András Z. Rónai

Comparative study on analgesic effect of Met5-enkephalin and related lipotropin fragments

HUGHES et al.1 have reported the isolation and structure of two pentapeptides from porcine brain with opiate agonist activity in isolated systems. The structure of one of these peptides, Met5-enkephalin, is identical with the sequence of pituitary β-lipotropin (β-LPH) between residues 61–65 (refs 2–4). To prove the biological correlation of brain enkephalin and pituitary β-LPH, a series of lipotropin fragments, LPH-(61–69)-5, LPH-(61–76)-6 and LPH-(61–91)-peptides7–10, have been isolated and shown to have opiate agonist activity in vitro. Only few and controversial data have been available so far on the analgesic effect in vivo of the above or similar lipotropin fragments. Enkephalins have recently been reported to induce analgesia in vivo11,12. Our preliminary data5,13, however, seemed to contradict these observations, rather suggesting that some larger fragment(s) of β-LPH may have analgesic properties. We have therefore compared the analgesic effects of Met5-enkephalin and some lipotropin fragments containing the complete structure of Met5-enkephalin at their NH2-terminus. The results show that the in vivo effect is a function of the length of the peptide chain, Met5-enkephalin being the least and LPH-(61–91)-peptide the most potent. During the preparation of this paper we have become aware of the recent observation of Bradbury and coworkers10,14 on a strong analgesic activity of LPH-(61–91)-peptide.

Attenuation of morphine tolerance and dependence by α-melanocyte stimulating hormone (α-MSH)

Abstract Repeated administration of morphine resulted in significant reduction of its analgesic potency. If 0.1 mg/kg α-MSH was coadministered, the tolerance development was attenuated, 1 mg/kg MIF (MSH release inhibiting factor), given simultaneously with morphine, did not affect tolerance. Injecting, however, MIF 1 hour prior to the daily opiate treatment resulted in accelerated development of tolerance supposedly by lowering the plasma α-MSH level at the time of morphine administration. Of the morphine abstinence symptoms the naloxone-induced jumping in morphine pretreated mice could not be modified either by α-MSH coadministration or by MIF pretreament, but the withdrawal body weight loss was found to be diminished by the former and increased by the latter peptide. The possible role of α-MSH in preventing the development of tolerance to the analgesic effect of endogenous opioid peptides is discussed.

Opioid agonist activity of β-lipotropin fragments: a possible biological source of morphine like substances in the pituitary

An endogenous peptide with opioid agonist activity, termed enkephalin, has been isolated from the porcine brain [ 1 ] , and more recently the primary structure of this pentapeptide has been elucidated: H-Tyr-GlyGly-Phe-Fe: -OH [2]. Hughes et al. [2] pointed out that the complete sequence of the major form of enkephalin, Met-enkephalin, is contained in the primary structure of pituitary /3-lipotropin between amino acid residues 61-65 [3-51. This sequence overlap raises the possibility that /I-lipotropin may be the biological precursor of enkephalin and/or other pituitary peptides with morphine-like activity. As an approach to this question the possible microheterogeneity of porcine /3-lipotropin at sequence position 65, and the opiate agonist activities of /3-lipotropin and some fragments of the hormone obtained by enzymic cleavages were investigated. The results of these studies are reported in this paper.

Diprotin A, an inhibitor of dipeptidyl aminopeptidase IV(EC 3.4.14.5) produces naloxone-reversible analgesia in rats.

The dipeptidyl aminopeptidase IV (DP IV) inhibitor Diprotin A produces a full, dose-dependent, short-lasting and naloxone-reversible analgesia in the rat tail-flick test when given intracerebroventricularly, with an ED50 of 295 nmol/rat but it has no direct opioid agonist activity in the longitudinal muscle strip of guinea-pig ileum bioassay. Two of the potential DP IV substrates, morphiceptin and endomorphin 1, identified recently in bovine brain were also analgesic given by similar route. The action of endomorphin 1 was more potent (ED50 = 7.9 nmol/rat) and slightly but significantly more sustained than that of Diprotin A. Diprotin A neither potentiated nor prolonged the effect of a marginally analgesic dose of endomorphin 1. The distinct time course and the lack of potentiation indicate that in the analgesic effect of Diprotin A in rats the protection of a brain Tyr-Pro-peptide other than endomorphin 1 is involved.

Alpha-2 adrenergic and opioid receptor-mediated gastroprotection

Clonidine inhibited the development of gastric mucosal lesions induced by either acidified ethanol or indomethacin. The ED50 values were: 7.1 and 5.2 microg x kg(-1) orally, respectively. The gastroprotective effect was antagonised by the pre-synaptic alpha-2 antagonist yohimbine, the more selective alpha-2 antagonist Ch-38083 and the pre-synaptic alpha-2B antagonist prazosin. Moreover, the non-selective opioid receptor antagonist naloxone, the delta receptor selective naltrindole also reversed the clonidine-induced mucosal protective action. Clonidine was also effective following intracerebroventricular administration with the ED50 of 37 ng/rat against ethanol-induced mucosal damage. Our results suggest that: 1) the gastroprotective effect of clonidine is likely to be mediated by alpha-2B adrenoceptor subtype; 2) there is an interaction between pre-synaptic alpha-2 adrenoceptors and opioid system; and 3) clonidine can induce gastroprotection by central mechanism.

Enkephalin-like character and analgesia

The opioid activities of enkephalin analogues bearing D- or L-aminopentane-sulfonic/phosphonic acid at position 5 were studied in vitro, in electrically stimulated longitudinal muscle strip of guinea-pig ileum and mouse vas deferens preparations and in vivo in the rat tail-flick test. Using their in vitro effects Met-enkephalin-like, beta-endorphin-like, (nor)morphine-like and derivatives of intermediate character could be differentiated. Correlating the in vitro activities with the analgesic activity in vivo it is concluded that the enkephalin-like character in a pentapetide may hinder the expression of analgesic activity, when the compounds are given into the cerebroventricular system.

STRUCTURE-FUNCTION RELATIONSHIPS IN LIPOTROPINS

Twelve years ago Li and co-workers’, 2 reported the discovery of a new lipolytic polypeptide of adenohypophysis. Because of the considerable fatmobilizing effect of this polypeptide it was designated as lipotropic hormone (LPH) or 1ipotropin.l The lipolytic activity however, has not been a specific property of lipotropin; on the contrary almost all the adenohypophyseal hormones have lipolytic side-effect in addition to their main biologic function. This fact may, at least in part, explain why comparatively less attention has been paid to lipotropin in the last ten years. During this period of time only Dr. C. H. Li’s laboratory and few additional ones, namely the authors’, Dr. M. Chretien’s, Dr. Y. Pankov’s and more recently Dr. P. J. Lowry’s group, made efforts to elucidate the structure and to understand the physiologic role of these curious polypeptides. After the discovery of enkephalins-two brain pentapeptides structurally related to lipotropin-at the end of 1975,s lipotropin, this so far “useless” polypeptide, suddenly became the focus of interest and excitement, and in turn, the knowledge of the chemistry and biology of lipotropins acquired a special value. This paper reviews the most important results achieved in our institute during the last few years, but mostly in 1976, as regards the relationships between the structure and different biologic functions of lipotropins.

Tetrapeptide-amide analogues of enkephalin: the role of C-terminus in determining the character of opioid activity.

Abstract The opioid activities of tetrapeptide-amide analogues of enkephalin /H-Tyr-D-Met-Gly-Phe-NH2; H-Tyr-D-Nle-Gly-Phe-NH2/ were studied in isolated, electrically stimulated longitudinal muscle strip of guinea-pig ileum and mouse vas deferens preparations in vitro and in vivo in the rat tail-flick test. Their effects were compared to those of the parent L-Pro5-NH2 containing analogues, and to other enkephalin derivatives substituted with D-Met in position 2 and L-amino/imino acids of different character in position 5. It was found that whilst the opioid receptor in mouse vas deferens preferred aliphatic residues of acidic character at the C-terminus of pentapeptides and was highly sensitive to the removal of C-terminal amino acid, the other systems were either much less responsive to these changes, or the effects were opposite to those found in mouse vas deferens.

Intracerebroventricular injection of clonidine releases β-endorphin to induce mucosal protection in the rat

The possibility that the endogenous opioid system could be involved in the central nervous system (CNS)-mediated gastroprotective effect of clonidine was investigated. Intracerebroventricularly (i.c.v.) injected clonidine (470 pmol/rat) inhibited the gastric mucosal lesions induced by (orally administered) acidified ethanol in a significant manner in the rat. The gastroprotective effect of the centrally administered clonidine was antagonised by i.c.v. or intracisternally (i.c.) administered presynaptic alpha-2 adrenoceptor antagonist, yohimbine; the non-selective opioid receptor antagonist, naloxone; and the delta opioid receptor antagonist naltrindole. These results suggest that an interaction between central alpha-2 adrenoceptors and endogenous opioid systems is involved in mediating the mucosal protective effect. beta-endorphin antiserum (i.c.) also antagonised the gastroprotection induced by intracerebroventricularly injected clonidine indicating that beta-endorphin release is likely to be a key factor in the gastroprotective effect of clonidine. Furthermore, the i.c.v. or i.c. injection of beta-endorphin produced a potent gastroprotection in the picomolar range. The mucosal protective effect of clonidine was abolished after vagotomy indicating that the central effect may be conveyed to the periphery by vagal efferents. Since atropine (1 mg/kg i.v.) failed to modify, but hexamethonium (10 mg/kg i.v.) antagonised the gastroprotective effect of clonidine, it would appear that in the periphery nicotinic, but not muscarinic, cholinergic receptors are likely to be involved in the mucosal protective effect of clonidine. In conclusion, clonidine (i.c.v.) induces gastroprotective action by releasing an endogenous opioid substance - most likely beta-endorphin - in the rat. The clonidine-induced central gastroprotection requires the integrity of vagal pathway; cholinergic nicotinic - but not muscarinic - receptors might mediate the effect in the periphery.

Enkephalin analogs containing amino sulfonic acid and amino phosphonic acid residues at position 5

Enkephalins, the endogenous opioid peptides: Tyr-Gly-Gly-Phe-Met and -Leu [ 11, have been known to show much higher agonist potency in the mouse vas deferens preparation (MVD) than in the guinea-pig ileum assay system (GPI) [2-61. The high MVD/GPI potency ratio is characteristic of enkephalins and of their synthetic analogs unless the pentapeptides are terminated by an amide function. For instance, potency ratios calculated for Met-enkephalin, its D-Ala2 analog and [D-Ala2, Met’]-enkeph~~ide were 9.0,8.4 and 2.0, respectively [2]. The MVDlGPI ratio for &endorphin, the 3 l-residue opioid peptide of the pituitary, is -1.2 and for nonpeptide narcotics, e.g., normorphine and morphine even smaller [2,4-61. Accordingly, the acidic COOH group at the C-terminus is essential for the ‘enkephalinoid’ character of pentapeptides. Starting from this consideration we synthesized some analogs of norleucine (Nle)-enkephahn in which the terminal COOH group is replaced by SOaH and POsH, group, respectively, i.e., Me’ is ~bstituted by a-amino~nt~e-s~fonic acid (NleS) and ff-amino-pentanephosphonic acid (MeP), respectively. The dissociation constant of a sulfonic acid or a phosphonic acid is known to be higher than that of the corresponding carboxylic acid. For instance, pKL values of taurine and p&nine are 1.5 and 3.6, respectively [7]. Similarly, pKH of glycine is 2.34 while the two pKH values of amino-methane-phosphonic acid are 1.85 and 5.35, respectively [S]. The more acidic terminal groups were expected to improve the enkephalin-like activity of the peptides. To study this question the opiate agonist potencies of the new compounds were