Thyrotropin-releasing hormone stimulates the release of melanotropin from frog neurointermediate lobes in vitro

The hypothalamus of Amphibia contains large amounts of tripeptide P-Glu-His-Pro-NH₂ (mammalian thyrotropin-releasing hormone, TRH). However, synthetic TRH is unable to stimulate thyrotropin release from frog pituitary gland. The recent discovery of TRH in the skin of the frog suggests a possible role of this peptide in skin-colour adaptation. Thus we have investigated the role of TRH upon melanotropin (α-MSH) release from perifused frog neurointermediate lobes. A dose related increase in α-MSH release was observed when TRH was added to the perifusion medium. Half-maximum stimulation occurred with the 1 × 10^?8M dose. Theophylline at a dose of 2 × 10^?3M strongly enhanced TRH-induced α-MSH release, indicating that cyclic AMP may be the second messenger. α-MSH releade was not modified by crude homogenates of rat hypothalamus but was significantly reduced when the hypothalamus extracts were preincubated with specific TRH antibodies. As far is known, these results provide the first evidence that P-Glu-His-Pro-NH₂ stimulates the release of α-MSH from frog neurointermediate lobes $\text{in vitro}$. The present findings suggest a possible feedback loop between skin TRH and pituitary MSH in Amphibia.     

Autonomous beta-endorphin secretion from the pituitary neurointermediate lobe: in vivo studies

The existence of independent control mechanisms of beta-endorphin (beta-EP) secretion from the anterior (AP) and intermediate (NIL) pituitary lobes is now ascertained. The aim of this study was to evaluate the effect of surgical separation from the hypothalamus of the two pituitary lobes on beta-EP secretion. Two experimental models of surgical hypothalamo-pituitary disconnection were used: 1) rats with ablation of the medial basal hypothalamus (MBH); 2) rats bearing two entire ectopic pituitaries or two anterior pituitaries (APs) only, transplanted under the kidney capsule. In rats with MBH-ablation plasma beta-EP levels were significantly higher than in sham-operated controls. Plasma beta-EP levels increased in rats transplanted with entire pituitaries 3 days after surgery and were still elevated after 1 week. In rats transplanted with APs only, no significant beta-EP changes in plasma were evident. In both experimental conditions no significant difference was present in beta-LPH plasma levels. Concentrations of beta-EP in the ectopic NILs decreased gradually after transplantation. In all these results indicate that that NIL but not the AP is capable, when is disconnected from the hypothalamus, or secreting autonomously beta-EP.     

Chronic ethanol treatment alters the biosynthesis of beta-endorphin by the rat neurointermediate lobe

Tolerance to ethanol was induced in male Sprague-Dawley rats (225-250 g) by chronic feeding with a liquid diet containing 6.5% ethanol (v/v). Control rats were pair-fed with a liquid diet in which the ethanol was replaced by an equicaloric concentration of sucrose. Immediately following sacrifice of the animals the neurointermediate lobes (NIL) were removed and incubated with [3H]phenylalanine. The biosynthesized proopiomelanocortin (POMC), beta-lipotropin (beta-LPH), and beta-endorphin (beta-EP) were purified by immunoprecipitation with an antiserum to beta-EP and analyzed by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. Alcohol treatment for 3 days had no effect on the degree of incorporation of [3H]phenylalanine into POMC, beta-LPH, and beta-EP but treatment for either 15 or 21 days increased the incorporation of [3H]phenylalanine into all three peptides. Ethanol treatment also increased the beta-endorphinlike immunoreactivity (beta-EPLIR) found in the incubation medium, but no significant change was observed in the beta-EPLIR extracted from the NIL either immediately after sacrifice or after 3 h of incubation of the NIL. However, a significant decrease of beta-EPLIR was found in the anterior lobes of rats treated with ethanol for 21 days. Furthermore, the beta-EPLIR in the serum of alcohol-treated rats was significantly higher than in the serum of their corresponding controls. These results indicate an effect of ethanol on the endorphin system and are consistent with the suggestion that endorphins may be mediators of some of the ethanol effects.     

Acetylation of alpha MSH and beta-endorphin by rat neurointermediate pituitary secretory granule-associated acetyltransferase

ACTH(1-8) and ACTH(9-13)NH2 were used as potential enzyme inhibitors to begin examining the relationship between the acetylation of ACTH- and beta-endorphin-related peptides. ACTH(1-8) was a potent inhibitor of the acetylation of both ACTH- and beta-endorphin-related peptides, whereas ACTH(9-13)NH2 was an effective inhibitor only of the acetylation of ACTH-related substrates. This inhibition pattern indicated that there may be an unusual interaction between some ACTH- and beta-endorphin-related peptides as substrates for the acetyltransferase. Utilizing HPLC to separate ACTH- and beta-endorphin-related peptides present in the same reaction mixture, ACTH(1-14) and beta-endorphin(1-27) at Km and saturating concentrations were used as substrates to examine the ability of one peptide substrate to affect the acetylation of the other. It was observed that the acetylation of ACTH(1-14), even at Km concentration, was relatively unaffected by the presence of beta-endorphin(1-27). However, the acetylation of beta-endorphin(1-27) was significantly reduced by the presence of ACTH(1-14). This preferential acetylation of ACTH-related peptides over the acetylation of beta-endorphin-related peptides might have physiological importance under some conditions.     

The benzodiazepine agonist clonazepam potentiates the effects of gamma-aminobutyric acid on alpha-MSH release from neurointermediate lobes in vitro

The action of the central-type benzodiazepine-receptor agonist clonazepam on alpha-MSH release has been studied in vitro using perifused frog neurointermediate lobes. High concentrations of clonazepam (3.16 X 10(-5) and 10(-4) M) caused an inhibition of alpha-MSH release and this effect was reversed by the central-type benzodiazepine-receptor antagonist Ro 15-1788. High doses of GABA (10(-5) and 10(-4) M) induced a biphasic effect on pars intermedia cells: a brief stimulation followed by a sustained inhibition of alpha-MSH secretion. Administration of clonazepam (10(-5) M) in the presence of various concentrations of GABA (10(-6) to 10(-4) M) led to a potentiation of both stimulatory and inhibitory phases of alpha-MSH secretion induced by GABA. Ro 15-1788 completely abolished the potentiating effect of clonazepam. Thus our results indicate that endogenous benzodiazepine receptors may modulate the effects of GABA on alpha-MSH secretion.     

Modulation of in vitro release of beta-endorphin from the separate lobes of the rat pituitary.

The rate of in vitro release of β-endorphin immunoreactivity from the anterior lobe of rat pituitary increased in response to hypothalamic extract and lys-vasopressin. Lys-vasopressin, at a low concentration, initiated a pronounced (5–6 fold) dose-dependent, parallel increase in the release of β-endorphin and ACTH from the anterior lobe. Corticosterone (5·10^?7 M) did not influence basal but could suppress such stimulated release. These stimulants did not, however, change the rate of release from the intermediate/posterior lobe.Chromatography of incubation media showed that β-endorphin and β-lipotropin were released in parallel from the anterior lobe but only β-endorphin from intermediate/posterior lobe tissue.These findings suggest that the β-endorphin pools in anterior and intermediate lobes differ both in their mechanism of release and in the regulation of this process.     

Benzodiazepine suppression of corticotropin-releasing factor (CRF)-induced beta-endorphin release from rat neurointermediate pituitary.

Dopamine and gamma-aminobutyric acid (GABA) inhibit POMC peptide release from the pituitary intermediate lobe, via interaction with D2 or GABA-A/benzodiazepine receptors. Here, we examined the effects of an antianxiety triazolobenzodiazepine, adinazolam, on corticotropin-releasing factor (CRF)-stimulated POMC peptide secretion from the rat neurointermediate pituitary. Neurointermediate lobes (NILS) were incubated with CRF (10(-7) M), then adinazolam (10(-8) or (10(-9) M) was added, with CRF remaining in the medium. Aliquots were removed at 15-min intervals and frozen for radioimmunoassay of beta-endorphin. Adinazolam alone did not significantly affect secretion as compared to controls or CRF alone. Adinazolam incubated with CRF led to significant inhibition of beta-endorphin secretion, as compared to CRF alone. In addition, adinazolam was as effective as dopamine or the CRF antagonist, alpha-helical CRF, in preventing CRF-induced beta-endorphin release. Adinazolam appears to act directly on the pituitary to suppress hormone release induced by a stress-related hypothalamic peptide.     

Dopamine-sensitive adenylate cyclase of the caudate nucleus of rats treated with morphine.

The addition of narcotic analgesics $\text{in vitro}$ to nerve ending preparations from rat caudate nucleus in an assay of adenylate cyclase activity (AC) resulted in an inhibition of basal AC only at drug concentrations of 10−⁴M or higher, and no inhibition of dopamine-stimulated (DA) AC at these drug concentrations. The acute administration of morphine at a moderately high dose (60 mg/kg) produced an increase in striatal cAMP levels, and increases in basal and DA-AC in caudate nerve-endings. In morphine-tolerant rats, striatal cAMP levels and basal AC were similar to control values, while DA-AC was elevated. These results suggest: (1) that opiates do not act directly on DA-AC, the ‘dopamine receptor’, and (2) that the observed behavioural DA sensitivity in tolerant animals may be produced by the DA-AC supersensitivity.     

Lack of antinociceptive cross-tolerance between intracerebroventricularly administered beta-endorphin and morphine or DPDPE in mice

Antinociceptive tolerance and cross-tolerance to intracerebroventricular (i.c.v.) beta-endorphin, morphine, and DPDPE (D-Pen2-D-Pen5-enkephalin) induced by a prior i.c.v. administration of beta-endorphin, morphine and DPDPE, respectively, were studied in mice. Acute tolerance was induced by i.c.v. pretreatment with beta-endorphin (0.58 nmol), morphine (6 nmol) and DPDPE (31 nmol) for 120, 180 and 75 min, respectively. Various doses of beta-endorphin, morphine or DPDPE were then injected. The tail-flick and hot-plate tests were used as antinociceptive tests. Pretreatment of mice with beta-endorphin i.c.v. reduced inhibition of the tail-flick and hot-plate responses to i.c.v. administered beta-endorphin, but not morphine and DPDPE. Pretreatment of mice with morphine i.c.v. reduced inhibition of the tail-flick and hot-plate responses to morphine but not beta-endorphin. Pretreatment of mice with DPDPE reduced inhibition of the tail-flick and hot-plate responses to DPDPE but not beta-endorphin. The results indicate that one injection of beta-endorphin, morphine or DPDPE induces acute antinociceptive tolerance to its own distinctive opioid receptor and does not induce cross-tolerance to other opioid agonists with different opioid receptor specificities. The data support the hypothesis that beta-endorphin, morphine and DPDPE produce antinociception by stimulating specific epsilon, mu- and delta-opioid receptors, respectively.     

Effects of morphine on adrenaline responses of uteri from progesterone or estradiol treated mice

1. The effect of a chronic morphine treatment on the in vitro contractile responses of the mouse uterus to adrenaline was studied. 2. Chronic morphine treatment induced a supersensitivity state in the uteri from both progesterone and estradiol treated mice. 3. The acute administration of morphine to the uteri from morphine tolerant-dependent and progesterone treated mice induced a further increase of the contractile effect of adrenaline. 4. Reserpine administration did not further increase the supersensitivity of the mouse uterus to adrenaline induced by a chronic morphine treatment. 5. Reserpine suppressed the acute effects of morphine in the uteri from tolerant-dependent mice.     

Methionine-enkephalin and beta-endorphin levels in spleen and thymus gland of morphine tolerant-dependent and abstinent rats

The effect of chronic administration of morphine and abrupt and naloxone-precipitated withdrawal on the levels of beta-endorphin and methionine-enkephalin in spleen, adrenals and thymus glands of Sprague-Dawley rats was determined. Rats were made tolerant to and dependent on morphine by subcutaneous implantation of 6 morphine pellets (75 mg morphine in each) during a 7-day period. The tolerant-dependent (with pellets intact) and abstinent (pellets removed 18 hours earlier) rats were sacrificed. In another group, rats with pellets intact were injected with naloxone and sacrificed 10 min later (precipitated abstinence). The weights of the tissues under any of the above treatments did not change nor did the levels of methionine-enkephalin and beta-endorphin in adrenals. The level of beta-endorphin was elevated in the spleen and thymus of morphine tolerant-dependent rats, while the levels of methionine-enkephalin in rats undergoing abrupt or naloxone-precipitated abstinence were significantly higher than in their respective placebo controls. The levels of methionine-enkephalin in the thymus gland of rats with placebo and morphine pellets left intact did not differ. It is concluded that in morphine tolerant-dependent rats the levels of beta-endorphin in spleen and thymus are elevated. During abrupt and naloxone-precipitated abstinence, the levels of methionine-enkephalin in the thymus gland are significantly elevated possibly due to an inhibition of their release. Since these opioid peptides have been implicated in immunomodulation, and alterations were seen in organs controlling immune function, the present results may be helpful in explaining altered immune function in morphine dependent and abstinent states.     

Increase of dopamine synthesis in synaptosomes from rats treated with neuroleptics or reserpine

Dopamine (DA) synthesis in rat striatum was increased three- to four-fold by in vivo treatment with gammabutyrolactone (GBL), reserpine, haloperidol and (-)sulpiride. DA synthesis in striatal synaptosomes (measured by formation of 14CO2 from labelled tyrosine) did not change after GBL and only doubled after reserpine and neuroleptic administration. The increase of synaptosomal DA synthesis was proportional to and probably due to kinetic activation of tyrosine hydroxylase which, after neuroleptic drugs, remained activated for at least 15 min in synaptosomal incubations at 37 degree C.     

The stimulatory effect of beta-endorphin on the plasma prolactin levels was diminished in the rats treated with 6-hydroxydopamine

Intraventicular injection of beta-endorphin (beta LPH_61^?91) in urethane anesthetized male rats led to a dose dependent increase of plasma prolactin levels. Intravenous injection of apomorphine completely abolished the stimulatory effect of beta-endorphin. Animals treated with 6-hydroxydopamine (6-OHDA) and 6-OHDA plus desmethylimipramine showed inhibition of beta-endorphin induced prolactin release. These results suggest that beta-endorphin presynaptically inhibits the activity of dopaminergic neurones, leading to the stimulation of plasma prolactin levels.     

Withdrawal and bidirectional cross-withdrawal responses in rats treated with adenosine agonists and morphine

The aim of this study was to investigate whether the A1/A2 receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), and the selective A1 agonist, N6-cyclopentyladenosine (CPA), induced physical dependence by quantifying specific antagonist-precipitated withdrawal syndromes in conscious rats. In addition, the presence of bidirectional cross-withdrawal was also investigated. The agonists were administered s.c. to groups of rats at 12 h intervals. Antagonists were administered s.c., 12 hours after the last dose, followed by observation and measurement of faecal output for 20 min. NECA (4 x 0.03 mg kg(-1), s.c) and CPA (4 x 0.03, 0.1 and 0.3 mg kg(-1), s.c.) induced physical dependence, as shown by the expression of a significant withdrawal syndrome when challenged with the adenosine A1/A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX, 0.1 mg kg(-1), s.c.) and the A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPDPX, 0.1 mg kg(-1), s.c.) respectively. The syndromes consisted of teeth chattering and shaking behaviours shown to occur in morphine-dependent animals withdrawn with naloxone viz, paw, body and 'wet-dog' shakes, but with the additional behaviours of head shaking and yawning. In further contrast to the opiate withdrawal syndrome, no diarrhoea occurred in the groups of animals treated with adenosine agonists and withdrawn with their respective antagonists. Bidirectional cross-withdrawal syndromes were also revealed when naloxone (3 mg kg(-1), s.c.) was administered to adenosine agonist pre-treated rats and adenosine antagonists were given to morphine pre-treated rats. This study provides further information illustrating that close links exist between the adenosine and opiate systems.     

Stimulation of the electron transport chain in mitochondria isolated from rats treated with mannoheptulose or glucagon

We investigated the kinetics of the mitochondrial respiratory chain, proton leak, and phosphorylating subsystems of liver mitochondria from mannoheptulose-treated and control rats. Mannoheptulose treatment raises glucagon and lowers insulin; it had no effect on the kinetics of the mitochondrial proton leak or phosphorylating subsystems, but the respiratory chain from succinate to oxygen was stimulated. Previous attempts to detect any stimulation of cytochrome c oxidase by glucagon are shown by flux control analysis to have used inappropriate assay conditions. To investigate the site of stimulation of the respiratory chain we measured the relationship between the thermodynamic driving force and respiration rate for the span succinate to coenzyme Q, the cytochrome bc1 complex and cytochrome c oxidase. Hormone treatment of rats altered the kinetics of electron transport from succinate to coenzyme Q in subsequently isolated mitochondria and activated succinate dehydrogenase. The kinetics of electron transport through the cytochrome bc1 complex were not affected. Effects on cytochrome c oxidase were small or nonexistent.     

Stimulation of beta-endorphin and beta-lipotropin release from the anterior but not the neurointermediate pituitary lobe in the rat after acute administration of serotonin-acting drugs

The respective contribution of the anterior (AP) and the neuro-intermediate (NIL) lobes of the pituitary gland to changes occuring in plasma β-endorphin (β-EP) and β-lipotropin (β-LPH) titers has been evaluated in the rat after administration of serotonin (5-HT)-acting drugs. β-EP-like immunoreactivity (β-EP-LI) was concurrently evaluated in the mediobasal hypothalamus (MBH). The administration of 50 mg/kg DL 5-hydorxytryptophan (5-HTP) or 12.5 mg/kg fluvoxamine, a 5-HT reuptake blocker, decreased markedly β-EP-LI in the AP and induced a striking rise in plasma β-EP and β-LPH concentrations. Combined administration of fluvoxamine and 5-HTP failed to potentiate the effect of individual treatments. Similarly, administration of 5.0 and 10 mg/kg quipazine, a 5-HT receptor agonist, evoked a marked decrease in β-EP-LI in the AP and a concomitant rise in β-EP and β-LPH concentrations in the plasma, while administration of 1.0 and 5 mg/kg of chlorophenylpiperazine, a weak 5-HT stimulant drug, did not alter the above indices. None of these treatments altered significantly β-EP-LI in the NIL and only the higher dose of quipazine increased it in the MBH. We conclude that brain serotonin neurons exert a stimulatory influence on β-EP and β-LPH release from the AP but, likely, not from the NIL and that hypothalamic endorphins are not implicated in the secretory events occuring at AP level after acute activation of 5-HT neurotransmission.     

Potentiation of beta-endorphin effects by cholecystokinin antiserum in rats

Intracerebroventricular administration of a C-terminal cholecystokinin (CCK) antiserum potentiated the analgesic and cataleptic effects of beta-endorphin. The results are opposite to those observed after injection of CCK-8. It was suggested that CCK-8 may play a physiological role antagonizing the action of beta-endorphin.