Met-enkephalin alteration in the rat during chronic injection of morphine and/or midazolam

doi:10.1016/S0006-8993(97)00875-5

Brain Research

Volume 775, Issues 1–2, 14 November 1997, Pages 119-126

https://doi.org/10.1016/S0006-8993(97)00875-5 Get rights and content

Abstract

We have recently reported that the short-acting anesthetic and analgesic drug midazolam can produce analgesia and decrease morphine tolerance and dependence in the rat by interacting with the opioid system. This study was designed to investigate the effect of midazolam, morphine, and both together on met-enkephalin levels in the rat. Male Sprague–Dawley rats were divided into four groups: (1) saline-saline; (2) saline-morphine; (3) midazolam-saline, and (4) midazolam-morphine groups. First, a saline or midazolam injection was given intraperitoneally and after 30 min a second injection of saline or morphine was given subcutaneously once daily for 11 days. Animals were sacrificed on the 11th day 60 min after the last injection to measure met-enkephalin by radioimmunoassay. Morphine tolerant animals showed a significant increase in met-enkephalin levels in the cortex (137%) and midbrain (89%), and a significant decrease in met-enkephalin levels in the pituitary (74%), cerebellum (34%) and medulla (72%). Midazolam treated animals showed a significant decrease in met-enkephalin levels in the pituitary (63%), cortex (39%), medulla (58%), kidneys (36%), heart (36%) and adrenals (43%), and a significant increase in met-enkephalin levels in the striatum (54%) and pons (51%). When morphine and midazolam were injected together, midazolam antagonized the increase in met-enkephalin levels in cortex and midbrain region and the decrease in met-enkephalin level in the medulla region observed in morphine tolerant animals. These results indicate that morphine tolerance and dependence is associated with changes in the concentration of met-enkephalin in the brain. Midazolam may inhibit morphine tolerance and dependence by reversing some of the changes induced in met-enkephalin levels in brain by morphine in morphine tolerant and dependent animals.

Introduction

One of the most important developments of recent years in neurobiology has been recognition of the widespread distribution of neuroactive peptides in the nervous system. Enkephalins are two small pentapeptides that bind to opioid receptors and produce analgesic effects when injected into animals 2, 4, 9, 19, 43. The control of pain by endogenous opioid peptides is far more complex than originally thought. Basically, opioid peptides control nociception at the level of the periaqueductal gray, the medulla, and the spinal cord 3, 43. The spinal modulation of nociception may involve either a segmental control system or a brainstem-spinal descending control system involving β-endorphin, enkephalins, and dynorphin. Immunocytochemical and radioimmunoassay techniques have been extensively used to map this neuronal network, within the brain and spinal cord, where they have shown a regional and distinct distribution of the major endogenous opioid peptide system 5, 11, 44.

Specific changes in the endogenous opioid peptide system occur as a result of chronic administration of opioid agonists or antagonists. Chronic administration of morphine inhibits the release and biosynthesis of β-endorphin by exerting a negative feedback on pro-opiomelanocortin neurons in the brain [7]and increases the concentration of FMRFamide in the cerebrospinal fluid, altering the balance of opiate and antiopiate peptides [25]. It is also reported that chronic administration of morphine, but not of naltrexone, enhances benzodiazepine binding and GABA_A receptor function [23]. In animal behavioral studies, opioid and benzodiazepine antagonists have overlapping effects. For example, the opioid antagonist naloxone blocks benzodiazepine induced analgesia in rats [30], while a benzodiazepine antagonist inhibits the respiratory depressant effects of opiates [26]. In addition, histochemical studies have also demonstrated that GABA and opioid peptides co-localize within neurons in several brain regions [20].

Midazolam, an ultrashort acting benzodiazepine, is used clinically for preoperative medication to induce general anesthesia [46]. Midazolam is also administered to supplement opioids or inhaled anesthetics during maintenance of anesthesia 37, 46. Recently, we observed that midazolam administration inhibits development of morphine tolerance and dependence in the rat [40]. We also observed that midazolam effects are mediated by alteration in the CNS β-endorphin [32]and dynorphin_1–13[33]levels. We now report that midazolam treatment also counteracts the changes in the CNS met-enkephalin levels induced by chronic morphine treatment.

Section snippets

Animals

Adult male Sprague–Dawley rats, weighing 250–275 g were used. They were kept on a normal day and night cycle, were housed in groups of three to four animals and given free access to food and water in the home cages.

Materials

3-[Iodotyrosyl-¹²⁵I]enkephalin (5-l-methionine) was obtained from Amersham (Arlington Heights, IL). Dextran T-70 was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Sodium chloride, octyl phenoxypolyethoxyethanol (Triton X-100), gelatin, sodium ethylmercurithiosalicylate

Effect of chronic treatment with morphine on met-enkephalin levels in pituitary gland, discrete brain regions, spinal cord and peripheral tissues in the rat

Morphine tolerant animals showed a significant decrease (74%) in met-enkephalin level in pituitary (F_1,15=63.21, P<0.01, Fig. 1), medulla (72%, F_1,15=35.52, P<0.01, Fig. 2) and cerebellum (34%, F_1,15=5.96, Fig. 3). A highly significant increase in met-enkephalin level was observed in the midbrain (89%, F_1,15=33.26, P<0.01, Fig. 3) and cortex (137%, F_1,15=17.17, P<0.01, Fig. 3). Morphine tolerance did not produce any change in the level of met-enkephalin in hypothalamus, striatum, lumbar region

Discussion

We have earlier reported that rats injected with morphine (10 mg/kg b.wt., once daily for 11 days) develop morphine tolerance and dependence. Injection of midazolam along with morphine delayed the development of morphine tolerance and dependence [40]. The present study is the first report to elucidate in detail the interaction between morphine and midazolam, a clinically used benzodiazepine receptor agonist, in altering the level of met-enkephalin in the CNS and peripheral tissues of rat.

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¹: Present address: St. Francis Hospital, DeMatteis Center, 100 Port Washington Blvd., Roslyn, NY 11576, USA.

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Research reportMet-enkephalin alteration in the rat during chronic injection of morphine and/or midazolam

Abstract

Introduction

Section snippets

Animals

Materials

Effect of chronic treatment with morphine on met-enkephalin levels in pituitary gland, discrete brain regions, spinal cord and peripheral tissues in the rat

Discussion

Eur. J. Pharmacol.

Behav. Brain Res.

Brain Res.

Eur. J. Pharmacol.

Biochem. Pharmacol.

Brain Res.

Prog. Brain Res.

Neurosci. Lett.

Brain Res.

Peptides

Life Sci.

Behav. Brain Res.

Brain Res.

Brain Res.

Brain Res. Bull.

Brain Res.

Brain Res.

Anal. Biochem.

Brain Res.

Peptides

Regul. Pept.

Tolerance of locus coeruleus neurons to morphine and suppression of withdrawal response by clonidine

Nature

Endogenous pain control systems: brainstem spinal pathways and endorphin circuitry

Annu. Rev. Neurosci.

Research report
Met-enkephalin alteration in the rat during chronic injection of morphine and/or midazolam