To investigate the functional role of different ␣ 1 -adrenergic receptor (␣ 1 -AR) subtypes in vivo, we have applied a gene targeting approach to create a mouse model lacking the ␣ 1b -AR (␣ 1b ؊͞؊). Reverse transcription-PCR and ligand binding studies were combined to elucidate the expression of the ␣ 1 -AR subtypes in various tissues of ␣ 1b ؉͞؉ and ؊͞؊ mice. Total ␣ 1 -AR sites were decreased by 98% in liver, 74% in heart, and 42% in cerebral cortex of the ␣ 1b ؊͞؊ as compared with ؉͞؉ mice. Because of the large decrease of ␣ 1 -AR in the heart and the loss of the ␣ 1b -AR mRNA in the aorta of the ␣ 1b ؊͞؊ mice, the in vivo blood pressure and in vitro aorta contractile responses to ␣ 1 -agonists were investigated in ␣ 1b ؉͞؉ and ؊͞؊ mice. Our findings provide strong evidence that the ␣ 1b -AR is a mediator of the blood pressure and the aorta contractile responses induced by ␣ 1 agonists. This was demonstrated by the finding that the mean arterial blood pressure response to phenylephrine was decreased by 45% in ␣ 1b ؊͞؊ as compared with ؉͞؉ mice. In addition, phenylephrine-induced contractions of aortic rings also were decreased by 25% in ␣ 1b ؊͞؊ mice. The ␣ 1b -AR knockout mouse model provides a potentially useful tool to elucidate the functional specificity of different ␣ 1 -AR subtypes, to better understand the effects of adrenergic drugs, and to investigate the multiple mechanisms involved in the control of blood pressure.

To explain how converting enzyme inhibition could improve the prognosis in cardiac insufficiency, the effect of converting enzyme inhibition (CEI) by S9490-3 (Perindoprii) treatment for 2 months (treated infarctions, 11= 18) on hormonal plasma variables and the quantitative and qualitative changes in myocardium were studied in an experimental model of left ventricular infarction in rats (untreated infarctions, 11= 18) and compared to a sham-operated control group (0= 15). Induction of myocardial infarction was associated with a transient decrease in blood pressure. CEI treatment maintained a lower blood pressure throughout the experimental period. Plasma renin concentration was not significantly increased in the untreated infarct group (155.4 ± 136.7 ng Al/ml/hr) as compared to the sham-operated group (47.6 ±15.9 ng Al/ml/hr). Plasma aldosterone did not change in the three experimental groups. The plasma level of immunoreactive atrial natriuretic factor increased in the untreated infarct group (185 ± 245 pg/ml) as compared with the control group (76 ± 40 pg/ml) and was normalized by CEI (66 ± 60 pg/ml). Body weight was slightly decreased in both treated and untreated infarct groups, whereas the heart weight was significantly increased in the untreated group (1,540 ±310 mg) and normalized by treatment (1,145 ±180 mg) as compared with sham-operated controls (1,071 ±80 mg). The combined atria and right ventricular mass was significantly increased in the untreated infarct group (660 ±210 mg) and decreased by treatment (443 ±106 mg) but was not completely normalized (controls, 343 ± 40 mg). Left ventricular isomyosin profiles were modified by myocardial infarction as compared with controls: V, form decreased from 62.4 ±9.4% in the sham-operated group to 41.6 ± 13.4% in the infarct group, and the V, form increased from 13.0 ± 4.7% in sham-operated animals to 27.4 ± 11.8% in untreated infarct animals. CEI treatment partially, but significantly, reversed this modification of the isomyosin profile (V,, 53.0 ±14.4%; V,, 17.5 ±8.0%). \blume density of collagen was significantly increased in the untreated infarct rats (4.14 ±0.81% versus 2.68 ± 0.49% in controls), and this was reversed by treatment (2.95 ± 0.66%). Messenger RNA encoding for atrial natriuretic factor, measured by dot blot hybridization, was significantly increased in both the atria and the ventricles in the untreated infarct group, and treatment by CEI partially reversed this increase. Thus, myocardial infarction profoundly modified several variables of peripheral circulation and quantitative and qualitative myocardial protein expression. CEI treatment largely reversed these changes but probably did not completely normalize them. (Circulation Research 1988; 62:641-650) v L eft ventricular myocardial infarction in rats' has been described as an experimental model of cardiac hypertrophy and overload that readily leads to cardiac insufficiency in relation to the size of the infarcted area." This experimental model has also been used to show that treatment by co...

Catecholamines as well as phorbol esters can induce the phosphorylation and desensitization of the ␣ 1B -adrenergic receptor (␣ 1B AR). In this study, phosphoamino acid analysis of the phosphorylated ␣ 1B AR revealed that both epinephrine-and phorbol ester-induced phosphorylation predominantly occurs at serine residues of the receptor. The findings obtained with receptor mutants in which portions of the C-tail were truncated or deleted indicated that a region of 21 amino acids (393-413) of the carboxyl terminus including seven serines contains the main phosphorylation sites involved in agonist-as well as phorbol ester-induced phosphorylation and desensitization of the ␣ 1B AR. To identify the serines invoved in agonist-versus phorbol ester-dependent regulation of the receptor, two different strategies were adopted, the seven serines were either substituted with alanine or reintroduced into a mutant lacking all of them. Desensitization is a general regulatory phenomenon of G protein-coupled receptors resulting in the attenuation of the receptor-mediated response. Two major patterns of desensitization referred to as homologous and heterologous desensitization can be distinguished. Homologous desensitization is defined as a rapid loss of responsiveness for a receptor repeatedly exposed to its specific agonist, whereas in heterologous desensitization stimulation of a receptor by an agonist can attenuate the response mediated by other receptors eliciting similar cellular effects (1).In the G protein-coupled receptor family (2), receptor desensitization has been extensively characterized for rhodopsin mediating phototransduction in retinal rod cells and for the ␤ 2 -adrenergic receptor (␤ 2 AR) 1 which mediates catecholamineinduced stimulation of adenylyl cyclase. The second messengerdependent cAMP-dependent protein kinase can phosphorylate and desensitize the ␤ 2 AR both in response to its agonist as well as to other agents increasing the cellular content of cAMP. On the other hand, a prominent role in homologous desensitization of rhodopsin and ␤ 2 AR is played by the second messengerindependent rhodopsin kinase (3) and ␤-adrenergic receptor kinase (␤ARK) (4), respectively. Once the receptor is occupied by the agonist, it is recognized by the kinase and becomes phosphorylated. The subsequent uncoupling of the receptor and G protein is then mediated by arrestin proteins, which specifically bind to the phosphorylated receptor (5, 6). Rhodopsin kinase and ␤ARK are members of the newly discovered family of G protein-coupled receptor kinases (GRK) (7). These protein kinases have the unique ability to recognize and phosphorylate their G protein-coupled receptor substrates predominantly in their active (i.e. agonist-occupied) conformations. Recently, we have provided evidence that the ␣ 1B AR coupled to Gq-mediated activation of phospholipase C can be phosphorylated by at least two types of protein kinases, a protein kinase C (PKC) upon its stimulation by phorbol esters (8) and protein kinases belonging to the GRK family o...

The endothelial angiotensin I-converting enzyme (ACE) is organized in two large homologous domains, each bearing a putative active site. However, only one of these sites is probably involved in catalyzing the conversion of angiotensin I into angiotensin II. The testicular form of ACE is equally active, encoded by the same gene, but translated from a shorter mRNA. Molecular cloning of the human testicular ACE cDNA indicates that the mRNA codes for 732 residues (vs 1306 in endothelium). The testicular transcript corresponds to the 3' half of the endothelial transcript and encodes one of the two homologous domains of endothelial ACE, preceded by a short specific sequence. This 5' specific sequence contains 228 nucleotides and encodes 67 amino acids, including the putative signal peptide followed by a serine/threonineenriched region, presumably glycosylated. The testicular transcript corresponds to the ancestral, non-duplicated form of the ACE gene. Since the carboxyl-terminal domain of the endothelial ACE is expressed in the testicular enzyme, it is likely that it bears the active site in both forms.

Using the yeast two-hybrid system, we identified the 2 subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the ␣ 1b -adrenergic receptor (AR). Direct association between the ␣ 1b -AR and 2 was demonstrated using a solid phase overlay assay. The ␣ 1b -AR/ 2 interaction occurred inside the cells, as shown by the finding that the transfected ␣ 1b -AR and the endogenous 2 could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the ␣ 1b -AR revealed that the binding site for 2 does not involve canonical YXX⌽ or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of 2 for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The ␣ 1b -AR specifically interacted with 2 , but not with the 1 , 3 , or 4 subunits belonging to other AP complexes. The deletion of the 2 binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.Desensitization to the effects of hormones and neurotransmitters is a fundamental regulatory mechanism of G proteincoupled receptor (GPCR) 1 function defined by a specific loss of responsiveness for those receptors that have been repeatedly stimulated by the agonist. Receptor desensitization results from the combination of multiple biochemical events occurring at different time frames: receptor-G protein uncoupling in response to receptor phosphorylation (seconds to minutes), internalization or endocytosis of cell surface receptors to intracellular compartments (minutes), and down-regulation of the total pool of receptors due to their decreased synthesis and/or increased degradation (hours) (1). A prominent role in homologous desensitization of GPCRs is played by G protein-coupled receptor kinases. Once the receptor is occupied by the agonist, it is recognized by the G protein-coupled receptor kinases and becomes phosphorylated (2). The subsequent uncoupling of the receptor from the G protein is then mediated by arrestin proteins, which preferentially bind to the agonist-occupied phosphorylated receptor (3). However, during the last decade, ␤-arrestins have emerged as key regulatory molecules controlling various steps of receptor desensitization. Beyond their role in physical uncoupling of GPCRs from the G proteins (3), it has been demonstrated that ␤-arrestins target GPCRs to the endocytic machinery (4). In fact, it is believed that for those GPCRs that internalize in a clathrin-dependent manner, like the ␤ 2 -adrenergic receptor (AR), targeting of the receptor-␤-arrestin complexes to clathrin-coated vesicles is mediated by a dual interaction of ␤-arrestin with both clathrin heavy chain and the ␤ 2 subunit of the heterotetrameric ...

A synthetic oligonucleotide probe complementary to messenger ribonucleic acid (mRNA) encoding for the C-terminal portion of atrial natriuretic factor (ANF) has been used to study the expression of the ANF gene in rat myocardium. Four experimental models were studied: binephrectomy (for 48 h); ligature of both ureters (for 48 h); deoxycorticosterone acetate-salt (for 3 wk); and aortocaval fistula (for 2 wk). Analysis of atrial RNA by gel-blot hybridization detected a single band, corresponding in length to that of mRNA coding for ANF. Such an mRNA was also detected in ventricular RNA but was 1/50th as abundant. In the four experimental groups ANF mRNA was increased significantly as compared with controls. In all rats there was no significant difference in the ANF mRNA content between the left and the right atrium. Each experimental condition was accompanied by a highly significant increase in ANF gene expression in the left ventricle, where all of the ventricular tissue could be recruited and with a negative gradient from the base to the apex of the left ventricle. These data were confirmed by in situ hybridization. Thus all of the atrial and ventricular myocardium can express the ANF gene. Recruitment increases in response to passive stretch of the cardiac chambers.

We provide the first evidence that point mutations can constitutively activate the L L 1 -adrenergic receptor (AR). Leucine 322 of the L L 1 -AR in the C-terminal portion of its third intracellular loop was replaced with seven amino acids (I, T, E, F, C, A and K) differing in their physico-chemical properties. The L L 1 -AR mutants expressed in HEK-293 cells displayed various levels of constitutive activity which could be partially inhibited by some beta-blockers. The results of this study might have interesting implications for future studies aiming at elucidating the activation process of the L L 1 -AR as well as the mechanism of action of beta-blockers.z 1999 Federation of European Biochemical Societies.

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