Potencies of normorphine and met-enkephalin in the vas deferens of different strains of mice.

The concentration of normorphine causing a 50 per cent inhibition (IC₅₀) of electrically induced twitches in the vas deferens from seven strains of mice varied over a 13-fold range, BALB/cKB being the most, C57BL/6J the least sensitive. There was no significant correlation between the IC₅₀'s of normorphine and met-enkephalin. In the sensitive BALB/cKB mice, both normorphine and met-enkephalin were more effective inhibiting contractions evoked by 0.1 Hz than by 0.01 or 1.2 Hz stimulation. This difference was not observed in the insensitive C57BL/6J mice. Naloxone was purely an antagonist against both normorphine and met-enkephalin in BALB/cKB mice but in low concentration it potentiated the inhibitory effect of both normorphine and met-enkephalin in C57BL/6J mice. Results suggest that qualitative differences in opiate receptors and differences in transmitter release mechanism contribute to the variable sensitivity to morphine of the vas deferens from different strains of mice.     

Amino-terminal extension analogs of methionine-enkephalin

Five NH₂-terminal extension analogs of methionine-enkephalin have been synthesized by solid phase methodology and their morphinomimetic activity determined in the guinea pig ileum-myenteric plexus preparation. Relative to methionine-enkephalin which corresponds to β-LPH-(61–65) and arbitrarily assigned a potency value of 100, β-LPH-(60–65), β-LPH-(59–65), β-LPH-(58–65), β-LPH-(57–65) and β-LPH-(56–65) have potencies of 90, 7, 4.4, 0.3 and 0.11 respectively.     

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.     

Comparative study on the effect of morphine and the opioid-like peptides in the vas deferens of rodents: species and strain differences, evidence for multiple opiate receptors

The effect of an opiate alkaloid and an opioid-like peptide was studied on the electrically evoked twitching of the vas deferens of 3 common laboratory rodents. Normorphine and the synthetic opioid peptide D-Alanine₂ methionine enkephalinamide (D-Ala₂) produced dose dependent inhibitions of the twitching response in the mouse vas deferens. In the rat vas, while β-endorphin (β-EP) caused an inhibitory effect in three strains of rats to a similar degree, morphine produced a dose related enhancement of the twitching. In the guinea pig, both morphine and β-EP caused an increased in the muscular twitch. The results are interpreted in terms of an heterogenous mixture of opiate receptors present in the vas deferens from these rodents. The mouse appears to contain mainly δ receptors while the rat has mostly ε receptors characterized by their specificity and sensitivity to the action of β-EP. The guinea pig vas deferens has apparently lost the sensitivity to the inhibitory influence of the opioids, suggesting the absence of μ or δ opiate receptors in this tissue.     

Evidence for morphine and morphine-like alkaloid responses resistant to naloxone blockade in the rat vas deferens.

The application of morphine or surrogates to the isolated rat vas deferens maintained at 37° C in Tyrode solution, produced an increase in the electrically induced muscular twitch. In contrast, leucine enkephalin or D-alanine₂methionine enkephalinamide produced a dose-dependent inhibition of the muscular twitch. The effect of morphine and derivatives was not antagonized by naloxone, but the depression caused by the opiate pentapeptides or β-Endorphin was readily antagonized and reversed by naloxone. Tolerance developed to the $\text{in vitro}$ effect of morphine; vasa deferentia obtained from tolerant-dependent rats were about six times less sensitive to the effect of morphine and about five times less sensitive to the depression caused by leucine enkephalin as compared to their respective paired, placebo implanted control rats.     

Endorphins in brains of decapitated and microwave-killed mice

The size distribution of opioid peptides (endorphins) in mouse brain was determined by gel filtration, using the inhibition of stereospecific ³H-etorphine binding to guinea pig brain membranes as a measure of endorphin activity. The largest endorphin was about the same size as β-LPH-(61–91), the next largest was about half this size and the smallest was the size of the pentapeptide enkephalins. There was no important difference in the size distribution between brains of mice killed by rapid microwave irradiation and those killed by decapitation. Thus, opioid peptides of all three size classes appear to be present in the brain $\text{in}$$\text{vivo}$.     

β-endorphin proteolysis by guinea-pig ileum myenteric plexus membranes: Increased γ-endorphin turnover after chronic exposure to morphine

The influence of chronic morphine exposure $\text{in vitro}$ on the biotransformation of β-endorphin (βE) was investigated using the myenteric plexus-longitudinal muscle of guinea-pig ileum. A membrane preparation was incubated with βE and the degradation of βE as well as the accumulation of several βE fragments in the incubation medium were followed with time. The levels of peptides were determined by specific radioimmunoassays after separation by high-pressure liquid chromatography. It was found that exposure to morphine did not affect the disappearance of βE, but altered the time course of accumulation of βE fragments. In fact, the accumulation of γ-endorphin, α-endorphin and des-tyrosine¹-α-endorphin was enhanced, while that of des-tyrosine¹-γ-endorphin was not changed. Additionally, the disappearance of γ-endorphin appeared to be stimulated by morphine exposure. These data provide evidence that the fragmentation of βE is changed by chronic morphine exposure in such a way that the turn-over of γ-endorphin is increased.     

Selective receptors for beta-endorphin on the rat vas deferens.

The isolated rat vas deferens, being insensitive to morphine, contains selective binding sites for β-end-orphin. A half-maximal inhibition of twitch tension evoked by electrical stimulation is established with 100 nM β-endorphin, while fragments of β-endorphin, that is, methionine-enkephalin, α- and γ-endorphin, are almost ineffective. The opiate alkaloid etorphine, a powerful inhibitor of guinea-pig ileum and mouse vas deferens, is 100-fold less potent on the rat vas deferens. The unique β-endorphin activity suggests very specific binding sites for this peptide, which cannot be related to the μ- or δ-receptors so far described for opiods on isolated preparations.     

Altered levels of β-endorphin fragments after chronic morphine treatment of guinea-pig ileum invitro and invivo

The isolated myenteric plexus-longitudinal muscle of the guinea-pig ilem (GPI) was used as testsystem to study the influence of chronic morphine treatment on the levels of enkephalins, β-endorphin and some of its fragments. The peptides were assayed by means of a combination of high pressure liquid chromatography and radioimmunoassays. It was found that the levels of methionine- and leucine-enkephalin and β-endorphin were not altered by chronic morphine treatment of guinea-pigs $\text{in}$$\text{vivo}$ nor in GPI exposed to morphine $\text{in}$$\text{vitro}$. However, the levels of some β-endorphin fragments i.c. γ-endorphin and des-tyrosine-γ-endorphin were elevated after morphine treatment $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ respectively. It is suggested that β-endorphin and its fragments are involved in homeostatic processes during development of opiate tolerance.     

Inhibition by β-carbolines of monoamine uptake into a synaptosomal preparation: Structure-activity relationships

The potency of a series of β-carboline compounds to inhibit ³H-serotonin (³H-5-HT) uptake into a synaptosomal suspension from mouse brain was studied. The $\text{in vitro}$ structure-activity study showed the tetrahydro-β-carbolines to be the most potent inhibitors compared to unsaturated β-carbolines. $\text{In vitro}$ inhibition of ³H-norepinephrine (³H-NE) and ³H-dopamine (³H-DA) uptake was determined for some tetrahydro-β-carbolines, and the degree of inhibition of uptake of these amines was less than that for ³H-5-HT (EC50s being 5–13 times those for ³H-5-HT). The tetrahydro-β-carbolines were also found to effectively inhibit ³H-5-HT but not ³H-NE or ³H-DA uptake when they were administered intra-peritoneally. These results suggest that the behavioral effects of the tetrahydro-β-carbolines which have been reported previously may be due to a relatively selective involvement of the serotonergic neurotransmitter system.     

Met-enkephalin immunoreactivity in blood platelets

Picogram amounts (50–150 pg/mg protein) of immunoreactive met-enkephalin material (met-enkephalin in IR) were detected by radioimmunoassay in human, rat and rabbit platelets. Characterization of this material by thin-layer chromatography, gel filtration chromatography and high-pressure liquid chromatography indicated that it behaves identically with synthetic met-enkephalin. No high molecular weight met-enkephalin IR could be detected in the platelet extracts, even after trypsin hydrolysis, using two antisera which are able to recognize some of the putative met-enkephalin precursors present in the adrenal gland or striatum. $\text{In vitro}$, thrombin released platelet met-enkephalin in IR concomitantly with 5-hydroxytryptamine (5-HT), suggesting a common subcellular localization, i.e. the 5-HT storing organelles, for met-enkephalin IR and the amine. $\text{In vivo}$, platelet met-enkephalin IR in the Sprague-Dawley rat was affected neither by adrenalectomy nor by hypophysectomy. Thirteen- and 18-week-old spontaneous hypertensive rats (SHR) had lower platelet concentrations of met-enkephalin in IR than age matched normotensive Wistar-Kyoto rats.     

Analgesic activity of intracerebroventricular administration of morphiceptin and β-casomorphins: Correlation with the morphine (μ) receptor binding affinity

Analgesic activities of morphiceptin, β-casomorphins, [D-Ala², D-Leu⁵] enkephalin and Sandoz peptide, FK 33–824, were examined by intracerebroventricular administration in rats. Their relative potencies $\text{in vivo}$ were compared with their receptor binding activities. The receptor binding affinities were determined from the competition curves against [³H]naloxone binding in the absence and presence of sodium ions for morphine (μ) receptors and against ¹²⁵I-[D-Ala², D-Leu⁵] enkephalin binding for enkephalin (δ) receptors. A good correlation between analgesic activity and morphine (μ) receptor but not enkephin (δ) receptor binding affinity was obtained. These data extend the hypothesis that morphine (δ) receptors mediate the major portion of the analgesic activity of opioids.     

Cardiovascular effects of peptides related to the enkephalins and β-casomorphin

In the urethane-anesthetized rat, intravenous injections of morphine produced a short-lasting fall in heart rate and blood pressure. The fall in heart rate (which is vagal in origin) was used here to bioassay peptides related to the enkephalins [-enk] and β-casomorphin [β-C, H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH]. A > 10% decrease in heart rate was used as a quantal index of response and the intravenous ED50 [μmol/kg] were estimated as: methionine-enk, 1.3 ± .24; leucine-enk, 1.5 ± .20; [D-Ala², D-Leu⁵]-enk, 0.45 ± .05; [D-Ala², NMePhe⁴, Met(0)⁵-ol]-enk, 0.0011 ± .0002; H-Tyr-Arg-OH, > 2.0; β-C (1–7), > 2.0; β-C(1–5), > 2.0; β-C(1–4), > 4.0; β-C(1–3), > 2.0; morphine sulfate, 0.11 ± .03; and human β-endorphin,, 0.07 ± .01. One β-C derivative, H-Tyr-Pro-Phe-Pro-NH₂ [β-C(1–4)-NH₂], was active at 0.32 ± .08. Naloxone pretreatment blocked the bradycardia produced by the enkephalins and β-C(1–4)-NH₂. The bioassay described here, based on heart rate, may prove to be useful for the rapid detection and estimation of the $\text{in vivo}$ pharmacological activities of new opioid peptides.     

Ozonolytic cleavage of authentic and pancreatic dolichyl mannopyranosyl phosphates: determination of sugar configuration in the fragments with alpha- and beta-mannosidases.

Exposure of authentic dolichyl α-D-[¹⁴C]mannopyranosyl phosphate ($\text{I}$) or calf pancreas dolichyl [¹⁴C]mannopyranosyl phosphate ($\text{II}$) to ozone at ?70° in pentane followed by treatment with triphenylphosphine gave water-soluble fragments in 65–95% yield. The radioactive products obtained were similar; the major fragment had a mobility on tlc greater than that of mannose but lower than that of citronellyl β-D-mannopyranosyl phosphate. The electrophoretic behavior of the fragments indicated that they possessed intact phosphodiester linkages. α-Mannosidase released [¹⁴C]mannose from the fragments of $\text{I}$ but not from the fragments of $\text{II}$; however, the latter were susceptible to β-mannosidase indicating that the pancreatic mannolipid contains a β-linked mannosyl residue.     

Highly potent side chain–main chain cyclized dermorphin–deltorphin analogues: An integrated approach including synthesis,bioassays, NMR spectroscopy and molecular modelling

Our continuing efforts to study structure–activity relationships of peptide opioids have resulted in the synthesis of a series of cyclic opioids related to dermorphins and deltorphins. The biological activities of the compounds have been determined and the conformational analyses carried out using ¹H-NMR spectroscopy and molecular modelling. The three compounds in the series Tyr-c[D -Orn-Phe-Ala], Tyr-c[D -Lys-Phe-Ala], and Tyr-c[A₂Bu-Phe-Ala-Leu] are cyclized via a lactam bridge from the side-chain of the residue at the second position with the carboxyl terminus of each compound. The molecules incorporate 12-, 13- and 14-membered rings, respectively. They include a phenylalanine at the third position which is a distinguishing characteristic of dermorphins and deltorphins. The guinea pig ileum and mouse vas deferens assays show that the compounds are highly active at both μ- and δ-opioid receptors. The compounds are all highly effective antinociceptive agents as measured by the intrathecal rat hot plate test. Conformational analyses of the molecules indicate that they can adopt topochemical arrays required for bioactivity at both μ- and δ-receptors which explains their high activity in both guinea pig ileum and mouse vas deferens in vitro assays. The results support our models for μ- and δ-receptor activity for constrained peptide opioids.     

Opiate receptor affinities and behavioral effects of enkephalin: Structure-activity relationship of ten synthetic peptide analogues

Synthetic met- and leu-enkephalin bind to rat brain opiate receptors with $\text{1}\text{2}$ and $\text{1}\text{7}$ the affinity of morphine. The aromatic hydroxyl moiety of the tyrosine residue is critical for receptor binding. Intracranial microinjection of met-enkephalin requires very high doses to produce an evanescent, naloxone reversible analgesia and stuperous immobility, presumably because of its rapid enzymatic degradation. Leu-enkephalin fails to elicit analgesia.     

Beta-endorphin. Biological activity of synthetic analogs with analgesia inhibiting property in rat vas deferens and guinea pig ileum assays

Three synthetic analogs of human beta-endorphin (beta h-EP) (I, [Gln8, Gly31]-beta h-EP-Gly-Gly-NH2; II, [Arg9,12,24,28,29]-beta h-EP and III, [Cys11,26, Phe27, Gly31]-beta h-EP), which have been shown to possess potent inhibiting activity to beta h-EP-induced analgesia, were assayed in rat vas deferens and guinea pig ileum bioassay systems. In the rat vas deferens assay, relative potencies of these analogs were beta h-EP, 100; I, 30; II, 40; III, 1, whereas in the guinea pig ileum assay: beta h-EP, 100; I, 184; II, 81; III, 163. From previous studies on their analgesia potency in mice and opiate receptor-binding activity in rat brain membranes, their activity in rat vas deferens correlates well with the analgesic potency and the activity from guinea pig ileum assay shows good correlations with that from the opiate receptor-binding assay.     

Actions of morphine on prostate gland fructose metabolism

Varying doses of morphine sulfate (10, 20 or 40 mg/kg daily × 10) were observed to suppress metabolic activities in the mouse prostate gland. Prostate gland fructose, an index of androgenic activity, was significantly reduced by these dose regimes of morphine (P < 0.01). Injections of morphine sulfate (20 mg/kg daily × 10) led to an inhibitition in the $\text{in vitro}$ synthesis of both fructose^?14C and sorbitol^?14C from glucose^?14C by the prostate gland, part of which may have been due to decreased uptake of glucose by the gland. The $\text{in vitro}$ assimilation of 2-deoxyglucose^?14C by the prostate was also reduced by morphine treatment. The $\text{in vitro}$ actions of morphine (2 × 10^?3M) on the metabolism of radioactive glucose by the mouse prostate gland likewise revealed a significant reduction in the formation of sorbitol^?14C, but no decrease in fructose^?14C formation. These results indicate that both the $\text{in vitro}$ and $\text{in vivo}$ actions of morphine can inhibit fructose metabolism in the prostate gland.     

Effect of clonidine on the chronic morphine tolerance and on the sensitivity of the smooth muscles in mice

Clonidine, when administered for prolonged period showed no tolerance to its analgesic activity. Prior exposure to clonidine attenuated the tolerance development to morphine-induced analgesia and the supersensitivity to acetylcholine (ACh) in ileum during chronic morphine treatment. Further, acute administration of lower doses of clonidine (upto 1 mg) produced supersensitivity in ileum to ACh while the higher dose (10 mg) induced subsensitivity. In vas deferens, clonidine in all the concentrations tested induced dose and time dependent supersensitivity to norepinephrine (NE) similar to that produced by morphine. Chronic clonidine treatment failed to alter the ACh responses in ileum while it produced supersensitivity to NE in vas deferens. The results suggest that clonidine and morphine possess comparable properties and the antagonism of chronic morphine tolerance by clonidine may be the therapeutic basis for its clinical application in the treatment of opiate addicts.     

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.