The effect of chronic treatment with imipramine on the G proteins mRNA level in the rat hippocampus--an interaction with a calcium channel antagonist.

The effect of long-term administration of imipramine (10 mg/kg po, twice daily, 30 days) with or without nifedipine (5 mg/kg ip, twice daily, 28 days) on the G protein alpha subunit, Gs alpha, Go alpha and Gi alpha mRNA levels was investigated in the rat hippocampus. An in situ hybridization histochemistry showed that imipramine decreased the Go alpha mRNA level in CA1 (by ca. 40%) and CA3 (by ca. 37%) hippocampal fields and, to a lesser extent, in the dentate gyrus (by ca. 25%), but had no effect on the Gs alpha and Gi alpha mRNA levels in those structures. Nifedipine decreased (by ca. 30%) the Gs alpha level in the studied fields of hippocampal formation, having no influence on the level of mRNA which codes other subunits of G protein. Coadministration of nifedipine and imipramine reversed the imipramine effect on Go alpha, but had no effect on the nifedipine-induced decrease at the Gs alpha mRNA level. These results suggest that inhibition of L calcium channels modifies the effect of imipramine at the level of intracellular signal transduction.

Differential effects of opioid receptor agonists on nociception and cAMP level in the spinal cord of monoarthritic rats.

Changes in functional responsiveness of spinal opioid receptors in monoarthritic rats were investigated at the behavioral and the molecular level. After intrathecal administration of morphine, D-Ala2-D-Leu5-enkephalin (DADLE), D-Pen2-D-Pen5-enkephalin (DPDPE) and dynorphin monoarthritic rats showed an enhanced antinociceptive response as measured by a tail-flick latency. No such changes were observed following administration of the selective kappa agonists U50,488H and U69,593. The opioid mu and delta receptor agonists (0.1-1.0 microM) inhibited the basal, as well as the forskolin-stimulated cAMP formation in spinal cord slices obtained from monoarthritic rats, whereas no significant changes were found in control animals. Higher concentrations of the mu and delta opioid receptor agonists were required to attenuate the cAMP level in spinal cord of control animals. The selective kappa agonists U50,488H and U69,593 did not influence the cAMP formation in monoarthritic or control animals. Additionally, we found that the GppNHp-stimulated level of cAMP was higher in the spinal cord slices of monoarthritic rats, which points to an enhanced responsiveness of the adenylate cyclase effector system to the action of this GTP analog. Our data suggest that the enhanced antinociceptive response to intrathecally administered opioids in monoarthritic rats may be connected with the increased sensitivity of adenylate cyclase to the inhibitory effects of mu and delta agonists.

Effects of morphine treatment on pro-opiomelanocortin systems in rat brain.

In previous studies to determine whether chronic opiate administration might negatively feedback upon endogenous opioid systems in the CNS, investigators found no changes in steady-state concentrations of opioid peptides following morphine pelleting. However, since only steady-state levels were measured, it was still not clear whether morphine treatment altered the release and/or biosynthesis of opioid-containing neurons. The goal of the present study was to assess the effects of chronic morphine pelleting on the dynamics of beta-endorphin (beta E) biosynthesis in rats. Hence, at several times during a 7-day morphine treatment, concentrations of total beta E-immunoreactivity (-ir), as well as chromatographically sieved forms of beta E, were determined by RIA, and mRNA levels of pro-opiomelanocortin (POMC) were measured by a solution phase protection assay using a mouse or rat POMC 32P-labelled riboprobe. Concentrations of total beta E-ir or different forms of beta E-ir peptides (i.e. beta-lipotropin, beta E1-31, or beta E1-27/beta E1-26) in the hypothalamus or midbrain following either 1 or 7 days of treatment were similar in morphine- and placebo-pelleted animals. However, a significant increase in total hypothalamic beta E-ir was observed following 3 days of morphine pelleting; chromatographic analyses indicated that this was primarily due to a selective increase in the opiate inactive forms of beta E, i.e. beta E1-27/beta E1-26. After 7 days of pelleting, morphine-treated animals tended to have lower POMC mRNA levels than those of placebo controls (20 to 50% decrease in different studies). The accumulation of hypothalamic beta E-ir at 3 days, and the apparent decline in POMC mRNA levels at 7 days, lend support to the hypothesis that morphine negatively feeds back upon POMC neurons in the brain by inhibiting beta E release and biosynthesis.

Involvement of endogenous opioid peptides in fenfluramine anorexia.

The effect of chronic fenfluramine (20 mg/kg, once daily) injections on the brain and peripheral immunoreactive (ir) dynorphin (DYN), alpha-neoendorphin (ANEO) and beta-endorphin (BE) was studied in rats. Fenfluramine injected repeatedly for 5 and 9 days induced anorexia. In the same animals there were no significant changes in the ir-DYN and ir-ANEO contents in the brain and pituitary. However, the ir-DYN and ir-ANEO contents in the gastrointestinal tract (duodenum) were markedly decreased after 5 and 9 days of fenfluramine injection. In contrast to ir-DYN and ir-ANEO, there was an increase in the hypothalamic and a decrease in the anterior lobe of pituitary ir-BE content. There was no significant change in the neurointermediate (NI) lobe of the pituitary. The results of our study suggest that part of the fenfluramine anorexia may be mediated by the peripheral prodynorphin and central beta-endorphin systems.

Brain and peripheral opioid peptides after changes in ingestive behavior.

The levels of dynorphin, alpha-neoendorphin and beta-endorphin immunoreactivity (ir-DYN, ir-alpha-NEO, ir-beta-E) were measured in the brain, pituitary and gut of rats subjected to a variety of manipulations. Water deprivation caused an increase in the ir-DYN and ir-alpha-NEO content in the hypothalamus and a decrease in the neurointermediate (NI) lobe of the pituitary. The ir-beta-E level decreased in the hypothalamus and anterior lobe of the pituitary, while it increased in the NI-pituitary. Food deprivation, as well as chronic fenfluramine (10-20 mg/kg) treatment increased, while acute muscimol (0.5 micrograms/10 microliter) treatment decreased the ir-beta-E, but not ir-DYN or ir-alpha-NEO content in the hypothalamus. The anterior pituitary content of ir-beta-E was increased after food deprivation and decreased after chronic fenfluramine treatment. However, the ir-DYN and ir-alpha-NEO contents in the duodenum were markedly increased after food deprivation, while chronic fenfluramine treatment led to a dramatic decrease in the ir-DYN content. These results suggest that the levels of opioid peptides in the brain, pituitary and gut may be differentially and independently affected by alteration of the ingestive behavior.

Distribution of immunoreactive alpha-neo-endorphin in the rat brain.

The distribution of immunoreactive alpha-neo-endorphin (alpha-Neo) in the rat brain was demonstrated by means of highly specific radioimmunoassay (RIA). The highest concentrations of immunoreactive (ir) alpha-Neo (100 pmol/g tissue) were found in the substantia nigra, the hypothalamic supraoptic nucleus (86 pmol/g) and the preoptic area (63 pmol/g); medium levels were found in the basal ganglia, mesencephalic reticular formation, nucleus accumbens and hippocampus. Other structures contained less alpha-Neo-ir. The possible anatomical interaction between alpha-Neo and monoaminergic nerve terminals or cell bodies in some structures is discussed.

The effect of food and water deprivation on post-stress analgesia in mice and levels of beta-endorphin and dynorphin in blood plasma and hypothalamus.

Pain sensitivity of food and/or water-deprived male mice was tested on a hotplate. The most pronounced analgesia ensued in animals given no food and water, and no food but water ad libitum, the least one in water-deprived mice. The magnitude of the rise in pain threshold depended on the duration of deprivation and was correlated with the increase in the blood plasma beta-endorphin level. In the hypothalamus beta-endorphin level increased after 72-h food deprivation only. The level of dynorphin remained unchanged. Naloxone (10 mg/kg) almost completely reversed food or water-deprivation induced analgesia.

Serotonergic regulation of the brain and gut beta-endorphin and dynorphin content in the rat.

A specific radioimmunoassay was used to measure immunoreactive dynorphin (ir-DYN) and beta-endorphin (ir-BE) in the brain, pituitary and gut, following a pharmacological manipulation of the serotonin system. Administration of the serotonin receptor agonist m-chlorophenylpiperazine (m-CPP, 2.5-5 mg/kg ip) or the serotonin releasing agent fenfluramine (20-40 mg/kg ip) induced a significant increase in the hypothalamic ir-BE content and a decrease in its anterior pituitary level. These effects were antagonized by cyproheptadine (1 mg/kg ip). Similar results were obtained after fluvoxamine (15 mg/kg ip), femoxetine (10 mg/kg ip) and 5-hydroxytryptophan (5-HTP 40-160 mg/kg ip). None of the above treatments altered significantly the ir-DYN content in the brain and pituitary. However, the gut ir-DYN level was dramatically decreased after m-CPP, fenfluramine, fluvoxamine, femoxetine and 5-HTP. The latter effect was antagonized by cyproheptadine. The obtained results suggest that serotonergic activation stimulates the release of beta-endorphin from the anterior pituitary and dynorphin from the gut, while the cerebral beta-endorphin system appears to be inhibited by activation of serotonin neurons.

Differential regulation of the brain and gut immunoreactive dynorphin by the serotonin system.

Administration of drugs such as fenfluramine, 20-40 mg/kg, and m-chlorophenylpiperazine (m-CPP), 2.5-5 mg/kg, which release serotonin or activate postsynaptic serotonin receptors, respectively, induced a dramatic decrease in the duodenal content of immunoreactive dynorphin (ir-DYN). The effect was antagonized by cyproheptadine, 1 mg/kg. Similarly, acute administration of the specific serotonin reuptake blockers fluvoxamine, 15 mg/kg, or femoxetine, 10 mg/kg, and 5-hydroxytryptophan (5-HTP), 40-160 mg/kg, evoked a marked decrease in the duodenal content of ir-DYN. A combined administration of fluvoxamine or femoxetine and 5-HTP failed to potentiate the effect of individual treatment. Only a higher dose of fenfluramine, 40 mg/kg, increased the ir-DYN content in the hypothalamus. These results suggest that the brain and gut ir-DYN is independently regulated by the serotonin system and that a serotonin mechanism might stimulate release of the gut dynorphin content.

Differential modulation of the beta-endorphin and dynorphin systems by serotonergic stimulation in the rat.

The serotonergic modulation of the brain, pituitary and gut beta-endorphin and dynorphin systems in the rat was determined pharmacologically. Acute administration of fenfluramine (20 mg/kg), m-chlorophenylpiperazine (m-CPP 2.5 mg/kg), fluvoxamine (15 mg/kg) and 5-hydroxytryptophan (5-HTP 160 mg/kg) increased immunoreactive (ir)beta-endorphin (beta E) in the hypothalamus and decreased it in the anterior lobe of the pituitary. That effect was antagonized by cyproheptadine (1 mg/kg). None of the treatments altered significantly ir-dynorphin (DYN) level in the hypothalamus and pituitary, however, ir-DYN in the gut was dramatically decreased after fenfluramine, m-CPP, fluvoxamine, femoxetine and 5-HTP, the latter effects being antagonized by cyproheptadine. The obtained results suggest that the serotonin system might stimulate the release of the anterior pituitary beta-endorphin and gut dynorphin pools, while the brain beta-endorphin system appears to be inhibited by activation of serotonin neurons.

Evidence for a role of the ventro-medial posterior hypothalamus in nociceptive processes in the rat.

Bilateral, radio-frequency destruction of the ventro-medial posterior hypothalamus (VMPH) resulted, as compared to sham-operated and control rats and evaluated in the tail-flick and vocalization tests, in a significant decrease in basal nociceptive threshold on day 4 post-surgery. By day 12, however, no significant difference between sham and lesioned rats was seen. At this time the antinociception elicited by either acute foot-shock or cold-water-immersion stress was profoundly attenuated. The antinociceptive response to various doses of morphine was not, in contrast, diminished. As established by use of radioimmunoassay, these lesions did not significantly alter hypothalamic levels of beta-endorphin, met-enkephalin, dynorphin or alpha-neo-endorphin. They did, however, produce a pronounced and significant fall in the hypothalamic content of substance P. These data are indicative that the VMPH may, via a mechanism not involving endorphins, be of importance in the determination of basal nociceptive threshold and in the generation of stress-, but not morphine-, evoked antinociception. The relationship of these findings to the interconnections of the VMPH, and to the possible significance of substance P and the pituitary in nociceptive processes, is discussed.

Endogenous opioids, circadian rhythms, nutrient deprivation, eating and drinking.

Immunoreactive (ir) beta-endorphin (b-END) and dynorphin (DYN) in rat brain and pituitary were measured after food and water deprivation and from brains taken during either day or night. In other rats, eating and drinking were measured following lesions in the arcuate n. Ir-DYN levels are higher in hypothalamus and lower in pituitary at night. Deprivation, particularly water deprivation, increases hypothalamic, day-time ir-DYN. Water deprivation decreases pituitary levels of ir-DYN. Arcuate-lesions, depleting both ir-b-END and ir-DYN, do not modify total daily intake of water or food but does modify circadian rhythmicity of eating and drinking. These data support the conclusion that b-END and DYN are involved in maintaining day-night patterns of eating and drinking.

Stress-induced release of brain and pituitary beta-endorphin: major role of endorphins in generation of hyperthermia, not analgesia.

The present paper examines the conjectured causal relationship between the alterations in brain, pituitary and plasma levels of endorphins and the antinociception (analgesia) and hyperthermia elicited by acute stress. A 5-min foot-shock instigated a significant depression in the levels of beta-endorphin immunoreactivity (beta-EI) in both the hypothalamus and periventricular beta-endorphinergic fibre-containing tissue. A large elevation in plasma levels of beta-EI, consisting of about 70% beta-endorphin (beta-EP), and 30% beta-lipotropin (beta-LPH) was associated with a significant reduction in the beta-EI content of both the anterior (AL) and neurointermediate (NIL) lobes of the pituitary. No concomitant changes in the levels of Met-enkephalin immunoreactivity (M-EI) in discrete areas of brain and pituitary were detectable. Application of a high (10 mg/kg) but not a low (1 mg/kg) dose of naloxone, prior to foot-shock, slightly reduced the increase in tail-flick latency evoked by this stress. In contrast, both of these doses strongly and dose-dependently attenuated the accompanying rise in core temperature (Tc). Chronic (approximately 30 day) morphine treatment resulted in a 45% decrease in the NIL content of beta-EI and a clear depression in its basal plasma levels, although a substantial post-stress rise in plasma beta-EI was still found: stress-induced analgesia (SIA) was enhanced, but the concurrent stress-induced hyperthermia (SIH), reduced in morphinized animals. These data demonstrate that stress produces a generalized mobilization of both central and pituitary pools of beta-EI, and indicate that endorphins may play a more important role in the mediation of changes in Tc than in the generation of the concomitant increase in nociceptive threshold, upon activation by stress.

Release of beta-endorphin from rat hypothalamus in vitro.

The rate of release of beta-endorphin-like immunoreactivity (beta-EI) from rat hypothalamic slices was increased 3- to 4-fold over the spontaneous release during exposure to a depolarizing concentration of potassium ions. This augmented outflow was abolished in the absence of calcium. The amount of beta-EI released from slices exposed to a second pulse of potassium was reduced by approximately 50% compared to that in the first. 70% of the potassium-induced released immunoreactive material migrated on a calibrated Sephadex G-50 column like synthetic human beta-endorphin. These findings are discussed in terms of a neuronal release of beta-endorphin and may be taken as supportive evidence for a neurotransmitter and/or neurohormonal role of beta-endorphin in the hypothalamus.