Eur. J. Biochem. 88,475-482 (1978)
Pyrimidine Nucleotide Biosynthesis and Turnover in Rat Skeletal Muscle and Liver Jens RASENACK, Junko NOWACK, and Karl DECKER Biochemisches Institut an der Medizinischen Fakultat, Albert-Ludwigs-Universitat, Freiburg i. Br. (Received February 3, 1978)
1. Pyrimidine nucleotide biosynthesis in skeletal muscle of rat hindquarter was studied with a hemoglobin-free constant-pressure perfusion system. The functional competence of this preparation was monitored by determinations of oxygen consumption, lactate/pyruvate ratio, glucose uptake and its stimulation by insulin, ATP and creatine phosphate contents of the muscle, the energy charge and the release of aldolase and creatine phosphokinase into the perfusate. 2. The time-dependence of NaHI4CO3 incorporation into the acid-soluble uracil nucleotides of muscle tissue was measured and the rate of de novo biosynthesis calculated to be 2.7 pmolx kg-' x h-'. 3. The half-life of the uracil nucleotides of skeletal muscle in vivo was estimated by pulse-labelling with [2-I4C]uridineand measuring the decrease of their specific radioactivity. It was found to be 19 h, corresponding to a turnover rate of the acid-soluble uracil nucleotides of 5 pmol x kg-' x h-'. 4. The influence of alimentary pyrimidines on uracil nucleotide biosynthesis in the perfused muscle was also investigated. In animals kept on a uridine-free diet for 7 days, the incorporation rate of labelled bicarbonate into acid-soluble uracil 5'-nucleotides was 30 % higher than in controls fed a commercial laboratory chow. 5. The half-life in vivo of the acid-soluble uracil nucleotides of rat liver was estimated from the decrease of their specific radioactivity following a [2-'4C]uridine pulse. It was found to be 15 h, corresponding to a turnover rate of about 80 pmol x kg-' x h-'. The regulation of pyrimidine nucleotide biosynthesis de novo in mammalian tissues has been studied extensively in vitro. It was concluded that the cytoplasmic glutamine-dependent carbamoyl phosphate synthase is subject to allosteric inhibition by UTP [l -71. The addition of uridine to superfused rat liver slices resulted in a reduced incorporation of NaH14C03into orotate [8,9], while the D-galactosamineinduced depletion of the intracellular UTP contents of perfused rat liver [lo] or hepatoma cells [ l l ] led to a dramatic increase of bicarbonate incorporation into the uracil nucleotides. Most of these investigations were done with liver, Abhreviution. CUMP, sum of all acid-soluble uracil 5'-nucleotides. Enzymes. Aldolase or fructose-I .6-bisphosphate D-glyCeraldehyde-3-phosphate-lyase(EC 4.1.2.13); carbamoyl-phosphate synthase (glutamine) or ATP : carbamate phosphotransferase (dephosphorylating, amido-transferring) (EC 2.7.2.9); carbonic anhydrase or carbonate hydro-lyase (EC 4.2.1.l); creatine phosphokinase or ATP: creatine N-phosphotransferase (EC 2.7.3.2); OMP decarboxylase or orotidine 5'-phosphate carboxylase (EC 4.1.1.23); OMP pyrophosphorylase or orotidine 5'-phosphate : pyrophosphate phosphoribosyl transferase (EC 2.4.2.10); phosphodiesterase I or oligonucleate 5'-nucleotidohydrolase (EC 3.1.4.1).
hepatoma or Ehrlich ascites cells, but little is known about pyrimidine nucleotide biosynthesis of muscle tissue. The activities of OMP pyrophosphorylase and OMP decarboxylase were hardly detectable in muscle extracts [12]. Th? possibility had to be considered that very little, if any, synthesis of pyrimidines de novo takes place in this tissue and that muscle depends on exogenous precursors which are provided by other organs or by the food. The following experiments were designed to measure uracil nucleotide biosynthesis de novo by the highly sensitive incorporation of labelled bicarbonate. To exclude interference by exogenous precursors or influences, the hemoglobinfree perfused rat hindquarter was used as muscle preparation. In addition, uracil nucleotide synthesis de novo was measured in muscle of animals kept on a uridine-free diet. The studies on the isolated muscle preparation were supplemented by measurements in vivo of half-lives and turnover rates of the soluble uracil nucleotide pools of rat skeletal muscle and liver in rats prelabelled with [2-14C]uridine. These data were compared with the values obtained with the perfused organs and found to be in reasonable agreement.
476
MATERIALS AND METHODS Female rats of the Wistar strain, weighing 150 to 170 g were used throughout. They had free access to water and to a commercial chow diet (Altromin R, Altrogge GmbH, Lage, Lippe) except in studies with a uridine-free diet. The latter diet was made up with reference to the Altromin standard diet; the pellets consisted of 22% egg albumin (purum), 53% starch (DAB 6), 10% sucrose, 3 % olive oil, 4 % cellulose, 6 % minerals and trace elements and 2 % vitamins. The uridine content of this diet was checked spectrophotometrically and found to be less than 0.1 nmol/g. Bovine serum albumin from C. Roth KG (Karlsruhe) used for muscle perfusions was pretreated with charcoal [13]. Uridine, pyruvate, enzymes and coenzymes were from Boehringer Mannheim GmbH (Mannheim). Dowex 1x10, 200-400 mesh was prepared for use by treatment with KOH and HC1 [14]; insulin (26 I.U./mg) was purchased from Sigma (St Louis, Mo., U.S.A.), thin-layer plates (Polygrani CEL 300) from Macherey and Nagel (Duren). All other reagents were of analytical grade (Merck AG, Darmstadt). [214C]Uridine (specific radioactivity 53.9 Ci x mmol-') was bought from CIS (Isotopendienst West, Sprendlingen) and sodium ['4C]bicarbonate (60.2 Ci x mol-', aqueous solution) from the Radiochemical Centre (Amersham).
Hindquarter Perfusion
The operating procedure was performed according to Ruderman et al. [I51 with the following modifications: the skin was removed in every case; uterus, sigmoid flexure, rectum and urinary bladder remained in situ with their vessels ligated. The anoxic interval was reduced to less than 5 s by the following method : a Brauniile (0.5, G 18, Braun Melsungen) was inserted into the abdominal aorta and filled retrograde from the tip with perfusion medium after removal of the trocar; it was then connected to the perfusion medium and the cannula pushed forward caudally for approximately 0.5 cm so that the tip of the Braunule was 0.5 - 1.O cm proximal of the aortic bifurcation. The vena cava was cannulated with another Brauniile (2.0, G 14). Its tip was positioned several millimetres proximal to that of the first. In every case, the animals were hemisected and the spinal cord destroyed. Finally, the hindquarter was placed horizontally in a closed chamber in order to prevent desiccation. The legs were elevated by wedges. The needles were held in place by clamps. The preparation was rinsed with 140 ml perfusion medium and then transferred into a temperature-controlled (37 "C) cabinet. The operation took 20 - 25 min; a total of 30 - 35 min passed until the hindquarter was connected to the perfusion circuit.
Pyrimidine Biosynthesis and Turnover in Muscle and Liver
The perfusion medium was composed of Krebs/ Henseleit/bicarbonate buffer containing 4 g of charcoal-treated bovine serum albumin per 100 ml, 5.5 mM glucose, 2 niM pyruvate and an amino acid mixture [16]. In some experiments, carbonic anhydrase was added to accelerate equilibration of added [14C]bicarbonate. The perfusate was gassed with humidified 02/c02(95/5). Perfusion apparatus and glassware were a modification of that described by Hems et al. [I71 and Teufel et al. [18]. The hydrostatic perfusion pressure was maintained by a gravity system of 65 cm. The oxygen tension of the venous medium was measured with a Clark's platinum electrode (Eschweiler & Co., Kiel). The volume of the recirculating perfusion medium was 150 ml. The experiments were started after a 10-min equilibration period. The [14C]bicarbonate incorporation experiments were performed in a closed gas volume of about 1.5 1. Analytical Methods
Glucose [19], lactate and pyruvate [20,21] in deproteinized perfusate were measured enzymatically. Muscle and liver samples were freeze-clamped and homogenized in about 5 vol. of cold 0.9 M HC104 by either the method of Potter-Elvehjem or by use of an Ultra-Turrax (Janke & Kunkel KG, Staufen). After neutralization with solid KHC03 and centrifugation, creatine phosphate [22], ATP [22,23], ADP and AMP [24] as well as CUMP [25] were measured enzymatically. Enzyme activities of fructose-l,6-bisphosphate aldolase and creatine phosphokinase were measured as described by Bergmeyer et al. [26] and Forster et al. [27]. Specific radioactivity of the bicarbonate in the perfusate was analysed as described by Pausch et al. [lo]. Radioactivity measurements were performed using a Tricarb liquid scintillation spectrometer 3380 (Packard Instruments). The scintillation solution [28] contained 4 "/, (w/v) silica powder. Radioactivity (in counts/min) was corrected for quenching and background by internal standardization. Radioactivity on chromatograms was detected with a radiochromatogram scanner (Frieseke & Hoepfner, Erlangen-Bruck). Sodium [ ''C]Bicarbonate Incorporation
The rate of bicarbonate incorporation into CUMP of the muscle was measured after injection of 1 mCi of an NaH14C03 solution into the perfusate 10 min after the hindquarter had been connected to the circuit. The decrease of the total radioactivity in the perfusion medium was measured [29] and, in some experiments, also the specific radioactivity of the precursor itself [lo]. At the times indicated about 6 g of muscle tissue of the thigh region was excised and freeze-clamped.
477
J. Rasenack, J. Nowack, and K. Decker
The tissue was homogenized in 5 vol. of cold 0.9 M perchloric acid with an Ultra-Turrax for 4 min in intervals of 30 s and neutralized with solid KHC03. The supernatant was treated with a snake venom phosphodiesterase I preparation [25] whose 5'-nucleotidase had been inactivated as described by Sulkowski and Laskowski [30]. The hydrolyzed supernatant was concentrated in vucuo to about 5 ml, adjusted to pH 6.5 and placed on an anion-exchange column (0.9 x 30 cm) of Dowex 1x10, formate form. A linear gradient of formic acid, 0.2- 4.0 M, with a total volume of 1200 ml was applied and 8-ml fractions collected. The absorption was monitored with an Uvicord I1 (LKB, Stockholm). Fractions containing UMP were pooled, concentrated in vacua and purified by chromatography on Whatman 3 MM sheets using an ethanol/ammonium acetatemixture (5/2, v/v) pH 7.5 [31]. The UMPcontaining band was detected with a radiochromatography scanner. It was eluted and rechromatographed on a cellulose thin-layer plate with isopropanol/5.5 M HCI (65/35, v/v) [32,33] or on a Whatman DE 81 ionexchange paper [34]. UMP was eluted and its specific radioactivity determined.
RESULTS Functional Criteriu of the Perfused Hindquarter
Immediately after the connection of the hindquarter to the open perfusion, the muscle became pale pink and the feet turned white indicating that most of the tissue was perfused. Usually only a small leakage of about one drop per minute occurred. It was primarily due to injured capillaries of the connective tissue as judged by perfusions with Evan's blue. The hindquarter was discarded if an edema developed at this stage of the procedure. The skin was removed to reduce the amount of non-muscular tissue which might contribute any pyrimidine precursors. According to Ruderman et al. [15] it is possible to calculate the approximate quantity of the perfused muscle on the basis of body weight. About 16.6% of a rat weighing 180 g are perfused if the operation is performed according to these authors. About 80 % of the hindquarter consist of muscle and fat, the remainder being skin and bone. As our perfusions were performed with the skin removed, which accounts for approximately lo%, there is a slight shift in favor of the muscle. The extent of the perfused tissue regions was checked by the distribution of added Evan's blue as described by Ruderman et al. [15]. As the perfusate was a hemoglobin-free Krebs/ Henseleit/bicarbonate buffer, the flow rate was of great importance to assure a sufficient oxygen supply. It is also a most useful indicator for the finctional integrity of the perfused hindquarter [15,35]. Gene-
rally, a flow rate through the hindquarter of more than 8 ml x min-' is held necessary [15,35 - 371 using an erythrocyte-containing perfusate. In the hemoglobin-free perfusion system, a higher rate of 14 k 1 ml x min-' was found necessary to provide enough oxygen. During the initial minutes after the connection to thecircuit, theflowratewasonly 1 0 - 1 2 m l x m i n ~ ' , but after 15 min it rose to 12- 15 ml x inin-' and remained constant for the rest of the perfusion. Also, the pH of the perfusate was constant during a 4-h period. The lactate/pyruvate ratio of the perfusion medium increased almost linearly with time reaching 22.7 at 4 11. The glucose uptake from the medium per 30 g of muscle was 1.7 pmol x min-' and almost linear with time. Insulin (1 I.U.jl50 ml) stimulated the uptake 3-fold to about 5.1 pmol x min-'. The higher stimulation of glucose uptake reported by others [15,35,36] might be attributed to higher glucose concentrations in the perfusate and to the intact innervation [15,35]. The rate of lactate production was 20 % higher in the presence of insulin (3.4 versus 2.8 pmol x min-' per 30 g) but this difference was only of marginal significance. The rate of lactate production is in good agreement with observations from skin-less preparations ~151. The oxygen consumption of 3 0 g of muscle increased during the initial 30 min from 6.7 _+ 0.9 ymol x min-' to 8.3 -9.0 pmol x min-'. These changes paralleled those of the flow rate. The oxygen consumption of the resting denervated muscle was similar to that of an unstimulated, innervated muscle [15]. Aldolase and creatine phosphokinase activities increased with time, although at a somewhat lower rate during the second half of the experimental period. Similar observations were reported by Tienhaus et al. [37]. The leakage of these enzymes did not correlate significantly with intracellular ATP and creatine phosphate levels and with the energy charge [38]which are considered as better criteria of tissue integrity. As shown in Table 1, ATP decreased during a 4-h period by less than 10% and creatine phosphate dropped to about 75% of the initial value. The energy charge remained constant during the whole period due to the concomitant reduction of ADP and AMP contents. Uracil Nucleotide Content of the Pecfused Muscle
The sum of the acid-soluble uracil nucleotides (CUMP) of muscular tissue is much smaller than that of liver. The hepatic content was found to be 1.27mmol per kg of wet liver tissue [39]. The corresponding figure for skeletal muscle freeze-clamped in sifu was 0.150 k 0.025 mmol x (kg wet wt)-' (n = 30) and it did not change significantly during the perfusion period. About 65% of muscle uracil nucleotides is represented by UTP UDP + UMP (0.098 mmol
+
Pyrimidine Biosynthesis and Turnover in Muscle and Liver
478
Table 1 . Parameters of the energy state of the perfused rat hindquarter Results are mean values k S.D. with the number of determinations in parentheses Time of perfusion
ADP
ATP
AMP
Creatine phosphate
Energy charge
16.8 k 0.9 15.8 I 2.0 12.2 f 2.0 12.3 k 0.9 12.3 & 1.9
0.95 0.01 (5) 0.94 k 0.01 (9) 0.95 +_ 0.01 (6) 0.94 k 0.01 (7) 0.94 k 0.01 (11)
~~
min
mmol x (kg wet wt)-' ~-
0 60 120 180 240
6.31 6.35 5.90 6.05 5.98
~
k 0.34 (5) f 0.48 & 0.54 f 0.64 f 0.26
(30) (8) (7) (12)
0.68 k 0.06 (5) 0.78 It 0.07 (9) 0.67 f 0.03 (7) 0.77 k 0.08 (7) 0.80 k 0.14 (11)
0.02 0.01 0.03 f 0.01 (9) 0.005 k 0.006 (6) 0.01 k 0.01 (7) 0.03 k 0.02 (11) ~
*
~
(5) (27) (7) (7) (12)
Table 2. Z U M P contents of muscle and liver, and weight gain after 4 days ( A ) and 24 days ( B ) ZUMP was measured enzymatically as described in Methods. The average daily food intake of a rat was 4.5g. The supplemented diet contained 300 mg uridine per kg of pelleted material. The results are given as mean values k S.D. (n) ~
Weight gain
ZUMP
Diet
~~
muscle
~
liver
~~~
of control
mmol x (kg wet wt)-' A
Diet without uridine Diet + uridine Controls (lab chow)
0.15 f 0.02 (6) 0.16 0.02 (6) 0.16 k 0.01 (4)
1.54 & 0.17 (5) 1.51 k 0.05 (6) 1.56 i 0.06 (4)
116 +_ 32 (6) 117 i 40 (6) 100 k 45 (4)
B
Diet without uridine Diet + uridine Controls (lab chow)
0.14 5 0.01 (5) 0.17 k 0.03 (4) 0.14 i 0.01 (4)
1.57 +_ 0.02 (4) 1.62 k 0.30 (3) 1.55 I 0.08 (4)
83 f 14 (6) 79 k 19 (6) 100 k 20 (4)
x kg-I). In liver, however, only about 20 % of CUMP are present as uridine phosphates. Muscle tissue is able to take up and phosphorylate added uridine [40]. Therefore, the possibility of an exclusively exogenous supply of uridine or cytosine precursors of uracil nucleotides of skeletal muscle was considered. The most likely sources are degradation products of nucleic acids and nucleotides contained in foodstuff or, possibly, an export of the nucleoside by the liver. The latter possibility was ruled out by analysis of the effluent of isolated perfused livers whose uracil nucleotide pools were prelabelled with ['4C]uridine (H. Groteluschen, unpublished data). To exclude nucleosides of alimentary origin as source of uracil nucleotides, animals were kept for various lengths of time on a diet free of pyrimidine bases (Table 2). The diet, however, did not influence the growth of the animals nor the CUMP content of their skeletal muscle. These results suggest that muscle tissue is able to synthesize pyrimidine nucleotides de novo. [14CjBicarbonateIncorporation into Uracil Nucleotides
The most sensitive method to establish the existence of de novo synthesis of pyrimidine nucleotides in an
intact organ was shown to be the incorporation of labelled bicarbonate into CUMP [lo]. With 1 mCi NaH14C03 per 3 50 ml of perfusate the specific radioactivity of HCO; could be kept at a satisfactory level only by use of a closed perfusion system with a constant gas volume of 1.4 1 (Fig. 1). The decrease of the specific radioactivity paralleled that of the total radioactivity in the perfusion medium. About 0.04 % of the applied radioactivity was recovered in the acid-soluble supernatant of the muscle and this uptake was essentially completed in less than 1 h. The specific radioactivity of CUMP increased as demonstrated in Fig. 2. This curve fits best a parabola, y = a 2 + bx, where y is specific radioactivity of CUMP at a given time (Cixmol-I); x is time (h); a is deceleration of the labelling rate (Ci x rno1-I x h-') and b is rate constant (h-I). The slope of the curve, y' = 2ax b, represents the rate of incorporation of labelled bicarbonate into the acid-soluble uracil nucleotides. As the specific radioactivity of the precursor at the start of the experiment, 0.267 Ci x mol-', was known and the CUMP content remained constant during the observation period, the incorporation rate of bicarbonate could be obtained by zero-time extrapolation as 2.7 pmol x kg-' x h-'. In another set of experiments, hindquarters of animals kept on a uridine-free diet for 7 days were
+
479
J. Rasenack, J. Nowack, and K. Decker
used. Since the tissue level of CUMP was the same as that of the control animals (Table 2), the incorporation rate was calculated from the data of Fig.2 as 3.5 pmolx k g - l x h - ' , i.e. 30% higher than in rats receiving a commercial diet. This indicates that pyrimidines supplied with the food can have a regulatory influence on the synthesis de novo.
0
1 2 3 Time of perfusion (h)
4
Fig. 1. D r c w u . of ~ tl?e rota/ iudiouctii,/lj,
Pyrimidine Nucleotide Biosynthesis and Turnover in Rat Skeletal Muscle and Liver Jens RASENACK, Junko NOWACK, and Karl DECKER Biochemisches Institut an der Medizinischen Fakultat, Albert-Ludwigs-Universitat, Freiburg i. Br. (Received February 3, 1978)
1. Pyrimidine nucleotide biosynthesis in skeletal muscle of rat hindquarter was studied with a hemoglobin-free constant-pressure perfusion system. The functional competence of this preparation was monitored by determinations of oxygen consumption, lactate/pyruvate ratio, glucose uptake and its stimulation by insulin, ATP and creatine phosphate contents of the muscle, the energy charge and the release of aldolase and creatine phosphokinase into the perfusate. 2. The time-dependence of NaHI4CO3 incorporation into the acid-soluble uracil nucleotides of muscle tissue was measured and the rate of de novo biosynthesis calculated to be 2.7 pmolx kg-' x h-'. 3. The half-life of the uracil nucleotides of skeletal muscle in vivo was estimated by pulse-labelling with [2-I4C]uridineand measuring the decrease of their specific radioactivity. It was found to be 19 h, corresponding to a turnover rate of the acid-soluble uracil nucleotides of 5 pmol x kg-' x h-'. 4. The influence of alimentary pyrimidines on uracil nucleotide biosynthesis in the perfused muscle was also investigated. In animals kept on a uridine-free diet for 7 days, the incorporation rate of labelled bicarbonate into acid-soluble uracil 5'-nucleotides was 30 % higher than in controls fed a commercial laboratory chow. 5. The half-life in vivo of the acid-soluble uracil nucleotides of rat liver was estimated from the decrease of their specific radioactivity following a [2-'4C]uridine pulse. It was found to be 15 h, corresponding to a turnover rate of about 80 pmol x kg-' x h-'. The regulation of pyrimidine nucleotide biosynthesis de novo in mammalian tissues has been studied extensively in vitro. It was concluded that the cytoplasmic glutamine-dependent carbamoyl phosphate synthase is subject to allosteric inhibition by UTP [l -71. The addition of uridine to superfused rat liver slices resulted in a reduced incorporation of NaH14C03into orotate [8,9], while the D-galactosamineinduced depletion of the intracellular UTP contents of perfused rat liver [lo] or hepatoma cells [ l l ] led to a dramatic increase of bicarbonate incorporation into the uracil nucleotides. Most of these investigations were done with liver, Abhreviution. CUMP, sum of all acid-soluble uracil 5'-nucleotides. Enzymes. Aldolase or fructose-I .6-bisphosphate D-glyCeraldehyde-3-phosphate-lyase(EC 4.1.2.13); carbamoyl-phosphate synthase (glutamine) or ATP : carbamate phosphotransferase (dephosphorylating, amido-transferring) (EC 2.7.2.9); carbonic anhydrase or carbonate hydro-lyase (EC 4.2.1.l); creatine phosphokinase or ATP: creatine N-phosphotransferase (EC 2.7.3.2); OMP decarboxylase or orotidine 5'-phosphate carboxylase (EC 4.1.1.23); OMP pyrophosphorylase or orotidine 5'-phosphate : pyrophosphate phosphoribosyl transferase (EC 2.4.2.10); phosphodiesterase I or oligonucleate 5'-nucleotidohydrolase (EC 3.1.4.1).
hepatoma or Ehrlich ascites cells, but little is known about pyrimidine nucleotide biosynthesis of muscle tissue. The activities of OMP pyrophosphorylase and OMP decarboxylase were hardly detectable in muscle extracts [12]. Th? possibility had to be considered that very little, if any, synthesis of pyrimidines de novo takes place in this tissue and that muscle depends on exogenous precursors which are provided by other organs or by the food. The following experiments were designed to measure uracil nucleotide biosynthesis de novo by the highly sensitive incorporation of labelled bicarbonate. To exclude interference by exogenous precursors or influences, the hemoglobinfree perfused rat hindquarter was used as muscle preparation. In addition, uracil nucleotide synthesis de novo was measured in muscle of animals kept on a uridine-free diet. The studies on the isolated muscle preparation were supplemented by measurements in vivo of half-lives and turnover rates of the soluble uracil nucleotide pools of rat skeletal muscle and liver in rats prelabelled with [2-14C]uridine. These data were compared with the values obtained with the perfused organs and found to be in reasonable agreement.
476
MATERIALS AND METHODS Female rats of the Wistar strain, weighing 150 to 170 g were used throughout. They had free access to water and to a commercial chow diet (Altromin R, Altrogge GmbH, Lage, Lippe) except in studies with a uridine-free diet. The latter diet was made up with reference to the Altromin standard diet; the pellets consisted of 22% egg albumin (purum), 53% starch (DAB 6), 10% sucrose, 3 % olive oil, 4 % cellulose, 6 % minerals and trace elements and 2 % vitamins. The uridine content of this diet was checked spectrophotometrically and found to be less than 0.1 nmol/g. Bovine serum albumin from C. Roth KG (Karlsruhe) used for muscle perfusions was pretreated with charcoal [13]. Uridine, pyruvate, enzymes and coenzymes were from Boehringer Mannheim GmbH (Mannheim). Dowex 1x10, 200-400 mesh was prepared for use by treatment with KOH and HC1 [14]; insulin (26 I.U./mg) was purchased from Sigma (St Louis, Mo., U.S.A.), thin-layer plates (Polygrani CEL 300) from Macherey and Nagel (Duren). All other reagents were of analytical grade (Merck AG, Darmstadt). [214C]Uridine (specific radioactivity 53.9 Ci x mmol-') was bought from CIS (Isotopendienst West, Sprendlingen) and sodium ['4C]bicarbonate (60.2 Ci x mol-', aqueous solution) from the Radiochemical Centre (Amersham).
Hindquarter Perfusion
The operating procedure was performed according to Ruderman et al. [I51 with the following modifications: the skin was removed in every case; uterus, sigmoid flexure, rectum and urinary bladder remained in situ with their vessels ligated. The anoxic interval was reduced to less than 5 s by the following method : a Brauniile (0.5, G 18, Braun Melsungen) was inserted into the abdominal aorta and filled retrograde from the tip with perfusion medium after removal of the trocar; it was then connected to the perfusion medium and the cannula pushed forward caudally for approximately 0.5 cm so that the tip of the Braunule was 0.5 - 1.O cm proximal of the aortic bifurcation. The vena cava was cannulated with another Brauniile (2.0, G 14). Its tip was positioned several millimetres proximal to that of the first. In every case, the animals were hemisected and the spinal cord destroyed. Finally, the hindquarter was placed horizontally in a closed chamber in order to prevent desiccation. The legs were elevated by wedges. The needles were held in place by clamps. The preparation was rinsed with 140 ml perfusion medium and then transferred into a temperature-controlled (37 "C) cabinet. The operation took 20 - 25 min; a total of 30 - 35 min passed until the hindquarter was connected to the perfusion circuit.
Pyrimidine Biosynthesis and Turnover in Muscle and Liver
The perfusion medium was composed of Krebs/ Henseleit/bicarbonate buffer containing 4 g of charcoal-treated bovine serum albumin per 100 ml, 5.5 mM glucose, 2 niM pyruvate and an amino acid mixture [16]. In some experiments, carbonic anhydrase was added to accelerate equilibration of added [14C]bicarbonate. The perfusate was gassed with humidified 02/c02(95/5). Perfusion apparatus and glassware were a modification of that described by Hems et al. [I71 and Teufel et al. [18]. The hydrostatic perfusion pressure was maintained by a gravity system of 65 cm. The oxygen tension of the venous medium was measured with a Clark's platinum electrode (Eschweiler & Co., Kiel). The volume of the recirculating perfusion medium was 150 ml. The experiments were started after a 10-min equilibration period. The [14C]bicarbonate incorporation experiments were performed in a closed gas volume of about 1.5 1. Analytical Methods
Glucose [19], lactate and pyruvate [20,21] in deproteinized perfusate were measured enzymatically. Muscle and liver samples were freeze-clamped and homogenized in about 5 vol. of cold 0.9 M HC104 by either the method of Potter-Elvehjem or by use of an Ultra-Turrax (Janke & Kunkel KG, Staufen). After neutralization with solid KHC03 and centrifugation, creatine phosphate [22], ATP [22,23], ADP and AMP [24] as well as CUMP [25] were measured enzymatically. Enzyme activities of fructose-l,6-bisphosphate aldolase and creatine phosphokinase were measured as described by Bergmeyer et al. [26] and Forster et al. [27]. Specific radioactivity of the bicarbonate in the perfusate was analysed as described by Pausch et al. [lo]. Radioactivity measurements were performed using a Tricarb liquid scintillation spectrometer 3380 (Packard Instruments). The scintillation solution [28] contained 4 "/, (w/v) silica powder. Radioactivity (in counts/min) was corrected for quenching and background by internal standardization. Radioactivity on chromatograms was detected with a radiochromatogram scanner (Frieseke & Hoepfner, Erlangen-Bruck). Sodium [ ''C]Bicarbonate Incorporation
The rate of bicarbonate incorporation into CUMP of the muscle was measured after injection of 1 mCi of an NaH14C03 solution into the perfusate 10 min after the hindquarter had been connected to the circuit. The decrease of the total radioactivity in the perfusion medium was measured [29] and, in some experiments, also the specific radioactivity of the precursor itself [lo]. At the times indicated about 6 g of muscle tissue of the thigh region was excised and freeze-clamped.
477
J. Rasenack, J. Nowack, and K. Decker
The tissue was homogenized in 5 vol. of cold 0.9 M perchloric acid with an Ultra-Turrax for 4 min in intervals of 30 s and neutralized with solid KHC03. The supernatant was treated with a snake venom phosphodiesterase I preparation [25] whose 5'-nucleotidase had been inactivated as described by Sulkowski and Laskowski [30]. The hydrolyzed supernatant was concentrated in vucuo to about 5 ml, adjusted to pH 6.5 and placed on an anion-exchange column (0.9 x 30 cm) of Dowex 1x10, formate form. A linear gradient of formic acid, 0.2- 4.0 M, with a total volume of 1200 ml was applied and 8-ml fractions collected. The absorption was monitored with an Uvicord I1 (LKB, Stockholm). Fractions containing UMP were pooled, concentrated in vacua and purified by chromatography on Whatman 3 MM sheets using an ethanol/ammonium acetatemixture (5/2, v/v) pH 7.5 [31]. The UMPcontaining band was detected with a radiochromatography scanner. It was eluted and rechromatographed on a cellulose thin-layer plate with isopropanol/5.5 M HCI (65/35, v/v) [32,33] or on a Whatman DE 81 ionexchange paper [34]. UMP was eluted and its specific radioactivity determined.
RESULTS Functional Criteriu of the Perfused Hindquarter
Immediately after the connection of the hindquarter to the open perfusion, the muscle became pale pink and the feet turned white indicating that most of the tissue was perfused. Usually only a small leakage of about one drop per minute occurred. It was primarily due to injured capillaries of the connective tissue as judged by perfusions with Evan's blue. The hindquarter was discarded if an edema developed at this stage of the procedure. The skin was removed to reduce the amount of non-muscular tissue which might contribute any pyrimidine precursors. According to Ruderman et al. [15] it is possible to calculate the approximate quantity of the perfused muscle on the basis of body weight. About 16.6% of a rat weighing 180 g are perfused if the operation is performed according to these authors. About 80 % of the hindquarter consist of muscle and fat, the remainder being skin and bone. As our perfusions were performed with the skin removed, which accounts for approximately lo%, there is a slight shift in favor of the muscle. The extent of the perfused tissue regions was checked by the distribution of added Evan's blue as described by Ruderman et al. [15]. As the perfusate was a hemoglobin-free Krebs/ Henseleit/bicarbonate buffer, the flow rate was of great importance to assure a sufficient oxygen supply. It is also a most useful indicator for the finctional integrity of the perfused hindquarter [15,35]. Gene-
rally, a flow rate through the hindquarter of more than 8 ml x min-' is held necessary [15,35 - 371 using an erythrocyte-containing perfusate. In the hemoglobin-free perfusion system, a higher rate of 14 k 1 ml x min-' was found necessary to provide enough oxygen. During the initial minutes after the connection to thecircuit, theflowratewasonly 1 0 - 1 2 m l x m i n ~ ' , but after 15 min it rose to 12- 15 ml x inin-' and remained constant for the rest of the perfusion. Also, the pH of the perfusate was constant during a 4-h period. The lactate/pyruvate ratio of the perfusion medium increased almost linearly with time reaching 22.7 at 4 11. The glucose uptake from the medium per 30 g of muscle was 1.7 pmol x min-' and almost linear with time. Insulin (1 I.U.jl50 ml) stimulated the uptake 3-fold to about 5.1 pmol x min-'. The higher stimulation of glucose uptake reported by others [15,35,36] might be attributed to higher glucose concentrations in the perfusate and to the intact innervation [15,35]. The rate of lactate production was 20 % higher in the presence of insulin (3.4 versus 2.8 pmol x min-' per 30 g) but this difference was only of marginal significance. The rate of lactate production is in good agreement with observations from skin-less preparations ~151. The oxygen consumption of 3 0 g of muscle increased during the initial 30 min from 6.7 _+ 0.9 ymol x min-' to 8.3 -9.0 pmol x min-'. These changes paralleled those of the flow rate. The oxygen consumption of the resting denervated muscle was similar to that of an unstimulated, innervated muscle [15]. Aldolase and creatine phosphokinase activities increased with time, although at a somewhat lower rate during the second half of the experimental period. Similar observations were reported by Tienhaus et al. [37]. The leakage of these enzymes did not correlate significantly with intracellular ATP and creatine phosphate levels and with the energy charge [38]which are considered as better criteria of tissue integrity. As shown in Table 1, ATP decreased during a 4-h period by less than 10% and creatine phosphate dropped to about 75% of the initial value. The energy charge remained constant during the whole period due to the concomitant reduction of ADP and AMP contents. Uracil Nucleotide Content of the Pecfused Muscle
The sum of the acid-soluble uracil nucleotides (CUMP) of muscular tissue is much smaller than that of liver. The hepatic content was found to be 1.27mmol per kg of wet liver tissue [39]. The corresponding figure for skeletal muscle freeze-clamped in sifu was 0.150 k 0.025 mmol x (kg wet wt)-' (n = 30) and it did not change significantly during the perfusion period. About 65% of muscle uracil nucleotides is represented by UTP UDP + UMP (0.098 mmol
+
Pyrimidine Biosynthesis and Turnover in Muscle and Liver
478
Table 1 . Parameters of the energy state of the perfused rat hindquarter Results are mean values k S.D. with the number of determinations in parentheses Time of perfusion
ADP
ATP
AMP
Creatine phosphate
Energy charge
16.8 k 0.9 15.8 I 2.0 12.2 f 2.0 12.3 k 0.9 12.3 & 1.9
0.95 0.01 (5) 0.94 k 0.01 (9) 0.95 +_ 0.01 (6) 0.94 k 0.01 (7) 0.94 k 0.01 (11)
~~
min
mmol x (kg wet wt)-' ~-
0 60 120 180 240
6.31 6.35 5.90 6.05 5.98
~
k 0.34 (5) f 0.48 & 0.54 f 0.64 f 0.26
(30) (8) (7) (12)
0.68 k 0.06 (5) 0.78 It 0.07 (9) 0.67 f 0.03 (7) 0.77 k 0.08 (7) 0.80 k 0.14 (11)
0.02 0.01 0.03 f 0.01 (9) 0.005 k 0.006 (6) 0.01 k 0.01 (7) 0.03 k 0.02 (11) ~
*
~
(5) (27) (7) (7) (12)
Table 2. Z U M P contents of muscle and liver, and weight gain after 4 days ( A ) and 24 days ( B ) ZUMP was measured enzymatically as described in Methods. The average daily food intake of a rat was 4.5g. The supplemented diet contained 300 mg uridine per kg of pelleted material. The results are given as mean values k S.D. (n) ~
Weight gain
ZUMP
Diet
~~
muscle
~
liver
~~~
of control
mmol x (kg wet wt)-' A
Diet without uridine Diet + uridine Controls (lab chow)
0.15 f 0.02 (6) 0.16 0.02 (6) 0.16 k 0.01 (4)
1.54 & 0.17 (5) 1.51 k 0.05 (6) 1.56 i 0.06 (4)
116 +_ 32 (6) 117 i 40 (6) 100 k 45 (4)
B
Diet without uridine Diet + uridine Controls (lab chow)
0.14 5 0.01 (5) 0.17 k 0.03 (4) 0.14 i 0.01 (4)
1.57 +_ 0.02 (4) 1.62 k 0.30 (3) 1.55 I 0.08 (4)
83 f 14 (6) 79 k 19 (6) 100 k 20 (4)
x kg-I). In liver, however, only about 20 % of CUMP are present as uridine phosphates. Muscle tissue is able to take up and phosphorylate added uridine [40]. Therefore, the possibility of an exclusively exogenous supply of uridine or cytosine precursors of uracil nucleotides of skeletal muscle was considered. The most likely sources are degradation products of nucleic acids and nucleotides contained in foodstuff or, possibly, an export of the nucleoside by the liver. The latter possibility was ruled out by analysis of the effluent of isolated perfused livers whose uracil nucleotide pools were prelabelled with ['4C]uridine (H. Groteluschen, unpublished data). To exclude nucleosides of alimentary origin as source of uracil nucleotides, animals were kept for various lengths of time on a diet free of pyrimidine bases (Table 2). The diet, however, did not influence the growth of the animals nor the CUMP content of their skeletal muscle. These results suggest that muscle tissue is able to synthesize pyrimidine nucleotides de novo. [14CjBicarbonateIncorporation into Uracil Nucleotides
The most sensitive method to establish the existence of de novo synthesis of pyrimidine nucleotides in an
intact organ was shown to be the incorporation of labelled bicarbonate into CUMP [lo]. With 1 mCi NaH14C03 per 3 50 ml of perfusate the specific radioactivity of HCO; could be kept at a satisfactory level only by use of a closed perfusion system with a constant gas volume of 1.4 1 (Fig. 1). The decrease of the specific radioactivity paralleled that of the total radioactivity in the perfusion medium. About 0.04 % of the applied radioactivity was recovered in the acid-soluble supernatant of the muscle and this uptake was essentially completed in less than 1 h. The specific radioactivity of CUMP increased as demonstrated in Fig. 2. This curve fits best a parabola, y = a 2 + bx, where y is specific radioactivity of CUMP at a given time (Cixmol-I); x is time (h); a is deceleration of the labelling rate (Ci x rno1-I x h-') and b is rate constant (h-I). The slope of the curve, y' = 2ax b, represents the rate of incorporation of labelled bicarbonate into the acid-soluble uracil nucleotides. As the specific radioactivity of the precursor at the start of the experiment, 0.267 Ci x mol-', was known and the CUMP content remained constant during the observation period, the incorporation rate of bicarbonate could be obtained by zero-time extrapolation as 2.7 pmol x kg-' x h-'. In another set of experiments, hindquarters of animals kept on a uridine-free diet for 7 days were
+
479
J. Rasenack, J. Nowack, and K. Decker
used. Since the tissue level of CUMP was the same as that of the control animals (Table 2), the incorporation rate was calculated from the data of Fig.2 as 3.5 pmolx k g - l x h - ' , i.e. 30% higher than in rats receiving a commercial diet. This indicates that pyrimidines supplied with the food can have a regulatory influence on the synthesis de novo.
0
1 2 3 Time of perfusion (h)
4
Fig. 1. D r c w u . of ~ tl?e rota/ iudiouctii,/lj,
