The French stroke program.

The French healthcare system is organised according to a political, geographical and demographic framework. In France today, less than 20 hospitals have dedicated intensive care units for acute stroke patients. In the 1999 national survey, 94% of stroke patients were admitted to public hospitals, 35% were treated in neurological wards and 4% had access to an intensive care stroke unit.

An intact N terminus of the gamma subunit is required for the Gbetagamma stimulation of rhodopsin phosphorylation by human beta-adrenergic receptor kinase-1 but not for kinase binding.

Cleavage after lysine 32 in the Ggamma2 subtype and after lysine 36 in the Ggamma3 subtype of purified mixed brain Gbetagamma by endoproteinase Lys-C blocks Gbetagamma-mediated stimulation of phosphorylation of rhodopsin in urea-extracted rod outer segments by recombinant human beta-adrenergic receptor kinase (hbetaARK1) holoenzyme while hbetaARK1 binding to rod outer segments is partially affected. This treatment does not attenuate the binding of the treated Gbetagamma to C-terminal fragments of hbetaARK1 containing the pleckstrin homology domain. Lys-C proteolysis also does not alter the association of the Gbetagamma with phospholipids, its ability to support pertussis toxin-catalyzed Galphao/Galphai ADP-ribosylation, or its ability to inhibit forskolin-stimulated platelet adenylate cyclase.

Expression of beta-arrestins and beta-adrenergic receptor kinases in the failing human heart.

The beta-adrenergic receptor system of the failing human heart is markedly desensitized. We have recently postulated that this desensitization may in part be caused by an increase in beta-adrenergic receptor kinase (beta ARK) expression. beta ARK is thought to effect desensitization by acting in concert with an inhibitor protein, called beta-arrestin.

Role of extracellular disulfide-bonded cysteines in the ligand binding function of the beta 2-adrenergic receptor.

Evidence is presented for a role of disulfide bridging in forming the ligand binding site of the beta 2-adrenergic receptor (beta AR). The presence of disulfide bonds at the ligand binding site is indicated by "competitive" inhibition by dithiothreitol (DTT) in radioligand binding assays, by specific protection by beta-adrenergic ligands of these effects, and by the requirement of disulfide reduction for limit proteolysis of affinity ligand labeled receptor. The kinetics of binding inhibition by DTT suggest at least two pairs of disulfide-bonded cysteines essential for normal binding.

Substitution of an extracellular cysteine in the beta 2-adrenergic receptor enhances agonist-promoted phosphorylation and receptor desensitization.

We constructed and expressed in a permanent cell line a beta 2-adrenergic receptor with a valine substitution for cysteine 184 of the second putative extracellular loop. The mutant receptor was partially uncoupled from adenylyl cyclase with impaired ability to form the high affinity agonist-receptor-G protein complex, yet displayed more rapid and extensive agonist-induced desensitization. The enhanced desensitization was accompanied by increased agonist promoted, but not cAMP promoted, receptor phosphorylation in intact cells.

Beta-adrenergic receptor kinase: primary structure delineates a multigene family.

The beta-adrenergic receptor kinase (beta-ARK), which specifically phosphorylates only the agonist-occupied form of the beta-adrenergic and closely related receptors, appears to be important in mediating rapid agonist-specific (homologous) desensitization. The structure of this enzyme was elucidated by isolating clones from a bovine brain complementary DNA library through the use of oligonucleotide probes derived from partial amino acid sequence. The beta-ARK cDNA codes for a protein of 689 amino acids (79.

Calculating receptor number from binding experiments using same compound as radioligand and competitor.

Saturation experiments using increasing concentrations of radioligand are commonly used to determine receptor number and affinity, but this protocol is not feasible in all situations. Alternatively, competitive binding experiments are often performed in which binding of a single concentration of radioligand is completed for by multiple concentrations of the same unlabelled ligand, but the analysis of such data has been difficult. Antonio DeBlasi and colleagues present here a simple method for calculating receptor number and affinity from competitive binding data.

Expression of a human cDNA encoding the beta 2-adrenergic receptor in Chinese hamster fibroblasts (CHW): functionality and regulation of the expressed receptors.

A human beta-adrenergic receptor cDNA was transfected and expressed in transformed Chinese hamster fibroblasts (CHW). The expressed receptor exhibited a typical beta 2-adrenergic selectivity for agonists and antagonists as assessed by radioligand binding and adenylate cyclase activation. Guanine nucleotide-sensitive high affinity binding of the agonist, isoproterenol, indicated effective coupling of the expressed receptor to a guanine nucleotide-regulatory protein.

Regulation of adrenergic receptor function by phosphorylation. I. Agonist-promoted desensitization and phosphorylation of alpha 1-adrenergic receptors coupled to inositol phospholipid metabolism in DDT1 MF-2 smooth muscle cells.

Continuous exposure of DDT1 MF-2 smooth muscle cells to 10-100 microM norepinephrine results in a dramatic attenuation of the ability of norepinephrine to stimulate inositol phospholipid hydrolysis via alpha 1-adrenergic receptors (alpha 1-AR). In addition to the functional desensitization, norepinephrine exposure also reduces the number of accessible cell surface alpha 1-AR as assayed by [3H]prazosin binding at 4 degrees C. Desensitization of the cells with norepinephrine results in an increase in the phosphorylation of the Mr 80,000 alpha 1-AR ligand binding peptide (2.

In vivo regulation of beta-adrenergic receptors on human mononuclear leukocytes: assessment of receptor number, location, and function after posture change, exercise, and isoproterenol infusion.

We studied the regulation of beta-adrenergic receptors in human mononuclear leukocytes (MNL). Total receptor number was determined as specific binding at 4 C of [3H] dihydroalprenolol or [125I]iodopindolol, and redistributed receptors were defined as those binding sites to which the hydrophilic antagonist CGP-12177 did not have access. Receptor function was assessed as cAMP accumulation stimulated by isoproterenol.

Desensitization and redistribution of beta-adrenergic receptors on human mononuclear leukocytes.

We have used intact human mononuclear leukocytes (MNL) to examine desensitization of beta-adrenergic receptors in normal mammalian cells. MNL were prepared and radioligand binding experiments were performed at 4 degrees C. At this temperature the ligand [125I]iodocyanopindolol ([125I]ICYP) identified the same number of receptors as at 37 degrees C, and the agonist isoproterenol competed for this binding with high affinity (dissociation constant, Ki = 20 nM).