Establishment Of Nuclear Envelope Associated Nucleoside Triphosphatase Activity Assay Method And Its Effect On The Restenosis

Objective: To establish a method to assay the nuclear envelope associated nucleoside triphosphatase (NTPase, EC 3.6.1.15) activity, and investigate the change of activity of NTPase in arteries during restenosis. Determined the relationship among the change of NTPase activity and the change of metabolism and proliferation of vascular smooth muscle cell (VSMC) after de-endothelialization.Methods:1. Establishment of NTPase assay method: (1) Isolated and purified of nuclear envelope of vascular wall from SD rat arteries. Arteries from rats were washed with STM buffer, minced and homogenized, filtered and collected by centrifugation. The pellet was washed once with STM buffer and washed twice with DS buffer and then dissolved in lysis buffer. Protein concentration was determined by Lowry method. (2) The method of NTPase activity assay: The concentration of inorganic phosphate was determined by spectrophotometer at 820nm. A standardization curve for concentration of phosphate was made by different concentration of KH2PO4. The activity of enzyme wasexpressed using nmol Pi. mg-1 Pr. min-1.2. Establishment of restenosis model: SD rats were divided into 5 groups at random (n=6), control group, 3 days after de-endothelialization, 7 days after de-endothelialization, 14 days after de-endothelialization and 21 days after de-endothelialization. The restenosis modal was established by balloon catheter denuding the arterial endothelium. Nuclear envelope was isolated and purified from arteries of rats, and the NTPase activity was determined by established method.3. Western blotting analysis of SM a -actin and osteopontin (OPN) expression at different time after de-endothelialization: The extracts from arteries were separated on 8% SDS-PAGE, and then blotted onto nitrocellulose membrane. The membrane was immunologically stained with and- a -actin or anti-OPN antibody.4. 5'-NT, ADA and SDH activity assay: The extracts from rat arteries were determined the activity of 5'-nucleotidase (5'-NT, EC3.1.3.5), succinate dehydrogenase (SDH, EC 1.3.99.1) and adenosine deaminase (ADA, EC 3.5.4.4) according to the manufacturer's instruction.Results:1.Establishment of NTPase activity assay: NTPase could hydrolyze nucleoside triphosphate and release inorganic phosphate. The absorbance at 820nm could bedetected by spectrophotometer after addition of acid molybdate solution and ascorbic acid. The NTPase activity was determined by comparing with standardization curve. The results showed that the variability among batchs was 4.7% and variability within batchs was 3.8%-4.72%. These suggested that this method was reliable and reproducible, so it could be used in NTPase activity assay.2.Establishment the restenosis model: Compared with smooth intimae and orderly arranged media VSMC of arteries in the normal rats, pathological changes of injured areas were first found in section of day 3 after de-endothelialization. On day 7 focal VSMC proliferation and disordered medial VSMC nuclei were showed in markedly hyperplastic intimae. On day 21, widespread intimae thickening and lumen stenosising were observed. At the same time, VSMC occupied the superiority in the thickened area. These results indicated that the model of rat vascular restenosis after de-endothelialization had been successfully established.3.The change of NTPase activity in rat arteries: Compared with control group, the activity of NTPase on 3 days after de-endothelialization elevated but had no significance (P>0.05). On day 7 the activity of NTPase was higher than that of the control (P<0.05). On day 14 and day 21, the NTPase activity was about 3 times as high as that of control (P0.05). The results showed that the change ofNTPase activity was associated with intimae thickening (r=0.918,P<0.05).4.Western blotting analysis of SM a -actin and OPN expression at different time after de-endothelialization: (1)Western blotting show...