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Studies on avian and mammalian heart tissues
Comp. Biochem. Physiol., 1969, Vol. 28, pp. 803 to 815. Pergamon Press. Printed in Great Britain
STUDIES ON AVIAN AND MAMMALIAN HEART TISSUES A. J. P A T E L * and C. V. R A M A K R I S H N A N Department of Biochemistry, Baroda University, Baroda, India (Received 11 June 1968)
Abstract--1. In a study of in vitro amino acid utilization avian heart tissues were found to utilize glutamate and alanine, and release glutamine, whereas mammalian tissues did the reverse. 2. These differences were attributable to the differences in the activities of L-glutamate : ammonia ligase, glutamine : glutamyl glutamyltransferase and alanine : 2-oxoglutarate aminotransferase in the above species. 3. Studies of heart tissue of cat, rabbit, rat, bat, pigeon, chick, sparrow, calotees, frog and fish showed a significant correlation between respiratory rate, heart beats/min, AMP aminohydrolase activity, L-glutamate:ammonia ligase and glutamate : NAD oxidoreductase activities together, and heart weight in relation to body weight. 4. The results support the view that L-glutamate:ammonia ligase and glutamate : NAD oxidoreductase serve as an ammonia removal mechanism in heart tissue. INTRODUCTION PRELIMINARY studies previously reported from this laboratory suggested differences in the utilization of glutamic acid and glutamine between avians and mammalian heart tissues cultivated in vitro in synthetic medium (Patel et al., 1965). Explants of the heart tissue of chick and pigeon when cultivated in a synthetic medium M150, devised by Morgan et al. (1955), were found to utilize glutamic acid and release glutamine whereas those of rat and rabbit were found to show the reverse phenomenon. Similar differences of a smaller order were found with regard to the utilization of alanine and aspartic acid; the avian tissues were found to utilize alanine and release aspartic acid, the reverse being true of mammalian tissues. T h e bat, a flying mammal, was found to resemble avians with regard to the utilization of glutamic acid and mammals with regard to that of aspartic acid. T h e avian heart tissues were found to show glutamine synthetase activity which was absent in the rat and rabbit but present in the bat. Glutamate-NAD oxidoreductase was found to be more active in mammalian heart. Studies carried out in this laboratory also showed the presence of L-glutamate : ammonia ligase in bird and bat heart tissues. In this connexion, birds have a big heart in relation to body weight because of the demands on this organ during flight *Present address: Medical Research Council Neuropsychiatric Research Unit, Carshalton, Surrey, U.K. 803
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A. J. PATEL AND C. V. RAMAKRISHNAN
(Spector, 1961 ; Walter & Sayles, 1961). T h e heart is a continuously active organ believed to be particularly sensitive to ammonia toxicity (Dukes, 1953; Walter & Sayles, 1961). T h e accumulation of ammonia in muscle during exercise because of A T P breakdown (White et al., 1964) results in muscular fatigue and the clearance of ammonia can take place during the period of resting, but such fatigue in cardiac muscle may be fatal. In this connexion, the brain is highly sensitive to ammonia toxicity (Seegmiller et al., 1954; McDermott & Adams, 1954) and L-glutamate : ammonia ligase is invariably present in the brain of animal species (Chung Wu, 1963). T h e presence of L-glutamate ammonia ligase in bird and bat heart and of glutamate : N A D oxidoreductase in the heart tissue of both mammals and avians raises the question whether these enzymes also provide a local machinery for the rapid removal of ammonia from heart tissue. T h e present paper reports a confirmation of the above findings under more controlled conditions including studies of free amino acids and enzymes in fresh tissue. T h e hypothesis regarding the role of glutamine synthetase and glutamateN A D oxidoreductase in ammonia removal was sought to be investigated by studying the interrelations between the following parameters in different species: activity of the heart as judged by heart weight in relation to body weight; heart beats/rain; respiratory activity as judged by oxygen uptake in vitro; the activities of Lg l u t a m a t e : a m m o n i a ligase and glutamate : N A D oxidoreductase which effect a removal of ammonia; A M P aminohydrolase, involved in ammonia formation from A T P breakdown; and A T P content of the heart tissues. Additional studies were made of the activity of the enzyme alanine : 2-oxoglutarate aminotransferase in different species because of differences found in the activity of this enzyme between avian and mammalian tissue. T h e species studied were: cat (Fells domestica), rabbit (Oryctolagus cuniculus), rat (Rattus norvegicus albinus), bat (Hipposiderons sperois), pigeon (Columba livia), chick (Gallus domesticus), house sparrow (Passer domesticus), calotees (Calotees versicolor), frog (Rana trigrina) and fish (Ophioce-
phalus punctalus). MATERIALS AND METHODS The methods followed and the conditions employed for the cultivation of heart tissues in vitro were essentially the same as those described previously (Patel et al., 1965) except for the following modifications: (i) the period of depletion was 8 hr as compared to 16 hr in the previous study; the modification was made in the light of subsequent studies which showed an 8-hr period of depletion to be compatible with maximum glucose utilization by the cultures, a longer period of depletion resulting in decreased glucose utilization; (ii) the period of cultivation was changed to 3 days as compared to 6 days in the original study in order to rule out the possibility of a change in the behaviour of the cultures after a change of medium on the fourth day; (iii) the biological status of the cultures at the end of cultivation was ascertained by determining their protein and DNA contents before and after cultivation; (iv) the optimum conditions for the enzyme assays were identified separately for avian and mammalian tissue; (v) the enzymes were assayed in fresh tissue instead of in the cultures and (vi) additional studies were made of the free amino acid contents of fresh tissue and the activity of the enzyme glutamine : glutamyl glutamyltransferase. Other details are summarized below.
AVIAN AND MAMMALIAN HEART TISSUES
805
Tissues Hearts of the species studied removed under aseptic conditions were used for these experiments. Glassware and chemicals Only Pyrex glassware and chemicals of research grade purity were used in the investigation. Media for the cultivation of heart explants The medium used was medium M150 devised by Morgan et al. (1955). Where so specified, omissions were made of individual constituents such as glutamine and alanine. The L-forms of leucine and glutamic acid were used at half the concentrations specified by Morgan et al. (1955) as the oL-forms were not available. Collection of cultures The culture fluid was collected as described previously (Patel et al., 1965). After removing the medium from the culture tubes, the explants were removed with the help of a stainless steel needle with a bent apex and collected in a small measuring cylinder containing 3"5 ml ice-cold 0"9% potassium chloride solution, p H 7"4. The solution and the explants were poured into a Potter-Elvejhem homogenizer and the cylinder rinsed with a further 0"5 ml of ice-cold potassium chloride solution and the washing added to the homogenizer. The explants were homogenized for 2 rain at 4,000 rev/min using a Teflon tissue grinder. All the operations were carried out at 0-4°C. The homogenate obtained was used for D N A and protein estimations. Chemical analysis Glucose and amino acids in culture fluid were determined as described previously (Patel et al., 1965) and protein by the method of Lowry et al. (1951). D N A was isolated by the method of Yamana& Sibatani (1960) and estimated by the diphenyl amino method (Ashwell, 1957) . For the estimation of free amino acids, tissue extracts were prepared by the method of Rajalakshmi et al. (1967), separated by the two-dimensional paper chromatographic method described by Hakkinen & Kulonen (1961), and estimated by the method described by Gothoskar et al. (1961). Enzyme assays The adult animals were killed by decapitation and their hearts removed. The fatty portions and blood were removed and the remaining tissue chopped to a fine pulp in a cold room at 0-4°C and ground in a chilled mortar with four times the volume of ice-cold 0"2 M potassium phosphate buffer, pH 7"0, for 15 man. The homogenate was used without centrifugation for the estimation of the activities of glutamine aminohydrolase, alanine : 2-oxoglutarate anainotransferase, aspartate : 2-oxoglutarate aminotransferase, alanine : oxaloacetate aminotransferase and glutamate : N A D oxidoreductase. For the estimation of L-glutamate : ammonia ligase and glutamine : glutamyl glutamyltransferase, the enzyme extract was prepared using 0"02 M potassium bicarbonate, pH 8"0, as grinding medium in place of potassium phosphate buffer. For the estimation of AMP aminohydrolase (E.C. 3.5.4.6) the enzyme extract was prepared using glass-distilled water as grinding medium. The dependence of enzyme activity on enzyme and substrate concentrations, pH and period of incubation was studied in the pigeon (avian), bat (flying mammal) and rabbit (mammal) and the optimum conditions worked out for the assay of different enzymes. For the assay of glutamine aminohydrolase (E.C. 3.5.1.2) the method described by Raina & Ramakrishnan (1964) was used except for the change that phosphate buffer, pH 7"5, was used instead of Tris buffer.
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For the assay of aminotransferases the method used was that described by Shah & Ramakrishnan (1963) except for the difference in the amount of the components added. For alanine : 2-oxoglutarate aminotransferase (E.C. 2.6.1.2) the assay system consisted of Tris buffer, pH 7-5, 50 bemoles; L-glutamate, 10 /zmoles; sodium pyruvate, 10/zmoles; pyridoxal phosphate, 0"08 bemole and enzyme preparation made to a final volume of 1"0 ml with water and incubated at 37°C for 30 min. For the assay of aspartate : 2-oxoglutarate aminotransferase (E.C. 2.6.1.1) and alanine : oxaloacetate aminotransferase (E.C. 2.5.1.12) the assay system consisted of Tris buffer, pH 7-5, 50 /zmoles; DL-aspartate, 20 bemoles; ketoacid, 10 /zmoles; pyridoxal phosphate, 0"04/zmoles and enzyme preparation made to a final volume of 1"0 ml with water and incubated at 37°C for 30 min. For the assay of L-glutamate : ammonia ligase (E.C. 6.3.1.2) the method of Gothoskar et al. (1960) was used except for the change that the concentrations of glutamate, MgSO4, NH2OH HC1, CSH and the incubation time were half of that used in the original method. For the assay of glutamine : glutamyl glutamyltransferase (E.C. 2.3.2.1) the method of T u r s h (1964) was used except for the change that the assay system contained double the concentrations of acetate buffer, glutamine, NH2OH HCI and half the concentration of A T P used in the original method. For the assay of g l u t a m a t e : N A D oxidoreductase (E.C. 1.4.1.2), the method of Srinivasamurthy & Swaminathan (1955) was used except for the change that the Tris buffer, pH 9"0 instead of pH 7'5, and triphenyl tetrazolium chloride instead of bromide were used. For the assay of AMP aminohydrolase (E.C. 3.5.4.6) the method described by Nikiforuk & Colowick (1955) was used. T h e assay system consisted of citrate NaOH buffer, pH 6"5, 50/~moles; AMP, 20 ftmoles; enzyme extract ,0"2 ml (0.1-0-6 mg protein) and water to a final volume of 2 ml. Protein contents of the enzyme extract were estimated by the method of Lowry et al. (1951). Estimation of A T P The animals were killed by decapitation and their hearts were removed. T h e blood and fatty portions were removed and approximately 1 g of the tissue was chopped to a fine pulp in the cold room at 0-4°C and ground with 10% solution of trichloroacetic acid in a mortar for about 15 rain. The homogenate was centrifuged at 7000 g at 2-4°C for 20 min and the clear supernatant collected in a beaker kept in ice. The residue was re-extracted in 5% trichloroacetic acid solution in the same manner. Both the supernatants were pooled together and neutralized by adding 10% sodium hydroxide. Equal volume of ice-cold alcohol was then added in order to precipitate the polysaccharides. After 3 hr the solution was centrifuged at 7000 g at 0-2°C for 20 min and the clear supematant collected in a beaker to which 1 ml of 25% barium acetate and another 2 vol. of alcohol were added and the pH adjusted to 8"2 using sodium hydroxide. This solution was kept overnight. The barium alcohol precipitate was collected and freed from barium by passing through Dowex 50W × 5, a cation exchange resin. This solution was used for the separation and estimation of A T P by the method of Albaum (1955). Measurement of respiration of heart tissue The standard Warburg technique, as described by Krebs (1950) and Umbreit et al. 1964), was used to measure oxygen consumption in vitro at 37°C. T h e animals were killed by decapitation and their hearts removed and placed immediately in cold salt solution as used by Krebs (1950). A small piece of tissue was then removed, transferred to a Petri dish kept in ice and finely sliced. The sliced material was quickly drained on filter paper and weighed on a torsion balance. About 100 nag of the wet tissue were transferred to the Warburg reaction flask having 3 ml of Krebs salt solution containing 5/~moles of A T P and 15 mg yeast extract. T h e amount of tissue slices was chosen so that
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AVIAN AND MAMMALIAN HEART TISSUES
oxygen uptake would be proportional to the same. In the centre well of the reaction flask 0.2 ml of 6 N potassium hydroxide was kept. A blank was run without the tissue. The gas phase was air. After addition of tissue slices, the flasks were attached to manometers and secured by rubber bands. The flasks were kept open to the atmosphere by opening the taps and then allowed to equilibrate at 37°C for 15 min. At the end of this period the taps were closed. The readings were taken every 10 min for 0"5 hr, the manometers being shaken all the time. The oxygen uptake of the slices was calculated in terms of kd of oxygen uptake/mg of dry tissue. Each experiment was carried out in duplicate and the values given are based on the mean of two trials. Values for the number of heart beats/rain were taken from available literature (Spector, 1961). RESULTS AND D I S C U S S I O N T h e data on the utilization of amino acids presented in T a b l e 1 confirm previous findings regarding the utilization of glutamic acid and alanine and non-utilization of glutamine and aspartic acid b y chick and pigeon heart tissue, and the reverse p h e n o m e n o n in the case of rat and rabbit heart tissue, with the bat sharing the characteristics of both. T A B L E 1 - - C H A N G E S I N AMINO ACID CONTENTS OF THE MEDIUM (M150) AFTER CULTIVATION OF PIGEON, CHICK, BAT~ RAT AND RABBIT HEART TISSUE EXPLANTS
Amino acid content (mg)/100 ml of medium (M150) After cultivation for 72 hr Amino acid L-Glutamine L-Glutamic acid nL-Aspartic acid DL-c~-Alanine
Initial 10-0 7-5 6"0 5"0
Pigeon
Chick
Bat
10"8+0"09 11"0+0"04 10"6+0"07 6"7+0"06 6"5 +0"14 6"7+0"09 6-3 + 0"05 6"3 + 0"06 5"8 + 0"04 4-8 + 0'01 4"8 + 0"03 5"4 + 0"03
Rat
Rabbit
9"1+0"04 8"1 +0"06 5"7 + 0"02 5"5 _+0"05
8-9+0'12 8"3 +0'05 5"6 + 0-07 5"7 + 0-05
Values are means of five individual experiments _+S.E. T h e s e observations are supported b y changes in glucose utilization and protein and D N A contents of the cultures with the omission of glutamine and alanine f r o m the m e d i u m (Tables 2-4). All the three parameters are affected to varying degrees b y the omission of alanine in the chick and pigeon tissue and b y that of glutamine in the rat and rabbit tissue. T h e bat tissue is not affected seriously b y either omission. It is interesting to note f r o m the data on free amino acid contents of the tissues studied (Table 5) a higher concentration of glutamic acid in pigeon, chick and bat tissue, whereas no appreciable difference is observed in the case of glutamine which would favour the enzyme reaction, glntamic acid to glutamine, should the enzyme be present in these tissues. M a m m a l i a n tissues have a higher concentration of alanine which perhaps accounts for their not being affected b y the omission of alanine. T h e high concentration of aspartic acid in pigeon and chick tissue
A. J. PATEL AND C. V. RAMAKRISHNAN
808
might account for the release of this amino acid by the cultures. It could be that aspartic acid in these tissues forms a reservoir from which glutamic acid may be formed. Glutamic acid, although present in high concentrations in pigeon, chick TABLE 2--GLucoSE
U T I L I Z A T I O N BY PIGEON, CHICK, BAT, RAT AND RABBIT HEART TISSUE
EXPLANTS CULTIVATED I N COMPLETE r ALANINE-DEFICIENT AND GLUTAMINE-DEFICIENT MEDIA
Glucose utilized (mg)/100 ml of the medium after cultivation for 72 hr in Heart tissue cultivated
Complete medium
Pigeon Chick Bat Rat Rabbit
6"0+-0"32 14'8+-0-75 5"9 +-0"17 7"3+-0"18 11"2+-0"61
Alanine-deficient Glutamine-deficient medium medium 4"0+-0"46 (33) 8"1+-0"64(45) 5.3 _+0"18 (10) 6"8+-0"17 (7) 10.7_+0"32(4)
4"6+-0"17 (23) 10"9+-0"32(26) 5"1 +-0-27 (13) 3"0+-0"17(59) 3"5_+0"17(68)
Values are means of three individual experiments + S.E. Values in parentheses show percentage decrease over complete medium. T A B L E 3 - - P R O T E I N CONTENT OF PIGEON~ CHICK, BAT, RAT AND RABBIT HEART TISSUE EXPLANTS AFTER CULTIVATION IN COMPLETE, ALANINE-DEFICIENT AND GLUTAMINE-DEFICIENT MEDIA
Protein content (mg/100 explants) After cultivation for 72 hr in Heart tissue cultivated Pigeon Chick Bat Rat Rabbit
Initial
Complete medium
Alanine-deficient medium
Glutamine-deficient medium
23"6+1"2
21"0+0-17 15"7+-0"19 21.3+-0.30 16.4+-0.38 14.6+-0.31
17"8+0"49(15) 12"9+0"33 (18) 20.8+-0.49(2) 16.4+-0.28 ( 0 ) 15.0+-0.44(0)
19"6+0"32(7) 14"2+-0"72( 9 ) 19.2+0.23(6) 12.4_+0.17 (24) 10'0+-0.14(31)
20"0+_0"17
22.9+-0.17 20.0+-0.7 19.7+-0.17
Values are means of three individual experiments + S.E. Values in parentheses show percentage decrease over complete medium. and bat tissue, is utilized by these tissues, whereas other amino acids present in high concentrations, such as aspartic acid in the case of pigeon and chick and alanine, in the case of bat, rat and rabbit, are released into the medium. Table 6 gives the activities of the enzymes in the different tissues. T h e most striking observation is the presence of L-glutamate : ammonia ligase in chick and pigeon heart and its absence in rat and rabbit heart confirming the presence of
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AVIAN AND MAMMALIAN HEART TISSUES
L - g l u t a m a t e : a m m o n i a ligase in avian heart tissues previously reported. I t is interesting to note that the enzyme is also present in the bat, a flying m a m m a l . I t is interesting to note that glutamine : glutamyl glutamyltransferase which is concerned with the further metabolism of glutamine shows greater activity in avians than in m a m m a l s with the bat showing an intermediate value. T h i s would TABLE 4---DNA
CONTENT I N PIGEON, CHICK~ BAT, RAT AND RABBIT HEART TISSUE EXPLANTS
AFTER CULTIVATION I N COMPLETE, ALANINE-DEFICIENT AND GLUTAMINE-DEFICIENT MEDIA
DNA content (/zg/100 explants) After cultivation for 72 hr in Heart tissue cultivated Pigeon Chick Bat Rat Rabbit
Initial
Complete medium
407_+4.6 482 + 5"5 387_+4-5 388 +-9-8 375 _+2.9
389_+12"1 429_+ 20"2 377_+ 7"5 385 + 8"7 337 + 3.5
Alanine-deficient medium
Glutamine-deficient medium
311+ 1"5(19) 343 + 4"0 (20) 376_+ 8 " 1 ( 0 ) 385 _+13"6 ( 0 ) 330 +_ 5.2 ( 2 )
366+14"2(6) 372 + 9"8 (13) 335+10.9(11) 250 + 3"8 (35) 223 + 16.8 (34)
Values are means of three individual experiments + S.E. Values in parentheses show percentage decrease over complete medium. T A B L E 5 - - F R E E AMINO ACID CONTENTS OF PIGEON, CHICK~ BAT, RAT AND RABBIT HEART TISSUE
/zg/g wet wt. of tissue Amino acid Glutamine Glutamic acid Asparticacid Alanine
Pigeon
Chick
Bat
Rat
860 + 38 1382 + 55 784+_23 131 +- 10
887 + 9 1500 _+10 759+41 116 + 7
807 + 15 966 + 45 185_+ 8 189_+ 5
788 + 32 821 _+32 300+- 7 171 _+ 8
Rabbit 795 + 9 548 + 14 192+-11 351 _+20
Values are means of three individual experiments + S.E. perhaps account for the fact that in spite of the higher activities of L-glutamate : a m m o n i a ligase in avians there is no great accumulation of glutamine. On the other hand, the enzyme concerned with the deamination of glutamine to glutamic acid is m o r e active in rat and rabbit heart than in pigeon and chick heart tissue. T h e lower activities of a l a n i n e : 2 - o x o g l u t a r a t e aminotransferase and alanine: oxaloacetate aminotransferase in avian heart and their high activities in m a m m a l i a n heart suggest the relatively p o o r ability of the former to f o r m alanine f r o m either glutamic acid or aspartic acid. T h i s would account for the requirement of alanine by avian heart and its release into the m e d i u m by m a m m a l i a n heart. T h e higher
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A. J. PATEL AND C. V. RAMAKRISHNAN
activity of glutamate : N A D oxidoreductase in mammalian heart tissue suggests a greater capacity of this tissue to synthesize glutamate from 2-oxoglutarate. These observations would account for the requirement of glutamic acid by pigeon and chick explants. Although the enzyme aspartate : 2-oxoglutarate aminotransferase is highly active in vitro in pigeon and chick tissues as well as in other tissues, in TABLE 6--SPECIFIC ACTIVITYOF CERTAINENZYMESOF AMINO ACID METABOLISMIN PIGEON~ CHICK, BA% RAT AND RABBITHEARTTISSUE Specific activity Enzyme
Pigeon
Chick
Glutamine aminohydrolase 0"044±0"002 0"038±0-002 Alanine : 2-oxoglutarate aminotransferase 0"038±0-0040"035±0-005 Aspartate : 2-oxoglutarate aminotransferase 40"0±2"0 23"0±3"3 Alanine : oxaloacetate aminotransferase 0-037 + 0"001 0.023 + 0"004 L-Glutamate: ammonia ligase 0.17+0.014 0.15+0.019 Glutamine : glutamyl glutamyltransferase 1.38 + 0"12 1"72± 0"26 Glutamate : NAD oxidoreductase 0"051 ± 0"005 0"054 ± 0"002
Bat
Rat
Rabbit
0"053±0"003 0.14±0"012 0.25+0"015 1.66±0"12
0.75±0"033 2"06±0"11
37.6±2"5
22"8±1"0
30-0±1"0
0.096 + 0"006 0-059 + 0.006 0"115 __+0.011 0.055+0.006
0"0
0"0
0.21 + 0.01
0.061 + 0.007 0.089 + 0.014
0.25 + 0.02
0.21 + 0"03
0.27 + 0-06
Values are means of five individual experiments + S.E.
vivo this may not be a favourable direction for the activity of this enzyme, as the tissue concentration of glutamic acid is m u c h higher than that of aspartic acid in the case of both. It would thus appear that pigeon and chick heart tissues have to depend on an exogenous supply of glutamic acid from the blood stream as the activities of glutamate : N A D oxidoreductase as well as glutamine aminohydrolase are relatively low. In mammals, however, this amino acid could be formed from either 2-oxoglutarate or glutamine as both glutamate : N A D oxidoreductase and glutamine aminohydrolase have a relatively high activity. T h e data on heart weight, body weight and other parameters measured in the different species studied and the average values reported in the literature for the n u m b e r of heart beats/min are given in Table 7. W h e n the data are considered in terms of activities/g of heart tissue, a significant correlation is observed between respiration, on the one hand, and the n u m b e r of heart beats/min and heart weight/100 g of body weight, on the other. This suggests that respiration proceeds at a faster rate in species where the heart is bigger in
7"45 3"63 0"65 0-068 2"81 1"20 0"20 0-099 1"03 0"015
Cat 2050 Rabbit 2575 Rat 246 Bat 10"3 Pigeon 262 Chick 602 Sparrow 1 6 " 7 Calotees 32 Frog 426 Fish 26
0-36 0"14 0"26 0"66 1"07 0"19 1"20 0"31 0"24 0"058
g/lO0 g bodywt, 120 205 328 750 170 312 804 64 35 39
Heart beats/ min* N.E. 5"70 8-01 2-44 8"13 1"52 6"96 4"43 0"60 N.E.
N.E. = Not estimated. Values are means of three or more individual experiments. * Spector (1961).
g
Heart wt.
0"0 0"0 0"0 5"48 26"75 4-14 46"92 0"0 0"0 0"0
27"72 44.24 31 "44 161"20 5-99 1"03 16"08 1.27 1"42 2"19
46"5 27"6 102"2 377"5 252"6 77"7 1673 811"9 338-4 66-7
Per whole heart/lO0 g body wt.
Alanine : Glutamate : L-Glutamate : 2-oxoglutarate N A D oxidoammonia aminoA M P aminoreductase ligase transferase hydrolase (units) (units) (units) (units)
PARAMETERS
936 353 583 2133 2869 533 4116 582 214 78
Oxygen uptake (~l/hr)
0"26 0"09 0"09 0"66 0"51 0"11 N.E. 0"17 0"18 N.E.
ATP (/~moles)
A C T I V I T I E S OF E N Z Y M E S I N HEART TISSUE OF D I F F E R E N T SPECIES I N R E L A T I O N TO BODY W E I G H T AS COMPARED TO O T H E R
Body Species wt. (g)
TABLE 7--TOTAL
OO
812
A.
J.
PATEL AND C. V. RAMAKRISHNAN
relation to body weight and blood circulation is more rapid in terms of number of heart beats/min. However, no values are available on the volume of blood pumped out of the heart during every heart beat either in absolute terms or in relation to body weight. AMP aminohydrolase activity, respiratory rate as well as ATP content are found to correlate with the size of the heart in relation to body weight. Further, AMP aminohydrolase activity and respiratory rate are themselves found to be significantly correlated with each other when the value for the frog which seems to be atypical is omitted. The question may be raised whether the observed correlations are not an artefact of considering both parameters in terms of units/whole heart 100 g of body weight. Such an approach is considered appropriate by other investigators such as Terroine (1960). The differences in the degree of correlation, as well as the absence of a positive correlation in the case of alanine : 2-oxoglutarate aminotransferase, suggest that this is not the case. The most interesting observation is, however, that when the activities of L-glutamate : ammonia ligase and glutamate : NAD oxidoreductase are considered together there is a highly significant correlation between the sums of the activities of these two enzymes, on the one hand, and respiratory rate and heart beats, on the other. It is considered legitimate to make such an addition since both enzymes measure the removal of ammonia in comparable terms. This supports the hypothesis regarding their possible role in detoxifying ammonia. A similar correlation is observed with the activity of AMP aminohydrolase (Table 8). The values for ATP content are in general lower than those reported in the literature, probably because in the present experiments ATP was assayed without the administration of saturated magnesium sulphate solution to the animal before sacrifice in order to avoid interference with activities of the other enzymes, so that some of the ATP must be presumed to have been broken down immediately after sacrifice. SUMMARY Studies on the amino acid utilization of explants of pigeon, chick, bat, rat and rabbit heart explants cultivated in Morgan's medium (Morgan eta/., 1955) for a period of 3 days confirmed previous observations of differences in amino acid utilization between these tissues. The avian tissues studied were found to utilize glutamic acid and alanine and release glutamine into the medium, whereas rat and rabbit tissues showed the reverse phenomenon. The bat tissue released both glutamine and alanine. These differences were associated with those in the activities of L-glutamate : ammonia ligase which was absent in the rat and rabbit; glutamine : glutamyl glutamyltransferase which showed greater activity in pigeon and chick and alanine: 2-oxoglutarate aminotransferase which showed greater activity in bat, rat and rabbit. The differences in enzyme activities are believed to account for those in amino acid utilization.
* Pearson's " r " was used (McNemar, 1955).
A M P aminohydrolase A M P aminohydrolase A T P content Glutamate : N A D oxidoreductase + L-glutamate : ammonia ligase Glutamate : N A D oxidoreductase + L-glutamate: ammonia ligase Alanine : 2-oxoglutarate aminotransferase
X
Heart wt. in relation to body wt. Heart wt. in relation to body wt.
A M P aminohydrolase
Respiration Heart wt. in relation to body wt. Heart wt. in relation to body wt.
Y
8 10
8
9 10 8
N
0.94 0.15
0.81
0.74 0.73 0.82
Product moment correlation * (r)
6.79 0.43
3.35
2.89 3.04 3.56
t
B E T W E E N THE VARIOUS PARAMETERS S T U D I E D I N HEART TISSUE OF D I F F E R E N T SPECIES
Variables correlated
TABLE 8--CORRELATION
< 0.001 >0.05
<0.05
< 0-05 <0.05 <0.05
Significance of r value of P
O0
t~ ~o
N
814
A.J. PATEL AND C. V. RAMAKRISHNAN
H e a r t tissues of different species were examined for possible correlations between enzymes concerned with a m m o n i a removal and the size and metabolic activity of the heart. T h e species studied were cat, rabbit, rat, bat, pigeon, chick, sparrow, calotees, frog and fish. A significant correlation was found between respiratory rate and A M P aminohydrolase with heart weight in relation to body weight, b o t h parameters being found to be higher in flying animals. T h e activities of L-glutamate : a m m o n i a ligase and glutamate : N A D oxidoreductase taken together were found to be correlated with respiration as well as heart beats/min. T h e results are consistent with the hypothesis that L-glutamate : ammonia ligase and glutamate : N A D oxidoreductase serve as a a m m o n i a - r e m o v a l mechanism in heart tissue. Alanine : 2-oxoglutarate aminotransferase showed a greater activity in m a m malians than in other species. REFERENCES ALBAUMH. G. (1955) Adenosine diphosphate, adenosine triphosphate. In Modern Methods of Plant Analysis (Edited by PAECHK. & TRaC~.YM. V.), Vol. IV, pp. 305-319. SpringerVerlag, Berlin. ASI-IWELLG. (1957) Colorimetric analysis of sugars. In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.), Vol. III, pp. 73-105. Academic Press, New York. CHtrNG WE (1963) Glutamine synthetase---I. A comparative study of its distribution in animals and its inhibition by DL-allo-8-hydroxylysine. Comp. Biochem. Physiol. 8, 335351. DUKESH. H. (1953) Anoxia. In The Physiology of Domestic ~lnimals, pp. 230-232. Comstock, New York. GOTHOSKAa B. P., RAINA P. N. & RAMAKRISHNANC. V. (1960) Glutamine synthetase in adult hen heart tissue. Biochim. biophys. Acta 37, 477-481. GOTHOSKARB. P., RAINA P. N. & RAMAKaISHNANC. V. (1961) Amino acid composition of the culture fluid during cultivation of chick and rat heart tissue explants in medium M150. Expl Cell. Res 24, 265-271. HAKKINEN H. M. & KULOr~N E. (1961) The effect of ethanol on the amino acids of the rat brain with reference to the administration of glutamine. Biochem. ft. 78, 588-593. K~BS H. A. (1950) Body size and tissue respiration. Biochim. biophys. Acta 4, 249-269. LOWRY O. H., ROSEBROUGHN. J., FAaa A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent, ft. biol. Chem. 193, 265-275. McDERMOTT W. V., Ja. & ADAMS R. D. (1954) Episodic stupor associated with an Eck fistula in the human with particular reference to the metabolism of ammonia, ft. Clin. lnvest. 33, 1-9. McNEMAR Q. (1955) Correlation: introduction and computation. In Psychological Statistics, pp. 115-121. John Wiley, New York. MOaCAN J. F., CAMPBELLM. E. & MOaTON H. J. (1955) The nutrition of animal tissues cultivated in vitro--I. A survey of natural materials as supplements to synthetic medium 199. ft. hath. Cancer lnst. 16, 557-567. NZKIFORUK G. & COLOWICK S. P. (1955) 5"-Adenylic acid deaminase from muscle. In Methods in Enzymology (Edited by COLOWICKS. P. & KAPLAN N. O.), Vol. II, pp. 469473. Academic Press, New York. PATELA. J., RAJALAKSHMIR. & RAMAKRISHN~C. V. (1965) Glutamine and alanine metabolism of avian and mammalian heart tissue explants cultivated in vitro in synthetic medium. In Tissue Culture (Edited by R . ~ a I s r r N A N C. V.), pp. 49--63. W. Junk, The Hague.
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815
RAINA P. N. & RAMAKRISHNANC. V. (1964) Comparative metabolic studies on normal and neoplastic rat l i v e r - - I V . Comparative studies on the activities of certain enzymes of amino acid metabolism in normal, neoplastic, newborn and regenerating liver. Oncologia 18, 14--20. RAJALAKSHMI R., ALL S. Z. & RAM~.RISHN&'~ C. V. (1967) Effect of inanition during the neonatal period on discrimination learning and brain biochemistry in the albino rat. 37. Neurochem. 14, 29-34. SEEGMILLER J. E., SCHWARTZ R. & DAVIDSON C. S. (1954) T h e plasma "ammonia" and glutamine content in patients with hepatic coma. j~. Clin. Invest. 33, 984-988. SHAH V. K. & RAMAKmSHN~'~C. V. (1963) Studies on acid metabolism in Aspargillus niger-II. Metabolic changes during citric acid utilization by Aspergillus niger. Enzymologia 26, 33-43. SPECTOR W. S. (1961) Heart rates. In Handbook of Biological Data (Edited by SPECTOR W. S.), pp. 277. Saunders, London. SRINIVASAMURTHY V. • SWAMINATHAN M . (1955) Colorimetric determination of dehydrogenase activities of liver using triphenyl tetrazolium bromide. Ind. j~. Physiol. Allied Sci. 9, 107-112. TERROIN~ T. (1960) T h e vitamin interrelations of ascorbic acid. In Worm Review of Nutrition and Dietetics (Edited by BOURNE G. H. & WILSON E. M. H.), Vol. II, pp. 105129. Pitman, London. TURSH G. P. (1964) Enzymes of glutamine metabolism in myocardium. Fedn Proc. 23, T.1305-1310. UMBREIT W. W . , BURroS R. H. & STANEFER J. F. (1964) T h e Warburg constant volume respirometer. In Manometric Techniques, pp. 1-18. Burgess, Minneapolis. WALTER H. E. & SAYLESL. P. (1961) T h e heart. In Biology of the Vertebrates, pp. 351-363. Macmillan, New York. WHITE A., HANDLER P. & SMITH E. L. (1964) Muscle. In Principles of Biochemistry, pp. 744-754. McGraw-Hill, New York. YAMANA Y. • SIBATANIA. (1960) Fractionation of ribonucleic acids with phenol. Biochim. biophys. _4cta 41, 295-303.
STUDIES ON AVIAN AND MAMMALIAN HEART TISSUES A. J. P A T E L * and C. V. R A M A K R I S H N A N Department of Biochemistry, Baroda University, Baroda, India (Received 11 June 1968)
Abstract--1. In a study of in vitro amino acid utilization avian heart tissues were found to utilize glutamate and alanine, and release glutamine, whereas mammalian tissues did the reverse. 2. These differences were attributable to the differences in the activities of L-glutamate : ammonia ligase, glutamine : glutamyl glutamyltransferase and alanine : 2-oxoglutarate aminotransferase in the above species. 3. Studies of heart tissue of cat, rabbit, rat, bat, pigeon, chick, sparrow, calotees, frog and fish showed a significant correlation between respiratory rate, heart beats/min, AMP aminohydrolase activity, L-glutamate:ammonia ligase and glutamate : NAD oxidoreductase activities together, and heart weight in relation to body weight. 4. The results support the view that L-glutamate:ammonia ligase and glutamate : NAD oxidoreductase serve as an ammonia removal mechanism in heart tissue. INTRODUCTION PRELIMINARY studies previously reported from this laboratory suggested differences in the utilization of glutamic acid and glutamine between avians and mammalian heart tissues cultivated in vitro in synthetic medium (Patel et al., 1965). Explants of the heart tissue of chick and pigeon when cultivated in a synthetic medium M150, devised by Morgan et al. (1955), were found to utilize glutamic acid and release glutamine whereas those of rat and rabbit were found to show the reverse phenomenon. Similar differences of a smaller order were found with regard to the utilization of alanine and aspartic acid; the avian tissues were found to utilize alanine and release aspartic acid, the reverse being true of mammalian tissues. T h e bat, a flying mammal, was found to resemble avians with regard to the utilization of glutamic acid and mammals with regard to that of aspartic acid. T h e avian heart tissues were found to show glutamine synthetase activity which was absent in the rat and rabbit but present in the bat. Glutamate-NAD oxidoreductase was found to be more active in mammalian heart. Studies carried out in this laboratory also showed the presence of L-glutamate : ammonia ligase in bird and bat heart tissues. In this connexion, birds have a big heart in relation to body weight because of the demands on this organ during flight *Present address: Medical Research Council Neuropsychiatric Research Unit, Carshalton, Surrey, U.K. 803
804
A. J. PATEL AND C. V. RAMAKRISHNAN
(Spector, 1961 ; Walter & Sayles, 1961). T h e heart is a continuously active organ believed to be particularly sensitive to ammonia toxicity (Dukes, 1953; Walter & Sayles, 1961). T h e accumulation of ammonia in muscle during exercise because of A T P breakdown (White et al., 1964) results in muscular fatigue and the clearance of ammonia can take place during the period of resting, but such fatigue in cardiac muscle may be fatal. In this connexion, the brain is highly sensitive to ammonia toxicity (Seegmiller et al., 1954; McDermott & Adams, 1954) and L-glutamate : ammonia ligase is invariably present in the brain of animal species (Chung Wu, 1963). T h e presence of L-glutamate ammonia ligase in bird and bat heart and of glutamate : N A D oxidoreductase in the heart tissue of both mammals and avians raises the question whether these enzymes also provide a local machinery for the rapid removal of ammonia from heart tissue. T h e present paper reports a confirmation of the above findings under more controlled conditions including studies of free amino acids and enzymes in fresh tissue. T h e hypothesis regarding the role of glutamine synthetase and glutamateN A D oxidoreductase in ammonia removal was sought to be investigated by studying the interrelations between the following parameters in different species: activity of the heart as judged by heart weight in relation to body weight; heart beats/rain; respiratory activity as judged by oxygen uptake in vitro; the activities of Lg l u t a m a t e : a m m o n i a ligase and glutamate : N A D oxidoreductase which effect a removal of ammonia; A M P aminohydrolase, involved in ammonia formation from A T P breakdown; and A T P content of the heart tissues. Additional studies were made of the activity of the enzyme alanine : 2-oxoglutarate aminotransferase in different species because of differences found in the activity of this enzyme between avian and mammalian tissue. T h e species studied were: cat (Fells domestica), rabbit (Oryctolagus cuniculus), rat (Rattus norvegicus albinus), bat (Hipposiderons sperois), pigeon (Columba livia), chick (Gallus domesticus), house sparrow (Passer domesticus), calotees (Calotees versicolor), frog (Rana trigrina) and fish (Ophioce-
phalus punctalus). MATERIALS AND METHODS The methods followed and the conditions employed for the cultivation of heart tissues in vitro were essentially the same as those described previously (Patel et al., 1965) except for the following modifications: (i) the period of depletion was 8 hr as compared to 16 hr in the previous study; the modification was made in the light of subsequent studies which showed an 8-hr period of depletion to be compatible with maximum glucose utilization by the cultures, a longer period of depletion resulting in decreased glucose utilization; (ii) the period of cultivation was changed to 3 days as compared to 6 days in the original study in order to rule out the possibility of a change in the behaviour of the cultures after a change of medium on the fourth day; (iii) the biological status of the cultures at the end of cultivation was ascertained by determining their protein and DNA contents before and after cultivation; (iv) the optimum conditions for the enzyme assays were identified separately for avian and mammalian tissue; (v) the enzymes were assayed in fresh tissue instead of in the cultures and (vi) additional studies were made of the free amino acid contents of fresh tissue and the activity of the enzyme glutamine : glutamyl glutamyltransferase. Other details are summarized below.
AVIAN AND MAMMALIAN HEART TISSUES
805
Tissues Hearts of the species studied removed under aseptic conditions were used for these experiments. Glassware and chemicals Only Pyrex glassware and chemicals of research grade purity were used in the investigation. Media for the cultivation of heart explants The medium used was medium M150 devised by Morgan et al. (1955). Where so specified, omissions were made of individual constituents such as glutamine and alanine. The L-forms of leucine and glutamic acid were used at half the concentrations specified by Morgan et al. (1955) as the oL-forms were not available. Collection of cultures The culture fluid was collected as described previously (Patel et al., 1965). After removing the medium from the culture tubes, the explants were removed with the help of a stainless steel needle with a bent apex and collected in a small measuring cylinder containing 3"5 ml ice-cold 0"9% potassium chloride solution, p H 7"4. The solution and the explants were poured into a Potter-Elvejhem homogenizer and the cylinder rinsed with a further 0"5 ml of ice-cold potassium chloride solution and the washing added to the homogenizer. The explants were homogenized for 2 rain at 4,000 rev/min using a Teflon tissue grinder. All the operations were carried out at 0-4°C. The homogenate obtained was used for D N A and protein estimations. Chemical analysis Glucose and amino acids in culture fluid were determined as described previously (Patel et al., 1965) and protein by the method of Lowry et al. (1951). D N A was isolated by the method of Yamana& Sibatani (1960) and estimated by the diphenyl amino method (Ashwell, 1957) . For the estimation of free amino acids, tissue extracts were prepared by the method of Rajalakshmi et al. (1967), separated by the two-dimensional paper chromatographic method described by Hakkinen & Kulonen (1961), and estimated by the method described by Gothoskar et al. (1961). Enzyme assays The adult animals were killed by decapitation and their hearts removed. The fatty portions and blood were removed and the remaining tissue chopped to a fine pulp in a cold room at 0-4°C and ground in a chilled mortar with four times the volume of ice-cold 0"2 M potassium phosphate buffer, pH 7"0, for 15 man. The homogenate was used without centrifugation for the estimation of the activities of glutamine aminohydrolase, alanine : 2-oxoglutarate anainotransferase, aspartate : 2-oxoglutarate aminotransferase, alanine : oxaloacetate aminotransferase and glutamate : N A D oxidoreductase. For the estimation of L-glutamate : ammonia ligase and glutamine : glutamyl glutamyltransferase, the enzyme extract was prepared using 0"02 M potassium bicarbonate, pH 8"0, as grinding medium in place of potassium phosphate buffer. For the estimation of AMP aminohydrolase (E.C. 3.5.4.6) the enzyme extract was prepared using glass-distilled water as grinding medium. The dependence of enzyme activity on enzyme and substrate concentrations, pH and period of incubation was studied in the pigeon (avian), bat (flying mammal) and rabbit (mammal) and the optimum conditions worked out for the assay of different enzymes. For the assay of glutamine aminohydrolase (E.C. 3.5.1.2) the method described by Raina & Ramakrishnan (1964) was used except for the change that phosphate buffer, pH 7"5, was used instead of Tris buffer.
806
A . J . PATELAND C. V. RAMAKRISHNAN
For the assay of aminotransferases the method used was that described by Shah & Ramakrishnan (1963) except for the difference in the amount of the components added. For alanine : 2-oxoglutarate aminotransferase (E.C. 2.6.1.2) the assay system consisted of Tris buffer, pH 7-5, 50 bemoles; L-glutamate, 10 /zmoles; sodium pyruvate, 10/zmoles; pyridoxal phosphate, 0"08 bemole and enzyme preparation made to a final volume of 1"0 ml with water and incubated at 37°C for 30 min. For the assay of aspartate : 2-oxoglutarate aminotransferase (E.C. 2.6.1.1) and alanine : oxaloacetate aminotransferase (E.C. 2.5.1.12) the assay system consisted of Tris buffer, pH 7-5, 50 /zmoles; DL-aspartate, 20 bemoles; ketoacid, 10 /zmoles; pyridoxal phosphate, 0"04/zmoles and enzyme preparation made to a final volume of 1"0 ml with water and incubated at 37°C for 30 min. For the assay of L-glutamate : ammonia ligase (E.C. 6.3.1.2) the method of Gothoskar et al. (1960) was used except for the change that the concentrations of glutamate, MgSO4, NH2OH HC1, CSH and the incubation time were half of that used in the original method. For the assay of glutamine : glutamyl glutamyltransferase (E.C. 2.3.2.1) the method of T u r s h (1964) was used except for the change that the assay system contained double the concentrations of acetate buffer, glutamine, NH2OH HCI and half the concentration of A T P used in the original method. For the assay of g l u t a m a t e : N A D oxidoreductase (E.C. 1.4.1.2), the method of Srinivasamurthy & Swaminathan (1955) was used except for the change that the Tris buffer, pH 9"0 instead of pH 7'5, and triphenyl tetrazolium chloride instead of bromide were used. For the assay of AMP aminohydrolase (E.C. 3.5.4.6) the method described by Nikiforuk & Colowick (1955) was used. T h e assay system consisted of citrate NaOH buffer, pH 6"5, 50/~moles; AMP, 20 ftmoles; enzyme extract ,0"2 ml (0.1-0-6 mg protein) and water to a final volume of 2 ml. Protein contents of the enzyme extract were estimated by the method of Lowry et al. (1951). Estimation of A T P The animals were killed by decapitation and their hearts were removed. T h e blood and fatty portions were removed and approximately 1 g of the tissue was chopped to a fine pulp in the cold room at 0-4°C and ground with 10% solution of trichloroacetic acid in a mortar for about 15 rain. The homogenate was centrifuged at 7000 g at 2-4°C for 20 min and the clear supernatant collected in a beaker kept in ice. The residue was re-extracted in 5% trichloroacetic acid solution in the same manner. Both the supernatants were pooled together and neutralized by adding 10% sodium hydroxide. Equal volume of ice-cold alcohol was then added in order to precipitate the polysaccharides. After 3 hr the solution was centrifuged at 7000 g at 0-2°C for 20 min and the clear supematant collected in a beaker to which 1 ml of 25% barium acetate and another 2 vol. of alcohol were added and the pH adjusted to 8"2 using sodium hydroxide. This solution was kept overnight. The barium alcohol precipitate was collected and freed from barium by passing through Dowex 50W × 5, a cation exchange resin. This solution was used for the separation and estimation of A T P by the method of Albaum (1955). Measurement of respiration of heart tissue The standard Warburg technique, as described by Krebs (1950) and Umbreit et al. 1964), was used to measure oxygen consumption in vitro at 37°C. T h e animals were killed by decapitation and their hearts removed and placed immediately in cold salt solution as used by Krebs (1950). A small piece of tissue was then removed, transferred to a Petri dish kept in ice and finely sliced. The sliced material was quickly drained on filter paper and weighed on a torsion balance. About 100 nag of the wet tissue were transferred to the Warburg reaction flask having 3 ml of Krebs salt solution containing 5/~moles of A T P and 15 mg yeast extract. T h e amount of tissue slices was chosen so that
807
AVIAN AND MAMMALIAN HEART TISSUES
oxygen uptake would be proportional to the same. In the centre well of the reaction flask 0.2 ml of 6 N potassium hydroxide was kept. A blank was run without the tissue. The gas phase was air. After addition of tissue slices, the flasks were attached to manometers and secured by rubber bands. The flasks were kept open to the atmosphere by opening the taps and then allowed to equilibrate at 37°C for 15 min. At the end of this period the taps were closed. The readings were taken every 10 min for 0"5 hr, the manometers being shaken all the time. The oxygen uptake of the slices was calculated in terms of kd of oxygen uptake/mg of dry tissue. Each experiment was carried out in duplicate and the values given are based on the mean of two trials. Values for the number of heart beats/rain were taken from available literature (Spector, 1961). RESULTS AND D I S C U S S I O N T h e data on the utilization of amino acids presented in T a b l e 1 confirm previous findings regarding the utilization of glutamic acid and alanine and non-utilization of glutamine and aspartic acid b y chick and pigeon heart tissue, and the reverse p h e n o m e n o n in the case of rat and rabbit heart tissue, with the bat sharing the characteristics of both. T A B L E 1 - - C H A N G E S I N AMINO ACID CONTENTS OF THE MEDIUM (M150) AFTER CULTIVATION OF PIGEON, CHICK, BAT~ RAT AND RABBIT HEART TISSUE EXPLANTS
Amino acid content (mg)/100 ml of medium (M150) After cultivation for 72 hr Amino acid L-Glutamine L-Glutamic acid nL-Aspartic acid DL-c~-Alanine
Initial 10-0 7-5 6"0 5"0
Pigeon
Chick
Bat
10"8+0"09 11"0+0"04 10"6+0"07 6"7+0"06 6"5 +0"14 6"7+0"09 6-3 + 0"05 6"3 + 0"06 5"8 + 0"04 4-8 + 0'01 4"8 + 0"03 5"4 + 0"03
Rat
Rabbit
9"1+0"04 8"1 +0"06 5"7 + 0"02 5"5 _+0"05
8-9+0'12 8"3 +0'05 5"6 + 0-07 5"7 + 0-05
Values are means of five individual experiments _+S.E. T h e s e observations are supported b y changes in glucose utilization and protein and D N A contents of the cultures with the omission of glutamine and alanine f r o m the m e d i u m (Tables 2-4). All the three parameters are affected to varying degrees b y the omission of alanine in the chick and pigeon tissue and b y that of glutamine in the rat and rabbit tissue. T h e bat tissue is not affected seriously b y either omission. It is interesting to note f r o m the data on free amino acid contents of the tissues studied (Table 5) a higher concentration of glutamic acid in pigeon, chick and bat tissue, whereas no appreciable difference is observed in the case of glutamine which would favour the enzyme reaction, glntamic acid to glutamine, should the enzyme be present in these tissues. M a m m a l i a n tissues have a higher concentration of alanine which perhaps accounts for their not being affected b y the omission of alanine. T h e high concentration of aspartic acid in pigeon and chick tissue
A. J. PATEL AND C. V. RAMAKRISHNAN
808
might account for the release of this amino acid by the cultures. It could be that aspartic acid in these tissues forms a reservoir from which glutamic acid may be formed. Glutamic acid, although present in high concentrations in pigeon, chick TABLE 2--GLucoSE
U T I L I Z A T I O N BY PIGEON, CHICK, BAT, RAT AND RABBIT HEART TISSUE
EXPLANTS CULTIVATED I N COMPLETE r ALANINE-DEFICIENT AND GLUTAMINE-DEFICIENT MEDIA
Glucose utilized (mg)/100 ml of the medium after cultivation for 72 hr in Heart tissue cultivated
Complete medium
Pigeon Chick Bat Rat Rabbit
6"0+-0"32 14'8+-0-75 5"9 +-0"17 7"3+-0"18 11"2+-0"61
Alanine-deficient Glutamine-deficient medium medium 4"0+-0"46 (33) 8"1+-0"64(45) 5.3 _+0"18 (10) 6"8+-0"17 (7) 10.7_+0"32(4)
4"6+-0"17 (23) 10"9+-0"32(26) 5"1 +-0-27 (13) 3"0+-0"17(59) 3"5_+0"17(68)
Values are means of three individual experiments + S.E. Values in parentheses show percentage decrease over complete medium. T A B L E 3 - - P R O T E I N CONTENT OF PIGEON~ CHICK, BAT, RAT AND RABBIT HEART TISSUE EXPLANTS AFTER CULTIVATION IN COMPLETE, ALANINE-DEFICIENT AND GLUTAMINE-DEFICIENT MEDIA
Protein content (mg/100 explants) After cultivation for 72 hr in Heart tissue cultivated Pigeon Chick Bat Rat Rabbit
Initial
Complete medium
Alanine-deficient medium
Glutamine-deficient medium
23"6+1"2
21"0+0-17 15"7+-0"19 21.3+-0.30 16.4+-0.38 14.6+-0.31
17"8+0"49(15) 12"9+0"33 (18) 20.8+-0.49(2) 16.4+-0.28 ( 0 ) 15.0+-0.44(0)
19"6+0"32(7) 14"2+-0"72( 9 ) 19.2+0.23(6) 12.4_+0.17 (24) 10'0+-0.14(31)
20"0+_0"17
22.9+-0.17 20.0+-0.7 19.7+-0.17
Values are means of three individual experiments + S.E. Values in parentheses show percentage decrease over complete medium. and bat tissue, is utilized by these tissues, whereas other amino acids present in high concentrations, such as aspartic acid in the case of pigeon and chick and alanine, in the case of bat, rat and rabbit, are released into the medium. Table 6 gives the activities of the enzymes in the different tissues. T h e most striking observation is the presence of L-glutamate : ammonia ligase in chick and pigeon heart and its absence in rat and rabbit heart confirming the presence of
809
AVIAN AND MAMMALIAN HEART TISSUES
L - g l u t a m a t e : a m m o n i a ligase in avian heart tissues previously reported. I t is interesting to note that the enzyme is also present in the bat, a flying m a m m a l . I t is interesting to note that glutamine : glutamyl glutamyltransferase which is concerned with the further metabolism of glutamine shows greater activity in avians than in m a m m a l s with the bat showing an intermediate value. T h i s would TABLE 4---DNA
CONTENT I N PIGEON, CHICK~ BAT, RAT AND RABBIT HEART TISSUE EXPLANTS
AFTER CULTIVATION I N COMPLETE, ALANINE-DEFICIENT AND GLUTAMINE-DEFICIENT MEDIA
DNA content (/zg/100 explants) After cultivation for 72 hr in Heart tissue cultivated Pigeon Chick Bat Rat Rabbit
Initial
Complete medium
407_+4.6 482 + 5"5 387_+4-5 388 +-9-8 375 _+2.9
389_+12"1 429_+ 20"2 377_+ 7"5 385 + 8"7 337 + 3.5
Alanine-deficient medium
Glutamine-deficient medium
311+ 1"5(19) 343 + 4"0 (20) 376_+ 8 " 1 ( 0 ) 385 _+13"6 ( 0 ) 330 +_ 5.2 ( 2 )
366+14"2(6) 372 + 9"8 (13) 335+10.9(11) 250 + 3"8 (35) 223 + 16.8 (34)
Values are means of three individual experiments + S.E. Values in parentheses show percentage decrease over complete medium. T A B L E 5 - - F R E E AMINO ACID CONTENTS OF PIGEON, CHICK~ BAT, RAT AND RABBIT HEART TISSUE
/zg/g wet wt. of tissue Amino acid Glutamine Glutamic acid Asparticacid Alanine
Pigeon
Chick
Bat
Rat
860 + 38 1382 + 55 784+_23 131 +- 10
887 + 9 1500 _+10 759+41 116 + 7
807 + 15 966 + 45 185_+ 8 189_+ 5
788 + 32 821 _+32 300+- 7 171 _+ 8
Rabbit 795 + 9 548 + 14 192+-11 351 _+20
Values are means of three individual experiments + S.E. perhaps account for the fact that in spite of the higher activities of L-glutamate : a m m o n i a ligase in avians there is no great accumulation of glutamine. On the other hand, the enzyme concerned with the deamination of glutamine to glutamic acid is m o r e active in rat and rabbit heart than in pigeon and chick heart tissue. T h e lower activities of a l a n i n e : 2 - o x o g l u t a r a t e aminotransferase and alanine: oxaloacetate aminotransferase in avian heart and their high activities in m a m m a l i a n heart suggest the relatively p o o r ability of the former to f o r m alanine f r o m either glutamic acid or aspartic acid. T h i s would account for the requirement of alanine by avian heart and its release into the m e d i u m by m a m m a l i a n heart. T h e higher
810
A. J. PATEL AND C. V. RAMAKRISHNAN
activity of glutamate : N A D oxidoreductase in mammalian heart tissue suggests a greater capacity of this tissue to synthesize glutamate from 2-oxoglutarate. These observations would account for the requirement of glutamic acid by pigeon and chick explants. Although the enzyme aspartate : 2-oxoglutarate aminotransferase is highly active in vitro in pigeon and chick tissues as well as in other tissues, in TABLE 6--SPECIFIC ACTIVITYOF CERTAINENZYMESOF AMINO ACID METABOLISMIN PIGEON~ CHICK, BA% RAT AND RABBITHEARTTISSUE Specific activity Enzyme
Pigeon
Chick
Glutamine aminohydrolase 0"044±0"002 0"038±0-002 Alanine : 2-oxoglutarate aminotransferase 0"038±0-0040"035±0-005 Aspartate : 2-oxoglutarate aminotransferase 40"0±2"0 23"0±3"3 Alanine : oxaloacetate aminotransferase 0-037 + 0"001 0.023 + 0"004 L-Glutamate: ammonia ligase 0.17+0.014 0.15+0.019 Glutamine : glutamyl glutamyltransferase 1.38 + 0"12 1"72± 0"26 Glutamate : NAD oxidoreductase 0"051 ± 0"005 0"054 ± 0"002
Bat
Rat
Rabbit
0"053±0"003 0.14±0"012 0.25+0"015 1.66±0"12
0.75±0"033 2"06±0"11
37.6±2"5
22"8±1"0
30-0±1"0
0.096 + 0"006 0-059 + 0.006 0"115 __+0.011 0.055+0.006
0"0
0"0
0.21 + 0.01
0.061 + 0.007 0.089 + 0.014
0.25 + 0.02
0.21 + 0"03
0.27 + 0-06
Values are means of five individual experiments + S.E.
vivo this may not be a favourable direction for the activity of this enzyme, as the tissue concentration of glutamic acid is m u c h higher than that of aspartic acid in the case of both. It would thus appear that pigeon and chick heart tissues have to depend on an exogenous supply of glutamic acid from the blood stream as the activities of glutamate : N A D oxidoreductase as well as glutamine aminohydrolase are relatively low. In mammals, however, this amino acid could be formed from either 2-oxoglutarate or glutamine as both glutamate : N A D oxidoreductase and glutamine aminohydrolase have a relatively high activity. T h e data on heart weight, body weight and other parameters measured in the different species studied and the average values reported in the literature for the n u m b e r of heart beats/min are given in Table 7. W h e n the data are considered in terms of activities/g of heart tissue, a significant correlation is observed between respiration, on the one hand, and the n u m b e r of heart beats/min and heart weight/100 g of body weight, on the other. This suggests that respiration proceeds at a faster rate in species where the heart is bigger in
7"45 3"63 0"65 0-068 2"81 1"20 0"20 0-099 1"03 0"015
Cat 2050 Rabbit 2575 Rat 246 Bat 10"3 Pigeon 262 Chick 602 Sparrow 1 6 " 7 Calotees 32 Frog 426 Fish 26
0-36 0"14 0"26 0"66 1"07 0"19 1"20 0"31 0"24 0"058
g/lO0 g bodywt, 120 205 328 750 170 312 804 64 35 39
Heart beats/ min* N.E. 5"70 8-01 2-44 8"13 1"52 6"96 4"43 0"60 N.E.
N.E. = Not estimated. Values are means of three or more individual experiments. * Spector (1961).
g
Heart wt.
0"0 0"0 0"0 5"48 26"75 4-14 46"92 0"0 0"0 0"0
27"72 44.24 31 "44 161"20 5-99 1"03 16"08 1.27 1"42 2"19
46"5 27"6 102"2 377"5 252"6 77"7 1673 811"9 338-4 66-7
Per whole heart/lO0 g body wt.
Alanine : Glutamate : L-Glutamate : 2-oxoglutarate N A D oxidoammonia aminoA M P aminoreductase ligase transferase hydrolase (units) (units) (units) (units)
PARAMETERS
936 353 583 2133 2869 533 4116 582 214 78
Oxygen uptake (~l/hr)
0"26 0"09 0"09 0"66 0"51 0"11 N.E. 0"17 0"18 N.E.
ATP (/~moles)
A C T I V I T I E S OF E N Z Y M E S I N HEART TISSUE OF D I F F E R E N T SPECIES I N R E L A T I O N TO BODY W E I G H T AS COMPARED TO O T H E R
Body Species wt. (g)
TABLE 7--TOTAL
OO
812
A.
J.
PATEL AND C. V. RAMAKRISHNAN
relation to body weight and blood circulation is more rapid in terms of number of heart beats/min. However, no values are available on the volume of blood pumped out of the heart during every heart beat either in absolute terms or in relation to body weight. AMP aminohydrolase activity, respiratory rate as well as ATP content are found to correlate with the size of the heart in relation to body weight. Further, AMP aminohydrolase activity and respiratory rate are themselves found to be significantly correlated with each other when the value for the frog which seems to be atypical is omitted. The question may be raised whether the observed correlations are not an artefact of considering both parameters in terms of units/whole heart 100 g of body weight. Such an approach is considered appropriate by other investigators such as Terroine (1960). The differences in the degree of correlation, as well as the absence of a positive correlation in the case of alanine : 2-oxoglutarate aminotransferase, suggest that this is not the case. The most interesting observation is, however, that when the activities of L-glutamate : ammonia ligase and glutamate : NAD oxidoreductase are considered together there is a highly significant correlation between the sums of the activities of these two enzymes, on the one hand, and respiratory rate and heart beats, on the other. It is considered legitimate to make such an addition since both enzymes measure the removal of ammonia in comparable terms. This supports the hypothesis regarding their possible role in detoxifying ammonia. A similar correlation is observed with the activity of AMP aminohydrolase (Table 8). The values for ATP content are in general lower than those reported in the literature, probably because in the present experiments ATP was assayed without the administration of saturated magnesium sulphate solution to the animal before sacrifice in order to avoid interference with activities of the other enzymes, so that some of the ATP must be presumed to have been broken down immediately after sacrifice. SUMMARY Studies on the amino acid utilization of explants of pigeon, chick, bat, rat and rabbit heart explants cultivated in Morgan's medium (Morgan eta/., 1955) for a period of 3 days confirmed previous observations of differences in amino acid utilization between these tissues. The avian tissues studied were found to utilize glutamic acid and alanine and release glutamine into the medium, whereas rat and rabbit tissues showed the reverse phenomenon. The bat tissue released both glutamine and alanine. These differences were associated with those in the activities of L-glutamate : ammonia ligase which was absent in the rat and rabbit; glutamine : glutamyl glutamyltransferase which showed greater activity in pigeon and chick and alanine: 2-oxoglutarate aminotransferase which showed greater activity in bat, rat and rabbit. The differences in enzyme activities are believed to account for those in amino acid utilization.
* Pearson's " r " was used (McNemar, 1955).
A M P aminohydrolase A M P aminohydrolase A T P content Glutamate : N A D oxidoreductase + L-glutamate : ammonia ligase Glutamate : N A D oxidoreductase + L-glutamate: ammonia ligase Alanine : 2-oxoglutarate aminotransferase
X
Heart wt. in relation to body wt. Heart wt. in relation to body wt.
A M P aminohydrolase
Respiration Heart wt. in relation to body wt. Heart wt. in relation to body wt.
Y
8 10
8
9 10 8
N
0.94 0.15
0.81
0.74 0.73 0.82
Product moment correlation * (r)
6.79 0.43
3.35
2.89 3.04 3.56
t
B E T W E E N THE VARIOUS PARAMETERS S T U D I E D I N HEART TISSUE OF D I F F E R E N T SPECIES
Variables correlated
TABLE 8--CORRELATION
< 0.001 >0.05
<0.05
< 0-05 <0.05 <0.05
Significance of r value of P
O0
t~ ~o
N
814
A.J. PATEL AND C. V. RAMAKRISHNAN
H e a r t tissues of different species were examined for possible correlations between enzymes concerned with a m m o n i a removal and the size and metabolic activity of the heart. T h e species studied were cat, rabbit, rat, bat, pigeon, chick, sparrow, calotees, frog and fish. A significant correlation was found between respiratory rate and A M P aminohydrolase with heart weight in relation to body weight, b o t h parameters being found to be higher in flying animals. T h e activities of L-glutamate : a m m o n i a ligase and glutamate : N A D oxidoreductase taken together were found to be correlated with respiration as well as heart beats/min. T h e results are consistent with the hypothesis that L-glutamate : ammonia ligase and glutamate : N A D oxidoreductase serve as a a m m o n i a - r e m o v a l mechanism in heart tissue. Alanine : 2-oxoglutarate aminotransferase showed a greater activity in m a m malians than in other species. REFERENCES ALBAUMH. G. (1955) Adenosine diphosphate, adenosine triphosphate. In Modern Methods of Plant Analysis (Edited by PAECHK. & TRaC~.YM. V.), Vol. IV, pp. 305-319. SpringerVerlag, Berlin. ASI-IWELLG. (1957) Colorimetric analysis of sugars. In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.), Vol. III, pp. 73-105. Academic Press, New York. CHtrNG WE (1963) Glutamine synthetase---I. A comparative study of its distribution in animals and its inhibition by DL-allo-8-hydroxylysine. Comp. Biochem. Physiol. 8, 335351. DUKESH. H. (1953) Anoxia. In The Physiology of Domestic ~lnimals, pp. 230-232. Comstock, New York. GOTHOSKAa B. P., RAINA P. N. & RAMAKRISHNANC. V. (1960) Glutamine synthetase in adult hen heart tissue. Biochim. biophys. Acta 37, 477-481. GOTHOSKARB. P., RAINA P. N. & RAMAKaISHNANC. V. (1961) Amino acid composition of the culture fluid during cultivation of chick and rat heart tissue explants in medium M150. Expl Cell. Res 24, 265-271. HAKKINEN H. M. & KULOr~N E. (1961) The effect of ethanol on the amino acids of the rat brain with reference to the administration of glutamine. Biochem. ft. 78, 588-593. K~BS H. A. (1950) Body size and tissue respiration. Biochim. biophys. Acta 4, 249-269. LOWRY O. H., ROSEBROUGHN. J., FAaa A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent, ft. biol. Chem. 193, 265-275. McDERMOTT W. V., Ja. & ADAMS R. D. (1954) Episodic stupor associated with an Eck fistula in the human with particular reference to the metabolism of ammonia, ft. Clin. lnvest. 33, 1-9. McNEMAR Q. (1955) Correlation: introduction and computation. In Psychological Statistics, pp. 115-121. John Wiley, New York. MOaCAN J. F., CAMPBELLM. E. & MOaTON H. J. (1955) The nutrition of animal tissues cultivated in vitro--I. A survey of natural materials as supplements to synthetic medium 199. ft. hath. Cancer lnst. 16, 557-567. NZKIFORUK G. & COLOWICK S. P. (1955) 5"-Adenylic acid deaminase from muscle. In Methods in Enzymology (Edited by COLOWICKS. P. & KAPLAN N. O.), Vol. II, pp. 469473. Academic Press, New York. PATELA. J., RAJALAKSHMIR. & RAMAKRISHN~C. V. (1965) Glutamine and alanine metabolism of avian and mammalian heart tissue explants cultivated in vitro in synthetic medium. In Tissue Culture (Edited by R . ~ a I s r r N A N C. V.), pp. 49--63. W. Junk, The Hague.
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815
RAINA P. N. & RAMAKRISHNANC. V. (1964) Comparative metabolic studies on normal and neoplastic rat l i v e r - - I V . Comparative studies on the activities of certain enzymes of amino acid metabolism in normal, neoplastic, newborn and regenerating liver. Oncologia 18, 14--20. RAJALAKSHMI R., ALL S. Z. & RAM~.RISHN&'~ C. V. (1967) Effect of inanition during the neonatal period on discrimination learning and brain biochemistry in the albino rat. 37. Neurochem. 14, 29-34. SEEGMILLER J. E., SCHWARTZ R. & DAVIDSON C. S. (1954) T h e plasma "ammonia" and glutamine content in patients with hepatic coma. j~. Clin. Invest. 33, 984-988. SHAH V. K. & RAMAKmSHN~'~C. V. (1963) Studies on acid metabolism in Aspargillus niger-II. Metabolic changes during citric acid utilization by Aspergillus niger. Enzymologia 26, 33-43. SPECTOR W. S. (1961) Heart rates. In Handbook of Biological Data (Edited by SPECTOR W. S.), pp. 277. Saunders, London. SRINIVASAMURTHY V. • SWAMINATHAN M . (1955) Colorimetric determination of dehydrogenase activities of liver using triphenyl tetrazolium bromide. Ind. j~. Physiol. Allied Sci. 9, 107-112. TERROIN~ T. (1960) T h e vitamin interrelations of ascorbic acid. In Worm Review of Nutrition and Dietetics (Edited by BOURNE G. H. & WILSON E. M. H.), Vol. II, pp. 105129. Pitman, London. TURSH G. P. (1964) Enzymes of glutamine metabolism in myocardium. Fedn Proc. 23, T.1305-1310. UMBREIT W. W . , BURroS R. H. & STANEFER J. F. (1964) T h e Warburg constant volume respirometer. In Manometric Techniques, pp. 1-18. Burgess, Minneapolis. WALTER H. E. & SAYLESL. P. (1961) T h e heart. In Biology of the Vertebrates, pp. 351-363. Macmillan, New York. WHITE A., HANDLER P. & SMITH E. L. (1964) Muscle. In Principles of Biochemistry, pp. 744-754. McGraw-Hill, New York. YAMANA Y. • SIBATANIA. (1960) Fractionation of ribonucleic acids with phenol. Biochim. biophys. _4cta 41, 295-303.