Addition Of Naloxone Research Articles

GABA A receptor antagonists reduce acute opiate withdrawal in isolated tissue

1. 1. The effect exerted by GABA, receptor agonists and antagonists on the acute opiate withdrawal induced by μ and k receptor agonists were investigated in vitro. 2. 2. Following a 4 min in vitro exposure to morphine (less selective μ agonist), DAGO (highly selective μ agonist) and U50-488H (highly selective k agonist) the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone. 3. 3. Bicuculline (1 × l0 5 − 5 × l0 −5 − 1 × l0 4M), a GABA A receptor antagonist, injected 10 min before or after the opioid agonists was able dose-dependently to antagonize the naloxone-induced contracture after exposure to p (morphine and DAGO) and k (U50-488H) opiate agonists. 4. 4. Furthermore, picrotoxin (1 × l0 5 − 5 × l0 −5 − 1 × l0 4M) ,an antagonist of GABA-linked chloride channels, was able to exert the same effects. 5. 5. Muscimol (1 × l0 5 − 5 × l0 −5 − 1 × l0 4M), a GABA A receptor agonist, was able to increase dose dependently both μ. and k opiate withdrawal. 6. 6. The data indicate that both GABA A receptor agonists and antagonists are able to control opiate withdrawal in vitro suggesting an important functional interaction between GABAergic system and the opioid withdrawal both at the μ. and k receptor level.

Modulation by opioid peptides of mechanosensory pathways supplying the guinea‐pig inferior mesenteric ganglion.

1. Radioimmunological techniques were used in isolated guinea-pig inferior mesenteric ganglion (IMG)-colon preparations to determine whether opioid peptides and neurotensin8-13 (NT8-13), the C-terminal region of NT1-13 recognized by neurotensin receptors, modulate distension-induced release of substance P (SP)- and vasoactive intestinal polypeptide (VIP)-like immunoreactive (LI) material. 2. Colonic distension significantly increased the amount of SP- and VIP-LI material released in the ganglionic superfusate. A low-Ca2+ (0.1 mM), high-Mg2+ (15 mM) solution blocked their release. 3. In vivo capsaicin pretreatment abolished release of SP-LI material during colonic distension but had no significant effect on distension-induced release of VIP-LI material. 4. The addition of [Leu5]enkephalin, [Met5]enkephalin, PL017 (a mu-receptor agonist) and DPDPE (a delta-receptor agonist) to the ganglion side of a two-compartment chamber blocked distension-induced release of SP-LI material. The addition of naloxone and ICI-174,864 (a delta-receptor antagonist) to the ganglion compartment reversed the inhibitory effect of the mu- and delta-receptor agonists. 5. Addition of [Leu5]enkephalin and [Met5]enkephalin to the ganglion compartment had no significant effect on release of VIP-LI material during colonic distension. 6. Addition of NT8-13 to the ganglion compartment significantly increased in the amount of SP-LI material released during colonic distension but had no affect on distension-induced release of VIP-LI material. 7. The results suggest the hypothesis that under in vivo conditions, enkephalinergic nerves decrease and neurotensinergic nerves increase the release of SP from peripheral branches of primary afferent sensory nerves.

Subanesthetic concentrations of lidocaine selectively inhibit a nociceptive response in the isolated rat spinal cord

Systemically administered local anesthetics are known to provide analgesia in a variety of pain states; however, the site of action and the mechanism by which these effects are produced remain in question. In the present study, the effects of low (subblocking for nerve conduction) concentrations of lidocaine on a spinal cord nociceptive potential were studied. Spinal cords were removed from neonatal rats and maintained in vitro. Lumbar dorsal and ipsilateral ventral roots were attached to suction electrodes for stimulation and recording, respectively. Following a stabilization period (60–120 min) with control measurements, each preparation was exposed to a single concentration of lidocaine (30–60 min) then returned to control perfusate for recovery (60–120 min). Data were digitized and integrals computed for both monosynaptic and slow ventral root potentials (VRP). Low concentrations of lidocaine produced a selective reduction in the magnitude of the slow-VRP. At lidocaine concentrations of 1–10 μg/ml (3.6–36 μM), the slow-VRP was reduced from 79% to 36% of control. Recovery to pre-exposure control levels was slow and sometimes not complete after 60–120 min in drug-free perfusate. The monosynaptic component of the VRP was unaffected by lidocaine at any concentration, suggesting that the depression of the slow-VRP cannot be attributed to simple conduction block. The addition of naloxone 0.1 μM to the perfusate had minimal effect on lidocaine-induced depression. Although resembling the selective effects of morphine, the antinociceptive effects of lidocaine do not appear to be primarily mediated through opiate receptors. Subblocking concentrations of tetrodotoxin, a selective sodium-channel blocker, did not mimic the effects of lidocaine. However, a subblocking concentration of benzonatate, an orally effective local anesthetic, did produce lidocaine-like selective effects on the slow-VRP. This study demonstrated that lidocaine at clinically relevant concentrations can selectively depress a well-characterized nociceptive response in the isolated rat spinal cord. We propose that the site of action for systemic lidocaine analgesia is, at least in part, at the level of the spinal cord.

Withdrawal contractures of guinea‐pig isolated ileum after acute activation of κ‐opioid receptors

1. The present study was undertaken to investigate firstly whether a brief exposure for 5 min of guinea-pig isolated ileum to the kappa-opioid agonist, U-50,488H produced a withdrawal contracture on addition of naloxone and secondly to ascertain whether the response was due to the activation of kappa-opioid receptors. 2. Naloxone (10(-6) M) did not elicit a response in preparations exposed to U-50,488H (5 x 10(-7) M-2 x 10(-6) M). However, after exposure to U-50,488H (5 x 10(-7) M), naloxone (10(-6) M) produced a strong contracture if the agonist was washed out 1 min before the addition of the antagonist. 3. The addition of naloxone (10(-6) M) to the ileum preparation exposed to U-50,488H (10(-7) M or lower) caused a response of similar intensity irrespective of whether the agonist had been washed out. 4. The selective kappa-opioid antagonist, nor-binaltorphimine (2.7 x 10(-9) M and 2.7 x 10(-7) M), injected before the opioid agonists, prevented the naloxone-induced contracture after exposure to U-50,488H (8 x 10(-8) M) but did not affect the contracture after exposure to morphine (5 x 10(-7) M). 5. Nor-binaltorphimine (2.7 x 10(-9) M) caused a contraction of the ileum preparation when injected 5 min after exposure to U-50,488H (8 x 10(-8) M) but not after morphine (5 x 10(-7) M). 6. The alpha 2-adrenoceptor agonist, clonidine (3 x 10-8 M) and the calcium channel blocker, nifedipine(3 x 10-8 M), injected 1 min before naloxone, blocked the ileum contraction to naloxone after exposure to U-50,488H (8 x 10-8 M). The results demonstrate that the stimulation of Kappa-opioid receptors can induce a similar dependence in guinea-pig ileum to that produced by activation of micro receptors.

Ethanol-induced increase in endogenous dopamine release may involve endogenous opiates.

The effect of opiate peptides on basal and potassium-stimulated endogenous dopamine (DA) release from striatal slices was studied in vitro. Dual stimulation of the striatal slices gave a reproducible increase in DA release that was calcium dependent. Addition of the delta-opiate receptor agonists Met5-enkephalin, [D-Ala2,D-Leu5]enkephalin (DADLE), and [D-Ser2]Leu-enkephalin-Thr (DSLET), increased the basal DA release without affecting potassium-stimulated release in a dose-dependent manner. The effect of DADLE was antagonized by the addition of naloxone. In contrast, the mu-opioid receptor agonist [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAGO) and the epsilon-opioid agonist beta-endorphin inhibited the stimulated DA release without changing the basal release. The inhibitory effect of DAGO on potassium-stimulated release was antagonized by naloxone. The addition of ethanol (75 mM) to the incubation media produced a delayed increase of both the basal and stimulated DA release. There was no change in stimulated DA release when the change in basal release was subtracted, suggesting that ethanol produced a dose-dependent, selective increase in basal DA release. Naloxone and the selective delta-opiate antagonist ICI 174864 inhibited the ethanol-induced increase in basal DA release. Naloxone and ICI 174864 added alone did not alter either basal or stimulated DA release. We therefore suggest that the ethanol-induced increase in basal DA release is an indirect effect involving an endogenous delta-opiate agonist.

Manifestations of acute opiate withdrawal contracture in rabbit jejunum after mu-, kappa- and delta-receptor agonist exposure.

1. Following a 5 min in vitro exposure to morphine (1.3 x 10(-7) M), U-50,488H (2.5 x 10(-8) M) and deltorphin (1.6 x 10(-8)-6.5 x 10(-9) M), the rabbit isolated jejunum exhibited a precipitated contracture after the addition of naloxone (2.75 x 10(-7) M). 2. The precipitated responses to U-50,488H and deltorphin but not to morphine were reproducible in the same tissue. 3. The precipitated contractures were blocked completely by tetrodotoxin (3 x 10(-7) M), partially by atropine (1.5 x 10(-7) M) and not affected by hexamethonium (1.4 x 10(-5) M). 4. Naloxone administration (2.75 x 10(-7) M) before the agonist prevented the development of the adaptive response to morphine and U-50,488H but not to deltorphin. 5. The selective antagonists norbinaltorphimine (2.7 x 10(-8)-2.7 x 10(-9) M) and naltrindole (1.1 x 10(-7) M) prevented the adaptive response development only to the respective agonists. 6. The opioid agonists partially inhibited the spontaneous activity of the tissue. This study has shown that independent activation of mu-, kappa- and delta-opioid receptors can induce dependence in this isolated tissue. Rabbit jejunum is a suitable tissue for studying the acute effects of opioids on the adaptative processes determined by their administration.

Effects of putative neurotransmitters and related drugs on withdrawal contractures of guinea‐pig isolated ileum following brief contact with [Met5] enkephalin

1. Brief exposure for 2 min of guinea-pig isolated ileum to [Met5]enkephalin (MEnk) and noradrenaline has been shown previously to produce withdrawal contractures on washout of the agonist or addition of naloxone (MEnk) or phentolamine (noradrenaline). 2. The present study was undertaken to investigate firstly, whether other putative neurotransmitters and/or related drugs which inhibit transmitter release also produced withdrawal responses following 2 min contact with the ileum and secondly, whether they affected the opioid withdrawal response. 3. Adenosine (1-5 microM), but not U-50,488H (1-5 microM), somatostatin (0.01-5 microM), ocreotide (1-5 microM), baclofen (1-25 microM) or dopamine (5, 50 microM), produced a contracture on washout following 2 min contact with the ileum. The adenosine (5 microM) washout contracture, in common with MEnk and noradrenaline washout contractures, was inhibited by the substance P antagonist, spantide (10 microM). 4. Added 30 s before washout at a concentration of 5 microM, noradrenaline, U-50,488H, adenosine, somatostatin and ocreotide significantly inhibited the washout withdrawal response following 2 min contact of the ileum with MEnk, 1 microM. A higher concentration of baclofen, 250 microM, also inhibited this response. 5. The naloxone (1 microM)-precipitated withdrawal response following contact of the ileum with MEnk, 1 microM, for 2 min, was inhibited only by noradrenaline (5 microM) and U-50,488H (5 microM). 6. It is concluded that during naloxone-precipitated opioid withdrawal an additional population of enteric motor neurones is recruited which is not involved in the washout withdrawal response and these neurones have less diversity of presynaptic receptors mediating inhibition of transmitter release than cholinergic motor neurones.

Naloxone's effect on the inotropic and chronotropic responses of isolated, electrically stimulated or spontaneously beating rat atria

Experiments were conducted to determine (i) how naloxone administration alone could modify the inotropic (in electrically stimulated (ES) rat atria) and both the inotropic and chronotropic responses (in spontaneously beating (SB) rat atria) isolated from normotensive and hypotensive (hemorrhaged) rats, and (ii) how naloxone administration would modify the inotropic and chronotropic responses of isolated rat atria previously administered an opiate agonist (morphine), a muscarinic agonist (carbachol), or an alpha- and beta-adrenergic agonist (noradrenaline). Naloxone (51-340 microM) added to ES atria caused a delayed but dose-related decrease in atrial tension (AT), whereas in SB atria, naloxone caused atrial heart rate (AHR) to fall and atrial tension (AT) to increase. Naloxone (68-340 microM), given to SB atria from acutely hypotensive rats, caused a similar increase in atrial tension as seen in the "normotensive" isolated (SB) atria and a similar decrease in atrial heart rate. Morphine sulphate (MS), 37-375 microM, administered to ES atria caused a delayed fall in AT; which was further decreased when naloxone (340 microM) was also added. In the SB atria, morphine caused a dose-related decrease in atrial heart rate whereas atrial tension increased. In SB preparations, atrial heart rate fell even further when naloxone was added to morphine compared with when morphine sulphate was given alone, whereas atrial tension was increased. Noradrenaline (3 or 12 microM) caused a positive, dose-related inotropic response in the ES atria, effects not influenced by the addition of naloxone. In the SB atria, naloxone caused no change in the dose-related increases in atrial tension and heart rate when combined with the lower dose of noradrenaline but decreased AT when combined with 12 microM noradrenaline, compared with when this dose of noradrenaline was given alone. Carbachol (683 nM-1.37 microM) caused a dose-related decrease in atrial tension in ES atria, which was reversed completely by the addition of naloxone. In SB atria, carbachol decreased both atrial tension and heart rate, and with the addition of naloxone (340 microM), a further slight drop in atrial heart rate occurred, but concurrently, a marked rise in atrial tension was observed. The results indicate that naloxone can act with receptors directly within atrial tissue to cause changes in atrial tension and heart rate. The comparable delayed responses of morphine and naloxone suggest their effects are mediated by nonopiate receptors which, in time, cause decreases in calcium influx into the atria.(ABSTRACT TRUNCATED AT 250 WORDS)

Untersuchungen zur Therapie des hypovolämischen Schocks bei Kühen mit rechtsseitiger Labmagenverlagerung*

In this trial, the effect of supplementary administration of the opioid antagonist Naloxone was tested, in comparison to infusion of Ringer's-solution only, in the treatment of hypovolemic shock in cows suffering from right-sided abomasal displacement. Twenty cows were selected by several criteria (pulse rate greater than 95/min; plasma chloride content less than 90 mmol/l; body temperature less than 39.5 degrees C) and alternatively assigned to the Naloxone group and the control group. All animals were treated surgically by the usual clinical method. After opening the abdominal cavity paralumbally from the right, intravenous administration of 13 l of Ringer's-solution was started and continued for about 5 hours. In addition, the cows of the experimental group received 0.75 g Naloxone as an intravenous bolus before, and 1 g Naloxone dissolved in 7 l fluid during the first hour of the permanent drip infusion. Further treatments (additional fluid therapy or administration of purgatives and ruminomotorics on the following days) were given according to the condition of the animal. The evaluation of efficacy was done in regard to the clinical (course of disease), and clinicochemical (e.g. hemogram, blood gas analysis, plasma chloride, plasma lactate) parameters as well as to blood pressure. No statistically significant differences were found between both groups. Only nine of the twenty cows were sent home cured, one patient of each group died one day after surgery, and the remaining 9 animals had to be sent to the slaughterhouse within 3 to 22 days after surgery after only transient improvement. These results suggest, that under the conditions described, conventional fluid therapy is equivalent to fluid therapy with addition of Naloxone. Hypoxic damage of the abomasum caused by its displacement and the subsequent hypovolemic shock are discussed as main causes for the poor postsurgical prognosis of right-sided abomasal displacement.

Enkephalinergic modulation of the dopamine system in the turtle retina

One subpopulation of amacrine interneurons in the turtle retina was shown to contain met-enkephalin by means of immunocytochemistry, and another was demonstrated to have a high-affinity uptake system for [3H]-dopamine by means of autoradiography. Although the amacrine soma size, density, and distribution of their neurites in IPL substrata was similar in retinas in which met-enkephalin and dopamine were localized, combined light microscope immunocytochemistry-autoradiography demonstrated that these two neurotransmitter systems did not coexist in the same cells. Because the two amacrine cell subtypes ramify in the same IPL substrata, neuronal interaction between them is possible. Release experiments showed that the potassium-induced release of [3H]-dopamine from the superfused turtle retina was reduced by 40% when enkephalin was added to the superfusate. The inhibition of [3H]-dopamine release could be blocked by the addition of naloxone. The addition of enkephalin had no effect of the potassium-induced release of [3H]-GABA from the superfused retina. These findings suggest that an enkephalinergic modulation of the dopaminergic amacrine cell system exists in the turtle retina.

Effects of Opioid Peptides on Thyrotropin-Releasing Hormone Release from the Rat Caecum in Vitro

Effects of opioid peptides (beta-endorphin, dynorphin (1-13). alpha-neoendorphin, beta-neoendorphin, leucine-enkephalin, methionine-enkephalin) on the release of thyrotropin-releasing hormone (TRH) from the rat caecum were studied in vitro. The rat caecum was incubated in medium 199 with 1.0 mg/ml of bacitracin (pH 7.4) (medium). The amount of TRH release from the rat caecum into the medium was measured by radioimmunoassay. The immunoreactive TRH (ir-TRH) release from the rat caecum was inhibited significantly in a dose-related manner with the addition of opioid peptides. The inhibitory effects of opioid peptides on ir-TRH release from the rat caecum were blocked with an addition of naloxone. The elution profile of acid-methanol-extracts of rat caecum on Sephadex G-10 was identical to that of synthetic TRH. The findings suggest that opioid peptides inhibit TRH release from the rat caecum in vitro.

Evidence for the involvement of opioid neuropeptides in the adherence and migration of immunocompetent invertebrate hemocytes.

Evidence for the participation of opioid neuropeptides in immunoregulatory activities, especially cellular adherence and migration, has been obtained in representatives of two phyla of invertebrates, the mollusc Mytilus edulis and the insect Leucophaea maderae. The injection of a synthetic analog of [Met]enkephalin [( D-Ala2,Met5]enkephalinamide, DAMA; 10(-6) M) had a stimulatory, naloxone-reversible effect on the directed migration of immunocompetent hemocytes. Incubation of hemolymph in the presence of exogenous or endogenous opioid material significantly enhanced the adherence of hemocytes on albumin-coated slides as demonstrated by use of indirect Zeiss-Zonax reflectance computer analysis. Conversely, hemocyte adherence was markedly reduced by the addition of naloxone (10(-8) M) to the incubation medium, either alone or in combination with DAMA. The antagonistic effects of naloxone on the stimulatory activities of opioids indicate that, like those previously reported in mammals, they are receptor-mediated. The presence of an endogenous [Met]enkephalin-like material was demonstrated in cell-free hemolymph as well as sequestered hemocytes by use of high-pressure liquid chromatography and radioimmunoassay. These results demonstrate that the capacity of immunocytes to release and respond to opioid neuropeptide messengers is not restricted to mammalian organisms but was developed early in the course of evolution.

The Effect of Artificial Sweetener on Insulin SecretionII. Stimulation of Insulin Release from Isolated Rat Islets by Acesulfame K (In Vitro Experiments)

The effect of the artificial sweetener Acesulfame K on insulin release in vitro was investigated. Pancreatic islets were obtained from Male Wistar rats. Acesulfame K produced a significant increase in insulin release from incubated islets. This effect was dose- and glucose-dependent. When islets were incubated with different amounts of Acesulfame K (2.5, 7.5 and 15 mM) and 15 mM glucose in the media for one hour, insulin concentrations were 140.2 +/- 21.1, 246.7 +/- 32.4 and 313.9 +/- 37.7 microU/ml, respectively. When 15 mM Acesulfame K was added to a media containing 0, 2.5, 5, 10 and 15 mM glucose, insulin release from incubated islets after 60 min were 25.6 +/- 6.4, 65.4 +/- 12.1, 109.0 +/- 10.0, 229.6 +/- 28.0 and 313.9 +/- 37.7 microU/ml. Incubating islets in the media containing arginine or acetylcholine increased insulin release significantly. However, when Acesulfame K was added to the media containing either arginine or acetylcholine, no further potentiating effect could be detected. The effect of Acesulfame K on insulin secretion was decreased by noradrenaline. However, the addition of naloxone, atropine and propranolol had no significant effect. Somatostatin inhibited insulin release from isolated pancreatic islets, but did not antagonize the action of Acesulfame K. When 2.5 mM Acesulfame K was added to a medium containing somatostatin, the inhibitory effect of somatostatin was totally neutralized. In a perifusion system, Acesulfame K stimulated both phases of insulin secretion. In conclusion, Acesulfame K acts directly on the pancreatic islets and potentiates glucose-induced insulin release.

Opioid peptides inhibit the release of noradrenaline from slices of rat medial preoptic area.

Previous circumstantial evidence suggested that endogenous opioid peptides inhibit an excitatory noradrenergic projection to the medial preoptic area (MPOA), and thereby suppress the activity of neurones containing luteinising hormone-releasing hormone and thus systemic concentrations of luteinising hormone (LH) itself. In this paper, we report that electrically stimulated release of 3H-Noradrenaline (3H-NA) from perifused slices of rat MPOA is diminished when opioid agonists are added to the incubation medium. Thus, morphine (10 microM), beta-Endorphin (1 microM) and met-Enkephalin (1 microM), but not Dynorphin A (1-8) (1 microM), caused a significant decrease in electrically stimulated 3H-NA release. The inhibition was reversed by addition of naloxone (10 microM) to the perifusion medium but 3H-NA release was unaffected by dopamine or acetylcholine (or their antagonists sulpiride and atropine, respectively), or serotonin, neurotensin, muscimol or bicuculline (the latter two being agonist and antagonist respectively for the GABA A receptor). Therefore, the experiments provide direct evidence that brain opioids modulate the noradrenergic input to MPOA neurones and support the hypothesis that this may be one mechanism for the regulation of LH secretion.

Pituitary secretions related to adrenocorticotropic hormone induce sensitivity of adipose tissue to the insulin-like actions of growth hormone.

In its initial encounter with growth hormone (GH) in vitro, epididymal fat excised from GH-deficient rats responds with an insulin-like increase in glucose metabolism. Tissues freshly excised from normal rats are refractory to the insulin-like effects of GH, but become sensitive immediately after surgical stress. Reversal of refractoriness is prevented by administration of the opioid antagonist, naloxone, just prior to stress, suggesting a possible role of beta-endorphin or related peptides. These experiments were undertaken to determine the source of these peptides which might equally well be released from the pituitary, adrenal medullae, or nerve endings in response to stress. Since adrenalectomy, like stress, also results in increased secretion of adrenocorticotropic hormone (ACTH) and related peptides, we studied the effects of GH on glucose oxidation in adipose tissue obtained from adrenalectomized rats and found a significant insulin-like response to GH in tissues studied 4 days after adrenalectomy. This effect was not due to GH deficiency, since plasma concentrations were only slightly reduced by adrenalectomy. Administration of naloxone (250 micrograms/rat), 30 or 60 min before sacrifice, or dexamethasone (100 micrograms/injection), 60 and 120 min before sacrifice, prevented a response to GH without affecting circulating levels of GH. The effects of adrenalectomy could not be reproduced by preincubation of adipose tissue from normal nonstressed rats with ACTH and beta-endorphin, but were duplicated by preincubation of adipose tissue for 15 min in medium in which pituitary glands had previously incubated in the presence of corticotropin-releasing hormone (0.1 microM) and arginine vasopressin (0.2 microM). Addition of naloxone (250 micrograms/ml) blocked this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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 proposal 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 millimicron 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.

In vitro effects of endogenous opiate peptides on thyrotropin function: Inhibition of thyrotropin-releasing hormone release and absence of effect on thyrotropin release

Thyrotropin-releasing hormone (TRH) or thyroid-stimulating hormone (TSH) was measured by radioimmunoassay in the incubation medium of rat hypothalami or anterior pituitary halves, respectively. We studied the effect of opioid peptide addition (10 −8 to 10 −6 M) on TRH or TSH release. α- or β-Endorphin decreased TRH release in a dose-dependent manner while only 10 −6 M Leu- or Met-enkephalin decreased TRH release. These inhibitory effects were prevented by addition of naloxone (10 −5 M). In the dose range used none of the opioid peptides modified TSH release. These results indicate that opioid peptides may play a role in the regulation of thyrotropin secretion via a hypothalamic action on TRH release.

Effect of insulin on secretion of bombesin-like immunoreactivity and gastrin from the isolated rat stomach in response to acetylcholine, VIP and leucine-enkephalin

Bombesin-like immunoreactivity (BLI), a putative peptidergic neurotransmitter of the gastrointestinal intrinsic nervous system is released from the isolated perfused rat stomach in response to the classical neurotransmitter acetylcholine and in response to other putative peptidergic neurotransmitters such as vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI) or growth hormone releasing factor (GRF). The secretion of BLI is modulated not only by gastric factors such as the intragastric pH but also by changes of perfusate glucose concentrations indicating that alterations of carbohydrate metabolism might have an effect on gastric neuroendocrine regulation. Since previous studies have shown that insulin, the major regulatory hormone of glucose metabolism, reduces gastric somatostatin and glucagon secretion it was of interest to determine the effect of insulin on gastric BLI and gastrin secretion. The experiments were performed in the isolated perfused rat stomach model. The addition of porcine insulin to the perfusate at concentrations of 50 and 100 μU/ml had no effect on basal BLI and gastrin secretion. The infusion of acetylcholine (2 × 10 −6M and 4 × 10 −6M) elicited a stimulation of BLI and gastrin secretion which was not altered by the addition of insulin (100 μU/ml). On the other hand, significant effects of insulin were observed during administration of the two putative peptidergic neuro-transmitters VIP and leu-enkephalin. The infusion of VIP at 10 −11M and 10 −8M had no effect on BLI and gastrin secretion in the absence of insulin, however, with the addition of insulin (100 μU/ml) the higher dose of VIP (10 −8M) elicited a significant stimulation of BLI secretion while both doses of VIP (10 −11M and 10 −8M) significantly increased gastrin release. Simlar to VIP the infusion of leu-enkephalin at doses of 10 −9M and 10 −6M had no effect on BLI and gastrin secretion in the absence of insulin. When insulin was added to the perfusate both doses of leu-enkephalin elicited a significant stimulation of BLI secretion while gastrin remained unchanged. The addition of the specific opiate receptor antagonist naloxone (10 −5M) did not block the effect of leu-enkephalin in the presence of insulin. In addition the effect of naloxone was also examined during cholinergic stimulation. The addition of naloxone (10 −5M) during the infusion of acetylcholine abolished the stimulatory effect on BLI secretion in the absence of insulin, whereas in the presence of insulin naloxone did not alter cholinergically-induced BLI secretion. In conclusion the present data demonstrate that in the rat stomach insulin has no effect on cholinergically-induced BLI and gastrin secretion but it augments BLI secretion in response to VIP and leu-enkephalin and gastrin release in response to VIP. This suggests that insulin has a modulatory effect on the action and release of putative peptidergic neurotransmitters of the rat peripheral autonomic nervous system. Furthermore, endogenous opioids apparently mediate the cholinergic stimulation of BLI secretion and insulin antagonizes the effect of the opiate-receptor antagonist naloxone via as yet unknown mechanisms.

Naloxone reduces release of creatine kinase in the isolated ischemic rat heart.

The effects of naloxone, propranolol, or both on the release of creatine kinase (CK) from the isolated ischemic rat heart were studied. Naloxone at concentrations of 1.1 and 3.6 mmole liter-1 in the perfusate at a rate of 1-2 ml min-1 reduced the release of CK from the isolated ischemic rat heart during myocardial ischemia in a dose-dependent manner. Propranolol at a concentration of 7 mumole liter-1 in the perfusate also reduced the release of CK. Addition of naloxone (1.1 mmole liter-1) to propranolol further reduced the release of CK. The effect of the joint administration of the two drugs seemed to be additive.

Desensitization of opioid-stimulated GTPase in neuroblastoma × glioma hybrid cells

NG108-15 cells were pretreated with the opioid peptide [D-Ala 2, D-Leu 5]enkephalin and the opioid-dependent low Km GTPase was assayed in membranes. Pretreatment resulted in a small decrease in basal GTPase activity and led to a concentration-dependent reduction in opioid-mediated stimulation of the enzyme. These effects were observed whether the agonist was present or absent throughout all the experimental procedures, but, in the second condition, the desensitization was smaller. The addition of naloxone had no effect on basal GTPase activity, in either control or pretreated cell membranes. Both Na + and Mg ++ were required for the opioid-induced stimulation of the GTPase. Mg ++ enhanced basal enzymatic activity in controls, whereas in membranes from pretreated cells, it produced an inhibition. Thus, desensitization of the opioid-dependent low Km GTPase occurs upon chronic opioid treatment and a Mg ++ regulatory site might be altered in the course of this process.