Mechanisms involved in the cardiovascular responses to opioid products of proenkephalin in the anaesthetised rat.

1. Cardiovascular effects of opioid peptide products of proenkephalin, [Met] enkephalin (ME), [Leu] enkephalin (LE), [Met] enkephalyl Arg6-Phe7 (MEAP) and [Met] enkephalyl Arg6-Gly7-Leu8 (MEAGL) have been studied in urethane-anaesthetised rats. 2. ME, LE, MEAP and MEAGL produced vasodepression and bradycardia mediated by mu-opioid receptors. 3. Atypical responses to MEAP were observed in a quarter of the animals studied showing tachycardia and pressor effects. This response was probably due to the release of the dipeptide Arg-Phe which exerted its effects at sympathetic ganglia. 4. Studies with the peptidase inhibitors captopril and bestatin showed a differential potentiation of the cardiovascular effects of the proenkephalin products by inhibition of angiotensin converting enzyme and aminopeptidase. 5. The effects of vagotomy, pithing and studies with atropine, and N-methyl levallorphan were used to demonstrate that, for all four proenkephalin peptides, cardiovascular effects were mediated by peripheral opioid receptors and transmission to the CNS via vagal afferents.     

Effect of [D-Ala2,Met5]enkephalinamide and [D-Ala2,D-Leu5]enkephalin on cholinergic and noradrenergic neurotransmission in isolated atria

In rabbit isolated atria, [D-Ala2,Met5]enkephalinamide and [D-Ala2,D-Leu5]enkephalin (0.1-3 microM) inhibited responses to cholinergic nerve stimulation in a concentration-dependent manner without affecting responses to exogenous acetylcholine. The inhibitory effect was blocked by the opiate receptor antagonist naloxone (1 microM). In rabbit atria in which the transmitter acetylcholine stores had been radioactively labelled by preincubating the tissue in [3H]choline, tetrodotoxin (100 ng/ml) significantly (P less than 0.001) blocked the stimulation-induced (2 Hz for 3 min) release of radioactivity. Both [D-Ala2,Met5]enkephalinamide and [D-Ala2,D-Leu5]enkephalin (0.3 and 1 microM) significantly decreased stimulation-induced radioactivity release and their effects were blocked by naloxone (1 microM). In rat isolated atria, [D-Ala2,Met5]enkephalinamide and [D-Ala2,D-Leu5]enkephalin (0.3-3 microM) inhibited responses to cholinergic nerve stimulation without affecting responses to exogenous acetylcholine. The inhibitory effect was blocked by naloxone (1 microM). In guinea-pig isolated atria, responses to cholinergic nerve stimulation were unaffected by the enkephalin analogues. In rabbit, rat and guinea-pig isolated atria, responses to noradrenergic nerve stimulation and exogenous noradrenaline were unaffected by the enkephalin analogues.     

Effect of bestatin and thiorphan on [Met5]enkephalin-Arg6-Phe7-induced analgesia

We investigated the effect of bestatin, a specific inhibitor of aminopeptidase and thiorphan, a specific inhibitor of enkephalinase A, on the analgesic effect induced by the intracerebral injection of heptapeptide [Met5]enkephalin-Arg6-Phe7 (MEAP) in cannulated rats. In contrast with the results obtained when [Met5]enkephalin (ME) was used, bestatin clearly potentiated the analgesic effect of MEAP, but thiorphan was totally ineffective. These observations indicate that the predominant inactivating mechanism for MEAP is the action of an aminopeptidase whereas this enzyme seems to be little involved in the catabolism of ME. The existence of two different catabolic pathways for MEAP and ME suggests that MEAP may act not only as a precursor of ME but also as an independent neuromodulator.     

Effects of proenkephalin products on rat cardiac and vascular tissue in-vitro

The cardiovascular effects of the four proenkephalin products [Met]-enkephalin (ME), [Leu]-enkephalin (LE), [Met]-enkephalyl-arg6phe7 (MEAP) and [Met]-enkephalyl-arg6-gly7-leu8 (MEAGL) have been studied on the isolated spontaneously beating rat atria and the perfused rat mesentery in-vitro. All four peptides (at concentrations up to 10(-6)M and in the presence of peptidase inhibitors) had no direct effect on atrial rate or contractility and did not alter responses to noradrenaline or field stimulation. In addition, the peptides had no effect on the perfusion pressure of the mesentery and did not alter vasoconstrictor responses to noradrenaline. The results show that proenkephalin products are without direct or modulating effects on atrial muscle or mesenteric vasculature of the rat, and suggest that there is no endogenous opioid control in these tissues.     

The enhancing effects of peptidase inhibitors on the antinociceptive action of [Met5]enkephalin-Arg6-Phe7 in rats

Previous in vitro studies have shown that the degradation of [Met(5)]enkephalin-Arg(6)-Phe(7) during incubation with cerebral membrane preparations is largely prevented by a mixture of three peptidase inhibitors: amastatin, captopril, and phosphoramidon. The present in vivo study shows that the inhibitory effect of [Met(5)]enkephalin-Arg(6)-Phe(7) administered intra-third-ventricularly on the tail-flick response was increased more than 1000-fold by the intra-third-ventricular pretreatment with three peptidase inhibitors. The antinociceptive effect produced by the [Met(5)]enkephalin-Arg(6)-Phe(7) in rats pretreated with any combination of two peptidase inhibitors was significantly smaller than that in rats pretreated with three peptidase inhibitors, indicating that any residual single peptidase could inactivate significant amounts of the [Met(5)]enkephalin-Arg(6)-Phe(7). The present data, together with those obtained from previous studies, clearly show that amastatin-, captopril-, and phosphoramidon-sensitive enzymes play important roles in the inactivation of endogenous opioid peptides, such as [Met(5)]enkephalin, [Met(5)]enkephalin-Arg(6)-Phe(7), [Met(5)]enkephalin-Arg(6)-Gly(7)-Leu(8), and dynorphin A (1-8), administered intra-third-ventricularly to rats.     

Alterations in [Met]- and [Leu]enkephalin and neurotensin content in basal ganglia induced by the long-term administration of dopamine agonist and antagonist drugs to rats

Treatment of rats for 18 months with trifluoperazine increased [Met⁵]- and [Leu⁵]enkephalin and neurotensin content in striatum and nucleus accumbens. However, in substantia nigra the content of [Met⁵]enkephalin was decreased, [Leu⁵]enkephalin unchanged and that of neurotensin increased. Administration of L-DOPA plus carbidopa, bromocriptine or pergolide for 12 months decreased [Met⁵]enkephalin (except bromocriptine) and neurotensin, but not [Leu⁵]enkephalin, levels in striatum. L-DOPA decreased and bromocriptine increased neurotensin levels in substantia nigra. But neurotensin levels in nucleus accumbens were unaffected.     

Effect of peptidase inhibitors on [Met5]enkephalin-Arg6-Phe7 and [Met5]enkephalin-Arg6-Gly7-Leu8-induced antinociception

The effect of the angiotensin converting enzyme inhibitor 2-[N-[(S)-1-carboxy-3-phenylpropyl]-L-alanyl]-(1S,3S,5S)-2- -azabicyclo(3.3.0)octane-3-carboxylic acid (Hoe 498 diacid) and the aminopeptidase inhibitor bestatin on antinociception induced in rats by intraventricular injections of [Met5]enkephalin-Arg6-Phe7 and [Met5]enkephalin-Arg6-Gly7-Leu8 was investigated. Potentiation and prolongation of the antinociception was observed after pretreatment with bestatin. Further potentiation of the antinociception elicited by the heptapeptide was obtained in rats pretreated with a combination of bestatin and Hoe 498 diacid. In contrast, antinociception elicited by the octapeptide was not potentiated further by pretreatment with the bestatin and Hoe 498 diacid combination.     

Conversion of [met5] -enkephalin-Arg6-Phe7 to [met5]-enkephalin by dipeptidyl carboxypeptidase

[Met⁵]-Enkephalin-Arg⁶-Phe⁷ can be hydrolyzed by a microsomal preparation of bovine striatum, which contains dipeptidyl carboxypeptidase, to release [met⁵]enkephalin. This reaction was inhibited by 10^?6 M SQ 20881 (PGlu-Gly-Leu-Pro-Pro-Gly-Pro-Pro-Ileu Pro-Prol) which has little or no effect in inhibiting the production of Tyr-Gly-Gly from [met⁵]-enkephalin. The conversion of [met⁵]-enkephalin-Arg-Phe⁷ to [met⁵]-enkephalin is not enhanced by 100 mM chloride ion.     

[Met5]enkephalin acts via delta-opioid receptors to inhibit pelvic nerve-evoked contractions of cat distal colon.

1 The effects of opioids on the sacral parasympathetic outflow to cat distal colon were studied in vitro using muscle strips orientated in the axis of the longitudinal muscle layer, with pelvic nerves attached. Electrical stimulation of the pelvic nerves evoked contractions that were blocked by atropine (1 X 10(-6) M) and tetrodotoxin (3 X 10(-7) M). 2 [D-Pen2, D-Pen5]enkephalin and [Met5]- and [Leu5]enkephalin caused concentration-dependent, reversible inhibition of pelvic nerve-evoked contractions, with IC50 values of 8.3 X 10(-10) M, 2.2 X 10(-9) and 2.1 X 10(-9) M respectively. 3 Morphine (1 X 10(-7)-1 X 10(-5) M) and [D-Ala2, MePhe4, Gly-ol5]enkephalin (1 X 10(-8)-1 X 10(-6) M) and U-50,488H (1 X 10(-8)-10(-6) M) were much less potent as inhibitors than [Met5]- or [Leu5]enkephalin. 4 Naloxone (1 X 10(-7) M), an antagonist at each of the three opioid receptor types, antagonized the effects of both [Met5]enkephalin and morphine. However, ICI 174,864, a specific delta-opioid receptor antagonist, antagonised the effects of [Met5]enkephalin only. 5 The inhibitory actions of [Met5]enkephalin were inversely related to frequency of pelvic nerve stimulation. Also, [Met5]enkephalin at a concentration (3 X 10(-9) M) which produced a large inhibition of neurogenic contractions, had no effect on contractions to exogenous acetylcholine. These results suggest a prejunctional site for inhibitory opioid receptors. 6 In summary, prejunctional inhibitory delta-opioid receptors are present on the sacral parasympathetic outflow to cat distal colon; kappa- and/or mu-opioid receptors may also be present, but appear to be of lesser importance.     

Effects of [Met5]enkephalin on regional blood flow and vascular resistance in rabbits

Methionine enkephalin [( Met5]enkephalin) has different cardiovascular effects, depending on species and routes of peptide administration. In anesthetized animals [Met5]enkephalin decreases blood pressure and heart rate. The site(s) and the mechanism of the hypotensive effects of the peptide are not known. The main purpose of this study was to test the hypothesis that [Met5]enkephalin dilates specifically a certain vascular bed which may account for the hypotensive effect of the peptide. Anesthetized male rabbits were instrumented for the measurement of blood pressure, heart rate, electrocardiogram and renal nerve activity. Four different microspheres (15 microns) were infused into the left ventricle in 10-15 s in either saline or with [Met5]enkephalin (1 mg/kg). Upon completion of the last microsphere injection the animals were killed and 35 tissues samples were taken for the determination of blood flow. Blood flows to many organs such as brain, glandular and cardiac tissues were not altered significantly by [Met5]enkephalin. However, [Met5]enkephalin increased blood flow to skeletal muscular bed. The increase in muscle blood flow was antagonized by naloxone, a specific opioid antagonist. These results suggest that the hypotensive action of [Met5]enkephalin in anesthetized rabbit is in part due to its vasodilatory effect in skeletal muscle.     

Effects of opioid drugs on capsaicin-sensitive neurones in guinea-pig atria

Transmural nerve stimulation of isolated guinea-pig atria in the presence of atropine induced a biphasic positive inotropic effect but only a slow increase in contractility (NANC response) in atria obtained from 6-hydroxydopamine-pretreated animals. The latter effect disappeared after exposure of the preparations to capsaicin. The effects of some opioid peptides were investigated on NANC responses. [D-Ala²,D-Leu⁵]enkephalin (DADLE) and [D-Ala²,N-Me-Phe⁴ Gly⁵-ol]enkephalin (DAGO, 0.1–10 μM) inhibited the cardiac response to transmural nerve stimulation in a dose-dependent and naloxone-sensitive manner. Dynorphin-(1–13) and morphine, at 10-fold higher concentrations (1–10 μM), reduced the response in a naloxone-sensitive manner. Naloxone alone however did not affect the response. Opioid peptides were not able to reduce the positive inotropic effect induced by calcitonin gene-related peptide (CGRP), or the increase in cardiac contractility produced by capsaicin. These results suggest that opioid receptors exert a modulatory role on peripheral terminals of capsaicin-sensitive sensory nerves.     

The effect of enkephalins and of beta-endorphin on the hypertensive response to physostigmine in the rat.

1 Intracarotid injection of [Leu]enkephalin and [Met]enkephalin produced a dose-dependent biphasic change in blood pressure of the rat consisting of an initial shortlasting fall followed by a longlasting increase of blood pressure. Naloxone consistently depressed or abolished the effects of enkephalins on blood pressure. 2 Intracarotid injection of beta-endorphin only occasionally produced a hypotension, or did not produce any change in the blood pressure of the rat. 3 All three opioids ([Leu]enkephalin, [Met]enkephalin and beta-endorphin) significantly depressed or abolished the hypertensive response to intravenous injection of physostigmine. This depressive action of opioids was easily reversed by naloxone. 5 It is concluded that opioids depress the central cholinergic link implicated in the hypertensive response to physostigmine most probably by inhibiting acetylcholine and/or noradrenaline release in the structures relevant for the action of physostigmine on blood pressure of the rat. This interaction is realized through the activation of opioid receptor(s).     

Characterization of opioid receptors modulating the function of capsaicin-sensitive neurons in guinea-pig atria

Transmural nerve stimulation of isolated atria, obtained from reserpine-pretreated guinea-pigs, in the presence of atropine and the β₁-adrenoceptor-blocking drug CGP 20712A, induced a positive inotropic effect. [D-Ala², N-Me-Phe⁴, Gly⁵-ol] enkephalin (DAGO), [D-Ala², D-Leu⁵]enkephalin (DADLE), morphine and dynorphin dose dependently reduced the cardiac response to transmural nerve stimulation. The δ receptor selective agonist [D-Pen², D-Pen⁵]enkephalin (DPDPE), and the κ receptor agonist, U50488, were unable to modify the response. The inhibitory effect of all the active opioid agonists was antagonized by naloxone but not by the selective δ and κ opioid receptor antagonists, ICI 174.864 and MR 2266. These results suggest the presence on sensory nerve terminals of inhibitory opioid receptors belonging to the μ, but not to the δ and κ subtypes.     

Studies on the effect of SCH-34826 and thiorphan on [Met5]enkephalin levels and release in rat spinal cord

SCH-34826 and thiorphan are inhibitors of the neutral endopeptidase (NEP; E.C. 3.4.24.11;) that cleaves the opiate peptides [Met5]- and [Leu5]enkephalin at the glycinylphenylalanine bond. These compounds were evaluated for their ability to affect the levels of [Met5]enkephalin-like immunoreactivity (MELI) in the brain and the spinal cord and the release into the extracellular space under resting and K+-evoked conditions. The results showed that oral administration of SCH-34826 (30-100 mg/kg p.o.) or thiorphan (10-30 mg/kg p.o.) had no effect on tissue levels of MELI. In contrast, both agents caused a dose-dependent increase in both the resting and the K+-evoked levels in spinal perfusates, which reached up to 10 times the control values. These data indicate that tissue (presumably intracellular) stores of [Met5]enkephalin are not affected by NEP inhibition and that it is the extracellular effects of the peptide that are potentiated by enzyme blockade. This agrees with the prior results demonstrating that NEP inhibitors require a nociceptive stimulus sufficient to release endogenous stores of [Met5]enkephalins for their actions to be observed.     

INHIBITION OF SYMPATHETIC NEUROTRANSMISSION BY THE OPIOID δ-RECEPTOR AGONIST DAMA IN THE PITHED RAT

1. The effects of [D-Ala2,Met5]enkephalinamide (DAMA), an analogue of [Met5]-enkephalin that acts selectively on opioid receptors of the delta-subtype, were studied on pressor responses elicited by sympathetic stimulation in pithed rats. 2. Intravenous injections of bolus doses of 0.1 mg/kg and 0.3 mg/kg of DAMA did not affect either the basal blood pressure or pressor responses to noradrenaline. 3. Pressor responses elicited either by electrical stimulation of the spinal sympathetic outflow or by stimulation of sympathetic ganglion cells with the muscarinic agonist McN-A-343 were reduced by DAMA. 4. Naloxone (1 mg/kg + 0.5 mg/kg per h) had no significant effect on the basal blood pressure or on pressor responses to spinal sympathetic stimulation, but antagonised the inhibitory effect of DAMA. 5. These results indicate that activation of opioid delta-receptors on sympathetic vasomotor nerve terminals can inhibit noradrenergic neurotransmission.     

Acute effects of D-1 and D-2 dopamine receptor agonist and antagonist drugs on basal ganglia [Met5]- and [Leu5]-enkephalin and neurotensin content in the rat.

The effects of acute systemic injection of the D-1 agonist SKF 38393 (2.5-20 mg/kg) or the D-1 antagonist SCH 23390 (0.25-2.0 mg/kg), and of the D-2 agonist quinpirole (0.12-1.0 mg/kg) or the D-2 antagonist sulpiride (25-100 mg/kg) on the neuropeptide content of rat basal ganglia were investigated. In striatum, the [Met5]- and [Leu5]-enkephalin content was unaffected by administration of SKF 38393 or SCH 23390. Quinpirole had no effect on [Met5]- and [Leu5]-enkephalin levels but sulpiride produced an increase in both [Met5]- and [Leu5]-enkephalin content. In the nucleus accumbens, SKF 38393 decreased and SCH 23390 increased [Met5]- and [Leu5]enkephalin levels. Quinpirole decreased [Met5]- and [Leu5]-enkephalin levels, while sulpiride decreased [Leu5]-enkephalin levels alone. The content of [Leu5]- but not [Met5]-enkephalin levels in the substantia nigra was increased by administration of SKF 38393, and decreased by SCH 23390. Quinpirole and sulpiride were without effect on the [Met5]- or [Leu5]-enkephalin content of substantia nigra. Neurotensin levels in striatum were increased by administration of SKF 38393 and decreased by SCH 23390. Similarly, quinpirole decreased the neurotensin content while sulpiride caused an increase. In the nucleus accumbens, the neurotensin content was not affected by administration of SKF 38393 but increased by SCH 23390. Neither quinpirole nor sulpiride altered neurotensin levels in the nucleus accumbens. Neurotensin levels in substantia nigra were unaffected by the administration of SKF 38393 and SCH 23390, or by quinpirole and sulpiride. These results indicate that acute administration of D-1 and D-2 agonist and antagonist drugs can alter the levels of [Met5]- and [Leu5]-enkephalin and neurotensin in basal ganglia. However, there are marked differences between brain regions in the regulation of peptide levels by acute D-1 and D-2 receptor occupation.     

Inhibitory effect of taurine on wet-dog shakes produced by [D-Ala2,Met5] enkephalinamide with reference to effects on hippocampal epileptic discharges

The effects of taurine on wet-dog shakes produced by [D-Ala2,Met5]enkephalinamide (DAME) were investigated in rats. Wet-dog shakes and epileptic discharges in the hippocampus were produced by intraventricular administration of 50 micrograms of DAME. Pretreatment with 10 microliter of taurine, given intraventricularly in a dose of 0.95 mumol, inhibited wet-dog shakes and epileptic discharges in the hippocampus. While the same dose of gamma-aminobutyric acid (GABA) also inhibited the wet-dog shakes, the same dose of L-leucine did not suppress them. These observations indicate that the inhibition of DAME-induced wet-dog shakes by taurine is associated with the suppression of seizure activities in the hippocampus. The possibility that taurine possesses an antagonistic action on opioid peptides is discussed.     

Analgesic activity and release of [MET5]enkephalin-Arg6-Gly7-Leu8 from rat spinal cord in vivo

Rat spinal cord contains the opioid peptide including [Met5]enkephalin-Arg6-Gly7-Leu8 (YGGFMRGL) and a higher molecular weight (HMW) immunoreactive peptide which is an N-terminal extended molecular form of YGGFMRGL. Since a high proportion of tissue immunoreactivity resides in the HMW component we have determined whether this form is released during perfusion of the spinal cord subarachnoid space in vivo while (1) electrically stimulating the sciatic nerves bilaterally or (2) superfusing with substance P. We have found that YGGFMRGL and the HMW immunoreactivity are released by both types of stimuli. The HMW material appeared to be the more stable of the two species of immunoreactivity; its presence in the superfusate was more consistently observed than that of YGGFMRGL itself. Injection of YGGFMRGL into the spinal subarachnoid space in chronically catheterized rats produced a suppression of the tail-flick response. This effect of YGGFMRGL was reversed by naloxone suggesting an action mediated by spinal opiate receptors. These data suggest that YGGFMRGL plays an integral role in the neurotransmission process between spinal neurons storing enkephalin and other neurons. The possibility that enkephalin-mediated neurotransmission includes multiple chemical signals can be entertained.     

Synthesis of 2-(4-substitutedbenzyl-[1,4]diazepan-1-yl)-n-(1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinolin-7-yl)acetamides as inotropic agents

In an attempt to search for more potent positive inotropic agents, a series of N-(4,5-dihydro-1-methyl-[1,2,4]triazolo[4,3-a]quinolin-7-yl)-2-(substitutedbenzyl-[1,4]diazepan-1-yl)acetamides were synthesized and evaluated for positive inotropic activity by measuring left atrium stroke volume in isolated rabbit heart preparations. Some of these derivatives exhibited favorable activity compared with the standard drug, milrinone, among which 2-(4-(4-methylbenzyl)-[1,4]-diazepan-1-yl)-N-(4,5-dihydro-1-methyl-[1,2,4]triazolo[4,3-a]quinolin-7-yl)acetamide (6m) was the most potent, increasing stroke volume by 8.38±0.16% (milrinone 2.45± 0.06%) at 3 x 10(-5) m. The chronotropic effects of those compounds having inotropic effects were also evaluated in this work.     

Preparation and opioid activities of N-methylated analogs of [D-Ala2, Leu5]enkephalin

Analogs of opioid pentapeptide [D-Ala², Leu⁵]enkephalin were prepared using two kinds of N-methylation reactions, namely quaternization and amide-methylation. Quaternization reaction with CH³I-KHCO³ in methanol was applied to the deprotected N-terminal group of the pentapeptide derivatives affording trimethylammonium group-containing analogs. [Me₃⁺ Tyr¹,D-Ala², Leu⁵]enkephalin and its amide were found to show opioid activity on guinea pig ileium assay only slightly lower than the parent unmethylated peptides. Application of amide-methylation reaction using CH₃I-Ag₂O in DMF to the protected pentapeptide yielded a pentamethyl derivative in which all of the five N atoms were methylated. Deprotection of the derivative gave pentamethyl analogs of [D-Ala², Leu⁵]enkephalin, which showed no significant activity on the guinea pig ileum assay and opiate-receptor binding assay.