Functional expression of the human serotonin 5-HT1A receptor in Escherichia coli. Ligand binding properties and interaction with recombinant G protein alpha-subunits.

Signaling through serotonin 5-HT1A receptors involves multiple pathways. We have investigated the functional coupling of the human 5-HT1A receptor to different G proteins using an in vitro reconstitution system based on the expression of recombinant receptor (r5-HT1A) and G alpha-subunits (rG alpha) in Escherichia coli. The r5-HT1A receptor was expressed by insertion in a vector allowing its active expression in E.

Gi alpha-1 expression in the human thyroid is regulated by TSH: loss of regulation in thyroid autonomous adenoma.

The molecular mechanisms underlying the development of endocrine active thyroid tumors are poorly understood. These tumors produce excess thyroid hormone, which then suppresses TSH (thyroid stimulating hormone) production. In the present report, we show that the expression of Gi alpha-1 is under control of TSH in the normal human thyroid.

Expression of two human beta-adrenergic receptors in Escherichia coli: functional interaction with two forms of the stimulatory G protein.

When expressed in Escherichia coli, the human beta 1- and beta 2-adrenergic receptors retain their ligand binding specificity. Their functional integrity was investigated by analyzing receptor-guanine nucleotide-binding regulatory (G) protein coupling by using two splice variants of the alpha subunit of the stimulatory G protein Gs synthesized in E. coli (rGs alpha-S and rGs alpha-L) and the beta gamma subunits of G protein purified from bovine brain.

Interactions of the bovine brain A1-adenosine receptor with recombinant G protein alpha-subunits. Selectivity for rGi alpha-3.

The ability of the bovine brain A1-adenosine receptor to discriminate between different G protein subtypes was tested using G protein alpha-subunits synthesized in Escherichia coli (rG alpha-subunits). When combined with a 3-fold molar excess of beta gamma-subunit purified from bovine brain and used at high concentrations, all three subtypes of rGi alpha (rGi alpha-1, rGi alpha-2, and rGi alpha-3) and rGo alpha were capable of reconstituting guanine nucleotide-sensitive high-affinity binding of the agonist radioligand (-)-N6-3-[125I] (iodo-4-hydroxyphenylisopropyl) adenosine ([125I]HPIA) to the purified A1-adenosine receptor (Kd approximately 1.2 nM).

Interactions of purified bovine brain A1-adenosine receptors with G-proteins. Reciprocal modulation of agonist and antagonist binding.

The bovine brain A1-adenosine receptor was purified 8000-fold by affinity chromatography on xanthine-amine-congener (XAC)-Sepharose. Addition of a 120-fold molar excess of a purified bovine brain G-protein preparation (Go,i a mixture of Go and Gi, containing predominantly Go) decreases the Bmax of the binding of the antagonist radioligand [3H]XAC to the receptor. This decrease is observed not only after insertion into phospholipid vesicles but also in detergent solution, and is reversed by GTP analogues.

Adenosine receptors mediate a pertussis toxin-insensitive prejunctional inhibition of noradrenaline release on a papillary muscle model.

The effects of adenosine receptor agonists and antagonists on field-stimulated release of radioactivity from superfused guinea-pig papillary muscles preincubated with [3H] noradrenaline were studied. N6-cyclopentyladenosine (CPA), N6-(R-phenylisopropyl)-adenosine, and 5'-N-ethylcarboxamidoadenosine caused concentration-dependent inhibition of evoked overflow with a rank order of potency typical for interaction of the compounds with the A1-subtype of adenosine receptors. Maximum inhibition was 80%.

Bioequivalence between two furosemide-spironolactone formulations: a pharmacokinetic and pharmacodynamic approach.

Two formulations of the drug combination furosemide (20 mg)-spironolactone (100 mg) were tested for bioequivalence in a randomized crossover trial in 12 healthy volunteers. By comparing the AUC values derived from drug serum concentrations, bioequivalence was only achieved for canrenone, the main metabolite of spironolactone, but not for furosemide. A significant difference in the tmax values indicates sustained release of furosemide from one of the formulations.

[Role of G protein-mediated signal transduction in molecular pharmacodynamics].

Hormones, neurotransmitter and autacoid receptors, localized on the plasma membrane, do not interact directly with their respective downstream effector (i.e., an ion channel and/or an enzyme that synthesizes a second messenger), but control their target systems via activation of an intermediary guanine nucleotide binding protein on G protein, which serves as signal transducer.

P2-, but not P1-purinoceptors mediate formation of 1, 4, 5-inositol trisphosphate and its metabolites via a pertussis toxin-insensitive pathway in the rat renal cortex.

1. The adenosine receptor (P1-purinoceptor) agonists N6-cyclopentyladenosine and N-5'-ethyl-carboxamidoadenosine at concentrations up to 10 mumols 1(-1) affected neither basal, nor noradrenaline- and angiotensin II-stimulated formation of inositol-1-phosphate, inositol-1,4-bisphosphate, and inositol-1,4,5-trisphosphate in slices of rat renal cortex. 2.

Mutations of GS alpha designed to alter the reactivity of the protein with bacterial toxins. Substitutions at ARG187 result in loss of GTPase activity.

We have introduced two types of mutations into cDNAs that encode the alpha subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase. The arginine residue (Arg187) that is the presumed site of ADP-ribosylation of Gs alpha by cholera toxin has been changed to Ala, Glu, or Lys. The rate constant for hydrolysis of GTP by all of these mutants is reduced approximately 100-fold compared with the wild-type protein.

G proteins control diverse pathways of transmembrane signaling.

Hormones, neurotransmitters, and autacoids interact with specific receptors and thereby trigger a series of molecular events that ultimately produce their biological effects. These receptors, localized in the plasma membrane, carry binding sites for ligands as diverse as peptides (e.g.

A different desensitization pattern of cardiac beta-adrenoceptor subtypes by prolonged in vivo infusion of isoprenaline.

(-)Isoprenaline was continuously administered to rats at a rate of 0.4 mg/kg/h for 7 days via subcutaneously (s.c.

Expression of Gs alpha in Escherichia coli. Purification and properties of two forms of the protein.

Cloning of complementary DNAs that encode either of two forms of the alpha subunit of the guanine nucleotide-binding regulatory protein (Gs) that stimulates adenylyl cyclase into appropriate plasmid vectors has allowed these proteins to be synthesized in Escherichia coli (Graziano, M.P., Casey, P.

The role of a low beta 1-adrenoceptor selectivity of [3H]CGP-12177 for resolving subtype-selectivity of competitive ligands.

On the basis of saturation binding studies on rat cardiac microsomes, which contained a mixed population of beta-adrenoceptor subtypes, [3H]CGP-12177 is presumed to be a non-selective beta-adrenergic radioligand. However, saturation binding studies carried out in the presence of subtype-saturating concentrations of the beta 2-selective antagonist ICI 118,551 and the beta 1-selective antagonist ICI 89,406, respectively, revealed a KD for beta 1-adrenoceptors of 0.33 +/- 0.

Binding of [3H]ouabain to endothelial cells derived from various vascular beds.

Binding experiments were performed with [3H]ouabain on plasma membranes derived from several types of isolated and cultivated endothelial cells. Identical saturation curves for [3H]ouabain binding to endothelial cells from pig aorta, caval vein, and pulmonary artery were obtained with a dissociation constant (KD) of 3.29 +/- 0.

Effects of ischemia on the canine myocardial beta-adrenoceptor-linked adenylate cyclase system.

Several studies indicate that myocardial ischemia causes a redistribution of beta-adrenergic receptors from a presumably intracellular compartment to the cell surface. However, a decreased adenylate cyclase and contractile responsiveness to beta-adrenergic stimuli has also been reported. The aim of the present study was to investigate possible ischemia-induced changes in myocardial beta-adrenoceptor coupling to adenylate cyclase.

Stimulation of adenylate cyclase activity via A2-adenosine receptors in isolated tubules of the rabbit renal cortex.

Adenylate cyclase activity in a tubular fraction obtained from rabbit renal cortex was stimulated by typical adenosine receptor agonists with a rank order of potency NECA (5'-(N-ethyl-carboxamido)-adenosine) (EC50 = 0.48 mumol/l) greater than R-PIA [(-)N6 (R-phenylisopropyl)-adenosine] (3.22 mumol/l).

Glomeruli and microvessels of the rabbit kidney contain both A1- and A2-adenosine receptors.

Rabbit renal cortices were fractionated by collagenase dispersion and glomeruli, microvessels and tubuli purified on a discontinuous sucrose gradient. Binding experiments with (-)[125I]N6-(4-hydroxyphenylisopropyl)-adenosine ([125I]HPIA) provided evidence for the presence of A1-adenosine receptors in the glomerular and microvascular fraction. With glomeruli, saturation isotherms for specific [125I]HPIA binding were mono-phasic with a KD of 1.

Cardiac ventricular beta 2-adrenoceptors in guinea-pigs and rats are localized on the coronary endothelium.

In mammalian heart tissue beta 2-adrenoceptors are known to coexist with beta 1-adrenoceptors. In the present study, evidence that beta 2-adrenoceptors in guinea-pig and rat ventricles are primarily localized on the coronary endothelium is provided by competition binding studies with the subtype-selective beta-adrenoceptor antagonists ICI 89.406 (beta 1-selective) and ICI 118.

Cardiac sarcolemmal purity is essential for the verification of adenylate cyclase inhibition via A1-adenosine receptors.

Inhibition of cardiac adenylate cyclase by adenosine receptor agonists was reinvestigated in a more homogeneous sarcolemmal vesicular preparation than used in a previous study. Microsomal particles obtained by differential centrifugation were further fractionated on a shallow density gradient of Percoll. Two populations of plasma membrane vesicles were partially resolved.

Two saturable recognition sites for (-) [125I]iodo-N6-(4-hydroxyphenyl-isopropyl)-adenosine binding on purified cardiac sarcolemma.

Analysis of (-) [125]iodo-N6-(4-hydroxyphenylisopropyl)-adenosine [( 125I]HPIA) binding to purified sarcolemmal preparations of guinea pig and bovine hearts revealed two classes of binding sites when unlabeled iodo-HPIA (100 mumol/l) was used as non-specific binding marker. In the presence of 1 mmol/l theophylline, however, only the high affinity component was detected. Adenosine receptor agonists caused biphasic displacement of [125I]HPIA binding, with a high affinity potency rank order typical of interaction with A1-adenosine receptors.