Studies on opioid receptor selectivity of β-endorphin antagonists

Opioid receptor selectivity of several β-endorphin (β-EP) analogs which antagonize β-EP-induced analgesia has been assessed using partially selective binding assays. Although the apparent affinity dissociation constant of β-EP in these assays varies from 0.2 to 360 nm, the potency of β-EP antagonists relative to β-EP remains largely unchanged. It is unlikely that differences in receptor affinities can account for the antagonist properties of these analogs in vivo.     

Excitatory effect of morphine and opioid peptides in the rat isolated colon

Morphine and the opioid peptides cause isolated segments of rat colon to contract and relax rhythmically. This study re-examines two hypotheses to explain this phenomenon: Release of 5-hydroxytryptamine (5-HT)/acetylcholine by morphine or inhibition of a tonically active non-adrenergic, non-cholinergic (NANC) inhibitory mechanism. Rhythmic contractions induced by morphine (5 x 10(-6) M) were naloxone sensitive (10(-6) M) but unaffected by methysergide (10(-6) M), atropine (10(-6) M) or pretreatment of rats with p-chlorophenylalanine (200 mg kg-1 i.p. for four days) which lowered the 5-HT level in the colon from 3.73 +/- 0.83 mg g-1 in controls to 0.41 +/- 0.06 mg g-1 (P less than 0.001). The pattern of rhythmic contractions produced by morphine was unlike those produced by 5-HT (5 x 10(-6) M), acetylcholine (5 x 10(-6) M) or potassium chloride (30 mM). Tetrodotoxin (10(-6) M), apamin (10(-8) M), clonidine (2 x 10(-8) M), phentolamine (10(-5) M) or oxprenolol (10(-5) M) caused rhythmic contractions which were unaffected by naloxone. Clonidine contractions were inhibited by yohimbine (10(-7) M) but not by prazosin (10(-6) M). Electrical field stimulation at the peak of a contraction induced by morphine, apamin or clonidine, produced an inhibitory response which was unaffected by atropine, phentolamine, propranolol and guanethidine (all 10(-5) M). It persisted in colon segments from the rats with reserpine or 6-hydroxydopamine. These results suggest that neither the 5-HT/acetylcholine hypothesis nor inhibition of the NANC mechanism adequately explains the excitatory effect of morphine in the rat colon.     

Temperature modulation of α- and β-adrenoceptors in the isolated frog heart

1. The effects of adrenaline on the isolated frog's heart at 27 degrees C are not antagonized by phentolamine (1.5 x 10(-6)M) but are abolished at 7 degrees C.2. At 27 degrees C isoprenaline was more potent than noradrenaline, but at 7 degrees C noradrenaline was more potent than isoprenaline.3. Phenoxybenzamine (1.5 x 10(-5)M) or dibenamine (1.5 x 10(-5)M) at 7 degrees C abolished the work output induced by adrenaline. When the temperature was raised to 24 degrees C, adrenaline caused an increase in work output.4. It is concluded that in the isolated frog heart there are at least two pools of adrenoceptors, the availability of which can be governed by temperature.     

Pharmacological properties of ββ-dimethylacryloylcholine and some other β-substituted acryloylcholines

The mammalian pharmacology of a new naturally occurring ester of choline, ββ-dimethylacryloylcholine (DMAC), has been studied, mainly in the cat, together with that of two synthetic β-substituted acryloylcholines, crotonoylcholine and pent-2-enoylcholine. Comparisons have been made with the reduced form of DMAC, isovalerylcholine, with another naturally ocurring β-substituted acryloylcholine, murexine (urocanoylcholine), and with suxamethonium. DMAC has been shown to be a ganglion stimulating and neuromuscular blocking agent generally similar, in potency and properties, to murexine. It is also a powerful respiratory stimulant. The other unsaturated esters behaved similarly but were less potent.     

阿片生长因子甲硫氨酸脑啡肽抗肿瘤作用研究进展

甲硫氨酸脑啡肽是一种内源性阿片生长因子,它除了具有阿片肽类镇痛、免疫调控等共性以外,还对细胞的增生和更新、肿瘤生长、伤口愈合、血管发生等具有比较强的抑制作用.甲硫氨酸脑啡肽可以作用于细胞的生长周期,从而达到抑制肿瘤细胞生长的作用.本文对甲硫氨酸脑啡肽的药理作用、抗肿瘤作用及其作用机制的研究现状进行综述.     

Selective ligands for opioid receptors: β-Cyclopropylalanyl containing analogs of enkephalin

The synthesis and resolution of the amino acid β-cyclopropylalanine (Cpr) and its incorporation into four enkephalin analogs is reported. The analogs prepared were: Tyr - l - Cpr - Gly - Phe - Pen (des - COOH - Nle = n - pentylamide = Pen) (l -Cpr²-Pen⁵-ENK), Tyr-d -Cpr-Gly-Phe-Pen (d -Cpr²-Pen⁵-ENK), l -Cpr-Tyr-d -Ala-Gly-Phe-Pen (l -Cpr⁰-d -Ala²-Pen⁵-ENK) and d -Cpr-Tyr-d -Ala-Gly-Phe-Pen (d -Cpr⁰-d -Ala²-Pen⁵-ENK). Each was tested for its ability to inhibit the field stimulated guinea pig ileum (GPI) and rat vas deferens (RVD) and the results compared to the effect d -Ala²-d -Leu⁵-enkephalin (DADLE) has on the same preparations. The results show that at concentrations up to 10^-5 m all four analogs, as well as DADLE, are full agonists on the GPI preparation. The concentrations necessary to produce a 50% inhibition of the twitch response were, DADLE, 3.5 °× 10^-8 m ; l - Cpr⁰-d -Ala²-Pen⁵-ENK, 6.0 × 10^-8 m ; d -Cpr²-Pen⁵-ENK, 1.1 × 10^-7 m ; l -Cpr²-Pen⁵-ENK, 1.2 × 10^-6 m and d -Cpr⁰-d -Ala²-Pen⁵-ENK, > 10^-5 m . On RVD a different result was observed with only DADLE (1.3 × 10^-6 m ) and l -Cpr⁰-Pen⁵-enkephalin (1.8 × 10^-6 m ) showing full agonist activity. d -Cpr²-Pen⁵-ENK was a partial agonist (29 · 5% inhibition of the twitch at 10^-5 m ) while d -Cpr⁰-d -Ala²-Pen⁵-ENK and l -Cpr²-Pen⁵-ENK did not inhibit the twitch at concentrations up to 10^-5 m . These compounds which were inactive or of low potency on each preparation were also tested as antagonists. Only d -Cpr²-Pen⁵-ENK was an antagonist (pA₂ = 6.09) versus DADLE on RVD while d -Cpr⁰-d -Ala²-Pen⁵-ENK was inactive as an antagonist on both GPI and RVD. d -Cpr²-Pen⁵-ENK, therefore, represents the first enkephalin analog to be categorized as a mixed agonist-antagonist.     

Synthesis and receptor binding activity of elephant β-endorphin,a β-endorphin homolog with highly potent analgesic activity#

Elephant β-endorphin and its analog, elephant β-endorphin(6-31) were synthesized by standard solid phase method. Receptor binding activity showed that elephant β-endorphin was five to six times more potent than human β-endorphin in its ability to bind to opiate receptors on rat brain membrane. In a previous study (Wong, C.-L., Wai, M.-K., Cheng, H.-C., Chung, D. & Yamashiro, D (1990) Clinical and Experimental Pharmacology and Physiology 16 , 33–37), tail flick test for intracerebroventricularly administered β-endor-phin showed that the antinociceptive potency of elephant β-endorphin was seven to eight times higher than that of human β-endorphin in mice. Results from both studies suggest that elephant β-endorphin was a much more potent antinociceptive agent than human β-endorphin in tail flick test and its higher analgesic activity might be due to its higher affinity for opiate receptors in the brain.     

Effect of centrally and peripherally administered β-endorphin on fooc intake in rats

A role for β-EP in the regulation of food intake has been suggested as a contributory factor in the obesity of some genetically obese animal models. Studies undertaken to determine whether continuous administration of β-EP could alter food intake in normal rats are described. The present studies demonstrated that continuous subcutaneous infusion with β-EP was ineffective in modulating food intake, but that acute intraperitoneal or intracerebroventricular administration stimulated food intake in previously food deprived or satiated animals, respectively. These results suggest that β-EP is not involved in the long-term regulation of food intake, but under certain conditions it may play some role in the regulation of individual meals. It is speculated that the latter activity may result from the action of other appetitive regulatory hormones.     

Effects of β-endorphin on the contraction and electrical activity of the isolated perfused rat heart

β-Endorphin at pharmacological doses reduced both the left ventricular systolic and diastolic pressures of the isolated rat heart. The reduction in the diastolic ventricular pressure was dose dependent. β-Endorphin also altered the electrical activities of the isolated heart, disturbing the normal electrocardiogram pattern. The degree of disturbance was also dose dependent. Naloxone itself did not produce any effect. However it antagonized the depressant effects and completely abolished the effect of β-endorphin on the electrical activity of the heart, indicating that β-endorphin acts via the naloxone sensitive receptors. The results suggest a possible regulatory role of intracardiac endogenous opioid peptides in the cardiac functions.     

Reversed phase thin-layer chromatography for the separation of β-endorphin, β-lipotropin and enkephalins

A novel, simple technique using reversed phase thin-layer chromatography has been developed to separate β-endorphin, β-lipotropin and Met-and Leuenkephalins. This method is useful for the rapid determination of the purity of these opioid peptides. The effectiveness of several solvent systems has been assessed. The resolution between β-endorphin and β-lipotropin achieved by this method exceeds that reported using high performance liquid chromatography or a variety of column techniques. This system also eliminates the problem of unrecognized loss of peptide due to binding on HPLC or other types of columns since the entire stationary phase is visualized using fluorescamine detection. Efficient, inexpensive and reliable simultaneous separation with visualization of several opioid peptides, their peptide precursors and their degradation products is now possible using the new technique. Such separations are required for many purposes including preparation of samples for radioimmunoassay of opioid peptides.     

Effect of catecholamines and angiotensin on extracellular water and cation movements and the effect of α and β-adrenoceptor blockade

1. The extracellular fluid volume (ECFV), as raffinose space, and its content of Na, K and Ca were measured in anaesthetized dogs in acute experiments before and during the vascular response to intravenous injections and infusions of noradrenaline, adrenaline, isoprenaline and angiotensin.2. In male dogs the effect of noradrenaline was unpredictable, the ECFV might increase or decrease. In female dogs noradrenaline caused an increase in the ECFV. The difference between the responses of the two sexes was statistically significant (P<0.001). After phentolamine, noradrenaline exerted no effect at all in either sex. After bretylium the results were like those in normal animals.3. In both males and females adrenaline generally induced an increase in the ECFV. After phentolamine, adrenaline decreased the ECFV in males and caused little change in females. The differences before and after blockade were statistically significant (P<0.001). After bretylium the results in both sexes were like those in normal animals.4. In both male and female dogs isoprenaline induced an increase in the ECFV and the results were the same as in the normal animals after both phentolamine and bretylium.5. In male dogs there was no change in the ECFV as a result of administering angiotensin, either alone or in the presence of phentolamine or bretylium. In normal females angiotensin induced a decrease in ECFV and the difference between the responses of the males and females was statistically significant (P<0.005). In females which had received either phentolamine or bretylium the results were indistinguishable from those in the males.6. Blockade of the beta-adrenoceptors with pronethalol in a few animals did not change the response to the drugs from those seen in normal animals.7. The cation content of the ECF changed in the same direction and to about the same extent as the water, except after noradrenaline when in some experiments the proportionate change in potassium concentration was considerably greater than that of the other substances.8. The inulin space and its Na and K content were measured in several dioestrous, oestrous and pro-oestrous rats and in normal and stilboestrol treated males before and after giving an intravenous injection of angiotensin. There was little difference between the results of control injections of 0.9% saline solution and of angiotensin in dioestrous and oestrous females and normal males. On the other hand, pro-oestrous females and stilboestrol treated males responded alike to angiotensin in the form of a decrease in ECFV which was statistically different from the responses in the other three groups (P<0.0005).9. It is suggested that the various results depend on two factors: the site of action of the drug-for example, whether it increases or decreases capillary pressure and therefore, fluid transfer-and also the sex of the animal. The ground substance of the small blood vessels is probably important in taking up and releasing fluid, and its capacity for so doing may well vary with the amount of available oestrogen. It appears that the effect of oestrogens and events at the alpha-adrenoceptor site are connected in some way.     

The effect of β-adrenoceptor blockade on human sweating

1. Changes in cutaneous water loss were followed by continuously monitoring total body weight loss.2. Sweating was induced in normal subjects by raising the environmental temperature or by subjecting them to the emotional stress of mental arithmetic.3. Propranolol in a dosage of 0.15 mg/kg body weight intravenously had no significant effect on either thermal or emotional sweating, whereas thermal sweating was completely blocked temporarily by administration of atropine 2.4 mg intravenously.4. It is concluded that beta-adrenoceptor blockade has no effect on physiological sweating in normal people.     

Protective Effects of the Hydroethanolic Extract of Fridericia chica on Undifferentiated Human Neuroblastoma Cells Exposed to α-Zearalenol (α-ZEL) and β-Zearalenol (β-ZEL)

Fridericia chica (Bignoniaceae) is a traditional medicinal plant. The aim of this research was to determine the protective effects of the hydroethanolic extract from the F. chica leaves (HEFc) against the cytotoxicity of zearalenone (α-ZEL) and β-ZEL on SH-SY5Y cells. Free radical scavenging activity of HEFc was evaluated using the DPPH method. The cytotoxicity of both zearalenone metabolites and HEFc was examined using MTT test, as was the cytoprotective effects of the HEFc on cells treated with these mycotoxins. The chemical composition of HEFc was determined using UPLC-QTOF-MS/MS. HEFc elicited good DPPH radical scavenging activity following a concentration-dependent relationship. Cells exposed to α-ZEL exhibited a viability ˂50% after 48 h of treatment (25 and 50 µM), while those exposed to β-ZEL showed viability ˂50% (100 µM) and ˂25% (25-100 µM) after 24 and 48 h of exposure, respectively. HEFc showed a significant increase in cell viability after exposure to α-ZEL (25 and 50 µM) and β-ZEL (6–100 µM) (p < 0.05). UPLC-QTOF-MS/MS analyses allowed the identification of 10 phytochemical components in the HEFc. In short, the hydroethanolic extract of F. chica grown in Colombian Caribbean can protect against the effects of mycotoxins and it is a valuable source of compounds with antioxidant properties.     

β-Adrenoceptor agonist activity of labetolol on the isolated uterus of the rat

Isoprenaline produced dose-dependent reductions of responses of the isolated uterus of the rat produced by an EC₈₀ of acetylcholine. Propranolol acted as a competitive antagonist to isoprenaline. Labetolol also reduced the acetylcholine-induced contractions but was much less potent than isoprenaline. The greatest reduction was smaller than that produced by isoprenaline. Propranolol antagonized the lower doses of labetolol. It is suggested that labetolol possesses partial agonist activity at the β-adrenoceptors of the rat isolated uterus.     

α- and β-Adrenoceptors in the detrusor muscle and bladder base of the pig and β-adrenoceptors in the detrusor muscle of man

1 The presence and type of adrenoceptors in the smooth muscle of the pig and human urinary bladder was assessed on the basis of the relative potency of α- and β-adrenoceptor agonists and antagonists.

2 In isolated, carbachol-contracted bladder strips from the pig detrusor muscle the relaxing potency of isoprenaline was four times that of salbutamol and ritodrine and thirty times that of noradrenaline.

3 Propranolol caused a parallel shift to the right of the noradrenaline dose-response curve which was not changed by phentolamine.

4 Propranolol and butoxamine showed, in contrast to practolol, a dose-dependent antagonism of the response to isoprenaline. A pA₂ value of 9.2 ± 0.2 and 6.8 ± 0.2 (mean ± s.e. mean) for the first two antagonists was calculated.

5 In the bladder base of the pig, propranolol caused a parallel shift to the right and phentolamine a shift to the left of the dose-response curve to noradrenaline.

6 In the human detrusor muscle the potency and maximum effect of isoprenaline and salbutamol were less than those in the pig detrusor muscle. The potency of isoprenaline was sixty times that of salbutamol.

7 Whereas a parallel shift to the right of the dose-response curve to isoprenaline was obtained with propranolol, no antagonism was obtained with butoxamine or practolol.

8 The results are interpreted as indicating the presence of β₂-adrenoceptors in the detrusor muscle of the pig and β-adrenoceptors with neither β₁- nor β₂-characteristics in the detrusor muscle of man. An indication of the presence of α-adrenoceptors in the bladder base but not in the detrusor muscle of the pig was obtained.

     

Inhibition of rabbit intestine mediated by α- and β-adrenoceptors

1. The effects of some alpha- and beta-adrenoceptor agonists and antagonists were studied on isolated segments of rabbit intestine in an attempt to characterize the two types of inhibitory response produced by sympathomimetic amines.2. Phenylephrine, an alpha-adrenoceptor agonist, produced an inhibition of rapid onset, from which recovery occurred despite the continued presence of the drug. On washout there was an overshoot in contraction height. Isoprenaline, a beta-adrenoceptor agonist, produced an inhibition of slow onset which was maintained throughout the presence of the drug and there was no overshoot on washout.3. Adrenaline resembled phenylephrine more closely than it resembled isoprenaline, in that it showed more affinity for alpha-adrenoceptors, whereas noradrenaline, and the transmitter released on periarterial nerve stimulation, behaved more like isoprenaline, although both types of receptor were affected.4. Adenosine-5'-triphosphate produced an inhibition resembling that produced by an alpha-adrenoceptor agonist, whereas the dibutyryl analogue of cyclic adenosine 3',5'-monophosphate (cyclic 3',5'-AMP) produced an inhibition resembling that produced by a beta-adrenoceptor agonist.5. In critical concentrations theophylline augmented and imidazole inhibited beta-adrenoceptor mediated responses, as well as responses to dibutyryl cyclic AMP. However, additional actions of theophylline and imidazole were also demonstrated.6. Responses mediated by alpha-adrenoceptors, but not those mediated by beta-adrenoceptors, were blocked by membrane stabilizers, quinidine being the most potent of those studied.7. The results are discussed in relation to the possible mechanisms of action of alpha- and beta-adrenoceptor agonists.     

Distribution of α- and β-adrenoceptors in human urinary bladder

1 The distribution of alpha- and beta-adrenoceptors in isolated preparations of human bladder neck and detrusor muscle has been studied.2 Adrenaline caused contraction of the bladder neck which was blocked by phenoxybenzamine but unaltered by propranolol.3 Isoprenaline caused relaxation of the bladder neck which was blocked by propranolol. High concentrations caused contraction which was enhanced by propranolol but blocked by phenoxybenzamine.4 Detrusor muscle was relaxed by isoprenaline and this effect was blocked by propranolol. Phenylephrine caused relaxation of detrusor which was unaffected by phenoxybenzamine; in some cases contraction was produced in the presence of propranolol.5 It is concluded that the bladder neck contains mainly alpha-receptors and the detrusor mainly beta-receptors but both regions posses both types of adrenoceptor.     

Release of β-lipotropin- and β-endorphin-like material induced by angiotensin in the conscious rat

1 Groups of mice were pretreated with the 5-hydroxytryptamine (5-HT) depletors, fenfluramine or p-chlorophenylalanine (PCPA), followed by pethidine or morphine. 2 Fenfluramine alone produced a short lasting analgesia but PCPA was without any effect. 3 Pethidine and morphine both increased hot plate reaction times measured after 30 min. 4 Pretreatment with PCPA attenuated morphine analgesia but did not affect pethidine analgesia. Fenfluramine did not alter the response to either analgesic. 5 PCPA produced a significant depletion of brain 5-HT levels which was not reversed by the analgesics. The fenfluramine-induced decrease in 5-HT was reversed by morphine but not by pethidine. 6 The results support the involvement of 5-HT in the antinociceptive action of morphine in the mouse.     

The effect of certain β-adrenoceptor antagonists on overdrive suppression

1. Propranolol, as the racemate and the (+)- and (-)-isomers (400 μg/l.) and practolol (50 mg/l.) were tested for their effects on atrial and ventricular rates and on the duration of overdrive suppression (ODI) in isolated perfused cat hearts with surgically-induced heart block.

2. Racemic propranolol and the (+)- and (-)-isomers prolonged ODI and slowed the rate of the ventricular pacemaker; the (+)- and (-)-isomers also reduced the rate of the atrial pacemaker. Practolol shortened ODI and increased the rate of the atrial and ventricular pacemakers.

3. The (+)- and (-)-isomers were more potent than the racemate; the (-)-isomer was more potent than the (+)-isomer. The results suggest there is a stereospecific mechanism involved in the biological distribution of propranolol.

4. Twenty-four hours after reserpine treatment (5 mg/kg, i.p.) practolol continued to increase the rate of the atrial and ventricular pacemakers but did not shorten ODI.

5. The mechanism by which these agents affect myocardial excitability and automaticity is discussed.