Biphasic effects of a potent enkephalin analogue (d-Met2,Pro5)-enkephalinamide and morphine on locomotor activity in mice

The effects of morphine and a potent enkephalin analogue on spontaneous locomotion have been compared in mice. In doses of 3–10 mg/kg SC both compounds induced a brief reduction of motor activity followed by a period of behavioral hyperactivity. Similar receptorial mechanisms are suggested in mediation of their motor effects.     

Morphine-like discriminative stimulus effects of opioid peptides: possible modulatory role ofd-Ala2-d-Leu5-enkephalin (DADL) and dynorphin A(1-13)

The role of the different opioid receptors was studied in rats trained to discriminate SC injections of 3.0 mg/kg morphine from saline by tests for generalization to graded doses of morphine and receptor-selective peptides administered into the lateral cerebral ventricle. Dose-dependent morphine-like stimulus effects were produced over a wide range of doses (0.001–30 g), depending upon ligand and animal, by morphine, by themu-selective peptides DAGO[d-Ala²-NMePhe⁴-Gly(ol)-enkephalin] and FK33824[d-Ala²,NMePhe⁴-Met(O)⁵-(ol)-enkephalin], and by thedelta-selective peptide, DADL[d-Ala²,d-Leu⁵enkephalin]. The order of relative potency of these substances was: FK33824>DAGO>morphine>DADL. In contrast, DPLPE[d-Pen²,l-Pen⁵)enkephalin], which has much greaterdelta receptor selectivity than does DADL, and dynorphin A(1-13) (0.1–10 g), akappa-receptor agonist, engendered choice responding appropriate for saline. When 1.0 g DADL, a dose lacking morphine-like discriminative effects, was administered concurrently with SC morphine, the stimulus effects of morphine were potentiated. Concurrent administration of 10 g dynorphin A(1-13) and morphine attenuated the stimulus effects of morphine inconsistently. These results support previous findings thatmu-opioid receptors are of primary importance in mediating the morphine-like discriminative effects of opioid peptides. They also suggest that morphine-like discriminative effects can be modulated by other types of opioid receptors.     

Peripheral effects of opioid drugs on capsaicin-sensitive neurones of the guinea-pig bronchus and rabbit ear

Summary The effect of a potent opioid agonist, [d-Met², Pro⁵]-enkephalinamide was investigated on two responses involving capsaicin-sensitive afferent neurones, namely, atropine-resistant contractions of the guinea-pig bronchus evoked by electrical field stimulation and the nociceptor stimulation to intraarterial injections of acetylcholine or capsaicin into the vascularly isolated rabbit ear. The hypotheses to be tested were whether (a) opioid receptor activation may inhibit mediator release from primary afferent neurones and (b) the opioid could exert an analgesic effect at a peripheral site of action. Non-cholinergic contractions of the guinea-pig isolated main bronchi due to electrical stimulation were concentration-dependently inhibited by [d-Met², Pro⁵]-enkephalinamide (10 nM–1 M). This effect was abolished by naloxone (1 M). Naloxone alone induced no change in the stimulation-evoked contractions of the bronchus, indicating that no endogenous opioid control was present. Substance P and neurokinin A induced bronchial contractions that were not influenced by [d-Met², Pro⁵]-enkephalinamide. This indicates that [d-Met², Pro⁵]-enkephalinamide inhibits electrically-evoked bronchial contractions by reduced mediator release from capsaicin-sensitive sensory nerve endings, since these contractions are most probably brought about by tachykinins, released from afferent neurones. Capsaicin-induced bronchial contractions were in contrast to electrical stimulation not influenced by [d-Met², Pro⁵]-enkephalinamide which suggests a different site of action. The activation of sensory neurones in the rabbit ear by i. a. injection of acetylcholine and capsaicin was not reduced under infusion of [d-Met², Pro⁵]-enkephalinamide (1 and 10 M) or lofentanil (1 and 10 M). The enhancement of the effect of acetylcholine by infusion of prostaglandin E₂ (0.15 M) also remained unchanged under infusion of 10 M [d-Met², Pro⁵]-enkephalinamide. A peripheral analgesic action of the two opioid agonists studied is therefore not indicated. Send offprint requests to F. Lembeck     

Selective potentiations in opioid analgesia following scopolamine pretreatment

The effects of the muscarinic receptor antagonist scopolamine upon analgesia induced by d-ala-d-leu-enkephalin (DADL), beta-endorphin (BEND) and morphine were examined. While scopolamine (10 mg/kg, IP) significantly potentiated the analgesic responses following DADL (40 g, ICV) and morphine (5 mg/kg, SC) on the jump test, it failed to alter significantly BEND (1 g, ICV) analgesia.     

Comparison of tolerance development and dependence capacities of morphine, β-endorphin,and [D-MET2, PRO5]-enkephalinamide

The tolerance-development capacities of -endorphin, [D-Met², Pro⁵]-enkephalinamide, and morphine were compared in rats, and the dependence capacity of morphine was compared with that of the enkephalin analogue in mice. Tolerance to the analgesic effect, as measured by the tail-flick test, developed somewhat more rapidly in the [D-Met², Pro⁵]-enkephalinamide-treated group than in the others. A similar relationship was found for the dependence capacity. Considering that the enkephalin analogue displayed the strongest analgesic activity, the wellknown correlation between antinociceptive and tolerance development/dependence capacities of opiates seems to be valid for opioid peptides as well.     

Behavioral activities of opioid peptides and morphine sulfate in golden hamsters and rats

The behavioral effects of -endorphin, [D-Ala², D-Leu⁵]-enkephalin and morphine were investigated in golden hamsters and in rats. In golden hamsters, -endorphin and [D-Ala², D-Leu⁵]-enkephalin induced loss of righting reflex, whereas morphine caused no such effect. Both opiate peptides and morphine caused the inhibition of tail-flick response and catalepsy in rats. -Endorphin was the most potent, followed by [D-Ala², D-Leu⁵]-enkephalin and then by morphine. The catalepsy induced in rats by [D-Ala², D-Leu⁵]-enkephalin was different from that of -endorphin and morphine in that it produced catalepsy without muscular rigidity. -Endorphin and [D-Ala², D-Leu⁵]-enkephalin caused hypothermia in golden hamsters; morphine was less active in altering the body temperature. -Endorphin caused hypothermia at high doses and hyperthermia at low doses in rats. These heterogenous behavioral responses indicate that multiple types of receptors mediate the effects of opiates in the central nervous system.     

Effects of D-Met2, Pro5-enkephalinamide on pain tolerance and some cognitive functions in man

The effects ofd-Met², Pro⁵-enkephalinamide (EA) on pain tolerance and some cognitive functions have been examined in healthy male volunteers. Dihydrocodeine (DC) was used as reference substance. Applying the submaximum effort tourniquet technique EA (10 mg SC) was found to elevate the pain threshold similarly to DC (20 mg SC). Neither DC nor EA impaired the performance in the symbol cancellation test, which quantitates the intensity of attention. In this assay rather a slight improvement was detected. In addition the short-term memory performance (Wechsler test) was also improved by EA and DC. No alteration was seen in the word fluency test, an indicator of long-term (semantic) memory. The data show that EA not only improves pain tolerance but some of its mental effects are similar to those of a classical morphine congener DC.     

Attenuation of some signs of opioid withdrawal by inhibitors of nitric oxide synthase

Effects of nitric oxide synthase (NOS) inhibitors (l-N ^G-nitroarginine,l-N ^G-nitroarginine methyl ester) on precipitated opioid withdrawal were studied in morphine-dependent rats given naloxone, in order to assess the involvement of nitric oxide (NO) in opioid dependence.l-N ^G-Nitroarginine (7.5 mg/kg, IP, 1 h before naloxone or b.i.d. on days 4–7 of an 8-day morphine treatment) reduced wet dog shakes and weight loss; when given by osmotic pumps (15 mg/kg per day), the drug reduced wet dog shakes but not weight loss.l-N ^G-Nitroarginine methyl ester (60 mg/kg, 1 h before naloxone) also reduced wet dog shakes and weight loss. The results indicate that NOS inhibitors warrant further study as potential treatment of the opioid withdrawal syndrome.Abstracts were presented at meetings of the Society for Neuroscience in New Orleans, La., November 10–15, 1991 and of the American Society for Pharmacology and Experimental Therapeutics in Orlando, Fla., August 10–18, 1992     

Effects of methysergide,bromocriptine and naloxone on prolactin,growth hormone and TSH release induced by d-Met2,Pro5-enkephalinamide in man

d-Met²,Pro⁵-enkephalinamide (EA) 10 mg, given SC, induced a dramatic rise in serum prolactin (PRL) and growth hormone (GH) levels in healthy male volunteers. The TSH content was also moderately elevated. Naloxone 0.8 mg administered IV abolished these effects. Bromocriptine 2.5 mg given per os also antagonized EA-induced PRL and TSH release but potentiated the GH surge. Methysergide 2.0 mg administered orally partially reversed EA-elicited PRL release, further augmented GH liberation and did not modify TSH output. The data indicate that inhibition of the dopaminergic tone and/or activation of certain serotonergic mechanisms play an important role in the EA-induced release of PRL and TSH. However, primarily other neurotransmitters might mediate the GH liberation elicited by this opioid peptide. Offprint requests to: J. Földes     

Opiate receptors in the rabbit iris

Summary Evidence pointing to the presence of opiate receptors in the rabbit iris was obtained in an in vivo study of the effects on the pupil of the intraocular injection of morphine (10–100 g) and d-Ala²-met-enkephalinamide (d-Ala-E) (5–50 g). Both opiates induced a significant dose-dependent decrease in pupillary size and an appreciable fluctuation of pupillary size. I.v. administration of naloxone (0.5 mg/kg) before or after injection of opiates prevented the miotic effect and the fluctuation of pupil size. The parallelism of the dose-response curves of opiates in the presence and absence of naloxone indicated competitive reversible antagonism.     

Withdrawal responses of guinea-pig isolated ileum following brief exposure to opiates and opioid peptides

Summary Withdrawal contractures following brief exposure of guinea-pig ileum to enkephalin analogues, dynorphin A-(1-13) and beta-endorphin and the opiate drugs morphine, ketocyclazocine, buprenorphine and MR2034 were investigated. Following 2 min contact with the ileum a withdrawal contracture was induced by washout of (Met⁵)- and (Leu⁵)-enkephalin and by several enkephalin analogues but not by washout of (D-Ala²,D-Leu⁵)-enkephalinamide or any of the other drugs tested. Addition of naloxone precipitated withdrawal to all opioids tested except the kappa receptor preferential drugs ketocyclazocine and MR2034 and the mu receptor partial agonist, buprenorphine.The heights of the withdrawal contractures to enkephalin analogues were found to be dependent on the concentration of agonist and of naloxone, and on the duration of the contact period of opioid with the ileum.The naloxone-precipitated withdrawal responses to morphine, dynorphin A-(1-13) and the washout withdrawal response to (Met⁵)-enkephalin were inhibited by substance P antagonists thus supporting the previous pro posal that substance P mediates the opiate withdrawal response.This study has shown that mu receptor agonists produced dependence in the guinea-pig ileum revealed by a withdrawal contracture on addition of naloxone, whereas dependence on kappa receptor agonists could not be revealed with naloxone. Since the guinea-pig ileum preparation has m and kappa receptors it was concluded that the endogenous opioid peptides, enkephalins, beta-endorphin and dynorphin A-(1-13), all induced dependence by acting on mu receptors in this preparation.     

Morphine,d-Pen2,d-Pen5 enkephalin and U50,488H differentially affect the locomotor activity and behaviours induced by quinpirole in guinea-pigs

The effects of morphined-Pen²,d-Pen⁵ enkephalin (DPDPE) and U50,488H on the behavioural syndrome elicited by the dopamine (DA) D-2 agonist quinpirole, were investigated. Morphine (1, 5 and 15 mg/kg SC) and morphine administered intracerebroventricularly (ICV) (2×5 µl, 10^–3 M; total dose = 10 nmol) produced piloerection and sedation. DPDPE-ICV (2×5 µl and 2×10 µl, 10–³ M; total doses = 10 and 20 nmol) produced piloerection and sedation similar to morphine. U50,488H (1 mg/kg SC) induced locomotor activity and some stereotyped behaviour, whereas U50,488H (5 and 10 mg/kg SC) induced muscle rigidity and dystonic-like movements. The locomotor and behavioural response elicited by quinpirole (3 mg/kg IP) was attenuated in guinea-pigs pretreated with morphine (1, 5 and 15 mg/kg SC), morphine-ICV (2 × 5 µl, 10–³ M), and DPDPE-ICV (2 × 5 µl and 2 × 10 µl, 10^–3 M). These effects were reversed by naloxone (15 mg/kg SC). U50,488H (1 mg/kg SC) increased the quinpirole-induced locomotor activity, whereas U50,488H (5 and 10 mg/kg SC) decreased the locomotor activity and stereotyped behaviours produced by quinpirole. These results indicate that the gross behavioural effects of mu, delta and kappa opioids differ in guinea-pigs compared to other rodent species, and suggest differential involvement of these opioid receptor subtypes with DA D-2 receptor-mediated activity.     

Effect of monoamine precursors on the forced-swimming test in mice

The antidepressant properties of monoamine precursors were evaluated by the forced-swimming test for mice developed by Porsolt et al. DOPA but not 5-hydroxy-tryptophan (5HTP) shortened immobility at doses that did not increase locomotor activity. Although l-threo-dihydroxyphenylserine (DOPS), an artificial norepinephrine (NE) precursor, did not change immobility in intact mice, DOPS significantly reduced immobility in mice pretreated with the selective NE neurotoxin DSP4. These results suggest possible antidepressant properties of DOPA and DOPS, the latter of which may act as an antidepressant in a certain NE-depleting condition.     

Kinetics of l-tryptophan in depressive patients: A possible correlation between the plasma concentrations of l-tryptophan and some psychiatric rating scales

The plasma concentration and flux of l-tryptophan are abnormal in primary depressive patients, according to the literature. The plasma concentrations of l-tryptophan over a 6-h period after ingestion of 5 g l-tryptophan were investigated and did not differ significantly between depressive patients and controls during the absorption, distribution, and elimination phases. There was no correlation between the plasma concentrations with anxiety or depression scores, or with the excretion in urine of 17-hydroxycorticosteroids and xanthurenic acid during the 24 h after l-tryptophan. Treatment with either 125 mg pyridoxine (three times daily with meals) and l-tryptophan (3 g at 10 PM) or with maprotiline (100 mg at 10 PM) had no influence on the plasma concentrations of l-tryptophan after 2 or 4 weeks of treatment. This excludes l-tryptophan deficiency as a pathogenic factor of depression in the patients studied. No kinetic differences could be demonstrated in the depressive patients, making differences in body compartments or flux of l-tryptophan unlikely to be of pathogenic importance to depression.     

Comparison of 7-nitroindazole with other nitric oxide synthase inhibitors as attenuators of opioid withdrawal

Previously, we demonstrated that two nonselective inhibitors of nitric oxide synthase (NOS),l-N ^G-nitroarginine (l-NNA) andl-N ^G-nitroarginine methyl ester (l-NAME), reduced some signs of morphine withdrawal in rats. The present work extended these studies to include 7-nitroindazole (7-NI), an inhibitor specific for cerebral NOS, andN(5)-(1-iminoethyl)-l-ornithine (l-NIO), a potent inhibitor of endothelial NOS. Behavioral effects of these four NOS inhibitors and clonidine, an ₂-adrenoceptor, agonist, on morphine withdrawal in rats were assessed. Rats received one 75-mg morphine pellet subcutaneously (SC). Three days later, NOS inhibitors were administered IP 1 h before withdrawal was precipitated with naloxone (0.5 mg/kg, SC) and scored. 7-NI,l-NIO,l-NAME andl-NNA produced dose-related decreases in weight loss, diarrhea, wet dog shakes and grooming. 7-NI also reduced mastication, salivation and genital effects. Clonidine produced effects similar to 7-NI. In awake, morphine-naive and morphine-dependent rats not subjected to withdrawal, 7-NI was the only NOS inhibitor that did not increase blood pressure. Because 7-NI attenuated more signs of opioid withdrawal thanl-NNA,l-NAME orl-NIO without causing hypertension, 7-NI appears to warrant further testing as a potential candidate for human use.Abstracts were presented at the annual meetings of the College on Problems of Drug Dependence, West Palm Beach, Fla., 18–23, June 1994; International Narcotics Research Conference, North Falmouth, Cape Cod, Mass., 16–21, July 1994; and a Satellite Symposium to IUPHAR, Montreal, Canada, 22–24, July 1994.     

Protriptyline induced inhibition of the in vivo 3H-noradrenaline synthesis from 3H-l-Dopa in the rat brain

Summary ³H-l-Dopa was given intraperitoneally, after a peripheral decarboxylase inhibitor Ro 4-4602 (50 mg/kg), to male Wistar rats and the effect of protriptyline pretreatment (10 mg/kg, 30 min) on the formation and metabolism of the brain ³H-catecholamines, dopamine and noradrenaline and all their metabolites was investigated.Protriptyline produced a strong decrease of labelled noradrenaline and its metabolites normetanephrine, free and conjugated 3-methoxy-4-hydroxyphenyl-eneglycol and 3,4-dihydroxyphenyleneglycol 60 and 120 min after ³H-l-Dopa. ³H-noradrenaline was also decreased 30 and 45 min after ³H-l-Dopa. In rats and mice the pretreatment with protriptyline (10 mg/kg, 30 min) induced also a significant decrease in brain ³H-noradrenaline but not ³H-dopamine synthesized from ³H-l-tyrosine. Protriptyline (10 mg/kg) produced no effect on endogenous dopamine and noradrenaline in the rat or mouse brain.The present findings strongly indicate that the acute treatment with protriptyline inhibits the ³H-noradrenaline formation from ³H-l-Dopa. This effect seems most likely to be related to an interaction of protriptyline with the precursor(s) ³H-l-Dopa or ³H-dopamine at sites of ³H-noradrenaline biosynthesis.A preliminary report of the present study was presented at the Scandinavian Pharmacological Society, June 1972, Uppsala, Sweden and the VIII C.I.N.P. Congress, August 1972, Copenhagen, Denmark.     

Evidence that the large neutral amino acidl-valine decreases electrically-evoked release of 5-HT in rat hippocampus in vivo

l-Valine competes with tryptophan for transport into the brain and has previously been shown to decrease brain 5-HT synthesis. In the present study, the effect ofl-valine on electrically evoked hippocampal 5-HT release was determined in the anaesthetized rat using microdialysis. In control animals two electrical stimulations of the dorsal raphe nucleus 120 min apart (S1 and S2, respectively) released similar amounts of 5-HT. In contrast, in animals which receivedl-valine (200 mg/kg) between stimulations, S2 released a significantly smaller amount of 5-HT than did S1, although basal 5-HT release was unchanged. The data demonstrate thatl-valine decreases the electrically-evoked release of 5-HT in hippocampus in vivo.     

Possible involvement of l-arginine-nitric oxide pathway in modulating regional blood flow to brown adipose tissue of rats

To evaluate whether the l-arginine-nitric oxide (NO) pathway is involved in the regulation of regional blood flow to brown adipose tissue (BAT), the effects of two specific NO synthase inhibitors, N^G-nitro-l-arginine methyl ester (l-NAME) and N^G-monomethyl-l-arginine (l-NMMA), on the blood flow to interscapular brown adipose tissue (IBAT) were studied in urethane-anesthetized rats. Regional blood flow in MAT was measured with laser-Doppler flowmetry.An intravenous injection of l-NAME and l-NMMA, but not of either d-enantiomer, caused a transient and dose-dependent increase in IBAT blood flow. Dose-response curves for these NO synthase inhibitors showed that l-NAME was more potent than l-NMMA in increasing IBAT blood flow. We also observed a concomitant pressor effect accompanied by a slight decrease in heart rate following intravenous injection of l-NAME and l-NMMA. An elevation of IBAT blood flow and blood pressure induced by both l-NAME and l-NMMA was reversed by l-arginine in an enantiomerically specific manner. The increase in IBAT blood flow induced by NO synthase inhibitors was of shorter duration and less sensitive to l-arginine than the increase in blood pressure.Our results show that the WAY blood flow is increased by inhibition of NO synthase and that the response of IBAT vasculature to NO synthase inhibitors is different from that of the resistance vessels which regulate blood pressure. The involvement of l-arginine-NO pathways in modulating microcirculation in IBAT is suggested. Correspondence to: Y. Uchida at the above address     

Neurochemical and behavioural investigations of the NMDA receptor-associated glycine site in the rat striatum: Functional implications for treatment of parkinsonian symptoms

The glutamatergic cortico-striatal and subthalamo-entopeduncular pathways are both overactive in parkinsonism. Previous behavioural investigations have shown that intra-entopeduncular injection of either NMDA-site or glycine-site antagonists results in alleviation of parkinsonian symptoms, although injection of the former is associated with the appearance of anaesthetic-like side effects. These behavioural differences may be mediated by action on different NMDA receptor subtypes. Recent neurochemical and molecular pharmacological studies have indicated the existence of NMDA receptor subtypes which display differential modulation by glycine. In the present study, three potential modes of NMDA antagonism were differentiated in vitro by effects on [³H]-glycine binding to striatal sections. Specific [³H]-glycine binding was totally displaced by the glycine partial agonist (R)-HA-966; the NMDA-site antagonistd-CPP had no effect; and the NMDA-site antagonistd-AP5 displaced [³H]-glycine binding in a subpopulation of glycine sites. The anti-parkinsonian effects of (R)-HA-966,d-CPP andd-AP5 were assessed by intra-striatal injection in reserpine-treated rats and 6-OHDA-lesioned rats. Injection of (R)-HA-966 andd-CPP resulted in alleviation of parkinsonian akinesia, although the latter elicited anaesthetic-like side effects;d-AP5 was ineffective as an anti-parkinsonian agent. (R)-HA-966 was also effective as an anti-parkinsonian agent when administered systemically in the reserpine-treated rat. These data suggest that different classes of NMDA antagonist mediate different behavioural responses within the parkinsonian striatum. The behavioural response produced may depend on the exact nature of the conformational change induced by the antagonist and the location of the subtype most sensitive to that class of compound. Selection of a specific mode of NMDA receptor antagonism or targeting of striatal NMDA receptor subtypes may form the basis of a novel therapeutic approach to Parkinson's disease.     

Studies on some metabolic effects of dopa and dopamine in the rat

Summary In male rats some metabolic effects of l-dopa and dopamine were compared with those of noradrenaline, adrenaline and tyramine by measuring the changes of plasma free fatty acids (FFA), plasma glycerol and plasma glucose as well as those of blood lactate and blood pyruvate. After intravenous injection of dopamine lactate and pyruvate concentrations were elevated maximally already within 5 min and returned to control levels after 10–20 min, i.e. at a time, when the levels of FFA, glycerol and glucose were maximally elevated in plasma. l-dopa had about 1/8 to 1/6 the potency of dopamine in producing these metabolic effects. The effects of dopamine were similar to those obtained with 1/20 the dose of noradrenaline, while adrenaline produced a more pronounced hyperglycaemic response than dopamine did when given in lipolytically equieffective doses.Pretreatment of the animals with phentolamine completely prevented the hyperglycaemic response to dopamine or noradrenaline without clearcut effects on the lipolytic effect of these catecholamines. Also, pretreatment with dihydroergotamine antagonized the hyperglycaemic effect of adrenaline and prevented that of dopamine and noradrenaline, while the effect of catecholamines on plasma glycerol concentration was not affected. However, the elevation in plasma FFA level induced by catecholamines was clearly antagonized by dihydroergotamine. The -adrenolytic drug Kö 592 had no effect on the hyperglycaemic effect of dopamine or noradrenaline, but antagonized the lipolytic effect of these amines. Pargyline enhanced the elevation of FFA and glycerol induced by dopamine or noradrenaline but reduced their hyperglycaemic effect. Chemical sympathectomy induced by pretreatment with 6-hydroxydopamine prevented the hyperglycaemic and lipolytic effects of tyramine, antagonized those of dopamine and potentiated the lipolytic response to noradrenaline. The effect of syrosingopine on the metabolic responses to the catecholamines was similar to that of 6-hydroxydopamine.Since the metabolic effects of dopamine were clearly antagonized by various - and -receptor-blocking agents and by chemical sympathectomy, we conclude that dopamine exerts its metabolic effects through a stimulation of - and -adrenoceptors and that part of this effect is mediated by a tyramine-like action of dopamine.