Characterization and inhibition of beta-adrenergic receptor kinase in intact myocytes.
OBJECTIVE
beta-Adrenergic receptor kinase (beta ARK) phosphorylates and thereby inactivates agonist-occupied beta-adrenergic receptors (beta AR). beta ARK is thought to play an important role in the regulation of cardiac function. Therefore, we studied beta ARK activation and its inhibition in intact smooth muscle cells and in cardiomyoblasts.


RESULTS
beta AR agonist-stimulated translocation of beta ARK was monitored by immunofluorescence labelling with specific antibodies and confocal laser scanning microscopy in DDT-MF 2 hamster smooth muscle cells and in H9c2 rat cardiomyoblasts. In unstimulated cells. beta ARK was mainly located in the cytosol. After beta AR agonist stimulation, the beta ARK signal was partially translocated to the membranes. Liposomal gene transfer of the COOH-terminus of beta ARK ('beta ARKmini') as a beta ARK inhibitor led to functional expression of this protein in both cell lines with high efficiency. Western blots with beta ARK antibodies showed a gene concentration-dependent immunoreactivity of the 'beta ARKmini' protein. 'beta ARKmini'-transfected myocytes demonstrated reduced membrane targeting of the beta ARK immuno-fluorescence signal. Additionally, the effect of 'beta ARKmini' on beta AR-induced desensitization of myocytic cAMP accumulation was investigated. In control cells, desensitization with isoproterenol led to a subsequent reduction of beta AR-induced cAMP accumulation. In 'beta ARKmini'-transfected myocytes, this beta AR-induced desensitization was significantly diminished, whereas normal beta AR-induced cAMP accumulation was unaffected. A gene concentration of 2 micrograms 'beta ARKmini' DNA/100,000 cardiomyoblasts, and of 0.7 microgram 'beta ARKmini' DNA/100,000 DDT-MF2 smooth muscle cells led to approximately 5.9- and approximately 5.6-fold overexpressions of 'beta ARKmini' vs. native beta ARK, respectively. These gene doses proved sufficient to attenuate beta-adrenergic desensitization significantly.


CONCLUSIONS
(1) beta ARK translocation was evidenced in DDT-MF2 smooth muscle cells and in cardiomyoblasts by confocal laser scanning microscopy. (2) Feasibility of 'beta ARKmini' gene transfer to myocytes was demonstrated, and necessary gene doses for beta ARK inhibition were titered. (3) Overexpression of 'beta ARKmini' functionally interacted with endogenous beta-adrenergic signal transduction, leading to sustained cAMP accumulation after prolonged beta-adrenergic stimulation.
Laugwitz
K L
KL
I. Medizinische Klinik, Klinikum rechts der Isar, Munich, Germany.
Kronsbein
K
K
Schmitt
M
M
Hoffmann
K
K
Seyfarth
M
M
Schömig
A
A
Ungerer
M
M
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D005434:Flow Cytometry; D007545:Isoproterenol / Q000494:pharmacology; D018613:Microscopy, Confocal; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology* / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013268:Stimulation, Chemical; D014178:Translocation, Genetic; D051552:beta-Adrenergic Receptor Kinases
D000318:Adrenergic beta-Agonists; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D007545:Isoproterenol