The Role Of Dopamine D₁-Like Receptors In The Autophagy Of VSMCs And Its Possible Mechanisms

Autophagy is a highly conservative process of cell protection.Recent studies have shown that autophagy plays a critical role in regulation of vascular function and cardiovascular disease.During the development of atherosclerosis,moderate autophagy can reduce the accumulation of lipid droplets in vascular smooth muscle cells,avoid the formation of foam cells derived from VSMCs,contribute to the maintenance of phenotype of VSMCs and inhibit the progression of atherosclerotic plaques and increase the stability of the plaque.Excessive autophagy may cause apoptosis of the cells,resulting in thinning of the fibrous cap and instability of the atherosclerotic plaque.Starvation,hypoxia,metabolic stress,cytokines,,active substances and drugs can affect cell survival,function and phenotypic transformation by regulating the level of autophagy in VSMCs.Understanding the autophagy and signaling pathways of VSMCs induced by various factors is of great significance for the prevention and treatment of cardiovascular diseases.Dopamine?DA?is a catecholamine neurotransmitter that acts on dopamine receptors and plays an important regulatory role in vasomotor,renal sodium water metabolism,and oxidative stress.At least five kinds of dopamine receptors are known at present,and can be classified into two types according to their structures and functions,a D₁ receptor which excites adenylate cyclase and a D₂ receptor which inhibits adenylate cyclase.Studies have confirmed that dopamine D₁ receptors are expressed on the surface of rat vascular smooth muscle,but its relationship with autophagy is still unclear.Objective:Study the role of dopamine D₁ receptors in autophagy of rat vascular smooth muscle cells and further explore its potential regulatory mechanisms.Methods:1.Culture rat vascular smooth muscle cells?VSMCs?.2.VSMCs were stimulated with dopamine D₁ receptor agonist?Fenoldopam?at a concentration of 0 uM,0.5 uM,1.0 uM,2.0 uM,4.0 uM,the autophagy-related protein LC3-I and LC3-II were detected by western blot.3.The VSMCs were stimulated with 1.0uMFenoldopam for 0h,3h,6h and 12h,the autophagy-related protein LC3-I and LC3-II were detected by western blot.4.Pretreatment of cells with inhibitor?Sch22390?followed by Fenoldopam stimulation of VSMCs,Western blot analysis of PKA-C-?,autophagy-related protein ATG5,LC3-I and LC3-II changes.5.After pretreatment of cells with PKA inhibitor?Rp-cAMP?,Fenoldopam was added to stimulate VSMCs,and the ratio of autophagy-related proteinsATG5,LC3-I and LC3-II were detected by western blot.Results:1.VSMCs were treated with Fenoldopamat 0uM,0.5uM,1.0uM,2.0uM,4.0uM for 3hours,LC3-II/LC3-I ratio gradually increased,the ratio increased significantly at 1.0uM?P<0.05?,and there was no significant difference in LC3-II/LC3-I ratio between 1.0uM and 2.0uM.2.VSMCs were treated with 1.0uM Fenoldopam for 0 h,3 h,6 h,12 h,the ratio of LC3-II/LC3-I increased with time,and reached the peak at 3 h?P<0.001?,the ratio decreased at 6h compared with 3h,and still increased at 12 h compared with the control group,3 h was significantly higher than that at 1 h?P<0.05?.3.Compared with the control group,the ATG5 and LC3-II/LC3-I ratios of the Fenoldopam group increased?P<0.01?,the autophagy of the SCH23390 group did not change significantly,SCH23390 pretreated VSMCs and then added Fenoldopam,the effects of autophagy in VSMCs decreased compared with the Fenoldopam group?P<0.05?.4.Compared with the control group,Fenoldopam stimulated vascular smooth muscle cells alone,and the expression of PKA-C-?did not change significantly?P>0.05?,there was no significant change in the VSMCs treated by the inhibitor SCH23390.5.Compared with the control group,Fenoldopam stimulated vascular smooth muscle cells alone,the ratio of LC3-II/LC3-I significantly increased?P<0.01?,the autophagy of the VSMCs was no significant change in the Rp-cAMP group,Rp-cAMP pretreated the VSMCs and then added Fenoldopam,the effect of Fenoldopam on autophagy was inhibited?P<0.01?.Conclusion:1.Activation of dopamine D₁ receptors can induce autophagy in vascular smooth muscle cells in a concentration-time dependent manner.2.Activation of dopamine D₁ receptors can induce autophagy in vascular smooth muscle cells by activating PKA signaling pathway.3.Activation of dopamine D₁ receptors may induce autophagy in vascular smooth muscle cells by regulating the allosteric expression of PKA rather than expression.