Inhibition of sarcotubular calcium transport by caffeine: Species and temperature dependence

566

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 45 776 I N H I B I T I O N OF SARCOTUBULAR CALCIUM T R A N S P O R T BY C A F F E I N E : SPECIES AND T E M P E R A T U R E D E P E N D E N C E FRANKLIN FUCHS Department of Physiology, University of Pittsburgh, School of Medicine, Pittsburgh, Pa. r 5 213 (U.S.a.)

(Received September I9th, 1968)

SUMMARY

The effect of caffeine on calcium transport b y isolated sarcotubular vesicles from rabbit and frog skeletal muscle was compared. At 25 ° the calcium uptake b y rabbit vesicles which sedimented at I5OO-lOOOO × g was only slightly inhibited b y caffeine (I-IO mM). Increasing the temperature to 37 o caused a marked increase in the caffeine sensitivity of the rabbit vesicles. The lighter vesicular fraction (IO 000-30 ooo × g) was relatively insensitive to caffeine at both temperatures. Frog vesicles at 25 ° were as sensitive to caffeine as the rabbit vesicles at 37 ° . This difference corresponds to the known differential sensitivity of amphibian and mammalian muscle to caffeine contracture. These results are consistent with the hypothesis that caffeine produces contracture through an inhibition of sarcotubular calcium transport.

INTRODUCTION Caffeine, at concentrations greater than about 2 mM, causes contracture of amphibian skeletal muscle 1. Since this contracture occurs without depolarization of the cell membrane 2 it has been inferred t h a t caffeine acts at some intermediate step in the excitation-contraction coupling process 1. The crucial step in the coupling process is the release of calcium from the sarcoplasmic reticuluml, 3. That caffeine might promote calcium release is suggested b y tracer studies showing an enhanced efflux of 45Ca from prelabelled frog muscles in the presence of caffeine 4. However, a t t e m p t s to demonstrate a direct effect of caffeine on sarcotubular calcium transport have not met with uniform success. HASSELBACH AND MAKINOSE5 and CARSTEN AND MOMMAERTS6, using oxalate to trap calcium inside the vesicles, failed to detect any appreciable effect of caffeine on ATP-dependent calcium uptake b y isolated sarcoplasmic reticulum. Recently, CARVALHO AND LEO7, extending earlier observations of HERZ AND WEBER 8, reported t h a t in the absence of oxalate the ATP-dependent calcium uptake consists of two fractions; one fraction is tightly bound to the vesicular membrane and is not affected b y caffeine while the other is a smaller, more labile fraction which is displaced b y caffeine. Their data suggest that the caffeine-sensitive component represents calcium which is translocated across the sarcotubular membrane and exists within the vesicle as Ca 2+. This interpretation raises the question of why caffeine does not affect calcium Biochim. Biophys. Acta, 172 (I969) 566-57 °

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uptake in the presence of oxalate, where it is clear that most of the calcium accumulated by the vesicles must be translocated across the membrane rather than adsorbed to it 3. One possible interpretation of these seemingly contradictory observations is that caffeine affects the efflux of calcium from the vesicle rather than active transport across the vesicular membrane. In the presence of oxalate most of the calcium would be trapped inside the vesicle as calcium oxalate and would not readily diffuse out in response, for instance, to an increase in membrane permeability, In evaluating the published data one must take into account the experimental materials which have been used. Previous investigators 5-7, with one exception s, have studied subcellular fractions derived from rabbit muscle. Moreover, these studies were conducted at temperatures (20-25 °) which are nonphysiological for mammalian muscle. In this temperature range mammalian muscle is highly resistant to the contracture-producing effects of caffeine9. Recently FRANK AND Buss 1° have shown that at the physiological temperature of 37 ° mammalian muscle is as sensitive to caffeine as amphibian muscle. Taking these facts into account I have reinvestigated the effect of caffeine on oxalate-dependent calcium transport by isolated sarcoplasmic reticulum of rabbit and frog with special emphasis on a possible relationship between temperature and caffeine inhibition. MATERIALS AND METHODS

Sarcotubular vesicles were prepared from the back and leg muscles of the rabbit and the leg muscles of the frog as described elsewhere n. Centrifugal fractions were collected at 15OO-lO ooo and 1o000-3o000 × g. Calcium uptake was measured by the Millipore filtration technique 1~, using filters of 0.45 t~ pore diameter. Incubations were carried out at either 25 or 37 ° in a buffer solution (see legend of Fig. I) to which ethyleneglycol-bis-(/g-aminoethyl ether)-N,N'-tetraacetic acid was added to adjust the free calcium concentration to a physiological level of about I /~M (ref. 13). In 1500-I0 O00xg ,of_2

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Fig. I. The effect of caffeine on calcium uptake by subcellular fractions from rabbit muscle. Incubation medium contained ioo mM KC1, 2o mM imidazole (pH 7.o), 4 mM MgCI~, 4 mM potassium oxalate, o.2o mM 45CAC12, 0.24 mM ethyleneglycol-bis-(~-aminoethyl ether)-N,N'tetraacetic acid, o.02 mg per ml protein, 5 mM ATP, and 0. 3 M sucrose. O, control; Q, IO mM caffeine.

Biochim. Biopkys. Acta, i72 (1969) 566--570

568

F. FUCHS

t h e e x p e r i m e n t s with r a b b i t vesicles o.3 M sucrose was a d d e d t o t h e i n c u b a t i o n m e d i u m to p r e v e n t t h e r a p i d d e t e r i o r a t i o n of t h e vesicles which g e n e r a l l y occurred at 37 ° (unpublished observation). R e c e n t w o r k of E. W. GERTZ AND F. N. BRIGGS (personal communication) has shown t h a t sucrose p r e v e n t s t h e release of lysosomal e n z y m e s which t a k e s place at e l e v a t e d t e m p e r a t u r e s . The sucrose h a d no effect on calcium u p t a k e at 25 ° . RESULTS AND DISCUSSION T h e effect Of ~o mM caffeine on t h e h e a v y ( 1 5 o o - I o o o o × g) a n d light (IOOOO30000 × g) vesicular fractions from r a b b i t muscle a t 25 a n d 37 ° is shown in Fig. I. I n a g r e e m e n t with previous workersS, 6, t h e r e was o n l y a slight effect on e i t h e r fraction at 25 °. Raising t h e t e m p e r a t u r e to 37 ° caused a m a r k e d increase in t h e s e n s i t i v i t y of t h e h e a v y fraction to caffeine inhibition. The degree of i n h i b i t i o n of calcium t r a n s p o r t was a linear function of t h e caffeine c o n c e n t r a t i o n over t h e c o n c e n t r a t i o n range of I - I O m M (Fig. 2). A t 25 ° t h e r e was an a v e r a g e i n h i b i t i o n of a b o u t IO % which was a p p a r e n t l y i n d e p e n d e n t of caffeine c o n c e n t r a t i o n . As i l l u s t r a t e d in Fig. 3, t h e h e a v y

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Fig. 2. The rate of calcium uptake by the heavy fraction (i 5OO-lOOOO × g) of rabbit muscle as a function of caffeine concentration. Measurements were made at 25 and 37 °, as indicated. Figures in parentheses indicate number of measurements, and vertical bars indicate standard error. See legend of Fig. i for experimental conditions. Fig. 3. The effect of caffeine on calcium uptake by subcellular fraction from frog muscle at 25 °. O, control; 0 , io mM caffeine. Experimental conditions were as in Fig. I except that the protein concentration was o.I mg per ml and the sucrose was omitted. gl anule fraction from frog muscle was as s t r o n g l y i n h i b i t e d at 25 o as t he c o r r e s p o n d i n g fraction from r a b b i t muscle at 37 ° . Here again, t h e l i g h t e r vesicles did n o t have t h e same s e n s i t i v i t y to caffeine. HERZ AND WEBER s, w o r k i n g w i t h an oxalate-free s y s t e m , also n o t e d t h a t caffeine caused a g r e a t e r d i s p l a c e m e n t of calcium from h e a v y vesicles (650-2000 × g) p r e p a r e d from frog muscle. H o w e v e r , this o b s e r v a t i o n raises t h e question of w h e t h e r t h e caffeine m i g h t n o t be affecting m i t o c h o n d r i a l calcium u p t a k e . T h e fact t h a t t h e calcium u p t a k e b y this p r e p a r a t i o n was s t r o n g l y s t i m u l a t e d b y o x a l a t e a n d unaffected b y azide a n d d i n i t r o p h e n o l would t e n d t o s u p p o r t t h e conclusion of previous workers 1.,1~ t h a t t h e calcium u p t a k e b y t h e h e a v y centrifugal Biochim. Biophys. Acta, i72 (1969) 566-57 °

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fraction of s k e l e t a l muscle is due p r e d o m i n a n t l y t o p a r t i c l e s of s a r c o t u b u l a r origin. F u r t h e r studies c o m b i n i n g tissue f r a c t i o n a t i o n a n d electron m i c r o s c o p y should be useful in localizing m o r e precisely t h e i n t r a c e l l u l a r site of a c t i o n of caffeine. I t will be n o t e d t h a t t h e r a t e s of calcium u p t a k e of t h e frog vesicles are considerable less t h a n t h o s e of t h e r a b b i t vesicles. This difference p r o b a b l y reflects differences in p u r i t y of t h e two p r e p a r a t i o n s r a t h e r t h a n an intrinsic difference in t h e calciumt r a n s p o r t system. T h e frog p r e p a r a t i o n s m a d e in this l a b o r a t o r y t e n d to be more labile t h a n t h e r a b b i t p r e p a r a t i o n s , a n d it m a y be t h a t b y t h e t i m e t h e y are a s s a y e d (usually t h e d a y a f t e r p r e p a r a t i o n ) , t h e y c o n t a i n a higher p r o p o r t i o n of d a m a g e d vesicles. Thus, w i t h r e g a r d to b o t h species difference a n d t e m p e r a t u r e v a r i a t i o n s , s e n s i t i v i t y of t h e i n t a c t muscle to caffeine is a s s o c i a t e d w i t h a c o r r e s p o n d i n g s e n s i t i v i t y of t h e c a l c i u m - t r a n s p o r t s y s t e m t o this c o m p o u n d . These d a t a p r o v i d e evidence t h a t caffeine p r o d u c e s c o n t r a c t u r e t h r o u g h an i n h i b i t i o n of s a r c o t u b u l a r calcium t r a n s p o r t . W h e t h e r caffeine also enhances t h e efftux of calcium from t h e vesicles c a n n o t be decided from t h e s e d a t a since t h e presence of o x a l a t e w o u l d p r e s u m a b l y obscure such an effect. T h e results of HERZ AND WEBER 8 a n d CARVALHOAND LEO 7 suggest t h a t such is t h e case, a l t h o u g h an effect on active u p t a k e in t h e i r e x p e r i m e n t s c a n n o t be excluded. I t would seem plausible to suppose t h a t caffeine m i g h t affect b o t h u p t a k e a n d release of calcium. As shown b y GUTMANN AND SANDOW9, u n d e r t e m p e r a t u r e conditions in which caffeine does n o t cause c o n t r a c t u r e of m a m m a l i a n muscle it does cause pot e n t i a t i o n of t w i t c h tension. This o b s e r v a t i o n would suggest t h a t caffeine p a r t i c i p a t e s in two processes, one of which is t e m p e r a t u r e d e p e n d e n t in m a m m a l i a n muscle. On t h e basis of studies w i t h i n t a c t frog muscle BIANCHI AND BOLTONls a n d BIANCHI1~ h a v e s u g g e s t e d t h a t caffeine enhances t h e release of calcium a n d also r e t a r d s t h e r a t e of r e a c c u m u l a t i o n . A t higher caffeine c o n c e n t r a t i o n s t h e l a t t e r effect would p r e d o m i nate, hence, t h e p r o l o n g e d c o n t r a c t u r e . T h e results r e p o r t e d here, as well as o t h e r d a t a in t h e l i t e r a t u r e , are c o m p a t i b l e w i t h such a view. ACKNOWLEDGEMENTS

This research was s u p p o r t e d b y research g r a n t A M - I o 5 5 I from t h e N a t i o n a l I n s t i t u t e s of H e a l t h . T h e a u t h o r is t h e recipient of a Lederle Medical F a c u l t y A w a r d . Miss J o LINGIS p r o v i d e d e x p e r t technical assistance.

REFERENCES i A. SANDOW,Pharmacol. Rev., 17 (1965) 265. 2 J. AXELSSONAND S. THESLEFF, Acta Physiol. Scan&, 44 (1958) 553 A. WEBER, in D. R. SANADI,Current Topics in Bioenergetics, Vol. i, Academic Press, New York, 1966, p. 2o3. 4 C. P. BIANCHI,J. Gen. Physiol., 44 (1961) 845. 5 W. HASSELBACHAND 1V[.!V[AKINOSE,in J. GERGELY,Biochemistry of Muscle Contraction, Little, Brown, Boston, 1964, p. 247. 6 M. E. CARSTENAND W. F. H. M. MOMMAERTS,J. Gen. Physiol., 48 (1964) 183. 7 A. P. CARVALHOAND A. ]3. LEO, J. Gen. Physiol., 5° (1967) 1327. 8 R. HERZ AND A. WEBER, Federation Proc., 24 (1965) 2o8. 9 E. GUTMANNAND A. SANDOW,Life Sci., 4 (1965) 1149' IO G. B. FRANKAND W. C. BUSS, Arch Intern. Pharmacodyn., 17o (1967) 343. i i F. FucHs, E. W. GERTZ AND F. N. BRIGGS,J. Gen. Physiol., 52 (1968) 955. Biochim. Biophys. Acta, i72 (1969) 566-57o

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A. MARTONOSI AND R. FERETOS, J. Biol. Chem., 239 (1964) 648. S. CBAB~R~EKAND A. E. MARTELL, Organic Sequestering Agents, Wiley, New York, 1959. A. S. FAIRHORST AND D. J. J~NDEN, J. Cellular Comp. Physiol., 67 (1966) 233. A. WEB~R, R. HERz AND I. REISS, Biochem. Z., 345 (1966) 329. C. P. BIANCHI AND T. C. BOLTON, J. Pharmacol. Exptl. Therap., 157 (1967) 388. C. P. BIANCm, Federation Proc., 27 (1968) 126.

Biochim. Biophys. Acta, 172 (1969) 566-570