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Potassium loss from rabbit myocardium during hypoxia: Evidence for passive efflux linked to anion extrusion
j Mol Cardio118, 389-399 (1986)
P o t a s s i u m Loss From Rabbit Myocardinm During Hypoxia: Evidence for Passive Efflux Linked to Anion Extrusion Achille Gaspardone, Kenneth I. Shine, Stephen R. Seabrooke and philip A. Poole-Wilson*
Cardiothoracic Institute, 2 Beaumont Street, London, UK ( Received 5 August 1985, accepted in revisedform 24 December 1985) A. GASPARDONE,K. I. SHINE, S. R. SEABROOKE,AND P. A. POOLE-WILSON.Potassium Loss from Rabbit Myocardium During Hypoxia: Evidence for Passive Effiux Linked to Anion Extrusion. journal of Molecularand Cellular Cardiology(1986) 18, 389 399. To determine the effects of permeant and impermeant anions and of osmolarity on potassium (K +) e~xc~ange, the net uptake and effiux of 42K+ were recorded in the isolated arterially perfused rabbit septum. Perfusion With solution made hyperosmolar by adding NaCl (30 mM) or sucrose (60 mM):cauSed similar ii6~r~s~'of '*2~K+ uptake which were reversible on returning to the control solution. WashoUf experiments showed that the loss of K + on returning to the control perfusate was due to a decreased inflffX probably mediated by inhibition of the sodium pump. The effects of anions were studied by replacing chloride in the control solution with the inert and impermeant substitute isethionate (114 mM) or by loading the myocardium with sodium dlmethyloxazeolidinedione" (NaDMO, 30 mM) under isosmotic.condition42 and switching tO a perfusate Containing sodium isethionate (30 mM). In both these conditions a reducuon of K content could be detected and was attributable to an increased efllux. During hypoxic substrate free perfusion K + loss was due to an increased efflux with no evidence for altered influx of potassium. The extrusion of accumulated anions from the myo~ardium could be the major determinant of the early potassium loss during hypoxia and ischaemia. KEy WORDS: Myocardium; Potassium; Perfused septum; Hypoxia; Anions; Osmolarity.
Introduction I n a n i m a l s a n d in m a n p o t a s s i u m is lost f r o m h y p o x i c [4, 18, 32, 33] or i s c h a e m i c m y o c a r d i u m [6, 10, 37, 41, 42, 45]. T h e increase o f the e x t r a c e l l u l a r p o t a s s i u m c o n c e n t r a t i o n in ischa e m i a ( [ K + ] 0 ) is c h a r a c t e r i s e d by a triphasic t i m e course [15, 45]. A n increase o f [ K +]0 is d e t e c t a b l e w i t h i n seconds after i n t e r r u p t i o n o f m y o c a r d i a l perfusion a n d reaches a p p r o x i m a t e l y 15 mmol/1 witfiin 10 mins. A f t e r a f u r t h e r 10 mins, d u r i n g w h i c h [ K + ] 0 is c o n s t a n t ( p l a t e a u phase), a second progressive increase of p o t a s s i u m occurs. T h e initial rise in [ K + ] o is r a p i d , reversible a n d r e p r o d u c i b l e [32]. T h e s e features suggest t h a t the e v e n t is f u n c t i o n a l in n a t u r e . I n c o n t r a s t the second
rise in [ K + ] 0 is associated w i t h irreversible loss o f m y o c a r d i a l f u n c t i o n a n d w i t h cell d e a t h [31, 32]. T h e increase o f [ K + ] o in hypoxic or ischaemic myocardium probably a c c o u n t s for m a n y of the initial c h a n g e s in the a c t i o n p o t e n t i a l [13, 17, 31, 43, 44]. T h e e a r l y p o t a s s i u m loss is d u e to a n i n c r e a s e d effiux [15, 32], is n o t closely linked to the e n e r g e t i c state o f the m y o c a r d i u m [14, 32], is n o t solely d u e to i n h i b i t i o n o f the s o d i u m p u m p [14, 15, 32, 37], a n d is n o t d u e to the cell m e m b r a n e b e c o m i n g m o r e p e r m e a b l e to p o t a s s i u m a l o n e [31]. A possible m e c h a n i s m w h i c h m i g h t e x p l a i n the loss o f p o t a s s i u m d u r i n g h y p o x i a a n d isc h a e m i a has b e e n suggested by the analysis o f
Achille Gaspardone's present address: Malattie Dell'Apparato Cardiovascolare, Cattedra II, Universita' di Roma, Policlinico Umberto 1, 00161 Rome, Italy. Kenneth Shine's present address: University of California Los Angeles, Center for the Health Sciences, Los Angeles, California 90024, USA. * To whom correspondence should be addressed. 0022-2828/86/040389 + 11 $03.00/0
9
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A. Gaspardone et al.
the ions in the venous effluent during restriction of coronary flow. The intracellular accumulation of lactate, phosphate and hydrogen ions in the myocardium and the efflux of the ions into the extracellular space [3, 20] occurs at the same time as the loss of potassium from the myocardium and the rise of [K+]0 [7]. The magnitude of the loss of lactate and phosphate ion is greater than the loss of potassium [3, 20]. Mainwood and Lucier suggested that the efflux of lactate in fatigued frog skeletal muscle was linked to the efflux of potassium [19]. During hypoxia and ischaemia weak acids are generated by glycolysis and from the breakdown of energy rich phosphates [23]. The protons so generated are buffered by fixed negative charges ofintracellular proteins [9]. As a result of the consequent rise in the intracellular concentration of freely movable anions an efflux of permeant anions with a cation to maintain cellular electroneutrality is to be expected [2, 14, 15, 35, 36]. Potassium is the commonest cation in the myocardium. The production of weak acids during hypoxia and ischaemia will also increase intracellular osmolarity [39] which is known to affect potassium exchange [5, 24-27, 38, 39]. We have investigated the effects of permeant and impermeant anions and hyperosmolarity on potassium fluxes in isolated heart muscle in an attempt to determine the relative importance of the two factors in causing potassium loss during hypoxia and ischaemia.
Materials and Methods
Perfused septal preparation The experimental preparation was the isolated arterially perfused interventricular septum of the rabbit, that has been described previously [16, 29]. Adult male New Zealand white rabbits, weighing 1.5 to 2 kg, were heparinised (10 000 U heparin) and anaesthetised (180 mg sodium pentobarbitone). The thorax was opened rapidly. The heart was excised and placed in warm oxygenated perfusate. A polyethylene cannula was inseted into the septal artery within 3 to 5 mins. The septum was dissected from the right and left ventricular
free walls and underperfused tissue excised. The base of the triangular piece of tissue was clamped between two opposing forceps mounted on geared slides with the cannula to the lower left. The apex was connected by a silk suture to a strain gauge transducer (UC 4, Statham Instruments, CA, USA). Tension was recorded (M4 Devices, Welwyn Garden City, UK). Septa were perfused with a roller-pump (Watson-Marlow, Falmouth, Cornwall) and flow was measured by timing and weighing effluent drops of constant size falling from a specially designed rod placed under the muscle. Flow was maintained constant in each experiment and averaged 1.9 ___0.2 ml/g/min (n = 58). The perfusate was warmed by means of a heating coil immediately adjacent to the cannula. The temperature of the tissue was measured by a needle thermister and kept constant during each experiment at 30.5 _+ 0.5~ (n = 58). Septa were stimulated by a pair of platinum electrodes with pulses of 10 V lasting 5 ms (S.R.I., Croydon, Surrey). The stimulation rate ranged from 85 to 95 beats/min and was kept constant during each experiment. Septa weighed between 0.54 and 1.1 g.
Solutions and chemicals The control perfusate contained (mM): NaC1, 114; KC1, 5; NaHCO3, 28; NaH2PO42H20 , 0.435; MgCI26H20 , 1 ; CaC12, 1.8; D-Glucose, 11.1 (Analar, BDH Chemicals, UK). 5,5-dimethyl-2,4-oxazolidinedione (DMO) was obtained from Eastman. Sodium isethionate was obtained from Sigma. In chloride depleted solutions an equimolar concentration of isethionate (114 and 30 mM) was used to maintain isomolarity. When D M O was used an appropriate reduction of the chloride concentration of the perfusate was made to avoid changes in osmolarity. Solutions were prepared with the constituents shown in Table 1. After equilibration of the solutions with 95% 0 2 - 5 % CO2, the final pH was 7.4. The hypoxic substrate-free solution contained an equimolar concentration of Dmannitol to replace glucose and was equilibrated with a gas mixture containing 95% N 2 and 5% CO 2. *2K+ (Radiochemical
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Centre, Amersham) was added directly to the experimental solutions.
Measurements and experimentalprocedures Developed tension (DT) and resting tension (RT) were continuously recorded (M4 Devices, Welwyn Garden City, UK) at a known stimulation rate and temperature. Muscles were only used when the initial developed tension was greater than 10 g at a resting tension of no greater than 5 g at 30.5~ and at a stimulation rate of 85 to 95 beats/rain. A period of at least 90 min was allowed for equilibration of the septa. Addition of the isotope to the perfusate had no effect on tension. Tissue 42K+ during uptake studies was recorded with a sodium iodide crystal (8.5 cm diameter) (Harshaw NaI, Harshaw Chemic B.V., Holland) and counted (J & P Engineering, Reading, UK). Septa were placed 3 to 6 mm from the NaI crystal. Effluent drops fell freely into a lead-shielded drop collector and were immediately returned to a reservoir 1 m away and behind 15 cm of lead. Counts per minute (ct/min) from septa were recorded over each minute, corrected for decay and background and plotted. Background counts remained constant and were measured at the end of the experiment after removal of the muscle from the experimental area. The technique has been described previously [16, 29]. In isotopic uptake experiments the tissue was labelled with 42K for 120 to 150 min at which time tissue radioactivity had reached about 95% of that at the asymptote. The specific activities of the 42K solutions used in any experiment were identical. At the end of each experiment radioactivity in both weighed samples of perfusate and the whole septa was measured (Tracerlab Spectro-Matic, MA, USA). The potassium concentration in mmol/kg wet tissue was calculated from the specific activity of the perfusate affd the activity of the~:muscle'at the end of the uptake studies. It was then possible to calibrate the uptake curve in terms of K + labelled with isotope. The absolute and percentage changes during interventions were calculated.. For isotopic washout experiments muscles were labelled with 42K+ containing solutions for 30 to 50 min. Washout was started with
non isotopic solution and after 20 min of perfusion, to permit accurate determination of the rate of potassium exchange, the experimental intervention was induced. During washout of 42K+ from a septum 4 or 5 effluent drops were collected in individual planchets. The time required for formation of each set of drops was recorded. The isotopic activity of each sample was counted (Model 3380 Tricarb, Packard) after the addition of 10 cm a of water by Cerenkov counting [8]. The counts were corrected for background and decay and expressed as ct/min of perfusate flow, to account for any slight changes in the flow rate. The value was plotted semilogarithmically against time from the start of the washout. The tissue activity was also monitored during washout experiments, corrected for background and decay, and plotted semilogarithmically against time from the start of the washout as ct/min. These procedures have been described previously [16, 29]. At the end of each experiment, septa were removed, excess fluid lightly blotted away and the tissue weighed. Total tissue water was obtained by drying to constant weight in an oven for 36 h at 95~ and reweighing. In washout experiments the net potassium loss due to increased efflux was calculated by integrating the area under the altered effluent curve during interventions [16]. A fall in the tissue content of 42K + could be due either to an increased efflux or a reduced uptake. If the fall in tissue 42K+ could be accounted for by the measured loss during a washout experiment then the fall of tissue 4eK+ was attributed to an increased efflux. Results are expressed as mean + SEM. Differences = betWeen groups of results were analysed using the unpaired Student's t test
Results
Potassium exchangeduring hypoxia The mean tissue potassium content of 12 septa perfused for 4 to 6 h was calculated to be 59.3 + 2.5 mmol K+/kg wet tissue. After hypoxia for 15 min the mean net loss of potassium detected in uptake experiments was 7.2 + 1.1 mmol K+/kg wet tissue ( n = 6) (Fig. 1). The effects of 15 rains of hypoxia on the efflux of potassium were evaluated in five
Potassium
,
Hyper NaCI HyperNoCI
,
i
Tension
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septa by inducing h y p o x i a d u r i n g the washout (Fig. 2). T h e increased efflux of 42K+ d u r i n g washout was calculated to represent 6.5__+0.1 mmol K + / k g wet tissue which was not significantly different from the net loss of potassium measured in the u p t a k e experiments (P = 0.57). Thus, the net potassium loss d u r i n g h y p o x i a could be a t t r i b uted to increased efflux o f t h e ion and not to a r e d u c e d uptake. ~,.~,~ After 15 min of h y p o x i a D T fell to 38 __+ 1.9% of the control. R T rose from 6.3 -t- 0.1 to 10.8 -I- 0.3 g (n = 11). R e o x y g e n ation i n d u c e d a r a p i d recovery in D T a n d a m a r k e d decrease in R T (Figs 1 and 2).
Potassium exchange duringperfusion with hyperosmolar NaCl (30 mM) and sucrose (60 raM) solutions After 15 min perfusion with h y p e r o s m o l a r NaC1 (30 mM) a m e a n net gain of 42K+ was observed and calculated to represent 5.9 __t_0.4 m m o l K + / k g wet tissue (n = 6). O n reperfusion with control solution (Fig. 3) recovery to the control steady state was achieved within 15 min. 1000 ['-
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20 mm FIGURE 1. Effects ofhypoxia on tissue 42K+ content and on mechanical function.
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Hypoxia
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Loss During Hypozia
T h e effects of 15 min perfusion with hyperosmolar NaC1 (30 mM) on the loss of potassium in the effluent were evaluated in four septa d u r i n g washout. All studies failed to reveal any change of potassium effiux d u r i n g h y p e r o s m o l a r perfusion (Fig. 4). T h e perfusion with h y p e r o s m o l a r NaC1 (30 mM) solution caused an i m m e d i a t e 12.4 _ 0.4% decline in D T followed by a progressive increase in D T up to 5 . 6 - t - 0 . 3 % a b o v e the control value. Reperfusion with control solution caused a 14.5_t_+0.3% increase in D T foll6wed by a progressive decline to the control level. These effects were reversible a n d r e p r o d u c i b l e (Figs 3 and 4). After 15 min of h y p e r o s m o l a r sucrose (60 mM) perfusion the m e a n net gain of potassium was 5.3 _+ 0.2 mmol K + / k g wet tissue (n = 6). T h e gain was reversed within 15 mins of perfusion with the control solution (Fig. 5). I n four washout studies no change of 42K efflux was observed (Fig. 6). Perfusion with h y p e r o s m o l a r sucrose (60 mM) caused an i m m e d i a t e decline in D T (13.8-t- 0.31) and in R T (4.9 _ 0.5%) followed by a progressive increase in D T to 14.5 __+0.3% above the control value. R e p e r -
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FIGURE 3. Perfusion with hyperosmolar NaC1 solution (see Table 1) causes a reversible net gain of tissue 42K+"
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FIGURE 2. Effects of 20 mins of hypoxia on 42K+ efflux. Simultaneous records of total tissue 42K+ (upper plot, right ordinate), effluent 42K+ activity (lower plot, left ordinate) and mechanical performance are presented.
7I
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FIGURE 4. Perfusion with hyperosmolar NaCl solution (see Table 1) does not alter a2 K + efflux.
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~o~i~ F I G U R E 5. Peffusion with hyperosmolar sucrose solution (see Table 1) causes a reversible net gain of tissue 42K+.
20 rain' FIGURE 7. Perfusion with isosmolar NaIse (sodium isethionate) solution (114 mM) (see Table 1) causes a reversiblenet lossof tissue 42K+. I000
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fusion with control solution caused a rapid increase in D T of 9.7 4- 0.4% followed by a progressive recovery to the control values (Figs 5 and 6).
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The effects of perfusion with sodium isethionate (NaIse, 114 mM) were evaluated in uptake studies. After 15 min the mean net toss of 42K+ was 5.4 _ 0.3 mmol K + / k g wet tissue (n = 6). The loss was reversed in 10 min (Fig. 7). In washout experiments the increased appearance of r in the effluent effiux (Fig. 8) was calculated to represent 4.6 4- 0.4 mmol K + / k g wet tissue (n = 5). The loss due to increased efflux was not significantly different from the net loss calculated from the uptake experiment (P = 0.115). Perfusion with isosmolar NaIse (114 mM) solution in seven experiments caused an increase in D T 7.8 + 0.4% and a decrease in R T 9.1 4-0.2%. These effects were rapidly reversible on reperfusion with control solution and reproducible (Figs 7 and 8). In four
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....................
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Potassium exchange during perfusion with isosmolar chloride depleted solutions (dValse 114 and 30 mM)
Hyper sucrose 60
.
1
IOmin F I G U R E 8. Perfusion with isosmolar NaIse solution (114 mM) (see Table 1) causes an immediate increase in 42K+ effiux.
experiments mechanical function was not affected by the intervention. In three uptake and four washout studies no alteration of K + exchange due to the presence of isosmolar NaIse (30 mM) solution (Figs 9 and 10) was observed. Perfusion with isosmolar NaIse (30 mM) solution had no effect on mechanical performance (Figs 9 and 10).
Potassium exchange duringperfusion with isosmolarNaDMO (30 mm) solution and isosmolar NaIse (30 mM) solution
In uptake studies muscles were labelled 7100 ~ durin perfusion with isosmolar N a D M O (30 t * mM). After labelling to asymptote, perfusion ~~ was switched to NaIse (30 raM). A mean net Tissue I0
•
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10 rain F I G U R E 6. Perfusion with hyperosmolar sucrose solution (see Table 1) does not alter 42K+ efflux.
F I G U R E 9. Peffusion with isosmolar NaIse solution (30 mM) (see Table 1) does not alter 42K+ uptake.
P o t a s s i u m L o s s During Hypoxia
....................
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jisoNa Ise 30 i
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FIGURE 10. Perfusion with isosmolar NaIse solution (30 mM) (see Table l) does not alter *2K+ efflux. so
~" 0
+
#
so
so
,
iso
asc
iso
so
NaDMO30 Na se3OINoDM030 NO Ise30 NaDMO3OIN~ Ise30 NoDM030
FIGURE 12. After loading the myocardium with '*2K+ in the presence ofisosmolar NaDMO (30 mM) the washout was started with isosmolar NaDMO (30 mM) unlabelled perfusate. After 20 mins the perfusate was switched to isosmolar NaIse (30 mM) solution. Perfusion with NaIse solution (30 mM) causes an immediate increase of*ZK + efllux.
25 E Tension
raM) solution. The eftlux of 42K+ was calculated to r e p r e s e n t 4 __+ 0.1 m m o l K + / k g wet
,Sg]
FIGURE 11. After loading the myocardium with • molar NaDMO solution (30 raM) (see Table 1) perfusion with isosmolar Nalse solution (30 raM) causes a reversible net loss of 42K + followed by recovery on return to perfusion with isosmolar NaDMO solution (30 raM). lOSS of 4.0 • 0.2 m m o l K + / k g wet tissue (n = 3) o c c u r r e d after 15 m i n (Fig. 11). T h e effects of 15 m i n perfusion with N a I s e (30 mM) on the effiux of p o t a s s i u m were e v a l u ated in five septa (Fig. 12). D u r i n g the labell i n g time a n d dae first p a r t o f w a s h o u t muscles were perfused with isosmolar N a D M O (30 TABLE 2.
tissue (n = 5) a n d was n o t significantly differe n t from the net loss observed in u p t a k e e x p e r i m e n t s (P = 0.82). N a I s e (30 raM) perfusion caused a 26 + 1% i m m e d i a t e increase in D T followed b y a progressive decline to the c o n t r o l value. O n reperfusion with N a D M O (30 raM) a r a p i d decline in D T 3 8 _ 2% was observed, followed b y a progressive recovery to c o n t r o l values. T h e effects were reversible a n d reprod u c i b l e (Figs 11 a n d 12). T h e effects of different i n t e r v e n t i o n s o n potassium e x c h a n g e are s u m m a r i s e d in T a b l e 2.
Potassium homeostasis 15 mins after the imposition of an intervention. Results are expressed in mmol/kg wet tissue (mean
• S.E.M.). Intervention
Uptake experiments K + loss (--) or gain ( + )
Hypoxia Isosmolar NaIse (t14 raM) Isosmolar D M O (30 mM)-Ise (30 mM) Hyperosmolar NaC1 (30 mM) Hyperosmolar sucrose (60 raM) Isosmolar Ise (30 mM)
Washout experiments K + efflux
-7.2 + 1 - 5 . 4 • 0.3
(n = 6) (n = 6)
6.5 • 0.14 4.6 • 0.4
(n = 5) (n = 5)
--4.0 • 0.2
(n = 3)
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0
(n = 4)
5.3 _ 0.16 (n = 6)
0
(n = 4)
0
(n = 3)
0
(n = 5)
Results are expressed in mmol/Kg wet tissue (mean -I- S.E.M.).
396
A. Gaspardone et aL
Discussion
The precise mechanism for the loss of potassium from ischaemic or hypoxic myocardium is not known. Several hypotheses have been put forward in recent years [31]. The loss has been attributed to inhibition of the sodium pump, acidosis, a selective increase in the permeability of the cell membrane, an A T P dependent potassium channel and the outward movement of intracellular anions. There are arguments against several of these mechanisms. The observation that the intracellular sodium remains unchanged at least over 15 mins ofischaemia in the guineapig heart [14] and that altering heart rate can cause the extracellular potassium both to rise and fall suggest that the sodium p u m p is at least partially intact [14, 15, 32]. Acidosis develops rapidly in both ischaemic and hypoxic myocardium but an imposed acidosis brought about by increasing the PCO z of the perfusate causes a gain of potassium, not a loss [29, 30]. Furthermore the alteration of potassium exchange in response to acidosis is not immediate but delayed for several minutes [29]. Potassium loss cannot be simply due to the cell membrane becoming more permeable to potassium. If that were so the membrane potential would initially become more negative and approach the equilibrium potential for potassium. In hypoxia and ischaemia the membrane is depolarised initially [3/] and the membrane potential only equals the potassium equilibrium potential later. The observation that ATP (adenosinetrisphosphate) is necessary to maintain a potassium channel closed has provided a basis for the proposition that potassium loss could be related to the energetic state of the cell [1, 12, 21], although other workers have been unable to link potassium efflux with the energetic state [14, 32]. A potassium channel regulated by ATP concentration has recently been described in guinea-pig ventricular cells. It was shown that depletion ofintracellular A T P induced a potassium outward current under a variety of experimental conditions [1, 21, 22] but only after a delay of several minutes. Potassium loss during hypoxia or ischaemia is an immediate event occurring within seconds
[15, 32, 37] and at that time there is almost no change in the total A T P content of the myocardium [11, 28]. Moreover even if a potassium channel were opened, it would cause the membrane potential to approach the equilibrium potential for potassium and not lead to immediate depolarisation as observed in hypoxia and ischaemia [3/]. We have tested two other possible mechanisms, first that the efflux of potassium is linked to the increase of osmolarity which occurs in ischaemic myocardium [39] and second that the potassium leaves the cell passively, accompanying the outward movement of anions such as lactate and phosphate [2, 15, 19]. During hypoxia or ischaemia weak acids will be generated intracellularly by the breakdown of high energy phosphates, glycolysis and glycogenolysis. Protons generated will be partly buffered by the negative charges on intracellular proteins leaving a permanent anion to diffuse out of the cell. In order to maintain approximate electroneutrality a cation, potassium, will also be lost from the cell. The experimental preparation used in the present study was the arterially perfused interventricular septum of the rabbit. Preliminary experiments on the effect of hypoxic substratefree perfusion (Figs 1 and 2) confirmed that the net loss of potassium was primarily due to an increased effiux ofpotassiurn and not to an alteration of influx because the observed efflux was not different from the observed net loss. This observation has been reported earlier by others [15, 32]. Substrate free hypoxic perfusion was used in this study because it mimicks some of the features of myocardial ischaemia and has been studied previously [32, 33, 44, 45]. The temperature in the present study was monitored at 30.5~ and the results did not differ materially from results obtained at higher temperatures
[32, 3~J. Myocardium perfused with hyperosmolar NaC1 solution (Fig. 3) showed an increased potassium uptake which was reversible on returning to the control solution. This finding confirms previous observations [38]. A similar result was obtained when the myocardium was perfused with hyperosmolar sucrose which cannot cross the cell membrane (Fig.
Potassium Loss During Hypoxia 5). The potassium uptake appears to be accounted for by an osmotic effect. Washout experiments (Figs 4 and 6) showed that the uptake was not due to altered efflux of potassium and must therefore be due to an increased influx. A possible mechanism is that water moves out from the cell increasing the intracellular sodium activity. The sodium pump is stimulated bringing about an increase of potassium influx. On returning to control perfusate the reverse occurs and the net loss of potassium is due to a fall of potassium influx. Alteration o f potassium exchange in response to a c h a n g e of extracellular osmolarity are manifest through changes of potassium influx and cannot therefore account for the observed net loss of potassium occurring in hypoxia (Fig. 1) or ischaemia which is due to an increased ettlux (Fig. 2). The increase of developed tension during perfusion with hyperosmolar sodium chloride or sucrose could be accounted for by the presumed elevation of intracellular sodium. The mechanism of the initial transient fall of tension is uncertain. The effects of small anions were studied by replacing NaC1 in the control solution with sodium isethionate at a constant osmolarity. Isethionic acid has been used as an inert impermeant substitute for extracellular chloride [34]. As a result of removing extracellular chloride and assuming the intracellular chloride concentration to be about 20 mM [40] a concentration gradient for chloride is to be expected causing chloride efflux. During perfusion with sodium isethionate (114 mM) a reduction in tissue K + was detected (Fig. 7). The net loss was due to an increased effiux of potassium (Fig. 8). The effect was concentration dependent in that perfusion with sodium isethionate (30 raM) had no effect on net potassium content or on potassium effiux (Figs 9 and 10). The effects of sodium isethionate (30 mM or 114 raM) on developed tension were small (Figs 7 to 10). Further evidence for the role of anions was obtained by loading the muscle with N a D M O (30 raM) under isosmotic conditions and switching to a perfusate containing sodium isethionate (30 rnM). D M O is an inert non-metabolised substance which rapidly enters the cell. The distribution of D M O is
397
pH dependent and the intracellular concentration at an extracellular pH of 7.4 and assumed intracellular pH of 7.1 will be about 14 mM. On switching to sodium isethionate a gradient for D M O is created which should cause D M O and K + to diffuse out of the cell. That was observed (Figs 11 and 12). The mechanical transients seen upon changing from D M O to ISE were reproducible (Figs 11 and 12). An immediate increase of tension occurred which returned within 10 mins to the control value. Since dae diffusable form of D M O is the H D M O entity these transients may reflect alterations in intracellular pH. Removal of extracellular D M O would generate a transient intracellular alkalosis while reintroduction of D M O into the perfusate would give rise to the opposite effect. The movements of 42K seen with these solution changes in the current studies are too rapid and too large to result solely from changes in pH [29]. In previous experiments an increase of Pco2 was shown to cause an uptake of potassium [29]. In the present experiments sodium D M O also caused an uptake. Important differences exist between these experiments despite the similar results. Equilibrium with a high Pco2 altered both extra and intracellular pH, the gain of potassium was delayed several minutes, H C O ~ generated intracellularly is affected by carrier mechanisms, acidosis is known to have many effects on cardiac function and developed tension was substantially reduced and did not return to control (compare with Fig. 11). O u r results show that changes ofosmolarity and of the intracellular concentration of anions can modify potassium exchange in the myocardium. The response to changes of extracellular osmolarity are due to alteration of potassium influx, probably linked to sodium pump activity. The efflux of potassium was not altered by changes ofperfusate osmolarity. The establishment of a gradient of anions between the intracellular and extracellular fluid caused a net loss of potassium due to an increased efflux. In the context of myocardial ischaemia other studies (personal communication, J. Weiss, UCLA, Los Angeles, USA) indicate that the ion which might be linked to such changes is unlikely to
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A. Gaspardone et al.
be lactate. D u r i n g b o t h h y p o x i a a n d isc h a e m i a , the p o t a s s i u m loss is d u e to a n i n c r e a s e d effiux w i t h o u t e v i d e n c e o f r e d u c e d s o d i u m p u m p activity. T h e loss o f u n d e f i n e d a n i o n s f r o m the m y o c a r d i u m c o u l d be the m a j o r d e t e r m i n a n t o f p o t a s s i u m loss.
Acknowledgements T h i s i n v e s t i g a t i o n was c o n d u c t e d d u r i n g the t e n u r e by A. G a s p a r d o n e o f a fellowship f r o m C . N . R . I t a l y a n d was s u p p o r t e d by the British Heart Foundation.
References 1 BEeriESt,M., POTT, L. K-channel activated by loss ofintracellular ATP in guinea-pig atrial cardioballs. J Physiol 348, 50 (1983). 2 BOYLE,P. J., CONWAV,E.J. Potassium accumulation in muscle and associated changes. J Physiol 100, 1 63 (1941). 3 CASE,R. B. Ion alterations during myocardial ischemia. Cardiology 56, 245-262 (1971/1972). 4 CONRAD,G, L., RAU, E. E., SHINE, K. I. Creatine kinase release, potassium-42 content, and mechanical performance in anoxic rabbit myocardium, J Clin Invest 64, 155-161 (1979). 5 EHARA,T., HASEGAWA,J. Effects ofhypertonic solution on action potential and input resistance in the guinea-pig ventricular muscle, jpn J Physio132, 151-167 (1983). 6 FRIEDRICH,R., HIRCHE,H., KEBEL, U., ZYLKA,g., Blssm, R. Changes ofextracellular Na +, K +, Ca + + and H + of the ischemic myocardium in pigs. Basic Res Cardio176, 453-456 ( 1981 ). 7 GARLmK,P. B., RADnA, G. K., SEELEY, P. J. Studies of acidosis in the ischemic heart by phosphorus nuclear magnetic resonance. Biochem J 184, 547-554 (1979). 8 GARRAHAN,P.J., GLYN, I. M. Measurement of 2*Na and 42K with a liquid-scintillation counting system without, added scintillator.J Physiot 186, 55 56 (1966). 9 HEISLER,N., PnPER, J. The buffer value of rat diaphragm muscle tissue determined by PCO 2 equilibration of homogenates. Respir Physio112, 169-178 ( 1971). 10 Htt.L,J. L., GEa'T~S,L. S. Effect of acute coronary artery occlusion on local myocardial extracellular K activity in swine. Circulation, 61,768-778 (1980). 11 JE~mNos, R. B., REIMER,K. A., HILL, M. L., MAYER,S. E. Total ischemia in dog hearts, in vitro. Comparison of high energy phosphate production, utilization, and depletion, and of adenine nucleotide catabolism in total ischemia in vitro vs. severe ischemia in vivo. Cite Res 49, 892-900 ( 1981 ). 12 KAKEt,M., NOMA,A., SmeASAKI,T. Properties of adenosine-triphosphate-regulated potassium channel in guineapig ventricular cells.J Physio1363, 441-462 (1985). 13 KODAMA,i., WILDE, A~,#ANSE,M.J., DURRER,D., YAMADA,K'. Combined effeets-o~bypoxia, hyperkalemia and acidosis on membrane action potential and excitability of guinea-pig ventricular muscle. J Mol Cell Cardiol, 16, 247-259 (1984). 14 KLEBRR,A. G. Resting membrane potential, extracellular potassium activity, and intracellular sodium activity during acute global ischemia in isolated perfused guinea pig hearts. Circ Res 52, 442-450 (1983). 15 KLEBER,A. G. Extracellular potassium accumulation in acute myocardiol ischemia. J Mol Cell Cardiol 16, 389 394 (1984). 16 LANOER,G. A., SERENA,S. D. Effects ofstrophanthidin upon contraction and ionic exchange in rabbit ventricular myocardium: relation to control of active state. J Mol Cell Cardiol 1, 65-90 (1970). 17 Locle, J. R. Enhancement of the vulnerability of the ventricle to fibrillation (VF) by regional hyperkalaemia. Cardiovasc Res 7, 501-507 (1973). 18 LOWRY,O. H., KRAVER,O., BAIRDHASa'tNGS,A., TUCKER,R. P. Effects ofanoxemia on myocardium of isolated heart of the dog. Proe Soc Exp Biol Med 49, 670~74 (1942). 19 MAINWOOD,G. W., LUeIERG. E. Fat!gue and recovery in isolated frog sartorius muscles: the effecs of bicarbonate concentration and associated potassium loss. Can J Physiol Pharmaco150, 132-142 (1972). 20 MATHUR,P, P,, CASE,R. B. Phosphate loss during reversible myocardial ischemia. J Mol Cell Cardiol 5, 375-393 (1973). 21 NOMA,A. ATP-regulated K channels in cardiac muscle. Nature 305, 147-148 (1983). 22 NOUA, A., SVIIBASAKI,T. Membrane current through adenosine-triphosphate-regulated potassium channels in guinea-pig ventricular cells. J Physio1363, 463-480 (1985). 23 OPtE, L. H. Metabolic regulation in ischemia and hypoxia. Circ Res 38, [Suppl I], t52-168 (1976). 24 PAOJS,E. Cat heart muscle in vitro. IIL The extracellular space.J Gen Physio146, 201 213 (1962). 25 PAGE,E., STORM,S. R. Cat heart muscle in vitro. IX. Cell ion and water contents in anisosmolal solutions. J Gen Physio149, 641-653 (1966). 26 PAOE,E., GROSSPAOE,E. Distribution of ions and water between tissue compartments in the perfused left ventricle of the rat heart. Circ Res 22, 435-446 (1968). 27 PINE, M. B., BROOKS,W. W., NOSTA,J. J., ABELMANN,W. H. Hydrostatic forces limit swelling of rat ventricular myocardium. AmJ Physio1241, H740-H747 (1981).
Potassium Loss During Hypoxla 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
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POOL, P. E., COVELL,J. W., CHIDSEY,C. A., BRAUNWALD,E. Myocardial high energy phosphate stores in acutely induced hypoxic heart failure. Circ Res 2, 221-229 (1966). POOLZ-WxLsON,P. A., LANCER, G. A. Effect ofpH on ionic exchange and function in rat and rabbit myocardium. AmJ Physio1229, 570-581 (1975). POOLE-WILsON,P. A., CAMERON, I. R. Intracellular pH and K of cardiac and skeletal muscle in acidosis and alkalosis. A m J Physio1229, 1305 1310 (1975). POOLE-WIcsoN,P. A. Potassium and the heart. Clin Endocrinol Metab 2, 249-268 (1984). RAu, E. E., SHINE, K. I., LANOER, G. A. Potassium exchange and mechanical performance in anoxic mammalian myocardium. AmJ Pbysio1232, H85-H94 (1977). RAt:, E. E., LAN~ER, G. A. Dissociation of energetic state and potassium loss from anoxic myocardium. Am J Physio1935, I-I537-H543 (1978). REMTVLLA,M. A., APVLEOARTH,D. A., CLARK, D. G., WILIaA~S, I. H. Analysis ofisethionic acid in mammalian tissues. Life Sci 20, 2079-2036 (1977). Roos, A. Intracellular pH and distribution of weak acids across cell membranes. A study of d- and l-lactate and of DMO in rat diaphragm.J Physio1249, 1-25 (1975). Roos, A., BORON,W. F. Intracellular pH. Physiol Rev 61,296~34 (1981). SFIXNE,K. I. Ionic events in ischemia and anoxia. AmJ P.atho1102, 256-261 (1981). TILIaSCH,J. H., LANOER, G. A. Myocardial mechanical responses and ionic exchange in high-sodium perfusate. Circ Res 34, 40 50 (I974). TRANVM-JENsEN,J.,JANsE, M.J., FXOLET,J. W. T., KRXEOER,W.J.G., NAUI,tANND' ALNONeOUaT, C., DURRER, D. Tissue osmolality, cell swelling, and reperfusion in acute regional myocardiol ischemia in the isolated porcine heart. Circ Res 49, 364-381 ( 1981 ). VAUOHAN-JoNEs,R. D. Regulation of chloride in quiescent sheep-heart Purkinje fibres studied using intracellular chloride and pH-sensitive microelectrodes. J Physiol 295, 111-137 (1979). WEI3B,S. C., RICKARDS, A. F., POOLE-WILsON, P. A. Coronary sinus potassium concentration recorded during coronary angioplasty, Br Heart J 50, 146-148 (1983). WI~oAN9,V., GooGI, M., MEESMANN,W., KESSLER, M., GREXTSCltUS,F. Extracellular potassium activity changes with canine myocardium after acute coronary occlusion and the influence of blockade. Cardiovasc Res 13, 297-302 (1979). WExss, J., StoNE, K. L Extracellular potassium accumulation during myocai-diol ischemia: implications for arrhythmogenesis. J Mol Cell Cardio113, 699-673 ( 1981). WEiss,J., StoNE, K. I. [K+]0 accumulation and electrophysiological alterations during early myocardiol ischemia. AmJ Physio1243, H318-H327 (1982). WExss,J. StoNE, K. I. Extracellular K + accumulation during myocardial ischemia in isolated rabbit heart. Am J Physio1242, H619-H628 (1982).
P o t a s s i u m Loss From Rabbit Myocardinm During Hypoxia: Evidence for Passive Efflux Linked to Anion Extrusion Achille Gaspardone, Kenneth I. Shine, Stephen R. Seabrooke and philip A. Poole-Wilson*
Cardiothoracic Institute, 2 Beaumont Street, London, UK ( Received 5 August 1985, accepted in revisedform 24 December 1985) A. GASPARDONE,K. I. SHINE, S. R. SEABROOKE,AND P. A. POOLE-WILSON.Potassium Loss from Rabbit Myocardium During Hypoxia: Evidence for Passive Effiux Linked to Anion Extrusion. journal of Molecularand Cellular Cardiology(1986) 18, 389 399. To determine the effects of permeant and impermeant anions and of osmolarity on potassium (K +) e~xc~ange, the net uptake and effiux of 42K+ were recorded in the isolated arterially perfused rabbit septum. Perfusion With solution made hyperosmolar by adding NaCl (30 mM) or sucrose (60 mM):cauSed similar ii6~r~s~'of '*2~K+ uptake which were reversible on returning to the control solution. WashoUf experiments showed that the loss of K + on returning to the control perfusate was due to a decreased inflffX probably mediated by inhibition of the sodium pump. The effects of anions were studied by replacing chloride in the control solution with the inert and impermeant substitute isethionate (114 mM) or by loading the myocardium with sodium dlmethyloxazeolidinedione" (NaDMO, 30 mM) under isosmotic.condition42 and switching tO a perfusate Containing sodium isethionate (30 mM). In both these conditions a reducuon of K content could be detected and was attributable to an increased efllux. During hypoxic substrate free perfusion K + loss was due to an increased efflux with no evidence for altered influx of potassium. The extrusion of accumulated anions from the myo~ardium could be the major determinant of the early potassium loss during hypoxia and ischaemia. KEy WORDS: Myocardium; Potassium; Perfused septum; Hypoxia; Anions; Osmolarity.
Introduction I n a n i m a l s a n d in m a n p o t a s s i u m is lost f r o m h y p o x i c [4, 18, 32, 33] or i s c h a e m i c m y o c a r d i u m [6, 10, 37, 41, 42, 45]. T h e increase o f the e x t r a c e l l u l a r p o t a s s i u m c o n c e n t r a t i o n in ischa e m i a ( [ K + ] 0 ) is c h a r a c t e r i s e d by a triphasic t i m e course [15, 45]. A n increase o f [ K +]0 is d e t e c t a b l e w i t h i n seconds after i n t e r r u p t i o n o f m y o c a r d i a l perfusion a n d reaches a p p r o x i m a t e l y 15 mmol/1 witfiin 10 mins. A f t e r a f u r t h e r 10 mins, d u r i n g w h i c h [ K + ] 0 is c o n s t a n t ( p l a t e a u phase), a second progressive increase of p o t a s s i u m occurs. T h e initial rise in [ K + ] o is r a p i d , reversible a n d r e p r o d u c i b l e [32]. T h e s e features suggest t h a t the e v e n t is f u n c t i o n a l in n a t u r e . I n c o n t r a s t the second
rise in [ K + ] 0 is associated w i t h irreversible loss o f m y o c a r d i a l f u n c t i o n a n d w i t h cell d e a t h [31, 32]. T h e increase o f [ K + ] o in hypoxic or ischaemic myocardium probably a c c o u n t s for m a n y of the initial c h a n g e s in the a c t i o n p o t e n t i a l [13, 17, 31, 43, 44]. T h e e a r l y p o t a s s i u m loss is d u e to a n i n c r e a s e d effiux [15, 32], is n o t closely linked to the e n e r g e t i c state o f the m y o c a r d i u m [14, 32], is n o t solely d u e to i n h i b i t i o n o f the s o d i u m p u m p [14, 15, 32, 37], a n d is n o t d u e to the cell m e m b r a n e b e c o m i n g m o r e p e r m e a b l e to p o t a s s i u m a l o n e [31]. A possible m e c h a n i s m w h i c h m i g h t e x p l a i n the loss o f p o t a s s i u m d u r i n g h y p o x i a a n d isc h a e m i a has b e e n suggested by the analysis o f
Achille Gaspardone's present address: Malattie Dell'Apparato Cardiovascolare, Cattedra II, Universita' di Roma, Policlinico Umberto 1, 00161 Rome, Italy. Kenneth Shine's present address: University of California Los Angeles, Center for the Health Sciences, Los Angeles, California 90024, USA. * To whom correspondence should be addressed. 0022-2828/86/040389 + 11 $03.00/0
9
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the ions in the venous effluent during restriction of coronary flow. The intracellular accumulation of lactate, phosphate and hydrogen ions in the myocardium and the efflux of the ions into the extracellular space [3, 20] occurs at the same time as the loss of potassium from the myocardium and the rise of [K+]0 [7]. The magnitude of the loss of lactate and phosphate ion is greater than the loss of potassium [3, 20]. Mainwood and Lucier suggested that the efflux of lactate in fatigued frog skeletal muscle was linked to the efflux of potassium [19]. During hypoxia and ischaemia weak acids are generated by glycolysis and from the breakdown of energy rich phosphates [23]. The protons so generated are buffered by fixed negative charges ofintracellular proteins [9]. As a result of the consequent rise in the intracellular concentration of freely movable anions an efflux of permeant anions with a cation to maintain cellular electroneutrality is to be expected [2, 14, 15, 35, 36]. Potassium is the commonest cation in the myocardium. The production of weak acids during hypoxia and ischaemia will also increase intracellular osmolarity [39] which is known to affect potassium exchange [5, 24-27, 38, 39]. We have investigated the effects of permeant and impermeant anions and hyperosmolarity on potassium fluxes in isolated heart muscle in an attempt to determine the relative importance of the two factors in causing potassium loss during hypoxia and ischaemia.
Materials and Methods
Perfused septal preparation The experimental preparation was the isolated arterially perfused interventricular septum of the rabbit, that has been described previously [16, 29]. Adult male New Zealand white rabbits, weighing 1.5 to 2 kg, were heparinised (10 000 U heparin) and anaesthetised (180 mg sodium pentobarbitone). The thorax was opened rapidly. The heart was excised and placed in warm oxygenated perfusate. A polyethylene cannula was inseted into the septal artery within 3 to 5 mins. The septum was dissected from the right and left ventricular
free walls and underperfused tissue excised. The base of the triangular piece of tissue was clamped between two opposing forceps mounted on geared slides with the cannula to the lower left. The apex was connected by a silk suture to a strain gauge transducer (UC 4, Statham Instruments, CA, USA). Tension was recorded (M4 Devices, Welwyn Garden City, UK). Septa were perfused with a roller-pump (Watson-Marlow, Falmouth, Cornwall) and flow was measured by timing and weighing effluent drops of constant size falling from a specially designed rod placed under the muscle. Flow was maintained constant in each experiment and averaged 1.9 ___0.2 ml/g/min (n = 58). The perfusate was warmed by means of a heating coil immediately adjacent to the cannula. The temperature of the tissue was measured by a needle thermister and kept constant during each experiment at 30.5 _+ 0.5~ (n = 58). Septa were stimulated by a pair of platinum electrodes with pulses of 10 V lasting 5 ms (S.R.I., Croydon, Surrey). The stimulation rate ranged from 85 to 95 beats/min and was kept constant during each experiment. Septa weighed between 0.54 and 1.1 g.
Solutions and chemicals The control perfusate contained (mM): NaC1, 114; KC1, 5; NaHCO3, 28; NaH2PO42H20 , 0.435; MgCI26H20 , 1 ; CaC12, 1.8; D-Glucose, 11.1 (Analar, BDH Chemicals, UK). 5,5-dimethyl-2,4-oxazolidinedione (DMO) was obtained from Eastman. Sodium isethionate was obtained from Sigma. In chloride depleted solutions an equimolar concentration of isethionate (114 and 30 mM) was used to maintain isomolarity. When D M O was used an appropriate reduction of the chloride concentration of the perfusate was made to avoid changes in osmolarity. Solutions were prepared with the constituents shown in Table 1. After equilibration of the solutions with 95% 0 2 - 5 % CO2, the final pH was 7.4. The hypoxic substrate-free solution contained an equimolar concentration of Dmannitol to replace glucose and was equilibrated with a gas mixture containing 95% N 2 and 5% CO 2. *2K+ (Radiochemical
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Centre, Amersham) was added directly to the experimental solutions.
Measurements and experimentalprocedures Developed tension (DT) and resting tension (RT) were continuously recorded (M4 Devices, Welwyn Garden City, UK) at a known stimulation rate and temperature. Muscles were only used when the initial developed tension was greater than 10 g at a resting tension of no greater than 5 g at 30.5~ and at a stimulation rate of 85 to 95 beats/rain. A period of at least 90 min was allowed for equilibration of the septa. Addition of the isotope to the perfusate had no effect on tension. Tissue 42K+ during uptake studies was recorded with a sodium iodide crystal (8.5 cm diameter) (Harshaw NaI, Harshaw Chemic B.V., Holland) and counted (J & P Engineering, Reading, UK). Septa were placed 3 to 6 mm from the NaI crystal. Effluent drops fell freely into a lead-shielded drop collector and were immediately returned to a reservoir 1 m away and behind 15 cm of lead. Counts per minute (ct/min) from septa were recorded over each minute, corrected for decay and background and plotted. Background counts remained constant and were measured at the end of the experiment after removal of the muscle from the experimental area. The technique has been described previously [16, 29]. In isotopic uptake experiments the tissue was labelled with 42K for 120 to 150 min at which time tissue radioactivity had reached about 95% of that at the asymptote. The specific activities of the 42K solutions used in any experiment were identical. At the end of each experiment radioactivity in both weighed samples of perfusate and the whole septa was measured (Tracerlab Spectro-Matic, MA, USA). The potassium concentration in mmol/kg wet tissue was calculated from the specific activity of the perfusate affd the activity of the~:muscle'at the end of the uptake studies. It was then possible to calibrate the uptake curve in terms of K + labelled with isotope. The absolute and percentage changes during interventions were calculated.. For isotopic washout experiments muscles were labelled with 42K+ containing solutions for 30 to 50 min. Washout was started with
non isotopic solution and after 20 min of perfusion, to permit accurate determination of the rate of potassium exchange, the experimental intervention was induced. During washout of 42K+ from a septum 4 or 5 effluent drops were collected in individual planchets. The time required for formation of each set of drops was recorded. The isotopic activity of each sample was counted (Model 3380 Tricarb, Packard) after the addition of 10 cm a of water by Cerenkov counting [8]. The counts were corrected for background and decay and expressed as ct/min of perfusate flow, to account for any slight changes in the flow rate. The value was plotted semilogarithmically against time from the start of the washout. The tissue activity was also monitored during washout experiments, corrected for background and decay, and plotted semilogarithmically against time from the start of the washout as ct/min. These procedures have been described previously [16, 29]. At the end of each experiment, septa were removed, excess fluid lightly blotted away and the tissue weighed. Total tissue water was obtained by drying to constant weight in an oven for 36 h at 95~ and reweighing. In washout experiments the net potassium loss due to increased efflux was calculated by integrating the area under the altered effluent curve during interventions [16]. A fall in the tissue content of 42K + could be due either to an increased efflux or a reduced uptake. If the fall in tissue 42K+ could be accounted for by the measured loss during a washout experiment then the fall of tissue 4eK+ was attributed to an increased efflux. Results are expressed as mean + SEM. Differences = betWeen groups of results were analysed using the unpaired Student's t test
Results
Potassium exchangeduring hypoxia The mean tissue potassium content of 12 septa perfused for 4 to 6 h was calculated to be 59.3 + 2.5 mmol K+/kg wet tissue. After hypoxia for 15 min the mean net loss of potassium detected in uptake experiments was 7.2 + 1.1 mmol K+/kg wet tissue ( n = 6) (Fig. 1). The effects of 15 rains of hypoxia on the efflux of potassium were evaluated in five
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septa by inducing h y p o x i a d u r i n g the washout (Fig. 2). T h e increased efflux of 42K+ d u r i n g washout was calculated to represent 6.5__+0.1 mmol K + / k g wet tissue which was not significantly different from the net loss of potassium measured in the u p t a k e experiments (P = 0.57). Thus, the net potassium loss d u r i n g h y p o x i a could be a t t r i b uted to increased efflux o f t h e ion and not to a r e d u c e d uptake. ~,.~,~ After 15 min of h y p o x i a D T fell to 38 __+ 1.9% of the control. R T rose from 6.3 -t- 0.1 to 10.8 -I- 0.3 g (n = 11). R e o x y g e n ation i n d u c e d a r a p i d recovery in D T a n d a m a r k e d decrease in R T (Figs 1 and 2).
Potassium exchange duringperfusion with hyperosmolar NaCl (30 mM) and sucrose (60 raM) solutions After 15 min perfusion with h y p e r o s m o l a r NaC1 (30 mM) a m e a n net gain of 42K+ was observed and calculated to represent 5.9 __t_0.4 m m o l K + / k g wet tissue (n = 6). O n reperfusion with control solution (Fig. 3) recovery to the control steady state was achieved within 15 min. 1000 ['-
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T h e effects of 15 min perfusion with hyperosmolar NaC1 (30 mM) on the loss of potassium in the effluent were evaluated in four septa d u r i n g washout. All studies failed to reveal any change of potassium effiux d u r i n g h y p e r o s m o l a r perfusion (Fig. 4). T h e perfusion with h y p e r o s m o l a r NaC1 (30 mM) solution caused an i m m e d i a t e 12.4 _ 0.4% decline in D T followed by a progressive increase in D T up to 5 . 6 - t - 0 . 3 % a b o v e the control value. Reperfusion with control solution caused a 14.5_t_+0.3% increase in D T foll6wed by a progressive decline to the control level. These effects were reversible a n d r e p r o d u c i b l e (Figs 3 and 4). After 15 min of h y p e r o s m o l a r sucrose (60 mM) perfusion the m e a n net gain of potassium was 5.3 _+ 0.2 mmol K + / k g wet tissue (n = 6). T h e gain was reversed within 15 mins of perfusion with the control solution (Fig. 5). I n four washout studies no change of 42K efflux was observed (Fig. 6). Perfusion with h y p e r o s m o l a r sucrose (60 mM) caused an i m m e d i a t e decline in D T (13.8-t- 0.31) and in R T (4.9 _ 0.5%) followed by a progressive increase in D T to 14.5 __+0.3% above the control value. R e p e r -
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7I
l" Tissue
"~ I0
i
Effluent
I
Tension
L
I
I0 min
FIGURE 4. Perfusion with hyperosmolar NaCl solution (see Table 1) does not alter a2 K + efflux.
m
A. Gaspardone e t aL
394
Hyper I sucrose I
401
Noise 114
-~ E2 o20
g +~
1 ~ "~"
Tension
Noise 114
t
5
O
~
........ ":'
~o~i~ F I G U R E 5. Peffusion with hyperosmolar sucrose solution (see Table 1) causes a reversible net gain of tissue 42K+.
20 rain' FIGURE 7. Perfusion with isosmolar NaIse (sodium isethionate) solution (114 mM) (see Table 1) causes a reversiblenet lossof tissue 42K+. I000
N__o,s_e,,__4,
fusion with control solution caused a rapid increase in D T of 9.7 4- 0.4% followed by a progressive recovery to the control values (Figs 5 and 6).
-
: ...........................................1 '~ Tension
The effects of perfusion with sodium isethionate (NaIse, 114 mM) were evaluated in uptake studies. After 15 min the mean net toss of 42K+ was 5.4 _ 0.3 mmol K + / k g wet tissue (n = 6). The loss was reversed in 10 min (Fig. 7). In washout experiments the increased appearance of r in the effluent effiux (Fig. 8) was calculated to represent 4.6 4- 0.4 mmol K + / k g wet tissue (n = 5). The loss due to increased efflux was not significantly different from the net loss calculated from the uptake experiment (P = 0.115). Perfusion with isosmolar NaIse (114 mM) solution in seven experiments caused an increase in D T 7.8 + 0.4% and a decrease in R T 9.1 4-0.2%. These effects were rapidly reversible on reperfusion with control solution and reproducible (Figs 7 and 8). In four
~_~ I0O
"""
....................
I0 9
Potassium exchange during perfusion with isosmolar chloride depleted solutions (dValse 114 and 30 mM)
Hyper sucrose 60
.
1
IOmin F I G U R E 8. Perfusion with isosmolar NaIse solution (114 mM) (see Table 1) causes an immediate increase in 42K+ effiux.
experiments mechanical function was not affected by the intervention. In three uptake and four washout studies no alteration of K + exchange due to the presence of isosmolar NaIse (30 mM) solution (Figs 9 and 10) was observed. Perfusion with isosmolar NaIse (30 mM) solution had no effect on mechanical performance (Figs 9 and 10).
Potassium exchange duringperfusion with isosmolarNaDMO (30 mm) solution and isosmolar NaIse (30 mM) solution
In uptake studies muscles were labelled 7100 ~ durin perfusion with isosmolar N a D M O (30 t * mM). After labelling to asymptote, perfusion ~~ was switched to NaIse (30 raM). A mean net Tissue I0
•
~-
Eo
+~
~0
~
~O :55
so I L No Ise 30
iso ~ No Ise 30
Iso ~ t No Ise 30
,
so
[[59]TensiOn +
I 1 - - 1
I
l Tension ~-z 25~-5g] ~
10 rain F I G U R E 6. Perfusion with hyperosmolar sucrose solution (see Table 1) does not alter 42K+ efflux.
F I G U R E 9. Peffusion with isosmolar NaIse solution (30 mM) (see Table 1) does not alter 42K+ uptake.
P o t a s s i u m L o s s During Hypoxia
....................
IOOO -
jisoNa Ise 30 i
x:
9
c
ooTo o 0 o o0 o o
_
o
O.
~
. ......................
395
O
•
~
.g
,oo
*'%
loot-
o
.................
-tlo
E m
~
~0
I
~
o
~
tO
I
a)
Tension
I rio mina
I0 rain
FIGURE 10. Perfusion with isosmolar NaIse solution (30 mM) (see Table l) does not alter *2K+ efflux. so
~" 0
+
#
so
so
,
iso
asc
iso
so
NaDMO30 Na se3OINoDM030 NO Ise30 NaDMO3OIN~ Ise30 NoDM030
FIGURE 12. After loading the myocardium with '*2K+ in the presence ofisosmolar NaDMO (30 mM) the washout was started with isosmolar NaDMO (30 mM) unlabelled perfusate. After 20 mins the perfusate was switched to isosmolar NaIse (30 mM) solution. Perfusion with NaIse solution (30 mM) causes an immediate increase of*ZK + efllux.
25 E Tension
raM) solution. The eftlux of 42K+ was calculated to r e p r e s e n t 4 __+ 0.1 m m o l K + / k g wet
,Sg]
FIGURE 11. After loading the myocardium with • molar NaDMO solution (30 raM) (see Table 1) perfusion with isosmolar Nalse solution (30 raM) causes a reversible net loss of 42K + followed by recovery on return to perfusion with isosmolar NaDMO solution (30 raM). lOSS of 4.0 • 0.2 m m o l K + / k g wet tissue (n = 3) o c c u r r e d after 15 m i n (Fig. 11). T h e effects of 15 m i n perfusion with N a I s e (30 mM) on the effiux of p o t a s s i u m were e v a l u ated in five septa (Fig. 12). D u r i n g the labell i n g time a n d dae first p a r t o f w a s h o u t muscles were perfused with isosmolar N a D M O (30 TABLE 2.
tissue (n = 5) a n d was n o t significantly differe n t from the net loss observed in u p t a k e e x p e r i m e n t s (P = 0.82). N a I s e (30 raM) perfusion caused a 26 + 1% i m m e d i a t e increase in D T followed b y a progressive decline to the c o n t r o l value. O n reperfusion with N a D M O (30 raM) a r a p i d decline in D T 3 8 _ 2% was observed, followed b y a progressive recovery to c o n t r o l values. T h e effects were reversible a n d reprod u c i b l e (Figs 11 a n d 12). T h e effects of different i n t e r v e n t i o n s o n potassium e x c h a n g e are s u m m a r i s e d in T a b l e 2.
Potassium homeostasis 15 mins after the imposition of an intervention. Results are expressed in mmol/kg wet tissue (mean
• S.E.M.). Intervention
Uptake experiments K + loss (--) or gain ( + )
Hypoxia Isosmolar NaIse (t14 raM) Isosmolar D M O (30 mM)-Ise (30 mM) Hyperosmolar NaC1 (30 mM) Hyperosmolar sucrose (60 raM) Isosmolar Ise (30 mM)
Washout experiments K + efflux
-7.2 + 1 - 5 . 4 • 0.3
(n = 6) (n = 6)
6.5 • 0.14 4.6 • 0.4
(n = 5) (n = 5)
--4.0 • 0.2
(n = 3)
4.0 • 0.1
(n = 5)
5.9 • 0.35 (n = 6)
0
(n = 4)
5.3 _ 0.16 (n = 6)
0
(n = 4)
0
(n = 3)
0
(n = 5)
Results are expressed in mmol/Kg wet tissue (mean -I- S.E.M.).
396
A. Gaspardone et aL
Discussion
The precise mechanism for the loss of potassium from ischaemic or hypoxic myocardium is not known. Several hypotheses have been put forward in recent years [31]. The loss has been attributed to inhibition of the sodium pump, acidosis, a selective increase in the permeability of the cell membrane, an A T P dependent potassium channel and the outward movement of intracellular anions. There are arguments against several of these mechanisms. The observation that the intracellular sodium remains unchanged at least over 15 mins ofischaemia in the guineapig heart [14] and that altering heart rate can cause the extracellular potassium both to rise and fall suggest that the sodium p u m p is at least partially intact [14, 15, 32]. Acidosis develops rapidly in both ischaemic and hypoxic myocardium but an imposed acidosis brought about by increasing the PCO z of the perfusate causes a gain of potassium, not a loss [29, 30]. Furthermore the alteration of potassium exchange in response to acidosis is not immediate but delayed for several minutes [29]. Potassium loss cannot be simply due to the cell membrane becoming more permeable to potassium. If that were so the membrane potential would initially become more negative and approach the equilibrium potential for potassium. In hypoxia and ischaemia the membrane is depolarised initially [3/] and the membrane potential only equals the potassium equilibrium potential later. The observation that ATP (adenosinetrisphosphate) is necessary to maintain a potassium channel closed has provided a basis for the proposition that potassium loss could be related to the energetic state of the cell [1, 12, 21], although other workers have been unable to link potassium efflux with the energetic state [14, 32]. A potassium channel regulated by ATP concentration has recently been described in guinea-pig ventricular cells. It was shown that depletion ofintracellular A T P induced a potassium outward current under a variety of experimental conditions [1, 21, 22] but only after a delay of several minutes. Potassium loss during hypoxia or ischaemia is an immediate event occurring within seconds
[15, 32, 37] and at that time there is almost no change in the total A T P content of the myocardium [11, 28]. Moreover even if a potassium channel were opened, it would cause the membrane potential to approach the equilibrium potential for potassium and not lead to immediate depolarisation as observed in hypoxia and ischaemia [3/]. We have tested two other possible mechanisms, first that the efflux of potassium is linked to the increase of osmolarity which occurs in ischaemic myocardium [39] and second that the potassium leaves the cell passively, accompanying the outward movement of anions such as lactate and phosphate [2, 15, 19]. During hypoxia or ischaemia weak acids will be generated intracellularly by the breakdown of high energy phosphates, glycolysis and glycogenolysis. Protons generated will be partly buffered by the negative charges on intracellular proteins leaving a permanent anion to diffuse out of the cell. In order to maintain approximate electroneutrality a cation, potassium, will also be lost from the cell. The experimental preparation used in the present study was the arterially perfused interventricular septum of the rabbit. Preliminary experiments on the effect of hypoxic substratefree perfusion (Figs 1 and 2) confirmed that the net loss of potassium was primarily due to an increased effiux ofpotassiurn and not to an alteration of influx because the observed efflux was not different from the observed net loss. This observation has been reported earlier by others [15, 32]. Substrate free hypoxic perfusion was used in this study because it mimicks some of the features of myocardial ischaemia and has been studied previously [32, 33, 44, 45]. The temperature in the present study was monitored at 30.5~ and the results did not differ materially from results obtained at higher temperatures
[32, 3~J. Myocardium perfused with hyperosmolar NaC1 solution (Fig. 3) showed an increased potassium uptake which was reversible on returning to the control solution. This finding confirms previous observations [38]. A similar result was obtained when the myocardium was perfused with hyperosmolar sucrose which cannot cross the cell membrane (Fig.
Potassium Loss During Hypoxia 5). The potassium uptake appears to be accounted for by an osmotic effect. Washout experiments (Figs 4 and 6) showed that the uptake was not due to altered efflux of potassium and must therefore be due to an increased influx. A possible mechanism is that water moves out from the cell increasing the intracellular sodium activity. The sodium pump is stimulated bringing about an increase of potassium influx. On returning to control perfusate the reverse occurs and the net loss of potassium is due to a fall of potassium influx. Alteration o f potassium exchange in response to a c h a n g e of extracellular osmolarity are manifest through changes of potassium influx and cannot therefore account for the observed net loss of potassium occurring in hypoxia (Fig. 1) or ischaemia which is due to an increased ettlux (Fig. 2). The increase of developed tension during perfusion with hyperosmolar sodium chloride or sucrose could be accounted for by the presumed elevation of intracellular sodium. The mechanism of the initial transient fall of tension is uncertain. The effects of small anions were studied by replacing NaC1 in the control solution with sodium isethionate at a constant osmolarity. Isethionic acid has been used as an inert impermeant substitute for extracellular chloride [34]. As a result of removing extracellular chloride and assuming the intracellular chloride concentration to be about 20 mM [40] a concentration gradient for chloride is to be expected causing chloride efflux. During perfusion with sodium isethionate (114 mM) a reduction in tissue K + was detected (Fig. 7). The net loss was due to an increased effiux of potassium (Fig. 8). The effect was concentration dependent in that perfusion with sodium isethionate (30 raM) had no effect on net potassium content or on potassium effiux (Figs 9 and 10). The effects of sodium isethionate (30 mM or 114 raM) on developed tension were small (Figs 7 to 10). Further evidence for the role of anions was obtained by loading the muscle with N a D M O (30 raM) under isosmotic conditions and switching to a perfusate containing sodium isethionate (30 rnM). D M O is an inert non-metabolised substance which rapidly enters the cell. The distribution of D M O is
397
pH dependent and the intracellular concentration at an extracellular pH of 7.4 and assumed intracellular pH of 7.1 will be about 14 mM. On switching to sodium isethionate a gradient for D M O is created which should cause D M O and K + to diffuse out of the cell. That was observed (Figs 11 and 12). The mechanical transients seen upon changing from D M O to ISE were reproducible (Figs 11 and 12). An immediate increase of tension occurred which returned within 10 mins to the control value. Since dae diffusable form of D M O is the H D M O entity these transients may reflect alterations in intracellular pH. Removal of extracellular D M O would generate a transient intracellular alkalosis while reintroduction of D M O into the perfusate would give rise to the opposite effect. The movements of 42K seen with these solution changes in the current studies are too rapid and too large to result solely from changes in pH [29]. In previous experiments an increase of Pco2 was shown to cause an uptake of potassium [29]. In the present experiments sodium D M O also caused an uptake. Important differences exist between these experiments despite the similar results. Equilibrium with a high Pco2 altered both extra and intracellular pH, the gain of potassium was delayed several minutes, H C O ~ generated intracellularly is affected by carrier mechanisms, acidosis is known to have many effects on cardiac function and developed tension was substantially reduced and did not return to control (compare with Fig. 11). O u r results show that changes ofosmolarity and of the intracellular concentration of anions can modify potassium exchange in the myocardium. The response to changes of extracellular osmolarity are due to alteration of potassium influx, probably linked to sodium pump activity. The efflux of potassium was not altered by changes ofperfusate osmolarity. The establishment of a gradient of anions between the intracellular and extracellular fluid caused a net loss of potassium due to an increased efflux. In the context of myocardial ischaemia other studies (personal communication, J. Weiss, UCLA, Los Angeles, USA) indicate that the ion which might be linked to such changes is unlikely to
398
A. Gaspardone et al.
be lactate. D u r i n g b o t h h y p o x i a a n d isc h a e m i a , the p o t a s s i u m loss is d u e to a n i n c r e a s e d effiux w i t h o u t e v i d e n c e o f r e d u c e d s o d i u m p u m p activity. T h e loss o f u n d e f i n e d a n i o n s f r o m the m y o c a r d i u m c o u l d be the m a j o r d e t e r m i n a n t o f p o t a s s i u m loss.
Acknowledgements T h i s i n v e s t i g a t i o n was c o n d u c t e d d u r i n g the t e n u r e by A. G a s p a r d o n e o f a fellowship f r o m C . N . R . I t a l y a n d was s u p p o r t e d by the British Heart Foundation.
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POOL, P. E., COVELL,J. W., CHIDSEY,C. A., BRAUNWALD,E. Myocardial high energy phosphate stores in acutely induced hypoxic heart failure. Circ Res 2, 221-229 (1966). POOLZ-WxLsON,P. A., LANCER, G. A. Effect ofpH on ionic exchange and function in rat and rabbit myocardium. AmJ Physio1229, 570-581 (1975). POOLE-WILsON,P. A., CAMERON, I. R. Intracellular pH and K of cardiac and skeletal muscle in acidosis and alkalosis. A m J Physio1229, 1305 1310 (1975). POOLE-WIcsoN,P. A. Potassium and the heart. Clin Endocrinol Metab 2, 249-268 (1984). RAu, E. E., SHINE, K. I., LANOER, G. A. Potassium exchange and mechanical performance in anoxic mammalian myocardium. AmJ Pbysio1232, H85-H94 (1977). RAt:, E. E., LAN~ER, G. A. Dissociation of energetic state and potassium loss from anoxic myocardium. Am J Physio1935, I-I537-H543 (1978). REMTVLLA,M. A., APVLEOARTH,D. A., CLARK, D. G., WILIaA~S, I. H. Analysis ofisethionic acid in mammalian tissues. Life Sci 20, 2079-2036 (1977). Roos, A. Intracellular pH and distribution of weak acids across cell membranes. A study of d- and l-lactate and of DMO in rat diaphragm.J Physio1249, 1-25 (1975). Roos, A., BORON,W. F. Intracellular pH. Physiol Rev 61,296~34 (1981). SFIXNE,K. I. Ionic events in ischemia and anoxia. AmJ P.atho1102, 256-261 (1981). TILIaSCH,J. H., LANOER, G. A. Myocardial mechanical responses and ionic exchange in high-sodium perfusate. Circ Res 34, 40 50 (I974). TRANVM-JENsEN,J.,JANsE, M.J., FXOLET,J. W. T., KRXEOER,W.J.G., NAUI,tANND' ALNONeOUaT, C., DURRER, D. Tissue osmolality, cell swelling, and reperfusion in acute regional myocardiol ischemia in the isolated porcine heart. Circ Res 49, 364-381 ( 1981 ). VAUOHAN-JoNEs,R. D. Regulation of chloride in quiescent sheep-heart Purkinje fibres studied using intracellular chloride and pH-sensitive microelectrodes. J Physiol 295, 111-137 (1979). WEI3B,S. C., RICKARDS, A. F., POOLE-WILsON, P. A. Coronary sinus potassium concentration recorded during coronary angioplasty, Br Heart J 50, 146-148 (1983). WI~oAN9,V., GooGI, M., MEESMANN,W., KESSLER, M., GREXTSCltUS,F. Extracellular potassium activity changes with canine myocardium after acute coronary occlusion and the influence of blockade. Cardiovasc Res 13, 297-302 (1979). WExss, J., StoNE, K. L Extracellular potassium accumulation during myocai-diol ischemia: implications for arrhythmogenesis. J Mol Cell Cardio113, 699-673 ( 1981). WEiss,J., StoNE, K. I. [K+]0 accumulation and electrophysiological alterations during early myocardiol ischemia. AmJ Physio1243, H318-H327 (1982). WExss,J. StoNE, K. I. Extracellular K + accumulation during myocardial ischemia in isolated rabbit heart. Am J Physio1242, H619-H628 (1982).