5′-Adenosine monophosphate and adenosine metabolism, and adenosine responses in mouse, rat and guinea pig heart

Comparative Biochemistry and Physiology Part A 130 Ž2001. 615᎐631

5⬘-Adenosine monophosphate and adenosine metabolism, and adenosine responses in mouse, rat and guinea pig heart John P. Headrick a,U , Jason Peart a , Ben Hack a , Bronwyn Garnham a , G. Paul Matherne b b

a NHF Research Centre, Griffith Uni¨ ersity Gold Coast Campus, Southport Queensland, Australia Department of Pediatrics and the Cardio¨ ascular Research Center, Uni¨ ersity of Virginia Health Sciences Center, Charlottes¨ ille, VA, USA

Received 30 October 2000; received in revised form 16 May 2001; accepted 30 May 2001

Abstract We examined myocardial 5⬘-adenosine monophosphate Ž5⬘-AMP. catabolism, adenosine salvage and adenosine responses in perfused guinea pig, rat and mouse heart. V O 2 increased from 71 " 8 ␮l O 2rmin per g in guinea pig to 138 " 17 and 221 " 15 ␮l O 2rmin per g in rat and mouse. V O 2rbeat was 0.42" 0.03, 0.50" 0.03 and 0.55" 0.04 ␮l O 2rg in guinea pig, rat and mouse, respectively. Resting and peak coronary flows were highest in mouse vs. rat and guinea pig, and peak ventricular pressures and Ca2q sensitivity declined as heart mass increased. Net myocardial 5⬘-AMP dephosphorylation increased significantly as mass declined Ž3.8" 0.5, 9.0" 1.4 and 11.0" 1.6 nmolrmin per g in guinea pig, rat and mouse, respectively.. Despite increased 5⬘-AMP catabolism, coronary venous wadenosinex was similar in guinea pig, rat and mouse Ž45 " 8, 69 " 10 and 57 " 14 nM, respectively.. Comparable venous wadenosinex was achieved by increased salvage vs. deamination: 64%, 41% and 39% of adenosine formed was rephosphorylated while 23%, 46%, and 50% was deaminated in mouse, rat and guinea pig, respectively. Moreover, only 35᎐45% of inosine and its catabolites derive from 5⬘-AMP Žvs. IMP. dephosphorylation in all species. Although post-ischemic purine loss was low in mouse Ždue to these adaptations., functional tolerance to ischemia decreased with heart mass. Cardiovascular sensitivity to adenosine also differed between species, with A 1 receptor sensitivity being greatest in mouse while A 2 sensitivity was greatest in guinea pig. In summary: Ži. cardiac 5⬘-AMP dephosphorylation, V O 2 , contractility and Ca2q sensitivity all increase as heart mass falls; Žii. adaptations in adenosine salvage vs. deamination limit purine loss and yield similar adenosine levels across species; Žiii. ischemic tolerance declines with heart mass; and Živ. cardiovascular sensitivity to adenosine varies, with increasing A 2 sensitivity relative to A 1 sensitivity in larger hearts. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Adenosine; Adenosine kinase; Adenosine deaminase; Ischemia; 5⬘-Nucleotidase; Purines; Metabolism; Myocardium

U

Corresponding author. Tel.: q61-7-5594-8292; fax: q61-7-5594-8908. E-mail address: [email protected] ŽJ.P. Headrick..

1095-6433r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 1 . 0 0 3 8 0 - 4

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1. Introduction Based upon well established principles of allometric scaling ŽEmmett and Hochachka, 1981; Pietschmann et al., 1982; Schmidt-Nielsen, 1984; Weibel et al., 1991; Dobson and Headrick, 1995; West et al., 1997., mass specific metabolic rate increases as body size decreases. Higher mass specific metabolic rates in smaller mammals necessitates higher cardiac output which is largely accomplished by allometric scaling of heart rate ŽSchmidt-Nielsen, 1984; Hamilton and Ianuzzo, 1991; Weibel et al., 1991; West et al., 1997.. The associated elevations in myocardial ATP demand must be compensated for by increased mitochondrial density ŽHoppeler and Kayar, 1988; Barth et al., 1992; Kim et al., 1994., oxidative and potentially glycolytic enzyme levels ŽCrabtree and Newsholme, 1972; Emmett and Hochachka, 1981; Blank et al., 1989; Burness et al., 1999.. The mechanisms responsible for this regulation of enzyme levels in response to tissue mass and metabolic rate are unclear. Enzyme synthesis can be translationally and post-translationally regulated ŽBurness et al., 1999., and chronic changes in metabolic activity ŽOrnatsky et al., 1995. and factors including O 2 availability ŽHochachka et al., 1997, 1998. may play a role. Given higher metabolic rates and oxidative enzyme levels in smaller hearts one might predict adaptations in the enzymes responsible for handling of adenine nucleotides and, importantly, the ubiquitous signaling compound adenosine. While adenine nucleotide and adenosine metabolism are crucial to myocardial homeostasis and to survival during and following ischemic or hypoxic insult, few have investigated the impact of body or heart mass on purine metabolism and physiology. In an early study Arch and Newsholme found that total myocardial activities of 5⬘-nucleotidase and adenosine deaminase were lower and adenosine kinase higher in mouse vs. rat ŽArch and Newsholme, 1978.. Such a difference might facilitate purine moiety preservation in the face of elevated mass specific metabolic rate, and may impact significantly on cardiac function and purine levels during ischemic or hypoxic insult. However, it is not known whether these differences in total enzyme activities, assessed in vitro, translate into differences in metabolic fluxes in vivo. The primary goal of the present study was to test whether myocardial 5⬘-AMP catabolism is

modified andror whether adenosine salvage is enhanced as heart mass declines and MV O 2 increases in hearts from three mammalian species of different sizes. In addition, we examined the relationship between metabolic rate, endogenous adenosine levels and cardiovascular sensitivity to adenosine. Based on the concept of symmorphosis ŽWeibel et al., 1991., sequential levels within the cardiovascular system should be functionally and structurally matched to each other and to metabolic rate. Increased mass specific myocardial metabolism must be matched by enhanced O 2 delivery which, in turn, can be met by enhanced O 2 transport by blood cells ŽWeibel et al., 1991., increased blood velocity ŽChen and Kaul, 1994., andror alterations in coronary vascular structure and function ŽPietschmann et al., 1982; Rakusan and Tomanek, 1986.. Since coronary vascular tone is responsive to extracellular adenosine, we wished to test the possibility that vascular responses to adenosine are modified in relation to myocardial metabolic rate in the three species studied.

2. Materials and methods 2.1. Langendorff-perfused mouse, rat and guinea pig hearts The following investigations conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health ŽNIH Publication No. 85-23, revised 1996.. Hearts were isolated from 7᎐12-week-old male and female wild-type C57rBL6 mice, Wistar rats and Hartley guinea-pigs anesthetized with sodium pentobarbital Ž60 mgrkg. administered intraperitoneally, as described previously ŽHeadrick et al., 1991, 1998a,b, 2000; Headrick, 1996a,b; Gauthier et al., 1998.. A thoracotomy was performed and hearts rapidly excised into ice-cold perfusion fluid. The aorta was rapidly cannulated and the coronary circulation perfused with modified Krebs᎐Henseleit buffer containing Žin mM.: NaCl, 120; NaHCO3 , 25; KCl, 4.7; CaCl 2 , 2.5; MgCl 2 1.2; KH 2 PO4 1.2, glucose, 15; and EDTA, 0.5. Unless otherwise stated all hearts were perfused at an aortic pressure of 90 mmHg. The perfusate was equilibrated with 95% O 2 , 5% CO 2 at 37⬚C to give a pH of 7.4 and PO 2 of ; 600 mmHg at the aortic cannula. Perfusate was fil-

J.P. Headrick et al. r Comparati¨ e Biochemistry and Physiology Part A 130 (2001) 615᎐631

tered Ž5.0 ␮m. immediately after preparation and was passed through an in-line 0.450 ␮m SterivexHV filter cartridge ŽMillipore, Bedford, MA, USA. in the perfusion apparatus to continuously remove microparticulates. Left ventricles were vented with polyethylene apical drains and hearts were instrumented for functional measurements as described below. The hearts were then immersed in warmed perfusate inside a waterjacketed bath maintained at 37⬚C. The temperature of coronary perfusion fluid was continuously assessed by a needle thermistor located at the entry into the aortic cannula, and the temperature of the water bath assessed using a second thermistor probe. Temperatures were recorded using a three-channel Physitemp TH-8 digital thermometer ŽPhysitemp Instruments Inc, Clifton, NJ, USA.. 2.2. Measurement of contractile function and MVO2 For assessment of isovolumic contractile function fluid-filled balloons constructed of polyvinyl chloride plastic film Žfor mice. and thin-walled latex Žfor rats and guinea pigs. were inserted into the left ventricle via the mitral valve. Balloons were connected to a P23 XL pressure transducer ŽViggo-Spectramed, Oxnard, CA, USA. by a fluid-filled polyethylene tube, permitting continuous measurement of left ventricular pressure. Balloon volume was increased to give end-diastolic pressures of ; 6 mmHg in all species. Coronary flow was continuously monitored via a cannulating Doppler flow-probe ŽTransonic Systems Inc, Ithaca, NY, USA. located in the aortic perfusion lines and connected to a T206 flowmeter ŽTransonic Systems Inc, Ithaca, NY, USA.. All functional data Žventricular and aortic pressures, coronary flow. were recorded at a sampling speed of 1 kHz on a 4rs MacLab data acquisition system ŽAD Instruments, Castle Hill, Australia. connected to an Apple 7300r180 computer. Ventricular pressure signals were digitally processed to yield peak systolic pressure, diastolic pressure, "d Prdt and heart rate. To estimate myocardial oxygen consumption ŽMV O 2 . cannulas constructed of polyethylene tubing were tied in place in the pulmonary artery in a sub-set of hearts from each species Ž n s 6 per species., as we have described previously ŽHeadrick, 1996b; Headrick et al., 1998b.. Arte-

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rial Žperfusate. and venous Žpulmonary arterial . fluid samples were acquired in gas-tight syringes and PO 2 values immediately measured on a Corning 278 blood-gas analyzer. MV O 2 was then calculated as: MVO 2 Ž ml O 2 r min per g. s Ž PO 2a y PO 2v . =coronary flow Ž ml r min per g. = cr760 where PO 2a and PO 2v are partial pressures of O 2 in arterial and venous effluent samples ŽmmHg., respectively, and c is the solubility coefficient for O 2 in Krebs Ž22.7 ␮l O 2ratm per ml at 37⬚C.. 2.3. Measurement of ¨ enous metabolites Coronary venous effluent was sampled and frozen at y80⬚C until analyzed by HPLC for adenosine, inosine, hypoxanthine, xanthine and urate as outlined by us previously ŽHeadrick, 1996a,b.. Normoxic efflux was calculated as the product of coronary flow Žmlrmin per g dry weight. = effluent concentrations Žnmolrml. whereas post-ischemic efflux was calculated as the product of concentration in the reperfusion effluent Žnmolrml. = reperfusion volume Žmlrg wet weight.. 2.4. Measurement of myocardial metabolites For determination of intracellular metabolic status total myocardial ATP, phosphocreatine ŽPCr., creatine, ADP and 5⬘-AMP levels were determined in hearts freeze-clamped in tongs cooled in liquid N2 Ž n s 7 for all species.. Powdered frozen tissue was extracted in 0.6 M perchloric acid and neutralized samples analyzed by HPLC as outlined by us in detail previously ŽHeadrick et al., 1991, 1998a,b; Dobson and Headrick, 1995; Headrick, 1996a,b.. All concentrations were expressed per gram wet weight. To assess differences in metabolically relevant free cytosolic levels of energy metabolites, a series of 31 P-NMR experiments were undertaken. Mouse, rat and guinea-pig hearts were perfused as already described and located within the bore of a 7 Tesla NMR magnet and 31 P-spectral data acquired as described by us previously. Briefly, consecutive 31 P-spectra were acquired at 121.47 MHz using a 90⬚ RF pulse applied every 2.0 s. Spectral

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width was 4 kHz and a total of 4000 data points were obtained. Individual spectra consisted of 160 signal averaged free-induction decays ŽFIDs. acquired over consecutive 5-min periods. FIDs were multiplied by a 25-Hz line broadening factor to improve spectral signal-to noise. 31 P-Spectral intensities were determined by integration and corrected for partial relaxation using spin-lattice relaxation times ŽT1 s. for ␤-ATP, phosphocreatine ŽPCr. and inorganic phosphate ŽPi . determined by us previously for guinea pig, rat and mouse hearts ŽHeadrick et al., 1991, 1998a,b; Dobson and Headrick, 1995; Headrick, 1996a,b.. To convert spectral intensities to metabolite concentrations, the total tissue ATP concentration determined in freeze-clamped tissues Žsee above. was assigned to the saturation corrected ␤-ATP intensity in baseline spectra. All saturation corrected intensities were then normalized against this ATP concentrationrintensity ratio ŽDobson and Headrick, 1995; Headrick, 1996a,b; Headrick et al., 1998a,b.. Intracellular pH ŽpH i . and free cytosolic Mg 2q ŽwMg 2q x i . were calculated from the chemical shifts of Pi relative to PCr, and the shifts of the ␣-P and ␤-P resonances of ATP as described in detail by us previously ŽDobson and Headrick, 1995; Headrick et al., 1998b.. Free cytosolic wADPx, w5⬘-AMPx and the phosphorylation ratio ŽwATPxrwADPx = wPi x. cannot be directly measured and are estimated from creatine kinase and adenylate kinase equilibria ŽDobson and Headrick, 1995; Headrick et al., 1998a,b.. Cytosolic wADPx was calculated as: w ADPx s Žw Cr x = w ATPx. r Žw PCr x = K ⬘ck . where wCrx was determined by subtraction of PCr from the total chemically measured creatine pool, and K ⬘ck is the observed equilibrium constant for creatine kinase, adjusted for measured pH i and wMg 2q x i as outlined in detail previously ŽDobson and Headrick, 1995; Headrick et al., 1998b.. Free cytosolic w5⬘-AMPx was calculated from the adenylate kinase equilibrium: w 5⬘ - AMPx s K ⬘ak = w ADPx 2r w ATPx where the observed equilibrium constant for adenylate kinase Ž K ⬘ak . was also adjusted for measured pH i and wMg 2q x i ŽDobson and Headrick, 1995; Headrick et al., 1998b.. The phosphorylation ratio ŽwATPxrwADPx = wPi x. was calcu-

lated from the free cytosolic wADPx and the 31 PNMR determined wATPxrwPi x ratio. The phosphorylation ratio was converted to mM units based on an estimated cytosolic volume for perfused myocardium of 0.47 mlrg wet weight ŽHeadrick et al., 1998b.. 2.5. Determination of hematocrit, red blood cell count and hemoglobin content Freshly drawn blood samples were collected by cardiac puncture from anesthetized animals. Hematocrit was estimated following centrifugation at 15 000 = g for 5 min. Red blood cell counts were made under a light microscope using a hemocytometer. Hemoglobin content was measured via a kit from Sigma Chemicals ŽSt Louis. employing a cyanmethemoglobin detection technique. Mean cell volume Ž␮l. was then determined as: hematocritrred blood cell count Ž10 6 . = 10. Mean cell hemoglobin concentration Ž%. was determined as: hemoglobin contentr hematocrit = 100. 2.6. Experimental protocol Normoxic metabolite levels, enzyme activities and purine efflux were assessed in mouse and rat hearts stabilized under aerobic conditions for a period of 30 min. To examine the impact of ischemia-reperfusion hearts were subjected to 20 min of global normothermic ischemia followed by 30 min of aerobic reperfusion. The Ca2q sensitivity of left ventricular contractile function was assessed in groups of mouse Ž n s 8. rat Ž n s 7. and guinea pig hearts Ž n s 7. initially stabilized for 30 min with control perfusate containing 2.5 mM total Ca2q and 0.5 mM EDTA. Hearts were then switched to perfusion with perfusate containing 3.50, 1.75, 1.25 and 0.75 mM total Ca2q for periods of 5 min. Free extracellular Ca2q values for each total Ca2q Žand 1.2 mM total Mg 2q . were calculated based on the method outlined by Fabiato Ž1988., employing log association constants of 7.7 for Ca2q and 5.8 for Mg 2q at a pH of 7.4 ŽDawson et al., 1991.. Contractile function was measured at the end of each 5-min period. Heart rate was stable over these protocols and is not reported. Perfusate wCa2q x required to produce 50% maximal activation of systolic pressure ŽEC 50 . was determined from individual concentration᎐response data Žexpressed as absolute

J.P. Headrick et al. r Comparati¨ e Biochemistry and Physiology Part A 130 (2001) 615᎐631

units. by fitting the following three-parameter logistic equation to data from each experiment: Developed pressure sAy

A 1 q Ž w Ca

2q x

r EC 50 .

slope factor

where A is the maximal response Žat infinite dose.. The equation was fit to raw data using the Statistica data analysis program ŽStatsoft, Tulsa, OK, USA., and individual EC 50 values derived from each fit. In order to determine absolute rates of myocardial adenosine formation in non-noxic myocardium, and to assess the relative activities of adenosine phosphorylation vs. deamination, groups of mouse Ž n s 6. rat Ž n s 7. and guinea pig hearts Ž n s 7. were perfused under aerobic conditions for 20 min after which they were switched to ventricular pacing slightly above the intrinsic rate observed in control hearts Ži.e. at 400 beatsrmin for mouse, 280 beatsrmin for rat, 210 beatsrmin in guinea pig.. After a further 10 min of stabilization at these rates the hearts were treated with 20 ␮M EHNA and 5 ␮M iodotubercidin to simultaneously block adenosine deaminase and adenosine kinase, respectively. After 10 min of drug infusion venous effluent samples were acquired and analyzed for purine catabolites. In order to verify approximate rates of adenosine deamination vs. dephosphorylation, a similar experiment was carried out with infusion of 20 ␮M EHNA alone in mouse Ž n s 8., rat Ž n s 7. and guinea pig hearts Ž n s 8.. Pacing was necessary in these studies as preliminary experiments revealed that heart rate dropped markedly upon treatment with EHNA and iodotubercidin, likely as a result of enhanced adenosine levels. EHNA and iodotubercidin are commonly employed to assess absolute rates of adenosine formation in different models ŽMeghji et al., 1988; Kroll et al., 1993; Deussen et al., 1999., and levels employed should maximally block adenosine deaminase and kinase ŽKroll et al., 1993.. To examine potential regulation of coronary vascular tone by enhanced adenosine levels during inhibition of catabolism, the relationship between coronary resistance and venous adenosine levels was assessed in control, EHNA and EHNA q iodotubercidin-treated hearts which were electrically paced as described above. To

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eliminate changes in flow and therefore rates of metabolite washout, the mouse, rat and guinea pig hearts were perfused at fixed flow rates of 22.0" 0.8, 18.1" 1.0, and 6.0" 0.2 mlrmin per g, respectively. Changes in coronary perfusion pressure were recorded. After a 25-min stabilization period baseline measurements were made and hearts were then treated with 20 ␮M EHNA Ž n s 6᎐8 for all species. or 20 ␮M EHNA plus 5 ␮M iodotubercidin Ž n s 7 for all species.. After 10 min of treatment venous effluent was sampled for analysis of purine levels and measurements of heart rate were made. To assess potential regulation of heart rate by elevated endogenous adenosine additional groups of mouse, rat and guinea pig hearts were permitted to beat at intrinsic heart rates. After a 25-min stabilization period baseline measurements were made and hearts were then treated with 20 ␮M EHNA Ž n s 7 for all species. or 20 ␮M EHNAq 5 ␮M iodotubercidin Ž n s 7 for all species.. Again, venous effluent was sampled for purine levels and measurements of heart rate were made. Relationships between adenosine levels and coronary resistance or heart rate were assessed by fitting the following single-receptor three-parameter logistic equation to the data for purine efflux or concentration and vascular resistance and heart rate: Response sA"

B slope factor Žw 1 q adenosine x r EC 50 .

where A is the response at zero concentration or dose, B is the response at infinite concentration or dose, and the EC 50 reflects the level of agonist mediating a 50% maximal response. Raw data were fit to the equation using the Statistica data analysis program ŽStatsoft, Tulsa, OK, USA.. Coefficients of determination Ž r 2 . were calculated. Responses to exogenously applied adenosine and the more stable analogue 2-chloroadenosine were acquired in separate groups of hearts. Specifically, electrically paced mouse Ž n s 7., rat Ž n s 8. and guinea pig hearts Ž n s 7. were treated with increasing concentrations of adenosine or 2-chloroadenosine and coronary flow responses assessed. In constant, flow perfused intrinsically beating mouse Ž n s 7., rat Ž n s 7. and guinea pig hearts Ž n s 7., heart rate responses to infused adenosine or 2-chloroadenosine were also assessed.

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Finally, in order to examine the impact of ischemia-reperfusion groups of mouse Ž n s 8. rat Ž n s 7. and guinea pig hearts Ž n s 8. were stabilized for 30 min after which baseline function measurements were made and the hearts were then subjected to 20 min of global normothermic ischemia followed by 30 min of aerobic reperfusion. 2.7. Metabolic fluxes for adenosine From the venous efflux values for adenosine and ⌺inosine q hypoxanthine q xanthine q urate in untreated hearts and hearts treated with EHNA " iodotubercidin it is possible to determine metabolic fluxes through key enzymes, as described in part by Chen and Gueron Ž1996.. Specifically, the decline in ⌺ inosine q hypoxanthine q xanthine q urate upon EHNA treatment reflects the flux of adenosine through adenosine deaminase Žto inosine., and should parallel any elevation in adenosine. Moreover, the additional elevation in adenosine efflux upon addition of iodotubercidin Žto EHNA-treated hearts . reflects the flux of adenosine through adenosine kinase Žto 5⬘-AMP., and adenosine formation in these hearts, in which adenosine deaminase and kinase are blocked, reflects net catabolism of 5⬘-AMP to adenosine. The efflux of ⌺inosine q hypoxanthine q xanthine q urate in these hearts approximates catabolism of IMP ŽChen and Gueron, 1996.. The metabolic fluxes shown in Fig. 3 were approximated in this manner from efflux rates for adenosine and ⌺inosine q hypoxanthine q xanthine q urate in all groups. 2.8. Statistical analysis All data are expressed as mean " S.E.M. Functional responses to ischemia-reperfusion were analyzed by multi-way analysis of variance for repeated measures. When significance was detected a Tukey’s HSD post-hoc test was employed for individual comparisons. Metabolite levels were compared by one-way analysis of variance, while purine effluxes and enzyme activities were compared by Student’s t-test. In all tests significance was accepted for P- 0.05.

3. Results 3.1. Cardiac function, metabolic rate and metabolic profiles for mouse, rat and guinea pig hearts Normoxic function for the three species studied is shown in Table 1. MV O 2 and heart rate rise dramatically as heart mass declines. However, V O 2rbeat increased as hearts mass fell, being ; 0.45, 0.50 and 0.55 ␮l O 2rg in guinea pig, rat and mouse, respectively. However, normalized to the amount of pressure development per heart beat Ži.e. aerobic efficiency., O 2 use was similar in hearts from all three species ŽTable 1.. Basal coronary flow increased as heart mass declined. Furthermore, heart rate, ventricular pressure development and contractility Žqd Prdt . were all greatest in the smaller hearts and declined as mass increased. Myocardial sensitivity to extracellular Ca2q was highest in mouse ŽEC 50 s 0.9" 0.2 mM. and rat ŽEC 50 s 1.2" 0.2 mM. and was significantly lower in guinea pig hearts ŽEC 50 s 2.1 " 0.3 mM. ŽFig. 1.. Similarly, the maximal inotropic response to Ca2q apparently declined as heart mass increased, being greatest in mouse Žpeak systolic pressure 187 " 11. vs. rat Ž137 " 8 mmHg. and guinea pig hearts Ž130 " 12 mmHg.. Under normoxic conditions steady-state levels of total myocardial ATP, ADP, AMP and PCr were comparable in hearts from mice, rats and guinea pigs ŽTable 2.. Similarly, free cytosolic metabolite levels, pH i , wMg 2q x i and the cytosolic phosphorylation did not differ substantially between species ŽTable 2.. 3.2. Purine efflux in normoxic mouse, rat and guinea pig hearts Normoxic efflux and levels of adenosine were not statistically different in rat and mouse hearts ŽTable 3.. However, levels of the remaining purine catabolites Ži.e. ⌺inosine q hypoxanthine q xanthine q urate. were lower in mouse vs. rat heart. Consequently, total purine efflux was significantly lower in mouse vs. rat under normoxic conditions ŽFig. 2.. As shown in Table 3 an increase in work rate during increased Ca2q resulted in increased overflow of adenosine and purines into the venous effluent. Importantly, despite the greater work rate and metabolic rate of smaller hearts, the purine moiety washout was not dissimilar between species.

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Table 1 Baseline functional parameters in Langendorff perfused isovolumically contracting mouse and rat hearts Mouse Ž n s 7. Heart rate Žbeatsrmin. Diastolic pressure ŽmmHg. Systolic pressure ŽmmHg. Rate᎐pressure product ŽmmHgrmin. qd Prdt ŽmmHgrs. yd Prdt ŽmmHgrs. MV O2 Ž␮l O2 rmin g. V O2 rbeat Ž␮l O2 rg. Aerobic efficiency ŽmmHgr␮l O2 . Coronary flow Žmlrmin per g. U

Rat Ž n s 8.

Guinea pig Ž n s 8.

396 " 10

271 " 17U

194 " 11

6"2

5"2

6"2

175 " 4

124 " 11

109 " 7U

54 443 " 6354

32 940 " 2355U

19 896 " 1722U †

6808 " 372

3605 " 322U

2970 " 207U †

5028 " 144

2981 " 294U

2392 " 155U †

218 " 15

138 " 17U

90 " 8U †

0.55" 0.04

0.50" 0.03

0.46" 0.03U

249 " 19

240 " 21

229 " 17

21.8" 1.7

17.5" 2.0U

6.5" 0.5U †

All data were measured after 30 min of aerobic perfusion at 80 mmHg in hearts beating at intrinsic rates. Data are means " S.E.M. P- 0.05 vs. mouse heart; †P- 0.05 guinea pig vs. rat heart.

Infusion of EHNA and iodotubercidin resulted in pronounced elevations in adenosine formation in hearts from all species ŽFig. 2.. Estimated net rates of adenosine formation were highest in mouse vs. rat and guinea pig. Total purine efflux was enhanced by EHNA and iodotubercidin in the three species due to the elevation in adenosine formation rather than formation of other

Fig. 1. Extracellular Ca2q sensitivities of ventricular pressure development in isovolumically functioning mouse Ž n s 8., rat Ž n s 7. and guinea pig Ž n s 7. hearts. All data are means " S.E.M. U P- 0.05 vs. mouse hearts.

catabolites. Infusion of 20 ␮M EHNA alone elevated adenosine efflux by ; 2500 pmolrmin per g in mouse hearts, by ; 3000 pmolrmin per g in rat hearts, and by ; 1300 pmolrmin per g in guinea pig hearts ŽFig. 2.. These elevations were Table 2 Myocardial adenine nucleotide levels and energy state in normoxic mouse, rat and guinea pig Mouse Ž n s 7.

Rat Ž n s 8.

Guinea pig Ž n s 8.

Total tissue metabolites ATP ADP 5⬘-AMP ⌺ Nucleotides PCr

25.8" 1.8 7.8" 0.6 1.8" 0.7 33.7 " 3.7 38.4" 4.0

27.6" 3.0 8.4" 0.6 1.2" 0.5 34.2" 2.8 39.2" 2.9

25.8" 3.2 4.9" 0.9 0.6" 0.2 31.2" 2.8 44.8" 5.0

Free cytosolic metabolites ATP ADP 5⬘-AMP wMg2q xi pHi wATPxrwADPx = wPi x

25.2" 1.1 0.04" 0.01 0.05" 0.01 0.6" 0.2 7.09" 0.06 57.3" 4.2

25.9" 0.9 0.05" 0.01 0.09" 0.01 0.8" 0.2 7.11" 0.04 47.0" 5.1

26.0" 1.2 0.05" 0.01 0.08" 0.01 0.6" 0.1 7.10" 0.03 55.4" 5.6

Total and free cytosolic tissue metabolite values were measured after 30 min normoxic perfusion and are expressed as ␮molrg dry weight. wATPxrwADPx = wPi x is given per mM units. Data are means " S.E.M. U P - 0.05 vs. mouse heart.

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Table 3 Venous purine metabolite levels ŽnM. in Langendorff perfused isovolumically contracting mouse and rat hearts Mouse Ž n s 7.

Rat Ž n s 8.

Guinea pig Ž n s 8.

Baselinea Adenosine ⌺Ino q Hypoq Xan q UA

57 " 14 252 " 31

69 " 10 942 " 128U

45 " 8 427 " 52

Ca2 q-stimulatedb Adenosine ⌺Ino q Hypoq Xan q UA

311 " 36† 1257 " 154†

391 " 45† 3568 " 428U †

277 " 33† 1874 " 205†

Significance: U P- 0.05 vs. mouse heart. †P- 0.05 vs. baseline. a Metabolite levels measured in venous effluent after 30 min of normoxic perfusion with 2.5 mM perfusate Ca2q. b Metabolite levels measured during 5 min of inotropic stimulation with 3.5 mM Ca2q. Data are means " S.E.M.

matched by reductions in remaining purine catabolites and total efflux was unaltered. Based on purine release profiles for untreated and treated normoxic hearts shown in Fig. 2, we estimate that 64% of adenosine formed in mouse hearts is rephosphorylated to 5⬘-AMP while 23% is deaminated to inosine Ž2.8:1. ŽFig. 3.. In contrast, in rat 41% of adenosine is phosphorylated and 46% deaminated Ž0.9:1., while in guinea pig 39% is rephosphorylated and 50% deaminated Ž0.8:1.. Moreover, efflux data indicate that 55%, 67% and 67% of inosine, hypoxanthine, xanthine and urate are derived from IMP catabolism rather than adenosine catabolism in mouse, rat and guinea pig, respectively. The relative rates of 5⬘AMP catabolism to adenosine vs. catabolism via IMP to inosine are 3.3:1, 0.7:1 and 1:1 in mouse, rat and guinea pig, respectively. Thus, the mouse has more than a threefold higher rate of 5⬘-AMP catabolism to adenosine vs. deamination to IMP and subsequent dephosphorylation to inosine. 3.3. Coronary and cardiac responses to endogenous and exogenous adenosine in mouse, rat and guinea pig hearts Heart rate, systolic pressure, rate-pressure product and "d Prdt were all significantly higher in mouse vs. rat vs. guinea pig hearts under basal conditions ŽTable 1.. However, coronary flow was comparable in hearts from the three species. As already noted, heart rate was observed to be depressed in preliminary studies of hearts treated with EHNA and iodotubercidin, necessitating pacing to ensure similar metabolic rates in treated vs. untreated hearts. Coronary flow increased significantly from 22.5" 0.9 mlrmin per g to 36.4"

1.4 mlrmin per g in mouse hearts, from 17.0" 2.1 mlrmin per g to 21.7" 2.5 mlrmin per g in rat hearts, and from 6.6" 0.5 mlrmin per g to 11.4" 0.9 mlrmin per g in guinea pig hearts. In parallel studies coronary vascular resistance was shown to correlate with alterations in adenosine levels during treatment with EHNA" iodotubercidin ŽFig. 4.. Similarly, in un-paced hearts, reductions in heart rate were also correlated with extracellular adenosine concentrations ŽFig. 4.. Coronary vascular sensitivity to endogenous adenosine appeared greatest in guinea pig ŽpEC 50 s 7.3. vs. rat ŽpEC 50 s 6.0. and mouse ŽpEC 50 s 6.8.. A 1 sensitivity to endogenous adenosine was similar in mouse and guinea pig ŽpEC 50 s s 6.3 and 6.8, respectively. and lower in the rat ŽpEC 50 s 5.5.. The above differences in apparent sensitivity to endogenous adenosine paralleled those for exogenously infused adenosine and the more stable analogue 2-chloroadenosine ŽFig. 5.. A 2 adenosine receptor sensitivities for adenosine and 2chloroadenosine were greatest in the guinea pig and lowest in mouse and rat ŽTable 4.. In direct contrast, A 1 adenosine receptor sensitivities were greatest in the mouse vs. rat and guinea pig. Due to these opposing changes in A 2 and A 1 sensitivity in the smaller heart, the A 2 :A 1 sensitivity ratio Žcalculated from EC 50 s for adenosine and 2-chloroadenosine. increased from 15 to 20 in the mouse, to 50᎐75 in rat and ) 170 in the guinea pig ŽTable 4.. 3.4. Sensiti¨ ity to ischemic insult in mouse, rat and guinea pig hearts A 20-min period of total global normothermic ischemia resulted in pronounced ischemic dias-

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Fig. 2. Normoxic adenosine and purine efflux from Ža. mouse, Žb. rat and Žc. guinea pig hearts. Hearts were untreated ŽControl, n s 7, 8 and 8 for mouse, rat and guinea pig hearts, respectively., treated with 20 ␮M EHNA alone ŽEHNA, n s 8, 7 and 8 for mouse, rat and guinea pig hearts, respectively., or treated with 20 ␮M EHNA and 5 ␮M iodotubercidin Ž n s 6, 7 and 7 for mouse, rat and guinea pig hearts, respectively.. All data are means " S.E.M. U P- 0.05 vs. untreated hearts; P0.05 vs. mouse hearts.

tolic contracture and impaired contractile function upon reperfusion in all species. Ischemic contracture development was extremely rapid and more pronounced in mouse vs. rat vs. guinea pig hearts ŽFig. 6.. Time to contracture Ždefined here as a rise in diastolic pressure of 5 mmHg. was

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265 " 32 s in mouse vs. a significantly longer value of 729 " 62 s in rat and 1158 " 150 in guinea pig Ž P- 0.05.. The latter value for guinea pigs represents a mean of only five hearts since the remainder did not actually achieve contracture during the ischemic insult. The peak contracture achieved during ischemia was 98 " 6 mmHg in mouse compared with a significantly lower value of 32 " 4 mmHg in rat and only 19 " 3 mmHg in guinea pig Ž P- 0.05.. Functional recovery during reperfusion was gradual in the three species ŽFig. 6.. Diastolic pressure remained significantly elevated only in mouse and rat, and systolic pressure was significantly depressed relative to pre-ischemia in mouse and rat but not guinea-pig. Expressed as % of pre-ischemia, developed pressure recovered to the lowest extent in mouse Ž48:0%. vs. rat Ž70 " 5%. vs. guinea pig Ž81 " 7%.. Heart rate recovered to within 95% of pre-ischemic levels in all species ŽFig. 6c.. Coronary flow initially displayed a hyperemic response followed by a gradual decline to levels which were slightly lower than pre-ischemia ŽFig. 6d.. During reperfusion purine efflux was markedly enhanced in all species ŽFig. 7.. As opposed to higher purine efflux in rat vs. mouse hearts under normoxic conditions, total purine efflux following 20 min ischemia was similar in mouse and rat hearts, and was significantly lower in guinea pig hearts ŽFig. 7.. A similar profile was observed for efflux of adenosine alone. Over the 30-min duration of reperfusion 25᎐30% of the nucleotide pool Žnormoxic ⌺ATPq ADPq AMP shown in Table 2. was lost as adenosine, inosine, hypoxanthine, xanthine and urate in mouse and rat hearts, and ; 12% was lost in guinea pig hearts.

4. Discussion In the present study we examined and compared cardiovascular function, 5⬘-A M P catabolism, adenosine handling and A 1 and A 2 adenosine receptor responses in mouse, rat and guinea pig hearts. Our data demonstrate that the rate of 5⬘-AMP catabolism to adenosine increases significantly as heart mass falls and metabolic rate increases. However, reasonably consistent extracellular adenosine levels are achieved across the three species due to adaptations in rates of adenosine phosphorylation relative to deamina-

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Fig. 3. Pathways of adenosine formation and metabolism in mouse, rat and guinea pig hearts. All numerical values are metabolic fluxes Žpmolrmin per g. approximated from the efflux data in Fig. 2 from hearts perfused with and without EHNA and iodotubercidin, as described in Section 2.

tion. Coronary and cardiac sensitivities to adenosine also change, with a decline in A 2 relative to

A 1 sensitivity in smaller hearts. Data also indicate that, despite adaptations conserving purine moieties under normoxic and post-ischemic conditions, the small and rapidly respiring mouse heart is functionally less tolerant of ischemic insult than larger rat and guinea pig hearts. 4.1. Contractile function in mouse, rat and guinea pig myocardium Baseline contractile function appears inversely related to heart mass, with peak ventricular pressures, qd Prdt and heart rate all higher in mouse Table 4 Coronary vascular and cardiac sensitivities ŽpEC 50 s. for adenosine and 2-chloroadenosine Mouse Ž n s 10.

Fig. 4. Relationship between extracellular adenosine and Ža. coronary resistance, and Žb. heart rate in normoxic mouse, rat and guinea pig hearts. Data are taken from control, 20 ␮M EHNA treated, or 20 ␮M EHNAq 5 ␮M iodotubercidin treated hearts Ž n s 6᎐8 in all groups.. Curves shown were fit to a standard dose᎐response relationship as outlined in Section 2.

Rat Ž n s 9.

Guinea pig Ž n s 9.

A1 pEC50 (M) Adenosine 2-Chloroadenosine

5.77" 0.07 4.34" 0.10U 4.80" 0.06U 6.50" 0.19 5.75" 0.07U 5.87" 0.06U

A2 pEC50 (M) Adenosine 2-Chloroadenosine

6.91" 0.16 6.19" 0.08U 7.68" 0.19U 7.70" 0.18 7.40" 0.12 8.17" 0.14U

A2 :A1 sensiti¨ ity ratio Adenosine 15 2-Chloroadenosine 17

71 45

762 175

pEC 50 values were estimated in hearts treated with exogenous adenosine or 2-chloroadenosine. The A 2 :A 1 sensitivity was calculated as the ratio of EC 50 values for A 2 vs. A 1 responses. Data are means " S.E.M. U P- 0.05 vs. mouse heart.

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Fig. 5. Concentration᎐response curves for adenosine and 2chloroadenosine mediated coronary vasodilation Ža. and bradycardia Žb. in mouse, rat and guinea pig hearts Ž n s 6᎐8.. All data are means " S.E.M.

and lowest in guinea pig ŽTable 1.. This is consistent with the data of Kim et al. Ž1994. who recently documented a relationship between mitochondrial density, oxidative capacity and contractile function in cardiac muscle. Mechanisms underlying a decline in contractility in relation to oxidative metabolism are unclear. However, we show that contractile sensitivity to extracellular Ca2q increases in smaller hearts ŽFig. 1.. While we do not directly measure intracellular Ca2q, our data are consistent with observations regarding intracellular Ca2q in individual species. Studies in guinea pig ŽStowe et al., 1999. and rat hearts ŽBrooks et al., 1994. show that peak contractile responses to Ca2q are achieved at ) 4 mM extracellular and ) 1 ␮M intracellular Ca2q, whereas studies in mouse heart ŽHampton et al., 1998. show peak pressures at ; 1.5 mM extracellular and - 0.8 ␮M intracellular Ca2q. These observations and our data support a reduction in myofibrillar Ca2q sensitivity from mouse to rat to

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guinea pig, providing a mechanism whereby contractile activity may be reduced as heart mass increases and MV O 2 declines ŽKim et al., 1994.. The molecular mechanism responsible for the reduction in Ca2q sensitivity is not evident from our data. However, Ca2q sensitivity depends Žamongst other things. on myosin ATPase activity, myosin heavy chain isozyme profiles, and sarcoplasmic reticulum ŽSR. Ca2q-ATPase activity. Smaller hearts with high beating rates possess significantly higher Ca2q-activated myofibrillar ATPase activities ŽRouslin and Broger, 1996.. Additionally, mouse myocardial myosin is primarily of the fast V1 isoform ŽNg et al., 1991. whereas there is a predominance of the slower V3 isoform in larger species ŽHamilton and Ianuzzo, 1991.. Indeed, analysis of myosin isoforms reveals a progressive decline in the V1:V3 isoform ratio from mouse to rat to guinea pig ŽReiser and Kline, 1998.. There is evidence, however, that myosin isoform distribution does not impact markedly on myofibrillar Ca2q sensitivity ŽJacob et al., 1986. or peak activity ŽRouslin and Broger, 1996.. However, SR Ca2q-ATPase activity changes inversely with heart mass ŽHamilton and Ianuzzo, 1991., and increased SR Ca2q-ATPase levels do enhance myocardial contractility ŽBaker et al., 1998.. 4.2. 5⬘-AMP catabolism to adenosine in mouse, rat and guinea pig myocardium Despite higher metabolic rates and levels of contractility in smaller hearts ŽTable 1., myocardial adenine nucleotide pools were comparable in all three species, consistent with the need to maintain a uniformly high ⌬GATP to support ionic homeostasis and contractile function. Similarly, despite different metabolic rates, extracellular adenosine levels were similar in mouse, rat and guinea pig hearts ŽTable 3, Fig. 2., and are comparable with levels in other species. Maintenance of extracellular adenosine concentration across species is in keeping with the proposed importance of the compound in regulation of coronary flow, heart rate, impulse conduction, ischemic tolerance and myocardial responsiveness to adrenoceptor stimulation ŽEly and Berne, 1992; Shryock and Belardinelli, 1999.. Conservation of adenosine concentration despite quite different metabolic rates may be achieved by modifying relative rates of 5⬘-AMP catabolism vs. adenosine rephosphorylation and

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Fig. 6. Functional responses to 20 min global ischemia and 30 min reperfusion in isovolumically contracting mouse Ž n s 7., rat Ž n s 7. and guinea pig hearts Ž n s 8.. Responses for Ža. left ventricular diastolic pressure, Žb. left ventricular developed pressure Ž% of pre-ischemia., Žc. heart rate Ž% of pre-ischemia. and Žd. coronary flow Ž% of pre-ischemia. are shown. All data are means " S.E.M. Note that all diastolic pressure and developed pressure values during ischemia and reperfusion differed significantly from pre-ischemia in all species Ž P- 0.05.. U P- 0.05 vs. mouse hearts. P- 0.05 vs. pre-ischemia in heart rate and coronary flow graphs.

deamination. Data acquired from control hearts and hearts treated with EHNA and iodotubercidin Žto block adenosine deamination and

Fig. 7. Purine efflux from mouse Ž n s 7., rat Ž n s 8. and guinea pig hearts Ž n s 8. during 30 min of reperfusion following 20 min global normothermic ischemia. All data are means " S.E.M. U P- 0.05 vs. mouse hearts.

rephosphorylation, respectively. reveal that total myocardial adenosine formation from 5⬘-AMP increases substantially with mass-specific metabolic rate ŽFigs. 2 and 3.. Thus, comparable adenosine levels must be achieved by adaptations in adenosine handling itself Žsee below.. Since there is evidence of lower total 5⬘-nucleotidase activity in mouse vs. rat heart ŽArch and Newsholme, 1978., our observation of greater 5⬘-AMP catabolism in the mouse suggests that: Ži. substrate delivery for 5⬘-nucleotidase must be greatly increased in mouse; Žii. the enzyme is allosterically activated to a greater degree in mouse; andror Žiii. changes in the isoform responsible for adenosine formation are not revealed by measurement of total 5⬘-nucleotidase activity. With respect to the first possibility, the K m for 5⬘-AMP is orders of magnitude higher than cytosolic w5⬘-AMPx and sub-

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strate delivery therefore limits adenosine formation ŽHeadrick et al., 1991, 1998b.. However, since free cytosolic 5⬘-AMP in mouse Ž0.5᎐1.0 ␮M. ŽHeadrick et al., 1998a. is similar to that in rat ŽHeadrick et al., 1998b. substrate delivery does not contribute to greater catabolism in mouse. With respect to allosteric regulation in vivo, although 5⬘-nucleotidase is modulated by Mg 2q, ATP, ADP and pH, we have previously shown that free Mg 2q, ADP and pH in the normoxic mouse heart ŽHeadrick et al., 1998a. are similar to values for rat and guinea pig heart Ži.e. ; 0.5 mM, 80᎐100 ␮M and 7.1, respectively. ŽHeadrick et al., 1991, 1998b., and we show here that pH and ATP, ADP and Mg 2q levels are similar in the three species under normoxic conditions ŽTable 2.. Differing allosteric modulation is therefore unlikely. The final possibility relates to the form of 5⬘-nucleotidase responsible for normoxic adenosine formation. Different cytosolic 5⬘-nucleotidase isoforms exist, possessing differing kinetic and allosteric properties. Since Arch and Newsholme Ž1978. only assessed total myocardial 5⬘-nucleotidase activity, it is feasible that levels of the isoform responsible for normoxic production in different species varies differently from total cytosolic activities. This remains to be directly tested.

cardium since inosine and its catabolites are not be as efficiently re-captured. Conversion of hypoxanthine to IMP and then 5⬘-AMP requires two phosphates from 5-phosphoribose 1-pyrophosphate and GTP, respectively, and results in ultimate hydrolysis of formed pyrophosphate. Adenosine is more economically and rapidly salvaged via adenosine kinase to 5⬘-AMP, using a single phosphate bond. Previous studies suggest that reducing adenosine deamination is functionally and metabolically beneficial in ischemic and hypoxic myocardium ŽBarankiewicz et al., 1997; Abd-Elfattah et al., 1998.. Indeed, reduced deamination and enhanced phosphorylation mouse leads to a lower overall purine washout vs. that for larger rat hearts ŽFig. 2.. Additionally, post-ischemic efflux is comparable in rat and mouse, and slightly lower in guinea pig ŽFig. 7.. Nonetheless, we show that functional tolerance to ischemia and reperfusion declines with heart mass, being lowest in the mouse ŽFig. 6.. We conclude therefore that functional tolerance in different sized hearts may correlate inversely with metabolic rate, but is not directly related to preservation of the myocardial purine pool.

4.3. Adenosine catabolism in mouse, rat and guinea pig myocardium

Some studies in animal and human myocytes indicate that myocardial 5⬘-AMP catabolism occurs primarily via 5⬘-nucleotidase with only a minor contribution from adenylate deaminase ŽMeghji et al., 1988; Smolenski et al., 1992; Kochan et al., 1994.. In contrast, we estimate that only 35᎐45% of extracellular inosine and its catabolites are derived from 5⬘-AMP vs. IMP dephosphorylation ŽFig. 3.. Treatment with EHNA and iodotubercidin, at concentrations predicted to maximally block adenosine deaminase and adenosine kinase ŽKroll et al., 1993., markedly elevated adenosine release with only modest relative changes in inosine, hypoxanthine, xanthine and urate release ŽFig. 2.. The extent of adenosine deaminase blockade is verified by the lack of change in inosine and catabolites in EHNA treated hearts when adenosine levels were further enhanced by iodotubercidin ŽFig. 2.. The present data are in reasonable agreement with recent data demonstrating that IMP catabolism is the single greatest source of extracellular purines in rat myocardium ŽChen and Gueron, 1996.. A

While resting levels of adenosine within the extracellular compartment were similar across the three species studied, resting levels of inosine, hypoxanthine, xanthine and urate were much lower in mouse vs. rat heart ŽTable 3, Fig. 2., and the pattern of efflux varied, with a greater percentage contribution from adenosine in mouse vs. rat and guinea pig Ždata not shown.. These profiles suggest lower relative rates of deamination in mouse, and this is verified in studies with EHNA and iodotubercidin. Based on the data shown in Fig. 2 and summarized in Fig. 3, the mouse possesses a greater capacity for adenosine salvage relative to deamination Ž2.8:1 vs. 0.9:1 in rat and 0.8:1 in guinea pig.. Differences between mouse and rat are consistent with total adenosine kinase and adenosine deaminase activities measured by Arch and Newsholme Ž1978.. Greater rephosphorylation vs. deamination may be beneficial in preserving nucleotides in rapidly respiring myo-

4.4. 5⬘-AMP catabolism to IMP and inosine in mouse, rat and guinea pig myocardium

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practical consequence of these observations is that summation of purine efflux to assess myocardial adenosine formation ŽHeadrick, 1996b. or energy state ŽZucchi et al., 1990. is invalid. The physiological importance of a high flux through IMP to inosine vs. 5⬘-AMP to adenosine is not clear. It has been suggested that conversion of 5⬘-AMP to IMP may be beneficial since IMP does not cross the sarcolemma and effectively ‘traps’ cellular 5⬘-AMP ŽChen and Gueron, 1996.. However, we show that a large amount of IMP is catabolized to diffusible products readily exiting the cell, and which are less effectively re-captured compared with adenosine. The value of IMP as a nucleotide ‘trap’ is therefore questionable. Indeed, enhancing 5⬘-AMP deamination to IMP vs. de-phosphorylation to adenosine does not protect the adenine nucleotide pool during and following ischemic insult ŽHohl, 1999.. 4.5. O2 deli¨ ery and A 1 and A 2 adenosine receptormediated responses in mouse, rat and guinea pig hearts The increase oxidative requirements of the heart in smaller species must be met in part by adaptations in O 2 carrying capacity of blood andror vascular function and structure ŽPietschmann et al., 1982.. We observe a trend towards reduced hemoglobin and red blood cell count as body size increases, which results in an increase in cell volume and decline in cell hemoglobin concentration as body size increases from mouse to guinea pig ŽTable 5.. Red blood cells in the mouse therefore appear to be smaller, more numerous, and loaded with higher levels of hemoglobin. These differences, together with a greater red blood cell velocity in smaller species ŽChen and Kaul, 1994., will facilitate O 2 delivery to tissues in smaller species. However, the changes in O 2 carrying capacity are modest and increased MV O 2 must additionally be met by enhanced coronary flow in small hearts. We show that basal and peak hyperemic coronary flows are significantly higher in mouse vs. rat and guinea pig ŽTable 1, Fig. 4., in keeping with normally tight coupling between coronary blood flow and myocardial V O 2 . Resting and dilated coronary resistances are lowest in the mouse vs. rat and guinea pig, and this must result from enhanced vascular relaxation andror increased vascular density. While there is relatively little data available re-

Table 5 Erythrocyte data for mouse, rat and guinea pig

Hematocrit Ž%. Hemoglobin Žgrdl. RBC Ž106rmm3 . Mean cell volume Žfl. Cell hemoglobin concentration Ž%.

Mouse Ž n s 10.

Rat Ž n s 9.

Guinea pig Ž n s 9.

41.0" 2.1 15.9" 1.4 9.5" 0.7 43.2" 0.4 38.8" 2.2

42.3" 2.9 15.0" 1.1 8.4" 0.9 50.4" 0.5U 35.4" 1.9

42.0" 3.0 12.9" 1.3 8.1" 0.7 51.9" 0.4U 30.6" 2.0U

Erythrocyte data were acquired in freshly drawn blood. Data are means " S.E.M. U P- 0.05 vs. mouse heart.

garding allometric variations in coronary density or function ŽWeibel et al., 1991., there is some evidence that coronary vascular density does correlate with mitochondrial density and oxidative capacity ŽSillau et al., 1990; Rakusan and Tomanek, 1986.. In contrast, some studies indicate that coronary vascular density does not differ substantially between species of differing sizes ŽRakusan et al., 1992.. If adenosine is to serve as a metabolically coupled signaling mechanism in hearts of different species, it is predicted that cardiovascular adenosine sensitivity should correlate with adenosine formation, and responses should be adjusted in keeping with metabolic rate. Adenosine does mediate substantial coronary dilation Žin addition to bradycardia. in all three species. However, the sensitivity of A 1 and A 2 adenosine receptormediated responses does not change predictably with MV O 2 or basal adenosine levels. Indeed, A 1 and A 2 receptor sensitivities changed in opposite directions as heart mass declined. As a result, the A 2 :A 1 sensitivity ratio increased from only 10 in mouse to ; 50 and 150 in rat and guinea pig hearts, respectively ŽTable 4.. Greater A 2 vs. A 1 sensitivity in large species reflects a logical hierarchy of cardiovascular adaptations to metabolic perturbation ᎏ as adenosine is released the heart initially responds with A 2-mediated coronary dilation, increasing energy supply. If energy state is not effectively normalized with increased O 2 delivery further elevations in adenosine will induce A 1-mediated bradycardia, reducing energy consumption. The change in magnitude of the A 2 :A 1 ratio suggests that the range over which hearts initially respond with dilation before down-regulation of energy demand is narrower in smaller hearts. This may reflect an adaptation to protect

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energy state in smaller species. moreover, this difference may limit peak heart rate during functional activation of hearts from smaller species. We have recently shown that peak chronotropic responses to adrenergic stimulation are inhibited by adenosine receptors in mouse and rat ŽGauthier et al., 1998; Headrick et al., 2000., and the increase in chronotropic relative to dilatory sensitivity to adenosine in smaller hearts may contribute to modest dynamic heart rate ranges observed in small mammals. Moreover, these data are consistent with the notion that peak heart rates in different species may be limited by O 2 delivery ŽLillywhite et al., 1999.. As noted by Lillywhite and colleagues, the evolution of the coronary circulation parallels, to some extent, the evolution of higher heart rates.

5. Conclusions In conclusion, the present study characterizes differences in 5⬘-AMP catabolism and adenosine handling in hearts from mouse, rat and guinea pig. We find that 5⬘-AMP catabolism increases in keeping with mass specific metabolic rate. However, adaptations in relative rates of adenosine rephosphorylation vs. deamination lead to preservation of purine moiety levels in smaller hearts. Additionally, we show that only 15᎐25% of normoxic extracellular purines arise from 5⬘-AMP catabolism to adenosine in all three species studied. Coronary flow is shown to be adapted appropriately to metabolic rate in the differing species, and cardiovascular A 1 and A 2 adenosine receptor sensitivities are altered in keeping with extracellular levels of adenosine achieved during functional stimulation. Interestingly, we find that A 1 and A 2 sensitivities appear to change in opposing ways such that the pronounced difference in vascular vs. chronotropic sensitivity the guinea pig is markedly reduced in the mouse. The physiological relevance of this latter adaptation is not clear, although the greater relative chronotropic vs. vasodilatory sensitivity to adenosine in smaller hearts may limit peak heart rates and work levels during cardiac stimulation in small species.

Acknowledgements This work was supported by grants from the

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National Heart Foundation of Australia ŽG95B 4260., the National Health and Medical Research Council of Australia Ž噛145310., and the National Institutes of Health ŽHL 59419.. References Abd-Elfattah, A.S., Jessen, M.E., Levken, J., Wechsler, A.S., 1998. Differential cardioprotection with selective inhibitors of adenosine metabolism and transport: role of purine release in ischemic and reperfusion injury. Mol. Cell. Biochem. 180, 179᎐191. Arch, J.R.S., Newsholme, E.A., 1978. Activities and some properties of 5⬘-nucleotidase, adenosine kinase and adenosine deaminase in tissues from vertebrates and invertebrates in relation to the control of the concentration and the physiological role of adenosine. Biochem. J. 174, 965᎐977. Baker, D.L., Hashimoto, K., Grupp, I.L. et al., 1998. Targeted expression of the sarcoplasmic reticulum Ca2q-ATPase increases cardiac contractility in transgenic mouse hearts. Circ. Res. 83, 1205᎐1214. Barankiewicz, J., Danks, A.M., Abushanab, E. et al., 1997. Regulation of adenosine concentration and cytoprotective effects of novel reversible adenosine deaminase inhibitors. J. Pharm. Exp. Ther. 283, 1230᎐1238. Barth, E., Stammler, G., Speiser, B., Schaper, J., 1992. Ultrastructural quantitation of mitochondria and myofilaments in cardiac muscle from 10 different animal species including man. J. Mol. Cell. Cardiol. 24, 669᎐681. Blank, S., Chen, V., Hamilton, N., Salerno, T.A., Ianuzzo, C.D., 1989. Biochemical characteristics of mammalian myocardia. J. Mol. Cell. Cardiol. 21, 367᎐373. Brooks, W.W., Bing, O.H., Litwin, S.E., Conrad, C.H., Morgan, J.P., 1994. Effects of treppe and calcium on intracellular calcium and function in the failing heart from the spontaneously hypertensive rat. Hypertension 24, 347᎐356. Burness, G.P., Leary, S.C., Hochachka, P.W., Moyes, C.D., 1999. Allometric scaling of RNA, DNA, and enzyme levels: an intraspecific study. Am. J. Physiol. Reg. Int. Comp. Physiol. 277, R1164᎐R1170. Chen, D., Kaul, D.K., 1994. Rheologic and hemodynamic characteristics of red cells of mouse, rat and human. Biorheology 31, 103᎐113. Chen, W., Gueron, M., 1996. AMP degradation in the perfused rat heart during 2-deoxy-D-glucose perfusion and anoxia. Part II: the determination of the degradation pathways using an adenosine deaminase inhibitor. J. Mol. Cell. Cardiol. 28, 2175᎐2182. Crabtree, B., Newsholme, E.A., 1972. The activities of phosphorylase, hexokinase, phosphofructokinase, lactate dehydrogenase and the glycerol 3-phosphate

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