It has been described that ageing induces changes in liver function and structure, such as a decrease in hepatic size, blood flow, metabolism of certain drugs and urea and cholesterol synthesis. The hepatocytes’ capacity for regeneration is also reduced. Proinflamatory molecule production could play an important role during ageing. The present study has investigated whether nitric oxide (NO) and carbon monoxide (CO) release is modified by age in isolated rat hepatocytes, and if these changes are accompanied by modifications in cGMP and lipid hydroperoxides (LPO) levels.
Forty male (n = 8 each group) and fifty-four female young (2 months old) and old (14, 18, 22 and 24 months old) Wistar rats were used. Half of the old female rats were ovariectomized after Equithesin anaesthesia (0.3 ml (100 g)-1 of weight; intraperitoneal) at 12 months of age (n = 6 for each group). After decapitation, hepatocytes were isolated and cultured in RPMI 1640 medium with serum, glutamine, antibiotics and insulin. After 24 h of culture, cells and medium were separately collected for measuring CO and NO release to the medium, and cGMP, phosphatidylcholine (PC) and LPO content of the cells. The results are presented as the mean ± standard error of the mean. Mean comparison was done by Friedman’s analysis of variance followed by a two-tailed Wilcoxon’s test for paired data; a confidence level of 95 % (P < 0.05) was considered significant. The experimental procedures employed in this study are in accordance with the principles and practices of the 1986 Animals Act, published in Spain (RD 223/1988).
Hepatocytes isolated from 14-month-old female rats released significantly higher amounts of NO (nmol (µg protein)-1) than young ones (1.54 ± 0.02 vs. 1.23 ± 0.02; P < 0.001), an increase that was more apparent in males (1.81 ± 0.03 vs. 1.24 ± 0.005; P < 0.001) and ovariectomized females. With age, NO production was further increased, and the increase was even higher in males and ovariectomized females (1.71 ± 0.05, 2.11 ± 0.03 and 2.01 ± 0.03; P < 0.001; females, males and ovariectomized females respectively, at 24 months). CO (nmol ml-1) release was significantly augmented with age (5.334 ± 0.23 vs. 1.92 ± 0.09; 24-month-old male vs. 2-month-old male; P < 0.001), although this increase occurred later in females compared to males and ovariectomized females (2.21 ± 0.05 vs. 5.334 ± 0.23 vs. 5.5 ± 0.5; 24-month-old female vs. 24-month-old male vs. 24-month-old ovariectomized female). As expected, the increase in NO and CO release was accompanied by an increase in intracellular cGMP (fmol (µg protein)-1) content (248 ± 2.1 vs. 46.6 ± 4.1; 24-month-old male vs. 2-month-old male; P < 0.001). Age also increased cellular LPO levels (5.6 ± 0.3 vs. 0.9 ± 0.05; 24-month-old male vs. 2-month-old male; P < 0.001), whereas PC (pmol (mg protein)-1 in 24 h) levels were decreased (5.82 ± 0.8 vs. 317 ± 7.1; 24-month-old male vs. 2-month-old male; P < 0.001).
These data suggest that the harmful effect of age on hepatic function could be partially due to an increase in proinflamatory molecule production, which could induce alterations in membrane lipids.
This work was supported by a grant of C.A.M. 8.5/0062/2001.