Characterization and inhibition of β-adrenergic receptor kinase in intact myocytes

Karl Ludwig Laugwitz, Kai Kronsbein, Manfred Schmitt, Katrin Hoffmann, Melchior Seyfarth, Albert Schömig, Martin Ungerer

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

Objectives: β-Adrenergic receptor kinase (βARK) phosphorylates and thereby inactivates agonist-occupied β-adrenergic receptors (βAR). βARK is thought to play an important role in the regulation of cardiac function. Therefore, we studied βARK activation and its inhibition in intact smooth muscle cells and in cardiomyoblasts. Methods and Results: βAR agonist- stimulated translocation of βARK 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, βARK was mainly located in the cytosol. After βAR agonist stimulation, the βARK signal was partially translocated to the membranes. Liposomal gene transfer of the COOH-terminus of βARK ('βARKmini') as a βARK inhibitor led to functional expression of this protein in both cell lines with high efficiency. Western blots with βARK antibodies showed a gene concentration-dependent immunoreactivity of the 'βARKmini' protein. 'βARKmini'-transfected myocytes demonstrated reduced membrane targeting of the βARK immuno-fluorescence signal. Additionally, the effect of 'βARKmini' on βAR-induced desensitization of myocytic cAMP accumulation was investigated. In control cells, desensitization with isoproterenol led to a subsequent reduction of βAR- induced cAMP accumulation. In 'βARKmini'-transfected myocytes, this βAR-induced desensitization was significantly diminished, whereas normal βAR-induced cAMP accumulation was unaffected. A gene concentration of 2 μg 'βARKmini' DNA/100 000 cardiomyoblasts and of 0.7 μg 'βARKmini' DNA/1000 000 DDT-MF2 smooth muscle cells led to ≃ 5.9-and ≃ 5.6-fold overexpressions of 'βARKmini' vs. native βARK, respectively. These gene doses proved sufficient to attenuate β-adrenergic desensitization significantly. Conclusions: (1) βARK translocation was evidenced in DDT-MF2 smooth muscle cells and in cardiomyoblasts by confocal laser scanning microscopy. (2) Feasibility of 'βARKmini' gene transfer to myocytes was demonstrated, and necessary gene doses for βARK inhibition were titered. (3) Overexpression of 'βARKmini' functionally interacted with endogenous β-adrenergic signal transduction, leading to sustained cAMP accumulation after prolonged β- adrenergic stimulation.

Original languageEnglish
Pages (from-to)324-333
Number of pages10
JournalCardiovascular Research
Volume35
Issue number2
DOIs
StatePublished - Aug 1997
Externally publishedYes

Keywords

  • Adenylyl cyclase
  • Gene transfer
  • Myocytes
  • β-Adrenergic receptor kinase

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