[Gene transfer of a beta-adrenergic receptor kinase inhibitor up-regulates the level of beta2-adrenergic receptor and cyclic adenosine monophosphate in the asthmatic murine lung].
OBJECTIVE
To investigate the effects of gene transfer of a beta-adrenergic receptor (beta(2)AR) kinase inhibitor (betaARKct) on pulmonary beta(2)-adrenergic receptor and cyclic adenosine monophosphate (cAMP) following beta(2)AR agonist treatment in asthmaticmice, and to analyze the relationship between the routes of gene delivery and the changes of beta(2)AR and cAMP.


METHODS
(1)Thirty-five BALB/c mice were sensitized and challenged by 10% and 5% ovalbumin respectively to establish the asthmatic model and then divided into 7 groups (5 each). Sterile saline was injected intramuscularly in the asthmatic control group (group B), while beta(2)AR agonist (salbutamol) was injected intramuscularly in other 5 asthmatic groups (group C, D, E, F, and G) so as to establish the asthmatic model with prolonged application of beta(2)AR agonist. In addition, 5 healthy mice served as the normal control group (group A). (2) The plasmid with the expression of betaARKct was constructed and gene transfer was performed through intravenous injection or intratracheal instillation in asthmatic mice. Intravenous injection with plasmid-betaARKct was performed in group D, but with plasmid-pcDNA 3.1 in group E. Intratracheal instillation with plasmid-betaARKct was performed in group F, but with plasmid-pcDNA 3.1 in group G. (3) The gene expression was measured with Western blot analysis. Radioimmunoassay was used to evaluate the changes of pulmonary beta(2)AR and cAMP.


RESULTS
(1) The expression of transferred betaARKct gene was detectable in the lungs. The level of betaARKct expression was higher in the lungs of mice receiving intratracheal plasmid (group F) than those receiving intravenous plasmid (group D). (2) The levels of beta(2)AR and cAMP in group A were (109 +/- 11) fmol/mg and (3.50 +/- 0.50) pmol/L respectively; The levels of beta(2)AR and cAMP in group B were (81 +/- 3) fmol/mg and (1.50 +/- 0.20) pmol/L respectively; the differences between the two groups were significant (all P < 0.05). The levels of beta(2)AR and cAMP in group C were (63 +/- 3) fmol/mg and (0.90 +/- 0.10) pmol/L respectively, and the differences were significant, as compared with those in group A (all P < 0.01). (3) The levels of beta(2)AR and cAMP in group D were (86 +/- 11) fmol/mg and (2.01 +/- 0.10) pmol/L respectively after using plasmid-betaARKct to the asthmatic mice treated with beta(2)AR agonist, and the differences were significant, as compared with those in group C (all P < 0.05). The levels of beta(2)AR and cAMP in group F were (107 +/- 13) fmol/mg and (3.30 +/- 0.20) pmol/L respectively, and the differences were significant, as compared with those in group C (all P < 0.05). The extent of up-regulation of beta(2)AR and cAMP was higher in group F than that in group D (all P < 0.05). The levels of beta(2)AR and cAMP were (70 +/- 11) fmol/mg and (1.30 +/- 0.10) pmol/L in group E, (67 +/- 4) fmol/mg and (3.30 +/- 0.20) pmol/L in group G, respectively, and there were no significant differences between group C and E or G (all P > 0.05).


CONCLUSIONS
The results indicated that there was down-regulation of beta(2)AR and cAMP in asthmatic mice (group B), especially in those treated with beta(2)AR agonist (group C). Gene transfer of betaARKct could inhibit the extent of the down-regulation of beta(2)AR and cAMP in asthmatic mice treated with beta(2)AR agonist. The route of gene delivery could also affect the degree of up-regulation of beta(2)AR and cAMP. Gene transfer of betaARKct may provide a novel approach to the therapeutic strategy for asthma.
Huang
Mao
M
Department of Respiratory Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
Wu
Yan
Y
Yao
Xin
X
Cha
Wang-Jian
WJ
Yin
Kai-Sheng
KS
D000818:Animals; D001249:Asthma / Q000235:genetics / Q000628:therapy*; D000242:Cyclic AMP / Q000378:metabolism*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D008168:Lung / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D010455:Peptides / Q000235:genetics*; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics* / Q000627:therapeutic use; D019076:Transgenes
D010455:Peptides; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; C445259:beta-adrenergic receptor kinase inhibitory peptide; D000242:Cyclic AMP