Pharmacological and biochemical characterization of the beta-adrenergic signal transduction pathway in different segments of the respiratory tract.
Although in the respiratory system there is great therapeutic interest in manipulating and understanding the beta-adrenoceptor-G-protein-adenylate cyclase (AC) signal transduction pathway, little is known on segmental differences among lung, bronchus, and trachea with regard to the receptor concentration and interaction to G-proteins and coupling to AC. In this study, patterns of distribution and absolute quantities of beta-adrenoceptor subtypes beta(1) and beta(2) were determined in membranes of equine lung parenchyma, bronchial and tracheal epithelium with the underlying smooth muscle by saturation and competition binding assays using the radioligand (-)-[125I]-iodocyanopindolol (ICYP). Additionally, the functional coupling of beta-adrenoceptors to G-proteins (assessed by beta-agonist competition binding in the presence and absence of GTP) as well as the coupling efficiency and biochemical activities of AC was investigated in each region. The specific ICYP binding was rapid, reversible, saturable with time and of high affinity. The radioligand binding identified more total beta-adrenoceptors in the lung than in bronchus or trachea (428+/-19, 162.4+/-4.8, 75.6+/-1.2 fmol/mg protein, respectively) with about 40% of receptors in the high affinity state. The beta(2)-adrenoceptor subtype predominated in all segments (approximately 74-80%), as the highly selective beta(2)-adrenoceptor antagonist ICI 118,551 was about 10,000 times more potent in inhibiting ICYP binding than was the beta(1)-selective adrenoceptor antagonist CGP 20712A, and beta-adrenoceptor agonists inhibited ICYP binding with an order of potency: (-)-isoprenaline>(-)-adrenaline>(-)-noradrenaline. The dissociation constant (K(d)) was higher in the trachea than in bronchus or lung (13.0+/-0.9 pM vs. 20.0+/-2.3 pM vs. 30.8+/-4.4 pM, P<0.05, respectively). The beta(2)-adrenoceptor-mediated AC response was tissue-dependent; stimulants acting on beta-adrenoceptor (isoproterenol), G-protein (GTP, NaF) and AC (forskolin, Mn(2+)) enhanced AC responses in all three regions, but the AC activity was higher in tracheal crude membranes than in bronchus or lung (trachea>>>bronchus>lung), hence, the number of beta(2)-adrenoceptors correlated inversely with the amount of AC. We conclude that (1) the stoichiometry of components within the pulmonary beta-adrenoceptor-G-protein complex is segment-dependent, and (2) the receptor number or AC activity is possibly the rate-limiting factor in the beta-adrenoceptor-G-protein-AC-mediated physiological responses. Thus, it is speculated that this could have important therapeutic consequences in beta-adrenoceptor agonist-induced receptor regulation in bronchial asthma.
Abraham
Getu
G
Faculty of Veterinary Medicine, Institute of Pharmacology, Pharmacy and Toxicology, Leipzig University, An den Tierkliniken 15, 04103 Leipzig, Germany. gabraham@rz.uni-leipzig.de
Kottke
Claudia
C
Dhein
Stefan
S
Ungemach
Fritz Rupert
FR
D000262:Adenylyl Cyclases / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology*; D000818:Animals; D001980:Bronchi / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006736:Horses; D020120:Iodocyanopindolol / Q000494:pharmacology*; D007545:Isoproterenol / Q000494:pharmacology; D008168:Lung / Q000201:enzymology / Q000378:metabolism; D011869:Radioligand Assay; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D012137:Respiratory System; D015398:Signal Transduction; D014132:Trachea / Q000378:metabolism
D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D011943:Receptors, Adrenergic, beta; D020120:Iodocyanopindolol; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol