.J Mol Cell Cardiol 22, 1051-1064
Calcium
Exchange in Rabbit Myocardium Hypoxia: Role of Sodium-Calcium Tom Crake
and Philip
During and After Exchange
A. Poole-Wilson
Department of Cardiac Medicine, National Heart and Lung Institute and National Dovehouse Street, London S W3 6LY, UK
Heart Hospital,
(Received 9 September 1989, accepted in revised form 6 Februar_v 1990) AND P. A. POOLE-WILSON. Calcium Exchange in Rabbit Myocardium During and After Hypoxia: Role of Sodium-Calcium Exchange. Journal of Molecular and Cellular Curdiolo~ (1990) 22, 105 l-1064. Calcium uptake was measured using “‘Ca’+ in the isolated and arterially perfused interventricular septum of the rabbit. Experiments were undertaken to determine whether calcium uptake on reoxygenation is linked to recovery of mechanical function and whether calcium uptake is through the sodium-calcium exchange mechanism. During substrate-free hypoxia for 45 min total tissue calcium remained unchanged but immediately upon reoxygenation there was a substantial net gain of calcium. Recovery of mechanical function upon reoxygenation was inversely related to the increase in tissue calcium. Activation of sodium-calcium exchange by perfusion with a lowsodium, zero-potassium, sucrose solution also increased tissue calcium and the relation to mechanical recovery was similar to that observed on reoxygenation. The sodium-calcium exchange mechanism was not affected by hypoxia and could be demonstrated during perfusion with a substrate-free hypoxic solution. Lithium (100 rnM! substitution for sucrose prevented the calcium influx induced by a low-sodium and zero-potassium perfusate under normoxic conditions. Lithium substitution early during hypoxia or on reoxygenation did not affect the increase in myocardial calcium on reoxygenation. Amiloride ( low4 M), presumed to inhibit sodium-hydrogen exchange during hypoxia, had no effect upon reoxygenation induced calcium uptake. It is concluded that the increase ill calcium uptake that occurs on reoxygenation after a period of substrate-free hypoxia is related to mechanical recovery. Sodium-calcium exchange may contribute to calcium uptake on reoxygenation in this experimental model but is not the major mechanism.
T. CRAKE
KEY WORDS:
Reoxygenation;
Sodium-calcium
exchange;
Introduction The sequenceof events resulting in cell necrosis during or after a period of ischaemia or hypoxia in heart muscle is relevant to current therapies for myocardial ischaemia, such as thrombolysis and angioplasty. Reperfusion (Shen and Jennings, 1972; Bourdillon and Pool-Wilson, 1981; Cheung et al., 1986) or reoxygenation (Nayler et al., 1979; Harding and Poole-Wilson, 1980) after a period of ischaemia or hypoxia is associated with an increase in tissuecalcium, diminished recovery of mechanical function and eventual necrosis of at least part of the myocardium. When experimental conditions, such as temperature and the availability of substrate, are manipulated so that recovery of mechanical function is complete, tissuecalcium does not increase (Nayler et al., 1979; Harding and * Please address all correspondence 0022-2828/90/101051
+ 14 $03.00/O
Myocardium.
Poole-Wilson, 1980; Poole-Wilson et al. 1 1984). Several authors have proposed a causal link between calcium accumulation and cell injury (Shen and Jennings, 1972; Nayler et al., 1979; Fleckenstein et al., 1984). The gain in tissue calcium is similar on reperfusion and reoxygenation and is a consequence of an increased calcium influx rather than a reduction of efflux (Harding and PooleWilson, 1980; Bourdillon and Poole-Wilson, 1982). Many mechanismsfor the gain in tissue calcium (Poole-Wilson et al., 1984) have been proposed including, sarcolemmal disruption (Steenbergen et al., 1985), lipid peroxidation (Mak et al., 1986), radical damage (Thompson and Hess, 1986) and sodium-calcium exchange (Grinwald, 1982; Renlund et al., 1984; Lazdunski et al., 1985). Intracellular sodium concentration doesnot
to: P. A. Poole-Wilson. (0 1990 Acadrmic
Press Limited
1052
T. Crake and P. A. Poole-Wilson
increaseearly during hypoxia when developed tension is partially reduced and resting tension unchanged (Nakaya et al., 1985; Guarnieri, 1987; Ellis and Noireaud, 1987) but after more prolonged hypoxia or ischemia intracellular sodium concentration is elevated (Fiolet et al., 1984; Wilde and Kleber, 1986; Poole-Wilson and Tones, 1986; Grinwald and Brosnaham, 1987; Guarnieri, 1987). The mechanismsfor the increaseof cellular sodium include inhibition of the sodium pump, increased permeability of the sarcolemma to sodium and stimulation of sodium-hydrogen exchange by an intracellular acidosis. Sodium-calcium exchange appears to be inhibited during prolonged hypoxia because total tissue calcium does not increase (Harding and Poole-Wilson, 1980) although the cytosolic calcium may be elevated late in hypoxia (Allen and Orchard, 1983; Allen and Smith, 1985; Guarnieri, 1987). On reoxygenation sodium-calcium exchange and sodium-hydrogen exchange could be reactivated and possibly account for the observed immediate uptake of calcium ( (Lazdunski et al., 1985; Allen and Orchard, 1987). Alteration of extracellular sodium is known to modify the recovery of myocardial function on reperfusion (Grinwald, 1982; Renlund et al., 1984). This study was designed to investigate whether a causal relationship exists between myocardial calcium uptake on reoxygenation and subsequent mechanical recovery and to elucidate the role of sodium-calcium exchange in an experimental model where resting tension increasesduring hypoxia, mechanical recovery on reoxygenation is incomplete and the uptake of calcium on reoxygenation has been well characterized.
temperature. A fine polythene cannula was inserted into the septal artery within 2 to 4 min of heart excision and the septum perfused with control solution. The septum was dissected from the right and left ventricular free walls and underperfused tissue was removed. The remaining triangular piece of tissuewas clamped at its basebetween opposing pairs of forceps and the apex wasattached to a tension transducer (UC4 Statham Instruments, Oxnard, CA). Septa were perfused with solution pumped by a peristaltic pump (Watson-Marlow, Falmouth, UK) and the flow, measured by timing and weighing effluent drops, was maintained constant for each experiment (mean 1.96 ) 0.07 ml/min/g wet tissue,range 1.28 to 2.64 mI/min/g) . Temperature was maintained constant (+0,5”C) during each experiment and was monitored by a thermistor embedded in the muscle. The perfusate was warmed by varying the current passedthrough a heating coil surrounding the perfusate tubing immediately prior to the cannula. Septa were paced throughout all experiments by a pair of platinum electrodes at 90 beats/min with pulses of 1OV lasting 5 ms (SRI, Croydon, UK). Solutions
The control solution contained (in mM): Na+ 142, K+ 5.0, Ca*+ 1.8; Mg*+ 1.0, Cl- 125, Hz Pod- 0.43, HCOs- 28, and D-ghCOSe 11.1 and was equilibrated with 95% Oz-5% CO*; pH was 7.39 at 35°C. The hypoxic substratefree perfusate contained an equimolar amount of n-mannitol to replace the glucose and was equilibrated with 95% Nz-5% CO*. LiCl (100 mM) replaced 100 mmol of NaCl in low Na+-Li+ solutions. LiCl (10Om~) and mannitol (10 mM) replaced 100 mmol of NaCl and 5 mmol of KC1 in low Na+-OK+-Li+ experiMaterials and Methods ments. Sucrose (210 mM) replaced 100 mmol Experimental preparation of NaCl and 5 mmol of KC1 in low Na+-OK+Experiments were performed on the isolated sucrose experiments. All chemicals were obarterially perfused interventricular septum of tained from Sigma, UK and amiloride from the rabbit (Harding and Poole-Wilson, 1980; Merck Sharp and Dohme (UK). Bourdillon and Poole-Wilson, 1981). Adult male New Zealand white rabbits were hepariIsotope uptake measurements nized and anaesthetized with sodium pentobarbitone. The hearts were removed and Septa were equilibrated for at least 60 min at immediately placed in a physiological solution 32°C. They were then warmed to 35°C for 30 bubbled with 95% Os-5% COz at room min before labelling was begun. Muscles were
Sodium-Calcium
Exchange
During
rejected if they did not develop at least -10 g of active tension with 10 g resting tension at 35°C. Isotope uptake was measured by placing the preparation 3 to 6 mm in front of a sodium iodide crystal (8 x 5 cm, type 1258/3E, Harshaw, Holland) attached to a counter (BIN, NM108, J and P Engineering, Reading, UK). The specific activity of 47Ca2+ (Radiochemical Centre, Amersham, UK) in all solutions in any one experiment was identical. Addition of isotope to the perfusate did not affect developed tension. EWuent drops fell freely into a lead shielded drop collector and were immediately removed from the experimental area to a reservoir over 1 m away behind a 15-cm lead shield. With this precaution the background counts remained constant during an experiment. Background counts were measured at the end of each experiment after removal of the septum from the apparatus. Septa weighed between 0.61 and 1.22 g (mean 0.88 + 0.15 g, n = 62). Analysis
Results Control experiments
Over 125 min of control perfusion (n = 4) resting tension remained constant and developed tension fell by 2.5 If: 0.8%. 47CaZ+ after 125 min perfusion was 16.0 + I.996 greater than that after 40 min perfusion (Table 1). EJect
1. Calcium
uptake
and
mechanical
(125
hypoxia and reoxygenation on 47Ca2 ’ uptake and mechanical function
function
Values
Control
of
The effects of 30 min (n = 4) and 45 min (n = 7) substrate-free hypoxia on mechanical function and 47 Ca2+ uptake (Fig. 1 and Table 1) were similar to those previously described (Harding and Poole-Wilson, 1980; PooleWilson et al., 1984). On exposure to hypoxic solution developed tension declined and was followed by a marked rise in resting tension during which time 47Ca2+ uptake remained constant or fell slightly. Reoxygenation after 30 min and 45 min hypoxia .resulted in an increased uptake of 47Ca + which was accompanied by a partial recovery of mechanical function (Fig. 1 and Table 1).
Uptake curves were normalized to the counts per minute at 40 min of labelling. Results are TABLE
1053
expressed as mean & S.E.M. Significance was tested using Student’s t-test for unpaired data.
data
of
Reoxygawtion
after 40 min labelling
Values after 40 min reoxygenation “{, Recovery DT
Increase in ct/min 47CaZ + as t/b of ct/min after 40 min labelling
n
RT
4
6.1 f
Li+”
4 4
6.6 f 1.2 6.5 + 1.2
18.3 f 17.9 f
1.1 1.6
13.7 f 1.5 14.3 + 1.7
53.5 f 7.1 51.5 f 6.2
45* 11 41 k6
min) min Li+b min) Li+” min) Li+d
7 6 6 8
6.8 6.3 6.0 6.3
17.3 17.7 18.2 16.7
0.6 1.0 0.7 0.5
19.9 18.2 17.9 19.3
30.9 20.7 15.9 24.8
73 40 69 79
5
6.4 + 1.7
1.6
24.5 + 3.0
min)
Hypoxia Hypoxia
(30 min) (30 min)
Hypoxia Hypoxia Hypoxia Hypoxia
(45 (45 (45 (45
Hypoxia (45 min) amiloride
k)
f + f f
0.3
0.4 0.3 1.1 0.3
DT
RT
k)
18.4 _+ 1.6
+ + f &-
20.2 f
k)
6.2 f 0.3
f + + +
1.2 2.0 1.6 1.2
97.5
+ 0.6
f + f +
6.4 6.1 3.1 5.1
14.9 + 2.4
16 + 2
* f + *
7 3’ 6 9
72 i
8
Results are expressed as mean f S.E.M. RT = resting tension. DT = developed tension, cpm = counts/min All interventions are beg-m after a 40 min labelling period. For control experiments measurements were made after 40 and 125 min of labelling. “lithium (100 mM) substitution 10 min before reoxygenation. b, ’ and d lithium (100 mM) substitution 15 min before hypoxia, and 40 and 10 min before reoxygenation, respectively. ‘P < 0.0 1 compared to hypoxia (45 min), ’ and d.
T. Crab
1054
and P. A. Poole-Wilson
hypoxia 1
no substrate
Mmutes
FIGURE 1. Theeffectofhypoxiaandsubstrate-free perfusion on 47Ca2+ uptake and tension. Developed tension fell, resting tension rose and on reoxygenation there was only partial recovery. During hypoxia, calcium uptake changed, partly due to alterations of the extracellular space. On reoxygenation an immediate uptake of calcium occurred. Mechanical function and tissue calcium
In 10 septa experimental conditions were chosen to increase calcium uptake by sodium-calcium exchange. The septa were perfused with a low Na+-OK+-sucrose solution for between 5 and 45 min after the initial labelling period. The perfusate was then switched back to the control solution.
5 0
Perfusion with the low Na+-OK+-sucrose solution caused a rapid increase in 47Ca2+ uptake (Fig. 2). This was accompanied by an initial transient increase in developed tension and a small fall in resting tension. Thereafter, a rapid rise in resting tension occurred and developed tension declined. On returning to the control perfusate, resting tension fell and
‘. 30
60
90
120
Minutes FIGURE
2. The effect of perfusion
of the septum
with a low Na+-OK+-sucrose
solution.
Sodium-Cal&sun
Exchange Dnring Reosygmation
there was a gradual recovery of developed tension. Mechanical recovery was inversely related to the increase of tissue 47Ca2+ (Fig. 3). A similar relationship was observed between recovery of mechanical function and tissue 47Ca2 + uptake on reoxygenation after hypoxia (Fig. 3).
“0 ; g 1 + ;
I 25-
1055
hypom-no
substrate
i
low No+-OK+-sucrose HM
20-
/~\~ .x-*
::---.a z
Hypoxia and sodium-calcium
0
exchange
Experiments were undertaken to investigate the effect of substrate-free hypoxia on sodium-calcium exchange. In four septa, after labelling for 40 min, substrate-free hypoxic solution was introduced. Net calcium uptake via sodium-calcium exchange was induced by exposure to an hypoxic, low Na+-OK+sucrose solution (Fig. 4). Tissue calcium increased and when substrate-free hypoxic solution was reintroduced tissue47Ca2+ ceased to rise. In three further septa net calcium uptake via dosium-calcium exchange was induced after the 40 min labelling period by perfusing with a low Na+-OK+-sucrose solution and substrate-free hypoxia was introduced while calcium influx was increased. Hypoxia did not affect the rate of increase in tissuecalcium (Fig. 5). These resultsshow that net calcium uptake via sodium-calcium exchange can be induced in responseto a change of the constituents of the extracellular fluid during substrate-free hypoxia and
0
60 l
;o
o 0 0 :
40
l
.
20
0
Oo
0
0
:
f 0 0
I 20
I 40 47,-.02+
I 60 -%
increase
I 80
I.
I
100
120
l
140
(ct/min)
FIGURE 3. The recovery ofdeveloped tension plotted against the increase of tissue calcium in control (A), hypoxic, substrate-free (0) or low Na+-OK+-sucrose conditions (0). The relationship was similar if the increase of tissue calcium was brought about by reoxygenation or sodium-calcium exchange.
I 30
I 60
I 90
I 120
Minutes
FIGURE 4. The effect of perfusion of the septum with a low Na+-OK+-sucrose solution during hypoxia. Hypoxia was begun at 40 min; a low Na+-OK+-sucrose perfusate was introduced at 50 min for 15 min and again at 85 min. The uptake of calcium could be stimulated during hypoxia. The uptake was slightly greater after the second exposure to low Na+-OK+-sucrose.
sodium-calcium exchange is not inhibited by short (20 min) periods of hypoxia. E#ect of lithium and low Na’ on 47Ca2+ uptake during normoxia
Four septa were perfused with a solution containing LiCl (100 mM) replacing an equimolar amount of NaCl for 30 min. Calcium uptake by the septum remained unchanged during this period and was similar to control experiments (Fig. 6). There wasan initial increasein developed tension followed by a small decline but developed tension remained above control whilst lithium was present. Return to the control perfusate resulted in a rapid fall in developed tension followed by recovery to control values. Experiments (Fig. 7) were undertaken to demonstrate that lithium does not substitute for Na+ in sodium-calcium exchange. After labelling, the septa were perfused with a low Na+-OK+-sucrose solution for 40 min during which time a marked increase in “Ca’+ uptake occurred. Associated with this was a lossof developed tension and an increase in resting tension (Fig. 7). The perfusate was then changed to low Na+-OK+-Li+ solution. The uptake of 47Ca2+ ceased and resting tension declined (Fig. 7). The perfusate was than ed back to low Na+-OK+-sucrose and 47Ca5+ uptake increased again as did resting tension.
T. Crake
1056
and P. A. Poole-Wilson
low Na+-OK+-sucrose
hypoxia-no
substrate
:....
0
I 60
I 30
-.
I 90
120
Minutes FIGURE 5. Calcium uptake was stimulated by perfusion Hypoxia had little or no effect on calcium uptake and tension
Eflect of lithium on 47Ca2+ uptake during hypoxia and reoxygenation
Three groups of experiments were performed (Figs 8-10). In the first group LiCl ( 100 mM) was substituted for 100 mM NaCl 15 min before the onset of substrate-free hypoxia and continued throughout the period of substratefree hypoxia and continued throughout the
of the septum record.
with
a low Na+-OK+-sucrose
solution.
period of substrate-free hypoxia and the first 20 min of reoxygenation (Fig. 8). In the second group lithium substitution was not made until the last 40 min of the hypoxic period and continued for 20 min of reoxygenation (Fig. 9). In the third group lithium substitution was begun 10 min before reoxygenation and continued for 20 min during
llthlum -
’
to-
(100 mm01 /I-‘)
’
“0x E E 2 -2
5-
N+ 3 t
4 l .
0
60 Minutes
FIGURE 6. Substitution of LiCl (100 mM) and NaCl (100 uptake but did cause a reversible increase of developed tension.
mM)
in the perfusate
had little
or no effect on calcium
low
Na+-OK+-
low
No+-
low Na+I OK+-sucrose
I sucrose
OK+-LI+
-
.*2
I:;.
Calcium
Exchange in Rabbit Myocardium Hypoxia: Role of Sodium-Calcium Tom Crake
and Philip
During and After Exchange
A. Poole-Wilson
Department of Cardiac Medicine, National Heart and Lung Institute and National Dovehouse Street, London S W3 6LY, UK
Heart Hospital,
(Received 9 September 1989, accepted in revised form 6 Februar_v 1990) AND P. A. POOLE-WILSON. Calcium Exchange in Rabbit Myocardium During and After Hypoxia: Role of Sodium-Calcium Exchange. Journal of Molecular and Cellular Curdiolo~ (1990) 22, 105 l-1064. Calcium uptake was measured using “‘Ca’+ in the isolated and arterially perfused interventricular septum of the rabbit. Experiments were undertaken to determine whether calcium uptake on reoxygenation is linked to recovery of mechanical function and whether calcium uptake is through the sodium-calcium exchange mechanism. During substrate-free hypoxia for 45 min total tissue calcium remained unchanged but immediately upon reoxygenation there was a substantial net gain of calcium. Recovery of mechanical function upon reoxygenation was inversely related to the increase in tissue calcium. Activation of sodium-calcium exchange by perfusion with a lowsodium, zero-potassium, sucrose solution also increased tissue calcium and the relation to mechanical recovery was similar to that observed on reoxygenation. The sodium-calcium exchange mechanism was not affected by hypoxia and could be demonstrated during perfusion with a substrate-free hypoxic solution. Lithium (100 rnM! substitution for sucrose prevented the calcium influx induced by a low-sodium and zero-potassium perfusate under normoxic conditions. Lithium substitution early during hypoxia or on reoxygenation did not affect the increase in myocardial calcium on reoxygenation. Amiloride ( low4 M), presumed to inhibit sodium-hydrogen exchange during hypoxia, had no effect upon reoxygenation induced calcium uptake. It is concluded that the increase ill calcium uptake that occurs on reoxygenation after a period of substrate-free hypoxia is related to mechanical recovery. Sodium-calcium exchange may contribute to calcium uptake on reoxygenation in this experimental model but is not the major mechanism.
T. CRAKE
KEY WORDS:
Reoxygenation;
Sodium-calcium
exchange;
Introduction The sequenceof events resulting in cell necrosis during or after a period of ischaemia or hypoxia in heart muscle is relevant to current therapies for myocardial ischaemia, such as thrombolysis and angioplasty. Reperfusion (Shen and Jennings, 1972; Bourdillon and Pool-Wilson, 1981; Cheung et al., 1986) or reoxygenation (Nayler et al., 1979; Harding and Poole-Wilson, 1980) after a period of ischaemia or hypoxia is associated with an increase in tissuecalcium, diminished recovery of mechanical function and eventual necrosis of at least part of the myocardium. When experimental conditions, such as temperature and the availability of substrate, are manipulated so that recovery of mechanical function is complete, tissuecalcium does not increase (Nayler et al., 1979; Harding and * Please address all correspondence 0022-2828/90/101051
+ 14 $03.00/O
Myocardium.
Poole-Wilson, 1980; Poole-Wilson et al. 1 1984). Several authors have proposed a causal link between calcium accumulation and cell injury (Shen and Jennings, 1972; Nayler et al., 1979; Fleckenstein et al., 1984). The gain in tissue calcium is similar on reperfusion and reoxygenation and is a consequence of an increased calcium influx rather than a reduction of efflux (Harding and PooleWilson, 1980; Bourdillon and Poole-Wilson, 1982). Many mechanismsfor the gain in tissue calcium (Poole-Wilson et al., 1984) have been proposed including, sarcolemmal disruption (Steenbergen et al., 1985), lipid peroxidation (Mak et al., 1986), radical damage (Thompson and Hess, 1986) and sodium-calcium exchange (Grinwald, 1982; Renlund et al., 1984; Lazdunski et al., 1985). Intracellular sodium concentration doesnot
to: P. A. Poole-Wilson. (0 1990 Acadrmic
Press Limited
1052
T. Crake and P. A. Poole-Wilson
increaseearly during hypoxia when developed tension is partially reduced and resting tension unchanged (Nakaya et al., 1985; Guarnieri, 1987; Ellis and Noireaud, 1987) but after more prolonged hypoxia or ischemia intracellular sodium concentration is elevated (Fiolet et al., 1984; Wilde and Kleber, 1986; Poole-Wilson and Tones, 1986; Grinwald and Brosnaham, 1987; Guarnieri, 1987). The mechanismsfor the increaseof cellular sodium include inhibition of the sodium pump, increased permeability of the sarcolemma to sodium and stimulation of sodium-hydrogen exchange by an intracellular acidosis. Sodium-calcium exchange appears to be inhibited during prolonged hypoxia because total tissue calcium does not increase (Harding and Poole-Wilson, 1980) although the cytosolic calcium may be elevated late in hypoxia (Allen and Orchard, 1983; Allen and Smith, 1985; Guarnieri, 1987). On reoxygenation sodium-calcium exchange and sodium-hydrogen exchange could be reactivated and possibly account for the observed immediate uptake of calcium ( (Lazdunski et al., 1985; Allen and Orchard, 1987). Alteration of extracellular sodium is known to modify the recovery of myocardial function on reperfusion (Grinwald, 1982; Renlund et al., 1984). This study was designed to investigate whether a causal relationship exists between myocardial calcium uptake on reoxygenation and subsequent mechanical recovery and to elucidate the role of sodium-calcium exchange in an experimental model where resting tension increasesduring hypoxia, mechanical recovery on reoxygenation is incomplete and the uptake of calcium on reoxygenation has been well characterized.
temperature. A fine polythene cannula was inserted into the septal artery within 2 to 4 min of heart excision and the septum perfused with control solution. The septum was dissected from the right and left ventricular free walls and underperfused tissue was removed. The remaining triangular piece of tissuewas clamped at its basebetween opposing pairs of forceps and the apex wasattached to a tension transducer (UC4 Statham Instruments, Oxnard, CA). Septa were perfused with solution pumped by a peristaltic pump (Watson-Marlow, Falmouth, UK) and the flow, measured by timing and weighing effluent drops, was maintained constant for each experiment (mean 1.96 ) 0.07 ml/min/g wet tissue,range 1.28 to 2.64 mI/min/g) . Temperature was maintained constant (+0,5”C) during each experiment and was monitored by a thermistor embedded in the muscle. The perfusate was warmed by varying the current passedthrough a heating coil surrounding the perfusate tubing immediately prior to the cannula. Septa were paced throughout all experiments by a pair of platinum electrodes at 90 beats/min with pulses of 1OV lasting 5 ms (SRI, Croydon, UK). Solutions
The control solution contained (in mM): Na+ 142, K+ 5.0, Ca*+ 1.8; Mg*+ 1.0, Cl- 125, Hz Pod- 0.43, HCOs- 28, and D-ghCOSe 11.1 and was equilibrated with 95% Oz-5% CO*; pH was 7.39 at 35°C. The hypoxic substratefree perfusate contained an equimolar amount of n-mannitol to replace the glucose and was equilibrated with 95% Nz-5% CO*. LiCl (100 mM) replaced 100 mmol of NaCl in low Na+-Li+ solutions. LiCl (10Om~) and mannitol (10 mM) replaced 100 mmol of NaCl and 5 mmol of KC1 in low Na+-OK+-Li+ experiMaterials and Methods ments. Sucrose (210 mM) replaced 100 mmol Experimental preparation of NaCl and 5 mmol of KC1 in low Na+-OK+Experiments were performed on the isolated sucrose experiments. All chemicals were obarterially perfused interventricular septum of tained from Sigma, UK and amiloride from the rabbit (Harding and Poole-Wilson, 1980; Merck Sharp and Dohme (UK). Bourdillon and Poole-Wilson, 1981). Adult male New Zealand white rabbits were hepariIsotope uptake measurements nized and anaesthetized with sodium pentobarbitone. The hearts were removed and Septa were equilibrated for at least 60 min at immediately placed in a physiological solution 32°C. They were then warmed to 35°C for 30 bubbled with 95% Os-5% COz at room min before labelling was begun. Muscles were
Sodium-Calcium
Exchange
During
rejected if they did not develop at least -10 g of active tension with 10 g resting tension at 35°C. Isotope uptake was measured by placing the preparation 3 to 6 mm in front of a sodium iodide crystal (8 x 5 cm, type 1258/3E, Harshaw, Holland) attached to a counter (BIN, NM108, J and P Engineering, Reading, UK). The specific activity of 47Ca2+ (Radiochemical Centre, Amersham, UK) in all solutions in any one experiment was identical. Addition of isotope to the perfusate did not affect developed tension. EWuent drops fell freely into a lead shielded drop collector and were immediately removed from the experimental area to a reservoir over 1 m away behind a 15-cm lead shield. With this precaution the background counts remained constant during an experiment. Background counts were measured at the end of each experiment after removal of the septum from the apparatus. Septa weighed between 0.61 and 1.22 g (mean 0.88 + 0.15 g, n = 62). Analysis
Results Control experiments
Over 125 min of control perfusion (n = 4) resting tension remained constant and developed tension fell by 2.5 If: 0.8%. 47CaZ+ after 125 min perfusion was 16.0 + I.996 greater than that after 40 min perfusion (Table 1). EJect
1. Calcium
uptake
and
mechanical
(125
hypoxia and reoxygenation on 47Ca2 ’ uptake and mechanical function
function
Values
Control
of
The effects of 30 min (n = 4) and 45 min (n = 7) substrate-free hypoxia on mechanical function and 47 Ca2+ uptake (Fig. 1 and Table 1) were similar to those previously described (Harding and Poole-Wilson, 1980; PooleWilson et al., 1984). On exposure to hypoxic solution developed tension declined and was followed by a marked rise in resting tension during which time 47Ca2+ uptake remained constant or fell slightly. Reoxygenation after 30 min and 45 min hypoxia .resulted in an increased uptake of 47Ca + which was accompanied by a partial recovery of mechanical function (Fig. 1 and Table 1).
Uptake curves were normalized to the counts per minute at 40 min of labelling. Results are TABLE
1053
expressed as mean & S.E.M. Significance was tested using Student’s t-test for unpaired data.
data
of
Reoxygawtion
after 40 min labelling
Values after 40 min reoxygenation “{, Recovery DT
Increase in ct/min 47CaZ + as t/b of ct/min after 40 min labelling
n
RT
4
6.1 f
Li+”
4 4
6.6 f 1.2 6.5 + 1.2
18.3 f 17.9 f
1.1 1.6
13.7 f 1.5 14.3 + 1.7
53.5 f 7.1 51.5 f 6.2
45* 11 41 k6
min) min Li+b min) Li+” min) Li+d
7 6 6 8
6.8 6.3 6.0 6.3
17.3 17.7 18.2 16.7
0.6 1.0 0.7 0.5
19.9 18.2 17.9 19.3
30.9 20.7 15.9 24.8
73 40 69 79
5
6.4 + 1.7
1.6
24.5 + 3.0
min)
Hypoxia Hypoxia
(30 min) (30 min)
Hypoxia Hypoxia Hypoxia Hypoxia
(45 (45 (45 (45
Hypoxia (45 min) amiloride
k)
f + f f
0.3
0.4 0.3 1.1 0.3
DT
RT
k)
18.4 _+ 1.6
+ + f &-
20.2 f
k)
6.2 f 0.3
f + + +
1.2 2.0 1.6 1.2
97.5
+ 0.6
f + f +
6.4 6.1 3.1 5.1
14.9 + 2.4
16 + 2
* f + *
7 3’ 6 9
72 i
8
Results are expressed as mean f S.E.M. RT = resting tension. DT = developed tension, cpm = counts/min All interventions are beg-m after a 40 min labelling period. For control experiments measurements were made after 40 and 125 min of labelling. “lithium (100 mM) substitution 10 min before reoxygenation. b, ’ and d lithium (100 mM) substitution 15 min before hypoxia, and 40 and 10 min before reoxygenation, respectively. ‘P < 0.0 1 compared to hypoxia (45 min), ’ and d.
T. Crab
1054
and P. A. Poole-Wilson
hypoxia 1
no substrate
Mmutes
FIGURE 1. Theeffectofhypoxiaandsubstrate-free perfusion on 47Ca2+ uptake and tension. Developed tension fell, resting tension rose and on reoxygenation there was only partial recovery. During hypoxia, calcium uptake changed, partly due to alterations of the extracellular space. On reoxygenation an immediate uptake of calcium occurred. Mechanical function and tissue calcium
In 10 septa experimental conditions were chosen to increase calcium uptake by sodium-calcium exchange. The septa were perfused with a low Na+-OK+-sucrose solution for between 5 and 45 min after the initial labelling period. The perfusate was then switched back to the control solution.
5 0
Perfusion with the low Na+-OK+-sucrose solution caused a rapid increase in 47Ca2+ uptake (Fig. 2). This was accompanied by an initial transient increase in developed tension and a small fall in resting tension. Thereafter, a rapid rise in resting tension occurred and developed tension declined. On returning to the control perfusate, resting tension fell and
‘. 30
60
90
120
Minutes FIGURE
2. The effect of perfusion
of the septum
with a low Na+-OK+-sucrose
solution.
Sodium-Cal&sun
Exchange Dnring Reosygmation
there was a gradual recovery of developed tension. Mechanical recovery was inversely related to the increase of tissue 47Ca2+ (Fig. 3). A similar relationship was observed between recovery of mechanical function and tissue 47Ca2 + uptake on reoxygenation after hypoxia (Fig. 3).
“0 ; g 1 + ;
I 25-
1055
hypom-no
substrate
i
low No+-OK+-sucrose HM
20-
/~\~ .x-*
::---.a z
Hypoxia and sodium-calcium
0
exchange
Experiments were undertaken to investigate the effect of substrate-free hypoxia on sodium-calcium exchange. In four septa, after labelling for 40 min, substrate-free hypoxic solution was introduced. Net calcium uptake via sodium-calcium exchange was induced by exposure to an hypoxic, low Na+-OK+sucrose solution (Fig. 4). Tissue calcium increased and when substrate-free hypoxic solution was reintroduced tissue47Ca2+ ceased to rise. In three further septa net calcium uptake via dosium-calcium exchange was induced after the 40 min labelling period by perfusing with a low Na+-OK+-sucrose solution and substrate-free hypoxia was introduced while calcium influx was increased. Hypoxia did not affect the rate of increase in tissuecalcium (Fig. 5). These resultsshow that net calcium uptake via sodium-calcium exchange can be induced in responseto a change of the constituents of the extracellular fluid during substrate-free hypoxia and
0
60 l
;o
o 0 0 :
40
l
.
20
0
Oo
0
0
:
f 0 0
I 20
I 40 47,-.02+
I 60 -%
increase
I 80
I.
I
100
120
l
140
(ct/min)
FIGURE 3. The recovery ofdeveloped tension plotted against the increase of tissue calcium in control (A), hypoxic, substrate-free (0) or low Na+-OK+-sucrose conditions (0). The relationship was similar if the increase of tissue calcium was brought about by reoxygenation or sodium-calcium exchange.
I 30
I 60
I 90
I 120
Minutes
FIGURE 4. The effect of perfusion of the septum with a low Na+-OK+-sucrose solution during hypoxia. Hypoxia was begun at 40 min; a low Na+-OK+-sucrose perfusate was introduced at 50 min for 15 min and again at 85 min. The uptake of calcium could be stimulated during hypoxia. The uptake was slightly greater after the second exposure to low Na+-OK+-sucrose.
sodium-calcium exchange is not inhibited by short (20 min) periods of hypoxia. E#ect of lithium and low Na’ on 47Ca2+ uptake during normoxia
Four septa were perfused with a solution containing LiCl (100 mM) replacing an equimolar amount of NaCl for 30 min. Calcium uptake by the septum remained unchanged during this period and was similar to control experiments (Fig. 6). There wasan initial increasein developed tension followed by a small decline but developed tension remained above control whilst lithium was present. Return to the control perfusate resulted in a rapid fall in developed tension followed by recovery to control values. Experiments (Fig. 7) were undertaken to demonstrate that lithium does not substitute for Na+ in sodium-calcium exchange. After labelling, the septa were perfused with a low Na+-OK+-sucrose solution for 40 min during which time a marked increase in “Ca’+ uptake occurred. Associated with this was a lossof developed tension and an increase in resting tension (Fig. 7). The perfusate was then changed to low Na+-OK+-Li+ solution. The uptake of 47Ca2+ ceased and resting tension declined (Fig. 7). The perfusate was than ed back to low Na+-OK+-sucrose and 47Ca5+ uptake increased again as did resting tension.
T. Crake
1056
and P. A. Poole-Wilson
low Na+-OK+-sucrose
hypoxia-no
substrate
:....
0
I 60
I 30
-.
I 90
120
Minutes FIGURE 5. Calcium uptake was stimulated by perfusion Hypoxia had little or no effect on calcium uptake and tension
Eflect of lithium on 47Ca2+ uptake during hypoxia and reoxygenation
Three groups of experiments were performed (Figs 8-10). In the first group LiCl ( 100 mM) was substituted for 100 mM NaCl 15 min before the onset of substrate-free hypoxia and continued throughout the period of substratefree hypoxia and continued throughout the
of the septum record.
with
a low Na+-OK+-sucrose
solution.
period of substrate-free hypoxia and the first 20 min of reoxygenation (Fig. 8). In the second group lithium substitution was not made until the last 40 min of the hypoxic period and continued for 20 min of reoxygenation (Fig. 9). In the third group lithium substitution was begun 10 min before reoxygenation and continued for 20 min during
llthlum -
’
to-
(100 mm01 /I-‘)
’
“0x E E 2 -2
5-
N+ 3 t
4 l .
0
60 Minutes
FIGURE 6. Substitution of LiCl (100 mM) and NaCl (100 uptake but did cause a reversible increase of developed tension.
mM)
in the perfusate
had little
or no effect on calcium
low
Na+-OK+-
low
No+-
low Na+I OK+-sucrose
I sucrose
OK+-LI+
-
.*2
I:;.
