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Adenine pool catabolism in the ischemic, the calcium-depleted ischemic, and the substrate free anoxic isolated rat heart: Relationship to contracture development
J. Mol Cell Cardio116, 1127-I 136 (1984)
A d e n i n e P o o l C a t a b o l i s m in the I s c h e m i c , t h e C a l c i u m D e p l e t e d I s c h e m i c , a n d t h e S u b s t r a t e Free A n o x i c I s o l a t e d R a t H e a r t : R e l a t i o n s h i p to C o n t r a c t u r e D e v e l o p m e n t S. M. Humphrey*, D. G. Holliss and R. N. Seelye Department of Pathology, University of Auckland Medical School, Auckland, New Zealand (Received 17 January 1984, accepted in revisedform 25 April 1984) S. M. HUMPHREY, D. G. HOLLISS and R. N. SEELYE. Adenine Pool Catabolism in the Ischemic, the CalciumDepleted Ischemic, and the Substrate Free Anoxic Isolated R a t Heart: Relationship to Contracture Development. Journal of Molecular and Cellular Cardiology (1984) 16, 1129-1138. Metabolic changes in the myocardial adenine and hypoxanthine pools of isolated rat hearts subjected to global ischemia, hypocalcemic global ischemia, and global substrate-free anoxia were compared. At timed intervals between 0 and 60 rain separate aliquots of extracts of the ventricles were used to determine either tissue pH, or the components of the adenine pool and their catabolites by reverse phase high performance liquid chromatography (HPLC). The coronary perfusate draining from anoxically perfused hearts was collected over perchloric acid, neutralised and chromatographed by HPLC. The development of left ventricular resting tension (contracture) was recorded in the three groups of hearts. After 60 min ischemia the major catabolites, (AMP, inosine and hypoxanthine) comprised 70% of the total pool (11, 7 and 4/2mol/g dry wt, respectively). After the same period of anoxia 50% of the total pool, comprising adenosine, inosine, hypoxanthine and uric acid in approximately equal proportions, was recovered from the coronary perfusate. The major products remaining in the tissue were IMP and, to a lesser extent A M P (8 and 5 #mol/g dry wt, respectively). Left ventricular contracture developed at different rates in the three groups of hearts but always correlated closely with the m a x i m u m rate of adenine pool catabolism. The loss of components from the tissue and the divergence in pathway from adenosine to I M P production which occurs during anoxic perfusion should possibly be considered when assessing the biochemical events occurring in regionally ischemic heart muscle with significant residual flow. KEY WORDS: Adenine nucleotides; Adenosine; Anoxia; Contracture; Inosine; Inosine monophosphate; Ischemia.
Introduction Myocardial ischemia which leads to the development of an infarct rarely involves a total cessation of blood flow to the affected area [4, 23]. For this reason, the relationship between loss of tissue high energy phosphate and the capacity of the myocardium to recover function has been evaluated after varying periods of both global ischemia (a total lack of flow and consequently of oxygen and nutrient) but also global anoxia (high flow but with similar lack of oxygen and nutrient) [5, 7, 9, 22]. However, a comprehensive study of adenine nucleotide catabolism with its associated increase in the components of the hypo-
xanthine pool, utilising the new and accurate technique of high performance liquid chromatography (HPLC), has been described only for the zero-flow ischemic situation [13, 14]. No similar H P L C study of adenine nucleotide loss for substrate-free anoxia has appeared in the literature. The available data relating to this situation are scattered and incomplete
[15, 25, 29]. An apparent lack of information also exists in relation to the association between high energy phosphate depletion and the deveiopment of contracture in substrate-free anoxia, although a close association has been described for ischemic hearts [6, 11, 19].
* To whom correspondence should be addressed at the Department of Pathology, University of Auckland Medical School, Private Bag, Auckland, New Zealand. 0022 2828/84/121127 + i0 $03.00/0
9 1984 Academic Press Inc. (London) Limited
1128
S.M. Humphrey et aL
This paper describes a comprehensive study undertaken to compare the effects of 60 min each of global substrate-free anoxia and ischemia on the metabolite changes (determined by H P L C ) and the associated changes in contracture in the isolated rat heart. The findings confirm that the maximum rate of high energy phosphate decline parallels the time course of contracture development in anoxia and in ischemia. However, the pattern of change in the various adenine pool catabolites differs considerably in the two situations.
15, 20, 30, 40 and 60 min of anoxia or ischemia (six hearts for each time interval).
M a t e r i a l s and M e t h o d s
Contracture measurement In each group, the development of contracture was measured by recording the pressure exerted on a balloon which was inflated with normal saline to just fill the left ventricular lumen. T h e balloon, which was inserted into the left ventricle via the left atrium prior to the commencement of either anoxia or ischemia was connected via the open end to a pressure transducer (Bell and Howell 4-422) and chart recorder as described previously [8, 10].
Male albino Wistar rats (280 to 320 g) in three groups of 48 were each lightly anesthetized with ether and injected with 200 I U of heparin via the femoral vein. Each heart was quickly excised and placed in ice-cold perfusion solution to arrest beating. The aorta was attached to a stainless steel cannula and the heart subjected to a non-recirculating Langendorff perfusion. All hearts were maintained at 37~ in a water-jacketed chamber and perfused for a 10-min equilibration period at a pressure of 100 cm H 2 0 with Krebs-Henseleit bicarbonate buffer (KHB) which contained 11 mt~ glucose. The perfusion fluid was gassed continuously with a mixture of 95% Ozand 5% CO 2. At the end of the equilibration period hearts from the first group were made globally anoxic by switching to a glucose-free perfusion fluid (KHB) maintained at a pressure of 100 cm HzO and gassed continuously with a mixture of 95% N 2 and 5% CO2. Hearts from the second group were made globally ischemic by clamping the aortic cannulae. In the third group (calcium-depleted ischemia), hearts were perfused for a further 10 min normoxic period with K H B in which the calcium content had been reduced from 2.5 mM to 0.05 m~ before being made globally ischemic as previously described for group I. The experimental period of anoxia or ischemia was maintained for 60 min during which time the development of contracture was recorded in six hearts from each group. Tissue p H and metabolite assays were performed on the remaining hearts, which were freeze-clamped in liquid nitrogen after 5, 10,
Frozen tissue samples were powdered in a percussion mortar maintained at the temperature of liquid nitrogen. For each heart three accurately weighed aliquots of powder were prepared: one was dried in an oven at 80~ for 24 h to obtain the tissue wet/dry ratio, whilst the other two were dispersed (100 mg/ml) in either ice-cold 6% perchloric acid (PCA), neutralized and centrifuged (4~ at 2500 g for metabolite determinations, or neutral 2 m~ iodoacetate for pH measurements by a method described in detail previously [28]. Ten microlitre samples of the neutralized PCA supernatant were analysed by high performance liquid chromatography using a reverse phase column (Zorbax ODS, 7 pm, Dupont Instruments, Wilmington, DE, USA) and a 254-nm wavelength detector to separate and quantitate successively, uric acid, inosine monophosphate (IMP), adenosine tri-, di-, and mono-phosphates (ATP), (ADP), (AMP), hypoxanthine, inosine and adenosine. Solvent A was an aqueous solution containing K H 2 P O 4 (0.022M), MgSO 4 (0.004M) and Trishydroxy methyl-aminomethane (TRIS) (0.004M) adjusted to pH 6.25. Solvent B was methanol : water 60 : 40. All metabolites were eluted within 20 min using a linear gradient of 3% to 80% Solvent B. The results are expressed throughout as #moles per gramme dry weight (/~mol/g dry wt). For the anoxically perfused group of hearts, in addition to tissue extracts, the coronary effluents were similarly assayed for adenine pool catabolites. Measured volumes of coro-
Metabolite measurement
Adenine Pool Catabolism
nary fluid draining from the right side of the heart were collected at timed intervals into ice-cold 60% PCA. The fluid was adjusted to a final concentration of 6% P C A before neutralization and c h r o m a t o g r a p h y as described above.
Results
T h e development of contracture in the three groups of hearts is shown in Figure l. In anoxic hearts, contracture began within 1 to 2 min of the onset of anoxia and rapidly increased to its m a x i m u m pressure of 50 4- 6 m m H g (mean __ SEM) by 6 min. In ischemic hearts the onset ofcontracture was delayed for 5 to 8 rain, developed at a rate slower than for anoxic hearts and reached a m a x i m u m level of only 30 + 3 m m H g by 30 min (Fig. 1). I n the calcium-depleted ischemic group, contracture did not develop to a measurable extent until 15 to 20 min of ischemia had elapsed. It then increased at a similar rate (in terms of percent of m a x i m u m per minute) to that seen in normo-calcemic ischemic hearts but reached a m a x i m u m pressure of only 8 +__2 m m H g . T h e results of the metabolite measurements are tabulated (Table 1). I n the two ischemic groups of hearts the total pool size did not alter significantly throughout the experiment (31.2 _+ 0.9 #mol/g dry wt for 96 hearts). In
50
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FIGURE 1. Graph showing the development of increasing resting tension (contracture) in hearts during 60 min of (a) global anoxia, (b) total global ischemia, and (c) total global ischemia after pre-ischemic perfusion with 0.05 mM calcium buffer. Values represent the means of six hearts and bars the standard errors of the means (S.E.M.).
and Contracture
I 129
the anoxic group, however, the total pool increased gradually during the 60-min experiment from 31.4 to 37.0. Since extremely high volumes (300 to 400 ml per heart) of coronary effluent fluid were collected during 60 min anoxia, the error associated with assaying the adenine pool catabolites from the perfusate was greater than that from the tissue assays. To allow for this, the rates of depletion and accumulation of metabolites were calculated from values normalized to the control value of 31.4 #mol/g dry wt. A comparison of the accumulation of adenine pool catabolites in the coronary fluid with their complementary tissue content during 60 rain anoxia is shown in Figure 2. Both adenosine and inosine were released at a constant rate of approximately 0.15 #mol/g dry wt/min for the first 30 min [Fig. 2(a)]. H y p o x a n t h i n e and uric acid accumulated at slower rates namely, 0.11, and 0.08 #mol/g dry wt/min respectively. Following 30 min of anoxia there was little change in either adenosine or uric acid, and only small increases in inosine and hypoxanthine during the period 30 to 60 min. This is reflected in the plot of total tissue metabolites removed from, or remaining in, the heart during anoxia [Fig. 2(b)]; more than 80% of the total change occurred during the first 30 min. Figure 3 depicts a composite of the changes in the major metabolites that occurred during 60 min of oxygen and nutrient depletion in all three groups of hearts. During the first 10 min of anoxia, [Fig. 3(a)] there was a rapid fall in the tissue content of combined (ATP + ADP) with a m a x i m u m rate of approx. 3 #mol/g dry wt/min. Associated with this fall were the m a x i m u m rates of accumulation of A M P , I M P , adenosine and [ H I ( [ H I = the combined values of inosine, hypoxanthine and Uric Acid) which were 2.0, 0.5, 0.8 and 0.5 #mol/g dry wt/min respectively. This phase of rapid change correlated with the development of contracture pressure (Fig. 4, Table 2), Between 10 and 30 min, the (ATP + ADP) moiety declined more slowly (0.2 #mol/g dry wt/min). T h e A M P level fell rapidly during this stage (0.5 #mol/g dry wt/min) and the [ H ] pool continued to accumulate at a similar rate. I M P also continued to accumulate, in contrast to adenosine which remained at a constant value for the remaining period of
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Time (min) FIGURE 2. Graphs showing the accumulation of the major products of adenine pool catabolism in rat hearts
subjected to 60 min of anoxia. (a) The solid line represents components accumulating in the tissue and the dotted line those removed in the coronary perfusate. (b) Changes in the level of the total adenine and hypoxanthine pools (ATP, ADP, AMP, adenosine, IMP, inosine, hypoxanthine and uric acid) in the tissue (O) and in the perfusate (0) during 60 rain anoxia. Values represent the means of six hearts and the bars the S.E.M. anoxia. After 30 min of anoxia the overall change in tissue metabolite level was small [Fig. 2(a)]. The rate of A M P decline was complementary to the rate of I M P accumulation during this period (0.09 #mol/g dry wt/min) and by 60 min the I M P reached 7 #mol/g dry wt to become the highest single component of the total pool. In the ischemic group of hearts the first approximately 15 min was associated with very little change [Fig. 3(b)]. This was followed by a rapid change in all moieties,
chiefly (ATP + ADP) decline (0.73 #mol/g dry wt/min) and associated rises in AMP, Adenosine and [-H] (0.54, 0.40 and 0.18 #mol/g dry wt/min respectively). In these hearts I M P did not rise by more than 2 #mol/g dry wt throughout 60 rain ofischemia. Again, the phase of rapid change (15 to 30 min) coincided with the development of contracture. Thereafter both the A M P and adenosine moieties declined from their peak value and the (ATP + ADP) and [ H I curves levelled off.
1132
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F I G U R E 3. Accumulation of the products of adenine pool catabolism in rat hearts during 60 min of (a) global anoxia, (b) total global ischemia and (c) total global ischemia after pre-ischemic perfusion with 0.05 mM calcium buffer. O , A M P ; I , [H] (sum ofinosine, hypoxanthine and uric acid); &, adenosine; 9 IMP; A T P 4-ADP. Values represent the means of six hearts and the bars the S.E.M.
(ATP + ADP) had not yet reached the low values observed for the other two groups. Results of plots relating increments in the -6 E 12 =L force of contracture to the metabolic tissue content of ATP, AMP, adenosine, and inosine at corresponding times for each of the three groups is shown in Figure 4. The tissue content of metabolites continued to change after contracture was completed particularly in the anoxic group (Fig. 4). However in all three groups of hearts a close correlation exists lO 20 30 40 50 60 between contracture and the fluxes of the Time(rain) adenine nucleotides, adenosine and inosine as The calcium-depleted ischemic group of indicated by a Pearson coefficient of correlahearts [Fig. 3(c)] showed a similar but tion (Table 2) performed on the data repextended first period of minor change fol- resented schematically in Figure 4. The change in the tissue pH during 60 lowed by a phase of rapid flux coincident with the development of contracture. The rates of minutes of anoxia and ischemia is shown in fall of (ATP + ADP) (0.55) and concomitant Figure 5. The continuously perfused hearts rises in AMP (0.42) adenosine (0.30) and [ H I maintained a pH of 7.36 throughout anoxia (0.13) were similar to those in the nor- whereas the ischemic hearts showed a rapid mocalcemic ischemic group. After the rapid early decline during the first 10 to 15 min phase in this group there was no further to pH 6.6, followed by a slower decrease to pH change in the level of adenosine and AMP 6.4 by 30 to 40 min of ischemia. Therevalues. [H] continued to rise and the after, the pH did not alter significantly.
A d e n i n e Pool C a t a b o l i s m and Contracture
1133
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F I G U R E 4. Graphs showing the relationships between the development of contracture (P) and the tissue content of the major metabolites during anoxia (left) ischemia (centre) and ischemia (right) after pre-ischemic perfusion with 0.05 mM calcium buffer. 0 , AMP; &, ADO, a d e n o s i n e ; . , INO, inosine.
Discussion The results of this investigation clearly indicate that, in both anoxically perfused and globally ischemic rat heart preparations, there is a very close association between the rapid phase of adenine pool catabolism and the development of contracture. This holds despite the existence of a 15-min difference in onset of the rapid phase under the two conditions. Delaying the onset and development of contracture by pre-perfusion with the 0.05 mM calcium solution delays proportionally the loss of (ATP + ADP) and the rise in AMP, adenosine, inosine, hypoxanthine and uric acid. The preservation of A T P and delay in the onset of contracture in the low-calcium group
may have been related to the cardioplegic effect of reduced calcium. Certainly contractility is stopped immediately subsequent to ischemia in this group whereas it continues for 1 to 2 min i n the normocalcemic ischemic hearts. This results in a reduced A T P consumption (1 ktmol/g dry wt/beat) during the first minutes of ischemia, however the tissue content of A T P remains essentially similar in the two ischemic groups even after 10 min. It is more likely therefore that the reduction of extracellular calcium availability conserves ATP by reducing calcium-mediated A T P hydrolysis associated with contracture. The lower coefficient of correlation observed for the anoxic group of hearts (Table 2) suggests that anoxic contracture may not
1134
S . M . Humphrey et aL (01 7.2
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Yimo(min) FIGURE 5. Graph showing the changes in tissue acidity of rat hearts during 60 min of (a) anoxia, (b) total global ischemia and (c) total global ischemia after pre-ischemic perfusion with 0.05 mM calcium buffer. Values represent the means of six hearts and the bars the S.E.M. be directly c o m p a r a b l e to ischemic contracture. W h e r e a s ischemic c o n t r a c t u r e p r o b a b l y represents the a c c u m u l a t i o n of rigor complexes arising as a result of A T P deficiency [_6, 11], i m p a i r e d calcium homeostasis m a y be the p r i m a r y cause of the early c o n t r a c t u r e in a n o x i a [20]. I n the latter case, however, this intracellular calcium-overload-contracture w o u l d r a p i d l y convert to r i g o r - c o n t r a c t u r e consequent upon A T P depletion which occurs within 3 to 5 rain of p e a k contracture. T h e p r o d u c t i o n and fate of A M P in the three groups is of interest. D u r i n g ischemia, the relatively small c h a n g e in A M P level prior to c o n t r a c t u r e indicates t h a t the catabolism of A T P proceeds u n i n h i b i t e d to adenosine. T h e r a p i d a c c u m u l a t i o n of A M P associated with
c o n t r a c t u r e d e v e l o p m e n t is therefore likely to be related to the m e t a b o l i c events taking place at the myofibrils. T h e depletion of creatine p h o s p h a t e early in ischemia and anoxia [_6, 19] restricts A D P to r e p h o s p h o r y l a t i o n (by the action of myokinase) at the expense of e q u i v a l e n t A M P production. T h e continuous cycling of this reaction will result in the r a p i d a c c u m u l a t i o n of only A M P . T h e a c c u m u l a tion of A M P , r a t h e r t h a n its d e p h o s p h o r y l a tion to adenosine, is most likely related to a shift of the r a t e - d e t e r m i n i n g step from adenosine d e a m i n a s e to 5'nucleotidase. This could be due simply to rate of p r o d u c t i o n being in excess of rate of d e p h o s p h o r y l a t i o n . I n a c tivation of the enzyme as tissue p H falls does not seem likely since the greatest decline in
TABLE 2. Relationship between the development of contracture and the tissue content of selected adenine pool constituents in the three groups of hearts. Correlation coefficient (r) Condition
ATP
AMP
Anoxia Ischemia Ca 2 +-Depleted Ischemia
0.88 0.98 0.94
0.80 a 0.98 0.93
Adenosine
Inosine
0.76 0.94 0.97
0.54 0.90 0.95
a This value relates only to the increase in the tissue content of
Adenine Pool Catabolism
pH occurred during the first 15 min of ischemia (Fig. 5) when A M P did not accumulate. Moreover, in all cases, the production of hypoxanthine pool metabolites appeared remarkably constant throughout ischemia and during the first 30 min of anoxia (by which :time nucleotide fluxes were 95% complete). Thus, the accumulation and depIetion of A M P did not appear to influence greatly the overall catabolic flux. During anoxia, the decline in A M P was equivalent to the production of IMP. The deamination of A M P to I M P rather than its dephosphorylation to adenosine has not previously been thought to be a n important pathway in heart tissue. The production of adenosine, and not IMP, is favoured during ischemia because decreasing A T P concentra+ tions will increase 5' nucleotidase activity [21] and decrease adenylate deaminase activity [2]. However, the factors involved in the regulation of adenylate deaminase activity appear to be altered during anoxia. We believe, therefore, that these results relating to AMP production and decay provide further support for the compartmentation of an A T P pool associated with the myofibrillar apparatus [1]. The AMP produced from this pool by the rapid actin/ myosin interaction during contracture development is either converted to I M P if flow is sufficient to minimise pH change, or remains fairly constant if flow is absent. Although it is known that there is an approximately 7-fold increase in A M P [12, 31] during ischemia its correlation with contracture has not been noted. The hitherto unexplained markedly late rise in A M P level (60 to 120 min) observed in dog heart slices made ischemic in vitro [12, 13] can be explained by its association with the development of contracture. This late A M P accumulation occurs long after the most rapid decline in A T P tissue content, but in precise agreement with the late time of contracture in this model [30]. The improved analytical technique of H P L C has led to much higher estimates of the cardiac adenine nucleotide pool catabolites. Values for normal cardiac adenosine content, for instance, have increased from 0~2 to 0.3 nmol/g [26] to 10 to 20 nmol/g [16]. Our control values for adenosine and inosine were similar in range to the results of Klabunde
and Contracture
1135
and colleagues [16]. However, these values may be elevated above in situ rat heart concentrations, owing to the unphysiological control conditions of the Langendorff perfusion technique. During the first 15 min of anoxia adenosine showed a significant accumulation in the tissue. Although it is not possible to determine precisely the intra- and extra-cellular concentration of adenosine from the results of these experiments, if the adenosine resided solely in the interstitial compartment (0.2 ml/g [3]) it would approximate to 2 to 3 #mol/ml. This would be over 1000 times the concentration in the perfusate (coronary flow rate was 12 ml/min after 15 min of anoxia) and yet its efflux into the coronary perfusate was the same as that for inosine which had a considerably lower tissue content. Thus, it is likely that the rapid accumulation of adenosine during early anoxia occurs within the cell and this adds support to the finding of Schutz et al. [27] that adenosine is predominantly formed intracellularly in the hypoxic heart. The most effective method of conserving the cellular adenine pool, and consequently the potentially rapid re-establishment of A T P levels, m a y therefore be by inhibition of the adenosine transport carrier [18, 27] rather than inhibition of the membrane-bound 5' nucleotidase enzyme. Further work is needed to evaluate this important possibility since conservation of the adenine pool appears to be synonymous with the post-ischemic recovery of functional and morphological integrity of the myocardium [9, 17, 24]. In conclusion, this investigation has shown that both the rates and the products of adenine nucleotide pool catabolism are highly dependent upon the specific type of oxygen deficiency (anoxia v. ischemia), however, in all cases the period of fastest metabolic flux was associated with the development of contracture.
Acknowledgements This study was supported by grants from the Medical Research Council of New Zealand and the National H e a r t Foundation of New Zealand. We gratefully acknowledge the assistance of Dr Lois Armiger, Miss Maree Morrison, and Mrs Gail van Veen in the preparation of this manuscript.
1136
S.M. Humphrey et al. References
1 BRICKNELL,O. L:, DARIES, P. S., OPIE, L. H. A relationship between adenosine triphosphate, glycolysis and ischaemic contracture in the isolated rat heart. J Mol Cell Cardio113, 941-945 ( 1981 ). 2 BURGER,R., LOWENSTEIN,J. M. Adenylate deaminase: III. Regulation ofdeamination pathways in extracts of rat heart and lung.J Biol Chem 242, 5281-5285 (1967). 3 FRANK,J. S., LANGER,G. A. The myocardial interstitium : its structure and its role in ionic exchange. J Cell Biol 60, 586-601 (1974). 4 HEARSE,D. J. Cardioplegia: the protection of the myocardium during open heart surgery: a review. J Physiol [Paris] 76, 751 768 (1980). 5 HEARSE,D.J., CHAIN, E. B. The role of glucose in the survival and 'recovery' of the anoxic isolated perfused rat heart. BiochemJ 128, 1125 1133 (1972). 6 HEARSE, D. J., GARLICK, P. B., HUMPHREY, S. M. Ischaemic contracture of myocardium. Mechanisms and prevention. AmJ Cardio139, 986-993 (1977). 7 HEARSE,D.J., STEWART,D. A. Functional recovery of the myocardium after elective cardiac arrest in the isolated rat heart. Lancet 1, 192 194 (1974). 8 HUMPHREY,S. M., GAVIN,J. B., HERDSON,P. B. The relationship ofischemic contracture to vascular reperfusion in the isolated rat heart.J Mol Cell Cardio112, 1397 1406 (1980). 9 HU~aVHREY,S. M., SEELYE, R. N. Improved functional recovery of ischemic myocardium by suppression of adenosine catabolism. J Thor Cardiovasc Surg 84, 16~22 (1982). 10 HUS~PHREY,S. M., THOMSON, R. W., GAVIN,J. B. The effect of an isovolumic left ventricle on the coronary vascular competence during reflow following global ischaemia in the rat heart. Circ Res 49, 786 791 ( 1981 ). 11 JARMAKANI,J., NAOATOMO,T., LANGER,G. A. The effect of calcium and high energy phosphate components on myocardial contracture in the newborn and adult rabbit.J Mol Cell Cardio110, 1017 1029 (1978). 12 JENNINGS,R. B., HAWKINS,H. K., LOWE,J. E., HILL, M. L., KLOTMAN,S., REIMER,K. A. Relation between high energy phosphate and lethal injury in myocardial ischemia in the dog. Am]" Patho192, 187-214 (1978). 13 JENNINGS,R. B., REIMER,K. A. Lethal myocardial ischemic injury. AmJ Patho1102, 241-255 (1981). 14 JENNINC;S,R. B., REIMER,K. A., HILL, M. L., MAYER,S. E. Total ischemia in dog hearts, in vitro I. Circ Res 49, 892-900 (1981). 15 KATORI,M., BERNE,R. M. Release of adenosine from anoxic hearts: Relationships to coronary flow. Circ Res 19, 420-425 (1966). 16 KLABUNDe,R. E., WINSER, C. L., Iro, C. S., MAYER, S. E. Measurement of adenosine and inosine in heart samples by high pressure liquid chromatography.J Mol Cell Cardiol 11,707 715 (1979), 17 KLONER,R. A., DEBOER,L. W. V., DARSEE,J. R., INOWALL,J. S., HALE, S., TUMAS,J., BRAUNWALD,E. Prolonged abnormalities of myocardium salvaged by reperfusion. Am J Physio1241, H591-H599 (1981 ). 18 KUBLER,W., SPIECKERMANN,P. G., BRETSCrINEIDER, H.J. Influence of dipyridamole (persantin) on myocardial adenosine metaboIism.J Mol Cell Cardiol I, 23 38 (1970). 19 LowE, J. E., JENNIN~S, R. B., REIMEV,,K. A. Cardiac rigor mortis in dogs. J Mol Cell Cardiol 11, 1017-1031 (1979). 20 NAYLER, W. G., POOLE-WILSON, P. A., WILLIAMS, A. Hypoxia and calcium. J Mol Cell Cardiol 11, 683-706 (1979). 21 NAtTO,Y., LOWENSTEIN,J. M. 5'nucleotidase from rat heart. Biochemistry 20, 5188-5194 (1981). 22 NEELY,J. R., ROVETTO, M. J., WHITMER,J. T., MORGAN,H. E. Effects ofischaemia on function and metabolism of the isolated working rat heart. AmJ Physio1225, 651-658 (1973). 23 OPIE, L. H. Effects of regional ischemia on metabolism of glucose and fatty acids. Relative rates of aerobic and anaerobic energy production during myocardial infarction and comparison with effects of anoxia. Circ Res 38, (Suppl 1), 52 74 (1976). 24 REmEL, D: K., ROVETTO, M. J. Myocardial adenosine salvage rates and restoration of ATP content following ischemia. AmJ Physio1237, H247-H252 (1979). 25 ROVETTO,M.J., WmTMER,J. T., NEELY,J. R. Comparison of the effects of anoxia and whole heart ischemia on carbohydrate utilization in isolated working rat hearts. Circ Res 32, 699 711 (1973). 26 Rumo, R., BERNE, R. M. Release of adenosine by the normal myocardium in dogs and its relationship to the regulation of coronary resistance. Circ Res 25, 407-415 (1969). 27 SCHUTZ,W., SCHRADER,J., GERLACH,E. Different sites of adenosine formation in the heart. Am J Physiol 240, H963-H970 (1981). 28 SEELYE,R. N., CARNELL,V. M., ARMIGER,L. C. A simple method for determining relative pH values and lactate levels in extracts of normal and ischemic or autolysing heart muscle. Biochem Med 16, 187-194 (1976). 29 VAN DeR LAARSE,A., GRAF-MINOR,M. L., WITTEVEEN,S.J. Release ofhypoxanthine from and enzyme depletion in rat heart cell cultures deprived of oxygen and metabolic substrates. Clin Chim Acta 91, 47 52 (1979). 30 VANDERWEE,n . A., HUMPHREY, S. M., GAVIN,J. B., ARMIGER,L. C. Changes in the contractile state, fine structure and metabolism of cardiac muscle ceils during the development of rigor mortis. Virchows Archly B (Cell Pathol) 35, 159 167 (1981). 31 VARY, T. C., ANGELAKOS,E. T., SHAFFER, S. W. Relationship between adenine nucleotide metabolism and irreversible ischemic tissue damage in isolated perfused rat hearts. Circ Res 45, 218 225 (1979).
A d e n i n e P o o l C a t a b o l i s m in the I s c h e m i c , t h e C a l c i u m D e p l e t e d I s c h e m i c , a n d t h e S u b s t r a t e Free A n o x i c I s o l a t e d R a t H e a r t : R e l a t i o n s h i p to C o n t r a c t u r e D e v e l o p m e n t S. M. Humphrey*, D. G. Holliss and R. N. Seelye Department of Pathology, University of Auckland Medical School, Auckland, New Zealand (Received 17 January 1984, accepted in revisedform 25 April 1984) S. M. HUMPHREY, D. G. HOLLISS and R. N. SEELYE. Adenine Pool Catabolism in the Ischemic, the CalciumDepleted Ischemic, and the Substrate Free Anoxic Isolated R a t Heart: Relationship to Contracture Development. Journal of Molecular and Cellular Cardiology (1984) 16, 1129-1138. Metabolic changes in the myocardial adenine and hypoxanthine pools of isolated rat hearts subjected to global ischemia, hypocalcemic global ischemia, and global substrate-free anoxia were compared. At timed intervals between 0 and 60 rain separate aliquots of extracts of the ventricles were used to determine either tissue pH, or the components of the adenine pool and their catabolites by reverse phase high performance liquid chromatography (HPLC). The coronary perfusate draining from anoxically perfused hearts was collected over perchloric acid, neutralised and chromatographed by HPLC. The development of left ventricular resting tension (contracture) was recorded in the three groups of hearts. After 60 min ischemia the major catabolites, (AMP, inosine and hypoxanthine) comprised 70% of the total pool (11, 7 and 4/2mol/g dry wt, respectively). After the same period of anoxia 50% of the total pool, comprising adenosine, inosine, hypoxanthine and uric acid in approximately equal proportions, was recovered from the coronary perfusate. The major products remaining in the tissue were IMP and, to a lesser extent A M P (8 and 5 #mol/g dry wt, respectively). Left ventricular contracture developed at different rates in the three groups of hearts but always correlated closely with the m a x i m u m rate of adenine pool catabolism. The loss of components from the tissue and the divergence in pathway from adenosine to I M P production which occurs during anoxic perfusion should possibly be considered when assessing the biochemical events occurring in regionally ischemic heart muscle with significant residual flow. KEY WORDS: Adenine nucleotides; Adenosine; Anoxia; Contracture; Inosine; Inosine monophosphate; Ischemia.
Introduction Myocardial ischemia which leads to the development of an infarct rarely involves a total cessation of blood flow to the affected area [4, 23]. For this reason, the relationship between loss of tissue high energy phosphate and the capacity of the myocardium to recover function has been evaluated after varying periods of both global ischemia (a total lack of flow and consequently of oxygen and nutrient) but also global anoxia (high flow but with similar lack of oxygen and nutrient) [5, 7, 9, 22]. However, a comprehensive study of adenine nucleotide catabolism with its associated increase in the components of the hypo-
xanthine pool, utilising the new and accurate technique of high performance liquid chromatography (HPLC), has been described only for the zero-flow ischemic situation [13, 14]. No similar H P L C study of adenine nucleotide loss for substrate-free anoxia has appeared in the literature. The available data relating to this situation are scattered and incomplete
[15, 25, 29]. An apparent lack of information also exists in relation to the association between high energy phosphate depletion and the deveiopment of contracture in substrate-free anoxia, although a close association has been described for ischemic hearts [6, 11, 19].
* To whom correspondence should be addressed at the Department of Pathology, University of Auckland Medical School, Private Bag, Auckland, New Zealand. 0022 2828/84/121127 + i0 $03.00/0
9 1984 Academic Press Inc. (London) Limited
1128
S.M. Humphrey et aL
This paper describes a comprehensive study undertaken to compare the effects of 60 min each of global substrate-free anoxia and ischemia on the metabolite changes (determined by H P L C ) and the associated changes in contracture in the isolated rat heart. The findings confirm that the maximum rate of high energy phosphate decline parallels the time course of contracture development in anoxia and in ischemia. However, the pattern of change in the various adenine pool catabolites differs considerably in the two situations.
15, 20, 30, 40 and 60 min of anoxia or ischemia (six hearts for each time interval).
M a t e r i a l s and M e t h o d s
Contracture measurement In each group, the development of contracture was measured by recording the pressure exerted on a balloon which was inflated with normal saline to just fill the left ventricular lumen. T h e balloon, which was inserted into the left ventricle via the left atrium prior to the commencement of either anoxia or ischemia was connected via the open end to a pressure transducer (Bell and Howell 4-422) and chart recorder as described previously [8, 10].
Male albino Wistar rats (280 to 320 g) in three groups of 48 were each lightly anesthetized with ether and injected with 200 I U of heparin via the femoral vein. Each heart was quickly excised and placed in ice-cold perfusion solution to arrest beating. The aorta was attached to a stainless steel cannula and the heart subjected to a non-recirculating Langendorff perfusion. All hearts were maintained at 37~ in a water-jacketed chamber and perfused for a 10-min equilibration period at a pressure of 100 cm H 2 0 with Krebs-Henseleit bicarbonate buffer (KHB) which contained 11 mt~ glucose. The perfusion fluid was gassed continuously with a mixture of 95% Ozand 5% CO 2. At the end of the equilibration period hearts from the first group were made globally anoxic by switching to a glucose-free perfusion fluid (KHB) maintained at a pressure of 100 cm HzO and gassed continuously with a mixture of 95% N 2 and 5% CO2. Hearts from the second group were made globally ischemic by clamping the aortic cannulae. In the third group (calcium-depleted ischemia), hearts were perfused for a further 10 min normoxic period with K H B in which the calcium content had been reduced from 2.5 mM to 0.05 m~ before being made globally ischemic as previously described for group I. The experimental period of anoxia or ischemia was maintained for 60 min during which time the development of contracture was recorded in six hearts from each group. Tissue p H and metabolite assays were performed on the remaining hearts, which were freeze-clamped in liquid nitrogen after 5, 10,
Frozen tissue samples were powdered in a percussion mortar maintained at the temperature of liquid nitrogen. For each heart three accurately weighed aliquots of powder were prepared: one was dried in an oven at 80~ for 24 h to obtain the tissue wet/dry ratio, whilst the other two were dispersed (100 mg/ml) in either ice-cold 6% perchloric acid (PCA), neutralized and centrifuged (4~ at 2500 g for metabolite determinations, or neutral 2 m~ iodoacetate for pH measurements by a method described in detail previously [28]. Ten microlitre samples of the neutralized PCA supernatant were analysed by high performance liquid chromatography using a reverse phase column (Zorbax ODS, 7 pm, Dupont Instruments, Wilmington, DE, USA) and a 254-nm wavelength detector to separate and quantitate successively, uric acid, inosine monophosphate (IMP), adenosine tri-, di-, and mono-phosphates (ATP), (ADP), (AMP), hypoxanthine, inosine and adenosine. Solvent A was an aqueous solution containing K H 2 P O 4 (0.022M), MgSO 4 (0.004M) and Trishydroxy methyl-aminomethane (TRIS) (0.004M) adjusted to pH 6.25. Solvent B was methanol : water 60 : 40. All metabolites were eluted within 20 min using a linear gradient of 3% to 80% Solvent B. The results are expressed throughout as #moles per gramme dry weight (/~mol/g dry wt). For the anoxically perfused group of hearts, in addition to tissue extracts, the coronary effluents were similarly assayed for adenine pool catabolites. Measured volumes of coro-
Metabolite measurement
Adenine Pool Catabolism
nary fluid draining from the right side of the heart were collected at timed intervals into ice-cold 60% PCA. The fluid was adjusted to a final concentration of 6% P C A before neutralization and c h r o m a t o g r a p h y as described above.
Results
T h e development of contracture in the three groups of hearts is shown in Figure l. In anoxic hearts, contracture began within 1 to 2 min of the onset of anoxia and rapidly increased to its m a x i m u m pressure of 50 4- 6 m m H g (mean __ SEM) by 6 min. In ischemic hearts the onset ofcontracture was delayed for 5 to 8 rain, developed at a rate slower than for anoxic hearts and reached a m a x i m u m level of only 30 + 3 m m H g by 30 min (Fig. 1). I n the calcium-depleted ischemic group, contracture did not develop to a measurable extent until 15 to 20 min of ischemia had elapsed. It then increased at a similar rate (in terms of percent of m a x i m u m per minute) to that seen in normo-calcemic ischemic hearts but reached a m a x i m u m pressure of only 8 +__2 m m H g . T h e results of the metabolite measurements are tabulated (Table 1). I n the two ischemic groups of hearts the total pool size did not alter significantly throughout the experiment (31.2 _+ 0.9 #mol/g dry wt for 96 hearts). In
50
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I0 O0
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:50
40
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FIGURE 1. Graph showing the development of increasing resting tension (contracture) in hearts during 60 min of (a) global anoxia, (b) total global ischemia, and (c) total global ischemia after pre-ischemic perfusion with 0.05 mM calcium buffer. Values represent the means of six hearts and bars the standard errors of the means (S.E.M.).
and Contracture
I 129
the anoxic group, however, the total pool increased gradually during the 60-min experiment from 31.4 to 37.0. Since extremely high volumes (300 to 400 ml per heart) of coronary effluent fluid were collected during 60 min anoxia, the error associated with assaying the adenine pool catabolites from the perfusate was greater than that from the tissue assays. To allow for this, the rates of depletion and accumulation of metabolites were calculated from values normalized to the control value of 31.4 #mol/g dry wt. A comparison of the accumulation of adenine pool catabolites in the coronary fluid with their complementary tissue content during 60 rain anoxia is shown in Figure 2. Both adenosine and inosine were released at a constant rate of approximately 0.15 #mol/g dry wt/min for the first 30 min [Fig. 2(a)]. H y p o x a n t h i n e and uric acid accumulated at slower rates namely, 0.11, and 0.08 #mol/g dry wt/min respectively. Following 30 min of anoxia there was little change in either adenosine or uric acid, and only small increases in inosine and hypoxanthine during the period 30 to 60 min. This is reflected in the plot of total tissue metabolites removed from, or remaining in, the heart during anoxia [Fig. 2(b)]; more than 80% of the total change occurred during the first 30 min. Figure 3 depicts a composite of the changes in the major metabolites that occurred during 60 min of oxygen and nutrient depletion in all three groups of hearts. During the first 10 min of anoxia, [Fig. 3(a)] there was a rapid fall in the tissue content of combined (ATP + ADP) with a m a x i m u m rate of approx. 3 #mol/g dry wt/min. Associated with this fall were the m a x i m u m rates of accumulation of A M P , I M P , adenosine and [ H I ( [ H I = the combined values of inosine, hypoxanthine and Uric Acid) which were 2.0, 0.5, 0.8 and 0.5 #mol/g dry wt/min respectively. This phase of rapid change correlated with the development of contracture pressure (Fig. 4, Table 2), Between 10 and 30 min, the (ATP + ADP) moiety declined more slowly (0.2 #mol/g dry wt/min). T h e A M P level fell rapidly during this stage (0.5 #mol/g dry wt/min) and the [ H ] pool continued to accumulate at a similar rate. I M P also continued to accumulate, in contrast to adenosine which remained at a constant value for the remaining period of
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subjected to 60 min of anoxia. (a) The solid line represents components accumulating in the tissue and the dotted line those removed in the coronary perfusate. (b) Changes in the level of the total adenine and hypoxanthine pools (ATP, ADP, AMP, adenosine, IMP, inosine, hypoxanthine and uric acid) in the tissue (O) and in the perfusate (0) during 60 rain anoxia. Values represent the means of six hearts and the bars the S.E.M. anoxia. After 30 min of anoxia the overall change in tissue metabolite level was small [Fig. 2(a)]. The rate of A M P decline was complementary to the rate of I M P accumulation during this period (0.09 #mol/g dry wt/min) and by 60 min the I M P reached 7 #mol/g dry wt to become the highest single component of the total pool. In the ischemic group of hearts the first approximately 15 min was associated with very little change [Fig. 3(b)]. This was followed by a rapid change in all moieties,
chiefly (ATP + ADP) decline (0.73 #mol/g dry wt/min) and associated rises in AMP, Adenosine and [-H] (0.54, 0.40 and 0.18 #mol/g dry wt/min respectively). In these hearts I M P did not rise by more than 2 #mol/g dry wt throughout 60 rain ofischemia. Again, the phase of rapid change (15 to 30 min) coincided with the development of contracture. Thereafter both the A M P and adenosine moieties declined from their peak value and the (ATP + ADP) and [ H I curves levelled off.
1132
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F I G U R E 3. Accumulation of the products of adenine pool catabolism in rat hearts during 60 min of (a) global anoxia, (b) total global ischemia and (c) total global ischemia after pre-ischemic perfusion with 0.05 mM calcium buffer. O , A M P ; I , [H] (sum ofinosine, hypoxanthine and uric acid); &, adenosine; 9 IMP; A T P 4-ADP. Values represent the means of six hearts and the bars the S.E.M.
(ATP + ADP) had not yet reached the low values observed for the other two groups. Results of plots relating increments in the -6 E 12 =L force of contracture to the metabolic tissue content of ATP, AMP, adenosine, and inosine at corresponding times for each of the three groups is shown in Figure 4. The tissue content of metabolites continued to change after contracture was completed particularly in the anoxic group (Fig. 4). However in all three groups of hearts a close correlation exists lO 20 30 40 50 60 between contracture and the fluxes of the Time(rain) adenine nucleotides, adenosine and inosine as The calcium-depleted ischemic group of indicated by a Pearson coefficient of correlahearts [Fig. 3(c)] showed a similar but tion (Table 2) performed on the data repextended first period of minor change fol- resented schematically in Figure 4. The change in the tissue pH during 60 lowed by a phase of rapid flux coincident with the development of contracture. The rates of minutes of anoxia and ischemia is shown in fall of (ATP + ADP) (0.55) and concomitant Figure 5. The continuously perfused hearts rises in AMP (0.42) adenosine (0.30) and [ H I maintained a pH of 7.36 throughout anoxia (0.13) were similar to those in the nor- whereas the ischemic hearts showed a rapid mocalcemic ischemic group. After the rapid early decline during the first 10 to 15 min phase in this group there was no further to pH 6.6, followed by a slower decrease to pH change in the level of adenosine and AMP 6.4 by 30 to 40 min of ischemia. Therevalues. [H] continued to rise and the after, the pH did not alter significantly.
A d e n i n e Pool C a t a b o l i s m and Contracture
1133
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Discussion The results of this investigation clearly indicate that, in both anoxically perfused and globally ischemic rat heart preparations, there is a very close association between the rapid phase of adenine pool catabolism and the development of contracture. This holds despite the existence of a 15-min difference in onset of the rapid phase under the two conditions. Delaying the onset and development of contracture by pre-perfusion with the 0.05 mM calcium solution delays proportionally the loss of (ATP + ADP) and the rise in AMP, adenosine, inosine, hypoxanthine and uric acid. The preservation of A T P and delay in the onset of contracture in the low-calcium group
may have been related to the cardioplegic effect of reduced calcium. Certainly contractility is stopped immediately subsequent to ischemia in this group whereas it continues for 1 to 2 min i n the normocalcemic ischemic hearts. This results in a reduced A T P consumption (1 ktmol/g dry wt/beat) during the first minutes of ischemia, however the tissue content of A T P remains essentially similar in the two ischemic groups even after 10 min. It is more likely therefore that the reduction of extracellular calcium availability conserves ATP by reducing calcium-mediated A T P hydrolysis associated with contracture. The lower coefficient of correlation observed for the anoxic group of hearts (Table 2) suggests that anoxic contracture may not
1134
S . M . Humphrey et aL (01 7.2
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Yimo(min) FIGURE 5. Graph showing the changes in tissue acidity of rat hearts during 60 min of (a) anoxia, (b) total global ischemia and (c) total global ischemia after pre-ischemic perfusion with 0.05 mM calcium buffer. Values represent the means of six hearts and the bars the S.E.M. be directly c o m p a r a b l e to ischemic contracture. W h e r e a s ischemic c o n t r a c t u r e p r o b a b l y represents the a c c u m u l a t i o n of rigor complexes arising as a result of A T P deficiency [_6, 11], i m p a i r e d calcium homeostasis m a y be the p r i m a r y cause of the early c o n t r a c t u r e in a n o x i a [20]. I n the latter case, however, this intracellular calcium-overload-contracture w o u l d r a p i d l y convert to r i g o r - c o n t r a c t u r e consequent upon A T P depletion which occurs within 3 to 5 rain of p e a k contracture. T h e p r o d u c t i o n and fate of A M P in the three groups is of interest. D u r i n g ischemia, the relatively small c h a n g e in A M P level prior to c o n t r a c t u r e indicates t h a t the catabolism of A T P proceeds u n i n h i b i t e d to adenosine. T h e r a p i d a c c u m u l a t i o n of A M P associated with
c o n t r a c t u r e d e v e l o p m e n t is therefore likely to be related to the m e t a b o l i c events taking place at the myofibrils. T h e depletion of creatine p h o s p h a t e early in ischemia and anoxia [_6, 19] restricts A D P to r e p h o s p h o r y l a t i o n (by the action of myokinase) at the expense of e q u i v a l e n t A M P production. T h e continuous cycling of this reaction will result in the r a p i d a c c u m u l a t i o n of only A M P . T h e a c c u m u l a tion of A M P , r a t h e r t h a n its d e p h o s p h o r y l a tion to adenosine, is most likely related to a shift of the r a t e - d e t e r m i n i n g step from adenosine d e a m i n a s e to 5'nucleotidase. This could be due simply to rate of p r o d u c t i o n being in excess of rate of d e p h o s p h o r y l a t i o n . I n a c tivation of the enzyme as tissue p H falls does not seem likely since the greatest decline in
TABLE 2. Relationship between the development of contracture and the tissue content of selected adenine pool constituents in the three groups of hearts. Correlation coefficient (r) Condition
ATP
AMP
Anoxia Ischemia Ca 2 +-Depleted Ischemia
0.88 0.98 0.94
0.80 a 0.98 0.93
Adenosine
Inosine
0.76 0.94 0.97
0.54 0.90 0.95
a This value relates only to the increase in the tissue content of
Adenine Pool Catabolism
pH occurred during the first 15 min of ischemia (Fig. 5) when A M P did not accumulate. Moreover, in all cases, the production of hypoxanthine pool metabolites appeared remarkably constant throughout ischemia and during the first 30 min of anoxia (by which :time nucleotide fluxes were 95% complete). Thus, the accumulation and depIetion of A M P did not appear to influence greatly the overall catabolic flux. During anoxia, the decline in A M P was equivalent to the production of IMP. The deamination of A M P to I M P rather than its dephosphorylation to adenosine has not previously been thought to be a n important pathway in heart tissue. The production of adenosine, and not IMP, is favoured during ischemia because decreasing A T P concentra+ tions will increase 5' nucleotidase activity [21] and decrease adenylate deaminase activity [2]. However, the factors involved in the regulation of adenylate deaminase activity appear to be altered during anoxia. We believe, therefore, that these results relating to AMP production and decay provide further support for the compartmentation of an A T P pool associated with the myofibrillar apparatus [1]. The AMP produced from this pool by the rapid actin/ myosin interaction during contracture development is either converted to I M P if flow is sufficient to minimise pH change, or remains fairly constant if flow is absent. Although it is known that there is an approximately 7-fold increase in A M P [12, 31] during ischemia its correlation with contracture has not been noted. The hitherto unexplained markedly late rise in A M P level (60 to 120 min) observed in dog heart slices made ischemic in vitro [12, 13] can be explained by its association with the development of contracture. This late A M P accumulation occurs long after the most rapid decline in A T P tissue content, but in precise agreement with the late time of contracture in this model [30]. The improved analytical technique of H P L C has led to much higher estimates of the cardiac adenine nucleotide pool catabolites. Values for normal cardiac adenosine content, for instance, have increased from 0~2 to 0.3 nmol/g [26] to 10 to 20 nmol/g [16]. Our control values for adenosine and inosine were similar in range to the results of Klabunde
and Contracture
1135
and colleagues [16]. However, these values may be elevated above in situ rat heart concentrations, owing to the unphysiological control conditions of the Langendorff perfusion technique. During the first 15 min of anoxia adenosine showed a significant accumulation in the tissue. Although it is not possible to determine precisely the intra- and extra-cellular concentration of adenosine from the results of these experiments, if the adenosine resided solely in the interstitial compartment (0.2 ml/g [3]) it would approximate to 2 to 3 #mol/ml. This would be over 1000 times the concentration in the perfusate (coronary flow rate was 12 ml/min after 15 min of anoxia) and yet its efflux into the coronary perfusate was the same as that for inosine which had a considerably lower tissue content. Thus, it is likely that the rapid accumulation of adenosine during early anoxia occurs within the cell and this adds support to the finding of Schutz et al. [27] that adenosine is predominantly formed intracellularly in the hypoxic heart. The most effective method of conserving the cellular adenine pool, and consequently the potentially rapid re-establishment of A T P levels, m a y therefore be by inhibition of the adenosine transport carrier [18, 27] rather than inhibition of the membrane-bound 5' nucleotidase enzyme. Further work is needed to evaluate this important possibility since conservation of the adenine pool appears to be synonymous with the post-ischemic recovery of functional and morphological integrity of the myocardium [9, 17, 24]. In conclusion, this investigation has shown that both the rates and the products of adenine nucleotide pool catabolism are highly dependent upon the specific type of oxygen deficiency (anoxia v. ischemia), however, in all cases the period of fastest metabolic flux was associated with the development of contracture.
Acknowledgements This study was supported by grants from the Medical Research Council of New Zealand and the National H e a r t Foundation of New Zealand. We gratefully acknowledge the assistance of Dr Lois Armiger, Miss Maree Morrison, and Mrs Gail van Veen in the preparation of this manuscript.
1136
S.M. Humphrey et al. References
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