- Email: [email protected]
Effects of ouabain and high K+ on respiration of turtle brain and urinary bladder in vitro
Camp. Biochem. Physiol., 1973, Vol. 45A, pp. 283 to 292. Pergamon Press. Printed in Great Britain
EFFECTS OF OUABAIN AND HIGH K+ ON RESPIRATION OF TURTLE BRAIN AND URINARY BLADDER
IN VITRO M. E. LEFEVRE The Mount Sinai Medical and Graduate Schools of the City University of New York, New York, N.Y. 10029, U.S.A. and The Medical Research Center, Brookhaven National Laboratory, Upton, N.Y. 11973, U.S.A. (Received 25 September 1972) Abstract-l. In agreement with findings on mammalian brain, respiration of turtle cerebral cortical slices was stimulated by ouabain and high K+ in the external medium. 2. The effects of 40 mM K+ and 10e4 M ouabain on brain respiration were not significantly additive. 3. The possibility that mitochondrial “Ca-respiration” accounts for both Kf-induced and ouabain-induced stimulation of brain slice respiration is discussed. 4. In contrast to brain, respiration of urinary bladder tissue was slightly depressed by 40 mM K+ in the external medium. 5. Ouabain (low4 M) depressed bladder oxygen consumption by approximately 20 per cent in the presence of both 2 and 40 mM K+. INTRODUCTION THE RESPIRATIONof slices of mammalian cerbral cortex is stimulated by increasing the concentration of potassium ion in the external medium (Ashford & Dixon, 1935; Dickens & Greville, 1935; Hertz, 1966; Ruscak & Whittam, 1967; Chan & Quastel, 1970). Maximal stimulation occurs at concentrations of Kf higher than those of ordinary Ringer solutions by a factor of S-10 and elevates respiration rate to as much as twice the resting rate. Several explanations have been advanced for the “potassium effect” on brain respiration. Most of these attribute the increased respiration to activation of ion transport (Ruscak & Whittam, 1967; Hertz, 1968) or of an enzyme system involved in carbohydrate metabolism (Kini & Quastel, 1959; Clark & Nicklas, 1970). The respiration of cortical slices is also stimulated by the cardiac glycoside ouabain (Schwartz, 1962; Bourke & Tower, 1966; Swanson & Ullis, 1966 ; Ruscak & Whittam, 1967) ; in this respect, brain differs from most other tissues (Wollenberger, 1947; LeFevre et al., 1970a). Ouabain-induced increase in brain slice oxygen uptake occurs only in the presence of Caa+ in bathing solutions (Schwartz, 1962; Bourke & Tower, 1966; Ruscak & Whittam, 1967) and has been explained as a mitochondrial response to free Caa+ “mobilized” by ouabain (Tower, 1969). If disparate processes cause the stimulation of respiration 283
284
M. E. LEFEVRE
by high K+ and by ouabain, these two agents might have additive effects. This possibility was tested in the experiments reported here. The effects of K+ and ouabain, separately and together, were investigated on the turtle brain. To gain further information on the effects of K+ and ouabain, experiments were performed on an unrelated tissue, the urinary bladder of the turtle. The bladder provides an interesting contrast to brain since both tissues, although different in function, are involved in processes which require large movements of ions (McIlwain, 1966; Gonzalez et al., 1967a, b). MATERIALS Preparation
AND METHODS
of tissues
Turtles (Pseudemys scripta, Mogul-Ed Co., Oshkosh, Wisconsin) were kept with access to fresh running water but without feeding. Brains were obtained through a diamondshaped incision in the top of the severed head. The bone and meninges were quickly removed and slices 050-0*75 mm in thickness were cut from the gray matter of the cerebral lobes with a razor. Brain slices showed a tendency to fragment during experiments, but, if handled carefully, could be kept intact for hours. Urinary bladders were dissected out, rinsed and cut into small pieces measuring approximately 0.75 mm in thickness. Oxygen consumption Oxygen consumption was measured polarographically using procedures and instrumentation described elsewhere (LeFevre, 1969; LeFevre et al., 1970b, c). Temperature was constant at 25°C. The amount of tissue in the 10 ml respirometer vessels was 0+4-0*08 g (dry weight). During the 60-90 min equilibration phase and the 4-5-hr experimental phase, airsaturated fluid was run into the respirometer approximately every 20 min. The first 5-7 min of each 20-min period were disregarded in calculating the rate of oxygen consumption to avoid error due to diffusional loss of oxygen from medium into tissues (LeFevre et al., 197Oc). Results were expressed as mean + S.E.M. Solutions Final concentrations in the basic Ringer solution were NaCl, 105 mM; NaHCO,, 3 mM; KCl, 2 mM; CaCl,, 2 mM; MgCl,, 1 mM; phosphate buffer, 0.8 mM; glucose, 5.5 mM; streptomycin sulfate, 5 mg%. Osmolality was approximately 225 and pH, 7% When K+ was varied, equivalent reciprocal changes were made in Na+ so that the osmolality was unchanged. In the experiments to be described, Ringer solutions are designated by their KC1 concentration; e.g. 40 mM Kf Ringer indicates a Ringer containing 40 mM KC1 and 67 mM NaCl. Ouabain tetrahydrate was obtained from Sigma Chemical Co., St. Louis, MO. RESULTS
High K+ concentrations In agreement with findings on mammalian brain, the respiration of turtle cortical slices was stimulated promptly by increasing K+. Figure 1 shows oxygen uptake iates during a 20-min period 1 hr after high K+ addition. The rates are expressed as a percentage of the mean rate of a 40-min control period. Tissue respiration’ increased to 123.7 f 5.6 per cent of control in 15 mM K+ Ringer (four experiments) and to 143.3 + 13 per cent of control in 30 mM K+ Ringer
RESPIRATION
OF TURTLE
BRAIN
AND
285
BLADDER
60
30
mM
K’
FIG. 1. Oxygen consumption of slices of turtle cerebral cortex as a function of K+ concentration. The rate after 1 hr in 15, 30 and 60 mM Kf Ringer is expressed as a percentage of the mean rate during a 4O-min control period in 2 mM K+ Ringer. Equivalent reciprocal changes were made in Na+ at each Kf concentration so that osmolality was unchanged. Vertical lines indicate one-half standard error.
Fi 0
80-
0" I
-40
I
I
I
I
I
I
I
-20
0
20
40
60
80
too
I 120
MINUTES FIG. 2. Effect of 100 mM K+ on oxygen consumption
of turtle cerebral cortical slices. At time 0 the ambient solution was changed from 2 mM Kf Ringer to 100 mM K+ Ringer. Bars give standard error. Number of experiments: 6.
286
M. E. LEFJWRE
(five experiments). Since respiration was 148 t_ 16.1 per cent of control after 1 hr in 60 mM K+ Ringer (five experiments), not much more stimulation was induced with 60 mM K+ than with 30 mM Kf. The stimulation was long sustained, lasting two or more hours with few exceptions. Figure 2 illustrates the response of brain slice oxygen consumption to the addition of 100 mM K+ Ringer (Na+ was 7 mM). This high K+ concentration caused a biphasic response illustrated by the sharp rise and subsequent decline. At 1 hr the rate was 93*1+ 4.8 per cent of initial and still declining. Ouabain The effects of ouabain in the range 1O-s-1O-3 M were tested in eight experiments. Figure 3 gives representative results: 10” M ouabain caused a slow rise in oxygen consumption of slices of turtle brain; 1O-5 M ouabain caused a sharper rise followed by a decline; 104 M ouabain caused a maximal rise also followed by a decline. The decline after 1O-5 and 1O-4 M ouabain was always present though variable in time of onset and rapidity. The effects of 10es M ouabain (not shown in Fig. 3) were similar to those of lO-4 M. 140 a $ -
130
b e 9
120
z 0 k I 2 5 ” 0”
110 100 90
*o_,
-6;O
-20
0
40
80
120
160
200
240
MINUTES
FIG. 3. Effect of varying concentrations of ouabain on oxygen consumption of turtle cerebral slices. Each curve is a single experiment. Ouabain was added at time 0. Ouabain concentrations: O, lO-4 M; A, 10es M; ?? , lObE M.
Forty mM K+ followed by ouabain Following a 60-90 min equilibration period in regular 2 mM K+ Ringer, the bathing solution was changed to 40 mM K+ Ringer. Ouabain ( lOA M) was added 135 min later and measurements were continued for an additional 120 min. Identical experiments were conducted on slices of urinary bladder tissue from the
RJSPIRATION
OF TURTLE
BRAIN
AND
BLADDER
287
same
turtles during the same time periods to characterize the response of bladder respiration to the same conditions. The results of twelve experiments are shown in Fig. 4. The maximal increase in response to 40 mM K+ in brain tissue occurred at approximately 45 min (Fig. 4). Respiration increased to 145 + 6.6 per cent of initial (I’< 0.001 for paired differences in rates at - 5 and + 45 min.) The addition of ouabain in the presence of 40 mM K+ produced a brief increase of borderline significance (O-02 < PC 0.05 for paired differences in rates at 130 and 150 min). Oxygen consumption continued at a high rate for the remainder of the experiment without marked changes in either direction. The behavior of turtle brain in the last stage of the experiment differs from that of rabbit brain in which K+-stimulated respiration declined after the addition of ouabain (Ruscak & Whittam, 1967). The respiration of bladder tissue, in contrast to that of brain, was slightly depressed by changing the ambient solution from 2 mM K+ Ringer to 40 mM K+ Ringer (Fig. 4). Oxygen uptake 75 min after the change was 88.8 f 2.2 per cent of initial (PC 0.02 for paired differences in rates at - 5 and + 75 min).
MINUTES
FIG. 4. Effect of 40 mM K+ followed by 10m4 M ouabain on oxygen consumption of slices of turtle cerebral cortex (0) and urinary bladder (A). At time 0 the bathing solutions were changed from 2 mM K+ Ringer to 40 mM K+ Ringer. At 135 min low4 M ouabain in 40 mM K+ Ringer was added. Bars give standard error. Number of experiments: cerebral cortex, 6; bladder, 6.
Ouabain in 40 mM K+ Ringer produced a further depression of bladder respiration (Fig. 4). Oxygen uptake 75 min after the addition of ouabain was 77.5 f 2.3 per cent of the pre-addition rate (PC 0.01 for paired differences in rates at 130 and 210 min).
M. E. LEFWRE
288
Ouabainfollowed by 40 mM K+ These experiments differed from those described previously in that the order of additions was reversed. The results of twelve experiments are shown in Fig. 5. The rise of respiration rate was slower with ouabain than with 40 mM K+, reaching its maximum at 105 min. The oxygen uptake rate increased to 137.0 + 4.3 per cent of initial (P-z 0.01 for paired differences in rates at -5 and + 105 min). The addition of 40 mM K+ produced a slight but not significant further increase (P> 0.05 for paired differences in rates at 130 and 150 min). It is possible that, in some cases, 40 mM K+ delayed a ouabain-induced late decline in respiration that would have occurred in its absence (see Fig. 3). The experimental results were more variable than those shown in Fig. 4 as shown by the relatively large standard error. Bladder respiration was depressed by 10M4M ouabain in 2 mM Kf Ringer (Fig. 5). The oxygen uptake rate 75 min after ouabain addition was 80.3 + 2.7 per cent of initial (PC 0.01 for paired differences in rates at - 5 and + 75 min.) The addition of 40 mM K+ to tissue already exposed to ouabain produced little change in bladder oxygen consumption. Respiration continued to be depressed during the last stage of the experiment with only a slight suggestion of recovery (Fig. 5). Despite reports of prevention or reversal of ouabain effects by high K+ (Charneck & Opit, 1968), this was not found for turtle bladder respiration in the ’
I
2 :mM k G
3.4
0
’
2mM
oUABA’N
IO-”
I
’
I
’
I
’
I
40mM
K+
M OUABAIN
lO-4
’
I
K+’
M OUABAIN
-
kl a 3.0 -
1
101 :40
0
40
80
120 MINUTES
160
::
200
0
240
FIG. 5. Effect of 10e4 ouabain followed by 40 mM K+ on oxygen consumption of slices of turtle cerebral cortex (a) and urinary bladder (A). At time 0 the bathing solutions were changed from 2 mM K+ Ringer to 2 mM K+ Ringer containing lo-’ M ouabain. At 135 min 40 mM K+ Ringer containing lob4 M ouabain was added. Bars give standard error. Number of experiments: cerebral cortex, 6; bladder, 6.
RESPIRATION
OF TURTLE
BRAIN
AND
BLADDER
289
experiments illustrated in Figs. 4 and 5. Depression of oxygen consumption 75 min after the addition of ouabain to bladder tissue in 2 mM K+ (19.7 per cent) was slightly less than that occurring 75 min after the addition of ouabain to bladder tissue in 40 mM K+ (22.5 per cent). DISCUSSION
Stimulation
of brain respiration
by high K+ and ouabain
The experiments reported here show that respiration of turtle brain was stimulated by high K+ in the external medium, the optimal concentration being 3060 mM. In this response, turtle brain resembles mammalian brain (Ashford & Dixon, 1935; Dickens & Greville, 1935; Hertz, 1966; Ruscak & Whittam, 1967; Chan & Quastel, 1970). The turtle brain response was rapid (Figs. 2 and 4) and, except at very high concentrations, long lasting. Also in agreement with findings on mammalian brain (Schwartz, 1962; Bourke & Tower, 1966; Swanson and Ullis, 1966; Ruscak & Whittam, 1967), respiration of turtle brain was stimulated by ouabain (Figs. 3 and 5). Since high K+ and ouabain produce similar effects in the early phase of their action and since their effects were not significantly additive (Figs. 4 and 5), it is pertinent to examine the possibility that the two agents stimulate by the same mechanism. This seems unlikely on the basis of commonly proposed explanations of high K+ action, i.e. activation of ion transport (Ruscak & Whittam, 1967; Hertz, 1968), or activation of pyruvate oxidation and acetyl-CoA production (Kini & Quastel, 1959; Chan & Quastel, 1970; Clark & Nicklas, 1970; Williamson et al., 1971), because ouabain inhibits ion transport (Schwartz, 1962; Whittam, 1962; Bourke & Tower, 1966) and depresses glycolysis in brain (Bourke & Tower, 1966; Rolleston & Newsholme, 1967). There is another mechanism of stimulation of respiration, however, in which the actions of high Kf and ouabain may be similar; this is the release of an endogenous uncoupler of mitochondrial respiration, specifically cytoplasmic Ca2+ (Lehninger et al., 1967; Rossi & Lehninger, 1963). Cytoplasmic Ca 2+ levels are important in the control of respiration (Kleinzeller, 1960; Vasington & Murphy, 1962; Bygrave, 1967) and are known to be affected by both high K+ and ouabain. For example, ouabain labilizes intracellular Ca2+ in heart (Besch & Schwartz, 1970; Lee et al., 1970) and increases cytoplasmic and mitochondrial calcium levels in brain slices (Tower, 1968). High K+ causes increased calcium uptake in some nervous and muscular tissues (Hodgkin & Keynes, 1957; Douglas & Poisner, 1964; Urakawa et al., 1970). Thus the possibility that calcium is the key to the stimulatory effects of ouabain and high ambient K+ deserves further investigation. The decline in respiration which follows the stimulatory phase of lo-4 and 10e5 M ouabain (Fig. 3) and 100 mM K+ (Fig. 2) may be a result of the cessation of ion transport (Ruscak & Whittam, 1967), or it may be due to supramaximal Ca2+ concentrations. Above an optimal level, intracellular Ca2+ ceases to stimulate mitochondrial respiration and becomes an inhibitor (Vasington & Murphy, 1962; Bygrave, 1967).
290
M. E. LEFEVREZ
Eflects of h&h K+ on tissues other than brain As first shown by Dickens & Greville (1935), the respiration of most tissues is not increased by exposure to high K+ in the external medium. The kidney cortex (Taggart et al., 1953) and, as shown in Fig. 4, the turtle urinary bladder are of the non-stimulated type. The respiration of muscle, on the other hand, is markedly stimulated by high Kf (Kernan, 1965). In frog skeletal muscle (Kernan, 1965; Van der Kloot, 1967) and guinea pig smooth muscle (Urakawa et al., 1968, 1969) the respiratory response has been dissociated from the contractile response to high ambient K+. Why high Kf stimulates the respiration of some tissues but not others is not clear. The apparent correlation of Kf-induced stimulation of respiration with excitability in nerve and muscle may not be meaningful since Hertz (1966) localized K+-responsive oxygen consumption in cat cortex to the non-excitable neuroglial cells. As discussed in the previous section, however, the effect of high Kf on the state of cellular Ca2+ may be determining. High K+ may stimulate respiration only when it increases Ca 2+ to optimal levels. Differences in calcium-binding components in different cell types could account for the variability of the response to high K+. Interpretation of the eflects of ouabain Inhibition of tissue respiration by ouabain is usually attributed to inhibition of Na-K-ATPase and subsequent cessation of Na+ and K+ transport. There is much evidence in favor of this mechanism (Charnock & Opit, 1968). The partial inhibition of turtle bladder respiration by ouabain (Figs. 4 and 5) probably is best explained on the basis of Na-K-ATPase inhibition (Solinger et al., 1968). However, experiments with brain respiration such as those reported here suggest that ouabain action is not simple. Brain tissue with its demonstrably great ion turnover and its abundant ouabain-inhibitable ATPase (McIlwain, 1966) responds to ouabain by an increase rather than a decrease in respiration (Figs. 3 and 5). These facts are fitted into the usual picture of ouabain action (inhibition of Na-KATPase) only with considerable difficulty (Swanson & Ullis, 1966; Ruscak & Whittam, 1967). Ouabain effects may require reinterpretation as this agent’s complex actions, particularly those on cellular Ca2+ levels, become better understood. Acknowledgements-The technical assistance of Ms. Leslie Dox in the work reported here is gratefully acknowledged. This study was supported by N.I.H. Research Grants Nos. AM 13953 and AM 13037, by NSF. Research Grant No. GB 7764 and by the U.S. Atomic Energy Commission. REFERENCES ASHFORDC. A. & DIXON K. C. (1935) The effect of potassium on the glucolysis of brain tissue with reference to the Pasteur effect. Biochem. J. 29, 157-168. BESZH H. R. & SCHWARTZA. (1970) On a mechanism of action of digitalis. ,J. Mol. Cell. Cardiol. 1, 195-199. BOURKER. S. & TOWER D. B. (1966) Fluid compartmentation and electrolytes of cat cerebral cortex in vitro. J. Neurochem. 13, 1099-l 117.
RESPIRATION OF TURTLEBRAINANDBLADDER
291
BYGRAVEF. L. (1967) The ionic environment and metabolic control. Nature, Lond. 214, 667-671. CHANS. L. & QUA~TELJ. H. (1970) Effects of neurotropic drugs on sodium influx into rat brain cortex in vitro. Biochem. Pharmuc. 19, 1071-1085. CHARNOCK J. S. & OPIT L. J. (1968). Membrane metabolism and ion transport. In Biological Basis of Medicine (Edited by BITTAR E. and BITTAR N.) pp. 69-103. Academic Press, New York. CLARK J. B. & NICKLASW. J. (1970) The metabolism of rat brain mitochondria. J. biol. Chem. 245, 4724-4731. DICKENS F. & GREVILLE G. D. (1935) The metabolism of normal and tumour tissue. Biochem. J. 29, 1468-1483. DOUGLASW. W. & POISNERA. M. (1964) Calcium movement in the neurohypophysis of the rat and its relation to the release of vasopressin. J. Physiol., Lond. 172, 19-30. GONZALEZC. F., SHAMOO Y. E. & BRODSKYW. A. (1967a) Electrical nature of active chloride transport across short-circuited turtle bladders. Am. J. Physiol. 212, 641-650. GONZALEZC. F., SHAMOOY. E., WYSSBRODH. R. & BRODSKYW. A. (1967b) Electrical nature of sodium transport across the isolated turtle bladder. Am. J. Physiol. 213, 333-340. HERTZ L. (1966) Neurological localization of potassium and sodium effects on respiration in brain. J. Neurochem. 13, 1373-1387. HERTZ L. (1968) Potassium effects on ion transport in brain. J. Neurochem. 15, l-16. HODGKINA. L. & KEYNES R. D. (1957) M ovement of labelled calcium in squid giant axons. J. Physiol., Lond. 138, 253-281. KERNANR. P (1965) Cell K. pp. 121-141 Butterworth, London. KINI, M. M. & QUA~TELJ. H. (1959) Carbohydrate-ammo-acid interrelations in brain cortex in vitro. Nature, Land, 184, 252-258. KLEINZELLERA. (1960) The role of potassium and calcium in the regulation of metabolism in kidney cortex slices. In Membrane Transport and Metabolism (Edited by KLEINZELLERA. & KOTYK A.), pp. 527-542. Academic Press, New York. LEE K. S., SHIN M. R., KANG D. H. & CHEN K. K. (1970) Studies on the mechanism of cardiac glycoside action. Biochem. Pharmac. 19, 1055-1069. LEFEVRE M. E. (1969) Calibration of the Clark oxygen electrode for use in aqueous solutions. J. appl. Physiol. 26, 844-846. LEFEVRE M. E., CRONKITEC. & BRODSKYW. A. (197Oa) Changes in oxygen consumption of isolated tissues after ouabain administration or substitution of choline for sodium in bathing solutions. Biochim. biophys. Acta 222, 212-215. LEFEVRE M. E., GENNAROJ. F. & BRODSKYW. A. (1970b). Properties of isolated mucosal and serosal fractions of turtle bladder. Am. J. Physiol. 219, 716-723. LEFEVRE M. E., WYSSBRODH. R. & BRODSKYW. A. (197Oc) Problems in the measurement of tissue respiration with the oxygen electrode. BioScience 20, 761-764. LEFEVRE M. E., GENNAROJ. F. & BRODSKYW. A. (1971) The isolated mucosa of turtle bladder. Anat. Rec. 171, 237-244. LEHNINGERA. L., CAROFOLIE. & ROSSI C. S. (1967) Energy-linked ion movements in mitochondrial systems. Adv. Enzymol. 29, 2.59-320. MCILWAIN H. (1966) Biochemistry and the Central Nervous System, pp. 49-77. 3rd edn., Churchill, London. ROLLE~TONF. S. & NEWSHOLMEE. A. (1967) Control of glycolysis in cerebral cortex slices. Biochem. J. 104, 524-533. ROSSIC. S. & LEHNINGERA. (1963) Stochiometric relationships between accumulation of ions by mitochondria and the energy-coupling sites in the respiratory chain. Biochem. 2.338, 698-713. RUSCAKM. & WHITTAMR. (1967) The metabolic response of brain slices to agents affecting the sodium pump. J. Physiol. Land. 190, 595-610.
292
M. E. LEFJNFUZ
SCHWAFCX A. (1962) Thee ffect of ouabain on potassium content, phosphoprotein
metabolism and oxygen consumption of guinea pig cerebral tissue. Biochem. Pharmac. 11, 389401. SOLINGER R. E., GONZALEZC. F., SHAMOOY. E., WY~SBRODH. R. & BRODSKYW. A. (1968) Effect of ouabain on ion transport mechanisms in the isolated turtle bladder. Am.J. Physiol. 215, 249-261. SWANSON P. D. & ULLIS K. (1966) Ouabain-induced changes in sodium and potassium content and respiration of cerebral cortex slices: dependence on medium calcium concentration and effects of protamine. r. Pharmac. exp. Ther. 153, 321-328. TAGGARTJ. V., SILVERMANL. & TRAYNERE. M. (1953) Influence of renal electrolyte composition on the tubular excretion of p-aminohippurate. Am. J. Physiol. 173, 345-350. TOWER D. B. (1968) Ouabain and the distribution of calcium and magnesium in cerebral tissues in vitro. Exp. Brain Res. 6, 273-283. TOWER D. B. (1969) In Handbook of Neurochemistry pp. 13-15, Vol. 1, (Edited by LAJTHA A.) Plenum Press, New York. URAKAWA N., IKEDAM., SAITOY. & SAKAIY. (1968) Effects of calcium depletion on oxygen consumption in guinea pig Taenia coli. Jap. J. Pharm. 18, 500-508. URAKAWAN., IKEDAM., SAITOY. & SAKAIY. (1969) Effects of factors inhibiting tension development on oxygen consumption of guinea pig Taenia coli in high K medium. Jap.J. Pharm. 19, 578-586. URAKAWA N., KARAKIH. & IKEDAM. (1970) Effects of ouabain and metabolic inhibiting factors on Ca distribution during K-induced contracture in guinea pig Taenia coli. 3ap.J. Pharm. 20,360-366. VAN DER KLOOT W. G. (1967) Potassium-stimulated respiration and intracellular calcium release in frog skeletal muscle. J. Physiol., Land. 191, 141-165. WHITTAM R. (1962) The dependence of the respiration of brain cortex on active cation transport. Biochem. J. 82, 205-212. VASINCTON F. D. & MURPHY J. V. (1962) Ca2+ uptake by rat kidney mitochondria and its dependence on respiration and phosphorylation. J. biol. Chem. 237, 2670-2677. WILLIAMSONJ. R., CLARK J. B., NICKLASW. J. & SAPERB. (1971) Control of glycolysis and oxidative metabolism in tissues. In Ion Homeostasis of the Brain (Edited by SIESJO B. K. & SORENSENS. C.), pp. 381-411. Academic Press, New York. WOLLENBERGER A. (1947) Metabolic action of the cardiac glycosides. y. Pharmac. 91,39-51. Key Word Index-Respiration; bolism; Pseudemys scripta.
ouabain; turtle bladder; intracellular Caa+; brain meta-
EFFECTS OF OUABAIN AND HIGH K+ ON RESPIRATION OF TURTLE BRAIN AND URINARY BLADDER
IN VITRO M. E. LEFEVRE The Mount Sinai Medical and Graduate Schools of the City University of New York, New York, N.Y. 10029, U.S.A. and The Medical Research Center, Brookhaven National Laboratory, Upton, N.Y. 11973, U.S.A. (Received 25 September 1972) Abstract-l. In agreement with findings on mammalian brain, respiration of turtle cerebral cortical slices was stimulated by ouabain and high K+ in the external medium. 2. The effects of 40 mM K+ and 10e4 M ouabain on brain respiration were not significantly additive. 3. The possibility that mitochondrial “Ca-respiration” accounts for both Kf-induced and ouabain-induced stimulation of brain slice respiration is discussed. 4. In contrast to brain, respiration of urinary bladder tissue was slightly depressed by 40 mM K+ in the external medium. 5. Ouabain (low4 M) depressed bladder oxygen consumption by approximately 20 per cent in the presence of both 2 and 40 mM K+. INTRODUCTION THE RESPIRATIONof slices of mammalian cerbral cortex is stimulated by increasing the concentration of potassium ion in the external medium (Ashford & Dixon, 1935; Dickens & Greville, 1935; Hertz, 1966; Ruscak & Whittam, 1967; Chan & Quastel, 1970). Maximal stimulation occurs at concentrations of Kf higher than those of ordinary Ringer solutions by a factor of S-10 and elevates respiration rate to as much as twice the resting rate. Several explanations have been advanced for the “potassium effect” on brain respiration. Most of these attribute the increased respiration to activation of ion transport (Ruscak & Whittam, 1967; Hertz, 1968) or of an enzyme system involved in carbohydrate metabolism (Kini & Quastel, 1959; Clark & Nicklas, 1970). The respiration of cortical slices is also stimulated by the cardiac glycoside ouabain (Schwartz, 1962; Bourke & Tower, 1966; Swanson & Ullis, 1966 ; Ruscak & Whittam, 1967) ; in this respect, brain differs from most other tissues (Wollenberger, 1947; LeFevre et al., 1970a). Ouabain-induced increase in brain slice oxygen uptake occurs only in the presence of Caa+ in bathing solutions (Schwartz, 1962; Bourke & Tower, 1966; Ruscak & Whittam, 1967) and has been explained as a mitochondrial response to free Caa+ “mobilized” by ouabain (Tower, 1969). If disparate processes cause the stimulation of respiration 283
284
M. E. LEFEVRE
by high K+ and by ouabain, these two agents might have additive effects. This possibility was tested in the experiments reported here. The effects of K+ and ouabain, separately and together, were investigated on the turtle brain. To gain further information on the effects of K+ and ouabain, experiments were performed on an unrelated tissue, the urinary bladder of the turtle. The bladder provides an interesting contrast to brain since both tissues, although different in function, are involved in processes which require large movements of ions (McIlwain, 1966; Gonzalez et al., 1967a, b). MATERIALS Preparation
AND METHODS
of tissues
Turtles (Pseudemys scripta, Mogul-Ed Co., Oshkosh, Wisconsin) were kept with access to fresh running water but without feeding. Brains were obtained through a diamondshaped incision in the top of the severed head. The bone and meninges were quickly removed and slices 050-0*75 mm in thickness were cut from the gray matter of the cerebral lobes with a razor. Brain slices showed a tendency to fragment during experiments, but, if handled carefully, could be kept intact for hours. Urinary bladders were dissected out, rinsed and cut into small pieces measuring approximately 0.75 mm in thickness. Oxygen consumption Oxygen consumption was measured polarographically using procedures and instrumentation described elsewhere (LeFevre, 1969; LeFevre et al., 1970b, c). Temperature was constant at 25°C. The amount of tissue in the 10 ml respirometer vessels was 0+4-0*08 g (dry weight). During the 60-90 min equilibration phase and the 4-5-hr experimental phase, airsaturated fluid was run into the respirometer approximately every 20 min. The first 5-7 min of each 20-min period were disregarded in calculating the rate of oxygen consumption to avoid error due to diffusional loss of oxygen from medium into tissues (LeFevre et al., 197Oc). Results were expressed as mean + S.E.M. Solutions Final concentrations in the basic Ringer solution were NaCl, 105 mM; NaHCO,, 3 mM; KCl, 2 mM; CaCl,, 2 mM; MgCl,, 1 mM; phosphate buffer, 0.8 mM; glucose, 5.5 mM; streptomycin sulfate, 5 mg%. Osmolality was approximately 225 and pH, 7% When K+ was varied, equivalent reciprocal changes were made in Na+ so that the osmolality was unchanged. In the experiments to be described, Ringer solutions are designated by their KC1 concentration; e.g. 40 mM Kf Ringer indicates a Ringer containing 40 mM KC1 and 67 mM NaCl. Ouabain tetrahydrate was obtained from Sigma Chemical Co., St. Louis, MO. RESULTS
High K+ concentrations In agreement with findings on mammalian brain, the respiration of turtle cortical slices was stimulated promptly by increasing K+. Figure 1 shows oxygen uptake iates during a 20-min period 1 hr after high K+ addition. The rates are expressed as a percentage of the mean rate of a 40-min control period. Tissue respiration’ increased to 123.7 f 5.6 per cent of control in 15 mM K+ Ringer (four experiments) and to 143.3 + 13 per cent of control in 30 mM K+ Ringer
RESPIRATION
OF TURTLE
BRAIN
AND
285
BLADDER
60
30
mM
K’
FIG. 1. Oxygen consumption of slices of turtle cerebral cortex as a function of K+ concentration. The rate after 1 hr in 15, 30 and 60 mM Kf Ringer is expressed as a percentage of the mean rate during a 4O-min control period in 2 mM K+ Ringer. Equivalent reciprocal changes were made in Na+ at each Kf concentration so that osmolality was unchanged. Vertical lines indicate one-half standard error.
Fi 0
80-
0" I
-40
I
I
I
I
I
I
I
-20
0
20
40
60
80
too
I 120
MINUTES FIG. 2. Effect of 100 mM K+ on oxygen consumption
of turtle cerebral cortical slices. At time 0 the ambient solution was changed from 2 mM Kf Ringer to 100 mM K+ Ringer. Bars give standard error. Number of experiments: 6.
286
M. E. LEFJWRE
(five experiments). Since respiration was 148 t_ 16.1 per cent of control after 1 hr in 60 mM K+ Ringer (five experiments), not much more stimulation was induced with 60 mM K+ than with 30 mM Kf. The stimulation was long sustained, lasting two or more hours with few exceptions. Figure 2 illustrates the response of brain slice oxygen consumption to the addition of 100 mM K+ Ringer (Na+ was 7 mM). This high K+ concentration caused a biphasic response illustrated by the sharp rise and subsequent decline. At 1 hr the rate was 93*1+ 4.8 per cent of initial and still declining. Ouabain The effects of ouabain in the range 1O-s-1O-3 M were tested in eight experiments. Figure 3 gives representative results: 10” M ouabain caused a slow rise in oxygen consumption of slices of turtle brain; 1O-5 M ouabain caused a sharper rise followed by a decline; 104 M ouabain caused a maximal rise also followed by a decline. The decline after 1O-5 and 1O-4 M ouabain was always present though variable in time of onset and rapidity. The effects of 10es M ouabain (not shown in Fig. 3) were similar to those of lO-4 M. 140 a $ -
130
b e 9
120
z 0 k I 2 5 ” 0”
110 100 90
*o_,
-6;O
-20
0
40
80
120
160
200
240
MINUTES
FIG. 3. Effect of varying concentrations of ouabain on oxygen consumption of turtle cerebral slices. Each curve is a single experiment. Ouabain was added at time 0. Ouabain concentrations: O, lO-4 M; A, 10es M; ?? , lObE M.
Forty mM K+ followed by ouabain Following a 60-90 min equilibration period in regular 2 mM K+ Ringer, the bathing solution was changed to 40 mM K+ Ringer. Ouabain ( lOA M) was added 135 min later and measurements were continued for an additional 120 min. Identical experiments were conducted on slices of urinary bladder tissue from the
RJSPIRATION
OF TURTLE
BRAIN
AND
BLADDER
287
same
turtles during the same time periods to characterize the response of bladder respiration to the same conditions. The results of twelve experiments are shown in Fig. 4. The maximal increase in response to 40 mM K+ in brain tissue occurred at approximately 45 min (Fig. 4). Respiration increased to 145 + 6.6 per cent of initial (I’< 0.001 for paired differences in rates at - 5 and + 45 min.) The addition of ouabain in the presence of 40 mM K+ produced a brief increase of borderline significance (O-02 < PC 0.05 for paired differences in rates at 130 and 150 min). Oxygen consumption continued at a high rate for the remainder of the experiment without marked changes in either direction. The behavior of turtle brain in the last stage of the experiment differs from that of rabbit brain in which K+-stimulated respiration declined after the addition of ouabain (Ruscak & Whittam, 1967). The respiration of bladder tissue, in contrast to that of brain, was slightly depressed by changing the ambient solution from 2 mM K+ Ringer to 40 mM K+ Ringer (Fig. 4). Oxygen uptake 75 min after the change was 88.8 f 2.2 per cent of initial (PC 0.02 for paired differences in rates at - 5 and + 75 min).
MINUTES
FIG. 4. Effect of 40 mM K+ followed by 10m4 M ouabain on oxygen consumption of slices of turtle cerebral cortex (0) and urinary bladder (A). At time 0 the bathing solutions were changed from 2 mM K+ Ringer to 40 mM K+ Ringer. At 135 min low4 M ouabain in 40 mM K+ Ringer was added. Bars give standard error. Number of experiments: cerebral cortex, 6; bladder, 6.
Ouabain in 40 mM K+ Ringer produced a further depression of bladder respiration (Fig. 4). Oxygen uptake 75 min after the addition of ouabain was 77.5 f 2.3 per cent of the pre-addition rate (PC 0.01 for paired differences in rates at 130 and 210 min).
M. E. LEFWRE
288
Ouabainfollowed by 40 mM K+ These experiments differed from those described previously in that the order of additions was reversed. The results of twelve experiments are shown in Fig. 5. The rise of respiration rate was slower with ouabain than with 40 mM K+, reaching its maximum at 105 min. The oxygen uptake rate increased to 137.0 + 4.3 per cent of initial (P-z 0.01 for paired differences in rates at -5 and + 105 min). The addition of 40 mM K+ produced a slight but not significant further increase (P> 0.05 for paired differences in rates at 130 and 150 min). It is possible that, in some cases, 40 mM K+ delayed a ouabain-induced late decline in respiration that would have occurred in its absence (see Fig. 3). The experimental results were more variable than those shown in Fig. 4 as shown by the relatively large standard error. Bladder respiration was depressed by 10M4M ouabain in 2 mM Kf Ringer (Fig. 5). The oxygen uptake rate 75 min after ouabain addition was 80.3 + 2.7 per cent of initial (PC 0.01 for paired differences in rates at - 5 and + 75 min.) The addition of 40 mM K+ to tissue already exposed to ouabain produced little change in bladder oxygen consumption. Respiration continued to be depressed during the last stage of the experiment with only a slight suggestion of recovery (Fig. 5). Despite reports of prevention or reversal of ouabain effects by high K+ (Charneck & Opit, 1968), this was not found for turtle bladder respiration in the ’
I
2 :mM k G
3.4
0
’
2mM
oUABA’N
IO-”
I
’
I
’
I
’
I
40mM
K+
M OUABAIN
lO-4
’
I
K+’
M OUABAIN
-
kl a 3.0 -
1
101 :40
0
40
80
120 MINUTES
160
::
200
0
240
FIG. 5. Effect of 10e4 ouabain followed by 40 mM K+ on oxygen consumption of slices of turtle cerebral cortex (a) and urinary bladder (A). At time 0 the bathing solutions were changed from 2 mM K+ Ringer to 2 mM K+ Ringer containing lo-’ M ouabain. At 135 min 40 mM K+ Ringer containing lob4 M ouabain was added. Bars give standard error. Number of experiments: cerebral cortex, 6; bladder, 6.
RESPIRATION
OF TURTLE
BRAIN
AND
BLADDER
289
experiments illustrated in Figs. 4 and 5. Depression of oxygen consumption 75 min after the addition of ouabain to bladder tissue in 2 mM K+ (19.7 per cent) was slightly less than that occurring 75 min after the addition of ouabain to bladder tissue in 40 mM K+ (22.5 per cent). DISCUSSION
Stimulation
of brain respiration
by high K+ and ouabain
The experiments reported here show that respiration of turtle brain was stimulated by high K+ in the external medium, the optimal concentration being 3060 mM. In this response, turtle brain resembles mammalian brain (Ashford & Dixon, 1935; Dickens & Greville, 1935; Hertz, 1966; Ruscak & Whittam, 1967; Chan & Quastel, 1970). The turtle brain response was rapid (Figs. 2 and 4) and, except at very high concentrations, long lasting. Also in agreement with findings on mammalian brain (Schwartz, 1962; Bourke & Tower, 1966; Swanson and Ullis, 1966; Ruscak & Whittam, 1967), respiration of turtle brain was stimulated by ouabain (Figs. 3 and 5). Since high K+ and ouabain produce similar effects in the early phase of their action and since their effects were not significantly additive (Figs. 4 and 5), it is pertinent to examine the possibility that the two agents stimulate by the same mechanism. This seems unlikely on the basis of commonly proposed explanations of high K+ action, i.e. activation of ion transport (Ruscak & Whittam, 1967; Hertz, 1968), or activation of pyruvate oxidation and acetyl-CoA production (Kini & Quastel, 1959; Chan & Quastel, 1970; Clark & Nicklas, 1970; Williamson et al., 1971), because ouabain inhibits ion transport (Schwartz, 1962; Whittam, 1962; Bourke & Tower, 1966) and depresses glycolysis in brain (Bourke & Tower, 1966; Rolleston & Newsholme, 1967). There is another mechanism of stimulation of respiration, however, in which the actions of high Kf and ouabain may be similar; this is the release of an endogenous uncoupler of mitochondrial respiration, specifically cytoplasmic Ca2+ (Lehninger et al., 1967; Rossi & Lehninger, 1963). Cytoplasmic Ca 2+ levels are important in the control of respiration (Kleinzeller, 1960; Vasington & Murphy, 1962; Bygrave, 1967) and are known to be affected by both high K+ and ouabain. For example, ouabain labilizes intracellular Ca2+ in heart (Besch & Schwartz, 1970; Lee et al., 1970) and increases cytoplasmic and mitochondrial calcium levels in brain slices (Tower, 1968). High K+ causes increased calcium uptake in some nervous and muscular tissues (Hodgkin & Keynes, 1957; Douglas & Poisner, 1964; Urakawa et al., 1970). Thus the possibility that calcium is the key to the stimulatory effects of ouabain and high ambient K+ deserves further investigation. The decline in respiration which follows the stimulatory phase of lo-4 and 10e5 M ouabain (Fig. 3) and 100 mM K+ (Fig. 2) may be a result of the cessation of ion transport (Ruscak & Whittam, 1967), or it may be due to supramaximal Ca2+ concentrations. Above an optimal level, intracellular Ca2+ ceases to stimulate mitochondrial respiration and becomes an inhibitor (Vasington & Murphy, 1962; Bygrave, 1967).
290
M. E. LEFEVREZ
Eflects of h&h K+ on tissues other than brain As first shown by Dickens & Greville (1935), the respiration of most tissues is not increased by exposure to high K+ in the external medium. The kidney cortex (Taggart et al., 1953) and, as shown in Fig. 4, the turtle urinary bladder are of the non-stimulated type. The respiration of muscle, on the other hand, is markedly stimulated by high Kf (Kernan, 1965). In frog skeletal muscle (Kernan, 1965; Van der Kloot, 1967) and guinea pig smooth muscle (Urakawa et al., 1968, 1969) the respiratory response has been dissociated from the contractile response to high ambient K+. Why high Kf stimulates the respiration of some tissues but not others is not clear. The apparent correlation of Kf-induced stimulation of respiration with excitability in nerve and muscle may not be meaningful since Hertz (1966) localized K+-responsive oxygen consumption in cat cortex to the non-excitable neuroglial cells. As discussed in the previous section, however, the effect of high Kf on the state of cellular Ca2+ may be determining. High K+ may stimulate respiration only when it increases Ca 2+ to optimal levels. Differences in calcium-binding components in different cell types could account for the variability of the response to high K+. Interpretation of the eflects of ouabain Inhibition of tissue respiration by ouabain is usually attributed to inhibition of Na-K-ATPase and subsequent cessation of Na+ and K+ transport. There is much evidence in favor of this mechanism (Charnock & Opit, 1968). The partial inhibition of turtle bladder respiration by ouabain (Figs. 4 and 5) probably is best explained on the basis of Na-K-ATPase inhibition (Solinger et al., 1968). However, experiments with brain respiration such as those reported here suggest that ouabain action is not simple. Brain tissue with its demonstrably great ion turnover and its abundant ouabain-inhibitable ATPase (McIlwain, 1966) responds to ouabain by an increase rather than a decrease in respiration (Figs. 3 and 5). These facts are fitted into the usual picture of ouabain action (inhibition of Na-KATPase) only with considerable difficulty (Swanson & Ullis, 1966; Ruscak & Whittam, 1967). Ouabain effects may require reinterpretation as this agent’s complex actions, particularly those on cellular Ca2+ levels, become better understood. Acknowledgements-The technical assistance of Ms. Leslie Dox in the work reported here is gratefully acknowledged. This study was supported by N.I.H. Research Grants Nos. AM 13953 and AM 13037, by NSF. Research Grant No. GB 7764 and by the U.S. Atomic Energy Commission. REFERENCES ASHFORDC. A. & DIXON K. C. (1935) The effect of potassium on the glucolysis of brain tissue with reference to the Pasteur effect. Biochem. J. 29, 157-168. BESZH H. R. & SCHWARTZA. (1970) On a mechanism of action of digitalis. ,J. Mol. Cell. Cardiol. 1, 195-199. BOURKER. S. & TOWER D. B. (1966) Fluid compartmentation and electrolytes of cat cerebral cortex in vitro. J. Neurochem. 13, 1099-l 117.
RESPIRATION OF TURTLEBRAINANDBLADDER
291
BYGRAVEF. L. (1967) The ionic environment and metabolic control. Nature, Lond. 214, 667-671. CHANS. L. & QUA~TELJ. H. (1970) Effects of neurotropic drugs on sodium influx into rat brain cortex in vitro. Biochem. Pharmuc. 19, 1071-1085. CHARNOCK J. S. & OPIT L. J. (1968). Membrane metabolism and ion transport. In Biological Basis of Medicine (Edited by BITTAR E. and BITTAR N.) pp. 69-103. Academic Press, New York. CLARK J. B. & NICKLASW. J. (1970) The metabolism of rat brain mitochondria. J. biol. Chem. 245, 4724-4731. DICKENS F. & GREVILLE G. D. (1935) The metabolism of normal and tumour tissue. Biochem. J. 29, 1468-1483. DOUGLASW. W. & POISNERA. M. (1964) Calcium movement in the neurohypophysis of the rat and its relation to the release of vasopressin. J. Physiol., Lond. 172, 19-30. GONZALEZC. F., SHAMOO Y. E. & BRODSKYW. A. (1967a) Electrical nature of active chloride transport across short-circuited turtle bladders. Am. J. Physiol. 212, 641-650. GONZALEZC. F., SHAMOOY. E., WYSSBRODH. R. & BRODSKYW. A. (1967b) Electrical nature of sodium transport across the isolated turtle bladder. Am. J. Physiol. 213, 333-340. HERTZ L. (1966) Neurological localization of potassium and sodium effects on respiration in brain. J. Neurochem. 13, 1373-1387. HERTZ L. (1968) Potassium effects on ion transport in brain. J. Neurochem. 15, l-16. HODGKINA. L. & KEYNES R. D. (1957) M ovement of labelled calcium in squid giant axons. J. Physiol., Lond. 138, 253-281. KERNANR. P (1965) Cell K. pp. 121-141 Butterworth, London. KINI, M. M. & QUA~TELJ. H. (1959) Carbohydrate-ammo-acid interrelations in brain cortex in vitro. Nature, Land, 184, 252-258. KLEINZELLERA. (1960) The role of potassium and calcium in the regulation of metabolism in kidney cortex slices. In Membrane Transport and Metabolism (Edited by KLEINZELLERA. & KOTYK A.), pp. 527-542. Academic Press, New York. LEE K. S., SHIN M. R., KANG D. H. & CHEN K. K. (1970) Studies on the mechanism of cardiac glycoside action. Biochem. Pharmac. 19, 1055-1069. LEFEVRE M. E. (1969) Calibration of the Clark oxygen electrode for use in aqueous solutions. J. appl. Physiol. 26, 844-846. LEFEVRE M. E., CRONKITEC. & BRODSKYW. A. (197Oa) Changes in oxygen consumption of isolated tissues after ouabain administration or substitution of choline for sodium in bathing solutions. Biochim. biophys. Acta 222, 212-215. LEFEVRE M. E., GENNAROJ. F. & BRODSKYW. A. (1970b). Properties of isolated mucosal and serosal fractions of turtle bladder. Am. J. Physiol. 219, 716-723. LEFEVRE M. E., WYSSBRODH. R. & BRODSKYW. A. (197Oc) Problems in the measurement of tissue respiration with the oxygen electrode. BioScience 20, 761-764. LEFEVRE M. E., GENNAROJ. F. & BRODSKYW. A. (1971) The isolated mucosa of turtle bladder. Anat. Rec. 171, 237-244. LEHNINGERA. L., CAROFOLIE. & ROSSI C. S. (1967) Energy-linked ion movements in mitochondrial systems. Adv. Enzymol. 29, 2.59-320. MCILWAIN H. (1966) Biochemistry and the Central Nervous System, pp. 49-77. 3rd edn., Churchill, London. ROLLE~TONF. S. & NEWSHOLMEE. A. (1967) Control of glycolysis in cerebral cortex slices. Biochem. J. 104, 524-533. ROSSIC. S. & LEHNINGERA. (1963) Stochiometric relationships between accumulation of ions by mitochondria and the energy-coupling sites in the respiratory chain. Biochem. 2.338, 698-713. RUSCAKM. & WHITTAMR. (1967) The metabolic response of brain slices to agents affecting the sodium pump. J. Physiol. Land. 190, 595-610.
292
M. E. LEFJNFUZ
SCHWAFCX A. (1962) Thee ffect of ouabain on potassium content, phosphoprotein
metabolism and oxygen consumption of guinea pig cerebral tissue. Biochem. Pharmac. 11, 389401. SOLINGER R. E., GONZALEZC. F., SHAMOOY. E., WY~SBRODH. R. & BRODSKYW. A. (1968) Effect of ouabain on ion transport mechanisms in the isolated turtle bladder. Am.J. Physiol. 215, 249-261. SWANSON P. D. & ULLIS K. (1966) Ouabain-induced changes in sodium and potassium content and respiration of cerebral cortex slices: dependence on medium calcium concentration and effects of protamine. r. Pharmac. exp. Ther. 153, 321-328. TAGGARTJ. V., SILVERMANL. & TRAYNERE. M. (1953) Influence of renal electrolyte composition on the tubular excretion of p-aminohippurate. Am. J. Physiol. 173, 345-350. TOWER D. B. (1968) Ouabain and the distribution of calcium and magnesium in cerebral tissues in vitro. Exp. Brain Res. 6, 273-283. TOWER D. B. (1969) In Handbook of Neurochemistry pp. 13-15, Vol. 1, (Edited by LAJTHA A.) Plenum Press, New York. URAKAWA N., IKEDAM., SAITOY. & SAKAIY. (1968) Effects of calcium depletion on oxygen consumption in guinea pig Taenia coli. Jap. J. Pharm. 18, 500-508. URAKAWAN., IKEDAM., SAITOY. & SAKAIY. (1969) Effects of factors inhibiting tension development on oxygen consumption of guinea pig Taenia coli in high K medium. Jap.J. Pharm. 19, 578-586. URAKAWA N., KARAKIH. & IKEDAM. (1970) Effects of ouabain and metabolic inhibiting factors on Ca distribution during K-induced contracture in guinea pig Taenia coli. 3ap.J. Pharm. 20,360-366. VAN DER KLOOT W. G. (1967) Potassium-stimulated respiration and intracellular calcium release in frog skeletal muscle. J. Physiol., Land. 191, 141-165. WHITTAM R. (1962) The dependence of the respiration of brain cortex on active cation transport. Biochem. J. 82, 205-212. VASINCTON F. D. & MURPHY J. V. (1962) Ca2+ uptake by rat kidney mitochondria and its dependence on respiration and phosphorylation. J. biol. Chem. 237, 2670-2677. WILLIAMSONJ. R., CLARK J. B., NICKLASW. J. & SAPERB. (1971) Control of glycolysis and oxidative metabolism in tissues. In Ion Homeostasis of the Brain (Edited by SIESJO B. K. & SORENSENS. C.), pp. 381-411. Academic Press, New York. WOLLENBERGER A. (1947) Metabolic action of the cardiac glycosides. y. Pharmac. 91,39-51. Key Word Index-Respiration; bolism; Pseudemys scripta.
ouabain; turtle bladder; intracellular Caa+; brain meta-