Nonuniform Distribution of Sodium in the Rat Hepatocyte G. H O O P E R and D. A. T. D I C K From the Department of Anatomy, University of Dundee, Dundee, Scotland
A B S X R AC T The volume of the nucleus, endoplasmic reticulum (including Golgi complex), mitochondria, and cytoplasmic ground substance was measured in rat hepatocytes by stereoiogical methods. The Na content was also measured by flame photometry. Variations in Na content correlated significantly with variations in volume of nucleus and endoplasmic reticulum. From the correlation parameters, Na concentrations were estimated as follows: nucleus, 108 raM; endoplasmic reticuium (ER) (including Golgi complex) 27 mM; cytoplasm (including mitochondria and remaining organelles) 16 raM.
Evidence r e g a r d i n g the distribution of Na in cells is inconclusive. M e a s u r e m e n t s o f intracellular Na activity have been recently reviewed (1). Zadunaisky (2) located Na in the transverse tubules o f frog muscle by the p y r o a n t i m o n a t e technique but the specificity o f this has been disputed (3, 4). In toad oocytes, the Na activity coefficient m e a s u r e d by cation-selective glass microelectrodes is m u c h lower t h a n that in bulk solutions o f the s a m e c o m p o s i t i o n (5); s o m e Na is not rei~laceable by Li a n d a u t o r a d i o g r a p h y shows that this fraction lies in the cytoplasm a n d not the nucleus a l t h o u g h p o o r resolution p r e v e n t e d m o r e exact location (6, 7, 8). Electron m i c r o p r o b e analysis has b e e n used to locate Na in a tissue such as f r o g skir~ (9) a l t h o u g h it has so far p r o v e d difficult to obtain g o o d intracellular localization. T h e m e t h o d o f the p r e s e n t study was to correlate liver Na concentration d e t e r m i n e d by flame p h o t o m e t r y with the v o l u m e o f various subcellular c o m p o nents o f the h e p a t o c y t e d e t e r m i n e d by stereological m e t h o d s in both control a n d p h e n o b a r b i t o n e - t r e a t e d rats. By relating changes in Na c o n c e n t r a t i o n to changes in the v o l u m e of c o m p o n e n t s , it is possible to m a k e estimates o f the Na concentration in each subcellular c o m p o n e n t . We have f o u n d that Na is not u n i f o r m l y distributed in the h e p a t o c y t e and that Na c o n c e m r a t i o n varies considerably f r o m one subcellular c o m p o n e n t to a n o t h e r . This work has b e e n briefly r e p o r t e d (10). MATERIALS
AND
METHODS
Eighteen Sprague-Dawley rats, weighing between 350 and 420 g, were used. Sixteen animals were paired as to age, weight, and sex; one of each pair was used as control and the other received phenobarbitone by injection (100 mg/kg daily for 4 days); there were two additional control animals (nos. 7 and 10). Laboratory diet and water were provided THE JOURNAL O r GENERAL PHYSIOLOGY " VOLUME 6 7 ,
1976
pages 469-474
469
470
T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M E
67
- 1976
ad libitum. Before sacrifice by ether inhalation on the 5th day animals were starved for 24 h to deplete liver glycogen. T h r e e to five milliliters of blood was taken before exsanguination and removal and weighing o f liver. For stereology blocks were fixed in buffered 4% glutaraldehyde, postfixed in Millonig's osmium fixative, dehydrated in changes of ethanol, and e m b e d d e d in araldite. Silver sections were stained with uranyl acetate and lead citrate. Water content of fresh slices of the same liver was determined by reweighing after heating at 105°C for 24 h. Sodium in similar fresh liver slices and in plasma was d e t e r m i n e d with an EEL flame p h o t o m e t e r (Evans Electroselenium Ltd., London) after digestion overnight in 0.15 M HNO3. Density was measured by flotation in sucrose solutions and was f o u n d to tie between 1.05 and 1.07 g/cm3; a value o f 1.06 g/cm 3 was used in calculations, Sampling o f blocks and recording o f micrographs was done by the methods of Weibei et al. (11), except that only magnifications x 2,500 and 10,000 were used. These were analyzed by means of a projection apparatus giving a secondary magnificatioo of x 5.5 or 0 (12). At x 2,500 between 1,573 and 3,360 points were counted and at × 10,000 between 359 and 1655 points, and from these volume densities were calculated (12). Extracellular Na was calculated from the stereologically determined extracellular space (control mean 0.103 cm3/cm 2 liver) assuming an Na concentration the same as that of the plasma; intracellular Na (control mean 21.4 mM) was calculated by subtraction from total Na. These values agree well with previous data of Williams and Woodbury (13) who found an extracellular space (l-h inulin space) of 9-12% and intracellular Na concentrations of 24.7 and 21.1 mmol/liter cell water in fasted male and female rats, respectively. RESULTS
Basic d a t a a r e s h o w n in T a b l e I. T h e o n l y statistically s i g n i f i c a n t e f f e c t o f p h e n o b a r b i t o n e was an i n c r e a s e in t h e specific w e i g h t o f t h e l i v e r a n d in t h e TABLE
I
E F F E C T OF P H E N O B A R B I T O N E ON LI V ER Control, m e a n ± SD (n = 10)
Liver specific weight (g/100 g body weight) Liver water concentration (g H20/g fresh liver) Total liver Na concentration (I.onol/cm 3) Plasma Na concentration (#raollcra a) Intracellular Na concentration (g:mol/cm 3) Volume density (cm3/cra 3 liver) Hepatocyte Nucleus Endoplasmic reticulum (including Golgi complex) Mitochondria Cytoplasmic ground substance (including remaining organelles) Littoral cells Extracellular space (including bile canaliculi) * Calculated by one-tail t test.
Phenobarbitone, mean±SD(n =
Significance o f
8)
difference*
2.52+_0.22
3.24±0.47
P < 0.0005
0.682±0.018
0.671±0.017
NS
36.7±3.4
34.6±3.5
NS
146.7±7.0
144.5±6.2
NS
21.4±4.8
NS
21.5+_5.4
0.882___0.041 0.060±0.046 0.144±0.040 0.209±0.038 0.472±0.037
0.896±0.027 0.051±0.012 0.202±0.051 0.189±0.024 0.457±0.054
NS NS P < 0.01 NS NS
0.014---0.008 0.013---0.005 NS 0.103---0.034 0.092---0.027 NS
471
G. HOOPER AND D. A. T. DICK Sodium in Rat Hepatocyte T A B L E II C O R R E L A T I O N OF V O L U M E D E N S I T Y OF O R G A N E L L E S W I T H I N T R A C E L L U L A R Na Volume density (cmSlem~ liver)
I ntracellular Na
Animal no.
Endoplasmic reticulum
Nuclei
Mitochondria
Cytoplasmic g r o u n d substance
g~dlon s
Control 1 2 3 4 5 6 7 8 9* 10
19.4 20.0 26.1 19.7 18.6 23.8 23.0 14.0 33.4 17.4
0.133 0.122 0.147 0.135 0.176 0.224 0.131 0.066 0.146 0.161
0.058 0.079 0.056 0.039 0.042 0.056 0.046 0.017 0.182 0.028
0.212 0.187 0.237 0.238 0.202 0.188 0.219 0.224 0.124 0.260
0.446 0.469 0.498 0.478 0.431 0.452 0.525 0.530 0.472 0.423
Phenobarbitone 1 2 3 4 5 6 8 9
23.5 22.2 21.2 23.5 28.8 16.7 22.1 12.9
0.181 0.157 0.230 0.286 0.244 0.218 0.131 0.170
0.056 0.046 0.067 0.054 0.064 0.041 0.032 0.049
0.170 0.178 0.233 0.162 0.214 0.177 0.190 0.189
0.495 0.489 0.383 0.429 0.403 0.469 0.549 0.442
Parameters of correlation with intracellular Na
b r P~t
÷31.6 +0.419
A B S X R AC T The volume of the nucleus, endoplasmic reticulum (including Golgi complex), mitochondria, and cytoplasmic ground substance was measured in rat hepatocytes by stereoiogical methods. The Na content was also measured by flame photometry. Variations in Na content correlated significantly with variations in volume of nucleus and endoplasmic reticulum. From the correlation parameters, Na concentrations were estimated as follows: nucleus, 108 raM; endoplasmic reticuium (ER) (including Golgi complex) 27 mM; cytoplasm (including mitochondria and remaining organelles) 16 raM.
Evidence r e g a r d i n g the distribution of Na in cells is inconclusive. M e a s u r e m e n t s o f intracellular Na activity have been recently reviewed (1). Zadunaisky (2) located Na in the transverse tubules o f frog muscle by the p y r o a n t i m o n a t e technique but the specificity o f this has been disputed (3, 4). In toad oocytes, the Na activity coefficient m e a s u r e d by cation-selective glass microelectrodes is m u c h lower t h a n that in bulk solutions o f the s a m e c o m p o s i t i o n (5); s o m e Na is not rei~laceable by Li a n d a u t o r a d i o g r a p h y shows that this fraction lies in the cytoplasm a n d not the nucleus a l t h o u g h p o o r resolution p r e v e n t e d m o r e exact location (6, 7, 8). Electron m i c r o p r o b e analysis has b e e n used to locate Na in a tissue such as f r o g skir~ (9) a l t h o u g h it has so far p r o v e d difficult to obtain g o o d intracellular localization. T h e m e t h o d o f the p r e s e n t study was to correlate liver Na concentration d e t e r m i n e d by flame p h o t o m e t r y with the v o l u m e o f various subcellular c o m p o nents o f the h e p a t o c y t e d e t e r m i n e d by stereological m e t h o d s in both control a n d p h e n o b a r b i t o n e - t r e a t e d rats. By relating changes in Na c o n c e n t r a t i o n to changes in the v o l u m e of c o m p o n e n t s , it is possible to m a k e estimates o f the Na concentration in each subcellular c o m p o n e n t . We have f o u n d that Na is not u n i f o r m l y distributed in the h e p a t o c y t e and that Na c o n c e m r a t i o n varies considerably f r o m one subcellular c o m p o n e n t to a n o t h e r . This work has b e e n briefly r e p o r t e d (10). MATERIALS
AND
METHODS
Eighteen Sprague-Dawley rats, weighing between 350 and 420 g, were used. Sixteen animals were paired as to age, weight, and sex; one of each pair was used as control and the other received phenobarbitone by injection (100 mg/kg daily for 4 days); there were two additional control animals (nos. 7 and 10). Laboratory diet and water were provided THE JOURNAL O r GENERAL PHYSIOLOGY " VOLUME 6 7 ,
1976
pages 469-474
469
470
T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M E
67
- 1976
ad libitum. Before sacrifice by ether inhalation on the 5th day animals were starved for 24 h to deplete liver glycogen. T h r e e to five milliliters of blood was taken before exsanguination and removal and weighing o f liver. For stereology blocks were fixed in buffered 4% glutaraldehyde, postfixed in Millonig's osmium fixative, dehydrated in changes of ethanol, and e m b e d d e d in araldite. Silver sections were stained with uranyl acetate and lead citrate. Water content of fresh slices of the same liver was determined by reweighing after heating at 105°C for 24 h. Sodium in similar fresh liver slices and in plasma was d e t e r m i n e d with an EEL flame p h o t o m e t e r (Evans Electroselenium Ltd., London) after digestion overnight in 0.15 M HNO3. Density was measured by flotation in sucrose solutions and was f o u n d to tie between 1.05 and 1.07 g/cm3; a value o f 1.06 g/cm 3 was used in calculations, Sampling o f blocks and recording o f micrographs was done by the methods of Weibei et al. (11), except that only magnifications x 2,500 and 10,000 were used. These were analyzed by means of a projection apparatus giving a secondary magnificatioo of x 5.5 or 0 (12). At x 2,500 between 1,573 and 3,360 points were counted and at × 10,000 between 359 and 1655 points, and from these volume densities were calculated (12). Extracellular Na was calculated from the stereologically determined extracellular space (control mean 0.103 cm3/cm 2 liver) assuming an Na concentration the same as that of the plasma; intracellular Na (control mean 21.4 mM) was calculated by subtraction from total Na. These values agree well with previous data of Williams and Woodbury (13) who found an extracellular space (l-h inulin space) of 9-12% and intracellular Na concentrations of 24.7 and 21.1 mmol/liter cell water in fasted male and female rats, respectively. RESULTS
Basic d a t a a r e s h o w n in T a b l e I. T h e o n l y statistically s i g n i f i c a n t e f f e c t o f p h e n o b a r b i t o n e was an i n c r e a s e in t h e specific w e i g h t o f t h e l i v e r a n d in t h e TABLE
I
E F F E C T OF P H E N O B A R B I T O N E ON LI V ER Control, m e a n ± SD (n = 10)
Liver specific weight (g/100 g body weight) Liver water concentration (g H20/g fresh liver) Total liver Na concentration (I.onol/cm 3) Plasma Na concentration (#raollcra a) Intracellular Na concentration (g:mol/cm 3) Volume density (cm3/cra 3 liver) Hepatocyte Nucleus Endoplasmic reticulum (including Golgi complex) Mitochondria Cytoplasmic ground substance (including remaining organelles) Littoral cells Extracellular space (including bile canaliculi) * Calculated by one-tail t test.
Phenobarbitone, mean±SD(n =
Significance o f
8)
difference*
2.52+_0.22
3.24±0.47
P < 0.0005
0.682±0.018
0.671±0.017
NS
36.7±3.4
34.6±3.5
NS
146.7±7.0
144.5±6.2
NS
21.4±4.8
NS
21.5+_5.4
0.882___0.041 0.060±0.046 0.144±0.040 0.209±0.038 0.472±0.037
0.896±0.027 0.051±0.012 0.202±0.051 0.189±0.024 0.457±0.054
NS NS P < 0.01 NS NS
0.014---0.008 0.013---0.005 NS 0.103---0.034 0.092---0.027 NS
471
G. HOOPER AND D. A. T. DICK Sodium in Rat Hepatocyte T A B L E II C O R R E L A T I O N OF V O L U M E D E N S I T Y OF O R G A N E L L E S W I T H I N T R A C E L L U L A R Na Volume density (cmSlem~ liver)
I ntracellular Na
Animal no.
Endoplasmic reticulum
Nuclei
Mitochondria
Cytoplasmic g r o u n d substance
g~dlon s
Control 1 2 3 4 5 6 7 8 9* 10
19.4 20.0 26.1 19.7 18.6 23.8 23.0 14.0 33.4 17.4
0.133 0.122 0.147 0.135 0.176 0.224 0.131 0.066 0.146 0.161
0.058 0.079 0.056 0.039 0.042 0.056 0.046 0.017 0.182 0.028
0.212 0.187 0.237 0.238 0.202 0.188 0.219 0.224 0.124 0.260
0.446 0.469 0.498 0.478 0.431 0.452 0.525 0.530 0.472 0.423
Phenobarbitone 1 2 3 4 5 6 8 9
23.5 22.2 21.2 23.5 28.8 16.7 22.1 12.9
0.181 0.157 0.230 0.286 0.244 0.218 0.131 0.170
0.056 0.046 0.067 0.054 0.064 0.041 0.032 0.049
0.170 0.178 0.233 0.162 0.214 0.177 0.190 0.189
0.495 0.489 0.383 0.429 0.403 0.469 0.549 0.442
Parameters of correlation with intracellular Na
b r P~t
÷31.6 +0.419
