Regulation Of Natriuretic Peptide Receptor-A Gene Expression And Guanylyl Cyclase-A Activity

Objective:The present study was designed to explore the chang of gene expression of atrial natriuretic peptide (ANP) receptors-A (NPR-A) and chang of guanylyl cyclase-A(GC-A) activity in two aspects: first, investgating effects of mechanical stretch on NPR-A gene expression and GC-A activity during cardiac myocytes hypertrophy caused by mechanical stretch, so as to clarify the mechanism of hypertrophy and the implication of this chang during myocardial cells hypertrophy. Secondly, investigating the chang of NPR-A protein and GC-A activity in heart and kidney of two-kidney, one-clip (2K1C) hypertensive rats, meanwhile extrinsic ANP is administered and investigating the modulations of NPR-A protein and GC-A activity effected by long-term extrinsic ANP administering. Methods:1. A mechanical device for stretching cultured myocardial cells from neonatal SD rat was designed. The cultured myocardial cells were subjected to static stretch and cyclic stretch stimulation at 1Hz respectively. After 12h, 24h, 48h, the photos of these cells were taken. NPR-A mRNA expression level after 3h, 6h, 12h, 24h, 48h was respectively determined by RT-PCR, at the same time the expression of NPR-A was respectively examined at above time by flow cytometer. After the cultured myocardial cells were stimulated by 10-7mol/L ANP for ten minutes, cGMP content was measured by radio immunity assay, and gained the values of GC-A activity . 2. Two-kidney, one-clip (2K1C) rats were used to establish the model of hypertension . The sustained-release system of recombinant human ANP(rhANP) in vivo was fabricated by implanting encapsulated hANP cDNA plasmid transfected cells into 2K1C hypertensive rats intraperitoneally 2 weeks post-2K1C operation.. The rats were divided into hypertensive group, hypertensive group treated with rhANP and normal control group, of 10 rats each group . After 6 weeks the rats were killed, heart and kidney tissues were taken out and paraffin-embedded,then sections of the heart and kidney were stained for NPR-A with immunity histochemical method. Analyzed content of NPR-A expression with image process software. Gained membrane preparation from homogenate of these tissues and measured concentration of membrane protein with Bradorf method. Next added 10-7mol/L ANP into membrane solution to stimulate cGMP production, then measured the quantity of cGMP production by radio immunity assay to calculat the enzyme activity of GC-A. Compared the collected data from membrane solution which been added ANP and membrane solution without ANP. Measured the enzyme activity of GC-A in heart and kidney tissue from every group rats. Results:1.After 24h static and 48h cyclic mechanical stretching, myocardial cells were obvious hypertrophy. Undertook 20% static stretch for 3h, NPR-A gene expression and the activity of GC increased. This effect got to top when stretch lasting 6 h. Then NPR-A gene expression and GC activity keep in a high level and decrease till 24 and 48 h, thewhole process shown a obvious time dependence. NPR-A gene expression and the activity of GC increased incardiac myocytes subjected to 20% cyclic stretch at 1Hz frequency for 6h,, then got to top at 12 h, decreased at 48 h. There was not marked difference in maxima response between the cardiomyocytes subjected to static stretch and cyclic stretch. 2. After 2K1C operation 2 weeks, the blood pressure increased in 2K1C hypertensive rats comparing with normal rats. It showed that NPR-A gene expression and the activity of GC-A in hypertensive rats decreased 8 weeks post-operation. For the rats treated with rhANP by implanting encapsulated hANP-producing cells, the NPR-A gene expression level and the activity of GC-A were almost normal. At the same time, the increase of blood pressure was blunted, the damages of heart and kindney caused by hypertension was also alleviated. Conclusion:1. During the hypertrophy of myocardial cells caused by mechanical stretch, the expression of NPR-A gene and GC-A activity increased ,followed by decresing. the expression of NPR-A gene and GC-A activity are reversely related with cardiomyocytes hypertrophy.It suggested that mechanical stretch can regulate expression of NPR-A gene and GC-A activity in time-dependent manner. 2. Expression of NPR-A protein and GC-A activity can be regulated by administering endogenous ANP chronically. This provides a theoretical basis for treatment of hypertension.