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Sodium entry into single striated muscle fibers Brigden, Malcolm Leslie 1969

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SODIUM ENTRY INTO SINGLE STRIATED MUSCLE FIBERS by Malcolm L e s l i e Brigden B . S c , U n i v e r s i t y o f M c G i l l , 1 9 6 5 A T h e s i s Submitted i n P a r t i a l F u l f i l m e n t of the Requirements f o r the Degree of MASTER OF SCIENCE i n the Department of ANATOMY We accept t h i s t h e s i s as conforming t o the standard r e q u i r e d from candidates f o r the degree of Master of S c i e n c e . THE UNIVERSITY OF BRITISH COLUMBIA October, 1 9 6 9 In presenting th i s thes i s in pa r t i a l f u l f i lment of the requirements fo r an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f ree l y ava i l ab le for reference and study. I fu r ther agree tha permission for extensive copying of th i s thes i s for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th i s thes i s fo r f i nanc ia l gain sha l l not be allowed without my wr i t ten permission. Department The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT - i i A review of the literature on sodium distribution in muscle has been presented including the evidence for heterogeneous distribution and the possible morphological sites of localization of this sodium. Special attention was paid to influx studies on crustacean muscle. The review reveals that while there is good evidence for a heterogeneous distribution, the morphological sites and their associated concentrations of sodium remain obscure. The experiments described in this thesis were performed on muscle fibers of the giant barnacle Balanus nubilis. Single muscle fibers of Balanus are a unique biological preparation due to their large size and ease of dissection. The major disadvantage to single fiber Balanus preparations is the extent of the extracellular space or cleft system. This cleft system contains approximately half the total fiber sodium in 6% of the fiber volume. The purpose of these experiments was to gain a 22 + picture of Na entry into these single striated muscle fibers with special emphasis on the role of the extracellular space. A histological study revealed that the cleft system was more extensive than had been reported. From measure-ments made on light and electron microscope pictures i t was concluded that no part of Balanus myoplasm was more than 1 - 2u from a 0.02u patent cl e f t . This study gave a picture - I l l 22 +• of the morphological pathway f o r Na d i f f u s i o n in the e x t r a c e l l u l a r space. Radioautography and radio isotope counting were two 22 + techniques used to examine Na exchange between the extra-c e l l u l a r space and the myoplasm. Since the e x t r a c e l l u l a r space had a sodium concen-t r a t i o n of 10 times the myoplasm, the c l e f t system should be adequately v i s u a l i z e d by radioautography. However, the 22 t section thickness (15;*) and the high maximum energy of Na (0.540MEV) l i m i t e d the resolution of the radioautograms to 15 - 17.5u. This resolution coupled with the extent of the c l e f t system prevented consistent radioautographic v i s u a l i -zation of discrete c l e f t s . Radioautographic analysis of f i b e r s with minimal 22 * exposure (0.5 minutes) to Na revealed a concentration gradient which could be used to define the e x t r a c e l l u l a r pathway and i t s d i f f u s i o n c o e f f i c i e n t f o r Na +. The radius of the f i b e r cross section was found to be a reasonable approxi-mation of the length of the pathway and the d i f f u s i o n of 22 *• Na in the e x t r a c e l l u l a r space along the pathway was as 22 + rapid as the s e l f d i f f u s i o n of Na in d i l u t e solution. A comparison of f i b e r s that had spent 1.5 minutes in the 22Na"*" bath with similar f i b e r s that had a 0.5 minute sucrose rinse revealed that the 0.5 minute sucrose rinse removed half of the 2 2 N a f from the e x t r a c e l l u l a r space. The 1.5 minute f i b e r s and a l l f i b e r s examined a f t e r longer 22 + periods in the Na bath revealed a homogeneous gram - i v d i s t r i b u t i o n i n radioautograms. This data c o n f l i c t e d with 22 + the rate of Na entry predicted by the 0 . 5 minute f i b e r s . Inappropriate a g i t a t i o n of the 1 . 5 minute f i b e r s was respon-s i b l e f o r the lack of agreement. 22 + A further radioautographic study of Na in f l u x with experimental times 5> 2 0 , 6 0 and 1B0 minutes was analyzed qu a n t i t a t i v e l y . To substantiate t h i s radioauto-graphic study an i n f l u x experiment was done with times 5 j 1 0 , 2 0 , 4 0 and 9 0 minutes. Each study demonstrated two compartments. An i n i t i a l r a p i d l y exchanging compartment with a half time of 6 - 1 0 minutes was i d e n t i f i e d as the e x t r a c e l l u l a r space. The e x t r a c e l l u l a r space contained approximately half the f i b e r sodium. Both studies detected a rnyoplasmic i n f l u x compartment which was s t i l l exchanging when the experiments were terminated. The calculated rate constant f o r rnyoplasmic exchange ( 5 . 6 x 10~^/minute) was in good agreement with the value of A l l e n and Hinke. In conclusion, a useful technique for the radio-autography of soluble substances was developed. Both a morphological and a physiological picture of the pathway 22 + f o r Na d i f f u s i o n in the e x t r a c e l l u l a r space was developed. The size and half time of loading of the e x t r a c e l l u l a r space was v e r i f i e d . The rnyoplasmic i n f l u x component was i d e n t i f i e d by two methods. From these studies emerges a more comprehensive picture of the e x t r a c e l l u l a r space as a pathway for d i f f u s i o n - V in Balanus muscle. The f a i l u r e to consider t h i s compartment in microinjection or f l u x studies may resu l t in ambiguous conclusions. TABLE OF CONTENTS - v i INTRODUCTION CHAPTER I GENERAL INTRODUCTION 1 CHAPTER I I LITERATURE REVIEW 4 A. E v i d e n c e f o r a heterogeneous d i s t r i b u t i o n o f sodium i n muscle 4 B. L o c a t i o n o f c o m p a r t m e n t a l i z e d sodium... 6 C. The s t a t e o f sodium i n B a l a n u s 11 RESULTS AND DISCUSSION CHAPTER I I I BARNACLE MUSCLE STRUCTURE 1 6 A. I n t r o d u c t i o n 16 B. Methods 19 C. R e s u l t s and D i s c u s s i o n 1 9 D. T a b l e s , I l l u s t r a t i o n s and F i g u r e s .... 23 CHAPTER IV RADIOAUTOGRAPHIC STUDIES OF 2 2Na+ INFLUX.. 2 9 A. I n t r o d u c t i o n 2 9 B. Methods 42 C. R e s u l t s 52 D. D i s c u s s i o n 58 E. T a b l e s , I l l u s t r a t i o n s and F i g u r e s .... 67 CHAPTER V INFLUX OF 2 2 N a + 108 A. I n t r o d u c t i o n 108* B. Methods 108 C. R e s u l t s 109 D. D i s c u s s i o n 110 E. T a b l e s , I l l u s t r a t i o n s and F i g u r e s .... 113 CONCLUSION CHAPTER V I GENERAL CONCLUSIONS 118 BIBLIOGRAPHY 124 - v i i LIST OF TABLES TABLE I Data f o r the r e s o l u t i o n curve of the 5 minute f i b e r 67 TABLE I I Data f o r the r e s o l u t i o n curve of the 20 minute f i b e r 69 TABLE I I I Data f o r the r e s o l u t i o n curve of the 60 minute f i b e r 71 TABLE IV Data f o r the r e s o l u t i o n curve o f the ISO minute f i b e r ' 73 TABLE V Data f o r the g r a i n d e n s i t y p r o f i l e graph of the 0 .5 minute f i b e r s 7S TABLE VI Data f o r the g r a i n d e n s i t y p r o f i l e graph o f the 1.5 minute f i b e r s 79 TABLE V I I Data f o r the g r a i n d e n s i t y p r o f i l e graph of the 1.5 minute f i b e r s with a 0 . 5 minute sucrose r i n s e SO TABLE V I I I A n a l y s i s o f v a r i a n c e f o r the g r a i n d e n s i t y p r o f i l e of a 0 .5 minute f i b e r c r o s s s e c t i o n $2 TABLE IX A n a l y s i s of v a r i a n c e f o r the g r a i n d e n s i t y p r o f i l e o f a 1.5 minute f i b e r c r oss s e c t i o n S3 TABLE X A n a l y s i s o f v a r i a n c e f o r the g r a i n d e n s i t y p r o f i l e of a 1.5 minute f i b e r c r o s s s e c t i o n with 0 . 5 minute sucrose r i n s e .... S4 TABLE XI Gr a i n counts f o r the l o n g term r a d i o -a u t o g r a p h i c i n f l u x study. 9 0 - 9 1 TABLE X I I Data f o r the g r a i n d e n s i t y p r o f i l e graph of the 5 minute f i b e r s 93 TABLE X I I I Data f o r the g r a i n d e n s i t y p r o f i l e graph of the 20 minute f i b e r s 94 TABLE XIV Data f o r the g r a i n d e n s i t y p r o f i l e graph o f the 60 minute f i b e r s 95 TABLE XV Data f o r the g r a i n d e n s i t y p r o f i l e graph of the ISO minute f i b e r s .1 96 - v i i i TABLE XVI A n a l y s i s of v a r i a n c e f o r the g r a i n d e n s i t y p r o f i l e of a 5 minute f i b e r c r o s s s e c t i o n • 9 ^ TABLE XVII A n a l y s i s of v a r i a n c e f o r the g r a i n d e n s i t y p r o f i l e o f a 5 minute f i b e r c r o s s s e c t i o n 9 9 TABLE XVIII A n a l y s i s o f v a r i a n c e f o r the g r a i n d e n s i t y p r o f i l e of a 5 minute f i b e r c r o s s s e c t i o n 1 0 0 TABLE XIX The e f f i c i e n c y of radioautograms 1 0 1 - 1 0 2 TABLE XX A comparison of experimental to t h e o r e t i -c a l d i s t a n c e o f 2 2 ^ a + d i f f u s i o n i n the 0.5 minute f i b e r c r o s s s e c t i o n s 1 0 3 - 1 0 4 TABLE XXI The t h e o r e t i c a l percent o f the f i b e r s u r f a c e area i n v o l v e d i n 2 2 N a + i n f l u x .... 1 0 5 - 1 0 6 TABLE XXII The time r e q u i r e d f o r the 2 2Na+ concen-t r a t i o n of the f i b e r center to equal one h a l f the c o n c e n t r a t i o n o f the p e r i p h e r y .. 1 0 7 TABLE XXIII Sample i n f l u x data f o r a ba r n a c l e 1 1 3 TABLE XXIV The bath s p e c i f i c a c t i v i t y 1 1 4 TABLE XXV The data f o r the i n f l u x graph 115-116 LIST OF ILLUSTRATIONS - i x I l l u s t r a t i o n 1 I l l u s t r a t i o n 2 I l l u s t r a t i o n 3 I l l u s t r a t i o n 4 I l l u s t r a t i o n 5 I l l u s t r a t i o n 6 I l l u s t r a t i o n 7 I l l u s t r a t i o n 8 I l l u s t r a t i o n 9 I l l u s t r a t i o n 10 I l l u s t r a t i o n 11 I l l u s t r a t i o n 12 I l l u s t r a t i o n 13 Electron micrograph of f e r r i t i n in a T tubule 23 Electron micrograph of horse radish peroxidase in T tubule 23 A low power photomicrograph of a f i b e r cut i n cross section demonstrating'the c l e f t system 24 A medium power photomicrograph of a f i b e r cut i n cross section demonstra-t i n g the c l e f t system 24 A high power photomicrograph of a f i b e r cut i n cross section demonstrat-ing the c l e f t system 25 An electron micrograph of a f i b e r cut in cross section demonstrating the c l e f t system 26 An electron micrograph of a f i b e r cut in cross section demonstrating the c l e f t system 27 An electron micrograph of a f i b e r cut in longitudinal section demonstrating the c l e f t system 28 A photomicrograph of a 0.5 minute f i b e r cross section with heterogeneous grain density 75 A higher power photomicrograph of the section i n i l l u s t r a t i o n 9 75 A photomicrograph of a 0^ .5 minute f i b e r cross section with heterogeneous grain density 76 A higher power photomicrograph of the section i n i l l u s t r a t i o n 11 76 A d a r k f i e l d photomicrograph showing the grain density of a cross section from a 1.5 minute f i b e r with a 0.5 minute sucrose rinse , 77 - X I l l u s t r a t i o n 1 4 I l l u s t r a t i o n 15 I l l u s t r a t i o n 1 6 I l l u s t r a t i o n 1 7 I l l u s t r a t i o n 1 8 I l l u s t r a t i o n 1 9 I l l u s t r a t i o n 2 0 I l l u s t r a t i o n 2 1 I l l u s t r a t i o n 2 2 I l l u s t r a t i o n 23 I l l u s t r a t i o n 2 4 A d a r k f i e l d photomicrograph showing the g r a i n d e n s i t y o f a 1.5 minute f i b e r c ross s e c t i o n 7 7 A d a r k f i e l d photomicrograph showing the g r a i n d e n s i t y o f a 5 minute f i b e r c r o s s s e c t i o n 8 5 A t r a n s m i t t e d l i g h t photomicrograph of the same s e c t i o n i n i l l u s t r a t i o n 1 5 8 5 A d a r k f i e l d photomicrograph showing the g r a i n d e n s i t y o f a 5 minute f i b e r c r o s s s e c t i o n 8 6 A t r a n s m i t t e d l i g h t photomicrograph o f the same s e c t i o n i n i l l u s t r a t i o n 1 7 8 6 A d a r k f i e l d photomicrograph showing the g r a i n d e n s i t y o f a 20 minute f i b e r c r o s s s e c t i o n 8 7 A t r a n s m i t t e d l i g h t photomicrograph of the same s e c t i o n i n i l l u s t r a t i o n 1 9 8 7 A d a r k f i e l d photomicrograph showing the g r a i n d e n s i t y of a 6 0 minute f i b e r c r o s s s e c t i o n 8 8 A t r a n s m i t t e d l i g h t photomicrograph of the same s e c t i o n i n i l l u s t r a t i o n 2 1 8 8 A d a r k f i e l d photomicrograph showing the g r a i n d e n s i t y o f a 1 8 0 minute f i b e r c r o s s s e c t i o n 8 9 A t r a n s m i t t e d l i g h t photomicrograph of the same s e c t i o n i n i l l u s t r a t i o n 23 8 9 - x i LIST OF FIGURES Figure 1 The resolution curve f o r a 5 minute f i b e r cross section .68 Figure 2 The resolution curve f o r a 20 minute f i b e r cross section 70 Figure 3 The resolution curve f o r a 60 minute f i b e r cross section 72 Figure 4 The resolution curve f o r a 180 minute f i b e r cross section 74 Figure 5 The cross section grain'density p r o f i l e f o r the short term radioautograms Si Figure 6 The 22j\ja+ i n f i u x curve derived from the long term radioautograms 92 Figure 7 The cross section grain density p r o f i l e f o r the long term radioautograms 97 Figure 8 The 22]\ja+ i n f j _ u x curve 117 Acknowledgements I would l i k e t o thank my t h e s i s s u p e r v i s o r , Dr. Hinke, f o r a d v i c e , encouragement and u n l i m i t e d freedom i n the p u r s u i t o f t h i s p r o j e c t . In a d d i t i o n t o a r r a n g i n g my stay i n Vancouver, Dr. and Mrs. Friedman d i d e v e r y t h i n g w i t h i n t h e i r power to make t h i s r e s e a r c h a meaningful and rewarding experience. C o n s i d e r a b l e a i d i n the development of the technique f o r s o l u b l e radioautography came from Dr. H a d f i e l d . Drs. Webber, S p i r a and Slonecker p r o v i d e d t h o u g h t f u l a d v i c e and c r i t i c i s m . T e c h n i c a l a s s i s t a n c e was generously g i v e n by Mr. Cox and Mrs. Blackbourn. L i t t l e would have been accomplished without the a s s i s t a n c e o f Mr. Crossen i n p r o c u r i n g s u p p l i e s and apparatus. My t y p i s t , Mrs. Peaker, k i n d l y prepared the manuscript i n my absence. - 1 CHAPTER I GENERAL INTRODUCTION The s t r i a t e d muscle f i b e r o f the gi a n t b a r n a c l e has become a v a l u a b l e i n v i t r o t e s t o b j e c t f o r those s t u d i e s i n muscle p h y s i o l o g y and b i o p h y s i c s which r e q u i r e e l e c t r o d e p e n e t r a t i o n s i n t o and chemical a n a l y s i s o f s i n g l e f i b e r s . I t s main advantage i s i t s unusual s i z e (about 1 mm diameter and 3 - 4 cm l o n g ) . A major disadvantage t o m u l t i f i b e r p r e p a r a t i o n s has been the r e l a t i v e l y l a r g e e x t r a c e l l u l a r space (15 - 20%), U n f o r t u n a t e l y t h i s problem must s t i l l be c o n s i d e r e d with s i n g l e f i b e r p r e p a r a t i o n s . A s i n g l e f i b e r from Balanus n u b i l i s has an ex t e n s i v e c l e f t system which makes up about 6% of the f i b e r volume. T h i s c l e f t system cannot be ign o r e d when one i s c o n s i d e r i n g Na + or C l ~ i o n d i s t r i b u t i o n i n the muscle f i b e r . For example, when a s i n g l e f i b e r i s bathed i n b a r n a c l e Ringer s o l u t i o n , c o n t a i n i n g 450 mM/L Na + and 540 mM/L CI", t h i s c l e f t system should c o n t a i n about h a l f of the t o t a l f i b e r sodium. C l e a r l y the e x t r a c e l l u l a r compartment can be a c o m p l i c a t i n g f a c t o r i n the study of i o n movements and d i s t r i b -u t i o n i n the f i b e r i n t e r i o r . M o r p h o l o g i c a l l y the c l e f t system i s profuse and branches i n th r e e dimensions through the muscle i n t e r i o r . P robably no pa r t of the myoplasm i s more than a few microns (u) from a branch c l e f t and the e x t r a c e l l u l a r space. - 2 The purpose o f these experiments was to re-examine the q u e s t i o n of Na* i o n exchange between the myoplasm and the e x t e r i o r of the f i b e r i n the l i g h t o f r e c e n t understanding o f the c l e f t system as a pathway f o r d i f f u s i o n . In t h i s study a t t e n t i o n was focused on the Na + i o n . The i s o t o p e Na1" was used to l a b e l the p o o l of Na + 22 *• i n the bath. P e n e t r a t i o n of a s i n g l e f i b e r by Na was f o l l o w e d by the technique of radioautography i n order to attempt some c o r r e l a t i o n o f 2 2 N a + d i s t r i b u t i o n with the morphology 22 + o f the f i b e r c r o s s s e c t i o n . In a d d i t i o n , the standard Na uptake experiment was performed with emphasis on short time i n t e r v a l s . The main c o m p l i c a t i o n i n u s i n g radioautography to study the d i s t r i b u t i o n of s o l u b l e ions such as sodium i s trans-l o c a t i o n of the i o n d u r i n g the experimental procedures. However, radioautography does permit i d e n t i f i c a t i o n of a compartment which may concentrate an i o n by l o c a l i z i n g the i s o t o p e i n r e l a t i o n to the morphology of the muscle f i b e r . T h i s i s v a l i d i f the d i s t a n c e between areas o f i s o t o p e l o c a l i z a t i o n i s not s m a l l e r than the r e s o l u t i o n of the r a d i o -a u t ographic t e c h n i q u e . Even i f p r e c i s e m o r p h o l o g i c a l l o c a l i z a t i o n i s i m p o s s i b l e , radioautography a t short time i n t e r v a l s can g i v e a v i s u a l p i c t u r e of the extent o f isotope p e n e t r a t i o n to the i n t e r i o r of the f i b e r . A l t e r n a t i v e l y at l o n g e r i n t e r v a l s , radioautograms may be analyzed q u a n t i t a t i v e l y p r o v i d e d extreme care i s taken to assure uniform p r o c e s s i n g . In t h i s context - 3 radioautograms can show rate of isotope entry into the f i b e r and therefore provide an alt e r n a t i v e method of i n f l u x analysis. These same radioautograms can also reveal whether the entry i s homogeneous. An i n f l u x study shows the rate of isotope entry into muscle f i b e r s . Complex or multicompartmental f l u x k i n e t i c s may be demonstrated. The size, rate constant, and h a l f loading time of the compartments represented can be calculated. However, the morphological lo c a t i o n of these compartments cannot be demonstrated. CHAPTER I I LITERATURE REVIEW Evidence For a Heterogeneous D i s t r i b u t i o n  of Sodium i n Muscle S e v e r a l d i f f e r e n t experimental approaches have i n d i c a t e d t h a t there e x i s t s a heterogeneous d i s t r i b u t i o n o f sodium i n muscle. These i n c l u d e m i c r o e l e c t r o d e s t u d i e s , NMR a n a l y s i s and i n f l u x - e f f l u x d a t a . A comparison of val u e s obtained by m i c r o e l e c t r o d e f o r the a c t i v i t y o f sodium i n myoplasm w i t h the t o t a l sodium content of the muscle f i b e r g i v e s a c t i v i t y v a l u e s lower than those of sodium s o l u t i o n s of equal s t r e n g t h . T h i s i n d i c a t e s a heterogeneous d i s t r i b u t i o n o f i n t r a c e l l u l a r sodium i n muscle. In Homarus muscle, Hinke (1) found a sodium a c t i v i t y o f 0.012 to 0.016 with a sodium c o n c e n t r a t i o n o f 0.055 moles/kg HgO. C a r c i n u s muscle had an a c t i v i t y c o e f f i c i e n t o f 0.0135 and sodium c o n c e n t r a t i o n of 0.0516 moles/kg H 2 O . McLaughlin and Hinke (2) r e p o r t e d t h a t a t y p i c a l s i n g l e muscle f i b e r o f the g i a n t b a r n a c l e Balanus n u b i l i s had a sodium c o n c e n t r a t i o n o f 0.070 moles/kg H^O. The a c t -i v i t y recorded by myoplasmic e l e c t r o d e f o r such f i b e r s was 0.007 moles/kg H 20. Lev (3) c a l c u l a t e d the a c t i v i t y c o e f f i c i e n t f o r Na + i n the myoplasm of f r o g muscle to be 0.190 to 0.197. T h i s suggested 7 0 % o f the f r o g muscle sodium was u n a v a i l a b l e to - 5 the microelectrode. Robertson (4) obtained evidence for a heterogeneous i n t r a c e l l u l a r sodium d i s t r i b u t i o n i n lobster muscle by comparing the sodium concentration i n intact muscle with the sodium concentration i n f l u i d extracted from the muscle by a tissue press. He found &2% of the i n t r a c e l l u l a r sodium was not extruded by the tissue press. Using nuclear magnetic resonance (NMR), Cope (5) observed 72% of the muscle sodium did not contribute to the sodium spectrum. He concluded that 60% to 80% of the muscle sodium existed i n a complexed state, or at least i n some state other than i n free solution. Conway (6) f i r s t suggested muscle sodium was divided into several compartments as a res u l t of i n f l u x -e f f l u x studies on amphibian muscle. Conway and Cary (7) using the frog sartorius preparation, examined the i n i t i a l r apid exchanges of sodium and potassium. They postulated a special f i b e r compartment larger than the e x t r a c e l l u l a r space, to explain the time course of sodium loss from muscles placed i n glucose solutions. The volume of the space was estimated to be 10% of f i b e r volume, while i t s sodium concentration was estimated to be O.OOct moles/kg H2O. Harris (8) a f t e r a study of the d i s t r i b u t i o n of chloride ion i n frog muscle postulated the existence of a small i n t r a - f i b e r compartment. The compartment would be acces s i b l e to a l l e x t r a c e l l u l a r ions and contain 15% of t o t a l c e l l water {12% of a l l volume). Such a compartment was necessary i f the Donnan equilibrium was to hold for chloride ions. - 6 Dunham and Gainer (9) recently found a non-exchangeable sodium compartment in lobster muscle. This compartment had a sodium concentration of 0.035, moles/kg H20, compared to a total myoplasmic sodium concentration of 0.090 moles/kg H 2 O . The chloride ion was compartmentalized in a fashion similar to sodium both in terms of the kinetics of efflux and of the concentration involved. Location of Compartmentalized Sodium A significant fraction of the total sodium appears complexed or compartmentalized. Where is this sodium located? Morphological studies have revealed a complex internal structure for muscle. Single muscle fibers have a number of compartments that are f l u i d f i l l e d , and some may be enclosed by membranes. These compartments include extra-cellular space, myoplasm, sarcoplasmic reticulum, nuclei and mitochondria. The exact extent and volume of the extracellular space is under considerable debate. Numerous morphological studies have shown that transverse tubules probably open to the extracellular space by contacting the sarcolemma (10,11). Alternative evidence that the transverse tubules are open to the fiber surface is drawn from attempts to explain the high membrane capacitance of muscle fiber compared to nerve c e l l . Falk and Flatt (12) f i r s t suggested a major part of the capacitance of a muscle fiber resides in the tubular membrane system. T h i s t u b u l a r c a p a c i t a n c e i s i n p a r a l l e l w ith the s u r f a c e c a p a c i t a n c e . T h i s r e q u i r e s the t u b u l a r and s u r f a c e membranes to be connected i n some way. E i s e n b e r g and Gage ( 1 3 ) developed a g l y c e r o l soaking technique f o r d i s r u p t i n g the t r a n s v e r s e t u b u l a r system. These authors found the muscle f i b e r c a p a c i t a n c e decreased t o the va l u e p r e d i c t e d f o r a simple tube. Zadunaisky ( 1 4 ) demonstrated h i g h sodium concen-t r a t i o n w i t h i n the t r a n s v e r s e t u b u l e s by a sodium pyroantimon-a t e p r e c i p i t a t i o n t e c h n i q u e . T h i s was c o n s i s t e n t w i t h con-n e c t i o n o f the t r a n s v e r s e t u b u l e s to the e x t r a c e l l u l a r space. However, Peachey ( 1 5 ) has shown t h i s space probably occupies o n l y 0 . 3 % o f f i b e r volume i n f r o g s a r t o r i u s . Thus, the p o t e n t i a l o f the t r a n s v e r s e t u b u l e f o r sodium storage i s l i m i t e d . Peachey ( 1 5 ) concluded the sarcoplasmic r e t i c u l u m r e p r e s e n t e d about 1 3 % o f c e l l volume of which the t e r m i n a l c i s t e r n a e comprised 4 % . B i r k s ( 1 6 ) had c a l c u l a t e d t h a t the sarcoplasmic r e t i c u l u m occupied 1 0 % of c e l l volume s i n c e the t e r m i n a l c i s t e r n a e c o n t a i n l a r g e l y s o l i d m a t e r i a l . Peachey ( 1 5 ) c o n s i d e r e d the s e r r a t e d appearance of the j u n c t i o n between the t r a n s v e r s e t u b u l e and t e r m i n a l c i s t e r n a e to r e p r e s e n t a low r e s i s t a n c e path. B i r k s ( 1 7 ) took the more extreme p o s i t i o n t h a t an a c t u a l communication e x i s t e d between t r a n s v e r s e t u b u l e and the t e r m i n a l c i s t e r n a e v i a m i c r o t u b u l e s . R e c e n t l y , B i r k s ( 1 6 ) has demonstrated t h a t the sar c o p l a s m i c r e t i c u l u m changes i n volume i n hyper- and - 8 hypotonic sucrose s o l u t i o n s . These changes i n volume were l i n e a r l y r e l a t e d w i t h a negative slope to the r e c i p r o c a l of the osmotic p r e s s u r e . To e x p l a i n these changes, B i r k s r e a f f i r m e d h i s c o n t e n t i o n t h a t the sarcoplasmic r e t i c u l u m i s e x t r a c e l l u l a r v i a i t s connection to the t r a n s v e r s e t u b u l e s . T h i s connection of the sarcoplasmic r e t i c u l u m to the t r a n s v e r s e t u b u l e s a l l o w s sucrose p e n e t r a t i o n . H a r r i s (8) had c o n s i d e r e d the connection of the sarcoplasmic r e t i c u l u m to the t r a n s v e r s e t u b u l e as p o s s i b l y patent because of the c l o s e agreement of the volume of the s a r c o -plasmic r e t i c u l u m {10% f i b e r volume) to the t h e o r e t i c a l e x t r a c e l l u l a r space he had p o s t u l a t e d {12% of f i b e r volume). A sarcoplasmic r e t i c u l u m communicating w i t h the e x t r a c e l l u l a r f l u i d should have a sodium c o n c e n t r a t i o n much h i g h e r than the myoplasm. Zadunaisky (14) was unable to show sodium p r e c i p i t a t i o n i n the sarcoplasmic r e t i c u l u m w i t h h i s technique. However, H e r l i h y , Conway and H a r r i n g t o n (18) u s i n g a d i f f e r e n t muscle f i x a t i o n procedure were s u c c e s s f u l . The muscle f i b e r s used by Zadunaisky may have been ina d e q u a t e l y p e n e t r a t e d by the pyroantimonate used f o r sodium p r e c i p i t a t i o n . Keynes and S t e i n h a r d t (19) have r e c e n t l y proposed the sarcoplasmic r e t i c u l u m to be a s o d i u m - f i l l e d membrane-bound compartment. Cope (20) has p o s t u l a t e d non-complexed sodium i n the s a r c o p l a s m i c r e t i c u l u m s i n c e f r e e z a b l e i n t r a -c e l l u l a r water i n s i n g l e c e l l s has been demonstrated to be l o c a t e d i n the r e g i o n of the sarcoplasmic r e t i c u l u m . - 9 These differences i n water structure might r e f l e c t d i f f e r -ences i n sodium coupling. Perhaps the most t e l l i n g argument against the sarcoplasmic reticulum as a sodium reservoir i s the known magnitude of i t s a f f i n i t y f o r calcium. Hasselbach (21) has shown the sarcoplasmic reticulum to have a calcium transporting mechanism more e f f i c i e n t than any other known ion transport system. It seems un l i k e l y that high concentr-ations of d i f f e r e n t cations would be sequestered in the same system. Further evidence against the sarcoplasmic reticulum as a non-complexed sodium compartment comes from the experiments of Hinke (22) dealing with the ef f e c t of hypertonic and hypotonic solutions on single muscle f i b e r s . These experiments indicated there was only one osmotically active compartment i n single muscle f i b e r s . This compart-ment was i d e n t i f i e d as the myoplasm. A sodium r i c h sarco-plasmic reticulum would have been detected as a second osmotically active compartment i n these experiments. The myoplasm may be the morphological s i t e responsible f o r the heterogeneous d i s t r i b u t i o n of sodium. Since approximately b\Q% of the s o l i d material of the myo-plasm i s protein, i t i s reasonable to examine the r e l a t i o n of sodium to the c o n t r a c t i l e proteins. It i s known that the a l k a l i metal cations l i k e sodium can form ion pairs , with charged groups on macromolecules. Such sodium i s said to be "bound". - 10 Szent-Gyorgyi (23) was the f i r s t to demonstrate that extracted myosin but not a c t i n "bound" sodium and potassium. This "binding" a b i l i t y of myosin was l a b i l e and appeared to depend on the secondary and t e r t i a r y structure of the myosin molecule. More recently Lewis and Saroff (24) found extracted myosin "bound" sodium p r e f e r e n t i a l l y over potassium. Fenn (25) used glycerinated f i b e r s to examine the binding of sodium and potassium. These f i b e r s have the c o n t r a c t i l e proteins s p a t i a l l y f i x e d i n a manner similar to the l i v i n g muscle f i b e r . The f i b e r s "bound" sodium p r e f e r e n t i a l l y over potassium. McLaughlin and Hinke (26) have pointed out that the data o f Lewis and Saroff and of Fenn predict that only as much sodium would be "bound" to myosin as i s free i n the myoplasm (20% t o t a l f i b e r sodium). The rest of the f i b e r sodium which re s u l t s i n the heterogeneous d i s t r i b u t i o n could be located elsewhere. A possible explanation i s that the s p a t i a l f i x a t i o n of the myosin molecule in the intact f i b e r greatly increases i t s sodium "binding" capacity. This theory i s supported by Szent-Gyorgyi*s observation of the l a b i l e nature of myosin binding. It i s also known that the binding capacity of ion exchange resins i s enhanced when the degree of cr o s s - l i n k i n g i s increased. Nuclei remain a possible s i t e f o r sodium "binding". Although evidence indicates n u c l e i can concentrate potassium - 11 i n p r e f e r e n c e to sodium (27), i t has a l s o been r e p o r t e d they c o n t a i n sodium a t three times the c o n c e n t r a t i o n o f the cytoplasm (28). A s i m i l a r s i t u a t i o n e x i s t s f o r m i t o c h o n d r i a . Although they have been shown to p r e f e r -e n t i a l l y concentrate potassium over sodium (29), t h e r e i s a l s o evidence of g r e a t e r i n t e r n a l sodium c o n c e n t r a t i o n i n mitochondria than i n the surrounding myoplasm (30). S t a t e o f Sodium i n Balanus What i s the s t a t e of sodium i n Balanus and s i m i l a r crustaceans? From r e c e n t i n f l u x and e f f l u x s t u d i e s on muscle f i b e r s o f Balanus n u b i l i s , A l l e n and Hinke (31) found t h a t i n t r a c e l l u l a r sodium i n Balanus was e a s i l y d i v i s i b l e i n t o two compartments. A r a p i d l y exchanging compartment was d e t e c t a b l e i n both i n f l u x and e f f l u x s t u d i e s and i d e n t i f i e d as f r e e rnyoplasmic sodium. A s l o w l y exchanging compartment was e v i d e n t o n l y i n i n f l u x experiments. A l l i n f l u x experiments were c o r r e c t e d f o r the e x t r a c e l l u l a r space which i n Balanus accounts f o r over h a l f of the t o t a l f i b e r sodium. The s i z e of the r a p i d l y exchanging compartment was about 60% o f t o t a l i n t r a c e l l u l a r sodium. T h i s value agreed w e l l w i t h the sodium e l e c t r o d e rnyoplasmic a c t i v i t y measurement. The second compartment r e p r e s e n t e d 40% of i n t r a c e l l u l a r (20% o f t o t a l f i b e r ) sodium and exchanged very s l o w l y . T h i s i n d i c a t e d b i n d i n g or compartmentalization of some s o r t . - 1 2 In p r e v i o u s s t u d i e s , McLaughlin and Hinke ( 2 6 ) estimated the average sodium c o n c e n t r a t i o n o f a s i n g l e f i b e r to be 0 . 0 7 0 moles/kg HgO. The f r e e c o n c e n t r a t i o n o f the myoplasm was 0 . 0 1 0 moles/kg H 2 0 {11+% t o t a l f i b e r sodium) w h i l e the e x t r a c e l l u l a r space accounted f o r 0 . 0 3 0 moles/kg H 2 0 {1+3% t o t a l f i b e r sodium). The sodium content of the e x t r a c e l l u l a r space was contained i n 6 - 1% o f t o t a l f i b e r volume. T h i s l e f t 0 . 0 3 0 moles/kg H 2 0 (1+3% t o t a l f i b e r sodium) e i t h e r compartmentalized i n o r g a n e l l e s i n the myoplasm or "bound" to the c o n t r a c t i l e p r o t e i n s . The s i z e o f t h i s compartment as deduced by m i c r o e l e c t r o d e s t u d i e s ( 4 3 % t o t a l f i b e r sodium) i s thus twice as l a r g e as the s l o w l y moving i n f l u x compartment of A l l e n and Hinke ( 3 D ( 2 0 % o f t o t a l f i b e r sodium). Thus, the s t a t e of sodium i n Balanus i s s i m i l a r to muscle i n g e n e r a l . There e x i s t s good evidence from f l u x s t u d i e s and m i c r o e l e c t r o d e measurements o f a heterogeneous sodium d i s t r i b u t i o n i n Balanus. A c e r t a i n f r a c t i o n of the t o t a l f i b e r sodium appears "bound" or compartmentalized. Where i n the muscle f i b e r i s t h i s sodium l o c a t e d ? In Balanus, mitochondria and n u c l e i occupy o n l y a s m a l l p o r t i o n of the f i b e r volume. The sarcoplasmic r e t i c u l u m i s not as e x t e n s i v e as i n the f r o g . However, the s a r c o p l a s m i c r e t i c u l u m would have to possess on l y h a l f the volume of the e x t r a c e l l u l a r space ( 3 - 4 % of t o t a l f i b e r volume) to account f o r 2 0 % of the t o t a l f i b e r sodium, (the compartment of A l l e n and Hinke or one h a l f the - 13 compartment of Hinke.) T h i s assumes the sodium c o n c e n t r a t i o n o f the sarcoplasmic r e t i c u l u m i s s i m i l a r to the c o n c e n t r a t i o n o f the e x t r a c e l l u l a r space. These volume requirements (3 - 4% of the t o t a l f i b e r volume) are not incompatible w i t h the e l e c t r o n microscope p i c t u r e o f the sarcoplasmic r e t i c u l u m o f Balanus. In an e a r l i e r s e c t i o n o f t h i s review, arguments a g a i n s t the sarcoplasmic r e t i c u l u m as a r e g i o n o f hig h sodium c o n c e n t r a t i o n were summarized. Thus, i t i s d o u b t f u l t h a t t h i s compartment i s wholly r e s p o n s i b l e f o r the heterogeneous sodium d i s t r i b u t i o n observed i n Balanus. However, the volume of the sarcoplasmic r e t i c u l u m would be adequate t o e x p l a i n sodium compartmentalization. Hinke and McLaughlin (32) have p o s t u l a t e d t h a t the f r a c t i o n of sodium i n Balanus f i b e r s i n a c c e s s i b l e to the m i c r o e l e c t r o d e i s "bound" to the c o n t r a c t i l e p r o t e i n s . In muscles heated to 40°C the mean sodium a c t i v i t y i n c r e a s e d 130%. T h i s was accompanied by an i r r e v e r s i b l e s h o r t e n i n g o f the f i b e r . The authors i n t e r p r e t e d the i n c r e a s e i n sodium a c t i v i t y as the r e l e a s e o f "bound" sodium from b i n d i n g s i t e s on myosin. In a subsequent paper McLaughlin and Hinke (26) demonstrated a decrease i n o p t i c a l d e n s i t y of s i n g l e muscle f i b e r s when these f i b e r s were p l a c e d i n sodium f r e e sucrose R i n g e r s s o l u t i o n . I t was p o s t u l a t e d t h a t "bound" sodium was r e l e a s e d from myosin. The authors' f i n a l c o n c l u s i o n was t h a t a minimum o f 20% of f i b e r sodium was bound to myosin. They b e l i e v e d t h a t s p a t i a l arrangements - 14 o f myosin i n v i v o r e s u l t e d i n myosin being the major sodium b i n d i n g s i t e . Other p e r t i n e n t s t u d i e s are those o f Shaw (33) on C a r c i n u s muscle. The morp h o l o g i c a l o r g a n i z a t i o n o f these muscle f i b e r s c l o s e l y resembles those of Balanus. Shaw demonstrated the uptake o f 2 % a + by C a r c i n u s muscle to be e x c e p t i o n a l l y r a p i d . Approximately 7 0 % o f the muscle sodium ( 0 . 0 3 5 moles/kg H 20) had exchanged with ^ N a * from the blood i n three minutes. He suggested a non-uniform sodium d i s t r i b u t i o n through the muscle f i b e r s with the bulk o f the sodium i n a s p e c i a l r e g i o n probably e x t r a -s arcoplasmic i n n a t u r e . The sodium c o n c e n t r a t i o n o f t h i s s p e c i a l r e g i o n would m i r r o r t h a t o f the b l o o d . The r e l a t i v e l y small volume of the r e g i o n would account f o r the f a c t t h a t measurement of sodium c o n c e n t r a t i o n on the b a s i s o f the f i b e r as a whole l e a d s to a value much lower than t h a t of bl o o d . The same i s t r u e f o r the e x t r a c e l l u l a r space of Balanus; h a l f the t o t a l f i b e r sodium i s i n the e x t r a c e l l u l a r space and i t s c o n c e n t r a t i o n m i r r o r s t h a t o f the b a t h i n g s o l u t i o n . In f u r t h e r experiments, Shaw (33) immersed C a r c i n u s muscles i n b a t h i n g media where sodium was p a r t i a l l y r e p l a c e d by de x t r o s e . The muscles l o s t h a l f t h e i r sodium very r a p i d l y probably from the e x t r a s a r c o p l a s m i c r e g i o n s . The myoplasmic sodium c o n c e n t r a t i o n f e l l from 0.060 - 0.0&0 moles/kg H 20 to 0.020 - 0.023 moles/kg H 20 i n ten minutes i n a bath having a sodium c o n c e n t r a t i o n of 0.027 moles/kg H 20. - 15 C a l d w e l l (34) has observed t h a t t h i s l a t t e r v a lue i s c l o s e to Hinke's m i c r o e l e c t r o d e value f o r f r e e rnyoplasmic sodium i n C a r c i n u s . He concluded that a f t e r t en minutes most of the sodium remaining i n the muscle i s unbound and i n the sarcoplasm. However, Hinke i n s t u d i e s on Balanus f i b e r s p l a c e d i n sodium f r e e s o l u t i o n s found t h a t sodium was removed p r o p o r t i o n a t e l y from the "bound" and rnyoplasmic f r a c t i o n s . T h i s r e s u l t e d i n the sodium content of myoplasm remaining c o n s t a n t . Only as a r e s u l t of a m i c r o e l e c t r o d e study would C a l d w e l l ' s c o n c l u s i o n be j u s t i f i e d . In c o n c l u s i o n there i s good evidence from s e v e r a l experimental techniques f o r a heterogeneous d i s t r i b u t i o n o f sodium i n muscle. The exact m o r p h o l o g i c a l l o c a t i o n of t h i s sodium e s p e c i a l l y the "bound" or compart-m e n t a l i z e d f r a c t i o n remains obscure. Crustaceans such as Balanus have an e x t e n s i v e e x t r a c e l l u l a r space which e q u i l i b r a t e s r a p i d l y with e x t e r n a l i o n s and c o n t a i n s a t l e a s t h a l f of the f i b e r sodium. A minimum of 20% of the t o t a l f i b e r sodium i n Balanus appears to be "bound" or compartmentalized but the l o c a t i o n o f t h i s f r a c t i o n remains u n c e r t a i n . CHAPTER I I I - 16 Barnacle Muscle S t r u c t u r e I n t r o d u c t i o n The e x i s t e n c e of c r o s s s t r i a t e d muscle f i b e r s o f u n u s u a l l y l a r g e s i z e i n the ba r n a c l e Balanus n u b i l i s was f i r s t r e p o r t e d by Hoyle and Smith (35). The muscle f i b e r s occur i n d i s c r e t e bundles having a m u l t i p l e f i b r i l l a r b a s a l a t t a c h -ment to the s h e l l and a 3 - 4mm tendon attachment to the scute. A bundle c o n t a i n s 30 - 40 f i b e r s with diameters r a n g i n g from 500 - 1,500LI. There i s l i t t l e c onnective t i s s u e between f i b e r s and they may be separated by s k i l l e d d i s s e c t i o n . These authors summarized the evidence t h a t these f i b e r s are i n f a c t d i s c r e t e s i n g l e muscle c e l l s . The h i s t o l o g y of i n d i v i d u a l f i b e r s was d e s c r i b e d by Hoyle and Smith i n a subsequent paper (36). The saroolemma and i t s connective t i s s u e forms a continuous r i n g around the circumference of the f i b e r and a l s o indents deeply i n t o the sarcoplasm. The connective t i s s u e i n v a g i n a t i o n s t h i n out r a p i d l y t o the p o i n t o f no l o n g e r being v i s i b l e with the l i g h t microscope. C l e f t s or channels enter the f i b e r from the p e r i p h e r y and become multibranched. They r a m i f y through the c e n t r a l p a r t o f the f i b e r . These c l e f t s a p p a r e n t l y are not l i n e d w i t h connective t i s s u e . When viewed i n l o n g i t u d i n a l s e c t i o n , l o n g c l e f t s running p a r a l l e l to the l o n g a x i s o f the f i b e r are conspicuous but do not course f o r more than a - 17 few m i l l i m e t e r s i n any i n s t a n c e . The b a s a l end of a f i b e r has g r e a t e r v a r i a b i l i t y i n i t s c l e f t system because of the m u l t i p l e attachment to the s h e l l . Most n u c l e i are s i t u a t e d a t the p e r i p h e r y o f the f i b e r subjacent to the sarcolemma. More c e n t r a l n u c l e i tend to be l o c a t e d on the margins of c l e f t s . An e l e c t r o n microscope d e s c r i p t i o n o f the c l e f t system o f Balanus was p r o v i d e d by S e l v e r s t o n (37). The c l e f t s appear as l o n g i t u d i n a l f l a t t e n e d i n f o l d i n g s o f both plasma membrane and f i b r o u s sheath of the f i b e r . The area o f the c l e f t system e f f e c t i v e l y i n c r e a s e s the f i b e r circum-f e r e n c e by about twenty times. S e l v e r s t o n estimated t h a t no p o r t i o n o f the c o n t r a c t i l e p r o t e i n s i s more than 20 - 50II from the plasma membrane of the sarcolemma. In the b a r n a c l e , t u b u l e s a r i s e from the sarcolemma both at the f i b e r s u r f a c e and from the s i d e s o f the c l e f t s . These t u b u l e s e v e n t u a l l y form d i a d i c c o n t a c t s with the sarcoplasmic r e t i c u l u m a t the j u n c t i o n o f the A and I bands. Hoyle (3&) c a l l e d the branches of the c l e f t s running to the sarcoplasmic r e t i c u l u m the T system. He d e s c r i b e d the T t u b u l e s as b e i n g remarkably f l a t t e n e d i n s e c t i o n . From the c l e f t these T t u b u l e s have o n l y 2u to t r a v e l to reach the most d i s t a n t p o i n t s of contact with the sarco p l a s m i c r e t i c u l u m . The contacts w i t h the sarcoplasmic r e t i c u l u m a re mainly d i a d i c and are formed where the t r a n s v e r s e , o b l i q u e or l o n g i t u d i n a l t u b u l e s come i n t o c l o s e r e l a t i o n with the sarcoplasmic r e t i c u l u m . Contact p o i n t s - 18 occur a l o n g the A band i n a d d i t i o n to those a t the A - I j u n c t i o n . The complex between the t u b u l e and the sarco-p l a s m i c r e t i c u l u m i s without p o r e s . Hoyle a l s o observed wide t r a n s v e r s e t u b u l e s making desmosone-like c o n t a c t s with the Z r e g i o n s where I f i l a m e n t s o v e r l a p between sarcomeres. S p i r a (39) has shown the edges of these T t u b u l e s appear patent a t the l e v e l of contact with the sarcoplasmic r e t i c u l u m . The t u b u l e s are approximately 0.02LI wide at t h i s p o i n t . In I l l u s t r a t i o n I the f e r r i t i n can be seen as b l a c k granules w i t h i n the edges o f the T tubule as i t approaches the s a r c o p l a s m i c r e t i c u l u m . The center does not appear open to f e r r i t i n . However, when horse r a d i s h peroxidase was used as a marker the e n t i r e T tubule appeared patent at the l e v e l of contact with the sarcoplasmic r e t i c u l u m . I l l u s t r a t i o n 2 shows such a t u b u l e . Note the t h i n l a y e r of horse r a d i s h peroxidase c o a t i n g the s i d e s of the t u b u l e (dark l i n e s ) w h ile a lumen i s c l e a r l y present ( l i g h t c e n t e r ) . In the l i g h t o f these r e s u l t s , the e x t r a c e l l u l a r space must be regarded as p e n e t r a t i n g to the l e v e l where these 0.02u t u b u l e s form d i a d s or t r i a d s w i t h the s a r c o p l a s m i c r e t i c u l u m . The t r a n s v e r s e t u b u l e s of amphibian muscle d e s c r i b e d by Peachey.(15) appear to be analogous to these f i n e t e r m i n a l c l e f t s . These are 0.08 microns i n diameter and patent u n t i l t h e i r t e r m i n a t i o n i n a . t r i a d s t r u c t u r e . - 19 Methods Since the c l e f t system p l a y s a major r o l e i n i n f l u x , f i r s t e f f o r t s were to f u r t h e r e l u c i d a t e i t s morph-olog y . With standard Haematoxylin and E o s i n s t a i n i n g the b r i g h t l y e o s i n o p h i l i c myoplasm makes i t d i f f i c u l t to see the margins of a c l e f t . An e f f e c t i v e s t a i n was found to be aqueous Haematoxylin f o l l o w e d by 0 . 2 % aqueous h y d r o c h l o r i c a c i d f o r d i f f e r e n t i a t i o n . T h i s l e f t the c l e f t system and n u c l e i w e l l s t a i n e d but faded the cytoplasm r e s u l t i n g i n good c o n t r a s t between c l e f t s and cytoplasm. As i n a l l subsequent experiments, care was taken to o b t a i n s e c t i o n s from the middle o f f i b e r s . A s e r i e s o f e l e c t r o n micrographs of Balanus was o b t a i n e d from Dr. A. S p i r a . R e s u l t s and D i s c u s s i o n The low, medium and o i l immersion photographs o f Balanus r e v e a l the complexity of the b r a n c h i n g of the c l e f t system. I l l u s t r a t i o n 3 i s a low power photomicrograph. Note the l a r g e c l e f t s which run from the edge of the f i b e r and t h e i r c l o s e l y a s s o c i a t e d n u c l e i . From these, s m a l l e r c l e f t s branch. These are not connected to the s u r f a c e . They may have a s s o c i a t e d n u c l e i . I l l u s t r a t i o n 4 i s a h i g h dry photomicrograph. Here three s m a l l e r c l e f t s with n u c l e i surround a t r i a n g u l a r area of myoplasm approximately $0p. a c r o s s . I l l u s t r a t i o n 5 i s an o i l immersion photomicrograph - 20 o f one s i d e o f the same a r e a . A l a r g e number of small c l e f t s a re seen. Although l a r g e r c l e f t s seem patent these small c l e f t s appear without v i s i b l e lumen. These l a r g e c l e f t s are seen to branch forming sm a l l e r c l e f t s . O v e r a l l t h e r e i s a complicated a r r a y o f branching and r a m i f i c a t i o n from l a r g e r t o smaller-and s m a l l e r c l e f t s . The s m a l l e s t c l e f t s i n t h i s photomicrograph probably r e p r e s e n t the c l e f t s from which the T t u b u l e s are seen to branch i n the e l e c t r o n micrographs. These c l e f t s as seen i n both the l i g h t and e l e c t r o n microscope, have the same dimensions ( 0 . 2 - 0.3ju). Measurements on t h i s photomicrograph r e v e a l no p a r t o f myoplasm more than 2 - 3)i from a 0.3p. c l e f t . T h i s i s a much s m a l l e r d i s t a n c e than t h a t r e p o r t e d by S e l v e r s t o n . I l l u s t r a t i o n s 6 and 7 are e l e c t r o n micrographs of a f i b e r cut i n c r o s s s e c t i o n . The bar i n the lower corner i s ly. i n l e n g t h . In both photographs there i s a 0 . 2 - 0.3>i c l e f t . In e l e c t r o n micrographs the c l e f t s appear to c o n s i s t o f e l e c t r o n dense m a t e r i a l t h a t s t a i n s s i m i l a r l y to basement membrane. T h i s i s c o n s i s t e n t with experiments by A l l e n (40) which demonstrated o i l c o u l d not enter the c l e f t system. From t h i s c l e f t a branch c l e f t (T tubule) l e a d s to a t r i a d . The l e n g t h o f t h i s T tubule i s between 1 - 2LI as s t a t e d by Hoyle. Thin sheets of sarcoplasmic r e t i c u l u m surround each m y o f i b r i l . T t u b u l e s contact t h i s s a r c o p l a s m i c r e t i c u l u m a t dia d s and t r i a d s a t the perimeter of the m y o f i b r i l s . S ince A, I and Z bands are seen i n the same t r a n s v e r s e s e c t i o n , the banding o f adjacent m y o f i b r i l s - 21 i s not always i n phase. I l l u s t r a t i o n 8 i s an e l e c t r o n micrograph of a l o n g i t u d i n a l s e c t i o n e d f i b e r . The bar i s l u i n l e n g t h . The t r i a d t e r m i n a t i o n s of the T t u b u l e s with the sarcoplasmic r e t i c u l u m are found along the f u l l l e n g t h of the A and I bands. Only i n the r e g i o n o f the Z band are no t e r m i n a t i o n s seen. M i t o c h o n d r i a are l o c a t e d between m y o f i b r i l s but are not numerous. By examining the l o c a t i o n of the d i a d and t r i a d t e r m i n a t i o n s of the T t u b u l e s i n r e l a t i o n to the m y o f i b r i l s i t i s p o s s i b l e to estimate the d i s t a n c e of any p a r t of the myoplasm from the e x t r a c e l l u l a r space. A s i n g l e m y o f i b r i l appears to have t r i a d t e r m i n a t i o n s on a l l s i d e s of the perimeter and a l o n g the f u l l l e n g t h except f o r a small area immediately under the Z band. M y o f i b r i l s are most f r e q u e n t l y quadrangular i n c r o s s s e c t i o n . One diameter i s u s u a l l y 1 - 2p. while the other i s 3 - 4}U I t i s reasonable to con-clude t h a t no p a r t of the myoplasm i s more than 1 - 2}i from the e x t r a c e l l u l a r space. In the area of the Z band, however, the d i s t a n c e may be somewhat g r e a t e r . In c o n c l u s i o n , the muscle f i b e r has a complex c l e f t system extending from the s u r f a c e and d i v i d i n g e x t e n s i v e l y i n three dimensions through the muscle i n t e r i o r t o the extent t h a t no p a r t of the myoplasm or c o n t r a c t i l e p r o t e i n s i s more than 1 - 2u from the c e l l membrane. This i s not unique to Balanus or to i n v e r t e b r a t e muscle. For - 22 example, i t has been demonstrated t h a t no p a r t o f mammalian c a r d i a c myoplasm i s more t h a n 2 - 3u from the e x t r a c e l l u l a r space (41). I l l u s t r a t i o n s 1 and 2 An e l e c t r o n micrograph of a T t u b u l e . Note the f e r r i t i n (black granules) i n the edges of the T t u b u l e as i t terminates i n c l o s e r e l a t i o n s h i p to the sarcoplasmic r e t i c u l u m as a t r i a d . The center of the T t u b u l e does not appear to be patent to molecules the s i z ' An e l e c t r o n micrograph o f a T t u b u l e . Note horse r a d i s h peroxidase c o a t i n g both s i d e s of the T t u b u l e (arrows) as i t meets the sarcoplasmic r e t i c u l u m . Unstained m a t e r i a l . M a g n i f i c a t i o n 34,300 x I l l u s t r a t i o n s 3 and 4 A photomicrograph of a f i b e r cut i n c r o s s s e c t i o n . Large c l e f t s extend from the s u r f a c e o f the f i b e r and have c l o s e l y a s s o c i a t e d n u c l e i . From these l a r g e r c l e f t s s m a l l e r c l e f t s are seen to branch. M a g n i f i c a t i o n 75 x A photomicrograph of a f i b e r cut i n c r o s s s e c t i o n . Three l a r g e c l e f t s d e f i n e a t r i a n g u l a r area o f myoplasm. Many s m a l l e r c l e f t s are seen i n the center o f t h i s a r e a . M a g n i f i c a t i o n 370 x I l l u s t r a t i o n 5 - 25 A photomicrograph o f a f i b e r cut i n c r o s s s e c t i o n . Note d i v i s i o n s and r a m i f i c a t i o n s o f the c l e f t s from a l a r g e r c l e f t at the s i d e and w i t h i n the myoplasm. L a r g e r c l e f t s can be seen s u b d i v i d i n g t o p r o g r e s s i v e l y s m a l l e r c l e f t s. M a g n i f i c a t i o n 1 , 8 0 0 x - 26 I l l u s t r a t i o n 6 An e l e c t r o n micrograph o f a f i b e r cut i n c r o s s s e c t i o n . A T t u b u l e (BC) branching from a c l e f t (C) l e a d s to a d i a d s t r u c t u r e . The sarcoplasmic r e t i c u l u m (SR) surrounds i n d i v i d -u a l m y o f i b r i l s as a t h i n f e n e s t r a t e d sheet. T t u b u l e s are i n c l o s e r e l a t i o n to the sarcoplasmic r e t i c u l u m a t diads (D) and t r i a d s (T) a t the perimeter of the m y o f i b r i l s . Banding o f adjacent m y o f i b r i l s i s not i n phase: A, I and Z bands are seen i n the same s e c t i o n . (A) (I) ( Z ) . S c a l e , Bar = l u . M a g n i f i c a t i o n 18,200 x - 27 I l l u s t r a t i o n 7 An e l e c t r o n micrograph of a f i b e r cut i n cross s e c t i o n . A T tubule (BC) branching from a c l e f t (C) l e a d s to a t r i a d s t r u c t u r e . The sarcoplasmic r e t i c u l u m (SR) surrounds i n d i v -i d u a l m y o f i b r i l s as a t h i n f e n e s t r a t e d sheet. T tubules ( u n l a b e l l e d ) are i n c l o s e r e l a t i o n to the sarcoplasmic r e t i c u l u m at diads and t r i a d s at the p e r i m e t e r of the myo-f i b r i l s . Banding of adjacent m y o f i b r i l s i s not i n phase: A, I ( u n l a b e l l e d ) and Z bands (Z) are seen i n the same s e c t i o n . A nucleus i s seen (N). S c a l e , Bar = l u . M a g n i f i c a t i o n 18,200 x An e l e c t r o n m i c r o g r a p h o f a l o n g i t u d i n a l l y s e c t i o n e d f i b e r . A c l e f t (C) and m i t o -c h o n d r i a (M) l i e between m y o f i b r i l s . G l ycogen i s abundant w i t h i n m y o f i b r i l s ( s m a l l a r r o w ) . A f u l l sarcomere i s l a b e l l e d from Z l i n e t o Z l i n e ( Z ) . The A and I bands o f t h i s sarcomere a r e marked. Note t h a t t h e t r i a d t e r m i n a t i o n s (T) o f t h e T t u b u l e s a r e p r e s e n t a l o n g t h e f u l l l e n g t h o f t h e A and I bands. None a r e seen i n the r e g i o n o f t h e Z l i n e . The Z l i n e s appear f e n e s t r a t e d a l l o w i n g communication o f the I bands o f a d j a c e n t s a r c o m e r e s . S c a l e , B ar = l u M a g n i f i c a t i o n 14|500 x . - 2 9 CHAPTER IV » 22 +• Radioautographic S t u d i e s o f Na I n f l u x I n t r o d u c t i o n General Theory and Terminology Radioautography may be d e f i n e d as a method f o r l o c a t i n g r a d i o a c t i v e substances by the use of m o d i f i e d photographic t e c h n i q u e s . Radioautographs may be used i n a q u a n t i t a t i v e as w e l l as i n a q u a l i t a t i v e manner p r o v i d -i n g procedures are r i g o r o u s l y s t a n d a r d i z e d to prevent v a r i a b i l i t y . L a t e n t Image Formation The photographic m a t e r i a l used i n t h i s study was Kodak A.R.-10 s t r i p p i n g f i l m , a 5>i l a y e r o f emulsion on a lOp. l a y e r of g e l a t i n base. When l i g h t o r i o n i z i n g r a d i a t i o n s t r i k e s a s i l v e r bromide (AgBr) c r y s t a l i n the emulsion, the c r y s t a l i s made s u s c e p t i b l e to c o n v e r s i o n to m e t a l l i c s i l v e r by the d e v e l o p e r . In photographic terms, the emulsion has a l a t e n t image which can be converted to a t r u e image by development. A v a r i e t y o f p h y s i c a l and b i o l o g i c a l agents may cause l a t e n t image for m a t i o n i n the emulsion. P h y s i c a l agents i n c l u d e mechanical p r e s s u r e , heat, l i g h t and i o n i z i n g r a d i a t i o n . Manual pressure can produce f i n g e r p r i n t s on r a d i o a u t o g r a p h s . Any r a d i o a u t o g r a p h i c technique must guard a g a i n s t undue pressure between s e c t i o n and emulsion d u r i n g the mounting p r o c e s s . T h i s w i l l cause i n a p p r o p r i a t e l a t e n t - 30 image f o r m a t i o n (pressure a r t i f a c t s ) . A p a r t of the back-ground o f any radioautograph i s caused by thermal a g i t a t i o n which r e s u l t s i n random l a t e n t image f o r m a t i o n i n the emulsion. B i o l o g i c a l m a t e r i a l s used i n radioautograms may cause the p r o d u c t i o n or d e s t r u c t i o n of l a t e n t images. These processes are r e s p e c t i v e l y c a l l e d p o s i t i v e and negative chemography. Chemography depends on the d i f f u s i o n o f molecules from the specimen i n t o the emulsion and the r e -a c t i o n o f these molecules with the s i l v e r h a l i d e c r y s t a l s . The r a t e s of both of these processes are temperature depend-ent. The p r o d u c t i o n o f l a t e n t images by i o n i z i n g r a d i a t i o n i s temperature independent. Exposing radioautographs a t about -20°C should d i s c r i m i n a t e a g a i n s t chemography. The amount of chemography may vary from experiment to experiment. and w i t h i n the same experiment. One s e r i e s o f s e c t i o n s may be a f f e c t e d by chemography or i t may vary from area t o area i n the same s e c t i o n . Rogers (42) has found negative chem-ography to be the s i n g l e most c o n s i s t e n t problem i n a wide v a r i e t y o f r a d i o a u t o g r a p h i c s t u d i e s . L a t e n t images may spontaneously r e v e r t over l o n g p e r i o d s of time. T h i s process i s c a l l e d l a t e n t image fade. I t occurs as a random process and i s on l y s i g n i f i c a n t when exposures are f o r s e v e r a l months. In c o n c l u s i o n , care must be taken t o prevent i n a p p r o p r i a t e l a t e n t image for m a t i o n i n the emulsion. L i g h t and mechanical p r e s s u r e must be a v o i d e d . During - 31 exposure the emulsion should be cooled to a v o i d thermal background and chemography. Exposure should be as short as p o s s i b l e to a v o i d l a t e n t image fade. C o n t r o l s should be used to d e t e c t the presence of p o s i t i v e or negative chemography. R e s o l u t i o n R e s o l u t i o n of radioautographs i s u s u a l l y d e f i n e d as the d i s t a n c e from the t i s s u e s e c t i o n edge (source) where the g r a i n d e n s i t y i s one h a l f the value d i r e c t l y over the t i s s u e s e c t i o n edge. P e l c (43) has shown f o r such high 22 + energy i s o t o p e s as Na T the best t h e o r e t i c a l r e s o l u t i o n i s equal to one h a l f the t h i c k n e s s of the emulsion and s e c t i o n . Thus f o r the experiments to be d e s c r i b e d w i t h f i v e microns o f emulsion and a f i f t e e n micron s e c t i o n , the best t h e o r -e t i c a l r e s o l u t i o n i s ten microns. Any s u b s t a n t i a l i n c r e a s e i n t h i s value i m p l i e s i s o t o p e t r a n s l o c a t i o n at some p o i n t i n the r a d i o a u t o g r a p h i c procedure. F a c t o r s e f f e c t i n g the r e s o l u t i o n i n d e c r e a s i n g order o f importance i n c l u d e the d i s t a n c e between the s e c t i o n and emulsion, the i s o t o p e used, the t h i c k n e s s of the s e c t i o n , the t h i c k n e s s of the emulsion, the l e n g t h of exposure, the s i z e of the emulsion s i l v e r h a l i d e c r y s t a l s and the s e n s i t i v i t y of the emulsion. An i s o t o p e emits beta p a r t i c l e s over a wide spectrum of e n e r g i e s . The path of the p a r t i c l e s i s unpre-d i c t a b l e w i t h many d i r e c t i o n changes. Sodium-22 emits a p o s i t r o n which behaves l i k e a h i g h energy beta p a r t i c l e u n t i l i t ends i t s b r i e f e x i s t e n c e by a n n i h i l a t i n g an o r b i t a l - 32 e l e c t r o n g i v i n g o f f two quanta o f gamma r a y s . With high energy i s o t o p e s the maximum energy of the p a r t i c l e s i s i n -creased. S i l v e r g r a i n s are produced a t g r e a t e r d i s t a n c e s from the t i s s u e s e c t i o n edge by h i g h e r energy beta p a r t i c l e s and the r e s o l u t i o n i s poorer. The great value of t r i t i u m i n radioautography i s the low maximum energy o f the p a r t i c l e s emitted (0.018 MEV). S e p a r a t i n g t i s s u e s e c t i o n and emulsion w i l l have more e f f e c t on r e s o l u t i o n than a l t e r i n g any other parameter. When the s e c t i o n i s i n d i r e c t contact with the emulsion the d i s t a n c e to g r a i n s immediately under the s e c t i o n i s much sm a l l e r than the d i s t a n c e to g r a i n s at some d i s t a n c e from the s e c t i o n edge. I f source and emulsion are separated by a t h i n l a y e r of i n e r t m a t e r i a l , g r a i n s a t some d i s t a n c e from the s e c t i o n edge are the same d i s t a n c e from the s e c t i o n as g r a i n s under the s e c t i o n . Grains i n the two areas now have equal p r o b a b i l i t i e s o f b e i n g h i t by beta p a r t i c l e s and the r e s o l u t i o n decreases. The t h i c k n e s s o f the s e c t i o n i s a s p e c i a l case of s e p a r a t i n g the s e c t i o n from the emulsion. A s e c t i o n may be regarded as a s e r i e s of l a y e r s . In e f f e c t the lower l a y e r s separate the upper l a y e r s from the emulsion. The r e s o l u t i o n of the e n t i r e s e c t i o n i s a composite o f the r e s o l u t i o n s of a l l the l a y e r s . T h i s r e s o l u t i o n i s poorer than the r e s o l u t i o n of a s i n g l e l a y e r i n d i r e c t c ontact with the emulsion. - 33 The e f f e c t o f i n c r e a s i n g the emulsion t h i c k n e s s i s s i m i l a r to the e f f e c t of i n c r e a s i n g the s e c t i o n t h i c k -ness. With a t h i n emulsion l a y e r i n d i r e c t contact with s e c t i o n the g r a i n d e n s i t y f a l l s o f f r a p i d l y from the s e c t i o n edge. As the emulsion t h i c k n e s s i s i n c r e a s e d the g r a i n d e n s i t y does not f a l l o f f a b r u p t l y . T h i s e f f e c t o f emulsion t h i c k n e s s on r e s o l u t i o n depends on the maximum energy o f the p a r t i c l e s emitted. With t r i t i u m the maximum t r a c k l e n g t h of the most e n e r g e t i c p a r t i c l e i s 3u. I n c r e a s i n g the emulsion t h i c k n e s s beyond 3^ w i l l not change the r e s o l u t i o n . For h i g h e r energy i s o t o p e s the emulsion t h i c k -ness i s c r i t i c a l . The l o n g e r exposure continues the g r e a t e r the p r o b a b i l i t y of m u l t i p l e h i t s by beta p a r t i c l e s of s i l v e r ' g r a i n s immediately under the s e c t i o n . M u l t i p l e h i t s are l e s s l i k e l y f o r g r a i n s s e v e r a l microns away from the s e c t i o n . I f exposure i s l o n g term, the i n c r e a s e i n g r a i n d e n s i t y immediately under the s e c t i o n w i l l not be p r o p o r t i o n a l to the i n c r e a s e i n g r a i n d e n s i t y s e v e r a l microns away from the s e c t i o n . For l o n g exposures m u l t i p l e h i t s can decrease r e s o l u t i o n i n t h i s manner. The s m a l l e r the s i z e of the s i l v e r h a l i d e c r y s t a l i n the emulsion, the more c l o s e l y the p o s i t i o n of the developed g r a i n corresponds to the t r a j e c t o r y of the p a r t i c l e . T h i s w i l l r e s u l t i n a small improvement i n r e s o l u t i o n . I f the emulsion i s r e l a t i v e l y i n s e n s i t i v e , the h i g h e s t energy beta p a r t i c l e s w i l l o n l y produce l a t e n t - 34 images a t some d i s t a n c e from the s e c t i o n . T h i s w i l l r e s u l t i n poor r e s o l u t i o n . T h i s i s a minor f a c t o r s i n c e i s o t o p e s emit b e t a p a r t i c l e s over a wide spectrum o f e n e r g i e s and e m u l s i o n s can be s e l e c t e d f o r v a r i o u s s e n s i t i v i t i e s . E f f i c i e n c y E f f i c i e n c y has been d e f i n e d f o r r a d i o a u t o g r a p h s as the number o f g r a i n s produced i n t h e volume o f e m u l s i o n under t h e s o u r c e p e r r a d i o a c t i v e d i s i n t e g r a t i o n i n t h e s o u r c e . M a j o r f a c t o r s i n f l u e n c i n g e f f i c i e n c y a r e energy o f t h e b e t a p a r t i c l e s e m i t t e d , t h i c k n e s s o f the e m u l s i o n and t h i c k n e s s o f t h e s o u r c e . The l o w e r t h e energy o f the b e t a p a r t i c l e e m i t t e d , t h e l e s s t h e chance o f t h e p a r t i c l e r e a c h i n g t h e em u l s i o n due t o s e l f a b s o r p t i o n i n the s e c t i o n . T h i s means i s o t o p e s l i k e t r i t i u m (0.018 MEV) w i t h low energy b e t a p a r t i c l e s t e n d t o have low e f f i c i e n c i e s . On the o t h e r hand, t h e l o w e r t h e energy o f t h e b e t a p a r t i c l e s t h a t r e a c h t h e e m u l s i o n , t h e h i g h e r t h e p r o b a b i l i t y o f t h e s e p a r t i c l e s c a u s i n g g r a i n f o r m a t i o n i n t h e e m u l s i o n . T h i s means i s o -22 * t o p e s o f h i g h energy l i k e Na (0.54 MEV) t e n d t o have low e f f i c i e n c i e s s i n c e p a r t i c l e s may pass c o m p l e t e l y t h r o u g h t h e e m u l s i o n w i t h o u t c a u s i n g l a t e n t image f o r m a t i o n . T h i s i s e s p e c i a l l y t r u e o f p a r t i c l e s o v e r 0.500 MEV. Herz's (44) t a b u l a t i o n o f g r a i n s produced i n A.R.-10 per b e t a p a r t i c l e e m i t t e d t o the e m u l s i o n f o r d i f f e r e n t i s o t o p e s , ranged from 2 g r a i n s p e r p a r t i c l e f o r carbon-14 (0.155 MEV) t o 0.8 g r a i n s p e r p a r t i c l e f o r phosphorus-32 (1.7 MEV). - 35 As the t h i c k n e s s o f the s e c t i o n i s i n c r e a s e d , s e l f a b s o r p t i o n o f p a r t i c l e s by the specimen i n c r e a s e s . A maximum p o i n t i s r e a c h e d where i n c r e a s i n g t h e t h i c k n e s s o f the s e c t i o n no l o n g e r produces an i n c r e a s e i n the l a t e n t images o f t h e e m u l s i o n . At t h i s p o i n t t h e s e c t i o n i s s a i d t o be i n f i n i t e l y t h i c k . When the s e c t i o n i s s u f f i c i e n t l y t h i n f o r s e l f a b s o r p t i o n t o be m i n i m a l i t i s s a i d t o be i n f i n i t e l y t h i n . At i n f i n i t e t h i c k n e s s s e l f a b s o r p t i o n by t h e s e c t i o n makes the number o f l a t e n t images formed i n t h e e m u l s i o n p r o p o r t i o n a l t o t h e i s o t o p e c o n c e n t r a t i o n o f t h e specimen but independent of i t s a b s o l u t e amount. The t h i c k -ness o f s e c t i o n s used i n t h e s e e x p e r i m e n t s ( 1 5 J - 0 p r o b a b l y r e p r e s e n t s i n f i n i t e t h i c k n e s s f o r ""Na7*. These r a d i o a u t o -g r a p h s , when t r e a t e d q u a n t i t a t i v e l y , a r e p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n o f t h e i s o t o p e i n the s e c t i o n , but a r e not an a c c u r a t e r e f l e c t i o n o f t h e a b s o l u t e amount. The e x t e n t t o w h i c h s e l f a b s o r p t i o n d e c r e a s e s t h e e f f i c i e n c y depends on t h e energy o f t h e i s o t o p e . H e n d l e r (45) has showi t h a t f o r carbon-14 (0.155 MEV) and 10p. s e c t i o n s , 30% o f t h e b e t a p a r t i c l e s do not r e a c h t h e e m u l s i o n . T r i t i u m (0.018 MB/") r e p r e s e n t s t h e extreme c a s e , where s e l f a b s o r p t i o n i s t h e s i n g l e most i m p o r t a n t f a c t o r i n e f f i c i e n c y . 22 -L. F o r h i g h energy i s o t o p e s l i k e Na , i n c r e a s i n g t h e t h i c k n e s s o f t h e e m u l s i o n w i l l i n c r e a s e t h e e f f i c i e n c y o f the r a d i o a u t o g r a p h s . T h i c k n e s s o f t h e e m u l s i o n i s t h e c r i t i c a l f a c t o r f o r q u a n t i t a t i v e work w i t h h i g h energy b e t a e m i t t e r s i f any comparison i s t o be made between - 3 6 specimens. L i q u i d emulsions produce a v a r i a b l e t h i c k n e s s f o r each batch of s l i d e s prepared. This was a major f a c t o r i n d e c i d i n g to use A.R.-10 s t r i p p i n g f i l m which Kodak claims to vary o n l y 1 0 % from the 5 ^ t h i c k n e s s . There are few r e l i a b l e estimates of e f f i c i e n c y i n the l i t e r a t u r e . For c a l c u l a t i n g the approximate exposure time and the minimum q u a n t i t y of i s o t o p e needed, Rogers ( 4 6 ) has estimated that f o r a 5 micron emulsion l a y e r and a 5 2 2 + micron s e c t i o n t h i c k n e s s , Na produced 2 0 to 3 0 g r a i n s i n the emulsion f o r a hundred d i s i n t e g r a t i o n s i n the specimen. Pr e v i o u s Techniques f o r S o l u b l e Radioautography Consider the problems posed by the radioautography with the h i g h l y s o l u b l e i o n 2 2 N a * . What i s needed are t h i n t i s s u e s e c t i o n s i n which i o n m i g r a t i o n i s i n h i b i t e d d u r i n g p r e p a r a t i o n . T h i s m i g r a t i o n can occur w i t h i n the s e c t i o n i t s e l f or l a t e r at the i n t e r f a c e between the emulsion and t i s s u e . The l o s s of o n l y small amounts o f r a d i o a c t i v i t y to s o l v e n t s or embedding media i n a r a d i o a u t o g r a p h i c procedure may seem t o l e r a b l e . However, t h i s i m p l i e s r e d i s t r i b u t i o n o f . the l a b e l w i t h i n the t i s s u e . A step which has l o s s of r a d i o -a c t i v i t y from the specimen i s i n v a l i d a t i n g . The c l a s s i c a l h i s t o l o g i c a l techniques of f i x a t i o n , d e h y d r a t i o n and wax embedding seem p e r f e c t l y s u i t e d to e x t r a c t l a b e l l e d m a t e r i a l . They are c l e a r l y inadequate. The same i s t r u e f o r any technique i n v o l v i n g the o v e r l a y i n g o f wet emulsion. Stumpf and Roth ( 4 7 ) have p o i n t e d out t h a t f i x a t i o n r e f e r s to the d e n a t u r a t i o n of p r o t e i n s and does not - 3 7 n e c e s s a r i l y imply b i n d i n g , t r a p p i n g or i m m o b i l i z i n g o f compounds a d m i n i s t e r e d as t r a c e r s . Kaminski ( 4 8 ) found with v a r i o u s f i x a t i v e s the amount of r a d i o a c t i v i t y l o s t i n f i x -a t i o n v a r i e d from 2 5 - 9 0 % f o r phosphorus-32 to 4 - 2 0 % f o r i o d i n e - 1 3 1 . F i x a t i o n may remove not o n l y the t r a c e r substance but s i g n i f i c a n t amounts of t i s s u e as w e l l . Merriam ( 4 9 ) r e p o r t e d t h a t 1 0 % formaldehyde f i x a t i o n e x t r a c t e d 4 % of the t o t a l p r o t e i n o f muscle and 6% of the t o t a l p r o t e i n o f l i v e r . Even formaldehyde vapour f i x a t i o n has been demon-s t r a t e d t o be capable of producing i s o t o p e t r a n s l o c a t i o n ( 4 7 ) . To date, r a p i d l y f r e e z i n g t i s s u e i n isopentane made v i s c o u s by l i q u i d n i t r o g e n seems the o n l y c e r t a i n way to immobilize i o n s . Appleton ( 5 0 ) has d e t e c t e d no r a d i o -a c t i v i t y i n l i q u i d isopentane used to f r e e z e t i s s u e samples 2 2 + c o n t a i n i n g h i g h c o n c e n t r a t i o n s o f Na T. Trump et a l ( 5 1 ) r e p o r t e d l o c a l i z e d d i s r u p t i o n s o f the c e l l u l a r membranes f o l l o w i n g the r a p i d f r e e z i n g of t i s s u e s i n v i s c o u s isopen-tane. T h i s c o u l d a l l o w the r a p i d i n f l u x o f e x t r a c e l l u l a r m a t e r i a l or the escape of c e l l u l a r c o n s t i t u e n t s . However, Smith ( 5 2 ) has shown membrane i n j u r y occurs f o l l o w i n g i c e c r y s t a l f o r m a t i o n , and t h e r e f o r e a f t e r f r e e z i n g has taken p l a c e . F u r t h e r movement of compounds w i t h i n the t i s s u e would be r e s t r i c t e d by the extreme r a p i d i t y of the f r e e z i n g . Trump et a l ( 5 1 ) demonstrated the c e n t r a l p a r t of a t i s s u e b l o ck 5 x 3 x 1mm reached - 1 7 5 ° C w i t h i n s i x seconds a f t e r immersion i n v i s c o u s isopentane. .-38 Proceeding from f r o z e n t i s s u e s e v e r a l approaches have been t r i e d . Wilske and Ross (53) took s m a l l f r o z e n s e c t i o n s and f r e e z e d r i e d them. These f r e e z e d r i e d s e c t i o n s were f i x e d i n formaldehyde vapour, and then embedded i n epon. Thin s e c t i o n s were cut with a g l a s s k n i f e and were dipped i n l i q u i d emulsion. The technique achieved good r e s u l t s with l a b e l l e d a s p i r i n . However, t r a n s l o c a t i o n of the h i g h l y s o l u b l e i o n 2 2 N a + could occur d u r i n g the epon embedding or when the s e c t i o n s were f l o a t i n g i n the boat of the g l a s s k n i f e . C u t t i n g f r o z e n s e c t i o n s i n a c r y o s t a t does ensure t h a t no t r a n s l o c a t i o n o c c u r s . U n f o r t u n a t e l y c r y o s t a t s e c t i o n s are not as s a t i s f a c t o r y as those prepared by wax embedding. Minute i c e c r y s t a l s may have formed d u r i n g the f r e e z i n g o f the t i s s u e g i v i n g a moth-eaten appearance to the s e c t i o n s a t h i g h m a g n i f i c a t i o n s . S e c t i o n s are f r a g i l e and f r e q u e n t l y may have t e a r s or w r i n k l e s . U l l b e r g (54) f i r s t suggested p o l y v i n y l c h l o r i d e backed tape to p i c k up s e c t i o n s and to press them onto emulsion coated s l i d e s . D i s s o l v i n g the tape i n acetone was necessary a t the end of exposure before development could proceed. Rabinow and Barry (55) m o d i f i e d t h i s technique by l e a v i n g the s e c t i o n s on the tape to f r e e z e dry o v e r n i g h t i n the c r y o s t a t . The f o l l o w i n g day s e c t i o n s were mounted at room temperature by p r e s s i n g the p o l y v i n y l tape a g a i n s t emulsion coated s l i d e s . D i f f i c u l t y i n removing the tape and l a c k of good s e c t i o n h i s t o l o g y on removal of the tape - 39 make these procedures cumbersome. The sandwich technique was f i r s t described by Kinter, Leape and Cohen (56). They simply placed frozen sections between a glass slide and an emulsion coated slide. The technique was later modified by coating the non-emulsion slide of the sandwich with Saran F-120 (Dow Chemicals)(57) . This was an attempt to prevent adhesion of the section to the glass slide at the termination of exposure. In both experiments i t was noted that permit-ting the section to thaw for only one second before contact-ing the emulsion allowed translocation of the label and prevented isotope localization to specific cellular structures. Although these authors reported no pressure artifacts, Stumpf and Roth (47) found such artifacts a major hazard of the sandwich technique. Horowitz and Finichel (58) coated the non-emulsion slide of the sandwich with teflon spray. These authors noted pressure artifacts and positive chemography in their radioautographs. Unfortunately they did not u t i l i z e controls to check the extent or consistency of these artifacts in their procedure. Roberts, Ciofalo and Martin (59) picked sections up on teflon slides. These sections were freeze dried at low pressure and then used to prepare a sandwich. The complications reported by these authors were pressure artifacts and d i f f i c u l t y in maintaining contact between section and emulsion on removal of the teflon slide. In summary, sandwich techniques appear promising - 40 since they allow no opportunity for translocation of the l a b e l . However, section adherence to the wrong s l i d e at the end of exposure and pressure a r t i f a c t s are major complications. In a recent paper Stumpf and Roth (47) evaluated a number of methods fo r t r e a t i n g frozen sections. Sections were cut only 0.5 - Ip. thick at -70°C using a s p e c i a l cryostat and a glass k n i f e . The following procedures were examined: (1) frozen sections were cut, freeze dried and mounted at room temperature by gentle manual pressure between t e f l o n support and emulsion coated s l i d e (a clamping technique previously used was rejected due to the production of pressure a r t i f a c t s ) ; (2) frozen sections were cut, mounted on thawed glass s l i d e s and dipped i n l i q u i d emulsion; (3) tissue sections were freeze dried, fixed i n the vapour phase, embedded in epoxy and dipped i n l i q u i d emulsion (similar to Wilske and Ross); (4) tissue sections were fixed in formalin, embedded i n p a r a f f i n and dipped i n l i q u i d emulsion. Method (1) gave good l o c a l i z a t i o n of the l a b e l with no evidence of translocation. Methods (2), (3) and (4) showed d i f f u s i o n of the l a b e l and loss of tissue r a d i o a c t i v i t y . Several of these techniques with obvious loss or trans-l o c a t i o n of the l a b e l gave reproducible r e s u l t s . Thus, r e p r o d u c i b i l i t y of r e s u l t s i s no c r i t e r i o n for accurate - 41 l o c a l i z a t i o n . In some sections excellent l a b e l l i n g of the lumen of a blood vessel was accompanied by spread or loss of isotope from adjacent structures. Therefore d i f f e r e n t i a l loss of a c t i v i t y can occur from adjacent s i t e s i n the same section. In a subsequent paper, Stumpf, Roth and Brown (60) u t i l i z e d the f i r s t method to determine the location of three l a b e l l e d e x t r a c e l l u l a r space markers ( i n u l i n , mannitol, sulphate) i n the superior c e r v i c a l sympathetic and nodose ganglia of cats. Except f o r a small quantity of i n t r a -neuronal a c t i v i t y there was good l o c a l i z a t i o n of these markers to the e x t r a c e l l u l a r space. The method of Stumpf and Roth shows promise but i s t e c h n i c a l l y d i f f i c u l t . Thin freeze dried sections have the consistency of fine powder and good h i s t o l o g i c a l preserv-ation i s d i f f i c u l t . The p o s s i b i l i t y of translocation or sublimation of the l a b e l i n the freeze drying of tissue must be considered. To date the' most promising technique i n i t s s i m p l i c i t y and r e l i a b i l i t y i s that of Appleton ( 6 l ) . He cut frozen sections i n darkness with safe l i g h t illumiration. To f a c i l i t a t e mounting a temperature d i f f e r e n t i a l was main-tained between tissue section and coverslip by storing the coverslips i n a separate container at -10°C. Mounting was accomplished by touching a c h i l l e d coverslip coated with A.R.-10 str i p p i n g f i l m to the section on the cryostat blade at -20°C. Appleton concluded that neither freeze s u b s t i t -ution i n absolute ethanol at dry ice temperature nor vacuum - 4 2 d e h y d r a t i o n a t low temperature showed any advantage over the storage of s e c t i o n s at - 2 5 ° C . In any case, s e c t i o n s are probably r a p i d l y dehydrated even a t - 2 5 ° C . In a subsequent paper Appleton ( 6 2 ) examined the 2 2 + r e s o l v i n g power o f h i s t e c h n i q u e f o r Na and A.R.-10 s t r i p p i n g f i l m at d i f f e r e n t temperatures. The best t h e o r -e t i c a l r e s o l u t i o n was 5 u . The r e s o l u t i o n achieved was 9 - 1 3 u , or 4 - % i g r e a t e r than the t h e o r e t i c a l r e s o l u t i o n . Appleton concluded t h a t i f t h i s d i f f e r e n c e was caused by d i f f u s i o n i t was o c c u r r i n g over the short d i s t a n c e o f 5 u . The optimum exposure temperature f o r A.R.-10 was found to be - 2 5 ° C by examining the s e n s i t i v i t y o f the f i l m at various -temperatures. At lower temperatures the s e n s i t i v i t y o f the emulsion decreased but the background d i d n o t . At the hi g h e r temperature of -10°C unmistakable a r t i f a c t s occurred under the s e c t i o n s . These a r t i f a c t s were probably due to p o s i t i v e chemography which i s temperature dependent. A p p l e t o n T s background f o r A.R.-10 and 2 2 N a + was 1 . 4 - 2 . 4 grains/lOO^i . Methods P r e p a r a t i o n of A.R.-—10 S t r i p p i n g F i l m A l l p r e p a r a t o r y steps were c a r r i e d out three f e e t from a Wratten s e r i e s 2 s a f e l i g h t . The f i l m i s packaged w i t h i t s g e l a t i n l a y e r mounted on g l a s s . Squares of f i l m were cut with a s c a l p e l and g e n t l y s t r i p p e d o f f the g l a s s . These squares o f f i l m were p l a c e d i n a water bath with the - 4 3 emulsion s i d e o f the f i l m f a c i n g upwards. The cut s e c t i o n s of f i l m f l o a t e d on the bath, imbibed water and s w e l l e d . Two percent by volume g l y c e r o l was added to the bath, f o l l o w i n g the recommendation of Rogers ( 4 6 ) . T h i s maintained the g e l a t i n i n a p l i a b l e s t a t e and allowed the subsequent adhesion between s e c t i o n and emulsion to be f i r m . A f t e r t hree minutes i n the bath the f i l m (emulsion s i d e up), was pi c k e d up on a s l i d e which was coated p r e v i o u s l y with g e l a t i n . The s l i d e p l u s f i l m was p l a c e d i n a l i g h t - t i g h t box c o n t a i n i n g s i l i c a g e l and allowed to dry o v e r n i g h t . D r i e d s l i d e s were s t o r e d i n l i g h t - t i g h t boxes a t - 2 5 P C . F r e e z i n g and Mounting o f Muscle F i b e r s A l l muscle f i b e r s used i n r a d i o a u t o g r a p h i c e x p e r i -ments were f r o z e n by immersion i n v i s c o u s isopentane (-155°C) c o o l e d by l i q u i d n i t r o g e n ( - 1 9 6 ° C ) . Although f r e e z i n g probably takes p l a c e i n s t a n t a n e o u s l y , f i b e r s were l e f t i n the f r e e z i n g mixture f o r 3 0 seconds before t r a n s f e r t o the c r y o s t a t (I.E.C. I n t e r n a t i o n a l H a r r i s C r y o s t a t , Model C.T.D.) f o r s e c t i o n i n g at - 2 5 ° C . T h i s c r y o s t a t was m o d i f i e d so the temperature s e n s i n g probe was at the blade l e v e l r a t h e r than at the c r y o s t a t bottom. When i n the c r y o s t a t , f i b e r s were handled o n l y with c h i l l e d instruments. C y l i n d r i c a l s e c t i o n s 3mm l o n g were cut from the cente r o f the muscle f i b e r s and mounted on the microtome chuck. The method of mounting these muscle c y l i n d e r s on the chuck i s c r i t i c a l to the whole procedure. I f the t i s s u e i s a l lowed to thaw while s i t t i n g i n a drop o f mounting - 4 4 media t r a n s l o c a t i o n of the l a b e l w i l l occur. A t h i c k s l u s h o f carboxymethyl c e l l u l o s e as recommended by U l l b e r g ( 5 4 ) was found to be s u p e r i o r to water or to d i l u t e g e l a t i n as a mounting media. T h i s m a t e r i a l i s i n s o l u b l e i n water. The water carboxymethyl c e l l u l o s e s l u s h must be s t i r r e d immediately before use s i n c e no l a s t i n g suspension w i l l form. Once a drop of the s l u s h i s d e p o s i t e d by Pasteur p i p e t t e the carbo-xymethyl c e l l u l o s e tends to s e t t l e out and water can be s u c t i o n e d back i n t o the p i p e t t e . Mounting was accomplished i n two stages. F i r s t the f r o z e n c y l i n d e r of muscle was taken w i t h p r e c o o l e d f o r c e p s and a p p l i e d to a drop of carboxymethyl c e l l u l o s e which was almost s o l i d l y f r o z e n on the chuck. I f the s e c t i o n sank more than 1mm i n t o the drop i t was d i s c a r d e d as u n s u i t -a b l e f o r subsequent s t e p s . F o r t u n a t e l y a drop blanches as i t f r e e z e s on the chuck so the proper moment f o r a p p l i c a t i o n of the muscle can be gauged. In the second stage the chuck wi t h drop and s e c t i o n was turned sideways i n order to i n s p e c t the mounting of the f i b e r c y l i n d e r . Carboxymethyl c e l l u l o s e was a p p l i e d to the s i d e s of the lower 2mm o f the f i b e r c y l i n d e r u s i n g the d e p o s i t i o n - s u c t i o n method. S e c t i o n s were taken o n l y from the top 1mm o f the muscle c y l i n d e r . T h i s was the o n l y p a r t of the c y l i n d e r which had not contacted mounting medium. Only with such an approach i s there reasonable hope of no t r a n s l o c a t i o n o c c u r r i n g i n the top m i l l i m e t e r o f the f i b e r c y l i n d e r . T h i s assumption i s j u s t i f i e d s i n c e - 45 thawing has not been v i s u a l l y detected and mounting media has not contacted the top 1mm of the muscle c y l i n d e r . The Technique f o r Radioautography The technique used f o r radioautography was a m o d i f i c a t i o n of Appleton's technique (6 l ). S l i d e s coated w i t h s t r i p p i n g f i l m (emulsion side up) were used r a t h e r than c o v e r s l i p s because of t h e i r l a r g e mass. Cooled s l i d e s r e q u i r e more heat to thaw than do cooled c o v e r s l i p s . Thus the r i s k of a c c i d e n t a l l y thawing s e c t i o n s i s l e s s w i t h cooled s l i d e s than w i t h c o v e r s l i p s . Another advantage i s t h a t s l i d e s are much e a s i e r to handle than c o v e r s l i p s under safe-l i g h t c o n d i t i o n s . I t was found to be d i f f i c u l t to c o n s i s t -e n t l y store s l i d e s at a higher temperature i n a separate container ( -15°C). A s p e c i a l p l e x i g l a s s c r y o s t a t s h e l f was b u i l t and s l i d e s were stored at c r y o s t a t temperature ( - 25°C). An arrangement of a s a f e - l i g h t and a mobile pole type of tungsten l i g h t was u t i l i z e d to a l l o w s e c t i o n i n g i n normal l i g h t . F i v e to s i x s e c t i o n s were cut at -25°C and the tungsten l i g h t switched o f f by simple overhead reach. Since the s a f e - l i g h t was l e f t continuously on, i t adequately i l l u m i n a t e d the c r y o s t a t blade three f e e t away. A s l i d e was now e x t r a c t e d from the s l i d e storage box on the c r y o s t a t s h e l f . The p r e v i o u s l y cut s e c t i o n s were brushed from the c r y o s t a t blade with a f i n e camel's h a i r brush onto t h i s s l i d e held below and p a r a l l e l to the blade. Next, the s l i d e w i t h the s e c t i o n s r e s t i n g on the emulsion was manipulated to ensure uniform a p p l i c a t i o n of the s e c t i o n s to the - 46 emulsion. Now the s l i d e with sections firmly adherent to the emulsion was placed i n the second l i g h t t i g ht box. Then the box was taped shut, sealed i n an a i r tight p l a s t i c bag and quickly transferred to the deep freeze (Viking V e r t i c a l Model V6305) for storage at -25°C. Appleton (63) has subsequently converted to a similar routine and no longer cuts sections under sa f e l i g h t i l l u m i n a t i o n . The manipulation to ensure adhesion f o r the duration of exposure consisted of gently pushing the sections against the emulsion with a t e f l o n plunger. This plunger was con-structed from the standard I.E.C. suction cup and a 1 inch square of t e f l o n . Control s l i d e s with non-radioactive speci-mens revealed that no s i g n i f i c a n t pressure a r t i f a c t s resulted from t h i s procedure. However, i t was impossible to consist-ently apply the same pressure to a series of s l i d e s . Thus, non uniform treatment of s l i d e s and tissue deformation were potent i a l problems. In the f i n a l technique, s l i d e s with the brushed on sections were inverted onto a 4 inch square o f t e f l o n . The square of t e f l o n was held f o r convenience by s i l i c o n grease to the top of a s l i d e box. This procedure created a temporary sandwich with emulsion, sections and t e f l o n sheet as the layers. The advantage of t h i s f i n a l procedure i s uniform gentle contact pressure ensured by the fact that only the weight of the s l i d e presses the sandwich together. Rogers (42) considers h i s variable success with the Appleton technique i s due to the f a i l u r e to achieve uniform contact between section and emulsion. - 4 7 Appleton ( 6 3 ) d i d not experience any s i g n i f i c a n t l o s s of s e c t i o n s from the emulsion with l o n g term exposures even up to one year. The l o s s o f s e c t i o n s from the emulsion was a major problem i n the course of the development of the technique r e p o r t e d here. The steps of the technique e s s e n t i a l to the p r e v e n t i o n of t h i s l o s s of s e c t i o n s were the a d d i t i o n of g l y c e r o l to the water bath p r i o r to the p r e p a r a t i o n of f i l m and the i n v e r s i o n o f emulsion and s e c t i o n s onto t e f l o n d u r i n g the mounting procedure. A l s o , f o r storage i t seemed advantageous to u t i l i z e g r a v i t y as a p o s i t i v e f a c t o r i n adhesion. A c c o r d i n g l y s l i d e s were p l a c e d i n the storage box so t h a t the emulsions with adherent s e c t i o n s a l l f a c e d the same d i r e c t i o n . To u t i l i z e g r a v i t y , boxes were s t o r e d on end r a t h e r than f l a t . Treatment and P r o c e s s i n g at the Termination of Exposure F i x a t i o n A l l s o l u t i o n s used i n f i x a t i o n and p r o c e s s i n g were kept a t 19°C to prevent s w e l l i n g of the g e l a t i n emulsion l e a d i n g to detachment of the s e c t i o n . P r e l i m i n a r y experiments i n d i c a t e d t h a t the most s u i t a b l e exposure time was 7 days. At t h e end o f exposure s l i d e s were removed from the deep f r e e z e and thawed f o r 2 minutes under a s a f e - l i g h t . The thawed s l i d e s were immersed i n 70% ethanol f o r 10 seconds i n o r d e r to prevent n e g a t i v e chemography due to d i f f u s i o n of s o l u b l e enzymes d u r i n g p r o c e s s i n g ( D a r z y n k i e w i c z ) ( 6 4 ) . A f t e r d r y i n g at room temperature i n a i r f o r 15 minutes f i x a t i o n was continued f o r 5 minutes i n 4 % b u f f e r e d f o r m a l -dehyde (phosphate b u f f e r PH7.4). S l i d e s were then washed f o r - 4 8 1 0 minutes i n water. Development A.R.-10 has an optimum development time of 5 - 6 minutes f o r maximum specimen g r a i n s with minimum background. However the g r a i n counting procedure allowed f o r a maximum of 4 0 - 5 0 grains/lOOu o f emulsion. T h i s g r a i n l e v e l was achieved i n the l o n g e s t l a b e l l e d specimens a t 4 minutes. Thus 4 minutes was the development time used. S l i d e s were p l a c e d i n Kodak D-19 developer f o r e x a c t l y 4 minutes w i t h a g i t a t i o n 5 times a t 1 minute i n t e r v a l s . A water stop bath was used f o r 3 0 seconds. Next s l i d e s were f i x e d i n Kodak F - 2 4 f i x e r (nonhardening) f o r 6 minutes w i t h a g i t a t i o n a t 1 minute i n t e r v a l s . S l i d e s were then p l a c e d i n water f o r 1 0 minutes and normal l i g h t i n g resumed. S t a i n i n g and Mounting The s t a i n i n g procedure was as f o l l o w s : H a r r i s Haematoxylin d i l u t e d 1.3 wit h water . . 1 5 minutes Wash i n water 1 9 C 3 0 seconds D i f f e r e n t i a t e i n 0 . 2 % aqueous HC1 2 0 seconds Blue i n water 19°C 1 0 minutes E o s i n 1% aqueous 2 0 seconds Wash i n water 19°C 3 0 seconds S l i d e s were than processed through graded a l c o h o l s to xy l e n e . The c o v e r s l i p was mounted with Permount. When the mount had hardened, excess f i l m was removed from the back o f the s l i d e with a r a z o r b l a d e . - 4 9 G r a i n Counting Procedures Grains were counted at a 1 , 0 0 0 power wi t h a Bausch. and Lo.mb b i n o c u l a r microscope ( r e s e a r c h model). G r a i n counts were performed with a i d of a 70y. square g r i d s u b d i v i d e d i n t o 4 9 s m a l l e r squares {lOp. s i d e s ) . Two types of g r a i n c ounting procedures were per-formed. In the f i r s t type the g r i d was moved a c r o s s the l o n g e s t diameter o f the f i b e r c r o s s s e c t i o n to determine a g r a i n d e n s i t y p r o f i l e . S i n c e the average f i b e r c r o s s s e c t i o n i s 1 , 0 0 0 - l , $ 0 0 u i n diameter, a minimum of 1 5 - 2 0 g r i d p o s i t i o n s were necessary to t r a v e r s e a cross s e c t i o n . 2 At each g r i d p o s i t i o n a d i a g o n a l row of seven IOOLI squares was counted. In t h i s way, the f u l l 70LI width o f the g r i d was r e p r e s e n t e d . The d i r e c t i o n of the d i a g o n a l was a l t e r e d f o r each new g r i d p o s i t i o n to a l l o w random sampling. The average background was removed from each g r i d p o s i t i o n by s u b t r a c t i n g the number o f g r a i n s a s s o c i a t e d with a g r i d d i a g o n a l of emulsion away from the specimen. The second type of g r a i n count was f o r r e s o l u t i o n . For t h i s d e t e r m i n a t i o n the edge o f s e c t i o n i s c o n s i d e r e d a p o i n t source. The g r i d was l o c a t e d w i t h the f i r s t l i r E of lOOii^ squares on the t i s s u e edge; the other squares extend-i n g out i n t o the emulsion. Counting the seven squares i n each row gave the v a r i a t i o n i n response of the emulsion as a f u n c t i o n of the d i s t a n c e from the source edge. Photography In photographing radioautographs the s t a i n e d t i s s u e - 50 s e c t i o n tends to obscure the s i l v e r g r a i n s s i n c e the s e c t i o n s i t s on top of the emulsion. The major photographic problem i s t h a t g r a i n s are l o c a t e d throughout the t h i c k n e s s o f t h e 22 + emulsion with such a high energy i s o t o p e as N a . Thus i t i s impo s s i b l e to have both g r a i n s and t i s s u e s e c t i o n i n focus i n one photograph u s i n g o r d i n a r y t r a n s m i t t e d l i g h t i l l u m i n -a t i o n . With d a r k f i e l d i l l u m i n a t i o n the s i l v e r g r a i n s appear as l i g h t specks on a dark background s i n c e they s c a t t e r l i g h t . The t i s s u e s e c t i o n absorbs l i g h t and i s dark. The s o l u t i o n was to take two photographs u s i n g both dark f i e l d and t r a n s m i t t e d i l l u m i n a t i o n . A Nikon b i n o -c u l a r microscope (Model SKE) equipped w i t h a Z e i s s Automatic Camera was used. . The g r a i n s were focused w i t h dark f i e l d i l l u m i n a t i o n and an exposure of 1 - 1.5 minutes was made with Adox KB-14 f i l m . The d a r k f i e l d condensor was removed and the t i s s u e s e c t i o n s h a r p l y f o c u s e d u t i l i z i n g normal t r a n s m i t t e d i l l u m i n a t i o n . A second exposure was made w i t h -out movement o f the t i s s u e s e c t i o n . Thus, the area o f the t i s s u e s e c t i o n photographed was the same f o r both exposures. By comparing the d a r k f i e l d photomicrograph ( g r a i n s i n focus) w i t h the t r a n s m i t t e d l i g h t photomicrograph ( s e c t i o n i n focus) the r e l a t i o n o f the g r a i n s to the c e l l u l a r morphology may be examined. Experimental Barnacle muscle f i b e r s were maintained i n b a r n a c l e R i n g e r ' s s o l u t i o n (In M / l i t r e 0.450 Na +, 0.008 K. +, 0.020 C a + + , 0.010 Mg + +, 0.518 C l + , 0.025 T r i s ) at 2°C. Barnacle Ringer - 5 1 was used f o r a l l l a b e l l e d and u n l a b e l l e d s o l u t i o n s i n i n f l u x experiments. Two b a s i c r a d i o a u t o g r a p h i c i n f l u x experiments were done. One was a q u a n t i t a t i v e i n f l u x study u t i l i z i n g times 5, 2 0 , 6 0 and ISO minutes. The second was a study o f the s h o r t e s t p o s s i b l e times i n which Na* p e n e t r a t i o n o f the f i b e r can be d e t e c t e d by the r a d i o a u t o g r a p h i c t e c h n i q u e . For t h i s l a t t e r study s i n g l e f i b e r s were l e f t a t t a c h e d to the b a s e p l a t e and handled i n the f o l l o w i n g manner. A. 1 cm loop o f number 4 s u r g i c a l s i l k was t i e d t o the f i b e r tendon. The f i b e r was momentarily suspended i n a i r from a hook a t t a c h e d to a micromanipulator. A 1 0 ml v i a l of l a b e l l e d R inger's s o l u t i o n ( s p e c i f i c a c t i v i t y 1 0 0 Lie/ml) was p o s i t i o n e d on a p l a t f o r m j a c k immediately below the b a s e p l a t e of the f i b e r . The suspended f i b e r was momentarily immersed i n the l a b e l l e d R inger's s o l u t i o n by r a i s i n g the j a c k . A f t e r removal the f i b e r was immediately f r o z e n by t h r u s t i n g i t i n t o v i s c o u s isopentane. The procedure, from immersion i n the bath to f r e e z i n g , was conducted e i t h e r a t 0.5 minutes or 1.5 minutes. Some o f the 1.5 minute f i b e r s were washed f o r 0.5 minutes i n i s o t o n i c sucrose b e f o r e f r e e z i n g . Three radioautographs each with 5 c r o s s s e c t i o n s were prepared from each f i b e r . For t h e q u a n t i t a t i v e study a group of e i g h t f i b e r s from the depressor muscle was d i s s e c t e d a p a r t l e a v i n g t h e b a s e p l a t e attachment i n t a c t . T h i s f i b e r and b a s e p l a t e p r e -p a r a t i o n was submerged in- Na*" l a b e l l e d R inger's s o l u t i o n - 52 ( s p e c i f i c a c t i v i t y lOOuc/ml) a t 2°G. At p e r i o d s o f 5, 20, 60 and 180 minutes, 1 - 2 f i b e r s were taken from the bath, r i n s e d f o r 0.5 minutes i n i s o t o n i c sucrose, and then q u i c k l y f r o z e n i n v i s c o u s isopentane. Three radioautographs each with 5 c r o s s s e c t i o n s were prepared from each f i b e r . • For both types of i n f l u x experiments, one r a d i o -autograph was prepared w i t h n o n - r a d i o a c t i v e f i b e r s e c t i o n s as a c o n t r o l f o r d e t e c t i o n of p o s i t i v e chemography. A l s o one normal radioautograph with r a d i o a c t i v e t i s s u e s e c t i o n s was "fogged" by exposing i t to room l i g h t . T h i s s l i d e was the c o n t r o l f o r n e g a t i v e chemography. R e s u l t s Table XI shows the average background was 1.5 grains/lOO^i f o r a t o t a l area o f 1.2 x 10 p. counted. The data f o r the r e s o l u t i o n study i s i n Tables I, I I , I I I and IV. In a d d i t i o n , F i g u r e s 1, 2, 3 and 4 show the r e s o l u t i o n o b t a i n e d with d i f f e r e n t time i n t e r v a l s of f i b e r immersion i n i s o t o p e . The response of the emulsion to i o n i z i n g r a d i a t i o n i s p l o t t e d as a . f u n c t i o n of the d i s t a n c e from the s e c t i o n edge. Zero i s the s e c t i o n edge. P l u s d i s t a n c e s extend on the emulsion under the s e c t i o n and minus d i s t a n c e s are on the emulsion away from the s e c t i o n edge. For the numerical value o f r e s o l u t i o n , the background i s s u b t r a c t e d from the number o f g r a i n s over the s e c t i o n edge and the r e s u l t d i v i d e d by two. T h i s value i n g r a i n s i s i n d i c a t e d by the h o r i z o n t a l dashed l i n e on the - 53 gra p h . The d i s t a n c e (u) a s s o c i a t e d w i t h t h i s h a l f v a l u e o f g r a i n s i s t h e r e s o l u t i o n . T h i s v a l u e i s i n d i c a t e d by the v e r t i c a l dashed l i n e . The r e s o l u t i o n v a l u e s o b t a i n e d range from 17.5^ a t f i v e m i n u t e s t o 15p. a t 180 m i n u t e s . Thus, s l i g h t l y b e t t e r r e s o l u t i o n was a c h i e v e d w i t h t h e l o n g e r f i b e r immersion. I l l u s t r a t i o n s 9 t o 14 a r e ph o t o m i c r o g r a p h s o f t h e s h o r t term i n f l u x f i b e r s . These photographs show h i g h e r g r a i n d e n s i t y p e r u n i t a r e a t h a n were d e t e c t e d i n t h e g r a i n c o u n t s . T h i s i s due t o t h e f a c t t h a t g r a i n s j u s t below t h e l i m i t s o f v i s i b i l i t y w i t h t r a n s m i t t e d l i g h t appear w i t h d a r k f i e l d i l l u m i n a t i o n . G r a i n counts made under dark f i e l d l i g h t i n g c o n d i t i o n s would be p r o p o r t i o n a l t o th o s e made w i t h t r a n s m i t t e d l i g h t . A l l p h o t o m i c r o g r a p h s a r e t a k e n w i t h d a r k f i e l d l i g h t i n g c o n d i t i o n s u n l e s s o t h e r w i s e i n d i c a t e d . The g r a i n count r e s u l t s f o r the s h o r t term experiment a r e g i v e n i n T a b l e s V, V I , and V I I . The g r a i n c o u n t s were p l o t t e d as a f u n c t i o n o f t h e d i s t a n c e from the c r o s s s e c t i o n edge i n F i g u r e 5. Zero i s t h e s e c t i o n edge. P l u s d i s t a n c e s extend on t h e e m u l s i o n under t h e s e c t i o n . Minus d i s t a n c e s e x t e n d on the e m u l s i o n away from t h e s e c t i o n edge. The l e f t h a l f o f t h e graph from 0 t o 140 i s a r e s o l u t -i o n curve s i m i l a r t o F i g u r e s 1, 2, 3 and 4. Each curve was f i t t e d t o a r e s o l u t i o n o f Y] ,5)x on t h i s l e f t h a l f o f t h e graph (t h e l a r g e s t r e s o l u t i o n number found e x p e r i m e n t a l l y ) . Each e x p e r i m e n t a l p o i n t r e p r e s e n t s t h e mean from 3 - 6 c r o s s s e c t i o n s from each o f 3 f i b e r s t a k e n from one b a r n a c l e . - 5 4 The r i g h t h a l f of the graph from 0 to 700^. i s one h a l f a c r o s s s e c t i o n diameter, s i n c e the average f i b e r c r o s s s e c t -i o n was 1 5 0 0 u i n diameter. The t h r e e curves are l a b e l l e d a c c o r d i n g t o the time i n t e r v a l from immersion to f r e e z i n g . Only the 0.5 minute graph appears to change s i g n i f i c a n t l y a l o n g the f i b e r r a d i u s . T h i s curve f a l l s from approximately 2 2 3 0 grains/lOO^i to 3 grains/lOOji i n a d i s t a n c e o f 400;a a l o n g the c r o s s s e c t i o n diameter. T h i s d i s t a n c e r e p r e s e n t s about . 2 7 o f the c r o s s s e c t i o n diameter o r .54 o f the c r o s s s e c t i o n r a d i u s . The 1.5 minute graph shows a s t r a i g h t l i n e r e p r e s e n t i n g - a b o u t 2 0 grains/lOOji . The 1 . 5 minute graph w i t h 0 . 5 minute r i n s e graph i s lower than the 1.5 minute graph. I t i s a s t r a i g h t l i n e r e p r e s e n t i n g approximately 5 g r a i n s / 1 0 0 ^ 2 , An a n a l y s i s of v a r i a n c e f o r one of the c r o s s s e c t i o n s r e p r e s e n t e d by these curves i s given i n Tables V I I I , IX and X. This a n a l y s i s shows t h e r e i s a very s i g -n i f i c a n t d i f f e r e n c e {1% l e v e l ) between the experimental p o i n t s of curve ( 1 ) . There i s no s i g n i f i c a n t d i f f e r e n c e a t the 5% l e v e l between the experimental p o i n t s o f curves ( 2 ) and ( 3 ) . I l l u s t r a t i o n s 1 5 to 2 4 are photomicrographs o f the l o n g term i n f l u x f i b e r s . A dark f i e l d and a t r a n s m i t t e d l i g h t microphotograph i s p r o v i d e d f o r each time i n t e r v a l . An examination of the r e l a t i o n s h i p s o f the c l e f t s and n u c l e i i n a p a i r o f photomicrographs r e v e a l s t h a t they are p i c t u r e s o f the same area of the t i s s u e s e c t i o n . By r e l a t i n g - 55 s p e c i f i c areas of the d a r k f i e l d to the t r a n s m i t t e d l i g h t microphotograph the r e l a t i o n s h i p o f the s i l v e r g r a i n s to the c l e f t system may be examined. In some areas o f v a r i o u s d a r k f i e l d photomicro-graphs g r a i n s are arranged i n s t r a i g h t l i n e or c i r c u l a r p a t t e r n s . When these areas of p a t t e r n e d g r a i n s are r e l a t e d to the t r a n s m i t t e d l i g h t photomicrograph a c l e f t o f the same shape may be v i s i b l e . Thus, these g r a i n p a t t e r n s prob-a b l y r e p r e s e n t areas of myoplasm with Na* c o n t a i n i n g c l e f t s . The g r a i n d e n s i t y o f the d a r k f i e l d photographs i s seen to i n c r e a s e s t e a d i l y from the 5 to the ISO minute f i b e r s . The g r a i n count v a l u e s f o r the l o n g term i n f l u x experiment are t a b u l a t e d i n Table X I . Each value f o r a f i b e r r e p r e s e n t s the average value o f two to three c r o s s s e c t i o n s . For each c r o s s s e c t i o n approximately 15 - 20 g r i d counts were made as has been d e s c r i b e d . Each g r i d 2 count comprised seven lOOu areas counted. Each f i b e r v a l u e thus r e p r e s e n t s the mean of 3 x 15 x 7 or 300 to 4 0 0 , 2 100LI areas counted. These f i b e r v a l u e s were p l o t t e d semi-l o g a r i t h m i c a l l y a g a i n s t time i n F i g u r e 6. T h i s graph shows the i n f l u x as determined by radioautography was d i v i s i b l e i n t o two phases. There was an i n i t i a l r a p i d phase r e p r e s e n t i n g the e x t r a c e l l u l a r space. H a l f of the sodium content of t h i s compartment had ente r e d by ten minutes. Therefore the h a l f time of t h i s compartment was ten minutes. The s i z e o f t h i s compartment - 56 was estimated by extrapolating the portion of the curve f o r the slow compartment to t = 0. No absolute statement as to compartment size can be made as the radioautographs are only proportional to the amount of r a d i o a c t i v i t y present i n the specimens. The reason i s tissue specimens were cut at i n f i n i t e thickness. The compartment size has 67% of grains present at 180 minutes. This implies that of a l l the l a b e l l e d sodium present at ISO minutes, over one h a l f had f i l l e d the e x t r a c e l l u l a r space. The more slowly exchanging compartment was s t i l l exchanging at 1&0 minutes when the experiment was termin-ated. It was not possible to calculate a rate constant f o r t h i s compartment but the slope of the l i n e was 0.0S compared to 1.5 f o r the slope of the l i n e for the rapid compartment. The slower compartment must represent myoplasmic exchange. Grain density p r o f i l e data for the long-term radioautographs i s given i n Tables XII, XIII, XIV and XV. These grain counts r e l a t e the grain density to the distance from the cross section edge. This data i s plotted i n Figure 7. Zero i s the section edge and p o s i t i v e distances extend on the emulsion under the section. Minus distances extend on the emulsion away from the section edge. The l e f t h a l f of the curve from 0 - 140 i s a resolution curve similar to Figure 5. The point i n the curves at 17.5p- on t h i s side of the graph i s f i x e d . This means each curve has a f i t t e d resolution of 17.5u. This i s j u s t i f i e d since 17.5u was the largest experimental resolution detected. Each experimental point - 5 7 i s the average of 3 . 5 cr o s s s e c t i o n s r e p r e s e n t i n g 3 f i b e r s from the same b a r n a c l e . The r i g h t h a l f of the graph from 0 - 700ju i s a s e c t i o n of r a d i u s s i n c e the average f i b e r c r o s s s e c t i o n was l , 5 0 0 i i i n diameter. The f o u r curves are l a b e l l e d a c c o r d i n g to time spent i n i s o t o p e p r i o r t o f r e e z i n g . The f i v e minute p l o t shows a s t r a i g h t l i n e r e p r e s e n t i n g about 7.5 grains/lOOu . The twenty minute p l o t i s a s t r a i g h t l i n e s i g n i f y i n g approximately 2 0 g r a i n s / p lOOu . The s i x t y minute p l o t i s a s t r a i g h t l i n e r e p r e s e n t -i n g about 3 0 grains/lOOu . The 1 $ 0 minute p l o t i s a l s o a s t r a i g h t l i n e s i g n i f y i n g approximately 4 0 grains/IOC^ . J u s t i f i c a t i o n f o r these values as s t r a i g h t l i n e p l o t s comes from the a n a l y s i s o f v a r i a n c e i n Tables XVI, XVII and XVIII . These analyses r e p r e s e n t a c r o s s s e c t i o n diameter f o r th r e e d i f f e r e n t 5 minute f i b e r s o f th r e e d i f -f e r e n t b a r n a c l e s . In each case the F t e s t i s not s i g n i f i c a n t . T h i s means t h e r e i s no s i g n i f i c a n t d i f f e r e n c e between the means of g r i d s counted a t d i f f e r e n t p o i n t s on the cr o s s s e c t i o n diameter. To put i t another way, the v a r i a t i o n between g r i d counts does not d i f f e r from the normal v a r i a t i o n w i t h i n a g r i d count. A l l g r i d counts belong to the same p o p u l a t i o n . T h e r e f o r e there i s a homogeneous d i s t r i b u t i o n o f g r a i n s a c r o s s a f i b e r c r o s s s e c t i o n . S i n c e t h i s was t r u e at 1 . 5 and 5 minutes, no a n a l y s i s o f v a r i a n c e was done f o r the l o n g e r f i b e r t i m e s . The s l i d e s with n o n - r a d i o a c t i v e s e c t i o n s d i d not r e v e a l any s i g n i f i c a n t g r a i n c o n c e n t r a t i o n s under the - 58 specimens. This means positive chemography was not occurring to any extent. It also implies that the technique did not produce pressure a r t i f a c t s which were masked i n the radio-active specimens. The s l i d e s with radioactive specimens "fogged" by exposure to l i g h t gave no indicat i o n of negative chemography. There was no fading of the emulsion under the section and the emulsion remained uniformly "fogged". Discussion The range of background found ( 1 . 4 to 1 . 7 grains/ 2 2 10On ) and the mean background ( 1 . 5 grains/lOOji agrees favourably with Appleton's ( 6 1 ) background values f o r Na* and A.R.-10 ( 1 . 4 to 2 . 4 grains/lOOu ). This implies adequate precautions against inappropriate latent image formation. It indicates l i g h t proof during preparation, mounting, exposure and processing of the radioautographs. Table XIX shows the calculated e f f i c i e n c y to be 1 3 % . This i s somewhat lower than the value predicted by Rogers ( 2 0 - 3 0 % ) . However Roger's ( 4 6 ) value was for an i n f i n i t e l y t h i n specimen (5p.). S e l f absorption by the thicker specimens (15>i) probably i s the major factor i n the v a r i a t i o n . The resolution obtained ( 1 5 - 17.5^u) exceeds the t h e o r e t i c a l resolution ( IOLI) by values si m i l a r to Appleton's ( 6 1 ) ( 4 - SLI) f o r 2 2 N a f . If d i f f u s i o n i s taking place during the technique i t only occurs over a distance of 5 - 7 « 5 u . This implies the technique developed i s com-parable to Appleton's i n i t s usefulness for the radioautography - 59 of soluble substances. No translocation occurs because i f i t did the resolution would be i n the v i c i n i t y of 70u as detected by Appleton f o r his unsuccessful methods. The c l e f t system has a sodium concentration 0.450 M/kg HgO), approximately ten times the myoplasm (0.040 M/kg HgO) . Because of t h i s concentration difference i t was o r i g i n a l l y hoped to v i s u a l i z e the c l e f t s by radio-autography as discrete structures i n r e l a t i o n to the myo-plasm. This proved r a r e l y possible. The grain patterns observed i n I l l u s t r a t i o n s 17 and 19 probably represent discrete c l e f t s . The morphological study revealed no part of the myoplasm more than 1 - 2p from a patent c l e f t . Thus to consistently resolve c l e f t s from myoplasm, a radioautographic technique must have a resolution of l u . Otherwise, grains produced over the myoplasm by isotope a c t u a l l y i n the c l e f t s r e s u l t s i n a homogeneous grain d i s t r i b u t i o n over both c l e f t and myoplasm. The profusion of the c l e f t system i n three dimen-sions means i t i s possible f o r the c l e f t - c l e f t distance to be smaller than the cleft-myoplasm distance. Thus, to be v i s u a l i z e d , the c l e f t must be immediately adjacent to the emulsion to allow optimum resolution and s u f f i c i e n t l y f a r from other c l e f t s to prevent c r o s s f i r e e f f e c t s . To allow v i s u a l i z a t i o n of the c l e f t s , there must be s u f f i c i e n t Na r (grains) to present a pattern. However, i f the grains are too numerous the pattern w i l l be obscured. - 60 Once the rnyoplasmic l o a d i n g i s unmasked by the s a t u r a t i o n o f the e x t r a c e l l u l a r space (20 minutes), the i n c r e a s i n g l o a d i n g of the myoplasm w i l l tend to obscure the c l e f t s . Thus, the r a r i t y of the v i s u a l i z a t i o n of c l e f t s as d i s c r e t e s t r u c t u r e s can be e x p l a i n e d both on the b a s i s of the t r a n s i e n t nature o f the optimum c o n d i t i o n s f o r the o b s e r v a t i o n and i n terms of the l i m i t s imposed by the r e s o l u t i o n . The f a c t t h a t the r e s o l u t i o n improved with the l o n g e r d u r a t i o n of f i b e r immersion i n i s o t o p e can be e x p l a i n e d on the b a s i s o f g r a i n counts. When counting 40 grains/lOOLi 2 over a s e c t i o n edge the presence or absence of one g r a i n w i l l have l e s s e f f e c t on the r e s o l u t i o n c a l c u l a t i o n than when the number i s 8 grains/lOOti . The short term r a d i o a u t o g r a p h i c study i l l u s t r a t e s the r a p i d i t y w i t h which i s o t o p e enters the e x t r a c e l l u l a r 22 4-space. At 0.5 minutes the Na -was d i s t r i b u t e d on a g r a d i e n t t r a v e r s i n g h a l f the f i b e r radius.. Table XX i s a comparison of the experimental 22 + d i s t a n c e along the f i b e r r a d i u s the Na had d i f f u s e d from the s e c t i o n edge (r) to the c a l c u l a t e d t h e o r e t i c a l d i s t a n c e the i s o t o p e had d i f f u s e d to have the observed c o n c e n t r a t i o n a t 0.5 minutes ( x ) . The equation of Crank (65) i s used f o r these c a l c u l a t i o n s . The use o f t h i s equation presumes that 22 +• d i f f u s i o n o f Na i n the e x t r a c e l l u l a r space i s a non-steady s t a t e o f one dimensional d i f f u s i o n from a source o f f i x e d c o n c e n t r a t i o n . The value of the d i f f u s i o n c o e f f i c i e n t used f o r sodium i s f o r s e l f d i f f u s i o n i n d i l u t e s o l u t i o n s . - 61 Therefore, these calculations also assume d i f f u s i o n of 2 2 N a + i n the e x t r a c e l l u l a r space i s equivalent i n rate to s e l f d i f f u s i o n i n d i l u t e solutions. The close agreement i n Table XX between the observed experimental distance (r) and the t h e o r e t i c a l calculated distance (x) indicates both of the assumptions are r a t i o n a l . Diffusion of Na i n the e x t r a c e l l u l a r space follows a r e l a t i v e l y straight pathway towards the f i b e r center. Thus the radius of the f i b e r cross section i s a reasonable approximation of t h i s p a t h w a y T h e increas-ing deviation of the experimental distance (r) from the t h e o r e t i c a l distance (x) as the f i b e r center i s approached i s p l a u s i b l e . The r e s u l t s also indicate that d i f f u s i o n of 22 + Na i n the e x t r a c e l l u l a r space i s equivalent i n rate to d i f f u s i o n i n d i l u t e solutions. Table XXI i s a c a l c u l a t i o n based on the 0.5 minute curve. It supplies information on the geometry of the pa th-2 2 + way f o r e x t r a c e l l u l a r d i f f u s i o n of Na . The equations of B u l l ( 6 6 ) were u t i l i z e d . These equations r e l a t e the experi-22 4-mental r a t i o of the Na concentrations at the section edge and at a given distance along the f i b e r radius to the f i b e r 2 2 4-surface area for Na T entry. The calculations show approximately 6 0 % of the 22 + f i b e r surface i s t h e o r e t i c a l l y involved i n Na entry of the e x t r a c e l l u l a r space. This implies the c l e f t system should occupy at least h a l f of the f i b e r surface. This i s not completely incompatible with the morphology. Examination of an area of the edge of the f i b e r cross section i n - 62 I l l u s t r a t i o n 3 reveals the c l e f t s comprise at least 20%. These c l e f t s do not a l l deeply penetrate the f i b e r . The c a l c u l a t i o n may be i n error by a factor of two to three. Table XXII i s a cal c u l a t i o n u t i l i z i n g the equation of Crank (65) and based on the 0.5 minute curve. This c a l -culation reveals 6.11 minutes i s required f o r the concen-22 + t r a t i o n of Na i n the f i b e r center to reach h a l f the concen-22 + t r a t i o n of Na T at the section edge. This value i s i n good agreement with the hal f time f o r loading of the e x t r a c e l l u l a r space calculated f o r the long term radioautographic experi-ment (10 minutes). The r e s u l t of the movement of t h i s 2 2 quantity of Na + to the center would be 15 grains/lOOu 2 of emulsion under the center of the cross section. The grain value under the center of the f i v e minute cross sections in the long term radioautographic study was h a l f of this predicted value (7.6 grains/100^ 2). This emphasizes that 22 +• the sucrose rinse must remove about h a l f the Na from the e x t r a c e l l u l a r space. In summary, these secondary calculations based on the data for the 0.5 minute grain density p r o f i l e curve 22 + provide a comprehensive picture of Na d i f f u s i o n i n the e x t r a c e l l u l a r space. The path of entry f o r 2 2Na* i s the c l e f t system which t h e o r e t i c a l l y occupies over 50% of the f i b e r surface area. The radius of the f i b e r i s a reasonable 22 +• approximation of the Na T d i f f u s i o n pathway. Diffus i o n of 22 + Na i s as rapid i n the e x t r a c e l l u l a r space as i n d i l u t e 2 2 solutions. The time required for the Na + concentration of - 63 the f i b e r center to reach h a l f the concentration of the f i b e r edge i s approximately 6 minutes. The 1.5 minute grain density p r o f i l e curve i s suspect since the grain count at the periphery of the f i b e r 2 cross section (20 grains/lOOu ) i s lower than the grain count f o r the 0.5 minute curve (30 grains/lOOu ). A possible explanation i s that the 0.5 minute f i b e r s and the 1.5 minute f i b e r s are from d i f f e r e n t barnacles. These l.;5 minute f i b e r s without sucrose rinse showed a homogeneous isotope d i s t r i b u t i o n . This i s d i f f i c u l t to explain since six minutes should be required f o r the center of the f i b e r to reach one half the isotope concentration of the edge of the f i b e r . This calculated time value thus 22 + predicts a nonhomogeneous Na d i s t r i b u t i o n f o r 1.5 minute cross sections although the tracer gradient would be le s s marked than f o r the 0.5 minute f i b e r s . Again, the o r i g i n of these 1.5 minute f i b e r s from a d i f f e r e n t barnacle than the 0.5 minute f i b e r s may be responsible. Another p o s s i b i l i t y i s a g i t a t i o n or contracture of the 1.5 minute f i b e r s . These f i b e r s spent a f u l l minute longer i n the isotope bath than the 0.5 minute f i b e r s and would be more susceptible to a g i t a t i o n . Agitation i s postulated because of i t s known effe c t on the 1.5 minute f i b e r s with sucrose rinse.. The 0.5 minute sucrose rinse should re s u l t i n a heterogeneous.distribution i n the 1.5 minute f i b e r s i f the k i n e t i c s were the same as f o r the loading of the 0.5 minute f i b e r . The homogeneity of the 1.5 minute f i b e r with sucrose - 6 4 rinse may be explained i n terms of d i f f e r e n t k i n e t i c s . The i n f l u x i s an exchange d i f f u s i o n with no manipulation of the f i b e r . The rinse process i s a d i f f u s i o n into a sodium free isotonic sucrose solution with vigorous f i b e r a g i t a t i o n . 22 + This vigorous agita t i o n probably disperses whatever Na gradient the rinse creates i n the f i b e r . S i m i l a r l y inapprop-r i a t e a g i t a t i o n while i n the bath or during handling could account for the homogeneous d i s t r i b u t i o n of the 1.5 minute f i b e r s without r i n s e . The 1.5 minute f i b e r s without sucrose rinse were equivalent i n grains to the 2 0 minute f i b e r s of the long 2 term experiment (approximately 2 0 grains/lOOp ). When 1.5 minute f i b e r s were washed fo r 0.5 minutes in isotonic sucrose t h e i r grain value was homogeneous representing 5 grains/lOOu . ? 2 This change from 2 0 grains/lOO^i to 5 grains/lOO}i implies that 0.5 minute sucrose rinse washes away at least half of 2 2 i n i t i a l f i b e r Na*". This i s supported by experimental r e s u l t s of Hinke ( 6 7 ) . He found Balanus f i b e r s without a 0.5 minute sucrose rinse had a sodium concentration of 9 0 - 1 2 0 moles/kg H 2 O . Following the sucrose rinse this value was 6 0 - 7 0 moles/kg HgO, or approximately 5 0 % of the i n i t i a l concentration. The fact the grain value of the 1.5 minute f i b e r s with rinse i s similar to the 5 minute f i b e r s with rinse also support the concept that much of the i n i t i a l 22Na+" i s washed out. It i s known that the e x t r a c e l l u l a r space takes twenty minutes to load. The twenty minute f i b e r s would have - 6 5 2 2 + the same amount of Na removed from the e x t r a c e l l u l a r space by the rinse as other f i b e r s with d i f f e r e n t durations 2 2 + of Na exposure. The grain value of the twenty minute 2 2 + f i b e r s a f t e r the removal of t h i s amount of Na was 2 0 2 grains/lOOu . Thus the e x t r a c e l l u l a r space f i l l s i n such a manner that the 0 . 5 minute rinse no longer removes a large f r a c t i o n of the l a b e l . In other words, there i s a portion of the e x t r a c e l l u l a r space that loads progressively and i s inaccessible to the h a l f minute r i n s e . This was noted by Hinke ( 6 7 ) who demonstrated prolonging the sucrose rinse beyond 0.5 minutes did not remove appreciably more sodium from the f i b e r . It i s tempting to postulate the small terminal c l e f t s and T tubules represent t h i s component of the e x t r a c e l l u l a r space. The sodium which the rinse removes i s in the larger c l e f t s . The long term i n f l u x study i d e n t i f i e d the loading of the e x t r a c e l l u l a r space as an i n i t i a l rapid phase. The h a l f time of t h i s compartment was ten minutes and i t con-tained about h a l f the f i b e r sodium. This i s i n good agree-ment with previous work by Hinke ( 6 S ) . The more slowly changing compartment had a smaller slope and was s t i l l exchanging when the experiment was terminated. This i s com-patible with unmasking of the rnyoplasmic exchange following the saturation of the c l e f t system. A similar interpretation was given to experimental r e s u l t s by All e n and Hinke ( 3 1 ) . It was unfortunate t h i s graph derived from radioautographs did not allow absolute calculations of the amount of sodium - 6 6 i n each compartment. To determine t h i s and to substantiate the radioautographic plot i t was decided to perform an i n f l u x experiment emphasizing the shorter time i n t e r v a l s . This i n f l u x experiment would have no subtraction fo r the e x t r a c e l l u l a r space since the radioautographic study made no correction for t h i s compartment. The p r o f i l e analysis curves f o r the long term experiment reveal homogeneous d i s t r i b u t i o n at each time 2 2 -f i n t e r v a l . The time required f o r the . Na concentration of the f i b e r center to reach half the concentration of the f i b e r edge i s 6 minutes. The sucrose rinse probably destroyed 2 2 + the small Na T gradient that would exist i n the f i v e minute f i b e r s . Longer times would have a homogeneous d i s t r i b u t i o n 22r- 4-of Na i n the e x t r a c e l l u l a r space. The amount of v a r i a t -ion between experimental points i s small but these are f i b e r s from the same barnacle. Generally in grain counting procedures (see Table XI) the amount of variation between barnacles was much greater than the amount of v a r i a t i o n between f i b e r s of the same barnacle. TABLE I Data for 5 Minute Resolution Curve Barnacle C Fiber 1 Section b Grain Counts Within a Grid Line .2 grains/lOOu' Grid Line Number Distance of Grid Line from Section Edge ( L I ) 0 16 13 16 8 13 9 10 10 7 9 11 9 6 8 7 20 4 5 5 4 5 4 3 4 30 4 3 3 2 3 3 3 40 2 1 2 2 2 1 3 Mean and S.E. 12.1 - 1.1 8.1 ± .6 4.3 * .3 3 - .2 1.9 Background Resolution Grain Value 1.5 grains/lOOu 2 12.1 - 1.5 - 5.3 grains 2 F i g u r e 1 TABLE II Data for 2 0 Minute Resolution Curve Barnacle C Fiber 1 Section b Grain Counts Within a Grid Line grains/lOOii Grid Line Number Distance of Grid Line from Section Edge (LI) Mean and S.E. 0 2 4 1 7 1 7 1 4 23 1 9 2$ 2 0 . 3 1 0 1 0 7 2 0 1 5 1 4 13 1 6 2 0 3 9 9 7 1 0 7 4 3 0 4 0 5 0 2 1 2 2 3 1 4 7 6 0 1 2 1 1 1 3 * 1 . 7 1 3 . 6 ± 1 . 4 8.3 - . 4 5 . 7 - .3 3 . 2 * .3 2 . 1 ±.3 1.3 -. Background 1.4 grains/100/i Resolution 2 0 . 3 - 1 . 4 • 9.4 grains Grain Value 2 1 ON vO Figure 2 TABLE III Data f o r 6 0 Minute Resolution Curve Barnacle B Fiber 2 Section b Grain Counts Within a Grid Line 2 g r a i n s / 1 0 0 ^ Grid Line Number 4 Distance of Grid Line from Section Edge (;a) 0 11 3 3 3 0 23 3 0 3 1 1 0 1 5 1 7 1 4 1 4 1 8 2 0 1 7 2 0 7 1 9 7 1 1 1 2 13 1 1 3 0 5 7 3 8 5 7 4 4 0 2 3 2 4 3 2 4 Mean and S.E. 2 6 . 6 * 2.4 1 6 . 4 * .8 1 1 . 4 - 1 . 4 5 . 6 - . 6 2 . 9 Background 1.8 grains/lOOLi 2 Resolution Grain Value 2 6 . 6 - 1 . 8 - 1 2 . 4 grains 2 Figure 3 TABLE IV Data for 1 8 0 Minute Resolution Curve Barnacle A Fiber 2 Section a Grain Counts Within a Grid Line grains/lOOu Grid Line Number 1 Distance of Grid Line from section 0 Edge (LI) 5 0 4 2 4 4 4 9 4 6 3 5 4 2 Mean and S.E. ^ 4 4 - 1 . 8 Background 2 3 4 5 1 0 2 0 3 0 4 0 2 8 2 0 6 6 2 4 1 3 7 5 2 9 15 9 4 3 4 1 5 5 5 3 0 1 8 1 0 3 2 7 21 8 5 1 9 1 2 7 4 2 7 . 3 - 1 . 7 1 6 . 3 ± 1 . 2 7 . 4 ± . 6 4 1.5 grains/lOOu Resolution . 4 4 - 1.5 = 2 1 . 2 grains Grain Value 2 Figure 4 I l l u s t r a t i o n s 9 and 10 - 75 A p h o t o m i c r o g r a p h o f a 0.5 minute f i b e r c r o s s s e c t i o n . The p e r i p h e r y i s h e a v i l y l a b e l l e d . T h i s l a b e l l i n g extends i n w a r d a p p r o x i m a t e l y h a l f o f t h e f i b e r r a d i u s . M a g n i f i c a t i o n 90x A p h o t o m i c r o g r a p h o f t h e same 0.5 minute f i b e r c r o s s s e c t i o n t a k e n a l o n g t h e a x i s o f the f i b e r d i a m e t e r . Note t h e heavy p e r i p h e r a l l a b e l l i n g . M a g n i f i c a t i o n 220 x - 76 I l l u s t r a t i o n s 11 and 12 A p h o t o m i c r o g r a p h o f a 0.5 minute f i b e r c r o s s s e c t i o n . Note t h a t t h e p e r i p h e r y i s h e a v i l y l a b e l l e d . T h i s l a b e l l -i n g has d e c r e a s e d t o background l e v e l s a t t h e f i b e r c e n t e r . M a g n i f i c a t i o n 90 x A p h o t o m i c r o g r a p h o f t h e same 0.5 minute f i b e r s t a k e n a l o n g t h e a x i s o f t h e f i b e r d i a m e t e r . M a g n i f i c a t i o n 220 x I l l u s t r a t i o n s 13 and 14 A ph o t o m i c r o g r a p h o f an a r e a o f a c r o s s s e c t i o n from a 1.5 minute f i b e r w i t h a 0.5 minute s u c r o s e r i n s e . The g r a i n d e n s i t y i s low. The s i l v e r g r a i n s appear homo-ge n e o u s l y d i s t r i b u t e d . M a g n i f i c a t i o n 185 x A ph o t o m i c r o g r a p h o f a 1.5 minute f i b e r c r o s s s e c t i o n . C o n t r a s t t h e g r a i n d e n s i t y t o t h e l o w e r d e n s i t y shown i n I l l u s t r a t i o n 1 3 . T h i s d i f f e r e n c e i n g r a i n d e n s i t y r e p r e s e n t s 22j^a+- removed by t h e s u c r o s e r i n s e . The l a b e l a ppears homogeneously d i s t r i b u t e d . M a g n i f i c a t i o n 185 x - 7 8 TABLE V Data f o r Grain Density P r o f i l e Graph 0.5. Minute Fibers Mean Grid Grain Density grains/lOOii 2 Distance {ix) of gr i d from section edge CS1 CS2 CS3 GS4 GS5 GS6 Average & S.E. 0 2 7 . 2 7 . 1 2 9 . 3 0 . 6 3 4 . 1 34.2 30.3 1.2 7 0 22.3 21.6 2 6 . 3 1 9 . 9 2 6 . 7 2 8 . 4 2 4 . 2 + 1.3 1 4 0 1 7 . 1 15. 1 9 . 3 15.1 24.3 21.8 18.8 + 1.4 210 10.9 14. 11.1 12.1 14.6 1 9 . 7 13.7 + 1.2 2 8 0 .5 . 1 6 . 7 7 . 1 7.9 7 . 3 6 . 9 6.8 ± .4 3 5 0 4. 5.9 5.1 5 . 4 4.7 6 . 1 5 . 2 + .3 4 2 0 2.9 4.4 3.6 3.3 2.6 3.9 3 . 5 + .3 4 9 0 2.3 3.9 3 . 6 3.4 2.1 2.7 3 . 0 t .3 5 6 0 2. 3 . 3 2.1 2.7 2.3 2.4 2.5 + .2 6 3 0 1-9 3.4 2.4 2.6 1.7 2.7 2.5 + .2 700 2. 1.6 2.6 2.8 3.3 2.4 2.4 + .2 TABLE VI Data for Grain Density P r o f i l e Graph 1.5 Minute Fibers Mean Grid Grain Density Grains/lOO>i 2 Distance (u) of g r i d from Average section edge CS1 CS2 CSJ. & S. E. 0 1 8 . 3 2 1 . 8 2 1 . 7 2 0 . 6 ± 1 7 0 2 2 . 3 19. 2 3 . 1 2 1 . 5 t 1 1 4 0 2 0 . 4 2 0 . 8 1 8 . 6 19.9 + .6 2 1 0 19.8 19.3 2 1 . 2 2 0 . 1 .5 280 2 2 . 1 2 0 . 1 20.9 2 1 . 0 •j- .5 3 5 0 2 2 . 2 19.4 2 0 . 4 2 0 . 7 .7 4 2 0 2 1 . 1 19.3 19.1 19.8 + .5 4 9 0 19.6 1 8 . 19.7 19.1 .5 5 6 0 19.4 17.9 2 1 . 6 19.6 + .9 6 3 0 19.7 19. 1 8 . 5 19.1 + .5 7 0 0 19.2 2 2 . 1 2 0 . 7 2 0 . 7 + .6 TABLE VII - 80 Data for Grain Density P r o f i l e Graph 1.5 Minute Fibers .5 Minute Rinse Mean Grid Grain Density grains/lOOLi Distance (u) of gr i d from section edge C S 1 0 5 . 7 0 5 . 1 1 4 0 5 . 4 2 1 0 5 . 7 2 8 0 7 . 3 5 0 4 . 7 4 2 0 4 . 7 490 5 . 4 5 6 0 5 . 7 6 3 0 6.3 7 0 0 5.3 Mean C S 2 CS? & S.E. 4.9 4 . 1 4.7 ± .3 4 . 4.3 4 . 5 ± .3 4 . 5 6.2 5.3 i .4 4.6 6.7 5.6 t .5 4.7 3.9 5.2 * .8 5 . 4.9 4.9 - .3 5 . 1 4.9 4.9 - .2 4.9 5.8 5 . 4 - .2 5 . 1 4.9 5.2 t .2 3.7 5.1 5 . 0 t .6 4.6 5.9 5.2 - .3 Figure 5 Cross Section Grain Density Profile 700 Distance (ya) from section edge i TABLE VIII Analysis of Variance for Grain Density P r o f i l e A. 0,5 Minute Cross Section Grain Counts Within Grid Position g r a i n s / l 0 0 ; i 2 Distance (_u) of gri d position from section edge Mean 0 7 0 1 4 0 2 1 0 2 8 0 3 5 0 4 2 0 * 4 9 0 5 6 0 6 3 0 3 5 • 28 1 8 1 2 9 6 4 ' 3 3 4 3 2 3 3 2 4 2 4 8 8 3 2 3 3 3 7 2 7 2 5 1 6 3 6 ' 5 4 2 3 3 3 2 5 22 1 7 . 6 5 4 2 ' 3 2 3 5 2 6 22 1 6 6- 5 4 3 - 1 2 3 4 3 2 2 2 2 2 7 6 4 4 3 4 3 6 2 8 22 2 0 9 7 3 1 2 3 3 4 . 6 2 8 . 4 ' 2 1 . 9 1 8 . 1 6.9 6 . 1 3-9 2 . 7 2 . 4 2 6 6 6 6 6 6 6 6 6 6 Source O f Variation Between g r i d positions Within g r i d positions Total Degrees of Freedom 9 6 0 6 9 Sum of Mean Squares Square 8 , 9 5 8 . 9 1 2 5 6 . 8 6 9 , 2 1 5 . 7 7 9 9 5 . 4 3 4 . 3 5 F. Ratio 2 2 8 . 7 * * F ( 9 , 6 0 degrees of freedom) must be greater than 2 . 7 2 to be s i g n i f i c a n t at the 1% l e v e l . TABLE, IX An Analysis of Variance for Grain Density P r o f i l e A 1.5 Minute Cross Section Grain Counts Within Grid Position grains/lOOix Distance (LI) of Mean edge 0 7 0 1 4 0 2 1 0 2 8 0 3 5 0 4 2 0 490 5 1 0 6 3 0 1 7 23 1 7 1 8 1 6 23 2 1 16 1 8 2 1 1 6 2 0 2 0 1 9 23 2 4 19 2 1 2 3 2 1 18 2 6 2 0 1 9 2 1 1 9 23 1 8 2 1 18 1 7 1 9 23 1 6 25 1 9 2 5 2 0 1 6 2 0 2 1 1 5 1 8 23 2 1 25 1 6 1 9 1 8 19 25 23 26 2 4 23 2 2 2 5 1 9 1 9 2 1 2 2 2 5 23 23 2 6 1 7 2 2 2 0 1 8 I 8 . 3 2 2 . 3 2 0 . 4 1 9 . ^ ' 2 2 . 1 2 2 . 2 2 1 . 8 1 9 . 6 2 0 I 8 . 7 of freedom 6 6 6 6 6 6 6 6 6 6 Source of Degrees of Sum of Mean Variation Freedom Squares Square F. Ratio Between g r i d positions Within gr i d positions Total 60 69 137.77 453.71 591.48 1 5 . 3 1 7 . 5 6 2 . 0 2 NS F ( 9 , 6 0 degrees of freedom) must be greater than 2 . 0 4 to be s i g n i f i c a n t at the 5% l e v e l . TABLE X Analysis of Variance for Grain Density P r o f i l e Distance (LI) of g r i d position from section edge Mean Degrees of freedom A 1.5 Minute Cross Section With 0.5 Minute Wash Grain Counts Within Grid P o s i t i o n gram S / I O O L I 2 o 70. 140 210 280 350 420 490 560 630 10 4 5 6 5 6 5 5 5 6 6 6 4 7 7 8 6 5 6 8 4 5 5 7 6 5 5 7 3 6 5 4 4 6 5 7 7 8 7 7 3 5 6 5 7 5 4 5 6 5 6 4 7 5 6 4 4 4 7 6 5 5 5 4 4 3 5 5 6 6 l A kA IA 5-7 5-6 5.4 5-1 5-6 5-7 6.2 6 6 6 6 6 6 6 6 6 6 Source of Variation Between grid positions Within grid positions Total Degrees of Freedom 60 69 Sum of Squares 11.42 108.07 119.49 Mean Square F. Ratio 1.27 1.80 .70 NS F (9> 60 degrees of freedom) must be greater than 2.04 to be s i g n i f i c a n t at the 5% l e v e l . I l l u s t r a t i o n s 15 and 16 A photomicrograph of an area of a 5 minute f i b e r c ross s e c t i o n . Note the low g r a i n d e n s i t y . The g r a i n s are homogeneously d i s t r i b u t e d . M a g n i f i c a t i o n 370 x A photomicrograph of the same area taken with t r a n s m i t t e d l i g h t . Magnification 370 x * I l l u s t r a t i o n s 17 and 18 - 86 A photomicrograph of an area of a 5 minute f i b e r cross s e c t i o n . The g r a i n d e n s i t y i s higher than the g r a i n density of the 5 minute f i b e r i n I l l u s t r a t i o n 15. The arrows demonstrate the s t r a i g h t l i n e type of g r a i n p a t t e r n . M a g n i f i c a t i o n 370 x A photomicrograph of the same area taken with t r a n s m i t t e d l i g h t . The arrows i l l u s t r a t e the c l e f t s to which the patterned l a b e l l i n g s correspond. M a g n i f i c a t i o n 370 x I l l u s t r a t i o n s 19 and 20 A photomicrograph of an area of a 20 minute f i b e r cross s e c t i o n . The c l u s t e r e d arrows i n d i c a t e two of the c i r c u l a r type of g r a i n p a t t e r n s i n c l o s e p r o x i m i t y to, a n u c l e u s . The s i n g l e arrow demonstrates a s t r a i g h t l i n e g r a i n p a t t e r n . M a g n i f i c a t i o n 370 x A photomicrograph of the same area taken with t r a n s m i t t e d l i g h t . Note the c i r c u l a r c l e f t s at the s i d e of the nucleus which correspond to the g r a i n p a t t e r n v i s i b l e i n the d a r k f i e l d photograph ( c l u s t e r e d arrows). The s i n g l e arrow demonstrates the c l e f t which corresponds to the s t r a i g h t l i n e g r a i n p a t t e r n . M a g n i f i c a t i o n 370 x I l l u s t r a t i o n s 21 and 22 - 88 A photomicrograph of an area of a 60 minute f i b e r cross section. The grain density has increased i n comparison with the 20 minute f i b e r cross section. Magnification 370 x A photomicrograph of the same area taken with transmitted l i g h t . Ice c r y s t a l damage probably caused by the freezing in isopentane has "pock marked" t h i s section to some extent. Magnification 370 x I l l u s t r a t i o n s 23 and 2 4 A photomicrograph of an area of a 1 & 0 minute f i b e r cross section. The grain density i s at a maximum, obscuring any possible grain patterns. Magnification 3 7 0 x A photomicrograph of the-same area taken with transmitted l i g h t . Magnification 3 7 0 x TABLE XI Grain Counts Average Time Fiber Mean Cross Section Background (Mins.) No. Grid Count grains/lOChu 2 grains/100^' C.S.a 1-A •6.3 2-A •6.2 1-B 10.6 2-B 7.1 1-C 6.0 2-C a.2 1-D 6.3 2-D 7.6 3-D 8.9 1-E 7-3 2-E 9.8 Mean and Standard Error C.S.b C.S.c Ave. 5.8 6.0 6.0 1.3 5.2 . 4.8 5.4 2.6 8.8 10.6 10.0 1.5 6.8 7.3 7.1 1.5 8.4 9.6 8.0 1.3 6.2 8.4 7.6 1.5 8.3 _ 7.3 1.4 7.2 - 7.4 1.3 8.3 - 8.6 1.7 7.0 I'2 1.5 7.9 — 8.9 1.5 7.6 ± 0.4 1- A' 2- A 19.2 16.1 17.0 15.8 17.9 15.8 18.0 1.6 1.2 1- B 2- B 19.4 22.0 22.0 21.3 16.6 20.2 19.3 21.2 1.6 1.5 1-C 20 2-C 26.4 22.8 23.0 19.9 19.5 21.3 23.0 . 21.3 1.5 1.2 1- D 2- D 21.7 21.2 21.4 20.1 — 21.6 20.7 1.7 1.2 1- E 2- E 3- E 24.2 26.4 26.1 25.6 25.8 25.9 - 24.9 26.1 26.0 1.6 \i Mean and.Standard Error 21.6 t 0.9 TABLE XI cont'd Grain Counts Average Time Fiber Mean Cross Section ? Background (Mins.) No. Grid Count grains/lOOjT grains/lOOu C.S.a C.S.b C.S.c Ave. 1- A 30.0 30.6 - 30.3 1.8 2- A 27.2 27.5 - 27.4 1.3 1- B 34.1 31.1 30.6 31.9 1.5 2- B 30.6 34.7 30.8 32.0 1.7 1-C 27.8 31.5 32.2 30.5 1.3 60 2-C 31.2 29.0 32.0 30.7 1.6 1- D 26.2 29.9 - 28.1 1.5 2- D 30.4 29.9 - 30.2 1.6 1- E 3 6 . 4 35.0 - 35.7 1.6 2- E 34.9 34.5 - 34.7 1.6 Mean and Standard Error 31.2 ± 0.8 1- A. 38.7 34.5 - 36.6 2.3 2- A 39.5 36.8 - 3 8 . 2 1.7 180 1-B 4 6 . 3 49.9 53.7 50.0 1.7 1-C 45.0 3 8 . 4 38.2 40.5 1.3 1-D 37.8 34.8 - 3 6 . 3 1.4 1-E 40.1 40.8 - 40.5 1.6 Mean and Standard Error 40.4 - 1.9 Figure 6 • _ 93' TABLE XII Data for Grain Density P r o f i l e Graph 5 Minute Fibers Mean Grid Grain Density grains/lOOu 2 Distance of g r i d section > (M) from edge C S 1 C S 2 C S 3 C S 4 CS5 Mean & S.E. 0 7 . 1 7 . 9 7 . 1 8 . 3 7 . 3 7 . 5 t .3 7 0 7 . 9 5 . 6 6 . 7 8 . 1 6 . 7 7 . 0 ± . 4 1 4 0 6 . 1 6.6 6.6 9 . 1 7 . 9 7 . 3 - . 4 2 1 0 6.6 7 . 4 6.9 8.7 8 . 1 7 . 5 * .3 280 7 . 9. 6 . 7 7 . 9 7 . 3 7 . 6 1 . 4 3 5 0 5 . 7 6 . 7 8 . 7 . 6 7 . 9 7 . 1 t . 4 420 6,4 8.7 7.3 9.4 8 . 4 8 . 0 ± . 4 4 9 0 6.6 8 . 3 6 . 9 9 . 7 . 9 7 . 7 - . 4 5 6 0 7 . 1 7 . 9 1 0 . 4 8.6 7 . 9 8 . 4 ± . 5 6 3 0 7 . 6 8.6 7 . 1 8.6 8 . 8 . 0 ± . 3 7 0 0 8 .1 6 . 1 ' 7 . 6 7 . 6 8 . 7 7 . 6 t . 4 TABLE XIII - 9 4 Data for Grain Density P r o f i l e Graph 2 0 Minute Fibers Mean Grid Grain Density grains/lOO^i Distance {p.) of g r i d from Mean Section edge CS1 CS2 CSJ. CS/j. CSJJ & S.E. 0 1 8 . 3 1 8 . 1 8 . 7 1 8 . 2 0 . 9 18.8. t .5 7 0 1 8 . 9 1 7 . 9 2 1 . 6 2 0 . 4 I6.4 1 9 . 0 i .8 140 17.9 1 9 . 7 1 7 . 3 1 8 . 1 2 1 . 4 1 8 . 9 - .7 2 1 0 19.5 1 9 . 1 2 0 . 1 7 . 6 1 7 . 1 1 8 . 7 - .5 2 8 0 2 0 . 1 8 . 19.6 1 8 . 6 18.3 1 8 . 9 i .3 3 5 0 19.3 1 7 . 3 1 9 . 7 1 6 . 1 2 0 . 3 1 8 . 5 - .7 4 2 0 19.4 1 7 . 1 2 0 . 5 1 8 . 7 1 6 . 2 1 8 . 4 i .6 4 9 0 1 7 . 1 8 . 1 2 0 . . 1 9 . 6 1 8 . 18.6 - .5 5 6 0 1 6 . 6 1 9 . 1 2 0 . 9 1 9 . 6 1 9 . 4 1 9 . 1 - .6 6 3 0 1 5 . 3 1 8 . 5 2 1 . 4 1 8 . 3 1 9 . 9 1 8 . 7 - .9 7 0 0 2 0 . 3 1 8 . 5 2 1 . 4 1 7 . 4 1 8 . 6 1 9 . 2 ± .6 - 9 5 TABLE XIV Data for Grain Density P r o f i l e Graph 60 Minute Fibers Mean Grid Grain Density grains/lOOji Distance (u) of g r i d from section edge 0 70 140 210 280 350 420 490 560 630 700 CS1 CS2 30.6 31.6 29.1 2 7 . 26.7 26. 30.3 28.9 29.4 29.8 29.3 29.9 27.9 26.8 30.9 29.8 29.9 27.8 27.5 26.3 29.3 3 2 . 9 CS3 CS4 2 9 . 3 2 8 . 0 3 2 . 9 3 2 . 3 2 . 9 3 2 . 30.7 32.3 31.7 3 1 . 9 3 3 . 1 3 0 . 6 3 1 . 9 3 2 . 5 31.2 28.7 3 1 . 3 3 0 . 5 31.9 33.1 3 0 . 9 2 8 . 6 Mean & S.E. 29.9 t .7 30.3 + 1.2 29.4 + 1.2 30.6 + .6 30.7 + .6 30.7 + .7 29.7 + .8 30.1 + .5 29.9 + .6 29.7 1.2 30.4 + .8 TABLE XV Data f o r Grain Density P r o f i l e Graph 180 Minute Fibers Mean Grid Grain Density grains/lOOja Distance {p.) of g r i d from section edge CS1 CS2 0 37.0 40,4 70 41.5 37.7 140 37.7 39.3 210 38,4 40.9 280 43 . 39,6 350 39.7 37.1 420 41,3 36.6 490 4 1 . 41.7 560 40,5 38.7 630 39.4 41.9 700 42.8 38,5 Mean c s ? CS4 & S . E . 3 9 . 6 38.8 39.0 ,6 40 . 38.4 39.4 + .6 3 6 . 7 39.9 38.4 .6 38,7 39.7 39.4 + .5 38. 40.9 40.4 + .9 43.1 38.3 39.6 + .1 43,4 41,7 40.7 1.3 37 . 40.6 40.1 + .9 39 . 37.4 38.9 f .6 38.1 40.7 40,0 f .7 37.3 39.5 39.5 f .8 Figure 7 40 Cross Section Grain Density Profile @ 180 minutes * i * I i I I I i I <*_^ 30 O O (|) 60 minutes i i • 1 1 f f ! ! i a O 20 @ 20 minutes "4 * ! i 5 i 1 i i £ 10 -! s * * r (T) 5 minutes I J-i i i — i 1 1 i i i i i_ 140 350 700 Distance (/a) from section edge vO -0 Distance (ja) of grid p o s i t i o n from section edge TABLE XVI Analysis of Variance for 5 Minute Cross Section Barnacle B Fiber 1 Cross Section b Grain Counts Within Grid Position grains/lOOu 2 Mean Degrees of freedom 0 7 0 140 210 280 350 420 490 560 630 700 770 840 910 980 1050 1120 1190 16 15 12 7 9 7 9 10 13 5 7 13 1 0 1 3 16 9 9 10 7 11 9 7 1 3 7 10 10 10 9 9 6 17 7 8 12 18 16 12 12 10 9 15 8 12 6 7 10 12 13 5 11 1 5 1 4 9 9 11 11 11 12 9 13 8 11 1 3 7 19 8 12 9 12 13 11 U 10 10 10 10 9 12 7 1 3 12 9 11 7 11 6 11 16 10 9 10 - 9 8 11 7 10 7 11 8 6 11 11 6 8 11 10 15 7 16 6 5 6 6 6 11 7 7 5 10 10 18 6 11 10 9 14 11.7 10.6 9.3 8.9 9.7 9 9.1 9.7 10 7.3- 11.3 2*Z 9.9 8.6 12 12 11.6 102 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 Source of Variation Between g r i d positions Within g r i d positions Total Degrees of Freedom 17 108 125 Sum of Squares 2 0 4 . 9 2 8 9 6 . 5 7 1 , 1 0 1 . 4 9 Mean Square 1 2 . 0 5 8 . 0 3 F. Ratio 1.45 NS F (17> 108 degrees of freedom) must be greater than 1.66 to be s i g n i f i c a n t at the 5% l e v e l . TABLE XVII Analysis of Variance for 5 Minute Cross Section Barnacle C Fiber 2 Cross Section b Grain Counts within Grid Position grains/lOO^i 2 Distance (u) of grid p o s i t i o n from section edge 0 7 0 1 4 0 2 1 0 2 8 0 3 5 0 4 2 0 4 9 0 5 6 0 6 3 0 7 0 0 7 7 0 8 4 0 9 1 0 9 8 0 7 7 7 1 0 1 1 7 8 8 8 9 1 0 8 8 9 8 8 7 7 7 7 1 1 9 1 0 5 1 0 1 0 1 4 1 2 8 6 7 5 6 1 0 1 0 8 9 6 1 0 7 9 1 1 5 8 8 4 9 7 8 9 8 1 0 7 7 6 7 9 6 8 9 5 1 1 9 5 8 9 1 0 6 6 8 7 6 7 7 7 8 9 7 6 7 9 1 1 8 7 9 8 7 8 1 0 7 5 7 5 7 7 9 8 8 9 5 7 7 1 0 9 Mean 6.3 7 . 6 7.2 7-3 8.4 8.4 9.4 7 - 6 _ 8 8.9 7 . 6 8 . 6 7 . 7 Degrees of freedom 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 Source of Variation Between g r i d positions Within g r i d positions Total Degrees of Freedom 1 4 9 0 1 0 4 Sum of Squares 6 0 . 2 1 2 6 0 . 8 3 3 2 1 . 0 4 Mean Square 4 . 3 0 2 . 9 3 F. Ratio 1 . 4 7 NS F ( 1 4 , 9 0 degrees of freedom) must be greater than 1 . 7 5 to be s i g n i f i c a n t at the 5% l e v e l . TABLE XVIII Analysis of Variance for 5 Minute Cross Section Barnacle A Fiber 1 Cross Section c Grain Counts within Grid Position g r a i n s / 1 0 0 ^ 2 Distance (p.) of g r i d p o sition from Mean Degrees of freedom 0 70 140 210 280 350 420 490 560 630 700 770 840 910 980 1050 11; 6 a 5 7 9 6 6 9 10 8 6 8 11 12 4 6 7 6 10 '5 6 7 5 6 7 8 9 7 9 10 11 7 7 7 10 9 4 7 5 7 12 6 7 6 9 6 6 6 8 8 7 7 7 5 6 9 4 7 5 4 7 10 5 a 8 11 9 5 9 6 5 5 7 6 6 8 8 11 5 6 5 7 8 6 5 5 10 6 4 5 7 9 7 7 a 12 7 10 6 a 9 4 7 5 10 a 7 9 12 7 6 a a 12 11 7 7 7. 7 7-1 7.9 5.7 6.1 7 8.3 7 7.1 8.1 8.1 7 .6 a . 7 8.1 7 .6 Zsifc 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 Source of Degrees of Variation Freedom Between grid positions 16 Within gr i d positions 102 Total 118 F ( 1 6 . 102 d than 1.75 to Sum of Mean Squares Square F. Ratio 8 6 . 6 4 5.41 1.44 NS 3 8 2 . 8 6 3 .75 4 6 9 . 5 0 agrees of freedom) must be greater be s i g n i f i c a n t at the 5% l e v e l . - 1 0 1 TABLE XIX C a l c u l a t i o n o f E f f i c i e n c y o f Radioautograms Measurements o f average f i b e r c r oss s e c t i o n r = 7 5 0 u = . 0 7 5 cm t h i c k n e s s = 1 5 u = . 0 0 1 5 cm Volume o f average f i b e r cross s e c t i o n = ( 3 . 1 4 x . 0 7 5 cm x . 0 7 5 cm x . 0 0 1 5 cm) = 2 . 6 5 x 1 0 ~ 5 cc Area of average f i b e r c r oss s e c t i o n = ( 3 . 1 4 x 750p. x 750;i) = 1 . 7 7 x 1 0 6 ; A 2 Average f i b e r i s 7 5 % water Average f i b e r cross s e c t i o n has (.75 x 2 . 6 5 x lO'^ccjHgO = 1 . 9 9 x 1 0 - 5 g m H 20 = 1 . 9 9 x 1 0 ~ % g H 2 0 Average f i b e r sodium c o n c e n t r a t i o n i s . 0 7 0 M/Kg.H 20 Average f i b e r cross s e c t i o n ( 1 . 9 9 x 1 0 " % g H 2 0 x 7 x 1 0 ~ 2 M Na) = 1 . 3 9 x 1 0 " 9 M Na At 180 minutes 78% f i b e r sodium has exchanged. So the average 180 minute cross s e c t i o n has ( 1 . 3 9 x 1 0 " 9 M Na x .78 = 1 . 0 8 x 1 0 " 9 M Na exchanged The average 180 minute f i b e r c r o s s s e c t i o n a f t e r 1 week exposure has 4 0 g r a i n s / l O O u 2 or ( 4 x 1 0 " 1 grains/>i 2 x 1 . 7 7 x 1 0 6 i i 2 ) = 7 . 0 8 x 10- 5 g r a i n s t o t a l - 102 TABLE XIX cont'd Calculation of E f f i c i e n c y of Radioautograms Bath has 100 pc/ml of 2 2 N a + Bath has 4.5 x 1 0 " % Na/ml So 4.5 x 1 0 " % Na has 100 jac 2 2 N a + 1 M Na has 2.22 x 105>ic 2 2 N a + The average 1 8 0 minute f i b e r cross section contains (1.08 x 10 - 9M Na x 2.22 x 10 5^c/M 2 2Na + = 2.4 x 10" V c 2 2 N a f 1 ;ac = 3.2 x 10*2 disintegrations per day = 2.24 x 10"^ disintegrations per week (dpw) The average 1 8 0 minute f i b e r cross section has (2.24 x 10 1 0(dpw/uc) x 2.4 x 1 0 " V c ) - 5.38 x 10 disintegrations i n a week The average 180 minute f i b e r cross section has produced 7.08 x 10^ grains in a week So the e f f i c i e n c y i s 7 . 0 8 x 10^ grains/disintegration = 13% 5 . 3 8 x 10 b The t h e o r e t i c a l e f f i c i e n c y according to Rogers 20 - 3 0 % - 103 TABLE XX Grain counts are per lOO^u2. The tissu e section i s assumed to be uniformly thick. So Grain counts per 100^2 also represent grain counts f o r a f i x e d volume. The e x t r a c e l l u l a r space represents 5% of f i b e r volume. The e x t r a c e l l u l a r space i s assumed to be equally d i s t r i b u t e d through the f i b e r cross section. So grain counts per 100)i2 also represent grain counts f o r a f i x e d volume of the e x t r a c e l l u l a r space. The number of grains per 100;a2 i s proportional to the amount of 22Na* within lOOu of t i s s u e . So grain counts per lOOji 2 also represent the concentration of 22jj a f f o r a f i x e c i volume of the e x t r a c e l l u l a r space. So grain counts per 100u2 may be used as r e l a t i v e concentrat-ions of 22j\ja<- present in the e x t r a c e l l u l a r space at the loc a t i o n of the area the count represents. A solution to Fick's Law f o r one dimentional d i f f u s i o n i n the non steady state from a source of fix e d concentration i s given by Crank (65). C = Co erfc x 2/ Dt where 22 . Co i s the concentration of Na r i n the e x t r a c e l l u l a r space at the f i b e r surface. This represents the source of f i x e d concentration = 30.3 grains/100^ 2 - 1 . 5 grains/lOOii 2 = 28.8 grains/lOOji 2 C i s the concentration of N^a"*" i n the e x t r a c e l l u l a r space at distance x from the f i b e r cross section edge at time t i s the time f o r d i f f u s i o n . = 30 seconds. x i s the distance the concentration C; of 2 2Na* has moved from the plane of Co at time t . D i s the d i f f u s i o n c o e f f i c i e n t f o r 22N a f . The value used i s f o r s e l f d i f f u s i o n i n di l u t e solutions = I.48 x 10-5cm 2sec-l. erfc i s the error function of x r i s the actual (experimental) distance along the f i b e r radius of the concentration C of 22N a* # TABLE XX - cont'd - 104 Table of values f o r the calculated distance (x) and the experimental distance (r) the concentration (C) of 22Nat moves from the plane of Co. r c* Z S X X grains/lOOu^ C/Co 2 /Dt x/r 70 22.7 0.79 0.19 80 1.14 140 17.3 0.60 0.37 156 1.11 210 .12.5 0.435 0.55 232 1.10 280 5.3 O.I84 0.94 396 1.41 350 3.7 0.129 1.075 453 1.29 420 2. 0.0695 1.285 452 1.29 490 1.5 0.052 1.375 580 1.18 * a l l values of C and Co corrected for background of 1.5 grains/lOOu 2. The values of C and Co are taken from Table XXI. Sample ca l c u l a t i o n . C = 22.7 grains/100^ 2 when x = BOy. and t = 30 seconds C = Co erfc x 2/Dt C/Co = .079, the error function of x. The value of x associated with t h i s value of the error function i s obtained from error function tables. It i s 0.19. — = 0.19 2/Dt x = 1.9 x 10-1 x 2y5t~ x = /3m 2/sec.sec x = 1.9 x 10-1 x 2 x / l . 4 8 x 10-5 x 30 = 1.9 x 10-1 x 2 x 2.11 x IO" 2 = 8.02 x 10-3Cm = .00802 cm = 80. 2p. - 10$ TABLE XXI A ca l c u l a t i o n of the t h e o r e t i c a l percent of f i b e r surface area involved i n 2 2Na+ i n f l u x . B u l l (66) gives equations (1) In A C = Dt and (2) B = f1_ - 1_\ A A Co \Y1 V J h where £ Co = Co - C at t = o AC = Co - C at t = t and C i s measured at x i n both cases. 22 +• Co i s the concentration of Na T i n the e x t r a c e l l u l a r space at the f i b e r surface. This represents the source of fixed concentration. = 28.8 grains/lOOu 2 C i s the concentration of 2 2Na* in the e x t r a c e l l u l a r space at distance h from the f i b e r cross section edge at time t . =4.8 grains/100^ 2. t i s the time f o r d i f f u s i o n . = 30 seconds. h i s the distance the concentration C of 2 2Na* has moved from the plane of Co at time t . = 380L! = .038cm D i s the d i f f u s i o n c o e f f i c i e n t for 2 2 N a + . The value used i s for s e l f d i f f u s i o n i n d i l u t e solutions. = 1.43 x 10-5cm2sec-!. Vi i s the volume of the 2 2Na* bath. = 10 ml. V 2 i s the volume of the f i b e r . = if r 2 h = 3.14 x .075cm x ,075cm x 3cm = 5.3 x 10- 2ml. A i s the area of the f i b e r surface involved i n the d i f f u s i o n . j3 i s a constant. - 1 0 6 TABLE XXI cont'd S u b s t i t u t i n g i n equation ( 1 ) 2.3 l o g 24 = -fi 3 0 x 1 . 4 8 x 1 0 - 5 fi = 0 . 1 8 4  3 0 x 1 . 4 8 x 1 0 - 5 fi ~ 4 1 5 P = /!_ - 1 _ \ A W v2>/ h \ L 0 5.3 x 10-2/ . 0 3 8 A = . 0 3 8 x 415 19 A = 0 . 8 3 c m 2 Surface area of f i b e r = 27Trh - 2 x 3 . 1 4 x . 0 7 5 c m x 3 c m - 1 . 4 1 c m 2 Percent of f i b e r surface area involved i n 2 2Na+ i n f l u x . 28~78 - 9A1 - 5 9 % 1 . 4 1 TABLE XXII - 107 A c a l c u l a t i o n of the time required f o r the Na* concen-t r a t i o n at the f i b e r center (C) to equal hal f the 2 2Na* concentration at the f i b e r edge (Co). Equation C = Co erfc x 2/Dt where C, Co e r f c , x, D and t are as defined i n Table XXI, C = 1/2 Co at x = ,07cm (average cross section radius) C/Co = . 5 , the error function of x. The value of x associated with t h i s value of the error function i s obtained from error function tables. It i s 0 . 4 7 5 . 0.07 - 0.475 2/Dt /Dt = .07 2 x . 4 7 5 = 7 x l Q - 2 ^ _ 9.5 x 10" 1 = 7 . 3 7 x IO' 2 Dt = 5.43 x 10"^ cm2/sec t = cm2 t = 5.43 x 10"^ 1.48 x 10-5 3 . 6 7 x IO 1 seconds 6.11 minutes. - 108 CHAPTER V I n f l u x o f 2 2Na+ I n t r o d u c t i o n The l o n g t e r m r a d i o a u t o g r a p h i c study r e v e a l e d two compartments. An i n i t i a l r a p i d compartment was i d e n t i -f i e d as the e x t r a c e l l u l a r space. The more s l o w l y e x c h a n g i n g compartment r e p r e s e n t i n g myoplasmic exchange o f 2 2Na+ was s t i l l e x c h a n g i n g when t h e experiment was t e r m i n a t e d . U n f o r t -u n a t e l y t h e graph d e r i v e d from t h e r a d i o a u t o g r a p h s d i d not a l l o w c a l c u l a t i o n o f the amount o f Na"1- i n each compartment. To d e t e r m i n e t h i s and v e r i f y t h e r a d i o a u t o g r a p h i c p l o t an i n f l u x experiment was performed u t i l i z i n g s h o r t time i n t e r v a l s . Methods The t o t a l t i me p e r i o d f o r i n f l u x was 90 m i n u t e s . Twenty f i b e r s were d i s s e c t e d but l e f t w i t h t h e i r b a s e p l a t e a t t a c h m e n t s i n t a c t . These f i b e r s and b a s e p l a t e were submerged i n 30 ml o f 22]\ja+ l a b e l l e d R i n g e r s s o l u t i o n o f a c t i v i t y 2uc/ml. At 5, 10, 20, 40 and 90 minutes f o u r f i b e r s were c u t from t h e b a s e p l a t e , removed from t h e b a t h i n g s o l u t i o n , r i n s e d f o r t h i r t y seconds i n i s o t o n i c s u c r o s e s o l u t i o n , g e n t l y b l o t t e d and p l a c e d i n a preweighed s t o p p e r e d b o t t l e . The wet weight o f t h e s e f i b e r s was dete r m i n e d and t h e y were l e f t t o d r y f o r 12 - 18 h o u r s . A f t e r d e t e r m i n a t i o n o f t h e d r y w e i g h t , f i b e r s were d i g e s t e d i n n i t r i c a c i d f o r 12 - 18 h o u r s , n e u t r a l i z e d w i t h ammonia and d i l u t e d up t o 10 m l . - 109 Half of t h i s solution was used f o r counting and half was used for sodium analysis (Unicam S.P.900 flame spectrophoto-meter) . Sample blanks were analyzed with each group of fi b e r s to correct f o r sodium contamination from solutions and from pyrex weighing b o t t l e s . Sample counting was performed on a Nuclear Chicago automatic gamma well counting system (Model 958). A single channel gamma radiation analyzer (Model 8725) measured gamma radiation over a 0.3MEV range centered at 1.28MEV, the p r i n c i p a l peak of Na . Background correction was made for a l l samples and care was taken to ensure constant sample geometry. Results Table XXIII gives sample i n f l u x data f o r a barnacle and Table XX1Y shows the s p e c i f i c a c t i v i t y of the bath. Table XXV shows the s p e c i f i c a c t i v i t i e s of f i b e r s and the percent l a b e l l i n g that had occurred at various times. No correction for the e x t r a c e l l u l a r space was made since none was made i n the radioautographic study. This data was plo t t e d semilogarithmically against time in Figure 8. This graph shows the i n f l u x of 2 2 N a + into the f i b e r was c l e a r l y d i v i s i b l e into two phases. The half time of loading f o r the i n i t i a l rapid phase was six minutes. The size of t h i s f i r s t compartment was estimated by extrapolating the portion of the curve f o r the slow compartment to t = 0. This i n i t i a l rapid compartment contained 44% of t o t a l f i b e r sodium. - 110 The more slowly exchanging compartment had a rate constant of 5.6 x 10-3/minute. This compartment was s t i l l exchanging when the experiment was terminated at 90 minutes. At t h i s time 70% of t o t a l f i b e r sodium had exchanged. Discussion The i n i t i a l r apidly exchanging compartment with ha l f time of s i x minutes and containing 44% of t o t a l f i b e r sodium represents the e x t r a c e l l u l a r space. .McLaughlin and Hinke (26) concluded the e x t r a c e l l u l a r space of an average f i b e r contained 0,030moles/kg H 2 O sodium (43% of t o t a l f i b e r sodium), so good agreement exists f o r the sodium content of the e x t r a c e l l u l a r space. The h a l f time determined f o r t h i s compartment l i e s midway between the half time f o r the ra p i d l y exchanging compartment detected i n the radioauto-graphic study (10 minutes) and the half time f o r sodium washout of the e x t r a c e l l u l a r space into sodium free sucrose Ringers (3 minutes) as detected by Hinke (68). The lower value of Hinke can be explained on the basis of the h a l f minute sucrose wash p r i o r to ion analysis removing sodium and making the h a l f time shorter than i n r e a l i t y . For i n f l u x studies the opposite effect occurs. After an i n f l u x of f i v e minutes a h a l f minute sucrose wash tends to decrease the amount of l a b e l l e d sodium i n the f i b e r and make the h a l f time of loading of the e x t r a c e l l u l a r space longer than i n r e a l i t y . The radioautographic study had fewer experimental points to allow sharp separation of the slow and fast compartments - I l l r e s u l t i n g in a longer estimation of half time. The more slowly exchanging compartment represents sodium exchange with the myoplasm. The rate constant f o r t h i s compartment {5*6 x 10"^/minute) i s in good agreement with the rate constant f o r rnyoplasmic exchange calculated by A l l e n and Hinke (3D (8.45 x 10"^/minute). The same authors concluded 50% of the rnyoplasmic sodium had exchanged at 90 minutes. McLaughlin and Hinke (26) have stated i n the average f i b e r the e x t r a c e l l u l a r space has 43% of total fib er sodium P.03 moles/kg H 2 O ) . If one assumes the e x t r a c e l l u l a r space i s f u l l y loaded at twenty minutes, the data of Al l e i and Hinke (31) would predict 72% of t o t a l f i b e r sodium exchanged at 90 minutes. When the experiment was terminated at 90 minutes, 71% of sodium had exchanged. In making a comparison of the i n f l u x curve (Figure $). with the curve obtained by quantitative radioauto-graphy (Figure 6) the following should be noted. Both curves reveal a fast and slow compartment. The calculated h a l f times f o r the i n i t i a l e x t r a c e l l u l a r space compartment are si m i l a r . Both studies demonstrate the e x t r a c e l l u l a r space contains approximately 50% of f i b e r sodium which can be mobilized i n 20 minutes. The slower rnyoplasmic component was s t i l l exchanging at the termination of both experiments. Absolute comparisons at experimental points cannot be made between curves since the curve obtained by radioautography was proportional to the concentration of r a d i o a c t i v i t y that was present i n the specimens but independent of the absolute amount. This i s due to the fact that the tissue sections f o r the radioautographs were cut at i n f i n i t e thick-ness. TABLE XXIII BARNACLE C Influx data Sodium Con- Total Fiber Fiber S p e c i f i c Time Fiber % Fiber water centration Sodium A c t i v i t y counts/ (Min.) No. Water Kg x IO"? M x 1 0 ~ 2 M x 1 Q - 7 Counts ' mole x 1 0 + 9 1-C 7 5 . 7 3 . 1 7 . 1 2 2 . 0 3 , 6 8 9 1 . 6 8 2-C 7 5 . 4 2 . 9 6.1 1 7 . 7 2 , 5 0 1 1 . 4 1 5 3-C 7 6 . 2 3 . 0 6.2 1 8 . 6 3 , 1 8 1 1 . 7 1 4-C 7 8 . 4 3 . 3 1 2 . 3 — — — 1-C 7 4 . 9 2.5 6.4 1 6 . 0 3 , 4 8 8 2 . 1 8 2-C 7 5 . 5 2 . 9 7 . 4 2 1 . 5 4 , 3 5 6 2.03 1 0 3-C 7 5 . 5 2 . 5 6 . 1 1 5 . 3 3 , 3 8 5 2 . 2 1 4-C 7 5 . 7 3 . 9 7 . 1 2 7 . 7 5 , 5 2 5 1 . 9 9 1-C 74.8 2.5 7.2 1 8 . 0 3 , 7 2 4 2 . 0 7 2-C 7 6 . 4 3 . 6 7 . 1 2 5 . 6 7 , 6 5 4 2 . 9 9 2 0 3-C 7 5 . 5 2 . 4 8.5 2 0 . 4 4 , 2 5 8 2 . 0 9 4-C 7 5 . 5 2 . 8 6.2 1 7 . 4 4 , 3 0 2 2 . 4 7 1-C. 7 5 . 7 2 . 2 8.4 1 8 . 5 5 , 6 6 1 3 . 0 6 2-C 7 4 . 8 2 . 9 7 . 1 2 0 . 6 4 , 9 3 3 2 . 3 9 4 0 3-C 7 5 . 4 2 . 9 6.3 1 8 . 3 5 , 1 9 6 2 . 8 4 4-C 7 5 . 2 2.5 9 . 9 2 4 . 8 6 , 7 4 6 2 . 7 2 1-C 7 5 . 4 2.6> 6.6 1 7 . 2 5 , 2 5 8 3 . 0 6 2-C 7 4 . 2 1 . 8 6.0 1 0 . 8 4 , 0 1 2 3 . 7 1 90 3-C 7 6 . 3 3.9- 7 . 1 2 7 . 7 5 , 1 0 0 2 . 6 6 4-C 6 9 . 3 2 . 1 1 1 . 6 - - -- 114 TABLE XXIY Calculation of Bath S p e c i f i c A c t i v i t y Counts from 1 ml bath sample Sodium concentration of sample Sodium content of sample Sp e c i f i c a c t i v i t y of bath 21.1743 x HP ° ' ^ 0 M / l i t e r 4.5 x 10~h M 21.1743 x 105 4.5 x 10-4 = 4.71 x 109 counts/mole TABLE XXV - 1 1 5 Influx Analysis Fiber S p e c i f i c Bath S p e c i f i c Time Fiber A c t i v i t y (Counts/ A c t i v i t y (Counts/ % (Mins.) No. Mole, x 10* Mole, x 109 L a b e l l i n g 1 - A 1 . 7 7 3 7 . 6 2- A 1 . 3 2 . 28.0 3 - A 1 . 3 6 2 8 . 9 4- A 0 . 8 2 1 7 . 4 5 1-B 1 . 1 2 4 . 7 1 2 3 . 9 2- B 1 . 6 2 3 4 . 5 3 - B 1 . 7 9 3 8 . 0 1 - C 1 . 6 8 3 5 . 6 2- C 1 . 4 1 3 0 . 0 3 - C 1 . 7 1 3 7 . 0 Mean and Standard Error 3 1 . 1 ± 2 . 0 1-A 2 . 3 9 5 0 . 8 2-A 2 . 5 1 5 3 . 3 3-A 2 . 0 3 4 3 . 1 4-A 2 . 0 9 4 4 . 4 1-B 2 . 5 2 5 3 . 5 2-B 2 . 1 9 4 . 7 1 4 6 . 6 3-B 1 . 9 9 4 2 . 4 4-B 1 . 8 1 3 8 . 3 1-C 2 . 1 8 4 6 . 3 2-C 2 . 0 3 4 3 . 1 3-C 2 . 2 1 4 7 . 0 4-C 1 . 9 9 4 2 . 4 Mean and Standard Error 4 5 . 9 * 1 . 3 - 1 1 6 TABLE XXV cont'd Influx Analysis Fiber S p e c i f i c Bath S p e c i f i c Time Fiber A c t i v i t y (Counts/ A c t i v i t y (Counts/ % (Mins.) No. Mole, x -10* Mole, x 1 0 9 L a b e l l i n g 1-A 1 . 9 4 4 1 . 3 2-A 2 . 9 6 6 3 . 0 1-B 3 . 0 4 6 4 . 6 2-B 2.15 4 5 . 8 2 0 3-B 1 . 7 9 4 . 7 1 3 8 . 0 4-B 2 . 8 5 6 O . 5 1-C 2 . 0 7 4 4 . 0 2-C 2 . 9 9 6 3 . 5 3-C 2 . 0 9 4 4 . 4 4-C 2 . 4 7 5 2 . 5 Mean and Standard Error 5 1 . 8 ± 3 . 1 1-A 3 . 2 0 6 8 . 1 2-A 2 . 7 5 5 8 . 5 3-A 2 . 8 9 6 1 . 6 1-B 2 . 9 2 6 2 . 0 4 0 2-B 2 . 6 6 4 . 7 1 5 6 . 7 3-B 2 . 7 3 5 7 . 9 1-C 3 . 0 6 65.O 2-C 2 . 3 9 5 0 . 9 3-C 2 . 8 4 6 O . 3 4-C 2 . 7 2 5 7 . 8 Mean and Standard Error 5 9 . 9 + 1 . 4 1-A 3 . 3 8 7 1 . 8 2-A 4 . 1 3 8 7 . 8 3-A 3 . 1 7 6 7 . 4 1-B 3 . 4 5 7 3 . 3 9 0 2-B 3 . 7 8 4 . 7 1 8 0 . 4 3-B 2 . 8 9 6I.4 1-C 3 . 0 6 6 4 . 9 2-C 3 . 7 1 7 8 . 8 3-C 2 . 6 6 . 5 6 . 4 Mean and Standard Error 7 1 . 4 ± 3 . 1 Figure 8 CHAPTER VI - 118 General Conclusion A useful technique for the radioautography of soluble substances was developed. This technique was simpler than many reported i n the l i t e r a t u r e . The r e s o l -ution achieved with the technique (15 - 17.5u) was compar-22 + able to that reported f o r Na with other types of t i s s u e . These low resolution values imply no s i g n i f i c a n t trans-lo c a t i o n of the l a b e l occurred throughout the technique. The background l e v e l s were equivalent to the values of Appleton (6l) for the same isotope and emulsion. The e f f i c i e n c y of these radioautographs compared favourably with the t h e o r e t i c a l e f f i c i e n c y f o r the p a r t i c u l a r emulsion, isotope and tissue u t i l i z e d . Useful information on the size and complexity of the e x t r a c e l l u l a r space of a single muscle f i b e r from the giant barnacle was obtained. The e x t r a c e l l u l a r space ( c l e f t and tubular system) i s more extensive than has been reported. The l i g h t photomicrographs revealed no part of the myoplasm was more than 2 - 3u from a 0.3>i c l e f t . The electron micro-scopic photographs indicated no part of the myoplasm was more than 1 - 2u from a t r i a d or diad contact of a tubule with the sarcoplasmic reticulum. These tubules were patent up to the diad (and triad) as revealed by f e r r i t i n and horse radish peroxidase uptake studies. The f i n a l picture of the c l e f t system was an invagination of the plasma membrane, di v i d i n g extensively in three dimensions and reducing in size down to - 1 1 9 T tubules. The l a t t e r make contact with the sarcoplasmic reticulum to form both diad and t r i a d junctions. The tubular system i s s u f f i c i e n t l y extensive so that no part of the myo-plasm i s more than' 2yx from e x t r a c e l l u l a r space. Valuable information on 22Na"*" entry into the extra-c e l l u l a r space was provided by the short term radioautographic study. The path of entry f o r 22fja+ i s the c l e f t system which t h e o r e t i c a l l y occupies over 50% of the f i b e r surface area. The radius of the f i b e r i s a reasonable approximation of the 2 2Na* d i f f u s i o n pathway. The rate of d i f f u s i o n of 2 2 N a + along t h i s pathway i s as rapid i n the e x t r a c e l l u l a r space 22 + as i n d i l u t e solutions. The time required f o r the Na concentration of the f i b e r center to reach h a l f the concen-t r a t i o n of the f i b e r edge by d i f f u s i o n along t h i s pathway i s approximately s i x minutes. The sucrose rinse data provided extra information on the complexity of the e x t r a c e l l u l a r space. The sucrose rinse washed at least half the ex t r a c e l l u l a r 2 2 N a + from the 1.5 minute f i b e r s . This 2 2Na* was thought to be washed from the larger c l e f t s . It was postulated that the agitat i o n of the f i b e r which accompanied the rinse destroyed whatever 2 2Na+ gradient the rinse created i n the f i b e r . The 2 2 N a + l e f t i n the smaller terminal c l e f t s following the rinse would explain the reduced but homogeneous grain d i s t r i b u t i o n of the 1.5 minute f i b e r s with sucrose r i n s e . The f i n e terminal c l e f t s involved may also include the T tubules. The loading of the e x t r a c e l l u l a r space i n the long term - 120 radioautographic study r e f l e c t e d increasing 2 2Na+ entry into these smaller c l e f t s inaccessible to the sucrose r i n s e . The f i n e d i v i s i o n s of the c l e f t system prevented consistent radioautographic v i s u a l i z a t i o n of discrete c l e f t s . The distance from the termination of the c l e f t s to the center of the myoplasm (1 - 2p.) was much less than the re s o l u t i o n of the technique (15 - 17.5u). Therefore, grains over the myoplasm caused by isotope present i n the c l e f t s resulted i n a homogeneous d i s t r i b u t i o n of grains over myo-plasm and c l e f t . The use of a lower energy isotope would not have helped appreciably (e.g. resolutions obtained with t r i t i u m are in the v i c i n i t y of 2 - kp.) . Radioautography at the electron microscope l e v e l i s required to v i s u a l i z e the c l e f t s consistently as d i s c r e t e l y l a b e l l e d structures. The sodium content of the e x t r a c e l l u l a r space was demonstrated by both the long term radioautographic study and the conventional i n f l u x study. Both techniques revealed that the e x t r a c e l l u l a r space contained approximately h a l f the f i b e r sodium. The h a l f time for exchange of t h i s Na-*" compartment was 6 - 1 0 minutes. Both studies also detected the same myoplasmic compartment which was s t i l l exchanging when the experiments were terminated. The c a l -culated rate constant for myoplasmic exchange was i n good agreement with the value of A l l e n and Hinke (31). This myoplasmic exchange i s intimately related to the e x t r a c e l l u l a r space. The myoplasmic exchange probably commences immediately o p when Na enters the c l e f t system and i t probably makes use - 121 of the f u l l area of the c l e f t surface. This rnyoplasmic loading i s masked by the f i l l i n g of the e x t r a c e l l u l a r space and does not become apparent u n t i l the l a t t e r i s saturated (20 minutes). From these studies emerges a more comprehensive picture of the e x t r a c e l l u l a r space as a pathway f o r d i f f u s i o n . A study of the morphology of the e x t r a c e l l u l a r space reveals an extensive c l e f t system with di f f u s e ramifications pene-t r a t i n g within 1 - 2LI of the myoplasm. This i s the morphol-og i c a l pathway f o r d i f f u s i o n . The data from the short term 22 + radioautographic study of Na i n f l u x gave a picture of the physiological pathway f o r d i f f u s i o n . The path of entry for 22 + Na i s the c l e f t system which t h e o r e t i c a l l y occupies over 50% of the f i b e r surface area. The radius of the f i b e r i s an approximation of the length of the pathway. Diffusion of 2 2Na* along the length of the pathway i s as rapid as s e l f -o p . d i f f u s i o n of Na T in d i l u t e solutions. The l i t e r a t u r e review summarized evidence f o r a heterogeneous sodium d i s t r i b u t i o n in muscle. Perhaps the e x t r a c e l l u l a r space i s the most important single Na* compart-ment of the single muscle since i t contains over half the f i b e r sodium at a concentration of 10 times the myoplasm in only 5 - 6% of f i b e r volume. In experiments reported here i t was concluded that the sucrose rinse removed half of the 22 + Na from the e x t r a c e l l u l a r space. This i s i n good agreement with data obtained by Hinke (67). However, McLaughlin and Hinke's (26) c a l c u l a t i o n of the sodium content of the - 122 e x t r a c e l l u l a r space (43% of t o t a l f i b e r sodium) was based on f i b e r s which had a sucrose r i n s e . This implies the extra-c e l l u l a r space before the 0.5 minute sucrose rinse contains about 60% of the t o t a l f i b e r sodium. These conclusions correlate well with the work of Shaw (33) on Carcinus. He found over 70% of the f i b e r sodium was i n an extrasarco-plasmic region and exchanged ra p i d l y . The experiments reported here provide no evidence as to the nature or loc a t i o n of the "bound" f r a c t i o n of sodium i n Balanus. With t h i s knowledge of the e x t r a c e l l u l a r space, i t i s apparent that microinjections or f l u x studies which do not allow for t h i s compartment could be erroneous. Bunch and Kallsen (69) i n a recent paper concluded the d i f f u s i o n rates of water, urea and glycerol were similar f o r barnacle myoplasm and d i l u t e solutions. Fibers were coated with o i l and the base plate rock was removed. This cut end of the f i b e r was immersed i n the isotope bath. Allen (40) has shown that o i l does not e f f e c t i v e l y enter the e x t r a c e l l u l a r space. Thus, t h i s procedure exposes both myoplasm and e x t r a c e l l u l a r space to the tracer bath. No separate consideration was made of the e x t r a c e l l -ular space. Therefore, t h i s experiment assumes d i f f u s i o n i n both the e x t r a c e l l u l a r space and the myoplasm i s equiva-lent to d i f f u s i o n in d i l u t e solutions, although the authors do not state t h i s . - 123 In experiments reported here i t was concluded that 22 + the rate of d i f f u s i o n of Na i n the e x t r a c e l l u l a r space was equivalent to the rate of s e l f d i f f u s i o n in d i l u t e solutions. The same should be true of larger non ionic molecules. Thus, f a i l u r e to consider the e x t r a c e l l u l a r space allows no clear conclusion of the rate of myoplasmic d i f f u s i o n to be made. In another paper, Bunch and Edwards (70) examined the e f f l u x of gl y c e r o l , urea, ASA and DMSO from barnacle muscle f i b e r s following microinjection. The l a b e l was injected as a 5y.l bolus through a 1300u needle. A. needle t h i s size would puncture both the e x t r a c e l l u l a r space and the myoplasm. Bunch compared the course of e f f l u x from the f i b e r to the e f f l u x that would be expected from an ideal i z e d cylinder. He concluded the f i b e r membrane exerted a retard-ing effect on the d i f f u s i o n of DMSO. No compensation f o r the e x t r a c e l l u l a r space was made. For l a b e l to diffuse out of the myoplasm i t would probably cross at least one c l e f t . Thus, the l a b e l may leave the f i b e r by d i f f u s i n g from the myoplasm adjacent to the f i b e r periphery or by d i f f u s i n g out along the e x t r a c e l l u l a r space pathway. Thus, Bunch and Edwards attributed part of the retarding effect on DMSO to the membrane when i t may be due to the e x t r a c e l l u l a r space. BIBLIOGRAPHY - 124 1. J.. A. M. Hinke. Nature. 184_, 1257 (1959). 2. S. G. A. McLaughlin and J. A. M. Hinke. Can. J. of Physiol, and Pharm. L^, 837 (1966). 3. A. A. Lev. Nature, 201, 1132 (1964). 4. J. D. Robertson. J. Exp. B i o l . 2$, 707 (1961). 5. F. W. 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