Experimental Study On Pathogenesis Of Rat Acute Lung Injury Participated By GRKs

In order to investigate the pathogenesis of acute lung injury(ALI) participated by G protein coupled receptors(GRKs), Western blot was first used to observe both the protein expression changes of GRK2, GRK3 in rat lung tissue of endotoxin induced ALI and the interfering action of anisodamine on the basis of establishing ALI rat model with lipopolysaccharide(LPS) originated from E. coli. Then, Rat pulmonary microvascular endothelial cells(PMVEC) were isolated and cultured in vitro. On the condition, Western blot was used again to observe the effects of LPS and tumor necrosis factorα(TNFα) on the protein expression of GRK2,GRK3 in rat PMVEC and the interfering action of anisodamine. RT-PCR was used to observe the effects of LPS on mRNA expression of GRK2,GRK3,β-arrestin1,β-arresti2 in rat PMVEC and the interfering action of anisodamine. Fura-2/AM and immunoradioactive assay were separately used to observe the effects of LPS, TNFα on intracellular concentration of calcium ion ([Ca2+]i) and cAMP in rat PMVEC, and the interfering action of anisodamine was observed, too. The study based on the past laboratory work was on the purpose of probing following problems:(1)the relation between GRKs changes and the down regulation of beta adrenoceptor in the lung tissue of LPS induced ALI;(2) the relation between GRKs changes and the down regulation of beta adrenoceptor in rat PMVEC injured by LPS and TNFα;(3)the relation among [Ca2+]i, cAMP changes and the down regulation of beta adrenoceptor in rat PMVEC, as well as the relation among [Ca2+]i, cAMP changes and pulmonary microvascular endothelium injury. Results:(1)Comparing with normal control group, the GRK2 contents in hybridization bands from rat lung tissue of LPS group and LPS+anisodamine group were significantly higher analyzed with Western blot. There was no significant difference between the GRK2 contents in hybridization bands of LPS group and LPS+anisodamine group. Western blot analyses for GRK3 expression in rat lung tissue were all negative repeated three times.(2) The GRK2 contents in hybridization bands from rat PMVEC of LPS group and LPS+anisodamine group were significantly higher than those in normal control group by the method of Western blot analysis; The GRK2 contents in hybridization bands of TNFαgroup and TNFα+anisodamine group were significantly higher than thosein normal control group, too. Western blot analyses for GRK3 expression in rat PMVEC were all negative repeated three times. No protein bands of GRK5,GRK6 found in SDS-PAGE of rat PMVEC lysate.(3) The GRK2 mRNA expressions in rat PMVEC of LPS group and LPS+anisodamine group were significantly higher than those in normal control group by the method of RT-PCR analysis. RT-PCR analysis results for GRK3 mRNA expression in rat PMVEC were negtive.(4)β-arrestin1,β-arrestin2 mRNA expression in rat PMVEC of LPS group and LPS+anisodamine group were significantly higher comparing with normal control group by the method of RT-PCR analysis.(5) [Ca2+]i in rat PMVEC of LPS group and LPS+anisodamine group were significantly higher than those in normal control group at time points of 15min and 90min. [Ca2+]i in rat PMVEC of TNFαgroup and TNFα+anisodamine group were also significantly higher than those in normal control group at time points of 15min and 90min.(6)The cAMP concentration in normal rat PMVEC was 0.473±0.054μμmol /40μl. The cAMP concentrations in rat PMVEC of LPS group and LPS+anisodamine group were significantly higher than those in normal control group at time points of 15min and 90min. The cAMP concentrations in rat PMVEC of TNFαgroup and TNFα+anisodamine group were also significantly higher than those in normal control group at time points of 15min and 90min.Conclusions: (1) The lung tissue of Wistar rat expresses GRK2,but not GRK3.(2)PMVEC of Wistar rat expresses GRK2 instead of GRK3,GRK5 or GRK6.(3)GRK2 is overexpressed in the lung tissue of endotoxin induced ALI rat, which may be one of the cardinal causes in beta adrenoceptor internalization and desensitization.