43

J. Physiol. (1977), 265, pp. 43-61 With 6 text-figurew Printed in Great Britain

MICROCALORIMETRIC DETERMINATION OF ENERGY EXPENDITURE DUE TO ACTIVE SODIUMPOTASSIUM TRANSPORT IN THE SOLEUS MUSCLE AND BROWN ADIPOSE TISSUE OF THE RAT

BY A. CHINET, T. CLAUSEN* AND L. GIRARDIER From the Department of Physiology, Medical School, 20, rue 4e l'Ecole-deMedecine 1211, Geneva 4, Switzerland and the *In8titute of Physiology, University of Arhu, Universitetsparken, DK-8000 Arhus C, Denmark

(Received 19 December 1975) SUMMARY

1. The resting heat production rate (E) of soleus muscles from young rats and brown adipose tissue from adult rats was measured by means of a perfusable heat flux microcalorimeter in the absence and presence of ouabain. In the soleus muscle, the acute response of E to ouabain was compared with the ouabain-suppressible components of 22Na-efflux and 42K-influx. 2. In standard Krebs-Ringer bicarbonate buffer, ouabain (10-3M) induced an immediate but transient decrease in E of around 5 %. Both in muscle and adipose tissue this was followed by a progressive rise in heat production rate. 3. When the medium was enriched with Mg (10 mM), ouabain produced a sustained decrease in E of the same magnitude as in the standard medium and the secondary rise was less marked or abolished. Under these conditions, in the soleus muscle, ouabain inhibited E? by 5 % (i.e. by 1-76 + 0-22 mcal.g wet wt.-'.min-'), 22Na-efflux by 58 % (0.187 + 0.013 ,mole. g wet wt. -. min-') and 42K-influx by 34 % (0.132 ± 0028 molel. g wet

w.l.min-1).

4. When the muscles were loaded with Na by pre-incubation in K-free Mg-enriched medium, the addition of K (3 mM) induced an immediate ouabain-suppressible increase in E of 2 98 + 0 33 mcal. g wet wt.- . minand a concomitant stimulation of 22Na-efflux of 0-388 + 0 136 molel. g wet wt.- . min-. 5. Maximum Na/ATP ratios for the active Na-K transport process were computed, with no assumption as to the in vivo free energy of ATP hydrolysis. These were 2 1, 1 9 and 2-3 under the conditions described in paragraphs (2), (3) and (4) respectively.

A. CHINET, T. CLAUSEN AND L. GIRARDIER 6. The calculated reversible thermodynamic work associated with active Na-K transport corresponded to 34 % of the measured ouabaininduced decrease in P. On the premise that the maximum efficiency of the cellular energy conservation processes is 65 %, this estimate indicates that the minimum energetic efficiency of ATP utilization by the active Na-K transport process in mammalian muscle is 52 %. 44

INTRODUCTION

It has been suggested that the active Na-K transport across the plasma membrane may be of importance in the hormonal regulation of thermogenesis (Girardier, Seydoux & Clausen, 1968; Herd, Horwitz & Smith, 1970; Ismail-Beigi & Edelman, 1970). This hypothesis has partly motivated the present work whose purpose was to obtain a quantitative estimate of the cellular energy requirement associated with this transport process under basal and slightly suprabasal metabolic conditions. This has often been done by measuring the oxygen uptake rate of tissues in the absence and presence of a cardiac glycoside. Since these compounds exert a very selective inhibitory effect on the active Na-K transport, the difference in metabolic activity observed may be interpreted as a measure of the overall energy requirement of the process. However, this approach does not take into account the possibility that progressive alterations in the distribution of Na and K across the plasma membrane can have secondary metabolic consequences. Thus, for instance, the Na gradient slowly decreases after the pump has been blocked. This gradient seems to represent a major energy source for the extrusion of Ca in several excitable tissues (Baker, 1972; Reuter, 1974). Ca, in turn, stimulates mitochondrial respiration (Chance, 1965). As proposed by several authors, ouabain may increase cytosolic Ca either from extracellular (Tower, 1968; Bailey & Sures, 1971) or cellular sources (Klaus & Lee, 1969; Langer & Serena, 1970; Nayler, 1973; Baker & Crawford, 1975). In any case, only the very early metabolic changes after inhibition of the active Na-K transport, when the ionic composition of the cytosol has not yet been significantly modified, may yield reliable information about the energy flow rate normally associated with the Na pump activity. Therefore, the acute effect of ouabain on the time course of the rate of heat production (thermogram) was examined. This was done by means of a newly designed flux microcalorimeter in perfused brown adipose tissue and muscles of the rat. Analysis of both thermogram and 22Na-efflux rate in the intact soleus muscle provided an estimate of the over-all energetic efficiency of active Na-K transport. Measurements were also performed during stimulation of this transport in order to overcome some of the difficulties related to

45 ENERGETICS OF ACTIVE Na-K TRANSPORT the secondary effects of ouabain, including the increase in spontaneous transmitter release from motor nerve endings in muscle (Katagi, 1927; Shigei, Imai & Murase, 1963; Birks, 1963; Elmqvist & Feldman, 1965; Baker & Crawford, 1975). The results suggest that the cost of the active Na-K transport was low under all conditions investigated. METHODS

Animals and tissue preparations. All experiments with rat soleus muscles and extensor digitorum longus muscles were performed using animals weighing 60-70 g. Male and female Wistar rats fed ad libitum were killed by decapitation and soleus muscles isolated as described elsewhere (Kohn & Clausen, 1971). Extensor digitorum longus muscles were dissected out after removal of the tibialis anterior muscle and the tendon retinacula at the front of the hind leg. In some experiments, soleus muscles were obtained from adult mice of the Swiss albino strain. Brown adipose tissue was prepared from male Sprague-Dawley rats (250-380 g), which had been housed at 23° C. For each experiment, one threadlike fragment, about 20 mm long and 1 mm thick, weighing between 10 and 25 mg, was excised from the ventral surface of one of the interscapular fat pads immediately after the animal had been decapitated. A rigid frame made of thin stainless steel thread prevented the preparation from furling and allowed its quick mounting and precise positioning in the calorimeter chamber. Incubation conditions. Unless otherwise specified, Krebs-Ringer bicarbonate buffered solution of the following composition (in mM) was used as the standard incubation medium: NaCl 116-8, NaHCO3 25, KCl 5 9, MgSO4 1-2, NaH2PO4 1-2, CaCl2 1P27. The medium was gassed continuously with a mixture of 95% 02 and 5 % CO2. In the calorimeter experiments the minimum flow velocity in the chamber was 1 mm- sec-1. In order to ensure sufficient oxygenation, all the experiments were performed at 300 C. Measurement of heat production. The microcalorimeter (Secfroid, S.A., Lausanne, Switzerland), which was designed in collaboration with G. Spinnler, is a thermic flux apparatus. A diagram of the experimental set-up is shown in Fig. 1. A differential calorimetric arrangement is provided by twin 1-8 ml. perfusable Tantalum chambers, one of which contains the preparation. Each chamber, the inside of which is cylindrical (7-4 mm diameter), is provided with a stopper and is placed between widely projecting thermal gradient layers made of semiconductor thermocouples mounted in series. Surrounding aluminium blocks function as a heat sink. The voltage difference between the two series of gradient layers (test chamber minus symmetrical control chamber) is proportional to the tissue heat production rate. A field effect transistor, powered by a constant voltage and mounted in a constant-current circuit, provides a calibrated heat source which allows determination of the proportionality constant at any perfusion flow rate. Since the calibrated source is situated slightly closer to the outlet of the chamber than the tissue preparation, a correction of the calibration factor was computed as a function of the perfusion flow rate. As this correction and the uncorrected factor vary in opposite directions, the over-all calibration factor (about 133 ,ucal . min-1.gV-1) increases by less than 3% when the flow rate is changed from 140 to 180 ml. h1. The signal is amplified (Keithley 150B Microvolt-Ammeter) and continuously recorded on paper (Honeywell Electronik 194 Lab/Test Recorder). Refined thermostating of a large water jacket (regulator action proportional to the error and its integral and differential values), thermal preequilibration of the perfusion medium (about 3 m long Tantalum tubing) and

46

A. CHINET, T. CLAUSEN AND L. GIRARDIER

efficient damping of residual temperature oscillations (large space, filled with polyurethane beads, between heat sink and water jacket) ensure a base-line stability such that errors due to slow random changes do not exceed 72 #scal . min-. In order to prevent any microbial heat source from developing in the course of an experiment, millipore filters (GS 0 22 #rm) were placed at the outlet of the two-channel perfusion

~~Time

das

mixtureX Eq3

0

ilk

t -

~~~~Recorder

>

pa

2

voltmser~~~~~~~~Fite

g

~~~~A-B Microvoitmeter

Thermal gradie'nt Sink

-

JC~~~~hermostatic jacket

--

Fig. 1. Diagram of the experimental setup. Chamber A of the heat flux microcalorimeter contains the perfused preparation; chamber B is the reference chamber. Krebs-Ringer bicarbonate medium was continuously pumped, filter sterilized, gassed (95% 02-5% C02) in a thin-layer tonometer and thermally equilibrated. For both chambers, the semiconductor thermocouple gradient layers are mounted in series, A in opposition to B. The over-all voltage difference A -B, which is proportional to the heat production rate of the preparation, was continuously recorded (thermogram) .

ENERGETICS OF ACTIVE Na-K TRANSPORT

47

pump. Day-to-day drift of the base line was avoided by systematically rinsing the perfusion circuits with a 1 % aqueous formaldehyde solution. Under these conditions, absolute heat production rates as low as 720 ,zcal. min- could be measured with a maximum error of 10%. Much smaller relative signals could be detected, however, provided they developed within minutes rather than hours after any change in the perfusion medium. The 90 % response time of the entire system to a sudden change in heat flow, as determined by the calibration source, was 77 sec. Introduction of a preparation into the test chamber entailed opening the thermostatic enclosure for about 30 sec. Sham introduction manoeuvres showed that the subsequent perturbation was over after 80 min. Measurement of 02 uptake rate. The basal rate of heat production was compared with the basal rate of 0, consumption in two ways: in one series of the experiments, the 02-uptake in the contralateral muscle was measured using an 02 cathode in a bubble-free liquid phase (Barde, Chinet & Girardier, 1975). In another series, direct comparison of heat production and 02-uptake was achieved by measuring Po. alternatively at the outlets of the twin calorimetric chambers using the same electrode and applying the Fick principle. The fluid distributor in front of the Clark electrode (Eschweiler catalogue no. Ea 1-AC) was switched from outside the thermostatic jacket at about 10 min intervals. The system was calibrated by injecting degassed water into the control channel at a precisely known flow rate (Braun Melsungen motor driven syringe) which was about 1 % of the tonometered medium flow rate. Measurements of Na-K-transport. These were performed as described elsewhere (Clausen & Kohn, 1977). Experimental details are given in the legends to figures and tables. All results were expressed per gram of the wet weight determined at the end of the experiments. Mean values ± s.E. of mean are given with the number of observations (n) in brackets. RESULTS

The rate of heat production (1) in the isolated rat soleus muscle was found to be stable for several hours, as recorded after the end of the 80 min perturbation due to the opening of the calorimeter. Occasional spontaneous fluctuations had a very slow time course and none had a large amplitude. In two experiments where E was recorded continuously for 9 hr without modification of the medium, the maximum deviation from the average value was 2-8 %. The rate of 02 uptake (M 02) was also constant for several hours. In the standard incubation medium (Krebs-Ringer), the basal E of the soleus, expressed in mcal .g wet wt.-'. min', was 37-7 + 0-9 (n = 23). In four experiments where E was compared withMo. measured in the contralateral muscle, a caloric equivalent of 114 + 7 kcal. mole-l was found (theoretical mean caloric equivalent for carbohydrate oxidation = 113 kcal per mole oxygen). As a further check of the oxygenation conditions in the calorimeter chamber, Mo0 and X9 were determined simultaneously using the same muscle. This yielded a caloric equivalent of 103 + 10 kcal .mole-' (n = 4). The metabolic rate of the isolated soleus falls within the ranges of values obtained by several investigators in other rat skeletal muscles (for references, see Kratzing, 1961).

A. CHINET, T. CLAUSEN AND L. GIRARDIER Basal1Pof the brown adipose tissue was 52-1 + 6-1 (n = 5). Measurements of 02 consumption performed under closely similar conditions yielded a caloric equivalent of 100 kcal . mole-' (theoretical mean caloric equivalent for lipid oxidation = 105 kcal per mole oxygen). Assuming a Q10 value of 2, the basal rate of 02 consumption of brown adipose tissue falls within the range of values obtained by other authors (for references, see Barde et al. 1975). Fig. 2 illustrates the time course of the effect of ouabain (10-3M) on X9 in four different preparations. It appears that in all instances, 48

10 F-

Ic C E (U

I 0 (U 4i

05 j-

D

0.0 L

I

-20

I

I

I

I

I

0 20 Time after addition of ouabain (min)

40

Fig. 2. The effect of ouabain on the time course of the heat production rate (E) in: A, a soleus muscle; B, a brown adipose tissue sample; C, an extensor digitorum longus muscle of the rat; and D, a soleus muscle of the mouse, re-drawn from original records. Standard Krebs-Ringer bicarbonate medium was used as perfusion fluid. Ouabain (10-3 M) was added at time zero and was then present throughout. The thermograms show the initial transitory inhibition of E induced by the glycoside and the consecutive increase in E.

ENERGETICS OF ACTIVE Na-K TRANSPORT 49 exposure of the preparation to the glycoside was immediately followed by a small transitory decrease in the rate of heat production. In brown adipose tissue and rat and mouse solei, E9 then rose to above the prior basal level whereas in another type of muscle, the extensor digitorum longus of the rat, this secondary increase was not nearly as pronounced. Other experiments showed that this biphasic response to ouabain was also obtained at lower concentrations of ouabain (10-5M for brown adipose tissue and 1O-4M for soleus). Control experiments performed without tissue in the calorimeter showed no detectable change in the base-line following the transition from normal medium to one containing 1O-3M ouabain. TABLE 1. The acute effect of ouabain (10-3 M) on heat production rate (E) in brown adipose tissue and soleus muscle of the rat AE AE (mcal. g wet wt.-' (% basal) min' Medium Preparation 4-7 + 03 2-46 ± 0-48 (3) Standard KrebsBrown adipose tissue of the rat Ringer 4.4 + 0.7 2-00 ± 037 (4) Mg-Krebs-Ringer, CaCl2 omitted 4.7 + 0*4 Standard KrebsSoleus muscle of 1*79± 0.19 (6) Ringer the rat 6.4 + 0.6 2*41 + 0.20 (7) Standard KrebsRinger, curare (10-5 M) 5-2 + 0 5 1-76 ± 0.22 (6) Mg-Krebs-Ringer

Maximum absolute and percentage decreases are expressed as means ( ± s.E. of mean) of individual values, with the number of observations indicated in brackets.

In all experiments the initial decrease in E2 induced by ouabain represented only a small percentage of the basal rate. Values obtained with rat soleus muscles and brown adipose tissue are shown in Table 1. The consecutive stimulatory effect of ouabain on X however raised the question as to whether, in its absence, the initial depressing effect would not have been larger. Therefore, attempts were made to prevent the secondary thermogenic effect. In brown adipose tissue slices, it has been shown that the release of noradrenaline from nerve endings elicits thermogenic effects which can be abolished either by prior reserpinization of the animals or by using a Mg-enriched, low-Ca medium (Barde et al. 1975). In brown adipose tissue isolated from reserpine treated rats (6 mg . kg body weight-', injected intraperitoneally 15 hr before the experiments), the calorigenic effect of ouabain was still present. However, when Ca was omitted from the medium and 15 mM-NaCl were replaced by 10 mM-MgCl2, the inhibitory effect of ouabain on E was sustained for at least 30 min and the secondary

50 A. CHINET, T. CLAUSEN AND L. GIRARDIER rise was less marked. Basal A in this medium was not significantly different from that measured in normal Krebs-Ringer, and the decrease in A was of the same magnitude (Table 1). In muscle, ouabain is known to facilitate the release of acetylcholine from motor nerve endings (see Introduction). D-tubocurarine (10-5 M) (Tubarine, Wellcome) did not prevent the calorigenic effect of ouabain but the initial drop of A was somewhat larger than in the absence of this neuromuscular blocker (Table 1). This agent had no effect on basal A. When the experiments were performed in a Mg-enriched medium (without curare) the inhibitory effect of ouabain on A was sustained for more than 1 hr. In soleus muscle, it was not necessary to omit Ca from the medium to obtain the sustained inhibitory effect of ouabain. Basal A in this medium was 325 + 0-9, i.e. not significantly different from the mean value in standard Krebs-Ringer and the decrease in A was of the same magnitude (Table 1). Attempts were also made to establish the relationships between the effects of ouabain on heat production and on the active Na-K transport in soleus muscle. Fig. 3 shows that in standard medium (Krebs-Ringer), both 22Na-efflux and A were clearly decreased within 5 min after the addition of ouabain (10-3M). However, at the time when it is evident that the inhibitory effect of ouabain on 22Na-efflux was complete, the decrease in A appears to be masked by the secondary rise. As the transient decrease in A induced by ouabain was slightly more pronounced in the presence of D-tubocurarine (10-rM), this drug was added to the incubation medium. D-tubocurarine produced no detectable change in the basal or the ouabain-suppressible components of 22Na-efflux. Since the calorigenic effect of ouabain could be suppressed by increasing the Mg concentration of the perfusion solution, it seemed appropriate to repeat the ion flux measurements in this modified medium. Fig. 4 shows that in this medium, the inhibitory effect of ouabain on A was maintained, also during the period when maximum inhibition of 22Naefflux can be ascertained. In Mg-Krebs-Ringer, the ouabain-suppressible components of 22Na-efflux and 42K-influx were somewhat smaller than in standard Krebs-Ringer (Table 2). In both media, the ratio between these two fluxes was close to 1*5. In order to analyse further the relationship between the ouabain sensitive Na-K transport and heat production, the free energy transmitted to Nla and K as they are actively transported across the plasma membrane (}v) was computed according to the formula:

iT

=

WNa+ WK = AJ22Na (-FEM+RT ln (Na0/Nai))

+ AJ42K (FEM -RT In

(K0/Ki)).

ENERGETICS OF ACTIVE Na-K TRANSPORT 51 EN, is the resting membrane potential and R, T and F have their usual significance. In Mg-Krebs-Ringer, Na0 is 130O1 mm and K., 5X9 mm. The values for Nai and K1 (respectively 18-6 and 168.6 mg) were determined A

6 E 5

Ouabain

(10-3M)

4 o_

___

2

________________

be 0

-2

.LIJ ~~~ ~~~~~~ ~~~ ~~~~~~ ~~~~~~~~~

-20

0

20

~~~

~~~

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

40

~~~B

0008 El

w 0.06

-

0

,U 004 0

0.02

C

._2

IL

0.00 L

-20

0 20 40 Time after addition of ouabain (min) Fig. 3. The effects of ouabain on heat production rate (E) and 22Na-efflux in rat soleus muscle. Standard Krebs-Ringer bicarbonate medium, to which 10-5 M D -tubocurarine was added in the calorimetry experiments. A, the change in E as compared to the basal level (AE)was read off the calorimeter tracings (n = 7) at various intervals of time after the addition of ouabain (10-3 M). B, the fraction of 22Na washed out per minute is shown as a func-

tion of time, both for controls (open circles) and for preparations exposed to ouabain (filled circles). Soleus muscles (n = 7) were loaded for 60 min with 22Na and the wash-out of isotopic Na was followed by transferring them through a series of tubes containing 3 ml. unlabelled medium.

as the concentrations in the cellular water space not available to sucrose (Clausen & Kohn, 1977) and AJ22Na and AJ42K denote the ouabainsuppressible components of the isotopic fluxes (Table 2, Mg-Krebs-

52 A. CHINET, T. CLAUSEN AND L. GIRARDIER Ringer). The resting membrane potential was assumed to be the same (-0-071 V) as in the standard Krebs-Ringer (T. Clausen and J. Flatman, in preparation). The W value 0 59 m-cal.g wet wt.-'. min-' was 6

E

-

A

r

4

Quabain (10-3M)

2

0

E

-

-

-

-

-

-

-

-

-

-2 I

-20

008

I

I

I

0

20

40

B -

E

0-06 0

U 0.,

004

-

z %.

0

0-02-

C

u..

0*00 L

-20

.0 20 Time after addition of ouabain (min)

40

Fig. 4. A, the effect of ouabain (10-3M) on heat production rate and B, 22Na-efflux in rat soleus muscle. Experimental conditions as described in the legend to Fig. 3, except that the medium was modified by replacing 15 mm of the NaCl by 10 mM-MgCl2 and that curare was omitted. (Same format as in Fig. 3.)

computed, using the converting factors 0-238 (calorie per joule) and 10-3 (m-mole per molele: W can be considered as the reversible thermodynamic work performed by the ouabain-sensitive Na-K pump per unit time. According to the definition of thermodynamic efficiency of a process (Wilkie, 1960), as applied to steady-state conditions (Wilkie, 1974),

ENERGETICS OF ACTIVE Na-K TRANSPORT 53 W is the numerator of a ratio defining the over-all efficiency (e) of the ouabain sensitive Na-K transport process: e= WI= A, where PA is the total rate of expenditure of Gibbs' free energy involved in the process and the concomitant series of oxidative and other metabolic reactions. Since the muscles were not storing any work internally by increasing the Na and K gradients in the steady-state, the measured ouabain-suppressible component of E (AP) can be considered the over-all TABLE 2. The ouabain-suppressible components of 22Na-efflux influx (AJ4SK) in rat soleus muscle

Medium

Standard Krebs-

(AJSN.) and 42K-

AJAXN, AJ42K AJ22NA molee . g wet wt.-1 molee . g wet wt.-I AJ4K min-)

min-)

AE

AJISN&

(kcal. mole-")

0-287 + 0-020 (7)

0-196 + 0-025 (4)

1-46

8-4

0-187+0-013 (11)

0-132+0-028 (4)

1-42

9-4

R~inger Mg-Krebs-

Ringer J.iswas calculated by multiplying the Na-content of the tissue water space not available to [14C]sucrose (7 -5 jumol. g wet wt.-1) with the difference between the rate coefficients for 22Na-washout in controls and in muscles which had been exposed for 20 min to ouabain (see Fig. 3). The amount of 42K taken up during a 10 min incubation period in the absence and presence of ouabain was determined and the difference (AJ42K) calculated on the basis of the specific activity of the 42K in the incubation medium.

enthalpy change per unit time associated with the process and its associated reactions. The comparisons between E and 02 consumption have shown that the metabolism was oxidative rather than anaerobic. Under these conditions the entropy change for substrate oxidation (multiplied by absolute temperature) is small compared with the enthalpy change (see Wilkie, 1960, Table 1) and the heat production rate is approximately equal to the Gibbs' free energy flow rate. Hence AP should be approximately equal to the total rate of free energy involved in the active Na-K transport process and its associated reactions, that is, to the over-all efficiency denominator (A. The ratio between JV and AP provides therefore an estimate of the over-all thermodynamic efficiency of the active Na-K transport and this was found to be 34 %. Another means of analysing the relationship between active Na-K transport and E9 was suggested by the observation that the addition of K to K-depleted soleus muscles induces a stimulation of Na efflux (Clausen

54 A. CHINET, T. CLAUSEN AND L. GIRARDIER & Kohn, 1977). In muscles which had been exposed for 150 min to K-free Krebs-Ringer, the wash-out of 22Na was found to proceed with a rate coefficient of around 0 035 min- (Fig. 5). This is slightly lower than the values obtained in the K-containing Mg-Krebs-Ringer (see Fig. 4) and consistent with earlier observations on the effect of K omission (Clausen & Hansen, 1974). The addition of KCl (3 mM) induced a prompt increase both in E and in 22Na-efflux (Fig. 5). In the presence of ouabain there was 6 E

K (3 mM)

A

4

I-

4)0

40

u

E

2

0

-l ,w,

K

-2 -20

C

008

0

4 40

20

B

E t-

006 0

004

T__ ---T

'U0o

z

- -

-

-I?-

__

-

I?

002

0 C 0 to U

U

._

0*00

L

-20

*

0

.

20 Time after addition of K (min)

.4

40

Fig. 5. A, the effect of K on heat production and B, 22Na-efflux in K-depleted rat soleus muscles. (Same format as in Fig. 3.) The muscles were perifused (or in the 22Na-efflux experiments, pre-incubated) for 150 min in K-free Mg-enriched solution. At the time indicated by the arrow, KCl (3 mM) was added to the incubation medium. The increase in E (as compared to the basal level) was read off the calorimeter tracings at various intervals after the addition of K. A correction was inade to take into account the small increase obtained in experiments performed in the presence of ouabain (10-3M). K was present throughout the period indicated by the horizontal bar.

ENERGETICS OF ACTIVE Na-K TRANSPORT 55 no significant stimulation of 22Na-efflux and the rise in 2 was considerably smaller. In a separate series of experiments, the ouabain-suppressible component of 42K-uptake was determined under similar conditions. In view of the transient nature of the K-induced stimulation, mean values for the ouabain-suppressible rise in X, 22Na-efflux and 42K-influx were calculated for the first 10 min after the addition of K (Table 3). The mean ratio between the suprabasal rates of 22Na-efflux and 42K-influx was 1-62, i.e. not markedly different from that obtained in the experiments where active Na-K transport was inhibited by ouabain. TABLE 3. The acute effects of K on E, 22Na-efflux and 42K-influx in rat soleus muscle (Mg-enriched medium) AE

At

AJASNa

(m-cal . g wet wt.-' . min-) 2 98± 0 33 (5)

(I mole . g wet wt.-' . min') 0-388 ± 0.136 (3)

AJ4SK (Pmole . g wet wt.-" . min-) 0 240 ± 0 023 (9)

AJssa

AJ22N

(kcal.

AJ.2K mole-") 1*62 7.7

Experimental conditions as described in the legend to Fig. 5. AE is the mean ouabain-suppressible increase in E as measured during the first 10 min after the addition of K (3 mM). AJa3Na was calculated by multiplying the Na-content of the tissue water space not available to [14C]sucrose (19.1 smole. g wet wt.-) with the difference between the rate coefficients for 22Na-washout measured in the presence and absence of K during the first 10 min after the addition of K.AJ42K was determined as the difference between the amount of 42K taken up in the absence and presence of ouabain (1I0M) during the first 10 min after the addition of KCl to muscles which had been pre-incubated for 150 min in K-free medium.

Since the membrane potential is likely to vary following the addition of K, the reversible thermodynamic work of the active Na-K transport could not be computed with satisfactory precision. It is apparent, however that the ratio between the rise in E and in J22Na(7 7 kcal. mole-') is not markedly different from that obtained under basal conditions (see Table 2). From the results presented in Table 1 and Figs. 3 and 4, it appears that the ouabain-suppressible fraction of the basal metabolic rate amounts to only 5-6 %. This is considerably lower than found in earlier studies, where ouabain could be shown to induce up to a 32 % decrease in the oxidation of glucose in the isolated rat hemidiaphragm (Clausen, 1966). It should be noted, however, that these latter results were obtained with a cut muscle preparation, whereas in the present study, intact muscles were used. For comparison, therefore, a number of experiments were performed in which soleus or extensor digitorum longus muscles were cut transversely at four levels so as to ensure that all fibres were leaky. In an earlier study, this was found to induce a loss of 90 % of the cellular K within 60 min (Clausen & Hansen, 1974).

A. CHINET, T. CLAUSEN AND L. GIRARD[ER From Fig. 6, it can be seen that both in soleus and extensor digitorum muscles, cutting led to an increase in P. The subsequent addition of ouabain (10-3M) produced a decrease of 14 and 21 % respectively, i.e. considerably more than obtained with the intact muscle. 56

60-

Ouabain (1O-3M)

*

0

co

E 0

co

20

0

e/

-20

0

20

60 Time after cut (min)

100

140

Fig. 6. The effect of mechanical damage on heat production in soleus and extensor digitorum longus muscles of the rat. Mg-enriched Krebs-Ringer medium. Following an equilibration period during which the E of the intact muscles was recorded, the muscles were cut transversally at four levels and then re-introduced into the calorimeter. When E had reached a new steady level, ouabain (10-3M) was added. Filled circles, soleus muscle; open circles, extensor digitorum longus muscle. DISCUSSION

The major advantages of the microcalorimeter used in the present study is that despite a relatively large perfusion rate, it has a high degree of stability together with a sensitivity and time resolution allowing the detection of small changes developing within minutes. The rates of heat production and 02 uptake measured in the calorimeter were steady for several hours in the absence of glucose and the corresponding caloric equivalent of 02 was compatible with a purely oxidative metabolism. The time course of the acute effects of ouabain on Na-efflux and A is compatible with the idea that inhibition of active Na-K transport leads to a concomitant decrease of the energy flow rate. The secondary rise in E, which was observed in all tissues studied (Fig. 2) may be the consequence of changes in membrane potential or the Na-K distribution across the

ENERGETICS OF ACTIVE Na-K TRANSPORT 57 plasma membrane, or both. This effect is reminiscent of the increased respiratory rates, with respect to controls, reported by several authors in ouabain-treated brain cortex slices (Schwartz, 1962; Swanson & McIlwain, 1965; Swanson & Ullis, 1966; Bourke & Tower, 1966; Ruscak & Whittam, 1967) and cardiac muscle (Wollenberger, 1947). The experiments with D-tubocurarine and those performed in reserpinized animals indicate that it cannot be accounted for, in our preparations, by transmitter release. The fact that this stimulatory effect is present in brain slices and in brown adipose tissue suggest that, in cardiac muscle and soleus, it may involve more than the contractile machinery. As discussed in the introduction, Ca2+-clearing processes, which require energy, may be of significance. This possibility is under further investigation. The isotopic flux measurements indicate that in soleus muscle, 10-3M ouabain produce maximum inhibition of the active Na-K-transport (Clausen & Kohn, 1977) but it cannot be excluded that part of the Na-Kactivated ATPase is not readily accessible to ouabain and, therefore, initially may be stimulated by the rise in the intracellular concentration of Na. However, this phenomenon should not be associated with any net transport of Na out of the cytoplasm and it would be difficult to explain how the addition of Mg might favour ouabain binding to such remote sites. In the present context it was of importance that by performing the experiments in a Mg-enriched medium it was possible to suppress or delay the secondary rise in B9. Furthermore, in this medium, the ratio between the initial decrease in 1 and the ouabain-suppressible component of 22Na-efflux was almost the same (9.4 kcal/mole) as in the standard Krebs-Ringer (8.4 kcal/mole). From this it seems reasonable to conclude that the initial drop in X following the addition of ouabain represents a good approximation to the total amount of energy involved per unit time, in active Na-K-transport and its associated reactions. In the standard medium experiments, it was not possible to safely relate A1 and the ouabain-suppressible component of 22Na-efflux because of the transient nature of the inhibitory effect of ouabain on E, together with the relatively long half-time for the wash-out of extracellular 22Na in the efflux experiments (Fig. 3). The comparison was done, therefore, for measurements performed in Mg-enriched medium (Fig. 4). Under these conditions, the sustained inhibitory effect of ouabain on E allowed the determination of the ratio between A1 and the ouabain-suppressible component of J22N. at a time when it could be ascertained that the effect of ouabain on22Naefflux was complete. Since this result was obtained through inhibition of the active Na-Ktransport, it was desirable to gain further information on the change in energy flow rate induced by stimulation of this transport process, through

A. CHINET, T. CLAUSEN AND L. GIRARDIER experiments in which ouabain plays a less critical role. In muscles which had been loaded with Na by pre-incubation in K-free medium, the addition of K induced a prompt rise in E, which was found to coincide with a stimulation of 22Na-efflux and 42K-influx. In these experiments, the ratio between the ouabain-suppressible increase in 1 and 22Na-efflux was 7X7 kcal/mole, which is in good agreement with the values obtained in the inhibition experiments with ouabain alone. 58

The transitory increase in E is reminiscent of the rise in heat production observed by Solandt (1936) in frog muscles exposed to buffer containing more than 8 mM-K. Others have shown that stimulation of 02 consumption is only produced when the extracellular concentration of K is increased to values above 7-5 mm (Hill & Howarth, 1957; Muller & Simon, 1960; Novotny & Vyskocil, 1966). Also in rat soleus muscles, the addition of 10 mM-K to the standard incubation medium induced a marked and ouabain-resistant increase in E. It should be noted that in contrast to these phenomena, the increase in E seen after the addition of K to Na-loaded soleus muscles was small and for its major part ouabain-suppressible.

Approximately 18-0 kcal of substrate oxidation enthalpy are involved, as a mean, in the formation of one mole ATP, no matter what part of the free energy is conserved in the phosphorylation reaction. As a matter of fact, this value is a minimum, as it was computed with the assumption that the maximum number of ATP molecules are formed during substrate oxidation (see Prusiner & Poe, 1968). The amount of energy represented by this minimum value is also very likely to be the maximum possible molar free energy of in vivo ATP hydrolysis. Thus, maximum Na/ATP ratios for the active Na-K transport process can be computed by simply dividing this figure (18-0 kcal.mole ATP-1) by the measured AJE/\J22Na ratios (Tables 1 and 2). Accordingly, at most 2-1, 1-9 and 2-3 sodium ions were actively transported per molecule of ATP used up in the process, under the respective conditions: (i) standard Krebs-Ringer + curare; (ii) Mg-enriched Krebs-Ringer; and (iii) activation of the Na-K pump induced in K-depleted muscles by re-admission of 3 mM-K. These values fall within the range reported for other tissues (Thomas, 1972; Casteels & Wuytack, 1975). The major single conclusion which may be drawn from the results presented in this report is that only 5-6 % of the total energy flux measured seems to be associated with active Na-K-transport under basal conditions. This differs from the results of earlier studies which indicated that, in the rat diaphragm, ouabain inhibited the conversion of glucose into lactate and CO2 by about 30 % (Clausen, 1966) and the 02 uptake by 16 % (Ismail-Beigi & Edelman, 1970). However, both these studies were performed with 'cut' hemi- or quarterdiaphragm preparations. It may thus be assumed that the active Na-K-transport was stimulated above its basal level due to Na leakage into the cytoplasm via the cut ends of the

ENERGETICS OF ACTIVE Na-K TRANSPORT 59 fibres. The present experiments with 'cut' soleus and extensor digitorum longus muscles support this contention. On the basis of measurements of 22Na-efflux and electrochemical gradients in rat diaphragm segments with intact fibres, Creese (1968) calculated that the reversible work required to extrude Na was 2S8 mcal. g- .minm-, which corresponded to 2 % of the total metabolism measured in the same preparation (Creese, Scholes & Whalen, 1958). If the present estimate (34 %) of the over-all energetic efficiency of active Na-K-transport is used, it can be calculated that the energetic cost of Na-extrusion in the intact diaphragm muscle preparation amounts to 5 9 % of the metabolic flow rate. This is entirely consistent with the values obtained in the present experiments with intact soleus muscles (Table 1). A further step in the analysis of the ouabain sensitive transport process is the subdivision of the over-all efficiency into that of the Na pump itself in utilizing ATP and that of the cellular energy conservation processes involved in building ATP. Under aerobic resting conditions, this involves estimating the maximum value of the molar free energy of ATP-hydrolysis in vivo (i.e. with the substrate and product concentrations actually existing inside the muscles). This value in mammalian tissue is not known but there are indications that, under the present experimental conditions, it can be more negative than the standard molar value by some 5 kcal (Klingenberg, 1961; Rosing & Slater, 1972; Nicholls, 1974). Thus the value - 1I1I7 kcal was chosen, which is tantamount to assuming a 65 % thermodynamic efficiency of ATP formation under conditions of fully controlled respiration. The efficiency of ATP utilization by the pump was then calculated as the ratio between the measured over-all efficiency (34 %) and the above value (65 %) and was found to be 52 %. This should be considered a minimum if the assumed value for the efficiency of ATP formation is taken as a maximum. It is noteworthy that Baker (1965) found a value of 80 % for the utilization of ATP for Na-extrusion in crab nerves. The skilled technical assistance of Miss Karin Skovgaard Sorensen and Miss Mette Bryderup is gratefully acknowledged. This investigation was supported by the Swiss National Science Foundation Grant No. 3.922.75 and by a grant from P. Carl Petersen's Fond. Travel expenses were covered by a grant from Hoffman-La Roche and Co., Basel. We would like to thank Dr David Hastings for valuable discussions and comments on the manuscript. REFERENCES & BAILEY, L. E. SURES, H. A. (1971). The effect of ouabain on the washout and uptake of calcium in the isolated cat heart. J. Pharmac. exp. Ther. 178, 259-270. BAER , P. F. (1965). Phosphorus metabolism of intact crab nerve and its relation to the active transport of ions. J. Physiol. 180, 383-423.

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