Mouse Vas Deferens Preparations Research Articles

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

Correlation between circular dichroism data and biological activities of 2, 5 substituted enkephalin analogues

The relative structural rigidity of enkephalin analogues characterized by the molar ellipticity data obtained from the circular dichroism spectra of peptides was correlated with the opioid agonist activities of compounds displayed in isolated, electrically stimulated longitudinal muscle strip of guinea-pig ileum and mouse vas deferens preparations. It was found that the so called μ receptors modelled by guinea-pig ileum preferred the analogues with high capacity to exist in folded form, whilst the so called δ receptors (mouse vas deferens) accepted flexible ligands as readily as rigid ones.

Further enhancement of analgesic activity: enkephalin analogs with terminal guanidino group

There is a great body of evidence that a basic amino-terminal is essential for the opioid activity of enkephalins, Tyr-Gly-Gly-Phe-Met and -Leu [ 1 I, and their analogs. Removal of the amino-group of Tyr results in a practically inactive peptide [2], as does its acetylation [3]. Introduction of two methylgroups into the terminal amino-moiety brings about a substantial loss in the biological activity [4], while mono-methylation decreases the potency in the mouse vas deferens preparation but enhances activity in the guinea-pig ileum [S]. It was tempting to speculate that interaction between the peptide N-terminus and its counterpart on the receptor (presumably a carboxyl-group) might be favourably influenced by replacing the terminal amino-group with a guanidino function. The resonance stabilized guanidinium cation has the capacity to interact.with some bidentate structure in the receptor site establishing hydrogen bridges in addition to the electrostatic interaction. One could anticipate that the improved binding properties of the peptide N-terminus would result in enhanced biological activity. It has also to be taken into account, however, that this replacement leads to a distal shift of the positive charge (~1.2 A). Such modification at the e-amino group of the lysine side chain in certain peptides, i.e., conversion of lysine into homoarginine residue, makes the lysine bond resistant to trypsin [6]. Thus trypsin seems to be unable to combine simultaneously with both the carbonyl-group and the positive charge of the basic residue when the distance between these is greater than occurs in a lysine or arginine residue. In this context one may recall that papain, which is less specific than trypsin, can hydrolyse homoarginine

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

The opioid activities of tetrapeptide-amide analogues of enkephalin /H-Tyr-D-Met-Gly-Phe-NH 2; H-Tyr-D-Nle-Gly-Phe-NH 2/ 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-Pro 5-NH 2 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.

A postsynaptic inhibitory histamine H2-receptor in the mouse isolated vas deferens

The effect of histamine on adrenergic neurotransmission in the mouse isolated vas deferens preparation was investigated. Concentrations of histamine ranging from 0.2 to 650 microM depressed, in a dose-related manner, not only the contractile response elicited by field stimulation but also the response caused by the addition of exogenous noradrenaline and acetylcholine. However, the release of [3H]-NA evoked by field stimulation or by high K+ remained unchanged in the presence of these concentrations of histamine. The inhibitory effect of histamine on the contractile responses caused by various stimuli was reduced or completely antagonized by cimetidine, a histamine H2-receptor antagonist but not by mepyramine, a conventional antihistamine. The inhibitory effect of histamine was found to be inversely proportional to both the Ca2+ concentration in the bathing medium and to the frequency of field stimulation. Further, the inhibitory effect of histamine was markedly reduced when Mg2+ was omitted from the bathing medium. It is concluded that the mouse vas deferens preparation contains a post-junctional inhibitory H2-receptor. The stimulation of H2-receptors by histamine inhibits the contractile response of the vas deferens, possibly by decreasing the availability of Ca2+ required for contraction by depressing the influx of Ca2+.

In vitro profile of some opioid pentapeptide analogues

The opiate agonist potentcy of thirteen synthetic enkephalin pentapeptides has been examined on the electrically stimulated guinea pig ileum and mouse vas deferens preparations in comparison with methionine and leucine enkephalins, β-endorphin and normorphine. Their antagonism by naloxone (Ke) was also assessed on each preparation. Our findings are compatible with, and are discussed in the context of, the hypothesis that these preparations possess at least two population of receptor.

Brain peptides with opiate antagonist action: Their possible role in tolerance and dependence

(1) Extracts of brain taken from rats injected with increasing doses of morphine acted like antagonists on the mouse vas deferens preparation in vitro and on morphine analgesia in vivo. (2) After electrophoretic fractionation, the antagonist properties were found among the basic peptides. One of these, Arg-Tyr-Gly-Gly-Phe-Met, was synthcsized and reproduced the analgesia-inhibiting effect of the brain extract. However, when tested on mouse vas deferens, it did not show antagonist properties but acted like enkephalin. (3) Quantitative estimation of the agonist and antagonist concentration in the brain of morphine-injected rats showed increase in both types of peptides during the first 12 days of treatment followed by a particularly marked fall of the agonists, far below the normal level. (4) Preliminary experiments have shown that the endogenous antagonists and the synthetic peptide can precipitate opiate withdrawal symptoms and may, therefore, be involved in the mechanism of dependence. (5) The results reported suggest the possibility that a shift in the balance of endogenous agonists and antagonists may contribute to the development of tolerance and dependence.

Human endorphin: Comparison with porcine endorphin, enkephalin and normorphine

We have extracted a material from human brain obtained at autopsy which has morphine-like activity on the mouse vas deferens preparation. This human endorphin behaved similarly on Sephadex G-15 and cation exchange columns as did porcine material and there were no qualitative differences in pharmacological activity between this material and porcine endorphin or enkephalin. Further purification of the human material is in progress. These results add further support to the suggestion that the stereospecific binding sites in opiate responsive species are receptors for an endogenous peptide ligand with a role in brain function.

Factors influencing the response of the mouse vas deferens preparation to noradrenaline.

Factors influencing the response of the mouse vas deferens preparation to noradrenaline.