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UBC Theses and Dissertations

The role of calcium and extracellular materials in the in vitro growth and contractility of carcinoma… Lee, Hong-Chao 1978

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THE ROLE OF CALCIUM AND EXTRACELLULAR MATERIALS IN THE IN VITRO GROWTH AND CONTRACTILITY OF CARCINOMA CELLS " b y HONG-CHAO^LEE B . S c . Fu - Jen C a t h o l i c U n i v e r s i t y , 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department o f Zoology We accept t h i s t h e s i s as conforming to the r e q u i r e d s tandard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1978 0 HONG-CHAO LEE , 1978 In presenting th i s thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the Library sha l l make it f ree l y a v a i l a b l e for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r l y purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or p u b l i c a t i o n of th is thes is fo r f i n a n c i a l gain shal1 not be allowed without my wr i t ten permission. Department of ZOOLOGY The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date APRIL 26, 1978. • 6 i i ABSTRACT I. E p i t h e l i a l morphogenesis i n many organs depends on c o o r d i n a t e d , m i c r o f i l a m e n t - m e d i a t e d c e l l u l a r c o n t r a c t i o n s . E f f e c t s of ionophores and Ion t r a n s p o r t i n h i b i t o r s have shown i n d i r e c t l y that these c o n t r a c -t i o n s are c a l c i u m dependent. To determine i f e x t r a c e l l u l a r c a l c i u m i s r e q u i r e d f o r c o n t r a c t i o n and to determine by a d i r e c t method whether e p i t h e l i a l c e l l c o n t r a c -t i o n i s a s s o c i a t e d w i t h an i n c r e a s e i n i n t r a c e l l u l a r c a l c i u m c o n t r a c -t i o n , c o n t r a c t i o n s were induced jLn v i t r o i n monolayers of an e p i t h e l i a l c e l l l i n e , C - 4 I I c e l l s , i n the absence of hetero logous t i s s u e s . C y t o -p l a s m i c m i c r o f i l a m e n t s have been i m p l i c a t e d as the c o n t r a c t i l e o r g a n e l l e s r e s p o n s i b l e f o r the c o n v o l u t i o n of C - 4 I I c e l l sheets i n t o s t r u c t u r e s resembl ing e p i t h e l i a . The "Lanthanum method", used to d i f f e r e n t i a t e between e x t r a c e l l u l a r and i n t r a c e l l u l a r c a l c i u m , developed o r i g i n a l l y f o r s t u d i e s of muscle c o n t r a c t i o n , was adapted to the study of c u l t u r e d e p i t h e l i a l c e l l s . The method i s based on removal of e x t r a -c e l l u l a r c a l c i u m by lanthanum and i n h i b i t i o n of c a l c i u m f l u x by lanthanum and c o l d . P r i o r to c o n t r a c t i o n , i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n s ranged from 0.22 to 0 .68 yg/mg p r o t e i n . These v a l u e s were i n f l u e n c e d by the p h y s i o l o g i c a l s t a t e of the c e l l s , by d i s s o c i a t i o n procedures used f o r s u b c u l t u r e and by the c o n c e n t r a t i o n of c a l c i u m i n the medium. Upon removal from the subst ra tum, c e l l sheets con t rac ted g r a d u a l l y and, c o n c o m i t a n t l y , there was a h i g h l y s i g n i f i c a n t i n c r e a s e i n i n t r a c e l l u l a r c a l c i u m (1.02 to 4 .33 yg/mg p r o t e i n ) . Ionophore A23187 d i d not i n c r e a s e i i i I | the r a t e of c o n t r a c t i o n . In Ca - f r e e medium the c e l l s d i d not c o n t r a c t but r e l e a s e d c a l c i u m to the medium. C o n t r a c t i o n was i n h i b i t e d by 10 ^ M L a C l ^ . These r e s u l t s show d i r e c t l y that c o n t r a c t i o n of e p i t h e l i a l c e l l s depends on i n f l u x of e x t r a c e l l u l a r c a l c i u m . These o b s e r v a t i o n s support the theory that c o n t r a c t i o n of non-muscle c e l l s may be c o n t r o l l e d by the c a l c i u m content of the c e l l s , as i s c o n t r a c t i o n i n muscle c e l l s . In v i v o , the i n t r a c e l l u l a r p o l a r i t y of e p i t h e l i a , i . e . the asymmet-r i c d i s t r i b u t i o n of o r g a n e l l e s a long the a p i c a l - b a s a l a x i s , may be induced by i n t e r a c t i o n s of the deve lop ing e p i t h e l i a w i t h hetero logous t i s s u e s and s p e c i a l i z e d components of e x t r a c e l l u l a r m a t e r i a l s ( e . g . b a s a l lamina) at the b a s a l s u r f a c e . The behav io r of the C-4II c u l t u r e s shows that such p o l a r i t y can a l s o be e s t a b l i s h e d autonomously by i s o -l a t e d e p i t h e l i a l c e l l p o p u l a t i o n s , i n v i t r o . To determine whether t h i s p o l a r i t y was a s s o c i a t e d w i t h an asymmetric d i s t r i b u t i o n of e x t r a c e l l u l a r g l ycosaminog lycans , g lucosamine i n c o r p o r a t i o n i n t o e x t r a c e l l u l a r m a t e r i a l s by C-4II c e l l s was examined a u t o r a d i o g r a p h i c a l l y . In a d d i t i o n , b a s a l lamina f o r m a t i o n was examined h i s t o c h e m i c a l l y and e l e c t r o n m i c r o s c o p i c a l l y . A l though C -4 I I c e l l s form b a s a l lamina i n v i v o , they d i d not form any i n v i t r o under the c o n d i t i o n s t e s t e d . A u t o r a d i o g r a p h i c s t u d i e s showed that there was an asymmetric d i s t r i b u t i o n of g lycosaminoglycans between the a p i c a l and b a s a l s i d e s of the c e l l s , i n v i t r o . I t f o l l o w s , that the es tab l i shment of p o l a r i t y i n c y t o f i l a m e n t d i s t r i b u t i o n and c o n t r a c t i l i t y i n these . c e l l s may depend on a g rad ien t i n amounts or c o n -c e n t r a t i o n of g l y c o s a m i n o g l y c a n - c o n t a i n i n g m a t e r i a l s , but not on the presence of a s t r u c t u r e d b a s a l l a m i n a . i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i i LIST OF FIGURES x ACKNOWLEDGMENTS x i i i INTRODUCTION 1 PART I. The Ro le of Ca lc ium i n E p i t h e l i a l C e l l C o n t r a c t i o n 11 MATERIALS AND METHODS 12 I. T i s s u e C u l t u r e 12 I I . E f f e c t of Ca lc ium Content of Test S o l u t i o n s on 12 E p i t h e l i a l C e l l C o n t r a c t i o n I I I . Measur ing C o n t r a c t i o n s 13 IV. L i g h t Microscopy 13 V. Ca lc ium D e t e r m i n a t i o n 14 1 . Measurement of Ca lc ium i n Test S o l u t i o n s 14 2 . Measurement of C e l l u l a r Ca lc ium 14 a . Removal of E x t r a c e l l u l a r Ca lc ium and Measurement 16 of the Time Required f o r L a - T r i s S o l u t i o n to D i s p l a c e E x t r a c e l l u l a r Ca lc ium i n C o n t r o l (Noncontracted) C u l t u r e s b. Measurement of T o t a l Ca lc ium 17 c . Measurement of I n t r a c e l l u l a r Ca lc ium  x~l d . Atomic A b s o r p t i o n Measurements x ^ Page V I . P r o t e i n Dete rminat ion 18 V I I . Ionophore A23187 19 1 . P r e p a r a t i o n of Stock S o l u t i o n 19 2. Exper imenta t ion on C - 4 I I C e l l Sheets 20 RESULTS 21 I. E f f e c t of Ca lc ium Content of Test S o l u t i o n s on 21 E p i t h e l i a l C e l l C o n t r a c t i o n I I . Ionophore A23187 28 I I I . Measurement of C e l l u l a r Ca lc ium 34 1 . Removal of E x t r a c e l l u l a r Ca lc ium and Measurement 34 of the Time Requirement f o r L a - T r i s S o l u t i o n to D i s p l a c e E x t r a c e l l u l a r Ca lc ium 2 . C o n t r o l C u l t u r e s i n T r i s S o l u t i o n v s . L a - T r i s S o l u t i o n 43 3 . Cont racted C e l l Sheets i n T r i s S o l u t i o n v s . L a - T r i s 43 S o l u t i o n 4 . I n f l u e n c e of Ca lc ium C o n c e n t r a t i o n i n the Medium on 47 C e l l u l a r Ca lc ium Content of C o n t r o l C u l t u r e s and Cont racted C e l l Sheets 5 . E f f e c t of C o n t r a c t i o n i n Waymouth's Medium on 52 I n t r a c e l l u l a r Ca lc ium Content IV. R e l a t i o n s h i p of I n t r a c e l l u l a r Ca lc ium to P r o t e i n 54 Content i n C o n t r o l C u l t u r e s V. I n f l u e n c e of C u l t u r e Age on C e l l u l a r Ca lc ium 58 and P r o t e i n Content V I . Changes i n I n t r a c e l l u l a r Ca lc ium w i t h C o n t r a c t i o n 58 V I I . E f f e c t of Lanthanum Ions on C o n t r a c t i o n of C e l l Sheets 63 V I I I . A r t i f i c i a l and B lank Background 67 v i DISCUSSION PART I I . The D i s t r i b u t i o n of E x t r a c e l l u l a r M a t e r i a l s i n C u l t u r e s of Carcinoma C e l l s MATERIALS AND METHODS I. . Autoradiography 1. C u l t u r e Techniques 2 . I sotope I n c o r p o r a t i o n 3 . L i g h t Microscopy and Autoradiography I I . H i s t o c h e m i s t r y 1. P e r i o d i c A c i d - S i l v e r Methenamine 2 . P e r i o d i c A c i d - S c h i f f S t a i n i n g ( P A S - S t a i n i n g ) I I I . Growth on C o l l a g e n Ge l 1. P r e p a r a t i o n of C o l l a g e n Ge l 2 . C u l t u r i n g of the C e l l s on C o l l a g e n Ge l 3 . E l e c t r o n Microscopy RESULTS I. Genera l Morphology I I . Autoradiography 1. 3 H-Glucosamine L a b e l l i n g 2 . 3 H - P r o l i n e L a b e l l i n g I I I . H i s t o c h e m i c a l and E l e c t r o n M i c r o s c o p i c Examinat ions IV. C u l t u r e on C o l l a g e n Ge l DISCUSSION SUMMARY BIBLIOGRAPHY v i i Page APPENDIX I . The Composition of Hanks Balanced S a l t S o l u t i o n and Calcium- and Magnesium-Free Hanks Balanced S a l t S o l u t i o n I I . P r e p a r a t i o n of C e l l s f o r E l e c t r o n Microscopy and L i g h t Microscopy I I I . Atomic Absorption Standards and S o l u t i o n s f o r Measuring Calcium i n Test S o l u t i o n s IV. Procedure f o r Measurement of C e l l u l a r Calcium V. Standard and Blank S o l u t i o n s f o r Measuring T o t a l and I n t r a c e l l u l a r Calcium VI. P r o t e i n Measurement w i t h the F o l i n Phenol Reagent V I I . L i n e a r Regression and C o r r e l a t i o n V I I I . General Procedure f o r L i q u i d Emulsion Autoradiography of the Unstained Epon Sections IX. General Guide f o r Autoradiography 138 138 139 140 141 144 146 149 152 154 v i i i LIST OF TABLES Table Page I. E f f e c t of Ca lc ium Content of Test S o l u t i o n s on E p i t h e l i a l C e l l C o n t r a c t i o n I I . Measurement of Calc ium Content of CMF Test S o l u t i o n s Incubated i n the Presence of C e l l Sheets I I I . Measurement of Ca lc ium Content of CF Test S o l u t i o n s Incubated i n the Presence of C e l l . S h e e t s IV. Atomic A b s o r p t i o n Readings of Ca lc ium of CF Test S o l u t i o n s i n the Presence of C -4 I I C e l l Sheets V. Atomic A b s o r p t i o n Readings of Ca lc ium of BSS Test S o l u t i o n s i n the Presence of C -4 I I C e l l Sheets V I . Atomic A b s o r p t i o n Readings of Ca lc ium of BSS Test S o l u t i o n s i n the Presence of C - 4 I I C e l l Sheets With and Without EtOH and Ionophore A23187 Ca lc ium Content of C -4 I I C e l l s ( C o n t r o l C u l t u r e s ) I n t r a c e l l u l a r Ca lc ium Content of C - 4 I I C e l l s ( C o n t r o l C u l t u r e s ) Measurement of Calc ium Content of C o n t r o l C u l t u r e s Incubated w i t h T r i s or L a - T r i s S o l u t i o n a t 4°C E f f e c t of Ca lc ium Content of Test S o l u t i o n on C e l l u l a r Ca lc ium Content and Degree of C o n t r a c t i o n of C e l l Sheets X I . E f f e c t of C o n t r a c t i o n i n Waymouth's Medium on I n t r a -c e l l u l a r Ca lc ium Content of C - 4 I I C e l l s V I I . V I I I . IX . X . 26 29 30 31 33 35 36 42 46 53 55 i x Tab le Page X I I . t - T e s t f o r Mean D i f f e r e n c e of C e l l u l a r Ca lc ium and 61 P r o t e i n between 8 to 10 Days i n C u l t u r e X I I I . I n t r a c e l l u l a r C a l c i u m / C e l l P r o t e i n (yg/mg) of C o n t r o l 64 C u l t u r e s and Cont racted C e l l Sheets a f t e r 30 Minutes i n 4°C L a - T r i s S o l u t i o n XIV. E f f e c t of Lanthanum Ions on C o n t r a c t i o n of C e l l Sheets 68 XV. E f f e c t of B lank S o l u t i o n s on Ca lc ium - 69 XVI . Sources of A r t i f i c i a l Background f o r Ca lc ium Content N 71 XV I I . C e l l u l a r Ca lc ium Content i n K idney , HeLa and C - 4 I I C e l l s 89 X V I I I . The D i s t r i b u t i o n of 3 H-Glucosamine L a b e l i n C -4 I I C u l t u r e s 101 XIX. The D i s t r i b u t i o n of 3 H - G l u c o s a m i n e - L a b e l / C e l l i n C - 4 I I 102 C u l t u r e s X LIST OF FIGURES F i g u r e Page 1-3 6 7 8 9 10 11 12 13 L i v i n g C u l t u r e s of C - 4 I I C e l l s and V e r t i c a l Sec t ions through Suspended, F i x e d Fragments of C - 4 I I Monolayers E l e c t r o n Micrograph of S e c t i o n through C e l l Sheet Suspended f o r 2 Hours i n C o n t r o l Medium E l e c t r o n Micrograph of S e c t i o n through C e l l Sheet Suspended f o r 2 Hours i n Medium w i t h 10 yg/ml C y t o c h a l a s i n B and 1.0% DMSO R e p r e s e n t a t i v e Standard Curve f o r Calc ium Dete rminat ion C e l l Sheets Incubated i n BSS, Standard O p t i c s C e l l Sheets Incubated i n CMF, Standard O p t i c s r The Time Requirement f o r L a - T r i s S o l u t i o n to D i s p l a c e E x t r a c e l l u l a r Ca lc ium i n C o n t r o l C u l t u r e s ( T r yps in D i s s o c i a t i o n ) The Time Requirement f o r L a - T r i s S o l u t i o n to D i s p l a c e E x t r a c e l l u l a r Ca lc ium i n C o n t r o l C u l t u r e s (Trypsin/EGTA D i s s o c i a t i o n ) The Time Requirement f o r L a - T r i s S o l u t i o n to D i s p l a c e E x t r a c e l l u l a r Ca lc ium i n Cont racted C e l l Sheets E f f e c t of T r i s S o l u t i o n on Ca lc ium Content of Cont racted C e l l Sheets E f f e c t of Ca lc ium C o n c e n t r a t i o n of Waymouth's Medium on C e l l u l a r Ca lc ium Content of C o n t r o l C u l t u r e s 15 22 24 37 39 44 48. 50 x i F i g u r e Page 14 R e l a t i o n s h i p of I n t r a c e l l u l a r Ca lc ium to C e l l 56 P r o t e i n ( C o n t r o l C u l t u r e s ) 15 R e l a t i o n s h i p of I n t r a c e l l u l a r Ca lc ium and C e l l P r o t e i n 57 to I n t r a c e l l u l a r C a l c i u m / C e l l P r o t e i n ( C o n t r o l C u l t u r e s ) 16 C e l l u l a r Ca lc ium and P r o t e i n Content i n E a r l y Stages 59 of C u l t u r e s (2 -5 Days) 17 C e l l u l a r Ca lc ium and P r o t e i n i n D i f f e r e n t C u l t u r i n g 60 Days (4 -10 Days) 18 Hemicyst and C u l t u r e of C -4 I I C e l l s 62 19 I n t r a c e l l u l a r Ca lc ium Content of E p i t h e l i a l C e l l 65 Sheets a f t e r D i f f e r e n t P e r i o d s of C o n t r a c t i o n 20-23 Autoradiography of C - 4 I I Monolayers w i t h 25 UgCi/ml 103 3 H-Glucosamine L a b e l l i n g ( C e l l s were l a b e l l e d before they became c o n f l u e n t ) 24-26 Autorad iography of C -4 I I Monolayers w i t h 25 yC i/ml 105 3 H-Glucosamine L a b e l l i n g ( C e l l s were l a b e l l e d a f t e r they became c o n f l u e n t ) 27 Autoradiography of 10 yC i/ml 3 H - P r o l i n e L a b e l l i n g 108 28 C o n t r o l Sec t ions of Mouse Kidney ( P A - S i l v e r Methenamine 108 S t a i n i n g ) 29 Monolayer of C - 4 I I C e l l s ( P A - S i l v e r Methenamine 1 ° 8 S t a i n i n g ) 30 , 31 V e r t i c a l S e c t i o n s through Fragments of C - 4 I I C e l l s H 2 Grown on C o l l a g e n Ge l ( P A - S i l v e r Methenamine S t a i n i n g ) x i i F i g u r e Page 32 , 33 V e r t i c a l S e c t i o n s through Fragments of C -4 I I C e l l s 114 Grown on C o l l a g e n Ge l ( P A - S i l v e r Methenamine S t a i n i n g , H igh M a g n i f i c a t i o n ) 34, 35 E l e c t r o n Micrograph of S e c t i o n through Fragments of 116 C -4 I I C e l l s Grown on C o l l a g e n Ge l 36 Standard Curve f o r P r o t e i n Dete rminat ion 147 37 L i n e a r Regress ion and C o r r e l a t i o n between C e l l u l a r 150 , P r o t e i n and I n t r a c e l l u l a r Ca lc ium x i i i ACKNOWLEDGMENTS I w ish to express my s i n c e r e a p p r e c i a t i o n t o : D r . N. Auersperg , Department of Zoology and Cancer Research C e n t r e , f o r her a d v i c e , gu idance , t o l e r a n c e and encouragement throughout t h i s i n -v e s t i g a t i o n . D r . B. D. R o u f o g a l i s , Department of P h a r m a c e u t i c a l S c i e n c e s , f o r h i s c r i t i c a l a d v i c e and i n t r o d u c t i o n to the "Lanthanum method" f o r measure-ment of c e l l u l a r c a l c i u m . D r s . C. V. F innegan and J . D. B e r g e r , Department of Zoology , f o r t h e i r i n t e r e s t i n t h i s i n v e s t i g a t i o n and f o r c r i t i c a l examinat ion of t h i s m a n u s c r i p t . D r . D. H. Copp, Department of P h y s i o l o g y , f o r the use of the J a r r e l l Ash N Atomsorb Model 280 Atomic A b s o r p t i o n Spectrophotometer . Dur ing the tenure of t h i s i n v e s t i g a t i o n , the author was the r e c i p i e n t of a Teaching A s s i s t a n t s h i p at Department of Zoology . Th is r e s e a r c h was suppor ed by grants from the N a t i o n a l Cancer I n s t i t u t e of Canada to Dr . N. A u e r s -p e r g . 1 INTRODUCTION Changes i n c e l l shape are f u n c t i o n a l components of many c e l l u l a r p r o -cesses such as c y t o k i n e s i s (Schroeder , 1 9 6 9 ) , c e l l movement (Goldman, 1971; Hay, 1973) , phagocy tos is and c e l l adhes ion (Reaven and S t a n t o n , 1973) , and egg c o r t e x c o n t r a c t i l i t y ( G i n g e l l , 1970) . Changes i n c e l l shape have a l s o been noted i n s e v e r a l developmental processes such as c leavage (Schroeder , . 1968; S z o l l o s i , 1970; Tucker', 1971) , p o l a r lobe f o r m a t i o n (Conrad, 1973) , and l a t e r s tages of embryogenesis ( B e r n f i e l d et a l . , 1973; H i l f e r , 1973; Spooner, 1973; B u r n s i d e , 1973; P e r r y , 1975) . In most of the above p r o c e s s e s , the changes i n c e l l shape have been a s s o c i a t e d w i t h at l e a s t one of the f o l l o w i n g f a c t o r s : (1) m i c r o f i l a m e n t s (2) m i c r o t u b u l e s and (3) e x t r a c e l l u -l a r m a t e r i a l s (mucopolysacchar ides and/or c o l l a g e n ) . Whi le i t seems c l e a r tha t these f a c t o r s are i n t i m a t e l y i n v o l v e d i n morphogenesis , t h e i r mode(s) of a c t i o n are not yet f u l l y d e f i n e d . C y t o k i n e s i s (Schroeder , 1969) , egg c o r t e x c o n t r a c t i l i t y ( G i n g e l l , 1970) , and morphogenesis of e p i t h e l i a (Spooner, 1973; B u r n s i d e , 1973; P e r r y , 1975) , a l l r e q u i r e c a l c i u m ions and the presence of m i c r o f i l a m e n t s at r e g i o n s of l o c a l i z e d c o n t r a c t i o n s . The r e l a t i o n s h i p of c a l c i u m to these a c t i v i t i e s i s not complete ly known. I t has been suggested tha t these o r g a n e l l e s cause c e l l u l a r c o n s t r i c t i o n and movement i n a manner s i m i l a r to the a c t i n and myosin f i l a m e n t s i n the s l i d i n g - f i l a m e n t h y p o t h e s i s proposed f o r muscle c e l l s (Huxley , 1973) . The ev idence f o r the presence of a c t i n and myosin- i n non-muscle c e l l s i s now widespread and has shown that they bear remarkably s i m i l a r p r o p e r t i e s to t h e i r analogs found i n s t r i a t e d 2 muscle ( P o l l a r d and Welh ing , 1974) . R e g u l a t i o n of the c o n t r a c t i o n of non -muscle c e l l s i s compl i ca ted and probab ly i n v o l v e s more than one mechanism ( H i t c h c o c k , 1977) . In v i v o and i n v i t r o s t u d i e s have shown that c a l c i u m may r e g u l a t e some of these m o t i l e systems i n a way s i m i l a r to c a l c i u m r e g u l a t i o n i n musc le : MgATP (adenosine 5 ' - t r i p h o s p h a t e ) and micromolar c o n c e n t r a t i o n s of f r e e c a l c i u m i o n are r e q u i r e d f o r f u n c t i o n a l a c t i n - m y o s i n i n t e r a c t i o n ( c o n t r a c t i o n ) to take p l a c e . Th is may be a t a r g e t f o r some of the i n t r a c e l l u l a r c a l c i u m . C o r t i c a l c o n t r a c t i o n s are induced i n Xenopus  l a e v i s eggs and embryos by c a l c i u m i n j e c t i o n s ( G i n g e l l , 1970) , and by t reatment w i t h the a n t i b i o t i c ionophore A23187. Th is ionophore promotes I | i n c r e a s e d membrane p e r m e a b i l i t y to d i v a l e n t c a t i o n s such as Ca , thereby a c t i v a t i n g the c o r t i c a l c o n t r a c t i o n system i n f r o g eggs (Schroeder and S t r i c k l a n d , 1974) . I t a l s o counte rac ts the i n h i b i t i o n of n e u r u l a t i o n by papaver ine i n Ambystoma maculatum embryos (Moran and R i c e , 1976) . In c o n t r a s t to ionophore A23187, papaver ine (a v a s o d i l a t o r ) has been repor ted to i n h i b i t the r e l e a s e of bound c a l c i u m (Imai and Takeda, 1967) . S a l i v a r y g land morphogenesis (Ash et^ a l . , 1973) and Ambystoma maculatum n e u r u l a t i o n (Moran and R i c e , 1976) do not proceed c h a r a c t e r i s t i c a l l y i n the presence of p a p a v e r i n e , a r e s u l t a s c r i b e d to i n t e r f e r e n c e w i t h c a l c i u m a c t i v a t i o n of m i c r o f i l a m e n t s . I f papaver ine treatment does not exceed 24 h o u r s , washing the embryos and r e t u r n i n g them to ionophore A23187 c o n t a i n i n g s p r i n g water r e s u l t s i n a r e i n i t i a t i o n of n e u r u l a t i o n . From these s t u d i e s , Moran and R i c e (1976) suggested the p o s s i b i l i t y t h a t s e l e c t i v e r e l e a s e of c a l c i u m ions may be i n v o l v e d i n the mechanism by which non-muscle c e l l s r e g u l a t e t h e i r movement. 3 Microtubules, l i k e microfilaments, are i n t r a c e l l u l a r organelles, re-quired during the process of morphogenesis, such as i n c e l l elongation during formation of neural plate (Burnside, 1973) and they have a role i n mitosis and meiosis (Weisenberg, 1973). Microtubules are thought to be responsible for the d i r e c t i o n a l i t y and s t r u c t u r a l s t a b i l i t y of moving c e l l s or tissues. Microtubules are s t r u c t u r a l l y associated with the moving elements (Tilney and Porter, 1965; Bi k l e et a l . , 1966; Freed and Lebowitz, 1970; Weisenberg, 1973), which might constitute an i n t r a c e l l u l a r transport system of widespread occurrence. B i d i r e c t i o n a l streaming i n neurons ap-pears to involve microtubules, and may play a part i n the rapid transport of materials along nerve processes (Lasek, 1967; Kreutzberg, 1969). Col-chicine (Borisy and Taylor, 1967; Ishikawa et^ a l . , 1968) and vinblastine (Bensch and Malawista, 1968) are i n h i b i t o r s which can cause depolymeriza-t i o n and block the assembly of microtubules. Ex t r a c e l l u l a r materials (ECM) can be considered a semisolid composite of materials on the outer surface of i n d i v i d u a l c e l l s or as an interface between c e l l s of metazoan organisms (Slavkin, 1972). In the past few years, there have been hundreds of reports concerning the regulation and function of e x t r a c e l l u l a r materials i n morphogenesis, such as glycosaminoglycans i n embryogenesis of salivary gland (Banerjee e_t a l . , 1977), collagen i n corneal d i f f e r e n t i a t i o n (Hay and Meier, 1976) and c e l l surface coat materials i n neurulation (Moran and Rice, 1975). In general, the e x t r a c e l l u l a r materials produced by c e l l s form their own external environment which may function i n the control of innumerable c e l l processes - i . e . , protecting, connecting, binding, organizing the c e l l s into a given functional system of tissue, organ or organism. 4 Most deve lop ing e p i t h e l i a r e q u i r e i n t e r a c t i o n s w i t h hetero logous t i s s u e s f o r morphogenesis , e . g . , mesenchyme i s r e q u i r e d f o r morphogenesis of mouse submandibular e p i t h e l i a ( B e r n f i e l d et a l . , 1973) . The r e s u l t i n g complex i t y of deve lop ing e p i t h e l i a l s t r u c t u r e s makes a n a l y s i s of morpho-g e n e t i c mechanisms d i f f i c u l t ; t h e r e f o r e , the present study was undertaken to examine some aspects of e p i t h e l i a l morphogenesis i n a s i m p l e r system. The c e l l l i n e C - 4 I I (Auersperg , 1969) was o r i g i n a l l y d e r i v e d from a s q u a m o u s - c e l l carc inoma. Over years i n c u l t u r e , c e l l s of t h i s l i n e s t i l l r e t a i n some of the c h a r a c t e r i s t i c s of normal e p i t h e l i a l b a s a l c e l l s , i n c l u d i n g the a b i l i t y to form b a s a l lamina i n v i v o and s imp le m u l t i c e l l u l a r s t r u c t u r e s i n v i t r o . I t has been demonstrated tha t fragments of c o n f l u e n t monolayers of l i n e C - 4 I I become convoluted i n response to removal from a s o l i d substratum ( F i g s . 1 -3 ) ,~and t h a t the s u b c e l l u l a r changes accompanying t h i s process resemble those d e s c r i b e d f o r c o n t r a c t i n g e p i t h e l i a i n v i v o , e . g . , d u r i n g a s c i d i a n metamorphosis (C loney , 1966) and i n v i t r o , e . g . , d u r i n g s a l i v a r y g land f o r m a t i o n (Spooner, 1973) . In c o n t r a s t to embryonic systems, the c o n t r a c t i o n of 0 4 1 1 c e l l s h e e t s . i s not dependent on the presence of any hetero logous t i s s u e s . C -4 I I c e l l s t h e r e f o r e represent a s imple system, c o n s i s t i n g of one c e l l - t y p e o n l y , f o r the a n a l y s i s of e p i t h e l i a l - c e l l c o n t r a c t i o n . Cytop lasmic m i c r o f i l a m e n t s have been i m p l i c a t e d as the c o n t r a c t i l e o r g a n e l l e s r e s p o n s i b l e f o r the i n v i t r o c o n v o l u t i o n of the C - 4 I I c e l l sheets ( F i g s . 4 & 5 ) . U l t r a s t r u c t u r a l s t u d i e s of C -4 I I c e l l c o n t r a c t i o n have shown t h a t dense m i c r o f i l a m e n t b u n d l e s , i . e . , the c o n t r a c t i l e o r g a n -i c 5 FIGUHES 1-3 Living cultures of C - t l l cells, standard optics. FlOURE 1 Confluent monolayer, growing on plastic substratum. X 1-0 FIUIKK 2 Fragments of monolayer suspended for $ hr in control medium. The cell sheets are convoluted. X 40. FlOURE 3 Fragments of monolayer suspended for i hr in medium with 10 ng 'ml of cytochalasin B and 1.0', of DMSO. The convolution seen in Fig. i is greatly reduced. X 40. FICI i iK-i1a-3d Vertical sections through suspended, fixed fragments of C-411 monolayers. Arrows point to the hasal surface, i.e. to the side of the cell sheets that was attached to the sulxtnitum. Kpon 81* emlH-dding, Toluidine tilue. FIUI-KKIO Fixation immediately upon detachment from the sul>stratum. The cells remain organized in a columnar monolayer. X 440. FIOI HK 2a Fixation after t hr of suspension in control medium (see Fig. i). The luual surface of the cell sheet* is contracted; the apieal surface it scalloped. X 440. 3a F ixation after .' hr of suspension in medium contaiiiing 10 jig ml of cytochabuiu B and \.V"e of l )MSO (see Fig. $). The (ell sheet is folded .lightly towards the apical side. X 440. (From Auersperg, 1972). 6 F i g . 4 E l e c t r o n micrograph of s e c t i o n through c e l l sheet suspended f o r 2 hr i n c o n t r o l medium (see F i g s . 2 , 2 a ) . At the con t rac ted b a s -a l s u r f a c e (B ) , the plasma membrane i s f o l d e d , and ad jacent to i t there i s a dense f i l a m e n t band ( s o l i d a r r o w s ) . At the a p i c a l s u r f a c e (A) , the c e l l s are j o i n e d by j u n c t i o n a l complexes (V) and form numer-ous m i c r o v i l l i . Most n u c l e i , p a r t i c u l a t e o r g a n e l l e s , and p e r i n u c l e a r f i l a m e n t bundles are o r i e n t e d v e r t i c a l l y . x 3000. (From Auersperg , 1972.) F i g . 5 E l e c t r o n micrograph of s e c t i o n through c e l l sheet suspended f o r 2 hr i n medium w i t h 10 yg/ml c y t o c h a l a s i n B and 1.0% DMSO (see F i g s . 3 , 3 a ) . There i s no m i c r o f i l a m e n t band at the b a s a l s u r f a c e (B) , i n t e r c e l l u l a r spaces are wider than i n F i g . 4 , and the a p i c a l s u r f a c e (A) shows m i c r o v i l l i and j u n c t i o n a l complexes (V ) , but i s f l a t r a t h e r than s c a l l o p e d . x 3600. (From Auersperg , 1972.) 7 8 e l l e s r e s p o n s i b l e f o r c o n t r a c t i o n , are l o c a t e d on the b a s a l s u r f a c e , i . e . , the c o n t r a c t i n g s i d e of the c e l l sheets that was at tached to the subst ratum, but not on the a p i c a l s u r f a c e which had been exposed to the c u l t u r e medium. C y t o c h a l a s i n B a l t e r e d the morphology of m i c r o f i l a m e n t s and prevented c e l l c o n t r a c t i o n (Auersperg , 1972) . The c r u c i a l p o s i t i o n i n g of these organ ized m i c r o f i l e m t n s i n a l o c a t i o n where a c o n t r a c t i o n cou ld produce the change i n c e l l shape i s i n keeping w i t h the hypothes is tha t these c e l l u l a r components are the c o n t r a c t i l e elements of non-muscular c e l l s (Wessel ls e_t a l . , 1971) . C o n s i s t e n t w i t h t h i s hypothes is i s the o b s e r v a t i o n tha t the m i c r o f i l a m e n t s of s a l i v a r y g land (Spooner e t a l . , 1973) , f i b r o b l a s t s (Goldman and K n i p e , 1972) , amoeba (Comi ly , 1973) , g l i a l c e l l s (Ludena and W e s s e l l s , 1973) , and metaphase c e l l s ( Ishikawa e_t al., 1969) a l l s e l e c t i v e l y b ind r a b b i t heavy meromyosin, a c a p a c i t y c h a r a c t e r i s t i c of muscle a c t i n . The mechanism of i n t e r a c t i o n between c a l c i u m and m i c r o f i l a m e n t s i n non -muscular c e l l s and, i n p a r t i c u l a r , i n c o n t r a c t i n g e p i t h e l i a i s not f u l l y d e f i n e d . The l a c k of a v a i l a b l e data l e d to the f i r s t p a r t of t h i s s tudy : To determine i f e x t r a c e l l u l a r c a l c i u m i s r e q u i r e d f o r c o n t r a c t i o n and to determine by a d i r e c t method whether e p i t h e l i a l c e l l c o n t r a c t i o n i s a s s o c i -ated w i t h an i n c r e a s e i n t o t a l i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n . Con-t r a c t i o n s were induced by detachment of c e l l sheets from the subst ra tum, and i n t r a - and e x t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n s were determined by atomic a b s o r p t i o n spect rophotometry . Because c o n t r a c t i o n s are induced i n monolayers of C -4 I I e p i t h e l i a l c e l l s i n the absence of hetero logous t i s s u e s , these c e l l s represent a s i m p l e r 9 system for the i s o l a t i o n of factors co n t r o l l i n g e p i t h e l i a l c e l l contrac-tion than embryonic systems. Due to many s i m i l a r i t i e s between ul t r a s t r u c -t u r a l changes accompanying the contraction of C-4II c e l l s and those i n other contracting e p i t h e l i a (e.g., during salivary gland formation), the observations on the control of C-4II c e l l contraction may be generally applicable to developing e p i t h e l i a i n vivo. The second part of this thesis deals with the possible role of extra-c e l l u l a r materials i n the establishment of p o l a r i t y , i . e . , the asymmetric d i s t r i b u t i o n of organelles along the apical-basal axis of C-4II c e l l s . In vivo, the i n t r a c e l l u l a r p o l a r i t y i n the d i s t r i b u t i o n and/or function of microfilaments required for asymmetric contractions as seen, e.g., i n salivary gland development, may be induced by interactions of the develop-ing e p i t h e l i a with heterologous tissues and specialized components of ex t r a c e l l u l a r materials (e.g., basal lamina) at the basal c e l l surface (Spooner and Wessels, 1970). The behavior of the C-4II cultures shows that such p o l a r i t y can also be established autonomously by isolated e p i t h e l i a l c e l l populations. I t was one object of this thesis to investigate how e p i t h e l i a l c e l l s alone i n v i t r o establish the p o l a r i t y between the apical and basal sides without the existence of heterologous tissues. Possibly, t h i s i s due to the d i f f e r e n t i a l concentration of the c e l l s ' own products at the c e l l u l a r periphery, i . e . , e x t r a c e l l u l a r materials secreted randomly by i n i t i a l l y nonpolar c e l l s may, upon adhesion, accumulate at high concen-trations at the interface with the substratum but not on the apical side. A p a r t i c u l a r complement of c o n t r a c t i l e microfilaments i n the basal cytoplasm of such adherent monolayer cultures might represent an i n t r a c e l l u l a r tens-i l e force which interacts with e x t r a c e l l u l a r adhesive factors i n the 10 maintenance of c o l o n i a l o r g a n i z a t i o n but c o n t r a c t s upon i n t e r f e r e n c e w i t h adhesion (Auersperg , 1972) . The c e l l s were examined e l e c t r o n - m i c r o s c o p i c a l l y and the d i s t r i b u t i o n of e x t r a c e l l u l a r g lycosaminoglycans of C -4 I I c e l l s i n c u l t u r e was examined a u t o r a d i o g r a p h i c a l l y and h i s t o c h e m i c a l l y , to determine : (1) whether an asymmetric d i s t r i b u t i o n of these m a t e r i a l s cou ld be demonstrated which cou ld subsequent ly be r e l e a t e d to p o l a r i t y i n c y t o f i l a m e n t d i s t r i b u t i o n and c o n t r a c t i l i t y , (2) whether C -4 I I c e l l s were capable of forming s t r u c t u r e d b a s a l lamina i n v i t r o . : 11 PART I THE ROLE OF CALCIUM IN EPITHELIAL CELL CONTRACTION 12 MATERIALS AND METHODS I. T i ssue C u l t u r e The c e l l l i n e C - 4 I I (Auersperg , 1969) was o r i g i n a l l y d e r i v e d from a human squamous c e r v i c a l carc inoma. The c u l t u r e s (passages 85-110) were mainta ined i n 30 ml p l a s t i c t i s s u e c u l t u r e f l a s k s (Corning P l a s t i c s ) i n Waymouth's medium MB752/1 w i t h 10% f e t a l c a l f serum, 100 u n i t s / m l of p e n i c i l l i n and 100 yg/ml of s t rep tomyc in a t 37°C i n a h u m i d i f i e d 5% C02/a i r i n c u b a t o r . The medium was changed every 2 -3 days , and the c e l l s were sub -c u l t u r e d every 7-10 days by d i s s o c i a t i o n i n 0.12% t r y p s i n and 0.025% EGTA i n Ca and Mg f r e e Hanks' ba lanced s a l t s o l u t i o n . ( In the e a r l i e s t exper iments , the c e l l s were s u b c u l t u r e d by d i s s o c i a t i o n i n 0.12% t r y p s i n / I | | | Ca and Mg f r e e Hanks' ba lanced s a l t s o l u t i o n . ) For c e l l sheet c o n t r a c t i o n s t u d i e s , c e l l s were grown on 60 x 15 mm t i s s u e c u l t u r e d i shes w i t h 2 mm g r i d s u n t i l they formed c o n f l u e n t mono-l a y e r s , u s u a l l y f o r 7-10 days . Changing of the medium was stopped 3 days b e f o r e the f o l l o w i n g exper iments . I I . E f f e c t of Ca lc ium Content of Test S o l u t i o n s on E p i t h e l i a l  C e l l C o n t r a c t i o n Conf luent c u l t u r e s were r i n s e d th ree t imes w i t h 5 ml of f r e s h Way-mouth's medium (37°C) . The monolayers were d i v i d e d i n t o 1 x 2 mm p i e c e s w i t h a s c a l p e l , f o l l o w i n g the g r i d des ign on the unders ide of the d i s h . The d i v i d e d monolayers were r i n s e d two t imes w i t h 5 ml (37°C) of one of the f o l l o w i n g s o l u t i o n s : (a) Hanks' ba lanced s a l t s o l u t i o n (BSS) (Appendix I ) , (b) c a l c i u m - f r e e and magnesium-free Hanks' ba lanced s a l t 13 s o l u t i o n (CMF) (Appendix I ) , (c) magnesium-free Hanks' ba lanced s a l t s o l u -t i o n (MF), (d) c a l c i u m - f r e e Hanks' ba lanced s a l t s o l u t i o n (CF) . F o l l o w i n g t h i s , the 1 x 2 mm fragments of c e l l sheets were detached from the p l a s t i c substratum w i t h a non -adhes ive p l a s t i c wedge, suspended i n a P e t r i d i s h i n the same t e s t s o l u t i o n used f o r r i n s i n g p r e v i o u s l y and a l lowed to c o n t r a c t at 37°C i n a h u m i d i f i e d atmosphere of 5% CO^ i n a i r f o r p e r i o d s of 5 min to 10 h r . I I I . Measur ing C o n t r a c t i o n s A f t e r the i n c u b a t i o n pe r iods the supernatant was removed and s t o r e d f o r measuring c a l c i u m c o n t e n t . The c e l l sheets were f i x e d i n 2.5% g l u t a r a l d e h y d e / M i l l o n i g ' s b u f f e r (Hayat, 1970) at 4°C f o r 1.5 h r . The lengths and w id ths of the fragments were measured at a m a g n i f i c a t i o n of x4 , u s i n g a L e i t z - W e t z l a r d i s s e c t i n g microscope w i t h an o c c u l a r micrometer . About 15-20 c e l l s h e e t s , randomly s e l e c t e d from those t h a t had not been m u t i l a t e d d u r i n g detachment from the p l a s t i c , were measured from each c u l t u r e d i s h . IV. L i g h t Microscopy A f t e r the dimensions were measured, the c e l l sheets were p o s t f i x e d i n c o l d 1% osmium t e t r o x i d e i n M i l l o n i g ' s b u f f e r f o r 15-30 m i n , washed i n M i l l o n i g ' s b u f f e r , dehydrated i n graded e thano ls from 50% to 100% and i n propy lene ox ide and embedded i n Epon 812 (Appendix I I ) . A f t e r p o l y m e r i z a -t i o n , sample sheets were cut out of the b l o c k and 0 . 5 - 1 . 0 y t h i c k s e c t i o n s were made p e r p e n d i c u l a r to the long a x i s of the c e l l sheets w i t h g l a s s 14 k n i v e s on a R e i c h e r t u l t r a m i c r o t o m e . The s e c t i o n s were t r a n s f e r r e d to g l a s s s l i d e s w i t h a f i n e w i r e l o o p , h e a t - f i x e d onto the s l i d e s and s t a i n e d w i t h 1% t o l u i d i n e b l u e i n 1% borax . V. Ca lc ium Dete rminat ion The c a l c i u m content of t e s t s o l u t i o n s and c e l l s was measured by atomic a b s o r p t i o n spect rophotometry . 1 . Measurement of Ca lc ium i n Test S o l u t i o n s S o l u t i o n s c o l l e c t e d as d e s c r i b e d i n S e c t i o n s I I and I I I were d i l u t e d i n 0.5% L a C l ^ by adding 0 .2 ml of the sample to 1.8 ml of the Lanthanum swamp (the Lanthanum s o l u t i o n has the a b i l i t y to suppress the i n t e r f e r e n c e by s u l f u r and phosphorus when measuring the c a l c i u m content ) ( i n s t r u c t i o n manual , J a r r e l l Ash C o . , 1969) . Standards between 0 and 5 .4 yg Ca/ml were prepared (Appendix I I I ) . The r e l a t i o n s h i p between a b s o r p t i o n as recorded i n the d e f l e c t i o n u n i t s and the content of c a l c i u m was l i n e a r over the range used i n t h i s study ( F i g . 6 ) . Samples were analyzed i n a J a r r e l l Ash Atomsorb Model 280 Atomic A b s o r p t i o n Spectrophotometer under the f o l l o w i n g c o n d i t i o n s . element wavelength lamp c u r r e n t f u e l ox idant expansion s c a l e Ca 422.7 mu 7 mAmp a c e t y l e n e a i r 3 11 p s i 50 p s i 2 . Measurement of C e l l u l a r Ca lc ium C e l l u l a r c a l c i u m (Ca) content was determined by a m o d i f i c a t i o n of the "Lanthanum (La) Method" of Van Breemen and McNaughton (1970) and James and 15 F i g . 6 Representative standard curve fo r calcium determination (for calcium concentrat ion In BSS) Number of de f lec t ions 16 Roufogalis (1977) (Appendix IV, V). The term "La resistant Ca" and "La displaceable Ca" have been used to denote the amount of Ca remaining i n the tissue and removed from the tissue respectively, with a Lanthanum (La) solution (Sutter and Kromer, 1975). Under the conditions described i n the present study, "La-Tris-resistant Ca" i s considered to represent i n t r a c e l l u l a r Ca. Although the term "La-Tris-resistant Ca" i s more exact, the term " i n t r a c e l l u l a r Ca" i s more convenient. Therefore the Ca remaining i n the c e l l s after washing with La-Tris solution w i l l be referred to as " i n t r a c e l l u l a r Ca." The term " t o t a l calcium" w i l l refer to the calcium content of the c e l l s including Ca displaceable by La-Tris solution ( i . e . , i n t r a c e l l u l a r plus c e l l coat and membrane bound Ca). La displaceable Ca, most of which i s bound to the c e l l coast and plasma membrane, w i l l be referred to as ext r a c e l l u l a r calcium. a. Removal of Ex t r a c e l l u l a r Calcium and Measurement of the Time  Required for La-Tris Solution to Displace E x t r a c e l l u l a r  Calcium i n Control (Noncontracted) Cultures Cultures i n duplicate were quickly rinsed with ice cold 10 mM LaCl^ i n 160 mM (isotonic) Tris-HCl solution at pH 7.4, bubbled with 100% 0^ (La-Tris solution). The monolayer fragments were removed by scraping them from the substratum with a rubber policeman or p l a s t i c wedge, pooled and suspended i n a 50 ml Pyrex centrifuge tube containing 50 ml La-Tris solution i n an ice bath. (Large clumps of c e l l sheets were mechanically separated into small c e l l groups and single c e l l s by Pasteur pipette.) The tubes were kept shaking on a Fisher Rotator i n the 4°C cold room for periods of 1 min to 20 hr. Following t h i s , the tubes were centrifuged 17 a t 1500 rpm (504 g) i n an I n t e r n a t i o n a l P o r t a b l e R e f r i g e r a t e d C e n t r i f u g e Model PR-2( IEC) f o r 5 m i n , washed once w i t h 20 ml i c e c o l d d i o n i z e d water and r e c e n t r i f u g e d at 1500 rpm f o r another 5 min ( t h i s procedure d i d not a f f e c t the v i a b i l i t y of the c e l l s as examined by e o s i n - e x c l u s i o n ) . Then, the remain ing Ca ug/mg p r o t e i n ( i n t r a c e l l u l a r ca lc ium) was determined as d e s c r i b e d i n Sec t ions V I , 2-b and 2 - c . L e v e l l i n g o f f of the c e l l u l a r Ca content f o l l o w i n g an i n i t i a l drop from the t o t a l Ca v a l u e was cons idered as an i n d i c a t i o n tha t a l l or most e x t r a c e l l u l a r Ca had been removed ( F i g s . 9 - 1 1 ) . b . Measurement of T o t a l Ca lc ium The same procedure , as i n S e c t i o n a , was f o l l o w e d , except f o r u s i n g 160 mM T r i s s o l u t i o n i n s t e a d of L a - T r i s s o l u t i o n . c . Measurement of I n t r a c e l l u l a r Ca lc ium L a - T r i s s o l u t i o n at 4°C e f f e c t i v e l y removed e x t r a c e l l u l a r c a l c i u m i n 30 min w h i l e p r e v e n t i n g l o s s of i n t r a c e l l u l a r c a l c i u m ( F i g s . 9 - 1 1 ) . C e l l sheets used f o r de te rmin ing i n t r a c e l l u l a r c a l c i u m of n o n c o n t r a c t -ed c o n t r o l c u l t u r e s were processed as i n S e c t i o n a , w i t h suspension i n 50 ml L a - T r i s s o l u t i o n i n an i c e bath kept constant at 30 m i n , and Ca and p r o t e i n contents measured as d e s c r i b e d i n the f o l l o w i n g s e c t i o n s . For d e t e r m i n i n g i n t r a c e l l u l a r c a l c i u m l e v e l s of c o n t r a c t e d c e l l s , 1 x 2 mm p i e c e s of c e l l sheets were incubated i n Hanks' BSS f o r 1 min to 2 hr to c o n t r a c t (see S e c t i o n I I ) , then they were processed as i n (a) of t h i s s e c t i o n to remove e x t r a c e l l u l a r c a l c i u m , aga in keeping the p e r i o d of s u s -pens ion i n L a - T r i s s o l u t i o n constant at 30 m i n . 18 d . Atomic A b s o r p t i o n Measurements A f t e r t reatment w i t h e i t h e r T r i s - s o l u t i o n or L a - T r i s - s o l u t i o n , the c e l l s were covered w i t h 0 . 5 ml of a 1 : 1 mix tu re of g l a c i a l a c e t i c a c i d and 3 M TCA (Sparrow and Johnstone , 1964) . The tubes were heated i n a b o i l i n g water bath u n t i l the mix tu re j u s t began to b o i l (about 20 m i n ) , and shaken g e n t l y to d i s s o l v e the c e l l s . Two ml of d e i o n i z e d water .was then added and the mix tu re was again heated n e a r l y to b o i l i n g p o i n t (about 20 m i n ) . The tubes were then a l lowed to c o o l at room temperature f o r 20-30 min w h i l e the p r o t e i n coagulated f i r m l y . A f t e r b r i e f a g i t a t i o n of the contents on a Vor tex m i x e r , 2 - 3 t i m e s , the tubes were c e n t r i f u g e d at 2500 rpm (1400 g , IEC Model PR-2) f o r 40 min and the supernatant s t o r e d . The p e l l e t was washed w i t h 0 . 5 ml d e i o n i z e d w a t e r , r e c e n t r i f u g e d at 2500 rpm f o r 40 min and used f o r p r o t e i n d e t e r m i n a t i o n . L a C l ^ ( 0 . 1 mmoles i n 0 . 5 ml) was added to the pooled supernatants ( to prevent Ca complexat ion w i t h phosphate) and the volumes were made up to 3 .5 ml w i t h d e i o n i z e d H^O. A J a r r e l l Ash Atomsorb Model 280 ( F i s h e r S c i e n t i f i c Co. ) Atomic A b s o r p t i o n Spectrophotometer was used to reco rd the c a l c i u m content under the same c o n d i t i o n s as d e s c r i b e d i n S e c t i o n V I . 1 except that the expansion s c a l e was changed from 3 to 8 . Standard s o l u t i o n s and b lanks conta ined s i m i l a r amounts of L a C l ^ ^ I ^ O , g l a c i a l a c e t i c a c i d and TCA. (Appendix V ) . V I . P r o t e i n Dete rminat ion (Appendix VI) The amount of c e l l p r o t e i n was determined by a m o d i f i c a t i o n of the methods of Oyama and Eagle (1956) and Lowry ert a l . (1951) . The c e l l 19 p r o t e i n (see S e c t i o n V) was d r i e d i n a d e s i c c a t o r and d i s s o l v e d i n 5 ml IN NaOH. P r o t e i n p r e c i p i t a t e d w i t h t r i c h l o r o a c e t i c - a c i d (TCA) d i s s o l v e d very p o o r l y i n Lowry 's Reagent C. However, i t d i s s o l v e d i n approx imate ly 1/2 h r . . i n IN NaOH at room temperature . A f t e r 1/2 hr or more, 45 ml of Lowry 's Reagent D was added, and reac ted w i t h a phenol ( F o l i n - C i o c a l t e a u ) reagent ( F i s h e r S c i e n t i f i c C o . ) . The o p t i c a l d e n s i t y of t h i s s o l u t i o n was read 30 min l a t e r at 660 mu u s i n g a Cary Record ing Quartz s p e c t r o -photometer Model I I (Cary Instruments I n c . , Monrov ia , C a l i f . ) . A mix tu re of 1 ml of w a t e r , Lowry 's Reagent C and phenol reagent served as a b l a n k . C r y s t a l l i n e bov ine serum albumin f r a c t i o n V. (Sigma Chemical C o . , S t . L o u i s ) served as the p r o t e i n s t a n d a r d . The amount of p r o t e i n per sample (2 c u l t u r e s ) was q u a n t i t a t e d as bov ine serum albumin e q u i v a l e n t . V I I . Ionophore A23187 1 . P r e p a r a t i o n of Stock S o l u t i o n The c a r b o x y l i c ionophorous a n t i b i o t i c , A23187, a g i f t from Dr . R. H a m i l l , E l y L i l l y and C o . , I n d i a n a p o l i s , I n d . , was d i s s o l v e d i n 95% e t h a n o l to g i ve a s tock s o l u t i o n of 5 mg/ml which was kept f r o z e n . The ionophore was added to the t e s t s o l u t i o n s j u s t be fo re the exper iments . The f i n a l c o n c e n t r a t i o n of e t h a n o l i n c u l t u r e medium d i d not exceed 0.5% ( v o l r v o l ) . In every experiment i n which the ionophore was used , the s o l u t i o n used i n the cor responding c o n t r o l experiment conta ined 0.5% EtOH. Th is c o n c e n t r a t i o n of the s o l v e n t d i d not a f f e c t the v i a b i l i t y of the c e l l s ( e o s i n - e x c l u s i o n t e s t ) . 20 2 . Exper imentat ion on C -4 I I C e l l Sheets As d e s c r i b e d i n S e c t i o n I I , 1-2 mm fragments of c e l l sheets were r i n s e d w i t h one of the f o l l o w i n g t e s t s o l u t i o n s , then detached from the g r idded p l a s t i c substratum w i t h a rubber pol iceman and resuspended i n the same t e s t s o l u t i o n as used f o r r i n s i n g . (1) BSS + ionophore A23187/95% e t h a n o l ( to 10 and 50 yg/ml ionophore/0.5% ethanol/BSS) (2) BSS (3) BSS + 95% e t h a n o l ( to 0 . 5 % , v o l : v o l ) (4) CF-BSS + ionophore A23187/95% e t h a n o l (1 % 10 yg/ml i n CF-BSS) (5) CF-BSS (6) CF-BSS + 95% e t h a n o l ( to 0 . 5 % , e thano l ) The pH of a l l media was kept constant at 7 . 2 - 7 . 4 . The suspended c e l l sheets were incubated f o r p e r i o d s from 30 min to 24 h r , the c e l l s were c o l l e c t e d by b r i e f c e n t r i f u g a t i o n and were f i x e d i n 2.5% g l u t a r a l d e h y d e / M i l l o n i g ' s b u f f e r at 4°C f o r 2 h r , then measured f o r w idths and l e n g t h s . Supernatants went through the c a l c i u m d e t e r m i n a t i o n as d e s c r i b e d i n S e c t i o n V. 21 RESULTS I. Effect of Calcium Content of Test Solutions on  E p i t h e l i a l C e l l Contraction This series of experiments was undertaken to determine whether C-4II c e l l s need ex t r a c e l l u l a r calcium for contraction as do some muscular sys-tems; or whether they are independent of e x t r a c e l l u l a r calcium as i n the case of.contraction i n eggs of Rana pipiens (Schroeder and Strickland, 1974) . This was done i n i t i a l l y by looking for differences of calcium content i n the suspending medium between control cultures and contracted c e l l sheets. 1 x 2 mm c e l l sheets started curling across the short axis within minutes upon removal from the substratum and suspension i n BSS. The degree of contraction increased gradually for 30 min after which time the sheets had formed tubular structures and contraction remained more or less constant (Fig. 7). The curling of the c e l l sheets was inhibited equally by calcium-and magnesium-free BSS (CMF) and by calcium-free BSS (CF). When comparing the degree of contraction between c e l l s i n BSS and CMF, the widths of c e l l sheets i n BSS were about 50% of those i n CMF (Table I ) . I n i t i a l l y , the 1 x 2 mm c e l l sheets i n CMF remained f l a t or wavy with no tubular forms as seen i n BSS (Fig. 8). I t was noted that the longer the c e l l sheets were l e f t i n CMF the more f r a g i l e they became, as indicated by their broken edges and the tendency of c e l l s to lose i n t e r c e l l u l a r adhesion and to become spherical (Fig. 8). No contraction occurred i n CMF and the c e l l s died i f they were l e f t for over 3 hr. The i n h i b i t i o n of contraction by lack 22 F i g u r e s la-Id C e l l sheets incubated i n BSS (37°C) , s tandard o p t i c s . F i g . 7a C o n t r o l c e l l sheet p r i o r to c o n t r a c t i o n . ><90. F i g . 7b A f t e r 3 minutes of i n c u b a t i o n , c e l l sheet s t a r t e d to c o n t r a c t a long the edges. M o r p h o l o g i c a l l y , i t s t i l l remained f l a t i n most areas w i t h l i t t l e * f o l d i n g at the edge. x90. F i g . 7c A f t e r 20 minutes of i n c u b a t i o n , the c e l l sheet was c o n v o l u t e d . x l 2 0 . F i g . 7d A f t e r 30 minutes of i n c u b a t i o n , the t u b u l a r s t r u c t u r e s were formed by r o l l i n g up of the sheets a long the shor t a x i s . At that t ime c e l l sheets reached the maximal degree of c o n t r a c -t i o n . x60. 23 24 F i g u r e s 8a -8c C e l l sheets incubated i n CMF (37°C) , s tandard o p t i c s . F i g . 8a A f t e r 5 minutes of i n c u b a t i o n , c e l l sheets remained f l a t . ><90. F i g . 8b A f t e r 30 minutes of i n c u b a t i o n , edges were f r a y e d . No c o n -t r a c t i o n was observed . x45 . F i g . 8c A f t e r 60 minutes of i n c u b a t i o n , most of the sheets became f r a g i l e as i n d i c a t e d by the broken edges and c e l l s tended to d i s s o c i a t e . No t u b u l a r s t r u c t u r e s were formed. x45. 25 26 Footnote f o r Table I: CMF : Ca"1"1", M g ^ - f r e e BSS BSS-A: 10 yg ionophore A23187/ml BSS/0.5% EtOH BSS-E : BSS/0.5% EtOH CF : C a ^ - f r e e BSS • A l l the samples ( c e l l sheets a f t e r i n c u b a t i o n i n 37°C t e s t s o l u t i o n f o r 40 min) had been f i x e d w i t h 2.5% g l u t a r a l d e h y d e (4°C) f o r 24 h r , then were measured at a m a g n i f i c a t i o n of x4 , u s i n g a L e i t z - W e t z l a r d i s s e c t i n g microscope w i t h an o c c u l a r micrometer . 27 T a b l e 1. E f f e c t o f c a l c i u m c o n t e n t o f t e s t s o l u t i o n s on e p i t h e l i a l c e l l c o n t r a c t i o n (Number o f s a m p l e s In b r a c k e t s ) T e s t s o l u t i o n Length o f ce 11 s h e e t ( mm ) Width o f ce11 s h e e t ( mm ) L e n g t h / W i d t h pH BSS 1.19 (20) 0 . 2 0 (20) 5 . 9 5 7 . 4 1.06 (20) 0 . 2 0 (20) 5 . 3 0 7 . 4 0 . 7 1 (20) 0 . 1 8 (20) 3 . 9 4 7 . 1 0 . 7 7 (20) 0 . 1 8 (20) 4 . 2 8 7 . 1 0 . 9 3 (20) 0 . 1 9 (20) 4 . 8 9 7 . 1 0 . 8 6 (20) 0 . 2 0 (20) 4 . 3 0 7 . 1 mean 0 .92±0.18 0 . 1 9 1 0 . 0 1 4 . 8 4 i 0 . 7 6 CMF' 0 . 8 6 (20) 0 . 3 4 (20) 2 . 5 3 7 . 0 0 . 9 3 (20) 0 . 3 9 (20) 2 . 3 8 7 . 0 . 0 . 8 0 (10) 0 . 2 7 (10) 2 . 9 6 7 . 0 0 . 9 9 (10) 0 . 2 7 (10) 3 . 6 7 7 . 0 0 . 9 6 (20) 0 . 3 0 (20) 3 . 2 0 7 . 0 1.06 (20) 0 . 4 4 (20) 2 . 4 1 7 . 0 1.02 (20) 0 . 3 6 (20) 2 . 8 3 7 . 0 1.06 (20) 0 . 3 8 (20) 2 . 7 9 7 . 0 mean 0 .96±0.09 0 . 3 4 t 0 . 0 6 2 . 8 2 1 0 . 4 4 BSS 1 .08 (20) 0 . 2 1 (20) 5 . 1 4 6 . 8 B S S - A 0 . 9 2 (20) 0 . 2 0 (20) 4 . 6 0 6 . 8 BSS -A 0 . 9 4 (20) 0 . 1 8 (20) 5 . 2 2 6 . 8 B S S - E 1 . 0 3 (20) 0 . 1 9 (20) 5 . 4 2 6 . 8 QF 1.00 (20) 0 . 4 1 (20) 2 . 4 4 6 . 8 C F - A 0 . 9 6 (20) 0 . 5 1 (20) 1 . 8 8 6 . 8 C F - E 1 . 0 3 (20) 0 . 4 8 (20) 2 . 1 4 6 . 8 28 of e x t r a c e l l u l a r c a l c i u m was c o r r e l a t e d w i t h a decrease i n the c a l c i u m content of c e l l s as measured by an i n c r e a s e i n c a l c i u m content i n CMF and CF t e s t s o l u t i o n s (Tables I I , I I I ) . . No s i g n i f i c a n t changes i n c a l c i u m content i n t e s t s o l u t i o n s were d e t e c t -ed when c e l l sheets were incubated f o r v a r i o u s t ime i n t e r v a l s (0-14 hr ) at 37°C e i t h e r i n BSS.or i n magnesium-free BSS (MF), i . e . , i n c a l c i u m -c o n t a i n i n g s o l u t i o n s (Tables IV, V ) . T h e o r e t i c a l l y , c o l d temperature (4°C) should be a b l e to b l o c k the c a l c i u m pump i n the c e l l membrane. Then, by p a s s i v e t r a n s p o r t mechanisms, c e l l s should be a b l e to take up more c a l c i u m from the medium at 4°C than at 37°C. As a r e s u l t , the c a l c i u m content l e f t i n the medium should be l e s s than the c o n t r o l v a l u e s . However, no s i g n i f i -cant d i f f e r e n c e s i n c a l c i u m content i n t e s t s o l u t i o n s were detected (Table I V ) , e i t h e r a t 37°C or 4°C. T h e r e f o r e , no uptake of e x t r a c e l l u l a r c a l c i u m by c o n t r a c t i n g c e l l s cou ld be shown by t h i s method. I I . Ionophore A23187 In 10 yg ionophore A23187/ml BSS/0.5% EtOH, c e l l sheets con t rac ted g r a d u a l l y as i n c o n t r o l BSS, but the speed of c o n t r a c t i o n appeared s lower than wi thout ionophore A23187 i n the f i r s t 15 m i n . However, by 40 m i n , they reached the same degree of c o n t r a c t i o n as d i d the c o n t r o l groups (Table I ) . 0.5% EtOH had no e f f e c t on c e l l v i a b i l i t y or on c o n t r a c t i o n over 40 m i n . 50 yg/ml ionophore A23187 caused the death of the c e l l s i n 40 min (eos in e x c l u s i o n t e s t ) , but 10 yg/ml o f , i o n o p h o r e A23187 had l i t t l e e f f e c t on c e l l v i a b i l i t y over that t ime p e r i o d . There was no s i g n i f i c a n t d i f f e r e n c e between BSS and ionophore A 2 3 1 8 7 - c o n t a i n i n g BSS i n the c a l c i u m . Measurement of calcium content of CMF test solutions In the presence of cell sheets experiment number incubation time (min) PH net reading a x slope tCa] ug/ml x d i 1ut i on factor (BSSxl 1.) (CMFx 2) x total volume (5ml) (ug) eel 1 prote In mg/culture Ca /cell released /protein 1 2 average ug/mg n mole/mg CMF-1 40' 7.4 7.0 7.0 7.0 0.027 0.19 6.38 1.90 3.50 0.54 13.50 CMF-2 40' 7.0 24 25 24.5 0.027 0.66 1.32 • 6.60 4.00 1.65 41.25 . CMF-3 40' 7.0 19 17 18 0.027 0.49 0.98 4.90 2.60 1.88 47.00 CMF-4 40* 7.0 23 22 22.5 0.027 0.61 1.22 6.10 3.15 1.94 48.50 CMF-5 40' 7.0 9 9.5 9.25 0.027 0.25 0.50 2.50 2.25. 1.11 27.75 CMF-6 40' 7.0 23 24 23.5 0.027 0.64 1.28 6.40 • 3.20 2.00 50.00 CMF-7 40" 7.0 28. 28 28 0.027 0.76 1.52 7.60 3.55 2.14 53.50 CMF-8 40' 6.7 18.5 17.5 18 0.024 0.43 0.86 4.30 3.20 1.34 33.50 'CMF-9 40' 6.8 4.5 5 4.75 0.024 0.11 0.22 1. 10 4.50 0.24 6.00 a: net reading = (exp. value + blank val ue )-(b lank value), blank values: 4,.5—6.0,v In-the presence- of CMFsoNtfon cells released Ca to the medium as Indicated by these positive values, b: read from standand curve. Table M l . Measurement of calcium content of CF test solutions In the presence of cel l sheets sample Incubation time (rain) PH read!na a Xi -X 2 x slope (0.024) x di1ution factor (BSSxM) (CMFx 2) (ug) x total voIume (5ml) eel 1 prote in (ma) Ca re leased/protein 1 2 (X :) uq/mq n rnole/mq CF CF-blank 40 6.8 6.8 37 6.5 38 6 37.5 6.25 31.25 0.75 1.5 7.5 4.70 1.60 39.89 CF-A-I b CF-A-2 CF-A-blank 40 40 6.8 6.8 6.8 29 37 5 27 38 5 28 37.5 5 23.0 32.5 0.552 0.78 1.104 1.56 5.52 7.8 3.90 3.90. 1.42 2.00 35.38 50.00 CF-E CF-E-b i ank 40 6.8 6.8 35 5 33 4 34 4.5 29.5 0.708 1.416 7.08 4.50 1.57 39.33 a: X 2 : mean of blank solutions b. CF-A : 10 ug ionopKore A23187/CF-BSS/0.5^'EtdH. 31 Footnote f o r Table IV: a . I ncubat ion t i m e : 40 min : c e l l sheets were: incubated i n 37°C M g ^ - f ree/BSS f o r 40 m i n . o J | 40-150* : c e l l sheets were incubated i n 37 C Mg„ - f r e e / B S S f o r 40 m i n , then at 4°C f o r another 150 m i n . 4 0 - 8 0 * - 1 0 : c e l l sheets were incubated i n 37°C M g ^ - f r e e / B S S f o r 40 m i n , at 4°C f o r 80 m i n , then i n 37°C f o r another 10 m i n . b. X : mean v a l u e of d u p l i c a t e c u l t u r e s . A l l the experiments were done at pH 7 . 2 . \ 32 Table IV. Atomic absorption readings of calcium in the presence of C-41I c e l l sheets of MF t e s t s o l u t i o n s Test - . - i s o l u t i o n Incubation • 11 me Read i ng ( X >b t t e s t MF-1 0 min 5 min 10 min 15 min 148 147 148 145 II 30 -min 40 min 151 148 p>0.05 50 min 150 100 min 150 150 min 150 10 hrs 151 MF-1-blank 148 MF-2 40 (min) 40 40-150 143 145 145 30 148 p>0.05 •i 30 30-150 148 148 15 141 „ 15 15-150* * 125 146 147 144 MF-2r-blank 140 MF-3 140 (min) 130 94 96 120 40-80 40-80 -10 40-45 -80 94 93 94 95 p>0.05 : MF-3-blank, 91 33 Table V. Atomic absorption readings of calcium of BSS test solutions In the presence of C-4II cel l sheets Test solutior i ncubation time a. pH « reading(X) t test BSS-1 150 min 7.2 106 • n 100 min II 101 II 50 min II 101.5 II 30 min II 97.5 it 15 min ti 99.5 P>0.05 n 10 min n 102.5 it 5 min ii 101 n 0 tnin 7.5 98.5 II 14 hr 6.8 102 II 14 hr 6.8 102 BSS-1-blank 7.5 101 BSS-2 150 min 7.2 98 II 100 min II 99 II 50 min II 92.5 II 30 min II 100.5 • it 15 min II 100.5 P>0.05 II 10 min it 99 n 5 min it 100.5 II 0 min 7.5 92 n 14 hr . 6.8 102 BSS-2-blank 7.5 99.5 BSS-3 40 min-1 7.1 138 II 40 min-2 '7.1 139.8 ti 40 min-3 7.1 138 • P>0.05 n 40 min-4 7.1 135 tt 40 min-5 7.1 137 BSS-3-blank 7.1 141 « X: mean value of duplicate cultures 3'4^  content of t e s t s o l u t i o n s (Table V I ) . There was no c o n t r a c t i o n over 40 min i n 10 yg/ml ionophore A23187/CF. The c e l l s r e l e a s e d the same amount of c a l c i u m to the CF t e s t s o l u t i o n s i n the presence or absence of ionophore A23187 (Table I I I ) . I I I . Measurement of C e l l u l a r Ca lc ium The above s t u d i e s showed no s i g n i f i c a n t d i f f e r e n c e s i n c a l c i u m content i n t e s t s o l u t i o n s between (1) BSS and ionophore/BSS, (2) CF and ionophore/ CF, (3) MF or BSS i n the presence or absence of c e l l s , i n v a r i o u s i n c u b a t i o n t i m e s - a n d temperatures . As i t seemed p o s s i b l e that the amount of c a l c i u m taken up by c o n t r a c t i n g c e l l s was too s m a l l to be detected by examining the suspending medium, the "Lanthanum method" of Van Breemen and McNaughton (1970) as m o d i f i e d by James and R o u f o g a l i s (1977) was employed. I t had been shown to be s u c c e s s f u l i n measuring changes i n i n t r a c e l l u l a r c a l c i u m i n c o n t r a c t i n g smooth muscle c e l l s . In the present s tudy , the method was m o d i f i e d f u r t h e r to adapt i t to c a l c i u m measurements i n c u l t u r e d e p i t h e l i a l c e l l s . 1 . Removal of E x t r a c e l l u l a r Ca lc ium and Measurement of the Time Requirement f o r L a - T r i s S o l u t i o n to D i s p l a c e E x t r a c e l l u l a r Ca lc ium In c o n t r o l c u l t u r e s , mean t o t a l c a l c i u m v a l u e s ranged from 1.38 yg/mg p r o t e i n i n e a r l y experiments to 3 .33 yg/mg p r o t e i n i n l a t e r experiments (Table V I I ) . I c e - c o l d L a - T r i s s o l u t i o n d i s p l a c e d most of the e x t r a c e l l u l a r c a l c i u m w i t h i n 5 min but, the re was much v a r i a t i o n u n t i l a f t e r 30 min ( F i g s . 9 & 1 0 ) . The l e v e l l i n g o f f of the c e l l u l a r c a l c i u m content at 30 min f o l l o w -i n g an i n i t i a l drop from the t o t a l c a l c i u m v a l u e was cons idered an i n d i c a -35 Table VI. Atomic absorption readings of calcium of.BSS t e s t s o l u t i o n s In the presence of C-4II c e l l sheets with .and without EtOH and ionophore A23187/EtOH Test so 1ution Incubation time (min) Reading-(X) b ' t t e s t BSS-A-1 1 5 171 BSS-A-2 15 172 BSS-A-3 40 169 BSS-A-4 40 171 p>0.05 BSS-A-5 40 167.5 BSS-A-6 40 169.5 BSS-A-blank 172 BSS-1 40 172 BSS-2 40 173 p>0.05 BSS-b lank - 173.5 BSS-E-1 40 170 BSS-E-2 40 170 p>0.05 BSS-E-blank 1^ 1 . a. BSS-A : 10 ug ionophore A23187/ml BSS/0.5? EtOH BSS-E : BSS/0.5? EtOH b. X :mean value of dupl icate cul tures 36 Table V I I . Ca lc ium content of C -4 I I c e l l s ( c o n t r o l c u l t u r e s ) (Number of o b s e r v a t i o n s in b r a c k e t s ) Sample C e l 1 u l a r compartment Ga1c i um/Ce11 p rote i n ug/mg nmole/mg E a r l y Tota 1 1.38±0.24 (12) 34.60+6.00 (12) exper iments ( t ryps i n) 1 n t r a c e 1 1 u 1 a r 0.26±0.10 (15) 6.52+2.50 (15) Ex t race 1 1 u l a r * 1.12±0.07 (12) 28.02+1.75 (12). La te r exper i ments ( t r y p s i n / EGTA) To ta l 3.33±0.40 (14) 83 .25110.00(14) 1 n t r a c e 1 1 u 1 a r 0.68+.0.24 (52) 17.0016,00 (52) Ex t race 1 1 ular*' 2.65±0.13 (14) 66.25+3.25 (14) L a t e r exper i ments ( t r y p s i n ) Tota 1 2 . 1 6 1 0 . 4 6 ( 4) 54.00+11.50( 4) 1ntrace11u1ar 0 . 2 2 1 0 . 0 7 ( 4) . 5 . 5 0 1 1 . 7 5 ( 4) Ext race11u1ar* 1 .9410.35 ( 4) 4 8 . 5 0 1 8 . 7 9 ( 4) * C a l c u l a t e d as t o t a l minus i n t r a c e l l u l a r . T r y p s i n : i n d i c a t e s t h a t c e l l s were d i s s o c i a t e d by 0 .12$ t r yps in/CMF. Trypsin/EGTA : I n d i c a t e s t h a t c e l l s were< d i s s o c i a t e d by 0 .12$ t r y p s i n - 0 . 0 2 5 $ EGTA/CMF. 37 F i g . 9 The t ime requirement f o r L a - T r i s s o l u t i o n to d i s p l a c e e x t r a -c e l l u l a r c a l c i u m i n c o n t r o l c u l t u r e s . Data c o l l e c t e d from e a r l y experiments i n which c e l l s were d i s s o c i a t e d w i t h 0.12% t r yps in/FCM. L e v e l l i n g o f f of the c e l l u l a r c a l c i u m content f o l l o w i n g an i n i t i a l drop from the t o t a l c a l c i u m i s cons idered as an i n d i c a t i o n tha t a l l or most of e x t r a c e l l u l a r c a l c i u m has been removed, and t h i s seems complete a f t e r 30 m i n . # : o b s e r v a t i o n s ; • : mean v a l u e . 9 1.5+ Totol calcium ? 1.0+ 5 U i < u < co < oc 0.5 » y © 0 5 10 20 -4- r— 30 40 MINUTES 50 60 120 39 F i g . 10 The t ime requirement f o r L a - T r i s s o l u t i o n to d i s p l a c e e x t r a c e l l u l a r c a l c i u m i n c o n t r o l c u l t u r e s . Data represent l a t e r experiments i n which c e l l s were d i s s o c i a t e d w i t h 0.12% t r y p s i n -0.025% EGTA/CMF. « : o b s e r v a t i o n s ; • : mean v a l u e . INTRACELLULAR CALCIUM ("9/019) 0* 41 t l o n tha t a l l or most e x t r a c e l l u l a r c a l c i u m had been removed. At t h i s p o i n t , the remain ing ( i n t r a c e l l u l a r ) c a l c i u m va lues ranged from 0.26 yg/mg p r o t e i n i n e a r l y experiments to 0 .68 yg/mg p r o t e i n i n l a t e r exper iments . Th is d i f f e r e n c e was s i g n i f i c a n t (p<0.05) . S i n c e , i n e a r l y exper iments , c e l l s at the t ime of s u b c u l t u r e were d i s s o c i a t e d by 0.12% t ryps in/CMF, w h i l e they were d i s s o c i a t e d by 0.12% t r y p s i n - 0 . 0 2 5 % EGTA/CMF i n l a t e r e x -pe r iments , i t seemed p o s s i b l e tha t the d i f f e r e n t d i s s o c i a t e d methods might have r e s u l t e d i n d i f f e r e n c e s i n c e l l u l a r c a l c i u m c o n t e n t . Th is was v e r i f i e d by a recent experiment where c e l l s were d i s s o c i a t e d by t ryps in/CMF. The i n t r a c e l l u l a r c a l c i u m c o n t e n t , 0 .22 yg/mg p r o t e i n , was s i g n i f i c a n t l y lower than tha t of concurrent c u l t u r e s d i s s o c i a t e d w i t h trypsin-EGTA/CMF and i n the same range as i n e a r l y experiments (0 .26 yg/mg p r o t e i n ) where t r y p s i n / CMF d i s s o c i a t i o n had been used (Tables VII, V I I I ) . There was no s i g n i f i c a n t d i f f e r e n c e i n i n t r a c e l l u l a r c a l c i u m content between i n t a c t c e l l sheets and sheets broken up m e c h a n i c a l l y i n t o s m a l l groups to i n c r e a s e contact between c e l l s u r f a c e s and L a - T r i s s o l u t i o n (p>0.05, Table V I I I ) . These r e s u l t s a l s o i n d i c a t e d tha t i n c o n t r o l c u l t u r e s , L a - T r i s s o l u t i o n at 4°C e f f e c t i v e l y removed e x t r a c e l l u l a r c a l c i u m i n 30 min w h i l e p r e v e n t i n g l o s s of i n t r a c e l l u -l a r c a l c i u m . For the con t rac ted c e l l s h e e t s , c e l l sheets were a l lowed to c o n t r a c t at 37°C i n BSS f o r 30 m i n , then shaken i n i c e - c o l d L a - T r i s s o l u t i o n f o r v a r i o u s t imes l i k e the c o n t r o l c u l t u r e s . The r e s u l t aga in showed that L a -T r i s s o l u t i o n removed most of the e x t r a c e l l u l a r c a l c i u m w i t h i n 5 m i n , and 20 to 30 min l a t e r completed the r e a c t i o n f o r c e l l sheets broken up i n t o 4 2 Table VIII. Intracellular calcium content of C-4II cells (control cultures) (Number of observations in brackets) SampIe Ca (ug) CeI 1 protein (mq) Ca/Cel1 protein uq/mq nmole/mq Early experiments (trypsin) Intact sheets* 1.33±0.50 (15) 5.10*0.72 (15) 0.26*0.10 (15) 6.50*2.50 (15) Later experiments (trypsin /EGTA) Broken up sheets** 2.35*0.72 (19) 3.15*0.48 (19) 0.75*0.20 (19) 18.75*4.25 (19) Intact sheets •2.46*0.97 (33) 3.84*0.90 (33) 0.64*0.26 (33) 16.00*6.50 (33) Mean 2.42±0.89 (52) 3.59*0.77 (52) 0.68*0.24 (52) 17.00*6.00 (52) Later experiments (trypsin) Broken up sheets 0.99±0.32 ( 4) 4.60*0.01 ( 4) 0.22*0.07 ( 4) 5.50*1.75 ( 4) _ * Intact, sheets : cell sheets were not further dissociated after separation from the plastic substratum-.. ** Broken up sheets : cell sheets were mechanically separated into small cell groups and/or single?,eel Is by Pasteur pipette when suspended in La-Tris solution. Trypsin : indicated that cel ls were dissociated by 0.12? trypsin/CMF. Trypsin/EGTA : indicated that cel ls were dissociated by 0.12? trypsin-0.025? EGTA/CMF. 43 s m a l l c e l l groups ( F i g . 1 1 ) . However, l i m i t e d data showed a l s o that L a -T r i s s o l u t i o n f o r 30 min r e s u l t e d i n more c a l c i u m i n i n t a c t sheets than i n broken up s h e e t s , suggest ing incomplete removal of e x t r a c e l l u l a r c a l c i u m . Th is r e s u l t i n d i c a t e d tha t L a - T r i s s o l u t i o n at 4°C e f f e c t i v e l y removed e x t r a c e l l u l a r c a l c i u m i n 30 min from c o n t r a c t e d , broken up c e l l s h e e t s , w h i l e p r e v e n t i n g l o s s of i n t r a c e l l u l a r c a l c i u m . The c a l c i u m content of c o n t r a c t e d c e l l sheets was h igher than i n the c o n t r o l c u l t u r e s ( F i g . 1 1 V (see a l s o S e c t i o n V I ) . 2 . C o n t r o l C u l t u r e s i n T r i s S o l u t i o n v s . L a - T r i s S o l u t i o n C e l l sheets were suspended i n i c e - c o l d T r i s s o l u t i o n broken up i n t o s m a l l c e l l g roups , and shaken at pH 7.4 f o r 30 min and 0 min ( i . e . , r i n s e d once w i t h i c e - c o l d T r i s s o l u t i o n and broken up w i thout f u r t h e r i n c u b a t i o n ) , then were e x t r a c t e d and c a l c i u m measured. A p a r a l l e l group of c u l t u r e s was processed s i m i l a r l y , u s i n g L a - T r i s s o l u t i o n i n s t e a d of T r i s s o l u t i o n . The r e s u l t s showed that w i thout Lanthanum, the c a l c i u m content i n the presence of i c e - c o l d T r i s s o l u t i o n f o r 30 min (0.93±0.12 yg/mg c e l l : p r o t e i n ) was not s i g n i f i c a n t l y d i f f e r e n t from 0 min (0.89±0.13 yg/mg p r o t e i n , p>0.05) . However, the c a l c i u m content f o l l o w i n g L a - T r i s e x t r a c t i o n (0.46 ±0.11 yg/mg p r o t e i n ) was s i g n i f i c a n t l y lower (Table I X ) . 3 . Cont racted C e l l Sheets i n T r i s S o l u t i o n v s . L a - T r i s S o l u t i o n C e l l sheets were a l lowed to c o n t r a c t f o r 30 min i n BSS (37 C ) , broken up and suspended i n 4°C i c e - c o l d T r i s s o l u t i o n f o r 0 to 120 m i n , then c a l -cium was e x t r a c t e d and measured. The r e s u l t s showed tha t the c a l c i u m content remained constant even a f t e r i n c u b a t i o n i n i c e - c o l d T r i s s o l u t i o n 44 F i g . 11 The t ime requirement f o r L a - T r i s s o l u t i o n to d i s p l a c e e x t r a -c e l l u l a r c a l c i u m i n c o n t r a c t e d c e l l s h e e t s . Data represent experiments in : 'which c e l l s were d i s s o c i a t e d w i t h 0.12% t r y p s i n - 0 . 0 2 5 % EGTA/CMF. • : i n t a c t c e l l s h e e t s ; • : broken up c e l l s h e e t s ; • : c o n t r o l c u l t u r e s (non -cont rac ted c e l l s h e e t s ) . INTRACELLULAR C A L C I U M ( ug/rag) /46 Table IX. Measurement of calcium content of control cultures Incubated with Tris or La-Tris'solution at 4°C (Number.of observations In brackets) ; " Sample (ug/mg) Ca/cel1 protein t-test Cell sheets in Tris solution for 30 min 0.93±0.12 (4) p>0.05 Cell sheets in Tris solution for 0 min* 0.89+0.13 (4) Cell sheets in Tris solution for 0 and 30 min ( mean ) 0.91+0.13 (8) p<0.05 Cell sheets in La-Tris solution.for 30 min 0.46+0.U (4) * 0 min cel l sheets were just rinsed once with without further Incubation. ice-cold Tris solution 47 f o r 120 m i n . In T r i s s o l u t i o n the c e l l u l a r c a l c i u m c o n c e n t r a t i o n s were about 2 .0 yg/mg c e l l p r o t e i n , which was h igher than i n L a - T r i s s o l u t i o n ( i . e . , 1.2 yg/mg c e l l p r o t e i n , 30 min a f t e r suspension) ( F i g . 1 2 ) . The r e s u l t s from the above experiments i n d i c a t e d tha t Lanthanum ions were necessary f o r removal of e x t r a c e l l u l a r c a l c i u m . 4. I n f l u e n c e of Ca lc ium C o n c e n t r a t i o n i n the Medium on C e l l u l a r Ca lc ium Content of C o n t r o l C u l t u r e s and Cont racted C e l l Sheets Th is experiment was done to see (1) i f doub l ing the amount of c a l c i u m i n the medium would a f f e c t the c e l l u l a r c a l c i u m c o n t e n t , (2) i f a d d i t i o n a l amounts of e x t r a c e l l u l a r c a l c i u m cou ld be removed by the amounts of L a n t h -anum ions (10 mM) used r o u t i n e l y . Monolayers were incubated i n 37°C I | Waymouth's medium c o n t a i n i n g 1.64 mM or 0 .82 mM ( c o n t r o l ) Ca ions f o r 30 m i n , then were suspended i n i c e - c o l d L a - T r i s s o l u t i o n at pH 7.4 f o r 30 m i n . The r e s u l t showed t h a t the t o t a l l e v e l s of c e l l u l a r c a l c i u m were h igher i n 1.64 mM Ca /Waymouth's medium (2.65±0.48*yg/mg p r o t e i n ) than i n I j 0 .82 mM Ca /Waymouth's medium (1.84±0.18 yg/mg p r o t e i n ) . The i n t r a c e l l u -l a r c a l c i u m ranged from 0.22±0.05 to 0 . 5 0 1 0 . 1 1 yg/mg p r o t e i n f o r 0 .82 mM and 1.64 mM/Waymouth's medium r e s p e c t i v e l y . Therefore i t seemed that the i n c r e a s e d c a l c i u m c o n c e n t r a t i o n i n the medium r e s u l t e d i n a h i g h e r t o t a l c a l c i u m content of the c e l l s and i n d o u b l i n g of the i n t r a c e l l u l a r c a l c i u m l e v e l s . However, i t appeared t h a t , i n terms of abso lu te v a l u e s , the L a -T r i s s o l u t i o n was ab le to remove most of the e x t r a c e l l u l a r c a l c i u m d u r i n g the p e r i o d of 30 min ( F i g . 1 3 ) . 48 F i g . 12 C e l l sheets c o n t r a c t e d f o r 30 min i n BSS (37°C) , broken up and suspended i n 4°C T r i s s o l u t i o n (pH 7.4) f o r 0 to 120 m i n . • : samples i n T r i s s o l u t i o n ; • : samples i n L a - T r i s s o l u t i o n . CELLULAR CALCIUM ( u g/mg) O — —• rO O 'o> O b. O O r I I ! 1—©33^-© 09 / C J + o o iv. t '4 © © e © • 0 / / / I f I I / \ \ \ \ \ • © © 67 50 F i g . 13 Measurement of c e l l u l a r c a l c i u m content of c o n t r o l c u l t u r e s by the "Lanthanum method" a f t e r exposure of the c e l l sheets to Waymouth's medium c o n t a i n i n g 1.64 mM Ca Ions f o r 30 min (A) as compared to c o n t r o l (B) i n normal Waymouth's medium I | c o n t a i n i n g 0.82 mM Ca i o n s . Data represent mean v a l u e of 4 o b s e r v a t i o n s . £~| : t o t a l c e l l u l a r c a l c i u m ; ^ : i n t r a c e l l u -l a r c a l c i u m ; b a r : s tandard d e v i a t i o n . C E L L U L A R C A L C I U M ( u g / m g ) _ co o -o o 52 C e l l sheets were a l lowed to c o n t r a c t i n 1.26 mM or 2.52 mM C a ' ' / B S S a t 37°C f o r 30 m i n , then processed to measure i n t r a c e l l u l a r c a l c i u m l e v e l s . The r e s u l t s showed t h a t the c a l c i u m content i n c r e a s e d from 2 . 4 1 ±0.47 to 4.66±0.10 yg/mg p r o t e i n as the c a l c i u m c o n c e n t r a t i o n i n the BSS was i n c r e a s e d from 1.26 mM to 2.52 mM (Table X ) . Th is cou ld mean that La was not capable of d i s p l a c i n g a l l of the e x t r a c e l l u l a r c a l c i u m when the c a l c i u m c o n c e n t r a t i o n was r a i s e d . However, i t cou ld a l s o mean that r a i s i n g the e x t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n drove more of the e x t r a -c e l l u l a r c a l c i u m i n t o the c e l l s from where i t cou ld not be r e a d i l y d i s -p l a c e d . Th is needs f u r t h e r i n v e s t i g a t i o n . N e v e r t h e l e s s , the c e l l sheets I | c o n t r a c t e d to s i m i l a r degree i n both 1.26 mM and 2.52 mM Ca - c o n t a i n i n g BSS as i n d i c a t e d by the i n s i g n i f i c a n t d i f f e r e n c e of the w i d t h of the c e l l sheets (Table X ) . 5 . E f f e c t of C o n t r a c t i o n i n Waymouth's Medium on I n t r a c e l l u l a r  Ca lc ium Content For most c o n t r a c t o n exper iments , c e l l sheets were a l lowed to c o n t r a c t i n BSS (37 C) which conta ined 1.26 mM of Ca i o n s , w h i l e the c o n t r o l I | c u l t u r e s were kept i n Waymouth's medium which conta ined 0 .82 mM of Ca i o n s . In v iew of the r e s u l t s d e s c r i b e d i n par t 4 of t h i s S e c t i o n , i t was necessary to determine whether or not the h igher c a l c i u m content i n c o n -t r a c t e d c e l l s compared to c o n t r o l c e l l s was due to the d i f f e r e n t c a l c i u m c o n c e n t r a t i o n of the medium. The f o l l o w i n g experiment was performed: Both c o n t r o l and exper imenta l c e l l sheets ( c o n t r a c t i n g ) were kept i n Waymouth's medium (37°C) f o r 30 m i n , then processed as u s u a l . The r e s u l t showed tha t i n t r a c e l l u l a r c a l c i u m was aga in h i g h e r i n c o n t r a c t i o n sheets Table X. Effect of calcium content of test solution on cellular calcium content and degree 6f contraction of cell sheets (Number of observations in brackets) Test solution Length of ce 1 1 sheet(mm) Width of ceI 1 sheet(mm) Length/Width. Intrace11ular calcium/cell protein(ug/mg) 1.26 mM Ca++/BSS 1.0710.08 (20) 0.1710.04 (20) 6.96 2.4110.47 (4) 2.52 mM Ca^/BSS 0.94+0.06 (20) 0.15+0.05 (20) 6.27 4.6610,10 (4) CMF* 0.9610.09(140) 0.3410.06(140) 2.82 All samples (cell sheets after Incubation In 37°C test solution) were fixed with 2.5% glutaraldehyde <4°C) for 1.5 hrs., then measured at a magnification of x4, using a Leltz-Wetzlar dissecting microscope with an occular micrometer. * Mean-± standard deviation of eight separate experiments (from Table I ) . 54 than i n the c o n t r o l c u l t u r e s (Table X I ) . T h e r e f o r e , the h i g h e r c a l c i u m content i n con t rac ted c e l l s compared to c o n t r o l c e l l s was not due to the d i f f e r e n t c a l c i u m c o n c e n t r a t i o n s of the two media . IV. R e l a t i o n s h i p of I n t r a c e l l u l a r Ca lc ium to P r o t e i n Content  i n C o n t r o l C u l t u r e s As shown i n F i g . 14, i n t r a c e l l u l a r c a l c i u m ranged from 0.55 to 4.75 yg per sample, and c e l l u l a r p r o t e i n ranged from 1.95 to 6.35 mg per sample. The r e l a t i o n s h i p between i n t r a c e l l u l a r c a l c i u m and c e l l u l a r p r o t e i n was not l i n e a r ; i t was of i n t e r e s t tha t (1) i n t r a c e l l u l a r c a l c i u m i n c r e a s e d i n p r o p o r t i o n to i n t r a c e l l u l a r c a l c i u m / c e l l p r o t e i n , (2) v a l u e s of i n t r a c e l l u -l a r c a l c i u m / c e l l p r o t e i n ranged from 0 .15 to 1.05 yg/mg but were independ-ent of c e l l u l a r p r o t e i n content (Fig' . 15) and (3) the r a t i o of i n t r a c e l l u -l a r c a l c i u m to c e l l p r o t e i n was always l e s s than 1.05 yg/mg. As mentioned i n S e c t i o n I I I , i t appeared t h a t changes i n d i s s o c i a t i o n method ( t r y p s i n v s . trypsin/EGTA) might a f f e c t the c e l l u l a r c a l c i u m c o n t e n t . The c o r r e l a -t i o n c o e f f i c i e n t was 0.57 and 0.49 (p<0.05) f o r t r y p s i n d i s s o c i a t e d c u l t -ures and trypsin/EGTA d i s s o c i a t e d c u l t u r e s r e s p e c t i v e l y ( c e l l sheets had been broken up i n t o s m a l l c e l l g roups ) . However, the c o r r e l a t i o n c o e f f i c i -ent was not s i g n i f i c a n t f o r t rypsin/EGTA d i s s o c i a t e d c u l t u r e s which had not been broken up i n t o s m a l l c e l l groups ( c o r r e l a t i o n c o e f f i c i e n t = 0 . 2 2 , p>0.05) (Appendix V I I ) . There was no s i g n i f i c a n t d i f f e r e n c e between the 3 p o p u l a t i o n s . 55 Table XI. E f f e c t of c o n t r a c t i o n in Waymouth's medium on i n t r a c e l l u l a r c a lcium content of C-4II c e l l s . Sample ug/mg ce11 p r o t e i n Contracted c e l l sheets ( i n t r a c e l l u l a r calcium) 2.1010.96 Control c u l t u r e s ( i n t r a c e l l u l a r calcium) 0.32+0.06 Data represent mean l standard d e v i a t i o n of 4 o b s e r v a t i o n s . Fig.14 Relationship of intracellular calcium to eel I protein (control cultures) lopen symbols are means') c4 a • • e sn a o o a * „ © oo a * o a a •• „ 9 a o . • 1 • * • .. • A • A a . A .. . CM A * A _ I . A © A A » 0j " A B -.Early experiments (trypsin dissociation) A :Later experiments (broken up cell sheets) (trypsin/EGTA dissociation) ©:Later experiments (Intact cell sheets) (trypsin/EGTA dissociation) .'I i l ! I I 1.0 2.0 3.0 4.0 5.0 6.0 INTRACELLULAR CALCIUM ( u g ) 57 Fig.15 Relationship of Intracellular calcium and cell protein to Intracellular calcium/cell protein (control cultures) 6.01 5.0 14.0 0 < u * 3.0 u 1.0 ft •9 0 " < » A f A A e A E B H fiij e B&m or B D 0.5 1.0 I N T R A C E L L U L A R C A L C I U M Z oi Q o. L U U 6.0 + 5.0 + 4.0 3-0+ 2.0+ 1.0 ° © H B B a H O O O B e a • B e c O O o • s " 1 A « A © A A A A A * A O A A 9 O o e OO o A -4-(ug/mg) 0.5 1.0 I N T R A C E L L U L A R C A L C I U M C E L L P R O T E I N (ug/mg) C E L L PROTE IN B : early experiments (trypsin dissociation) A : later experiments- broken up cell sheets (trypsin/EGTA dissociation) 9 : later experiments- intact cel l sheets (trypsin/EGTA dissociation) 58 V. I n f l u e n c e of C u l t u r e Age on C e l l u l a r Ca lc ium and P r o t e i n Content Two separate experiments were done to observe the r e l a t i o n s h i p of c e l l -u l a r c a l c i u m and p r o t e i n content to d i f f e r e n c e s i n c u l t u r e age. S e r i e s of c u l t u r e s were set up, the medium was changed every two days , and i n t r a -c e l l u l a r c a l c i u m was . e x t r a c t e d and measured a f t e r d i f f e r e n t t ime i n t e r v a l s . The r e s u l t s showed t h a t (1) c e l l u l a r p r o t e i n i n c r e a s e d w i t h c u l t u r i n g t i m e , (2) the i n t r a c e l l u l a r c a l c i u m content (yg and yg/mg c e l l p r o t e i n ) was very v a r i a b l e w i t h i n 4 days a f t e r s u b c u l t u r e , then dropped between day 4 and 6, u n t i l , 8 days a f t e r s u b c u l t u r e , the i n t r a c e l l u l a r c a l c i u m l e v e l became lower and more or l e s s constant ( F i g s . 16 , 1 7 ) . The b i g v a r i a t i o n between i n t r a c e l l u l a r c a l c i u m l e v e l s i n e a r l y s tages of c u l t u r e s when c e l l s were i n e x p o n e n t i a l phase may be r e l a t e d to the f a c t tha t c e l l s were i n d i f f e r e n t p h y s i o l o g i c a l c o n d i t i o n s . A f t e r 8 to 10 days i n c u l t u r e , the i n t r a c e l l u l a r c a l c i u m l e v e l became more or l e s s constant and no s i g n i f i c a n t d i f f e r e n c e s i n c a l c i u m content were found (Table X I I ) . Th is t ime (7 to 8 days a f t e r subcu l tu re ) corresponded to the t ime when C - 4 I I c e l l s became c o n f l u e n t and crowded, and s t a r t e d to form hemicysts ( F i g . 1 8 ) . Though more experiments are necessary f o r d e f i n i t i v e c o n c l u s i o n s , the r e s u l t s suggested that the i n t r a c e l l u l a r c a l c i u m l e v e l s i n o l d c u l t u r e s were more constant than i n younger c u l t u r e s . V I . Changes i n I n t r a c e l l u l a r Ca lc ium w i t h C o n t r a c t i o n C e l l sheets were a l lowed to c o n t r a c t f o r 1 to 120 min i n 37°C BSS, then the e x t r a c e l l u l a r c a l c i u m was removed by i c e - c o l d L a - T r i s s o l u t i o n I n t r a c e l l u l a r caIcIum/ceI I protein Cug/mg) i n t r a c e l l u l a r calcium (ug) c e l l protein (mg) e — Of— i n t r a c e l l u l a r ca lx:i um/ce 11 protein (ug/mg) i n t r a c e l l u l a r calcium (ug) ce11 protein (mg) 61 • • I. t-test for mean difference of c e l l u l a r calcium andproteln between 8 to 10 days tn-cuIture V \ c u l ' u e 1 ays | 8 9 <u\ 3 \ ^ * V) — > . * Ca p rote i n Ca/protein Ca protein I Ca/protein = 0 • o P>0.05 P>0.05 o protein P<0.05 P<0.05 protein 'protein P>0.05 P>0.05 ro O ro o P>0.05 protein P>0.05 c +-8 8 P>0.05 * Sample : data from Table XII ,group-B Ca : Intracellular calcium (ug) Protein :- eel lular protein ("ng;) Ca/protein : Intracellular calcium/protein (ug/mg) 62 F i g . 18 H emicyst (arrowhead) i n c u l t u r e o f C - 4 I I c e l l s . I n c e n t r a l a r e a , c e l l s form a p a v e m e n t - l i k e p a t t e r n w i t h p r o m i n e n t i n t e r c e l l u l a r spaces and l i t t l e o v e r l a p p i n g . On t h e above r e g i o n , c e l l s f orm clumps ( s t a r ) w i t h o v e r l a p p i n g . C e l l s were d i s s o c i a t e d by t r y p s i n method, s t a n d a r d o p t i c s . x400. 63 treatment for 30 min. The results showed that c e l l s took up calcium from BSS i n large amounts within the f i r s t 3 min. After 30 to 120 min, the c e l l s s t i l l contained more calcium than the controls, but the amounts were , smaller compared, to the f i r s t 3 min (Table X I I I ) . In a l l experiments the i n t r a c e l l u l a r calcium content i n contracted c e l l sheets was always higher than i n the controls (Table XIII and Fig..19). The i n t r a c e l l u l a r calcium content of contracted c e l l sheets was s i g n i f i c a n t l y higher i f the sheets were kept intact rather than broken up i n La-Tris solution (p<0.05). The c e l l sheets, after 3 min i n the presence of BSS at 37°C, morpho-l o g i c a l l y looked l i k e controls, i . e . , they s t i l l remained mostly f l a t , though after 30 min they had reached the maximal degree of contraction (Fig. 7). In other words, i n the f i r s t 30 minutes, the degree of contrac-t i o n of c e l l sheets increased with incubation time. However, the c e l l s contained more calcium i n the f i r s t 3 min than 30 min after contraction (Fig. 19). There was no s i g n i f i c a n t difference of calcium content between intact and broken up c e l l sheets i n control cultures (Table X I I I ) . Data i n Table XIII and Fig. 19 were pooled from 7 separate experiments. Without , exception, the i n t r a c e l l u l a r calcium content of contraction c e l l sheets was always s i g n i f i c a n t l y higher than i n control cultures. VII. Effect of Lanthanum Ions on Contraction of C e l l Sheets 1 x 2 mm c e l l sheets were allowed to incubate for 35 min with 5 ml O | | (37 C) of one of the following solutions: (1) 1.26 mM (Ca )/BSS, (2) 1.26 mM (Ca^/BSS/lO^M LaCl3«6H 0, (3) 1.26 mM (Ca4^)/BSS/10~4M LaCl '6H 0, (4) CMF. The c e l l sheets were incubated, then fixed i n Table XIII. Intracellular calcium/cell protein (ug/mg) of control cultures and contracted cel l sheets after 30 minutes In 4°C La-Tris solution (Number of observations In brackets) N^amp le ContractIohv time (min) /X. Broken up ce11 sheets Intact ce11 sheets Significance of difference (Intact vs. broken up eel 1 sheets) 0 (control)* 0.58±0.10 (4) 0.66+0.26 (16) p>0.05 1 4.33±0,95 ( 6) — 3 4.22±0.28 ( 6) — . 5 — 3.87+0.76 ( 7) — 10 1.36±0.01 (2) 3.27+0.75 ( 6) p<0.05 15 — 3.16+0.62 ( 3) — 20 — 2.86±0.81 ( 4) — • 30 1.02+0.14 (8) 2.28+0.60 (16) p<0.05 40 1.10±0;04 (2) 2.74+0.63 ( 2) p<0.05 120 1.36±0.03 (2) 2.70+0.08 ( 2) p<0.05 The data are a composite of 7 separate experiments. * 0 min (control) = no contraction. 65 Fig. 19 I n t r a c e l l u l a r calcium content of e p i t h e l i a l c e l l sheets after different periods of contraction. C e l l sheets were allowed to contract for 1 to 120 min, then suspended i n ice-cold La-Tris solution for 30 min to remove ex t r a c e l l u l a r calcium. Data c o l l e c t -ed from 7 separate experiments (each symbol represents duplicate cultures). A : broken up c e l l sheets; # : intact c e l l sheets; A : control cultures (broken up c e l l sheets); o : control cultures (intact c e l l sheets). 19 C O N T R A C T I O N T I M E (m inu tes ) 67-2.5% g l u t a r a l d e h y d e / M i l l o n i g ' s b u f f e r at 4°C f o r 1.5 hours and t h e i r l engths and w id ths measured. The r e s u l t s showed tha t Lanthanum ions i n h i b i t e d the c o n t r a c t i o n of I | c e l l s h e e t s . The w id ths of c e l l sheets i n the presence of 1.26 mM Ca /BSS (0.17±0.04 mm) were about 50% of those i n CMF (0.34±0.06 mm). However, the w id ths of c e l l sheets i n 1.26 mM C a ^ / B S S / l O ^ M L a C l 3 - 6 H 2 0 (0.25±0.04 mm) and 1.26 mM C a ^ / B S S / l o " ^ L a C l ^ F ^ O (0.32±0.06 mm) were 73.5% and 94.1% of those i n CMF (Table XIV) . The r e s u l t s i n d i c a t e d tha t Lanthanum ions i n h i b i t e d the c o n t r a c t i o n of the c e l l sheets by a mechanism, very l i k e l y , of removal of e x t r a c e l l u l a r c a l c i u m ions from the c e l l s and/or p r e v e n t i o n of i n f l u x of e x t r a c e l l u l a r c a l c i u m ions i n t o the c e l l s . V I I I . A r t i f i c i a l and B lank Background To determine whether v a r i a t i o n s i n c a l c i u m readings were due to a r t i -f a c t s , s e v e r a l c o n t r o l s were a n a l y s e d . 50 ml L a - T r i s s o l u t i o n was p r o c e s s -ed as f o r measurement of c e l l u l a r c a l c i u m except tha t there were no c e l l s . In a d d i t i o n , the f o l l o w i n g were a l s o examined: (1) i c e - c o l d L a - T r i s s o l u t i o n ; (2) g l a c i a l a c e t i c a c i d and t r i c h l o r o a c e t i c a c i d (GAA-TCA); (3) i c e - c o l d L a - T r i s s o l u t i o n p l u s GAA-TCA; (4) a l l of the p reced ing w i t h and w i thout aluminum f o i l cover (aluminum f o i l had been used to cover the sample d u r i n g the processes of measuring c e l l u l a r c a l c i u m ) . The r e s u l t s showed t h a t L a - T r i s s o l u t i o n as w e l l as GAA-TCA s o l u t i o n gave background readings of c a l c i u m contents rang ing from 0.24 to 0 . 5 8 , w i t h a mean of 0.44±0.13 yg (Table XV) . Table XIV. Effect of lanthanum ,ons on contraction of cell sheets Wumber of observations In brackets) Test solution Length of eel 1 sheets(mm) Width eel 1 sheets(mm) Length/Width 1.26 mM Ca++/BSS • 1.07±0.08 (20) 0.17+0.04 (20) 6.29 1.26 mM Ca++/BSS/10-iM LaCI,-6H90 0.89+0.06 (20) 0.25+0.11 (20) 3.56 1.26 mM CaT"7BSS/10"4M LaCly6H 20 CMF* 1.13±0.03 (20) 0.96+0.09(140) 0.32+0.06 (20) 0.34+0.06(.140> 3.53 2.82 O N co All the samples (cell sheets after incubation in 37°C test solution for 35 min) were fixed with 2.5$ glutaraldehyde (4°C) for 1.5 hrs, then measured at a magnification of x4, using a Leltz-Wetzlar dissecting microscope with an occular micrometer. * CMF : from Table I. 69 Table XV. E f f e c t of b lank s o l u t i o n s on c a l c i u m (Number of o b s e r v a t i o n s in b r a c k e t s ) Experiments* Ca content (ug) (mean) j L a - T r i s s o l u t i o n l-g~l ™ p n -0.24 (2) jgj L a - T r i s s o l u t i o n ^ 0 .54 (2) j-j L a - T r i s s o l u t i o n ^ 0 .45 (4) TCA-GAA s o l u t i o n 0 .42 (2) TCA-GAA p l u s aluminum f o i l 0 . 4 8 (2) L a - T r i s s o l u t i o n 0 . 5 8 (2) L a - T r i s s o l u t i o n p l u s aluminum f o i l 0 .37 (2) mean ± s tandard d e v i a t i o n 0.44±0.13 (16) * Arrow —^s- i n d i c a t e s t h a t the sample was processed as f o r measuring i n t r a c e l l u l a r c a l c i u m in c e l l s (Appendix IV) ; absence of arrow i n d i c a t e s t h a t the sample was read d i r e c t l y w i t h o u t f u r t h e r p r o c e s s i n g . : c e n t r i f u g e tube | | : P e t r i d i sh J : scraped o f f w i th p l a s t i c wedge. 70 In order to examine i f any factors other than the blank solution could produce an a r t i f i c i a l background i n calcium determinations, the following experiments were done i n the absence of c e l l s : (1) Waymouth's medium with and without serum was kept i n culture dishes for the same periods as were cultures, then the medium was discarded and the dishes processed l i k e the cultures (Appendix IV). ^ (2) The surfaces of the tissue culture dishes were scraped with rubber policeman or p l a s t i c wedges i n the presence of ice-cold La-Tris solution, then went through the rest of the procedure. (3) BSS was kept i n tissue culture dishes for 2 hours, the substratum scraped with rubber policeman, the medium discarded, La-Tris solution added to the sediment and the rest of the procedure completed. The results showed that components of rubber policeman and, rarely Waymouth's medium with and without serum could be the sources of a r t i f i c i a l background. The precipitate of 10% f e t a l c a l f serum-Waymouth's medium collected from scraping by rubber policeman, contained 1.97±0.97 yg calcium and 0.32±0.14 mg protein (Table XVI). No s i g n i f i c a n t difference of calcium content was found between the precipitate of 10% f e t a l calf serum-Waymouth's medium and Waymouth's medium (p>0.05). Serum was suspected to be a source of a r t i f i c i a l background because i t has been reported that culture media .with serum can form precipitates on the substratum i n the presence or absence of c e l l s (Stamatoglon, 1977). The precipitate i s mainly glycopro-t e i n which may have some role i n mediating c e l l adhesion. Furthermore, a model has been presented according to which calcium ions may cross-link substrate-attached materials, and the c e l l glycocalyx (Culp, 1974). 71 Table XV I . Sources of a r t i f i c ia l background for calcium content (Number of observations in brackets) Experiments* Calcium content (ug! observations X±S.D. I \Q% FCS-WM rinse with Lar-Tris • * L a - T r i s Jp*-1.18,3.09,2.02,0.88 3.47,1.60,1.57** 1.97±0.97 (7) WM -> rinse with La-Tris -+ La-Tris 1.81,1.23 1.52+0.40 (2) La-Tris -^»-1.07,0.27,0.36,0.33 0.33,0.21,0.21,0.74 0.44±0.30 (8) La-Tris 0.33,0.81,0.33 0,49+0.28 (3) t f^10? FCS-WM -+ rinse with La-Tris -> ^ La-Tris*» - 0.76,0.39 0.58±0.24 (2) "^j^ WM •> rinse with La-Tris -»• La-Tr Is 0.25,0.28,0.56,0.57 0.98,0.63,0.25,0.28 0.70,0.61,0.24,0.30 0.54 0,48±0.23 (13) ( (^ WM -+ rinse with Tris -+ Tris .0.28,0.39,0.53,0.36 0.36,0.41,0.24 0.37+0.09 (7) [-^ j BSS ^-/^l •»• a d d La-Tris 0.36,0.33,0.33 0.34±0.01 (3) La-Tris(scrape 100 times) with/or without small pieces of rubber policeman 2.01,1.42,1.23 1.55±0.41 (3) Mean va1uelstandard deviation I X + S.D. ) 0.82+0.32 (48) * FCS: fetal calf serum WM : Waymouth's medium I j : Petric dish : centrifuge tube f :scraped with rubber policeman t{ -.scraped with plastic wedge (\ :discard supernatant Arrow See footnote of Table 16. The protein conteat in this experiment was found to be 0,32+0.14 mg (4). 72 In separate exper iments , a rubber pol iceman was rubbed a g a i n s t the w a l l of c e n t r i f u g e tubes 100 to 150 t imes i n the presence of i c e - c o l d L a -T r i s s o l u t i o n , w i t h or w i thout a d d i t i o n of t i n y p i e c e s ( 0 . 1 mm) of the rubber m a t e r i a l , and was processed as f o r measuring c a l c i u m . The r e s u l t showed tha t components of rubber pol iceman cou ld produce a r t i f a c t u a l read ings ( 1 . 5 5 1 0 . 4 1 y g ) . BSS and the p l a s t i c wedge seemed to have no e f f e c t on the r e a d i n g s . These experiments showed tha t (1) the b l a n k s o l u t i o n , L a - T r i s and/or GAA-TCA s o l u t i o n , gave a background read ing (blank background) which was, however, i n c o r p o r a t e d i n the s t a n d a r d s , and (2) components of rubber pol iceman and Waymouth's medium produced an a d d i t i o n a l a r t i f i c i a l background. T h i s cou ld e x p l a i n some of the i n d i v i d u -a l h igher read ings tha t occur red randomly throughout t h i s s tudy . 73 DISCUSSION It has become apparent during the past few years that close s i m i l a r i -t i e s exist i n a number of instances between some of the proteins d i r e c t l y involved i n contraction i n s t r i a t e d muscle, and proteins present i n certain non-muscle c e l l s i n which movements occur. The question therefore arises whether a l l these systems share a common basic mechanism, and i f so, whether the current picture of the well known contracting mechanism i n str i a t e d muscle can cast any l i g h t on these other motile mechanisms. This question has become an important topic i n recent research. The c r u c i a l positioning of the organized microfilaments i n a location where a contraction could produce changes i n c e l l shape has led to the hypothesis that these c e l l u l a r components are the contractile elements of non-muscular c e l l s (Wessells e_t a l . , 1971) . The correlations from cytochalasin B experiments greatly strengthen the case: As the m i c r o f i l a -ment morphology i s altered or disrupted by cytochalasin B, an i d e n t i f i a b l e -contractile process i s concomitantly i n h i b i t e d ; furthermore, when the drug i s removed, the microfilaments reappear and the previously inhibited con-t r a c t i l e a c t i v i t y i s resumed (Wessells et a l , , 1971). Si m i l a r l y , the microfilaments located on the basal surface, i . e . , the side of e p i t h e l i a l c e l l sheets attached to the substratum, have been implicated as the con-t r a c t i l e organelles responsible for the i n v i t r o convolution of the C-4II c e l l sheets (Auersperg, 1972). Microfilaments appear to be s i m i l a r to muscle F-actin by several c r i t e r i a i n addition to their size (4-7 nm) and morphology (Goldman e_t a l . , 1976). They bind rabbit s k e l e t a l muscle heavy meromyosin (HMM) (Huxley, 1963; Ishikawa et a l . , 1969; Goldman, 1975). In addition, several f l u o r -74 escence microscope techniques ( e . g . , a c t i n ant ibody or f l u o r e s c e i n - l a b e l l e d HMM) i n d i c a t e that a c t i n i s a component of m i c r o f i l a m e n t s (Lazar ides and Weber, 1974; Goldman et a l . , 1975) . A c t i n - c o n t a i n i n g m i c r o f i l a m e n t s are i n v o l v e d i n many k i n d s of c e l l m o t i l i t y and are u s u a l l y found i n c l o s e a s s o c i a t i o n w i t h c e l l membranes. Examples i n c l u d e c y t o k i n e s i s (Schroeder , 1973; P e r r y e_t ' a l . , 1971) , amoeboid movement ( P o l l a r d and K o r n , 1973 a , b ) , c h l o r o p l a s t s t reaming (Kersey , 1974; P a l e v i t z and H e p l e r , 1975) , m o t i l i t y of c u l t u r e d f i b r o b l a s t s ( c e l l adhesion to a subst ra tum, c e l l l o c o m o t i o n , membrane r u f f l i n g ) , and m a i n t e n -ance of c e l l shape (Wessel ls ejt a l . , 1973; Miranda e_t a l . , 1974) , morpho-g e n e t i c movements i n embryonic e p i t h e l i a (Spooner e_t a l . , 1973) , p l a t e l e t c o n t r a c t i o n ( Z u c k e r - F r a n k l i n and Grusky, 1972) , the g e n e r a t i o n of the acrosomal porcess ( T i l n e y , 1975)., and endocy tos is and e x o c y t o s i s i n macro-phages ( A x l i n e and Reaven, 1974; Orr and A l l i s o n , 1972) . An obvious f u n c -t i o n of the a s s o c i a t i o n of a c t i n - c o n t a i n i n g m i c r o f i l a m e n t s w i t h c e l l mem-branes may be that membranes serve as attachment s i t e s f o r a c t i n f i l a m e n t s i n non-muscle m o t i l e systems. Th is a s s o c i a t i o n cou ld a l s o f u l f i l l a b a s i c requirement of actomyosin m o t i l e systems, i . e . , a c t i n and/or myosin must be anchored i f t h e i r i n t e r a c t i o n i s to r e s u l t i n the g e n e r a t i o n of f o r c e (Mooseker, 1976) . There i s reasonable avidence t h a t myos in , t ropomyosin and t ropon ins may be components of plasma membrane or r e l a t e d membranes of i n t r a c e l l u l a r v e s i c l e s , i . e . , r a t b r a i n s y n a p t i c v e s i c l e s (Korn , 1976) . Bes ides membrane-associated a c t i n - c o n t a i n i n g m i c r o f i l a m e n t s , f r e e a c t i n has now been i d e n t i f i e d i n the cytoplasm of approx imate ly 50 e u k a r y o t i c c e l l types (animals and p l a n t s ) as w e l l as i n p r o t i s t s . In most c a s e s , 75 the a ctin has been isolated from the c e l l s and shown to activate the Mg-ATPase of muscle heavy meromyosin. The cytoplasmic actin also resembles muscle ac t i n i n i t s v i s c o s i t y , amino acid composition (including the pres-ence of 3-methylhistidine, a rare amino acid found only i n muscle) and filament ultrastructure (Gordon et^ a l . , 1976). In addition, myosins have been found i n a variety of non-muscular c e l l s (Weber et a l . , 1974; Hitchcock, 1977). Like muscle myosins, cytoplas-mic myosins have ATPase a c t i v i t y which i s the energy-transducing enzyme for c e l l m o t i l i t y (Pollard, 1977). An a - a c t i n i n - l i k e protein, which i s a major protein of the Z-line of muscle, has also been found i n many non-muscle c e l l s , e.g., i n t e s t i n a l e p i t h e l i a l c e l l s (Schloomeyer et a l . , 1974; Lazarides and Burridge, 1975; Mooseker et a l . , 1976). The presence of actin and myosin i n a large number of vertebrate and invertebrate c e l l s (Pollard and Weihing, 1974) suggests that these proteins play a fundamental role i n c e l l functions. Moreover, the d i v e r s i t y of location of these proteins within a given c e l l implies that they may func-tion i n more than one c e l l u l a r process during the c e l l ' s l i f e cycle, although most of the evidence for their p a r t i c i p a t i o n has been circumstan-t i a l (Adelstein, 1975). In addition to function, control of actin-myosin interaction i n non-muscle c e l l s i s of current i n t e r s t . Szent-Gybrgyi (1975) reviewed factors controlling calcium s e n s i t i v i t y ( i . e . , conditions under which calcium activates the actin-activated ATPase a c t i v i t y of myosin) i n both vertebrate and invertebrate muscle c e l l s . Both actin-associated (troponin-tropomyosin) and myosin-associated (myosin l i g h t chains) regulation, as well as mixed controls, were found to ex i s t . Proteins very similar to tropomyosin have 76 been i s o l a t e d and c h a r a c t e r i z e d from p l a t e l e t s (Cohen and Cohen, 1972) and b r a i n (F ine e t a l . , 1973) . L a z a r i d e s (1975) has shown that tropomyosin i s l o c a l i z e d i n the a c t i n - c o n t a i n i n g m i c r o f i l a m e n t s . Suggest ive evidence f o r t r o p o n i n - l i k e p r o t e i n s has been r e p o r t e d by Cohen e_t a l . (1973) and Nachmias and Asch (1974) , i n p l a t e l e t s and the s l i m e mold Physarum. M y o s i n -a s s o c i a t e d r e g u l a t i o n has not yet been demonstrated i n non-muscle systems ( H i t c h c o c k , 1977) . A l though there i s much evidence f o r m u s c l e - l i k e p r o -t e i n s produc ing c o n t r a c t i o n i n non-muscular c e l l s , there i s l a c k of knowledge about the c o n t r o l of c o n t r a c t i o n i n these types of c e l l s . The requirement of muscle f o r c a l c i u m i o n has been known s i n c e 1883 when Stan ley R inger found that i t was an e s s e n t i a l i o n i c i n g r e d i e n t i n the b a t h i n g medium of hear t muscle ( R i n g e r , 1883) . The cur rent s l i d i n g f i l a -ment theory f o r muscle c o n t r a c t i o n has p o s t u l a t e d c a l c i u m as a c o n t r o l l i n g f a c t o r i n the i n i t i a t i o n of muscle c o n t r a c t i o n . Calc ium content i n the sarcoplasm i s r e g u l a t e d by the sa rcop lasmic r e t i c u l u m (Huxley, 1973) . In a r e l a x e d s t r i a t e d m u s c l e , the c o n c e n t r a t i o n of f r e e c a l c i u m i s kept at a —7 —8 very low v a l u e (probably l e s s than 10 to 10 M) by the a c t i o n of the c a l c i u m pump i n the sa rcop lasmic r e t i c u l u m . Upon a c t i v a t i o n , c a l c i u m i s —6 r e l e a s e d so t h a t the l e v e l of f r e e c a l c i u m r i s e s (probably to 10 to 10 **M) and attachment of myosin to a c t i n can take p l a c e . In v e r t e b r a t e s t r i a t e d musc le , t h i s change i s e f f e c t e d by the t r o p o m y o s i n - t r o p o n i n system i n the a c t i n - c o n t a i n i n g f i l a m e n t s (Ebashi and Endo, 1968) , which prevents attachment of myosin i n the absence of f r e e c a l c i u m , but a l l o w s i t to take p l a c e i n i t s p resence , p o s s i b l y by a s t e r i c b l o c k i n g mechanism dependent on changes i n p o s i t i o n of t ropomyos in . In mol luscan musc le , however, r e g u l a t i o n i s a f f e c t e d by changes i n the myosin molecules to 77 which calcium ions become bound upon activation (Kendrick-Jones'et a l . , 1970). The d i s t r i b u t i o n of these two types of regulation, or combination of them, between species i s a subject of very active current research (Szent-Gyorgyi, 1975). Nevertheless, both systems so far investigated share the common feature that they are operated by changes i n the concentra-t i o n of free calcium ions over the c r i t i c a l range of 10 ^  to 10 "*M. Thus the c r u c i a l s p a t i a l arrangement of the actin and myosin filaments would be dependent on the presence of calcium ions i n the sarcoplasm. In s t r i a t e d muscle the concentration of calcium i n the sarcoplasm i s regulated mainly by the sarcoplasmic reticulum. However, i n smooth muscle where this c e l l u -l a r component i s poorly developed, the calcium must come p a r t i a l l y from the e x t r a c e l l u l a r f l u i d (Imai and Takeda, 1967; James and Roufogalis, 1977). In smooth muscle the mitochondria, c e l l membrane (sarcolemma and surface microvesicles) and perinuclear sac have been postulated as additional controls of the inte r n a l calcium concentrations (Popescu ejt a l . , 1974). Non-muscular c e l l s are similar to smooth muscle c e l l s with respect to lacking a highly specialized sarcoplasmic reticulum for calcium transport and sequestering. I t has been hypothesized (Popescu, 1974) that i n smooth muscle an increase i n calcium concentration during a c t i v i t y may result from (1) an in f l u x of calcium ions into the c e l l from the extr a c e l l u l a r space (Durbin and Jenkinson, 1961; Edman and Schild, 1962), (2) the translocation of calcium ions located i n and/or near the c e l l membrane (Lullman, 1970; Chang and Triggle, 1973; Hurwitz et d . , 1973), (3) the release of calcium ions that are sequestered i n some i n t r a c e l l u l a r storage s i t e s , thought to be the sarcoplasmic reticulum, mitochondria, vesicles and perinuclear sac 78 (Devine et a l . , 1972; Popescu et a l . , 1974; C a r a f o l i ejt a l . , 1975; Robert-son, 1976). However, the evidence for each of these hypotheses i s quite circumstantial," and they remain c o n t r o v e r s i a l subjects. The r e l a t i v e con-t r i b u t i o n of these sources may vary i n d i f f e r e n t tissues and under d i f f e r -ent experimental conditions ( C a r a f o l i et a l , , 1975). By an i n d i r e c t method ( i n h i b i t i o n of c o n t r a c t i l i t y by Lanthanum), Burt and Berns (1977) showed that normal beating and the associated action potentials of cultured neonatal rat v e n t r i c u l a r c e l l s are dependent on the a v a i l a b i l i t y of e x t r a c e l l u l a r calcium ions. Myocardial c e l l s , which also lack a highly developed sarcoplasmic reticulum system, thus provide a d d i t i o n a l evidence for a c l e a r l y s i m i l a r calcium dependent c o n t r a c t i l i t y . By way of i n d i r e c t evidence, e x t r a c e l l u l a r calcium has also been shown to be needed for non-muscular c e l l contraction. G a i l e_t a l . (1973) found that f i b r o b l a s t m o t i l i t y was prevented by lowering the e x t r a c e l l u l a r -3 -7 calcium concentration from 10 M to about 10 M, and t h i s e f f e c t was r e v e r s i b l e . The i s o l a t i o n of myosin and a c t i n from f i b r o b l a s t s (Adelstein, 1975) and the strong morphological evidence for a c t i n filaments i n f i b r o -b l a s t s (Ishikawa et^ a l . , 1969) supports the idea that t h i s type of i n h i b -i t i o n i s due to i n h i b i t i o n of muscle-like contraction. Ash £t a l . (1973) found that the formation of indentations of c l e f t s i n the contour of the developing mouse s a l i v a r y gland was prevented by the absence of e x t r a c e l l u -l a r calcium. This study was also correlated with the i d e n t i f i c a t i o n of microfilament bands inthe co n s t r i c t e d edge of the wedge-shaped c e l l s of the developing c l e f t s during normal development and the s e l e c t i v e binding of heavy meromyosin by these microfilaments (Spooner et a l . , 1973). 79 Recently, with X-ray probe microanalysis, Rice and Moran (1977) have shown j | that calcium (Ca ) levels are considerably higher i n the neural f o l d region than i n the rest of the embryo of the amphibian Amby stoma macula turn.' The involvement of microfilaments i n the formation of the neural f o l d had also been demonstrated by Burnside (1973). The C-4II c e l l s , i n keeping with many other types of carcinoma c e l l s , have poorly developed endoplasmic reticulum and few mitochondria (Auersperg, 1969). They thus lack the organelles which act as calcium stores, as well as sequestering mechanisms, i n such highly di f f e r e n t i a t e d contractile c e l l s as muscle. Therefore, C-4II c e l l s were expected to be more dependent on ext r a c e l l u l a r calcium for contraction. I t was shown i n the present study that they contracted gradually i n the presence of BSS and, concomitantly, there was a highly s i g n i f i c a n t increase i n i n t r a c e l l u l a r calcium. The increased i n t r a c e l l u l a r calcium i n these c e l l s may either act on the organized cont r a c t i l e microfilaments or be required for the organization and/or activation of the actomyosin system i n the cytoplasm and thereby induce c o n t r a c t i l i t y . In calcium-free BSS, the c e l l s did not contract and they released calcium to the medium. These results show d i r e c t l y that contraction of e p i t h e l i a l c e l l s also depends on i n f l u x of ex t r a c e l l u l a r calcium. Schroeder and Strickland (1974) demonstrated that the c o r t i c a l contrac-t i o n i n eggs of Rana pipiens, e l i c i t e d by ionophore, was independent of ex t r a c e l l u l a r calcium. The i n j e c t i o n of the calcium-specific chelating agent EGTA (ethylenebis(oxytheylenenitrilo) tetraacetic acid), was shown to i n h i b i t the contraction induced by the ionophore A23187. The results 80 suggested that eggs might have s u f f i c i e n t i n t e r n a l c a l c i u m s t o r e s to a l l o w the c o n t r o l of c o n t r a c t i o n by s e l e c t i v e s e q u e s t e r i n g of c a l c i u m . Th is concept was supported by the d e t e c t i o n of l o c a l i z e d c a l c i u m c o n c e n t r a t i o n i n the area of the p r o s p e c t i v e furrow of the c leavage of sea u r c h i n eggs (T imourian e_t a l . , 1974) . The r e s e a r c h i n the area of non-muscular c e l l c o n t r a c t i o n s , c i t e d above, and t h i s study s t r o n g l y support the hypothes is that non-muscular c e l l c o n t r a c t i o n , s i m i l a r to muscle c e l l c o n t r a c t i o n , may be c o n t r o l l e d by c a l c i u m . The a c t u a l o r i g i n of the r e q u i r e d c a l c i u m seems to d i f f e r i n non-muscular c e l l as shown by the requirement of e x t r a c e l l u l a r c a l c i u m i n C - 4 I I c e l l sheet c o n t r a c t i o n , s a l i v a r y g land morphogenesis and f i b r o b l a s t m o t i l i t y i n c o n t r a s t to c l e a v i n g eggs that r e q u i r e on ly t h e i r normal i n t e r n a l c a l c i u m content . Lack of c a l c i u m , i n t h i s study as w e l l as i n other s t u d i e s , has been a s s o c i a t e d w i t h the i n h i b i t i o n of c o n t r a c t i o n , but the l i g h t m i c r o s c o p -i c examinat ion of the C -4 I I c e l l sheets exposed to c a l c i u m - f r e e BSS i n d i c a t e s tha t t h i s t reatment a l s o has o ther e f f e c t s on the c e l l s . The change of the c u b o i d a l or columnar shape to the s p h e r i c a l shape a s s o c i a t e d w i t h a l o s s i n i n t e r c e l l u l a r adhesion as w e l l as c e l l u l a r c a l c i u m , i s s i m i l a r to the e f f e c t of c a l c i u m d e p r i v a t i o n on c e l l u l a r adhesion and membrane s t a b i l i t y i n many systems as rev iewed by Mannery (1966) . Ca lc ium has been shown to promote and m a i n t a i n i n t e r c e l l u l a r adhesion ( C u r t i s , 1967; W e i s s , 1967 c) as w e l l as the adhesion to c e r t a i n s u b s t r a t a (Weiss, 1960; T a k e i c h i and Okada, 1972) . E p i t h e l i a incubated i n c a l c i u m - f r e e medium, become much more f r a g i l e and d i s s o c i a t e d than the c o n t r o l s (Ash et d , , 1973) . For t h i s r e a s o n , the i n h i b i t i o n of c o n t r a c t i o n by l a c k of 81 ext r a c e l l u l a r calcium could be a secondary effect due to c e l l damage. However, i n this study, by measuring and comparing the i n t r a c e l l u l a r calcium content between contracted c e l l sheets and control cultures, d i r e c t , quantitative evidence was provided to show that calcium i s a controlling factor of e p i t h e l i a l c e l l contraction. No s i g n i f i c a n t effects of contracting C-4II c e l l s on the calcium content of test solutions were found, because the changes were too small to be detected by that method. However, the "Lanthanum method" was quite successful i n comparing the t o t a l and i n t r a c e l l u l a r calcium content of control cultures and contracted c e l l sheets. I | | Lettvin et a l . (1964) predicted that Lanthanum ions (La ), by I | | | virtue of an i o n i c radius si m i l a r to Ca and a higher valence than Ca , I | . w i l l bind at s u p e r f i c i a l l y located Ca site s i n a less reversible manner I j than does Ca . This was v e r i f i e d for s i t e s on lobster axon membrane (Takata et a l . , 1966). and the negative groups of a phospholipid a r t i f i c i a l membrane (Van Breemen et a l . , 1969). The high a f f i n i t y Of La''' for membrane binding sit e s was of p a r t i c u l a r interest when related to evidence that the d i s t r i b u t i o n of La''' i n many different preparations was confined to membrane areas contiguous with the ex t r a c e l l u l a r space (Revel and Karnovsky, 1967; Brightman and Reese, 1969; Payton ejt a l . , 1969; Zacks, 1970; Garant, 1972; Lane and Treherne, 1972). The use of Lanthanum as a tool for the study of changes i n c e l l u l a r I | Ca i n smooth muscle has been proposed by Van Breemen and co-workers (Van Breemen et a l . , 1970 & 1972). Their idea, which they have termed "the Lanthanum method" ("La method") i s based upon the assumptions that a 82 s u f f i c i e n t l y high concentration of ex t r a c e l l u l a r La w i l l (1) displace I | | [ and replace e x t r a c e l l u l a r Ca , (2) block Ca f l u x into c e l l s , (3) block I | c e l l u l a r Ca e f f l u x , and (4) not enter the c e l l i n appreciable quantities I [ to displace or a l t e r c e l l u l a r Ca d i s t r i b u t i o n . On this basis, tissues could be exposed to a variety of stimulatory agents or conditions i n the presence of h5Ca and subsequently placed into washout solutions containing a concentration of La' high enough to replace a l l e x t r a c e l l u l a r or super-f i c i a l Ca and to prevent any further uptake or ef f l u x of c e l l u l a r k5Ca. In this manner, effects on c e l l u l a r 4 5Ca uptake that were obscured by much larger quantities of ex t r a c e l l u l a r h5Ca can be detected. Experimental v e r i f i c a t i o n of the "La method" has been presented by Van Breemen jilt a l . (1973) but c r i t i c i s m s of the method have been pointed out by Hodgson e_t a l ; (1972), Freeman and Daniel (1973) and Weiss (1974). The l a t t e r author also points out that although not perfect, the "La method" appears to I | provide a more precise approach than use of a procedure such as Ca de-l [ p l e t i o n , whether accomplished by ionic v a r i a t i o n or by addition of Ca -chelating agents. Hodgson e_t al_. (1972) reported that mitochondria and sarcoplasmic reticulum fractions isolated from ^ " L a C l ^ treated rat myometrial c e l l s contained 1 4 0 L a . From this they questioned the assumption that La''' i s only confined to the ex t r a c e l l u l a r space and limited i n i t s action to the c e l l surface. However, the electron dense La''' ions ( c o l l o i d a l Lanthanum) only stained the ex t r a c e l l u l a r space and outer surfaces of c e l l membranes (Doggenweiler and Frenk, 1965; Revel and Karnovsky, 1967; Overton, 1968). Using LaCl^ rather than c o l l o i d a l Lanthanum, Langer and Frank (1972) did not observe La''' to penetrate the 83 plasma membrane of cultured myoblast or fibroblast c e l l s . Recently, Ma and Bose (1977) observed that when 10 mM LaCl^ was used to seal vascular smooth muscle c e l l s (rabbit aorta), La did not appear i n the cytoplasm. These three-dimensional staining studies could weaken the argument of Hodgson e_t a l . (1972) that La penetrated the c e l l . Freemen and Daniel III | | (1973) reported that 2 mM La could displace Ca from ext r a c e l l u l a r I | s i t e s and i n h i b i t i n f l u x of e x t r a c e l l u l a r Ca while they questioned I | whether the blockade of c e l l u l a r Ca e f f l u x was complete. In fact, the I | | concentration'of La appears rather c r i t i c a l . Casteels et a l . (1972) investigated the Lanthanum concentrations required to cause various effects and concluded that 10 mM LaCl^ blocked transmembrane calcium movements whereas the 2 mM LaCl^j as previously used i n the "La method" was not quite s u f f i c i e n t . Calcium e f f l u x was inh i b i t e d by 87% by 2 mM LaCl^. Besides the lower concentration of Lanthanum, Van Breemen e_t a l . (1973) also attributed the residual 13% calcium loss from vascular smooth muscle to displacement from damaged c e l l s . La can enter c e l l s damaged by mechanic-a l injury or enzymatic degradation (Revel and Karnovsky, 1967; Overton, 4-t-1968). Others f e e l that La does not block Ca e f f l u x e f f e c t i v e l y at 37 C but that 10 mM L a C l 0 i n a washing solution at 4 C i s more effective I | (James and Roufogalis, 1977). Ca e f f l u x i s greatly reduced at low temperature (4°C) (Goodford, 1965; Borle, 1972). In fact, a l l active transport a c t i v i t y i s almost inhi b i t e d at 4°C (Setekleiv, 1970; Tomita, 1970) and passive leakage of ions across the membrane should also be reduced especially with the additional membrane-stabilizing effect of I | | 10 mM LaClg (Weiss, 1974). Weiss (1974) examined the effects of La on .84 smooth muscle by postulating that La''' displaced C a ^ from surface s i t e s , bound firmly at these s i t e s , and exerted a s t a b i l i z i n g action that pre-vented inward release of Ca from less accessible membrane s i t e s . Van Breemen (1969) explained the blockage to be the result of unavailable fixed negative s i t e s for Ca transport; whenever these site s were associ-ated with La they were unable to mediate Ca transport. Blockade of membrane Ca transport by La has been shown d i r e c t l y i n (1) skeletal muscle, e.g., barnacle muscle fib e r s (Hagiwara and Takahashi, 1967) and frog sartorius muscle (Weiss, 1970), (2) nerve c e l l s , e.g., squid axon (Van Breemen et a l . , 1970), (3) cardiac muscle, e.g., rabbit heart muscle (Langer and Frank, 1972), (4) smooth muscle, e.g., i n t e s t i n a l (Weiss, 1969), uterine (Goodman and Weiss, 1971) and vascular smooth muscle (Van Breemen, 1969), and (5) a r t i f i c i a l membranes (Van Breemen, 1969). Based on the above experimental evidence, i t was f e l t that at 4°C, an isotonic Tris-HCl solution (pH 7.4) containing 10 mM LaCl^ could be used to wash off ex t r a c e l l u l a r Ca while retaining most of the i n t r a c e l l u l a r 4+ Ca (James and Roufogalis, 1977). The t o t a l and i n t r a c e l l u l a r calcium could be extracted from the c e l l s (or tissues) and measured by atomic absorption spectrophotometry. In summary, the general theories upon which the modified "La method" was based were: (1) 10 inM La w i l l displace and replace e x t r a c e l l u l a r Ca (2) 10 mM La w i l l s t a b i l i z e the membrane and block i n f l u x of Ca into the c e l l . I | | | | (3) 10 mM La w i l l block e f f l u x of c e l l u l a r Ca 85 (4) A c t i v e t r a n s p o r t w i l l be reduced at 4 C. (5) P a s s i v e f l u x e s w i l l be reduced at 4°C. J (6) P e r m e a b i l i t y to L a ' ' ' i s ve ry low and w i l l be even l e s s a t 4°C and t h e r e f o r e i t should not a f f e c t i n t r a c e l l u l a r i o n s . The present study was compl i ca ted by the f a c t that the exper imenta l m a t e r i a l c o n s i s t e d of m u l t i c e l l u l a r e p i t h e l i a l sheets w i t h narrow i n t e r -c e l l u l a r spaces . I t was i m p o r t a n t , t h e r e f o r e , to show that apparent d i f f e r e n c e s i n i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n between c o n t r o l c u l t u r e s I | | and c o n t r a c t e d c e l l sheets were not due to reduced access of La ions to i n t e r c e l l u l a r spaces i n the c o i l e d , c o n t r a c t e d sheets and to r e s u l t i n g incomplete removal of e x t r a c e l l u l a r c a l c i u m . There fo re , c a l c i u m l e v e l s were compared between i n t a c t c e l l sheets and sheets broken up i n t o s m a l l c e l l groups and s i n g l e c e l l s by p i p e t t i n g i n 10 mM L a - T r i s s o l u t i o n . In c o n t r o l c u l t u r e s , i n both i n t a c t sheets and c e l l sheets broken up i n t o s m a l l c e l l groups, 10 mM L a - T r i s s o l u t o n a t 4°C e f f e c t i v e l y removed e x t r a c e l l u l a r c a l c i u m i n 30 min w h i l e p r e v e n t i n g l o s s of i n t r a c e l l u l a r c a l c i u m . In c o n t r a c t e d sheets tha t were broken up, L a ' ions a l s o d i s -I | p l a c e d the e x t r a c e l l u l a r Ca ions i n 30 m i n . However, l i m i t e d data j j | suggest tha t i t may take a longer t ime f o r La ions to d i s p l a c e e x t r a -I | c e l l u l a r Ca ions of i n t a c t c o n t r a c t e d s h e e t s , p o s s i b l y due to mechanica l s h i e l d i n g . T h e r e f o r e , the use of broken up c e l l sheets xvas adapted as the r o u t i n e method. Mechan ica l b r e a k i n g up of c e l l sheets i n t o s m a l l clumps by Pas teur p i p e t t e i n the presence of 4°C 10 mM L a - T r i s s o l u t i o n (pH 7.4) d i d not a f f e c t the i n t e g r i t y and v i a b i l i t y of the c e l l s as examined by e o s i n - ' e x c l u s i o n t e s t . Th is suggests that mechanica l s e p a r a t i o n 86 d i d not cause damage of c e l l s tha t would r e s u l t i n l e a k i n g of i n t r a c e l l u l a r Ca i o n s . L e v e l l i n g o f f of the c e l l u l a r c a l c i u m content a f t e r 30 min f o l l o w i n g an i n i t i a l drop from the t o t a l c a l c i u m v a l u e i n the presence of L a - T r i s s o l u t i o n was cons idered as an i n d i c a t i o n that a l l or most e x t r a -c e l l u l a r c a l c i u m had been removed. The h igher c e l l u l a r content which p e r s i s t e d i n the presence of i c e - c o l d T r i s s o l u t i o n f o r up to 120 m i n , compared to that i n L a - T r i s s o l u t i o n ( R e s u l t s , S e c t i o n I I I ) , as w e l l as the i n h i b i t o r y e f f e c t of L a ' i ons on c o n t r a c t i o n ( R e s u l t s , S e c t i o n V I I ) , g i ve f u r t h e r support to the assumption tha t L a ' ' ' i ons d i s p l a c e d e x t r a c e l l u l a r Ca ions i n C - 4 I I c e l l s h e e t s . o The c e l l s h e e t s , 3 min a f t e r i n i t i a t i o n of c o n t r a c t i o n , m o r p h o l o g i c a l l y s t i l l resembled c o n t r o l s , i . e . , they were most l y s t i l l f l a t , and reached the maximal degree of c o n t r a c t i o n on ly a f t e r 30 m i n . Yet the c e l l s c o n t a i n -ed more c a l c i u m i n the f i r s t 3 min than a f t e r 30 min of c o n t r a c t i o n (Table X I I I ) . These r e s u l t s suggest tha t the c e l l s take up l a r g e amounts of c a l c i u m i n the f i r s t 3 min and that t h i s c a l c i u m i n i t i a t e s and/or s t i m u l a t e s I | c o n t r a c t i o n (some Ca ions might a l s o be r e l e a s e d from i n t r a c e l l u l a r s t o r e s ) . A f t e r the i n i t i a l p e r i o d o f c o n t r a c t i o n , the c e l l s seem to g r a d u a l l y r e l e a s e c a l c i u m to the medium. Shanes (1961) observed c a l c i u m i n f l u x i n c o n t r a c t i n g f r o g r e c t u s abdominis musc le , and a l s o found that enhanced i n f l u x might not be mainta ined d u r i n g pro longed s t i m u l a t i o n but might decrease w i t h t i m e . The i n a c t i v a t i o n of the c a l c i u m cur ren t may prevent the i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n from i n c r e a s i n g to a p o i n t where i t would be d e l e t e r i o u s to the c e l l (Shanes, 1961) . The C -4 I I c e l l s have p o o r l y developed endoplasmic r e t i c u l u m and few m i t o c h o n d r i a ; they 87 thus lack the organelles which act as calcium stores and as a sequestering mechanism. So, the C-4II c e l l s are more dependent on e x t r a c e l l u l a r c a l -cium for contraction and once they have taken up s u f f i c i e n t extracellular calcium to stimulate contraction, then, i n order to prevent deleterious effects, prolonged and enhanced i n f l u x of calcium i s not maintained, but rather, calcium i s released to the medium. The fact that the highest c e l l u l a r calcium content was reached i n the f i r s t 3 min of contraction, when morphologically the c e l l sheets s t i l l remained f l a t , provides further evidence that the high calcium levels of contracted c e l l sheets were not an a r t i f a c t due to incomplete replacement of e x t r a c e l l u l a r calcium by Lanthan-um. Ionophore A23187 has been reported as a mobile c a r r i e r which f a c i l i -l | tates the movements of Ca ions across b i o l o g i c a l membranes (Reed and Lardy, 1972; Wong et a l . , 1973; Case et a l . , 1974). There was no s i g n i f i -cant difference i n calcium content of test solutions between BSS and iono-phore A23187/BSS i n the presence of c e l l s . Ionophore did not increase contraction - rather, the speed of contraction appeared slower i n ionophore A23187/BSS than i n BSS i n the f i r s t 15 min. I t i s possible that i t takes some time for ionophore A23187 to enter the c e l l membrane before i t functions as c a r r i e r of Ca ions movement. Since control c e l l sheets take up large amounts of calcium i n the f i r s t 3 min of contraction, i t i s possible that any effect of ionophore A23187 i s masked by these amounts. Blank solution, La-Tris and/or GAA-TCA ( g l a c i a l acetic a c i d - t r i c h l o r o -acetic acid) give a background reading (blank background, Table XV). Since the blank i s incorporated i n the standards, i t should not contribute 88 to the abso lu te c a l c i u m v a l u e s o b t a i n e d . Components of the rubber p o l i c e -man used to d i s l o d g e the c e l l s and Waymouth's medium produce sporad ic a r t i f a c t u a l read ings (Table XVI) which may be r e s p o n s i b l e f o r some of the v a r i a t i o n observed i n t h i s s tudy . The growth stage a f f e c t e d the i n t r a c e l l u l a r c a l c i u m l e v e l s : the i n t r a -c e l l u l a r c a l c i u m l e v e l s i n c o n f l u e n t s t a t i o n a r y c u l t u r e s were lower and more constant than i n younger, r a p i d l y growing c u l t u r e s . V a r i a b l e c a l c i u m content i s l i k e l y due to d i f f e r e n t p h y s i o l o g i c a l c o n d i t i o n s . For example, c a l c i u m has a r o l e i n the o r g a n i z a t i o n of the m i t o t i c apparatus ( H a r r i s , 1975) . Th is i s a n ' i n t e r e s t i n g t o p i c f o r f u r t h e r i n v e s t i g a t i o n s . C e l l u l a r c a l c i u m content of n e r v e , muscle or other v a r i o u s t i s s u e s i s u s u a l l y expressed i n nmole/Kg wet weight or umole/g dry we ight . There fo re , i t was d i f f i c u l t to compare the r e s u l t s of t h i s study w i t h other s t u d i e s . However, s t u d i e s have been r e p o r t e d where c e l l u l a r c a l c i u m i s expressed as nmole/mg c e l l p r o t e i n : i n HeLa c e l l s ( B o r l e , 1968) and i n k idney c e l l s ( B o r l e , 1972) . The c e l l p o r t e i n was determined by a m o d i f i c a t i o n of the methods of Oyama and Eagle (1956) and Lowry et_ a l . (1951) , and c a l c i u m was determined by an automatic f l u o r o m e t r i c t i t r a t i o n method. The c a l c i u m content of C -4 I I c e l l s i s very c l o s e to the v a l u e s of k idney and HeLa c e l l s (Table X V I I ) . Bes ides d i f f e r e n t p h y s i o l o g i c a l c o n d i t i o n s , d i f f e r e n t c u l t u r i n g methods a l s o a f f e c t e d the c a l c i u m content of the c e l l s (Table X V I I ) . The b a s i s of the t r y p s i n v s . t rypsin/EGTA e f f e c t on c e l l u l a r c a l c i u m content awai ts f u r t h e r i n v e s t i g a t i o n . 89 Table XVII. Ce11ular caleium content In kidney, HeLa and C-4II c e l l s 1ntrace11u1ar ca1ci um Total calcium Sample nmole/mg c e l l p r o t e i n nmole/mg c e l l p r o t e i n ki dney ce11 (Borle,1972) . 4.16-4.90 100-125 HeLa ce11 (Borle,1968) 5.74±0.30 43.52±2.62 C-4 II ce11 ( t r y p s i n d i s s o c i a t i o n ) 6.25±2;50 39.45±6r00 C-4 II eel 1 ( t r y p s i n/EGTA d i s s o c i a t i o n ) T7.00±6.00 83.25±10.00 90 In t h i s study, the "Lanthanum method" provided a successful way for measuring the i n t r a c e l l u l a r calcium content of cultured c e l l s . D i f f e r e n t p h y s i o l o g i c a l conditions as w e l l as c u l t u r i n g methods may a f f e c t the calcium content of C-4II c e l l s . In a l l experiments, the i n t r a c e l l u l a r calcium content i n contracted c e l l sheets was always higher than i n cont r o l cultures. In conjunction with the morphological changes, t h i s indicates that contraction of C-4II c e l l sheets i s dependent on i n f l u x of e x t r a c e l l u l a r calcium. Influx of e x t r a c e l l u l a r calcium into these c e l l s may ei t h e r act on the organized c o n t r a c t i l e microfilaments or organize and/or activa t e the actomyosin system i n the cytoplasm and thereby induce c o n t r a c t i l i t y . 9 1 PART II THE DISTRIBUTION OF EXTRACELLULAR MATERIALS IN CULTURES OF CARCINOMA CELLS 92 MATERIALS AND METHODS -I. Autoradiography 1 . C u l t u r e Techniques C u l t u r e s of the l i n e C -4 I I were grown on s t e r i l i z e d , 25 mm p l a s t i c t i s s u e c o v e r s l i p s (Lux S c i e n t i f i c C o r p o r a t i o n , Cat . #5415) i n p l a s t i c P e t r i d i shes i n Waymouth's medium MB752/1 w i t h 10% f e t a l c a l f serum, 100 y/ml of p e n i c i l l i n and 100 yg/ml of s t rep tomyc in a t 37°C i n a h u m i d i f i e d 5% CO2/ a i r i n c u b a t o r , c u l t u r e d and mainta ined as d e s c r i b e d i n P a r t I. 2 . Isotope I n c o r p o r a t i o n Monolayered c u l t u r e s were incubated i n 2 .0 ml 10% f e t a l c a l f serum/ Waymouth's medium c o n t a i n i n g 12 .5 - 50 uC i/ml of [6 - 3 H]g lucosamine (10.13 Ci/mmole, New E n g l a n d - N u c l e a r , Bos ton , Mass . ) or [ 2 , 3 - 3 H ] p r o l i n e (24.5 C i / mmole, New England N u c l e a r , Bos ton , Mass . ) f o r 2 hr to 8 days . A f t e r l a b e l l i n g , the c u l t u r e s were washed 3 t imes f o r 5 min i n 2 ml of Waymouth's medium (37°C) . The supernatant was removed, and c o v e r s l i p s w i t h the c e l l sheets were f i x e d i n 2.5% g l u t a r a l d e h y d e / M i l l o n i g ' s b u f f e r a t pH 7 . 3 - 7 . 4 f o r 1 .5 hr at 4°C. 3 . L i g h t Microscopy and Autoradiography A f t e r f i x a t i o n i n 2.5% g l u t a r a l d e h y d e / M i l l o n i g ' s b u f f e r f o r 1 .5 h r , the c u l t u r e s were washed 3 t imes i n M i l l o n i g ' s b u f f e r , 5 min each t i m e , a t 4°C, then p o s t f i x e d i n c o l d 1% O s O ^ / M i l l o n i g ' s b u f f e r f o r 15 m i n , washed i n M i l l o n i g ' s b u f f e r , and dehydrated i n graded e thano ls from 50% to 100%. F o l l w o i n g t h i s , p a r t of the monolayer fragments (Group-1) were removed by s c r a p i n g them from the p l a s t i c c o v e r s l i p s w i t h a non -adhes ive p l a s t i c wedge 93 into a 1 ml beaker, dehydrated i n propylene oxide and embedded i n layers of Epon 8.2, 3 or 4 mm thick. This material served to provide sections through the c e l l s . Sections, 0.2 to 2.0 u thick, were made perpendicular to the substratum. ' The exact position and orientation of the c e l l s within these areas were determined by l i g h t microscopic examination of sections stained with 1% toluidine blue/1% borax. Sections were cut with glass knife on a Reichert ultramicrotome. The rest of the monolayers with the p l a s t i c coverslips (Group-2), went through 4 changes i n 100% ethanol, ethanol/epon and then were embedded i n epon 812 as shown i n Appendix VIII. The sample could be dried i n a i r after 100% ethanol processed for autoradi-ography by standard o p t i c a l observation. These coverslips provided autoradiographs of complete c e l l s and i n t e r c e l l u l a r materials i n intact monolayers. Again, sections, 0.2 to 2.0 u thick, were made perpendicular to the substratum. Then, epon sections were placed on 0.5% gelatin-0.05% chrome alum coated s l i d e s , dried and coated with NTB-3 emulsion (Eastman Kodak Corp., Rochester, N. Y.), dried, stored i n a light-proof black s l i d e box and exposed for 2 to 4 weeks, fi x e d , then mounted for observation (Appendices VIII and IX). The following variables were examined to establish optimal conditions: 1) Thickness of sections (0.2 to 2.0 u). 2) Duration of exposure of emulsion (1-4 weeks). I I . Histochemistry 1. Periodic Acid-Silver Methenamine (A modification of Rambourg and Leblond's method, 1967.) a. Solutions and Reagents: 94 - Methenamine (hexamethylenetetramine) - f r e s h l y made 3% s o l u t i o n i n d i s t i l l e d wate r . - Sodium bora te - 2% s o l u t i o n i n d i s t i l l e d wate r , which cou ld be kept f o r 1 week at room temperature . - S i l v e r n i t r a t e - f r e s h l y made 5% s o l u t i o n i n d i s t i l l e d water . - P e r i o d i c a c i d - 1% s o l u t i o n i n d i s t i l l e d water which cou ld be kept i n d e f i n i t e l y at room temperature . - Gold c h l o r i d e - 0.05% i n d i s t i l l e d w a t e r , which cou ld be kept f o r 3 months i n a l i g h t - p r o o f c o n t a i n e r a t 4°C. - Sodium t h i o s u l f a t e - 5% s o l u t i o n i n d i s t i l l e d water which cou ld be kept i n d e f i n i t e l y a t room temperature . S i lver"methenamine s o l u t i o n : added 5 ml of s i l v e r n i t r a t e to 45 ml methen-amine s o l u t i o n . A wh i te p r e c i p i t a t e appeared which cou ld be d i s s o l v e d by s h a k i n g . The f i n a l s o l u t i o n was obta ined by adding 5 ml sodium borate s o l u t i o n , which should be p e r f e c t l y c l e a r . A f t e r thorough m i x i n g , the f i n a l s o l u t i o n was f i l t e r e d through Whatman paper No. 1 and kept i n a brown b o t t l e . Th is s o l u t i o n had to be f r e s h l y prepared be fo re u s i n g . b. Glassware The g lassware used i n t h i s method was washed i n s u l f o c h r o m i c mix tu re and thoroughly r i n s e d w i t h tap and d i s t i l l e d water j u s t be fo re use . The dimensions of the 60 x 15 mm g l a s s P e t r i d i shes were c r i t i c a l s i n c e the su r face - vo lume- temperatu re r a t i o of the s i l v e r methenamine s o l u t i o n must be kept constant to avo id p r e c i p i t a t i o n dur ing h e a t i n g (Rambourg, 1967) . 95 c . Procedure Epon s e c t i o n s , 0 .2 to 2 .0 u, were s t o r e d by f l o a t i n g on d i s t i l l e d w a t -er i n 60 x 15 mm g l a s s P e t r i d i s h e s . For s t a i n i n g , the s e c t i o n s were passed from s o l u t i o n to s o l u t i o n w i t h a p l a t i n u m loop (which d i d not r e a c t w i t h s i l v e r ) . The f i r s t step was to f l o a t s e c t i o n s f o r 1 hr on 1% aqueous p e r i o d i c a c i d at room temperature . They were r i n s e d by s e v e r a l b r i e f passages on d i s t i l l e d water baths and a 30 min s tay on the l a s t of these b a t h s . The s e c t i o n s were then taken to the f r e s h l y prepared s i l v e r methenamine s o l u -t i o n i n a 62-64°C oven. Th is should be done i n a dark room. A f t e r 60 min a l l s e c t i o n s were removed from the s t a i n i n g s o l u t i o n , q u i c k l y r i n s e d on d i s t i l l e d water and t r a n s f e r r e d to a second bath of s i l v e r methenamine. A g a i n , s t a i n i n g was a l lowed to proceed i n a 62-64°C oven. The s e c t i o n s were examined every 30 min u n t i l they showed a ye l low-brown t i n g e . U s u a l -l y 2 to 2 .5 hr i n s i l v e r methenamine s o l u t i o n was enough f o r 0 . 5 to 2 .0 u t h i c k epon s e c t i o n s . A l l s e c t i o n s then f l o a t e d tw ice on d i s t i l l e d water . 3 to 5 min on 0.05% go ld c h l o r i d e , 5 min on 3% sodium t h i o s u l p h a t e and f i n a l l y were r i n s e d on two baths of d i s t i l l e d water . The s e c t i o n s were t r a n s f e r r e d onto s l i d e s , d r i e d , and mounted w i t h DePeX or Eupona l . C o n t r o l s e c t i o n s were a l s o u s e d , which were not t r e a t e d w i t h p e r i o d i c a c i d be fo re be ing taken to the s i l v e r s o l u t i o n . Mouse k idney was used as the c o n t r o l t i s s u e to show p o s i t i v e s t a i n i n g of b a s a l l a m i n a . 2. P e r i o d i c A c i d - S c h i f f S t a i n i n g ( P A S - S t a i n i n g ) (A m o d i f i c a t i o n of the method of Neva la inen et^ a l . , 1972.) 96 a . Reagents and S o l u t i o n s (a) S c h i f f s Reagent: (Chayen et a l . , P r a c t i c a l H i s t o c h e m i s t r y [1973] , p. 5 8 ) . 1 g b a s i c f u c h s i n ( F i s h e r S c i e n t i f i c Co. ) was d i s s o l v e d i n 200 ml b o i l i n g d i s t i l l e d w a t e r , shaken and coo led to 50°C and f i l t e r e d . Then 3 ml of I N HCl and 3 g of potass ium m e t a b i s u l p h i t e were added. This s o l u t i o n was a l lowed to s tand f o r 24 hr i n a d a r k - c o l o r e d , stoppered b o t t l e . Dur ing t h i s t ime the b i s u l p h i t e d e c o l o r i z e d the f u c h s i n , i . e . , i t formed the c o l o r l e s s S 0 2 - p a r a r o s a n i l i n e complex ( l e u c o - b a s i c f u c h s i n ) . Then, 0 . 5 g of d e c o l o r i z i n g a c t i v a t e d c h a r c o a l ( N o r i t , F i s h e r S c i e n t i f i c Co. ) was added to remove i m p u r i t i e s and o ther c o l o r e d m a t t e r . The reagent was then shaken w e l l f o r about 1 m i n , and f i l t e r e d r a p i d l y (to avo id r e -c o l o r i z i n g the l e u c o - b a s i c f u c h s i n ) through Whatman No. 1 f i l t e r paper . The s o l u t i o n should be c o l o r l e s s and c l e a r , and i t cou ld be s t o r e d f o r months i n a w e l l - s t o p p e r e d , dark c o l o r e d b o t t l e . (b) S 0 2 - w a t e r : 25 ml 1 N h y d r o c h l o r i c a c i d and 50 ml 0.5% aqueous s o l u t i o n of potass ium m e t a b i s u l p h i t e , mixed immediately be fo re u s e . b. Procedure 0 . 5 to 2 .0 u t h i c k epon s e c t i o n s were p laced on 0.5% g e l a t i n - 0 . 0 5 % chrome alum coated s l i d e s , d r i e d , o x i d i z e d f o r 10 min i n 1% p e r i o d i c a c i d , washed tw ice i n d i s t i l l e d w a t e r , s t a i n e d f o r 40 min i n S c h i f f ' s reagent , r i n s e d i n S02~water, d i s t i l l e d water and 70% e t h a n o l , then mounted i n DePeX. Mouse k idney was used as the c o n t r o l t i s s u e to show p o s i t i v e s t a i n i n g of b a s a l l a m i n a . 97 I I I . Growth on Collagen Gel 1. Preparation of Collagen Gel (Leighton eit a l . , 1967) A 1% suspension of native bovine collagen i n 25% methanol and 0.25% cyanoacetic acid was obtained from Ethicon, Inc., Somerville, New Jersey, diluted i n 0.5% acetic acid to make a concentration of 0.1% and spread into 35 mm Lux tissue culture dishes or Falcon bacteriological P e t r i dishes over the growth surface at a' r a t i o of 0.1 ml/cm2. Then, the dishes were exposed to NH^ OH fumes for 1 hr, washed thoroughly with Hanks' balanced s a l t solution and then stored i n fresh Hanks' solution, pH 6.8 to 7.0 at 4°C u n t i l ready for use. 2. Culturing Of the Cells on Collagen Gel About 1-2 x 10 c e l l s i n 10% f e t a l calf serum/Waymouth's medium were added to each 35 mm collagen coated dish and cultured as described previous-i y . 3. Electron Microscopy (Appendix II) For electron microscopy, c e l l s were fi x e d , dehydrated and embedded as described i n Section I. Preceding thin sectioning, the exact position and orientation of the c e l l s within epon were determined by l i g h t microscopic examination of 0.5-1.0 y sections stained with 1% toluidine blue/10% borax. Silver-gold sections were mounted on carbon-coated copper grids, stained with uranyl acetate (K and K Lab, Inc., Plainsview, N. Y.) (Watson, 1958) and lead c i t r a t e (Venable and Coggeshall, 1965) and examined with a Hitachi HS-7S electron microscope. 98 RESULTS I. Genera l Morphology C e l l s formed p o l y g o n a l , p a v e m e n t - l i k e c o l o n i e s w i t h i n two days i n c u l t u r e . Seven days l a t e r , they became c o n f l u e n t , and were spot ted w i t h round, b l i s t e r - l i k e s t r u c t u r e s r e f e r r e d to as hemicysts ( F i g . 18) or domes and g e n e r a l l y cons idered as ev idence of s e c r e t o r y a c t i v i t y (Le ighton et a l . , 1969; Auersperg , 1969; P i c k e t t e t a l . , 1975) . Th ick s e c t i o n s of Epon embedded c u l t u r e s showed that dur ing the e a r l y l o g a r i t h m i c growth phase, C -4 I I c e l l s were f l a t , t h i n and o r i e n t e d p a r a l l e l to the growth s u r f a c e , and as they became crowded, c e l l s i n most areas assumed rhomboidal or columnar shapes so that t h e i r main a x i s and the i n t e r c e l l u l a r spaces became v e r t i c a l l y o r i e n t e d and the c o l o n i e s remained monolayered. Dur ing the s t a t i o n a r y phase, the i n t e r c e l l u l a r spaces expanded which r e s u l t e d i n s e p a r a t i o n of ad jacent c e l l s ( F i g s . 1 & l a ) . U l t r a s t r u c t u r a l l y , the c e l l s showed the s u r f a c e p o l a r i t y c h a r a c t e r i s -t i c of t r a n s p o r t i n g e p i t h e l i u m . The i r a p i c a l s u r f a c e s , f a c i n g the medium, bore m i c r o v i l l i , and t i g h t j u n c t i o n s as w e l l as j u n c t i o n a l complexes j o i n e d ad jacent c e l l s . I n t r a c e l l u l a r l y they e x h i b i t e d a h i g h n u c l e a r : c y t o p l a s m i c r a t i o . In reg ions of m u l t i l a y e r i n g , c e l l s between the s u r f a c e l a y e r and substratum were u n p o l a r i z e d and l a c k e d t i g h t j u n c t i o n s . No b a s a l lamina was formed i n any of the p r e p a r a t i o n s . M i c r o f i l a m e n t s cou ld be found both i n a p i c a l and b a s a l s i d e s . The C - 4 I I c e l l s , i n keeping w i t h many other types of carcinoma c e l l s , have p o o r l y developed endoplasmic r e t i c u l u m and few m i t o c h o n d r i a (Auersperg , 1969) . 99 I I . Autoradiography Glycosaminoglycans (GAG) have been shown by autoradiography to be a s s o c i a t e d w i t h some embryonic basement membranes, e . g . , embryonic mouse s a l i v a r y g land (Banerjee e_t a l . , 1977) , embryonic c h i c k c o r n e a l e p i t h e l i u m (Hay and M e i e r , 1976) and embryonic r a t p a r i e t a l y o l k sac (Minor et a l . , 1976) . The re fo re , a u t o r a d i o g r a p h i c s t u d i e s of 3 H - g l u c o s a m i n e - and 3 H - p r o l i n e - l a b e l l e d c u l t u r e s were used to determine the p a t t e r n s of d e p o s i t i o n of e x t r a c e l l u l a r m a t e r i a l s and accumulat ion of b a s a l l a m i n a , i f any. These s t u d i e s , combined w i t h u l t r a s t r u c t u r a l and h i s t o c h e m i c a l s t u d i e s were to determine (1) the r o l e of e x t r a c e l l u l a r m a t e r i a l s i n the d e t e r m i n a t i o n of the p o l a r i t y of e p i t h e l i a l c e l l s when the c e l l s were c u l t u r e d ±a v i t r o i n the basence of hetero logous t i s s u e s ; (2) whether e p i t h e l i a l c e l l s i n v i t r o were ab le to form b a s a l lamina as occur red i n v i v o , i . e . , when grown i n hamster cheek pouches (Auersperg , 1969) . 1 . 3 H-Glucosamine L a b e l l i n g The f o l l o w i n g experiments were performed i n order to determine (1) whether the^ 3 H - g l u c o s a m i n e , i n c o r p o r a t e d i n t o e p i t h e l i a l c e l l s d u r i n g a shor t p u l s e , was depos i ted on the b a s a l s i d e , t h e r e f o r e p o s s i b l e c o n t r i b -u t i n g to b a s a l l a m i n a , (2) whether the components tha t were l a b e l l e d dur ing a shor t p u l s e remained l o c a l i z e d w i t h i n the c e l l s , (3) whether there was a d i f f e r e n t i a l g r a d i e n t among d i f f e r e n t s i t e s of the c e l l s , and, i f any, at what stage t h i s asymmetric d i s t r i b u t i o n was formed. Group 1 : C e l l s were l a b e l l e d w i t h 25 ]aei 3 H-g lucosamine/ml 10% f e t a l c a l f serum-Waymouth's medium f o r 3 hours a f t e r s u b c u l t u r e , then chased f o r 8 days w i t h u n l a b e l l e d medium. 100 Group 2: Cells were labelled for 48 hours ( u n t i l they had settled down, i . e . , attached to the coverslips), then chased for 6 days. Group 3: Cells were labelled for 8 days after subculture. Group 4: Cells grew for 6 days u n t i l confluence, were l a b e l -led for 3 hours thereafter, then chased for 6 days. Group 5: Cells grew for 6 days, were labelled for 48 hours, then chased for 4 days. Group 6: Cells grew for 6 days, were then labelled for 6 days. The results showed that during pulse l a b e l l i n g of a l l of these cultures, the d i s t r i b u t i o n of isotopic grains concentrated at the basal side of the c e l l s did not resemble structured basal lamina as i s the case i n salivary gland (Banerjee et^ a l . , 1977). Most iostopic grains (Table XVIII) were located next to c e l l s (80.4-95.4%). Less than 10% were over the c e l l s which indicates that the glucosamine lab e l was transported out of the i n t r a c e l l u l a r area into the e x t r a c e l l u l a r coat. In Groups 1, 2, 4 and 5, the glucosamine l a b e l from pulsing and chasing might represent the true ex t r a c e l l u l a r components of the c e l l s (Figs. 20-22, 24, 25). In a l l of these s i x groups, the amounts of isotopic l a b e l l i n g on the basal sides were higher than that on the apical sides. There was a s i g n i f i c a n t difference i n the d i s t r i b u t i o n of 3H-glucosamine l a b e l l i n g between apical and basal sides i n Groups 1 and 3, while the i n s i g n i f i c a n t difference of the Group 2 might be due to the small sampling resulting i n large standard deviation values (Table XVIII). In Groups 1-3 (cultures which had been labelled i n younger stages), there were constant ratios of the number of grains i n Table XVI11.The Distribution of 3H-glucosamine Label (x + S.D.) i n C-4II Cultures Experi- Basal side Apical side Intercellular Extracellular^ Intracellular Total C e l l Total mental group 3 No. (x) % d No. (x) 7. No. (x) 7. No. (x) % No. (x) . grain No. (x) No. (x) Grains/ c e l l t - t e s t c Group 1 (5 ) e 20.4+4.3 22.7 14.0+1.6 15.6 50.6±18.8 56.2 85±18.8 94.4 5.0±1.9 5.6 90±19.5 105 1.2±0.4 P<0.05 Group 2 (2) 38.5±21.9 25.7 23.5±12.0 15.7 79.5±12.0 53.0 141.5±21.9 94.3 8.5+3.5 5.7 150±25.5 106 1.4±0.3 P>0.05 Group 3 (3) 193±37.0 23.4 134.7+22.6 16.3 434+24.0 52.7 761.7±51.0 92.4 §1.7*3.1 7.5 823.3+52.4 44 18.7+.2.0 P<0.05 Group 4 (2) 24±7.1 9.3 12±0 4.6 211+14.1 81.5 247±7.1 95.4 .12+2.8 4.6 259±9.9 70 3.7+.0.6 P>0.05 Group 5^ (1) 71 19.2 6.2 16.8 219 59.2 352 95.1 18 4.9 370 62 6.0 Group 6 (1) 176 10.1 147 8.4 1253 71.9 1576 90.4 167 9.6 1743 99 ' 17.6 o Group 1 - Cells labelled for 3 hr after subculture, then chased for 8 days with unlabelled medium. Group 2 - Cells labelled for 48 hr (until cells had settled down), then chased for 6 days. Group 3 - Cells labelled for 8 days after subculture. Group 4 - Cells grew for 6 days until confluence, labelled for 3 hr, then chased for 6 days. Group 5 - Cells grew for 6 days until confluence, labelled for 48 hr, then chased for 4 days. Group 6 - Cells grew for 6 days until confluence, labelled for 6 days. ''Extracellular grain no. • (basal side + apical side + intercellular) grain no. Ct-test ~ comparison of the means of grain numbers between basal and apical sides. • percentage of the total grain numbers. eBracket indicates numbers of observations in each experimental group (from different epon sections). Each experimental group stands for 1 culture of cells which were grown on plastic coverslips in medium-containing Petri dishes. .The basal sides were very difficult to see in this group. TABLE XIX. The Distribution of ^H-glucosamine-label/cell i n C4II cultures* Experi-nental Basal Apical group" side. side Inter-cellular Extra-cellular Intra-cellular Total Apical/ b a s al c Inter-Basal/ Apical/ cellular/ extra- «xtra- extra-cellular cellular cellular 1 0.19 0.13 • 0.48 . 0.81 0.05 1.2 0.68 0.23 0.16 0.59 2 0.36 0.22 0.75 1.33 0.08 1.4 • . 0.61 0.27 0.17. 0.56 3 4.39 3.06- 9.86 17.31 1.40 18.7 0.70 0.25 0.18 0.57 A 0.34 " 0.17 3.01 3.53- . . 0.17 3.7 0.50 0.10 0.05 . 0.85 6 1.78 1.48 • _ 12.66 15.92 1.69 . . 17.6 0.83- 0.11 . 0.09 0.80 ^ o t a l label/number of cells per microscopic section. Group 5 was omitted because of inadequate data. See Table 8 for details. cNunber of grains in apical side per cell/number of grains i n basal side per c e l l . O 103 Figs. 20-23 Autoradiography of C-4II monolayers with 25 uCi/ml 3H-glucosamine l a b e l l i n g ( c e l l s were labelled before they become confluent). x500. Figs. 20, 21 Cells were labelled for 3 hr after subculture, then chased for 8 days with unlabelled medium. Very few isotopic grains can be observed. Fig. 21 shows the structure of a hemicyst. Plas-t i c coverslip i s shown on the bottom. (Group 1.) Fig. 22 Cells labelled for 48 hr ( u n t i l c e l l s settled down), then chased for 6 days. Dark f i e l d photography. More isotopic grains than i n Figs. 20 & 21. (Group 2.) Fig. 23 Cells labelled for 8 days after subculture. The number of iostopic grains i s greatly increased. Section i s taken from a multilayered area; dead c e l l s and debris are enclosed inside the c e l l mass. (Group 3.) 104 105 F i g s . 24-26 Autoradiography of C -4 I I monolayers w i t h uCi/ml 3 H-g lucosamine l a b e l l i n g ( c e l l s were l a b e l l e d a f t e r they became c o n f l u e n t ) . *500. F i g s . 24, 25 C e l l s grew f o r 6 days , l a b e l l e d f o r 48 h r , then chased f o r 4 days. (Group 4 . ) F i g . 26 C e l l s grew f o r 6 days , were then l a b e l l e d f o r 6 days . More i s o t o p i c g r a i n s are concent ra ted on the b a s a l s i d e . (Group 6 . ) 106 107 a p i c a l s i d e / c e l l and b a s a l s i d e / c e l l to t o t a l e x t r a c e l l u l a r / c e l l , i . e . , 0 . 1 6 - 0 . 1 8 and 0 . 2 3 - 0 . 2 7 (Table X I X ) . C e l l c u l t u r e s s y n t h e s i z e d more e x t r a -c e l l u l a r m a t e r i a l s as they became crowded. In Groups 4 , 6 ( c u l t u r e s which had been l a b e l l e d a f t e r c o n f l u e n c e ) , the r a t i o s of the number of g r a i n s i n a p i c a l s i d e / c e l l and b a s a l s i d e / c e l l to t o t a l e x t r a c e l l u l a r / c e l l , 0 . 0 5 - 0 . 0 9 and 0 . 1 0 - 0 . 1 1 , were s m a l l e r than i n Groups 1 - 3 . However, the r a t i o s of g r a i n s of i n t e r c e l l u l a r / e x t r a c e l l u l a r were h igher i n Groups 4 , 6 ( 0 . 8 0 -0 . 85) than Groups 1 -3 ( 0 . 5 6 - 0 . 5 9 ) , i n d i c a t i n g tha t more 3 H-g lucosamine was i n c o r p o r a t e d i n t o the i n t e r c e l l u l a r compartment when the c u l t u r e became c o n f l u e n t . In Groups 3 and 6 , f o l l o w i n g cont inuous p u l s i n g w i thout c h a s i n g , there were more or l e s s cont inuous l a y e r s of i o s t o p i c g r a i n s a long the b a s a l s i d e s of the c e l l s . 2 . 3 H - P r o l i n e L a b e l l i n g One experiment was done to a s c e r t a i n whether 3 H - p r o l i n e l a b e l l i n g cou ld demonstrate the b a s a l l a m i n a . C e l l s , 48 hours a f t e r s u b c u l t u r e , were l a b e l l e d w i t h 10 Uci 3 H - p r o l i n e / m l 10% f e t a l c a l f serum-Waymouth's medium f o r 4 days. The c e l l s were fed at 48 hour i n t e r v a l s . The r e s u l t showed tha t 3 H - p r o l i n e l a b e l l i n g was d i s t r i b u t e d randomly throughout the c e l l s , 1 . e . , e x t r a c e l l u l a r l y as w e l l as i n t r a c e l l u l a r l y . No b a s a l lamina fo rma-t i o n was found i n t h i s experiment ( F i g . 2 7 ) . The p a t t e r n of the d i s t r i b u -t i o n of 3 H - p r o l i n e l a b e l l i n g needs f u r t h e r i n v e s t i g a t i o n to conf i rm the r e s u l t s . 108 F i g . 27 Autoradiography of 10 y.Ci/ml 3 H - p r o l i n e l a b e l l i n g . No b a s a l lamina can be observed , the i s o t o p i c g r a i n s are randomly d i s -t r i b u t e d throughout the c e l l s . x500. F i g . 28 C o n t r o l s e c t i o n of mouse k i d n e y . The b a s a l lamina has been s t a i n e d to be a dense l a y e r i n b r o w n - y e l l o w i s h c o l o r by PA -s i l v e r methenamine s t a i n i n g ( b a s a l lamina i s i n d i c a t e d by a r r o w s ) . x450. F i g . 29 Monolayer of C - 4 I I . No b a s a l lamina can be demonstrated by P A - s i l v e r methenamine s t a i n i n g . M i c r o v i l l i are l o c a t e d on the a p i c a l s i d e , i . e . , f a c i n g the medium. ( M i c r o v i l l i are i n d i c a t e d by a r rows . ) x750. 109 110 I I I . Histochemical and Electron Microscopic Examinations Although the basal lamina i n the control sections of mouse kidney stained as a dense layer i n brown-yellowish color (Fig. 28), there was no basal lamina i n C-4II c e l l s (Fig. 29). Nuclei, n u c l e o l i , pigment and glycoprotein materials could be stained c l e a r l y i n the C-4II c e l l s . The periodic acid-Schiff method was also not able to demonstrate basal lamina i n C-4II c e l l s . However, from time to time, there was a discontinuous, amorphous material along the basal side of the c e l l s which could corres-pond to the autoradiographic observations. The glycoprotein materials on the apical side i n the region of m i c r o v i l l i could also be demonstrated. However, with this method i t was not possible to demonstrate any di f f e r e n t -i a l gradient of the c e l l s . These results confirm previous u l t r a s t r u c t u r a l studies (Auersperg, 1969) which showed that c e l l s when grown i n vivo, i n hamster cheek pouches, formed basal lamina but did not form i t i n v i t r o . IV. Culture on Collagen Gel" The foregoing results suggested that, i n v i t r o , C-4II c e l l s could not form basal lamina when they were grown on p l a s t i c substratum. I t seemed possible that C-4II c e l l s act l i k e embryonic corneal e p i t h e l i a l c e l l s and need induction and stimulation by the presence of a collagenous substratum (Hay and Meier, 1976) . Michalopoulos e_t a l . (1975) reported that primary c e l l cultures of adult rat l i v e r epithelium had morphological and functional features resembling l i v e r i n vivo when grown on f l o a t i n g collagen membranes. A si m i l a r phenomenon had been observed i n mouse mammary epithelium ( i . e . , I l l the normal mouse mammary e p i t h e l i u m cou ld form b a s a l lamina j in v i t r o on ly when grown on f l o a t i n g c o l l a g e n g e l ) (Emerman and P i t e l k a , 1977) . F o l l o w -i n g these methods, C -4 I I c e l l s were grown on f l o a t i n g c o l l a g e n g e l to observe whether the c o l l a g e n m a t e r i a l cou ld induce the fo rmat ion of s t r u c t u r e d b a s a l l a m i n a . C - 4 I I c e l l s s t a r t e d to s e t t l e down on the c o l l a g e n g e l 2 hours a f t e r s e e d i n g . Twenty hours l a t e r the g e l on the edge of the P e t r i d i shes was f l o a t i n g i n the medium. The g e l w i t h the c e l l s was m e c h a n i c a l l y f reed from the p l a s t i c substratum by Pas teur p i p e t t e to f l o a t i n the- medium. When examined 20 hours l a t e r , those g e l s b e a r i n g c e l l s began to s h r i n k or c o n t r a c t i n w i d t h . F l o a t i n g g e l s d i d not c o n t r a c t un less there were c e l l s on them. C o n t r a c t i o n cont inued f o r approx imate ly 7 days . As g e l s c o n t r a c t -ed they l o s t t ransparency and i t became d i f f i c u l t to observe the l i v i n g c e l l s w i t h the i n v e r t e d mic roscope . C e l l s d i d not form hemicysts when grown on c o l l a g e n g e l . The c e l l s became rounder , formed m u l t i p l e l a y e r s , and were c u b o i d a l to columnar i n shape ( F i g s . 3 0 - 3 3 ) . In some reg ions the c e l l sheets formed f o l d s e n c l o s i n g i s o l a t e d c e l l s and d e b r i s and some c e l l s i n f i l t r a t e d the c o l l a g e n m a t r i x . PAS and P A - s i l v e r methenamine s t a i n i n g d i d not demonstrate b a s a l l a m i n a . A c i d mucopolysacchar ides ( t o l u i d i n e b l u e s t a i n i n g ) as w e l l as g l y c o p r o t e i n m a t e r i a l s ( P A - s i l v e r methenamine s t a i n i n g ) were d i s t r i b u t e d throughout the c e l l s i n c l u d i n g a p i c a l and b a s a l s i d e s . No s p e c i a l p a t t e r n showed a g r a d i e n t d i f f e r e n c e i n the c e l l s . U l t r a s t r u c t u r a l l y , the a p i c a l border has numerous long m i c r o v i l l i and t y p i c a l j u n c t i o n a l complexes between c e l l s ( F i g . 3 4 ) . M i c r o f i l a m e n t s were l o c a t e d on both a p i c a l and b a s a l s i d e s ( F i g . 3 5 ) . No b a s a l lamina cou ld be demonstrated e l e c t r o n m i c r o s c o p i c a l l y . 112 F i g s . 3 0 , 31 V e r t i c a l s e c t i o n s through fragments of C - 4 I I c e l l s grown on c o l l a g e n g e l . The c e l l s form m u l t i p l e l a y e r s and are c u b o i d a l to columnar i n shape. In some reg ions the c e l l sheets form f o l d s e n c l o s i n g i s o l a t e d . c e l l s and d e b r i s , and some c e l l s i n f i l t r a t e d the c o l l a g e n m a t r i x . No b a s a l lamina can be found. P A - s i l v e r methenamine s t a i n i n g . ( F i g . 30, x430; F i g . 3 1 , x l 6 0 . ) 113 31 114 F i g s . 32 , 33 Enlargement of F i g . 3 1 . No b a s a l lamina can be found. In some reg ions the c e l l sheets form f o l d s e n c l o s i n g i s o l a t e d c e l l s and d e b r i s . C e l l u l a r d e b r i s and fragments of c o l l a g e n m a t r i x are found w i t h i n sheets of C -4 I I c e l l s . x i 2 0 0 . 115 116 Figs. 34, 35 Electron micrograph of sections through fragments of C-4II c e l l s grown on collagen g e l . F i g . 34 Colony of c e l l s and loosely overlapping edges of adjacent c e l l s . x51,000. F i g . 35 Basal c o r t i c a l cytoplasm. Region near the plasma membrane shows c o r t i c a l filaments (arrowhead) which are the main machinery for the contraction of C-4II c e l l s . x47,200. 117 118 I t was concluded t h a t , a l though C - 4 I I c e l l s form b a s a l lamina i n v i v o , they do not form any i n v i t r o under the c o n d i t i o n s t e s t e d . No s t r u c t u r e d b a s a l lamina cou ld be demonstrated a u t o r a d i o g r a p h i c a l l y , h i s t o c h e m i c a l l y or e l e c t r o n m i c r o s c o p i c a l l y . A u t o r a d i o g r a p h i c s t u d i e s showed that there was an asymmetric d i s t r i b u t i o n of e x t r a c e l l u l a r g lycosaminoglycans between a p i c a l . a n d b a s a l s i d e s , and as c u l t u r e s aged, there was a r e d i s t r i b u t i o n of l a b e l of e x t r a c e l l u l a r m a t e r i a l ( 3 H - g l y c o s a m i n e ) , w i t h a h igher p r o p o r t i o n of l a b e l going to i n t e r c e l l u l a r spaces i n o l d e r c u l t u r e s . 119 DISCUSSION The molecular mechanisms which u n d e r l i e the development of the charac-t e r i s t i c morphology of t i s s u e s and organs are poorly understood. Substan-t i a l evidence supports the view, however, that e x t r a c e l l u l a r m a t e r i a l s a s s o c i a t e d w i t h the t i s s u e surface are i n v o l v e d i n e p i t h e l i a l morphogene-s i s . With regard to the b a s a l lamina, f o r example, an i n t a c t lamina i s r e q u i r e d f o r p r o l i f e r a t i o n and o r i e n t a t i o n of c e l l s i n the epidermis (Wessells, 1967) and developing tooth ( S a l v k i n , 1974), and removal of the lamina causes a l o s s of l o b u l a r morphology of the submandibular gland ( B e r n f i e l d et a l . , 1973). Embryonic b a s a l lamina may c o n t a i n glvcosamino-glycan (GAG) ( B e r n f i e l d et a l . , 1973; Hay and Meier, 1974), c o l l a g e n (Hay, 1973; Minor et a l . , 1976) and by analogy w i t h a d u l t organs, l i k e l y contains other components, e.g., g l y c o p r o t e i n ( K e f a l i d e s , 1973). Studies on the t i s s u e of o r i g i n of the e p i t h e l i a l b a s a l lamina have revealed that the e p i t h e l i a l c e l l s themselves are the predominant source (Minor e_t a l . , 1976; Hay and Meier, 1976; Banerjee et a l . , 1977). F u n c t i o n a l l y , t h i s e x t r a c e l l u l a r m a t e r i a l i s thought to have a r o l e i n the support of over-l y i n g c e l l s , i n the r e g u l a t i o n of the i n t e r a c t i o n s of a d j o i n i n g t i s s u e s , and as a semipermeable f i l t e r f o r s o l u t e s i n t i s s u e f l u i d s , although the evidence f o r that i s q u i t e c i r c u m s t a n t i a l ( S l a v k i n , 1972). The c e r v i c a l carcinoma C-4II c e l l s form b a s a l lamina i n v i v o (Auers-perg, 1969), w h i l e i n v i t r o , no b a s a l lamina could be demonstrated auto-r a d i o g r a p h i c a l l y , h i s t o c h e m i c a l l y , or e l e c t r o n m i c r o s c o p i c a l l y , even when C-4II c e l l s were grown on c o l l a g e n g e l under co n d i t i o n s where b a s a l lamina 120 f o r m a t i o n i s induced i n c u l t u r e d mammary e p i t h e l i u m (Emerman and P i t e l k a , 1977) . I t i s c o n c e i v a b l e tha t organ s p e c i f i c d i f f e r e n c e s e x i s t i n the amount of e p i t h e l i a l l y d e r i v e d c o l l a g e n and mucopo lysacchar ides , i n i t s r a t e s of d e p o s i t i o n i n t o l a m i n a , or the r a t e and ex tent of i t s d e g r a d a t i o n , any of which would a f f e c t the r e s u l t of t h i s s tudy . For example, normal mammary e p i t h e l i u m can be main ta ined and m o r p h o l o g i c a l d i f f e r e n t i a t i o n induced when i t i s grown on c o l l a g e n g e l , i n c l u d i n g fo rmat ion of b a s a l l a m i n a , w h i l e mammary adenocarcinoma c e l l s that form lamina i n v i v o l a c k the a b i l i t y to form b a s a l lamina i n v i t r o (Emerman and P i t e l k a , p e r s o n a l communicat ion) . A u t o r a d i o g r a p h i c s t u d i e s show that there i s a s i g n i f i c a n t d i f f e r e n c e i n the d i s t r i b u t i o n of 3 H-g lucosamine l a b e l l i n g between a p i c a l and b a s a l s i d e s , i . e . , the amount of g r a i n s on the b a s a l s i d e was h igher than on the a p i c a l s i d e . Though the exper imenta l groups w i l l need to be expanded f o r d e f i n i t i v e c o n c l u s i o n s , the r e s u l t s s t r o n g l y suggest . t h a t there i s an asymmetric d i s t r i b u t i o n of e x t r a c e l l u l a r g lycosaminoglycans between a p i c a l and b a s a l s i d e s which might be r e s p o n s i b l e f o r the d e t e r m i n a t i o n of p o l a r i t y of carc inoma c e l l s i n the absence of connect i ve t i s s u e i n v i t r o . These m a t e r i a l s cou ld subsequent ly be r e l a t e d to p o l a r i t y i n cy top lasmic m i c r o -f i l a m e n t d i s t r i b u t i o n and c o n t r a c t i l i t y of the C -4 I I c e l l s . The asymmetric d i s t r i b u t i o n of e x t r a c e l l u l a r m a t e r i a l s between the two s i d e s of the c e l l s may be the r e s u l t of d i f f e r e n t i a l r a t e s of s e c r e t i o n , or of t r a p p i n g of m a t e r i a l on the b a s a l s i d e which i s not exposed to the c u l t u r e medium, i . e . , the re cou ld be l o s s of m a t e r i a l s i n t o the medium on the a p i c a l s i d e . 121 As c u l t u r e s aged there was a r e d i s t r i b u t i o n of l a b e l of e x t r a c e l l u l a r m a t e r i a l s ( 3 H - g l u c o s a m i n e ) , w i t h a h i g h e r p r o p o r t i o n of l a b e l go ing to i n t e r c e l l u l a r spaces r e l a t i v e to a p i c a l and b a s a l s i d e s i n o l d e r c u l t u r e s . I t has been shown that u n d i f f e r e n t i a t e d c e r v i c a l carcinoma c e l l s i n v i v o s e c r e t e e x t r a c e l l u l a r m a t e r i a l s and i t was suggested that (1) these mater -i a l s might be s t i m u l a t o r y or even r e q u i r e d f o r the a b i l i t y J of the c e l l s to i n v a d e , (2) such s e c r e t i o n by cancer c e l l s i n t o i n t e r c e l l u l a r spaces might f a c i l i t a t e t h e i r d i s p e r s i o n under n u t r i t i o n a l l y unfavorab le c i rcumstances (Auersperg e_t a l . , 1973) . The r e s u l t s of t h i s study support the suggest ion that crowding i s a s s o c i a t e d w i t h i n c r e a s e d s e c r e t i o n of g lycosaminoglycans i n t o i n t e r c e l l u l a r spaces , thus c o n t r i b u t i n g to c e l l s e p a r a t i o n and d i s p e r s i o n . 122 SUMMARY 1 . 1 x 2 mm c e l l sheets c u r l e d ac ross the shor t a x i s w i t h i n minutes i n C a ^ - c o n t a i n i n g medium at 37°C upon removal from the subst ratum. The degree of c o n t r a c t i o n i n c r e a s e d g r a d u a l l y f o r 30 minutes a f t e r which t ime the degree of c o n t r a c t i o n remained more or l e s s c o n s t a n t . 2. The c u r l i n g of the c e l l sheets was i n h i b i t e d by Ca - f r e e medium; t h i s i n h i b i t i o n was c o r r e l a t e d w i t h a decrease i n the c a l c i u m content I j of c e l l s as measured by an i n c r e a s e i n c a l c i u m content i n Ca - f r e e t e s t s o l u t i o n s . I | 3 . The method of measuring the c a l c i u m c o n c e n t r a t i o n i n Ca - c o n t a i n i n g t e s t medium cou ld not de tec t any s i g n i f i c a n t d i f f e r e n c e s of c a l c i u m content i n the presence of c e l l s h e e t s , be fo re and a f t e r c o n t r a c t i o n . 4 . The "Lanthanum method" was s u c c e s s f u l i n measuring i n t r a c e l l u l a r c a l c i u m . I s o t o n i c L a - T r i s s o l u t i o n at 4°C (pH 7.4) c o n t a i n i n g 10 mM L a C l ^ e f f e c t i v e l y removed e x t r a c e l l u l a r c a l c i u m i n 30 minutes w h i l e p r e v e n t i n g l o s s of i n t r a c e l l u l a r c a l c i u m of c o n t r o l c u l t u r e s and c o n t r a c t e d , broken up c e l l s h e e t s . However, i t seemed that longer [ j | | | p e r i o d s are needed f o r La ions to d i s p l a c e e x t r a c e l l u l a r Ca ions of i n t a c t c o n t r a c t e d c e l l s h e e t s . Th is was p o s s i b l e due to mechanica l s h i e l d i n g . 5 . Components of (1) the rubber pol iceman used to d i s l o d g e c e l l s , and (2) Waymouth's medium produced s p o r a d i c a r t i f i c i a l c a l c i u m readings and may be r e s p o n s i b l e f o r some of the v a r i a b i l i t y of the data on c a l c i u m c o n c e n t r a t i o n s . 123 D i f f e r e n t p h y s i o l o g i c a l c o n d i t i o n s , d i s s o c i a t i o n methods as w e l l as the c a l c i u m c o n c e n t r a t i o n of the medium a f f e c t e d the c a l c i u m content of C -4 I I c e l l s . The i n t r a c e l l u l a r c a l c i u m content was always h igher i n c o n t r a c t e d c e l l sheets (1 .02 to 4 .33 yg/mg p r o t e i n ) than i n c o n t r o l c u l t u r e s (0 .22 to 0.68 yg/mg p r o t e i n ) , which i n d i c a t e d tha t d u r i n g c o n t r a c t i o n , c e l l sheets took up c a l c i u m from the e x t r a c e l l u l a r environment (medium). A l though i t has been shown that C -4 I I c e l l s form b a s a l lamina i n v i v o , they d i d not form them i n v i t r o under the c o n d i t i o n s t e s t e d . No s t r u c t u r e d b a s a l lamina cou ld be demonstrated a u t o r a d i o g r a p h i c a l l y , h i s t o c h e m i c a l l y or e l e c t r o n m i c r o s c o p i c a l l y . A u t o r a d i o g r a p h i c s t u d i e s showed that as c e l l c u l t u r e s aged, there was a r e d i s t r i b u t i o n of l a b e l between the a p i c a l , b a s a l and i n t e r c e l l u l a r compartments of e x t r a c e l l u l a r m a t e r i a l , w i t h a h igher p r o p o r t i o n of l a b e l go ing to i n t e r c e l l u l a r spaces i n o l d e r c u l t u r e s . Th is p a t t e r n supports the s u g g e s t i o n , based on the u l t r a s t r u c t u r e of c e r v i c a l carcinoma b iopsy specimens, that crowding i s a s s o c i a t e d w i t h i n c r e a s e d s e c r e t i o n of g lycosaminoglycans i n t o i n t e r c e l l u l a r spaces , thus c o n -t r i b u t i n g to c e l l s e p a r a t i o n and d i s p e r s i o n . There i s an asymmetric d i s t r i b u t i o n of e x t r a c e l l u l a r g lycosaminoglycans between a p i c a l and b a s a l s i d e s of the c e l l s , which may be r e s p o n s i b l e f o r the d e t e r m i n a t i o n of p o l a r i t y of carcinoma c e l l s i n the absence of connect i ve t i s s u e i n v i t r o . Th is may subsequent ly be r e l a t e d to p o l a r i t y i n c y t o p l a s m i c m i c r o f i l a m e n t d i s t r i b u t i o n and c o n t r a c t i l i t y . 124 BIBLIOGRAPHY Adelstein, R. S. (1975). 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The compos i t ion of c a l c i u m - ' a n d maqnesiurn-free Hanks balanced s a l t s o l u t  (q/l i t e r ) KCI 0 .40 KH oP0„ 0.06 2 4 ' NaCl 8 .00 N a 2 H P 0 4 . 7 H 2 0 0.09 NaHCC 3 0 .30 Glucose • " 1.00 Phenol Red 0.01 A v a i l a b l e from Grand Is land B i o l o g i c a l Company, Cat . #418. 139 APPENDIX II P r e p a r a t i o n of C e l l s f o r E l e c t r o n Microscopy and L i g h t Mic roscopy (a) F i x a t i o n 1. R inse c u l t u r e g e n t l y w i t h Waymouth medium (37°C) 2 . F i x in 2.5% gIutaraIdehyde/MiI I o n i g ' s b u f f e r , 1.5 h r , 4°C. 3 . Wash wi th Mi I lontg 's b u f f e r 3 t imes f o r 5 min , 4°C. 4 . P o s t f i x in 1% OsO^/Mi I l o n i g ' s b u f f e r , 15-30 min , 4°C. 5 . Rinse in M i l l o n i g ' s b u f f e r 2 t imes f o r 5 min , 4°C. (b) Dehydrat ion and Embedding I . 50$ EtOH, 5 min , 4°C 2 . 70% EtOH, 5 m i n , 4°C 3 . 90% EtOH, 5 m i n , room temperature 4. 100% EtOH, 10 m i n , room temperature 5 . 100% EtOH, 10 min , room temperature 6 . 100% EtOH:propylene ox ide ( 1 : 1 ) , 10 min , room temperature 7. Propylene o x i d e , 10 m i n , room temperature 8 . Propylene o x i d e , 10 m i n , room temperature 9 . Propylene o x i d e : Epon ( 1 : 1 ) , I h r , room temperature 10. Propylene ox iderEpon ( 1 : 3 ) , I h r , room temperature I I . Epon, I h r . 12. Fresh Epon, 24 hr a t 37°C oven, then o v e r n i g h t at 56°C oven Epon s o l u t i o n Stock s o l u t i o n A = Epon 812 Resin ' 62 ml Dodecenyl S u c c i n y l Anhydr ide (DDSA) 100 ml Stock s o l u t i o n B = Epon 812 Resin 100 ml ' / Nadic Methyl Anhydride (NMA) . 89 ml Working s o l u t i o n = Mix s o l u t i o n A and B ( 1 : 1 . 5 ) , then add 1.5% by volume, the a c c e l e r a t o r DimethyI aminomethyI phenol ( D M P - 3 0 - t r i s ) 140 Append i x II I Atomic absorp t ion s tandards and s o l u t i o n s f o r measuring c a l c i u m in t e s t s o l u t i o n s 3 Fi nal ca1c i um c o n c e n t r a t i o n of s tandard un/ml Stock s o l u t i o n m|b Lanthanum swamp m l c CaCI 2 concent ra t i on ug/ml d 5.400 1 .2 0 . 8 15.000 4.500 1 .0 1 .0 12.500 3.600 0 . 8 1.2 10.000 2 .700 0 . 6 1 .4 7.500 1 .800 0 . 4 1 .6 5.000 0.900 0 . 2 1.8 2 .500 0.450 0 . 1 1.9 1 .250 0.225 0 . 0 5 1 .95 0.625 Sample s o l u t i o n p r e p a r a t i on : ' (1) BSS: 0 . 2 (2) MF-BSS: 0 ml .2 samp 1e + \ A m 1 sample + 1 ml BSS b lank 1.8 ml MF-BSS blank (1/10 d i l u t i o n ) (3) CF -BSS: h m sample + 1 ml CF-BSS b lank (1/2 di l u t i o n ) (4) CMF-BSS: I ml sample + I ml CMF-BSS b lank ^Stock s o l u t i o n was prepared as 25 yg CaCI 2/ml b lank s o l u t i o n . °Lanthanum swamp : 0 .5$ LaCI^.H^O Blank s o l u t i o n s were prepared as f o l l o w s : (1) B S S - b l a n k : 100 ml BSS + 900 ml 0.5% LaCI 3 /H 2 0 (2) CF-BSS b l a n k : 100 ml CF-BSS + 900 ml 0.5% L a C I 3 / H 2 0 (3) MF-BSS b l a n k : 100 ml MF-BSS + 900 ml 0.5% LaC I 3 /H 2 0 (4) CMF-BSS b l a n k : 100 ml CMF-BSS + 900 ml 0.5% L a C I 3 / H 2 0 d ( C a ) = (CaCI 2 ) x 36% 141 Appendix IV Procedure f o r measurement of ceI IuIar caIc ium 1. RemovaI of ex t raceI IuI a r ca Ic i um a) Contro l c u I t u r e s d u p l i c a t e c u l t u r e s grown to confIuency in 10% f e t a i c a l f serum-Waymouth's medium in 60 x 15 mm t i s s u e c u l t u r e d ishes + d i s c a r d the medium, r i n s e once wi th i c e - c o l d L a - T r i s s o l u t i o n scrape o f f the monolayer w i th p l a s t i c wedge and suspend c e l l s in a 50 ml Pyrex c e n t r i f u g e tube c o n t a i n i n g 50 ml L a - T r i s s o l u t i o n in an i ce bath* keep the tubes shaking in a F i s h e r Rotator in the c o l d room f o r 30 min + c e n t r i f u g e a t 1500 rpm, 5 min a t 4°C ( I n t e r n a t i o n a l P o r t a b l e C e n t r i f u g e Model PR-2) wash once wi th 20 ml i ce c o l d d e i o n i z e d water , use Pasteur p i pe t te 4-r e c e n t r i f u g e a t 1500 rpm, 5 min + d i s c a r d supernatant + the p e l l e t i s l e f t f o r measuring i n t r a c e l l u l a r c a l c i u m and p r o t e i n de te rminat ion *Ce l l sheets were broken up in to small c e l l groups wi th a Pasteur p i p e t t e o r , a l t e r n a t i v e l y , l e f t i n t a c t . 142 Contracted c e l l sheets d u p l i c a t e c u l t u r e s grown to conf luency in \0% c a l f serum-Waymouth ' s medium in 60 x 15 mm t i s s u e c u l t u r e d ishes wi th 2 mm g r i d s + d i s c a r d the medium, r i n s e 2 t imes wi th Waymouth's medium (37°C) d i v i d e monolayers in to 1 x 2 mm p ieces ( in the l a s t Waymouth's med i um) + r i n s e 2 t imes wi th BSS add 5 ml BSS, scrape o f f 1 x 2 mm c e l l sheets w i th a . 2 mm p l a s t i c wedge, and incubate a t 37 C f o r 30 min + suspend the c e l l sheets in 50 ml i c e - c o l d L a - T r i s s o l u t i o n in an i ce bath keep the tubes in F i s h e r Rotator and go through the same process as desc r ibed f o r c o n t r o l c u l t u r e s / 143 Measurement of i n t r a c e l l u l a r c a l c i u m c e l l p e l l e t s from P a r t 1 i n 50 ml c e n t r i f u g e tube + add 0 . 5 ml of a mix ture of g l a c i a l a c e t i c a c i d and 3 M TCA (1:1) heat the tubes in a b o i l i n g water bath u n t i l the mixture j u s t beg to boi I (20 min) shake g e n t t y ' t o d i s s o l v e the c e l l s add 2 ml d e i o n i z e d water heat the mixture f o r another 20 min in a b o i l i n g water bath + cool the tube a t room temperature f o r 20-30 min w h i l e the p r o t e i n coagu la tes f i r m l y b r i e f l y a g i t a t e the contents on a Vortex mixe r , 2 - 3 t imes + c e n t r i f u g e a t 2500 rpm f o r 40 min , s t o r e supernatant wash the p e l l e t once wi th 0 . 5 ml de ion i zed water , use Pasteur p i p e t t e and r e c e n t r i f u g e a t 2500 rpm f o r 40 min . Poolwashing wi th supernatant + "supernatants f o r c a l c i u m measurement + p r e c i p i t a t e f o r p r o t e i n dete rminat ion 144 Appendix V Standard and b1ank s o l u t i o n s f o r measuring t o t a l and i n t r a c e l l u l a r C a a Ca1ci um concent ra t i on yg/ml Worki ng ml s o l u t i o n b s o l u t i o n Blank s o l u t i o n 0 • ml 1 .500 0 . 5 A 1 .0 1 . 125 0 . 5 B 0 . 5 0 .750 0 . 5 B 1.0 0 .450 0 . 5 C 0 . 5 0 .300 0 . 5 C 1 .0 0 . 150 0 . 5 D 1 . 0 0.075. 0 .25 D 1 .25 0.030 0 . 1 D 1.4 a Sample p r e p a r a t i o n : 3 .5 ml sample der i ved from ce 1 1 u'lar ca 1 c i um, Working s o l u t i o n s were prepared as f o l l o w s : Stock s o l u t i o n : 25 yg CaCI 2 /ml in b lank s o l u t i o n Stock s o l u t i o n b lank s o l u t i o n Ca c o n c e n t r a t i o n ^ S o l u t i o n ml ml ug/ml A 1.0 1.0 4.50 B 0 . 5 1.5 . 2 .25 C 0 . 2 1.8 0 .90 D 0.1 1.9 0 .45 c B l a n k s o l u t i o n : 50 ml TCA & g l a c i a l a c e t i c a c i d + 50 ml 0 . 2 M LaC 1 3 / H 2 0 + 250 ml d e i o n i z e d H 2 0 S o l u t i o n s I. L a C I 3 - T r i s s o l u t i o n ( in i c e bath) - I Jl p r e p a r a t i o n : 10 mM LaCI 3 (3.5336 g of L a C I 3 - 6 H 2 0 ) in I I o f 160 mM T r i s (19.382 g/l 1), s t i r red we I I , g a s s i n g wi th 100% 0 2 t o prevent La p r e c i p i t a t i o n , ad justed pH t o 7 .4 by adding IN H C l . 145 2 . 1:1 mixture of g l a c i a l a c e t i c a c i d - 3 M TCA : 49.017 g TCA/100 ml r \ £ (3 M) + 100 ml g l a c i a l a c e t i c a c i d 3 . 0.1 m moles of L a C I 3 : 6 H 2 0 : 0 . 5 ml of 0 . 2 M (70.67 g/ l£ H 2 0) of LaCI •6 H 2 0 s o l u t i o n d ( C a ) = (CaCI 2) x 36$ 146 Appendix VI P r o t e i n measurement wi th the F o l i n phenol reagent (Oyama and E a g l e , I966; Lowry et al. , 195 I ). Reagents - d o w r y ' s Reagent) Reagent A. N a 2 C 0 3 - 20 g ) NaOH 4 g ) d i s s o l v e d in I l i t e r H 2 0 NaK t a r t r a t e 0 . 2 g ) B. CuSC\ 5H 2 0 - 0 . 5 g in 100 ml H 2 0 C. Prepared f resh d a i l y , by mix ing 50 p a r t s A and I p a r t B. D. the same as Reagent C except f o r omiss ion of NaOH IN Phenol Reagent (s tock was 2N, a v a i l a b l e form F i s h e r S c i e n t i f i c C o . , 2 - f o l d d i I u t i o n wi th H 2 0 t o make i t IN) Method f o r the s tandard curve 1. Standard s o l u t i o n s of p r o t e i n (BSA f r a c t i o n V) were made up between 25 and 250 yg/ml of H 2 0 . 2 . One ml of the p r o t e i n s o l u t i o n was d i s s o l v e d in 5 ml of Reagent C. 3 . The mixture was shaken we l l and a l lowed t o s tand f o r 10 min. 4 . 0 . 5 ml of IN Phenol Reagent was added r a p i d l y . The rap id admixture of the c o l o r reagent w i th the s o l u t i o n was e s s e n t i a l . 5 . The c o l o r was read a f t e r 30 minutes a t 660 mu. (Co lo r was s t a b l e f o r 2 hr ) As shown in F i g . 36 , the r e l a t i o n s h i p between o p t i c a l d e n s i t y and the content of p r o t e i n was l i n e a r over 25-250 yg BSA/ml H 2 0 . Method f o r p r o t e i n de te rminat ion of samples 1. C e l l s were d i s s o l v e d in 5 ml 1 N NaOH a t room temperature . 2 . 45 ml of Reagent D was added, mixed w e l l , and a l lowed t o stand f o r 10 m i n . 3 . One mi of t h i s s o l u t i o n was d i s s o l v e d in 4 ml Reagent C and mixed w e l l . 4 . I ml H 2 0 was added, mixed w e l l . Fig.36 Standard curve for protein determination (each point Is the mean value of duplicate readings! 148 5 . 0 . 5 ml of 1 N Phenol Reagent was added r a p i d l y . 6 . A f t e r 30 min , the o p t i c a l d e n s i t y of the mixture was read a t 660 mu and compared wi th the standard curve t o determine the p r o t e i n c o n t e n t . Whenever the p r o t e i n content of the samples was determined, standards between 25 and 250 yg/ml were run c o n c u r r e n t l y . B l a n k s : 1 ml H ? 0 + 5 ml Reagent C + 0 . 5 ml Phenol Reagent. 149 ( Appendix VII L inear r e g r e s s i o n and c o r r e l a t i o n (Data from F i g . 14) 1. Regress ion l i n e and c o r r e l a t i o n c o e f f i c i e n t ( r ) ( F i g . 37) X : i n t r a c e l l u l a r c a l c i u m (ug) ; Y : c e l l u l a r p r o t e i n (mg). T r y p s i n d i s s o c i a t e d c u l t u r e s : Y = 0 . 7 3 9 X + 4.062 r = 0.572 (1) Tryps i n/EGTA d i s s o c i a t e d c u l t u r e s : Y = 0;325X + 2 .386 (broken up c e l l sheets ) r = 0 486 (2) Tryps i n/EGTA d i s s o c i a t e d c u l t u r e s : Y = 0.218X + 3.308 ( i n t a c t c e l l sheets ) r = 0 216 '"^ 2 . S i g n i f i c a n c e of the c o r r e l a t i o n c o e f f i c i e n t r (1) t - t e s t t , = / . 2 n-2 / 1 - r n -2 T r y p s i n d i s s o c i a t e d c u l t u r e s : = C 5 7 2 _ = > = 2 . 1 1 ; s i g n i f i c a n t (4) •1-0.572* + 9 b d f = 1 7 ' a = 0 - 0 5 , 19-2 T ryps i n/EGTA d i s s o c i a t e d c u l t u r e s (broken UD c e l l sheets ) : t = ° - 4 8 6 = 2 .29 > t . , 1 7 n n = = 2.11 ; s i g n i f i c a n t (5) 19-2 Tryps i n/EGTA d i s s o c i a t e d c u l t u r e s ( i n t a c t eel I- sheets ) : + = % = = = : = K + t a b df=31;a=0.05 = 2 ' ° 3 ; " o t s i g n i f i c a n t - ^ ) / 1 - 0 . 2 ; 1 6 Z 3 3 - 2 (2) X - t e s t Z.= - k l o g (1 + r> - ' l o g <1-r>} i 2 e e o , , . „ 2 ' {E(n . - 3 ) ' Z . } 2 Xz = E ( n . - 3 ) Z. - i ^ ^ ' 7 ' '. Z ( n . - 3 ) X 2 =2.27 < X + K A* o - n n«T 5 - 9 9 ' n o + s i g n i f i c a n t A tab df=2;a=0.05 t h e r e f o r e , r]f r^, r^, are from the same popuI a t i o n . - ( 7 ) Fig.37 Linear regression and correlation o •Js>-X 0 1 2 3 4 5 INTRACELLULAR CALCIUM ,(ug) 151 (3) Z-test / i + r nj-3 n2-3 ; Zx * ' j {loge(1+r) - (1-r)} r i and r 2 . Z =.0.65 - 0.5308 0.3371 < 1.96 ; not significant 19-3 19-3 rj and r 3 Z = 0.65-0.2176 19-3 33-3 = 1.3396 < 1.96 ; not significant r 2 and r 3 Z = 0.5038-0.2176 = 1 ' 0 ! 2 < 1 , 9 6 ; n o + significant — i — 19-3 33-3 therefore, there are non-significant di fference_s between each two groups 3. Combine n , r? , r$ into an estimate of r --(8) r. x n. x Z. ^ n .-3 X (n.-3 )-Z. x ^ 2 (n .-3)-Z.. ^ x corrected z: x (n.-3)-Z'. x i 0.572 19 0.650 16 10.406 6.768 0.562 8.986 0.486 19 0.466 16 7.456 3.474 0.425 6.794 0.216 33 0.220 30 6.584 1.445 0.213 6.378 71 62 24.447 11.688 1.199 22.157 Average Z = £ ( V 3 ) ' Z t : = 0.394 * P + a b z = 0 394 = °- 3 7 5 Z(n.-3) 1 Z'. = Z.-x x 2(n.-1) x ; Average Z' I(n.-3)*Z'. r x E(n.-3) = 0.357 rtab Z=0.357 = ° - 3 4 3 (10) t-test t= • = 3.032 > t L ^ „ ^ ftC = 1.99 . significant -(11) JyZp.— + a b df=69;a=0.05 , a n-2 152 Appendix VIII General Procedure f o r L i q u i d Emulsion Autorad iography of the Unsta ined Epon S e c t i o n s - C4II C e l l L ines C4I I c u l t u r e s on p l a s t i c c o v e r s l i p s incubated in 12.5 ^ 50 uCi/ml [ 3 H]g lucosamine o r 10 uCi/ml [ 3 H ] p r o l i n e in 10$ FCS-Waymouth medium, t o t a l volume, 2 m l . 4- a t 37°C, 5 $ C 0 2 i n c u b a t o r f o r 2 hr 8 days , remove f r e e 3 H - g I u c o s a m i n e , wash 3 t imes w i t h I X Waymouth's medium 4- 2 ml/each t ime (37°C) . f i x i n 2 . 5 $ gl u t a r a l dehyde/Mi M o n - i g ' s b u f f e r 1.5 h r , 4°C 4-wash w i t h M i l l o n i g ' s b u f f e r 3 t i m e s , 5 min/each t i m e , 4°C +' p o s t f i x in \% O s 0 4 / M i I l o n i g ' s b u f f e r , 15 m i n , 4°C 4-r i n s e in M i l l o n i g ' s b u f f e r 2 t i m e s , 5 min/each t i m e , 4°C 4-50$ EtOH, 5 m i n , 4°C 4-70$ EtOH, 5 m i n , 4°C 4-90$ EtOH, 5 m i n , room temperature 4-100$ EtOH, 10 min,room temperature (Group- I ) scrape some of the c e l l s o f f covers l i p and s e t t l e in I ml smal l beaker (Group-2) 4 changes in 100$ EtOH, 10 min/ 6zZh 100$ EtOH, 10 m i n , room temperature 4-100$ EtOH/propyIene ox ide ( 1 : 1 ) , 10 min , room temperature 4-propy lene o x i d e , 10 min , room temperature 4- • ' • propy lene o x i d e , 10 m i n , room temperature 4-P.O./Epon ( 1 : 1 ) , I h r , room temperature EtOH/Epon ( 1 : 1 ) , I f i r , 4- 4- room temp. P.O./Epon ( 1 : 3 ) , I h r , room temperature EtOH/Epon ( 1 : 3 ) , I h r , room temp. covers l i p wi th remai n i ng ce I I s a t tached 153 Epon, o v e r n i g h t in 37°C oven + f resh Epon, 24 h r , in 56 C oven 4-s e c t i oni ng 4 put s e c t i o n s on g e l a t i n - c h r o m e alum coated s l i d e s , d r i e d and coated wi th NTB-3 e m u l s i o n , s tay a i r dry f o r 30 min 4- 0 s t o r e and expose in the darkroom at 4 C f o r 2 -4 weeks 4-deve lop , f i x , mount and observe Sample a f t e r t h i s step can be d r i e d , c o a t e d . w i t h NTB-3 e m u l s i o n , s to red and xposed, deve loped , f ixe'd and mounted f o r s t a n d a r d - o p t i c a l o b s e r v a t i o n . 154 Appendix IX General Guide f o r Autoradiography 1) P r e p a r a t i o n of s l i d e s : The s l i d e s on.which s e c t i o n s are t o be mounted should be e j t h e r a l c o h o l c leaned or p r e f e r a b l y a c i d washed and r i n s e d many t imes wi th d i s t i l l e d water , then dipped once i n - a n aqueous mixture of 0.5% g e l a t i n - 0 . 5 % chrome alum. The t h i n g e l a t i n f i l m prov ides a good adhesive su r face f o r both s e c t i o n s and e m u l s i o n . Th is t h i n f i l m a l s o reduces b a c k s c a t t e r from the g l a s s , thus reducing background s t a i n s (Gahan, 1972). The coated s l i d e s are then p laced v e r t i c a l l y on a support with the bottom p a r t r e s t i n g on t i s s u e paper t o absorb the excess g e l a t i n and chrome alum. The s l i d e s are d r i e d o v e r n i g h t in a dust f r e e c a b i n e t . The top of each s l i d e i s l a b e l l e d wi th a diamond marking . p e n c i I , and marked on the back of the s l i d e wi th a masking tape to t e l l which s i d e i s up or down in the darkroom. On the lower t w o - t h i r d s of the s l i d e s , w i t h i n an area t h a t i s at l e a s t 4 mm from each s i d e and 10 mm from the bottom of the s l i d e s , 2 -4 small r i n g s wi th 3 mm diameter are marked as the p laces to put the epon s e c t i o n s (Diagram 1) . 2) P o s i t i o n of the L i g h t M i c r o s c o p i c S e c t i o n s (Loop Method, Gahan, 1972) While f l o a t i n g in the boat of the microtome k n i f e , small r ibbons of s e c t i o n s a re manipulated w i t h a h a i r u n t i l they are a l l concent ra ted in a s u r f a c e area of less than 3 mm d iameter . D e i o n i z e d , d i s t i l l e d water i s used in c o n t a c t w i th the s e c t i o n s . The group of s e c t i o n s i s p icked up wi th a 3 mm diameter loop of f i n e copper w i re and t r a n s f e r r e d t o the g e l a t i n , coated s l i d e in the area which has been l a b e l l e d in advance. Furthermore, 155 leve l of geI at in/chrome alum l e v e l of emuls ion T i s s u e s e c t i o n s in f r o n t a . b. Diagram 1. a) Appearance of microscope s l i d e c a r r y i n g LM s e c t i o n s before emuls ion c o a t i n g , showing the he ight of the s u p p o r t i n g f i l m and the l o c a t i o n of 4 groups of LM s e c t i o n s , b) The same s l i d e a f t e r emuls ion c o a t i n g , showing the he ight of the emuls ion c o a t i n g , c) Schematic r e p r e s e n t a t i o n showing the sequence of laye rs in a LM microscope a u t o r a d i o g r a p h y . covers I ip immersion o i I Emulsion laye r t i s s u e s e c t i o n s Ge la t in/chrome alum c o a t i n g Mic roscope s I i d e c . 156 the excess of d i s t i l l e d water around the s e c t i o n s in the loop must be immediately removed. Th is i s done by p l a c i n g a t h i n wedge of f i l t e r paper from above the edge of the loop, and soaking up the water as the g e l a t i n membrane. 3) Emulsion C o a t i n g : The emulsion c o a t i n g i s c a r r i e d out in an e n t i r e l y l i g h t - p r o o f d a r k -room which can be mainta ined a t s tandard i zed atmosphere c o n d i t i o n s . One hour before m e l t i n g the e m u l s i o n , a d j u s t the temperature to 25°C and the r e l a t i v e humidi ty t o approx imate ly 75$. A s a f e l i g h t w i th f i l t e r Wratten s e r i e s No. 2 i s used f o r a l l emuls ions . A. Choice of emuls ion : NTB-3 emuls ions are a v a i l a b l e in a homogenous gel form (NTB stands f o r Nuc lear t r a c k b e t a ) . The average g r a i n s i z e in these emuls ions i s 0 . 3 ym. There are four k inds of emuls ions produced by Eastman Kodak Co. such a s : NTB, NTB-2, NTB-3 and NTB-4. The emulsions d i f f e r from one another mainly in t h e i r s e n s i t i v i t y to b e t a - r a d i a t i o n s , the i n c r e a s i n g numbers denot ing i n c r e a s i n q s e n s i t i v i t y to these r a d i a t i o n s . T r i t i u m has an E of M ' max 18 Kev, and NTB-3 emulsion i s the most s u i t a b l e one f o r l i g h t m i c r o s c o p i c l i q u i d emuls ion auto rad iography . The emulsion should be s to red a t 4°C in a c l e a n , f u m e - f r e e , dry r e f r i g e r a t o r and f a r away from any p o s s i b l e source of r a d i a t i o n . B. M e l t i n g of emuls ion : 1. In the " c o a t i n g room", prewarm the Kodak NTB-3 emulsion at 43-44°C in a water -bath f o r 45 minutes . 157 2 . D i l u t e the emuls ion wi th d i s t i l l e d water (43 -44 w C) to the volume 1 : 1 , s t i r then s low ly and g e n t l y . (Background might be enhanced by f requent warming -coo l ing and general a g i t a t i o n of the e m u l s i o n . ) 3 . To t e s t i f the emulsion i s mixed w e l l , d ip a c l e a n e d , warm s l i d e , he ld v e r t i c a l l y i n t o the emulsion f o r one second. S lowly withdraw the s l i d e , s t i l l h o l d i n g i t in a v e r t i c a l p o s i t i o n , and examine i t under the s a f e l i g h t . If t h e r e are ho bubbles and the emulsion has f lowed evenly over the s l i d e (g ive a f i n a l t h i c k n e s s of 3 -4 ym, which i s uniform [Gahan, 1972]) , then the emulsion i s ready. C. D ipp ing of s l i d e s i n t o e m u l s i o n : 1. Ho ld ing the s l i d e s , d ip them s l o w l y i n t o the c o n t a i n e r of emuls ion (43°C) . A f t e r a 1-second d i p , withdraw the s l i d e s in a v e r t i c a l p o s i t i o n . 2 . A l l ow the excess emulsion t o d r a i n by dabbing the end of the s I i des on paper toweI . 3 . Hang the s l i d e s over the support of the rack , l e t the s l i d e s dry f o r about 30 minuteswf th the s a f e l i g h t turned o f f (Diagram 2 ) . Note: (1) Dry ing a t low temperature w i l l r e s u l t in an uneven c o a t , w h i l e d r y i n g too r a p i d l y a t low humidi ty w i l l cause s t r e s s f o r c e s in the emulsion and r e s u l t a n t clumps of g r a i n s a t the s u r f a c e s and w i t h i n the t i s s u e ( Leb Iond e t a_{_., 1963). (2) Di I uted emu I s ion (emu I s ion :d i s t . water = 1:1) can do about 150-200 s l i d e s per h a l f emuls ion or 300-400 s l i d e s per whole emu Is i o n . 4) Storage and Exposure: 1. A f t e r d r y i n g , the coated s l i d e s are put i n t o a ' I i g h t - p r o o f b l a c k s l i d e box, s to red wi th c e l l s s i d e f a c i n g up. 158 Microscope s Iide Emulsion layer •Tissue s e c t i o n s Support of the rack ?Paper towel Diagram 2 . Black s i i d e box Store w i t h eel Is f a c i n g up Blank s i i d e j D r i e r i t e bag Diagram 3 . 2. To minimize the fading of the l a t e n t image which i s b e l i e v e d t o be caused by o x i d a t i o n in the presence of water vapor, the s l i d e box contains a t i s s u e paper bag with 10 g of the i n d i c a t i n g d e s i c c a n t D r i e r i t e . 3. Seal the s l i d e box with black adhesive tape and keeps standing on edge a in a low humidity r e f r i g e r a t o r (4 C), so t h a t the s l i d e s are exposed in the h o r i z o n t a l p o s i t i o n with the emulsion f a c i n g up and the D r i e r i t e bag i s on the bottom of the s l i d e box (Diagram 3 ) , 4. The s l i d e s are stored and exposed f o r 1-4 weeks. 5) Photographic Processing: Development and F i x a t i o n Procedures: A f t e r the a p p r o p r i a t e exposure time, put the s l i d e s v e r t i c a l l y t o the s t a i n -ing g l a s s t r a y and go through the f o l l o w i n g s : 1. In Kodak developer D-19 f o r 3 minutes. 2. D i s t i l l e d water f o r 1 minute. 3. Kodak f i x e r f o r 10 minutes. 159 4. Kodak hypocIear ing agent f o r 1 minute . 5 . R inse in a slow stream of running water f o r 20 minutes . Be sure to keep a l l the temperature of these s o l u t i o n s a t I9-20°C! 6) Mounting of P r e p a r a t i o n s : 1. Mount the s l i d e s wi th immersion o i l and observe them under the Normasky i n t e r f e r e n c e c o n t r a s t microscope . 2 . A more permanent mounting r e q u i r e s s e a l i n g the edges of the c o v e r s l i p w i th n a i l p o l i s h to prevent the loss of immersion o i I . 

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