Pseudomonas exotoxin A prevents beta-adrenoceptor-induced upregulation of Gi protein alpha-subunits and adenylyl cyclase desensitization in rat heart muscle cells.
Exposure of rat heart muscle cells to noradrenaline (1 microM) for 48 hr led to a decrease in the number of beta 1-adrenoceptors of 50% and a concomitant decrease in adenylyl cyclase stimulation by isoprenaline and forskolin of about 60 and 30%, respectively. In addition, the levels of two inhibitory guanine nucleotide-binding protein (Gi protein) alpha-subunits (Gi alpha 40 and Gi alpha 41) were increased in membranes of noradrenaline-treated cells. Evidence is presented that noradrenaline induces this increase by activation of beta-adrenoceptors. First, the noradrenaline action was mimicked by the beta-adrenoceptor agonist isoprenaline. Second, beta-adrenoceptor blockade by timolol but not alpha-adrenoceptor blockade by prazosin prevented the noradrenaline-induced up-regulation of Gi alpha proteins. Furthermore, timolol but not prazosin abolished the noradrenaline-induced down-regulation of beta 1-adrenoceptors and the decreases in receptor-dependent (isoprenaline) and -independent (forskolin) adenylyl cyclase stimulation. The specific protein synthesis inhibitor Pseudomonas exotoxin A was used to study whether the noradrenaline-induced up-regulation of Gi alpha subunits depends on increased synthesis of these proteins. This toxin inhibits peptide chain elongation by ADP-ribosylating elongation factor 2. Treatment of rat heart muscle cells with Pseudomonas exotoxin A (1 ng/ml) completely prevented the noradrenaline-induced increase in Gi alpha proteins, measured by both pertussis toxin-catalyzed ADP-ribosylation and immunoblotting with anti-Gi alpha antibodies. Most importantly, Pseudomonas exotoxin A also completely prevented the noradrenaline-induced decrease in forskolin-stimulated adenylyl cyclase activity. Furthermore, the noradrenaline-induced decrease in isoprenaline-stimulated adenylyl cyclase activity was significantly attenuated by the toxin, although the down-regulation of beta 1-adrenoceptors caused by noradrenaline treatment was not affected. The data presented suggest that prolonged activation of beta-adrenoceptors in rat heart muscle cells, in addition to causing a receptor down-regulation, induces the synthesis of Gi alpha proteins, which then apparently mediate a decreased adenylyl cyclase responsiveness. The data, additionally, suggest that the synthesis of Gi alpha proteins is under control of the activity of the adenylyl cyclase system and that altered levels of these proteins may play a major role in long term regulation of signal transduction by this enzyme.
Reithmann
C
C
Pharmakologisches Institut der Universität Heidelberg, Im Neuenheimer Feld, Federal Republic of Germany.
Gierschik
P
P
Müller
U
U
Werdan
K
K
Jakobs
K H
KH
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000319:Adrenergic beta-Antagonists / Q000378:metabolism; D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D005098:Exotoxins / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006321:Heart / Q000187:drug effects; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D009130:Muscle, Smooth / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009638:Norepinephrine / Q000494:pharmacology; D011412:Propanolamines / Q000378:metabolism; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000502:physiology*; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose; D000262:Adenylyl Cyclases; D000319:Adrenergic beta-Antagonists; D001427:Bacterial Toxins; D005576:Colforsin; D005098:Exotoxins; D019204:GTP-Binding Proteins; D007545:Isoproterenol; D046911:Macromolecular Substances; D009243:NAD; D009638:Norepinephrine; D011412:Propanolamines; D011943:Receptors, Adrenergic, beta; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C033394:CGP 12177