Influence Of LPS On Contractility Of Pulmonary Artery Smooth Muscle Cells And It's Mechanisms

OBOJECTIVESeptic shock is characterized by systemic hypotension and early elevation of pulmonary artery pressure, even pulmonary artery hypertension. The aim of this work was to investigate whether high level lipopolysaccharide (LPS) can exert influence on the contractility of pulmonary artery smooth muscle cells (PASMC).METHODSPASMCs were enzymatically isolated and observed under inverted microscope. Drugs were delivered in HEPES-buffered salt solution by persistent perfusion. Longitudinal changes of cells and time-needed for completely contraction were used to assess influences of LPS on the contractility of PASMCs.80mM high K+-containing physiological saline solution(HKSS),10 u M phenylephine(PE) and 10 nM endothelin-1 (ET-1) were used to evaluate the contractility of LPS-pretreated PASMCs.RESULTS(1)The survival rate of PASMCs isolated from acute enzyme digestion were over 95% showed by Trypan Blue Staine experiment, and the purity of PASMCs were 97.7±1.78% assessed by Immunol Fluorence Staining of smooth muscle specific a-actin. (2) Contractile response of PASMCs can not be induced directly by 10μg/ml of LPS. (3) 80Mm HKSS can excite PASMCs and cause contractile response. 10μg/ml LPS pretreated PASMCs showed more sensitivity to 80mM HKSS. They had higher contract amplitude in the time point of 2.5 min(68.43±1.46 vs 47.70±5.70, P<0.001),5 min(75.42±0.87 vs 63.45±3.65, P<0.01),10 min(80.23±0.57 vs 74.01±2.17, P<0.05) after excited by HKSS. They also expended less time to complete 50% of contraction response(1.03±0.10 vs 2.38±0.36, P<0.01), as well as 90% of it(4.04±0.31 vs 7.14±0.75, P<0.001). (4)Neither 10μM norlmM of PE can excite a contractile response on the PASMCs with or without pretreatment of LPS. (5)PASMCs can develop intensive contraction response in about 1 min after excited by 10nM of ET-1, which were free from the impact of LPS-pretreatment (P>0.05).CONCLUSIONSPretreatment with 10μg/ml of LPS can not directly cause contractile response in PASMCs, but it had a role on the PASMCs to enhance their sensitivity to 80mM HKSS, while it had no impact on the contractile response excited by ET-1.OBOJECTIVEThe aim of this research was to investigate whether high level lipopolysaccharide (LPS) can exert influence on the [Ca2+]i of pulmonary artery smooth muscle cells (PASMC) and involvement of store-operated Ca2+ channels.METHODSReal-time PCR was used to detect the mRNA expressions of TRPC1,3-6, which is highly presumed to be the molecular bases for SOC, in pulmonary artery smooth muscle treated with or without 10μg/ml of LPS. Calcium fluorescent probe and laser confocal microscopy were used to access the influence of LPS on the [Ca2+]i evoked by vasoconstrictors, as well as the effect of calcium release and calcium entry. SKF-96365(a non-selective SOC channel inhibitor),2-APB (a selective SOC channel inhibitor), nifedipine(a selective voltage-operated Ca2+ channel inhibitor), and BQ-123 (a selective ET-1 receptor inhibitor) were used to assess the role of SOC in the excitement of PASMCs.RESULTS(1) The mRNAs of TRPC1,3-6 expressed non-significantly in the smooth muscle cells among rat's pulmonary artery, cervical artery and tail artery, but mRNAs of TRPC1(1.21E-05±0.01E-05 vs 3.82E-05±0.46E-05, P<0.001),TRPC3(1.34E-05±0.17E-05 vs 6.29E-05±0.15E-05, P<0.001), and TRPC4(1.36E-05±0.15E-05 vs 7.46E-05±0.11E-05, P<0.001) were greatly up-regulated in pulmonary artery pretreated with LPS. (2) Both of mRNAs of ETA-R(1.31E-05±0.23E-05 vs 5.26E-05±0.15E-05, P<0.01) and ETB-R(1.44E-05±0.10E-05 vs 3.43E-05±0.20E-05, P<0.001) were up-regulated, while that of al-adrenergic receptor (3.16E-05±0.10E-05 vs 0.72E-05±0.29E-05, P<0.001) was down-regulated in pulmonary artery pretreated with LPS. (3) [Ca2+]i had no detectable changes between PASMCs pretreated with or without LPS(0.01±0.01 vs 0.01±0.02, P>0.05). PASMCs pretreated with LPS had a significant higher [Ca2+]i after evoked by 80mM HKSS (0.95±0.06 vs 1.25±0.10, P<0.05) and ET-1 (1.56±0.07 vs 1.98±0.09, P<0.05) respectively. (4) Contrasted with PASMCs pretreated without LPS, calcium release was decreased (1.36±0.07 vs 1.66±0.06, P<0.01) while calcium entry was enhanced (1.32±0.10 vs 1.06±0.07, p<0.05) in PASMCs pretreated with LPS. (5) To block the calcium entry by SOC inhibitors caused a significant inhibition on PASMCs stimulated by 10nM of ET-1 (73.0% and 70.8%).CONCLUSIONS[Ca2+]i had no detectable changes between PASMCs pretreated with or without LPS, but PASMCs pretreated with LPS did had a significant higher [Ca2+]i after evoked by 80mM HKSS or ET-1, which caused by enhancement of calcium entry. LPS may change PASMCs'contractility by up-regulating expression of SOC(TRPC), as well as it's activity.