Functional characterization of substance P receptors in the rabbit ear artery

Summary Rabbit isolated ear arteries were perfused at a constant flow and stimulated with field pulses (5 Hz, 5 impulses). Different tachykinins and capsaicin depressed stimulation-induced vasoconstriction, substance P (SP) being the most potent inhibitor. The rank order of potency of the tachykinins was, SP physalaemin eledoisin>SP-methylester; that of SP and its C-terminal fragments, SPSP-(2–11)SP-(4–11)>SP-(6–11). SP-(1–9) was inactive. The SP antagonist (Arg⁵,d-Trp^7,9,Nle¹¹)SP-(5–11) 10 mol/l shifted the concentration-response curve of SP to the right (pA₂=5.43), whereas it did not reduce the action of capsaicin. Another SP antagonist (d-Pro⁴,d-Trp^7,9,10)SP-(4–11) 10 mol/l failed to affect the SP depression. Neither antagonist changed vasoconstriction by itself. Pretreatment of the arteries with a mixture of yohimbine, propranol, atropine, diphenhydramine, burimamide, methysergide and indomethacin, all 1 mol/l, did not influence the effect of SP or capsaicin. Only the inhibition by SP, but not that by capsaicin was abolished after mechanical destruction of the endothelium. SP, physalaemin and eledoisin, all 3 mol/l, reduced vasoconstriction by noradrenaline or histamine; capsaicin 30 mol/l depressed noradrenaline-induced vasoconstriction. In arteries preincubated with³H-noradrenaline, electrical stimulation (1 Hz, 120 pulses) triggered an increase in the outflow of tritium and evoked vasoconstriction. SP 1 mol/l did not change either basal or stimulation-evoked tritium outflow, whereas it reduced vasoconstriction. In conclusion, SP depresses nerve stimulation-induced vasoconstriction solely by changing smooth muscle contractility; the receptor activated seems to belong to the SP-P type. Under the conditions of these experiments there is no indication for a similar effect of endogenous SP.     

Involvement of substance P in the excitatory action of GABAA agonists on cholinergic neurons in the guinea-pig ileum

Summary 1. The possible involvement of substance P (SP) in cholinergic contractions induced by GABA_A agonists in the guinea-pig ileum was further investigated. 2. Responses evoked by 3-aminopropane sulphonic acid (3-APS) or muscimol consisted of a rapid phasic contraction followed in 70% of preparations by a tonic contraction, usually smaller in amplitude but considerably longer in duration. Phasic and tonic components were sensitive to bicuculline, neurogenic (cholinergic) in nature and susceptible to desensitization. 3. Capsaicin (0.2 M) pretreatment and SP receptor desensitization caused by 3 different priming SP concentrations (10 nM, 30 nM, 100 nM), depressed both components of the 3-APS-induced response, the magnitude of antagonism being greater for tonic contractions. Similar findings were obtained by using 10 M (d-Pro⁴, d-Trp^7,9)SP-(4–11), even though the degree of antagonism caused by this SP antagonist was consistently lower. 4. These results indicate that depression of SP receptor function achieved by three different procedures decreases cholinergic contractile responses to GABA_A agonists in the guinea-pig ileum. This provides further support for the hypothesis that GABA_A receptor activation evokes both direct and indirect stimulation of enteric cholinergic neurons and that SP and/or a related peptide play an important role in mediating the indirect component of the cholinergic response. Send offprint requests to M. Tonini at the above address     

Role of substance P in the antidromic vasodilation,neurogenic plasma extravasation and disruption of the blood-aqueous barrier in the rabbit eye

Summary The involvement of substance P (SP) in the antidromic vasodilation, neurogenic plasma extravasation and disruption of the blood-aqueous barrier has been investigated in the rabbit eye. The distribution, inactivation and effects of exogenous SP in the anterior uvea after injection into the anterior chamber were studied and the results were compared with data obtained on endogenous SP. About 2% of intracamerally injected ¹²⁵I-tyr-8-SP was recovered from the ciliary processes. Out of this 1–3% was found to be biologically active. Thirteen to 24% of endogeneous SP in the anterior uvea was detected in the ciliary processes. Intracameral injection of 50–100 g SP in indomethacin pretreated rabbits had practically no effect on ocular blood flow, capillary permeability to Evans blue or the bloodaqueous barrier. Continuous infusion of 0.8–4.0 g/min SP for 30 min into the common carotid artery had no significant effect on capillary permeability or the blood-aqueous barrier. In contrast to these findings, nociception accomplished with topical application of 1% neutral formaldehyde for 4 min in indomethacin pretreated rabbits induced a marked increase in blood flow in the anterior uvea plasma extravasation, disruption of the blood-aqueous barrier and an increase in the intraocular pressure. These effects could not be blocked by the SP antagonist analog (d-Pro², d-Phe⁷, d-Trp⁹)-SP. The topical pulse treatment with formaldehyde, although inducing longlasting irritation, did not significantly decrease the amount of endogenous SP in the anterior uvea as studied 30–60 min after application of the irritant. It is concluded that in the rabbit anterior uvea, which is practically devoid of mast cells, it is questionable whether SP is causatively involved in the antidromic vasodilation, neurogenic plasma extravasation and disruption of the blood-aqueous barrier. The same may also be true for the nonhistaminergic part of the corresponding vascular responses in the skin.     

The sympathetic axons innervating the sinus node of the rabbit possess presynaptic opioid к- but not μ- or δ-receptors

Summary The postganglionic sympathic nerves of rabbit isolated hearts were stimulated with pulses delivered at 5 Hz and train durations of 1–5 s. Ethylketocyclazone 0.01–1 mol/l and fentanyl 1 and 10 mol/l but not morphine 1 and 10 mol/l, Met-enkephalin 1 and 4 mol/l or d-Ala², d-Leu⁵-enkephalin 0.5 and 5 mol/l diminished the stimulation-evoked increase in heart rate. The effect of ethylketocyclazocine 0.1 mol/l was antagonized by naloxone 1 and 10 mol/l. In contrast, the effect of fentanyl was not changed by naloxone 10 mol/l. Ethylketocyclazocine 0.03 and 1 mol/l did not reduce the tachycardia elicited by exogenous noradrenaline. The results suggest that, under in vitro conditions, only presynaptic opioid - but not - or -receptors inhibit the release of noradrenaline from the sympathetic neurones innervating the sinus node.     

Intranigral application of substance P antagonists prevents the haloperidol-induced activation of striatal tyrosine hydroxylase

Summary The effect of intranigral injections of stable substance P analogs with antagonist properties, namely [D-Pro², D-Trp^7,9]-substance P and [D-Pro², D-Phe⁷, D-Trp⁹]-substance P, on the activity of the nigrostriatal dopaminergic pathway was studied. Dopaminergic neural activity was evaluated by measuring changes in striatal tyrosine hydroxylase activity in response to systemic treatment with haloperidol. When injected into the substantia nigra of normal rats, neither of the two substance P analogs influenced the V _max of striatal tyrosine hydroxylase or the affinity of the enzyme for its pteridine cofactor. However, when applied into the substantia nigra 10 min before systemic haloperidol, 20 g of either substance P analog was able to prevent the haloperidol-induced activation of striatal tyrosine hydroxylase. The effect was not altered by concurrent microinjection of the GABA-antagonist bicuculline. These results suggests that nigral substance P plays an important role in the activation of nigrostriatal dopamine neurons produced by haloperidol. Thus, both substance P and GABA may reciprocally and independently regulate the activity of nigrostriatal dopamine neurons in response to changes in dopamine receptor activity.     

Opioid peptides decrease noradrenaline release and blood pressure in the rabbit at peripheral receptors

Summary Effects of dynorphin-(1–13), Leu⁵-enkephalin,d-Ala²,d-Leu⁵-enkephalin (DADLE), and for comparison bremazocine, on plasma noradrenaline concentration and mean arterial pressure (MAP) were studied in pithed rabbits. In the first series of experiments, the sympathetic outflow was stimulated electrically via the pithing rod at 2 Hz twice for 3 min each (S₁, S₂). Drugs were administered before S₂. Bremazocine 10 g/kg+2g/kg/h and 100 g/kg+20 g/kg/h, dynorphin 1 and 3 g/kg/min, Leu⁵-enkephalin 100 g/kg/min and DADLE 10 and 30 g/kg/min all diminished the electrically-evoked increase in plasma noradrenaline and MAP. The effects were antagonized by naloxone. In the second series, an infusion of noradrenaline (2 g/kg/min) was given twice for 3 min each (N₁, N₂). Drugs were administered before N₂. Bremazocine 100 g/kg+20 g/kg/h slightly enhanced the pressor effect of exogenous noradrenaline, whereas dynorphin 3 g/kg/min, Leu⁵-enkephalin 100 g/kg/min and DADLE 30 g/kg/min caused no significant change. In the third series, the sympathetic outflow was stimulated continuously at 2 Hz, and the interaction of dynorphin and DADLE was studied. Dynorphin 1 g/kg/min and DADLE 10g/kg/min initially decreased MAP to a similar extent. The effect of DADLE faded with time. When, during continuous infusion of DADLE 10 g/kg/min, and after return of MAP to the pre-DADLE level, dynorphin 1 g/kg/min or DADLE 10 g/kg/min was infused additionally, the effect of dynorphin was unchanged, whereas that of DADLE was almost abolished. We conclude that the opioid peptides as well as bremazocine decrease action potential-evoked release of noradrenaline and, secondarily, blood pressure. They act at peripheral sites, presumably prejunctional opioid receptors at postganglionic sympathetic axons. Dynorphin on the one hand, and Leu⁵-enkephalin and DADLE on the other hand, appear to act at different receptors, dynorphin probably at a - and DADLE and Leu⁵-enkephalin at a -receptor.     

Enhancement of δ- but not μ-opiate agonist binding by calcium

Summary Present evidence for distinction of 2 types of opiate receptor sites in rat brain homogenates originates from different relative affinities of morphine-like alkaloids and enkephalins to -or enkephalin and - or morphine-receptor sites. We now report that Ca²⁺ in a physiological dose range (0.5–3 mM) enhances the binding of ³H-enkephalin in hypotonically treated rat brain membranes, whereas specific binding of ³H-morphine-like alkaloids is not affected. Furthermore, the potency of [d-Ala², d-Leu⁵]-enkephalin to inhibit [³H]-diprenorphine and [³H]-ethylketazocine binding increased in the presence of Ca²⁺, whereas an increase in potency of [d-Ala², d-Leu⁵]-enkephalin to inhibit binding of -receptor ligands was not observed. Kinetic analysis revealed that Ca²⁺ decreased the rate of dissociation of [d-Ala², d-Leu⁵]-enkephalin without affecting the rate of association, thereby increasing the affinity. However, in saturation binding studies, performed in diencephalic membranes, in which [d-Ala², d-Leu⁵]-enkephalin binds predominantly to -receptors, Ca²⁺ also increased the binding affinity of [³H]-[d-Ala², d-Leu⁵]-enkephalin. Double reciprocal analysis suggested a mixed competitive-noncompetitive type of inhibition of [d-Ala², d-Leu⁵]-enkephalin binding by dihydromorphine. Thus, the interactions of - and -opiate ligands with -receptors may involve topographically different, but closely related binding sites, located on a single receptor molecule.Abbreviations DADL [d-Ala², d-Leu⁵]-enkephalin - DHM dihydromorphine - met-enkephalin methionine-enkephalin - leu-enkephalin leucine-enkephaline - FK 33-824 [d-Ala², MePhe⁴, Met(O)-ol]-enkephalin - EGTA ethyleneglycol-bis-(-aminoethylether) N, N'-tetraacetic acid - TRIS Tris (hydroxymethyl)-aminomethan     

The effect of opiates on arterial baroreceptor reflex function in the rabbit

Summary The contribution of exogenous and endogenous opioid peptides to the central modulation of the baroreceptor reflex was investigated in rabbits.Baroreceptor sensitivity was assessed in pentobarbitone anaesthetised animals by measuring heart period in response to rises in arterial pressure after bolus intravenous injections of phenylephrine and falls induced by intravenous sodium nitroprusside and controlled haemorrhage. The slope of the linear relationship between arterial pressure and heart period was used as an index of baroreflex sensitivity.Thirty minutes after the intracisternal administration of 50 g/kg RX783016 (a -opiate receptor agonist) baroreceptor sensitivity was reduced to all three methods of blood pressure manipulation. Ketazocine (50 g/kg) a -opiate agonist and [d-Ala ² , d-Leu ⁵ ] enkephalin (1 g/kg) a -opiate agonist, 15 min after intracisternal injection caused an increase in baroreflex sensitivity in response to a rise in pressure and a reduction in response to a fall.Intravenous injection of naloxone (80 g/kg) caused an increase in varoreflex gain. However, a higher dose (200 g/kg) was required to attenuate the effects of RX783016 and [d-Ala ² , d-Leu ⁵ ] enkephalin but not ketazocine. No change in baseline arterial pressure or heart rate occurred after the opiates or naloxone. It appears that exogenous and endogenous opiates modify baroreceptor reflex function, through a mechanism which involves central opiate receptors of the - and -types.     

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     

Photoelectric measurement of neurogenic vasoconstriction in jejunal branches of the rabbit mesenteric artery reveals the presence of presynaptic opioid δ-eceptors

Summary The perivascular nerves of rabbit mesenteric arteries were stimulated with 15 pulses at 2 Hz, and decreases in external diameter were measured by means of a photoelectric device. Both extra- and intraluminally added [Met⁵]-enkephalin 1 mol/l depressed vasoconstriction, although with the second mode of application a larger inhibition occurred. Therefore, in the subsequent experiments all opioids were added into the lumen. [Met⁵]enkephalin 0.1 mol/l had no effect. [d-Pen², l-Pen⁵]enkephalin 3 mol/l was less potent than [Met⁵]enkephalin 1 mol/l. ICI 174864 1 mol/l was also without effect when given alone, but antagonized the action of [Met⁵]enkephalin 1 mol/l.Ethylketocyclazocine, dynorphin A(1–13), normorphine and DAGO, all 1 mol/l, were ineffective. [Met⁵]enkephalin 1 mol/l did not change the vasoconstriction evoked by the application of noradrenaline (0.1 –3 mol/l). It is concluded that in the mesenteric artery action potential-induced transmitter release, and in consequence vasoconstriction can be inhibited by the activation of presynaptic opioid -receptors. Send offprint requests to P. Illes at the above address     

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.     

Tachykinin antagonists inhibit the morphine withdrawal response in guinea-pigs

Summary This study was an investigation of the effects of the tachykinin antagonists, spantide and (d-Pro⁴, d-Trp^{7, 9, 10})substance P 4-11, injected intracerebroventricularly (ICV), on the locomotor and behavioural responses of guinea-pigs to substance P (SP) injected ICV and to naloxone-induced morphine withdrawal. SP, 50 nmol, produced increased locomotor activity and behaviour that mimicked the response induced by injection of naloxone hydrochloride, 15 mg/kg, in guinea-pigs treated 2 h previously with morphine sulphate, 15 mg/kg. Spantide or (d-Pro⁴, d-Trp^{7, 9, 10})SP4-11, 10 nmol, reduced the locomotor and behavioural responses to SP and to morphine withdrawal. The results support the suggestion that SP or a related tachykinin might be a mediator of the opioid withdrawal response in the central nervous system as has been proposed for the enteric nervous system.     

β-endorphin-sensitive opioid receptors in the rat tail artery

Summary Isolated tail arteries of rats were perfused and field-stimulated every 2 min with 2 pulses at 1 Hz. Different opioid peptides depressed the contractile responses to stimulation; their concentration-response curves showed a maximum at about 40% inhibition. The rank order of potency of the peptides was -endorphin (IC₅₀ = 97 nmol/1) BAM-22P > FK-33824 > DAGO > [d-Ala²,d-Leu⁵]-enkephalin metorphamide > dynorphin A-(1-13) [Met⁵]enkephalin. All these substances have in common a certain activity at opioid -receptors, although the enkephalins are preferential -, and the dynorphins preferential -agonists. However, the selective -agonist [d-Pen²,l-Pen⁵]enkephalin was ineffective at up to 10 mol/l, and the -agonists ethylketocyclazocine and U-50488 acted only at concentrations higher than 3 mol/l. Whereas the effects of -endorphin, DAGO and [d-Ala²,d-Leu⁵]enkephalin could be reduced by the -preferential antagonist naloxone, the effects of ethylketocyclazocine and U-50488 were not changed. The -selective antagonist ICI 174864 did not influence the action of [d-Ala²,d-Leu⁵]enkephalin. Naloxone in a concentration (1 mol/l) which nearly abolished the effect of DAGO 3 mol/l, slightly enhanced responses to stimulation. Neither -endorphin nor DAGO influenced vasoconstriction evoked by the application of noradrenaline or adenosine triphosphate; U-50488 reduced it. In arteries preincubated with [³H]noradrenaline DAGO depressed, whereas naloxone enhanced the tritium overflow and vasoconstriction evoked by field stimulation (0.4 Hz, 24 pulses every 14 min). In addition, naloxone antagonized the effect of DAGO. We suggest that the axon terminals of postganglionic sympathetic neurones in the rat tail artery possess -endorphin-sensitive opioid receptors of the -type. The activation of these receptors by exogenous or endogenous opioids inhibits the release of the neuroeffector transmitter.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 325) Send offprint requests to P. Illes at the above address     

Different types of opioid receptors mediating analgesia induced by morphine,DAMGO, DPDPE,DADLE and β-endorphin in mice

Summary The effects of intracerebroventricular (i.c.v.) administration of d-Phe-Cys-Tyr-d-Try-Orn-Thr-Pen-Thr-NH₂ (CTOP), a selective mu-opioid receptor antagonist, (Allyl)2-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174864) and (N,N-Bisallyl-Tyr-Gly-Gly--(CH₂S)-Phe-Leu-OH (ICI 154129), selective delta-opioid receptor antagonists on blocking analgesia induced by -endorphin, morphine, d-Ala²-NMePhe⁴-Gly-ol-enkephalin (DAMGO), d-Ala²-d-Leu⁵-enkephalin (DADLE) and d-Pen²-enkephalin (DPDPE) administered i.c.v. were studied in male ICR mice. The analgesia was assessed by the tail-flick and paw-licking (hot-plate) tests. The potencies of opioid agonists injected i.c.v. for producing analgesia were DAMGO > DADLE > -endorphin > morphine > DPDPE. Intracerebroventricular administration of CTOP (0.05 g) selectively antagonized inhibition of the tail-flick and paw-licking response induced by morphine, DAMGO or DADLE but not -endorphin or DPDPE. ICI 174864 (5 g) and ICI 154129 (5 g) injected i.c.v. selectively antagonized analgesia induced by DPDPE or DADLE but not -endorphin, morphine or DAMGO injected i.c.v. These results indicate that analgesia induced by morphine and DAMGO is mediated by the stimulation of mu-opioid receptors while analgesia induced by DPDPE is mediated by the stimulation of delta-opioid receptors. DADLE-induced analgesia is mediated by the stimulation of both mu- and delta-opioid receptors. Analgesia induced by -endorphin is mediated by neither munor delta-opioid receptors.Abbreviations i.c.v. intracerebroventricular - i.t. intrathecal - CTOP d-Phe-Cys-Tyr-d-Try-Orn-Thr-Pen-Thr-NHZ - DAMGO d-Ala²-NMePhe²-Gly-ol-enkephalin - DADLE d-Ala²-d-Leus-enke-phalin - DPDPE dd-Pen²-dd-Pen⁵-enkephalin - ICI 174864 (Allyl)2Tyr-Aib-Aib-Phe-Leu-OH - ICI 154129 (N,N-Bisallyl-Tyr-Gly-Gly-(CH₂S)-Phe-Leu-OH     

Roles ofδ andμ opioid receptors in mediating the effects of enkephalins on avoidance conditioning

The effects on one-way active avoidance conditioning of pre-training, systemic administration of the selective -receptor agonist [d-Ala²,N-Me-Phe⁴, Glyol]enkephalin (DAGO), and the selective -receptor antagonist (d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP), were determined in Swiss-Webster mice. A low dose of DAGO (0.92 µg/kg) moderately enhanced avoidance acquisition, whereas a 100 µg/kg dose of CTOP more dramatically impaired acquisition. However, the avoidance-enhancing dose of DAGO significantly increased locomotor activity as measured in a separate group of mice in the avoidance chamber, and the avoidance-impairing dose of CTOP significantly decreased activity. Under these same training conditions, earlier studies (Schulteis et al. 1988; Schulteis and Martinez 1990) demonstrated that enkephalins impaired avoidance learning, and selective-receptor antagonists such as ICI 174,864 enhanced learning; in contrast to the present study, both of these effects were dissociated from performance effects such as alterations in locomotor activity. Taken together, the results suggested that the effects of enkephalins were mediated by the-, but not -, class of opioid receptor.     

A method to estimate the potency of the μ-component of an opioid having mixedμ-, andκ- and/orδ-agonist activities

The SC administration of either typical-agonists such as morphine, pethidine, fentanyl and levorphanol or a mixed- and-agonist like [d-Ala²,d-Leu⁵]-enkephalin to 10-day-old rats produced loss of righting reflex. Additionally, the loss of righting reflex induced by these opioid agonists was antagonized by naloxone, an opioid antagonist having a preference for-receptors, but by neither nor-binaltorphimine nor naltrindole, a specific- or-antagonist, respectively, indicating that the loss of righting reflex was produced by the interaction of an opioid with-receptors. Moreover, the potency of each opioid agonist relative to that of morphine estimated by the present in vivo method was similar to that determined by the traditional in vitro isolated preparation. In contrast to-agonists, neither typical-agonists such as U-50, 488H, ketocyclazocine, pentazocine and butorphanol, nor a selective-agonist like [d-Pen²,d-Pen⁵]-enkephalin affected the righting reflex of 10-day-old rats, indicating that-agonists, but neither- nor-agonists, produced the naloxone-reversible loss of righting reflex in infant rats. By employing the present in vivo method to estimate the-agonist activity of an opioid with mixed agonist activities, it was indicated that the-agonist activity of ethylketocyclazocine, which had been employed as a representative-agonist, was essentially the same as that of morphine, a representative-agonist.     

Differential effect of stimulation strength in mouse vas deferens on inhibition of neuroeffector transmission by receptor type selective opioids

Summary In the mouse isolated vas deferens the amplitude of excitatory junction potentials (e.j.p.s) recorded intracellularly from smooth muscle cells varied with the strength of stimulation. Receptor type selective opioids were tested in this preparation. The -agonist normorphine (2,000 nmol/l) reduced the amplitude of e.j.p.s and shifted the stimulus-response curve in a parallel way to the right. By contrast, the -agonist U-50488 (1,000 nmol/l) and the -agonist [d-Ala²,d-Leu⁵]-enkephalin (2 nmol/l) caused a nonparallel shift of the curve. In addition, opioids having a lower selectivity for one type of receptor were also used. The preferential -agonists ethylketocyclazocine (40 nmol/l) and dynorphin A_1–13 (100 nmol/l) produced parallel and nonparallel shifts, respectively. Thus, normorphine and ethylketocyclazocine were more effective in depressing e.j.p.s evoked by low intensities of stimulation than those evoked by high intensities of stimulation. U-50488, dynorphin A_1–13 and [d-Ala²,d-Leu⁵]-enkephalin caused an equal depression of e.j.p.s evoked by either intensity of stimulation. The preferential - and -antagonists naloxone (1,000 nmol/l) and ICI 154129 (10,000 nmol/l), reversed the action of the respective agonists normorphine and [d-Ala²,d-Leu⁵]-enkephalin. In addition, ICI 154129 (10,000 nmol/l) reversed the action of dynorphin A_1–13, as well. The preferential -antagonist MR-2266 (1,000 nmol/l) prevented the effect of both ethylketocyclazocine and U-50488. It is concluded that under the conditions of these experiments normorphine and ethylketocyclazocine acted at -, U-50488 at -, and dynorphin A_1–13 and [d-Ala²,d-Leu⁵]-enkephalin at -receptors. In the mouse vas deferens more excitable fibres are probably equally sensitive to all three types of opioids, whereas less excitable fibres seem to be more sensitive to - and -, than to -agonists.     

Antinociceptive activity of glycosidic enkephalin analogues

The antinociceptive activity of two new enkephalin analogues:N ^1.5-(-d-glucopyranosyl)[d-Met², Pro⁵]enkephalinamide andN ^1.5-(-d-galactopyranosyl)[d-Met², Pro⁵]enkephalinamide was assessed using the tail immersion and paw pressure behavioural tests. Both enkephalin analogues appear to be more active than morphine when injected either into the fourth ventricle or intrathecally; the galactose analogue is more than 5000 times more active than morphine when injected into the fourth ventricle. The analgesic effects produced by the analogues are partially reversed by SC naloxone (0.1 mg/kg) and totally reversed when the dose of naloxone used was 1 mg/kg, suggesting that the analogues act upon more than one type of opiate receptor (/).     

Response of rat small intestinal active aldohexose transport to elevation of mucosal cyclic AMP by forskolin and 3-isobutyl-1-methylxanthine in vitro

Summary The phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine (IBMX) was able to elevate rat small intestinal cyclic AMP levels to 300% of basal values. Active jejunal d-glucose transport was enhanced parallel to the rise of intracellular cyclic AMP levels to 140% of control values at 100 mol/l IBMX. Transport parameters, as determined in a three compartment model in vitro using a dual label method, indicate increased uphill glucose transport at the site of the brush border membrane, higher intracellular accumulation of the sugar, with unchanged passive permeabilities. Phlorizin-inhibited d-glucose transport and l-glucose transfer in the rat were not affected by the persisting cyclic AMP elevation produced by IBMX. Stimulating effects could also be demonstrated with d-galactose as a substrate. IBMX 100 mol/l also increased active d-glucose as well as 3-O-methylglucose transport in mouse jejunum.Stimulatory effects on intestinal hexose transport and mucosal cyclic AMP levels were also found with the adenylate-cyclase activator forskolin. In the present study, forskolin effects on jejunal mucosal cyclic AMP levels were enhanced in the presence of 100 mol/l IBMX, resulting in a 20-fold increase compared to controls at 20 mol/l forskolin. The concentration response for the effect of forskolin in the presence of 100 mol/l IBMX on d-glucose transport did not produce a significant increase compared to transport stimulation with IBMX alone. At higher concentrations of forskolin however, glucose transport decreased to levels well below the IBMX controls.The elevation of cellular cyclic AMP levels had no effects on passive permeability.Both IBMX 100 mol/l as well as forskolin 20 mol/l inhibited rat jejunal net fluid transport by 40%, combination of both agents resulted in a 55% reduction of net fluid absorption in everted sacs of rat jejunum.These results indicate a functional relationship between jejunal mucosal cyclic AMP levels and active hexose absorption different from the inhibitory role of cyclic AMP in intestinal fluid transport.A preliminary account of this work has been given at the 26th Spring Meeting (March 1985) of the Deutsche Pharmakologische Gesellschaft in Mainz, FRG     

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.