- Email: [email protected]
The effect of age on the biliary transport maximum (Tm) of bile salts in rats
Arch. GerontoL Geriatr., 14 (1992) 101-115
101
© 1992 Elsevier Science Publishers B.V. All rights reserved. 0167-4943/92/$05.00
AGG 00427
The effect of age on the biliary transport maximum (Tm) of bile salts in rats Setsuko Kanai, Yuko Sato and Kenichi Kitani Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho. ltabashi-ku, Tokyo, Japan (Received 16 April 1991; revised version received 23 September 1991; accepted 26 September 1991)
Summary Age-related differences in the biliary transport maxima (Tm) values for taurocholate (TC) and tauroursodeoxycholate (TUDC) were examined in female Fischer-344 rats as well as Wistar-derived rats of both sexes. The Tm values for TUDC were more than two times higher than corresponding values for TC in young (3-month-old) rats of both sexes. Tm values for both bile salts tended to decline with age, demonstrating a significant negative correlation between the Tm (#mol/min per g liver) and rat age (months) for all rat groups. The decline in Tm value was, however, dominant in the first year with little significant change after 1 year. The results in the present study coupled with our previous observations for Tm values of sulfobromophthalein (BSP) and conjugated BSP support our hypothesis that the transport capacity for bile canalicular membrane generally declines with age in rats. Bile salts; Biliary transport maxima (Tm); Taurocholate; Tauroursodeoxycholate; Rats; Age
Introduction A number of past studies have reported an age-dependent decrease in various hepatic functions (detoxifying functions in particular) in experimental animals. However, results are very variable depending on species, strain and sex of animals. Accordingly, the generalization of results on the same parameter is often difficult. For example, microsomal monooxygenase functions have been reported to decline drastically in male rats and in certain mouse strains of both sexes, whereas in female rat livers these functions are known to remain very stable (Fujita et al., 1982; Kamataki et al., 1985; for review, see Kitani, 1984, 1986, 1988). Several hypotheses have been proposed as underlying mechanisms for an age-dependent decline in microsomal monooxygenase functions (for review, see Kitani, 1988). However, the Correspondence to: S. Kanai, Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo, Japan.
102 significance of such hypotheses may be quite limited in terms or mechanisnls for cellular aging, if similar changes are not generally found. Alterations in the activities of phase I1 reactions such as glutathione S-transferases and sulfotransferases again appear to be variable depending on the sex and species (Kitahara et al., 1982: Fujita et al., 1985; Galinsky et al., 1986, 1990; Carrillo et al., 1989, 1990, 1991: for review, see Kitani, 1988). Thus, age-related changes in many of both phase I and phase II reactions in rat liver appear to be regulated by humoral factors specific to male rats and do not appear to be a general phenomenon caused by aging per se. Summary of available data so far suggests that these detoxifying functions of the liver by means of biotransformation barely decline with aging per se, which also agrees with recent studies on human livers (James et al., 1982; Boobis and Davis, 1984: Matsubara et al., 1986) as well as non-human primate livers (Sutter et al., 1985: Maloney el al., 1986). These studies all reported very stable liver function during aging. There is another efficient detoxifying system in the liver which is not via biotransformation, namely the hepatobiliary transport system. Through this system, a variety of endo- and exogenous toxic substances are efficiently eliminated from the body (for review, see Klaassen and Watkins, 1984). Classically, the biliary excretory system has been classified into three categories, namely for organic anions, cations and neutral compounds (Schanker, 1968). These pathways were believed to be separate from each other in terms of carrier systems. Among the three, the hepatobiliary transport system for organic anions is believed to handle the greatest number of endogenous substances, such as bilirubin, and exogenous ones, such as indocyanine green, sulfobromophthalein and biligraphin, and many kinds of metabolites of exogenous chemicals and pharmaceuticals (Klaassen and Watkins, 1984). In earlier studies on female rats (De-Leeuw lsrael, 1969) as well as on humans (Thompson and Williams, 1965; Skaunic et al., 1970), it has been suggested that the biliary transport maximum (Tin) of sulfobromophthalein remains unchanged with age. In these studies, however, Tm values were indirectly determined. In contrast, subsequent studies in our laboratory which determined Tm directly have provided substantial evidence that the BSP T m actually declines with age in rats, regardless of the sex or strain of animals (Kitani et al., 1978, 1981, Kanai et al.. 1985). Furthermore, a more recent study indicates that the l"m of BSP conjugated with glutathione also declines with age in both male and female Fischer-344 rats (Kanai et al., 1988). From these studies, it has been suggested that the canalicular membrane functions may decline with age (Kitani, 1988). In order to determine whether or not the declining Tm value for BSP and conjugated BSP is a more general phenomenon, it is desirable to examine changes of T m values during aging for other substances having different systems. Bile salts are endogenous metabolic products from cholesterol in the liver and are efficiently excreted into the bile, providing the primary driving force for a fraction of bile water termed 'bile-salt-dependent bile'. Furthermore, it is well known that many bile salts are reabsorbed in the intestine and taken up by the liver undergoing an efficient entero-hepatic circulation. The mechanisms of the hepatobiliary transport of bile salts are not fully understood. However, it is generally believed that the hepatic uptake and biliary excretory systems are separate. Furthermore, both
103 systems are known to be a saturable and energy requiring process, suggesting that active transport is the most likely mechanism (Boyer, 1980; Erlinger, 1981). In a recent study in our laboratory, it was clearly shown that the hepatic uptake rate of taurocholic acid declines in a linear fashion with age. (Ohta and Kitani, 1990). The Vmax value for taurocholic acid by isolated hepatocytes nearly halved at 24 months of rat age in comparison to the young adult value at 4 months. Biliary Tm values for bile salts are known to be several fold lower than corresponding hepatic uptake maxima values, so that the former is the rate-limiting step for the entire transport system for bile salts through the liver. Although bile salts are anions, carrier systems for bile salts and other anion dyes such as BSP are known to be different (Schanker, 1968; Klaassen and Watkins, 1984). We thought that bile salts would be an ideal substance to test our hypothesis that canalicular membrane functions decline with age. Except for our previous study on taurocholate uptake (Ohta and Kitani, 1990), to our knowledge, there is only one previous report with regard to the hepatobiliary transport function change with age for a bile salt, which indicated that the biliary transport function is more severely affected by aging than the hepatic uptake function for taurocholic acid (Kroker et al., 1980). In the present study, two different bile salts (i.e., taurocholate (TC) and tauroursodeoxycholate (TUDC)) were examined in three different rat groups (Wistar-derived rats of both sexes and female Fischer-344 rats. Unlike TC, T U D C is barely found in the natural rat bile. However, we found previously that this bile salt has a Tm value more than two-fold higher than that of TC in rats of both sexes (Kitani and Kanai, 1981; Kitani et al., 1986). Furthermore, the very low hepatotoxicity of T U D C makes this bile salt an ideal bile salt for assessing the liver function. Materials and Methods Male and female Wistar rats and female Fischer-344 (F-344) rats of various ages (3 to 30 months old) were used. Wistar rats were bred in our aging farm under SPF conditions. F-344 rats were purchased from Japan Charles River Co. (Atsugi) in SPF conditions at the age of 4 weeks. Both strains of rats were raised in the institute's aging farm in SPF conditions until use. The husbandry conditions, survivals and pathologies in the later period of their lives have been reported elsewhere (Nokubo, 1985; Tokyo Metropolitan Inst Gerontol., 1989). Tm values for two different bile salts, TC and TUDC, were examined in each group of animals by the method previously reported in detail (Kitani and Kanai, 1981; Kitani et al., 1986). In brief, under pentobarbital anesthesia (Nembutal, Abbott, North Chicago, IL), the common bile duct was cannulated with PE-50 (for males) or PE-10 (for females) tubing (Clay-Adams, Parsippany, N J). Basal bile was collected for 10-20 min. Thereafter, one of the two bile salt solutions was continuously infused through a jugular vein catheter by a Harvard Pump (Harvard Apparatus, Millis, IL). Infusion rates varied depending on the sex and age of animals, but generally, the infusion rate was raised stepwise every 20 min until the bile flow rate began to decline. Bile was collected at 10-min intervals in stock tubes and weighed. Bile samples were frozen and kept at -70°C until the determination of the bile salt concentration in the bile. Chemical purities and sources of bile salts used have
104
been described previously (Kitani et al., 1986). The bile salt solutions were prepared by dissolving sodium salts of these bile salts in 3'7,, bovine serum albumin solution, and the pH (7.4) and osmolality (300 mOsm/kg) were adjusted to physiological levels, as reported previously (Kitani and Kanai, 1981; Kitani et al., 1986). During experiments, the body temperature was continuously monitored by a rectal probe and maintained at 37-37.5°C by a heating lamp. The total bile salt concentration in the bile was determined enzymatically using 3c~-hydroxysteroid dehydrogenase (Worthinton Biochem., Freehold, N J). The total biliary excretion rate of a bile salt was determined as the product of the total bile salt concentration and bile volume, the latter being estimated gravimetrically by assuming that the specific gravity of bile is 1.0. The Tm value of each animal was determined as an average of the three highest excretion rates which were obtained usually just before the drop of bile flow and bile salt excretion rate (see Figs. 1 and 2). The significance of the difference in values among rat groups of different ages was analyzed by means of a one-way analysis of variance (ANOVA). When the difference was found to be significant with respect to the animal's age, Scheffe's test was applied to evaluate the significance of the difference between values of any set of Tm values of two different age groups. Linear regression analysis was also applied to evaluate the age dependency of the value. P values lower than 0.05 were judged to be significant. Results
Table I summarizes the bile flow and total bile salt excretion rate in the basal bile before bile salt infusion. The bile flow as well as total bile salt excretion rate did not show any significant difference among different age groups (ANOVA). Figures 1 and 2 show representative Tm studies in female F-344 rats of two different ages for TC (Fig. 1) and T U D C (Fig. 2). In all studies, the bile flow and bile salt excetion rate began to increase immediately after the start of bile salt infusions.
3-month-old
29-month-old
Infusion rate
0.2
0.6
pmol/min/lOOg b.wt. 1.0 - 1.2
0,8
]
- - ]1.4
0.2
0.4
0.6
0.8
,,,.;
g
1.0
10
0
30 Time,
60
90
120
0
30
60
90
rain
Fig. 1, Representative T m studies for taurocholate in young and old female Fischer-344 rats.
105 3-month-old Infusion rate L 1.2 1.6 , 2.0
pmol/min/lOOg b.wt, 2.4
2.6
2.8
3.0
3.2
3.4]
3.0 J~
29-month-old
0
~o..q --
2.0
..o~"°"e"°"
®
•~_
t._._.,y"
1.o
E
H,, 0
30 Time.
60 rain
90
120
150
180
0
, 30
60
,1i 90
120
Fig. 2. Representative T m studies for tauroursodeoxycholate in young and old female Fischer-344 rats.
With the increase in the infusion rate, the bile flow and bile salt excretion rate reached a plateau and thereafter, they began to decline. Figures 3-5 show the distribution of Tm values in rats of different groups for TC and TUDC. Table II summarizes Tm values as well as bile flow rate and bile salt concentration in the bile during the Tm period in rats of different ages. In general, Tm values expressed per gram liver weight were 2- to 3-fold higher for T U D C than that of TC in young rat groups. Tm values at young age in female Wistar rats tended to be higher than corresponding male rat values in most studies. It is also noted in Table II that the bile salt concentration in the bile during Tm period also generally decreased with age of animals. Tm values were significantly different with respect to rat age as analyzed by one-way ANOVA. The Tm values in older rat groups were significantly lower than respective Tm values in the youngest rat groups (Scheffe's test, P < 0.05) and Tm values tended to decrease with rat age. However, in most rat groups, the decline in Tm values was most pronounced in the first year of rat life and after 12 months, the changes with age were negligible. In fact, the difference in Trn values between 12- to 13- and 24-month-old rats were all insignificant, as analyzed by Scheffe's test, except for the group of Wistar females given TC infusion which showed an intermediate Tm value in the middle-aged rats. Furthermore, in the oldest group for each study, Tm values of several rats were considerably higher than the average values for the group next to the oldest (Figs. 3-5). Accordingly, the average Tm values in the oldest group were somewhat higher than those for the groups next to the oldest in most studies. Regression parameters obtained from t h e linear-regression analysis of the relation between the Tm value and rat age are shown in Table III. A significant negative correlation existed between the Tm value
(3) (6) (13-13.5) (24) (29)
Y-I Y-2 M O-1 0-2
O
(3) (12-13) (25)
Y M O
6 6 7
6.1
4. 5.96 ± 9.15" ± 16.0" ± 38.5* ± 30.6*
± 8.8* ± 16.5" ± 27.8* ± 23.6*
6.4
174.8 ± 9.5 239.2 ± 28.9* 300.0 + 18.0"
175.8 ± 9.6 218.8 ± 11.1" 3 1 4 . 0 ± 46.0*
519.3 ± 38.7* 4 7 8 . 5 ± 27.9*
291.9 4.
290.5 ± 3.94 510.0 ± 15.8" 443.7 ± 54.7*
175.5 207.5 247.6 295.0 322.0
206.4 248.8 287.9 281.7
175.0 ±
4. 4. ± ± ±
4. ± + ± 0.23 0.67* 0.59* 0.68* 1.73'
0.25* 0.62* 0.54* 0.40*
5.03 ± 0.28 7.32 ± 0.68* 8.40 ± 0.48*
4.97 ± 0.49 7.00 ± 0.88* 9.42 ± 1.54"
15.95 ± 1.13" 16.84 ± 1.95"
9.16 ± 0.64
9.56 ± 1.19 16.18 4- 2 . 1 3 " 15.53 ± 1.83"
5.06 6.15 6.76 8.15 10.42
5.90 6.78 7.63 9.02
4.93 + 0.34
L i v e r wt.
(g)
B o d y wt.
(g)
a N u m b e r in p a r e n t h e s i s i n d i c a t e s the a g e in m o n t h o f e a c h g r o u p . *Significantly different f r o m c o r r e s p o n d i n g v a l u e s in the y o u n g e s t g r o u p ( P < 0.05). Y, y o u n g ; M, m i d d l e - a g e d ; O, old.
(3) (13) (25-26)
Y M O
5 4 5
7 6
Female Wistar
10
(3)
6 4 6
6 5 5 6 5
5 4 7 3
7
(12) (24)
(3) (13) (25-26.5)
n
f
Y M 9
Male Wistar
(6) (12) (24) (29)
(3) a
Y-2 M O-1 0-2
Y-I
Female F-344
Rat groups
Bile flow a n d bile salt e x c r e t i o n r a t e o f ' b a s a l bile' in r a t s o f d i f f e r e n t a g e s
TABLE 1
± ± ± +
0.42 0.13 0.40 0.10
0.38 0.29 0.19 0.40 0.18
2.33 ± 0.39 2.12 ± 0.23 1.99 + 0.14
2.16 + 0.67 1.90 ± 0.06 1.63 ± 0.17
2.23 ± 0.33 1.56 4. 0.18 1.54 4- 0.22
2.04 ± 0.21 1.52 ± 0.13 1.52 ± 0.17
1.76 ± 0.54
2.08 1.94 1.96 1.84
+ ± ± ± + ± + + ± +
+ ± ± + ± 0.024 0.005 0.019 0.005 0.013
0.019 0.021 0.015 0.021 0.007
0.065 ± 0.011 0.079 + 0.012 0.064 + 0.022
0.083 + 0.02 0 . 0 6 9 + 0.01 0.045 ± 0.02
0.050 ± 0.006 0.056 ± 0.025
0.068 ± 0.025
0.048 ± 0.017 0.043 ± 0.015 0 . 0 5 6 + 0.025
0.068 0.048 0.072 0.056 0.068
0.066 0.071 0.077 0.076 0.064
(/~mol/min/g)
(tA/min/g)
2.26 2.09 2.00 1.94 1.90
Basal bile salt excretion rate
Basal bile flow rate
TUDC TUDC TUDC
TC TC TC
TUDC TUDC
TUDC
TC TC TC
TUDC TU DC TUDC TUDC TUDC
TC TC TC TC TC
Infusion
107
10)r°u's°de°x'ch°''t' 0.8 0 . 6 ~
Taurocholate
•
0,4
i
|
•
•
E
0.2
0.2
"~
r= -0.66 i
i
i
Age 3 6 (months)
i
i
i
12
24
30
r= -0.79 *
i
3 6
i
i
i
12
24
30
Fig. 3. Scattergram of bile salt Tm values in female Fischer-344 rats of different ages.
and the age of animals for all rat groups. Rates of decrease in Tm values with age (% per month) calculated from the linear regression analysis are also shown in Table III. The rate of decrease ranged from 1.3% to 3.0% per month for all rat groups.
Discussion The results of the present study revealed that, first of all the basal bile flow as well as total bile salt excretion rate stayed fairly stable with aging in all three groups of rats (male and female Wistar, and female F-344 rats) (Table I). These observations essentially agree with our previous observations in rats of different strains and sexes (Kitani et al., 1978, 198 l; Kanai et al., 1985). Furthermore, regardless of the bile salt species tested and sexes of animals, there was a general tendency of decline in bile salt Tm with age in rats when the parameter was expressed per gram liver tissue. Hardison et al. (1981) suggested that secretory maxima values for bile salts as defined here as 'T m' is a compromise between their hepatocellular toxicity and the capacity of the transport system (a true Tm by the definition of Hardison et al. (1981)), and that practically the secretory maximum (T m in the present manuscript) is determined by the degree of bile salt cytotoxicity (Hardison et al., 1981). However, his thesis is probably an overemphasis on the role of membrane toxicity of a bile salt, since we subsequently found that the Tm of TUDC which is least cytotoxic among
108
Tauroursodeoxycholate
1.0
| |
0.8
0.6
Taurocholate
.o
0.4
0.4
.E
E "6 E "! 0.2
0.2
|
....,
••
r: -0.50
r: -0.80 i
Age 0 3 (months)-
=
i
12
24
0 3
12
24
Fig. 4. Scattergram of bile salt Tm values in male Wistar rats of different ages.
known bile salts was much lower than those for TC and even taurochenodeoxcholate (a far more toxic bile salt than TUDC) in hamsters (Kitani et al., 1986). Especially in the case of TUDC, the cytotoxicity may not play any significant role, since this bile salt was found to be even cytoprotective rather than cytotoxic (Kitani and Kanai, 1982; Kitani et al., 1985). Thus, the age-dependent decline for the Tm of T U D C shown in this study can be interpreted rather straightforwardly as due primarily to changes in membrane carrier with age as will be discussed later. In the case of TC Tin, we may need to consider the vulnerability of bile canalicular membrane to a bile salt toxicity in interpreting the data. However, as is evident in Figs. 1 and 2, much smaller amounts of bile salts were infused in older animals in all studies. Thus, a bile salt load to hepatocytes and eventually to bile canalicular membranes, is considerably lower in older animals than young ones. Furthermore, the bite salt concentrations in the bile during the Tm period were all consistently lower in older animals, suggesting that the lower Tm values in older animals are not due to a higher bile salt loading to hepatocytes and eventually to canalicular membranes in our experimental design. It remains unknown whether the age increases the membrane vulnerability to TC. If this is a
109 Tauroursodeoxycholate o
1.2
1.0
0.8
." 0.61 ee
]¢ "=
Taurocholate r: -0.70
0.4
0.2
Age 3 (months)
12
24
i
i
i
3
12
24
Fig. 5. Scattergram of bile salt Tm values in female Wistar rats of different ages.
contributing factor for our observation, however, a sharper decline in Tm value may be expected to occur with TC than with TUDC, which was not the case at least for female Fischer rats and Wistar male rats. Furthermore, Abernathy et al. (1980), showed a more resistant membrane quality to cytotoxycities of erythromycin and chlorpromazine in aged rat hepatocytes. Based on the above considerations, we interprete that the decline of Tm values with age is primarily due to the quantitative (and/or) qualitative alteration of membrane carriers for bile salt transport into bile canaliculi. Significantly higher Tm values for T U D C than TC in young rats are consistent with our previous results in male and female Wistar-derived rats (Kitani and Kanai, 1981; Kitani et al., 1986). This tendency was maintained until very old age of animals, although the difference in Tm values between the two bile salts became smaller as the age advanced. Although there are various hypotheses concerning the greater Tm value for T U D C (Hardison et al., 1981), the true reason for this difference remains unknown• The transport systems (carrier proteins, in particular) for TC and T U D C may possibly be different, since when these two bile salts are
6 4 6
10 7 6
Male Wistar Y (3) M (13) O (25-26.5)
Y M O
(3) (12-131 (25)
Y M O
6 6 6
TUDC TUDC TUDC
TC TC TC
TUDC TUDC TUDC
TC TC TC
TUDC TUDC TUDC TUDC TUDC
TC TC TC TC TC
Infusion
± 44± ±
± 4± ± ± 0,83 0.38* 0.52 0.48* 0.91"
0.46 0.25 0.27* 0.57* 0.23
6.96 ± 0,67 4,49 + 0.37* 4,97 ± 0,67*
3.66 ± 0.28 2.89 ± 0.16" 2.50 ± 0.28*
6.19 ± 1,38 2.74 4- 0.22* 3.27 ± 0,61"
4.07 ± 0.38 1.92 ± 0.11" 2.88 ± 0.43*
6,82 5.16 4.05 4,12 3.31
4,13 3.45 2.74 3.03 3.16
Bile flow rate (/~l/min per g)
aNumber in parenthesis indicates the age in m o n t h of each group, *Significantly different from c o r r e s p o n d i n g values in the y o u n g e s t g r o u p ( P < 0.05). Y, young; M, middle-aged; O, old.
(3) (13) (25-26)
Y M O
Female Wistar
5 4 5
6 5 5 6 5
(3} (6) (13-13,51 (24) (29)
Y-I Y-2 M O-1 0-2
(3) (12) (24)
7 5 4 7 3
n
Female F-344 Y-I (3) a Y-2 (6) M (121 O-1 (24) 0-2 (29)
Rat group
6.33 5.93 4,40 5.45 2,39
6.73 2.06* 6.93* 162.73 ± 8,1l* 131.72 ± 7.09* 128.95 ± 21.13
93.40 ± 74.44 ± 56.76 ±
160.08 ± 6,67 124.61 ± 16.43" 122.99 ± 8.85*
3.45 2.70 9.00
± 5.65 ± 7.01 ± 7.30 ± 6,57* ± 11.72"
± ± 44±
72.59 ± 72.43 ± 69.60 ±
128,57 120.68 117.28 113.43 105.10
80.51 78.62 73.27 70.65 71.91
Bile salt cone. in the bile ( m M )
Bile flow, bile salt c o n c e n t r a t i o n and e x c r e t i o n rate at the T m p e r i o d in rats o f different ages
T A B L E II
44± ± 4-
± ± ± ± ± 0,108 0,025* 0,072* 0,068* 0.128"
0.046 0.030 0.022* 0.039* 0.020*
1.13(I ± 0.099 0.590 ± 0.050* 0,646 ± 0,149"
0.342 ± 0.045 0.215 ± 0.015" 0.141 ± 0.020*
0.944 ± 0.155 0.340 ± 0.036* 0.398 ± 0.084*
0.296 ± 0.030 0.139 ± 0.012" 0.204 ± 0.057*
0.876 0.621 0,476 0.469 0.36(/
0.331 0.272 0.204 0,213 0.226
Bile salt excretion rate (Tnl) ( # m o l / m i n per g)
TC TUDC TC TUDC TC TUDC
Female F-344
× x × × × ×
10 -3 10 -2 10 -3 10 -2 10 -3 10 -2
0.311 0.797 0.273 0.954 0.356 1.059
(ttmol/min per g)
(/~mol/min per g per month)
-4.0 -1.5 -3.58 -2.9 -8.75 -1.98
ba
aa
trrhe rate of decrease of Tm with age (% per month).
ay = a x 4- b, (x, rat age in month; y, Trn value in ttmol/min per g liver).
Female Wistar
Male Wistar
Bile salts
Rat groups
Regression coefficients for the relationship between bile salt Trn value and rat age
TABLE III
1.3 1.9 1.3 3.0 2.4 1.8
(% per month)
a/b b
26 27 16 21 14 19
-0.664 -0.790 -0.496 -0.804 -0.935 -0.700
<0.005 <0.005 <0.05 < 0.0005 < 0.0005 < 0.0005
112
simultaneously infused, there is no competitive inhibition in their biliary transport process (Kitani and Kanai, 1982; Kitani et al., 1985). Instead of inhibition, TUDC tended to facilitate the transport of TC. This circumstantial evidence favors the view that the transport system for these two bile salts may not be the same. Despite the possible difference in carrier proteins for these two different bile salts tested, the manner of the age-dependent decline of Tm values was quite similar for these two bile salts. Furthermore, the declining tendency with age for the T m value of these bile salts is similar to that in our previous studies on Tm values for BSP (Kitani et al., 1978, 1981; Kanai et al., 1985) and its gluthathione conjugate (Kanai et al., 1988). Although the possible involvement of membrane vulnerability is not excluded in a rigorous sense especially for strong toxins such as TC as discussed above, such a decline in Tm values with age is usually explained as due to the age-dependent decrease in the carrier unit number for individual materials. Transport systems (more specifically, carrier proteins and energy for a driving force) for bile salts and the above cholephilic dyes are believed to be different from each other (Schanker, 1968; Klaassen and Watkins, 1984). Despite these possible differences in carrier proteins and driving forces for the biliary transport of these materials, the declining tendency for Tm values with age was very similar. These results suggest that there might be some common factor(s) other than the decrease in carrier unit number for individual substances, which regulates in general the declining transport functions through the bile canalicular membrane with age. As one of the candidates for such factor(s), we suggested the declining mobility of membrane proteins in the bile canaliculus (Kanai et al., 1988). Although no direct proof for such a mechanism is at present available, our recent studies using the fluorescence recovery after photobleaching (FRAP) technique have shown that the lateral mobility (average lateral diffusion constant) of proteins in the surface membrane of hepatocytes (predominantly of sinusoidal domain) declines almost linearly with age in both male and female rats of different strains (F-344 and Wistar-derived rats) (Zs.-Nagy et al., 1986; Kitani et al., 1988) and mice (Zs.-Nagy et al., 1989). Furthermore, we have shown a striking linear relationship between the diffusion constant of proteins and hepatic uptake rates of ouabain (Ohta et al., 1988) and more recently of TC (Ohta and Kitani, 1990). From these studies, we proposed that the decreasing membrane protein mobility with age may be a significant factor regulating the surface membrane functions mediated by membrane proteins (Ohta and Kitani, 1990: Kitani et al., 1991). Because the membrane surface area of the canalicular domain is very small relative to that of the sinusoidal domain of hepatocytes, our FRAP technique cannot be directly applied to assess the physico-chemical properties of canalicular membrane proteins of rat hepatocytes. Despite these difficulties, future studies are expected to test our hypothesis wherein the plasma membrane protein mobility also declines in the canalicular domain, as has been shown for sinusoidal membranes in hepatocytes, and that it is (at least partially) regulating the transport of materials through canalicular membranes. Rates of decrease per month in TC uptake (2.1%) (Ohta and Kitani, 1990), ouabain uptake (2.4%) (Ohta et al., 1989) and initial 10-min excretion of ouabain (2.2-2.8%) (Sato et al., 1987; Ohta et al., 1989: Kitani et al., 1991) are very close to
113 values observed for the decline of Tm values with age in our present study. Previously found values are thought to reflect the function of sinusoidal membranes (Sato et al., 1987; Ohta et al., 1989; Ohta and Kitani, 1990; Kitani et al., 1991), while values found in the present study reflect that of canalicular membranes. The striking similarities for these values may be only a coincidence, but it is also possible that it is the reflection of some common causal mechanism as suggested above. Except for the group of female Wistar rats given TC infusion which showed an age-dependent gradual decline in Tm value, most other groups showed almost comparable values for middle-aged (12-month-old) and old (24-month-old or older) rat groups. This may suggest that the Tm value declines relatively early in the life of animals. Furthermore, unusually high Tm values close to those in the youngest rat groups were observed in some rats in the oldest groups (e.g., TC Tm in male Wistar rats). In general, the scattering of each Tm value was narrower in the middle-aged rats than in the old rats. Similar phenomena have been observed in previous studies for many different kinds of functional parameters of the liver (van Bezooijen et al., 1977; Kitani et al., 1981; Kanai et al., 1985; Zs.-Nagy et al., 1986; Sato et al., 1987; Ohta et al., 1988; Ohta and Kitani, 1990). Since Tm values for bile salts were presented per unit liver weight, it is unlikely that higher Tm values in some of the oldest rats compared with those of middleaged rats were due to an enlargement of the liver in old age. As a possible explanation for such phenomena, two hypotheses have been proposed (Sato et al., 1987). One is an actual increase in liver functions with advancing age of animals possibly as a compensatory response to other changes occurring in the late life of an animal. For example, the increase in the albumin synthesis rate in rat liver in old age was explained to be a response to the enhanced urinary loss of albumin in old age (van Bezooijen et al., 1977). The other possibility is that this is due to the survival of animals that have maintained exceptionally good liver functions up to their old age (i.e., survival selection). In previous studies, the biliary excretion of i.v. injected ouabain tended to be higher in the oldest rat groups (29-31-months-old) than in the second-oldest group (Sato et al., 1987; Ohta et al., 1988). Thus, the latter possibility (i.e., selection by survival of animals) could not be excluded in these studies. In the present study, the reversed difference (i.e., higher Tm values in old groups) was observed between middle-aged (12-month-old) and old (24-month-old) rats. Since survival rates of these rats are all more than 90% at 24 months of age (Nokubo, 1985; Tokyo Metropolitan Inst. Gerontol., 1989), it is difficult to explain such a high value in old rat groups as due to survival selection. Thus, the former possibility that livers of some old rats become hyperactive must be considered, although not directly proven. Only future studies in a longitudinal scheme can directly answer this question. In summary, the Tm values for both TC and T U D C tended to decline in both male and female rats, suggesting that some common mechanism may underlie the age-induced declines of biliary Tm for bile salt, BSP and conjugated BSP as demonstrated in our previous studies (Kanai et al., 1985, 1988).
Acknowledgement The skillful secretarial assistance of Ms. K. Tagami is gratefully acknowledged.
114
References Abernathy, C.O., Lukacs, L. and Zimmerman, H.J. (1980): Cytotoxic effects oferythromycin estolate and chlorpromazine on hepatocytes isolated from rats of varying ages: A brief note. Mech. Ageing Dev., 12, 1-6. Boobis, A.R. and Davis, D.S. ( 1984): Human cytochromes P-450. Xenobiotica, 14, 151-185. Boyer, J.L. (1980): New concepts of mechanisms of hepatocyte bile formation. Physiol. Rev, 60, 303-326. Carrillo, M.-C., Kitani, K., Kanai, S., Sato, Y., Nokubo, M., Ohta, M. and Otsubo, K. (1989): Differences in the influence of diet on hepatic glutathione S-transferase activity and glutathione content between young and old C57 black female mice. Mech. Ageing Dev., 47, 1-15. Carrillo, M.-C., Nokubo, M., Sato, Y., Kanai, S., Ohta, M. and Kitani, K. (1990): Effect of protein-free diet on activities and subunits of glutathione S-transferase in livers of young and aged female rats. Mech. Ageing Dev., 56, 237-251. Carrillo, M.-C., Nokubo, M., Kitani, K., Sato, K. and Satoh, K. (1991): Age-related alterations of subunits of hepatic glutathione S-transferases in male and female F-344 rats. Biochim. Biophys. Acta, 1077, 325-331. Erlinger, S. (1981): Hepatocyte bile secretion Current views and controversies. Hepatology, 1, 352-359. De Leeuw-lsrael, F.R., Hollander, C.F. and Arp-Neefjes, J.M. (1969): Hepatic storage and maximal biliary excretion of bromsulphalein (BSP) in young and old rats. J. Gerontol.. 24, 140-142. Fujita, S., Uesugi, T., Kitakawa, H., Suzuki, T. and Kitani, K. (1982): Hepatic microsomal monooxygenase and azoreductase activities in aging Fischer-344 rats. Importance of sex difference for aging study. In: Liver and Aging .- 1982, Liver and Drugs, pp. 55-71. Editor: K. Kitani. Elsevier/NorthHolland Biomedical Press, Amsterdam. Fujita, S., Kitagawa, H., Ishizawa, H., Suzuki, T. and Kitani, K. (1985): Age associated alterations in hepatic glutathione-S-transferase activities. Biochem. Pharmacol., 34, 3891-3894. Galinsky, R.E., Kane, R.E., Franklin and M.R. (1986): Effect of aging on drug metabolizing enzymes important in acetaminophen elimination. J. Pharmacol. Exp. Ther., 237, 107-113. Galinsky, R.E., Johnson, D.H., Kane, R.E. and Franklin, M.R. (1990): Effect on aging on hepatic biotransformation in female Fischer 344 rats: Changes in sulfotransferases activities are consistent with known gender-related changes in pituitary growth hormone secretion in aging animals. J. Pharmacol. Exp. Ther., 255, 577-583. Hardison, W.G.M., Hatoff, D.E., Miyai, K. and Weiner, R.G. (1981): Nature of bile acid maximum secretory rate in the rat. Am. J. Physiol., 241, (Gastrointest. Liver Physiol. 4) G337-G344. James, O.F.W., Rawlines, M.D. and Woodhouse, K. (1982): Lack of ageing effect on human microsomal monooxygenase activities and on inactivation pathways for reactive metabolic intermediates. In: Liver and Aging 1982, Liver and Drugs, pp. 395-408. Editor: K. Kitani. Elsevier/North Holland, Amsterdam. Kamataki, T., Maeda, K., Shimada, M., Kitani, K., Nagai, T. and Kato, R. (1985): Age-related alteration in the activities of drug-metabolizing enzymes and contents of sex-specific forms of cytochrome P-450 in liver microsomes from male and female rats. J. Pharmacol. Exp. Ther., 233, 222-228. Kanai, S., Kitani, K., Fujita, S. and Kitagawa, H. (1985): The hepatic handling of sulfobromophthalein in aging Fischer-344 rats: in vivo and in vitro studies. Arch. Gerontol. Geriatr., 4, 73-85. Kanai, S., Kitani, K., Sato, Y. and Nokubo, M. (1988): The age-dependent decline in the biliary transport maximum of conjugated sulfobromophtbalein in the rat. Arch. Gerontol. Geriatr., 7, 1-8. Kitahara, A., Ebina, T., Ishikawa, T., Soma, Y., Sato, Y. and Kanai, S. (1982): Changes in activities and molecular forms of rat hepatic drug-metabolizing enzymes during aging. In: Liver and Aging 1982, Liver and Drugs, pp. 135-142. Editor: K. Kitani. Elsevier/North Holland Biomedical Press, Amsterdam. Kitani, K. (1984): The effect of age on hepatic metabolism of xenobiotics. In: Foreign Compound Metabolism, pp. 275-287. Editors: J. Caldwell and G. Paulson. Taylor and Francis, London. Kitani, K. (1986): Hepatic drug metabolism in the elderly. Hepatology, 6, 316-319. Kitani, K. (1988): Drugs and the ageing liver. Life Chem. Rep., 6, 143-230. Kitani, K. and Kanai, S. (1981): Biliary transport maximum of tauroursodeoxycholate is twice as high as that of taurocholate in the rat. Life Sci., 29, 269-275.
115 Kitani, K., and Kanai, S. (1982): Tauroursodeoxycholate prevents taurocholate induced cholestasis in the rat. Life Sci., 30, 515-523. Kitani, K., Kanai, S. and Miura, A. (1978): Hepatic metabolism of sulfobromophthalein (BSP) and indocyanine green (ICG) in aging rats. In: Liver and Aging - - 1978, pp. 145-156. Editor: K. Kitani. Elsevier-North Holland, Amsterdam. Kitani, K., Zurcher, C. and Van Bezooijen, C.F.A. (1981): The effect of aging on the hepatic metabolism of sulfobromophthalein in BN/Bi female and Wag/Rij male and female rats. Mech. Ageing Dev., 7, 381-393. Kitani, K., Ohta, M. and Kanai, S. (1985): Tauroursodeoxycholate prevents biliary protein excretion induced by other bile salts in the rat. Am. J Physiol., 248, G407-C417. Kitani, K., Kanai, S., Ohta, M. and Sato, Y. (1986): Differing transport maxima values for taurine conjugated bile salts in rats and hamsters. Am. J. Physiol. 251, G852-G858. Kitani, K., Zs.-Nagy, I., Kanai, S., Sato, Y. and Ohta, M. (1988): Correlation between the biliary excretion of ouabain and the lateral mobility of hepatocyte membrane proteins in the rat. The effects of age and spironolactone pretreatment. Hepatology, 8, 125-131. Kitani, K., Ohta, M., Kanai, S., Sato, Y., Nokubo, M., and Zs.-Nagy, i. (1991): Age-induced restricted mobility of proteins in hepatocyte surface membranes: A possible determinant of membrane transport function. In: Liver and Aging - - 1990, pp. 305-318. Editor: K. Kitani. Elsevier Science Publishers, B.V., Amsterdam. Klaassen, C.D. and Watkins, J.B. 3rd. (1984): Mechanisms of bile formation, hepatic uptake and biliary excretion. Pharmacol. Rev., 36, 1-67. Kroker, R., Hegner, D. and Anwer, M.S. (1980): Altered hepatobiliary transport of taurocholic acid in aged rats. Mech. Ageing Dev., 12, 367-373. Maloney, A., Schmucker, D.L., Vessey, D.S. and Wang, R.K. (1986): The effects of aging on the hepatic microsomal mixed-function oxidase system of male and female monkeys. Hepatology, 6, 282-287. Matsubara, T., Yamada, N., Mitomi, T., Yokoyama, S., Fukiishi, Y., Hasegawa, Y. and Nishimura, H. (1986): Cytochrome P-450-dependent monooxygenase activities in prenatal and postnatal human livers: Comparison of human liver 7-alkoxycoumarin O-deethylases with rat liver enzymes. Jpn. J. Pharmacol., 40, 389-398. Nokubo, M. (1985): Physical-chemical and biochemical differences in liver plasma membranes in aging F-344 rats. J. Gerontol., 40, 409-414. Ohta, M., Kanai, S., Sato, Y. and Kitani, K. (1988): Age-dependent decrease in the hepatic uptake and biliary excretion of ouabain in rats. Biochem. Pharmacol., 37, 935-942. Ohta, M., and Kitani, K.(1990): Age-dependent decrease in the hepatic uptake of taurocholic acid resembles that for ouabain. Biochem. Pharmacol., 39, 1223-1228. Sato, Y., Kanai, S., and Kitani, K. (1987): Biliary excretion of ouabain in aging male and female F-344 rats. Arch. Gerontol. Geriatr., 6, 141-152. Schanker, L.S. (1968): Secretion of organic compounds. In: Handbook of Physiology, Section 6, Vol. 5, pp. 2433-2449. Am. Physiol. Soc., Washington D.C. Skaunic, V., Nerad, V. and Fedrichova, M. (1970): Mechanismus poklesu relativni stradaci schopnosti jater pro bromsulftalein s rostoucim vekem II Vyznam snizeneho jaterniho prutoku krve. Cs Gastroenterologie, 24, 206-215. Sutter, M.A., Gibson, G., Williamson, L.S., Strong, R., Pickham, K. and Richardson, A. (1985): Comparison of the hepatic mixed function oxidase systems of young, adult and old non-human primates (Macaca nemestrina). Biochem. Pharmacol., 34, 2983-2987. Thompson, E.N. and Williams, R. (1965): Effect of age on liver function with particular reference to bromsulphalein excretion. Gut, 6, 266-269. Tokyo Metropolitan Institute of Gerontology (1989): Data book for aging farm 11. Pathology and Blood Chemistry 1989. Van Bezooijen, C.F.A., Gvell, T. and Knook, D.L. (1977): The effect of age on protein synthesis by isolated liver parenchymal cells. Mech. Ageing Dev., 6, 293-304. Zs.-Nagy, I., Kitani, K., Ohta, M. and lmahori, K. (1986): Age-dependent decrease of the lateral diffusion constant of proteins in the plasma membrane of hepatocytes as revealed by fluorescence recovery after photobleaching in tissue smears. Arch. Gerontol. Geriatr., 5, 131-146. Zs.-Nagy, 1., Kitani, K. and Ohta, M. (1989): Age-dependence of the lateral mobility of proteins in the plasma membrane of hepatocytes of C57BL/6 mice: FRAP studies on liver smears. J. Gerontol., 44, B83-87.
101
© 1992 Elsevier Science Publishers B.V. All rights reserved. 0167-4943/92/$05.00
AGG 00427
The effect of age on the biliary transport maximum (Tm) of bile salts in rats Setsuko Kanai, Yuko Sato and Kenichi Kitani Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho. ltabashi-ku, Tokyo, Japan (Received 16 April 1991; revised version received 23 September 1991; accepted 26 September 1991)
Summary Age-related differences in the biliary transport maxima (Tm) values for taurocholate (TC) and tauroursodeoxycholate (TUDC) were examined in female Fischer-344 rats as well as Wistar-derived rats of both sexes. The Tm values for TUDC were more than two times higher than corresponding values for TC in young (3-month-old) rats of both sexes. Tm values for both bile salts tended to decline with age, demonstrating a significant negative correlation between the Tm (#mol/min per g liver) and rat age (months) for all rat groups. The decline in Tm value was, however, dominant in the first year with little significant change after 1 year. The results in the present study coupled with our previous observations for Tm values of sulfobromophthalein (BSP) and conjugated BSP support our hypothesis that the transport capacity for bile canalicular membrane generally declines with age in rats. Bile salts; Biliary transport maxima (Tm); Taurocholate; Tauroursodeoxycholate; Rats; Age
Introduction A number of past studies have reported an age-dependent decrease in various hepatic functions (detoxifying functions in particular) in experimental animals. However, results are very variable depending on species, strain and sex of animals. Accordingly, the generalization of results on the same parameter is often difficult. For example, microsomal monooxygenase functions have been reported to decline drastically in male rats and in certain mouse strains of both sexes, whereas in female rat livers these functions are known to remain very stable (Fujita et al., 1982; Kamataki et al., 1985; for review, see Kitani, 1984, 1986, 1988). Several hypotheses have been proposed as underlying mechanisms for an age-dependent decline in microsomal monooxygenase functions (for review, see Kitani, 1988). However, the Correspondence to: S. Kanai, Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo, Japan.
102 significance of such hypotheses may be quite limited in terms or mechanisnls for cellular aging, if similar changes are not generally found. Alterations in the activities of phase I1 reactions such as glutathione S-transferases and sulfotransferases again appear to be variable depending on the sex and species (Kitahara et al., 1982: Fujita et al., 1985; Galinsky et al., 1986, 1990; Carrillo et al., 1989, 1990, 1991: for review, see Kitani, 1988). Thus, age-related changes in many of both phase I and phase II reactions in rat liver appear to be regulated by humoral factors specific to male rats and do not appear to be a general phenomenon caused by aging per se. Summary of available data so far suggests that these detoxifying functions of the liver by means of biotransformation barely decline with aging per se, which also agrees with recent studies on human livers (James et al., 1982; Boobis and Davis, 1984: Matsubara et al., 1986) as well as non-human primate livers (Sutter et al., 1985: Maloney el al., 1986). These studies all reported very stable liver function during aging. There is another efficient detoxifying system in the liver which is not via biotransformation, namely the hepatobiliary transport system. Through this system, a variety of endo- and exogenous toxic substances are efficiently eliminated from the body (for review, see Klaassen and Watkins, 1984). Classically, the biliary excretory system has been classified into three categories, namely for organic anions, cations and neutral compounds (Schanker, 1968). These pathways were believed to be separate from each other in terms of carrier systems. Among the three, the hepatobiliary transport system for organic anions is believed to handle the greatest number of endogenous substances, such as bilirubin, and exogenous ones, such as indocyanine green, sulfobromophthalein and biligraphin, and many kinds of metabolites of exogenous chemicals and pharmaceuticals (Klaassen and Watkins, 1984). In earlier studies on female rats (De-Leeuw lsrael, 1969) as well as on humans (Thompson and Williams, 1965; Skaunic et al., 1970), it has been suggested that the biliary transport maximum (Tin) of sulfobromophthalein remains unchanged with age. In these studies, however, Tm values were indirectly determined. In contrast, subsequent studies in our laboratory which determined Tm directly have provided substantial evidence that the BSP T m actually declines with age in rats, regardless of the sex or strain of animals (Kitani et al., 1978, 1981, Kanai et al.. 1985). Furthermore, a more recent study indicates that the l"m of BSP conjugated with glutathione also declines with age in both male and female Fischer-344 rats (Kanai et al., 1988). From these studies, it has been suggested that the canalicular membrane functions may decline with age (Kitani, 1988). In order to determine whether or not the declining Tm value for BSP and conjugated BSP is a more general phenomenon, it is desirable to examine changes of T m values during aging for other substances having different systems. Bile salts are endogenous metabolic products from cholesterol in the liver and are efficiently excreted into the bile, providing the primary driving force for a fraction of bile water termed 'bile-salt-dependent bile'. Furthermore, it is well known that many bile salts are reabsorbed in the intestine and taken up by the liver undergoing an efficient entero-hepatic circulation. The mechanisms of the hepatobiliary transport of bile salts are not fully understood. However, it is generally believed that the hepatic uptake and biliary excretory systems are separate. Furthermore, both
103 systems are known to be a saturable and energy requiring process, suggesting that active transport is the most likely mechanism (Boyer, 1980; Erlinger, 1981). In a recent study in our laboratory, it was clearly shown that the hepatic uptake rate of taurocholic acid declines in a linear fashion with age. (Ohta and Kitani, 1990). The Vmax value for taurocholic acid by isolated hepatocytes nearly halved at 24 months of rat age in comparison to the young adult value at 4 months. Biliary Tm values for bile salts are known to be several fold lower than corresponding hepatic uptake maxima values, so that the former is the rate-limiting step for the entire transport system for bile salts through the liver. Although bile salts are anions, carrier systems for bile salts and other anion dyes such as BSP are known to be different (Schanker, 1968; Klaassen and Watkins, 1984). We thought that bile salts would be an ideal substance to test our hypothesis that canalicular membrane functions decline with age. Except for our previous study on taurocholate uptake (Ohta and Kitani, 1990), to our knowledge, there is only one previous report with regard to the hepatobiliary transport function change with age for a bile salt, which indicated that the biliary transport function is more severely affected by aging than the hepatic uptake function for taurocholic acid (Kroker et al., 1980). In the present study, two different bile salts (i.e., taurocholate (TC) and tauroursodeoxycholate (TUDC)) were examined in three different rat groups (Wistar-derived rats of both sexes and female Fischer-344 rats. Unlike TC, T U D C is barely found in the natural rat bile. However, we found previously that this bile salt has a Tm value more than two-fold higher than that of TC in rats of both sexes (Kitani and Kanai, 1981; Kitani et al., 1986). Furthermore, the very low hepatotoxicity of T U D C makes this bile salt an ideal bile salt for assessing the liver function. Materials and Methods Male and female Wistar rats and female Fischer-344 (F-344) rats of various ages (3 to 30 months old) were used. Wistar rats were bred in our aging farm under SPF conditions. F-344 rats were purchased from Japan Charles River Co. (Atsugi) in SPF conditions at the age of 4 weeks. Both strains of rats were raised in the institute's aging farm in SPF conditions until use. The husbandry conditions, survivals and pathologies in the later period of their lives have been reported elsewhere (Nokubo, 1985; Tokyo Metropolitan Inst Gerontol., 1989). Tm values for two different bile salts, TC and TUDC, were examined in each group of animals by the method previously reported in detail (Kitani and Kanai, 1981; Kitani et al., 1986). In brief, under pentobarbital anesthesia (Nembutal, Abbott, North Chicago, IL), the common bile duct was cannulated with PE-50 (for males) or PE-10 (for females) tubing (Clay-Adams, Parsippany, N J). Basal bile was collected for 10-20 min. Thereafter, one of the two bile salt solutions was continuously infused through a jugular vein catheter by a Harvard Pump (Harvard Apparatus, Millis, IL). Infusion rates varied depending on the sex and age of animals, but generally, the infusion rate was raised stepwise every 20 min until the bile flow rate began to decline. Bile was collected at 10-min intervals in stock tubes and weighed. Bile samples were frozen and kept at -70°C until the determination of the bile salt concentration in the bile. Chemical purities and sources of bile salts used have
104
been described previously (Kitani et al., 1986). The bile salt solutions were prepared by dissolving sodium salts of these bile salts in 3'7,, bovine serum albumin solution, and the pH (7.4) and osmolality (300 mOsm/kg) were adjusted to physiological levels, as reported previously (Kitani and Kanai, 1981; Kitani et al., 1986). During experiments, the body temperature was continuously monitored by a rectal probe and maintained at 37-37.5°C by a heating lamp. The total bile salt concentration in the bile was determined enzymatically using 3c~-hydroxysteroid dehydrogenase (Worthinton Biochem., Freehold, N J). The total biliary excretion rate of a bile salt was determined as the product of the total bile salt concentration and bile volume, the latter being estimated gravimetrically by assuming that the specific gravity of bile is 1.0. The Tm value of each animal was determined as an average of the three highest excretion rates which were obtained usually just before the drop of bile flow and bile salt excretion rate (see Figs. 1 and 2). The significance of the difference in values among rat groups of different ages was analyzed by means of a one-way analysis of variance (ANOVA). When the difference was found to be significant with respect to the animal's age, Scheffe's test was applied to evaluate the significance of the difference between values of any set of Tm values of two different age groups. Linear regression analysis was also applied to evaluate the age dependency of the value. P values lower than 0.05 were judged to be significant. Results
Table I summarizes the bile flow and total bile salt excretion rate in the basal bile before bile salt infusion. The bile flow as well as total bile salt excretion rate did not show any significant difference among different age groups (ANOVA). Figures 1 and 2 show representative Tm studies in female F-344 rats of two different ages for TC (Fig. 1) and T U D C (Fig. 2). In all studies, the bile flow and bile salt excetion rate began to increase immediately after the start of bile salt infusions.
3-month-old
29-month-old
Infusion rate
0.2
0.6
pmol/min/lOOg b.wt. 1.0 - 1.2
0,8
]
- - ]1.4
0.2
0.4
0.6
0.8
,,,.;
g
1.0
10
0
30 Time,
60
90
120
0
30
60
90
rain
Fig. 1, Representative T m studies for taurocholate in young and old female Fischer-344 rats.
105 3-month-old Infusion rate L 1.2 1.6 , 2.0
pmol/min/lOOg b.wt, 2.4
2.6
2.8
3.0
3.2
3.4]
3.0 J~
29-month-old
0
~o..q --
2.0
..o~"°"e"°"
®
•~_
t._._.,y"
1.o
E
H,, 0
30 Time.
60 rain
90
120
150
180
0
, 30
60
,1i 90
120
Fig. 2. Representative T m studies for tauroursodeoxycholate in young and old female Fischer-344 rats.
With the increase in the infusion rate, the bile flow and bile salt excretion rate reached a plateau and thereafter, they began to decline. Figures 3-5 show the distribution of Tm values in rats of different groups for TC and TUDC. Table II summarizes Tm values as well as bile flow rate and bile salt concentration in the bile during the Tm period in rats of different ages. In general, Tm values expressed per gram liver weight were 2- to 3-fold higher for T U D C than that of TC in young rat groups. Tm values at young age in female Wistar rats tended to be higher than corresponding male rat values in most studies. It is also noted in Table II that the bile salt concentration in the bile during Tm period also generally decreased with age of animals. Tm values were significantly different with respect to rat age as analyzed by one-way ANOVA. The Tm values in older rat groups were significantly lower than respective Tm values in the youngest rat groups (Scheffe's test, P < 0.05) and Tm values tended to decrease with rat age. However, in most rat groups, the decline in Tm values was most pronounced in the first year of rat life and after 12 months, the changes with age were negligible. In fact, the difference in Trn values between 12- to 13- and 24-month-old rats were all insignificant, as analyzed by Scheffe's test, except for the group of Wistar females given TC infusion which showed an intermediate Tm value in the middle-aged rats. Furthermore, in the oldest group for each study, Tm values of several rats were considerably higher than the average values for the group next to the oldest (Figs. 3-5). Accordingly, the average Tm values in the oldest group were somewhat higher than those for the groups next to the oldest in most studies. Regression parameters obtained from t h e linear-regression analysis of the relation between the Tm value and rat age are shown in Table III. A significant negative correlation existed between the Tm value
(3) (6) (13-13.5) (24) (29)
Y-I Y-2 M O-1 0-2
O
(3) (12-13) (25)
Y M O
6 6 7
6.1
4. 5.96 ± 9.15" ± 16.0" ± 38.5* ± 30.6*
± 8.8* ± 16.5" ± 27.8* ± 23.6*
6.4
174.8 ± 9.5 239.2 ± 28.9* 300.0 + 18.0"
175.8 ± 9.6 218.8 ± 11.1" 3 1 4 . 0 ± 46.0*
519.3 ± 38.7* 4 7 8 . 5 ± 27.9*
291.9 4.
290.5 ± 3.94 510.0 ± 15.8" 443.7 ± 54.7*
175.5 207.5 247.6 295.0 322.0
206.4 248.8 287.9 281.7
175.0 ±
4. 4. ± ± ±
4. ± + ± 0.23 0.67* 0.59* 0.68* 1.73'
0.25* 0.62* 0.54* 0.40*
5.03 ± 0.28 7.32 ± 0.68* 8.40 ± 0.48*
4.97 ± 0.49 7.00 ± 0.88* 9.42 ± 1.54"
15.95 ± 1.13" 16.84 ± 1.95"
9.16 ± 0.64
9.56 ± 1.19 16.18 4- 2 . 1 3 " 15.53 ± 1.83"
5.06 6.15 6.76 8.15 10.42
5.90 6.78 7.63 9.02
4.93 + 0.34
L i v e r wt.
(g)
B o d y wt.
(g)
a N u m b e r in p a r e n t h e s i s i n d i c a t e s the a g e in m o n t h o f e a c h g r o u p . *Significantly different f r o m c o r r e s p o n d i n g v a l u e s in the y o u n g e s t g r o u p ( P < 0.05). Y, y o u n g ; M, m i d d l e - a g e d ; O, old.
(3) (13) (25-26)
Y M O
5 4 5
7 6
Female Wistar
10
(3)
6 4 6
6 5 5 6 5
5 4 7 3
7
(12) (24)
(3) (13) (25-26.5)
n
f
Y M 9
Male Wistar
(6) (12) (24) (29)
(3) a
Y-2 M O-1 0-2
Y-I
Female F-344
Rat groups
Bile flow a n d bile salt e x c r e t i o n r a t e o f ' b a s a l bile' in r a t s o f d i f f e r e n t a g e s
TABLE 1
± ± ± +
0.42 0.13 0.40 0.10
0.38 0.29 0.19 0.40 0.18
2.33 ± 0.39 2.12 ± 0.23 1.99 + 0.14
2.16 + 0.67 1.90 ± 0.06 1.63 ± 0.17
2.23 ± 0.33 1.56 4. 0.18 1.54 4- 0.22
2.04 ± 0.21 1.52 ± 0.13 1.52 ± 0.17
1.76 ± 0.54
2.08 1.94 1.96 1.84
+ ± ± ± + ± + + ± +
+ ± ± + ± 0.024 0.005 0.019 0.005 0.013
0.019 0.021 0.015 0.021 0.007
0.065 ± 0.011 0.079 + 0.012 0.064 + 0.022
0.083 + 0.02 0 . 0 6 9 + 0.01 0.045 ± 0.02
0.050 ± 0.006 0.056 ± 0.025
0.068 ± 0.025
0.048 ± 0.017 0.043 ± 0.015 0 . 0 5 6 + 0.025
0.068 0.048 0.072 0.056 0.068
0.066 0.071 0.077 0.076 0.064
(/~mol/min/g)
(tA/min/g)
2.26 2.09 2.00 1.94 1.90
Basal bile salt excretion rate
Basal bile flow rate
TUDC TUDC TUDC
TC TC TC
TUDC TUDC
TUDC
TC TC TC
TUDC TU DC TUDC TUDC TUDC
TC TC TC TC TC
Infusion
107
10)r°u's°de°x'ch°''t' 0.8 0 . 6 ~
Taurocholate
•
0,4
i
|
•
•
E
0.2
0.2
"~
r= -0.66 i
i
i
Age 3 6 (months)
i
i
i
12
24
30
r= -0.79 *
i
3 6
i
i
i
12
24
30
Fig. 3. Scattergram of bile salt Tm values in female Fischer-344 rats of different ages.
and the age of animals for all rat groups. Rates of decrease in Tm values with age (% per month) calculated from the linear regression analysis are also shown in Table III. The rate of decrease ranged from 1.3% to 3.0% per month for all rat groups.
Discussion The results of the present study revealed that, first of all the basal bile flow as well as total bile salt excretion rate stayed fairly stable with aging in all three groups of rats (male and female Wistar, and female F-344 rats) (Table I). These observations essentially agree with our previous observations in rats of different strains and sexes (Kitani et al., 1978, 198 l; Kanai et al., 1985). Furthermore, regardless of the bile salt species tested and sexes of animals, there was a general tendency of decline in bile salt Tm with age in rats when the parameter was expressed per gram liver tissue. Hardison et al. (1981) suggested that secretory maxima values for bile salts as defined here as 'T m' is a compromise between their hepatocellular toxicity and the capacity of the transport system (a true Tm by the definition of Hardison et al. (1981)), and that practically the secretory maximum (T m in the present manuscript) is determined by the degree of bile salt cytotoxicity (Hardison et al., 1981). However, his thesis is probably an overemphasis on the role of membrane toxicity of a bile salt, since we subsequently found that the Tm of TUDC which is least cytotoxic among
108
Tauroursodeoxycholate
1.0
| |
0.8
0.6
Taurocholate
.o
0.4
0.4
.E
E "6 E "! 0.2
0.2
|
....,
••
r: -0.50
r: -0.80 i
Age 0 3 (months)-
=
i
12
24
0 3
12
24
Fig. 4. Scattergram of bile salt Tm values in male Wistar rats of different ages.
known bile salts was much lower than those for TC and even taurochenodeoxcholate (a far more toxic bile salt than TUDC) in hamsters (Kitani et al., 1986). Especially in the case of TUDC, the cytotoxicity may not play any significant role, since this bile salt was found to be even cytoprotective rather than cytotoxic (Kitani and Kanai, 1982; Kitani et al., 1985). Thus, the age-dependent decline for the Tm of T U D C shown in this study can be interpreted rather straightforwardly as due primarily to changes in membrane carrier with age as will be discussed later. In the case of TC Tin, we may need to consider the vulnerability of bile canalicular membrane to a bile salt toxicity in interpreting the data. However, as is evident in Figs. 1 and 2, much smaller amounts of bile salts were infused in older animals in all studies. Thus, a bile salt load to hepatocytes and eventually to bile canalicular membranes, is considerably lower in older animals than young ones. Furthermore, the bite salt concentrations in the bile during the Tm period were all consistently lower in older animals, suggesting that the lower Tm values in older animals are not due to a higher bile salt loading to hepatocytes and eventually to canalicular membranes in our experimental design. It remains unknown whether the age increases the membrane vulnerability to TC. If this is a
109 Tauroursodeoxycholate o
1.2
1.0
0.8
." 0.61 ee
]¢ "=
Taurocholate r: -0.70
0.4
0.2
Age 3 (months)
12
24
i
i
i
3
12
24
Fig. 5. Scattergram of bile salt Tm values in female Wistar rats of different ages.
contributing factor for our observation, however, a sharper decline in Tm value may be expected to occur with TC than with TUDC, which was not the case at least for female Fischer rats and Wistar male rats. Furthermore, Abernathy et al. (1980), showed a more resistant membrane quality to cytotoxycities of erythromycin and chlorpromazine in aged rat hepatocytes. Based on the above considerations, we interprete that the decline of Tm values with age is primarily due to the quantitative (and/or) qualitative alteration of membrane carriers for bile salt transport into bile canaliculi. Significantly higher Tm values for T U D C than TC in young rats are consistent with our previous results in male and female Wistar-derived rats (Kitani and Kanai, 1981; Kitani et al., 1986). This tendency was maintained until very old age of animals, although the difference in Tm values between the two bile salts became smaller as the age advanced. Although there are various hypotheses concerning the greater Tm value for T U D C (Hardison et al., 1981), the true reason for this difference remains unknown• The transport systems (carrier proteins, in particular) for TC and T U D C may possibly be different, since when these two bile salts are
6 4 6
10 7 6
Male Wistar Y (3) M (13) O (25-26.5)
Y M O
(3) (12-131 (25)
Y M O
6 6 6
TUDC TUDC TUDC
TC TC TC
TUDC TUDC TUDC
TC TC TC
TUDC TUDC TUDC TUDC TUDC
TC TC TC TC TC
Infusion
± 44± ±
± 4± ± ± 0,83 0.38* 0.52 0.48* 0.91"
0.46 0.25 0.27* 0.57* 0.23
6.96 ± 0,67 4,49 + 0.37* 4,97 ± 0,67*
3.66 ± 0.28 2.89 ± 0.16" 2.50 ± 0.28*
6.19 ± 1,38 2.74 4- 0.22* 3.27 ± 0,61"
4.07 ± 0.38 1.92 ± 0.11" 2.88 ± 0.43*
6,82 5.16 4.05 4,12 3.31
4,13 3.45 2.74 3.03 3.16
Bile flow rate (/~l/min per g)
aNumber in parenthesis indicates the age in m o n t h of each group, *Significantly different from c o r r e s p o n d i n g values in the y o u n g e s t g r o u p ( P < 0.05). Y, young; M, middle-aged; O, old.
(3) (13) (25-26)
Y M O
Female Wistar
5 4 5
6 5 5 6 5
(3} (6) (13-13,51 (24) (29)
Y-I Y-2 M O-1 0-2
(3) (12) (24)
7 5 4 7 3
n
Female F-344 Y-I (3) a Y-2 (6) M (121 O-1 (24) 0-2 (29)
Rat group
6.33 5.93 4,40 5.45 2,39
6.73 2.06* 6.93* 162.73 ± 8,1l* 131.72 ± 7.09* 128.95 ± 21.13
93.40 ± 74.44 ± 56.76 ±
160.08 ± 6,67 124.61 ± 16.43" 122.99 ± 8.85*
3.45 2.70 9.00
± 5.65 ± 7.01 ± 7.30 ± 6,57* ± 11.72"
± ± 44±
72.59 ± 72.43 ± 69.60 ±
128,57 120.68 117.28 113.43 105.10
80.51 78.62 73.27 70.65 71.91
Bile salt cone. in the bile ( m M )
Bile flow, bile salt c o n c e n t r a t i o n and e x c r e t i o n rate at the T m p e r i o d in rats o f different ages
T A B L E II
44± ± 4-
± ± ± ± ± 0,108 0,025* 0,072* 0,068* 0.128"
0.046 0.030 0.022* 0.039* 0.020*
1.13(I ± 0.099 0.590 ± 0.050* 0,646 ± 0,149"
0.342 ± 0.045 0.215 ± 0.015" 0.141 ± 0.020*
0.944 ± 0.155 0.340 ± 0.036* 0.398 ± 0.084*
0.296 ± 0.030 0.139 ± 0.012" 0.204 ± 0.057*
0.876 0.621 0,476 0.469 0.36(/
0.331 0.272 0.204 0,213 0.226
Bile salt excretion rate (Tnl) ( # m o l / m i n per g)
TC TUDC TC TUDC TC TUDC
Female F-344
× x × × × ×
10 -3 10 -2 10 -3 10 -2 10 -3 10 -2
0.311 0.797 0.273 0.954 0.356 1.059
(ttmol/min per g)
(/~mol/min per g per month)
-4.0 -1.5 -3.58 -2.9 -8.75 -1.98
ba
aa
trrhe rate of decrease of Tm with age (% per month).
ay = a x 4- b, (x, rat age in month; y, Trn value in ttmol/min per g liver).
Female Wistar
Male Wistar
Bile salts
Rat groups
Regression coefficients for the relationship between bile salt Trn value and rat age
TABLE III
1.3 1.9 1.3 3.0 2.4 1.8
(% per month)
a/b b
26 27 16 21 14 19
-0.664 -0.790 -0.496 -0.804 -0.935 -0.700
<0.005 <0.005 <0.05 < 0.0005 < 0.0005 < 0.0005
112
simultaneously infused, there is no competitive inhibition in their biliary transport process (Kitani and Kanai, 1982; Kitani et al., 1985). Instead of inhibition, TUDC tended to facilitate the transport of TC. This circumstantial evidence favors the view that the transport system for these two bile salts may not be the same. Despite the possible difference in carrier proteins for these two different bile salts tested, the manner of the age-dependent decline of Tm values was quite similar for these two bile salts. Furthermore, the declining tendency with age for the T m value of these bile salts is similar to that in our previous studies on Tm values for BSP (Kitani et al., 1978, 1981; Kanai et al., 1985) and its gluthathione conjugate (Kanai et al., 1988). Although the possible involvement of membrane vulnerability is not excluded in a rigorous sense especially for strong toxins such as TC as discussed above, such a decline in Tm values with age is usually explained as due to the age-dependent decrease in the carrier unit number for individual materials. Transport systems (more specifically, carrier proteins and energy for a driving force) for bile salts and the above cholephilic dyes are believed to be different from each other (Schanker, 1968; Klaassen and Watkins, 1984). Despite these possible differences in carrier proteins and driving forces for the biliary transport of these materials, the declining tendency for Tm values with age was very similar. These results suggest that there might be some common factor(s) other than the decrease in carrier unit number for individual substances, which regulates in general the declining transport functions through the bile canalicular membrane with age. As one of the candidates for such factor(s), we suggested the declining mobility of membrane proteins in the bile canaliculus (Kanai et al., 1988). Although no direct proof for such a mechanism is at present available, our recent studies using the fluorescence recovery after photobleaching (FRAP) technique have shown that the lateral mobility (average lateral diffusion constant) of proteins in the surface membrane of hepatocytes (predominantly of sinusoidal domain) declines almost linearly with age in both male and female rats of different strains (F-344 and Wistar-derived rats) (Zs.-Nagy et al., 1986; Kitani et al., 1988) and mice (Zs.-Nagy et al., 1989). Furthermore, we have shown a striking linear relationship between the diffusion constant of proteins and hepatic uptake rates of ouabain (Ohta et al., 1988) and more recently of TC (Ohta and Kitani, 1990). From these studies, we proposed that the decreasing membrane protein mobility with age may be a significant factor regulating the surface membrane functions mediated by membrane proteins (Ohta and Kitani, 1990: Kitani et al., 1991). Because the membrane surface area of the canalicular domain is very small relative to that of the sinusoidal domain of hepatocytes, our FRAP technique cannot be directly applied to assess the physico-chemical properties of canalicular membrane proteins of rat hepatocytes. Despite these difficulties, future studies are expected to test our hypothesis wherein the plasma membrane protein mobility also declines in the canalicular domain, as has been shown for sinusoidal membranes in hepatocytes, and that it is (at least partially) regulating the transport of materials through canalicular membranes. Rates of decrease per month in TC uptake (2.1%) (Ohta and Kitani, 1990), ouabain uptake (2.4%) (Ohta et al., 1989) and initial 10-min excretion of ouabain (2.2-2.8%) (Sato et al., 1987; Ohta et al., 1989: Kitani et al., 1991) are very close to
113 values observed for the decline of Tm values with age in our present study. Previously found values are thought to reflect the function of sinusoidal membranes (Sato et al., 1987; Ohta et al., 1989; Ohta and Kitani, 1990; Kitani et al., 1991), while values found in the present study reflect that of canalicular membranes. The striking similarities for these values may be only a coincidence, but it is also possible that it is the reflection of some common causal mechanism as suggested above. Except for the group of female Wistar rats given TC infusion which showed an age-dependent gradual decline in Tm value, most other groups showed almost comparable values for middle-aged (12-month-old) and old (24-month-old or older) rat groups. This may suggest that the Tm value declines relatively early in the life of animals. Furthermore, unusually high Tm values close to those in the youngest rat groups were observed in some rats in the oldest groups (e.g., TC Tm in male Wistar rats). In general, the scattering of each Tm value was narrower in the middle-aged rats than in the old rats. Similar phenomena have been observed in previous studies for many different kinds of functional parameters of the liver (van Bezooijen et al., 1977; Kitani et al., 1981; Kanai et al., 1985; Zs.-Nagy et al., 1986; Sato et al., 1987; Ohta et al., 1988; Ohta and Kitani, 1990). Since Tm values for bile salts were presented per unit liver weight, it is unlikely that higher Tm values in some of the oldest rats compared with those of middleaged rats were due to an enlargement of the liver in old age. As a possible explanation for such phenomena, two hypotheses have been proposed (Sato et al., 1987). One is an actual increase in liver functions with advancing age of animals possibly as a compensatory response to other changes occurring in the late life of an animal. For example, the increase in the albumin synthesis rate in rat liver in old age was explained to be a response to the enhanced urinary loss of albumin in old age (van Bezooijen et al., 1977). The other possibility is that this is due to the survival of animals that have maintained exceptionally good liver functions up to their old age (i.e., survival selection). In previous studies, the biliary excretion of i.v. injected ouabain tended to be higher in the oldest rat groups (29-31-months-old) than in the second-oldest group (Sato et al., 1987; Ohta et al., 1988). Thus, the latter possibility (i.e., selection by survival of animals) could not be excluded in these studies. In the present study, the reversed difference (i.e., higher Tm values in old groups) was observed between middle-aged (12-month-old) and old (24-month-old) rats. Since survival rates of these rats are all more than 90% at 24 months of age (Nokubo, 1985; Tokyo Metropolitan Inst. Gerontol., 1989), it is difficult to explain such a high value in old rat groups as due to survival selection. Thus, the former possibility that livers of some old rats become hyperactive must be considered, although not directly proven. Only future studies in a longitudinal scheme can directly answer this question. In summary, the Tm values for both TC and T U D C tended to decline in both male and female rats, suggesting that some common mechanism may underlie the age-induced declines of biliary Tm for bile salt, BSP and conjugated BSP as demonstrated in our previous studies (Kanai et al., 1985, 1988).
Acknowledgement The skillful secretarial assistance of Ms. K. Tagami is gratefully acknowledged.
114
References Abernathy, C.O., Lukacs, L. and Zimmerman, H.J. (1980): Cytotoxic effects oferythromycin estolate and chlorpromazine on hepatocytes isolated from rats of varying ages: A brief note. Mech. Ageing Dev., 12, 1-6. Boobis, A.R. and Davis, D.S. ( 1984): Human cytochromes P-450. Xenobiotica, 14, 151-185. Boyer, J.L. (1980): New concepts of mechanisms of hepatocyte bile formation. Physiol. Rev, 60, 303-326. Carrillo, M.-C., Kitani, K., Kanai, S., Sato, Y., Nokubo, M., Ohta, M. and Otsubo, K. (1989): Differences in the influence of diet on hepatic glutathione S-transferase activity and glutathione content between young and old C57 black female mice. Mech. Ageing Dev., 47, 1-15. Carrillo, M.-C., Nokubo, M., Sato, Y., Kanai, S., Ohta, M. and Kitani, K. (1990): Effect of protein-free diet on activities and subunits of glutathione S-transferase in livers of young and aged female rats. Mech. Ageing Dev., 56, 237-251. Carrillo, M.-C., Nokubo, M., Kitani, K., Sato, K. and Satoh, K. (1991): Age-related alterations of subunits of hepatic glutathione S-transferases in male and female F-344 rats. Biochim. Biophys. Acta, 1077, 325-331. Erlinger, S. (1981): Hepatocyte bile secretion Current views and controversies. Hepatology, 1, 352-359. De Leeuw-lsrael, F.R., Hollander, C.F. and Arp-Neefjes, J.M. (1969): Hepatic storage and maximal biliary excretion of bromsulphalein (BSP) in young and old rats. J. Gerontol.. 24, 140-142. Fujita, S., Uesugi, T., Kitakawa, H., Suzuki, T. and Kitani, K. (1982): Hepatic microsomal monooxygenase and azoreductase activities in aging Fischer-344 rats. Importance of sex difference for aging study. In: Liver and Aging .- 1982, Liver and Drugs, pp. 55-71. Editor: K. Kitani. Elsevier/NorthHolland Biomedical Press, Amsterdam. Fujita, S., Kitagawa, H., Ishizawa, H., Suzuki, T. and Kitani, K. (1985): Age associated alterations in hepatic glutathione-S-transferase activities. Biochem. Pharmacol., 34, 3891-3894. Galinsky, R.E., Kane, R.E., Franklin and M.R. (1986): Effect of aging on drug metabolizing enzymes important in acetaminophen elimination. J. Pharmacol. Exp. Ther., 237, 107-113. Galinsky, R.E., Johnson, D.H., Kane, R.E. and Franklin, M.R. (1990): Effect on aging on hepatic biotransformation in female Fischer 344 rats: Changes in sulfotransferases activities are consistent with known gender-related changes in pituitary growth hormone secretion in aging animals. J. Pharmacol. Exp. Ther., 255, 577-583. Hardison, W.G.M., Hatoff, D.E., Miyai, K. and Weiner, R.G. (1981): Nature of bile acid maximum secretory rate in the rat. Am. J. Physiol., 241, (Gastrointest. Liver Physiol. 4) G337-G344. James, O.F.W., Rawlines, M.D. and Woodhouse, K. (1982): Lack of ageing effect on human microsomal monooxygenase activities and on inactivation pathways for reactive metabolic intermediates. In: Liver and Aging 1982, Liver and Drugs, pp. 395-408. Editor: K. Kitani. Elsevier/North Holland, Amsterdam. Kamataki, T., Maeda, K., Shimada, M., Kitani, K., Nagai, T. and Kato, R. (1985): Age-related alteration in the activities of drug-metabolizing enzymes and contents of sex-specific forms of cytochrome P-450 in liver microsomes from male and female rats. J. Pharmacol. Exp. Ther., 233, 222-228. Kanai, S., Kitani, K., Fujita, S. and Kitagawa, H. (1985): The hepatic handling of sulfobromophthalein in aging Fischer-344 rats: in vivo and in vitro studies. Arch. Gerontol. Geriatr., 4, 73-85. Kanai, S., Kitani, K., Sato, Y. and Nokubo, M. (1988): The age-dependent decline in the biliary transport maximum of conjugated sulfobromophtbalein in the rat. Arch. Gerontol. Geriatr., 7, 1-8. Kitahara, A., Ebina, T., Ishikawa, T., Soma, Y., Sato, Y. and Kanai, S. (1982): Changes in activities and molecular forms of rat hepatic drug-metabolizing enzymes during aging. In: Liver and Aging 1982, Liver and Drugs, pp. 135-142. Editor: K. Kitani. Elsevier/North Holland Biomedical Press, Amsterdam. Kitani, K. (1984): The effect of age on hepatic metabolism of xenobiotics. In: Foreign Compound Metabolism, pp. 275-287. Editors: J. Caldwell and G. Paulson. Taylor and Francis, London. Kitani, K. (1986): Hepatic drug metabolism in the elderly. Hepatology, 6, 316-319. Kitani, K. (1988): Drugs and the ageing liver. Life Chem. Rep., 6, 143-230. Kitani, K. and Kanai, S. (1981): Biliary transport maximum of tauroursodeoxycholate is twice as high as that of taurocholate in the rat. Life Sci., 29, 269-275.
115 Kitani, K., and Kanai, S. (1982): Tauroursodeoxycholate prevents taurocholate induced cholestasis in the rat. Life Sci., 30, 515-523. Kitani, K., Kanai, S. and Miura, A. (1978): Hepatic metabolism of sulfobromophthalein (BSP) and indocyanine green (ICG) in aging rats. In: Liver and Aging - - 1978, pp. 145-156. Editor: K. Kitani. Elsevier-North Holland, Amsterdam. Kitani, K., Zurcher, C. and Van Bezooijen, C.F.A. (1981): The effect of aging on the hepatic metabolism of sulfobromophthalein in BN/Bi female and Wag/Rij male and female rats. Mech. Ageing Dev., 7, 381-393. Kitani, K., Ohta, M. and Kanai, S. (1985): Tauroursodeoxycholate prevents biliary protein excretion induced by other bile salts in the rat. Am. J Physiol., 248, G407-C417. Kitani, K., Kanai, S., Ohta, M. and Sato, Y. (1986): Differing transport maxima values for taurine conjugated bile salts in rats and hamsters. Am. J. Physiol. 251, G852-G858. Kitani, K., Zs.-Nagy, I., Kanai, S., Sato, Y. and Ohta, M. (1988): Correlation between the biliary excretion of ouabain and the lateral mobility of hepatocyte membrane proteins in the rat. The effects of age and spironolactone pretreatment. Hepatology, 8, 125-131. Kitani, K., Ohta, M., Kanai, S., Sato, Y., Nokubo, M., and Zs.-Nagy, i. (1991): Age-induced restricted mobility of proteins in hepatocyte surface membranes: A possible determinant of membrane transport function. In: Liver and Aging - - 1990, pp. 305-318. Editor: K. Kitani. Elsevier Science Publishers, B.V., Amsterdam. Klaassen, C.D. and Watkins, J.B. 3rd. (1984): Mechanisms of bile formation, hepatic uptake and biliary excretion. Pharmacol. Rev., 36, 1-67. Kroker, R., Hegner, D. and Anwer, M.S. (1980): Altered hepatobiliary transport of taurocholic acid in aged rats. Mech. Ageing Dev., 12, 367-373. Maloney, A., Schmucker, D.L., Vessey, D.S. and Wang, R.K. (1986): The effects of aging on the hepatic microsomal mixed-function oxidase system of male and female monkeys. Hepatology, 6, 282-287. Matsubara, T., Yamada, N., Mitomi, T., Yokoyama, S., Fukiishi, Y., Hasegawa, Y. and Nishimura, H. (1986): Cytochrome P-450-dependent monooxygenase activities in prenatal and postnatal human livers: Comparison of human liver 7-alkoxycoumarin O-deethylases with rat liver enzymes. Jpn. J. Pharmacol., 40, 389-398. Nokubo, M. (1985): Physical-chemical and biochemical differences in liver plasma membranes in aging F-344 rats. J. Gerontol., 40, 409-414. Ohta, M., Kanai, S., Sato, Y. and Kitani, K. (1988): Age-dependent decrease in the hepatic uptake and biliary excretion of ouabain in rats. Biochem. Pharmacol., 37, 935-942. Ohta, M., and Kitani, K.(1990): Age-dependent decrease in the hepatic uptake of taurocholic acid resembles that for ouabain. Biochem. Pharmacol., 39, 1223-1228. Sato, Y., Kanai, S., and Kitani, K. (1987): Biliary excretion of ouabain in aging male and female F-344 rats. Arch. Gerontol. Geriatr., 6, 141-152. Schanker, L.S. (1968): Secretion of organic compounds. In: Handbook of Physiology, Section 6, Vol. 5, pp. 2433-2449. Am. Physiol. Soc., Washington D.C. Skaunic, V., Nerad, V. and Fedrichova, M. (1970): Mechanismus poklesu relativni stradaci schopnosti jater pro bromsulftalein s rostoucim vekem II Vyznam snizeneho jaterniho prutoku krve. Cs Gastroenterologie, 24, 206-215. Sutter, M.A., Gibson, G., Williamson, L.S., Strong, R., Pickham, K. and Richardson, A. (1985): Comparison of the hepatic mixed function oxidase systems of young, adult and old non-human primates (Macaca nemestrina). Biochem. Pharmacol., 34, 2983-2987. Thompson, E.N. and Williams, R. (1965): Effect of age on liver function with particular reference to bromsulphalein excretion. Gut, 6, 266-269. Tokyo Metropolitan Institute of Gerontology (1989): Data book for aging farm 11. Pathology and Blood Chemistry 1989. Van Bezooijen, C.F.A., Gvell, T. and Knook, D.L. (1977): The effect of age on protein synthesis by isolated liver parenchymal cells. Mech. Ageing Dev., 6, 293-304. Zs.-Nagy, I., Kitani, K., Ohta, M. and lmahori, K. (1986): Age-dependent decrease of the lateral diffusion constant of proteins in the plasma membrane of hepatocytes as revealed by fluorescence recovery after photobleaching in tissue smears. Arch. Gerontol. Geriatr., 5, 131-146. Zs.-Nagy, 1., Kitani, K. and Ohta, M. (1989): Age-dependence of the lateral mobility of proteins in the plasma membrane of hepatocytes of C57BL/6 mice: FRAP studies on liver smears. J. Gerontol., 44, B83-87.