Influence of morphine and naloxone on the release of noradrenaline from rat cerebellar cortex slices

Summary In slices of rat cerebellar cortex preincubated with (-)-³H-noradrenaline, the influence of morphine and naloxone on the efflux of tritium was investigated. The spontaneous outflow was not changed by 10^–5 M of either morphine or naloxone. On the other hand, morphine caused a concentration-dependent decrease of the overflow, of tritium evoked by electrical field stimulation. Naloxone did not change the stimulation-induced overflow, but prevented its inhibition by morphine. It is concluded that morphine, through an action on opiate receptors located on cerebellar noradrenergic neurones, inhibits the secretion of the transmitter in response to nerve impulses.     

Influence of fentanyl,levorphanol and pethidine on the release of noradrenaline from rat brain cortex slices

Summary In slices of rat brain cortex preincubated with (–)-³H-noradrenaline, the influence of fentanyl, levorphanol and pethidine on the efflux of tritium was investigated. The spontaneous outflow of tritium was not changed by low, and was accelerated by high concentrations of the drugs. The overflow of tritium evoked by electrical stimulation at 3 Hz was diminished by 10^–8–10^–7 M fentanyl and by 10^–7–10^–6 M levorphanol, but was augmented by 10^–5 M levorphanol. Naloxone prevented the inhibitory effect of fentanyl and levorphanol. In contrast to fentanyl and levorphanol, pethidine did not decrease, but at concentrations of 10^–6–10^–5 M greatly increased the stimulation-induced overflow of tritium. However, the increase was abolished, and the stimulation-evoked overflow slightly reduced, after the re-uptake of noradrenaline had been blocked by cocaine. It is concluded that fentanyl, levorphanol and pethidine share with morphine the ability to inhibit the release of transmitter from cerebrocortical noradrenaline neurones evoked by nerve impulses.     

Morphine tolerance and dependence in noradrenaline neurones of the rat cerebral cortex

Summary By subcutaneous implantation of 2 or 13 morphine pellets (75 mg morphine/pellet), rats were made tolerant to, and dependent on narcotic analgesics. Occipital cortex slices from dependent animals and placebo-implanted controls were incubated with (-)-³H-noradrenaline and subsequently superfused with physiological salt solution. The accumulation of ³H-noradrenaline was not changed by pretreatment with 2, but was slightly decreased by pretreatment with 13 morphine pellets. The overflow of tritium evoked by electrical field stimulation was higher in slices from morphine-implanted rats than in those from placebo controls. Morphine and levorphanol, added in vitro, inhibited the stimulation-induced overflow of tritium at, similar concentrations and to a similar degree in slices from morphine-and placebo-pretreated animals. —It is concluded that, during chronic treatment with morphine, an adaptation takes place in the brain to compensate for the acute effect of narcotic analgesics, i.e. inhibition of the release of noradrenaline by nerve impulses. The chain of events from the drug-receptor interaction to the depression of the release process can be excluded as substrate of this adaptation. During with-drawal, the compensatory changes provoke an enhanced increase of extracellular noradrenaline during nerve impulses.     

Effects of morphine on kinetics of 14C-dopamine in rat striatal slices

Summary The uptake and release of ¹⁴C-dopamine and the effect of morphine on these processes were studied in slices of rat striata. The uptake of the labelled dopamine was completed after 30 min. Low temperature, DNP and ouabain inhibited the accumulation of dopamine. Morphine (1.2 and 3.5×10^–5 M) did not affect the uptake of labelled dopamine, but it slowed the K⁺-induced release of ¹⁴C-dopamine. Naloxone significantly inhibited this effect of morphine. Our results demonstrate a direct and specific action of morphine on dopaminergic neurones of rat brain.     

Effect of desipramine on rat cortex slices incubated with 3H-dopamine

Rat cortex slices were incubated with ³H-dopamine (³H-DA), and the effect of desipramine (DMI) was studied on the accumulation of ³H-DA and ³H-noradrenaline (³H-NA) in the tissue and the concentrations of ³H-DA and ³H-NA in the incubation medium. This effect was compared with the effect of cocaine, reserpine and FLA 63 studied by the same technique, in order to elucidate the mechanisms of action of DMI.All the drugs induced a significant decrease of ³H-NA accumulation in the cortical tissue. The ³H-DA retention varied: cocaine and reserpine caused a significant decrease, FLA 63 a marked, significant increase, and DMI a slight, but significant increase. In the incubation medium, ³H-DA significantly increased after DMI, cocaine and reserpine, and remained unchanged after FLA 63. In all experiments ³H-NA in the medium was very low.It is suggested that DMI in cortex slices may exert a double mechanism of action on the final step in the noradrenaline biosynthesis: 1. an inhibition of the ³H-DA uptake at the level of the noradrenergic cell membrane, and 2. an inhibition of the intraneuronal transport of ³H-DA to sites where it is converted to ³H-NA, concomitant with an increased intraneuronal ³H-DA accumulation. Other possibilities are discussed.     

Lack of effect of morphine in reducing the release of labelled acetylcholine from brain slices stimulated electrically

Morphine in low concentration (0.3–30 μM) failed to reduce the evoked release of [³H]-ACh from slices of cortex, hippocampus or striatum stimulated at low or high frequencies. Reduced ACh release observed in vivo following morphine administration is probably an indirect action of morphine on the activity of cholinergic neurons.     

Facilitation of noradrenaline release from rat brain slices by \-adrenoceptors

The present study examined whether stimulation of \-adrenoceptors facilitated noradrenaline release in the rat brain. Electrical stimulation-evoked overflow of tritium from rat cerebral cortical, hypothalamic and hippocampal slices labelled with ³H-noradrenaline was measured during superfusion for 100 min. Tissue slices were electrically simulated (1 Hz, 20 mA, 2 ms, 2 min), at 20(S1) and 70(S2) min after the onset of superfusion. The nonselective \-adrenoceptor agonist isoproterenol (0.1 – 10 nM) enhanced stimulation-evoked overflow of tritium from slices of cerebral cortex, hypothalamus and hippocampus in a concentration-dependent manner; mean S2/S1 ratios with 10 nM isoproterenol were 161 +- 11%, 142 +- 15% and 143 - 12% of control, respectively, in the three brain regions. The facilitatory effect of isoproterenol in cerebral cortical slices was antagonized by propranolol (50 nM), a nonselective \sb-adrenoceptor antagonist, and by the \sb₁- and \sb₂-selective adrenoceptor antagonists ICI 89,406 (1 nM) and ICI 118,551(1 nM), respectively. The \sb₁- and \sb₂-selective adrenoceptor agonists prenalterol and albuterol (0.1 \2- 10 nM), respectively, also increased stimulation-evoked overflow of tritium from cerebral cortical slices; these effects were antagonized by \sb-adrenoceptor antagonists. These findings suggest that stimulation of \sb-adrenoceptors enhance noradrenaline release from rat cerebral cortical, hypothalamic and hippocampal slices; this release mechanism appears to involve both \sb₁- and \sb₂-adrenoceptor subtypes. These facilitating presynaptic receptors may be involved in mediating the antidepressant-like behavioral effects of \sb₂-adrenoceptor agonists.     

Noradrenaline release from slices of the thalamus of normal and morphine-dependent rats

Summary The effect of morphine on potassium-induced stimulation of (³H)-noradrenaline release from slices of the rat thalamus was investigated. The in vitro addition of morphine (10^–6 M) significantly depressed potassium-induced tritium overflow by 42% and this was prevented by the prior addition of naloxone (3×10^–6 M) to the medium. The stimulation-evoked overflow of tritium from slices of the thalamus of morphine-dependent rats was not significantly different from normal controls. Addition of naloxone (10^–5 M) 10 min before exposure of the tissues to 20 mM K⁺ significantly enhanced noradrenaline release from dependent slices. The results suggest that the basic release mechanism may have adapted to the continuous presynaptic inhibition of release by morphine.     

Cyclic AMP facilitates the electrically evoked release of radiolabelled noradrenaline,dopamine and 5-hydroxytryptamine from rat brain slices

Summary The adenylate cyclase activator forskolin as well as 8-bromo-cyclic AMP enhanced the electrically evoked release of³H-noradrenaline and³H-5-hydroxytryptamine from superfused rat neocortical slices and that of³H-dopamine from neostriatal slices with comparable EC₅₀'s of about 0.5 and 50 M, respectively, without affecting spontaneous tritium efflux. The phosphodiesterase inhibitor ZK 62771 (3–100 M) also enhanced³H-noradrenaline and³H-dopamine release but slightly reduced³H-5-hydroxytryptamine release. However, this drug profoundly enhanced spontaneous tritium release in the latter case. The facilitatory effect of forskolin (0.3 M) on the release of the amine neurotransmitters was potentiated in the presence of ZK 62771 (30 M). Therefore, cyclic AMP appears to exert a general facilitatory effect on the release of these biogenic amines from central nerve terminals.     

Differential antagonism by naloxone of inhibitory effects of haloperidol and morphine on brain self-stimulation

Haloperidol (0.16 mg/kg) or morphine sulfate (40 mg/kg), injected subcutaneously, completely suppressed bar-pressing for brain self-stimulation in rats implanted with electrodes in the lateral hypothalamus. Haloperidol also caused catalepsy and ptosis while morphine produced catatonia with exophthalmia. Naloxone in a dose (5 mg/kg) which was ineffective when given alone, differentially reversed the morphine-effects but was without any reversing influence on the actions of haloperidol.Visiting pharmacologist from Department of Pharmacology and Psychology, University of Rhode Island, Kingston, R. I., U.S.A.     

Comparison of subcutaneous and spinal subarachnoid injections of morphine and naloxone on analgesic tests in the rat

Small catheters were chronically implanted subdurally in the rat so that repeated microinjections could be made into the subarachnoid space at the lumbar area. Morphine, injected intrathecally (i.th.) produced analgesia as measured by the tail-flick test at doses $\text{1}\text{100}$ of those by the subcutaneous (s.c.) route. Analgesia from i.th. morphine was reversed by either i.th. or s.c. injected naloxone. The dose of naloxone by the i.th. route was about $\text{1}\text{30}$ of that by the s.c. route. However, i.th. injection was no more effective than s.c. injection of naloxone in reversing analgesia produced by s.c. injection of morphine. When [³H]-labelled naloxone was injected s.c. or i.th. in the above experiment of morphine antagonism, there was a more rapid entry of the labelled material in the brain by the i.th. route of administration. The results raise questions on the relative importance of the spinal mechanism of analgesia produced by s.c. injections of morphine. Analgesia was also measured by the tail-shock vocalization test in which morphine producd a dose-dependent elevation of shock threshold at s.c. doses above those prolonging tail-flick latencies. Morphine injected i.th. at doses above those which elevated tail-flick latency produced hypersensitivity, hyper-reflexia, and convulsive seizure of the hindquarters. The spinal analgesic effect of morphine, when administered localy, appears to have a low ceiling of efficacy.     

A cumulative dose-response technique for the characterization of presynaptic receptors modulating [3H]noradrenaline release from rat brain slices

A cumulative dose-response technique was developed for the characterization of presynaptic receptors involved in the modulation of [³H]noradrenaline (NA) release from rat hippocampus slices, using continuous K⁺ (20 mM) depolarization. The results obtained with this technique were compared with those obtained using a repetitive K⁺ stimulation procedure. The release of [³H]NA induced by continuous K⁺ stimulation as well as that caused by repetitive K⁺ stimulation was strongly Ca²⁺-dependent and consisted for more than 90% of unmetabolized [³H]NA. Using continuous K⁺ stimulation it was demonstrated that the presynaptic inhibition of ³H-NA release by exogenous NA reached a maximum 10 min after addition of NA. The inhibitory effect of NA appeared to be independent of the time of addition, suggesting that the sensitivity of the presynaptic α-adrenoceptors remained unchanged during the experiment. Cumulative dose-response curves were recorded by the successive addition, at 10 min intervals, of increasing concentrations of NA. It was shown that continuous stimulation and repetitive K⁺ stimulation were basically similar with regard to the characteristics of the resulting [³H]NA release as well as its presynaptic α-adrenoceptor-mediated modulation by exogeneous NA. However, the cumulative dose-response technique, which can be carried out only using continuous K⁺ stimulation, makes it possible to determine more rapidly and also more accurately the apparent affinities and intrinsic activities of drugs towards receptors involved in the modulation of neurotransmitter release from brain slices.     

Acetylcholine release from the brain of unanaesthetized cats following habituation to morphine and during precipitation of the abstinence syndrome

The rate of release of central acetylcholine (ACh) was determined in unanaesthetized cats by perfusing the sensorimotor cortex or the lateral ventricles with a neostigmine-containing solution by means of push-pull cannulae.Cats were made physically dependent on morphine over a 10-day schedule of daily increasing doses of the narcotic. No difference in both cortical and ventricular ACh release was noted between these morphine-dependent animals (determined following the last dose of morphine-20 mg/kg i.p.) and the same animals prior to the injection of any morphine. When the abstinence syndrome was precipitated in these habituated animals (0.2 mg/kg of naloxone, i.p.), both cortical and ventricular ACh release was enhanced, coincident with behavioural agitation of the animal. While the behavioural symptoms of the abstinence syndrome persisted, this increased release of ACh was only of short duration.In non-dependent cats, a 1-h localized perfusion of the sensorimotor cortex with a morphine-containing solution (10^–4 or 5×10^–4 g/ml) had no effect on either cortical ACh release or the behaviour of the animal.These results raise questions concerning the hypothesis that a cholinergic mechanism governs the occurrence of the morphine abstinence syndrome in cats.     

Differential control of Ca2+-dependent [3H]noradrenaline release from rat brain slices through presynaptic opiate receptors and alpha-adrenoceptors

The inhibitory actions of the Ca2+ antagonist Cd2+, morphine and noradrenaline (exogenously added + endogenously released) on electrically evoked release of [3H]noradrenaline from superfused rat neocortical slices were strongly reduced when release was enhanced by 4-aminopyridine. In the presence of 4-aminopyridine the release inhibiting effects of these drugs were restored by lowering the extracellular Ca2+ concentration. When release was enhanced by prolonging the pulse duration, only the release inhibiting effect of noradrenaline was reduced but the effects of Cd2+ and morphine were unchanged. Irrespective of the pulse duration, blockade of presynaptic alpha-adrenoceptors with phentolamine did not affect the release inhibiting effects of Cd2+ and morphine. The inhibitory effects of morphine and noradrenaline remained unchanged in Cl--free medium. Furthermore, these drugs strongly reduced the [3H]noradrenaline release induced by 20 mM K+ in the presence of tetrodotoxin. The results suggest that activation of presynaptic opiate-receptors inhibits Ca2+ entry through voltage-sensitive Ca2+ channels, whereas presynaptic alpha-adrenoceptors affect a step in the secretory process subsequent to Ca2+ influx. Moreover, the involvement of (direct) changes in Na+, K+ or Cl- permeability appears unlikely for both receptor systems.     

The action of morphine and naloxone on acid secretion by the rat isolated stomach

The action of morphine and naloxone on acid secretion by the rat isolated stomach has been studied. Morphine (10^?7 to 10^?4 M) had no effect upon spontaneous acid secretion. Morphine (10^?6 M) did not modify the acid output in response to sub-maximal stimulation by pentagastrin, histamine, bethanechol or isoprenaline. Naloxone (10^?6 M) was without effect on the response to pentagastrin or histamine. Our results suggest that opiate receptors do not modify acid secretion in this preparation.     

Effect of intracerebroventricular administration of morphine upon intestinal motility in rat and its antagonism with naloxone

Morphine, intracerebroventricularly (i.c.v.) or intraperitoneally (i.p.) administered to rats, inhibited intestinal propulsion as tested by a charcoal meal. Such an inhibition was shown to be linearly related to the log of administered doses for both routes of administration and the two linear regressions are parallel, so that morphine was calculated to be 206 times more potent when administered i.c.v. than i.p. A dose of morphine fully active by the i.c.v. route was completely inactive when injected by the i.v., i.p., i.m. and s.c. routes. Naloxone, administered i.c.v., blocked the antipropulsive effect of morphine i.c.v. or i.p. The pA2 of naloxone versus morphine, both administered i.c.v. was determined and calculated to be 7.14 (6.76-7.62).     

W-7 at calmodulin-antagonistic concentrations facilitates noradrenaline release from rat brain cortex slices

Slices from rat brain occipital cortex were preincubated with [3H]noradrenaline then superfused and stimulated electrically. W-7, 10 mumol/l, enhanced the stimulation-evoked overlow. Binding experiments with W-7 evidenced weak displacement of [3H]yohimbine and no displacement of [3H]prazosin. Release experiments with the addition of clonidine, cocaine or phentolamine provided no evidence for major interference of W-7 with alpha-adrenergic or uptake mechanisms. Facilitation could have been due to inhibition of a calmodulin-dependent enzyme which could not be identified from the present results.     

Behavioral effects of morphine and naloxone following chronic morphine administration

The effects of morphine, naloxone, and combinations of these drugs were examined in squirrel monkeys under shock-postponement schedules. In the absence of a lever press, shocks could be presented every 4s, and each response postponed shock for 20s. Acutely, morphine (0.10–3.00 mg/kg) produced not only overall response-rate decreases, but also increases in the number of shocks, whereas naloxone (0.10–30.00 mg/kg) had little effect on responding. When given in combination with morphine, several doses of naloxone antagonized the rate-reducing and shock-increasing effects of morphine. Daily administration of morphine resulted in a substantial decrease in the number of shocks received and a moderate attenuation of the rate-decreasing effects of morphine (tolerance). Lower doses substituted for the fixed daily dose resulted in a smaller effect on behavior than under acute administration. Naloxone given in combination with the daily morphine dose or substituted for the daily administration of morphine, produced effects similar to those seen prior to chronic drugging. Thus, behavioral effects of naloxone were not altered even though tolerance to morphine was observed. Larger doses of naloxone continued to antagonize the effects of morphine for at least 24h. No signs of physical dependence were noted when naloxone was administered or when administration of morphine ended.