15. 7. 1977

Specialia

equivocal t h a n o v i p o s i t i o n d a t a 6, suggesting a m u c h h i g h e r level of specificity for l a r v a l b e h a v i o u r . This is c o n s i s t e n t w i t h t h e e x p l o i t a t i o n of a v a r i e t y of foods b y a c o s m o p o l i t a n species since, following h a t c h i n g , larvae can

7

i

899

m o v e to t h e m o s t beneficial resources available to t h e m . H o w e v e r , in m a n y r a r e e n d e m i c species, q u i t e specific o v i p o s i t i o n s t i m u l i p r e s u m a b l y occur b e c a u s e of h i g h l y specific larval resource e x p l o i t a t i o n , o f t e n i n v o l v i n g p a r t s of p a r t i c u l a r p l a n t species 7. T h e k n o w n ecological differences b e t w e e n t h e s e 2 species are p r i n c i p a l l y q u a n t i t a t i v e r a t h e r t h a n q u a l i t a t i v e , b o t h species a p p a r e n t l y using r a t h e r similar resources 8 w i t h t h e single e x c e p t i o n of e t h a n o l . H o w e v e r , since s t u d i e s c o n c e n t r a t i n g on larvae are rare, f u t u r e r e s e a r c h could alter t h i s s i t u a t i o n . The c o m p a r a t i v e s t u d y of resource u t i l i z a t i o n b y l a r v a e of d i f f e r e n t D r o s o p h i l a species rem a i n s a n o p e n field, a n d d e t a i l e d i n v e s t i g a t i o n s in p a r t i c ular of sibling species m a y yield i n t e r e s t i n g i n f o r m a t i o n concerning evolutionary divergence.

6 1 5

2 3 4 5

D. simulans

3

i

i

I

i

i

15

30

60

90

120

6 7

Time (minutes)

Mean number of larvae out of i0 on ethanol containing agar up to 8

120 min ~or the 2 sibling species.

~Adenylate

'cyclase

activation

by trypsin

We are grateful to Drs Ian R. Bock, Robert M. Cook and Lee Ehrman for their comments on the manuscript. The work was supported in part by the Australian Research Grants Committee. J . A . MeKenzie and P. A. Parsons, Oecologia 70, 373 (1972). J.A. MeKenzie, Oecologia 15, 1 (1974). D. Sewell, B. Burner and K. CoImolly, Genet. Res. 24,163 (1975). M. Libion-Mannaert, J. Delcour, M. C. Deltcombe-Lietaert, N. Lenelle-Montfort and A. Elens, Experientia 32, 22 (1976). S. King, R. F. Rockwell and J. Grossfield, Genetics 83, 539 (1976). H.L. Carson, D. E. Hardy, H. T. Spieth and W. S. Stone, in: Essays in Evolution and Genetics in Honor of Theodosius Dobzhansky, p. 437. Ed. M. K. Hecht and W. C. Steere. AppletonCentury-Crofts, New York 1970. P.A. Parsons, Q. Rev. Biol. 50, 151 (1975).

in KB cell cultures

A n n i e G u i r a u d - S i m p l o t e t L. C o l o b e r t

Laboratoire de Chimie Biologique et Mddicale, Universitd Claude-Bernard, 8, avenue Rocke/eller, F - 6 9 3 7 3 L y o n Cddex 2 (France), I December 1976 S u m m a r y . T r y p s i n c o m m o n l y used for cell dispersion increases a d e n y l a t e cyclase a c t i v i t y of IKB cells. I t a c t s on c a t a lytic r e c e p t o r s , since t h e a p p a r e n t Km for A T P is lowered, a n d it alters t h e d e p e n d e n c e of a d e n y l a t e cyclase on Mg++ ions. E v i d e n c e has a c c u m u l a t e d s u g g e s t i n g t h a t cyclic A M P can influence cell divisions in a n u m b e r of cell t y p e s i n c u t t u r e l , L C e r t a i n s agents, such as pronase, w h i c h are k n o w n to a c t on t h e cell surface, can i n i t i a t e cell divisions. B o m b i c k a n d B u r g e r S h a v e s h o w n t h a t t h e a c t i o n of s u c h a g e n t s can b e b l o c k e d b y t h e a d d i t i o n of d i b u t y r y l cyclic AMP. It seems t h a t extracellular conditions m a y control cell divisions b y a f f e c t i n g t h e m e m b r a n a l c o m p o n e n t s of t h e cyclic A M P s y s t e m , i.e. t h e a d e n y l a t e cyclase. This p a p e r describes t h e effects on a d e n y l a t e cyclase of proc e d u r e s c o m m o n l y used to d e t a c h cells from flasks d u r i n g cell cultures. Materials and methods. K B cells were g r o w n in 75 c m c u l t u r e surface F a l c o n flasks c o n t a i n i n g 20 m l of E a g l e ' s m i n i m a l essential s u p p l e m e n t e d w i t h 10% calf serum, 0.25% s o d i u m b i c a r b o n a t e a n d 0.005% a u r e o m y c i n (pH = 7.1). A p p r o x i m a t e l y 8 • l 0 s cells were seeded f r o m c o n f l u e n t cells d i s p e r s e d b y one of t h e 3 c o m m o n l y used m e t h o d s : s c r a p i n g w i t h glass t a w s , t r e a t m e n t w i t h 0.25 % t r y p s i n f o r 2 min, or t r e a t m e n t w i t h 2.5% e t h y l e n e d i a m i n e t e t r a a c e t i c s o d i u m salt (EDTA) for 10 min.

Changes of m e d i a for t h e feeding of c u l t u r e were m a d e 42 h later. I n t h e s e c o n d i t i o n s , c o n f l u e n c y is o b t a i n e d 48 h later. F o r cell n u m e r a t i o n s , cell v i a b i l i t y was r o u t i n e l y t e s t e d b y cosine d y e exclusion. F o r a d e n y l a t e cyclase a s s a y cells were h a r v e s t e d b y s c r a p i n g in 25 m M Tris-HC1 p H = 7.6 s u p p l e m e n t e d w i t h 1 m M MgCI~. a n d 250 m M saccharose, b r o k e n a t 4~ a n d c e n t r i f u g a t e d a t 600• for 10 min, t h e pellet w a s r o u t i n e l y used for assays. 3 c u l t u r e flasks were pooled for e a c h e n z y m e a c t i v i t y d e t e r m i n a t i o n . A s s a y r e a c t i o n c o n s t i t u e n t s 4 included 2 m M (ea~p)_ATP 1 ~Ci, 1 m g / m l c r e a t i n e p h o s p h o kinase, 20 m M c r e a t i n e p h s o p h a t e , 1 m g / m l b o v i n e s e r u m a l b u m i n e , 10 m M MgCt 2 a n d e n z y m e ( a p p r o x i m a t e l y

1 2 3 4

J. Otten, G. Johnson and I. Pastan, Biochem. biophys. Res. Commun. 44, 1192 (1971). E. Rozengurt and A. Pardee, J. Cell Physiol. 80, 273 (1972). B.M. Bombik and M. M. Burger, Exp. Cell Res. 80, 88 (1973). A. Guiraud-Simplot and L. Colobert, C. r. Acad. Sci, Paris 283, 93 (1976).

900

Specialia

400 [xg p r o t e i n ) in a final v o l u m e of 100 ~xl. R e a c t i o n s were i n i t i a t e d b y a d d i t i o n of e n z y m e a t 4~ were incub a t e d a t 37~ for 10 m i n in a s h a k i n g w a t e r - b a t h a n d were i m m e r s e d in boiling w a t e r for 3 min. W e a d d e d 100 ~1 s t o p solution c o n t a i n i n g 20 m M A T P a n d 6 m M (83H)-cyclic A M P (5000 cpm) as c h r o m a t o g r a p h i c tracer. The c A M P f o r m e d was isolated a c c o r d i n g t o t h e m e t h o d of W h i t e 5. The r e c o v e r y of t h e carrier (88H)-cyclic A M P v a r i e d f r o m 50% to 60%. I m m e r s i o n was carried o u t a n d s t o p solution w a s a d d e d prior t o t h e a d d i t i o n of e n z y m e in b l a n k r e a c t i o n s and b l a n k values were s u b t r a c t e d f r o m e a c h e x p e r i m e n t va/ae. All e n z y m e a s s a y s were carried o u t in duplicate. A d e n y l a t e cyclase activities e x p r e s s e d as p m o l e s of cyclic A M P f o r m e d d u r i n g t h e r e a c t i o n were linear w i t h r e s p e c t t o t i m e of i n c u b a t i o n u p to 20 m i n a n d to p r o t e i n c o n c e n t r a t i o n u p to 1 m g p r o t e i n . A c t i v i t i e s were r o u t i n e l y e x p r e s s e d as p m o l e s of cyclic A M P f o r m e d per m i n u t e p e r m g p r o t e i n ( p m o l e s / m i n / m g prot.). Results. Figure 1 s h o w s i n c r e a s e d a d e n y l a t e cyclase a c t i v i t y following t r a n s f e r of K B cells r e m o v e d f r o m c u l t u r e flasks w i t h t r y p s i n . This effect lasts several hours. S c r a p i n g or E D T A , used i n s t e a d of t r y p s i n to r e m o v e cells, does n o t cause such a c t i v i t i o n . T h e n u m e r a t i o n of K B ceils does n o t s h o w s t i m u l a t i o n of cell divisions a f t e r t r y p s i n t r e a t m e n t , in t h e used c u l t u r e c o n d i t i o n s . I n o r d e r t o c h a r a c t e r i z e f u r t h e r t h e effect of t r y p s i n on t h e s t i m u l a t i o n of a d e n y l a t e cyclase, t h e activities were d e t e r m i n e d in t h e p r e s e n c e of increasing A T P c o n c e n t r a t i o n s w i t h c o n s t a n t MgC12 c o n c e n t r a t i o n (figure 2, A) a n d in t h e p r e s e n c e of increasing MgC12 c o n c e n t r a t i o n s wittl c o n s t a n t A T P c o n c e n t r a t i o n (figure 2, B). A d e n y l a t e cyclase of cells d e t a c h e d b y t r y p s i n s h o w s an a p p a r e n t Km for A T P lower t h a n t h a t of cells d e t a c h e d b y s c r a p i n g or E D T A (Km = 0.5 m M i n s t e a d of K ~ = 0.7 mM). T h e d e p e n d e n c e of a d e n y l a t e cyclase on Mg++ ions is also different. W i t h t h e a s s a y m e t h o d we used, t h e r e is no s i g n i f i c a n t a c t i v i t y in cells d e t a c h e d b y s c r a p i n g or E D T A w h e n MgClz c o n c e n t r a t i o n is lower t h a n 2 mM, b u t we o b t a i n e d 15% of m a x i m a l a c t i v i t y in cells d e t a c h e d b y t r y p s i n . This effect is n o t a b l e because Mg++ concent r a t i o n in c u l t u r e m e d i u m is 2.5 mM.

v~

30 N

?

? 20 O.

4

-~

10(1

i0

18 25

42 44 52

66

0

Fig. 1. This data shows the effect of the method used to disperse cells before seeding, on adenylate cyclase activity over a period of culture. Number of living cells (O 9 A), adenylate cyclase activity (9 [] A), cell dispersion by scraping ([B 9 EDTA (& A), trypsin (9 0).

2

100 %

50

~

~

i

~

c--

~o

Z~

30

MgCtz(mM)

Fig. 2. Confluent cells from 10 culture flasks were pooled and ade-

nylate cyclase activity was assayed ill triplicate. A shows the relative activities as a function of ATP concentrations with 10 mM MgCI=, the smallest panel shows the corresponding Lineweaver et Burk plots. B shows relative activities as a function of MgCI= concentrations with 2 mM ATP. Cell dispersion by scraping ([]), trypsin (O), EDTA (9

Discussion. T r y p s i n is k n o w n t o decrease t h e a c t i v i t y of cyclic nucleos p h o s p h o d i e s t e r a s e l o c a t e d ill p l a s m a m e m b r a n e 0, b u t also to cause a d e c r e a s e in t h e cyclic A M P level 7 a n d s t i m u l a t i o n of cell divisions a, s. T h e r e is a d i s c r e p a n c y b e t w e e n t h e s e findings a n d our reslalts conc e r n i n g t h e influence of t r y p s i n on a d e n y l a t e cyclase a c t i v i t y . R y a n e t al. 9 s u g g e s t t h a t cyclic A M P m a y be s y n t h e t i z e d a t a r a p i d r a t e b u t it m a y also be lost b y t h e cell v e r y q u i c k l y b e c a u s e t r y p s i n p r o d u c e s i n c r e a s e d p e r m e a b i l i t y of cells 1~ 11; t h e r e f o r e a d e c r e a s e in cellular cyclic A M P m a y occur. I n fact, t h e levels of cyclic A M P in c u l t u r e m e d i u m should h a v e b e e n d e t e r m i n e d before a n d a f t e r t r y p s i n t r e a t m e n t to c o n f i r m t h i s h y p o t h e s i s . F i g u r e i s h o w s t h a t t h e r e is n o s t i m u l a t i o n of cell divisions a f t e r t r y p s i n t r e a t m e n t , a l t h o u g h such s t i m u l a t i o n is generally a t t r i b u t e d t o t h e a c t i o n of p r o t e a s e s on cells. This r e a c t i o n m i g h t be d i f f e r e n t in special t r a n s f o r m e d cells Such as K B cells, or m i g h t v a r y w i t h t i m e a n d a c c o r d i n g to t h e c o n d i t i o n s of t h e t r y p s i n t r e a t m e n t . A d e n y l a t e cyclase a c t i v i t y is e n h a n c e d b y t r y p s i n w h e n used for only 2 rain, t h e r e f o r e a n e w e n z y m e s y n t h e s i s does n o t seem to be p r o b a b l e . T r y p s i n a c t s on t h e c a t a l y t i c r e c e p t o r since t h e a p p a r e n t Km for A T P is lowered (figure 2, A), f r o m 0.7 t o 0.5 mM, b u t t h e real s u b s t r a t e of t h i s e n z y m e is k n o w n to be M g - A T P , t h e n t h e d e p e n d e n c e on Mg++ ions m a y be a f f e c t e d n o t o n l y b y a m o d i f i c a t i o n of t h e r e g u l a t o r y r e c e p t o r s on w h i c h

5 6 7

9

13

72h

Time

9

ATP(mM)

10 11 12 0

. . . . . .

1(

o

2

i

A

5C

8

Adenylate cyclase activation by trypsin in KB cell cultures.

15. 7. 1977 Specialia equivocal t h a n o v i p o s i t i o n d a t a 6, suggesting a m u c h h i g h e r level of specificity for l a r v a l b e h...
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