The .8-adrenergic receptor kinase (.BARK) is the prototypical member of the family of cytosolic kinases that phosphorylate guanine nucleotide binding-protein-coupled receptors and thereby trigger uncoupling between receptors and guanine nucleotide binding proteins. Herein we show that this kinase is subject to phosphorylation and regulation by protein kinase C (PKC). In cell lines stably expressing GIBadrenergic receptors, activation of these receptors by epinephrine resulted in an activation of cytosolic f3ARK. Similar data were obtained in 293 cells transiently coexpressing a1B-adrenergic receptors and 8ARK-1. Direct activation of PKC with phorbol esters in these cells caused not only an activation of cytosolic ,BARK-1 but also a translocation of .8ARK immunoreactivity from the cytosol to the membrane fraction. A PKC preparation purified from rat brain phosphorylated purified recombinant pARK-1 to a stoichiometry of 0.86 phosphate per 8ARK-1. This phosphorylation resulted in an increased activity of .8ARK-1 when membrane-bound rhodopsin served as its substrate but in no increase of its activity toward a soluble peptide substrate. The site of phosphorylation was mapped to the C terminus of f8ARK-1. We conclude that PKC activates DARK by enhancing its translocation to the plasma membrane.Signaling via guanine nucleotide binding (G)-protein-coupled receptors is initiated by binding of agonist ligands and is mediated by the activation of G proteins, which involves binding of GTP by the a subunit and its dissociation from the f,y-subunit complex (1, 2). Both the a subunit and the 13y-subunit complex can then regulate effector molecules and thereby produce intracellular signals (3,4). This signaling pathway is subject to a variety of regulatory mechanisms that alter the expression and the function of the proteins involved. One of the key regulatory processes is the desensitization of such receptors in response to prolonged or repeated agonist exposure. Various mechanisms contribute to receptor desensitization (5, 6). The most rapid and probably quantitatively the most important one (7,8) is triggered by phosphorylation of the receptors by members of the family of G-protein-coupled receptor kinases. Of the six members of this family cloned to date (9), the f3-adrenergic receptor kinase-1 (,3ARK-1; ref. 10) has been studied as the prototypical member that mediates receptor desensitization. Desensitization by this kinase occurs as a two-step process: (i) The kinase translocates from the cytosol to the plasma membrane (11) and phosphorylates several serine and threonine residues in the cytoplasmic portions of the receptors. (ii) Members of another family of cytosolic proteins, the arrestins, bind to the phosphorylated receptors and thereby impair the receptor-Gprotein interaction (12)(13)(14). Much interest has recently been concerned with the mode of translocation of P3ARK to the plasma membrane. Three anchoring points have been identiThe publication costs of this article were defrayed in part by page charge pa...

A1 adenosine receptors are in general coupled to inhibition of adenylyl cyclase, but have more recently been reported to be capable of also activating phospholipase C. The present study was done in order to investigate whether these different effects can be elicited by a single A1 receptor, or whether A1 receptor subtypes have to be invoked. The cDNA of a rat brain A1 adenosine receptor was stably expressed in CHO-cells, resulting in clones with varying receptor densities; a clone expressing 1.9 pmol receptors/mg membrane protein was used for further characterization. The ligand binding properties of the expressed receptors were typical for the rat A1 adenosine receptor. A1 receptor agonists caused a concentration-dependent inhibition of adenylyl cyclase activity in the membranes, with maximal inhibition by 70%. A1 receptor stimulation also caused concentration-dependent stimulation of inositol phosphate generation in these cells, with maximal effects of 300%. Both adenylyl cyclase inhibition and enhancement of inositol phosphate generation were essentially abolished after pretreatment of the cells with pertussis toxin. These results indicate that a single A1 adenosine receptor can couple to two effector pathways, and that both effectors are activated via pertussis toxin sensitive G proteins.

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Tocolysis with a beta-adrenergic receptor agonist is the most common approach to premature labor management after the 25th wk of pregnancy. However, prolonged treatment is associated with a marked loss of efficacy. The biochemical mechanisms involved remain unclear. This study was undertaken to investigate the effect of fenoterol on beta-adrenergic receptor signal transduction in human myometrium. Myometrial biopsy specimens were obtained from 40 women at cesarean section between the 25th and 34th wk of pregnancy. Nineteen patients had received no tocolysis (controls, group I) and 21 had been treated with fenoterol (<48 h in 10, group II; > or = 48 h in 11, group III). As methods we used membrane preparation, adenylyl cyclase assay and cAMP RIA. Adenylyl cyclase activity was determined by the measurement of cAMP levels to evaluate signal transduction after stimulation of beta-adrenergic receptors with isoproterenol, G protein with GTP, and adenylyl cyclase with forskolin. The functional activity of GTP-binding regulatory proteins (G(s)) and adenylyl cyclase was not altered by fenoterol treatment. In the control group, the increase in adenylyl cyclase activity in response to GTP plus isoproterenol was greater than in response to GTP alone. The increase was reduced by 50% in group II and was insignificant in group III. There was no correlation between gestational age and basal adenylyl cyclase activity. Intravenous tocolysis with the beta2-adrenergic receptor agonist fenoterol leads to complete desensitization of the beta-adrenergic receptor system. In addition to the known reduction in receptor number (down-regulation) as underlying mechanism, uncoupling of the receptor from the stimulatory G protein G(s) was identified.

979Die Diets-Aider-Additionen von Benzvalen (4) an 3,5-Dichlor-, 3,4,5-Trichlor-und Tetrachlor-apyron liefern die ö-Lactone 8-10, welche beim Erhitzen Kohlendioxid abspalten und in die chlor- brings about the aromatizations of 11-13 with elimination of hydrogen chloride and formation of the chloro-substituted benzobenvalenes 19-ll. The treatmcnt of 10 with sodium methoxide Ieads to methyl benzobenzvalenecarboxylate 27. Several oxidizing reagents transform the dihydropyridazines 28 and 30 into the diazabenzobenzvalenes 29 and 31, respectively. Probably promoted by an acid catalysis this dehydrogenation of 28 is accomplished even by air, which to a considerable extent gives rise also to cis-3,4-dibenzoyltricyclo[3.1.0.0 2 · 6 ]hexane (32), however. -