Presence and function of ß-adrenergic receptors in primary equine bronchial epithelia cells.

The ß-adrenergic receptor (ß-AR) plays an important role in regulating a variety of cell and organ functions in different animal species and is an important target in asthma pathogenesis and therapy. The ß-AR expression and function in equine bronchial epithelial cells (EBEC) were not known but innervation and significant decrease in receptor level were reported in the equine bronchial tissues from asthmatic horses. 125I-iodocyanopindolol (ICYP) binding studies were undertaken in primary freshly isolated and cultured EBEC to identify the presence of the ß-ARs. The receptor distribution was assessed using subtype-selective ß-AR antagonists (ICI 118 551 (ß2) and CGP 20712A (ß1). The ß-AR function was confirmed by measuring the agonist-induced intracellular cAMP accumulation in freshly isolated and cultured EBEC. In both freshly isolated and cultured EBEC, the specific ICYP binding was saturable and of high affinity. The maximal receptor density (Bmax) was 9763 ± 140 binding sites/cell (mean ± SEM, n = 7) and 10575 ± 194 binding sites/cell (mean ± SEM, n = 5) in freshly isolated and cultured EBEC, respectively. The receptor affinity to the ligand (KD) was also not different between the two cell conditions. ICI 118.551 displaced ICYP with 25 000-fold higher affinity than CGP 20712A. Moreover, in both fresh isolated and cultured EBEC, cAMP-accumulation was stimulated with a rank-order of potency of isoproterenol > adrenaline > noradrenaline. These results highlight the ß2-AR to be a key subtype in both freshly isolated and cultured primary EBEC.

Design, synthesis, and functional assessment of Cmpd-15 derivatives as negative allosteric modulators for the ß2-adrenergic receptor.

The ß2-adrenergic receptor (ß2AR), a G protein-coupled receptor, is an important therapeutic target. We recently described Cmpd-15, the first small molecule negative allosteric modulator (NAM) for the ß2AR. Herein we report in details the design, synthesis and structure-activity relationships (SAR) of seven Cmpd-15 derivatives. Furthermore, we provide in a dose-response paradigm, the details of the effects of these derivatives in modulating agonist-induced ß2AR activities (G-protein-mediated cAMP production and ß-arrestin recruitment to the receptor) as well as the binding affinity of an orthosteric agonist in radio-ligand competition binding assay. Our results show that some modifications, including removal of the formamide group in the para-formamido phenylalanine region and bromine in the meta-bromobenzyl methylbenzamide region caused dramatic reduction in the functional activity of Cmpd-15. These SAR results provide valuable insights into the mechanism of action of the NAM Cmpd-15 as well as the basis for future development of more potent and selective modulators for the ß2AR based on the chemical scaffold of Cmpd-15.

Expression and functional coupling of liver ß2 - adrenoceptors in the human hepatocellular carcinoma.

AIM: Hepatocellular carcinoma (HCC) is one of the most common cancers and a leading cause of death worldwide. There are now multiple lines of evidence demonstrating that the ß-adrenoceptor ( ß-AR) signaling plays an important role in the progression and metastasis of cancer and may become a novel target for cancer therapy. Little information exists regarding the status of ß-ARs and their postreceptor intracellular signaling cascade in the development of human HCC. This study was conducted to detect the expression signal transduction of the ß-ARs in liver membranes obtained from patients with HCC and elucidate their possible implication on HCC development. METHODS: The ß-AR density and subtype distribution were determined by receptor binding studies. Protein levels of the ß(2)-AR and G(s)(α) protein were determined by Western blot analysis. The receptor coupling efficiency and biochemical activities of the adenylate cyclase(AC) was also determined. RESULTS: In HCC liver membranes, the ß(2)-AR density was higher than the density in the nonadjacent nontumor liver membranes. The ß(2)-AR protein expression was 1.5-fold increased as compared with nonmalignant controls, and positively correlated with the receptor density. The G s protein expression as well as the receptor, AC and G protein-stimulated activation of the cAMP formation was reduced in HCC. CONCLUSION: The ß(2)-AR was upregulated in human HCC. Despite this upregulation of the receptor,there was an altered postreceptor signal transduction in HCC liver. The mechanisms responsible for this change in the growth of HCC and the nature of this alteration remain unclear.

Identification and characterization of ß-adrenergic receptors in isolated primary equine tracheal epithelial cells.

Responses and functions of airway epithelial cells are stimulated by ß2-agonists via the ß2-adrenergic receptors (ß2-ARs)-G(s)-protein-cAMP-system, thus, affecting airway inflammation such as in asthma and equine recurrent airway obstruction (RAO). Though horses can be used as large animal model for human asthma, evaluation of the expression and functions of the ß-AR system in primary equine airway epithelial cells has not been yet carried out. Thus, for the first time, we determined the ß-AR density and subtype distribution by [¹²5I]-iodocyanopindolol (ICYP) binding, examined ß-AR function by cAMP assay as well as their expression by western blot analysis and immunocytochemical staining in primary equine tracheal epithelial cells (ETEC). Cells were collected from 19 horses and cultured subsequently. The specific ICYP binding was saturable and of high affinity: in freshly isolated cells the receptor density (B(max)) and ICYP affinity (K(D)) for ß-ARs were 12727 ± 883 binding sites/cell and 31.78 ± 6.57 pM, respectively, and in cultured ETEC 3730 ± 212 binding sites/cell and 15.26 ± 3.37 pM, respectively. The ß-AR subtype assessed by ß1-selective (CGP 20712A) and ß2-selective (ICI 118.551) adrenergic receptor antagonists demonstrated that the ß2-AR subtype predominated (>95%) in both cell populations (p < 0.001). The ß-AR agonists increased cAMP formation with a rank order of potency: isoproterenol > epinephrine > norepinephrine. ICI 118.551 (100 nM) significantly blocked (p < 0.05) isoproterenol-induced cAMP accumulation but not CGP 20712A (300 nM). Western blot analyses and immunocytochemical staining further indicated the expression of the ß(2)-AR subtype in both cell preparations. Our data indicate that in acutely dissociated and primary cultured ETEC the ß(2)-AR-AC system is expressed, but varies considerably between the two preparations.

Control of yeast mating signal transduction by a mammalian beta 2-adrenergic receptor and Gs alpha subunit.

To facilitate functional and mechanistic studies of receptor-G protein interactions, [corrected] the human beta 2-adrenergic receptor (h beta-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h beta-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h beta-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h beta-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by beta-adrenergic receptor agonists was achieved in cells coexpressing h beta-AR and a mammalian G protein (Gs) alpha subunit-demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.

Alveolar liquid clearance in lung injury: Evaluation of the impairment of the ß2-adrenergic agonist response in an ischemia-reperfusion lung injury model.

While alveolar liquid clearance (ALC) mediated by the ß2-adrenergic receptor (ß2-AR) plays an important role in lung edema resolution in certain models of lung injury, in more severe lung injury models, this response might disappear. Indeed, we have shown that in an ischemia-reperfusion-induced lung injury model, ß2-agonists do not enhance ALC. The objective of this study was to determine if downregulation of the ß2-AR could explain the lack of response to ß2-agonists in this lung injury model. In an in vivo canine model of lung transplantation, we observed no change in ß2-AR concentration or affinity in the injured transplanted lungs compared to the native lungs. Furthermore, we could not enhance ALC in transplanted lungs with dcAMP + aminophylline, a treatment that bypasses the ß2-adrenergic receptor and is known to stimulate ALC in normal lungs. However, transplantation decreased αENaC expression in the lungs by 50%. We conclude that the lack of response to ß2-agonists in ischemia-reperfusion-induced lung injury is not associated with significant downregulation of the ß2-adrenergic receptors but is attributable to decreased expression of the ENaC channel, which is essential for sodium transport and alveolar liquid clearance in the lung.

Colorectal cancer metastases affect the biochemical characteristics of the human liver beta-adrenoceptor-G-protein-adenylate cyclase system.

The sympathetic-catecholamine system is involved in the regulation of hepatic metabolic pathways mainly through cAMP-linked beta2-adrenoceptors (beta2-ARs) in humans and to a lesser extent through cAMP-independent mechanisms, but no information is available about the possible biochemical changes of beta2-ARs and their signalling pathways in human colorectal cancer (CRC) and colorectal cancer hepatic metastases (CRCHM). Changes in density and distribution of beta-ARs as well as in post-receptor signalling components were studied in membranes of human liver with CRCHM, and for comparison, in membranes of nonadjacent, non-metastatic human liver (NA-NM) obtained from 13 patients, using binding and competition binding studies. Studies were also carried out using normal and cancerous human colon tissues. In CRCHM, the density of beta-ARs (B(max)) was significantly reduced, compared to NA-NM liver tissues (40.09+/-2.83 vs. 23.09+/-3.24 fmol/mg protein; P<0.001). A similar decrease in the beta-AR density was observed in the colon with primary colorectal cancer compared to healthy colon (37.6+/-2.2 vs. 23.8+/-3.5 fmol/mg protein), whereas the affinity of ICYP binding to the receptor remained unaffected. Desensitized beta-ARs were uncoupled from stimulatory G-protein (G(S)), as total density of beta-adrenoceptors in the high affinity state was significantly reduced. Concomitantly, CRCHM elicited decrease in the catalytic adenylate cyclase (AC) activity (cAMP formation) in response to isoproterenol plus GTP or forskolin or NaF. In NA-NM and CRCHM liver, the inhibition-concentration curves of ICI 118.551 showed the presence of a homogeneous population of the beta2-AR subtypes. Neither the binding patterns nor the inhibition constant (K(i)) of ICI 118.551 were altered in CRCHM. In CRCHM, the hepatic beta-AR-G-protein(s)-AC signalling system was markedly impaired, thus, these changes may well influence beta-AR-mediated functions in both organs.

Chronic nonocclusive coronary artery constriction in rats. Beta-adrenoceptor signal transduction and ventricular failure.

To determine the effects of chronic coronary artery constriction on the relationship between cardiac function and regulation of beta-adrenoceptor signal transduction, the left main coronary artery was narrowed in rats and the animals were killed 5 mo later. An average reduction in coronary luminal diameter of 44% was obtained and this change resulted in an increase in left ventricular end-diastolic pressure and a decrease in positive and negative dP/dt. Significant increases in left and right ventricular weights indicative of global cardiac hypertrophy were observed. Radioligand binding studies of beta-adrenoreceptors, agonist-stimulated adenylate cyclase activity, and ADP ribosylation of 45-kD substrate by cholera toxin were all depressed in the failing left ventricle. In contrast, in the hypertrophic non-failing right ventricle, beta-adrenoreceptor density was preserved and receptor antagonist affinity was increased. In spite of these findings at the receptor level, agonist stimulated cyclic AMP generation was reduced in the right ventricular myocardium. The quantity of the 45-kD substrate was also decreased. In conclusion, longterm nonocclusive coronary artery stenosis of moderate degree has profound detrimental effects on the contractile performance of the heart in association with marked attenuation of adrenergic support mechanisms.

Beta-adrenergic neuroeffector abnormalities in the failing human heart are produced by local rather than systemic mechanisms.

In order to investigate the general cause of beta-adrenergic receptor neuroeffector abnormalities in the failing human heart, we measured ventricular myocardial adrenergic receptors, adrenergic neurotransmitters, and beta-adrenergic receptor-effector responses in nonfailing and failing hearts taken from nonfailing organ donors, subjects with endstage biventricular failure due to idiopathic dilated cardiomyopathy (IDC), and subjects with primary pulmonary hypertension (PPH) who exhibited isolated right ventricular failure. Relative to nonfailing PPH left ventricles, failing PPH right ventricles exhibited (a) markedly decreased beta 1-adrenergic receptor density, (b) marked depletion of tissue norepinephrine and neuropeptide Y, (c) decreased adenylate cyclase stimulation in response to the beta agonists isoproterenol and zinterol, and (d) decreased adenylate cyclase stimulation in response to Gpp(NH)p and forskolin. These abnormalities were directionally similar to, but generally more pronounced than, corresponding findings in failing IDC right ventricles, whereas values for these parameters in nonfailing left ventricles of PPH subjects were similar to values in the nonfailing left ventricles of organ donors. Additionally, relative to paired nonfailing PPH left ventricles and nonfailing right ventricles from organ donors, failing right ventricles from PPH subjects exhibited decreased adenylate cyclase stimulation by MnCl2. These data indicate that: (a) Adrenergic neuroeffector abnormalities present in the failing human heart are due to local mechanisms; systemic processes do not produce beta-adrenergic neuroeffector abnormalities. (b) Pressure-overloaded failing right ventricles of PPH subjects exhibit decreased activity of the catalytic subunit of adenylate cyclase, an abnormality not previously described in the failing human heart.

Terbutaline-induced desensitization of human lymphocyte beta 2-adrenoceptors. Accelerated restoration of beta-adrenoceptor responsiveness by prednisone and ketotifen.

We investigated, in 36 healthy volunteers, the effects of prednisone and ketotifen on recovery of lymphocyte beta 2-adrenoceptor density (determined by (-)-125iodocyanopindolol binding) and responsiveness (assessed by lymphocyte cyclic AMP [cAMP] responses to 10 microM (-)-isoprenaline) after desensitization by the beta 2-agonist terbutaline. Terbutaline (3 X 5 mg/d) decreased lymphocyte beta 2-adrenoceptor density by approximately 40-50%; concomitantly, lymphocyte cAMP responses to 10 microM (-)-isoprenaline were significantly reduced. After withdrawal of terbutaline beta 2-adrenoceptor, density and responsiveness gradually increased, reaching predrug levels after 4 d. Prednisone (1 X 100 mg orally) accelerated beta 2-adrenoceptor recovery; only 8-10 h after administration of the steroid beta 2-adrenoceptor density and cAMP responses to (-)-isoprenaline had reached values not significantly different from pretreatment levels. Similar effects were obtained with ketotifen (2 mg; thereafter 2 X 1 mg/d for 4 d): 24 h after application of the drug beta 2-adrenoceptor density and cAMP responses to (-)-isoprenaline had reached pretreatment levels. Furthermore, ketotifen simultaneously applied with terbutaline completely prevented terbutaline-induced decrease in lymphocyte beta 2-adrenoceptor density and responsiveness. Prednisone (1 X 100 mg orally) or ketotifen (2 mg; thereafter 2 X 1 mg/d for 2 d) had no significant influence on lymphocyte beta 2-adrenoceptor density in healthy volunteers not pretreated with terbutaline, but shifted the ratio high-to-low affinity state of the lymphocyte beta 2-adrenoceptor toward high affinity state. We conclude that glucocorticoids as well as ketotifen can accelerate recovery of density and responsiveness of lymphocyte beta 2-adrenoceptors desensitized by long-term treatment with beta 2-agonists. Such an effect may have clinical implications for preventing tachyphylaxis of asthmatic patients against therapy with beta 2-agonists.

Mechanisms underlying regional differences in lipolysis in human adipose tissue.

Catecholamine-induced lipolysis was investigated in nonobese females and males. Isolated subcutaneous adipocytes were obtained from the abdominal and gluteal regions. The lipolytic effect of noradrenaline was four to fivefold more marked in abdominal adipocytes than in gluteal fat cells. This regional difference was more apparent in females than in males. No site differences were observed when lipolysis was stimulated with agents acting at different postreceptor levels. The beta-adrenergic lipolytic sensitivity was 10-20 times greater in abdominal adipocytes from both sexes than in gluteal adipocytes. Abdominal adipocytes from females showed a 40 times lower alpha 2-adrenergic antilipolytic sensitivity than did gluteal adipocytes, but the adenosine receptor sensitivity was similar in both sites. Beta-receptor affinity for agonists displayed no site or sex variation. Abdominal adipocytes showed a twofold increased beta-adrenoceptor density than did gluteal cells from both sexes. The alpha 2-adrenoceptor density was similar in all regions, but in females the affinity of clonidine for these sites was 10-15 times lower in the abdominal fat cells compared with gluteal cells. In conclusion, regional differences in catecholamine-induced lipolysis are regulated at the adrenoceptor level, chiefly because of site variations in beta-adrenoceptor density. Further variations in the affinity properties of alpha 2-adrenergic receptor in females may explain why the regional differences in catecholamine-induced lipolysis are more pronounced in women than in men.

Functional beta-adrenergic receptor signalling on nuclear membranes in adult rat and mouse ventricular cardiomyocytes.

OBJECTIVE: We sought to determine if different beta-adrenergic receptor (betaAR) subtypes, and their associated signalling machinery, are functionally localized to nuclear membranes. METHODS: Employing enriched nuclear preparations, we assayed the specific presence of betaAR by measuring 125I-cyanopindolol (CYP) binding, Western blotting, confocal microscopy and functional assays. RESULTS: Western blots of rat heart nuclear fractions and confocal immunofluorescent analysis of adult rat and mouse ventricular cardiomyocytes displayed the presence of beta 1AR and beta 3AR but, surprisingly, not the beta 2AR on nuclear membranes. Nuclear localization of downstream signalling partners Gs, Gi and adenylyl cyclases II and V/VI was also demonstrated. The functional relevance of nuclear betaAR was shown by receptor-mediated stimulation of adenylyl cyclase activity by isoproterenol but not the beta 3AR-selective agonist CL 316243 in enriched nuclear preparations. We also examined the effect of subtype-selective ligands on the initiation of RNA synthesis in isolated nuclei. Both isoproterenol and another beta 3AR-selective agonist, BRL 37344, increased RNA synthesis which was inhibited by pertussis toxin (PTX). Neither a beta 1AR-selective agonist, xamoterol, nor a beta 2AR-selective agonist, procaterol, was able to stimulate transcription. However, both CGP 20712A and ICI 118,551 blocked isoproterenol-mediated effects to varying extents. PTX treatment also revealed that nuclear betaAR may be coupled to other signalling pathways in addition to Gi, as stimulation under these conditions reduced initiation of transcription below basal levels. CONCLUSION: These results highlight differential subcellular localization for betaAR subtypes and indicate that betaAR may have specific roles in regulating nuclear function in cardiomyocytes.

Comparison of three radioligands for the labelling of human beta-adrenoceptor subtypes.

We have compared the ability of three radioligands, [(125)I]-cyanopindolol, [(3)H]-CGP 12,177 and [(3)H]-dihydroalprenolol, to label the three human beta-adrenoceptor subtypes. Saturation and competition binding experiments were performed using membrane preparations from Chinese hamster ovary cells stably transfected with the three subtypes. While [(3)H]-CGP 12,177 had very similar affinity for beta(1)- and beta(2)-adrenoceptors (about 40 pM), [(125)I]-cyanopindolol and [(3)H]-dihydroalprenolol had 4- to 6-fold higher affinity for beta(2)- as compared to beta(1)-adrenoceptors (10 vs 45 and 187 vs 1,021 pM, respectively). The affinity of [(125)I]-cyanopindolol at beta(3)-adrenoceptors was considerably lower (440 pM) than at the other two subtypes. The beta(3)-adrenoceptor affinity of [(3)H]-CGP 12,177 and [(3)H]-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 [(3)H]-CGP 12,177 and [(3)H]-dihydroalprenolol, respectively). We conclude that all three radioligands are ill-suited to label beta(3)-adrenoceptors, particularly in preparations co-expressing multiple subtypes. In the absence of alternatives, [(125)I]-cyanopindolol appears the least unsuitable to label beta(3)-adrenoceptors. There is a need for high-affinity radioligands which are either selective for beta(3)-adrenoceptors or reasonably non-selective among all three beta-adrenoceptor subtypes.

Extract of Lycopus europaeus L. reduces cardiac signs of hyperthyroidism in rats.

Extracts from the plant Lycopus europaeus L. are traditionally used in mild forms of hyperthyroidism. High doses caused a reduction of TSH or thyroid hormone levels in animal experiments, whereas in hyperthyroid patients treated with low doses of Lycopus an improvement of cardiac symptoms was reported without major changes in TSH or thyroid hormone concentrations. Lycopus extract was tested in thyroxine treated hyperthyroid rats (0.7 mg/kg BW i.p.). Co-treatment with an hydroethanolic extract from L. europaeus L. started one week later than T4-application and lasted 5.5 weeks. As reference substance atenolol was used. The raised body temperature was reduced very effectively even by the low dose of the plant extract, whereas the reduced gain of body weight and the increased food intake remained unaffected by any treatment. No significant changes of thyroid hormone concentrations or TSH levels were observed. Lycopus extract and atenolol reduced the increased heart rate and blood pressure. The cardiac hypertrophy was alleviated significantly by both treatment regimes. beta-Adrenoceptor density in heart tissue was significantly reduced by the Lycopus extract or the beta-blocking agent showing an almost equal efficacy. Although the mode of action remains unclear, these organo-specific anti-T4-effects seem to be of practical interest, for example in patients with latent hyperthyroidism.

Prolonged treatment with the beta3-adrenergic agonist CL 316243 induces adipose tissue remodeling in rat but not in guinea pig: 1) fat store depletion and desensitization of beta-adrenergic responses.

Beta3-adrenergic agonists have been considered as potent antiobesity and antidiabetic agents mainly on the basis of their beneficial actions discovered twenty years ago in obese and diabetic rodents. The aim of this work was to verify whether prolonged treatment with a beta3-adrenergic agonist known to stimulate lipid mobilisation, could promote desensitization of beta-adrenergic responses. Wistar rats and guinea pigs were treated during one week with CL 316243 (CL, 1 mg/kg/d) by implanted osmotic minipumps. In control animals, beta3-adrenergic agonists were lipolytic in rat but not in guinea pig adipocytes. CL-treatment did not alter body weight gain in both species, but reduced fat stores in rats. Lipolysis stimulation by forskolin was unmodified but responses to beta1-, beta2- and beta3-agonists were reduced in visceral or subcutaneous white adipose tissues of CL-treated rats. Similarly, the beta3-adrenergic-dependent impairment of insulin action on glucose transport and lipogenesis in rat adipocytes was diminished after CL-treatment. In rat adipocytes, [125I]ICYP binding and beta3-adrenoceptor mRNA levels were reduced after sustained CL administration. These findings show that CL 316243 exerts (beta3-adrenergic lipolytic and antilipogenic effects in rat adipocytes. These actions, which are likely involved in the fat depletion observed in rat, also lead to the desensitization of all beta-adrenergic responses. Therefore this desensitization, together with the lack of slimming action in guinea pig, seriously attenuates the usefulness of beta3-agonists as antiobesity agents, and may explain why such agonists have not been conducted to a widespread clinical use.

Increased number of beta-adrenoceptors in hepatocytes from rats treated with 2-acetylaminofluorene.

Treatment of rats with chemical carcinogens, including 2-acetylaminofluorene (2-AAF), leads to a strong increase in the hepatic catecholamine-sensitive adenylate cyclase activity. The present study was undertaken to investigate the mechanism for the development of this increase. We report that hepatocytes isolated from rats which had been fed 2-AAF (0.025% w/w) for 8-12 weeks had an increased number of beta-adrenoceptors, as determined by [3H]dihydroalprenolol binding to whole cells and [125I]iodocyanopindolol binding to washed particles. For both ligands the number of binding sites was about 4-fold higher in hepatocytes from 2-AAF-treated rats than in those from controls. The adenylate cyclase activity of the carcinogen-fed animals showed both a general increase manifested in the basal level (2-fold) and in the activities obtained by stimulation with guanine nucleotides (2-3-fold), cholera toxin (1.5-fold), and glucagon (1.3-fold) and a selective, larger increase in the beta-adrenoceptor-linked activity (7-fold increment of the isoproterenol-sensitive activity). The results indicate that the number of hepatocyte beta-adrenoceptors increases during 2-AAF carcinogenesis. This may, at least in part, explain the rise in catecholamine-sensitive adenylate cyclase activity.

Beta-adrenergic mitogenic signal transduction in peripheral lung adenocarcinoma: implications for individuals with preexisting chronic lung disease.

Peripheral adenocarcinoma (PAC) of the lung has increased dramatically over the last 20 years and is today the leading histological type of lung cancer in smokers and nonsmokers in industrialized countries. There is no apparent explanation for the steep rise in the number of individuals developing this cancer type. Using assays for the assessment of cell proliferation, receptor binding, and production of cyclic AMP (cAMP), we have identified a beta-adrenergic receptor-mediated mitogenic pathway, which activates cAMP down-stream, in cell lines derived from human peripheral adenocarcinomas that express features of Clara cells. Agonists of beta-adrenergic receptors strongly stimulated cell proliferation, whereas antagonists of this receptor and its associated second messenger, cAMP, were potent inhibitors of this effect. Agonists of beta-adrenergic receptors are the active ingredients of many decongestants and bronchodilators, and such medications are, therefore, likely to stimulate this pathway in vivo. Patients suffering from chronic upper and lower respiratory tract diseases and treated with such medications over many years may, therefore, be at a higher risk than the average population to develop PAC, particularly when simultaneously exposed to carcinogenic environmental factors such as smoking. Because the incidence of chronic respiratory tract diseases has risen in industrialized countries during the same time frame as PAC, a potential etiological link between the therapy of such nonneoplastic diseases with beta-adrenergic agonists and the risk for PAC should be investigated.

Altered adrenergic response and specificity of the receptors in rat ascites hepatoma AH130.

Adenylate cyclase activation through adrenergic receptors in rat ascites hepatoma (AH) 130 cells in response to adrenergic drugs was studied, and receptor binding and displacement were compared with those of normal rat hepatocytes. Epinephrine (Epi) and norepinephrine (NE) activated AH130 adenylate cyclase about half as much as isoproterenol (IPN) but equaled IPN after treatment with the alpha-antagonist phentolamine or islet-activating protein (IAP). The three catecholamines in hepatocytes were similar regardless of phentolamine or IAP. These catecholamines activated adenylate cyclase in order of IPN greater than NE greater than Epi in AH130 cells but IPN greater than Epi greater than NE in hepatocytes. We then used the alpha 1-selective ligand [3H]prazosin, the alpha 2-selective ligand [3H]clonidine, and the beta-ligand [125I]iodocyanopindolol [( 125I]ICYP), and found that AH130 cells had few prazosin-binding sites, about eight times as many clonidine-binding sites with high affinity, and many more ICYP-binding sites than in hepatocytes. The dissociation constant (Ki) of the beta 1-selective drug metoprolol by Hofstee plots for AH130 cells was lower than that for hepatocytes. The inhibition of specific ICYP binding by the beta 2-selective agonist salbutamol for AH130 cells gave only one Ki value which was much higher than both high and low Ki values of the drug for hepatocytes. These findings indicate that the alpha- and beta-adrenergic receptors in hepatocytes are predominantly alpha 1-type and beta 2-type, but that those in AH130 cells are predominantly alpha 2-type and beta 1-type, and the low adrenergic response of AH130 cells is due to the dominant appearance of alpha 2-adrenergic receptors, linked with the inhibitory guanine-nucleotide binding regulatory protein, instead of alpha 1-adrenergic receptors, and beta 1-adrenergic receptors with low affinity for the hormone.

Demonstration of human platelet beta-adrenergic receptors using 125I-labeled cyanopindolol and 125I-labeled hydroxybenzylpindolol.

The radioiodinated pindolol analogs 125I-labeled cyanopindolol ([125I]CYP) and 125I-labeled hydroxybenzylpindolol ([125I]HBP) have been used to study binding to human platelet beta-adrenergic receptors. [125I]CYP binds to a saturable class of binding sites on platelet membranes with a dissociation constant (Kd) of 14 +/- 3 pM and maximal binding capacity (Bmax) of 18 +/- 4 fmol/mg protein. Binding of [125I]CYP is reversible and is characterized by forward and reverse rate constants of 1.8 . 10(7) s-1 . M-1 and 3.8 . 10(-4) s-1, respectively. [125I]HBP binds to a saturable class of platelet membrane sites with a Kd of 50 +/- 10 pM and Bmax of 32 +/- 6 fmol/mg protein. [125I]HBP also binds to a saturable class of sites on intact platelets with a Kd of 58 +/- 14 pM and Bmax of 24 +/- 4 molecules per platelet. Binding of [125I]CYP and [125I]HBP is stereospecifically inhibited by propranolol and epinephrine; the (-) stereoisomers are at least 50-times more potent than the (+) stereoisomers. Binding of both radioligands is inhibited by adrenergic ligands with a potency order of propranolol much greater than isoproterenol greater than epinephrine greater than practolol greater than norepinephrine greater than phenylephrine. These observations indicate that [125I]CYP and [125I]HBP bind to platelet sites which have the pharmacological characteristics of beta-adrenergic receptors but which are not typical of either the beta 1 or beta 2 sub-type.

Characterization of beta-adrenergic receptor subtypes in androgen-induced mouse kidney hypertrophy using a new high-affinity ligand, [125I]iodocyanopindolol.

In fully developed androgen-induced hypertrophy of female mouse kidney, beta-adrenergic receptors per unit membrane protein were increased approx. 2.5-fold, as measured by the binding of [125I]iodocyanopindolol, with no change in apparent dissociation constants (Kd range 20-25 pM). Membrane protein relative to total kidney protein, Na+/K+-dependent ATPase (EC 3.6.1.3) and 5'-nucleotidase (EC 3.1.3.5) activities and cholesterol content per unit membrane protein did not differ significantly in preparations from control and treated animals. The binding of iodocyanopindolol to kidney membranes was characterized with respect to association and dissociation kinetics, and also in regard to the less-specific contributions of other major catecholamine or indolamine receptors, using mixtures of the corresponding specific competitors. beta 1-selective drugs, practolol and metoprolol, and beta 2-selective agents, IPS-339 and zinterol, were competed with iodocyanopindolol to assess the receptor type specificity, and the ensuing binding profiles were dissected by a nonlinear regression analysis as described by Munson, P.J. and Rodbard, D. (Anal. Biochem. (1982) 107, 220-239). Most of the androgen-induced beta-adrenergic receptors had the binding properties corresponding to beta 2-subtype. No consistent increase in the density of beta 1-adrenergic receptors could be shown.