Vasopressin stimulates release of β-lipotropin and β-endorphin in conscious rats as measured by radioimmunoassay of unextracted plasma

Summary Using a newly developed radioimmunoassay to determine the -endorphin-like immunoreactivity (-EI) in unextracted plasma, the effect of vasopressin injections on plasma -EI was investigated in conscious rats. Arginine vasopressin caused a dose-dependent increase of plasma -EI from 34.5±7.8 fmol ml^–1 (n=6) in vehicle-treated animals to 205.0±36.1 fmol ml^–1 (n=7) after injection of the highest vasopressin dose employed (486 ng/100 g b.w.). In view of the appreciable cross-reactivity of -lipotropin (-LPH) in the radioimmunoassay used, plasma was extracted and subjected to gel chromatography on a Sephadex G-50 column. On average, about 70% of the -EI co-eluted with human -LPH and about 30% with human -endorphin in plasma extracts obtained from both control and vasopressin-treated rats. No peripheral conversion of human -LPH occurred under the experimental conditions, since after i.v. bolus injection of human -LPH 97% of the -EI comigrated with human -LPH during gel filtration. A similar blood pressure increase to that induced by the vasopressin injections, when elicited by noradrenaline or angiotensin II i.v., was not followed by an elevation of plasma -EI.These data indicate that vasopressin stimulates -lipotropin and -endorphin release into the systemic circulation in vivo.     

Radioimmunoassay of β-endorphin basal and stimulated levels in extracted rat plasma

Summary A sensitive radioimmunoassay for -endorphin is described. Antibodies against human -endorphin which exhibit a high avidity for the C-terminal of the peptide were raised in rabbits following the injection of thyroglobulin-coupled human -endorphin (h-E) as immunogen. Methionineenkephalin, - -endorphin, as well as ACTH peptides did not cause interference in the radioimmunoassay. -Lipotropin, however, showed a 50% cross-reactivity. The sensitivity of the assay is 25 pg/0.5 ml tube volume for -endorphin. -Endorphin was extracted with a high recovery from the rat plasma using silicic acid and -endorphin levels as low as 100 pg/ml could be measured.Basal levels of -endorphin-like immunoreactivity in plasma of rats were about 400 pg/ml. -Endorphin levels in adrenalectomized rats and in animals chronically treated with the cortisol synthesis blocker metyrapone were found to be markedly increased (about 7-fold). Exposure of the rats to electrically induced foot-shocks caused a similar increase of immunoreactive -endorphin in plasma. A significant increase was also seen after insulin injection.     

Studies on the antinociception effect of melatonin and its mechanism in the rat

The antinociception effect of melatonin（Mel） and its mechanisms were studied on the trauma- pain model in the rat. A trauma - pain model was established in Wistar rats by combining right - hind limb amputation with 50℃ tail - flick test. The effects of Mel on central sensitization were studied through measuring the changes of the expression of c - fos （ an indicator of nociceptive transmission at the spinal level ） , subtance P（SP） and brain - derived neurotrophic factor （BDNF） in the spinal cord using immunohistochemistry. In addition, the contents of nitric oxide （NO） in brain and spinal tissues were also observed. The results showed that the immunoreactivity of BDNF and c - fos in spinal dorsal horn in injuried rats began to increase at the first day,     

Effects of acute ethanol on corticotropin-releasing hormone and β-endorphin systems at the level of the rat central amygdala

Rationale  

The endogenous opioid and corticotropin-releasing hormone (CRH) systems, present in the central amygdala (CeA), are implicated in alcohol consumption.     

A microdialysis profile of β-endorphin and catecholamines in the rat nucleus accumbens following alcohol administration

Rationale Alcohol stimulates the release of dopamine in the nucleus accumbens (NACB) of rats, mice and humans. There is evidence to suggest that the activation of beta-endorphin (β-EP) in the mesolimbic pathway by alcohol and other drugs of abuse may be associated with the rise in dopamine levels in the NACB. Objectives The present studies investigate whether the release of β-EP in the NACB is (1) dependent on the dose of alcohol that is administered, and (2) associated with changes in the extracellular concentrations of the catecholamines dopamine and norepinephrine, and the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in the NACB. Methods Male Sprague-Dawley rats were implanted with a microdialysis probe positioned in the shell region of the NACB. Artificial cerebrospinal fluid was pumped at a rate of 2.3 μl/min in awake and freely moving animals and the dialysate was collected at 30-min intervals. After a baseline period, rats were injected intraperitoneally with either physiological saline or one of three doses of alcohol: 0.8, 1.6, or 2.4 g ethanol/kg body weight. The dialysates collected were analyzed with radioimmunoassay, to estimate the content of β-EP; and high performance liquid chromatography, to estimate the content of dopamine, norepinephrine, DOPAC and HVA. Results Alcohol induced a dose-dependent increase in the extracellular levels of β-EP and dopamine. However, elevations in the extracellular levels of norepinephrine, DOPAC and HVA did not reach significance. The largest increase in β-EP and dopamine was observed with the 2.4 g/kg dose. Conclusion The alcohol-induced release of β-EP and dopamine in the NACB is dose-dependent, where the highest dose resulted in more pronounced concentrations in the dialysate. Furthermore, the increase in the extracellular levels of dopamine appeared to occur at an earlier time point following alcohol administration, than for β-EP. These results suggest that alcohol stimulates dopamine and β-EP in the NACB, but probably does so via independent mechanisms.     

The effect of δ 9-tetrahydrocannabinol and LSD on the acquisition of an active avoidance response in the rat

The course of active avoidance learning of rats in a symmetrical Y-maze under the influence of 1, 3, and 9 mg/kg of ⁹-THC i.p., and 5, 20, and 100 g/kg of LSD was investigated. ⁹-THC in a dosage of 1 mg/kg had no effect on avoidance learning. Three and to a lesser extent 9 mg/kg produced more rapid learning with a significantly better performance. Learning under ⁹-THC proved to be statedependent. The withdrawal of ⁹-THC caused a decrease in the avoidance rate, which was dependent on the dosage. Upon renewal of the THC doses, the animals reattained their earlier performance. In the course of the experiment there was rapid tolerance development, especially of the sedative properties of THC.LSD retarded the rate of acquisition of the active avoidance response. Whereas the control animals displayed over 80% successful active avoidance from the 14th session onwards, this was achieved by the LSD groups only after the 20th session. However, in contrast to the control group the LSD animals were able to increase their avoidance rate to over 90%, and this was maintained to the end of the experiment (a total of 24 sessions with LSD). The sudden withdrawal of LSD produced a fall in avoidance rate, which was dependent on the previous training dosage; as with ⁹-THC state-dependent learning can also be assumed for LSD.     

Naloxone and β-endorphin alter the effects of post-training epinephrine on memory

These experiments examined the involvement of opioid peptides in the memory-modulating effects of post-training epinephrine (Epi). Mice were trained on inhibitory avoidance (IA) and Y-maze discrimination (YMD) tasks and given post-training injections followed by retention tests 24 h later. In the IA task retention was enhanced by low doses of Epi and impaired by high doses. In both tasks, naloxone facilitated retention and blocked the memory-impairing effects of Epi. These findings are consistent with other evidence suggesting that the memory-impairing effects of -endorphin are mediated by the release of opioid peptides. Previous studies have shown that a novel exploratory experience given 1 h prior to training blocks the release of brain -endorphin and blocks the memory-enhancing effects of post-training naloxone. In the present study we found that a novel experience given 1 h prior to training blocked the memory-impairing effect of post-training Epi otherwise obtained in both tasks. The effects of a low, memory-enhancing dose of Epi appear not to involve the release of opioid peptides: a low dose of Epi blocked the memory-impairing effect of -endorphin. Further, low doses of Epi and naloxone, which were ineffective when administered alone, significantly enhanced retention when administered together. We interpret these findings as indicating that the memory-enhancing and memory-impairing effects of Epi are mediated by different mechanisms.     

Effects of some GABA-mimetic drugs on the antinociceptive activity of morphine and β-endorphin in rats

Summary Intracerebroventricular administration of muscimol, a potent GABA-receptor agonist, counteracted the antinociceptive effect of morphine or -endorphin in rats as measured by the tail flick method. Muscimol's activity was reversed by bicuculline. Isoguvacine, another GABA agonist, as well as nipecotic acid and guvacine, two inhibitors of neuronal and glial uptake of GABA, also antagonized morphine's antinociceptive effect. A role of the central GABA-ergic system in mediating opiate antinociception is proposed.This study was supported by C.N.R. grant no. CT 77.01401.04     

Inhibition of acetylcholine turnover in rat hippocampus by intraseptal injections of β-endorphin and morphine

Summary Intraseptal administration of morphine (70 nmol) or -endorphin (0.7 nmol) reduced the rate of acetylcholine (ACh) turnover (TR_ACh) in rat hippocampus but not in striatum or cortex. These intraseptal injections failed to modify the ACh content and did not elicit analgesia. Naltrexone (15 mol/kg, i.p.) completely antagonized the decrease of hippocampal TR_ACh elicited by the two opiate receptor agonists. Furthermore, intraseptal injections of naltrexone partially blocked the decrease in hippocampal TR_ACh induced by intraperitoneal administration of morphine (70 mol/kg, i.p.). These data suggest that opiate agonists decrease hippocampal TR_ACh by regulating septal cholinergic neurons, and that this effect is not associated with analgesia.     

The effect of selective α1- and α2-adrenoceptor stimulation on intraocular pressure in the conscious rabbit

Summary The influence of topically applied selective ₁- and ₂-adrenoceptor agonists on intraocular pressure and the diameter of the pupil was investigated in conscious rabbits. Selective stimulation of the ₁-subtype of receptors induced an elevation in intraocular pressure, accompanied by mydriasis, whereas stimulation of the ₂-subtype caused a marked and dose-dependent ocular hypotensive response, which was blocked by the selective ₂-adrenoceptor antagonist yohimbine. ₂-Agonists induced neither macroscopic ocular side effects, nor an effect on the pupil size. Possibly, the subclass of ₂-adrenoceptor stimulating drugs represent a group of new antiglaucomatous agents.     

Protective effect of Puerarin on β-amyloid-induced neurotoxicity in rat hippocampal neurons

Puerarin (CAS Number 3681-99-0), a major isoflavone glycoside purified from Pueraria lobata, was reported to possess antioxidative and estrogen-like biological activities. Recent studies showed that puerarin protects different cell types from damage caused by a variety of toxic stimuli. In the present study, we investigated the neuroprotective effect of puerarin against Aβ25-35-induced neurotoxicity in cultured hippocampal neurons, as well as the underlying mechanism(s). Following exposure of cells to Aβ25-35, cell survival and glutathione peroxidase (GSH-Px) and catalase (CAT) activities were reduced while production of reactive oxygen species (ROS) was increased. Preincubation of the cells with puerarin prior to Aβ25-35 exposure increased cell survival and GSH-Px and CAT activities and decreased ROS production. It was previously shown that overactivation of glycogen synthase kinase-3β (GSK-3β) is implicated in Aβ-induced cell death. In this study, Aβ25-35 treatment is found to increase GSK-3β activity and pretreatment with puerarin preventesAβ-induced activation of GSK-3β based on Western blot analysis. In addition, puerarin is shown to activate protein kinase B (PKB)/Akt, an important upstream kinase of GSK-3β, possibly promoting subsequent GSK-3β inhibition. Our data suggest that puerarin attenuates cell death induced by Aβ25-35 via various mechanisms, which might be beneficial for the treatment of Alzheimer's disease.     

Plasma concentration and vascular effect of β-endorphin in spontaneously hypertensive and Wistar Kyoto rats

Summary In order to find out whether -endorphin (-E) is involved in the development of hypertension, we performed two series of experiments. Firstly, spontaneously hypertensive rats (SHR) and their normotensive Wistar Kyoto controls (WKY) were submitted to ether stress. Plasma concentrations of -endorphin-like immunoreactivity (-EI), adrenocorticotropin (ACTH) and -melanotropin (-MSH) were measured by radioimmunoassay. The basal concentration of -EI was similar in WKY and SHR, whereas WKY had higher levels of ACTH and lower levels of -MSH than SHR. In both strains acute stress enhanced the plasma concentration of -EI to the same extent and with a similar time-course. The increase of plasma -El coincided with a rise in ACTH but not -MSH. Gel chromatography of -EI revealed that plasma extracts contain similar amounts of -lipotropin- (-LPH) and -E-sized immunoreactive components, and that acute stress elevated both forms of -El. Secondly, isolated tail arteries of SHR and WKY were perfused and field stimulated with two pulses at 1 Hz. -E depressed stimulation-evoked vasconstriction with the same potency in both strains. Thus, basal and stress-induced levels of -EI did not differ in SHR and WKY. Moreover, in the tail artery of both strains the sensitivity of presynaptic opioid receptors towards -E was almost identical. If the -E sensitivity of these receptors in other arteries of WKY and SHR is also similar, a major role of the circulating peptide in the development of hypertension is rather unlikely.This work was partly supported by the Deutsche Forschungsgemeinschaft (SFB 325) Send offprint requests to B. Bucher at the above address     

Excitatory and depressant effects of Δ 9-tetrahydrocannabinol and cannabidiol on cortical evoked responses in the conscious rat

The influences of ⁹-tetrahydrocannabinol (THC) and cannabidiol on electrically evoked cortical potentials of conscious rats with chronically implanted electrodes were investigated. Specifically, the cannabinoids' effects on a transcallosal evoked response were compared with those of ethosuximide, phenytoin, and pentylenetetrazol. THC produced dose-related opposite effects: Low doses increased the amplitude of the response, whereas higher doses reduced the response. Other drugs that can cause or exacerbate seizures, i. e., phenytoin and pentylenetetrazol, also increased the amplitude of the cortical response. In contrast, cannabidiol, over a wide dosage range, caused only depression. Ethosuximide, like cannabidiol, elicited a depressant effect. The data indicate that under the conditions of the present investigation, cannabidiol shares electrophysiological properties with ethosuximide but not with phenytoin, and that cannabidiol is a relatively selective, centrally acting drug. In addition, our findings support the suggestion that augmentation of neurotransmission in central pathways may contribute to the convulsant actions of THC, and the cannabinoids' depressant effects may, at least partially, account for their anticonvulsant actions.     

Opposite interactions between α- and β-endorphin fragments with dopamine mediated responses on the rat rectum in vitro

Summary Dopamine causes a dose-dependent contraction of the rat rectum in vitro followed by a relaxation. This contraction can be inhibited by apomorphine and phenylephrine. This inhibition can be attenuated by the -endorphin (E) fragments 2–17 (des-Tyr¹--endorphin, DTE) and 6-17 (des-enkephalin--endorphin, DEE). E 6-17 seems to be the shortest sequence with full activity in this respect since a shorter fragment (E 10-17) was less effective. The atypical neuroleptics oxypertine, sulpiride, and clozapine, the classic neuroleptic haloperidol and metoclopramide have a similar action to DEE. The peptides and atypical neuroleptics do not affect the dopamine response per se while the classic neuroleptics haloperidol and metoclopramide enhance the dopamine response.The effects of the -type endorphins are opposite to those of the -type endorphins, since des-Tyr¹--endorphin (DTE, E 2-16) and des-enkephalin--endorphin (DEE, E 6-16) enhance the phenylephrine-induced decreased responsiveness to dopamine. Structure-activity studies revealed that the active moiety of the -endorphin fragments probably resides in the 6–9 region. In addition the -type endorphins directly inhibit the dopamine response.It is concluded that the rat rectum may be used to analyse neuroleptic-like action. In this model - and -endorphin fragments may directly or indirectly influence the interaction of dopamine with the rectum. Because of the strong similarities between the effects of -type endorphins and that of neuroleptics the results support the purported neurolepticlike action of -type endorphins. The influence of -type endorphins and -type endorphins on the apomorphine or phenylephrine induced decreased responsiveness to dopamine, although opposite, seems to be mediated by an influence on different dopamine sensitive systems.     

Possible activation of intracellular β-adrenoceptors by extraneuronally accumulated isoprenaline in perfused rat heart

Summary Two rat hearts were perfused in series by a modified Loewi's method. The recipient heart was perfused with the perfusate collected from the donor heart. 1. After perfusion with ³H-isoprenaline in the presence of tropolone, an inhibitor of catechol-O-methyltransferase, the donor heart was washed out with amine-free medium containing tropolone and corticosterone. The heart rate of the recipient heart increased after the change to the perfusate from the donor heart during the wash-out. After wash-out the heart rate of the donor heart (which had accumulated 43.4 pmol. g^{–1 3}H-isoprenaline) was higher than that of the recipient heart (which had accumulated 0.44 pmol · g^{–1 3}H-isoprenaline), and the rates of efflux of ³H-isoprenaline from both hearts were similar. 2. After perfusion with ³H-isoprenaline and corticosterone in the absence of tropolone, the enhanced heart rate of the donor heart decreased during wash-out with amine-free medium in the presence of corticosterone. The heart rate of the recipient heart increased after the medium change to the perfusate from the donor heart, and the heart rates in both hearts were similar after wash-out. Only small amounts of ³H-isoprenaline remained in both hearts after wash-out, and the rates of efflux of ³H-isoprenaline from both hearts were similar. 3. After perfusion with ³H-isoprenaline in the presence of tropolone, the effects of propranolol and atenolol on the heart rate during wash-out with amine-free medium containing tropolone and corticosterone were compared. The inhibitory effect of propranolol on the heart rate was significantly greater than that of atenolol. 4. These findings provide pharmacological evidence for functionally effective intracellular -adrenoceptors in the rat heart, because the heart with high extraneuronal accumulation of isoprenaline had a higher heart rate than that with low accumulation, although the concentration of isoprenaline in the extracellular space was similar and because a lipophilic -adrenoceptor blocking agent (propranolol) inhibited the heart rate more effectively than a hydrophilic -adrenoceptor blocking agent (atenolol) did. Send offprint requests to K. Kurahashi at the above address     

Chronic administration of ethanol on pituitary and hypothalamic β-endorphin in rats and golden hamsters

The effect of chronic exposure to ethanol on hypothalamic and pituitary endorphin levels of rats and golden hamsters was studied. In rats, chronic ethanol consumption caused a decrease in the level of immuno-reactive (i.r.) β-endorphin (β-EP) in the pituitary but an increase of i.r. β-EP in the hypothalamus. The Met-enkephalin level in the hypothalamus of ethanol-treated rats remained the same as the control. The body weights, as well as food and liquid intake, of ethanol-treated rats were observed to be lower than the controls. In golden hamsters, i.r. β-EP level in the hypothalamus and pituitary remained unchanged with chronic ethanol consumption. The body weight and liquid intake of golden hamsters also remained the same as the controls. Since the changes of pituitary and hypothalamic β-EP after ethanol administration were found only in rats but not in golden hamsters, it is likely that the effects of ethanol observed in rats are not specific.     

Mediation of β-endorphin in andrographolide-induced plasma glucose-lowering action in type I diabetes-like animals

In the present study, we investigated the mechanism(s) for glucose-lowering action of andrographolide in streptozotocin-induced diabetic rats (STZ-diabetic rats). Andrographolide lowered plasma glucose concentrations in a dose-dependent manner and increased plasma beta-endorphin-like immunoreactivity (BER) dose-dependently in diabetic rats. Both of these responses to andrographolide were abolished by the pretreatment of animals with prazosin or N-(2 -(2-cyclopropylmethoxy) ethyl) 5-choro-alpha-dimethyl-1H-indole-3-thylamine (RS17053) at doses sufficient to block alpha(1)-adrenoceptors (ARs). Also, andrographolide enhanced BER release from isolated rat adrenal medulla in a concentration-related manner that could be abolished by alpha(1)-ARs antagonists. Bilateral adrenalectomy in STZ-diabetic rats eliminated the activities of andrographolide, including the plasma glucose-lowering effect and the plasma BER-elevating effect. Andrographolide failed to lower plasma glucose in the presence of opioid mu-receptor antagonists and in the opioid mu-receptor knockout diabetic mice. Treatment of STZ-diabetic rats with andrographolide resulted in the reduced expression of phosphoenolpyruvate carboxykinase (PEPCK) in liver and an increased expression of the glucose transporter subtype 4 (GLUT 4) in soleus muscle. These effects were also blocked by opioid mu-receptor antagonists. In conclusion, our results suggest that andrographolide may activate alpha(1)-ARs to enhance the secretion of beta-endorphin which can stimulate the opioid mu-receptors to reduce hepatic gluconeogenesis and to enhance the glucose uptake in soleus muscle, resulting in a decrease of plasma glucose in STZ-diabetic rats. However, the roles of other endogenous opioid peptides or the mixture of several opioid peptides in the activation of opioid mu-receptors associated with the plasma glucose-lowering action of andrographolide, should be considered and need more investigation in the future.     

Interactions of carbon tetrachloride and promethazine in the rat—I: Effects of promethazine on the concentrations of carbon tetrachloride in blood and liver,and on the production of chloroform

The effects of Promethazine (PM, 78 μmoles/kg body wt, i.p.) on the concentrations of CCl₄ in samples of blood and liver of male, fasted rats after oral dosing with CCl₄ have been determined. With an administered dose of CCl₄ of 13 moles/kg body wt the concentrations of CCl₄ in the blood and liver were measured using gas chromatography with a flame ionisation detector. It was found that Promethazine delayed absorption of CCl₄ from the gastro-intestinal tract by approximately 2 hr as judged by blood levels of CCl₄; the maximum blood concentration (C_max) and the total absorption of ccl₄ (assessed by the area under the plot of blood concentration vs time during the first 6 hr after administration of CCl₄) were not significantly changed by Promethazine treatment. Liver and blood measurements were carried out on each rat in this series and the ratio of the CCl₄-concentrations in liver: blood were found to lie within the range 8–12 when studied in the absence of Promethazine treatment.Gas chromatography with electron capture was used on serial samples of blood from the same rat to measure CCl₄ and CHCl₃ concentrations following oral administration of 13, 6.5 or 1.3 mmoles CCl₄/kg body wt. The increase in blood CCl₄ levels at all doses of CCl₄ administered was delayed by about 2 hr by administration of Promethazine. The maximum blood concentrations of CCl₄ and total amount absorbed (judged by area under the plot of blood concentration vs time) were dose-related to the amount of CCl₄ administered with or without Promethazine administration. Blood concentrations of CHCl₃ were relatively constant over the range of CCl₄-doses used indicating that the metabolic production rate of CHCl₃ is saturated at rather low doses of CCl₄ administered.     

Comparison of the effects of semicarbazide and β-aminopropionitrile on the arterial extracellular matrix in the Brown Norway rat

To investigate a putative role for semicarbazide-sensitive amine oxidase (SSAO) in arterial extracellular matrix (ECM) organization, we compared arteries of growing Brown Norway (BN) rats after chronic administration of semicarbazide (SCZ) and β-aminopropionitrile (BAPN), two inhibitors with different properties and relative specificities for SSAO and lysyl oxidase (LOX). The BN model is particularly well adapted to evaluating effects of toxic compounds on the arterial elastic network. We measured aortic LOX and SSAO activities and quantified several ECM parameters. After a pilot study comparing doses previously studied and testing for additivity, we studied low and high equimolar doses of SCZ and BAPN. Both compounds similarly inhibited LOX, whereas SCZ inhibited SSAO far more effectively than BAPN. Both decreased carotid wall rupture pressure, increased tail tendon collagen solubility, decreased aortic insoluble elastin (% dry weight) and dose-dependently increased defects in the internal elastic lamina of abdominal aorta, iliac and renal arteries. Our results suggest that either these effects are mediated by LOX inhibition, SCZ being slightly more effective than BAPN in our conditions, or SSAO acts similarly to and in synergy with LOX on ECM, the greater SCZ effect reflecting the simultaneous inhibition of both enzymes. However, the high SCZ dose increased aortic collagen and ECM proteins other than insoluble elastin markedly more than did equimolar BAPN, possibly revealing a specific effect of SSAO inhibition. To discriminate between the two above possibilities, and to demonstrate unequivocally a specific effect of SSAO inhibition on ECM formation or organization, we must await availability of more specific inhibitors.