Gary L. Stiles

Mechanisms of membrane-receptor regulation. Biochemical, physiological, and clinical insights derived from studies of the adrenergic receptors.

VIRTUALLY all hormones and drugs initiate their biologic actions by binding to specific cellular recognition sites, termed receptors. Receptor binding is followed by alterations of cellular metabol...

The role of receptor structure in determining adenosine receptor activity.

Adenosine produces a wide variety of physiological effects through the activation of cell surface adenosine receptors (ARs). ARs are members of the G-protein-coupled receptor family, and currently, four subtypes, the A1AR, A2AAR, A2BAR, and A3AR, are recognized. This review focuses on the role of receptor structure in governing various facets of AR activity. Ligand-binding properties of ARs are primarily dictated by amino acids in the transmembrane domains of the receptors, although a role for extracellular domains of certain ARs has been suggested. Studies have identified certain amino acids conserved amongst AR subtypes that are critical for ligand recognition, as well as additional residues that may differentiate between agonist and antagonist ligands. Receptor regions responsible for activation of Gs have been identified for the A2AAR. The location of these intracellular sites is consistent with findings described for other G-protein-coupled receptors. Site-directed mutagenesis has been employed to analyze the structural basis for the differences in the kinetics of the desensitization response displayed by various AR subtypes. For the A2AAR and A3AR, agonist-stimulated phosphorylation of the AR, presumably via a G-protein receptor kinase, has been shown to occur. For these AR subtypes, intracellular regions or individual amino acids that may be targets for this phosphorylation have been identified. Finally, the role of A1AR gene structure in regulating the expression of this AR subtype is reviewed.

Human cardiac beta-adrenergic receptors: Subtype heterogeneity delineated by direct radioligand binding

Human myocardial beta-adrenergic receptors were directly identified and characterized using the high affinity antagonist radioligand [125I]iodocyanopindolol. Beta 1 and beta 2 adrenergic receptors were found to coexist in both the left ventricle and right atrium. The relative proportions of the two receptor subtypes were determined by the use of competition radioligand binding and computer modelling techniques employing the subtype selective agents atenolol (beta 1 selective) and zinterol (beta 2 selective). The left ventricle contains 86 +/- 1% beta 1 and 14 +/- 1% beta 2 adrenergic receptors while the right atrium contains 74 +/- 6% beta 1 and 26 +/- 6% beta 2 adrenergic receptors. The direct demonstration of beta 2 adrenergic receptors in the human heart, with a higher proportion in the right atrium agrees with pharmacologic data and supports the notion that chronotropic effects of adrenergic agonists in man may be mediated by both beta 1 and beta 2 adrenergic receptors.

Adenosine receptors: structure, function and regulation

Abstract Adenosine receptors may be divided into two subtypes termed A 1 and A 2 which inhibit or stimulate, respectively, the enzyme adenylate cyclase. Adenosine is capable of regulating the physiological function of most tissues and recent evidence suggests that the receptors mediating these effects are dynamically regulated under both normal and pathophysiologic conditions. In this review, Gary Stiles highlights the recent findings concerning the identification of the structure of adenosine receptors, their mechanism of transmembrane signalling, and the modes of receptor regulation.

Multiple components of the A1 adenosine receptor-adenylate cyclase system are regulated in rat cerebral cortex by chronic caffeine ingestion.

The effects of chronic caffeine on the A1 adenosine receptor-adenylate cyclase system of rat cerebral cortical membranes were studied. Caffeine treatment significantly increased the number of A1 adenosine receptors as determined with the A1 adenosine receptor antagonist radioligand [3H]xanthine amine congener (XAC). R-PIA (agonist) competition curves constructed with [3H]XAC were most appropriately described by a two affinity state model in control membranes with a KH of 2.1 +/- 0.8 and a KL of 404 +/- 330 nM with 50 +/- 4% of receptors in the high affinity state (%RH). In contrast, in membranes from treated animals, there was a marked shift towards the high affinity state. In three of seven animals all of the receptors were shifted to a unique high affinity state which was indistinguishable from the KH observed in membranes from control animals. In four of seven animals the %RH increased from 50 to 69% with KH and KL indistinguishable from the control values. Thus, the agonist specific high affinity form of the receptor was enhanced following caffeine treatment. Maximal inhibition of adenylate cyclase activity in cerebral cortical membranes by R-PIA (1 microM) was significantly increased by 28% following caffeine treatment, consistent with an increased coupling of receptor-Gi protein with adenylate cyclase. Importantly, the quantity of Gi (alpha i) in rat cerebral cortex, determined by pertussis toxin-mediated labeling, was also increased to 133% of control values by this treatment. Thus, multiple components and interactions of the A1 adenosine receptor-adenylate cyclase complex are regulated by caffeine. These changes are likely compensatory measures to offset blockade of A1 receptors in vivo by caffeine and lead to a sensitization of this inhibitory receptor system.

A1 adenosine receptor-G protein coupling in bovine brain membranes: effects of guanine nucleotides, salt, and solubilization

Abstract: The effects of guanine nucleotides, NaCl, and solubilization on the interaction of antagonists and agonists with the A1 adenosine receptor of bovine brain membranes were studied using the high‐affinity antagonist radioligand [3H]xanthine amine congener ([3H]XAC). In membranes, guanine nucleotides and NaCl had no effect on [3H]XAC saturation curves. Using agonist (R)‐phenylisopropyladenosine (R‐PIA) competition curves versus [3H]XAC, it was demonstrated that agonists could differentiate two affinity states having high and low affinity for agonist and that guanine nucleotides shifted the equilibrium to an all‐low‐affinity state that was indistinguishable from the low‐affinity state in the absence of guanine nucleotides. In contrast, NaCl decreased agonist affinity by a distinctly different mechanism characterized by a parallel rightward shifted agonist curve such that R‐PIA still recognized two affinity states albeit of lower affinity than in the absence of salt. R‐PIA competition curves in the presence of both guanine nucleotides and salt were still shallow but were shifted far to the right, and two very low affinity states were discerned. On solubilization, guanine nucleotides in a reversible, concentration‐dependent manner increased antagonist ([3H]XAQ but not agonist (R‐N6‐−[3H]phenyl‐isopropyladenosine) binding. This was consequent to a change in maximal binding capacity. R‐PIA competition curves (versus [3H]XAC) in solubilized preparations demonstrated that agonist could still differentiate two agonist specific affinity states which were modulated by guanine nucleotides. In the presence of guanine nucleotides all the receptors were shifted to a uniform low‐affinity state. In contrast, NaCl had no effect on agonist affinity as determined by agonist competition curves in a solubilized receptor preparation. This is firm evidence that guanine nucleotides and NaCl must decrease agonist affinity by distinct mechanisms.

Photoaffinity labelling of mammalian beta-adrenergic receptors: metal-dependent proteolysis explains apparent heterogeneity.

The beta-adrenergic receptors in membranes from rat and hamster lungs have been studies using the photoaffinity label p-azido-m-[125I]-iodobenzyl-carazolol. Previous work with several beta adrenergic photoaffinity probes has suggested heterogeneity of the labelled beta adrenergic receptor peptides with 2-3 receptor peptides generally being identified. We now report that rat and hamster lung membranes prepared either in the presence or absence of protease inhibitors reveal striking differences in the ratios of photoaffinity labelled peptides. In the rat lung the inclusion of protease inhibitors in the membrane preparation changes the ratios of the 64,000, 53,000 and 44,000 molecular weight peptides from 28:42:30 to 72:16:12. Similarly, in hamster lung membranes there is evidence of multiple photoaffinity labelled peptides in preparations without protease inhibitors while only one peptide (app. Mr = 64,000) is labelled in preparations with protease inhibitors. Of the inhibitors tested EDTA and EGTA were the most active in preventing appearance of multiple labelled peptides suggesting that metal-dependent proteolysis may be involved in the generation of apparent receptor peptide heterogeneity.

Thyroid hormone modulation of agonist - beta-adrenergic receptor interactions in the rat heart

Abstract We have investigated alterations in beta-adrenergic receptors in rat myocardial membranes derived from hypothyroid and hyperthyroid animals. (-)Isoproterenol competition curves with (-)[3H]dihydroalprenolol revealed that isoproterenol binds to the beta-adrenergic receptor with two distinct affinity states having high (KH) and low (KL) dissociation constants. In the presence of guanine nucleotides the isoproterenol competition curve steepened and had a higher EC50 (50% displacement). This was due to a transition of the high affinity state to a uniformly low affinity state. Using computer modeling of these competition curves, we have demonstrated that in hyperthyroidism, the isoproterenol curve in the absence of guanine nucleotides is shifted to the left with the EC50 changing from 180 ± 40 to 80 ± 20 nM (p There was no significant alteration in any of these parameters in hypothyroid animals. Receptor number was decreased in hypothyroidism, 16 ± 3 fmol/mg protein (p In the rat heart agonist affinity and receptor number are modulated in hyperthyroidism, but only receptor number in hypothryoidism. Thus thyroid hormone can modify not only receptor number but agonist affinity as well.

Adenosine receptors: Clinical implications and biochemical mechanisms

Adenosine receptors comprise a group of cell surface receptors which mediate the physiological effects of adenosine. These receptors were originally classified by Burnstock as P1 and P2 purinergic receptors, depending on their preference for adenosine or adenine nucleotides [1]. The relative potencies for P1 sites are adenosine ≥ AMP ≥ ADP ≥ ATP, while relative potencies for P2 sites are ATP ≥ ADP ≥ AMP ≥ adenosine [1]. Further distinctions between P1 and P2 sites can be made by the use of selective antagonists. For example, methylxan-thines, such as theophylline and 3-isobutyl-1-methylxanthine, are potent inhibitors of P1 receptors, but are without effect on P2 sites. These latter sites are selectively blocked by quinidine and high concentrations of 2-substituted imidazoline compounds such as antazoline and phentolamine, and 2’2-dipyridilisatogen.

Clinical relevance of exercise-induced S-T segment elevation

Patients with exercise-induced S-T elevation or S-T depression were evaluated with demographic, treadmill and angiographic data. When 541 patients with S-T depression were compared with 109 patients with S-T elevation, a greater proportion of the former had chest pain (71 versus 58 percent) and a normal-sized ventricle (86 versus 61 percent) with normal wall motion (54 versus 30 percent). A greater proportion of patients with S-T elevation had had a previous myocardial infarction (61 versus 33 percent). Among patients without prior infarction (360 with S-T depression and 42 with S-T elevation), these differences disappeared. In this group of 42 patients with S-T elevation, 83 percent had a normal-sized ventricle, 64 percent had normal contractility and none had a ventricular aneurysm; the severity of coronary disease and ventricular dysfunction did not differ from the severity in patients with S-T depression. Thus, in patients without prior myocardial infarction, the cause of the development of S-T elevation or S-T depression during exercise does not appear to be related to the severity of the coronary lesions, ventricular function or wall abnormalities at rest. In patients with prior myocardial infarction, exercise-induced S-T elevation appears to be a marker of depressed left ventricular function.