Levels Of Endogenous Opioid Peptides Research Articles

Increased Cardiovascular Disease Risk in Adult Offspring with Prenatal Opioid Exposure Is Associated with Endogenous Opioid Peptide Dysregulation

Parental drug abuse and dysfunctional households during childhood increase the risk for cardiovascular disease (CVD) later in life. Chronic opioid exposure and opioid use disorder (OUD) are associated with a 2-fold increased risk of CVD and a 16% increase in coronary artery disease. OUD among pregnant women is an understudied area related to the opioid epidemic, causing a 5-fold increase in neonatal opioid withdrawal syndrome (NOWS) incidence. In former studies, we subjected female rats to two models of maternal opioid exposure: 1) morphine sulfate during gestation or 2) fentanyl citrate during preconception and gestation. At birth, female and male opioid-exposed offspring showed normal birth weight compared with vehicle-exposed offspring while displaying NOWS-like somatic withdrawal signs after parturition. As adults, in utero opioid-exposed offspring displayed increased mean arterial pressure, sympathetic activation, and CVD risk factors. Prenatal exposure to opioids has been shown to dysregulate the endogenous opioid peptides (EOPs) and alter behavioral outcomes later in life, but the transplacental effects of opioids on blood pressure regulation remain understudied. Therefore, this study aimed to assess whether adult offspring with prenatal opioid exposure display changes in 1) the expression of the EOP proenkephalin (PENK) in brain regions known to control the sympathetic outflow (Paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM)), heart, kidneys, and liver; 2) the plasma levels of EOPs (PENK, enkephalin, prodynorphin and dynorphin); and 3) the fentanyl plasma clearance (pharmacokinetics). Protein was extracted from tissues for western blot analysis using the primary antibody for PENK (1:1000, Invitrogen). In opioid-exposed offspring, PENK protein expression was reduced in RVLM (0.55±0.06 vs. 0.36±0.01, AU) and PVN (1.56±0.09 vs. 1.08±0.08, AU), showing a ~35% and ~31% reduction, respectively (n=4-5, p<0.05). In the heart, PENK protein expression was not statistically different in opioid-exposed offspring compared with vehicle-exposed offspring. However, in the kidneys (0.72±0.09 vs. 1.16±0.10, AU) and liver (0.40±0.06 vs. 0.60±0.05, AU), PENK protein expression showed a 61% and 50% increase only in male opioid-exposed offspring compared to vehicle-exposed offspring (n=6-8, p<0.05). None of the plasma circulating EOP levels measured by ELISA were affected by prenatal opioid exposure. Fentanyl clearance in plasma (0, 15, 30, 60, 120, 360,1440 mins) following a fentanyl injection (20ug/kg, sc.) was not different between adult vehicle and opioid-exposed offspring, indicating potential pharmacodynamic alterations secondary to prenatal opioid exposure. As EOPs exert inhibitory effects on adrenergic signaling, our data indicate that prenatal exposure to opioids may induce a permanent downregulation of EOPs in the central nervous system and thus contributing to increasing the sympathetic tone and desensitize the acute depressor responses to opioid peptides, suggesting that EOPs may be critical for blood pressure regulation. However, peripheral expression of PENK seems to reflect pathophysiological alterations in different tissues since elevated PENK level is used in clinical settings as a marker to assess the severity of tissue damage in the liver and kidneys. This study was funded by 1013177615 (NRPA-UK), 1013400001 (SUGAR-UK) and 1013171360 (DPNS-UK). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cannabinoid Receptor-2 Activation in Keratinocytes Contributes to Elevated Peripheral β-Endorphin Levels in Patients With Obstructive Jaundice.

Cholestatic diseases are often accompanied by elevated plasma levels of endogenous opioid peptides, but it is still unclear whether central or peripheral mechanisms are involved in this process, and little is known about the change of pain threshold in these patients. The purpose of this study was to determine the preoperative pain threshold, postoperative morphine consumption, and central and peripheral β-endorphin levels in patients with obstructive jaundice. This study also tests the hypothesis that activation of the cannabinoid receptor-2 (CB2R) in skin keratinocytes by endocannabinoids is the mechanism underlying circulating β-endorphin elevation in patients with obstructive jaundice. The electrical pain thresholds, 48-hour postoperative morphine consumption, concentrations of β-endorphin in plasma and cerebrospinal fluid, skin and liver β-endorphin expression, and plasma levels of endocannabinoids were measured in jaundiced (n = 32) and control (n = 32) patients. Male Sprague-Dawley rats and human keratinocytes (human immortalized keratinocyte cell line [HaCaT]) were used for the in vivo and in vitro experiments, respectively. Mechanical and thermal withdrawal latency, plasma level, and skin expression of β-endorphin were measured in CB2R-antagonist-treated and control bile duct-ligated (BDL) rats. In cultured keratinocytes, the effect of CB2R agonist AM1241-induced β-endorphin expression was observed and the phosphorylation of extracellular-regulated protein kinases 1/2, p38, and signal transducer and activator of transcription (STAT) pathways were investigated. This study found (1) the plasma level of β-endorphin (mean ± standard error of the mean [SEM]) was 193.9 ± 9.6 pg/mL in control patients, while it was significantly increased in jaundiced patients (286.6 ± 14.5 pg/mL); (2) the electrical pain perception threshold and the electrical pain tolerance threshold were higher in patients with obstructive jaundice compared with controls, while the 48-hour postoperative morphine consumption was lower in the jaundiced patients; (3) there was no correlation between plasma β-endorphin levels, electrical pain thresholds, and 48-hour postoperative morphine consumption in patients with obstructive jaundice; (4) the plasma level of the endogenous cannabinoid anandamide was increased in the jaundiced patients; (5) CB2R antagonist treatment of the BDL rats reduced β-endorphin levels in plasma and skin keratinocytes, while it did not alter the nociceptive thresholds in BDL and control rats; (6) the endocannabinoid anandamide-induced β-endorphin synthesis and release via CB2R in cultured keratinocytes; and (7) phosphorylation of extracellular-regulated protein kinases 1/2 is involved in the CB2R-agonist-induced β-endorphin expression in keratinocytes. CB2R activation in keratinocytes by the endocannabinoid anandamide may play an important role in the peripheral elevation of β-endorphin during obstructive jaundice.

Phase II double-blind randomised controlled trial of exogenous administration of melatonin in chronic pain (DREAM—CP): a study protocol

IntroductionChronic pain is prevalent, and approximately half of patients with chronic pain experience sleep disturbance. Exogenous melatonin is licensed to treat primary insomnia and there is some evidence for analgesic...

Endogenous peripheral pain regulatory systems in orofacial pain patients

IntroductionInitiation of pulpal pain begins with the release of bacterial toxins and inflammatory mediators that either activate or sensitize nociceptors thereby activating the trigeminal pain pathway. In addition, pain perception quickly triggers inhibitory signals that are often mediated by endogenously produced analgesic substances such as opioid peptides. The balance between inflammatory mediators and endogenously released analgesics may contribute to clinical conditions such as symptomatic versus asymptomatic pulpitis. We hypothesized that patients with asymptomatic periradicular pathosis have increased levels of the opioid peptides compared to patients with symptomatic periradicular pathosis. In addition, we also proposed to study the immune cell type that is responsible for the release the opioid peptides in periradicular lesions. Material and methodFifty tissue biopsy samples obtained from patients who have received endodontic microsurgery and assigned to either symptomatic and asymptomatic groups (n = 25/group) based on their pre-operative clinical periradicular testings. Immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and Fluorescence-Activated Cell Sorting (FACS) were performed to determine the expressions, levels and the cellular sources of opioid peptides, namely; β-endorphin (END), met-enkephalin (ENK) and dynorphin A (DYN), respectively. Radioimmunoassay (RIA) was performed to determine the functional role of conditioned media (CM) from asymptomatic and symptomatic patients on regulation of trigeminal (TG) neuronal activity in vitro. ResultsCompared to symptomatic group, patients with asymptomatic lesions showed 3-fold increase in END levels only. Macrophages showed the highest expression of END, DYN and ENK. This study shows that the application of CM from asymptomatic and symptomatic patients triggered differential regulation of TG neuronal activity. ConclusionEndogenous opioid peptides are present in periapical lesions. The higher level of endogenous opioid peptides in asymptomatic periapical lesions could explain the lack of symptoms in asymptomatic lesions.

Episodic Ethanol Exposure in Adolescent Rats Causes Residual Alterations in Endogenous Opioid Peptides.

Adolescent binge drinking is associated with an increased risk of substance use disorder, but how ethanol affects the central levels of endogenous opioid peptides is still not thoroughly investigated. The aim of this study was to examine the effect of repeated episodic ethanol exposure during adolescence on the tissue levels of three different endogenous opioid peptides in rats. Outbred Wistar rats received orogastric (i.e., gavage) ethanol for three consecutive days per week between 4 and 9 weeks of age. At 2 h and 3 weeks, respectively, after the last exposure, beta-endorphin, dynorphin B and Met-enkephalin-Arg6Phe7 (MEAP) were analyzed with radioimmunoassay. Beta-endorphin levels were low in the nucleus accumbens during ethanol intoxication. Remaining effects of adolescent ethanol exposure were found especially for MEAP, with low levels in the amygdala, and high in the substantia nigra and ventral tegmental area three weeks after the last exposure. In the hypothalamus and pituitary, the effects of ethanol on beta-endorphin were dependent on time from the last exposure. An interaction effect was also found in the accumbal levels of MEAP and nigral dynorphin B. These results demonstrate that repeated episodic exposure to ethanol during adolescence affected opioid peptide levels in regions involved in reward and reinforcement as well as stress response. These alterations in opioid networks after adolescent ethanol exposure could explain, in part, the increased risk for high ethanol consumption later in life.

РІВЕНЬ ГОРМОНІВ СТРЕС-ЛІМІТУЮЧОЇ СИСТЕМИ У ВАГІТНИХ З ПСИХОЕМОЦІЙНИМИ ПОРУШЕННЯМИ, ОБУМОВЛЕНИМИ ТРИВОЖНІСТЮ

Мета дослідження – визначити рівень гормонів стрес-лімітуючої системи у вагітних з урахуванням їх психоемоційного стану.Матеріали та методи. Обстежено 86 жінок у ІІ та на початку ІІІ триместру вагітності. До основної групи включені 60 вагітних із середнім та високим рівнями ситуативної тривожності, а також 26 вагітних із рівнем тривоги 30 балів і нижче, що свідчить про низький рівень СТ (група порівняння). Для оцінки ситуативної тривожності та особистісної тривожності використовували шкалу Ч. Д. Спілбергера, яка адаптована Ю. Л. Ханіним. Кількісну оцінку концентрації гормонів у плазмі крові визначали методом імуноферментного аналізу на апараті «SIRIO S». Для визначення рівня пролактину (Пр) використовували тест-систему Monobind INC (США), кортизолу (К) та інсуліну (Ін) – тест-системи DRG (Німеччина). Рівень ендогенних опіоїдних пептидів (β-ендорфіну) визначали з використанням тест-системи Peninsula Laboratories, LLC (США).Результати дослідження та їх обговорення. Групи дослідження були розділені залежно від рівня СТ. Встановлено, що середній показник СТ у групі порівняння склав (26,3±1,2) бала та був статистично достовірно нижчим (p<0,05) за середній показник в основній групі ((37,9±1,4) бала). Досліджуючи концентрацію Пр, встановлено, що взаємовплив із тривожністю підтверджується наявністю позитивного кореляційного зв’язку між його рівнем та СТ (r=+0,690, p<0,05), а також менш значиму з ОТ (r=+0,254, p<0,05) відповідно. При оцінці рівня Пр було встановлено статистично достовірно вищий (p<0,05) серед вагітних із середньо-високим рівнем тривожності 136,2 (118,0; 151,6) нг/мл порівняно з жінками з низьким її рівнем 93,7 (67,4; 110,7) нг/мл. При порівнянні концентрації К у вагітних із середньо-високим рівнем СТ – 416,0 (368,2; 560,0) нг/мл встановлено статистично достовірне (p<0,05) його переважання порівняно з жінками, які мали низький її рівень – 343,2 (304,6; 370,5) нг/мл. Взаємозв’язок рівня СТ та К у вагітних із високим та середнім її рівнями підтверджується також наявністю позитивного кореляційного зв’язку (r=+0,704, p<0,05). У вагітних із низьким рівнем СТ таких зв’язків не встановлено. При оцінці рівня Ін встановлено, що він не мав зв'язку з тривожністю, а також із терміном гестації та віком вагітних. Так, рівень Ін серед вагітних із середньо-високим рівнем тривожності склав 11,5 (9,1; 16,8) мкл Од/мл, а в групі жінок із низьким її рівнем – 12,2 (9,8; 21,9) мкл Од/мл та був статистично не достовірним (p>0,05). Однак розрахунок К/Ін індексу, як маркера стресу, дозволив встановити його статистично достовірне (p<0,05) переважання в групі вагітних із середньо-високим рівнем 39,4 (23,2; 54,9) порівняно з жінками з низьким 24,6 (16,02; 34,1) її рівнем. При оцінці β-ендорфіну не було встановлено статистично достовірної різниці (p>0,05) між показниками у вагітних із середньо-високим – 1,2 (0,6; 3,6) нг/мл та низьким – 1,4 (0,7; 3,0) нг/мл рівнями СТ. Вірогідно, це свідчить про відсутність виразного впливу означеного нейропептиду на формування емоційного стану в жінок під час вагітності.Висновки. Оцінка рівня пролактину, як ендогенного анксіолітика, дозволила встановити статистично достовірне (р<0,05) його зростання у вагітних із середньо-високим рівнем СТ у порівнянні з групою жінок із низьким її рівнем. Зростання рівня пролактину у вагітних із низьким рівнем СТ залежить від терміну гестації (r=+0,665, p<0,05) та не має зв’язку як з рівнем СТ, так і ОТ. Серед вагітних із середнім та високим рівнями СТ мала місце протилежна картина – негативний зв’язок із терміном вагітності (r = -0,338, p>0,05) та позитивний – із рівнем СТ (r = +0,576, p<0,05). Порівняння рівня інсуліну та β-ендорфіну у вагітних з урахуванням рівня СТ не дозволило встановити статистично значимої різниці (p>0,05). Однак визначення К/Ін індексу, як маркера стресу, дозволило встановити його статистично достовірне (p<0,05) переважання в групі вагітних із середньо-високим рівнем, порівнюючи з вагітними, які мали низький рівень СТ.

Role of Endogenous Opioid System in Ischemic-Induced Late Preconditioning.

BackgroundOpioid receptors (OR) are involved in myocardial late preconditioning (LPC) induced by morphine and δ1-opioid receptor (δ1-OR) agonists. The role of OR in ischemic-induced LPC is unknown. We investigated whether 1) OR are involved in the trigger and/or mediation phase of LPC and 2) a time course effect on the expression of different opioid receptors and their endogenous ligands exists.MethodsMale Wistar rats were randomly allocated to four groups (each group n = 8). Awake animals were ischemic preconditioned by a 5 minutes coronary occlusion. 24 hours later, anesthetized animals underwent 25 minutes coronary occlusion followed by 2 hours of reperfusion. The role of OR was investigated by treatment with intraperitoneal naloxone (Nal) 10 minutes prior to LPC (Nal-LPC; trigger phase) or 10 min prior to sustained ischemia (LPC-Nal; mediation phase).ResultsLPC reduced infarct size from 61±10% in controls to 25±9% (P<0.001). Naloxone during trigger or mediation phase completely abolished LPC-induced cardioprotection (59±9% and 62±9%; P<0.001 vs. LPC). 8, 12 and 24 hours after the ischemic stimulus, expression of δ-OR in the heart was increased, whereas μ-opioid receptor (μ-OR) and κ-opioid receptor (κ-OR) were not. Plasma concentrations of β-endorphin and leu-enkephalin but not dynorphin were increased by LPC.ConclusionIschemic LPC is triggererd and mediated by OR. Expression of δ-OR and plasma levels of endogenous opioid peptides are increased after ischemic LPC.

Role of endogenous opioid peptides in the infarct size-limiting effect of adaptation to chronic continuous hypoxia

AimsThe objective of this study was to examine the involvement of endogenous opioid peptides and opioid receptor (OR) subtypes in the cardioprotective effect of adaptation to chronic hypoxia in rats. Main methodsRats were exposed to continuous normobaric hypoxia (CNH; 12% oxygen) for 3 weeks. Myocardial ischemia was induced by 20-min coronary artery occlusion followed by 3-h reperfusion in anesthetized open-chest animals. Various OR antagonists were administered to rats prior to ischemia. The size of myocardial infarction and the incidence of ischemic ventricular arrhythmias were assessed. Myocardial and plasma concentrations of opioid peptides (met-enkephalin, β-endorphin, and endomorphins) were determined. Key findingsAdaptation to CNH significantly increased myocardial and plasma concentrations of opioids, potentiated their further elevation by ischemia/reperfusion, and reduced myocardial infarct size, but it did not affect the incidence of ischemic arrhythmias. The infarct size-limiting effect of CNH was abolished by OR antagonists naltrexone (non-selective), naloxone methiodide (non-selective peripherally acting), TIPP[ψ] (δ-OR), naltriben (δ2-OR), or CTAP (μ-OR), while BNTX (δ1-OR) and nor-binaltorphimine (κ-OR) had no effect. SignificanceThe results suggest that the infarct size-limiting effect afforded by adaptation to CNH is mediated by activation of peripheral δ2- and μ-ORs by elevated levels of endogenous opioid peptides.

Hormonal regulation of delta opioid receptor immunoreactivity in interneurons and pyramidal cells in the rat hippocampus

Clinical and preclinical studies indicate that women and men differ in relapse vulnerability to drug-seeking behavior during abstinence periods. As relapse is frequently triggered by exposure of the recovered addict to objects previously associated with drug use and the formation of these associations requires memory systems engaged by the hippocampal formation (HF), studies exploring ovarian hormone modulation of hippocampal function are warranted. Previous studies revealed that ovarian steroids alter endogenous opioid peptide levels and trafficking of mu opioid receptors in the HF, suggesting cooperative interaction between opioids and estrogens in modulating hippocampal excitability. However, whether ovarian steroids affect the levels or trafficking of delta opioid receptors (DORs) in the HF is unknown. Here, hippocampal sections of adult male and normal cycling female Sprague-Dawley rats were processed for quantitative immunoperoxidase light microscopy and dual label fluorescence or immunoelectron microscopy using antisera directed against the DOR and neuropeptide Y (NPY). Consistent with previous studies in males, DOR-immunoreactivity (-ir) localized to select interneurons and principal cells in the female HF. In comparison to males, females, regardless of estrous cycle phase, show reduced DOR-ir in the granule cell layer of the dentate gyrus and proestrus (high estrogen) females, in particular, display reduced DOR-ir in the CA1 pyramidal cell layer. Ultrastructural analysis of DOR-labeled profiles in CA1 revealed that while females generally show fewer DORs in the distal apical dendrites of pyramidal cells, proestrus females, in particular, exhibit DOR internalization and trafficking towards the soma. Dual label studies revealed that DORs are found in NPY-labeled interneurons in the hilus, CA3, and CA1. While DOR colocalization frequency in NPY-labeled neuron somata was similar between animals in the hilus, proestrus females had fewer NPY-labeled neurons that co-labeled with DOR in stratum oriens of CA1 and CA3 when compared to males. Ultrastructural analysis of NPY-labeled axon terminals within stratum radiatum of CA1 revealed that NPY-labeled axon terminals contain DORs that are frequently found at or near the plasma membrane. As no differences were noted by sex or estrous cycle phase, DOR activation on NPY-labeled axon terminals would inhibit GABA release probability equally in males and females. Taken together, these findings suggest that ovarian steroids can impact hippocampal function through direct effects on DOR levels and trafficking in principal cells and broad indirect effects through reductions in DOR-ir in NPY-labeled interneurons, particularly in CA1.

Variations in opioid peptide levels during the estrous cycle in Sprague–Dawley rats

The estrous cycle, with its various hormonal conditions, may provide us with the means of understanding how endocrine states relate to opioid mechanisms. There has been increasing experimental support for interaction between sex steroids and opioid peptides in the central nervous system. Here, we describe fluctuations in endogenous brain immunoreactive (ir) peptide levels during various phases of the estrous cycle in the female Sprague-Dawley rat. Ir levels of dynorphin A, dynorphin B, Leu-enkephalin-Arg(6), Met-enkephalin-Arg(6)Phe(7) and nociceptin/orphanin FQ were measured in the pituitary gland and in 10 areas of the brain during the diestrus, proestrus and estrus phase. In several areas of the brain, basal levels of endogenous opioid peptides showed variation during the course of the estrous cycle. Significant differences were found between the diestrus state and the proestrus and/or estrus conditions, particularly in the nucleus accumbens, caudate putamen and the substantia nigra. The ir levels of the endogenous peptide nociceptin/orphanin FQ became altered in only one of the areas measured, indicating less variance during the estrous cycle. Correlation analyses revealed that significant associations between dynorphin A or dynorphin B and Leu-enkephalin-Arg(6) were found more often during estrus than during the diestrus and proestrus conditions. The ratio between the ir levels of Leu-enkephalin-Arg(6), a cleavage product of the enzymatic conversion of dynorphin peptides into shorter peptides in vivo, and dynorphin peptides was calculated. The significantly lower ratio between Leu-enkephalin-Arg(6) and dynorphin B in diestrus than in proestrus and estrus also indicates cyclic fluctuations in the enzymatic cleavage of dynorphin. These findings are discussed in relation to the possible role of interactions between sex steroids and opioid peptide mechanisms during the normal estrous cycle.

THE ROLE OF THE OPIOIDERGIC SYSTEM IN A FAMILIAL MODEL OF DILATED CARDIOMYOPATHY

Opioidergic signaling has recently been determined to negatively regulate cardiac function. Opioid receptor signaling has also been shown to be cardioprotective against ischemia-reperfusion injury. However, whether this signaling is altered in cardiac pathology is unknown. Using a hamster model of dilated cardiomyopathy (Bio14.6) and the appropriate controls (F1B), we investigated alterations in levels of endogenous opioid peptides, as well as functional changes using the perfused, work-performing heart setup. We saw no differences in opioid peptide levels at any stage of pathology. However, we observed an altered response to kappa and delta opioid receptor stimulation at different stages of pathology. Delta receptor stimulation caused a significantly more robust response in Bio14.6 than F1B hamsters at 1 month, prior to pathology, however there was no difference in the response at later, pathological, time points. Kappa receptor stimulation was similar at pre- and early-pathology time points but was significantly more efficacious and potent in Bio14.6 at the heart failure time point. That opioidergic stimulation is altered at multiple time points during the progression to heart failure suggests it may be a critical component of the failing heart phenotype.

Maternal Separation Alters Acquisition of Ethanol Intake in Male Ethanol‐Preferring AA Rats

Background: Prolonged daily maternal separation can increase the risk for developing substance abuse, whereas brief maternal separation has been reported to induce positive behavioral effects, decrease voluntary ethanol intake and induce long‐lasting changes in brain opioid peptides. The ethanol‐preferring AA (Alko, Alcohol) rats have altered basal levels of endogenous opioid peptides that may relate to their high voluntary ethanol intake. The purpose of this study was to investigate whether maternal separation could affect acquisition of ethanol intake in AA rats.Methods: The rat pups were exposed to 15 min (MS15) or 360 min (MS360) of maternal separation during postnatal day 1‐21, while control rats were exposed to normal animal facility rearing. As adults, the male rats were gradually introduced to increasing concentrations of ethanol. Furthermore, the effect of restraint stress on voluntary ethanol intake was investigated.Results: The MS15 rats reached a high voluntary ethanol intake later than MS360 and control rats. The MS15 rats had a lower ethanol intake and preference at 8% ethanol compared to MS360 rats and lower ethanol intake compared to control rats. MS15 rats also had a lower 10% ethanol intake in comparison with MS360 rats. Restraint stress decreased the ethanol intake in MS15 and MS360 rats, whereas the ethanol intake in control rats was unaffected.Conclusions: We have previously shown that prolonged periods of maternal separation in Wistar rats result in an increased ethanol intake later in life. This was not repeated in this study, using AA rats with an inherent high ethanol intake. However, it is shown that brief maternal separation can delay acquisition of high ethanol intake and in addition decrease voluntary ethanol intake and preference in AA rats. Maternal separation for 15 min is therefore suggested to protect against high voluntary ethanol intake later in life.

Clonidine attenuates naloxone-induced opioid-withdrawal syndrome in cholestatic mice.

Cholestasis is associated with elevated plasma level of endogenous opioid peptides. Naloxone-precipitated withdrawal syndrome has been described in a mouse model of acute cholestasis. Thus we aimed at determining whether central noradrenergic hyperactivity is involved in manifestation of naloxone-precipitated withdrawal syndrome in mice with obstructive cholestasis. Acute cholestasis was induced by bile duct resection in mice and physical dependence was observed by precipitating a withdrawal syndrome with naloxone (2 mg/kg, intraperitoneally) 5 days after induction of cholestasis. Administration of clonidine (0.1 mg/kg, intraperitoneally), an alpha2-adrenoceptor agonist, 15 min. before naloxone injection significantly alleviates withdrawal severity in cholestatic mice. However, pretreatment of animals with yohimbine (3 mg/kg, intraperitoneally), an alpha2-adrenoceptor antagonist, 15 min. before clonidine blocked this ameliorative effect of clonidine. The results of this study support the evidence for involvement of the alpha2-adrenoceptors in the withdrawal syndrome of cholestasis in a mouse model.

Clonidine Attenuates Naloxone‐Induced Opioid‐Withdrawal Syndrome in Cholestatic Mice*

Abstract: Cholestasis is associated with elevated plasma level of endogenous opioid peptides. Naloxone‐precipitated withdrawal syndrome has been described in a mouse model of acute cholestasis. Thus we aimed at determining whether central noradrenergic hyperactivity is involved in manifestation of naloxone‐precipitated withdrawal syndrome in mice with obstructive cholestasis. Acute cholestasis was induced by bile duct resection in mice and physical dependence was observed by precipitating a withdrawal syndrome with naloxone (2 mg/kg, intraperitoneally) 5 days after induction of cholestasis. Administration of clonidine (0.1 mg/kg, intraperitoneally), an α2‐adrenoceptor agonist, 15 min. before naloxone injection significantly alleviates withdrawal severity in cholestatic mice. However, pretreatment of animals with yohimbine (3 mg/kg, intraperitoneally), an α2‐adrenoceptor antagonist, 15 min. before clonidine blocked this ameliorative effect of clonidine. The results of this study support the evidence for involvement of the α2‐adrenoceptors in the withdrawal syndrome of cholestasis in a mouse model.

The role of minoxidil on endogenous opioid peptides in the spinal cord: a putative co-agonist relationship between K-ATP openers and opioids

ATP-gated K + channel openers produce antinociception that is attenuated by opioid receptor antagonists, indicating K-ATP openers produce antinociception, in part, via the release of endogenous opioid peptides. Utilizing the spinal perfusion method, male Sprague–Dawley rats were administered minoxidil intrathecally (i.t.) at doses ranging from 12.5 to 200 μg/rat for 3 min, tested for antinociception using the tail-flick test, and perfused with artificial cerebrospinal fluid (aCSF) to collect endogenous opioid peptides. Endogenous opioid peptide levels were measured by radioimmunoassay. Naltrindole, a δ-opioid receptor antagonist, at 4 mg/kg, subcutaneously (s.c.), blocked minoxidil-induced antinociception. β-Funaltrexamine, a μ-opioid receptor antagonist, at 100 μg/rat, partially blocked minoxidil, whereas the κ-opioid receptor antagonist nor-binaltorphimine, at a dose of 100 μg/rat, did not attenuate minoxidil. Although antagonists of the μ- and δ-opioid receptor attenuated minoxidil-induced antinociception, there was no increase in β-endorphin, an endogenous ligand with affinity for both μ- and δ-opioid receptors or [Leu 5]enkephalin, an endogenous ligand with affinity for δ-opioid receptors.

Enkephalinergic Neurons in the Periaqueductal Gray and Morphine Withdrawal

The effects of opioid (e.g., morphine) withdrawal on levels of endogenous opioid peptides and their mRNA in the various brain regions have been studied. However, the role of this opioidergic mechanism in the mediation of opioid withdrawal is not fully understood. Preproenkephalin (PPE) mRNA in the caudal periaqueductal gray (cPAG), an important brain region in opioid withdrawal, is increased by both opioid antagonist (naloxone)-precipitated and spontaneous morphine withdrawal, but not by various other stresses in rats, indicating a role of endogenous enkephalins in the cPAG in morphine withdrawal. In addition, PPE mRNA levels in the cPAG increase in the course of the dissipation of morphine withdrawal, and they are returned to the control levels after disappearance of morphine withdrawal signs. Local administration of an enkephalin analog or peptidase inhibitors into the cPAG suppresses morphine withdrawal signs. These facts suggest that enkephalinergic neurons in the PAG may have a critical role in the recovery phase of morphine withdrawal. Recently, an involvement of transcription factors in morphine withdrawal has been suggested. Thus, the possible role of transcription factors in the regulation of PPE gene expression in the cPAG during morphine withdrawal is also discussed.

Brain Opioid Receptor Binding of [ 3H]CTOP and [ 3H]Naltrindole in Alcohol-Preferring AA and Alcohol-Avoiding ANA Rats

We compared μ- and δ-opioid receptor distributions between the brains of alcohol-preferring Alko, Alcohol (AA) and alcohol-avoiding Alko, Non-Alcohol (ANA) rat lines, using autoradiography on brain sections with μ- and δ-opioid receptor antagonist ligands [ 3H]CTOP and [ 3H]naltrindole, respectively. The labeling patterns of the ligands were consistent with the known opioid receptor distributions in both rat lines and no major genetic differences were found between the lines. However, the binding density of μ- and δ-opioid receptors differed slightly in several brain areas: in the AA brain sections, limbic areas, such as hippocampus and amygdala, showed decreased μ- and δ-opioid receptor binding, whereas the striatal patches were larger and the substantia nigra showed higher binding density of the μ-receptors compared to the ANA sections. The small differences observed between the rat lines could be due to adaptations to altered endogenous opioid peptide levels or neural circuits, and associated with the differences in alcohol drinking or other behaviors.

The Effects of Intrathecally Administered FK480, a Cholecystokinin-A Receptor Antagonist, and YM022, a Cholecystokinin-B Receptor Antagonist, on the Formalin Test in the Rat

Cholecystokinin (CCK) is located in the brain and the spinal cord, and CCK antagonist is reported to enhance the analgesic effect of morphine.It has been suggested that, during inflammation, the level of endogenous opioid peptides increases in the spinal cord. Intrathecally administered CCK antagonist may have some analgesic effect during inflammation via the activated spinal opioid system. To gain a better understanding of the roles of CCK-A and CCK-B receptors in spinal nociceptive transmission during inflammation, this study evaluated the effects of intrathecally administered FK480 (a CCK-A receptor antagonist) and YM022 (a CCK-B receptor antagonist). Inflammation was induced by paw formalin injection (formalin test) in rats. The subcutaneous injection of formalin into the hind paw evoked biphasic flinching (Phase 1, 0-9 min; Phase 2, 10-60 min) of the injected paw. Drugs were administered intrathecally 10 min before (pretreatment) or 7 min after (posttreatment) the formalin injection. Neither pretreatment nor posttreatment with FK480 has any effect on the formalin test. Pretreatment, but not posttreatment, with YM022 depressed the Phase 1 and Phase 2 flinching behavior in a dose-dependent manner, and this YM022 effect was stereospecific and was not antagonized by naloxone. These data indicate that a CCK-B receptor antagonist, but not a CCK-A receptor antagonist, produces an antinociceptive effect in the rat formalin test. This effect of a CCK-B receptor antagonist was not mediated by the spinal opioid receptor activation. (Anesth Analg 1996;83:107-13)

The effects of intrathecally administered FK480, a cholecystokinin-A receptor antagonist, and YM022, a cholecystokinin-B receptor antagonist, on the formalin test in the rat.

Cholecystokinin (CCK) is located in the brain and the spinal cord, and CCK antagonist is reported to enhance the analgesic effect of morphine. It has been suggested that, during inflammation, the level of endogenous opioid peptides increases in the spinal cord. Intrathecally administered CCK antagonist may have some analgesic effect during inflammation via the activated spinal opioid system. To gain a better understanding of the roles of CCK-A and CCK-B receptors in spinal nociceptive transmission during inflammation, this study evaluated the effects of intrathecally administered FK480 (a CCK-A receptor antagonist) and YM022 (a CCK-B receptor antagonist). Inflammation was induced by paw formalin injection (formalin test) in rats. The subcutaneous injection of formalin into the hind paw evoked biphasic flinching (Phase 1, 0-9 min; Phase 2, 10-60 min) of the injected paw. Drugs were administered intrathecally 10 min before (pretreatment) or 7 min after (posttreatment) the formalin injection. Neither pretreatment nor posttreatment with FK480 has any effect on the formalin test. Pretreatment, but not posttreatment, with YM022 depressed the Phase 1 and Phase 2 flinching behavior in a dose-dependent manner, and this YM022 effect was stereospecific and was not antagonized by naloxone. These data indicate that a CCK-B receptor antagonist, but not a CCK-A receptor antagonist, produces an antinociceptive effect in the rat formalin test. This effect of a CCK-B receptor antagonist was not mediated by the spinal opioid receptor activation.

The effects of alcoholism on the hypothalamic-pituitary-adrenal axis: interaction with endogenous opioid peptides.

Abnormal baseline hypothalamic-pituitary-adrenal axis function and dexamethasone suppressibility seen in withdrawing alcoholics returns to normal on abstinence, but some studies report blunting of the ACTH response to CRH persisting during the early abstinence phase. Reduced central levels of endogenous opioid peptides have been postulated to have an aetiological role in alcohol addiction. To evaluate hypothalamic-pituitary-adrenal axis function in a group of recently abstinent alcoholics using basal hormone data, naloxone (an opioid receptor antagonist), and ovine CRH. Nine alcoholics (age 41.4 +/- 3.1 years) studied more than one week after the acute withdrawal period but within 6 weeks of cessation of drinking, and nine age and sex matched non-alcoholic controls. Cortisol, ACTH, CRH and AVP levels were measured every 20 minutes for 2 hours between 0900 and 1100h Twenty mg naloxone i.v. was administered at 1100h (0 minutes) and further samples for the above hormones were taken at 15, 30, 45, 60, 90 and 120 minutes. On a separate occasion, again at 1100h, oCRH 1 microgram/kg (n = 7 alcoholics, n = 6 controls) was administered, with samples for cortisol, ACTH and AVP taken at the same times. Results were examined by analysis of variance for repeated measures (ANOVA), while incremental hormone response and area under the secretory curve (AUC) in alcoholics versus controls were compared by the two-tailed Student's t-test. Linear regression analysis was carried out to examine the relation between basal cortisol and hormone responses to naloxone and oCRH. Basal hormone levels did not differ between the groups. The alcoholics had a blunted ACTH incremental response to naloxone (11.4 +/- 3.0 vs 21.1 +/- 2.5 pmol/l, P < 0.05) but the cortisol response was not significantly different (205 +/- 51 vs 305 +/- 42 nmol/l, P = 0.15). The alcoholics also had a blunted ACTH incremental response to oCRH (28.7 +/- 4.2 vs 41.2 +/- 3.7 pmol/l, P = 0.052) and by ANOVA a significant main effect of group (alcoholic vs control) was seen (P < 0.02) for the ACTH response to oCRH. There was no difference between the groups in the cortisol incremental response to oCRH. In the control subjects, a negative correlation was found between basal cortisol and the cortisol increment (r = -0.82, P < 0.05) and ACTH increment (r = -0.81, P = 0.052) following oCRH, while in contrast, basal cortisol correlated positively with cortisol increment (r = 0.72, P < 0.05) following naloxone. There was also a trend for basal cortisol to correlate positively with ACTH increment following naloxone in the controls (r = 0.63, P < 0.07). In the alcoholics, the normal negative effect of basal cortisol on the cortisol increment after oCRH was reversed, with a positive correlation between basal cortisol and cortisol increment (r = 0.75, P = 0.05). Recently abstinent alcoholics with normal basal HPA axis hormone levels have a blunted ACTH response to naloxone and oCRH. While reduced levels of central endogenous opioid peptides may be a factor in the blunted ACTH response to naloxone in the alcoholics, it is proposed that the alcoholics have reduced pituitary responsiveness to CRH. This may be via a direct pituitary effect of the chronic ethanol exposure or by a reduction in hypothalamic-hypophyseal vasopressin levels.