Rotational behaviour produced by intranigral injections of bovine and human β-casomorphins in rats

The biological activity of -casein derived -casomorphin peptides was evaluated by injecting bovine -casomorphin-5 (Tyr-Pro-Phe-Pro-Gly), the homologous sequence in human -casein (Tyr-Pro-Phe-Val-Glu) and the corresponding N-terminal tetrapeptides into the left substantia nigra of rats. Their ability to produce rotational behaviour was compared to that produced by three reference compounds, morphine, d-ala² d-leu⁵enkephalin and U50,488H, ligands for , and types of opioid receptors, respectively. The relative potencies of -casomorphins and morphine were compared to those tested in two in vitro assays for opioid activity: (1) inhibition of the electrically induced contraction of the isolated myenteric plexus-longitudinal muscle of the guinea-pig ileum and (2) displacement of ³H-dihydromorphine binding to brain membranes. The same ranking order of potency was found in all three assays, the peptides from human -casein being about 10-fold less potent than those from bovine -casein. The effects of both morphine and bovine -casomorphin-5 in producing rotational behaviour were antagonized by naloxone; however, approximately 10-fold more naloxone was required to antagonize the -casomorphin-5 effect than that of morphine. The present data are discussed in the light of the recent observation that high concentration of -casomorphin-like peptides are found in the cerebrospinal fluid and plasma of women with postpartum psychosis.     

Specificity evaluation of antibodies against human β3-adrenoceptors

β(3)-Adrenoceptors are a promising drug target for the treatment of urinary bladder dysfunction, but knowledge about their expression at the protein level and their functional role is limited, partly due to a lack of well validated tools. As many antibodies against G-protein-coupled receptors, including those against β(3)- and other β-adrenoceptor subtypes, lack selectivity for their target, we have evaluated the specificity of five antibodies raised against the full-length protein of the human β(3)-adrenoceptor (H155-B01), its N-terminus (LSA4198 and TA303277) and its C-terminus (AB5122, Sc1472) in immunoblotting and immunocytochemistry. Our primary test system were Chinese hamster ovary cells stably transfected to express each of the three human β-adrenoceptor subtypes at near physiological levels (100-200?fmol/mg protein). None of the five antibodies exhibited convincing target specificity in immunoblotting with Sc1472 apparently being least unsuitable. In immunocytochemistry, LSA4198 and Sc1472 appeared most promising, exhibiting at least some degree of specificity. As these two antibodies have been raised against different epitopes (N- and C-terminus of the receptor, respectively), we propose that concordant staining by both antibodies provides the most convincing evidence for β(3)-adrenoceptor labelling in cyto- or histochemistry studies.     

Synthesis of β-hydroxy-propenamide derivatives and the inhibition of human dihydroorotate dehydrogenase

Novel beta-hydroxy propenamides as analogues of the active metabolite of leflunomide (A 771726) were synthesized and evaluated for their inhibitory activity on dihydroorotate dehydrogenase (DHODH) in an investigation into their immunosuppressive activity. Compounds 2a, 3a, and 3h were approximately 4-40 times more potent than leflunomide in their activity while they were-less active than A 771726.     

Subsensitivity of human β-adrenergic adenylate cyclase after salbutamol treatment of depression

Although numerous studies have suggested that depression may be associated with a reduction in synaptic noradrenaline in the brain, direct -adrenergic receptor agonists have only recently been tested in the treatment of depression. Moreover, newer theories of antidepressant action suggest that a reduction in -adrenergic receptor sensitivity is a better correlate of antidepressant treatment than noradrenaline turnover changes. Eleven depressed patients were treated with salbutamol, a -2-adrenergic agonist, and -2-adrenergic receptor sensitivity was evaluated before, during, and after treatment. -Adrenergic receptor sensitivity was evaluated by measuring the plasma cyclic AMP increase after an IV dose of salbutamol. The -adrenergic agonist exhibited antidepressant efficacy and induced subsensitivity of the -adrenergic adenylate cyclase with a time course paralleling the antidepressant effects. The results support the concept that receptor sensitivity changes occur during antidepressant therapy.     

Comparison of three radioligands for the labelling of human β-adrenoceptor subtypes

We have compared the ability of three radioligands, [¹²⁵I]-cyanopindolol, [³H]-CGP 12,177 and [³H]-dihydroalprenolol, to label the three human β-adrenoceptor subtypes. Saturation and competition binding experiments were performed using membrane preparations from Chinese hamster ovary cells stably transfected with the three subtypes. While [³H]-CGP 12,177 had very similar affinity for β₁- and β₂-adrenoceptors (about 40 pM), [¹²⁵I]-cyanopindolol and [³H]-dihydroalprenolol had 4- to 6-fold higher affinity for β₂- as compared to β₁-adrenoceptors (10 vs 45 and 187 vs 1,021 pM, respectively). The affinity of [¹²⁵I]-cyanopindolol at β₃-adrenoceptors was considerably lower (440 pM) than at the other two subtypes. The β₃-adrenoceptor affinity of [³H]-CGP 12,177 and [³H]-dihydroalprenolol was so low that it could not be estimated within the tested range of radioligand concentrations (up to 4,000 pM and 30,000 pM for [³H]-CGP 12,177 and [³H]-dihydroalprenolol, respectively). We conclude that all three radioligands are ill-suited to label β₃-adrenoceptors, particularly in preparations co-expressing multiple subtypes. In the absence of alternatives, [¹²⁵I]-cyanopindolol appears the least unsuitable to label β₃-adrenoceptors. There is a need for high-affinity radioligands which are either selective for β₃-adrenoceptors or reasonably non-selective among all three β-adrenoceptor subtypes.     

Inhibition of human and rat 11β-hydroxysteroid dehydrogenases activities by bisphenol A

Bisphenol A (BPA) is a potential endocrine disruptor. It has been shown that it can interfere with steroid biosynthesis and metabolism. However, the mechanism is unclear. The objective of the present study is to investigate the effects of BPA on two isoforms of 11β-hydroxysteroid dehydrogenases (11β-HSD1 and 11β-HSD2) in human and rat tissues. Human liver, rat testis microsomes as well as rat adult Leydig cells were used for measurement of 11β-HSD1 activity, and human and rat kidney microsomes for 11β-HSD2 activity. BPA inhibited human and rat 11β-HSD1 activities with the half maximal inhibitory concentrations (IC₅₀s) of 14.81 ± 0.06 μM (mean ± SEM) for human and 19.39 ± 0.09 μM for rat enzyme, respectively. BPA inhibited rat 11β-HSD1 activity in intact rat Leydig cells. BPA also weakly inhibited both human and rat 11β-HSD2 activities. At 100 μM, BPA inhibited human and rat enzymes by 51.16% and 41.61%, respectively. In conclusion, BPA is an inhibitor for both 11β-HSD1 and 11β-HSD2, with selectivity against the type I enzyme.     

Effect of thiols on β2-adrenoceptors in human mononuclear leucocytes

Summary The effect of the disulfide reducing agent dithiothreitol (DTT) and other thiols on binding of the gb-adrenoceptor antagonist (–)-¹²⁵iodocyanopindolol (¹²⁵ICYP) to human mononuclear leucocytes (MNL) was investigated. Saturation experiments and dissociation kinetics revealed two classes of specific ¹²⁵ICYP binding sites, one of high and the other of low affinity, respectively. In intact MNL DTT caused a decrease in specific binding. This was due almost selectively to a decrease in the affinity of high affinity binding sites, which decreased gradually in a concentration-dependent manner to the affinity of low affinity binding sites. In MNL membranes DTT decreased not only the affinity but also the number of high affinity binding sites. The DTT effect was completely reversible by simple reoxidation on air. The structural isomers (±)-DTT, (–)-DTT and dithioerythritol revealed identical effects on specific binding, whereas the monothiols mercaptoethanol and -monothioglycerol, having a lower redox potential, were considerably less effective. In the same concentration range that influenced specific binding, DTT stimulated intracellular cAMP production. These results suggest functionally important disulfide bridges which regulate the affinity of -adrenoceptor binding sites in human MNL. They stabilize the receptor in a high affinity state; their reduction causes the conversion of the high affinity state into a low affinity state in a process associated with stimulation of adenylate cyclase. Available evidence indicates that a similar transformation is made by -adrenoceptor agonists. Consequently low affinity ¹²⁵ICYP binding sites preexistent in untreated cells could represent a reduced receptor state resulting from agonist-receptor interaction in vivo. Send offprint requests to J. Remien at the above address     

Penetration of β-naphthylamine and o-toluidine through human skin in vitro

Several aromatic amines (AAs) are known to be carcinogens for humans. AAs are considered to be substantially absorbed through the skin. However, the database for dermal absorption of AAs in general is limited and no specific studies on dermal absorption of β-naphthylamine (BNA) and o-toluidine (OT) have been published. In the present study using diffusion cells, we investigated dermal penetration of BNA and OT through human skin. We have demonstrated that both AAs penetrate through human skin fast (lag time: ∼1.2 vs. 0.8 h) and in high percentages (54 vs. 50%, respectively, of the applied dose within 24 h). A skin notation is therefore justified for these substances.     

Synthesis and antimicrobial activities of highly functionalised novel β-lactam and thiazolidine-grafted tetrahydrobenzothiophenes

A series of biologically active N-(3-chloro-2-oxo-4-phenylazetidin-1-yl)/3-N-(4-oxo-2-arylthiazolidin-3-yl)-2-(methylsulfonamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamides derivatives have been synthesised in good yield. The structures of compounds have been established on the basis of IR, ¹H, ¹³C NMR, and elemental analysis. The structure–activity relationships have been studied by screening of antimicrobial activity over a representative panel of bacterial and fungal strains using two-fold serial dilution method. All these synthesised compounds exhibited significant activities against all bacterial and fungal strains.     

Stable overexpression of human β2-adrenergic receptors in mammalian cells

Summary Human ₂-adrenergic receptors were overexpressed in chinese hamster ovary (CHO) and human epitheloid carcinoma (HeLa) cells. Stable expression in these cells was achieved by transfection of a vector containing the cDNAs for the human ₂-adrenergic receptor as well as for dihydrofolate reductase. By stepwise increases of the concentration of methotrexate — an inhibitor of dihydrofolate reductase — the expression in CHO cells could be increased to levels of almost 200 pmol/mg membrane protein, which is more than 1% of the total membrane protein. In contrast, overexpression of the receptors in HeLa cells by the same technique led to cell death.The receptors produced in overexpressing CHO cells were correctly processed and were fully functional with respect to their ligand binding and signalling properties. The adenylyl cyclase activity of membranes from these cells responded with extremely high sensitivity to the -adrenergic receptor agonist isoproterenol. The receptors could be purified from these membranes to apparent homogeneity by solubilization and chromatography on a single affinity column. Thus, the expression system described here allows the preparation of human ₂-adrenergic receptors in quantities sufficient for pharmacological and biochemical investigations.     

Anti-oxidative and anti-aging activities of 2-O-α-glucopyranosyl-L-ascorbic acid on human dermal fibroblasts

A stable ascorbic acid derivative, 2-O-α-glucopyranosyl-l-ascorbic acid (AA-2G), was evaluated and compared with ascorbic acid for its protective effect against cellular damage and senescence induced by hydrogen peroxide (H(2)O(2)). Pretreatment with AA-2G for 72 h promoted the proliferation of normal human dermal fibroblasts (NHDF) and protected against cell damage induced by H(2)O(2). In contrast, ascorbic acid increased the proliferation and protected against cell damage, only when culture medium containing ascorbic acid was replaced every 24 h during the pretreatment period. These results suggest that the effect of AA-2G is longer-lasting compared to that of ascorbic acid. Senescence associated-β-galactosidase (SA-β-gal) activity, a classical biomarker of cellular senescence, was increased in H(2)O(2)-exposed NHDF cells, but pretreatment or posttreatment with ascorbic acid or AA-2G significantly inhibited the increase in SA-β-gal levels. AA-2G was more potent than ascorbic acid in down-regulating SA-β-gal activity. Expression of SIRT1, which has attracted attention as an anti-aging factor in recent years, was significantly decreased in H(2)O(2)-exposed NHDF cells compared to untreated cells. However, pretreatment NHDF cells with AA-2G before H(2)O(2) exposure significantly inhibited this decrease in SIRT1 expression, whereas ascorbic acid had no effect. After H(2)O(2) exposure, the expression levels of p53 and p21 were increased in NHDF cells and pretreatment with AA-2G inhibited this increase. Together, these results suggest that AA-2G protects dermal fibroblasts from oxidative stress and cellular senescence. These characteristics indicate that AA-2G could become a promising material for its anti-aging properties.     

Immunomodulatory and immunorestorative activities of β-d-glucan-rich extract and polysaccharide fraction of mushroom,Pleurutus tuberregium

Abstract

Context: Some edible mushrooms are reputed to possess useful medicinal properties which are related to their ability to modulate the protective responses of the immune system.

Objective: This study explored the immunomodulatory and immunorestorative properties of a hot aqueous extract (APTR) and of a β-d-glucan-enriched polysaccharide fraction (BGP) of a local oyster mushroom Pleurutus tuberregium (Fr.) Singer (Pleurotaceae).

Materials and methods: Immunomodulatory activities were investigated by assessing specific and none-specific immune responses in immunocompetent and immunosuppressed mice; as well as in vitro in culture of RAW264.7 macrophages stimulated with BGP.

Results: In a homologous prime-boost immunization schedule, oral supplementation with APTR (100, 200, or 400?mg/kg) and BGP (100 or 200?mg/kg) resulted in significantly higher titers of total IgG, IgG₁, and IgG_2a by as much as 2–4-folds compared with the levels in untreated control mice. The mean hemagglutination (HA) titer in immunized mice that were treated with dexamethasone (DEX; 5?mg/kg) was significantly (p?<?0.05) lower than the titer in groups that did not receive dexamethasone; however, short-term alternate day administration of APTR (200?mg/kg) to mice that had been immunosuppressed with 5?mg DEX/kg produced significant increases in secondary anti-SRBC antibody compared with the mean titer of mice immunized and treated with DEX alone. In in vitro studies, stimulation of RAW264.7 macrophages with BGP caused significant increases in iNO and TNF-α expression, and phagocytic functions of the cell.

Conclusion: Taken together, the results of these studies showed that P. tuberregium imparts immunostimulatory and immunorestorative effects that could be explained, in part, by the actions of its β-d-glucan constituent(s) on macrophages.     

Distinct down-regulation of cardiac β1- and β2-adrenoceptors in different human heart diseases

Summary Cardiac -adrenoceptor density and ₁- and ₂-subtype distribution were examined in human left ventricular myocardium from transplant donors serving as controls and from patients with mitral valve stenosis, aortic valve stenosis, idiopathic dilated cardiomyopathy, and ischaemic cardiomyopathy respectively. The total -adrenoceptor density was similar in transplant donors and patients with moderate heart failure (NYHA II–III) due to mitral valve stenosis, but was markedly reduced in all forms of severe heart failure (NYHA III–IV) studied. A reduction of both ₁- and ₂-adrenoceptors was found in patients with severe heart failure due to mitral valve stenosis or ischaemic cardiomyopathy. In contrast, a selective down-regulation of ₁-adrenoceptors with unchanged ₂-adrenoceptors and hence a relative increase in the latter was observed in idiopathic dilated cardiomyopathy and aortic valve stenosis. It is concluded that the extent of total -adrenoceptor down-regulation is related to the degree of heart failure. Selective loss of ₁-adrenoceptors is not specific for idiopathic dilated cardiomyopathy but also occurs in aortic valve stenosis. Changes in ₁- and ₂-subtype distribution are rather related to the aetiology than to the clinical degree of heart failure. Send offprint requests to M. Steinfath at the above address     

Assessment of a novel β2-adrenoceptor agonist, trantinterol, for interference with human liver cytochrome P450 enzymes activities

The effect of a novel β₂-adrenoceptor agonist, trantinterol on the activities of cytochrome P450 (CYP450) was investigated with human liver microsomes and human cryohepatocytes in order to assess the potential for drug-drug interactions. The ability of trantinterol to inhibit CYP450 activities was evaluated in vitro in human liver microsomes. Trantinterol did not inhibit CYP2C19, CYP2D6, and CYP3A4/5 (IC₅₀ > 100 μM). It acted as a weak inhibitor of CYP1A2 and CYP2C9 with IC₅₀ of 70.8 and 81.9 μM, respectively. No time-dependent inhibitions were observed in the present research. To evaluate CYP450 induction, human cryohepatocytes (n = 3) were used and treated once daily for 3 days with trantinterol (0.01, 0.1, and 1 ng/ml), after which CYP450 activities were measured. At concentration of 0.01 ng/ml, which is close to the C_max at maximal recommended doses (50 μg), trantinterol was about 8% as effective as omeprazole (CYP1A2 inducer) only with donor 2. At concentration of 1 ng/ml, trantinterol was about 3.6 ± 3.1% as effective as rifampin (CYP3A4/5 inducer). These in vitro results indicated that, at pharmacological relevant concentrations, trantinterol will not produce clinically significant CYP450 inhibition or induction.     

Functional effects of polyamines via activation of human β1- and β2-adrenoceptors stably expressed in CHO cells

Polyamines mediate acute metabolic effects and cardiac hypertrophy associated with β-adrenoceptor stimulation. They may also modulate β-adrenoceptors, causing functional responses in rat atria and tracheal smooth muscle. The aim of this study was to determine whether polyamines interact with human β(1)- and β(2)-adrenoceptors and the functional consequences of such an interaction. Chinese hamster ovary (CHO) cells stably transfected with human β(1)- and β(2)-adrenoceptors were used to evaluate the effect of polyamines binding to β-adrenoceptors, cAMP production and morphological changes, which were pharmacologically validated by investigating the effects of the β-adrenoceptor agonists, isoproterenol and salbutamol. Polyamines interacted with human β(1)- and β(2)-adrenoceptors, as shown by the displacement of [(125)I]iodocyanopindolol in the binding assay. Putrescine showed higher affinity to β(1)- than β(2)-adrenoceptors. Spermidine and spermine produced partial displacement (approximately 50%) and, at the highest concentration, the effect was reversed. Putrescine and spermine acutely increased cAMP and, in a serum-free medium, induced a stellate-like form in cells, which was inhibited by propranolol, a β-blocker. A 10 to 15 h incubation with putrescine produced a spindle-like form and spatial organization via β-adrenoceptor activation, evidenced by the antagonizing effect by propranolol and lack of effect in wild-type CHO cells. Additionally, it decreased cell proliferation independently of β-adrenoceptor activation. Spermine caused cell death via fetal bovine serum-dependent and -independent mechanisms. The results suggest that putrescine may act as a non-selective and low affinity agonist of human β(1)- and β(2)-adrenoceptors, eliciting morphological changes. These findings may be of importance in physiology and in diseases involving β-adrenoceptor functionality.     

Agonist effects of zinterol at the mouse and human β3-adrenoceptor

The present study investigates the action of zinterol at β₃-adrenoceptors. We used mouse primary brown adipocytes and Chinese hamster ovary (CHO-K1) cells expressing the mouse or human β₃-adrenoceptor. Zinterol was a full agonist at increasing cyclic AMP levels in primary brown adipocytes (which express β₁- and β₃-adrenoceptors but not β₂-adrenoceptors), and this effect was almost totally abolished in adipocytes derived from β₃-adrenoceptor knock-out (KO) mice. Zinterol was also a full agonist at increasing another biological end-point, glucose uptake in brown adipocytes. This effect was reduced in adipocytes derived from β₃-adrenoceptor KO mice, with the remaining response sensitive to β₁-adrenoceptor antagonism. To determine whether the effect of zinterol on β₃-adrenoceptors in primary brown adipocytes can be replicated in a recombinant system, we used CHO-K1 cells expressing the mouse or human β₃-adrenoceptor. Zinterol was a full agonist at mouse and human receptors with respect to increasing cyclic AMP levels, with pEC₅₀ values similar to that of the selective β₃-adrenoceptor agonist (R, R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-dicarboxylate (CL316243) at the mouse receptor. At the human receptor, zinterol was more potent at increasing cyclic AMP levels than CL316243. In cytosensor microphysiometer studies, zinterol was a full agonist for increases in extracellular acidification rates at the mouse and human β₃-adrenoceptor. Therefore, we have shown that zinterol is a potent, high-efficacy β₃-adrenoceptor agonist at the endogenous mouse β₃-adrenoceptor in primary brown adipocytes and at the cloned mouse and human β₃-adrenoceptor expressed in CHO-K1 cells. Zinterol is therefore one of few β-adrenoceptor agonists with high potency and efficacy at the human β₃-adrenoceptor.     

Effects of β2-agonist- and dexamethasone-treatment on relaxation and regulation of β-adrenoceptors in human bronchi and lung tissue

1. Long-term treatment with β₂-adrenoceptor agonists can lead to a decreased therapeutic efficacy of bronchodilatation in patients with obstructive pulmonary disease. In order to examine whether or not this is due to β-adrenoceptor desensitization, human bronchial muscle relaxation was studied in isolated bronchial rings after pretreatment with β₂-adrenoceptor agonists. Additionally, the influence of pretreatment with dexamethasone on desensitization was studied.
2. The effect of β₂-agonist incubation alone and after coincubation with dexamethasone on density and affinity of β-adrenoceptors was investigated by radioligand binding experiments.
3. In human isolated bronchi, isoprenaline induces a time- and concentration-dependent β-adrenoceptor desensitization as judged from maximal reduction in potency by a factor of 7 and reduction of 73±4% in efficacy of isoprenaline to relax human bronchial smooth muscle.
4. After an incubation period of 60 min with 100 μmol l⁻¹ terbutaline, a significant decline in its relaxing efficacy (81±8%) and potency (by a factor 5.5) occurred.
5. Incubation with 30 μmol l⁻¹ isoprenaline for 60 min did not impair the maximal effect of a subsequent aminophylline response but led to an increase in potency (factor 4.4).
6. Coincubation of dexamethasone with isoprenaline (120 min; 30 μmol l⁻¹) preserved the effect of isoprenaline on relaxation (129±15%).
7. In radioligand binding experiments, pretreatment of lung tissue for 60 min with isoprenaline (30 μmol l⁻¹) resulted in a decrease in β-adrenoceptor binding sites (B_max) to 64±1.6% (P<0.05), while the antagonist affinity (K_D) for [³H]-CGP-12177 remained unchanged.
8. In contrast, radioligand binding studies on lung tissue pretreated with either dexamethasone (30 μmol l⁻¹) or isoprenaline (30 μmol l⁻¹) plus dexamethasone (30 μmol l⁻¹) for 120 min did not lead to a significant change of B_max (160±22.1% vs 142.3±28.7%) or K_D (5.0 nmol l⁻¹ vs 3.5 nmol l⁻¹) compared to the controls.
9. In conclusion, pretreatment of human bronchi with β-adrenoceptor agonists leads to functional desensitization and, in lung tissue, to down-regulation of β-adrenoceptors. This effect can be counteracted by additional administration of dexamethasone. Our model of desensitization has proved useful for the identification of mechanisms of β-adrenoceptor desensitization and could be relevant for the evaluation of therapeutic strategies to counteract undesirable effects of long-term β-adrenoceptor stimulation.

     

Crotoxin: Novel activities for a classic β-neurotoxin

Crotoxin, the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, was the first snake venom protein to be purified and crystallized. Crotoxin is a heterodimeric β-neurotoxin that consists of a weakly toxic basic phospholipase A₂ and a non-enzymatic, non-toxic acidic component (crotapotin). The classic biological activities normally attributed to crotoxin include neurotoxicity, myotoxicity, nephrotoxicity and cardiotoxicity. However, numerous studies in recent years have shown that crotoxin also has immunomodulatory, anti-inflammatory, anti-microbial, anti-tumor and analgesic actions. In this review, we describe the historical background to the discovery of crotoxin and its main toxic activities and then discuss recent structure-function studies and investigations that have led to the identification of novel pharmacological activities for the toxin.     

Regional quantification of muscarinic acetylcholine receptors and β-adrenoceptors in human airways

BACKGROUND AND PURPOSE Muscarinic acetylcholine receptors (mAChRs) and β-adrenoceptors in the airways and lungs are clinically important in chronic obstructive pulmonary disease (COPD) and asthma. However, the quantitative and qualitative estimation of these receptors by radioligand binding approaches in human airways has not yet been reported because of tissue limitations. EXPERIMENTAL APPROACH The regional distribution and relative proportion of mAChR and β-adrenoceptor subtypes were evaluated in human bronchus and lung parenchyma by a tissue segment binding method with [(3)H]-N-methylscopolamine ([(3)H]-NMS) for mAChRs and [(3)H]-CGP-12,177 for β-adrenoceptors. Functional responses to carbachol and isoprenaline were also analysed in the bronchus. KEY RESULTS The M(3) subtype predominantly occurred in the bronchus, but the density decreased from the segmental to subsegmental bronchus, and was absent in lung parenchyma. On the other hand, the M(1) subtype occurred in the lung only, and the M(2) subtype was distributed ubiquitously in the bronchus and lungs. β(2)-adrenoceptors were increased along the airways, and their densities in the subsegmental bronchus and lung parenchyma were approximately twofold higher than those of mAChRs in the same region. β(1)-adrenoceptors were also detected in lung parenchyma but not in the bronchus. The muscarinic contractions and adrenoceptor relaxations in both bronchial regions were mediated through M(3)-mAChRs and β(2)-adrenoceptors, respectively. CONCLUSIONS AND IMPLICATIONS From the present radioligand binding approach with intact tissue segments, we constructed a distribution map of mAChRs and β-adrenoceptors in human bronchus and lung parenchyma for the first time, providing important evidence for future pharmacotherapy and new drug development for respiratory disorders.