222
P. ARESE, A. BOSIA, and L. ROSSINI
isotonic Ringer solution. Injection rates of 0.5 ml/30 sec, 1 ml/60 sec or 1 ml/120
sec were chosen. After 30, 60 or 120 sec the ventricles were cut, quick-frozen,
and stored in liquid nitrogen. The following substrates were determined in the
neutralized perchloric acid extracts, according to BERGMEYER [2]: glucose (GLU),
glucose-1-P (G1P), glucose-6-P (G6P), fructose-6-P (F6P), fructose diphosphate
(FDP) , dihydroxyacetone-P (DAP), glyceraldehyde-3-P (GAP), a-glycero-P
(a-GP), pyruvate (P), lactate (L), oxaloacetate (OAA), malate (M), a-ketoglutarate (a-KG), P-creatine (PC), creatine (Cr), ATP, ADP, and AMP. Beside the
18 substrates, the following 17 derived parameters were also taken into account:
mass-action ratios of the phosphoglucomutase (G 1P IG6P), phosphohexose isomerase (F6P IG6P), triosephosphate isomerase (DAP IGAP), phosphofructokinase
(1. FDP X ADP/F6P X ATP; 2.FDP X Cr/F6P X PC), aldolase (1. DAp 2 /FDP; 2.
GAP X DAP/FDP), adenyl ate kinase (AMP X ATP/ADp2), creatine kinase (Cr X
ATP/PC X ADP); redox ratios LIP, a-GP/DAP, M/OAA; ratios PC/Cr, ATPI
ADP; the sums PC + Cr, ATP + ADP + AMP, and L + P. The 18 substrates
or the 35 parameters were subjected to stepwise discriminant analysis [5], carried
out on an IBM 7094 computer. Three main variance factors were considered:
different times of injection, varying amounts and composition of injected fluids.
Every group (4 X Ca++, 0.1 % ouabain and related controls at 30, 60 and 120 sec)
included 125 hearts. As 20-25 frozen hearts were pooled for each extract, every
group was the result of 5 to 6 distinct experiments.
Results
Control conditions (Fig. 1) Comparison of control steady-state levels
showed that most glycolytic and citric-acid-cycle intermediates, as well as
AMP, are 3 to 6 times higher in the frog than in the rat [12,13]. Close correspondence was found only for eX-GP, DAP and ATP. The redox ratios LIP,
a-GP IDAP and M/OAA are apparently not kept at equilibrium through a
common NAD-pool. Whereas the phosphoglucomutase reaction was near
to thermodynamic equilibrium (mass-action ratio in vivo 0.09, Keq: 0.05)
clear shifts were observed for the two isomerase-catalyzed steps. Owing to
the compartmentation of ATP in muscle tissues [9, 10], the phosphofructokinase equilibrium constant was also computed using the PCfCr-ratio [4].
Both results showed a clear shift from thermodynamic equilibrium. With
respect to mammalian heart, A TP level in the frog was constantly higher
than PC; the steady-state equilibrium constant for the creatine-kinase reaction was lower than in mammalian heart (frog: 3.06; rat: 8.1 [13]) and
virtually the same as in rat skeletal muscle [9]. Multivariate analysis on
hearts injected with different amounts of Ringer-frog solution for 30, 60 or
120 sec showed clear wash-out effects for glucose, pyruvate, eX-ketoglutarate,
a-glycero-P, and creatine.
Effect of ionic modifications (Fig. 2). A 30 sec infusion with calciumenriched Ringer solution (Ca++ = 7.2 mM) caused shifts in the metabolite
pattern. The following changes were relevant: (1) slowing down of glucose
entry into the cell, (2) activation of phosphofructokinase, (3) increase of
two NAD-linked redox ratios, and (4) negative PC and ATP balance. No
P. ARESE, A. BOSIA, and L. ROSSINI
isotonic Ringer solution. Injection rates of 0.5 ml/30 sec, 1 ml/60 sec or 1 ml/120
sec were chosen. After 30, 60 or 120 sec the ventricles were cut, quick-frozen,
and stored in liquid nitrogen. The following substrates were determined in the
neutralized perchloric acid extracts, according to BERGMEYER [2]: glucose (GLU),
glucose-1-P (G1P), glucose-6-P (G6P), fructose-6-P (F6P), fructose diphosphate
(FDP) , dihydroxyacetone-P (DAP), glyceraldehyde-3-P (GAP), a-glycero-P
(a-GP), pyruvate (P), lactate (L), oxaloacetate (OAA), malate (M), a-ketoglutarate (a-KG), P-creatine (PC), creatine (Cr), ATP, ADP, and AMP. Beside the
18 substrates, the following 17 derived parameters were also taken into account:
mass-action ratios of the phosphoglucomutase (G 1P IG6P), phosphohexose isomerase (F6P IG6P), triosephosphate isomerase (DAP IGAP), phosphofructokinase
(1. FDP X ADP/F6P X ATP; 2.FDP X Cr/F6P X PC), aldolase (1. DAp 2 /FDP; 2.
GAP X DAP/FDP), adenyl ate kinase (AMP X ATP/ADp2), creatine kinase (Cr X
ATP/PC X ADP); redox ratios LIP, a-GP/DAP, M/OAA; ratios PC/Cr, ATPI
ADP; the sums PC + Cr, ATP + ADP + AMP, and L + P. The 18 substrates
or the 35 parameters were subjected to stepwise discriminant analysis [5], carried
out on an IBM 7094 computer. Three main variance factors were considered:
different times of injection, varying amounts and composition of injected fluids.
Every group (4 X Ca++, 0.1 % ouabain and related controls at 30, 60 and 120 sec)
included 125 hearts. As 20-25 frozen hearts were pooled for each extract, every
group was the result of 5 to 6 distinct experiments.
Results
Control conditions (Fig. 1) Comparison of control steady-state levels
showed that most glycolytic and citric-acid-cycle intermediates, as well as
AMP, are 3 to 6 times higher in the frog than in the rat [12,13]. Close correspondence was found only for eX-GP, DAP and ATP. The redox ratios LIP,
a-GP IDAP and M/OAA are apparently not kept at equilibrium through a
common NAD-pool. Whereas the phosphoglucomutase reaction was near
to thermodynamic equilibrium (mass-action ratio in vivo 0.09, Keq: 0.05)
clear shifts were observed for the two isomerase-catalyzed steps. Owing to
the compartmentation of ATP in muscle tissues [9, 10], the phosphofructokinase equilibrium constant was also computed using the PCfCr-ratio [4].
Both results showed a clear shift from thermodynamic equilibrium. With
respect to mammalian heart, A TP level in the frog was constantly higher
than PC; the steady-state equilibrium constant for the creatine-kinase reaction was lower than in mammalian heart (frog: 3.06; rat: 8.1 [13]) and
virtually the same as in rat skeletal muscle [9]. Multivariate analysis on
hearts injected with different amounts of Ringer-frog solution for 30, 60 or
120 sec showed clear wash-out effects for glucose, pyruvate, eX-ketoglutarate,
a-glycero-P, and creatine.
Effect of ionic modifications (Fig. 2). A 30 sec infusion with calciumenriched Ringer solution (Ca++ = 7.2 mM) caused shifts in the metabolite
pattern. The following changes were relevant: (1) slowing down of glucose
entry into the cell, (2) activation of phosphofructokinase, (3) increase of
two NAD-linked redox ratios, and (4) negative PC and ATP balance. No