The biliary excretion of intravenously injected ouabain and the diffusion constant of the lateral mobility of hepatocyte plasma membrane proteins were examined in control (saline-treated) and spironolactone-treated Wistar male rats of different ages (4, 14 to 15 and 24 months old). The biliary excretion of ouabain progressively decreased with age in control rats, the first 10-min biliary recovery in 24-month-old animals being one-third that of the youngest rats (4-month-old). The oral administration of spironolactone for 4 days (10 mg per 100 gm body weight on the first day and 20 mg per 100 gm body weight for the successive 3 days) caused a marked increase in the biliary recovery of ouabain in all age groups. Similarly, the average lateral diffusion constant of hepatocyte plasma membrane proteins as measured by fluorescence recovery after photobleaching showed a linear decrease with age, as was previously observed with F-344 rats of both sexes. Markedly and significantly (30 to 40%) higher diffusion constants were observed in rats pretreated with spironolactone for all three age groups, compared with the respective control values of corresponding ages. The parallelism between ouabain excretion and protein diffusion (i.e., a decrease with age and an increase with spironolactone pretreatment) suggests that the lateral mobility of proteins in the hepatocyte plasma membrane is a candidate mechanism for regulating ouabain excretion through the liver into the bile, most probably by regulating the hepatic uptake process for ouabain.

Aging↗

PubMedDate not supplied

The effect of idebenone on the lateral mobility of proteins in hepatocyte plasma membrane of old rats as revealed by fluorescence recovery after photobleaching (FRAP) technique using an endogenous label.

The average lateral diffusion constant of proteins (D) in the cell membrane of hepatocytes has been measured in liver smears by fluorescence recovery after photobleaching (FRAP) based on the so-called peroxide-induced autofluorescence (PIAF) deriving from the oxidation of riboflavin bound to membrane proteins. It has been shown before that D displays a significant negative linear age-correlation. The in vivo effect of idebenone was tested on this parameter. Old (23.7 months at killing) male Fischer 344 rats received 50 mg of the drug per kg body weight per os through gastric tube, suspended in 5% gum arabic solution (Verum I group) or the same dose and form of idebenone completed by 80 mg centrophenoxine per kg body weight (Verum II group) for 35 days. D was measured on a double blind basis in hepatocyte plasma membrane of the Verum or Placebo (5% gum arabic, only) groups. A significant increase of D (29.6%) was induced by idebenone in the Verum I group; nevertheless, even the Placebo group displayed a moderate, but statistically significant increase of D (10.7%). The Verum II group showed a total of 39.6% increase in the value of D. The loss of body weight being characteristic for these animals at the age of around 2 years was between 0.87 and 1.06 g/day, whereas in other experiments higher values (1.77 g/day) were observed in the controls.

Journal Article↗

PubMedDate not supplied

The protease inhibitor leupeptin induces several signs of aging in brain, retina and internal organs of young rats.

The protease inhibitor leupeptin was administered to brain, retina and internal organs of young rats for up to two weeks in order to determine if specifically decreased proteolysis could cause symptoms of cellular aging in a variety of tissues. Electron microscopy showed that leupeptin induced the formation of dense substances with fine morphologies similar to and, in many cases, apparently identical with those of natural lipofuscin from aged tissues. Leupeptin also caused increased immunoreactivity to ubiquitin in cerebellar Purkinje cells and presumed Bergmann glia perikarya of brain tissue as well as in hepatocytes of liver tissue. Both of these effects were found in aged tissues as well. Finally, both leupeptin treatment and normal aging led to the onset of immunoreactivity in Purkinje cells to antibodies to the abnormal tau molecule of paired helical filaments from Alzheimer's disease brain. Together, these results indicate that inhibition of thiol (and possibly some serine) proteases by leupeptin is sufficient to cause obvious morphological manifestations of aging in several tissues, and are thus consistent with the hypothesis that lipofuscinogenesis as well as a build-up of ubiquilinated proteins with age is caused by decreased or defective proteolysis. These effects are likely secondary to the mechanism(s) interfering with proteolysis itself.

Journal Article↗

PubMedDate not supplied

Aging of the liver: facts and theories.

Although most theories of aging assume that cellular functions decline with aging, many intracellular functions in the liver, such as enzyme activities, stay fairly stable in old age. This does not appear to be an antifact caused by in vitro experimental design, since in vivo pharmacokinetic data also demonstrate that most, if not all, biotransformation capacities of the liver remain stable during the aging process, if we take the decline in liver volume with age into account. Thus, many theories to explain the decline in cellular functions during aging appear to be based on erroneous assumptions. The stability of cellular function in old age does not necessarily mean, however, that all cellular functions are identical for young and old organisms. Once unfavorable conditions, such as malnutrition, infection, etc., are involved, the response of the liver is quite different for young and old subjects, demonstrating a more efficient and versatile response in young animal livers in comparison to old livers. Large differences in enzyme activities between young and old organisms appear during stress and especially during recovery from stress. Accordingly, any aging theory needs to explain a potential difference in liver functions (such as response capability) rather than the difference in basal functions. In contrast to rather stable intracellular functions, the uptake function of the hepatocyte surface membranes was found to be progressively decreased with age. This was shown for at least two different types of carrier systems in the surface membranes. Although the decrease of carrier unit number for these substances remains a possible causal factor, we suggest that the decline in hepatic uptake with age is at least partially the result of a gradual decrease in the mobility of surface membrane proteins, which can be shown by the fluorescence recovery after photobleaching (FRAP) technique. Theories of aging need to be elaborated on the basis of unbiased observations on the actual manifestations of cellular aging.

Journal Article↗

PubMedDate not supplied

Effect of leupeptin on the lateral mobility of proteins in the plasma membrane of hepatocytes of C57BL/6 mice: FRAP studies on liver smears.

Four groups of male C57BL/6 mice (ages 2, 3.8, 11.8-12.5 and 24 months) were administered leupeptin at 5 mg/100 g body weight/day via intraperitoneally implanted osmotic minipumps. Both untreated and saline-treated mice served as controls. The time and dose-dependence of the effects of leupeptin on the lateral mobility of proteins in hepatocyte surface membranes were analyzed first in mice treated for 2, 5, 8, 11 and 14 days, respectively, by using the fluorescence recovery after photobleaching (FRAP) technique. The age dependence of the response to this treatment was also studied after 14 days of treatment in mice of various ages. The average lateral diffusion constant (D) and the fractional recovery (FR) were measured, and D x FR calculated. Leupeptin treatment at a daily dose of 5 mg/100g for 2 weeks increased plasma glutamic-pyruvlc transaminase levels 2-fold in all age groups. Leupeptin treatment caused a linear increase of D and a decrease of FR with respect to the duration of the treatment in adult mice. In all age groups about 30% of the membrane proteins became immobile after 14 days of treatment, whereas the still mobile fraction displayed a large increase of D. The values of D x FR in the leupeptin-treated groups were slightly higher than those in the untreated mice until the age of 15 months but decreased after this age. The results are interpreted in terms of the known inhibitory effect of leupeptin on cytoplasmic and lysosomal thiol proteases, causing a general slowing down of the protein turnover and, specifically, of proteins in the hepatocyte membrane.

Journal Article↗