S H O R T C O M M U N I C A T I O N S 2 1 3

sc 43020

Potassium contractures and creatine phosphate breakdown in frog muscle

I t is well known that on increase of the external K + muscle fibers show a sustained depolarization and undergo a transient contracture. For high concentrations of K +, the duration of the contraction bears an inw~rse relation to the K + concentration 1. The metabolic rate of muscle, as determined by rates of heat production 2 and of oxygen consumption 3 is also increased by elevated K + levels; the increase is not maintained, and the duration of the transient is inversely related to the K ÷ concentration for heat production and possibly for oxygen consumption. The time scales of the decay of these increases are different, but this in part reflects differences in experimental conditions. The results cited were consistent with the view that relaxation of the muscle in elevated K + might be due to depletion of high energy phosphate stores, and we are reporting measurements of the creatine phosphate levels following exposure to high K +.

Paired sartorius muscles were carefully dissected from the frog, Rana pipiens. The muscles were examined carefully and used only if there were no signs of injury. The muscles were stored overnight in aerated frog Ringer at 4 ° before use. The muscles were placed in the appropriate solution without being held in any way, so that in high K + the muscles tended to contract almost to a cylinder. In the high K + solution, K ÷ completely replaced Na + in the Ringer.

Following the experimental treatment, the muscles were dried rapidly on a cold cellulose tissue, and plunged into petroleum ether cooled with solid CO 2 to about - - 7 o°. They were then weighed rapidly, extracted with perchloric acid and analyzed 4 for total creatine and ereatine phosphate by the method of ENNOR 5.

T A B L E I

CHANGE IN CREATINE PHOSPHATE/TOTAL CREATINE UNDER VARIOUS EXPERIMENTAL CONDITIONS

Treatment Creatine phosphate[total creatine

Control Experimental Temperature Control Experimental

3 m i n R i n g e r 3 m i n K + o ° o . 8 1 8 o . 6 5 i 0 . 8 2 0 o . 6 4 6 0 . 7 9 9 o . 6 4 3

6 m i n K + 6 r a i n K + o ° 0 . 5 0 5 0 . 6 3 3 15 m i n R i n g e r o . 6 1 3 0 . 6 9 4

0 . 5 o 6 0 . 5 8 8

i m i n R i n g e r I m i n K + 19.5 ° o . 7 3 8 0 . 5 7 7 o .781 0 . 4 6 7 o . 7 9 0 0 . 5 3 0

2 m i n K + 2 m i n K + 19.5 ° 0 . 3 2 4 o . 4 1 8 7 m i n R i n g e r 0 . 3 3 9 0 . 3 6 0

o . 3 4 6 0 . 3 3 8

At o ° in isotonic KC1 the contracture is largely over in 2-3 min. However, after 3 min in high K +, the creatine phosphate level had dropped to only about 80 % of the control level (Table I). Exposure to K ÷ for a total oI 6 min reduced the creatine

B i o c h i m . B i o p h y s . Ac ta , 88 (1964) 2 1 3 - 2 1 5

214 SHORT COMMUNICATIONS

phosphate to about 66 % of the control level (on the assmnption that creatine phos- phate/total creatine of the control muscles at o ° was true for the other muscles). There was some restoration of the creatine phosphate lost with the 6-rain exposure to K + during a I5-min soak in Ringer at o °.

At I9.5 °, the K + contracture is much shorter in duration, lasting less than I min. Exposure to high K + for I rain reduced the creatine phosphate to about 68 % of the control level; after 2 rain it was reduced to less than 50 %. A 7-rain soak in Ringer following 2 rain in K + was without significant effect on the creatine phosphate level. I t should be noted that during a 7-min soak there is considerable recovery of the ability to give a second contracture on addition of high K +.

The concentrations of ATP in the muscle extracts were measured by the firefly enzyme method 8. For five pairs of muscles at o ° and I9.5 °, the average change in ATP concentration in high potassium was - -1 .6% (range --13.2 to +14. 5 %). Therefore there seems to have been no significant change in the ATP level following exposure to high potassium.

At o°, the average loss of ereatine phosphate in 3 rain was 5.94/~moles/g (range 4.28 to 7.22), with the assumption that creatine phosphate/total creatine is the same in paired muscles 4. Under isotonic conditions, with no load, the loss of creatine phosphate is 0.235 #mole/g per twitchL Thus during 3 rain in K + the net breakdown of creatine phosphate was as great as during about 25 twitches. The actual breakdown inay have been greater, for in the absence of iodoacetic acid and in the presence of 02, there could have been some resynthesis of creatine phosphate.

The results also indicate a temperature dependence of the rate of creatine phos- phate breakdown in high K +. At I9.5 °, the average loss of creatine phosphate was 4.16/zmoles/g in I rain (range 4.11 to 4.26), compared with 5.97 #moles/g in 3 rain at o °. If the rate of creatine phosphate breakdown is assumed to be linear with time, the Q10 is 1.5. The ratio of heat production in a toad sartorius muscle during a tetanus at 16.6 ° (stimulation frequency 25/see) to that at o ° (stimulation frequency Io/sec) is about 7.2 (HILLS). If the heat production per twitch is assumed to be independent of the frequency of stimulation, the QJ0 is 1. 9.

It seems clear from the results that the relaxation of the muscle in elevated K + is not due to exhaustion of energy stores. Some other mechanism must be involved to explain the relaxation, perhaps a model involving the exhaustion of an activator released by depolarization as suggested by HODC, KIN AND HOROWlCZ 1.

This research was supported by grants NB-o2712 and RG3723 from the National Institutes of Health. The technical assistance of Miss D. J. HARDY is gratefully acknowledged.

Department of PlLvsiology, University of Minnesota, Minneapolis, Minn. and Department of Biophysics, .Johns Hopkins University, Baltimore, Md. (U.S.A.)

CHARLES EDWARDS F. D. CARLSON

1 A. L. HOI)GKIN AND I ). HOROWICZ, d r. Physiol., 153 (196o) 386. C. G. SMITH AND D. Y. SOLANDT, J. Physiol., 93 (1938) 305 •

3 A. H . HEGNAUER, \ ¥ . O. ]_?ENN AND D. M. COBB, J. Cellular Comp. Physiol., 4 (1934) 505 . 4 F. 1). CARLSO1N" AND A. SIGER, d r. Gen. Physiol., 44 (196°) 33. 5 A. H . ENNOR, in S. P. COLOWICK AND •. KAPLAN, 2lfethods i~ Enzymologv, Vol. 3, A c a d e m i c

Press , N e w Y o r k , 1957, p. 85o 856.

Biochim. Bi@hvs. dcla, 88 (1964) 2 1 3 - 2 i 5

SHORT COMMUNICATIONS 215

6 t3. L. STREHLER AND W. D. MCELROY, in S. P. COLOWICK AND •. KAPLAN, Methods in Enzymol- ogy, Vol. 3, Academic Press, New York, 1957, p. 871-873.

2 F. D. CARLSON, D. J. HARDY AND D. R. WILKIE, J. Gen. Physiol., 46 (1963) 851. 8 A. V. HILL, jr. Physiol., 159 (1961) 518.

Received March 3ist, 1964 Biochim. Biophys. Acta, 88 (1964) 213-2i 5

sc 43021

Length, tension and metabolism during short isometric contractions of frog sartorius muscles

Recent experiments with frog rectus abdominis muscles treated with DNP have shown that phosphorylcreatine is cleaved during single isotoniO -4 and short isometric contractions 3-5 at o °. In working contractions the breakdown of phosphorylcreatine is directly proportional to the external work performed, whereas in isometric con- tractions it is directly related to the duration of the stimulation and the muscle length, and hence to the developed tension. MAR]~CHAL AND MOMMAERTS 6 studied 3-i2o-sec isometric contractions of sartorius muscles and found that inorganic phosphate and creatine increase with time. They did not obtain differences that were correlated with the length of the muscle, but the experiments were not regarded as final. This paper presents results which show such differences with frog sartorius muscles performing short isometric contractions at o °. Both sartorii were separated and dissected from female Rana pipiens, tied by a thread at each end, and then allowed to rest in an oxygenated bicarbonate saline solution 8, ~ for several hours at room temperature. The muscles were then suspended between an immobile support and a jeweler's chain

1 . 2 0

I O0

~, o . e o

o --~ 0 . 6 0

_z ~ 0 4 0

0 . 2 0

T

0 5 , o :5

D U R A T I O N OF S T I M U L A T I O N I S E C )

Fig. I. Relation between breakdown of ATP and format ion of inorganic phospha te and durat ion of single isometric contract ions of frog sar tor ius muscles at o °, The general experimental procedures were as described in the text. The muscles frozen after 0.56, 0.70 and 1.15 sec of s t imulat ion were incubated for 4 ° rain at o ° in b icarbonate saline solution containing 0.38 mM F D N B ; the muscles frozen after 1.5o sec of s t imulat ion were unt rea ted muscles incubated for 5 min in FDNB-free b icarbonate saline at o °. All muscles were s t imulated at their rest length as measured in the frog. Q, ~ A T P ; O, z~inorganic phospha te ± S.E. Each point represents 8-13 muscle pairs. The dashed line describes the t ime course of tension development ; m a x i m u m tension was abou t 65 g for sartorii

weighing abou t 80 mg and abou t 3-4 cm long.

Biochim. Biophys. Hera, 88 (1964) 215-217