Calcium adenosine 5' triphosphatase enzyme levels in pancreatic langerhans ıslet cells of experimental diabetic rat models

7. ABSTRACT This study was carried out to determine Ca+2 ATPase activity in pancreatic islet cells of streptozocin (STZ) induced diabetic rats. Wistar rats of both sexes, which were kindly provided by Experimental Surgery Research Center of Çukurova University, were ased in the experriments. In the first step, experimental diabetes was induced. With this purpose, rats were given 65 mg/kg STZ. Blood and urine glucose levels daily water consumption, urinary outputs and changes in body weights measured in control and test groups. Pathological examinations were performed at pathology department of Çukurova University. Blood glucose levels were 104+9 and 407+41 mg/dl in the control and diabetic rats, respectively. Pellet and water consumption were significantly different between both groups. Urinary glucose level was estimated as 0,064+0,07 in the controls where as it increased to 5,6+0,82 mg/day in the diabetic rats. Urinary output also increased in diabetic rats (9,5 ±1,3 ml/day and 17,5 ±1,40 ml/day for the controls and diabetics, respectively). Diabetic rats gained weight less than the controls did. In diabetic rats, histopathological examination revealed reduction in islet cell number, and thickening of basal membrane 4 weeks after administration of STZ. In the second step, pancreatic islets were purified by addition of 7 mg collagenase oud collected under light-microspose. In the third step, adenosin triphosphatase was purified from islets cells. Microsomal fraction was isolated by differential centrifugation following which spesific enzyme activity was determined in various fractions such as nucleus, mitochondria, granule microsomes. The highest enzyme activity was retained in the microsomal fraction. For this reason, the conditions for preserving the enzyme kinetics were investigated in microsomal fraction. To determine the optimal conditions for storing the enzyme, isolated islet cells the enzyme purified from islet cells were kept at room temperature, - 4 °C, + 4 °C and - 70 °C for 1 month. Specific activity of the enzyme was measured every week. No activity was found after keeping the samples at room temperature. Enzyme activity was 6,2 fimol Pi/mg protein/hour initially, 5,2 /xmdl Pi/mg protein/hour and 5,6 fimol 72Pi/mg protein/hour later keeping at - 75 and - 20 °C respectively. In the fourth stop in vitro Ca+2 ATPase activity was measured in various concentrations of glucose, calcium, magnesium, EDTA, ATP. Ca-ATPase activity was 5,9+0,34 in glucose lacking-medium. When glucose concentration was raised to 16 mM, Ca ATPase activity fell to 1,1+0,05 ^mol Pi/ml prot./ hours. The highest enzyme activity was found in the presence of 0,15 mMol Ca+2, 6 mMol Mg+2 0,1 mMol EDTA, 3 mM ATP. Furthermore, we also measured blood levels a-tokoferol and malondialdehyde (MDA), an end-product of lipid peroxidation. MDA levels increased and a-tokoferol levels decreased significant in the test group its compared with the control groups. According to our results, enhanced lipid peroxidation may impair membrane structure and integrity. Increase in glucose concentration may alter phospholipid turn-over as weel as fatty acid composition of the membrane and thus inhibit Ca+2 ATPase activity. Key words: Streptezotocin, diabetic rats, pancreas, Ca++ATPase 73