[Role of salbutamol in inducing apoptosis of cultured human airway smooth muscle cells]

Abstract

Objective: To study the effect of salbutamol on inducing apoptosis of cultured human airway smooth muscle cells (ASMCs).

Methods: Human ASMCs were isolated and cultured in DMEM containing 10% fetal bovine serum. Cells of Passes 4 approximately 6 were used in experiments. The cells were cultured with salbutamol, cromakalim, 8-Br-cAMP for 24 or 48 hours. Morphological changes were observed by light microscopy and electron microscopy. DNA fragmentation was analyzed by agarose gels. SP immunohistological staining method was performed to detect the changes of expressions of p53, Bcl-2 and Bax gene. The percentage of apoptosis cell was detected by situ end labeling technique (TUNEL) of fragmental DNA.

Results: (1) Salbutamol or 8-Br-cAMP decreased the number of viable cells. At 48 hour, at 300 micromol/L, number of viable cells was the lowest. (2) Human ASMCs incubated with salbutamol (100 micromol/L or 300 micromol/L) for 48 h revealed morphological features of apoptosis (cellular shrinkage, condensation of chromatin). (3) Agarose gel electrophoresis showed a characteristic "ladder" of DNA bands representing integer multiples of the internucleosomal DNA length (about 180 approximately 200 bp). (4) The expression of p53 or Bax gene in salbutamol or 8-Br- cAMP group was significantly higher than that in control group, but the expression of BCl-2 gene was lower than that in control group. (5) The TUNEL indicated: apoptotic positive rate of human ASMCs was significantly different following 100 micromol, 300 micromol salbutamol or 100 micromol 8-Br-cAMP treatment (q = 24.04, 58.47, 27.78 respectively, P < 0.0001), but cromakalim, propranolol before salbutamol not affected basal level of apoptotic cell (q = 0.12, 0.52 respectively, P = 0.932, 0.717 respectively), as compared with the controls.

Conclusions: (1) Salbutamol induced apoptosis in human ASMCs in vitro in time and concentration dependent manner. (2) A cAMP-protein kinase A pathway is necessary and sufficient for salbutamol induced apoptosis. (3) beta(2) adrenergic agonist or cAMP analogue may prevent airway remodeling in asthma.