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Effect of epinephrine of glycogen phosphorylase-α in various preparations of rat liver
Comp. Biochem. Physiol. Vol.96B, No. 1, pp. 113-118, 1990 Printed in Great Britain
0305-0491/90$3.00+ 0.00 © 1990PergamonPress plc
EFFECT OF EPINEPHRINE ON GLYCOGEN PHOSPHORYLASE-a IN VARIOUS PREPARATIONS OF RAT LIVER FREDERICKC. KAUFFMAN,*~MICHAELWHITTAKER,*MOSTAFAZ. BADR~" and RONALDG. THURMANt *Department of Pharmacology and Toxicology, Rutgers University, College of Pharmacy, Piscataway, NJ 08854, USA (Tel: 201 932 6900); and tDepartment of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27514, USA (Received 29 September 1989)
Abstract--1. Glycogen phosphorylase-a, a commonly used index of cytosolic free calcium, was compared in various preparations of rat liver in the absence and presence of 0.1/aM epinephrine. 2. Total phosphorylase in isolated perfused livers and freshly-isolatedhepatocytes were the same as that observed in liver in situ; however, phosphorylase-a was 50% higher in perfused liver and 80% higher in hepatocytes than activities measured in situ. Total phosphorylase was reduced approximately 50% in hepatocytes maintained in primary culture for 24 hr. 3. Epinephrine increased phosphorylase-a approximately 2-fold in livers perfused for 30 min but only about 20% in hepatocytes incubated for 30 min. After 90 min of perfusion or incubation, epinephrine increased phosphorylase-a nearly 4-fold in perfused livers and only 30% in isolated hepatocytes. The results suggest that amounts of free calcium and calcium-dependent coupling of adrenergic receptors to phosphorylase-a differ markedly between the intact liver and isolated hepatocytes.
INTRODUCTION It is well established that many hormones including alpha adrenergic agonists, vasopressin and angiotensin II increase intracellular calcium in liver (Hutson et al., 1976; Charest et al., 1983; Carafoli, 1987). Changes in intracellular calcium have been measured directly using a variety of calcium-sensitive dyes (Tsien and Peonie, 1986; Scarpa, 1985; Monk et al., 1988) and indirectly via measurement of glycogen phosphorylase-a (Williamson et al., 1985; Long and Moore, 1986; Mirabelli, 1986). Measurement of phosphorylase-a provides a measure of cytosolic Ca 2+ because the inactive "b" form of this enzyme is converted to the active phosphorylated form by phosphorylase kinase, an enzyme stimulated directly by Ca 2+. In intact hepatocytes, phosphorylase-a activity increases in response to intracellular Ca 2÷ over the range of 10-350 nM (Long and Moore, 1986; Hock et al., 1987). Thus, alterations in this activity are a sensitive index of changes in intracellular Ca 2+ and correlate with fluorescence of calcium-sensitive dyes (Carafoli, 1987; Monk et al., 1988). Isolated rat hepatocytes and perfused livers have been used widely to study Ca2+-dependent responses (Hutson et al., 1976; Charest et al., 1983; Carafoli, 1987; Berridge and Irvine, 1984; Williamson et al., 1985); however, basal amounts of free Ca 2+ and variation in the response of these experimental systems to hormones that increase Ca 2+ may differ. Accordingly, we compared the effect of epinephrine on phosphorylasea in isolated perfused livers, isolated hepatocytes and isolated hepatocytes maintained in primary culture. :[:To whom all correspondence should be addressed.
Isolated hepatocytes allow uniform access of substrate and provide an experimental system where actins of toxic agents can be examined in the absence of other cell types and changes in the microcirculation. Transient increases in calcium in single cells may also be measured in isolated hepatocytes (Monk et al., 1988). In contrast, the perfused liver provides a system where serosal and canalicular surfaces of hepatocytes are maintained and nearly physiological gradients of oxygen and nutrients occur across the hepatic lobule. Data obtained in this study suggest that marked differences in basal calcium levels and their response to epinephrine exist in these two experimental systems. MATERIALS AND METHODS
Preparation and incubation of hepatocytes Male Sprague-Dawley rats weighing 150-300g and having free access to water and laboratory chow were used in all experiments. Hepatocytes were isolated by perfusion with calcium-free medium followed by brief perfusion with collagenase using a modification of the method described by Berry and Friend (1969). Rats were anesthetized with sodium pentobarbital (75 mg/kg i.p.) prior to isolation of bepatocytes in Krebs-Henseleit bicarbonate buffer containing 50#M EDTA and 12.5#M HEPES, pH7.4 (Krebs and Henseleit, 1932). Buffers used for the isolation of hepatocytes were maintained at 37°C and equilibrated with 95% 02:5% CO2 using a water-jacketed oxygenator (Krebs et al., 1973). The liver was perfused for 5 min with 100 ml of calcium-free buffer containing 0.5 mM EGTA, 250 U heparin and 0.02% bovine serum albumin. Perfusion was continued in a recirculating system with 100 ml of buffer containing 50 U/ml collagenase (Cooper Biochemicals) for 15 min. The liver was then removed and minced in buffer containing 0.02% bovine serum albumin. Hepatocytes were
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FREDERICKC. KAUFFMANet al.
collected following two successive filtrations through nylon mesh (Nitex 3-70-T and 3-305-T), and were washed twice in buffer by centrifugation at 500g for 3min and resuspended in Krebs-Henseleit bicarbonate buffer. Viable hepatocytes were isolated by centrifugation through a 30% Percoll gradient (Dalet et al., 1982), and resuspended at 1 x 107cells/ml oxygenated Krebs-Henseleit bicarbonate buffer containing 50#M EDTA and 2% bovine sertim albumin. Cells were kept on ice and bubbled gently with 95% 02: 5% CO2 until utilized for experiments. Hepatocytes isolated in this manner were 95-97% viable as indexed by Trypan Blue exclusion. Hepatocytes (1 x 106 cells/ml) were incubated at 37°C in 4 ml Krebs-Henseleit bicarbonate buffer containing 50 # M EDTA, 0.2% bovine serum albumin and 12.5 mM HEPES, pH 7.4, in a shaking water bath under a 95% 02:5% CO2 atmosphere. At various times, 0.5 ml samples were removed and the cells were pelleted by centrifugation at 15,000g for 15 sec. The supernatant was analyzed for glucose and lactate (see below) and the pellet was frozen in a dry ice/methanol bath and stored at -70°C until analyzed for glycogen phosphorylase.
Primary culture of hepatocytes Isolated hepatocytes were cultured on collagen-coated 38.5 mm 2 plastic tissue culture dishes (0.6 mg collagen/dish) for 24 hr in Williams E medium containing 2 mM glutamine, 2#g/ml insulin, 100U penicillin/ml, 100/tg/ml streptomycin and 10% fetal calf serum. After incubation for 2 hr the media was changed and non-adherent hepatocytes were removed. At 24hr, cells were washed once with 3 ml Krebs-Henseleit bicarbonate buffer containing 50 # M EDTA, 0.2% bovine serum albumin, and 12.5 mM HEPES, pH 7.4, prior to incubation at 37°C for 90 min under a 95% 02:5% CO2 atmosphere. The buffer was then removed and cells were harvested and homogenized in 250#1 of 50 mM glycylglycine, pH 7.8, containing 100 rnM NaF and 20 mM EDTA to inhibit phosphatase and phosphorylase kinase, respectively (Davis and Kauffman, 1986). Phosphorylase-a was measured as described below. Due to the presence of collagen in the cultures, it was not possible to express activity from cultures on the basis of protein content; consequently, DNA was measured fluorometrically (Thomas and Farquhar, 1978) using calf thymus DNA as a standard. Phosphorylase activity was converted to activity on a protein basis using a DNA:protein ratio of 13.4#g DNA/mg protein determined from measurements on freshly isolated hepatocytes.
Liver perfusion Livers from male Sprague-Dawley rats weighing 180-220g were perfused in the anterograde direction at 37°C with Krebs-Henseleit bicarbonate buffer in a nonrecirculating system as described previously (Scholz et al., 1973). Livers were perfused at a flow rate of 3.5-4 ml g wet wt -1 min -I with buffer equilibrated with 95% 02:5% CO2. At various intervals, aliquots of effluent perfusate were collected for analysis of glucose and lactate (see below) and livers were freeze-clamped with an aluminum mallet chilled with liquid nitrogen. Frozen livers were stored at - 70°C for subsequent analysis of glycogen phosphorylase.
Measurement of glycogen phosphorylase Hepatocytes (approximately 4 mgwetwt) or samples of frozen liver (approximately 50 mg wet wt) were homogenized in 100 #1 or 500 pl, respectively, of 50 mM glycylglycine, pH 7.8, containing 0.5mM EDTA and 50raM NaF. Glycogen phosphorylase-a was measured by incubating samples (10-I00 #g protein/ml reaction mixture) in 1 ml of 50 mM imidazole, pH 7.5, containing 50 mM NaF, 5 mM K2HPO 4, 2 mM MgC12, 0.5mM EDTA, 0.5 mM dithiothreitol, 0.5mM caffeine, 0.1 mM NADP +, 0.08% glycogen, 0.02% bovine serum albumin, 0.5/~M glucose-l,6-diphos-
phate, 1.6 #g/ml glucose-6-phosphate dehydrogenase and 0.5 #g/ml phosphoglucomutase (Lowry et al., 1967). The assay was initiated by addition of tissue homogenate, and reduction of NADP + was monitored fluorometrically (340~420 nm). Since phosphorylase-b in liver is not fully activated by the addition of Y-AMP (Rall and Sutherland, 1962), total glycogen phospborylase was assayed after converting phosphorylase-b to the a form by incubating samples in 50 p l of 50raM Tris-HC1, pH 8.1, containing 15 mM MgC12, 5mM ATP, 0.1 mM CaC12, 0.08% glycogen and 5#g/ml phosphorylase kinase for 40min at 37°C and measuring phosphorylase-a as described above.
Analyses of biochemical intermediates Glucose and lactate were measured fluorometrically by direct enzymatic procedures (Lowry and Passonneau, 1972). Rates of glucose and lactate production by the perfused liver were calculated from the concentration of metabolite in the perfusate, the flow rate, and the liver wet weight. In isolated hepatocytes, rates of glucose and lactate production were calculated from rates of accumulation of intermediates in the media. Protein was determined according to the method of Lowry et al. (1951) using bovine serum albumin as a standard. RESULTS
Comparison o f glycogen phosphorylase in different liver preparations Glycogen phosphorylase activities measured in various preparations of rat liver are described in Table 1. Total phosphorylase activity in situ, in perfused liver and in freshly isolated hepatocytes was essentially the same indicating that phosphorylase-a was not lost during perfusions of liver or preparation of hepatocytes. Total activity of phosphorylase was reduced more than 50% in hepatocytes maintained in primary culture for 24 hr. Activity of the active form of the enzyme, phosphorylase-a, was significantly higher in freshly isolated hepatocytes compared to perfused liver or the liver in situ. Although total phosphorylase declined significantly in hepatocytes maintained in culture, the fraction of total enzyme in the a form (per cent of total phosphorylase) was essentially the same as the liver in situ. Phosphorylase-a in freshly isolated hepatocytes and perfused liver measured as a function of time of incubation or perfusion is depicted in Fig. 1. Phosphorylase-a activity was not significantly different in isolated hepatocytes and perfused livers at the beginning of the experiment but changes with time differed dramatically in the two preparations. In hepatocytes, phosphorylase-a increased initially by nearly 30% and then declined over 2 hr of incubation and was the same after 90 min of incubation as in freshly isolated hepatocytes. In contrast, phosphorylase-a activity in perfused livers declined gradually over the course of the experiment to values that were only about 40% of those noted in freshly isolated liver.
Glucose and lactate production by perfused livers and isolated hepatocytes Production of glucose and lactate, the products of glycogen breakdown, differed significantly when perfused livers and isolated hepatocytes were compared. In general, the production of glucose and lactate by the perfused liver followed the activity of
Glycogen phosphorylase-a in rat liver 12 ~~
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Fig. 1. Phosphorylase-a in isolated perfused livers and hepatocytes as a function of time. Incubations or perfusions were performed at 37°C for the time periods indicated. Values of phosphorylase-a activity for perfused livers are the average + SEM of four livers freeze-clamped at each time interval indicated. Phosphorylase activity was measured as described in Methods. Values for isolated hepatocytes are averages + SEM of samples obtained from four experiments. Total phosphorylase did not change significantly over the time course of the experiment and was 9.7 + 0.7 and 13.9 + 1.3 nmol min- 1mg prot- ~at 30 min in perfused livers and isolated hepatocytes, respectively. phosphorylase-a, i.e. production declined steadily over the time course of the perfusion (Fig. 3). Rates of glucose output were higher in isolated hepatocytes than in perfused livers at the outset of the incubation but declined rapidly during 30 min of incubation to values below those observed in the perfused liver. Despite the rapid decline in rates of glucose production in hepatocytes, rates of lactate output remained relatively constant during the first 30 min of incubation. Subsequently, lactate production by hepatocytes declined rapidly to values which were only around 10% of values observed in perfused livers. Comparison of rates of glucose plus lactate output with measured activities of phosphorylase-a at various time intervals indicated that rates of glucose and lactate output relative to activities ofphosphorylase-a were much higher in perfused livers than in isolated hepatocytes (Fig. 4). Effect of epinephrine on phosphorylase activity in various liver preparations Responses of perfused liver and hepatocytes to 0.1/~M epinephrine at 30 and 90 min of incubation is shown in Fig. 1. Epinephrine, in liver was added to the preparations 5 min before the measurement of phosphorylase-a and total phosphorylase at 30 and 90 min. At 30 min, basal activities of phosphorylase-a in perfused livers were about 40% of values for total phosphorylase and about 60% of total in isolated hepatocytes (Fig. 4). Addition of epinephrine increased phosphorylase-a to approximately 90% of total in perfused livers and to about 80% in isolated hepatocytes. Increases in phosphorylase-a occurred in the absence of changes in cyclic AMP which were 162 + 29 nmol/kg wet tissue in control livers and 177 _ 20 nmoi/kg wet tissue in livers exposed to 0.1 #M epinephrine (n = 4 in each group). Because basal activity of phosphorylase was much higher in hepatocytes than the perfused liver, the magnitude of the response to epinephrine was significantly different
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in the two preparations. At 30min, epinephrine increased phosphorylase-a activity by about 50% in the perfused liver and only about 25% of basal activity in isolated hepatocytes. Differences between perfused livers and isolated hepatocytes were even more apparent at later periods of perfusion or incubation. At 90 min, treatment of hepatocytes with 0.1/~M epinephrine increased phosphorylase-a to a value that was only about 65% of total (Fig. 4). In contrast, the response of the perfused liver to added epinephrine was greater than that observed at 30min, i.e. epinephrine increased phosphorylase-a to values around 90% of the total activity. The poor response of hepatocytes maintained in culture was similar to values observed in freshly isolated hepatocytes. Addition of epinephrine caused phosphorylase-a to increase by only about 20%0 to roughly 65% of total values in hepatocytes maintained in primary culture and transferred to Krebs-Henseleit bicarbonate buffer for 90 min. DISCUSSION Basal activities of phosphorylase-a in isolated hepatocytes and perfused liver There is considerable evidence indicating that isolated hepatocytes are more permeant to extracellular calcium than the intact organ (Kleineke and Stratman, 1974; Dubinsky and Cockrell, 1975; Kleineke and Soling, 1985). Measurement of phosphorylase-a activity in isolated hepatocytes and perfused livers are in accord with this data. Primary cultures of bepatocytes resemble the liver in situ in terms of the fraction of phosphorylase existing in the active form; however, cultured cells contained only about 50% o f total enzyme observed in situ. This decline in activity of phosphorylase during cell culture resembles changes in activities of other enzymes noted in hepatocytes maintained in primary culture. For example, dramatic decreases in the cytochrome P-450 system occur in hepatocytes during the first 24 hr in primary culture (Guzelian et al., 1977; Dickins and Peterson, 1980; Acosta et al., 1987). Decreases in the P-450 system in cultured hepatocytes involves activation of heme oxygenase (Acosta et al., 1987). Mechanisms underlying the loss of phosphorylase in hepatocytes kept in primary culture (Table 1) may involve activation of calcium sensitive proteases that degrade phosphorylase in plated cells. A striking difference between isolated hepatocytes and perfused livers is the production of glucose and lactate (Fig. 2). Depletion of carbohydrate reserves occurred more rapidly in isolated hepatocytes than perfused livers as indexed by the rapid decline in rates of glucose and lactate production by isolated cells. High initial rates of glucose and lactate output in hepatocytes is best explained by elevated activity of phosphorylase-a (Fig. 1). Higher rates of lactate output by the perfused liver compared to isolated hepatocytes could (Fig. 3) involve more efficient energy production by the intact liver, i.e. glycolysis is much faster in the intact liver compared to isolated hepatocytes. Alternatively, substrates produced in upstream periportal regions of the liver lobule (e.g. succinate, ct-ketoglutarate) could be consumed more
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FREDERICK C. KAUFFMAN el al. Table 1. Glycogen phosphorylase activity in various preparations of rat liver Liver Perfused
In situ
Phosphorylase-a Phosphorylase-b Total phosphorylase % Phosphorylase-a
4.03 + 6.03 + 10.15 + 39.7 +
0.68 0.25 1.01 4.0
5.97 + 0.99 5.11 + 1.21 10.99 + 1.64 54.3 __. 10.0
Hepatocytes Freshly isolated Primary culture 7.49 + 5.20 + 12.22 + 61.3 +
0.56* 1.73 1.05 11.3
2.73 _+ 0.17"* 3.18 _+ 0.15 5.91 _+ 0.24* 46.2 + 2.5
Values are averages + SEM for three-six preparations. Livers from sodium pentobarbital-anesthetized rats were perfused rapidly in situ with Krebs-Henseleit biocarbonate buffer and immediately removed and homogenized in 10 vols of 20 mM glycylglycine, pH 7.8, containing 20 mM EDTA and 100 mM NaF. Perfused livers were freeze clamped 1 min after isolation and initiation of perfusion. Hepatocytes were homogenized immediately following isolation. Primary cultures of bepatocytes were prepared on plastic culture dishes and maintained 24 hr in vitro as described in Methods. Data were analyzed statistically using Student's t-test. *P < 0.05 (freshly isolated hepatocytes versus in situ). **P < 0.01 (primary culture versus in situ).
efficiently in downstream areas in perfused liver than by dilute suspension of hepatocytes. Differences in patterns of glucose and lactate output by isolated hepatocytes and perfused liver emphasize that hepatic structure and hemodynamics are important in the regulation of aerobic glycolysis and gluconeogenesis. The rapid loss of glycogen in hepatocytes is noteworthy because this may also contribute to the fragility of isolated hepatocytes. Depletion of glycogen is known to enhance the sensitivity of liver to toxic chemicals and hypoxia (Bradford et al., 1986).
Effect of epinephrine on isolated hepatocytrs and perfused livers Addition of epinephrine caused more robust increases in phosphorylase-a activity in perfused 100 I-
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livers than in isolated hepatocytes. Addition of 0.1/~M epinephrine, which is well established to stimulate phosphorylation of phosphorylase-b in liver (Garrison, 1978), increased phosphorylase-a in the intact liver to a much greater extent than in isolated hepatocytes (Fig. 2). Phosphorylase-a activity increased nearly 3-fold in isolated perfused livers and only 20% in isolated hepatocytes exposed to 0.1/~ M epinephrine at 30 min of perfusion (Fig. 2). Differences in the response of isolated hepatocytes and perfused livers to epinephrine was even more apparent at 90 min where phosphorylase-a increased more than 3-fold in perfused livers but only about 30% in isolated hepatocytes treated with epinephrine. The lower responses of phosphorylase-a to epinephrine in isolated hepatocytes does not involve loss of phosphorylase from the isolated cells because total activity of this enzyme did not change over the course of the experiment (see legend Fig. 2). Addition of epinephrine to perfused livers at 90 min increased phosphorylase-a to the same level noted at 30 min, about 90% of total whereas the magnitude of the response of phosphorylase-a in hepatocytes to 180
40 120 a_
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Perfused Isolated Plated Liver HepatocytesHepatocytes
Fig. 2. Response of various preparations of liver cells to epinephrine. Basal values for phosphorylase-a (open bars) and epinephrine-stimulated activity (solid bars) were determined in perfused livers, freshly isolated hepatocytes in suspension or plated hepatocytes as described in Methods. Perfused livers and cells were perfused or incubated for either 30 or 90 min prior to the addition of epinephrine. Perfused livers and cells were perfused or incubated for either 30 or 90 min prior to the addition of epinephrine. Epinephrine (0.I #M) was added 5 min before livers were freeze-clamped or hepatocytes were sampled. Values are averages + SEM of four perfused livers sampled at each time point or samples obtained from these preparations of hepatocytes or six 35 mm culture plates of hepatocytes. Total phosphorylase measured in perfused livers and isolated hepatocytes did not change significantly over the time course of the experiment. Total phosphorylase measured in perfused livers at 30 and 90min was 9.7 +0.7 and 8.8 _ 0.9 nmol min- I mg prot- I , respectively. Values in hepatocytes were 13.9+ 1.3 and 13.4+ 1.2 at 30 and 90rain, respectively.
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Minutes Fig. 3. Time course of glucose and lactate output from isolated hepatocytes and perfused livers. Experimental conditions as in Fig. 1. Glucose (Panel A) and lactate (Panel B) were measured in effluent perfusate or incubation medium as described in methods. Rates of metabolite production by hepatocytes are based on amounts of glucose and lactate produced over 15 min intervals. Rates in the perfused liver are based on amounts of glucose or lactate measured in the effluent perfusate, the flow rate and the liver wet weight.
Glycogen phosphorylase-a in rat liver 90
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to epinephrine and possibly other hormones that act via calcium-dependent mechanisms.
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Acknowledgement--Supported, in part, by USPHS Grant
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REFERENCES
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Fig. 4. Relationship between phosphorylase-a activity and glycogenolysis in isolated hepatocytes and perfused livers. Glycogenolysis, [glucose plus 2 x lactate production (Fig. 2)], is compared with phosphorylase-a activity (data from Fig. 1) in perfused livers or hepatocytes determined at the same time points.
epinephrine at 9 0 m i n remained low and only increased to about 60% of total activity (Fig. 2). Hepatocytes that were plated and maintained in primary culture resembled freshly isolated hepatocytes and displayed a diminished response to epinephrine. The diminished effect of epinephrine on phosphorylase-a in isolated hepatocytes most likely involves alterations in calcium status. First, the total content of calcium is probably higher in hepatocytes than perfused livers at the outset of perfusion or incubation. In addition to increases in total calcium in hepatocytes, it has been suggested that depletion of a hormone responsive Ca 2÷ pool occurs in this preparation (Kleineke and Soling, 1985; Joseph and Williamson, 1983). In line with this possibility is the finding that the content of mitochondrial relative to non-mitochondrial calcium is greatly elevated in hepatocytes compared to the intact liver (Kleineke and Soling, 1985; Joseph and Williamson, 1983). Mechanisms coupling membrane receptors to intracellular second messenger system may be compromised during prolonged incubations of hepatocytes in vitro due to this redistribution of calcium. Higher concentrations of Ca 2+ in hepatocytes compared to perfused livers may also lead to increased protein phosphatase activity and dephosphorylation of phosphorylase-a. Phosphatase 2B is activated by concentrations of calcium considerably greater than that required to activate phosphorylase kinase (Blackmore et al., 1983). It is clear from many studies that intracellular free calcium is critical in intracellular regulation of metabolism and cell structure. The finding that basal phosphorylase-a, its change with time and its response to epinephrine differed dramatically in isolated perfused livers and hepatocytes indicate that mechanisms linking activation of epinephrine receptors to phosphorylase activates differ in these preparations. Differences reported above emphasize that the structural organization of the intact liver is an important determinant of the response of this organ
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0305-0491/90$3.00+ 0.00 © 1990PergamonPress plc
EFFECT OF EPINEPHRINE ON GLYCOGEN PHOSPHORYLASE-a IN VARIOUS PREPARATIONS OF RAT LIVER FREDERICKC. KAUFFMAN,*~MICHAELWHITTAKER,*MOSTAFAZ. BADR~" and RONALDG. THURMANt *Department of Pharmacology and Toxicology, Rutgers University, College of Pharmacy, Piscataway, NJ 08854, USA (Tel: 201 932 6900); and tDepartment of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27514, USA (Received 29 September 1989)
Abstract--1. Glycogen phosphorylase-a, a commonly used index of cytosolic free calcium, was compared in various preparations of rat liver in the absence and presence of 0.1/aM epinephrine. 2. Total phosphorylase in isolated perfused livers and freshly-isolatedhepatocytes were the same as that observed in liver in situ; however, phosphorylase-a was 50% higher in perfused liver and 80% higher in hepatocytes than activities measured in situ. Total phosphorylase was reduced approximately 50% in hepatocytes maintained in primary culture for 24 hr. 3. Epinephrine increased phosphorylase-a approximately 2-fold in livers perfused for 30 min but only about 20% in hepatocytes incubated for 30 min. After 90 min of perfusion or incubation, epinephrine increased phosphorylase-a nearly 4-fold in perfused livers and only 30% in isolated hepatocytes. The results suggest that amounts of free calcium and calcium-dependent coupling of adrenergic receptors to phosphorylase-a differ markedly between the intact liver and isolated hepatocytes.
INTRODUCTION It is well established that many hormones including alpha adrenergic agonists, vasopressin and angiotensin II increase intracellular calcium in liver (Hutson et al., 1976; Charest et al., 1983; Carafoli, 1987). Changes in intracellular calcium have been measured directly using a variety of calcium-sensitive dyes (Tsien and Peonie, 1986; Scarpa, 1985; Monk et al., 1988) and indirectly via measurement of glycogen phosphorylase-a (Williamson et al., 1985; Long and Moore, 1986; Mirabelli, 1986). Measurement of phosphorylase-a provides a measure of cytosolic Ca 2+ because the inactive "b" form of this enzyme is converted to the active phosphorylated form by phosphorylase kinase, an enzyme stimulated directly by Ca 2+. In intact hepatocytes, phosphorylase-a activity increases in response to intracellular Ca 2÷ over the range of 10-350 nM (Long and Moore, 1986; Hock et al., 1987). Thus, alterations in this activity are a sensitive index of changes in intracellular Ca 2+ and correlate with fluorescence of calcium-sensitive dyes (Carafoli, 1987; Monk et al., 1988). Isolated rat hepatocytes and perfused livers have been used widely to study Ca2+-dependent responses (Hutson et al., 1976; Charest et al., 1983; Carafoli, 1987; Berridge and Irvine, 1984; Williamson et al., 1985); however, basal amounts of free Ca 2+ and variation in the response of these experimental systems to hormones that increase Ca 2+ may differ. Accordingly, we compared the effect of epinephrine on phosphorylasea in isolated perfused livers, isolated hepatocytes and isolated hepatocytes maintained in primary culture. :[:To whom all correspondence should be addressed.
Isolated hepatocytes allow uniform access of substrate and provide an experimental system where actins of toxic agents can be examined in the absence of other cell types and changes in the microcirculation. Transient increases in calcium in single cells may also be measured in isolated hepatocytes (Monk et al., 1988). In contrast, the perfused liver provides a system where serosal and canalicular surfaces of hepatocytes are maintained and nearly physiological gradients of oxygen and nutrients occur across the hepatic lobule. Data obtained in this study suggest that marked differences in basal calcium levels and their response to epinephrine exist in these two experimental systems. MATERIALS AND METHODS
Preparation and incubation of hepatocytes Male Sprague-Dawley rats weighing 150-300g and having free access to water and laboratory chow were used in all experiments. Hepatocytes were isolated by perfusion with calcium-free medium followed by brief perfusion with collagenase using a modification of the method described by Berry and Friend (1969). Rats were anesthetized with sodium pentobarbital (75 mg/kg i.p.) prior to isolation of bepatocytes in Krebs-Henseleit bicarbonate buffer containing 50#M EDTA and 12.5#M HEPES, pH7.4 (Krebs and Henseleit, 1932). Buffers used for the isolation of hepatocytes were maintained at 37°C and equilibrated with 95% 02:5% CO2 using a water-jacketed oxygenator (Krebs et al., 1973). The liver was perfused for 5 min with 100 ml of calcium-free buffer containing 0.5 mM EGTA, 250 U heparin and 0.02% bovine serum albumin. Perfusion was continued in a recirculating system with 100 ml of buffer containing 50 U/ml collagenase (Cooper Biochemicals) for 15 min. The liver was then removed and minced in buffer containing 0.02% bovine serum albumin. Hepatocytes were
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collected following two successive filtrations through nylon mesh (Nitex 3-70-T and 3-305-T), and were washed twice in buffer by centrifugation at 500g for 3min and resuspended in Krebs-Henseleit bicarbonate buffer. Viable hepatocytes were isolated by centrifugation through a 30% Percoll gradient (Dalet et al., 1982), and resuspended at 1 x 107cells/ml oxygenated Krebs-Henseleit bicarbonate buffer containing 50#M EDTA and 2% bovine sertim albumin. Cells were kept on ice and bubbled gently with 95% 02: 5% CO2 until utilized for experiments. Hepatocytes isolated in this manner were 95-97% viable as indexed by Trypan Blue exclusion. Hepatocytes (1 x 106 cells/ml) were incubated at 37°C in 4 ml Krebs-Henseleit bicarbonate buffer containing 50 # M EDTA, 0.2% bovine serum albumin and 12.5 mM HEPES, pH 7.4, in a shaking water bath under a 95% 02:5% CO2 atmosphere. At various times, 0.5 ml samples were removed and the cells were pelleted by centrifugation at 15,000g for 15 sec. The supernatant was analyzed for glucose and lactate (see below) and the pellet was frozen in a dry ice/methanol bath and stored at -70°C until analyzed for glycogen phosphorylase.
Primary culture of hepatocytes Isolated hepatocytes were cultured on collagen-coated 38.5 mm 2 plastic tissue culture dishes (0.6 mg collagen/dish) for 24 hr in Williams E medium containing 2 mM glutamine, 2#g/ml insulin, 100U penicillin/ml, 100/tg/ml streptomycin and 10% fetal calf serum. After incubation for 2 hr the media was changed and non-adherent hepatocytes were removed. At 24hr, cells were washed once with 3 ml Krebs-Henseleit bicarbonate buffer containing 50 # M EDTA, 0.2% bovine serum albumin, and 12.5 mM HEPES, pH 7.4, prior to incubation at 37°C for 90 min under a 95% 02:5% CO2 atmosphere. The buffer was then removed and cells were harvested and homogenized in 250#1 of 50 mM glycylglycine, pH 7.8, containing 100 rnM NaF and 20 mM EDTA to inhibit phosphatase and phosphorylase kinase, respectively (Davis and Kauffman, 1986). Phosphorylase-a was measured as described below. Due to the presence of collagen in the cultures, it was not possible to express activity from cultures on the basis of protein content; consequently, DNA was measured fluorometrically (Thomas and Farquhar, 1978) using calf thymus DNA as a standard. Phosphorylase activity was converted to activity on a protein basis using a DNA:protein ratio of 13.4#g DNA/mg protein determined from measurements on freshly isolated hepatocytes.
Liver perfusion Livers from male Sprague-Dawley rats weighing 180-220g were perfused in the anterograde direction at 37°C with Krebs-Henseleit bicarbonate buffer in a nonrecirculating system as described previously (Scholz et al., 1973). Livers were perfused at a flow rate of 3.5-4 ml g wet wt -1 min -I with buffer equilibrated with 95% 02:5% CO2. At various intervals, aliquots of effluent perfusate were collected for analysis of glucose and lactate (see below) and livers were freeze-clamped with an aluminum mallet chilled with liquid nitrogen. Frozen livers were stored at - 70°C for subsequent analysis of glycogen phosphorylase.
Measurement of glycogen phosphorylase Hepatocytes (approximately 4 mgwetwt) or samples of frozen liver (approximately 50 mg wet wt) were homogenized in 100 #1 or 500 pl, respectively, of 50 mM glycylglycine, pH 7.8, containing 0.5mM EDTA and 50raM NaF. Glycogen phosphorylase-a was measured by incubating samples (10-I00 #g protein/ml reaction mixture) in 1 ml of 50 mM imidazole, pH 7.5, containing 50 mM NaF, 5 mM K2HPO 4, 2 mM MgC12, 0.5mM EDTA, 0.5 mM dithiothreitol, 0.5mM caffeine, 0.1 mM NADP +, 0.08% glycogen, 0.02% bovine serum albumin, 0.5/~M glucose-l,6-diphos-
phate, 1.6 #g/ml glucose-6-phosphate dehydrogenase and 0.5 #g/ml phosphoglucomutase (Lowry et al., 1967). The assay was initiated by addition of tissue homogenate, and reduction of NADP + was monitored fluorometrically (340~420 nm). Since phosphorylase-b in liver is not fully activated by the addition of Y-AMP (Rall and Sutherland, 1962), total glycogen phospborylase was assayed after converting phosphorylase-b to the a form by incubating samples in 50 p l of 50raM Tris-HC1, pH 8.1, containing 15 mM MgC12, 5mM ATP, 0.1 mM CaC12, 0.08% glycogen and 5#g/ml phosphorylase kinase for 40min at 37°C and measuring phosphorylase-a as described above.
Analyses of biochemical intermediates Glucose and lactate were measured fluorometrically by direct enzymatic procedures (Lowry and Passonneau, 1972). Rates of glucose and lactate production by the perfused liver were calculated from the concentration of metabolite in the perfusate, the flow rate, and the liver wet weight. In isolated hepatocytes, rates of glucose and lactate production were calculated from rates of accumulation of intermediates in the media. Protein was determined according to the method of Lowry et al. (1951) using bovine serum albumin as a standard. RESULTS
Comparison o f glycogen phosphorylase in different liver preparations Glycogen phosphorylase activities measured in various preparations of rat liver are described in Table 1. Total phosphorylase activity in situ, in perfused liver and in freshly isolated hepatocytes was essentially the same indicating that phosphorylase-a was not lost during perfusions of liver or preparation of hepatocytes. Total activity of phosphorylase was reduced more than 50% in hepatocytes maintained in primary culture for 24 hr. Activity of the active form of the enzyme, phosphorylase-a, was significantly higher in freshly isolated hepatocytes compared to perfused liver or the liver in situ. Although total phosphorylase declined significantly in hepatocytes maintained in culture, the fraction of total enzyme in the a form (per cent of total phosphorylase) was essentially the same as the liver in situ. Phosphorylase-a in freshly isolated hepatocytes and perfused liver measured as a function of time of incubation or perfusion is depicted in Fig. 1. Phosphorylase-a activity was not significantly different in isolated hepatocytes and perfused livers at the beginning of the experiment but changes with time differed dramatically in the two preparations. In hepatocytes, phosphorylase-a increased initially by nearly 30% and then declined over 2 hr of incubation and was the same after 90 min of incubation as in freshly isolated hepatocytes. In contrast, phosphorylase-a activity in perfused livers declined gradually over the course of the experiment to values that were only about 40% of those noted in freshly isolated liver.
Glucose and lactate production by perfused livers and isolated hepatocytes Production of glucose and lactate, the products of glycogen breakdown, differed significantly when perfused livers and isolated hepatocytes were compared. In general, the production of glucose and lactate by the perfused liver followed the activity of
Glycogen phosphorylase-a in rat liver 12 ~~
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Fig. 1. Phosphorylase-a in isolated perfused livers and hepatocytes as a function of time. Incubations or perfusions were performed at 37°C for the time periods indicated. Values of phosphorylase-a activity for perfused livers are the average + SEM of four livers freeze-clamped at each time interval indicated. Phosphorylase activity was measured as described in Methods. Values for isolated hepatocytes are averages + SEM of samples obtained from four experiments. Total phosphorylase did not change significantly over the time course of the experiment and was 9.7 + 0.7 and 13.9 + 1.3 nmol min- 1mg prot- ~at 30 min in perfused livers and isolated hepatocytes, respectively. phosphorylase-a, i.e. production declined steadily over the time course of the perfusion (Fig. 3). Rates of glucose output were higher in isolated hepatocytes than in perfused livers at the outset of the incubation but declined rapidly during 30 min of incubation to values below those observed in the perfused liver. Despite the rapid decline in rates of glucose production in hepatocytes, rates of lactate output remained relatively constant during the first 30 min of incubation. Subsequently, lactate production by hepatocytes declined rapidly to values which were only around 10% of values observed in perfused livers. Comparison of rates of glucose plus lactate output with measured activities of phosphorylase-a at various time intervals indicated that rates of glucose and lactate output relative to activities ofphosphorylase-a were much higher in perfused livers than in isolated hepatocytes (Fig. 4). Effect of epinephrine on phosphorylase activity in various liver preparations Responses of perfused liver and hepatocytes to 0.1/~M epinephrine at 30 and 90 min of incubation is shown in Fig. 1. Epinephrine, in liver was added to the preparations 5 min before the measurement of phosphorylase-a and total phosphorylase at 30 and 90 min. At 30 min, basal activities of phosphorylase-a in perfused livers were about 40% of values for total phosphorylase and about 60% of total in isolated hepatocytes (Fig. 4). Addition of epinephrine increased phosphorylase-a to approximately 90% of total in perfused livers and to about 80% in isolated hepatocytes. Increases in phosphorylase-a occurred in the absence of changes in cyclic AMP which were 162 + 29 nmol/kg wet tissue in control livers and 177 _ 20 nmoi/kg wet tissue in livers exposed to 0.1 #M epinephrine (n = 4 in each group). Because basal activity of phosphorylase was much higher in hepatocytes than the perfused liver, the magnitude of the response to epinephrine was significantly different
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in the two preparations. At 30min, epinephrine increased phosphorylase-a activity by about 50% in the perfused liver and only about 25% of basal activity in isolated hepatocytes. Differences between perfused livers and isolated hepatocytes were even more apparent at later periods of perfusion or incubation. At 90 min, treatment of hepatocytes with 0.1/~M epinephrine increased phosphorylase-a to a value that was only about 65% of total (Fig. 4). In contrast, the response of the perfused liver to added epinephrine was greater than that observed at 30min, i.e. epinephrine increased phosphorylase-a to values around 90% of the total activity. The poor response of hepatocytes maintained in culture was similar to values observed in freshly isolated hepatocytes. Addition of epinephrine caused phosphorylase-a to increase by only about 20%0 to roughly 65% of total values in hepatocytes maintained in primary culture and transferred to Krebs-Henseleit bicarbonate buffer for 90 min. DISCUSSION Basal activities of phosphorylase-a in isolated hepatocytes and perfused liver There is considerable evidence indicating that isolated hepatocytes are more permeant to extracellular calcium than the intact organ (Kleineke and Stratman, 1974; Dubinsky and Cockrell, 1975; Kleineke and Soling, 1985). Measurement of phosphorylase-a activity in isolated hepatocytes and perfused livers are in accord with this data. Primary cultures of bepatocytes resemble the liver in situ in terms of the fraction of phosphorylase existing in the active form; however, cultured cells contained only about 50% o f total enzyme observed in situ. This decline in activity of phosphorylase during cell culture resembles changes in activities of other enzymes noted in hepatocytes maintained in primary culture. For example, dramatic decreases in the cytochrome P-450 system occur in hepatocytes during the first 24 hr in primary culture (Guzelian et al., 1977; Dickins and Peterson, 1980; Acosta et al., 1987). Decreases in the P-450 system in cultured hepatocytes involves activation of heme oxygenase (Acosta et al., 1987). Mechanisms underlying the loss of phosphorylase in hepatocytes kept in primary culture (Table 1) may involve activation of calcium sensitive proteases that degrade phosphorylase in plated cells. A striking difference between isolated hepatocytes and perfused livers is the production of glucose and lactate (Fig. 2). Depletion of carbohydrate reserves occurred more rapidly in isolated hepatocytes than perfused livers as indexed by the rapid decline in rates of glucose and lactate production by isolated cells. High initial rates of glucose and lactate output in hepatocytes is best explained by elevated activity of phosphorylase-a (Fig. 1). Higher rates of lactate output by the perfused liver compared to isolated hepatocytes could (Fig. 3) involve more efficient energy production by the intact liver, i.e. glycolysis is much faster in the intact liver compared to isolated hepatocytes. Alternatively, substrates produced in upstream periportal regions of the liver lobule (e.g. succinate, ct-ketoglutarate) could be consumed more
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FREDERICK C. KAUFFMAN el al. Table 1. Glycogen phosphorylase activity in various preparations of rat liver Liver Perfused
In situ
Phosphorylase-a Phosphorylase-b Total phosphorylase % Phosphorylase-a
4.03 + 6.03 + 10.15 + 39.7 +
0.68 0.25 1.01 4.0
5.97 + 0.99 5.11 + 1.21 10.99 + 1.64 54.3 __. 10.0
Hepatocytes Freshly isolated Primary culture 7.49 + 5.20 + 12.22 + 61.3 +
0.56* 1.73 1.05 11.3
2.73 _+ 0.17"* 3.18 _+ 0.15 5.91 _+ 0.24* 46.2 + 2.5
Values are averages + SEM for three-six preparations. Livers from sodium pentobarbital-anesthetized rats were perfused rapidly in situ with Krebs-Henseleit biocarbonate buffer and immediately removed and homogenized in 10 vols of 20 mM glycylglycine, pH 7.8, containing 20 mM EDTA and 100 mM NaF. Perfused livers were freeze clamped 1 min after isolation and initiation of perfusion. Hepatocytes were homogenized immediately following isolation. Primary cultures of bepatocytes were prepared on plastic culture dishes and maintained 24 hr in vitro as described in Methods. Data were analyzed statistically using Student's t-test. *P < 0.05 (freshly isolated hepatocytes versus in situ). **P < 0.01 (primary culture versus in situ).
efficiently in downstream areas in perfused liver than by dilute suspension of hepatocytes. Differences in patterns of glucose and lactate output by isolated hepatocytes and perfused liver emphasize that hepatic structure and hemodynamics are important in the regulation of aerobic glycolysis and gluconeogenesis. The rapid loss of glycogen in hepatocytes is noteworthy because this may also contribute to the fragility of isolated hepatocytes. Depletion of glycogen is known to enhance the sensitivity of liver to toxic chemicals and hypoxia (Bradford et al., 1986).
Effect of epinephrine on isolated hepatocytrs and perfused livers Addition of epinephrine caused more robust increases in phosphorylase-a activity in perfused 100 I-
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livers than in isolated hepatocytes. Addition of 0.1/~M epinephrine, which is well established to stimulate phosphorylation of phosphorylase-b in liver (Garrison, 1978), increased phosphorylase-a in the intact liver to a much greater extent than in isolated hepatocytes (Fig. 2). Phosphorylase-a activity increased nearly 3-fold in isolated perfused livers and only 20% in isolated hepatocytes exposed to 0.1/~ M epinephrine at 30 min of perfusion (Fig. 2). Differences in the response of isolated hepatocytes and perfused livers to epinephrine was even more apparent at 90 min where phosphorylase-a increased more than 3-fold in perfused livers but only about 30% in isolated hepatocytes treated with epinephrine. The lower responses of phosphorylase-a to epinephrine in isolated hepatocytes does not involve loss of phosphorylase from the isolated cells because total activity of this enzyme did not change over the course of the experiment (see legend Fig. 2). Addition of epinephrine to perfused livers at 90 min increased phosphorylase-a to the same level noted at 30 min, about 90% of total whereas the magnitude of the response of phosphorylase-a in hepatocytes to 180
40 120 a_
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Perfused Isolated Plated Liver HepatocytesHepatocytes
Fig. 2. Response of various preparations of liver cells to epinephrine. Basal values for phosphorylase-a (open bars) and epinephrine-stimulated activity (solid bars) were determined in perfused livers, freshly isolated hepatocytes in suspension or plated hepatocytes as described in Methods. Perfused livers and cells were perfused or incubated for either 30 or 90 min prior to the addition of epinephrine. Perfused livers and cells were perfused or incubated for either 30 or 90 min prior to the addition of epinephrine. Epinephrine (0.I #M) was added 5 min before livers were freeze-clamped or hepatocytes were sampled. Values are averages + SEM of four perfused livers sampled at each time point or samples obtained from these preparations of hepatocytes or six 35 mm culture plates of hepatocytes. Total phosphorylase measured in perfused livers and isolated hepatocytes did not change significantly over the time course of the experiment. Total phosphorylase measured in perfused livers at 30 and 90min was 9.7 +0.7 and 8.8 _ 0.9 nmol min- I mg prot- I , respectively. Values in hepatocytes were 13.9+ 1.3 and 13.4+ 1.2 at 30 and 90rain, respectively.
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Minutes Fig. 3. Time course of glucose and lactate output from isolated hepatocytes and perfused livers. Experimental conditions as in Fig. 1. Glucose (Panel A) and lactate (Panel B) were measured in effluent perfusate or incubation medium as described in methods. Rates of metabolite production by hepatocytes are based on amounts of glucose and lactate produced over 15 min intervals. Rates in the perfused liver are based on amounts of glucose or lactate measured in the effluent perfusate, the flow rate and the liver wet weight.
Glycogen phosphorylase-a in rat liver 90
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to epinephrine and possibly other hormones that act via calcium-dependent mechanisms.
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Acknowledgement--Supported, in part, by USPHS Grant
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REFERENCES
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Glucose plus Lactate Production (p.mol/g/hr)
Fig. 4. Relationship between phosphorylase-a activity and glycogenolysis in isolated hepatocytes and perfused livers. Glycogenolysis, [glucose plus 2 x lactate production (Fig. 2)], is compared with phosphorylase-a activity (data from Fig. 1) in perfused livers or hepatocytes determined at the same time points.
epinephrine at 9 0 m i n remained low and only increased to about 60% of total activity (Fig. 2). Hepatocytes that were plated and maintained in primary culture resembled freshly isolated hepatocytes and displayed a diminished response to epinephrine. The diminished effect of epinephrine on phosphorylase-a in isolated hepatocytes most likely involves alterations in calcium status. First, the total content of calcium is probably higher in hepatocytes than perfused livers at the outset of perfusion or incubation. In addition to increases in total calcium in hepatocytes, it has been suggested that depletion of a hormone responsive Ca 2÷ pool occurs in this preparation (Kleineke and Soling, 1985; Joseph and Williamson, 1983). In line with this possibility is the finding that the content of mitochondrial relative to non-mitochondrial calcium is greatly elevated in hepatocytes compared to the intact liver (Kleineke and Soling, 1985; Joseph and Williamson, 1983). Mechanisms coupling membrane receptors to intracellular second messenger system may be compromised during prolonged incubations of hepatocytes in vitro due to this redistribution of calcium. Higher concentrations of Ca 2+ in hepatocytes compared to perfused livers may also lead to increased protein phosphatase activity and dephosphorylation of phosphorylase-a. Phosphatase 2B is activated by concentrations of calcium considerably greater than that required to activate phosphorylase kinase (Blackmore et al., 1983). It is clear from many studies that intracellular free calcium is critical in intracellular regulation of metabolism and cell structure. The finding that basal phosphorylase-a, its change with time and its response to epinephrine differed dramatically in isolated perfused livers and hepatocytes indicate that mechanisms linking activation of epinephrine receptors to phosphorylase activates differ in these preparations. Differences reported above emphasize that the structural organization of the intact liver is an important determinant of the response of this organ
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