CELL BIOCHEMISTRY AND FUNCTION
VOL.9: 9-12 (1991)
Efflux of Inorganic Phosphate from Rat Hepatocytes: Lack of Effect of Insulin MUKESH PARKASH AND PETER J. BUTTERWORTH Department of Biochemistry, King's College (London), Campden Hill Road, London W8 7AH, U K
Efflux of Pi from rat hepatocytes suspended in phosphate free-medium was studied by chemical assay of released Pi and by monitoring the loss in radioactivity of cells pre-labelled with ["PI-Pi. The release follows first-order kinetics fairly closely with a rate constant of 7 x 10e3min- approximately. Insulin at a concentration of ~ O - * Mhad no effect on the rate of Pi release and it is concluded therefore, that the insulin-stimulatedaccumulation of Pi described in the literature is the result of hormone action on Pi uptake by liver rather than on its release. KEY WORDS-
Phosphate; liver; hepatocytes; insulin.
INTRODUCTION It was shown nearly 70 years ago that injection of insulin into rabbits caused a rapid fall in blood phosphate Concentration' and it was suggested that insulin stimulates uptake of Pi by the tissues. A number of more recent have tended to support the belief that the hormone promotes accumulation of Pi by the tissues and it has been suggested that the mode of action involves a decrease of the efflux rate from tissues such as heart muscle and Apart from its importance as a constituent of biological molecules including nucleic acids, phospholipids and metabolic intermediates, phosphate is a substrate and/or allosteric effector for many metabolic reactions and most significantly, it is required for ATP synthesk6 Given the central role of the liver in metabolism it is likely that Pi handling in this tissue is under biological control. The hormones insulin and glucagon regulate intermediary metabolism in liver and therefore there is a likelihood that Pi homeostasis in liver is also influenced by these hormones. We report the results of some experiments performed with rat hepatocytes in order to test the effect of insulin on liver phosphate metabolism. MATERIALS AND METHODS Isolation of Hepatocytes
Livers removed from fed Sprague-Dawley rats (weighing 100-150 g) were cut into thin slices and 0263-6484/9 1/010009-04 $05.00 1991 by John Wiley & Sons, Ltd.
c i
digested with collagenase and hyaluronidase dissolved in Krebs-Ringer bicarbonate medium (KRB) using the method described by Fry.' The cells were harvested by centrifugation (MSE Centaur bench centrifuge) at 2000rpm for 30s and bovine serum albumin was added to the final suspension in KRB to a concentration of 1 mgml-'. The Pi concentration in KRB is 1.18 mM. A preparation of cells is obtained within 100min of killing the rat by cervical dislocation. The viability of the hepatocytes, as determined by exclusion of trypan blue, was greater than 90 percent. In some preparations 150 pCi of carrier-free C3*P]-Pi was added at the digestion stage so that the cells were exposed to labelled phosphate for 45 min. Effux of Pi
Freshly isolated cells were suspended in Pi-free KRB and centrifuged for 30s at 2000rpm. The supernatant was removed and the cells were resuspended in Pi-free KRB before centrifugation. The whole washing process was repeated once more and then the cells were suspended finally in Pi-free medium containing 1 mg ml-' of bovine serum albumin. The cells were incubated at 37°C with gentle shaking and under an atmosphere of 95 per cent 0,: 5 per cent C 0 2 . For non-labelled cells, duplicate samples of 0-5 ml were removed from the incubations at timed intervals up to 90 min and the
10
M. PARKASH AND P. J. BUTTERWORTH
supernatant separated by rapid microcentrifugation. A portion of supernatant (0.3 ml) was added to an equal volume of ice-cold trichloroacetic acid (5 per cent). Precipitated protein was removed by centrifugation and 0.2ml of the supernatant was assayed for Pi colorirnetrically.' When the incubations contained labelled cells, duplicate samples (0.3 ml) were removed at timed intervals and the cells were separated from the bathing medium by rapid centrifugation through 0.45 ml of silicone oil/ dinonyl phthalate into 0.05 ml of 3 M perchloric acid.g A 0.1 mi aliquot of the complete incubation medium was transferred to 4 ml of scintillation fluid for determination of radioactivity. The perchloric acid (cell layer) from each sample was also counted as was a 0.1 ml aliquot of the supernatant remaining above the silicone oil layer.
I
Prorein Assay
A Biuret method was used' with bovine serum albumin as standard. RESULTS AND DISCUSSION The pool of Pi within cells exchanges readily with a fraction of the cellular organic phosphate pool and also with Pi in the external medium." The analysis of data obtained in experiments in which the efflux of C3'P]-Pi from prelabelled cells is monitored can be complex because the specific radioactivity of the cellular Pi is changing throughout the experiment consequent to inflow of unlabelled phosphate from outside and by flux to and from the organic phosphate ~ 0 0 1 . ~In' order to simplify the analysis we chose to study efflux under conditions of zero external Pi and following lengthy preincubations to bring the specific radioactivity of cellular Pi and organic phosphate to a uniform state. This condition can be represented as follows: G
0
0.3
30 60 MINUTES
90
r
0.2
-
a0 t
a
U
0
k
Organic P =:Pi' -+ Pio where the superscripts i and o refer to intracellular and extracellular respectively, G is the flux between cellular Pi and organic phosphate and k is the rate constant for efflux of Pi across the plasma membrane. If the specific radioactivity of exchangeable phosphate within the cell has reached a uniform level, Pi' is at a steady state and Pi" is zero, efflux measurements provide information about k directly.
0
30
60 MINUTES
90
Figure 1. Release of phosphate from hepatocytes suspended in Pi-free medium. At the indicated times, the Pi content of the medium was determined colorimetrically. (a) Results from four experiments using different batches of cells provided the data points (Mean +/- SEM). (b) Semi-logarithmic plot of the Means shown in a. A correlation coefficient r, of 0.993 indicates that the efflux obeys first order kinetics.
I1
PHOSPHATE EFFLUX FROM HEPATOCYTES
Figure l a shows the results of Pi efflux from hepatocytes. The data were obtained by chemical assay of Pi and therefore should be free of the complications that can arise using labelled cells (see above). The data are well fitted by a first order plot (Figure Ib) with an efflux constant of 7.35 x loT3min-'. When the rate of efflux was determined by monitoring the time-dependent loss of radioactivity of pre-labelled hepatocytes, the results were somewhat anomalous in that a reproducible sudden fall in the degree of labelling occurs followed by a 'regain' and subsequent loss that follows first-order kinetics (Figure 2). The initial loss may represent the removal of C32PJ-Pi that is non-covalently attached to the outer face of the plasma membrane. The regain is difficult to explain on this basis unless there is some internalization of this (assumed) noncovalently bound material during incubation in Pifree medium. The rate constant for the first order and agrees reasonably well phase is 6.35 x with the value obtained in the experiment of Figure 1. The rate constant is about 20-fold less than for Pi efflux across the basolateral membrane of chick kidney proximal tubule cells.12 The faster rate of the latter process presumably reflects the functional requirement of allowing Pi reabsorbed from the
"
t
1
I 0
I
I
30 60 MINUTES
1
90
Figure 2. Decrease in the 32Pcontent of prelabelled cells on incubation in Pi-free medium. At each time point the radioactivity, expressed as counts per minute, is represented as a percentage of the radioactivity at the onset of the incubation. A,cells incubated in Pi-free medium (r = 0.977 for 30-90 min period); 0, cells incubated in Pi-free medium containing !0-8M insulin ( r = 0.926 for 30-60 min period).
'r rn 0 T-
X I
i
-
a U
0
30
60
90
MINUTES Figure 3. Efflux of 32Pfrom pre-labelled cells. The radioactivity of the medium is expressed as counts per minute. A,cells incubated in Pi-free medium. 0,cells incubated in Pi-free insulin. The data show the means medium containing 1 0 - 8 ~ +/--SEM of three experiments performed with different batches of cells.
glomerular filtrate to be returned to the plasma at a significant rate. When IO-*M insulin was present during the efflux period the first order rate constant calculated from data in Figure 2 is 9.3 x 10-3min-1. This value does not differ significantly from the nonstimulated state and we conclude that insulin does not decrease the rate of efflux of Pi from hepatocytes. Figure 3 shows the effect of insulin on release of 32Pi from prelabelled cells into the bathing medium and it seems that insulin has no effect on Pi efflux. It must be concluded that the stimulation of the accumulation of Pi by liver that has been reported in the literature represents an action on the uptake mechanism into the cells rather than on a release process. ACKNOWLEDGEMENT The expenses of this investigation were defrayed in part by a grant from the Wellcome Trust awarded to P.J.B. REFERENCES 1.
Wigglesworth, V. B., Woodrow, C. E., Smith, W. and Winter, L. B. (1922). On the effect of insulin on blood phosphate. J. Physiol. (Land), 51,447-449.
12 2. Hepp, D., Challoner, D. R. and Williams, R. H. (1968). Studies on the action of insulin in isolated adipose tissue cells. J. Bid. Chem., 243, 4020-4026. 3. Medina, G . and Illingworth, J. (1980). Some factors affecting phosphate transport in a perfused heart preparation. Biochem. J., 188,297-311. 4. Medina, G . and Illingworth, J. (1984). Some hormonal eflects on myocardial phosphate efflux. Biochem. J., 244, 153-162. 5. De Venanzi, F., Pena, F., Jimenez, V. 0.and De Alvarado, H. (1974). Effect of glucagon, epinephrine, cyclic 3'3AMP, N6-2'-0-dibutyryl cyclic 3',5'-AMP and insulin upon phosphate exchange of the isolated perfused fed rat liver. Endocrinol., 741, 741 -748. 6. Sestoft, L. and Bartels, P. D. (1981). Regulation of metabolism by inorganic phosphate. In: Shorf-term Regularion of Liver Merabolism (Hue, L. and Van der Werve, G., eds) Elsevier/North Holland Biomedical Press: Amsterdam, New York and Oxford. 7. Fry, J. R. (1981). Preparation of mammalian hepatocytes. Methodr Enzymol., 77, 130- 137.
M.PARKASH AND P. J. BUTTERWORTH 8. Plummer, D. T. (1987). An Introduction to Practical Biochemistry, 3rd edn, McGraw-Hill Book Co: U.K. 9. Jahan, M. and Butterworth, P. J. (1988). Study of the mechanism by which the Na'-Pi co-transporter of mouse kidney proximal tubule cells adjusts to phosphate depletion. Biochem. J., 252, 105-109. 10. Kemp, G . J., Bevington, A., Khodja, D. and Russell, R. G . G . (1988). Net fluxes of orthophosphate across the plasma membrane in human red cells following alteration of pH and extracellular Pi concentration. Biochim. Biophys. Acfa, 969, 148-157. 11. Kemp, G . J., Bevington, A. and Russell, R. G. G. (1988). Theoretical interpretation of isotope labelling experiments in cells in which the label is chemically incorporated: the example of orthophosphate. J. Theor. Biol., 134, 351-364. 12. Myint, S . and Butterworth, P. J. (1989). Phosphate transport across the basolateral membrane of chick kidney proximal tubule cells. Cell Biochem. Funcr., 7, 43-49. Received 10 August 1990 Accepted 21 August 1990
VOL.9: 9-12 (1991)
Efflux of Inorganic Phosphate from Rat Hepatocytes: Lack of Effect of Insulin MUKESH PARKASH AND PETER J. BUTTERWORTH Department of Biochemistry, King's College (London), Campden Hill Road, London W8 7AH, U K
Efflux of Pi from rat hepatocytes suspended in phosphate free-medium was studied by chemical assay of released Pi and by monitoring the loss in radioactivity of cells pre-labelled with ["PI-Pi. The release follows first-order kinetics fairly closely with a rate constant of 7 x 10e3min- approximately. Insulin at a concentration of ~ O - * Mhad no effect on the rate of Pi release and it is concluded therefore, that the insulin-stimulatedaccumulation of Pi described in the literature is the result of hormone action on Pi uptake by liver rather than on its release. KEY WORDS-
Phosphate; liver; hepatocytes; insulin.
INTRODUCTION It was shown nearly 70 years ago that injection of insulin into rabbits caused a rapid fall in blood phosphate Concentration' and it was suggested that insulin stimulates uptake of Pi by the tissues. A number of more recent have tended to support the belief that the hormone promotes accumulation of Pi by the tissues and it has been suggested that the mode of action involves a decrease of the efflux rate from tissues such as heart muscle and Apart from its importance as a constituent of biological molecules including nucleic acids, phospholipids and metabolic intermediates, phosphate is a substrate and/or allosteric effector for many metabolic reactions and most significantly, it is required for ATP synthesk6 Given the central role of the liver in metabolism it is likely that Pi handling in this tissue is under biological control. The hormones insulin and glucagon regulate intermediary metabolism in liver and therefore there is a likelihood that Pi homeostasis in liver is also influenced by these hormones. We report the results of some experiments performed with rat hepatocytes in order to test the effect of insulin on liver phosphate metabolism. MATERIALS AND METHODS Isolation of Hepatocytes
Livers removed from fed Sprague-Dawley rats (weighing 100-150 g) were cut into thin slices and 0263-6484/9 1/010009-04 $05.00 1991 by John Wiley & Sons, Ltd.
c i
digested with collagenase and hyaluronidase dissolved in Krebs-Ringer bicarbonate medium (KRB) using the method described by Fry.' The cells were harvested by centrifugation (MSE Centaur bench centrifuge) at 2000rpm for 30s and bovine serum albumin was added to the final suspension in KRB to a concentration of 1 mgml-'. The Pi concentration in KRB is 1.18 mM. A preparation of cells is obtained within 100min of killing the rat by cervical dislocation. The viability of the hepatocytes, as determined by exclusion of trypan blue, was greater than 90 percent. In some preparations 150 pCi of carrier-free C3*P]-Pi was added at the digestion stage so that the cells were exposed to labelled phosphate for 45 min. Effux of Pi
Freshly isolated cells were suspended in Pi-free KRB and centrifuged for 30s at 2000rpm. The supernatant was removed and the cells were resuspended in Pi-free KRB before centrifugation. The whole washing process was repeated once more and then the cells were suspended finally in Pi-free medium containing 1 mg ml-' of bovine serum albumin. The cells were incubated at 37°C with gentle shaking and under an atmosphere of 95 per cent 0,: 5 per cent C 0 2 . For non-labelled cells, duplicate samples of 0-5 ml were removed from the incubations at timed intervals up to 90 min and the
10
M. PARKASH AND P. J. BUTTERWORTH
supernatant separated by rapid microcentrifugation. A portion of supernatant (0.3 ml) was added to an equal volume of ice-cold trichloroacetic acid (5 per cent). Precipitated protein was removed by centrifugation and 0.2ml of the supernatant was assayed for Pi colorirnetrically.' When the incubations contained labelled cells, duplicate samples (0.3 ml) were removed at timed intervals and the cells were separated from the bathing medium by rapid centrifugation through 0.45 ml of silicone oil/ dinonyl phthalate into 0.05 ml of 3 M perchloric acid.g A 0.1 mi aliquot of the complete incubation medium was transferred to 4 ml of scintillation fluid for determination of radioactivity. The perchloric acid (cell layer) from each sample was also counted as was a 0.1 ml aliquot of the supernatant remaining above the silicone oil layer.
I
Prorein Assay
A Biuret method was used' with bovine serum albumin as standard. RESULTS AND DISCUSSION The pool of Pi within cells exchanges readily with a fraction of the cellular organic phosphate pool and also with Pi in the external medium." The analysis of data obtained in experiments in which the efflux of C3'P]-Pi from prelabelled cells is monitored can be complex because the specific radioactivity of the cellular Pi is changing throughout the experiment consequent to inflow of unlabelled phosphate from outside and by flux to and from the organic phosphate ~ 0 0 1 . ~In' order to simplify the analysis we chose to study efflux under conditions of zero external Pi and following lengthy preincubations to bring the specific radioactivity of cellular Pi and organic phosphate to a uniform state. This condition can be represented as follows: G
0
0.3
30 60 MINUTES
90
r
0.2
-
a0 t
a
U
0
k
Organic P =:Pi' -+ Pio where the superscripts i and o refer to intracellular and extracellular respectively, G is the flux between cellular Pi and organic phosphate and k is the rate constant for efflux of Pi across the plasma membrane. If the specific radioactivity of exchangeable phosphate within the cell has reached a uniform level, Pi' is at a steady state and Pi" is zero, efflux measurements provide information about k directly.
0
30
60 MINUTES
90
Figure 1. Release of phosphate from hepatocytes suspended in Pi-free medium. At the indicated times, the Pi content of the medium was determined colorimetrically. (a) Results from four experiments using different batches of cells provided the data points (Mean +/- SEM). (b) Semi-logarithmic plot of the Means shown in a. A correlation coefficient r, of 0.993 indicates that the efflux obeys first order kinetics.
I1
PHOSPHATE EFFLUX FROM HEPATOCYTES
Figure l a shows the results of Pi efflux from hepatocytes. The data were obtained by chemical assay of Pi and therefore should be free of the complications that can arise using labelled cells (see above). The data are well fitted by a first order plot (Figure Ib) with an efflux constant of 7.35 x loT3min-'. When the rate of efflux was determined by monitoring the time-dependent loss of radioactivity of pre-labelled hepatocytes, the results were somewhat anomalous in that a reproducible sudden fall in the degree of labelling occurs followed by a 'regain' and subsequent loss that follows first-order kinetics (Figure 2). The initial loss may represent the removal of C32PJ-Pi that is non-covalently attached to the outer face of the plasma membrane. The regain is difficult to explain on this basis unless there is some internalization of this (assumed) noncovalently bound material during incubation in Pifree medium. The rate constant for the first order and agrees reasonably well phase is 6.35 x with the value obtained in the experiment of Figure 1. The rate constant is about 20-fold less than for Pi efflux across the basolateral membrane of chick kidney proximal tubule cells.12 The faster rate of the latter process presumably reflects the functional requirement of allowing Pi reabsorbed from the
"
t
1
I 0
I
I
30 60 MINUTES
1
90
Figure 2. Decrease in the 32Pcontent of prelabelled cells on incubation in Pi-free medium. At each time point the radioactivity, expressed as counts per minute, is represented as a percentage of the radioactivity at the onset of the incubation. A,cells incubated in Pi-free medium (r = 0.977 for 30-90 min period); 0, cells incubated in Pi-free medium containing !0-8M insulin ( r = 0.926 for 30-60 min period).
'r rn 0 T-
X I
i
-
a U
0
30
60
90
MINUTES Figure 3. Efflux of 32Pfrom pre-labelled cells. The radioactivity of the medium is expressed as counts per minute. A,cells incubated in Pi-free medium. 0,cells incubated in Pi-free insulin. The data show the means medium containing 1 0 - 8 ~ +/--SEM of three experiments performed with different batches of cells.
glomerular filtrate to be returned to the plasma at a significant rate. When IO-*M insulin was present during the efflux period the first order rate constant calculated from data in Figure 2 is 9.3 x 10-3min-1. This value does not differ significantly from the nonstimulated state and we conclude that insulin does not decrease the rate of efflux of Pi from hepatocytes. Figure 3 shows the effect of insulin on release of 32Pi from prelabelled cells into the bathing medium and it seems that insulin has no effect on Pi efflux. It must be concluded that the stimulation of the accumulation of Pi by liver that has been reported in the literature represents an action on the uptake mechanism into the cells rather than on a release process. ACKNOWLEDGEMENT The expenses of this investigation were defrayed in part by a grant from the Wellcome Trust awarded to P.J.B. REFERENCES 1.
Wigglesworth, V. B., Woodrow, C. E., Smith, W. and Winter, L. B. (1922). On the effect of insulin on blood phosphate. J. Physiol. (Land), 51,447-449.
12 2. Hepp, D., Challoner, D. R. and Williams, R. H. (1968). Studies on the action of insulin in isolated adipose tissue cells. J. Bid. Chem., 243, 4020-4026. 3. Medina, G . and Illingworth, J. (1980). Some factors affecting phosphate transport in a perfused heart preparation. Biochem. J., 188,297-311. 4. Medina, G . and Illingworth, J. (1984). Some hormonal eflects on myocardial phosphate efflux. Biochem. J., 244, 153-162. 5. De Venanzi, F., Pena, F., Jimenez, V. 0.and De Alvarado, H. (1974). Effect of glucagon, epinephrine, cyclic 3'3AMP, N6-2'-0-dibutyryl cyclic 3',5'-AMP and insulin upon phosphate exchange of the isolated perfused fed rat liver. Endocrinol., 741, 741 -748. 6. Sestoft, L. and Bartels, P. D. (1981). Regulation of metabolism by inorganic phosphate. In: Shorf-term Regularion of Liver Merabolism (Hue, L. and Van der Werve, G., eds) Elsevier/North Holland Biomedical Press: Amsterdam, New York and Oxford. 7. Fry, J. R. (1981). Preparation of mammalian hepatocytes. Methodr Enzymol., 77, 130- 137.
M.PARKASH AND P. J. BUTTERWORTH 8. Plummer, D. T. (1987). An Introduction to Practical Biochemistry, 3rd edn, McGraw-Hill Book Co: U.K. 9. Jahan, M. and Butterworth, P. J. (1988). Study of the mechanism by which the Na'-Pi co-transporter of mouse kidney proximal tubule cells adjusts to phosphate depletion. Biochem. J., 252, 105-109. 10. Kemp, G . J., Bevington, A., Khodja, D. and Russell, R. G . G . (1988). Net fluxes of orthophosphate across the plasma membrane in human red cells following alteration of pH and extracellular Pi concentration. Biochim. Biophys. Acfa, 969, 148-157. 11. Kemp, G . J., Bevington, A. and Russell, R. G. G. (1988). Theoretical interpretation of isotope labelling experiments in cells in which the label is chemically incorporated: the example of orthophosphate. J. Theor. Biol., 134, 351-364. 12. Myint, S . and Butterworth, P. J. (1989). Phosphate transport across the basolateral membrane of chick kidney proximal tubule cells. Cell Biochem. Funcr., 7, 43-49. Received 10 August 1990 Accepted 21 August 1990
