Hypoxanthine phosphoribosyltransferase activity in tissues and hypoxanthine concentrations in plasma and CSF of the horse in comparison with other species

Comp. Biochem. Physiol. Vol. 97B, No. 3, pp. 591-596, 1990 Printed in Great Britain

0305-0491/90 $3.00+ 0.00 Pergamon Press pie

H Y P O X A N T H I N E PHOSPHORIBOSYLTRANSFERASE ACTIVITY IN TISSUES A N D HYPOXANTHINE CONCENTRATIONS IN PLASMA A N D CSF OF THE HORSE IN COMPARISON WITH OTHER SPECIES R. A. HARKNESS,*§ G. M. McCREANOR,* J. ALLSOP,* D. H. SNOW,t R. C. HARRIS,~" P. O. ROSSDALE~ and J. C. OUSEY:~ *Division of Inherited Metabolic Diseases, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex HAl 3UJ, UK; tDepartment of Comparative Physiology, The Animal Health Trust, Snailwell Road, Newmarket, Suffolk CB8 7DW, UK; and ~Beaufort Cottage Stables, High Street, Newmarket, Suffolk CB8 8JS, UK (Received 9 May 1990)

Al~tract--1. Plasma hypoxanthine and xanthine concentrations are very low in the horse and low in rat, mouse and greyhound compared to concentrations in beagles, man, sheep and rabbit. 2. Activities in erythrocytes of the main enzyme metabolizing hypoxanthine, hypoxanthine phosphoribosyltransferase, show a similar pattern (Tax et al., 1976, Comp. Biochem. Physiol. $4B, 209-212); thus low activities have been found where plasma concentrations were low. 3. Hypoxanthine phosphoribosyltransferase activities in horse tissue other than erythrocytes are similar to those in man and rabbit with high activities in brain; this enzyme may therefore be functionally important in equine brain.

INTRODUCTION

The enzyme converting hypoxanthine, hyp, to IMP, hypoxanthine (guanine) phosphoribosyltransferase (EC 2.4.2.8), HPRT, was discovered about 20 years before its functional and quantitative importance was recognized from the discovery of the widespread effects of its deficiency in man. The enzyme is important because more than 95% of the hyp pool is recycled in man as part of a purine nucleotide cycle. HPRT deficiency, by blocking such purine salvage increases hypoxanthine conversion to urate causing gout. In addition, there is generalized growth failure, neurological dysfunction and testicular atrophy (Watts et al., 1987; Harkness et al., 1988). The main physiological substrate for HPRT is hyp; the other substrate of HPRT, guanine, is readily converted to xanthine by a high affinity and widespread enzyme guanine aminohydrolase (EC 3.5.4.3) (Kuzmits et al., 1980). Xanthine is not a substrate of HPRT (Kelley et al., 1967). In man, injected guanine is rapidly excreted as urate (see Harkness et al., 1988). From the above and other evidence, intracellular concentrations of hyp are dependent on HPRT activity. Since hyp crosses plasma membranes easily probably due to its low polarity (Whittam, 1960; Murray, 1971) extracellular concentrations are closely related to intracellular concentrations (Harkness et al., 1983). Under steady state conditions intraceilular concentrations of hyp will be related to HPRT activity. Until the development of high per§Present address: Department of Clinical Biochemistry, Institute of Child Health, 30 Guilford Street, London WC1N 1EH. 591

formance liquid chromatography there was very little information on hyp concentrations. Widely differing activities of HPRT and related enzymes are known in a number of tissues in a variety of species (e.g. Tax et al., 1976; Adams and Harkness, 1976). The virtual absence of HPRT and adenosine deaminase (EC 3.5.4.4) activity in erythrocytes from the horse is remarkable. In addition erythrocyte concentrations of phosphoribosylpyrophosphate, which together with hyp are substrates for HPRT, are high (Tax and Veerkamp, 1978); this is found also in HPRT deficiency in man. However, the structural gene for HPRT was present and active in the horse since leucocytes show enzyme activity (Tax et aL, 1976). In order to establish the presence or absence of the main substrate of HPRT, the plasma concentrations of hyp and related compounds were therefore measured in horses and five other species partly selected on the basis of their varying erythrocyte HPRT activities (Tax et al., 1976). Equine CSF was also examined. In addition, HPRT activities were measured in a variety of equine tissues including brain and compared to those in man and rabbit. Preliminary studies were also carried out to examine the effects of acute exercise in horses on plasma hyp concentrations. MATERIALS AND METHODS

Hypoxanthine, xanthine, xan, and uridine, urd, and cytidine in plasma and CSF were measured by high performance liquid chromatography (Simmonds and Harkness, 1981). Precautions necessary in sampling to avoid cellular lysis and leakage raising concentrations have been reviewed (Harkness, 1988). Extracts were analyzed within weeks of

R. A. HARKNESSet al.

592

receipt but were stable at -20°C for at least 2 years. Samples of equine tissues were obtained from thoroughbred racehorses euthanased and brought to the Animal Health Trust for autopsy. Storage was at -20°C wrapped closely in aluminium foil and sealed in tubes to avoid dehydration. Activities of HPRT were measured by the method of Craft et al. (1970) and adenine phosphoribosyltransferase (EC 2.4.2.7), APRT, activities by the method of Dean et al. (1968). Both procedures used a final concentration of 3.3 mM TTP to inhibit 5'-nucleotidase present in all tissues except erythrocytes (Gutensohn and Guroff, 1972). Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. Exercise studies were carried out on four thoroughbred horses using a motorized treadmill (Sato, Sweden). Each horse was exercised on 3-4 separate occasions for 2 min at speeds of 8, 10, 12 and 14 msec -t against an incline of 5°. In each case the test exercise was preceded by a "warm up" exercise consisting of a 4 min walk, 1.6 msec -I, plus a 4 min trot, 3.2 msec-~, then rapid acceleration to cantering speeds. Blood was taken 12 rain after the end of exercise from the jugular vein into EDTA as an anticoagulant; centrifugal separation of plasma was performed within 30 min of sampling. Muscle biopsy studies had established that ATP depletion of skeletal muscle occurred in the horse at the higher levels of exercise used (Snow et al., 1985; Harris et al., 1987). Greyhounds were sampled at the Animal Health Trust, beagles at a pharmaceutical research laboratory and Texel sheep by Professor H. Britton. All other blood samples were obtained in the Division of Comparative Medicine, Clinical Research Centre. The strains were New Zealand White rabbits, Sprague-Dawley rats and T.O. mice. All procedures were in accordance with standard veterinary practice. CSF samples from foals and donkeys were obtained (Dr P. D. Rossdale) by an already described method (Ross et al., 1982).

RESULTS

Plasma concentrations o f hyp and related compounds in six species The resting concentrations in plasma of the purine bases of hyp and xan with the major circulating pyrimidine nucleoside urd are shown in Table 1. Inosine is included as a check for the absence of cellular lysis which causes marked elevations (Harkness et al., 1984). Large species variations in hyp concentrations were found with around 50 nmol/l or less in horse, mouse, rat and greyhound, about 2/~mol/l in beagles and man (Simmonds and Harkness, 1981) and about 10 in rabbits and 20 # m o l / l in sheep. Our values for the rabbit are similar to those of Eels and Spector (1983). Concentrations of xan show a similar but less marked pattern to hyp with higher concentrations in rabbit and sheep. The plasma concentrations of the pyrimidine nucleoside urd, varied

from 1 to 15 #mol/l in horse, mouse, rabbit, man and sheep with < 1 #mol/i in greyhounds and beagles. Concentrations of the pyrimidine nucleoside cytidine varied from 1 to 15 #mol/l, in rat, rabbit and sheep with lower but detectable amounts in greyhounds which were not, however, measurable. N o estimates appeared justifiable in the other species studied. In rats, mice and horses, somewhat similar results for urd and cytidine have been obtained by Moyer et al. (1981). The sensitivity of the method used was variably reduced by contaminants present in extracts; the extent of such contamination showed species variations (Table 1). C S F concentrations o f hyp and related compounds in horse and sheep The concentrations in C S F are shown in Table 2; the pattern is similar to that in the plasma obtained at rest with hyp concentration low in horse and high in sheep and with less marked differences in xan concentrations. In contrast, as in the plasma, urd concentrations were similar; cytidine was measurable in sheep C S F with a mean (SEM) concentration of 5.2 (1.8)/~mol/1 n = 6. C S F concentrations in eight donkeys were similar to those in horses being undetectable for hyp and xan and with mean (SEM) levels of urd being 10.2 (2.9)/~mol/1. However, a C S F sample taken during a postmortem examination of a foal contained hyp 10.3, xan 8.7, urd 22.8/~mol/1. Although these changes are less marked than in man (Harkness and Lund, 1983), they are raised. The horse brain thus possesses the necessary enzymes to produce hyp during severe A T P depletion. Exercise to, at or near fatigue raises plasma hyp and xan in horses Pilot studies of the effect of 2 min measured exercise on plasma hyp and xan concentrations measured 12 min after the end of exercise are shown in Fig. 1 for four separate thoroughbreds. Plasma concentrations of hyp and xan rose from concentrations 50 nmol/1 to 5-10 #mol/1 only after intense exercise resulting in A T P depletion in skeletal muscle (Snow et al., 1985; Harris et al., 1987). This shows that equine skeletal muscle possesses and uses the enzymes for producing hyp during A T P depletion. H P R T activities in equine tissues The H P R T activities in equine tissues other than erythrocytes were similar to those in man and the rabbit with high activities in brain (Table 3). In the same set of equine tissue samples adenine phosphoribosyltransferase, A P R T , activities

Table 1. Plasma concentrations (#tool/l) of hypoxanthine, xanthine, uridine, inosine and cytidine in six species Mean (SEM) No. of individuals hyp xan urd ino Cytidine Horse 7* ND ND 6.9 (0.3) ND Rat 5 0.04 (0.01) 0.07 (0.03) 0.9 (0.03) ND 12.7 (2.1) Mouse 4 <0.15 <0.33 12.6(2.9) <0.43 Greyhound I1 < 0.08 < 0.17 0.18 (0.8) 0.7 (0.5) Beagle 10 2.1 (0.3) <0.17 ND ND Rabbit 4 9.1 (1.5) 2(0.03) 3.6(0.4) 0 8.1 (0.9) Sheep 3~" 18.8 (7.9) 5.2 (1.6) 3.5 (1.5) 0.9 (0.3) 3.7 (0.4) ND is <0.1 ~mol/l unless specified by less than a figure. *32, t41 samples.

Hypoxanthine concentrations and HPRT activities Effect of exercise on horses

Table 2. CSF concentrations (/zmol/l) of hypoxanthinc, xanthine and uridine in horses and sheep Horses 1983 1984 1985 Sheep

No. of individuals 15 14 16 3*

hyp

[Mean (SEM)] xan

urd

1.5 (0.6) 1.1 (0.3) 1.7 (0.4) 6.3 (0.8)

8.6 (4.7) 9 (0.9) 7.5 (0.9) 4.3 (0.5)

0.6 0.5 (0.1) 0.5 (0.2) 16.7 (2.8)

593

8

O

.__q E

"16 samples. lnosine was not detected <0.1 ttmol/l.

0..a

(nmolhr -j mgprot 1) were measured (Table 4). Equine pituitary had an HPRT activity, m e a n + SEM (n), of 1 3 3 _ 3 7 (6) and an APRT activity of 32 + 11 (6) nmol hr-l mg prot-1. Activities in rats and mice are also shown in Tables 3 and 4. For the slow growing SVG strains of rats activities of HPRT were lower in brain mean (SEM) 127 _+ 5 (4) and liver 147 _ 27 (4) using a similar assay method (Adams and Harkness, 1976). Erythrocyte HPRT activities in our strain of mice were 2.6+0.3 (21) nmol hr -1 mg prot -~ which is similar to but somewhat lower than the results of Tax et al. (1976). Erythrocyte APRT activity in the mice was 2.9 _ 0.4 (6). DISCUSSION

The overall physiological function of HPRT was initially believed to include the transport of purines from the liver, a site of high de novo synthesis, to the brain a site of high ATP turnover (Murray, 1971). Radioactive hyp incorporated into rabbit erythrocytes could be recovered from nucleotides, RNA and DNA in brain, liver, kidney, skeletal muscle and testes of this species (Adams and Harkness, 1973); the exchange of preformed purines as bases or nucleosides between cells now appears to be a general mechanism. This could not be demonstrated in the early experiments presumably because it is difficult to load brains with labelled compounds. These early proposals may require further revision. Purine synthesis de novo has now been shown to be widespread and quantitatively uniform in tissues including brain (see Allsop and Watts, 1986). Moreover, a constant supply of hyp for HPRT is provided by the operation of a purine nucleotide cycle which has been demonstrated in skeletal muscle (Lowenstein, 1972) and a range of cultured cells with genetic or enzyme competitor induced blocks

_'-

:

<° 10

8

12

14

Velocity (m/sec)

Fig. 1. The effect of measured exercise for 2 min in four horses on plasma concentrations of hyp and xan sampled 12 rain after the exercise. (Hershfield and Seegmiller, 1977; Barankiewicz et al., 1982; Willis et al., 1984). In man hyp output is increased with increasing ATP turnover (McCreanor and Harkness, 1987). The horse appears to have largely lost the transport of preformed purines in plasma and erythrocytes. The activities of enzymes involved in a purine nucleotide cycle HPRT, adenosine deaminase and purine nucleoside phosphorylase are all very low in equine erythrocytes which also have a low ATP concentration relative to other species (Prankerd, 1961). In addition, in extensive studies (unpublished observations) we have never detected hyp in horse plasma at rest or even at exercise levels below those producing ATP depletion in skeletal muscle. It would seem reasonable to expect that a large flux through a small plasma pool would occasionally produce high levels. The possibility exists that the horse has lost the erythrocyte transport system and leakage of hyp from cells at low levels of ATP turnover, during its selection for muscular exercise (Harkness, 1986). To examine this possibility further we compared the greyhound with the beagle (Ryder, 1985). Consistent with the above suggestion, the greyhound has low concentrations of hyp in plasma relative to beagles;

Table 3. HPRT activities in tissues of adult horse, man, rabbit and rat Tissue Brain cerebral cortex basal ganglia cerebellar cortex Thymus Liver Kidney Heart Skeletal muscle

Horse 289 (3) 182 (3) 247 _+ 72 (9) 433 (1) 130 5:17 (9) 88 5:17 (9) 79 _+ 15 (6) 38 _+ 8 (7)

*Adams and Harkness (1976). tAdams and Harkness (1973). :~Allsop and Watts (1980). §Children. NA not measured.

[Mean _+ SEM (n) Rabbitt 294 5:58 (4) 230 (2) 193 5:24 (3) NA 29 + 4 (3)~ NA 53 _+ 14 (4)~ NA 71 + 11 (6) 86(2) 42 _+ 16 (4) 15(2) 38 _+9 (5)~ NA 13 _+ 3 (4)~ 14(2) Man*

nmol hr-1 mg prot -I ] Rat~: Mice (M) 394 + 26 (14) 417 5:34 (14) 4225:20(14) NA 3735:21(10) 625:5(10) NA NA

1230 + 96 NA NA NA 451_+43 305_+27 NA 11 5:2.5(15)

R. A. HARKNESSet al.

594

Table 4. A P R T activities in tissues of horse, man, rat and mouse [Mean + SEM (n) nmol h r - ] mg prot 11 Horse Man* Rat

Tissue Brain Cerebral cortex Basal ganglia Cerebellar cortex Thymus Liver Kidney Heart Skeletal muscle

Mouse 136 _+ 6 (12)

34 (3) 38 (3) 38 + 10 (9) 51 79+21(9) 48+20(9) 42 + 10 (6) 27 + 10 (7)

13 ___(4) 6 + (3) NA 96 + 38 (4) 89+39(6) 42+7(3) 17, 50 120, 19

101 +_ 7 (14) 134 _+ (14) 126 _4-9 (14) 26+3(10) 42+3(10) NA NA

25+3(12) 24+3(12) NA NA

*Adams and Harkness (1976), children, somewhat higher values were found in adults.

the ranges did not overlap. Our evidence on resting concentrations of hyp in plasma and existing data on erythrocyte HPRT activities suggested a possible link. In Fig. 2 resting plasma hyp concentrations for a species are plotted against erythrocyte HPRT activities for that species, using the data of Tax et al. (1976). Increasing plasma hyp is associated with increasing erythrocyte HPRT activities. A linear correlation was positive and significant, r = 0.623 P < 0.01. Greyhounds and beagles are shown separately since the plasma hyp values did not overlap. The operation of a purine nucleotide cycle as a part of the normal function of cells should be distinguished from events following ATP depletion which is essentially abnormal in most tissues except skeletal muscles. Our evidence shows that skeletal muscle and brain ATP depletion in the horse is associated with hyp release despite the normally very low concentrations of hyp in extracellular fluids (Table 1, 2; and Fig. 1). This is consistent with the findings in other species. ATP depletion increases hypoxanthine concentrations in many other species including rat (Osswald lO 000 • Rabbit Sheep

Doq (beaqte) Man 1000

T

Dog (greyhound)

100 -

Mouse •

Rat Horse

1-

I l

I 2

I r I 3 4 5

r 10

I F I I 21) 30 4050

1 100

Erythrocyte HPRT activity n m o t / h per mg protein

Fig. 2. The relationship between resting plasma hyp concentrations (nmol/l) and erythrocyte HPRT activities (Tax et al., 1976) in horse, rat, greyhound and beagle, mouse, man, sheep and rabbit. Both variables are plotted on log scales.

et al., 1977), guinea pig (Schrader et al., 1977), cat (Kleihues et al., 1974), mice (Warnick and Lazarus,

1981), pig (de Jong and Goldstein, 1974), dog (Saugstad et al., 1977), sheep (Thiringer, personal communi-

cation) and man (Harkness, 1988). The mechanisms involved in the preservation of relative ATP concentration have been reviewed (Atkinson, 1977). In man, levels of exercise below those causing muscle ATP depletion produce a curvilinear increase in hyp output (Ketai et al., 1987; McCreanor and Harkness, 1987). In addition the functional defects due to HPRT deficiency in man only become obvious with increased ATP use by tissues (Watts et al., 1987; Harkness et al., 1988). Since brain has a high turnover of ATP the accumulation of hyp due to a lack of HPRT is more obvious in the extracellular fluid of brain, CSF, than in plasma. Thus HPRT appears to operate and be functionally important to an extent dependent on ATP turnover. Control and function o f H P R T tissues

activities in equine

It seems unlikely that the transcriptional and translocational controls mediated by 5'- and Y-flanking sequencies (Stout and Caskey, 1985; Patel et al., 1986; Kim et al., 1986; Proudfoot, 1989) are mainly responsible for the low levels of HPRT and ADA in equine erythrocytes (McGuire et al., 1976; Adams and Harkness, 1976; Tax and Veerkamp, 1979). The mechanisms reducing HPRT in the mature equine erythrocyte are probably similar to those reducing the closely linked enzyme adenosine deaminase during erythrocyte maturation from the reticulocyte to the mature erythrocyte (Denton et al., 1975). This appears to be mediated by selective proteolysis. Since HPRT deficiency may cause megaloblastic anaemia and HPRT is expressed in many tissues including the shorter lived leucocytes (Tax et al., 1976; Adams and Harkness, 1976) it is probable that the early stages of erythrocyte development show HPRT activities similar to leucocytes, a general house keeping level. Differences between species must be subtle since the mouse HPRT differs from the human by only seven amino acid residues resulting in 95% homology. However, small differences in the N-terminal amino acid sequence in mice can cause selective proteolysis in reticulocytes (Johnson et al., 1988) possibly via conjugation with ubiquitin (Finley et al., 1987). Unstable HPRT proteins due to mutation are known in man (Wilson et al., 1986) and may have been produced in mice by deletion of the first five exons (Hooper et al., 1987).

Hypoxanthine concentrations and HPRT activities For H P R T a largely intracellular role seems probable. The apparently futile purine nucleotide cycle may be necessary to ensure a balanced supply of nucleotides essential for enzymic activities (Atkinson, 1977) and accurate R N A synthesis. The balanced supply of deoxynucleotides may be dependent on a pyrimidine deoxyribonucleotide cycle (Reichard, 1988) and can be necessary to ensure the fidelity of D N A synthesis (Bradley and Sharkey, 1978; Fersht, 1979). REFERENCES

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