Muscarinic receptor subtypes in bovine adrenal medulla

Muscarinic receptors in bovine adrenal medullary microsomes were characterized by radioligand binding assay, using l-[3H]quinuclidinyl benzilate (QNB), a muscarinic antagonist. Specific [3H]QNB binding to microsomes was rapid, reversible, saturable and of high affinity. Saturation experiments revealed a single class of binding sites for the radioligand with a maximum number of binding sites and an apparent dissociation constant of 162.6 fmoles/mg protein and 40.3 pM respectively. According to computer-assisted nonlinear regression analysis, however, drug/[3H]QNB competition curves indicated the presence of at least two affinity sites for muscarinic agonists (acetylcholine, carbamylcholine, oxotremorine), with a high (K1) and a low (K2) affinity (e.g. K1 = 664.8 nM and K2 = 36.5 microM for acetylcholine). The two affinity sites for acetylcholine showed only minimal regulation by magnesium and guanosine 5'-triphosphate. Furthermore, the presence of two affinity sites was suggested for the antagonists pirenzepine and gallamine, but not for atropine and pilocarpine. The K1 and K2 values for pirenzepine were 23.7 and 429 nM, respectively, with 54.5% of total sites having a high affinity. These results indicate that at least two distinct subtypes of muscarinic receptors exist in the bovine adrenal medulla and that they are distinguished by their relative binding affinity for muscarinic agonists and antagonists. The receptors are predominantly composed of the affinity state termed M1, as described for the receptors of sympathetic ganglia.     

κ阿片受体分子模拟及其与非肽类κ阿片激动剂的作用

目的：研究了κ阿片受体及其与非肽类激动剂的作用机制。方法;以细菌视紫红质为模板,模建κ阿片受体七个跨膜区的三维结构,将五个高活性非肽类激动剂对接以螺旋区内,研究作用机制。结果：（１）四氢吡咯环氮原子与Ａｓｐ１３８羧基成氢键;（２）乙酰胺羰基氧与受体Ｓｅｒ１８７间存在在氢键作用;（３）与乙酰胺相连的疏水基团处于由Ｖａｌ２３９,Ｖａｌ１２３６,Ｐｈｅ２３５,Ｖａｌ２３２,Ｌｅｕ１８６和Ｔｒｐ１８３构成     

Molecular modeling on kappa opioid receptor and its interaction with nonpeptide kappa opioid agonists 1

目的：研究κ阿片受体及其与非肽类激动剂的作用机制．方法：以细菌视紫红质为模板,模建κ阿片受体七个跨膜区的三维结构;将五个高活性非肽类激动剂对接到螺旋区内,研究作用机制．结果：（１）四氢吡咯环氮原子与Ａｓｐ１３８羧基成氢键;（２）乙酰胺羰基氧与受体Ｓｅｒ１８７间存在氢键作用;（３）与乙酰胺相连的疏水基团处于由Ｖａｌ２３９、Ｖａｌ２３６、Ｐｈｅ２３５、Ｖａｌ２３２、Ｌｅｕ１８６和Ｔｒｐ１８３构成的疏水区域内;（４）激动剂的四氢吡咯环为Ｉｌｅ２９０、Ａｓｐ１３８、Ｉｌｅ１９４、Ｉｌｅ１３５和Ｃｙｓ１３１残基包围．结论：模型将有助于设计新型高效安全的κ阿片受体激动剂．     

Cholinergic muscarinic receptors of bovine adrenal medulla

Muscarinic receptor binding sites were identified on bovine adrenal medulla membranes, using [³H]-quinuclidinyl benzylate. A Scatchard analysis revealed a k_d of 0.07 nM and a Bmax of 3 fmol/mg prot. Muscarinic receptors of the medulla may, in part, be responsible for the release of catecholamines and the modulation of the cyclic nucleotide content of chromaffin cells.     

Norbinaltorphimine: antagonist profile at kappa opioid receptors

The pharmacological profile of the opioid antagonist norbinaltorphimine has been characterised in vitro and in vivo. In vitro, norbinaltorphimine reversibly antagonised the effects of kappa agonists with pA2 values of 10.2-10.4. Norbinaltorphimine was much less potent as an antagonist at mu and delta receptors, pA2 values were 7.4-7.6 and 7.6-7.8, respectively. In all cases Schild slopes were unity. In vivo, norbinaltorphimine was an effective antagonist only at high dose levels and was not very selective between mu and kappa. The results indicate that in vitro norbinaltorphimine is a potent selective kappa antagonist; however, this antagonist profile is not maintained in vivo.     

Characterization of kappa opioid receptors in the rabbit ear artery

Quantitative characterization of the kappa opioid receptor in the rabbit ear artery was carried out using three kappa-selective agonist compounds, dynorphin-(1-13), U-69593 and ethylketocyclazocine. Kinetic analysis was performed using the antagonist, MR 2266. Two other in vitro preparations were studied for comparison: the mouse was deferens and rabbit was deferens. To avoid mu receptor action in the mouse was deferens the irreversible mu receptor antagonist, beta-funaltrexamine, was used. It was demonstrated that, using the highly selective kappa agonist compound U-69593, Ke values for MR 2266 obtained in the three assay systems were not significantly different. These results suggest that kappa receptors present in these three tissues share identical properties.     

Extraordinary postmortem stability of kappa opioid receptors in guinea-pig brain

Postmortem delay can be an important variable in biochemical studies on autopsy tissue. We subjected guinea-pig brains to gradual cooling, simulating temperature conditions of human postmortem brains, in order to assess the sensitivity of kappa receptors to postmortem degradation. Kappa receptor specific binding was defined as the (-)-[3H]ethylketocyclazocine bound in the presence of 100 nM D-ala2-D-leu5-enkephalin and 30 nM morphine. Postmortem delays of up to 16 h did not alter the affinity or density of kappa binding sites. The remarkable stability of kappa receptors may greatly facilitate the study of this opioid receptor subtype in human brain.     

Tetranitromethane modification of the muscarinic receptors in bovine adrenal medulla

Muscarinic receptor binding was examined in bovine adrenal medullary microsomes following exposure to tetranitromethane (TNM) that modifies tyrosine and cysteine residues in proteins. The TNM (10-100 microM) treatment of adrenal medullary microsomes caused a concentration-dependent and irreversible reduction in the maximum number of binding sites (Bmax) for l-(3H)quinuclidinyl benzilate (QNB), with a slight increase in the equilibrium dissociation constant (KD). Typically, about a 36% decrease and a 1.3-fold increase in the corresponding values were obtained at 50 microM of TNM. The alteration in the Bmax was partially prevented by atropine but not carbamylcholine, and it was not reversed by subsequent treatment with dithiothreitol, a disulfide reducing agent. The change in the KD was unaffected by these agents. The TNM (50 microM) treatment also caused a slight decrease in the affinity of atropine and pirenzepine (for both the high and low affinity sites), and it caused a slight decrease in the affinity of carbamylcholine at the high affinity site, with a large loss of the low affinity site. Thus, the results indicate that TNM causes a loss of muscarinic binding sites and a decrease in the binding affinity of muscarinic receptors in bovine adrenal medulla, probably through modifications of functional groups such as tyrosine residues.     

Cardiovascular responses to kappa opioid agonists in intact and adrenal demedullated rats

The effects of three kappa opioid agonists namely, bremazocine, tifluadom and U-50,488H were studied on blood pressure and heart rate in urethane-anesthetized normal and bilateral adrenal demedullated rats. Bremazocine (0.2, 0.4 and 0.6 mg/kg i.v.) produced a dose-dependent decrease in heart rate, while only 0.4 mg/kg bremazocine produced marked hypotension. The effect appeared to be long lasting because even at 60 min following drug administration the decreases in both heart rate and blood pressure continued. Bilateral adrenal demedullation did not change bremazocine-induced fall in blood pressure but the bradycardia was partially blocked. Tifluadom (0.1-0.4 mg/kg i.v.) produced an initial arrest of heart beat followed by bradycardia which recovered in about 60 min. Except for a very transient fall soon after drug administration no significant effect was observed on blood pressure. In adrenal demedullated rats, tifluadom induced initial arrest of heart was not affected but the subsequent bradycardia was blocked. U-50,488H (0.2, 0.4 and 0.6 mg/kg i.v.) produced dose-dependent bradycardia and hypotension both of which were blocked following bilateral adrenal demedullation. Naltrexone methylbromide (MRZ 2663 BR), a quaternary opioid antagonist, injected 5 min prior to U-50,488H, blocked its cardiovascular effects. The results suggest that kappa opioid agonists given i.v. depress cardiovascular system and these effects are mediated through the adrenal medulla and peripheral opioid receptors. The differential effects of kappa opioid agonists on blood pressure and heart rate suggest that either the three kappa agents interact differentially at the kappa opioid receptors or the subtypes of receptors for the kappa opioid exist.     

Evidence for muscarinic receptors in the adrenal medulla of the dog

1. The release of catecholamines from the adrenal medulla by the cholinergic drugs was monitored by the perfusion pressure rise in autoperfused and sympathetically denervated hindlimbs of the dog.2. Acetylcholine, dimethylphenylpiperazinium (DMPP) and methacholine, given in relatively small doses into the aorta proximal to the blood supply to the adrenal glands, caused a marked rise in perfusion pressure which was due to the release of catecholamines from the adrenal medulla.3. Atropine (1 mg/kg intravenously) blocked the pressor response to methacholine only. The ganglion-blocking agents (mecamylamine 4-5 mg/kg or hexamethonium 10 mg/kg) given subsequently blocked the pressor responses to the other two cholinergic drugs. The ganglion-blocking agents, when given before atropine, blocked the pressor action only of DMPP. These agents partly depressed, rather than potentiated, the pressor response to methacholine.4. The results suggest that muscarinic as well as nicotinic receptors are present in the adrenal medulla of the dog.5. Neither atropine nor ganglion-blocking drugs alone reduced the pressor response to acetylcholine. It is postulated that the blockade of one set of receptors makes more acetylcholine available for the other set of receptors and so inactivation of the former receptors are compensated by the increased release through the activation of the latter's.     

Bremazocine-induced backwards walking behavior in rats is mediated via opioid kappa receptors

Bremazocine dose-dependently induced backwards walking behavior in rats after its SC injection. Only the (-) but not the (+) enantiomer induced backwards walking. Pretreatment with either naloxone or MR 2266 reduced the bremazocine-induced backwards walking. MR 2266 was at least ten times more potent than naloxone. These findings suggest that bremazocine-induced backwards walking is mediated via an agonistic action of the drug with opioid kappa receptors. The data may contribute to the discussion concerning opioid kappa receptors and the psychotomimetic effects of some opioid analgesic drugs.     

用分子模构法建立κ阿片受体激动剂的药效基团

目的：建立非肽类κ阿片受体激动剂的药效基团。方法：从ＭＤＬ ＭＤＤＲ数据库中选出五个高活性非肽类κ阿片受体激动剂，以四氢吡咯环Ｎ原子和乙酰胺基团为叠加点，用分子模构法建立非肽类κ阿片受体激动剂的药效基团。结果：四氢吡咯环、乙酰胺的羰基和与惭酰胺相连的疏水基团为非肽类κ阿片受体激动剂共同结构特征。推测受体Ａｓｐ１３８与甲氢吡咯环的Ｎ原子构成氢键，Ｓｅｒ１８７可能与激动剂的乙酰胺羰基以氢键形式相作用。     

Angiotensin II receptor subtypes and biological responses in the adrenal cortex and medulla.

Angiotensin II (AII) receptor subtypes and their potential coupling mechanisms were studied using recently developed peptide and nonpeptide antagonists in rat and bovine adrenal zona glomerulosa cells, as well as in membranes prepared from rat and bovine adrenal cortex and medulla. Comparison of the potencies of these novel antagonists to displace 125I-[Sar1,Ile8]AII from its binding sites revealed two distinct AII binding sites in membranes prepared from rat adrenal capsules (zona glomerulosa) and from rat adrenal inner zones containing the medulla. About 85% of the binding sites of the glomerulosa zone and 30% of those of the inner zones were of the AT1 subtype, with relative affinities for the nonpeptide antagonists Dup 753 and PD 123177 and the peptide antagonist CGP 42112A in the order of Dup 753 much greater than CGP 42112A greater than PD 123177. In contrast, the relative binding potencies for the other (AT2) population of binding sites were CGP 42112A greater than PD 123177 much greater than Dup 753. Neither AII nor its peptide antagonist [Sar1,Ile8]AII could distinguish between the two sets of binding sites. The effects of the new antagonists on functional responses of rat adrenal glomerulosa cells demonstrated that both AII-stimulated aldosterone production and the AII-induced inhibition of adrenocorticotropic hormone-stimulated cAMP formation were mediated by the AT1 receptor subtype. In bovine adrenals, only AT1 receptors were detected in membranes prepared from the cortex and the medulla, as well as in cultured glomerulosa cells. The relative inhibitory potency of Dup 753 was lower by an order of magnitude at bovine than at rat AT1 receptors. The inhibition of AII-induced aldosterone production by the various antagonists was closely correlated with their inhibitory potencies on 125I-[Sar1,Ile8]AII binding to bovine glomerulosa cells. These data suggest that the known effects of AII in adrenal glomerulosa cells are mediated through the AT1 receptor subtype and that the distribution and/or specificity of the AT2 receptors shows marked species variations.     

Local administration of mu or kappa opioid agonists attenuates capsaicin-induced thermal hyperalgesia via peripheral opioid receptors in rats

Rationale: By acting on peripheral opioid receptors, opioid agonists can attenuate nociceptive responses induced by a variety of agents. Objectives: This study was conducted to characterize capsaicin-induced thermal hyperalgesia in rats and to evaluate the hypothesis that local administration of either mu or kappa opioid agonists (fentanyl and U50,488, respectively) can attenuate capsaicin-induced nociception. Methods: Capsaicin was administered s.c. in the tail of rats to evoke a nociceptive response, which was measured by the warm-water tail-withdrawal procedure. Either fentanyl or U50,488 was co-administered with capsaicin in the tail to evaluate local antinociceptive effects. In addition, the local antagonism study was performed to confirm the site of action of both opioid agonists. Results: Capsaicin (0.3–10 μg) dose dependently produced thermal hyperalgesia manifested as reduced tail-withdrawal latencies in 45°C water. Co-administration of either fentanyl (0.32–3.2 μg) or U50,488 (10–100 μg) with capsaicin (3 μg) attenuated capsaicin-induced hyperalgesia in a dose-dependent manner. Furthermore, this local antinociception was antagonized by small doses (10–100 μg) of an opioid antagonist, quadazocine, applied s.c. in the tail. However, the locally effective doses of quadazocine, when applied s.c. in the back (i.e., around the scapular region), did not antagonize either fentanyl or U50,488. Conclusions: In this experimental pain model, activation of peripheral mu or kappa opioid receptors can attenuate capsaicin-induced thermal hyperalgesia in rats. It supports the notion that peripheral antinociception can be achieved by local administration of analgesics into the injured tissue without producing central side effects. Received: 10 May 1999 / Final version: 18 August 1999     

Ontogenetic studies on mu, delta and kappa opioid receptors in rat brain

The ontogenetic pattern of multiple opioid binding sites in rat brain from birth until weaning has been investigated. [3H]-dihydromorphine ([3H]-DHM)3 [3H]-D-Ala2-D-Leu5-enkephalin ([3H]-DADLE) and [3H]-dynorphin A (1-8) ([3H]-DYN) as markers of mu (mu), delta (delta) and Kappa (kappa) sites were utilized respectively. The analysis of the kinetic parameters of [3H]-DHM binding shows that, at birth, mu sites possess an affinity similar to that of adult animals, and a density of 50%, which reaches 80% of the adult value at day 4. On the contrary, [3H]-DADLE binding in the first post-natal days shows low affinity and low density and delta-sites do not reach values comparable to the adult ones until the second week of life. The kinetic parameters of [3H]-DYN binding are almost undetectable during the preweanling period, due to the very low density of kappa sites at this stage of life. Displacement studies with mu-, delta- and kappa-selective ligands show that the Ki values on [3H]-DHM binding sites were similar in 4 day old and adult animals for all the tested compounds, whereas Ki values on [3H]-DADLE and [3H]-DYN binding sites reflected an immaturity of delta and kappa receptors. In conclusion, our data suggest that multiple opioid receptors follow different ontogenetic patterns. In the first stages of life only mu receptors are almost mature and possibly mediate endogenous opioid actions and exogenous opiate pharmaco-toxicological effects.     

Involvement of kappa opioid receptors in formalin-induced inhibition of analgesic tolerance to morphine in mice

This study examined the role of kappa opioid receptors (KOR) in the mechanism underlying tolerance to the analgesic effects of morphine induced by chronic pain. The analgesic effect of morphine (10 mg kg(-1)), estimated by the tail-flick test in mice, gradually decreased during repeated daily morphine treatment. A significant decrease in the analgesic effect of morphine was seen on the fifth day of repeated morphine treatment compared with the first day. Chronic pain was induced by subcutaneous administration of 2% formalin into the dorsal part of the left hind paw, which significantly inhibited development of tolerance to morphine analgesia. The effect of formalin-induced pain on inhibition of morphine tolerance was reversed by the KOR antagonist nor-binaltorphimine. Furthermore, an antisense oligodeoxynucleotide, but not a missense oligodeoxynucleotide, against KOR completely suppressed the inhibitory effect of formalin-induced pain on morphine tolerance. Naltrindole, an antagonist of delta opioid receptor, did not affect chronic-pain-induced tolerance to morphine. Our findings show that the inhibitory effect of chronic pain on analgesic tolerance to morphine is mediated by KOR rather than delta opioid receptors.     

Pharmacological characterization and autoradiographic localization of dopamine receptors in the rat adrenal medulla

The pharmacological profile and the anatomical localization of dopamine D₁-like and D₂-like receptors were studied in sections of rat adrenal medulla, with radioligand binding and autoradiographic techniques, respectively. [³H]([R]-(+)-chloro-2,3,4,5-tetrahydro-5-phenyl-1H-3benzazepin-al hemimaleate) (SCH 23390) was used as a ligand for dopamine D₁-like receptors and [³H]spiperone was used as a ligand for dopamine D₂-like receptors. Radioligand binding and light microscope autoradiography did not show specific [³H]SCH 23390 binding in sections of rat adrenal medulla. This suggests that rat adrenal medulla does not express dopamine D₁-like receptors. [³H]Spiperone was specifically bound to sections of rat adrenal medulla. The binding was time-, temperature- and concentration-dependent, with a dissociation constant (K_d) of 1.05 nM and a maximum density of binding sites (B_max) of 100.2 ± 3.8 fmol/mg tissue. The pharmacological profile of [³H]spiperone binding to rat adrenal medulla was similar to that displayed by neostriatum, which is known to express dopamine D₂ receptors. Light microscope autoradiography showed the accumulation of specifically bound [³H]spiperone as silver grains within sections of adrenal medulla. Silver grains were found primarily over the cellular membrane of chromaffin cells. The above data indicate that chromaffin cells of the rat adrenal medulla express dopamine receptors belonging to the dopamine D₂ receptor subtype. These receptors are probably involved in the modulation of catecholamine release from chromaffin cells, as documented by functional studies.     

Comparative neuroanatomical distribution of the kappa and mu opioid receptors in guinea pig brain sections

The opiate family of receptors is believed to consist of the mu, delta, kappa and sigma subtypes. Autoradiographic studies have been conducted to define the distribution of the mu and delta receptors. However, due to the lack of a selective ligand definitive studies have not been reported for the kappa receptor. Recently such a ligand was discovered and characterized. Results are presented in which the distribution of kappa receptors in guinea pig brain slices were determined using autoradiographic techniques and its distribution compared to that of a typical mu ligand. The results demonstrate numerous differences between the two opioid systems. The kappa receptors predominate in the prefrontal cortex, the nucleus accumbens, and deep cortex; whereas the mu receptors have a more intense, broader and different distribution.     

Pharmacological activation of kappa opioid receptors: aversive effects in adolescent and adult male rats

Rationale

The dynorphin (DYN)/kappa opioid receptor (KOR) system is involved in the dysphoric properties of drugs of abuse. Given that adolescents show reduced sensitivity to aversive effects of many drugs, alterations in the DYN/KOR system may contribute to the prevalence of drug use during adolescence.

Objectives

The present study was designed to assess dysphoric properties of a selective kappa agonist, U62,066, in adolescent and adult rats using both conditioned taste aversion (CTA) and conditioned place aversion (CPA) paradigms.

Methods

For CTA, water-restricted rats were administered U62,066 following 30 min access to a saccharin solution, with subsequent saccharin consumption used to index aversion. For CPA, animals were allowed access to both compartments of a two-compartment chamber for a 15-min pre- and post-conditioning test. For conditioning, subjects were administered U62,066 prior to confinement to one side of the chamber and saline prior to confinement to the other side for a total of four pairings.

Results

Overall, adolescents displayed reduced sensitivity to the kappa agonist relative to adults. Adults demonstrated taste aversions to the 0.2 and 0.3 mg/kg doses of U62,066, whereas adolescents did not display aversions to any tested doses. Adults demonstrated a place aversion to the 0.1 and 0.2 mg/kg dose of U62,066 when paired with the preferred side of the conditioning chamber. Adolescents did not display aversions to any of the doses tested.

Conclusions

Reduced sensitivity to DYN/KOR system activation during adolescence may be a contributing factor to the age-typical insensitivity to aversive properties of drugs commonly abused by adolescents.