- Email: [email protected]
[4] Transport of alanine across hepatocyte plasma membrane
[4]
HEPATOCYTE ALANINE TRANSPORT
31
sic membrane proteins with molecular weights of 54,000 and 49,000. Similar results were obtained when labeling was performed on intact hepatocytes followed by membrane isolation and SDS-gel analysis. The specificity of the labeling reaction was assessed by carrying out the photolysis of the 7-ADTC-membrane complex in the presence of taurocholic acid. As shown in Fig. 3, the presence of the natural substrate resulted in a large decrease in the labeling of the 68,000, 54,000, and 49,000 proteins. These results suggest a high degree of labeling specificity. Further support for the specificity of this labeling reaction is obtained from studies with hepatoma tissue culture (HTC) cells which have been shown to lack a functional bile acid transport system.4 When photolysis of this cell system was performed in the presence of 7-ADTC, no significant incorporation of radioactivity could be detected (Fig. 3), further supporting the validity of the labeling results. These studies indicate that the photoreactive derivative of taurocholic acid (7-ADTC) is a substrate for the hepatocyte bile acid transport system and specifically recognizes and covalently labels two intrinsic membrane proteins in purified sinusoidal plasma membranes as well as in intact hepatocytes. Subsequent studies using monoclonal antibodies ~2have demonstrated that the 49 K protein represents the Na+-dependent bile acid transport system. Acknowledgments This researchwas supportedby National Institutesof Health Grant DK-25836. J2M. Ananthanarayanan,P. yon Dippe, and D. Levy,J. Biol. Chem. 263, 8338 (1988).
[4] T r a n s p o r t o f A l a n i n e a c r o s s H e p a t o c y t e Plasma Membranes By JOHN D. McGIVAN
The metabolism of alanine to urea and glucose is a major metabolic function of the liver. The initial step in alanine metabolism is the Na+-de pendent transport of alanine across the cell membrane, and this process has been widely investigated. Alanine transport in liver is inducible in starvation and is subject to both short- and long-term control by hormones. There is evidence that the transport of alanine at physiological METHODS IN ENZYMOLOGY, VOL. 174
Copyright© 1989by AcademicPress, Inc. All rightsof reproductionin any form reserved.
32
TRANSPORT IN SUBCELLULAR ORGANELLES OF ANIMALS
[4]
concentrations is an important rate-determining step in alanine metabolism. The general properties of the transport of alanine and other amino acids in hepatocytes have been reviewed.l,2 The purpose of this chapter is to review the methods which have been used for the study of alanine transport in isolated hepatocytes and in liver plasma membrane vesicles. The principles of the approaches described can be applied to study the transport of other naturally occurring amino acids in suspensions of hepatocytes and in other cell systems. This chapter does not consider the study of the transport of amino acid analogs, the identification of individual transport systems, or the measurement of transport in cultured hepatocytes; these important subjects are dealt with elsewhere in this volume.
Transport of Alanine in Isolated Hepatocytes The routine isolation of hepatocytes by collagenase digestion of rat liver is described in detail elsewhere. 3 The hepatocytes are washed and suspended in Krebs-Henseleit bicarbonate-buffered medium containing 2% dialyzed bovine serum albumin. The presence of bicarbonate in the medium is essential, since its omission greatly reduces the rate of alanine transport? The cells can be used for transport studies after a short preincubation in the incubation medium, but it is sometimes convenient to store the suspension on ice for up to 2 hr before use. In this case the cells should be incubated at 37 ° for 20 rain before transport studies are initiated in order to allow the reestablishment of ion gradients across the membrane. For the accurate measurement of alanine transport in hepatocytes, there are three major requirements. First, if radioactive techniques are to be used, the metabolism ofalanine must be inhibited so that the radioactivity in the cells accurately represents the internal concentration of alanine. This can be achieved by the addition of 0.5 m M aminooxyacetate, which is a specific inhibitor of transaminase enzymes and does not reduce the ATP content of the cells. It is not necessary to add such an inhibitor if the amino acid used is metabolized only very slowly (as is the case with leucine) or if the incubation period is less than about 1 min (when little metabolism will have occurred). Second, since the cell pellet on centrifugation is contaminated with a considerable quantity of extracellular water, a marker of the extracellular space should be included. Inulin, polyethylene glycol, or sucrose are suitable compounds to use for this purpose. Third, the method of termination of the transport reaction by separation of cells from the meM. A. Shotwell, M. S. Kilberg, and D. L. Oxender, Biochim. Biophys. Acta 737, 267 (1983). 2 M. S. Kilberg, E. F. Barber, and M. E. Handlogten, Curr. Top. Cell. Regul. 25, 133 (1985). 3 M. S. Kilberg, this series. 4 j. D. McGivan, Biochem. J. 182, 697 (1979).
[4]
HEPATOCYTE ALANINE TRANSPORT
33
dium must also achieve the rapid deproteinization of the cells to prevent any possible metabolism of the substrate, and this is commonly done by centrifuging the cells through a mixture of silicone oil and dinonyl phthalate into perchloric acid. A suitable protocol for the measurement of alanine transport into hepatocytes is as follows:5 Tubes for a Beckman Microcentrifuge Model B are prepared by placing 50/zl perchloric acid (15%, v/v) in the bottom of the tube and layering 0.1 ml of a mixture (1 : 1, v/v) of silicone fluid MS550 and dinonyl phthalate above this. The hepatocyte suspension containing 10- 12 mg cell protein/ml is incubated with an equal volume of KrebsHenseleit bicarbonate medium containing [3H]inulin together with the appropriate concentration of alanine, [~4C]alanine, and 0.5 m M aminooxyacetate. Transport is terminated by layering a 0.25-ml aliquot of the incubation (containing not more than 3 mg of protein) above the silicone oil layer and centrifuging for a minimum of 10 sec. After withdrawal of a sample of the supernatant layer for counting, the tube is frozen and cut through the silicone oil layer with a sharp knife. The entire pellet is shaken with scintillation fluid, and the ~4C and 3H in the pellet and supernatant samples are determined by dual-label scintillation counting. The intracellular alanine is calculated as the total alanine in the pellet minus that in the extracellular water. If it is required to find the intracellular concentration of alanine, measurements of the internal volume of the cells must be performed in parallel using [carboxy-~4C]inulin and 3H20. The internal volume of hepatocytes determined by this method is 1.8- 2.0/d/mg protein.
Role of Alanine Transport in Regulation of Alanine Metabolism The transport of alanine across the cell plasma membrane is the first reaction in alanine metabolism, and it is of importance to determine whether there are conditions under which the rate of transport is rate-limiting for the subsequent metabolism of this amino acid. Identification of transport as a rate-limiting step requires the fulfilment of a number of criteria: (1) The rate of transport, measured in the absence of metabolism, must approximate the rate of alanine metabolism at the same alanine concentration. (2) Specific inhibitors of transport must inhibit the rate of metabolism with the same inhibitor concentration dependence. (3) The steady-state concentration ratio of intracellular to extracellular alanine during alanine metabolism should be low and should be greatly increased by the addition of aminooxyacetate to inhibit metabolism. (4) Stimulation of transport should lead to an increase in the intracellular to extracellular alanine concentration ratio associated with an increase in the rate of 5 S. K. Joseph, N. M. Bradford, and J. D. McGivan, Biochem. J. 176, 827 (1978).
34
TRANSPORT IN SUBCELLULAR ORGANELLES OF ANIMALS
[4]
alanine metabolism. If any of the criteria fail to apply under particular conditions, then alanine transport under those conditions is unlikely to exert a significant controlling influence on metabolism. In order to study this problem in isolated hepatocytes in suspension, alanine metabolism is best measured by the disappearance of substrate rather than by the appearance of products since the endogenous rate of formation of glucose in hepatocytes is considerable. Alanine is conveniently estimated using the NAD-linked bacterial enzyme alanine dehydrogenase. 6 After deproteinization of the cell suspension and removal of the precipitated protein by centrifugation, the supernatant is neutralized by the addition of a small volume of 3 M KOH or 3 M K2CO3. After addition of the appropriate buffer and alanine dehydrogenase, NAD + reduction is measured by spectrophotometric or fluorometric techniques depending on the sensitivity required. For the measurement of intracellular alanine concentrations during metabolism, the cell suspension (12 mg protein/ml) is incubated with the appropriate concentration of alanine in the absence of aminooxyacetate together with 3H20 and [carboxy-~4C]inulin as markers of the total and extracellular spaces, respectively. Aliquots (0.8 ml) of the cell suspension are layered on to 0.5 ml of silicone oil/dinonyl phthalate (l : l, v/v) which is itself layered above 0.1 ml of 150/o perchloric acid in a tube for the Eppendorf Model 3200 bench centrifuge. The reaction is terminated by the initiation of centrifugation. A sample of the supernatant is immediately acidified and radioactivity and alanine are assayed in the neutralized extract. An aliquot of the acid layer is withdrawn, neutralized with a small volume of K2CO 3 and similarly assayed for radioactivity and alanine. From these data, the extracellular and intracellular concentrations of alanine can be calculated. In this type of investigation, alanine transport can be inhibited by titration with ouabain or by reducing the extracellular sodium concentration. Transport can be stimulated by the addition of cyclic AMP. Simultaneous measurements of the rate of metabolism and of changes in the alanine distribution can then be made. Such investigations can also be carried out using the hepatocyte perifusion apparatus of van der Meet and Tager. 7 In a perifusion system a true steady state can be obtained at a constant low external alanine concentration. Centrifugal filtration techniques similar to those described above can be used for the measurement of intracellular alanine concentration. Using such a system, Sips e t al. s showed that the intracellular concentration of alanine remained low on increasing the extracellular concentration of 6 D. H. Williamson, O. Lopes Vereira, and B. Walker, Biochem. J. 104, 497 (1967). 7 R. van der Meet and J. M. Tager, FEBSLett. 67, 36 (1976). s H. J. Sips, A. K. Groen, andJ. M. Tager, FEBSLett. 119, 271 (1980).
[4]
HEPATOCYTE ALANINE TRANSPORT
35
alanine in the physiological range, and the addition of aminooxyacetate greatly increased the intracellular to extracellular concentration ratio. Using these various approaches, data from several laboratories indicate that alanine transport limits the rate of alanine metabolism at low alanine concentrations (<0.5 raM). s-m There is less agreement about the limitation of metabolism by transport at higher concentrations of alanine. Influence o f Cell Plasma Membrane Potential on Alanine Transport
Na + - alanine cotransport is an electrogenic process. Accordingly both the steady-state concentration ratio of alanine in the absence of metabolism as well as the kinetics of alanine transport should be influenced by the cell membrane potential. It is therefore important to be able to observe the effect of varying the cell membrane potential on the initial rate of transport. The measurement of cell membrane potential is isolated hepatocytes is not straightforward. Direct electrophysiological measurements involving the impalement of cells with microelectrodes has not so far yielded satisfactory results. In a number of cell types, the cell membrane potential has been calculated from the distribution ratio of permeant cations or from the absorption of fluorescence of cationic dyes. This approach is not feasible in hepatocytes, which contain large numbers of mitochondria that accumulate such permeant cations to a high intramitochondrial concentration. In hepatocytes, such measurements tend to reflect the mitochondrial membrane potential rather than that of the cell plasma membrane. In principle, the membrane potential can be calculated from the distribution of permeant anions, since these are not accumulated by the mitochondria. Thiocyanate has been used for this purpose by Hoek et al., ~ but these authors found it necessary to correct for the assumed binding of thiocyanate to intracellular constituents. Recently, Edmondson et al. ~2 have also used thiocyanate distribution as an indicator of cell membrane potential. In this study, digitonin was used to disrupt the cells and measure the cytosolic space. The thiocyanate technique remains somewhat unsatisfactory because of the various necessary correction factors involved and because of the lack of independent confirmation that changes in thiocyanate distribution in fact accurately measure changes in membrane potential. An alternative to the use of thiocyanate is to calculate the membrane potential from the distribution of chloride using 36C1-. It has been shown in 9 j. D. McGivan, J. C. Ramsell, and J. H. Lacey, Biochim. Biophys. Acta 644, 295 (1981). ~0p. Fafarnoux, C. Remesy, and C. Demigne, Biochem. J. 210, 645 (1983). ~l j. B. Hoek, D. G. Nicholls, and J. R. WiUiamson, J. Biol. Chem. 255, 1458 (1980). 12 j. W. Edmondson, B. A. Miller, and L. Lumeng, Am. J. Physiol. 249, G427 (1985).
36
TRANSPORT IN SUBCELLULAR ORGANELLES OF ANIMALS
[4]
this laboratory that chloride distribution in hepatocytes is not affected by inhibitors which inhibit chloride transport systems in other cell types. The membrane potential calculated from chloride distribution under a number of different conditions is equal to the potential measured directly using microelectrodes in perfused liver under the same conditions.la It therefore appears that chloride distributes passively across the cell membrane according to the membrane potential. Using this technique, a good correlation between cell membrane potential and initial rate of alanine transport can be demonstrated, and this relationship is independent of the mechanism by which the potential is varied. Chloride distribution can be measured by incubating cells in the presence of 36C1- together with [3H]inulin to measure the extracellular space. Dual-channel scintillation counting for 3H plus 36C1 is achieved as for aH plus 14C, but quench curves appropriate for 36C1 rather than ~4C must be used. While the use of chloride to determine the membrane potential may be preferable to that of thiocyanate, it must be recognized that the validity of both methods requires further independent justification. Alanine Transport in Liver Plasma Membrane Vesicles
The study of transport in plasma membrane vesicles allows the investigation of electrogenicity of the transport process and its ion dependence and obviates problems arising from possible substrate metabolism. Demonstration of transport in membrane vesicles is also a necessary prerequisite for the eventual identification of the carrier protein. Alanine transport in liver membrane vesicles was first reported by van Amelsvoort et al., ~4 and a modification of their method which is routinely used in this laboratory is detailed below. A single rat liver is homogenized in a medium containing 0.25 M sucrose, 10 m M K + - H E P E S , and 0.2 m M CaC12 at pH 7.4 and 4 ° initially by 6 strokes of a Teflon/glass Dounce homogenizer. The homogenate is diluted to 200 ml with the same medium and rehomogenized in 30-ml aliquots using a glass-glass homogenizer with approximately 20 strokes. This second homogenization is critical if satisfactory intact vesicles are to be obtained. After filtration through muslin to remove particles of fat, 1 m M EDTA (final concentration) is added. The homogenate is centrifuged at 1000 g for 10 min, and the pellet is discarded. The supernatant from the centrifugation is recentrifuged at 20,000 g for 30 rain, and the pellet is resuspended using the glass-glass homogenizer in 10 ml of homogenization medium to which 1 m M EDTA has been added. 13 N. M. Bradford, M. R. Hayes, and J. D. McGivan, Biochim. Biophys. Acta 845, 10 (1985). 14j. M. M. van Amelsvoort, H. J. Sips, and K. van Dam, Biochem. J. 174, 1083 (1978).
[4]
HEPATOCYTE ALANINE TRANSPORT
37
Discontinuous sucrose gradients are prepared in tubes for the 3 × 25 ml swing-out head of the M.S.E. 65 ultracentrifuge. Each gradient consists of a lower layer of l0 ml of 46.5% sucrose (w/v) and an upper layer of l0 ml of 21.5% (w/v) sucrose, each solution being made up in l0 m M K+-HEPES, pH 7.5. Three milliliters of suspension is layered on top of each gradient, and the tubes are centrifuged at 23,000 rpm (50,000 g) for 2.5 h at 4 °. The material at the interface between the two sucrose solutions is collected and diluted at least 4 times with the original homogenization medium (containing no EDTA). The resulting suspension is centrifuged at 100,000 g for 40 min, and the pellet is resuspended in approximately 2 ml of homogenization medium using a syringe. The membrane suspension is rapidly frozen in small volumes in liquid nitrogen, and can be kept frozen for some weeks before use in transport experiments. This preparation routinely yields approximately 10 mg of protein, and the specific activity of the plasma membrane marker 5'-nucleotidase is increased 10-fold over the original homogenate. For measurement of transport, 20/tl of membrane suspension is added to an equal volume of incubation medium to give the following final concentrations: 0.25 M sucrose, 10 m M K+-HEPES, 0.2 m M CaC12, 5 m M MgCI2, 100 m M KCNS or 100 m M NaCNS, and 0.1 m M [3H]alanine at pH 7.5. A suitable specific activity for such experiments is 100 Ci/ mol, i.e., approximately 200 dpm/pmol. The reaction is terminated by the addition of 1 ml of ice-cold "stopping" solution containing 0.25 M sucrose, 10 m M K+-HEPES, 0.2 m M CaC12, plus 0.2 M NaC1 at pH 7.5. The diluted suspension is immediately filtered through millipore filters (HAWP 0.45 #m) and washed with 2 × 1 ml of stopping solution. The filters are dissolved in a suitable scintillator for measurement of radioactivity. In this method, the NaCNS provides the necessary sodium and electrical gradient for Na+-alanine cotransport. Parallel experiments using KCNS provide a measure of binding of alanine to the membranes, retention of residual radioactivity on the membrane filters, plus any Na+-inde pendent alanine transport. As measured by the above method, the maximum uptake of alanine should be 0.25- 0.3 nmol/mg protein at an external alanine concentration of 0.1 raM, and this should be attained in 30 sec. Lower values indicate a less than satisfactory membrane preparation. After the vesicles have been thawed the transport activity progressively declines. The transport of alanine in this stem has been extensively investigated? 4- ~6
15 H. J. Sips, J. H. H. van Amelsvoort, and K. van Dam, Eur. J. Biochem. 105, 217 (1980). 16 H. J. Sips, and K. van Dam, J. Membr. Biol. 62, 231 (1981).
38
TRANSPORT IN SUBCELLULAR ORGANELLES OF ANIMALS
[4]
Approaches to Identification of Protein(s) Responsiblefor Alanine Transport in Liver Cell Membrane The alanine transport protein or proteins in liver plasma membranes have not yet been identified. The identification of such proteins would be of great interest, in particular because this would allow a detailed investigation of the hormonal induction of synthesis of transport proteins and their insertion into the plasma membrane. The successful identification of various transport proteins in mammalian cell membranes has relied mainly on the use of specific tight-binding inhibitors which can be used to label the transport protein. 17 No such inhibitor has so far been identified for the alanine carder in liver. Potential inhibitors of transport are best assessed by a study of their action on alanine transport in plasma membrane vesicles rather than intact cells, since inhibition in cells may be due to nonspeeific effects of the inhibitors on cell ATP levels. Using this approach, it has been shown that various sulfhydryl-blocking reagents (e.g., mersalyl, N-ethylmaleimide 18) inhibit alanine transport in membrane vesicles. The inhibition is due to an effect of the reagents on the carrier molecule rather than to nonspecific membrane damage. However, progress toward carrier identification is dependent on the unambiguous demonstration of protection of the cartier against the inhibitor by high concentrations of substrate, and this has proved difficult to achieve. A second approach to the identification of the transport protein involves reconstitution of various liver cell membrane fractions into artificial phospholipid membranes. Although such reconstitution has been reported for the alanine-transporting systems of kidney brush border membranes 19 and Ehrlich ascites cells, 2° the methods used do not produce satisfactory results when applied to liver. Modifications of these methods may be appropriate for the liver system, but this has not yet been demonstrated. In principle, a combination of labeling and reconstitution should lead to the identification of the alanine carrier in liver cell membranes, and other possible approaches to the problem exist. At present, however, the literature contains few reports of work in this area on liver amino acid transport system. ~7G. Semenza, M. Kessler, M. Hosang, J. Weber, and U. Schmidt, Biochim. Biophys. Acta 779, 343 (1984). ~s M. R. Hayes and J. D. MeGivan, Biochem. J. 214, 489 (1983). ~9H. Koepsell, K. Korn, D. Ferguson, H. Menuhr, D. OUig, and W. Haase, J. Biol. Chem. 259, 6548 (1984). 2o j. I. McCormick, D. Tsang, and R. M. Johnstone, Arch. Biochem. Biophys. 231, 355 (1984).
HEPATOCYTE ALANINE TRANSPORT
31
sic membrane proteins with molecular weights of 54,000 and 49,000. Similar results were obtained when labeling was performed on intact hepatocytes followed by membrane isolation and SDS-gel analysis. The specificity of the labeling reaction was assessed by carrying out the photolysis of the 7-ADTC-membrane complex in the presence of taurocholic acid. As shown in Fig. 3, the presence of the natural substrate resulted in a large decrease in the labeling of the 68,000, 54,000, and 49,000 proteins. These results suggest a high degree of labeling specificity. Further support for the specificity of this labeling reaction is obtained from studies with hepatoma tissue culture (HTC) cells which have been shown to lack a functional bile acid transport system.4 When photolysis of this cell system was performed in the presence of 7-ADTC, no significant incorporation of radioactivity could be detected (Fig. 3), further supporting the validity of the labeling results. These studies indicate that the photoreactive derivative of taurocholic acid (7-ADTC) is a substrate for the hepatocyte bile acid transport system and specifically recognizes and covalently labels two intrinsic membrane proteins in purified sinusoidal plasma membranes as well as in intact hepatocytes. Subsequent studies using monoclonal antibodies ~2have demonstrated that the 49 K protein represents the Na+-dependent bile acid transport system. Acknowledgments This researchwas supportedby National Institutesof Health Grant DK-25836. J2M. Ananthanarayanan,P. yon Dippe, and D. Levy,J. Biol. Chem. 263, 8338 (1988).
[4] T r a n s p o r t o f A l a n i n e a c r o s s H e p a t o c y t e Plasma Membranes By JOHN D. McGIVAN
The metabolism of alanine to urea and glucose is a major metabolic function of the liver. The initial step in alanine metabolism is the Na+-de pendent transport of alanine across the cell membrane, and this process has been widely investigated. Alanine transport in liver is inducible in starvation and is subject to both short- and long-term control by hormones. There is evidence that the transport of alanine at physiological METHODS IN ENZYMOLOGY, VOL. 174
Copyright© 1989by AcademicPress, Inc. All rightsof reproductionin any form reserved.
32
TRANSPORT IN SUBCELLULAR ORGANELLES OF ANIMALS
[4]
concentrations is an important rate-determining step in alanine metabolism. The general properties of the transport of alanine and other amino acids in hepatocytes have been reviewed.l,2 The purpose of this chapter is to review the methods which have been used for the study of alanine transport in isolated hepatocytes and in liver plasma membrane vesicles. The principles of the approaches described can be applied to study the transport of other naturally occurring amino acids in suspensions of hepatocytes and in other cell systems. This chapter does not consider the study of the transport of amino acid analogs, the identification of individual transport systems, or the measurement of transport in cultured hepatocytes; these important subjects are dealt with elsewhere in this volume.
Transport of Alanine in Isolated Hepatocytes The routine isolation of hepatocytes by collagenase digestion of rat liver is described in detail elsewhere. 3 The hepatocytes are washed and suspended in Krebs-Henseleit bicarbonate-buffered medium containing 2% dialyzed bovine serum albumin. The presence of bicarbonate in the medium is essential, since its omission greatly reduces the rate of alanine transport? The cells can be used for transport studies after a short preincubation in the incubation medium, but it is sometimes convenient to store the suspension on ice for up to 2 hr before use. In this case the cells should be incubated at 37 ° for 20 rain before transport studies are initiated in order to allow the reestablishment of ion gradients across the membrane. For the accurate measurement of alanine transport in hepatocytes, there are three major requirements. First, if radioactive techniques are to be used, the metabolism ofalanine must be inhibited so that the radioactivity in the cells accurately represents the internal concentration of alanine. This can be achieved by the addition of 0.5 m M aminooxyacetate, which is a specific inhibitor of transaminase enzymes and does not reduce the ATP content of the cells. It is not necessary to add such an inhibitor if the amino acid used is metabolized only very slowly (as is the case with leucine) or if the incubation period is less than about 1 min (when little metabolism will have occurred). Second, since the cell pellet on centrifugation is contaminated with a considerable quantity of extracellular water, a marker of the extracellular space should be included. Inulin, polyethylene glycol, or sucrose are suitable compounds to use for this purpose. Third, the method of termination of the transport reaction by separation of cells from the meM. A. Shotwell, M. S. Kilberg, and D. L. Oxender, Biochim. Biophys. Acta 737, 267 (1983). 2 M. S. Kilberg, E. F. Barber, and M. E. Handlogten, Curr. Top. Cell. Regul. 25, 133 (1985). 3 M. S. Kilberg, this series. 4 j. D. McGivan, Biochem. J. 182, 697 (1979).
[4]
HEPATOCYTE ALANINE TRANSPORT
33
dium must also achieve the rapid deproteinization of the cells to prevent any possible metabolism of the substrate, and this is commonly done by centrifuging the cells through a mixture of silicone oil and dinonyl phthalate into perchloric acid. A suitable protocol for the measurement of alanine transport into hepatocytes is as follows:5 Tubes for a Beckman Microcentrifuge Model B are prepared by placing 50/zl perchloric acid (15%, v/v) in the bottom of the tube and layering 0.1 ml of a mixture (1 : 1, v/v) of silicone fluid MS550 and dinonyl phthalate above this. The hepatocyte suspension containing 10- 12 mg cell protein/ml is incubated with an equal volume of KrebsHenseleit bicarbonate medium containing [3H]inulin together with the appropriate concentration of alanine, [~4C]alanine, and 0.5 m M aminooxyacetate. Transport is terminated by layering a 0.25-ml aliquot of the incubation (containing not more than 3 mg of protein) above the silicone oil layer and centrifuging for a minimum of 10 sec. After withdrawal of a sample of the supernatant layer for counting, the tube is frozen and cut through the silicone oil layer with a sharp knife. The entire pellet is shaken with scintillation fluid, and the ~4C and 3H in the pellet and supernatant samples are determined by dual-label scintillation counting. The intracellular alanine is calculated as the total alanine in the pellet minus that in the extracellular water. If it is required to find the intracellular concentration of alanine, measurements of the internal volume of the cells must be performed in parallel using [carboxy-~4C]inulin and 3H20. The internal volume of hepatocytes determined by this method is 1.8- 2.0/d/mg protein.
Role of Alanine Transport in Regulation of Alanine Metabolism The transport of alanine across the cell plasma membrane is the first reaction in alanine metabolism, and it is of importance to determine whether there are conditions under which the rate of transport is rate-limiting for the subsequent metabolism of this amino acid. Identification of transport as a rate-limiting step requires the fulfilment of a number of criteria: (1) The rate of transport, measured in the absence of metabolism, must approximate the rate of alanine metabolism at the same alanine concentration. (2) Specific inhibitors of transport must inhibit the rate of metabolism with the same inhibitor concentration dependence. (3) The steady-state concentration ratio of intracellular to extracellular alanine during alanine metabolism should be low and should be greatly increased by the addition of aminooxyacetate to inhibit metabolism. (4) Stimulation of transport should lead to an increase in the intracellular to extracellular alanine concentration ratio associated with an increase in the rate of 5 S. K. Joseph, N. M. Bradford, and J. D. McGivan, Biochem. J. 176, 827 (1978).
34
TRANSPORT IN SUBCELLULAR ORGANELLES OF ANIMALS
[4]
alanine metabolism. If any of the criteria fail to apply under particular conditions, then alanine transport under those conditions is unlikely to exert a significant controlling influence on metabolism. In order to study this problem in isolated hepatocytes in suspension, alanine metabolism is best measured by the disappearance of substrate rather than by the appearance of products since the endogenous rate of formation of glucose in hepatocytes is considerable. Alanine is conveniently estimated using the NAD-linked bacterial enzyme alanine dehydrogenase. 6 After deproteinization of the cell suspension and removal of the precipitated protein by centrifugation, the supernatant is neutralized by the addition of a small volume of 3 M KOH or 3 M K2CO3. After addition of the appropriate buffer and alanine dehydrogenase, NAD + reduction is measured by spectrophotometric or fluorometric techniques depending on the sensitivity required. For the measurement of intracellular alanine concentrations during metabolism, the cell suspension (12 mg protein/ml) is incubated with the appropriate concentration of alanine in the absence of aminooxyacetate together with 3H20 and [carboxy-~4C]inulin as markers of the total and extracellular spaces, respectively. Aliquots (0.8 ml) of the cell suspension are layered on to 0.5 ml of silicone oil/dinonyl phthalate (l : l, v/v) which is itself layered above 0.1 ml of 150/o perchloric acid in a tube for the Eppendorf Model 3200 bench centrifuge. The reaction is terminated by the initiation of centrifugation. A sample of the supernatant is immediately acidified and radioactivity and alanine are assayed in the neutralized extract. An aliquot of the acid layer is withdrawn, neutralized with a small volume of K2CO 3 and similarly assayed for radioactivity and alanine. From these data, the extracellular and intracellular concentrations of alanine can be calculated. In this type of investigation, alanine transport can be inhibited by titration with ouabain or by reducing the extracellular sodium concentration. Transport can be stimulated by the addition of cyclic AMP. Simultaneous measurements of the rate of metabolism and of changes in the alanine distribution can then be made. Such investigations can also be carried out using the hepatocyte perifusion apparatus of van der Meet and Tager. 7 In a perifusion system a true steady state can be obtained at a constant low external alanine concentration. Centrifugal filtration techniques similar to those described above can be used for the measurement of intracellular alanine concentration. Using such a system, Sips e t al. s showed that the intracellular concentration of alanine remained low on increasing the extracellular concentration of 6 D. H. Williamson, O. Lopes Vereira, and B. Walker, Biochem. J. 104, 497 (1967). 7 R. van der Meet and J. M. Tager, FEBSLett. 67, 36 (1976). s H. J. Sips, A. K. Groen, andJ. M. Tager, FEBSLett. 119, 271 (1980).
[4]
HEPATOCYTE ALANINE TRANSPORT
35
alanine in the physiological range, and the addition of aminooxyacetate greatly increased the intracellular to extracellular concentration ratio. Using these various approaches, data from several laboratories indicate that alanine transport limits the rate of alanine metabolism at low alanine concentrations (<0.5 raM). s-m There is less agreement about the limitation of metabolism by transport at higher concentrations of alanine. Influence o f Cell Plasma Membrane Potential on Alanine Transport
Na + - alanine cotransport is an electrogenic process. Accordingly both the steady-state concentration ratio of alanine in the absence of metabolism as well as the kinetics of alanine transport should be influenced by the cell membrane potential. It is therefore important to be able to observe the effect of varying the cell membrane potential on the initial rate of transport. The measurement of cell membrane potential is isolated hepatocytes is not straightforward. Direct electrophysiological measurements involving the impalement of cells with microelectrodes has not so far yielded satisfactory results. In a number of cell types, the cell membrane potential has been calculated from the distribution ratio of permeant cations or from the absorption of fluorescence of cationic dyes. This approach is not feasible in hepatocytes, which contain large numbers of mitochondria that accumulate such permeant cations to a high intramitochondrial concentration. In hepatocytes, such measurements tend to reflect the mitochondrial membrane potential rather than that of the cell plasma membrane. In principle, the membrane potential can be calculated from the distribution of permeant anions, since these are not accumulated by the mitochondria. Thiocyanate has been used for this purpose by Hoek et al., ~ but these authors found it necessary to correct for the assumed binding of thiocyanate to intracellular constituents. Recently, Edmondson et al. ~2 have also used thiocyanate distribution as an indicator of cell membrane potential. In this study, digitonin was used to disrupt the cells and measure the cytosolic space. The thiocyanate technique remains somewhat unsatisfactory because of the various necessary correction factors involved and because of the lack of independent confirmation that changes in thiocyanate distribution in fact accurately measure changes in membrane potential. An alternative to the use of thiocyanate is to calculate the membrane potential from the distribution of chloride using 36C1-. It has been shown in 9 j. D. McGivan, J. C. Ramsell, and J. H. Lacey, Biochim. Biophys. Acta 644, 295 (1981). ~0p. Fafarnoux, C. Remesy, and C. Demigne, Biochem. J. 210, 645 (1983). ~l j. B. Hoek, D. G. Nicholls, and J. R. WiUiamson, J. Biol. Chem. 255, 1458 (1980). 12 j. W. Edmondson, B. A. Miller, and L. Lumeng, Am. J. Physiol. 249, G427 (1985).
36
TRANSPORT IN SUBCELLULAR ORGANELLES OF ANIMALS
[4]
this laboratory that chloride distribution in hepatocytes is not affected by inhibitors which inhibit chloride transport systems in other cell types. The membrane potential calculated from chloride distribution under a number of different conditions is equal to the potential measured directly using microelectrodes in perfused liver under the same conditions.la It therefore appears that chloride distributes passively across the cell membrane according to the membrane potential. Using this technique, a good correlation between cell membrane potential and initial rate of alanine transport can be demonstrated, and this relationship is independent of the mechanism by which the potential is varied. Chloride distribution can be measured by incubating cells in the presence of 36C1- together with [3H]inulin to measure the extracellular space. Dual-channel scintillation counting for 3H plus 36C1 is achieved as for aH plus 14C, but quench curves appropriate for 36C1 rather than ~4C must be used. While the use of chloride to determine the membrane potential may be preferable to that of thiocyanate, it must be recognized that the validity of both methods requires further independent justification. Alanine Transport in Liver Plasma Membrane Vesicles
The study of transport in plasma membrane vesicles allows the investigation of electrogenicity of the transport process and its ion dependence and obviates problems arising from possible substrate metabolism. Demonstration of transport in membrane vesicles is also a necessary prerequisite for the eventual identification of the carrier protein. Alanine transport in liver membrane vesicles was first reported by van Amelsvoort et al., ~4 and a modification of their method which is routinely used in this laboratory is detailed below. A single rat liver is homogenized in a medium containing 0.25 M sucrose, 10 m M K + - H E P E S , and 0.2 m M CaC12 at pH 7.4 and 4 ° initially by 6 strokes of a Teflon/glass Dounce homogenizer. The homogenate is diluted to 200 ml with the same medium and rehomogenized in 30-ml aliquots using a glass-glass homogenizer with approximately 20 strokes. This second homogenization is critical if satisfactory intact vesicles are to be obtained. After filtration through muslin to remove particles of fat, 1 m M EDTA (final concentration) is added. The homogenate is centrifuged at 1000 g for 10 min, and the pellet is discarded. The supernatant from the centrifugation is recentrifuged at 20,000 g for 30 rain, and the pellet is resuspended using the glass-glass homogenizer in 10 ml of homogenization medium to which 1 m M EDTA has been added. 13 N. M. Bradford, M. R. Hayes, and J. D. McGivan, Biochim. Biophys. Acta 845, 10 (1985). 14j. M. M. van Amelsvoort, H. J. Sips, and K. van Dam, Biochem. J. 174, 1083 (1978).
[4]
HEPATOCYTE ALANINE TRANSPORT
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Discontinuous sucrose gradients are prepared in tubes for the 3 × 25 ml swing-out head of the M.S.E. 65 ultracentrifuge. Each gradient consists of a lower layer of l0 ml of 46.5% sucrose (w/v) and an upper layer of l0 ml of 21.5% (w/v) sucrose, each solution being made up in l0 m M K+-HEPES, pH 7.5. Three milliliters of suspension is layered on top of each gradient, and the tubes are centrifuged at 23,000 rpm (50,000 g) for 2.5 h at 4 °. The material at the interface between the two sucrose solutions is collected and diluted at least 4 times with the original homogenization medium (containing no EDTA). The resulting suspension is centrifuged at 100,000 g for 40 min, and the pellet is resuspended in approximately 2 ml of homogenization medium using a syringe. The membrane suspension is rapidly frozen in small volumes in liquid nitrogen, and can be kept frozen for some weeks before use in transport experiments. This preparation routinely yields approximately 10 mg of protein, and the specific activity of the plasma membrane marker 5'-nucleotidase is increased 10-fold over the original homogenate. For measurement of transport, 20/tl of membrane suspension is added to an equal volume of incubation medium to give the following final concentrations: 0.25 M sucrose, 10 m M K+-HEPES, 0.2 m M CaC12, 5 m M MgCI2, 100 m M KCNS or 100 m M NaCNS, and 0.1 m M [3H]alanine at pH 7.5. A suitable specific activity for such experiments is 100 Ci/ mol, i.e., approximately 200 dpm/pmol. The reaction is terminated by the addition of 1 ml of ice-cold "stopping" solution containing 0.25 M sucrose, 10 m M K+-HEPES, 0.2 m M CaC12, plus 0.2 M NaC1 at pH 7.5. The diluted suspension is immediately filtered through millipore filters (HAWP 0.45 #m) and washed with 2 × 1 ml of stopping solution. The filters are dissolved in a suitable scintillator for measurement of radioactivity. In this method, the NaCNS provides the necessary sodium and electrical gradient for Na+-alanine cotransport. Parallel experiments using KCNS provide a measure of binding of alanine to the membranes, retention of residual radioactivity on the membrane filters, plus any Na+-inde pendent alanine transport. As measured by the above method, the maximum uptake of alanine should be 0.25- 0.3 nmol/mg protein at an external alanine concentration of 0.1 raM, and this should be attained in 30 sec. Lower values indicate a less than satisfactory membrane preparation. After the vesicles have been thawed the transport activity progressively declines. The transport of alanine in this stem has been extensively investigated? 4- ~6
15 H. J. Sips, J. H. H. van Amelsvoort, and K. van Dam, Eur. J. Biochem. 105, 217 (1980). 16 H. J. Sips, and K. van Dam, J. Membr. Biol. 62, 231 (1981).
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TRANSPORT IN SUBCELLULAR ORGANELLES OF ANIMALS
[4]
Approaches to Identification of Protein(s) Responsiblefor Alanine Transport in Liver Cell Membrane The alanine transport protein or proteins in liver plasma membranes have not yet been identified. The identification of such proteins would be of great interest, in particular because this would allow a detailed investigation of the hormonal induction of synthesis of transport proteins and their insertion into the plasma membrane. The successful identification of various transport proteins in mammalian cell membranes has relied mainly on the use of specific tight-binding inhibitors which can be used to label the transport protein. 17 No such inhibitor has so far been identified for the alanine carder in liver. Potential inhibitors of transport are best assessed by a study of their action on alanine transport in plasma membrane vesicles rather than intact cells, since inhibition in cells may be due to nonspeeific effects of the inhibitors on cell ATP levels. Using this approach, it has been shown that various sulfhydryl-blocking reagents (e.g., mersalyl, N-ethylmaleimide 18) inhibit alanine transport in membrane vesicles. The inhibition is due to an effect of the reagents on the carrier molecule rather than to nonspecific membrane damage. However, progress toward carrier identification is dependent on the unambiguous demonstration of protection of the cartier against the inhibitor by high concentrations of substrate, and this has proved difficult to achieve. A second approach to the identification of the transport protein involves reconstitution of various liver cell membrane fractions into artificial phospholipid membranes. Although such reconstitution has been reported for the alanine-transporting systems of kidney brush border membranes 19 and Ehrlich ascites cells, 2° the methods used do not produce satisfactory results when applied to liver. Modifications of these methods may be appropriate for the liver system, but this has not yet been demonstrated. In principle, a combination of labeling and reconstitution should lead to the identification of the alanine carrier in liver cell membranes, and other possible approaches to the problem exist. At present, however, the literature contains few reports of work in this area on liver amino acid transport system. ~7G. Semenza, M. Kessler, M. Hosang, J. Weber, and U. Schmidt, Biochim. Biophys. Acta 779, 343 (1984). ~s M. R. Hayes and J. D. MeGivan, Biochem. J. 214, 489 (1983). ~9H. Koepsell, K. Korn, D. Ferguson, H. Menuhr, D. OUig, and W. Haase, J. Biol. Chem. 259, 6548 (1984). 2o j. I. McCormick, D. Tsang, and R. M. Johnstone, Arch. Biochem. Biophys. 231, 355 (1984).