine-enkephalin, normorphine and morphine are equipotent in depressing electrically evoked contractions; leucine-enkephalin has about 25% of the activity. The mouse vas deferens is more sensitive to the enkephalins which are 30 to 60 times more potent than morphine. Fragments of beta-lipotropin61-91 (beta-endorphin) having sequences up to LPH76 are more potent in the mouse vas deferens than in the guinea-pig ileum but beta-endorphin is about equipotent in the two preparations. None of the peptides has antagonist activity. Methionine-enkephalin and normorphine are equipotent in inhibiting [3H]-naloxone binding by homogenate of guinea-pig brain in the absence of Na+ while leucine-enkephalin has only 25% of this activity. In the guinea-pig ileum, naloxone antagonises normorhine and the enkephalins equally well whereas in the mouse vas deferens about ten times more naloxone is required for the enkophalins that for normorphine. Methionine-enkephalin depresses output of acetylcholine in the guinea-pig ileum and of noradrenaline in the mouse vas deferens.

Assessment in the guinea-pig ileum and mouse vas deferens of benzomorphans which have strong antinociceptive activity but do not substitute for morphine in the dependent monkey.

1 Four benzomorphans which have potent antinociceptive activity in the hot‐plate and writhing tests in the mouse but do not suppress or precipitate withdrawal symptoms in the morphine‐dependent monkey, have been examined for their pharmacological actions in the guinea‐pig ileum and mouse vas deferens. 2 In the guinea‐pig ileum their agonist potencies are 1.5 to 400 times greater than that of normorphine or morphine whereas in the mouse vas deferens their potencies relative to morphine are 0.3 to 100. They exhibit no antagonist activity in either preparation. Benzomorphans which substitute for morphine in the morphine‐dependent monkey do not show such differences between their relative potencies in the guinea‐pig ileum and mouse vas deferens. 3 The relative potencies of the four benzomorphans to inhibit stereospecific [3H]‐dihydromorphine binding by membrane fragments from rat brain, are more closely related to their relative agonist potencies in the mouse vas deferens than to those found in the guinea‐pig ileum. 4 In order to antagonize the agonist actions of these benzomorphans, naloxone is required in concentrations which are 3 to 7 times higher than those needed for the antagonism of normorphine or morphine or of benzomorphans which suppress abstinence in morphine‐dependent monkeys. 5 It may be possible to use the three assays, namely, ratio of relative agonist potency in mouse vas deferens to that in guinea‐pig ileum, ratio of relative agonist potency to relative affinity to opiate receptors and the concentration of naloxone required for antagonism, for the prediction of the potential of new compounds to produce physical dependence.

Opioid activities of fragments of β-endorphin and of ites leucine65-analogue. Comparison of the binding properties of methionine- and leucine-enkephalin

For characterisation in vitro, four parallel assays were used: the guinea-pig ileum and mouse vas deferens as pharmacological models at 36 degrees C and the inhibition of binding of [3H]-naltrexone, [3H]-leucine-enkephalin and [3H]-methione-enkephalin at 0 degrees C. The Leu65-analogue of beta-andorphin and its fragments (61-65, 61-76 and 61-77) have a lower affinity to the [3H]-naltrexone binding site of mu-receptors than the corresponding Met65-peptides wereheas no such difference was found for the [3H]leucine-enkephalin binding sites or delta-receptors. When the binding of [3H]-methionine-enkephalin or [3H]-leucine-enkephalin was inhibited by cold ligands interacting with delta-, mu-, or kappa-receptors, no evidence was obtained for more than one type of delta-binding site.

Stereospecific increase by narcotic antagonists of evoked acetylcholine output in guinea-pig ileum

Abstract In the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum, naloxone (30–100 nM) increases the output of acetylcholine evoked by electrical field stimulation at 0.017 Hz and to a lesser extent also at 10 Hz. The stereospecific requirements for this effect were studied with three pairs of optical isomers of antagonists of the benzomorphan series. The (−)-isomer of β-9-methyl-5-phenyl-2-allyl-2′-hydroxy-6,7-benzomorphan (GPA 1843) which had no agonist activity, had an effect similar to naloxone whereas the (+)-isomer was inactive in this respect. The (−)-isomer of antagonists with even weak agonist activity gave variable results. It is assumed that naloxone antagonises the action of enkephalin which has been shown to be present in the guinea-pig ileum. It is recommended to establish the stereospecificity of an antagonist action in order to exclude pharmacological effects not due to interaction with opiate receptors.

Structure‐activity relationships of methionine‐enkephalin

KHAZAN, N. & COLASANTI, B. (1971). Psychopharmacologia, 22, 56-63. KHAZAN, N. & SAWYER, C. H. (1964). Ibid., 5, 457-466. KHAZAN, N., WEEKS, J. R. & SCHROEDER, L. A. (1967). J. Pharmac. exp. Ther., 155, 521-531. MCMILLAN, D. E., WOLF, P. S. & CARCHMAN, R. A. (1970). Zbid., 175, 443-458. MORETON, J. E., ROEHRS, T. & KHAZAN, N. (1974). Pharmacologist, 16, 248. COMMUNICATIONS, J. Pharm. Pharmac., 1976, 28, 660

FACTORS INFLUENCING THE RELEASE OF ACETYLCHOLINE FROM THE MYENTERIC PLEXUS OF THE ILEUM OF THE GUINEA-PIG AND RABBIT

1 The effects of electrical stimulation, changes in external ion concentrations and various drugs on acetylcholine release from the myenteric plexus were measured by bioassay in the presence of physostigmine and by recording the responses of the longitudinal muscle. In preparations from the guinea‐pig, the acetylcholine output per pulse increased with decreasing frequency of stimulation and reached its maximum at a frequency of 0.017 Hz (1/min) and thus ensured that the output per unit of time was constant at frequencies below 0.5 Hz. Spontaneous release was suppressed during stimulation at 0.017 Hz. 2 In the rabbit, the fractional acetylcholine release was lower than in the guinea‐pig. The output per pulse increased with decreasing frequency of stimulation but at a lesser rate, with the effect that the output per unit time decreased between 0.5 and 0.017 Hz. 3 In the guinea‐pig, reduction of the Ca2+ concentration, addition to the bath fluid of Mn2+, ganglion‐blocking drugs, morphine and catecholamines reduced output more at low than at high frequencies of stimulation. In the rabbit, acetylcholine output was less sensitive to changes in Ca2+ concentration and insensitive to Mn2+ and morphine. 4 In the guinea‐pig, morphine and catecholamines depressed both the contractile response and acetylcholine output whereas Mn2+ in concentrations up to 125 μm, bretylium and ganglion‐blocking drugs depressed only acetylcholine output. 5 In preparations from the guinea‐pig, drugs blocking noradrenergic neurones or α‐adrenoceptors, e.g. bretylium, phenoxybenzamine, thymoxamine and phentolamine, increased acetylcholine output during stimulation at high (1.5 to 10 Hz) but not at low frequencies. 6 The implications of these findings for the release of acetylcholine from different pools in the heterogeneous myenteric plexus are considered. The possible errors, introduced by the effects of physostigmine, on the size of the acetylcholine pools and on the transmission of impulses within the myenteric plexus are discussed.

THE INHIBITORY EFFECTS OF PRESYNAPTIC α‐ADRENOCEPTOR AGONISTS ON CONTRACTIONS OF GUINEA‐PIG ILEUM AND MOUSE VAS DEFERENS IN THE MORPHINE‐DEPENDENT AND WITHDRAWN STATES PRODUCED in vitro

1 Isolated ilea from guinea‐pigs implanted with morphine pellets were stimulated coaxially, either with or without morphine present in the bath fluid, and the longitudinal contractions recorded. 2 In the absence of morphine the inhibitory effects of the presynaptic α‐adrenoceptor agonists, clonidine and oxymetazoline were much reduced and the dose‐response curve was flat. This state of ‘withdrawal’ was readily reversed by morphine and levorphanol but not its inactive (+)‐isomer, dextrorphan. 3 The κ‐agonists, ketazocine and ethylketazocine, also restored the effects of clonidine as did the opioid peptides Tyr‐d‐Ala‐Gly‐Phe‐d‐Leu, acting preferentially on δ‐receptors, and Tyr‐d‐Ala‐Gly‐MePhe‐Met(O)‐ol, acting mainly on μ‐receptors. 4 The inhibitory effects of adrenaline and adenosine 3′,5′‐diphosphate were reduced at low but not at high concentrations. 5 In contrast, the inhibitory effect of clonidine on the electrically evoked contractions of vasa deferentia from mice implanted with morphine pellets was not abolished by the lack of morphine in the bath fluid or by addition of naloxone. 6 A possible explanation is suggested for the loss of the inhibitory effects of presynaptic α‐adrenoceptor agonists in the withdrawn state of the dependent ileum.

Rates of onset and offset of action of narcotic analgesics in isolated preparations.

In isolated preparations of the myenteric plexus--longitudinal muscle of the guinea-pig ileum and the mouse vas deferens, the rates of onset and offset of action of narcotic analgesics are inversely related to lipid solubility; this effect is probably due to drug binding at secondary lipid-rich binding sites. These findings are in contrast to observations in vivo by A. Herz and his colleagues, who showed that the rates of onset of action and of recovery are directly related to lipid solubility because of the presence of lipid-rich barriers. In view of the opposite effects, an optimum may be expected when a drug has a degree of lipid solubility which ensures rapid penetration of the blood--brain barrier without seriously slowing down of receptor association and dissociation. From the interaction of rapidly acting quaternary antagonists, e.g. N-methylnalorphinium, with slowly acting agonists, e.g. methadone, it is concluded that at least a part of the receptor site is on the surface of the cell membrane, and the possibility of an allosteric agonist--antagonist interaction is considered.

An analysis of the phenomenon of acute tolerance to morphine in the guinea-pig isolated ileum.

1 The observations which Paton (1957) interpreted as ‘acute tolerance’ and ‘dependence’ have been confirmed for coaxially stimulated segments of guinea‐pig ileum and extended to the contractions evoked by field stimulation in the myenteric plexus‐longitudinal muscle preparation. Evidence is adduced that the morphine receptors of the myenteric plexus are not involved in the two phenomena. 2 The contraction of the longitudinal muscle depressed by low concentrations of morphine, or levorphanol, can be restored to control level not only by high concentrations of morphine but also by levorphanol and equally well by its (+)‐isomer, dextrorphan, which does not fulfil the stereospecific requirements of the morphine receptor. Acetylcholine output was not increased. 3 When, after restoration of the twitch by high concentrations of morphine, the drug is washed out, contractions become depressed. This effect cannot be due to ‘dependence’ because either morphine or its antagonist, naloxone, restore the twitch again. 4 In the concentrations used, morphine, levorphanol and dextrorphan inhibit the Cholinesterase of homogenates of the myenteric plexus‐longitudinal muscle preparation by 10‐15%, Since a concentration of physostigmine which causes a similar inhibition also restores the twitch, it is concluded that the described phenomena are best explained by the anticholinesterase effects of the drugs.