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UBC Theses and Dissertations

Calcitonin and electrolyte homeostasis 1972

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CALCITONIN AND ELECTROLYTE HOMEOSTASIS by V a l e r i e Rosemary Walker B.A., U n i v e r s i t y of B r i t i s h Columbia, 1959 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Ph.D. in the department of Physiology We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1972 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u rposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depa rtment The U n i v e r s i t y o f B r i t i s h Columbia V a n c o u v e r 8, Canada i A b s t r a c t The e f f e c t s o f c a l c i t o n i n on plasma and u r i n a r y e l e c t r o l y t e s were s t u d i e d i n r a t s and sheep. I . T w i c e - d a i l y a d m i n i s t r a t i o n o f salmon c a l c i t o n i n (250 mU(50 n g ) / lOOg r a t , i . p . ) t o young r a t s o v e r a p e r i o d o f 10 days r e s u l t e d i n i n c r e a s e s i n u r i n e volume and t h e e x c r e t i o n o f Na, Ca, P, and Mg. S i n g l e i n j e c t i o n s o f g raded doses o f salmon c a l c i t o n i n (100-2000 mU(20-400 n g ) / l 0 0 g r a t , i . p . ) r e s u l t e d i n d o s e - r e l a t e d i n c r e a s e s i n u r i n e volume and Na e x c r e t i o n o v e r a s i x - h o u r p e r i o d . P h o s p h a t e e x c r e t i o n was a l s o found t o i n c r e a s e . However, i n c o n t r a s t t o t h e c h r o n i c s t u d i e s , n e t d e c r e a s e s i n t h e e x c r e t i o n o f Ca and Mg were f o u n d . S i g n i f i c a n t d e c r e a s e s i n plasma Ca, i n o r g a n i c P, and Mg were found one hour a f t e r i n j e c t i o n o f 100 mU(20 ng) salmon c a I c i t o n i n / I O O g r a t , i . v . No s i g n i f i c a n t d e c r e a s e s i n plasma Na and K were o b s e r v e d . The u r i n a r y e l e c t r o l y t e changes t h a t o c c u r r e d f o l l o w i n g c a l c i t o n i n a d m i n i s t r a t i o n a l s o o c c u r r e d i n t h y r o p a r a t h y r o i d e c - t o m i z e d r a t s and r a t s p r e t r e a t e d , w i t h l a r g e doses o f DOCA (I mg/IOOg r a t , 1. m.), and were not a s s o c i a t e d w i t h i n c r e a s e s i n t h e e x c r e t i o n o f endogenous c r e a t i n i n e . 2. A one-hour i n f u s i o n o f Ca (10 mg Ca/kg) i n i n t a c t c o n s c i o u s sheep was accompanied by a r a p i d r i s e i n plasma Ca l e v e l s w h i c h r a p i d l y r e t u r n e d t o normal when t h e i n f u s i o n was s t o p p e d . Plasma P l e v e l s i m m e d i a t e l y d e c r e a s e d f o l l o w i n g t h e s t a r t o f t h e Ca i n f u s i o n . F o l l o w i n g t h y r o i d e c t o m y ( T X ) , Ca i n f u s i o n r e s u l t e d i n a s i g n i f i c a n t l y g r e a t e r i n c r e a s e i n plasma Ca (A2.31+0.10 mg%) as compared t o i n t a c t sheep (Al.60+0.07 mg%), and t h e r e t u r n t o w a r d s normal l e v e l s was d e l a y e d . i i Plasma P l e v e l s i n t h e TX sheep r o s e f o l l o w i n g the s t a r t o f t h e i n f u s i o n , and a t 1/2 hour p o s t i n f u s i o n were s i g n i f i c a n t l y h i g h e r (A+0.42+0.07 mg%) t h a n plasma P i n t h e i n t a c t sheep (A-0.66+0.22 mg%). In i n t a c t sheep, i n c r e a s e s i n u r i n e volume and t h e e x c r e t i o n o f Na, Ca, P, and Mg were o b s e r v e d d u r i n g and f o l l o w i n g t h e Ca i n f u s i o n . The TX sheep showed no i n c r e a s e i n u r i n e volume, and d e s p i t e a l a r g e r i n c r e a s e i n Ca e x c r e t i o n , t h e e x c r e t i o n o f Na i n t h e TX sheep was s i g n i f i c a n t l y l e s s t h a n t h a t found i n t h e i n t a c t a n i m a l s . 3. Volume e x p a n s i o n i n i n t a c t r a t s (5 ml 0.9% NaCI/IOOg r a t , i . p . ) was f o l l o w e d by s i g n i f i c a n t i n c r e a s e s i n u r i n e volume and t h e e x c r e t i o n o f Na, K, C a , P, and Mg, but t h e s e changes d i d not o c c u r i n TX r a t s i n t h e t h r e e h o urs f o l l o w i n g t h e s a l i n e l o a d . D e x t r a n i n f u s i o n (6% G e n t r a n 75 i n 0.9% NaCl) i n i n t a c t sheep r e s u l t e d i n a s u s t a i n e d f a l l i n plasma Ca l e v e l s . When d e x t r a n was i n f u s e d i n t o TX sheep, plasma Ca f e l l i n i t i a l l y but r e t u r n e d t o normal w i t h i n t h r e e h o u r s . In both g r o u p s o f sheep, t h e volume e x p a n s i o n r e s u l t e d i n a s i m i l a r i n c r e a s e i n Na e x c r e t i o n . However, an i n c r e a s e i n Ca e x c r e t i o n d i d not accompany t h e n a t r i u r e s i s i n t h e TX sheep, w h i c h was i n c o n t r a s t t o t h e f i n d i n g s i n t h e i n t a c t a n i m a l s . 4. A d m i n i s t r a t i o n o f p a r a t h y r o i d e x t r a c t t o TPTX r a t s (100 U/IOOg r a t , s .c.) d e c r e a s e d t h e u r i n a r y e x c r e t i o n o f Na, Ca, and Mg, and i n c r e a s e d t h e e x c r e t i o n o f P. In s i m i l a r s t u d i e s u s i n g h i g h l y - p u r i f i e d PTH (3000 U/mg), t h e e x c r e t i o n o f Ca and Mg a l s o d e c r e a s e d and t h e e x c r e t i o n o f K as w e l l as P was found t o i n c r e a s e . However, no s i g n i f i c a n t d e c r e a s e i n Na e x c r e t i o n was f o u n d . These s t u d i e s i n d i c a t e d t h a t exogenous a d m i n i s t r a t i o n o f i i i salmon c a l c i t o n i n a l t e r s the e x c r e t i o n of Na, Ca, P, and Mg in the u r i n e , in a d d i t i o n t o decreasing the l e v e l s of Ca, P, and Mg in the plasma. Infusion of Ca s a l t s in i n t a c t sheep r e s u l t e d in c e r t a i n changes in both plasma and u r i n a r y e l e c t r o l y t e s which were not found f o l l o w i n g thyroidectomy i n these animals. Since e l e v a t i o n of plasma Ca l e v e l s would increase c i r c u l a t i n g l e v e l s of c a l c i t o n i n in i n t a c t but not TX sheep, endogenous r e l e a s e of c a l c i t o n i n was implicated in the e l e c t r o l y t e changes t h a t were observed. C a l c i t o n i n may a l s o be i m p l i c a t e d in the plasma and u r i n a r y changes t h a t occur f o l l o w i n g volume expansion s i n c e removal of the c a I c i t o n i n - c o n t a i n i n g "C" c e l l s a l t e r e d the p a t t e r n of c e r t a i n e l e c t r o l y t e changes t h a t occurred in the i n t a c t animals f o l l o w i n g volume expansion. Studies with various PTH preparations suggested t h a t c a l c i t o n i n and PTH may be a c t i n g i n a n t a g o n i s t i c ways in the renal handling of Na, Ca, and Mg. i v TABLE OF CONTENTS Page ABSTRACT i LIST OF TABLES v i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS i x INTRODUCTION I GENERAL METHODS AND MATERIALS 8 SPECIFIC METHODS AND RESULTS 14 I. EXOGENOUS CALCITONIN ADMINISTRATION 14 A. Studies on Rats During Chronic A d m i n i s t r a t i o n of C a l c i t o n i n 14 B. Studies on Rats Following S i n g l e I n j e c t i o n s of C a l c i t o n i n 19 1. In t a c t r a t s 19 2. Thyroparathyroidectomized r a t s 31 I I . CALCIUM INFUSION STUDIES (ENDOGENOUS CALCITONIN RELEASE) 37 Plasma and U r i n a r y E l e c t r o l y t e Changes in Conscious Sheep Following Calcium Infusion 38 I I I . EXTRACELLULAR VOLUME EXPANSION 58 A. Urin a r y E l e c t r o l y t e Changes in Rats FoI lowing a S a I i n e Load 58 I. The e f f e c t s of s a l i n e loading in i n t a c t , TX, and TPTX r a t s 58 TABLE OF CONTENTS (continued) Page 2. The e f f e c t s of PTH ( L i l l y PTE) on the response of TPTX r a t s to s a I i n e load i ng 63 B. The E f f e c t of PTH on Plasma Ca and U r i n a r y E l e c t r o l y t e s 67 C. Plasma and Ur i n a r y E l e c t r o l y t e Changes in Conscious Sheep Following Dextran Infusion 76 DISCUSSION I. EXOGENOUS CALCITONIN ADMINISTRATION 92. A. Chronic Studies 92 B. Acute Studies 93 1. Plasma changes 93 2. Urine changes 95 I I . CALCIUM INFUSION STUDIES (ENDOGENOUS CALCITONIN RELEASE) 102 I I I . EXTRACELLULAR VOLUME EXPANSION 107 A. Volume Expansion i n Rats 107 B. PTH and Renal E l e c t r o l y t e Changes 109 C. Volume Expansion in Sheep 116 1. Plasma changes 116 2. Urine changes I 17 SUMMARY AND CONCLUSIONS 123 I. Exogenous C a l c i t o n i n A d m i n i s t r a t i o n 125 I I . Calcium Infusion Studies (Endogenous C a l c i t o n i n Release) 125 I I I . C a l c i t o n i n and Volume Homeostasis 126 BIBLIOGRAPHY 128 LIST OF TABLES The e f f e c t o f salmon c a l c i t o n i n on u r i n a r y e l e c t r o l y t e s i n d e x t r o s e - i n f u s e d r a t E f f e c t s o f salmon c a l c i t o n i n on u r i n a r y e l e c t r o l y t e e x c r e t i o n i n i n t a c t r a t s Plasma e l e c t r o l y t e changes f o l l o w i n g salmon c a l c i t o n i n i n i n t a c t r a t s The e f f e c t s o f s i m u l t a n e o u s a d m i n i s t r a t i o n o f DOCA and c a l c i t o n i n on u r i n a r y e l e c t r o - l y t e e x c r e t i o n i n i n t a c t r a t s E f f e c t o f c a l c i t o n i n on u r i n a r y e l e c t r o l y t e e x c r e t i o n i n r a t s o f d i f f e r e n t ages U r i n a r y e l e c t r o l y t e e x c r e t i o n i n TPTX and TX r a t s f o l l o w i n g salmon c a l c i t o n i n The e f f e c t o f c a l c i t o n i n on u r i n a r y e l e c t r o l y t e e x c r e t i o n i n TPTX and i n t a c t r a t s C o n t r o l u r i n a r y e l e c t r o l y t e e x c r e t i o n i n TPTX, TX, and i n t a c t r a t s d u r i n g a 24- hour c o l I e c t i o n Plasma changes f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX + CT sheep Changes i n u r i n e volume f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX + CT sheep Changes i n o s m o l a l o u t p u t f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX + CT sheep U r i n a r y P e x c r e t i o n f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX + CT sheep Changes i n u r i n a r y e l e c t r o l y t e e x c r e t i o n f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX + CT sheep VI I LIST OF TABLES ( c o n t i n u e d ) T a b l e Page XIV E f f e c t s o f s a l i n e l o a d i n g on u r i n a r y e l e c t r o l y t e e x c r e t i o n i n i n t a c t , TX, and TPTX r a t s 60-61 XV The e f f e c t o f PTE on u r i n a r y e l e c t r o l y t e e x c r e t i o n f o l l o w i n g a s a l i n e l o a d i n TPTX r a t s 65 XVI E f f e c t o f T C A - p r e c i p i t a t e d PTH and L i l l y PTE on t h e r e s p o n s e o f i n t a c t r a t s t o s a l i n e l o a d i n g 69-70 XVII Log-dose r e s p o n s e t o L i l l y PTE i n TPTX r a t s 71 X V I I I Log-dose r e s p o n s e t o h i g h l y - p u r i f i e d PTH i n TPTX r a t s 72 X I X Plasma changes f o l l o w i n g d e x t r a n i n f u s i o n i n i n t a c t , TX, and TX + CT sheep 79 XX Changes i n u r i n e volume f o l l o w i n g d e x t r a n i n f u s i o n i n i n t a c t , TX, and TX + CT sheep 82 XXI Changes i n o s m o l a l o u t p u t f o l l o w i n g d e x t r a n i n f u s i o n i n i n t a c t , TX, and TX + CT sheep 83 XXI I C l e a r a n c e o f Ca f o l l o w i n g d e x t r a n i n f u s i o n i n i n t a c t , TX, and TX + CT sheep 86 X X I I I U r i n a r y P e x c r e t i o n f o l l o w i n g d e x t r a n i n f u s i o n i n i n t a c t , TX, and TX + CT sheep 87 XXIV Changes i n u r i n a r y e l e c t r o l y t e e x c r e t i o n f o l l o w i n g d e x t r a n i n f u s i o n i n i n t a c t , TX, and TX + CT sheep 89 v i i i LIST OF FIGURES F i g u r e Page 1 T i m e - c o u r s e o f a c t i o n o f a s i n g l e i n j e c t i o n o f salmon c a l c i t o n i n (250 mU/100 g body w e i g h t , i . p . ) on plasma Ca l e v e l s i n s i x - w e e k - o l d r a t s . 15 2 U r i n e volume changes i n r a t s t r e a t e d c h r o n i c a l l y w i t h salmon c a l c i t o n i n (250 mU/100 g body w e i g h t , i . p . t w i c e d a i l y ) . 16 3 The e f f e c t o f c h r o n i c t r e a t m e n t w i t h salmon c a l c i t o n i n (250 mU/100 g body w e i g h t , i . p . , t w i c e d a i l y ) on u r i n a r y e l e c t r o l y t e e x c r e t i o n i n young r a t s . 18 4 E l e c t r o l y t e changes i n 460-g r a t (age s e v e n months) g i v e n 500 mU c a l c i t o n i n , i . v . d u r i n g d e x t r o s e ( 2 . 5 $ ) - s a l i n e (75 meq/l) i n f u s i o n g i v e n a t a r a t e o f 3 m l / h r . 21 5 U r i n a r y changes i n young r a t s i n r e s p o n s e t o g r a d e d doses o f salmon c a l c i t o n i n . 24 6 Compa r i s o n o f a b s o l u t e changes i n u r i n a r y e l e c t r o l y t e e x c r e t i o n i n r e s p o n s e t o a s i n g l e i n j e c t i o n o f c a l c i t o n i n (100 mU/100 g body w e i g h t , i . p . ) i n t o non-DOCA t r e a t e d and DOCA t r e a t e d r a t s . 29 7 Changes i n plasma Ca l e v e l s f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX sheep i n f u s e d w i t h salmon c a l c i t o n i n (2 U / k g / h r ) . 40 8 Changes i n plasma P l e v e l s f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX sheep i n f u s e d w i t h salmon c a l c i t o n i n (2 U / k g / h r ) . 43 9 Changes i n plasma Mg l e v e l s f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX sheep i n f u s e d w i t h salmon c a l c i t o n i n (2 U/kg/hr). 44 I X LIST OF FIGURES ( c o n t i n u e d ) F i g u r e Page 10 Changes i n u r i n e volume and o s m o l a l o u t p u t f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX sheep i n f u s e d w i t h salmon c a l c i t o n i n (2 U / k g / h r ) . 45 11 Changes i n U.. V f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX sheep i n f u s e d w i t h salmon c a l c i t o n i n (2 U / k g / h r ) . 49 12 Changes i n Up V f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX sheep i n f u s e d w i t h salmon c a l c i t o n i n (2 U / k g / h r ) . 50 13 Changes i n U pV d u r i n g t h e p e r i o d o f Ca i n f u s i o n i n i n d i v i d u a l i n t a c t , TX, and TX sheep i n f u s e d w i t h salmon c a l c i t o n i n (2 U / k g / h r ) . 51 14 Changes i n U^ V f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX sheep i n f u s e d w i t h salmon c a l c i t o n i n (2 U / k g / h r ) . 52 15 Changes i n U^V and U„ ^ V f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX sheep i n f u s e d w i t h salmon c a l c i t o n i n (2 U/kg/hr). 54 16 Changes i n u r i n a r y e l e c t r o l y t e e x c r e t i o n i n i n t a c t , TX, and TPTX r a t s d u r i n g t h e f i r s t t h r e e h o u r s f o l l o w i n g a s a l i n e l o a d (.0.9% N a C l , 5ml/100 g body w e i g h t , i . p . ) . 62 17 The e f f e c t o f L i l l y PTE (100 U/IOOg body w e i g h t , s.c.) on t h e r e s p o n s e o f TPTX r a t s t o a s a l i n e l o a d as compared t o t h e r e s p o n s e i n TX r a t s w i t h f u n c t i o n i n g p a r a - t h y r o i d t r a n s p l a n t s . 66 18 T i m e - c o u r s e o f a c t i o n o f L i l l y PTE (100 U/I00 g body w e i g h t , s.c.) on plasma Ca l e v e l s i n TPTX r a t s . 74 19 Log-dose r e s p o n s e t o L i l l y PTE i n TPTX r a t s . 75 20 Changes i n plasma Ca l e v e l s f o l l o w i n g a 15-minute d e x t r a n i n f u s i o n i n i n t a c t , TX, and TX sheep t r e a t e d w i t h salmon c a l c i t o n i n (2 X 50 U). 77 X LIST OF FIGURES ( c o n t i n u e d ) F i g u r e Page 21 Changes i n plasma P and plasma Mg f o l l o w i n g a 15-minute d e x t r a n i n f u s i o n i n i n t a c t , I X , and TX sheep t r e a t e d w i t h salmon c a l c i t o n i n (2 X 50 U ) . 80 22 Changes i n u r i n a r y volume and U V osm f o l l o w i n g a 15-minute d e x t r a n i n f u s i o n i n i n t a c t , TX, and TX sheep t r e a t e d w i t h salmon c a l c i t o n i n (2 X 50 U ) . 81 23 Changes i n U N g V and U ^ V f o l l o w i n g a 15- m i n u t e d e x t r a n i n f u s i o n i n i n t a c t , TX, and TX sheep t r e a t e d w i t h salmon c a l c i t o n i n (2 X 50 U ) . 85 24 Changes i n U^ V f o l l o w i n g a 15-minute d e x t r a n i n f u s ? o n i n i n t a c t , TX, and TX sheep t r e a t e d w i t h salmon c a l c i t o n i n (2 X 50 U ) . 88 25 Changes i n U^V and ^rreaf ^ f o l l o w i n g a 15-minute d e x t r a n i n f u s i o n i n i n t a c t , TX, and TX sheep t r e a t e d w i t h salmon c a l c i t o n i n (2 X 50 U). 91 x i AcknowIedgements The a u t h o r w i s h e s t o e x t e n d s p e c i a l t h a n k s t o h e r Committee f o r t h e i r c o n t i n u e d i n t e r e s t i n t h e e x p e r i m e n t a l s t u d i e s and f o r t h e i r h e l p i n p r e p a r a t i o n of t h e t h e s i s . The g u i d a n c e and d i r e c t i o n o f Dr. R. K e e l e r , t h e Committee C h a i r m a n , i n t h e a p p r o a c h t o r e n a l p h y s i o l o g y i s g r e a t l y a p p r e c i a t e d . Many t h a n k s a r e a l s o e x t e n d e d t o Dr. J . Ledsome, Mr. K. Henze, and Mr. G. S c h u l z f o r i n s t r u c t i o n and a s s i s t a n c e w i t h s u r g i c a l t e c h n i q u e s . To her R e s e a r c h S u p e r v i s o r , Dr. D.H. Copp, f o r h i s c o n t i n u a l i n s p i r a t i o n and d i r e c t i o n , and t o t h e "Lab" f o r t h e i r c o n s t a n t s u p p o r t and f e l l o w s h i p , t h e a u t h o r i s d e e p l y i n d e b t e d . INTRODUCTION The e l e c t r o l y t e s i n t h e e x t r a c e l l u l a r f l u i d (ECF) a r e m a i n t a i n e d a t c o n s t a n t l e v e l s d e s p i t e wide a l t e r a t i o n s i n m i n e r a l i n t a k e o r e x c r e t i o n . T h i s c o n s t a n c y depends on t h e e x i s t e n c e o f e f f i c i e n t h o m e o s t a t i c mechanisms, many o f which a r e h u m o r a l . T h i s t h e s i s i s p r i m a r i l y c o n c e r n e d w i t h t h e r o l e o f c a l c i t o n i n as one of t h e hormones w h i c h c a n m o d i f y t h e l e v e l s o f c e r t a i n body e l e c t r o l y t e s i n t h e ECF. However, c o n s i d e r a t i o n o f t h e a c t i o n o f p a r a t h y r o i d hormone (PTH) i s a l s o i n c l u d e d i n t h i s i n v e s t i g a t i o n s i n c e i t i s becoming i n c r e a s i n g l y a p p a r e n t t h a t c a l c i t o n i n and PTH can a c t i n a n t a g o n i s t i c ways. Bone i s a t a r g e t s i t e f o r both o f t h e s e hormones and s e r v e s as a s t o r e - house o r r e s e r v e s u p p l y o f m i n e r a l w h i c h , i n c o n t r a s t t o t h e f l u c t u a t i n g s o u r c e o f m i n e r a l o b t a i n e d from d i e t a r y s o u r c e s , i s c o n s t a n t l y a v a i l a b l e t o t h e body. Most of t h e body's Ca (99%) and i n o r g a n i c P (85%), 50% o f t h e t o t a l body Na and Mg, and a b o u t 8% o f t h e K i n t h e body i s i n bone. Some o f t h i s m i n e r a l i s r e a d i l y e x c h a n g e a b l e , and r e l e a s e o f t h e more f i r m l y bound f r a c t i o n can a l s o o c c u r c o i n c i d e n t w i t h f a c t o r s w h i c h may i n i t i a t e bone breakdown o r r e s o r p t i o n . Hormones w h i c h c a n i n f l u e n c e e i t h e r t h e r e s o r p t i o n o r a c c r e t i o n o f bone i n f l u e n c e t h e movement o f t h o s e m i n e r a l s found i n bone. The p a r a t h y r o i d g l a n d s have a c r i t i c a l r o l e i n m a i n t a i n i n g a normal plasma Ca l e v e l . P a r a t h y r o i d hormone from t h e c h i e f c e l l s o f t h e s e g l a n d s i s s e c r e t e d i n r e s p o n s e t o a h y p o c a l c e m i c s t r e s s and a c t s by e n h a n c i n g 2 o s t e o l y s i s ( I ) , r e s u l t i n g i n t h e r e l e a s e o f Ca from bone t o t h e ECF. Hypomagnesemia was a l s o shown i n i r i v i t r o s t u d i e s t o c a u s e r e l e a s e o f t h i s hormone ( 2 ) , and PTH a d m i n i s t r a t i o n r e s u l t s i n movement o f Mg as w e l l as Ca from bone t o ECF ( 3 , 4 ) . Ph o s p h a t e i s a l s o r e l e a s e d from bone by PTH. However, t h e o b s e r v e d e f f e c t o f a lowered plasma P l e v e l f o l l o w i n g PTH a d m i n i s t r a t i o n i s s e c o n d a r y t o t h e p h o s p h a t u r l c a c t i o n o f t h i s hormone ( 5 , 6 ) . P a r a t h y r o i d hormone has a l s o been i m p l i c a t e d i n t h e movement o f Na from bone i n r e s p o n s e t o Na d e p l e t i o n ( 7 ) . Thus, i t would appear t h a t PTH i s one o f t h e m a j o r hormones which c a n i n i t i a t e t h e movement o f e l e c t r o l y t e s from t h e bone p o o l . The second major hormone i n v o l v e d i n t h e dynamics o f bone m i n e r a l i s c a l c i t o n i n . T h i s was d i s c o v e r e d more r e c e n t l y t h a n PTH a l t h o u g h i t i s an o l d e r hormone p h y l o g e n e t i c a l l y . C a l c i t o n i n i s d e s c r i b e d as a hormone t h a t d e p r e s s e s bone r e s o r p t i o n ( 8 ) . I t has been r e p o r t e d t o enhance o s t e o b l a s t i c a c t i v i t y and t h u s may a l s o be i n v o l v e d i n m i n e r a l a c c r e t i o n and f o r m a t i o n o f new bone ( 9 ) . I t i s r e l e a s e d i n r e s p o n s e t o an e l e v a t e d plasma Ca l e v e l , d e p r e s s e s bone r e s o r p t i o n , and t h u s r e d u c e s t h e i n p u t o f bone Ca t o t h e ECF so t h a t plasma Ca l e v e l s a r e r e t u r n e d t o normal ( 1 0 ) . D u r i n g r e c e n t y e a r s , t h e i n v o l v e m e n t o f c a l c i t o n i n i n phosphate h o m e o s t a s i s has a l s o been r e c o g n i z e d . The hypophosphatemia which i s o b s e r v e d f o l l o w i n g c a l c i t o n i n t r e a t m e n t i s a p p a r e n t l y a d i r e c t a c t i o n o f t h e hormone and i s n o t s e c o n d a r y t o PTH s t i m u l a t i o n ( I I - 1 4 ) . The r o l e o f c a l c i t o n i n i n Mg h o m e o s t a s i s i s s t i l l n ot c l e a r . T h e r e i s some e v i d e n c e t h a t i t a c t s i n a s i m i l a r way i n t h e c o n t r o l o f t h i s i o n a s i t does w i t h Ca, t h a t i s by l o w e r i n g plasma Mg l e v e l s as a r e s u l t o f d e c r e a s i n g bone r e s o r p t i o n ( 4 , 15). O t h e r s have found no plasma Mg l o w e r i n g e f f e c t o f c a l c i t o n i n ( 1 6 - 18). N e v e r t h e l e s s , t h e f a c t t h a t a Mg l o a d r e s u l t s i n a h y p o c a l c e m i c r e s p o n s e , prov ided t h e 3 c a I c i t o n i n - c o n t a i n i n g "C" c e l l s a r e i n t a c t , s u g g e s t s t h a t c a l c i t o n i n may be i n v o l v e d i n Mg m e t a b o l i s m as w e l l ( 1 9 ) . Thus t h e r e a r e two m a j o r hormonal s y s t e m s , PTH and c a l c i t o n i n , w h i c h a p p e a r t o be w o r k i n g i n o p p o s i t e d i r e c t i o n s w i t h r e s p e c t t o bone r e s o r p t i o n and bone a c c r e t i o n . The duaI-hormonaI c o n c e p t (20) o f t h e c o n t r o l o f plasma Ca by PTH and c a l c i t o n i n i s now w e l l a c c e p t e d . However, t h e i n t e r r e l a t i o n - s h i p o f t h e s e two hormones i n t h e c o n t r o l o f t h e o t h e r m a j o r e l e c t r o l y t e s found i n bone i s f a r from c l e a r . B e s i d e s t h e i n p u t o f m i n e r a l from t h e bone p o o l , t h e ECF a l s o r e c e i v e s an i n p u t o f m i n e r a l from t h e g u t v i a i n t e s t i n a l a b s o r p t i o n . Once t h e e l e c t r o l y t e s a r e i n t h e ECF, t h e y c a n e i t h e r be t a k e n up by c e l l s o r bone, o r c a n be l o s t from t h e body v i a v a r i o u s e x c r e t o r y mechanisms. An e f f i c i e n t c o n t r o l mechanism must t h e r e f o r e have n o t o n l y some i n f l u e n c e on t h e i n p u t t o t h e ECF, but a l s o some i n f l u e n c e on t h e o u t p u t from i t . P a r a t h y r o i d hormone r a i s e s plasma Ca and Mg t o normal l e v e l s by i n c r e a s i n g t h e r a t e o f bone r e s o r p t i o n and by d e c r e a s i n g t h e l o s s o f Ca ( 2 1 , 5, 6, 22-26) and Mg (24-26) i n t h e u r i n e . An i n c r e a s e d r e a b s o r p t i o n o f t h e s e i o n s from t h e k i d n e y t u b u l e , under t h e i n f l u e n c e o f PTH, would p r o t e c t t h e bone from undue l o s s o f m i n e r a l and would h e l p r e s t o r e normal l e v e l s o f Ca and Mg i n t h e ECF. The p h o s p h a t u r i c e f f e c t o f PTH has been known f o r many y e a r s . A l b r i g h t and E l l s w o r t h (27) f i r s t o b s e r v e d t h a t a p h o s p h a t u r i a o c c u r r e d i m m e d i a t e l y f o l l o w i n g PTH t r e a t m e n t . T h i s i n t u r n r e s u l t e d i n a hypophosphatemia w h i c h t h e y e r r o n e o u s l y c o n s i d e r e d as t h e i n d i r e c t c a u s e o f t h e Ca r e l e a s e from bone. Numerous s t u d i e s s i n c e t h e n , s t i m u l a t e d by t h e i n v e s t i g a t i o n s o f B a r n i c o t (28) and G a i l l a r d ( 2 9 ) , have shown t h a t t h e a c t i o n o f PTH on bone i s a d i r e c t one. However, t h e m a j o r e f f e c t on phosphate e x e r t e d by PTH i s not v i a bone but by k i d n e y . 4 The r o l e o f t h e k i d n e y as a p o s s i b l e t a r g e t o r g a n o f c a l c i t o n i n i s o n l y j u s t becoming r e c o g n i z e d . C a l c i t o n i n d e c r e a s e s t h e amount o f Ca i n t h e ECF by i n h i b i t i n g bone r e s o r p t i o n a n d ^ s has been s u g g e s t e d , by d e c r e a s i n g t h e r e a b s o r p t i o n o f t h i s i o n from t h e k i d n e y t u b u l e ([4, 3 0 ) . C a l c i t o n i n may a l s o d e c r e a s e t h e r e a b s o r p t i o n o f Mg by t h e k i d n e y (30) which would enhance t h e plasma Mg l o w e r i n g e f f e c t which i s sometimes o b s e r v e d f o l l o w i n g c a l c i t o n i n t r e a t m e n t . C a l c i t o n i n has a l s o been shown t o c a u s e a p h o s p h a t u r i c r e s p o n s e . S i n c e t h i s e f f e c t i s a l s o f ound i n p a r a t h y r o i d e c t o m i z e d a n i m a l s , i t i s n o t s e c o n d a r y t o h y p o c a I c e m i c s t i m u l a t i o n o f t h e p a r a t h y r o i d g l a n d s ( 1 1 - 1 4 ) . The o b s e r v a t i o n t h a t c a l c i t o n i n a d m i n i s t r a t i o n i s f o l l o w e d by a n a t r i u r e t i c r e s p o n s e (31, 14, 32, 16) has i m p l i c a t e d t h i s hormone i n Na h o m e o s t a s i s as w e l l . The changes i n t h e r e n a l e x c r e t i o n o f c e r t a i n e l e c t r o l y t e s f o l l o w i n g e i t h e r PTH o r c a l c i t o n i n a d m i n i s t r a t i o n i n d i c a t e t h a t t h e k i d n e y i s a second m a j o r t a r g e t o r g a n o f t h e s e hormones. The a p p a r e n t changes i n r e a b s o r p t i v e c a p a c i t y o f t h e k i d n e y t u b u l e c e l l s t o c e r t a i n i o n s f o l l o w i n g i n t e r v e n t i o n o f e i t h e r hormone can enhance o r m o d i f y t h e plasma changes t h a t o c c u r a s a r e s u l t o f t h e a c t i o n o f PTH o r c a l c i t o n i n on bone. However, t h e e x p e r i - mental e v i d e n c e i s f a r from c o m p l e t e f o r c l a r i f y i n g t h e a c t i o n o f PTH o r c a l c i t o n i n on t h e k i d n e y . The p a r t i c i p a t i o n o f PTH and c a l c i t o n i n i n Na h o m e o s t a s i s has n o t been i n t e n s i v e l y e x p l o r e d . A l d o s t e r o n e and t h e r e n i n - a n g i o t e n s i n system a r e c o n s i d e r e d t o be t h e p r i m a r y hormones c o n c e r n e d w i t h t h e m a i n t e n a n c e o f a normal amount o f Na i n t h e ECF. A l t e r a t i o n s i n t h e c i r c u l a t i n g l e v e l s o f a l d o s t e r o n e , i n a d d i t i o n t o changes i n g l o m e r u l a r f i l t r a t i o n r a t e (GFR), hemodynamics, o r " S t a r l i n g f o r c e s " w i t h i n t h e k i d n e y a r e t h o u g h t t o be t h e 5 major c o n t r o l l i n g mechanisms i n Na r e g u l a t i o n ( 3 3 - 3 5 ) . However, t h i s e x p l a n a t i o n f o r t h e c o n t r o l o f body Na i s not e n t i r e l y s a t i s f a c t o r y . When a l l t h e s e v a r i a b l e s a r e c o n t r o l l e d , an i n c r e a s e d l o s s o f Na c a n s t i l l o c c u r i n r e s p o n s e t o ECF volume e x p a n s i o n ( 36, 3 7 ) , and e v i d e n c e from c r o s s - c i r c u l a t i o n (38, 39) and o t h e r s t u d i e s s u g g e s t s t h a t t h e f a c t o r ( s ) i n v o l v e d i s humoral i n n a t u r e . A l s o , a N a - l o s i n g f a c t o r has been found i n t h e plasma and u r i n e o f s a l t - l o a d e d man and sheep w h i c h c a u s e d a n a t r i u r e s i s when i n j e c t e d i n t o a s s a y r a t s ( 4 0 ) . T h e r e f o r e , a s e a r c h f o r f u r t h e r c o n t r o l s ystems i n t h e r e g u l a t i o n o f t h i s i m p o r t a n t i o n i s i n d i c a t e d . The o b s e r v a t i o n t h a t a n a t r i u r e s i s a ccompanies t h e a d m i n i s t r a t i o n o f c a l c i t o n i n s u g g e s t s t h e v a l u e o f an i n t e n s i v e s t u d y o f t h i s hormone and i t s r e l a t i o n t o Na c o n t r o l . A p a r a l l e l i s m seems t o e x i s t i n t h e r e n a l hand I ing o f Ca and Na ( 4 1 - 4 4 ) , and a t l e a s t i n some p a r t o f t h e nephron Ca and Na may s h a r e a common r e a b s o r p t i v e pathway ( 4 5 ) . However, i t i s not known i f h o r m o n a l l y - i n d u c e d changes i n t h e r e n a l h a n d l i n g o f Ca a r e p a r a l - l e l e d by s i m i l a r changes i n Na. A g r e a t d e a l o f e x p e r i m e n t a l work has been performed i n an a t t e m p t t o u n d e r s t a n d t h e a c t i o n o f PTH on t h e k i d n e y . Many o f t h e f i n d i n g s r e s u l t i n g f rom t h i s work have been r e v i e w e d by Rasmussen ( 4 6 ) . These s t u d i e s have c o n c e n t r a t e d on t h e C a , P, and Mg changes t h a t o c c u r f o l l o w i n g PTH a d m i n i s t r a t i o n , o r t h e c l i n i c a l a b n o r m a l i t i e s t h a t r e s u l t from m a l f u n c t i o n o f t h e p a r a t h y r o i d g l a n d . However, p o s s i b l e i n v o l v e m e n t o f PTH i n r e g a r d t o o t h e r e l e c t r o l y t e s has not been w i d e l y i n v e s t i g a t e d . The s t u d i e s r e p o r t e d i n t h i s t h e s i s e x p l o r e t h e e l e c t r o l y t e changes t h a t o c c u r i n plasma and u r i n e f o l l o w i n g exogenous c a l c i t o n i n a d m i n i s t r a t i o n , o r d u r i n g maneuvers d e s i g n e d t o promote endogenous c a l c i t o n i n r e l e a s e . 6 I t was hoped t h a t a c o n c u r r e n t s t u d y o f plasma and u r i n e f o l l o w i n g c a l c i t o n i n i n v o l v e m e n t would l e a d t o a g r e a t e r u n d e r s t a n d i n g o f t h e mechanism o f a c t i o n o f t h i s hormone. These i n v e s t i g a t i o n s were prompted by t h e a u t h o r ' s o b s e r v a t i o n t h a t a p r o f o u n d d i u r e t i c r e s p o n s e t o salmon c a l c i t o n i n o c c u r r e d d u r i n g a 10-day p e r i o d o f t r e a t m e n t i n r a t s . Repeat s t u d i e s c o n f i r m e d t h e s e i n i t i a l o b s e r v a t i o n s and showed t h a t t h e d i u r e t i c r e s p o n s e was accompanied by a n a t r i u r e s i s and a l s o changes i n o t h e r u r i n a r y e l e c t r o l y t e s . E a r l i e r s t u d i e s by A r d a i l l o u _£+£_[_• (14) and Rasmussen e t a j _ . (31) w i t h p o r c i n e c a l c i t o n i n r e v e a l e d a n a t r i u r e t i c r e s p o n s e f o l l o w i n g hormone a d m i n i s t r a t i o n , but a t t h a t t i m e no i n v e s t i g a t i o n o f t h e r e n a l e f f e c t s o f salmon c a l c i t o n i n had been r e p o r t e d . These i n i t i a l f i n d i n g s w i t h salmon c a l c i t o n i n l e d t o f u r t h e r i n v e s t i - g a t i o n s i n o r d e r t o e s t a b l i s h : (a) Was t h e e f f e c t o f t h e hormone on t h e k i d n e y a d i r e c t one o r was i t , f o r e xample, r e l a t e d t o r e l e a s e o f PTH i n r e s p o n s e t o h y p o c a l c e m i a ? (b) Was t h e plasma Ca I eve I p e r se t h e i n i t i a t i n g f a c t o r ? ( c ) Were t h e o b s e r v e d r e n a l r e s p o n s e s a r e s u l t o f changes i n GFR? (d) V/ere changes In mi nera l o c o r t i c o i d a c t i v i t y i m p l i c a t e d i n any way? (e) Would e l e c t r o l y t e changes s i m i l a r t o t h o s e found f o l l o w i n g exogenous c a l c i t o n i n t r e a t m e n t o c c u r w i t h endogenous I y - r e I eased hormone i n r e s p o n s e t o e l e v a t e d plasma Ca l e v e l s which a r e known t o i n c r e a s e r e l e a s e o f c a l c i t o n i n ( 4 7 , 4 8 ) ? ( f ) If t h e c a I c i t o n i n - c o n t a i n i n g "C" c e l l s a r e removed, a r e t h e e l e c t r o l y t e changes t h a t o c c u r f o l l o w i n g Ca s t r e s s a l t e r e d ? 7 (g) Is the n a t r i u r e s i s which accompanies the increased Ca e x c r e t i o n f o l l o w i n g Ca s t r e s s (49-54) r e l a t e d to c a l c i t o n i n s e c r e t i o n ? (h) i s c a l c i t o n i n involved in the n a t r i u r e s i s and c a l c i u r e s i s t h a t occur f o l l o w i n g volume expansion (41, 43, 55, 44, 56)? The experiments reported in t h i s t h e s i s were designed in an attempt t o answer these q u e r i e s . 8 GENERAL METHODS AND MATERIALS ExperimentaI an imaIs and d i e t s Rats: Male r a t s of the Long-Evans s t r a i n were used in a l l r a t s t u d i e s and were supplied by Blue Spruce Farms, Charles R i v e r , Ont. They were maintained on Purina Rat Chow and water and were without food during c o l l e c t i o n periods unless otherwise s t a t e d . Sheep: Female lambs, f i v e - s i x months of age, were obtained l o c a l l y and were studied during the ensuing six-month p e r i o d . These animals were given f r e e access t o crushed o a t s , Omolene ( P u r i n a ) , a l f a l f a hay, and water a t a I I times except f o r 24 hours p r i o r t o and during experimental periods when food was removed. An i o d i z e d s a l t l i c k was a l s o provided. Both r a t and sheep d i e t s contained Vitamins A and D in amounts considered adequate f o r normal n u t r i t i o n and metabolism as recommended by the Food and N u t r i t i o n Board, N.R.C., Canada. Procedures f o r pIasma and u r i n e c o l l e c t i o n s Rats: Blood samples were obtained e i t h e r from the t a i l v e i n o r by c a r d i a c puncture, depending on the number of analyses t o be performed. Urine was c o l l e c t e d w h i l e animals were housed in metabolism cages (Acme Metal Products, C i n c i n n a t i , Ohio) or in some s t u d i e s by f r a c t i o n a l c o l l e c t i o n v i a a bladder cannula. Sheep: Blood samples were obtained from a c a t h e t e r (Abbocath 18-guage i v . c a t h e t e r , Abbott L a b o r a t o r i e s , Montreal, Que.) i n s e r t e d i n t o the l a t e r a l 9 saphenous v e i n . The c a t h e t e r allowed frequent blood sampling and was removed a t the end of each experiment. A bladder c a t h e t e r (Bardex Foley C a t h e t e r , 14 FR, C R . Bard I n t e r n a t i o n a l L t d . , England) was a l s o i n s e r t e d p r i o r t o each experiment and u r i n e was c o l l e c t e d during 15-minute i n t e r v a l s w i t h uninterrupted flow f o l l o w i n g an e q u i l i b r a t i o n period of one hour. These c a t h e t e r s were held in p o s i t i o n by i n f l a t i o n of an i n t e r n a l 5-cc ba I I con. S u r g i c a l Procedures Rats: A l l surgery was performed with the a i d of a d i s s e c t i n g microscope while the animals were anaesthetized with ether. Thyroparathyroidectomy involved the removal of both lobes of the t h y r o i d with the attached para- t h y r o i d s and the connecting isthmus. Thyroidectomy included the sep a r a t i o n of the s u p e r i o r parathyroids from underlying t h y r o i d t i s s u e on each lobe and t h e i r immediate t r a n s p l a n t a t i o n deep i n t o the muscle mass of the p o s t e r i o r aspect of the t h i g h . The t h y r o i d t i s s u e was then completely removed. Control animals were subjected t o sham surgery which simply c o n s i s t e d of exposure of the t h y r o i d complex. The e f f e c t i v e n e s s of the surgery was determined two weeks a f t e r surgery by plasma Ca measurements f o l l o w i n g o v e r n i g h t s t a r v a t i o n . Those animals with plasma Ca l e v e l s below 7.5 mg% were considered s u c c e s s f u l l y thyroparathyroideetornized (TPTX), and the t r a n s p l a n t animals with plasma Ca l e v e l s g r e a t e r than 9.0 mg% were considered e f f e c t i v e l y thyroidectomized (TX). Plasma Ca l e v e l s in i n t a c t c o n t r o l animals were a l s o determined a t t h i s time. Those r a t s with t h y r o i d s removed were immediately s t a r t e d on t h y r o x i n e (Synthroid-Sodiurn Levothyroxine, U.S.P., F l i n t L a b o r a t o r i e s , A l l i s t o n , Ont.) 10 w h i c h t h e y o b t a i n e d from t h e i r d r i n k i n g w a t e r a t a c o n c e n t r a t i o n o f 100 u g / l . T h i s dose was based on a s t u d y by Kennedy and Talmage ( 5 7 ) . Sheep: T h y r o i d e c t o m y i n sheep was performed under h a l o t h a n e - n i t r o u s o x i d e a n a e s t h e s i a f o l l o w i n g i n i t i a l i n d u c t i o n w i + n p e n t o t h a I Sodium ( A b b o t t L a b o r a t o r i e s , M o n t r e a l , Que.) w h i c h e n a b l e d i n s e r t i o n o f an e n d o t r a c h e a l t u b e . S i n c e t h e p a r a t h y r o i d s i n sheep a r e c o m p l e t e l y s e p a r a t e * from t h e t h y r o i d g l a n d , t h y r o i d e c t o m y was p e rformed w i t h o u t t h e n e c e s s i t y o f p a r a - t h y r o i d t r a n s p l a n t s and c o n s i s t e d o f removal o f t h e two t h y r o i d l o b e s and t h e c o n n e c t i n g more caudaI I y - p I a c e d i s t h m u s . F o l l o w i n g s u r g e r y , t h e a n i m a l s were g i v e n r e p l a c e m e n t t h y r o x i n e ( S y n t h r o i d - S o d i u r n L e v o t h y r o x i n e , U.S.P., F l i n t L a b o r a t o r i e s , A l l i s t o n , Ont.) i n a d a i l y o r a l dose o f I mg ( 5 8 ) . Hormones and I n f u s a t e s C a I c i t o n i n : P u r e salmon c a l c i t o n i n (Armour P h a r m a c e u t i c a l Co., Kankakee, I I I . ) h a v i n g an a c t i v i t y o f 5000 MRC U/mg was used f o r a l l c a l c i t o n i n a d m i n i s t r a t i o n s t u d i e s . The a c t i v i t y o f t h e hormone was d e t e r m i n e d by b i o a s s a y i n young r a t s a c c o r d i n g t o a m o d i f i c a t i o n o f t h e method o f Kumar e t a I. (59 ) i n w h i c h t h e h y p o c a l c e m i c r e s p o n s e was compared t o t h a t o f M e d i c a l R e s e a r c h S t a n d a r d (MRC) B o f p o r c i n e c a l c i t o n i n . The v e h i c l e used f o r c a l c i t o n i n was I % NaAc i n 0.\% g l y c i n e , pH 4.6. P a r a t h y r o i d Hormone: B o v i n e PTH was used f o r a l l PTH a d m i n i s t r a t i o n s t u d i e s and was o b t a i n e d i n v a r i o u s d e g r e e s o f p u r i t y . L i l l y P a r a t h y r o i d E x t r a c t (PTE) ( P a r a - t h o r - m o n e , U.S.P., E l i L i l l y Co. Canada, L t d . , T o r o n t o , O n t . ) , O c c a s i o n a l l y , t h e s m a l l i n f e r i o r p a r a t h y r o i d s may be i n c o r p o r a t e d i n t h e t h y r o i d mass. However, t h e p r e d o m i n a n t s u p e r i o r p a r a t h y r o i d s a r e q u i t e d i s t i n c t and a r e found h i g h up i n t h e thymus t i s s u e . 11 T C A - p r e c i p i f a t e d PTH (240 U/mg) and a f u r t h e r h i g h l y p u r i f i e d p r e p a r a t i o n (3000 U/mg), k i n d l y p r o v i d e d by Dr. Na d i n e W i l s o n o f t h i s l a b o r a t o r y , were used f o r t h e d i f f e r e n t i n v e s t i g a t i o n s . B i o a s s a y s f o r PTH were based on t h e method o f C a u s t o n e t a I . ( 6 0 ) . D e s o x y c o r t i c o s t e r o n e a c e t a t e (DOCA): P e r c o r t e n ( C i b a Co., L t d . , D o r v a l , Que.), a s y n t h e t i c m i n e r a l o c o r t i c o i d , was used t o s i m u l a t e t h e a c t i v i t y o f a l d o s t e r o n e . D e x t r a n : 6% G e n t r a n 75 i n 0.9% NaCl s o l u t i o n ( T r a v e n o l L a b o r a t o r i e s , I n c . , Mo r t o n G r o v e , I I I . ) was used f o r t h e volume e x p a n s i o n s t u d i e s i n sheep. D e x t r o s e - S a I ? n e : 5% d e x t r o s e and 0.9$ N a C l , U.S.P. ( B a x t e r L a b o r a t o r i e s , M a l t o n , Ont.) was used as t h e i n f u s i o n v e h i c l e i n t h e C a C ^ s t u d i e s i n sheep. CaCI ̂  s o l u t i o n : C a C ^ was d i s s o l v e d i n d e x t r o s e - s a l i n e s o l u t i o n t o g i v e a dose of 10 mg Ca/kg/hour. Plasma and u r i n e a n a l y s e s B l o o d samples were c o l l e c t e d i n h e p a r i n i z e d t u b e s , i m m e d i a t e l y c e n t r i f u g e d , and t h e plasma s e p a r a t e d . C a l c i u m ( C a ) , i n o r g a n i c phosphorus ( P ) , magnesium (Mg), sodium ( N a ) , and p o t a s s i u m (K) c o n c e n t r a t i o n s were d e t e r m i n e d t h e same day and o s m o l a l i t y was a l s o measured i n some s t u d i e s . I n d i v i d u a l t i m e d u r i n e samples were c o l l e c t e d and t h e volume measured. The a n a l y s e s were c a r r i e d o u t i m m e d i a t e l y f o l l o w i n g c o l l e c t i o n . In a d d i t i o n t o t h e a n a l y t i c a l p r o c e d u r e s w h i c h were c a r r i e d o u t on plasma s a m p l e s , u r i n e a n a l y s e s a l s o i n c l u d e d a measurement o f endogenous c r e a t i n i n e e x c r e t i o n ( U r , V ) . T h i s measurement gave an i n d i c a t i o n o f changes i n 12 GFR and a l s o o f c o m p l e t e n e s s o f b l a d d e r e m p t y i n g . U r i n e o s m o l a l c o n c e n t r a t i o n s were a l s o d e t e r m i n e d i n some s t u d i e s . Ana I y t i c a I Methods C a I c i u r n : Plasma Ca was measured on t h e T e c h n i c o n Auto A n a l y z e r , Method N-31 P, m o d i f i e d by Newsome ( 6 1 ) . U r i n e Ca c o n c e n t r a t i o n s were measured by A t o m i c A b s o r p t i o n S p e c t r o p h o t o m e t r y u s i n g t h e J a r r e l l - A s h Model 280 Atom- s o r b . P h o s p h o r u s : Both plasma and u r i n e samples were measured on t h e T e c h n i c o n A u t o A n a l y z e r , Method N-4 b w h i c h i s based on t h e method o f F i s k e and Sub- barow ( 6 2 ) . Magnesium: Plasma and u r i n e samples were measured by A t o m i c A b s o r p t i o n S p e c t r o p h o t o m e t r y u s i n g t h e J a r r e l l - A s h Model 280 Atomsorb. Sodium and P o t a s s i u m : Plasma and u r i n e samples were measured by e m i s s i o n on I n s t r u m e n t a t i o n L a b o r a t o r i e s Model 143 f l a m e p h o t o m e t e r . C r e a t i n i ne: C r e a t i n i n e was e s t i m a t e d a s t h e a l k a l i n e p i c r a t e , t h e method b e i n g m o d i f i e d f o r use on automated c o l o r i m e t r i c a n a l y z e r s ( C a r l o Erba A u t o m a t i c A n a l y z e r , Model CLA 1510). OsmolaI i t y : O s m o l a l i t y o f plasma and u r i n e samples was measured by f r e e z i n g p o i n t d e p r e s s i o n u s i n g t h e Osmette P r e c i s i o n Osmometer, Model 2007 P r e c i s i o n Systems, I n c . S t a t i s t i c a I Methods S t u d e n t ' s t t e s t f o r s m a l l , u n p a i r e d g r o u p s was used f o r s t a t i s t i c a l e v a l u a t i o n o f t h e d a t a . Where £ v a l u e s of <_ 0.050 were f o u n d , t h e r e s u l t s were c o n s i d e r e d s i g n i f i c a n t l y d i f f e r e n t . 13 Abbrev i a t i o n s Clearance of x; the number of ml of plasma completely c l e a r e d of substance x_ per minute: C = U V = Urine c o n c e n t r a t i o n X Volume of u r i n e excreted (ml) X minute Plasma c o n c e n t r a t i o n x_ CT C a l c i t o n i n ECF E x t r a c e l l u l a r f l u i d GFR Glomerular f i l t r a t i o n r a t e mg^ mg per 100 ml s o l u t i o n Na/Creat. The r a t i o of sodium t o endogenous c r e a t i n i n e in the u r i n e . This r a t i o g i v e s an approximation of the f r a c t i o n a l e x c r e t i o n of sodium, assuming plasma sodium and c r e a t i n i n e c o n c e n t r a t i o n s are unchanged during the experimental p e r i o d . Na/K The r a t i o of sodium t o potassium in the u r i n e . P The chemical a b b r e v i a t i o n f o r phosphorus i s used when r e f e r r i n g t o plasma and u r i n a r y phosphate s i n c e the chemical a n a l y s i s measures in o r g a n i c phosphorus. PTE L i l l y p arathyroid e x t r a c t . PTH P a r a t h y r o i d hormone. This term i s used when r e f e r r i n g g e n e r a l l y to p a r a t h y r o i d hormone. TCA-precipitated PTH T r i c h l o r o a c e t i c - a c i d p r e c i p i t a t e d parathyroid hormone. The a b b r e v i a t i o n s PTE and T C A - p r e c i p i t a t e d PTH (240 U/mg) , in a d d i t i o n to the term h i g h l y - p u r i f i e d PTH (3000 U/mg) are used then comparing the responses to hormone preparations of varying degrees of p u r i t y . U V The r a t e of e x c r e t i o n of a given substance x in the u r i n e : U V = u r i n e x x c o n c e n t r a t i o n X Volume of u r i n e excreted X time '. x 14 SPECIFIC METHODS AND RESULTS I. EXOGENOUS CALCITONIN ADMINISTRATION A. Stud i es on R a t s P u r i ng C h r o n i c Adrni ri i s t r a t i o n o f C a I c i t o n i n Methods T h i r t y - d a y - o l d r a t s were i n d i v i d u a l l y housed i n m e t a b o l i s m c a g e s f o r 10 days and u r i n e was c o l l e c t e d o v e r 24-hour p e r i o d s . They had f r e e a c c e s s t o f o o d and w a t e r d u r i n g t h i s t i m e p e r i o d . Ten r a t s were each g i v e n 250 mU salmon c a l c i t o n i n i . p . i n v e h i c l e (\% NaAc i n 0.\% g l y c i n e , pH 4.6) e v e r y 12 h ours and a c o n t r o l group o f 10 a n i m a l s was g i v e n v e h i c l e o n l y a t t h e same t i m e . A p r e l i m i n a r y s t u d y on r a t s o f a s i m i l a r age was pe r f o r m e d i n o r d e r t o d e t e r m i n e t h e h y p o c a l c e m i c r e s p o n s e t o t h i s dose o f hormone and t o d e t e r m i n e t h e c o n s t a n c y o f t h e h y p o c a l c e m i a d u r i n g a 10-day p e r i o d . R e s u l t s In F i g . I i t c a n be seen t h a t salmon c a l c i t o n i n e x e r t s a l o n g - l a s t i n g h y p o c a l c e m i c r e s p o n s e f o l l o w i n g a s i n g l e i n j e c t i o n o f t h e hormone. T w i c e - d a i l y a d m i n i s t r a t i o n o f c a l c i t o n i n m a i n t a i n e d a h y p o c a l c e m i c s t a t e t h r o u g h o u t t h e e x p e r i m e n t a l p e r i o d a s r e v e a l e d by random s p o t c h e c k s . The p o l y u r i a which accompanied c h r o n i c a d m i n i s t r a t i o n °^ t h e hormone was t h e i n i t i a l o b s e r v a t i o n t h a t i n d i c a t e d a p o s s i b l e i n v o l v e m e n t o f c a l c i t o n i n i n r e n a l f u n c t i o n . In some i n s t a n c e s , 75-g r a t s e x c r e t e d a s much a s 45-50 ml o f u r i n e p e r day. F i g u r e 2 shows t h e p e r s i s t e n t and p r o g r e s s i v e p o l y u r i a d u r i n g t h e 10-day p e r i o d o f t r e a t m e n t w i t h salmon c a l c i t o n i n , t h e d i f f e r e n c e i n u r i n e volume between t h e c a I c i t o n i n - t r e a t e d I 5 I I . O T 10.0 - £ 9.0-1 E 8.0- O o E 7.0-1 CO o 0_ 6.0 H Mean ± S E M 5.0 J —i 1 — : — r 2 4 6 Time in Hours - i 8 F i g I . T i m e - c o u r s e o f a c t i o n o f a s i n g l e i n j e c t i o n o f salmon c a l c i t o n i n (250 ml)/100 g body w e i g h t , i . p . ) on plasma Ca l e v e l s i n s i x - w e e k - o l d r a t s . Each p o i n t r e p r e s e n t s t h e Mean +_ SEM o f f i v e a n i m a I s . 1 6 26 -, «- 22-O I 2 3 4 5 6 7 8 9 10 Time in Days F i g 2. Urine volume changes in r a t s t r e a t e d c h r o n i c a l l y with salmon c a l c i t o n i n (250 mU/100 g body weight, i . p . t w i c e d a i l y ) , (n = 10 r a t s per group.) 17 and c o n t r o l a n i m a l s b e i n g s i g n i f i c a n t l y d i f f e r e n t from Day 2. U r i n a r y e x c r e t i o n o f Na ( U ' ^ V ) , Ca < U C g V ) , Mg <UM V ) , and P (UpV) were a l s o found t o i n c r e a s e i n t h e c a I c i t o n i n - t r e a t e d r a t s as i s seen i n F i g . 3. An i n i t i a l t r a n s i e n t i n c r e a s e i n U N g V was f o l l o w e d by an e l e v a t e d mean o u t p u t from Day 4 - Day 10. S i g n i f i c a n t i n c r e a s e s i n U^ gV were found t o o c c u r from Day 4 o f t r e a t m e n t , whereas t h e g r e a t e s t i n c r e a s e i n U ^ V was found t o o c c u r from Day 2 - Day 7. An i n c r e a s e d UpV i n t h e c a I c i t o n i n - t r e a t e d r a t s o c c u r r e d o n l y d u r i n g t h e f i r s t f i v e days o f t r e a t m e n t . T h i s s t u d y i n d i c a t e d t h a t c h r o n i c i n young r a t s c o u l d i n c r e a s e t h e normal e l e c t r o l y t e s as w e l l as u r i n e volume. t r e a t m e n t e x c r e t i o n w i t h salmon c a l c i t o n i n o f c e r t a i n u r i n a r y f 18 ~ 6 0 i 4 0 > 2 0 X 3 cr eg 3 o Si a O 0 n C a l c i t o n i n V e h i c l e C l O O O n a- S 4 0 0 - — 0 J l U i I l w i i I 2 3 4 5 6 7 8 9 10 T ime in Days F i g 3. The e f f e c t of c h r o n i c treatment with salmon c a l c i t o n i n (250 mU/100 g body weight, i . p . , twi c e d a i l y ) on u r i n a r y e l e c t r o l y t e e x c r e t i o n in young r a t s . Mean outputs are shown from groups of ten r a t s . S i g n i f i c a n t increases in e l e c t r o l y t e e x c r e t i o n are i n d i c a t e d by an a s t e r i s k (*). 19 B. Stud ies on Rats Fo l l o w i n g Si ngIe I n j e c t i o n s of C a I c i t o n i n I. Intact Rats Methods In order t o determine the time-course of the u r i n a r y e l e c t r o l y t e changes t h a t occur f o l l o w i n g a s i n g l e i n j e c t i o n of c a l c i t o n i n , urine was c o l l e c t e d during timed i n t e r v a l s from i n d i v i d u a l r a t s i n which the bladder was cannulated and a constant i n f u s i o n of 2.5$ dextrose o r dextrose (2.5$)-saI ine(75 meq/l) a t a r a t e of 3 ml/hour was given v i a the external j u g u l a r v e i n . These r a t s were r e s t r a i n e d in a holding device during the c o l l e c t i o n period and were allowed to e q u i l i b r a t e f o r several hours f o l l o w i n g the s t a r t of the i n f u s i o n before c a l c i t o n i n was given. In some of these s t u d i e s , the animals were pre-loaded with large doses of DOCA (1-1.5 mg/rat). Since the major e l e c t r o l y t e changes appeared to occur w i t h i n a s i x - h o u r period f o l l o w i n g hormone a d m i n i s t r a t i o n , t h i s period was chosen f o r u r i n e c o l l e c t i o n i n subsequent s t u d i e s . Graded doses of c a l c i t o n i n were then given t o separate groups of r a t s and one group was given v e h i c l e o n l y . E l e c t r o l y t e changes were measured at these v a r i o u s dose l e v e l s i n a d d i t i o n t o changes in endogenous c r e a t i n i n e e x c r e t i o n . Plasma e l e c t r o l y t e l e v e l s were a l s o determined in a f u r t h e r s e r i e s of experiments with animals starved 12 hours p r i o r to hormone a d m i n i s t r a t i o n . Blood samples were obtained by c a r d i a c puncture on separate groups of 10-11 r a t s a t one hour and three hours a f t e r hormone i n j e c t i o n . Control groups were given v e h i c l e and were bled a t s i m i l a r times. 20 In o r d e r t o d e t e r m i n e i f changes i n m i n e r a l o c o r t i c o i d l e v e l s c o u l d b l o c k o r p o t e n t i a t e t h e n a t r i u r e t i c r e s p o n s e t o c a l c i t o n i n , DOCA was i n j e c t e d i n t o g r o u p s o f i n t a c t r a t s w i t h and w i t h o u t c a l c i t o n i n a d m i n i s t r a t i o n . A p p r o p r i a t e c o n t r o l g r o u p s were f o l l o w e d s i m u l t a n e o u s l y . The e f f e c t of age on t h e u r i n a r y r e s p o n s e t o c a l c i t o n i n was a l s o s t u d i e d s i n c e i t i s known t h a t t h e h y p o c a l c e m i c e f f e c t o f c a l c i t o n i n i s f a r g r e a t e r i n young r a p i d l y - g r o w i n g a n i m a l s t h a n i n a d u l t s ( 6 3 ) . In t h i s s t u d y , g r o u p s o f 5-week-old and 28-week-old r a t s were g i v e n c a l c i t o n i n , and u r i n e was c o l l e c t e d o v e r a p e r i o d o f 24 h o u r s i n s i x - h o u r i n t e r v a l s . C o n t r o l g r o u p s o f t h e same age d i f f e r e n c e were g i v e n v e h i c l e o n l y . R e s u I t s As c a n be seen i n T a b l e I, and i n a n o t h e r e x p e r i m e n t shown g r a p h i c a l l y i n F i g . 4, a n a t r i u r e t i c r e s p o n s e o c c u r r e d i n t h e f i r s t h a l f hour f o l l o w i n g hormone a d m i n i s t r a t i o n . A t t h i s t i m e , u r i n e volume, UpV, and t h e r a t i o s o f Na/K and N a / C r e a t . were a l s o i n c r e a s e d . However, a g e n e r a l d e c r e a s e i n U Q 3 V and U^gV was f o u n d . These r e s p o n s e s were most marked a t a b o u t | 1/2-2 h o urs f o l l o w i n g c a l c i t o n i n a d m i n i s t r a t i o n . In c o m p a r i n g t h e changes i n L)Q a v and U ^ V i n T a b l e I and F i g . 4 f o l l o w i n g c a l c i t o n i n a d m i n i s t r a t i o n , i t i s seen t h a t t h e d e c r e a s e i n t h e e x c r e t i o n o f t h e s e two i o n s was immediate i n t h e t h r e e - m o n t h - o l d r a t s t u d i e d i n T a b l e I, whereas a t r a n s i e n t i n c r e a s e i n t h e e x c r e t i o n o f Ca and Mg was o b s e r v e d i n t h e seven-month-old r a t s t u d i e d i n F i g . 4. F i g 4. E l e c t r o l y t e changes in 460-g r a t (age seven months) given 500/mll c a l c i t o n i n , i . v . during d e x t r o s e ( 2 . 5 % ) - s a I i n e (75 meq/l) i n f u s i o n given a t a r a t e of 3ml/hour. TABLE I The effect of salmon calcitonin on urinary electrolytes in dextrose-infused rat* Time Vol/lOOg/hr Na K Ca P Mg Creat. Na/K Na/Creat. (hr) (ml) yequiv/lOOg/hr ug/lOOg/hr Control •2h ~ 2 0.594 38.0 69.5 14.4 250 121.3 309 0.547 0.123 -2 - 1»5 1.862 7.4 46.6 9.2 279 100.5 205 0.160 0.036 IH - 1 2.683 13.4 37.6 14.5 215 102.0 188 0.357 0.071 -1 - *t 2.972 17.8 47.6 16.7 208 136.7 208 0.375 0.085 -H - 0 1.538 23.1 89.2 11.8 461 152.3 200 0.258 0.115 Calcitonin 0 - 1.241 54.6 120.4 8.8 918 114.2 211 0.453 0.258 H - 1 1.958 68.5 101.8 7.5 1410 76.4. 215 0.673 0.318 1 - Us 2.220 62.2 66.6 6.2 1043 42.2 178 0.933 0.350 1*5 - 2 1.556 82.5 70.0 4.9 902 56.0 187 1.177 0.441 2 - 2H 1.792 52.0 52.0 6.9 860 35.8 179 1.000 0.290 21j - 3 1.136 36.4 37.5 5.1 608 40.9 148 0.969 0.246 3 - 3>5 2.194 35.1 39.5 3.9 899 43.9 198 0.888 0.177 3*s " 4 1.503 37.6 33.1 8.1 736 76.7 188 1.136 0.200 4 - 4*5 1.294 38.8 33.6 8.4 725 88.0 181 1.153 0.214 4% - 5 2.281 25.1 25.1 10.3 707 93.5 205 1.000 0.122 5 - SH 1.040 46.8 36.4 10.5 385 171.6 198 1.285 0.236 5H - 6 0.760 52.4 41.0 10.1 502 126.9 190 1.277 0.275 6 - 6H 1.084 41.2 41.2 10.0 650 124.7 173 1.000 0.238 It 9t?/ h r a +' 3rT-! S ;.W e-!?^' , 2 2 9 9 > P r e + r e a + e d w i + h 1-5 mg DOCA, i.m. and i n f u s e d w i t h 2.5% d e x t r o s e a t 3ml/hour. C a l c i t o n i n (250 mU, i . v . ) g i v e n a t t i m e 0 hour. 23 Changes i n u r i n a r y e x c r e t i o n o v e r a s i x - h o u r p e r i o d a r e shown i n F i g . 5 where r e s p o n s e s t o 0, 10, 100, 500 and 2000 ml) c a l c i t o n i n p e r 100 g body w e i g h t a r e i n d i c a t e d . I t w i l l be noted t h a t i n c r e a s e s i n u r i n e volume and t h e e x c r e t i o n o f Na were r e l a t e d t o t h e does o f hormone g i v e n . I n c r e a s e s i n P e x c r e t i o n and d e c r e a s e s i n t h e e x c r e t i o n o f Ca and Mg were a l s o f o u n d . However, a t t h e h i g h e s t dose l e v e l (2000 mU) t h e r e d u c t i o n , i n t h e e x c r e t i o n o f Ca and i n p a r t t h a t o f Mg was overcome. A n a l y s i s o f a n o t h e r s t u d y a t t h e 100 mU dose l e v e l i n T a b l e II shows an i n c r e a s e i n t h e Na/K r a t i o w h i c h i n v a r i a b l y o c c u r r e d f o l l o w i n g c a l c i t o n i n t r e a t m e n t s i n c e t h e i n c r e a s e i n Na e x c r e t i o n was accompanied by l i t t l e o r no i n c r e a s e i n K e x c r e t i o n . The r a t i o o f N a / C r e a t . , w h i c h g i v e s an i n d i c a t i o n o f t h e f r a c t i o n a l e x c r e t i o n o f Na, a l s o i n c r e a s e d f o l l o w i n g hormone a d m i n i s t r a t i o n . S i g n i f i c a n t d e c r e a s e s i n t h e plasma c o n c e n t r a t i o n s o f Ca, P, and Mg were found f o l l o w i n g c a l c i t o n i n a d m i n i s t r a t i o n (100 mll/100 g body w e i g h t , i . v . ) a t one hour as i s seen i n T a b l e I I I . Plasma Na and K c o n c e n t r a t i o n s were no t changed. The d e c r e a s e i n plasma Ca and P was a l s o s i g n i f i c a n t a t t h r e e h o urs a f t e r i n j e c t i o n o f t h e hormone a l t h o u g h plasma Mg l e v e l s a t t h i s t i m e were not s i g n i f i c a n t l y d i f f e r e n t from t h e v e h i c I e - i n j e c t e d c o n t r o l s . T a b l e IV compares t h e e l e c t r o l y t e o u t p u t s i n r a t s w i t h and w i t h o u t DOCA a d m i n i s t r a t i o n . I t a l s o compares t h e r e l a t i v e r e s p o n s e s when c a l c i t o n i n was g i v e n i n a d d i t i o n t o DOCA t r e a t m e n t . I t can be seen t h a t DOCA cau s e d a s i g n i f i c a n t d e c r e a s e i n V, accompanied by a s l i g h t d e c r e a s e i n U^V. However, t h e e x c r e t i o n o f Ca, P, and Mg was u n a f f e c t e d by t h e DOCA. When c a l c i t o n i n was a d m i n i s t e r e d a t t h e same t i m e a s DOCA, t h e a b s o l u t e i n c r e a s e i n U., V v/as v i r t u a l l y t h e same a s t h a t found i n t h e non-DOCA, c a l c i t o n i n -Na ' t r e a t e d r a t s (non-DOCA, c a I c i t o n i n - t r e a t e d : 11.4 uequiv/1OOg/hr; DOCA, 2 4 F i g 5. Urinary changes in young r a t s in response to graded doses of salmon c a l c i t o n i n administered i . p . Urine was c o l l e c t e d over a s i x - h o u r p e r i o d . Each bar i n d i c a t e s the Mean + SEM in s i x r a t s . TABLE I I E f f e c t s o f salmon c a l c i t o n i n on u r i n a r y e l e c t r o l y t e e x c r e t i o n i n i n t a c t r a t s * T reatment Volume Na K Ca P Mg C r e a t . Na/K Na/Creat. ml/IOOg/hr uequiv/IOOg/hr ug/IOOg/hr V e h i c l e 0.264 27.3 41.3 12.1 414 86.4 120 0.658 0.225 (9) +0.054 +3.1 +2.2 +1.1 +29 +8.3 +4 C a l c i t o n i n 0.542 57.9 36.9 6.6 455 19.2 130 1.569 0.440 (12) +0.059 +5.7 +1.5 +1.0 +29 +3.7 +4 p <0.005 <0.00l NS <0.005 NS <0.00l NS *Age o f r a t s , 3 months. U r i n e c o l l e c t e d o v e r s i x hours f o l l o w i n g i n j e c t i o n of salmon c a l c i t o n i n , 100 mU/100 g body w e i g h t , I.p. Mean + SEM; n = number i n p a r e n t h e s i s . TABLE I I I Plasma e l e c t r o l y t e changes f o l l o w i n g salmon c a l c i t o n i n i n i n t a c t r a t s * T reatment Na Ca meg/ I mg% One-hour samp Ie V e h i c l e (10) C a l c i t o n i n (10) 141 + 1 138+1 NS 4.1+0.2 3.8+0.I NS 8.98+0.14 7.19+0.15 <0.00l 7.41+0.26 5.31+0.19 <0.00l 2.339+0.077 2.027+0.083 <0.025 Three-hour samp Ie V e h i c l e ( I I ) Ca I c i t o n i n ( I I ) 128+1 129+1 NS 4.1+0.I 4.0+0.I NS 8.55+0.14 6.14+0.06 <0.00l 8.55+0.14 6.22+0.08 <0.00l 2.096+0.077 I.983+0.054 NS *Age o f r a t s , 26 days. Dose o f c a l c i t o n i n , 100 mU/100 g body w e i g h t , i . v . A n i m a l s b l e d by c a r d i a c p u n c t u r e and s t a r v e d 12 hours p r i o r t o s t u d y . Mean +_ SEM; n_ = number i n p a r e n t h e s i s . TABLE IV The e f f e c t s o f s i m u l t a n e o u s a d m i n i s t r a t i o n o f DOCA and c a l c i t o n i n on u r i n a r y e l e c t r o l y t e e x c r e t i o n i n i n t a c t r a t s * T r e a tment Vo1ume ml/IOOg/hr Na K usqu i v/1 OOg/hr Ca P Mg yg/lOOg/hr C r e a t . Na/K Na/Creat V e h i c l e 0.239 22.6 49. 1 18.0 226 94. 7 146 0.484 0.155 (6) +0.025 +3.2 +5. 5 + 1 .8 +58 +3. 5 +8 C a l c i t o n i n 0.318 34.0 51 . 6 6.9 468 63. 5 162 0.689 0.216 (6) +0.041 +4.6 +3. 3 + 1 .5 +56 + 12. 9 + 1 1 DOCA + 0.205 8.6 42. 3 20.8 308 91 . 4 134 0.201 0.067 V e h i c l e ( 6 ) +0.035 + 1 .4 +2. 4 +6.9 + 19 +9. 6 +8 DOCA + 0.201 18.7 50. 0 7.8 435 47. 4 147 0.367 0.126 Ca1c i t o n1n +0.035 +3.7 +3. 4 +2.9 +39 +8. 8 +4 (6) *Age o f r a t s , two months. DOCA, I mg/100 g body w e i g h t , i.m.; C a l c i t o n i n , 100 mU/IOOg body w e i g h t , I. p. U r i n e c o l l e c t i o n o v e r s i x ho u r s . Mean + SEM; n = number i n p a r e n t h e s i s . 28 ca I c i t o n i n - t r e a t e d ; 10.1 uequ i v/1 0 0 g / h r ) . Changes i n U^gV, ^ ^ V , and UpV i n DOCA-treated r a t s g i v e n c a l c i t o n i n were not a p p r e c i a b l y d i f f e r e n t f r om t h o s e found i n non-DOCA, c a I c i t o n i n - t r e a t e d a n i m a l s . The a b s o l u t e changes i n u r i n a r y e l e c t r o l y t e e x c r e t i o n i n t h e non-DOCA and DOCA-treated r a t s g i v e n c a l c i t o n i n a r e shown i n F i g . 6. In T a b l e V, i t i s seen t h a t t h e n a t r i u r e t i c r e s p o n s e t o a s i n g l e i n j e c t i o n o f c a l c i t o n i n was much l a r g e r d u r i n g t h e s i x - h o u r p e r i o d f o l l o w i n g a d m i n i s t r a t i o n o f t h e hormone i n t h e 5-week-old r a t s t h a n i t was i n t h e 28-week-old r a t s . A l s o d u r i n g t h i s t i m e p e r i o d t h e % drop i n U^ aV and V was g r e a t e r i n t h e younger r a t s . However, i n t h e s e s t u d i e s on r a t s o f d i f f e r e n t ages t h e u r i n a r y changes i n r e s p o n s e t o c a l c i t o n i n a d m i n i s t r a t i o n p e r s i s t e d f o r a l o n g e r p e r i o d o f t i m e i n t h e o l d e r r a t s so t h a t t h e more a c u t e and immediate r e s p o n s e t o t h e hormone i n t h e younger r a t s was b a l a n c e d by a more p r o l o n g e d r e s p o n s e i n t h e o l d e r a n i m a l s . In summary, t h e e x p e r i m e n t s i n i n t a c t r a t s g i v e n a s i n g l e i n j e c t i o n o f c a l c i t o n i n i n d i c a t e d t h a t : (a) I n c r e a s e s i n u r i n e volume, l l ^ V , and U^V o c c u r r e d f o l l o w i n g a s i n g l e i n j e c t i o n o f c a l c i t o n i n , but i n c o n t r a s t t o t h e f i n d i n g s i n t h e c h r o n i c s t u d i e s , t h e r e were net d e c r e a s e s i n U n V and U., V. ' Ca Mg (b) The c hanges i n u r i n a r y volume and U^ gV were r e l a t e d t o t h e dose of c a l c i t o n i n g i v e n . ( c ) The plasma changes w h i c h o c c u r r e d w i t h i n t h e t i m e i n t e r v a l of t h e u r i n a r y c o l l e c t i o n s i n d i c a t e d s i g n i f i c a n t d e c r e a s e s i n plasma Ca, P, and Mg. However, a s i g n i f i c a n t d e c r e a s e i n plasma Mg was not found a t t h r e e hours a f t e r i n j e c t i o n o f hormone, and no s i g n i f i c a n t changes i n plasma Na Ca Mg -Z 12- SZ o> O O V > 10- 8 - 6 - cr £ 10- \ >§'. = i : <u 2 • =3. 12- la- s' 6 4- + 2- [ ] Non-DOCA treated DOCA treated - 2 4 6 8 10 12- 14 — 2 5 0 \ 200 a. o 150 3 O <J \ 100' CP _5 5 0 - I 6 0 - , " 5 0 - JZ > \ 4 0 - f O 3 Q 3 0 - <3 o> 2 0 - — + 10 - - 10- 2 0 - 3 0 - 4 0 - 5 0 - I Comparison o f a b s o l u t e changes i n u r i n a r y e l e c t r o l y t e e x c r e t i o n i n r e s p o n s e t o a s i n g l e i n j e c t i o n o f c a l c i t o n i n (100 mU/100 g body w e i g h t , i . p . ) i n t o non-DOCA t r e a t e d and DOCA t r e a t e d r a t s . (Compiled from T a b l e IV) Table V Effect of calcitonin on urinary electrolyte excretion in rats of different ages* Age (wks) 28 Weight (g) Treatment Time (hr) Volume ml/lOOg/hr Na K Ca P Mg Creat yequiv/10 Og/hr ug/lOOg/hr 474 Vehicle 0-6 0.195 13.8 24.5 11.1 223 16.1 115 +18 (5) +0.024 +1.7 + 2.0 + 1.9 + 18 + 4.3 +2 6-12 0.119 4.5 11.1 5.3 127 17 .4 101 +0.410 +0.68 + 1.6 + 1.1 + 20 + 4.8 +11 12-24 0.151 4.2 6.3 3.4 181 8.1 92 +0.350 + 0.54 + 1.4 +0.6 + 25 +1.4 +7 460 Calcitonin 0-6 0.299 24.3 26.5 5.4 322 2.6 128 +23 (5) +0.019 +3.5 + 1.5 +0.8 +18 + 0.3 + 11 6-12 0.162 12.1 8.0 3.4 189 1.7 90 +0.029 +1.5 + 1.1 + 0.7 + 22 +0.3 + 8 12-24 0.371 18.4 4.9 3.7 190 16.6 121 +0.124 + 5.2 + 1.4 + 0.5 + 4 2 + 2.5 +11 135 Vehicle 0-6 0.314 20.0 41.0 68.6 362 79.6 122 +6 (5) +0.055 +1.4 + 3.2 +18.4 + 66 +14.3 + 5 6-12 0.256 9.5 24.3 18.8 352 58.9 95 +0.061 +0.4 + 4.0 +4.4 + 58 +8.3 +7 12-24 0.244 11.2 15.6 4.6 508 22.2 87 +0.041 + 1.7 + 1.7 +1.9 + 32 + 9 . 4 + 6 130 Calcitonin 0-6 0.704 63.8 50.9 21.7 525 9.1 123 +6 (5) +0.086 + 5.3 +3.7 + 4.3 +25 +2.5 +11 6-12 0.314 15.3 19.5 5.4 509 16.3 102 +0.105 + 1.2 + 2.5 + 1.1 +66 + 5.9 +11 12-24 0.377 7.7 14.8 10.8 769 59.0 98 +0.071 +2.1 + 2.7 + 3.7 + 119 +18.5 + 8 0.567 0.437 0.800 0.915 1.633 5.208 0.502 0.447 0.790 1.272 0.826 0.498 ^ C a l c i t o n i n , 100 mU/IOOg, i . p . g i v e n a t t i m e 0 hour. Mean +_ SEM; n = number i n p a r e n t h e s i s . 31 Na and K were found a t e i t h e r one o r t h r e e h ours a f t e r i n j e c t i o n o f c a l - c i t o n i n . (d) The a b s o l u t e i n c r e a s e i n V i n r e s p o n s e t o c a l c i t o n i n was u n a f f e c t e d by l a r g e d oses o f t h e s y n t h e t i c m i n e r a l o c o r t i c o i d , DOCA. (e) C a l c i t o n i n caused a g r e a t e r and more immediate r e s p o n s e i n t h e changes i n t h e e x c r e t i o n o f Na, Ca, P and Mg i n young r a t s t h a n i t d i d i n o l d e r r a t s . 2. T h y r o p a r a t h y r o i d e c t o m i z e d r a t s S i n c e h y p o c a l c e m i a i s known t o s t i m u l a t e PTH r e l e a s e , and s i n c e c a l c i t o n i n i n t h e i n t a c t r a t s induced a l o n g - a c t i n g h y p o c a l c e m i c r e s p o n s e , e x p e r i m e n t s i n TPTX r a t s were c a r r i e d o u t i n o r d e r t o d e t e r m i n e whether t h e e l e c t r o l y t e changes i n t h e u r i n e f o l l o w i n g c a l c i t o n i n t r e a t m e n t were a p r i m a r y r e s p o n s e t o t h e hormone o r were perhaps s e c o n d a r y t o p a r a t h y r o i d s t i m u l a t i o n . Were t h e y r e l a t e d t o t h e a l t e r e d plasma Ca l e v e l p e r se? Methods Groups o f r a t s were t h y r o p a r a t h y r o i d e c t o m i z e d and t h y r o i d e c t o m i z e d a c c o r d i n g t o S u r g i c a l P r o c e d u r e s i n t h e GENERAL METHODS s e c t i o n . S i n c e b o t h g r o u p s o f r a t s were w i t h o u t t h y r o i d t i s s u e , and t h u s l a c k e d "C" c e l l s , t h e o n l y d i f f e r e n c e between them was t h e p r e s e n c e i n t h e TX group o f f u n c t i o n i n g p a r a t h y r o i d t r a n s p l a n t s , a s i n d i c a t e d by t h e a b i l i t y o f t h e s e r a t s t o m a i n t a i n a normal plasma Ca l e v e l . T h us, t h e TX a n i m a l s were an a p p r o p r i a t e c o n t r o l group f o r t h e TPTX r a t s . B o t h g r o u p s were t h e n g i v e n c a l c i t o n i n and t h e i r u r i n a r y e l e c t r o l y t e o u t p u t s were 32 measured o v e r a p e r i o d o f s i x h o u r s . A f u r t h e r c o m p a r i s o n o f t h e r e s p o n s e t o c a l c i t o n i n was made between TPTX and i n t a c t r a t s . C o n t r o l u r i n e o u t p u t s d u r i n g v a r i o u s t i m e p e r i o d s from 3-24 hours were a l s o examined i n t h e TPTX and TX r a t s i n o r d e r t o d e t e r m i n e i f an a l t e r e d p a t t e r n o f e l e c t r o l y t e e x c r e t i o n was e v i d e n t i n t h e s e two g r o u p s w i t h w i d e l y d i f f e r e n t r e s t i n g plasma Ca l e v e l s . R e s u l t s T a b l e VI shows t h e e f f e c t s o f c a l c i t o n i n on u r i n a r y e l e c t r o l y t e e x c r e t i o n i n TPTX and TX r a t s . I t i s e v i d e n t from t h i s s t u d y t h a t no r e d u c t i o n i n t h e n a t r i u r e t i c r e s p o n s e o c c u r r e d i n t h e TPTX r a t s d e s p i t e t h e a b s e n c e o f t h e p a r a t h y r o i d g l a n d s . In f a c t , an even g r e a t e r r e s p o n s e was f o u n d . The p o t e n t i a t i o n o f t h e n a t r i u r e t i c r e s p o n s e i n TPTX r a t s , as compared t o t h o s e r a t s h a v i n g p a r a t h y r o i d t i s s u e (TX and i n t a c t ) , was a c o n s i s t e n t f i n d i n g i n t h e s e s t u d i e s . The p a t t e r n o f changes i n o t h e r u r i n a r y p a r a m e t e r s i n t h e TPTX r a t s i n r e s p o n s e t o c a l c i t o n i n a d m i n i s t r a t i o n was s i m i l a r t o t h a t found i n t h e TX and i n t a c t r a t s a s i s seen i n T a b l e s VI and V I I . Thus i n a l l t h r e e g r o u p s o f r a t s (TPTX, TX, and i n t a c t ) , t h e d i r e c t i o n o f t h e e l e c t r o l y t e c h anges f o l l o w i n g exogenous c a l c i t o n i n was u n a l t e r e d by t h e p r e s e n c e o r abse n c e o f t h e p a r a t h y r o i d g l a n d s . As shown i n T a b l e V I I I , d e s p i t e t h e a l t e r e d r e s t i n g plasma Ca l e v e l s i n t h e TPTX r a t s (plasma Ca: 5.78+0.32 mg%) a s compared t o t h e normal plasma Ca l e v e l s i n t h e TX and i n t a c t r a t s (plasma Ca, TX: 10.13+0.12 mg%; i n t a c t : 10.20+0.13 mg%), t h e e x c r e t i o n o f Na and K o v e r a 24-hour p e r i o d was r e m a r k a b l y s i m i l a r i n t h e t h r e e g r o u p s o f r a t s . TABLE VI Urinary electrolyte excretion in TPTX and TX rats following salmon calcitonin* Group Plasma Ca Volume Na K Ca P Mg Creat. Na/K Na/Creat. mg% ml/10Og/hr yequiv/lOOg/hr ug/lOOg/hr TPTX 6.16 1st 3 hr ±0.14 Vehicle 0. 507 43 .3 57. ,6 71. .5 136 101. .2 139 0. 773 0. .312 (8) ±0. 073 ±8 .3 ±8, .2 ±16, .4 ±23 ±16, .8 ±14 Calcitonin 0. 536 81 .6 77. ,9 36. .3 356 53, .2 133 1. 064 0, .611 (10) ±0. 067 ±10 .5 ±8. .0 ±8, .6 ±38 ±9, .5 ±11 2nd 3 hr Vehicle 0. 236 24 .9 27. ,1 39. ,7 51 50. .6 87 0. 963 0. ,286 ±0. 036 ±5 .5 ±5. ,5 ±6. ,8 ±10 ±10. ,8 ±17 Calcitonin 0. 633 93 .1 46. .5 13. .6 317 41. ,5 106 1. 970 0. ,835 ±0. 079 ±15 .8 ±5. ,3 ±1. .7 ±38 ±7, ,9 TX _10.12 1st 3 hr ±0.33 Vehicle 0. 413 33 .9 57. ,9 88. .1 196 133. ,2 148 0. 479 0. 229 (7) ±0. 070 ±10 .4 ±13. .7 ±30. .7 ±70 ±16. ,5 ±20 Calcitonin 0. 252 48 .3 49. ,6 37. .0 257 43, .0 116 0. 964 0. ,399 (7) ±0. 031 ±7 .9 ±6, .6 ±10. .6 ±41 ±4, .9 ±11 2nd 3 hr Vehicle 0. 219 12 .5 23. .8 29. .3 83 63, .6 125 0. 539 0. ,100 ±0. 061 ±5 .5 ±6. .3 ±9, .2 ±23 ±14, .4 ±23 Calcitonin 0. 333 59 .3 31. .7 7. .6 213 43, .5 116 1. 800 0. .492 ±0. 044 ±11 .4 ±4. ,3 ±0. .8 ±31 ±7. .4 ±7 *Age o f r a t s , 2 1/2 months. C a l c i t o n i n , 100 mU/100 g body w e i g h t , i . p . Mean + SEM; n_ = number i n p a r e n t h e s i s . TABLE VII The e f f e c t o f c a l c i t o n i n on u r i n a r y e l e c t r o l y t e e x c r e t i o n i n TPTX and i n t a c t r a t s * Group Vo1ume Na K Ca P Mg C r e a t . Na/K Na/Creat m l/IOOg/hr uequiv/lOOg/hr y g / l OOg/hr TPTX Veh i c 1 e (5) 0.408 to.066 44.3 +6.7 61 .2 +2.6 51.4 + 10.5 124 +27 68.0 + 12.4 161 +7 0.716 0.271 Ca1c i t o n I n (6) 0.570 to. 074 62.5 +6.7 55.0 +4.3 1 1 .9 +3.3 205 +55 39.6 +6.4 128 + 12 1 . 169 0.495 1 n t a c t V e h i c l e (5) 0.469 to.049 40.9 + 10.3 67.4 +5. 1 26.2 +7.7 433 +63 85.9 + 16.9 165 +3 0.575 0.247 Ca Ic i t o n i n (5) 0.428 to.064 52.9 +7.2 69. 1 +4.8 4.3 +0.45 599 +35 62.3 + 15.6 163 + 15 0.761 0.329 *Age of r a t s , I 1/2 months. C a l c i t o n i n , 100 mU/IOOg body w e i g h t , i . p . U r i n e c o l l e c t e d o v e r s i x h o u r s . Mean + SEM; n = number i n p a r e n t h e s i s . TABLE V I I I C o n t r o l u r i n a r y e l e c t r o l y t e e x c r e t i o n i n TPTX, TX, and i n t a c t r a t s d u r i n g a 24-hour c o l l e c t i o n * Group Plasma Ca Volume ml/IOOg/hr Na uequ iv/1OOg/hr Ca Mg C r e a t . Na/K Na/Creat. yg/IOOg/hr TPTX (12) 5.78 tO. 32 0.257 +0.031 16.7 + 1 .7 26.0 + 1 .9 47.7 +4.4 120 + 10 61 . I +5.4 I 18 +3 0.647 0.140 TX (6) 10. 13 +0. 12 0.459 +0.109 15.4 +3.2 22.7 +3.1 36.7 + 10.9 206 +27 51 .6 +8.2 129 + 10 0.650 0.114 I n t a c t (12) 10.20 tO. 13 0.153 tO.017 17.4 + 1.5 33.4 + 1.5 14.5 + 1 .9 306 +27 102.3 + 18.7 I 10 +3 0.523 0.153 *Age o f r a t s , two months. A n i m a l s were w i t h o u t food d u r i n g c o l l e c t i o n p e r i o d . Mean +_ SEM; _n = number i n p a r e n t h e s i s . 36 The e x c r e t i o n of Ca over a 24-hour period in the TPTX r a t s was s i m i l a r to t h a t found in the TX r a t s as i s seen in Table VIII i n s p i t e of the lower plasma Ca l e v e l in the TPTX group. However, the e x c r e t i o n of P was less in the TPTX group as compared to e i t h e r the TX or the i n t a c t groups where para t h y r o i d t i s s u e was present. In summary: (a) The presence of f u n c t i o n i n g p a r a t h y r o i d t i s s u e was not e s s e n t i a l f o r the increase in the e x c r e t i o n of Na and P and the decrease in the e x c r e t i o n of Ca and Mg observed in young r a t s f o l l o w i n g a s i n g l e i n j e c t i o n of c a l c i t o n i n . (b) An a l t e r e d r e s t i n g plasma Ca l e v e l d i d not a f f e c t the net e x c r e t i o n of Na and K over a 24-hour p e r i o d . 37 I I . CALCIUM INFUSION STUDIES (ENDOGENOUS CALCITONIN RELEASE) In the r a t experiments, plasma s t u d i e s were n e c e s s a r i l y performed s e p a r a t e l y from the u r i n e s t u d i e s because of the trauma involved in blood sampling and the e f f e c t t h i s might have on kidney f u n c t i o n . A l s o , the s i z e of the r a t imposed a l i m i t on the number of blood samples t h a t could be taken from any one animal in a s i n g l e study. It seemed of great advantage to be abl e to f o l l o w simultaneous changes in plasma and u r i n e s i n c e t h i s would enable a c l e a r e r concept of the r e l a t i o n s h i p between the two and show how changes in one parameter might be r e f l e c t e d by a l t e r a t i o n s in the o t h e r . Thus, a l a r g e r animal was thought t o o f f e r d i s t i n c t advantages. The sheep was chosen not o n l y f o r i t s s u i t a b l e s i z e but a l s o because i t s t h y r o i d gland i s separate from the large s u p e r i o r p a r a t h y r o i d s . Therefore, surgery i n these animals would not r e q u i r e t r a n s p l a n t a t i o n of the parathyroids in those experiments where thyroidectomy was r e q u i r e d . In a d d i t i o n , in the la r g e r animal, any manipulations regarding c a t h e t e r i z a t i o n f o r sampling or i n f u s i o n could be performed without the use of anaesthesia so t h a t the animals could be studied f o r long periods of time in a conscious s t a t e . The r a t s t u d i e s a l l involved an i n v e s t i g a t i o n of the e l e c t r o l y t e changes f o l l o w i n g i n t e r v e n t i o n of exogenous c a l c i t o n i n from another c l a s s of animal. The sheep s t u d i e s were designed i n order to assess the e f f e c t s of endogenous c a l c i t o n i n s e c r e t i o n . 38 Since the i n f u s i o n of Ca s a l t s i s known to cause a p r o p o r t i o n a l increase in c i r c u l a t i n g c a l c i t o n i n l e v e l s (47, 48), a comparison of the plasma and u r i n a r y e l e c t r o l y t e changes in i n t a c t and thyroidectomized animals f o l l o w i n g a Ca s t r e s s should i n d i c a t e the degree of involvement of endogenous c a l c i t o n i n in the responses found. PIasma and Ur? nary E l e c t r o l y t e Changes in Conscious Sheep Fol l o w i n g CaIciurn Infusion Methods A l l sheep s t u d i e s were performed w h i l e the animals were l o o s e l y r e s t r a i n e d on a stand by a shoulder and hip harness. This allowed c o n s i d e r a b l e freedom of movement and the animals were q u i e t and relaxed during the 6-8 hour period of the experiment. F o l l o w i n g preparations according t o procedures in the GENERAL METHODS s e c t i o n , a maintenance i n f u s i o n of d e x t r o s e - s a l i n e was given a t a r a t e of 23 ml/hour which balanced the mean hourly u r i n e outputs determined in p r e l i m i n a r y c o n t r o l c o l l e c t i o n s . T h i s i n f u s i o n r a t e was maintained throughout the experiment. Blood and u r i n e c o l l e c t i o n s began a t the completion of a one-hour e q u i l i b r a t i o n p e r i o d , blood samples being taken a t the midpoint of each 15-minute u r i n e c o l l e c t i o n . A f t e r a c o n t r o l c o l l e c t i o n period of I 1/4 hours, Ca was added t o the in f u s a t e ( C a C ^ in d e x t r o s e - s a l i n e ) in a c o n c e n t r a t i o n t o g i v e 10 mg Ca/kg body weight f o r one hour. Blood and u r i n e c o l l e c t i o n s then continued f o r a f u r t h e r three hours f o l l o w i n g completion of the one-hour Ca i n f u s i o n so t h a t 39 t h e t o t a l c o l l e c t i o n t i m e was 5 1/4 h o u r s . C a t h e t e r s were t h e n removed and i n d i v i d u a l sheep were g i v e n a 10-day t o 2-week r e c o v e r y b e f o r e b e i n g used a g a i n . Upon c o m p l e t i o n o f s t u d i e s i n i n t a c t sheep, t h e same a n i m a l s were t h y r o i d e c t o m i z e d and s u b j e c t e d t o a f u r t h e r s e r i e s o f Ca i n f u s i o n s . The f i n a l s e r i e s o f e x p e r i m e n t s i n c l u d e d t h e a d d i t i o n o f salmon c a l c i t o n i n , g i v e n a t a r a t e o f 2 U/kg body w e i g h t / h o u r f o r a two-hour p e r i o d b e g i n n i n g a t t h e s t a r t o f t h e Ca i n f u s i o n i n t h e TX sheep. R e s u I t s PI asma changes F o l l o w i n g t h e s t a r t o f t h e Ca i n f u s i o n i n t h e i n t a c t sheep, plasma Ca l e v e l s i m m e d i a t e l y r o s e w i t h an i n c r e a s e o f 1.60+0.07 mg% a f t e r one hour o f t h e i n f u s i o n . As i s seen i n F i g . 7, t h e plasma Ca l e v e l s r a p i d l y r e t u r n e d t o normal v a l u e s when t h e i n f u s i o n was s t o p p e d . C a l c i u m i n f u s i o n i n t h e TX sheep was f o l l o w e d by a s i g n i f i c a n t l y g r e a t e r a b s o l u t e r i s e i n plasma Ca l e v e l s o f 2.31+0.10 mg%. The r e t u r n o f plasma Ca t o b a s e - l i n e l e v e l s was d e l a y e d i n t h i s group and was n o t a c h i e v e d d u r i n g t h e e x p e r i m e n t a l p e r i o d . When salmon c a l c i t o n i n was i n f u s e d f o r two h o u r s b e g i n n i n g a t t h e s t a r t o f t h e Ca i n f u s i o n i n t h e TX sheep, t h e a b s o l u t e r i s e i n plasma Ca l e v e l s was l e s s , a s compared t o t h e TX sheep w i t h o u t c a l c i t o n i n , and t h e r e t u r n t o b a s e - l i n e c o n t r o l l e v e l s was a c c e l e r a t e d . Plasma Ca l e v e l s a t 1,2, and 4 h o u r s a f t e r t h e s t a r t o f t h e Ca i n f u s i o n i n t h e t h r e e g r o u p s o f sheep a r e shown i n T a b l e IX. 40 F i g 7. Changes in plasma Ca l e v e l s f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX sheep infused with salmon c a l c i t o n i n (2 U/kg/hr). Changes in plasma Ca expressed as mg$ changes from b a s e - l i n e c o n t r o l values. (Mean values are i n d i c a t e d ; f o r n see Table IX.) TABLE IX Plasma changes f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX + CT sheep Time a f t e r s t a r t o f Ca i n f u s i o n ( h r ) : 1/2 A Plasma Ca I n t a c t (5) TX (6) TX + CT (4) £ I n t a c t vs TX I.60 + 0.07 2.31 + 0.10 I.82 + 0.26 <0.001 0.03 + 0.18 I.42 + 0.18 0.83 + 0.30 <0.001 -0.08 + 0.03 0.52 + 0.30 -0.32 + 0.31 A Plasma P I n t a c t (4) TX (5) TX + CT (4) £ I n t a c t vs TX TX vs TX+CT -0.24 + 0.15 0.18 + 0.07 •0.38 + 0.12 O.050 <0.005 -0.66 + 0.22 0.42 + 0.07 -0.73 + 0.19 <0.005 <0.00I -0.71 + 0.27 0.36 + 0.07 -0.86 + 0.30 <0.005 <0.005 -I.29 + 0.21 -0.80 + 0. 19 -I.42 + 0.45 A Plasma Mg I n t a c t (3) TX (6) TX + CT (4) £ TX vs TX+CT I n t a c t vs TX+CT -0.108 + 0.027 -0.096 + 0.055 -0.218 + 0.072 -0.224 + 0.040 -0.251 + 0.073 -0.498 + 0.092 -0.201 + 0.050 -0.261 + 0.097 -0.563 + 0.063 <0.050 <0.010 mg% changes from b a s e l i n e c o n t r o l v a l u e s a r e shown. Mean +_ SEM; n_ = number In p a r e n t h e s i s . >+2 As t h e plasma Ca l e v e l s were r i s i n g i n t h e i n t a c t sheep, plasma P l e v e l s were found t o f a l l a s i s shown i n F i g . 8. However, i n t h e TX a n i m a l s , t h i s d e c r e a s e i n plasma P l e v e l s was d e l a y e d and was p r e c e d e d by a s l o w r i s e r e a c h i n g a peak a b o u t 1/2 t o 3/4 hour a f t e r t h e end o f t h e Ca i n f u s i o n . Plasma P l e v e l s i n t h e TX sheep showed an i n c r e a s e o f +0.42+0.07 mg? a t t h i s t i m e as compared t o t h e i n t a c t group where l e v e l s showed a d e c r e a s e o f -0.66+0.22 mg%. When c a l c i t o n i n was i n f u s e d i n t h e TX group d u r i n g and f o l l o w i n g t h e Ca i n f u s i o n , an immediate f a l l i n plasma P was o b s e r v e d which was s i m i l a r t o t h a t seen i n t h e i n t a c t sheep. T a b l e IX shows t h e a b s o l u t e changes i n plasma P l e v e l s i n t h e t h r e e g r o u p s o f sheep a t I , I 1/2, 2,and 4 hours f o l l o w i n g t h e s t a r t o f t h e Ca i n f u s i o n . In F i g . 9, changes i n plasma Mg l e v e l s a r e shown f o l l o w i n g Ca i n f u s i o n . I t i s seen t h a t t h e f a l l i n plasma Mg w h i c h o c c u r r e d f o l l o w i n g t h e s t a r t o f t h e Ca i n f u s i o n i n t h e i n t a c t sheep was s i m i l a r t o t h a t found i n t h e TX sheep. O n l y when c a l c i t o n i n was i n f u s e d i n t h i s l a t t e r g r oup were s i g n i f i c a n t l y g r e a t e r d e c r e a s e s i n plasma Mg l e v e l s f o und ( T a b l e I X ) . U r i n e changes In r e s p o n s e t o t h e Ca i n f u s i o n , t h e i n t a c t sheep showed a d i u r e t i c r e s p o n s e w h i c h was n o t o b s e r v e d i n t h e TX a n i m a l s . In f a c t , t h e TX sheep put o u t l e s s u r i n e t h a n t h e y d i d d u r i n g t h e p r e - i n f u s i o n p e r i o d . These changes a r e shown i n F i g . 10 and T a b l e X where t h e d a t a were t r e a t e d a c c o r d i n g t o changes i n u r i n e volume from t h e mean o f b a s e - l i n e c o n t r o l l e v e l s . The i n f u s i o n o f c a l c i t o n i n i n t h e TX sheep was accompanied by an i n c r e a s e i n u r i n e volume. 43 F i g 8. Changes i n plasma P l e v e l s f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX sheep i n f u s e d w i t h salmon c a l c i t o n i n (2 U / k g / h r ) . Changes i n plasma P e x p r e s s e d a s mg% changes from b a s e - l i n e c o n t r o l v a l u e s . (Mean v a l u e s a r e i n d i c a t e d ; f o r n_ see T a b l e IX.) 44 intact TX TX + Calcitonin o +( Time in Hours F i g 9. Changes i n plasma Mg l e v e l s f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX sheep i n f u s e d w i t h salmon c a l c i t o n i n (2 U / k g / h r ) . Changes i n plasma Mg e x p r e s s e d a s mg% changes from b a s e - l i n e c o n t r o l v a l u e s . (Mean v a l u e s a r e i n d i c a t e d ; f o r n_ see T a b l e IX.) 45 jcoci,|- • f i t ' 1 L Calcitonin J |CoGlt | (K3 mg Ca/fco/w) Time in Hours F i g 10. Changes in u r i n e volume (upper t r a c e ) and osmolal output (lower t r a c e ) f o l l o w i n g Ca i n f u s i o n in i n t a c t , TX, and TX sheep infused with salmon c a l c i t o n i n (2 U/kg/hr). Changes from b a s e - l i n e c o n t r o l outputs are i n d i c a t e d . (Mean + SEM; f o r n see Tables X and XI.) TABLE X Changes i n u r i n e volume f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX t CT sheep* Time a f t e r s t a r t o f Ca i n f u s i o n ( h r ) : 0 - I - 2 2 - 3 Changes i n volume o u t p u t from b a s e - l i n e c o n t r o l v a l u e s ( m l / h r ) I n t a c t (3) TX (6) TX t CT (4) I 1.0 + 3.2 -8.3 + 3.I 4.3 t I.9 24.I + 6.8 -15.4 + 5.9 3.8 t 3.3 27.0 + 21.1 -19.5 + 6.6 - 2.5 + 2.3 -19.3 + 4.7 - 2.7 t |.5 £ I n t a c t v s TX TX vs TX+CT I n t a c t v s TX+CT <0.0I0 <0.025 <0.005 <0.050 <0.050 <0.050 <0.050 *Mean +_ SEM; n_ = number i n p a r e n t h e s i s . 47 The d i u r e s i s o b s e r v e d i n t h e i n t a c t sheep was accompanied by a l a r g e i n c r e a s e i n o s m o l a l o u t p u t a s i s seen i n F i g 10 and T a b l e X I . The i n c r e a s e i n o s m o l a l o u t p u t i n t h e TX sheep was c o n s i d e r a b l y l e s s by c o m p a r i s o n , but was enhanced ( d u r i n g t h e p e r i o d o f t h e Ca i n f u s i o n ) by c a l c i t o n i n . The changes i n u r i n a r y e l e c t r o l y t e e x c r e t i o n a r e shown i n F i g s I I , 12, 14, and 15 where 15-minute changes a r e r e c o r d e d . Changes i n U V a r e shown i n F i g 13 and T a b l e X I I . P Changes i n U ^ V a p p e a r e d t o r e f l e c t t h e plasma Ca l e v e l s . The amount o f Ca e x c r e t e d by t h e TX sheep was g r e a t e r t h a n t h a t e x c r e t e d by t h e i n t a c t o r TX+CT g r o u p s a s i s seen i n T a b l e X I I I . Not o n l y was t h e h o u r l y o u t p u t i n t h e TX group g r e a t e r , but a l s o t h e h y p e r c a I c i u r i a was o f l o n g e r d u r a t i o n . I n c r e a s e s i n U^ gV i n t h e TX sheep d i d n o t p a r a l l e l t h e enhanced U^ gV found i n t h i s g r o u p . In f a c t , U ^ V i n t h e TX a n i m a l s was l e s s t h a n i h a t found i n t h e i n t a c t sheep and s i g n i f i c a n t l y so d u r i n g t h e p e r i o d o f t h e Ca i n f u s i o n (p_<0.005). In two s t u d i e s w i t h t h e TX s h e e p , a l a r g e i n c r e a s e i n U ^ V was accompanied by no change i n U ^ V . R e s t i n g UpV l e v e l s v a r i e d c o n s i d e r a b l y , not o n l y between t h e d i f f e r e n t sheep b u t a l s o i n t h e i n d i v i d u a l sheep from day t o day. A b s o l u t e changes i n r e s p o n s e t o t h e Ca i n f u s i o n a l s o showed a w i d e v a r i a t i o n . T h e r e f o r e , i t was i m p o s s i b l e t o group t h e d a t a from any one s e r i e s o f e x p e r i m e n t s . O u t p u t s from i n d i v i d u a l s t u d i e s a r e shown, t h e r e f o r e , i n o r d e r t o o b t a i n some c o n c e p t o f t h e d i r e c t i o n o f changes t h a t o c c u r r e d f o l l o w i n g Ca i n f u s i o n r a t h e r t h a n an a b s o l u t e q u a n t i t a t i v e e v a l u a t i o n . F i g 13 shows t h e changes i n U pV t h a t o c c u r r e d d u r i n g t h e p e r i o d o f t h e Ca TABLE XI Changes In o s m o l a l o u t p u t f o l l o w i n g Ca I n f u s i o n i n i n t a c t , TX, and T X + C T sheep* Time a f t e r s t a r t o f Ca I n f u s i o n ( h r ) : 0 - I - 2 2 - 3 3 - 4 Changes i n U V from 3 osm b a s e - 1 i n e c o n t r o I v a l u e s (mOsm/hr) I n t a c t (3) TX (6) TX + CT (4) 6.88 + I.69 0.43 + 0.72 4.20 + 2.08 8.76 + 2.57 I .73 + 0.27 I .23 + I.40 7.05 + 2.93 0.92 + 0.70 - I .71 + 2.65 0.98 + I.62 -2.09 + 2.14 p_ I n t a c t vs TX <0.005 I n t a c t vs TX+CT <0.005 <0.050 <0.050 *Mean + SEM; n_ = number i n p a r e n t h e s i s . 49 Fig I I . Changes in V f o l l o w i n g Ca i n f u s i o n in i n t a c t , TX, and TX sheep infused with salmon c a l c i t o n i n (2 U/kg/hr). Changes from b a s e - l i n e c o n t r o l outputs are i n d i c a t e d . (Mean vaIues are shown; f o r n see Table X I I I . ) 50 F i g 12. Changes i n UQ V f o l l o w i n g Ca i n f u s i o n in i n t a c t , TX, and TX sheep infused with salmon c a l c i t o n i n (2U/kg/hr). Changes from b a s e - l i n e c o n t r o l outputs are i n d i c a t e d . (Mean values are shown; f o r n see Table X I I I . ) 51 intact TX TX + F i g 13. 52 Intact T X T X + Ca lc i ton in CaCl2 + l C a C I 2 O + l 2 T ime in Hours - l C o C I 2 ( I O m g C a / k g / h r ) + 1 F i g 14. Changes i n V f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX sheep ? n f u s e d w i t h salmon c a l c i t o n i n (2 U/kg/hr). Changes from b a s e - l i n e c o n t r o l o u t p u t s a r e i n d i c a t e d . (Mean v a l u e s a r e shown; f o r n see T a b l e X I I I . ) TABLE. XI I U r i n a r y P e x c r e t i o n f o l l o w i n g c a l c i u m i n f u s i o n i n I n t a c t , TX, and TX + CT sheep Control Calcium infusion Post infusion Time (hr) - 1 - 0 0 - 1 1 - 2 2 - 3 3 - 4 UpV (mg/hr) Intact 0 . 1 8 2 0 . 4 2 9 0 . 9 5 1 t 0 . 2 6 9 f 0 . 2 0 2 3 . 0 4 1 6 . 2 1 6 f 6 . 3 3 0 + 0 . 0 5 5 0 . 0 7 3 0 . 0 5 7 t 0 . 0 6 0 0 . 0 7 2 t 0 . 0 7 5 0 . 0 9 7 + 0 . 0 8 4 t 0 . 0 7 7 + 0 . 0 6 6 + TX 2 . 6 0 1 4 . 8 6 7 1 2 . 4 9 1 + 8 . 2 9 9 t 1 . 8 3 6 0 . 1 8 2 0 . 1 7 6 0 . 2 0 3 0 . 1 7 7 0 . 1 1 1 + 0 . 3 8 3 0 . 3 4 7 + 1 . 4 1 1 + 0 . 5 9 6 t 0 . 2 9 5 1 . 3 7 1 1 . 2 9 6 + 1 . 5 1 7 t 0 . 9 2 9 + 1 . 2 1 4 0 . 1 1 4 0 . 1 1 2 0 . 1 0 3 0 . 1 0 1 0 . 0 9 9 0 . 0 9 7 0 . 1 1 3 * 0 . 1 0 2 f 0 . 1 0 8 t 0 . 1 1 3 t TX + CT 0 . 0 8 4 0 . 1 0 4 i 0 . 0 7 4 0 . 0 6 5 0 . 0 6 4 + 3 4 . 1 1 9 3 5 . 6 4 3 i 2 9 . 8 2 6 2 1 . 1 2 3 + 1 8 . 2 1 0 + 0 . 1 1 6 0 . 1 3 2 t 0 . 1 0 9 0 . 1 1 0 0 . 1 0 2 0 . 0 6 5 0 . 0 9 4 t 0 . 0 4 7 0 . 0 5 1 0 . 0 7 9 f 0 . 0 8 9 0 . 0 9 0 t 0 . 0 8 5 0 . 0 7 1 + 0 . 0 9 1 + Arrows indicate either increase (+) or decrease (+) from control period values. 54 E > x => & <J © O 20 + 10 0 - 10 20 - 30 - + 500 0 - 500 Intact I I I I CaCI; I TX CaCI2 —i O +1 2 Time in Hours TX +• Calcitonin r Calcitonin -| CaCU (lOmgCa/kg/hr) F i g 15. Changes i n U KV (upper t r a c e ) and U „ r e a + V ( l o w e r t r a c e ) f o l l o w i n g Ca i n f u s i o n i n i n t a c t , TX, and TX sheep i n f u s e d w i t h salmon c a l c i t o n i n (2 U / k g / h r ) . Changes from base- l i n e c o n t r o l o u t p u t s a r e i n d i c a t e d . (Mean v a l u e s a r e shown; f o r n see T a b l e X I I I . ) 55 TABLE XIII Changes in urinary electrolyte excretion following Ca infusion in intact, TX, and TX + CT sheep* Time after start of Ca infusion (hr); 0 - I 2 - 3 AU V (meq/hr) Intact (3) TX (6) TX t CT (4) p_ I ntact vs TX I.64+0.40 0.45+0.09 0.98+0.28 <0.005 2.34+0.87 I.08+0.25 I.39+0.50 2.42+1.27 I.04+0.31 I.13+0.35 I.45+0.65 I.88+0.81 AUCgV (mg/hr) Intact (3) TX (6) TX+CT (4) 0.64+0.44 2.87+2.54 0.74+0.27 2.12+0.49 2.74+0.47 3.47+1.17 0.91+0.38 3.19+0.97 I.34+0.76 I.37+0.56 0.08+0.33 AUMgV (mg/hr) Intact (5) TX (6) TX+CT (4) p_ I ntact vs TX 5.48+0.80 2.66+0.50 3.82+0.76 <0.025 9.31+1.98 8.96+1.I 9 8.97+2.31 4.89+0.94 6.83+1.82 4.35+1.98 I.24+0.52 3.65+1.72 -0.25+0.67 AlO (meq/hr) IN Intact (5) TX (6) TX+CT (4) -0.13+0.13 -0.69+0.21 -0.04+0.43 -0.41+0.28 -I.45+0.36 -I.22+0.29 -0.37+0.35 -I .52+0.38 -I.86+0.55 -0.68+0.43 -I.31+0.35 -I.89+0.63 ^Changes in output from base-line control values are shown. Mean +_ SEM; n = number in parenthesis. 56 i n f u s i o n . T a b l e X I I shows t h e h o u r l y o u t p u t s b e f o r e , d u r i n g and f o l l o w i n g t h e p e r i o d o f t h e Ca i n f u s i o n . Whereas UQQV i n t h e t h r e e g r o u p s o f sheep appeared t o r e f l e c t t h e plasma Ca c h a n g e s , t h i s was not o b s e r v e d when r e l a t i n g UpV t o plasma P ch a n g e s . In t h e f i r s t , s e c o n d , and t h i r d h o urs f o l l o w i n g t h e s t a r t o f t h e Ca i n f u s i o n , t h e i n t a c t sheep showed an i n c r e a s e i n P e x c r e t i o n as i s seen i n T a b l e X I I I . T h i s o c c u r r e d d e s p i t e t h e d e c r e a s e d plasma P l e v e l s t h a t were found i n t h i s group a t t h i s t i m e . I n c r e a s e s i n U pV were a l s o f ound i n t h e TX+CT sheep when plasma P l e v e l s were f a l l i n g . However, t h i s o c c u r r e d o n l y d u r i n g t h e p e r i o d o f t h e Ca i n f u s i o n . In t h e TX sheep, no c o n s i s t e n t change i n U^V was found d u r i n g t h e p e r i o d o f t h e Ca i n f u s i o n , and i n f o u r o f t h e s i x s t u d i e s , a s l i g h t d r o p was f o u n d as i s seen i n F i g 13. Plasma P l e v e l s i n t h e TX sheep d u r i n g t h i s t i m e were j u s t b e g i n n i n g t o r i s e , and i n t h e second hour f o l l o w i n g t h e s t a r t o f t h e Ca i n f u s i o n , t h e h y p e r p h o s p h a t e m i a was r e f l e c t e d by a t r a n s i e n t i n c r e a s e i n P e x c r e t i o n ( T a b l e X I I ) . The i n c r e a s e i n U„ V wh i c h o c c u r r e d i n t h e i n t a c t a n i m a l s Mg i n t h e f o u r h ours f o l l o w i n g t h e s t a r t o f t h e Ca i n f u s i o n was s i m i l a r t o t h a t found i n t h e TX sheep ( F i g . 14). However, d u r i n g t h e a c t u a l p e r i o d o f t h e Ca i n f u s i o n , U ^ V i n t h e TX g r o u p s was s i g n i f i c a n t l y l e s s t h a n t h a t found i n t h e i n t a c t sheep. When c a l c i t o n i n was g i v e n t o t h e TX sheep, U ^ V was i n c r e a s e d d u r i n g t h i s same t i m e p e r i o d a s i s seen i n Tab Ie X I I I . 57 D u r i n g p r e l i m i n a r y c o n t r o l u r i n e c o l l e c t i o n s i n i n t a c t and TX sheep, I O was found t o d e c r e a s e o v e r a p e r i o d o f 4-5 h o u r s . The d e c r e a s e i n t h e e x c r e t i o n o f t h i s i o n i n t h e two g r o u p s d u r i n g c o n t r o l s t u d i e s was s i m i l a r . The i n f u s i o n o f Ca i n t h e i n t a c t sheep a p p e a r e d t o have no e f f e c t on t h e normal p a t t e r n o f K e x c r e t i o n i n t h i s g r o u p , and t h e a d m i n i s t r a t i o n o f exogenous c a l c i t o n i n d i d n o t a f f e c t t h e m agnitude o f t h e d e c r e a s e i n U^V i n t h e TX g r o u p . F i g u r e 15 shows t h e mean changes i n U^V i n t h e t h r e e g r o u p s o f sheep. These changes were n o t s i g n i f i c a n t . T h i s f i g u r e a l s o shows changes i n t h r e a t V and, as can be s e e n , no c o n s i s t e n t changes were o b s e r v e d i n r e s p o n s e t o Ca i n f u s i o n i n t h e t h r e e g r o u p s o f sheep. 58 I I I . EXTRACELLULAR VOLUME EXPANSION A n a t r i u r e t i c and d i u r e t i c r e s p o n s e i s commonly o b s e r v e d f o l lov/ing a s a l i n e l o ad and can o c c u r a p p a r e n t l y w i t h o u t any c o n c o m i t a n t i n c r e a s e i n GFR ( 6 4 ) . I t has been s u g g e s t e d by some ( 6 5 , 38, 66, 36, 39, 67) t h a t a hormone may be i m p l i c a t e d i n some way i n t h i s r e s p o n s e . C a l c i t o n i n a d m i n i s t r a t i o n a l s o r e s u l t s i n a n a t r i u r e t i c and d i u r e t i c r e s p o n s e . T h e r e - f o r e , i t seemed p o s s i b l e t h a t t h e r e m i g h t be an a s s o c i a t i o n between t h i s hormone and t h e r e s p o n s e t o s a l i n e l o a d i n g . S a l i n e l o a d i n g a TPTX r a t r e s u l t e d i n a t y p i c a l n a t r i u r e s i s (32) w h i c h s u g g e s t e d a t f i r s t t h a t c a l c i t o n i n was not i n v o l v e d . However, t h e more d e f i n i t i v e e x p e r i m e n t o f s a l i n e l o a d i n g an a n i m a l i n t h e a b s e n c e o f t h e m a j o r s o u r c e o f c a l c i t o n i n had n o t been p e r f o r m e d . T h e r e f o r e , a s e r i e s o f e x p e r i m e n t s was c a r r i e d o u t i n o r d e r t o compare t h e r e s p o n s e s f o l l o w i n g s a l i n e l o a d i n g i n i n t a c t , TX, and TPTX r a t s t o see i f any a I t e r a t i o n i n Na e x c r e t i o n o c c u r r e d i n t h o s e a n i m a l s w i t h o u t c a l c i t o n i n . A f u r t h e r s e r i e s o f e x p e r i m e n t s was c a r r i e d o u t on c o n s c i o u s i n t a c t and TX sheep t o compare t h e e l e c t r o l y t e changes i n both plasma and u r i n e i n r e s p o n s e t o volume e x p a n s i o n by d e x t r a n l o a d i n g . A. Ur i nary El e c t r o l y t e Changes i n R a t s FoI lo w i ng a_ Sa I i ne Load I. The e f f e c t s o f s a l i n e l o a d i n g i n i n t a c t , TX, and TPTX r a t s . Methods Groups o f i n t a c t , TX, and TPTX r a t s were g i v e n an i . p . l o a d o f s a l i n e (5 ml 0.9$ NaCI/100 g body w e i g h t ) and i m m e d i a t e l y p l a c e d 59 i n m e t a b o l i s m c a g e s f o r u r i n e c o l l e c t i o n d u r i n g two c o n s e c u t i v e t h r e e - hour p e r i o d s . R e s u l t s T a b l e XIV shows t h e u r i n a r y e l e c t r o l y t e changes f o l l o w i n g a s a l i n e l o a d i n i n t a c t , TX, and TPTX r a t s . A g r a p h i c a l r e p r e s e n t a t i o n o f t h i s d a t a i s shown i n p a r t i n F i g 16 where t h e a b s o l u t e changes i n e l e c t r o l y t e e x c r e t i o n a r e shown. (The a b s o l u t e changes were d e t e r m i n e d by c o m p a r i s o n w i t h e l e c t r o l y t e e x c r e t i o n i n non-loaded c o n t r o l a n i m a l s . ) Both i n t a c t and TPTX r a t s responded t o t h e s a l i n e load by i n c r e a s i n g u r i n e volume and U N g V i n t h e f i r s t t h r e e h o urs f o l l o w i n g l o a d i n g . However, t h e s e changes i n o u t p u t i n i n t a c t and TPTX r a t s were v e r y d i f f e r e n t from t h o s e found i n t h e TX a n i m a l s where u r i n e volume and U^gV were not s i g n i f i c a n t l y a l t e r e d d u r i n g t h i s t i m e p e r i o d . D u r i n g t h e second t h r e e - h o u r c o l l e c t i o n p e r i o d , both i n t a c t and TPTX r a t s showed a c o n t i n u e d i n c r e a s e i n u r i n e volume and U.. V. Na D u r i n g t h i s same t i m e p e r i o d , TX a n i m a l s a l s o showed a l a r g e i n c r e a s e i n u r i n e volume and U.. V. Na The t r a n s i t o r y r e d u c t i o n ( d u r i n g t h e f i r s t t h r e e h o urs f o l l o w i n g a s a l i n e l o a d ) i n u r i n e volume and V i n t h e TX r a t s as compared t o i n t a c t and TPTX r a t s was a c o n s i s t e n t f i n d i n g i n a l l l o a d i n g s t u d i e s i n t h e s e a n i m a l s . Groups o f r a t s t h y r o i d e c t o m i z e d i n J u n e , November, and A p r i l a l l showed t h i s t y p e of r e s p o n s e , t h e r e f o r e i t c o u l d n o t be a t t r i b u t e d t o e i t h e r one p a r t i c u l a r " b a t c h " o f r a t s , nor c o u l d i t be. a t t r i b u t e d t o s e a s o n a l v a r i a t i o n . TABLE XIV E f f e c t s o f s a l i n e l o a d i n g on u r i n a r y e l e c t r o l y t e e x c r e t i o n i n i n t a c t , TX, and TPTX r a t s * Volume Na K Ca P Mg G r e a t . Na/K Na/Creat. ml/IOOg/hr uequ iv/1OOg/hr yg/IOOg/hr 1st 5 hr I n t a c t Non-loaded (10) 0.255 tO.026 31 .9 +4.3 62.6 +4.8 38.7 +5.6 191 +30 102.3 + 10.5 162 +8 0.502 0.193 S a 1 i n e load (10) 0.567 tO.084 84.4 +9.6 85.4 +6.7 98.4 + 13.4 359 +57 152.9 + 13.8 175 +5 1 .01 1 0.485 £ <0.005 <0.00l <0.025 <0.00l <0.025 <0.0I0 NS TX Non-loaded (10) 0.307 +0.047 37.6 +5.1 60.2 +5.2 48.7 +8.4 158 +31 86. 1 + 16.9 150 +7 0.615 0.243 Sa1i ne load (10) 0.271 +0.037 41.1 +3.7 47.8 +3.7 59.9 +9.6 99 + 13 61 .9 + 1 1 .6 125 +5 0.917 0.333 £ NS NS NS NS NS NS <0.0I0 TPTX Non-loaded (5) 0.199 +0.025 34.5 +6.3 32.0 +4.0 38.4 +5.8 46 + 14 39.8 + 10.3 107 +7 1 . 1 17 0.324 Sa1Ine load (6) 0.481 +0.103 82.2 + 17.9 47.3 +4.7 63.8 +9. 1 76 + 16 60. 1 + 10.7 132 + 13 1 .702 0.621 £ <0.050 < 0.050 < 0.050 NS NS NS NS TABLE XIV (continued) Group Vo1ume Na K Ca P Mg Creat. Na/K Na/Creat, ml/IOOg/hr uequ i v/1OOg/hr ug/lOOg/hr 2nd 3 hr 1ntact Non-loaded 0. 167 +0.031 14.1 +2.7 29.3 +3.4 12.9 +2.8 172 + 14 59.4 +9.6 1 18 +9 0.464 0. 1 16 Sa1ine load 0.686 +0.114 89.5 + 1 1 .0 48.4 +6.0 53.9 +9.2 338 +57 103.4 + 13.4 158 +7 1 .892 0.557 £ <0.00l <0.00l <0.025 <0.00l <0.025 <0.025 <0.005 TX Non-loaded 0.229 +0.031 26.6 +4.5 36.3 +3.8 23.1 +4.5 169 +28 61 .6 +9.0 138 +8 0.765 0. 196 Sa1i ne load 0.527 +0.062 56.4 +8.3 42.8 +5.1 64.7 +9.3 193 +25 . 64.3 + 12.4 143 +7 1 .303 0.385 <0.00l <0.0I0 NS <0.00l NS NS NS TPTX Non-loaded 0. 189 +0.036 20.0 +2.3 24.8 +6. 1 52.8 +9.1 19 +9 63.5 +2.1 132 + 14 0.940 0.160 Sa1i ne load 0.752 +0.186 82.7 + 14.1 35.6 +5.0 91 .6 + 10.6 33 + 12 87.1 +5.6 150 + 12 2.281 0.553 £ <0.025 <0.005 NS <0.025 NS <0.010 NS *Age of r a t s , two months. Five ml 0.9% saline/IOOq body weight, i . p . was, admjnistered p r i o r t o s t a r t of 1st 3-hr c o l l e c t i o n . Mean + SEM; n_ = number in parenthesis. Changes in u r i n a r y e l e c t r o l y t e e x c r e t i o n in i n t a c t , TX, and TPTX r a t s during the f i r s t three hours f o l l o w i n g a s a l i n e load (0.9% NaCl, 5ml/l00g body weight, i . p . ) . (Compiled from Table XIV.) 63 S i g n i f i c a n t increases in U^V, U^V, LLV, and u^gV accompanied the n a t r i u r e t i c and d i u r e t i c responses i n the i n t a c t r a t s in the f i r s t three hours f o l l o w i n g the s a l i n e load. However, i n the TPTX r a t s no s i g n i f i c a n t increases in IL V, U V, and U.. V were found when ur i n e volume, a Ca ' p ' Mg U^gV, and U^V were s i g n i f i c a n t l y increased. In TX r a t s , no s i g n i f i c a n t changes i n any of the u r i n a r y e l e c t r o l y t e s measured were observed i n the f i r s t three hours f o l l o w i n g the s a l i n e load. In the second three-hour period in the TX r a t s , however, a s i g n i f i c a n t increase in LL.gV accompanied the increases in u r i n e volume and u N g V t h a t were found a t t h i s time. 2. The e f f e c t s of PTH ( L i l l y PTE) on the response of TPTX r a t s t o s a I i n e loading Methods Thyroparathyroidectomized (TPTX) r a t s were d i v i d e d i n t o t h r e e groups where the responses t o s a l i n e loading were compared with and without the a d d i t i o n of PTE given a t the same time as the s a l i n e load. The t h i r d group of TPTX r a t s was given PTE o n l y . A second s e r i e s of experiments compared the r e l a t i v e responses f o l l o w i n g a s a l i n e load in those animals having f u n c t i o n a l p a r a t h y r o i d t i s s u e (TX r a t s ) with TPTX r a t s given PTE. L i l l y PTE was used i n a l l PTH a d m i n i s t r a t i o n s t u d i e s in t h i s s e c t i o n , given as a s i n g l e i n j e c t i o n of 100 U/100 g body weight, s.c. 64 R e s u l t s In T a b l e XV i t i s seen t h a t t h e a d m i n i s t r a t i o n o f PTE t o s a l i n e - loaded TPTX r a t s r e s u l t e d i n a r e d u c t i o n i n u r i n e volume and t h e e x c r e t i o n o f Na, s i m i l a r t o t h a t e x h i b i t e d by t h e TX a n i m a l s ( f u n c t i o n i n g p a r a t h y r o i d t r a n s p l a n t s ) d u r i n g t h e f i r s t t h r e e hours f o l l o w i n g a s a l i n e l o a d . L i l l y PTE i n non-loaded r a t s reduced U^ gV t o a b o u t l / 6 t h t h e normal Na o u t p u t i n t h e f i r s t t h r e e h o urs f o l l o w i n g i n j e c t i o n . As was o b s e r v e d i n e x p e r i m e n t s i n p a r t I o f t h i s S e c t i o n , t h e r e d u c t i o n i n U^gV wh i c h o c c u r r e d i n t h e f i r s t t h r e e hours o f u r i n e c o l l e c t i o n was f o l l o w e d by an i n c r e a s e d U.. V. Na F i g u r e 17 compares t h e r e s p o n s e t o a s a l i n e l o a d i n TX r a t s w i t h t h a t i n TPTX r a t s g i v e n PTE. Both o f t h e s e g r o u p s were w i t h o u t c a l c i t o n i n , one h a v i n g f u n c t i o n a l p a r a t h y r o i d t r a n s p l a n t s and t h e o t h e r r e p l a c e m e n t t h e r a p y w i t h e x t r a c t o f t h e p a r a t h y r o i d g l a n d s ( P T E ) . The r e s p o n s e s o f t h e s e two g r o u p s a r e compared t o t h o s e found i n i n t a c t ( b o t h t h y r o i d and p a r a t h y r o i d t i s s u e ) and TPTX r a t s (no s o u r c e o f c a l c i t o n i n o r p a r a t h y r o i d t i s s u e ) . As i s seen i n F i g 17, t h o s e r a t s w i t h PTH ( e i t h e r endogenous o r exogenous) and no c a l c i t o n i n showed a t r a n s i e n t r e d u c t i o n o f U ^ V f o l l o w i n g ECF volume s t r e s s . TABLE XV The e f f e c t o f PTE on u r i n a r y e l e c t r o l y t e e x c r e t i o n f o l l o w i n g a s a l i n e l oad i n TPTX r a t s * T reatment Volume 'ml/IOOg/hr Na K yequiv/IOOg/hr ,Na/K S a I i n e load (5) 1st 3 hr 2nd 3 hr 0.402+0.109 0.42|t0.044 S a l i n e load + PTE (6) 1st 3 hr 2nd 3 hr Non-loaded + PTE (6) 0.227+0.044 0.602+0.094 1st 3 hr 2nd 3 h r 0.123+0.020 0.372+0.031 49.0+14.7 60.3+ 8.0 17.0+ 4.4 87.1+18.6 5.8+ I.4 60.3+10.8 38.3+4.4 40.3+5.5 28.1+5.7 49.2+7.7 22.6+3.6 61.9+9.2 I .284 I .628 0.614 I .924 0.261 0.600 *Age of r a t s , 2 1/2 months. L i l l y PTE, 100 U/100 g body w e i g h t , s . c . g i v e n a t same t i m e as s a l i n e l o a d , 5 ml 0.9% NaCI/100 g body w e i g h t , i . p . Mean + SEM; n_ = number i n p a r e n t h e s i s . c \ d> O O 60 20 100 60- 20 Z3 cr 3 100 60 20- lOO-i 60- 20- N o n - L o a d e d S a l i n e - S a l i n e - L o a d e d L o a d e d 0 - 3 h r s . 3 - 6 h r s . I n t a c t TPTX TX TPTX + PTE wi th S a l i n e l o ad The e f f e c t of L i l l y PTE (100 U/100 g body weight, s.c.) on the response of TPTX r a t s t o a sal ine load as compared to the response in TX r a t s with f u n c t i o n i n g p a r a t h y r o i d transpI ants.(VaIues shown are Mean + SEM; n = e i g h t r a t s per group.) 67 B. The E f f e c t o f PTH oh Plasma Ca and U r i n a r y E l e c t r o l y t e s The f i n d i n g s from t h e e x p e r i m e n t s i n p a r t A o f t h i s s e c t i o n s u g g e s t e d t h a t t h e t r a n s p l a n t e d p a r a t h y r o i d t i s s u e o r t h e p a r a t h y r o i d e x t r a c t ( L i l l y PTE) was i n v o l v e d i n t h e r e d u c t i o n i n Na e x c r e t i o n , o r a n t i n a t r i u r e t i c a c t i v i t y . I t seemed a p p r o p r i a t e t o s t u d y t h e r e l a t i v e e f f e c t s o f p u r i f i e d PTH p r e p a r a t i o n s w i t h t h a t o f L i l l y PTE i n o r d e r t o c l a r i f y whether i t was PTH i t s e l f o r some o t h e r s u b s t a n c e produced by t h e t r a n s p l a n t e d p a r a t h y r o i d t i s s u e o r p r e s e n t i n t h e PTE w h i c h was r e s p o n s i b l e f o r t h e a n t i n a t r i u r e s i s . Methods In o r d e r t o s t u d y t h e r e l a t i v e e f f e c t s o f p u r i f i e d PTH (TCA- p r e c i p i t a t e d PTH) w i t h t h a t o f L i l l y PTE, t h r e e g r o u p s o f i n t a c t r a t s were p r e - l o a d e d w t t h s a l i n e and a t one hour a f t e r l o a d i n g were i n j e c t e d w i t h e i t h e r T C A - p r e c i p i t a t e d PTH ( a c t i v i t y , 240 U/mg), L i l l y PTE, o r v e h i c l e . U r i n e o u t p u t s were d e t e r m i n e d a t h o u r l y i n t e r v a l s f o r f o u r hours f o l l o w i n g t h e s a l i n e l o a d . S e r i a l d i l u t i o n s o f L i l l y PTE and h i g h l y - p u r i f i e d PTH ( a c t i v i t y , 3000 U/mg) were t h e n made and g i v e n t o TPTX r a t s so t h a t t h e u r i n a r y r e s p o n s e s t o g r a d e d doses o f t h e hormone c o u l d be d e t e r m i n e d . In a f u r t h e r s e r i e s o f e x p e r i m e n t s , t h e t i m e - c o u r s e o f a c t i o n o f L i l l y PTE on plasma Ca l e v e l s was s t u d i e d i n o r d e r t o compare t h i s w i t h t h e t h r e e - h o u r t i m e p e r i o d d u r i n g which an i n h i b i t i o n o f U^gV o c c u r r e d . Groups o f TPTX r a t s were g i v e n a s i n g l e s . c . i n j e c t i o n o f L i l l y PTE (100 U/100 g body w e i g h t ) and were b l e d a t v a r i o u s t i m e i n t e r v a l s f o r d e t e r m i n a t i o n o f plasma Ca 68 l e v e l s . Graded doses of L i l l y PTE were a l s o g i v e n t o TPTX r a t s i n o r d e r t o compare t h e magnitude o f t h e h y p e r c a I c e m i c r e s p o n s e w i t h t h a t o f t h e Uk, V f o l l o w i n g d i f f e r e n t d o s e s of t h e hormone. Na 3 R e s u l t s Both p u r i f i e d PTH ( T C A - p r e c i p i t a t e d PTH; a c t i v i t y , 240 U/mg) and L i l l y PTE caused a r e d u c t i o n i n U N g V f o l l o w i n g a s a l i n e l o a d a s i s seen i n T a b l e X V I . In a d d i t i o n , d e c r e a s e s i n U„ V and U.. V were found t o La Mg o c c u r w i t h both hormone p r e p a r a t i o n s . In t h e t h i r d hour f o l l o w i n g hormone i n j e c t i o n , however, t h e d e c r e a s e i n LL.gV and V was n o t a p p a r e n t i n t h e 'TCA' g r o u p , a l t h o u g h i t was s t i l l e v i d e n t i n t h e ' L i l l y ' g r o u p . T a b l e XVII shows t h e i n c r e a s i n g r e d u c t i o n i n U ^ V w i t h i n c r e a s i n g d oses o f L i l l y PTE. T h i s T a b l e a l s o shows t h e d e c r e a s e d U Q 3 V and U ^ V and t h e p h o s p h a t u r i c e f f e c t t h a t n o r m a l l y o c c u r s f o l l o w i n g PTH a d m i n i s t r a t i o n . T a b l e X V I I I shows t h e r e s p o n s e o f TPTX r a t s t o graded doses o f h i g h l y - p u r i f i e d PTH ( a c t i v i t y , 3000 U/mg). I t i s seen t h a t no s i g n i f i c a n t d e c r e a s e i n U., V o c c u r r e d f o l l o w i n g a d m i n i s t r a t i o n o f h i g h l y - p u r i f i e d PTH, i n Na c o n t r a s t t o t h e f i n d i n g s w i t h s i m i l a r doses o f L i l l y PTE. S i g n i f i c a n t i n c r e a s e s In K e x c r e t i o n were f o u n d , however, w i t h h i g h l y - p u r i f i e d PTH and i n c r e a s e s i n P, and d e c r e a s e s i n Ca and Mg e x c r e t i o n were a l s o found in t h i s s t u d y . The changes i n u r i n a r y e l e c t r o l y t e e x c r e t i o n t h a t were found f o l l o w i n g a d m i n i s t r a t i o n o f h i g h l y - p u r i f i e d PTH o c c u r r e d w i t h o u t a p p a r e n t changes i n GFR s i n c e u C r e a t V was n o t a l t e r e d i n t h e t h r e e d ose r a n g e s . A c o n t r o l group of t h r e e r a t s was g i v e n L i l l y PTE (100 U/100 g body w e i g h t ) i n t h e same e x p e r i m e n t shown i n T a b l e X V I I I i n o r d e r t o compare TABLE XVI E f f e c t o f T C A - p r e c i p i t a t e d PTH and L i l l y PTE on t h e r e s p o n s e o f i n t a c t r a t s t o s a l i n e l o a d i n g * Group Time ( h r ) ^ : — / l A n ^ — ^.f^'/u Na/K Na/Creat. K yequiv/IOOg/hr yg/IOOg/hr Veh i c I e TCA** L i I l y t • 1 - 0 it 50.0t_7.2 49.8+ 3.3 53.5t 4.5 63.5+12.3 55.5+ 4.3 63.0t 4.1 157+ 6 166+10 159+12 0.823 0.908 0.865 0.314 0.304 0.349 Veh i c I e TCA L i I l y 0 - I it 79.5+13.5 26.3+ 3.9 11.5+ I.2 57.0+10.8 37.0+ 3.5 16.5+ 0.5 191+ 8 142+ 3 70+ 2 I .418 0.710 0.713 0.416 0. 186 0. 170 Veh i c I e TCA L i I l y - 2 II ti 43.3+ 3.9 21.0+ 4.9 5.9+ 1.0 37.5+ 5.5 23.0+ 4.7 32.3+ 2.5 I 62+10 130+21 164+13 I .208 0.908 0. 178 0.269 0. 168 0.036 V e h i c l e TCA LI I l y 48.3+ 6.8 37.5+ 3.8 8.0+ 0.9 39.3+ 9.I 34.3+ 3.8 35.8+ 6.5 I 55+18 146+18 152+12 I .388 I . 100 0.260 0.325 0.270 0.054 TABLE XV! ( c o n t i n u e d ) Group Time ( h r ) Ca yg/IOOg/hr Mg V e h i c l e TCA L i I l y I - 0 II II 8.4+2.6 16.4+2.6 7.0+1.6 67.8+16.3 95.6+14.0 52.2+ 9.2 V e h i c l e TCA L i l l y 0 - I it Veh i c I e TCA L i I l y I - 2 II 10.2+3.0 5.0+0.3 2.6+0.5 66.0+ 6.9 47.0+ 7.5 29.4+ 5.I 4.8+1.4 2.7+0.9 I.0+0.02 46.8+ 6.0 40.2+ 6.7 30.6+ 2.8 V e h i c l e TCA L i I l y 2 - 3 6.2+2.4 8.6+2.6 I.0+0.02 51.0+1 I.0 68.4+13.4 41.0+ 9.I *Mean w e i g h t of r a t s , 345 g. Time - I h r : 10 ml 0.9% N a C I / r a t , i . p . Time 0 h r : T C A - p r e c i p i t a t e d PTE, PTE, o r v e h i c l e (0.2% phenol i n 20 meq/l s a l i n e ) . * * T C A - p r e c i p i t a t e d b o v i n e PTH, 200 U / r a t , s . c . i n v e h i c l e . + L i I l y PTE, 200 U / r a t , s . c . Mean + SEM; n = f o u r r a t s p er gr o u p . TABLE XV I I Log-dose response t o L i l l y PTE i n TPTX r a t s * Dose PTE Volume Na K Ca P Mg C r e a t . Na/K Na/Creat. U/100 g ml/I OOg/hr ueqii i v / 1 OOg/hr '. yg/IOOg/hr I (5) 0.573 28.6 48.6 101.2 221 113.9 114 0.587 0.251 tO.122 +6.0 +6.4 +13.7 +23 +11.2 +9 10 (5) 0.379 19.0 49.8 43.4 498 79.3 113 0.295 0.168 +0.134 +6.2 +11.5 +14.8 +77 +37.3 +19 100 (5) 0.223 10.3 34.7 23.4 332 43.7 98 0.263 0.105 +0.044 +3.1 +6.5 +4.8 +37 +7.3 +8 *Age of r a t s , two months. U r i n e c o l l e c t e d o v e r a t h r e e - h o u r p e r i o d f o l l o w i n g s . c . i n j e c t i o n o f L i l l y PTE, s e r i a l l y d i l u t e d w i t h w a t e r . Mean + SEM; n = number i n p a r e n t h e s i s . TABLE XV I I I Log-dose response t o h i g h l y p u r i f i e d PTH i n TPTX r a t s * Dose PTH Volume Na K Ca P Mg C r e a t . .. , - U/IOOg ml/IOOg/hr uequiv/IOOg/hr yg/IOOg/hr N a / K Na/Creat. I (5) 0.325 +0.088 31 .7 +7.8 28.4 + 1 .8 13.2 +0.9 174 + 17 69.7 +8.2 163 + 13 .085 0. 198 10 (6) 0.252 +0.025 29.5 +2.5 40.9 +2.6 13.4 +4.5 574 +63 65.7 +9.0 160 +5 0.721 0. 185 100 (6) 0.260 +0.059 26.8 +5.9 42.4 +4.7 5.3 + 1 .2 759 +42 37.6 +7.0 169 + 1 I 0.600 0. 156 L i l l y PTE 100 (3) 0.105 8.1 23.4 4.1 435 11.7 125 +0.024 +3.2 +4.3 +0.5 +64 +3.1 +16 0.350 0.065 *Age of r a t s , f o u r months. U r i n e c o l l e c t e d o v e r a t h r e e - h o u r p e r i o d f o l l o w i n g s . c . i n j e c t i o n o f h i g h l y p u r i f i e d PTH ( a c t i v i t y , 3000 U/mg) i n 0.5 ml v e h i c l e (20 meq/l s a l i n e ) , o r L i l l y PTE (100 U/IOOg, s.c.) Mean + SEM; n = number i n p a r e n t h e s i s . 73 r e s p o n s e s under i d e n t i c a l c o n d i t i o n s . Here i t i s seen t h a t ^Qreaf V was reduced as compared t o t h e u Q r e a + v ' n + n e r a t s g i v e n h i g h l y - p u r i f i e d PTH. The e x c r e t i o n o f Na, K, C a , P, and Mg was a l s o l e s s w i t h t h e L i l l y PTE by c o m p a r i s o n w i t h t h e 100 U dose range o f h i g h l y - p u r i f i e d PTH. The h y p e r c a I c e m i c r e s p o n s e t o a g i v e n dose o f L i l l y PTE (100 U/100 g body w e i g h t ) i s a l o n g - l a s t i n g one a s i s shown i n F i g 18 where plasma Ca l e v e l s d i d n o t d e c r e a s e u n t i l 10-12 hours a f t e r i n j e c t i o n o f t h e hormone. A l i n e a r r e l a t i o n s h i p was found between t h e plasma Ca l e v e l s and t h e i r i n a r y e x c r e t i o n o f Na, and t h e l o g - d o s e o f L i l l y PTE, plasma Ca l e v e l s r i s i n g and U N g V f a l l i n g w i t h i n c r e a s i n g d oses o f t h e hormone a s i s seen i n F i g 19. 74 F i g 18. T i m e - c o u r s e o f a c t i o n o f L i Ily PTE (100 U/100 g body w e i g h t , s.c.) on plasma Ca l e v e l s i n TPTX r a t s . (Each p o i n t r e p r e s e n t s Mean +_ SEM from s e p a r a t e g r o u p s o f f i v e r a t s . ) 75 F i g 19. Log-dose r e s p o n s e +o L i l l y PTE i n TPTX ra+s. (Each p o i n t r e p r e s e n t Mean +_ SEM from f i v e r a t s . ) 76 C. PIasma and U r i nary E l e c + r o l y + e Changes i n Consc i o u s Sheep FoI Iow i ng Dex+ran I n f u s i o n Methods The sheep were p r e p a r e d f o r t h e v o l u m e - e x p a n s i o n s t u d i e s w i t h d e x t r a n u s i n g a s i m i l a r p r o t o c o l t o t h a t used i n t h e C a - i n f u s i o n s t u d i e s (see S e c t i o n I I , M e t h o d s ) , w i t h t h e e x c e p t i o n t h a t t h e y were n o t g i v e n a d e x t r o s e - s a l i n e i n f u s i o n . F o l l o w i n g a c o n t r o l plasma and u r i n e c o l l e c t i o n o v e r a p e r i o d o f I 1/4 h o u r s , d e x t r a n i n a volume e q u i v a l e n t t o \% body w e i g h t was i n f u s e d o v e r a p e r i o d o f 15 m i n u t e s . Plasma and u r i n e samples were c o l l e c t e d f o r a f u r t h e r 2 3/4 hours so t h a t t h e e n t i r e c o l l e c t i o n p e r i o d c o v e r e d 4 1/4 h o u r s . C a t h e t e r s were t h e n removed and t h e i n d i v i d u a l sheep were g i v e n a 10-day t o 2-week r e c o v e r y b e f o r e b e i n g used a g a i n . T h i s e x p e r i m e n t was performed w i t h t h e i n t a c t sheep and t h e n was r e p e a t e d i n t h e TX a n i m a l s . The f i n a l s e r i e s o f e x p e r i m e n t s was c a r r i e d o u t i n t h e same TX sheep w i t h t h e a d d i t i o n o f salmon c a l c i t o n i n w h i c h was a d m i n i s t e r e d i n two doses o f 50 U e a c h . The f i r s t dose was g i v e n a t t h e c o m p l e t i o n o f t h e 15-minute d e x t r a n i n f u s i o n , and t h e second one hour l a t e r . R e s u I t s PI asma changes F o l l o w i n g t h e c o m p l e t i o n o f t h e 15-minute d e x t r a n i n f u s i o n , plasma Ca l e v e l s were d e c r e a s e d as i s seen i n F i g 20. In t h e i n t a c t sheep, t h i s d e c r e a s e was m a i n t a i n e d t h r o u g h o u t t h e e x p e r i m e n t a l p e r i o d . A t t h r e e h o u rs p o s t i n f u s i o n , t h e change i n plasma Ca l e v e l was -0.72t0.22 mg%. In 77 F i g 20. Changes i n plasma Ca l e v e l s f o l l o w i n g a 15-minute d e x t r a n i n f u s i o n i n i n t a c t , TX, and TX sheep t r e a t e d w i t h salmon c a l c i t o n i n (2 X 50 U ) . Changes i n plasma Ca l e v e l s e x p r e s s e d as mg% changes from b a s e - l i n e c o n t r o l v a l u e s . (Mean v a l u e s a r e i n d i c a t e d ; f o r n_ see T a b l e X I X . ) 78 t h e TX sheep, however, t h e plasma Ca l e v e l s g r a d u a l l y r e t u r n e d t o normal and by t h r e e hours f o l l o w i n g t h e d e x t r a n l o a d showed a change o f o n l y -0.18+0.I I mg% from normal b a s e - l i n e l e v e l s as i s seen i n T a b l e X I X . The plasma Ca l e v e l s i n t h e i n t a c t and TX sheep a t t h i s t i m e were s i g n i f i c a n t l y d i f f e r e n t (p_<0.050). When c a l c i t o n i n was g i v e n t o t h e TX sheep, plasma Ca l e v e l s showed a p r o g r e s s i v e f a l l a s i s seen i n F i g 20. Plasma P and Mg changes a r e shown i n F i g 21. F o l l o w i n g t h e d e x t r a n i n f u s i o n , b oth plasma P and Mg l e v e l s d e c r e a s e d i n t h e i n t a c t sheep and were m a i n t a i n e d a t t h i s lower l e v e l d u r i n g t h e t h r e e - h o u r p e r i o d f o l l o w i n g t h e l o a d . Plasma P i n t h e TX a n i m a l s showed a p r o g r e s s i v e f a l l i n t h e hour f o l l o w i n g c o m p l e t i o n o f t h e d e x t r a n i n f u s i o n . A s i m i l a r f a l l i n plasma P was o b s e r v e d when t h e s e a n i m a l s were g i v e n c a l c i t o n i n b u t was c o n t i n u e d f o r a l o n g e r p e r i o d . Plasma Mg changes i n t h e TX and T X + C T sheep were v e r y s i m i l a r b u t showed a s l i g h t l y g r e a t e r f a l l i n l e v e l s t h a n was found i n t h e i n t a c t a n i m a l s . T h i s change was n o t s i g n i f i c a n t ( T a b l e X I X ) . U r i n e changes S i m i l a r i n c r e a s e s i n u r i n e volume and o s m o l a l o u t p u t were o b s e r v e d i n t h e i n t a c t and TX sheep f o l l o w i n g d e x t r a n i n f u s i o n , a s i s seen i n F i g 22 and T a b l e s XX and X X I . However, when c a l c i t o n i n was g i v e n t o t h e TX a n i m a l s , a l a r g e r i n c r e a s e i n u r i n e volume and o s m o l a l o u t p u t was o b s e r v e d . The changes i n U M V i n t h e t h r e e g r o u p s o f sheep r e f l e c t e d t h e o s m o l a l changes t h a t were o b s e r v e d , a s i s shown i n F i g 23. The a d m i n i s t r a t i o n o f c a l c i t o n i n was f o l l o w e d by a g r e a t e r i n c r e a s e i n Na e x c r e t i o n es compared t o t h a t o b s e r v e d i n t h e i n t a c t and TX sheep ( T a b l e X X I V ) . TABLE XIX Plasma changes f o l l o w i n g d e x t r a n i n f u s i o n i n I n t a c t , TX, and TX + CT sheep* Time a f t e r s t a r t o f . ,. d e x t r a n load ( h r ) : APIasma Ca (mg$) I n t a c t (3) TX (6) TX + CT (3) £ I n t a c t vs TX TX vs TX+CT I n t a c t vs TX+CT -0.72+0.06 -0.95+0.04 -0.88+0.21 <0.025 -0.58+0.07 -0.66+0. 15 -].25+0.22 <0.050 -0.60+0.15 -0.56+0.13 -I.72+0.36 <0.0I0 <0.050 -0.72+0.22 -0.18+0.I I - I .97+0.34 <0.050 < 0*001 <0.050 APIasma P (mg$) I n t a c t (3) TX (6) TX + CT (3) £ I n t a c t vs TX -0.20+0.15 -0.38+0.12 -0.20+0.15 -0.40+0.06 -0.97+0.16 -0.37+0.16 <0.050 -0.35+0.10 -I.23+0.22 -0.68+0.23 <0.050 -0.70+0.15 -I.26+0. 19 -I.03+0.27 APIasma Mg (mg%) I n t a c t (3) TX (6) T X + C T (3) -0.16+0.02 -0.20+0.03 -0.22+0.03 -0.17+0.03 -0.29+0.09 -0.24+0.03 -0.21+0.05 -0.34+0.09 -0.35+0.04 -0.22+0.08 -0.32+0.09 -0.29+0.01 * D e x t r a n (6% Gentran 75 i n 0.9$ N a C l ) , \% body w e i g h t , i n f u s e d o v e r 15 min. Changes i n plasma l e v e l s from b a s e - l i n e c o n t r o l l e v e l s . Mean + SEM; n = number In p a r e n t h e s i s . Intoct T X T X + C a l c i t o n i n T i m e in Hours Changes i n plasma P and plasma Mg f o l l o w i n g a 15-minute d e x t r a n i n f u s i o n i n i n t a c t , TX, and TX sheep t r e a t e d w i t h salmon c a l c i t o n i n (2 X 50 U). Changes i n plasma l e v e l s e x p r e s s e d as mg% changes from b a s e - l i n e c o n t r o l v a l u e s . (Mean v a l u e s a r e i n d i c a t e d ; f o r n see T a b l e XIX.) Intact T X T X + C a l c i t o n i n tt I I I I I I 0 A Dextran 2 +1 tt I I I I A Dextran n 1 1 O + l 2 3 T i m e in H o u r s I I I l tt t + 4. ^ C a l c i t o n i n Dextran Changes i n u r i n a r y volume (upper t r a c e ) and U V ( l o w e r t r a c e ) f o l l o w i n g a 15-minute d e x t r a n i n f u s i o n i n i n t a c t , TX, and TX sheep t r e a t e d w i t h salmon c a l c i t o n i n (2 X 50 U ) . Changes from b a s e - l i n e c o n t r o l v a l u e s a r e i n d i c a t e d . ( V a l u e s e x p r e s s e d as Mean + SEM; f o r n see T a b l e s XX and XXI.) TABLE XX Changes i n u r i n e volume f o l l o w i n g d e x t r a n i n f u s i o n i n I n t a c t , TX, and TX t CT sheep* Time a f t e r s t a r t of d e x t r a n load ( h r ) : 0 - Changes i n u r i n e volume from b a s e l i n e c o n t r o l v a l u e s ( m l / h r ) I n t a c t (5) TX (6) TX t CT (3) £ TX vs TX+CT 30.6+18.9 37.1+15.9 49.0+38.3 *Mean + SEM; n = number i n p a r e n t h e s i s . I - 2 2 - 3 38.7+10.5 44.3+14.3 69.9+29.I 18.3+5.3 15.3+4.8 35.8+2.3 <0.025 TABLE XXI Changes i n os m o l a l o u t p u t f o l l o w i n g d e x t r a n i n f u s i o n i n i n t a c t , TX, and TX + CT sheep* Time a f t e r s t a r t of d e x t r a n load ( h r ) : I - 2 2 - 3 Changes i n o s m o l a l o u t p u t from b a s e - l i n e c o n t r o l v a l u e s (mOsm/hr) I n t a c t (5) TX (6) TX + CT (3) I I.3+2.3 10.5+2.7 17.3+8.0 8.4+4.1 13.4+6.7 25.6+7.5 5.5+2.6 6.5+1.7 I I.9+4.4 *Mean + SEM; n = number i n p a r e n t h e s i s . oo UJ 84 A c a l c i u r i a was found t o accompany t h e n a t r i u r e t i c r e s p o n s e t o t h e d e x t r a n i n f u s i o n i n t h e i n t a c t sheep as i s shown i n F i g 23. However, t h i s was n o t o b s e r v e d i n t h e TX sheep d e s p i t e a n a t r i u r e s i s o f s i m i l a r m a gnitude t o t h a t found i n t h e i n t a c t a n i m a l s . When c a l c i t o n i n was a d m i n i s t e r e d t o t h e TX sheep, a c a l c i u r i a was o b s e r v e d a l t h o u g h i t was o f s m a l l e r m a g n i t u d e t h a n t h a t found i n t h e i n t a c t a n i m a l s . The I n c r e a s e i n Ca e x c r e t i o n i n t h e T X + C T sheep o c c u r r e d d e s p i t e t h e s i g n i f i c a n t l y g r e a t e r f a l l i n plasma Ca wh i c h was found i n t h i s g r o u p . The c l e a r a n c e o f Ca i n t h e i n t a c t and TX + CT g r o u p s was found t o i n c r e a s e i n r e s p o n s e t o t h e d e x t r a n i n f u s i o n , as i s seen i n T a b l e X X I I . In t h e TX sheep, c l e a r a n c e r a t e s d i d not change. C o n t r o l UpV and a b s o l u t e changes i n UpV showed c o n s i d e r a b l e v a r i a t i o n . O u t p u t s from i n d i v i d u a l sheep a r e shown,, t h e r e f o r e , and a r e compared t o c o n t r o l o u t p u t s b e f o r e t h e d e x t r a n i n f u s i o n i n T a b l e X X I I I . In f o u r o f t h e f i v e s t u d i e s i n t h e i n t a c t sheep, an i n c r e a s e i n UpV was o b s e r v e d f o l l o w i n g t h e d e x t r a n i n f u s i o n . When c a l c i t o n i n was g i v e n t o t h e TX sheep, a p h o s p h a t u r i c r e s p o n s e was a l s o s e e n . In t h e TX sheep w i t h o u t c a l c i t o n i n , v e r y l i t t l e change was o b s e r v e d i n U^V i n f o u r o f t h e s t u d i e s , and an i n c r e a s e i n U V was found i n t h e r e m a i n i n g two P a l t h o u g h t h i s was o n l y t r a n s i e n t i n one o f t h e s e a n i m a l s . Changes i n O^gV ' n + n e t h r e e g r o u p s o f sheep were f a r b e t t e r d e f i n e d as i s seen i n F i g 24. A ma g n e s u r i a was o b s e r v e d i n t h e i n t a c t a n i m a l s f o l l o w i n g t h e d e x t r a n i n f u s i o n . T h i s r e s p o n s e was d i m i n i s h e d i n t h e TX sheep b u t was r e s t o r e d i n p a r t when t h e s e a n i m a l s were g i v e n c a l c i t o n i n . T a b l e XXIV shows t h e h o u r l y changes i n Mg e x c r e t i o n i n t h e t h r e e g r o u p s o f sheep, i n a d d i t i o n t o changes i n U.. V, Up V, and U KV. 85 | Intact TX TX + Calcitonin > 250-1 Time in Hours F i g 23. Changes i n V (upper t r a c e ) and I L ^ V ( l o w e r t r a c e ) f o l l o w i n g a iB-minute d e x t r a n i n f u s i o n i n i n t a c t , TX, and TX sheep t r e a t e d w i t h salmon c a l c i t o n i n (2 X 50 U ) . Changes from b a s e - l i n e c o n t r o l v a l u e s a r e i n d i c a t e d . (Mean v a l u e s a r e shown; f o r n see T a b l e XXIV.) TABLE XXI I C l e a r a n c e o f Ca f o l l o w i n g d e x t r a n i n f u s i o n i n i n t a c t , TX, and TX + CT sheep* Time ( h r ) C o n t r o l - 1 - 0 P o s t I n f u s i o n * * 0 - I - 2 2 - 3 C l e a r a n c e o f Ca (ml/min) I n t a c t (3) TX (6) TX t CT (3) 0.124+0.066 0.086t0.034 0.081+0.039 0.272t0.09l 0.091+0.023 0.158+0.063 0.379+0.125 0.081+0.023 0.138+0.039 0.237+0.052 0.079+0.025 0.062+0.022 * C l e a r a n c e , [ U r i n e ] X V o l . ( m l ) / m i n LPIasmaJ D e x t r a n i n f u s i o n g i v e n a t t i m e 0 hour o v e r a p e r i o d o f 15 min. Mean + SEM; n = number i n p a r e n t h e s i s . TABLE XXI I I Urinary P excretion following dextran infusion in intact, TX, and TX + CT sheep Time (hr): Control Post infusion* 0 0-1 1-2 2 - 3 U V (mg/hr) Intact TX TX t CT 0.107 0. 165 + 0.131 t 0.073 + 0.1 12 0.222 + 0.1 14 t 0.096 0.134 0.272 t 0.248 t 0. 147 t 0.142 0.993 t 2.926 t 3.657 t 20.772 20.027 19.517 8.561 + 0.092 0.089 0.098 t 0.082 0.097 0.1 13 t 0.091 0.081 + 0.237 0.232 0.21 1 + 0. 153 0.278 0.265 0.200 0.210 1 .289 2.337 t 3.120 + 6.012 t 17.865 25.223 + 14.853 17.025 0.023 0.097 t 0.100 + 0.064 t 0.046 0.080 t 0.050 + 0.056 + 0.516 1 .791 t 6.645 t 6.813 t 0.615 0.758 t 0.579 0. 146 *Dextran load given at time 0 hr over a period of 15 minutes. Arrows indicate either increase ( t) or decrease ( +) from control period value. 88 I n t a c t TX T X + C a l c i t o n i n e \ CTJ •/ D e x t r a n +1 tt I I I I I I I l y D e x t r a n ~i 1 1 O + l 2 3 T i m e in H o u r s I I I I I L tt t L C a l c i t o n i n /. D e x t r a n 2 — r + i F i g 24. Changes in V f o l l o w i n g a 15-minute dextran i n f u s i o n i n i n t a c t , TX, and TX sheep t r e a t e d with salmon c a l c i t o n i n (2 X 50 U). Changes from b a s e - l i n e c o n t r o l values are i n d i c a t e d . (Mean values are shown; f o r n see Table XXIV). 89 TABLE XXIV Changes i n u r i n a r y e l e c + r o l y + e e x c r e t i o n f o l l o w i n g dex+ran i n f u s i o n i n i n t a c t , TX, and TX + CT sheep * Time a f t e r s t a r t o f d e x t r a n i n f u s i o n ( h r ) 0 - I - 2 2 - 3 A U N gV (meq/hr) I n t a c t (4) TX (6) TX t CT (3) 3.27+0.74 3.23+1.24 6.10+3.64 4.92+1.84 4.88+1.64 10.74+3.94 3.03+1.42 3.65+1.02 6.44+1.82 A U C gV (mg/hr) I n t a c t (5) IX (6) TX + CT (3) £ I n t a c t v s TX 0.50+0.I 3 -0.02+0.10 0.33+0.37 <0.025 0.77+0.60 -0.09+0.13 0.19+0.20 0.25+0.19 -0.09+0.I I -0.19+0.16 A U M gV (mg/hr) I n t a c t (5) TX (6) T X + C T (3) 2.02+0.73 I.21+0.54 2.47+1.37 2.32+1.22 0.94+0.68 I.78+0.23 0.45+0.61 0.20+0.42 -0.98+0.13 AU KV (meq/hr) I n t a c t (5) TX (6) TX + CT (3) £ I n t a c t v s TX+CT 0.14+0.15 -0.15+0.13 -0.06+0.18 -0.34+0.14 -0.47+0.20 -0.83+0.18 -0.35+0.29 -0.85+0.28 -I.64+0.08 <0.025 ^Changes i n o u t p u t from b a s e l i n e c o n t r o l v a l u e s a r e shown. D e x t r a n i n f u s e d o v e r a p e r i o d o f 15 m i n u t e s from t i m e 0 hour. Mean + SEM; n = number i n p a r e n t h e s i s . 90 Decreases in U^V were observed in a l i three groups of sheep during the period of u r i n e c o l l e c t i o n . The t r a n s i e n t increase in U^V f o l l o w i n g the dextran i n f u s i o n p a r a l l e l e d the t r a n s i e n t increase i n IL V observed a t t h i s time as i s seen in Fig 25. 91 I n t a c t TX T X + C a l c i t o n i n => "5 cr =3, <3 r 500 n •500 I I I I I I I I ft £ D e x t r a n C a l c i t o n i n — t -+ 1 ^ D e x t r a n -1 + i T i m e in H o u r s F i g 25. Changes in U^V (upper t r a c e ) and l - ' r ; r e a + V (lower t r a c e ) f o l l o w i n g a 15-minute dextran i n f u s i o n i n i n t a c t , TX, and TX sheep t r e a t e d with salmon c a l c i t o n i n (2 X 50 U). Changes from b a s e - l i n e c o n t r o l values are i n d i c a t e d . (Mean values are shown: f o r n see Table XXIV.) 92 DISCUSSION I. EXOGENOUS CALCITONIN ADMINISTRATION A. C h r o n i c S t u d i e s The f i n d i n g o f a p o t e n t n a t r i u r e t i c and d i u r e t i c e f f e c t o f salmon c a l c i t o n i n i n young r a t s d u r i n g c h r o n i c a d m i n i s t r a t i o n o f t h e hormone was o f c o n s i d e r a b l e i n t e r e s t . A t t h a t t i m e , no s t u d i e s on t h e u r i n a r y changes t h a t o c c u r f o l l o w i n g c h r o n i c t r e a t m e n t w i t h c a l c i t o n i n had been r e p o r t e d , and no e v i d e n c e was a v a i l a b l e t o show t h a t salmon c a l c i t o n i n c o u l d e x e r t such p r o f o u n d a l t e r a t i o n s i n u r i n a r y e x c r e t o r y p a t t e r n s . The n a t r i u r e t i c and d i u r e t i c r e s p o n s e , i n a d d i t i o n t o t h e i n c r e a s e d o u t p u t o f o t h e r e l e c t r o l y t e s w h i c h i n c l u d e d C a, P, and Mg, s u g g e s t e d t h a t t h e hormone, b e s i d e s i t s known e f f e c t on bone, may be e x e r t i n g a r e n a l e f f e c t which p e r s i s t s w i t h c o n t i n u e d a d m i n i s t r a t i o n o f t h e hormone. R e c e n t s t u d i e s i n r a t s by S^rensen and H i n d b e r g (68) s u p p o r t t h e s e f i n d i n g s . They found t h a t p o r c i n e c a l c i t o n i n i n a dose o f 100 mU/100 g body w e i g h t e v e r y t h r e e h o urs f o r t h r e e days c a u s e d a p r o g r e s s i v e i n c r e a s e i n u r i n e volume, and t h e e x c r e t i o n o f Na, C a , P, and Mg. They a t t r i b u t e d t h e s e e f f e c t s t o a d i r e c t a c t i o n o f c a l c i t o n i n on t h e r e n a l t u b u l e s . B i j v o e t e t a]_. (69) showed t h a t t h e c o n t i n u a l i n f u s i o n o f p o r c i n e c a l c i t o n i n i n man (63 MRC U/day) d u r i n g a p e r i o d o f 6 t o 39 days r e s u l t e d i n a t r a n s i e n t d i u r e s i s and n a t r i u r e s i s which was accompanied by w e i g h t l o s s i n t h e f i r s t two d a y s . I t would a p p e a r t h a t t h e hormone was e x e r t i n g a c o n t ? nued e f f e c t on Na and volume, however, s i n c e i t was o b s e r v e d t h a t a l d o s t e r o n e s e c r e t i o n r a t e s and plasma r e n i n a c t i v i t y were above normal 93 d u r i n g t h e e x p e r i m e n t a l p e r i o d . T h i s i n c r e a s e i n comp e n s a t o r y mechanisms c o u l d o b s c u r e a c o n t i n u e d a c t i o n o f t h e hormone on t h e k i d n e y . B i j v o e t e t a_l_.also o b s e r v e d an i n c r e a s e i n Ca and F e x c r e t i o n and r u l e d o u t t h e i n t e r v e n t i o n o f PTH s i n c e no i n c r e a s e i n e x c r e t i o n o f cAMP was o b s e r v e d . I t would a p p e a r from t h e s e c h r o n i c s t u d i e s i n r a t and man t h a t c a l c i t o n i n i s c a p a b l e o f c a u s i n g a volume s t r e s s w h i c h p e r s i s t s as long a s t h e hormone i s b e i n g a d m i n i s t e r e d . In man (69) and p o s s i b l y r a t s , mechanisms may be evoked i n o r d e r t o compensate f o r t h i s c o n d i t i o n . In a d d i t i o n t o t h e e f f e c t s o f t h i s hormone on u r i n a r y Na and volume d u r i n g c h r o n i c a d m i n i s t r a t i o n o f c a l c i t o n i n , l o s s o f o t h e r e l e c t r o l y t e s w h i c h i n c l u d e s Ca, P, and Mg c a n a l s o o c c u r and t h e s e e f f e c t s a p p e a r t o be l e s s r e a d i l y compensated f o r . B. A c u t e Stud i e s I. Plasma changes The h y p o c a l c e m i c e f f e c t o f c a l c i t o n i n i s w e l l documented and i s t h e b a s i s f o r t h e b i o a s s a y o f t h i s hormone (59). The r e s p o n s e i s dose-dependent and i s r e l a t e d t o t h e age o f t h e a n i m a l and more d i r e c t l y t o t h e r a t e o f bone t u r n o v e r (62). In T a b l e I I I , a p r o g r e s s i v e d r op i n plasma Ca l e v e l s i s shown from 0 t o 3 hours w h i c h was h i g h l y s i g n i f i c a n t (jp<0.00l). S i n c e t h i s r e s p o n s e was found a t t h e dose o f hormone used f o r many o f t h e s i x - h o u r u r i n a r y s t u d i e s (100 mll/100 g body w e i g h t ) , t h e r a t s were i n a h y p o c a l c e m i c s t a t e d u r i n g t h e p e r i o d s o f u r i n e c o l l e c t i o n . The h i g h l y s i g n i f i c a n t d e c r e a s e i n plasma P a t one and t h r e e h o u r s a s shown i n T a b l e I I I (p_<0.00l) i s c o n s i s t e n t w i t h t h e hypophosphatemic e f f e c t o f c a l c i t o n i n o b s e r v e d by o t h e r w o r k e r s (70, I I , 7 1 ) . The 94 hypophospha+ernic e f f e c t i s a p p a r e n t l y not a b o l i s h e d by nephrectomy o r p a r a t h y r o i d e c t o m y as shown by Kennedy e t a I . ( 7 1 ) , and t h u s a p p e a r s t o be a d i r e c t e f f e c t of t h e hormone on bone. T a b l e I I I a l s o shows a d e c r e a s e i n plasma Mg. C o n s i s t e n t d e c r e a s e s i n plasma Mg have not a l w a y s been found f o l l o w i n g c a l c i t o n i n t r e a t m e n t by o t h e r w o r k e r s . S t u d i e s i n man by S«5rensen e t a I. (72) r e v e a l e d a hypo- magnesemic e f f e c t w i t h p o r c i n e c a l c i t o n i n w h i c h was more pronounced i n h y p e r c a l c e m i c p a t i e n t s t h a n i n normocaIcemic p a t i e n t s . Haas e t a I.(50) a l s o found a d e c r e a s e i n plasma Mg i n h y p o p a r a t h y r o i d p a t i e n t s . N e g a t i v e r e s u l t s were r e p o r t e d by B i j v o e t e t a I . ( 7 3 ) . N i e l s e n e t a l . ( l 5 ) u s i n g s y n t h e t i c p o r c i n e c a l c i t o n i n o b s e r v e d a s i g n i f i c a n t hypomagnesemic e f f e c t i n young r a t s (70-110 g) as d i d P a l m i e r i e t a I . ( 4 ) u s i n g p a r t i a l l y p u r i f i e d p o r c i n e c a l c i t o n i n . These f i n d i n g s were i n c o n t r a s t t o t h o s e o f A l d r e d e t a I.(16) u s i n g salmon c a l c i t o n i n i n 220-250 g r a t s . W i l l jams e t a I.(18) u s i n g s y n t h e t i c human and salmon c a l c i t o n i n a l s o r e p o r t e d no hypomagnesemic r e s p o n s e t h r e e hours f o l l o w i n g t h e s t a r t o f a one-hour i n f u s i o n o f hormone. These d i f f e r e n c e s i n r e s p o n s e t o c a l c i t o n i n t r e a t m e n t may be r e l a t e d t o t h e age o f t h e animal and t h u s t h e r a t e of bone r e m o d e l l i n g , a hypomagnesemic e f f e c t b e i n g more o b v i o u s i n t h o s e a n i m a l s where bone t u r n o v e r i s o c c u r r i n g a t a f a s t e r r a t e . In a d d i t i o n , t h e t i m e a t w h i c h t h e b l o o d sample i s t a k e n f o l l o w i n g hormone a d m i n i s t r a t i o n may be an i m p o r t a n t f a c t o r . The d a t a i n T a b l e I I I were d e r i v e d from s t u d i e s i n t h r e e - w e e k - o l d r a t s ( c a . 40 g body w e i g h t ) from which samples were t a k e n a t one and t h r e e hours a f t e r a s i n g l e i n j e c t i o n o f hormone. O n l y a t one hour was t h e plasma Mg d e c r e a s e s i g n i f i c a n t (p <0.025). 95 S i g n i f i c a n t d e c r e a s e s i n plasma Na o r K i n r e s p o n s e t o c a l c i t o n i n t r e a t m e n t have not been c o n s i s t e n t l y o b s e r v e d and t h i s i s i n agreement w i t h d a t a shown i n T a b l e I I I . The d e c r e a s e i n plasma l e v e l s o f Ca, P, and Mg c o u l d r e a d i l y be a c c o u n t e d f o r by t h e i n h i b i t i o n o f bone r e s o r p t i o n w h i c h o c c u r s f o l l o w i n g c a l c i t o n i n a d m i n i s t r a t i o n . The l o w e r i n g o f plasma P c o u l d , i n a d d i t i o n , be a r e s u l t o f a g e n e r a l movement o f t h i s i o n i n t o s o f t t i s s u e s a s has been s u g g e s t e d by TaImage and A n d e r s o n (74). An i n c r e a s e d l o s s o f t h e s e i o n s from t h e k i d n e y i n r e s p o n s e t o c a l c i t o n i n c o u l d enhance t h e plasma changes f o u n d . 2. U r i n e changes Young r a t s t r e a t e d w i t h s i n g l e i n j e c t i o n s o f salmon c a l c i t o n i n e x h i b i t e d a marked n a t r i u r e t i c and d i u r e t i c r e s p o n s e . I n c r e a s e s i n P e x c r e t i o n were a l s o o b s e r v e d a t t h i s t i m e . However, Ca and Mg o u t p u t s were found t o d e c r e a s e , a l t h o u g h i n some f r a c t i o n a l - c o l l e c t i o n s t u d i e s , t h i s d e c r e a s e was preceded by a t r a n s i t o r y i n c r e a s e i n LL^V and U ^ V . The changes i n t h e e x c r e t i o n o f u r i n a r y e l e c t r o l y t e s f o l l o w i n g s i n g l e d o s e s o f c a l c i t o n i n were found t o o c c u r w i t h i n t h e range o f a s i x - h o u r p e r i o d , and t h e mag n i t u d e o f some o f t h e changes t h a t o c c u r r e d was r e l a t e d t o t h e dose o f hormone g i v e n a s i s seen i n F i g 5. The d e c r e a s e i n U_ V and U,, V wh i c h was o b s e r v e d i n t h e a c u t e Ca Mg s t u d i e s was a t v a r i a n c e w i t h f i n d i n g s from t h e c h r o n i c s t u d i e s where U^ aV and L)^ V i n c r e a s e d . I t i s p o s s i b l e t h a t t h e n e t changes found i n t h e a c u t e s t u d i e s were s e c o n d a r y t o t h e e f f e c t s o f t h e hormone on bone. 96 The decreased plasma Ca l e v e l , as a r e s u l t of decreased bone r e s o r p t i o n , would r e s u l t in a decreased f i l t e r e d load* of Ca (assuming GFR unchanged). This would mask any decreases in t u b u l a r r e a b s o r p t i o n of Ca t h a t might occur. If decreases in the f i l t e r e d load of Ca were s m a l l e r , the c a l c i u r i c e f f e c t of the hormone would become evident as was suggested by the f i n d i n g s of S«5rensen and Hindberg (67). They found t h a t during c h r o n i c a d m i n i s t r a t i o n of porcine c a l c i t o n i n , the hypocalcemic e f f e c t of the hormone was reduced a f t e r the f i r s t day of treatment, and as plasma Ca l e v e l s returned towards normal, the i n i t i a l decrease in u r i n a r y Ca e x c r e t i o n was replaced by an increase in the e x c r e t i o n of t h i s i o n . Decreases in the f i l t e r e d load of Mg could a l s o mask any decrease in r e a b s o r p t i o n of t h i s ion by the renal t u b u l e s . Since Mg i s normally reabsorbed a t a maximal r a t e (25), very smaI I decreases i n f i l t e r e d load could be r e f l e c t e d by large decreases in U^gV. The increase in Mg e x c r e t i o n which was found during c h r o n i c c a l c i t o n i n a d m i n i s t r a t i o n may be explained i n a way s i m i l a r to t h a t described above f o r Ca. Although the net e x c r e t i o n of Ca and Mg was reduced i n the acute s t u d i e s , i t w i l l be noted, as shown i n F i g 5, t h a t large doses of c a l c i t o n i n tended to reverse the t r e n d . In a d d i t i o n , as mentioned above, a bi phasic response in the output of these ions v/as observed i n some f r a c t i o n a I - c o l I e c t i o n s t u d i e s . In t h i s instance, the t r a n s i t o r y increase in e x c r e t i o n of Ca and Mg may have r e f l e c t e d a d i r e c t a c t i o n of c a l c i t o n i n ^ f i l t e r e d load = the amount of a given substance f i l t e r e d through the glomerulus in a given time: u I t r a f i I t r a b l e c o n c e n t r a t i o n X GFR. 97 on t h e r e n a l t u b u l e s i n d e c r e a s i n g r e a b s o r p t i o n o f t h e s e i o n s b e f o r e d e c r e a s e s i n f i l t e r e d l o a d were s u f f i c i e n t t o o b s c u r e t h e e f f e c t . The u r i n a r y e l e c t r o l y t e changes t h a t o c c u r r e d f o l l o w i n g a s i n g l e i n j e c t i o n o f c a l c i t o n i n c a n not be a c c o u n t e d f o r by an i n c r e a s e i n GFR s i n c e t h e y o c c u r r e d when endogenous c r e a t i n i n e e x c r e t i o n was unchanged. T h i s i s i n agreement w i t h t h e f i n d i n g s o f Rasmussen e t a I . ( 3 1 ) who found no i n c r e a s e i n endogenous c r e a t i n i n e e x c r e t i o n o r i n u l i n e x c r e t i o n ( i n r a t s r e c e i v i n g a c o n s t a n t i n f u s i o n o f i n u l i n ) i n r e s p o n s e t o c a l c i t o n i n . SgSrensen e t a I . (72) i n man a l s o found no change i n uQ r e a^. V wh i c h t h e y c o u l d a t t r i b u t e t o c a l c i t o n i n t r e a t m e n t . In s t u d i e s on r a t s , A l d r e d e t a I.(16) found I L , V e l e v a t e d a s d i d W i l l i a m s e t a l . ( l 8 ) . However, when C r e a t . W i l l i a m s e t a I.gave e i t h e r s y n t h e t i c human o r salmon c a l c i t o n i n i n doses w h i c h c a u s e d a s i m i l a r r i s e i n .̂ V, Na e x c r e t i o n was i n c r e a s e d by t h e salmon c a l c i t o n i n b u t not by t h e human p e p t i d e . A r d a i l l o u e t a I . (14) and Haas e t a_l_. (30) found GFR, a s i n d i c a t e d by c l e a r a n c e o f i n u l i n o r c r e a t i n i n e , u n a l t e r e d by c a l c i t o n i n a d m i n i s t r a t i o n when i n c r e a s e s i n e l e c t r o l y t e e x c r e t i o n were found (14, 3 0 ) . I t i s l i k e l y t h a t t h e changes i n e l e c t r o l y t e e x c r e t i o n o b s e r v e d f o l l o w i n g c a l c i t o n i n t r e a t m e n t were p r i m a r i l y a r e f l e c t i o n o f changes i n t h e r e a b s o r p t i v e c a p a c i t i e s o f t h e r e n a l t u b u l e s , a l t h o u g h t h e s e s t u d i e s c a n n o t l o c a l i z e any p a r t i c u l a r a~ea o f t h e nephron where t h e s e changes m i g h t have o c c u r r e d . The DOCA s t u d i e s d i d s u g g e s t t h a t t h e Na e f f e c t e x e r t e d by c a l c i t o n i n might- be o c c u r r i n g a t a s i t e i n t h e nephron p r o x i m a l t o t h e s i t e o f a c t i o n o f t h i s s y n t h e t i c m i n e r a l o c o r t i c o i d . T h i s was i n d i c a t e d by t h e f i n d i n g s t h a t when c a l c i t o n i n was g i v e n t o t h e DOCA-treated r a t s , 9a an increase in U..V was found (Table IV). An increased d e l i v e r y of Na t o t h i s d i s t a l s i t e could r e s u l t in an increased exchange f o r K (75). The DOCA s t u d i e s a l s o revealed t h a t c a l c i t o n i n can counteract the N a - r e t a i n i n g e f f e c t of large doses of m i n e r a l o c o r t i c o i d . Thus, i t can be ruled out t h a t changes in m i n e r a l o c o r t i c o i d a c t i v i t y are r e s p o n s i b l e f o r the changes i n U^gV t h a t were observed. The increase in V f o l l o w i n g c a l c i t o n i n treatment in DOCA-treated or non-treated r a t s was v i r t u a l l y the same, and the changes in the e x c r e t i o n of Ca, P, and Mg were unaltered by DOCA pretreatment. P a r a t h y r o i d hormone causes a decrease in the reabs o r p t i o n of phosphate from the kidney tu b u l e as demonstrated by micropuncture s t u d i e s (76) and an increased r e a b s o r p t i o n of Ca and Mg (23-25, 77). An increase in UpV and a decrease in U^gV and U^gV i s subsequently observed. I t i s u n l i k e l y , however, t h a t changes observed in i n t a c t r a t s f o l l o w i n g c a l c i t o n i n a d m i n i s t r a t i o n could be secondary t o s t i m u l a t i o n of the para t h y r o i d glands as a r e s u l t of the hypocalcemia s i n c e an increase in UpV and a decrease in UQ aV and W^V, ' n a d d i t i o n t o a n a t r i u r e t i c and d i u r e t i c response, were a l s o found in animals with t h e i r p a r a t h y r o i d glands removed (TPTX r a t s ) . In f a c t , an even g r e a t e r n a t r i u r e t i c response was c o n s i s t e n t l y observed f o l l o w i n g c a l c i t o n i n a d m i n i s t r a t i o n t o TPTX r a t s as compared t o e i t h e r TX or i n t a c t r a t s where p a r a t h y r o i d f u n c t i o n was normal. It i s p o s s i b l e t h a t the decreasing plasma Ca le v e l which was found during the period of n a t r i u r e s i s f o l l o w i n g c a l c i t o n i n a d m i n i s t r a t i o n might in i t s e l f be r e s p o n s i b l e f o r the increase in U^gV- However, t h i s i s i n d i r e c t c o n f l i c t with the f i n d i n g s of o t h e r s . V/olf and B a l l (49) 99 showed t h a t an i n c r e a s i n g plasma Ca l e v e l r e s u l t e d i n a prompt i n c r e a s e i n t h e r a t e o f Na e x c r e t i o n i n t h e dog. T h i s was a l s o o b s e r v e d by L e v i t t e t aj _ . (78) i n man and monkey. L e v i t t ' e t a_|_. a l s o d e m o n s t r a t e d t h a t a f a l l i n plasma Ca induced by Na EDTA was a s s o c i a t e d w i t h an immediate d e c r e a s e i n Na e x c r e t i o n . I f a lowered plasma Ca I eve I p e r se c o u l d a f f e c t Na e x c r e t i o n , t h i s would have been a p p a r e n t i n t h e s t u d i e s w i t h t h e TPTX r a t s w i t h plasma Ca l e v e l s r a n g i n g from 5-7 mg% as compared t o TX o r i n t a c t r a t s w i t h normal plasma Ca l e v e l s o f a p p r o x i m a t e l y 10 mg%. In T a b l e V I I I , i t i s seen t h a t V i n t h e t h r e e g r o u p s o f r a t s was n o t a p p r e c i a b l y d i f f e r e n t o v e r a 24-hour p e r i o d . In a d d i t i o n , i n s t u d i e s i n man where plasma Ca l e v e l s were not s i g n i f i c a n t l y a l t e r e d f o l l o w i n g c a l c i t o n i n a d m i n i s t r a t i o n , a n a t r i u r e s i s was s t i l l o b s e r v e d ( 7 9 , 14, 17). The e l e c t r o l y t e changes t h a t were o b s e r v e d i n t h e a c u t e s t u d i e s w i t h r a t s were i n g e n e r a l agreement w i t h t h o s e o b s e r v e d by Rasmussen e t a I.(51) i n 1967. However, t h e s e w o r k e r s o b s e r v e d a m i n i m a l n a t r i u r e t i c e f f e c t u s i n g h i g h doses (ca_. 70 U/kg body w e i g h t / h o u r ) o f p o r c i n e c a l c i t o n i n i n r a t s . A r d a i l l o u e t a I.(14) i n s t u d i e s i n man u s i n g p o r c i n e c a l c i t o n i n a t much lower doses ( c a . 6 mil/ kg body w e i g h t / h o u r ) r e p o r t e d a much g r e a t e r n a t r i u r e t i c r e s p o n s e i n a d d i t i o n t o i n c r e a s e s i n u p V , U^gV, and U C | V . Independent o b s e r v a t i o n s by A l d r e d e t aj _ . (16) w i t h salmon c a l c i t o n i n s u b s t a n t i a t e d o u r f i n d i n g s (32) and e s t a b l i s h e d t h e d r a m a t i c e f f e c t t h a t salmon c a l c i t o n i n has on Na and w a t e r e x c r e t i o n . R e c e n t s t u d i e s by W i l l i a m s e t a_l_. ( 1 8 ) , c o m p a r i n g t h e r e l a t i v e e f f e c t s o f s y n t h e t i c human and salmon c a l c i t o n i n i n r a t s , d e m o n s t r a t e d t h e f a r g r e a t e e r r e n a l 100 e f f e c t o f t h e salmon p r e p a r a t i o n on a w e i g h t b a s i s as compared t o t h a t o f human c a l c i t o n i n . T h i s t h e y a t t r i b u t e d t o not o n l y d i f f e r e n c e s i n s t r u c t u r e o f t h e two p e p t i d e s , but a l s o t o t h e g r e a t e r s t a b i l i t y o f t h e salmon p r e p a r a t i o n . In l i m i t e d c o m p a r a t i v e s t u d i e s i n man, Haas e t a I.(30) a l s o d e m o n s t r a t e d a more pronounced r e n a l e f f e c t w i t h s y n t h e t i c salmon c a l c i t o n i n t h a n t h a t o b t a i n e d w i t h e i t h e r t h e p o r c i n e o r human p e p t i d e s . I t would a p p e a r from t h e s e s t u d i e s i n r a t s , and f r o m t h e many r e c e n t i n v e s t i g a t i o n s i n b o t h man and a n i m a l s , t h a t c a l c i t o n i n c a n c a u s e p r o f o u n d changes i n t h e u r i n a r y e x c r e t i o n o f Na, C a , P, and Mg. These changes a r e not dependent on c o n c o m i t a n t changes i n GFR, a l d o s t e r o n e o r p a r a t h y r o i d a c t i v i t y , and may r e f l e c t a d i r e c t a c t i o n o f t h e hormone on t h e r e n a l t u b u l e s . R e c e n t s t u d i e s by Marx and Aur b a c h (80) l o c a l i z e s p e c i f i c b i n d i n g s i t e s f o r c a l c i t o n i n i n t h e k i d n e y , c o n c e n t r a t e d i n t h e i n n e r c o r t e x o r o u t e r m e d u l l a . These a u t h o r s a l s o f ound t h a t salmon c a l c i t o n i n had a h i g h e r a f f i n i t y t h a n t h e mammalian c a l c i t o n i n s f o r t h e s e t i s s u e r e c e p t o r s . The a b s o l u t e changes e x e r t e d by c a l c i t o n i n on t h e r e n a l e x c r e t i o n o f c e r t a i n e l e c t r o l y t e s a p p e a r t o be r e l a t e d t o t h e p a r t i c u l a r c a l c i t o n i n u s e d , t h e d u r a t i o n o f a d m i n i s t r a t i o n , and t o t h e r a t e o f bone t u r n o v e r t h a t i s f o u n d , t h e e f f e c t s b e i n g more pronounced i n younger a n i m a l s where bone t u r n o v e r i s o c c u r r i n g a t a g r e a t e r r a t e . As shown i n T a b l e V, an immediate and g r e a t e r change i n u r i n a r y e l e c t r o l y t e s was o b s e r v e d i n 5-week-old r a t s as compared t o t h e 28-week-old r a t s . T h i s i n c r e a s e d r e n a l r e s p o n s e i n t h e young r a p i d l y g r o w i n g a n i m a l i s c o n s i s t e n t w i t h t h e g r e a t e r h y p o c a l c e m i c r e s p o n s e w h i c h i s a l s o found i n t h e young r a t ( 6 3 ) . I t w i l l be o b s e r v e d , however, t h a t t h e r e n a l e f f e c t i n t h e o l d e r r a t s was more 101 prolonged and t h i s tended to compensate f o r the smaller immediate r e s - ponse (as compared to the younger r a t s ) . 102 I I . CALCIUM INFUSION STUDIES (ENDOGENOUS CALCITONIN RELEASE) Changes i n plasma c o n c e n t r a t i o n s o f C a , P, and Mg have been o b s e r v e d f o l l o w i n g exogenous a d m i n i s t r a t i o n o f c a l c i t o n i n a s w e l l a s by induced r e l e a s e o f endogenous c a l c i t o n i n f o l l o w i n g t r e a t m e n t w i t h Ca ( 8 1 , 82, 47, 83, 84, 57) o r Mg s a l t s ( 1 9 ) . However, a p o s s i b l e c o r r e l a t i o n between i n d u c e d changes i n t h e c i r c u l a t i n g l e v e l s o f endogenous c a l c i t o n i n and t h e e x c r e t i o n o f u r i n a r y e l e c t r o l y t e s has no t been i n v e s t i g a t e d . There i s some q u e s t i o n whether p h y s i o l o g i c a l r e l e a s e o f t h e a n i m a l ' s own hormone c a n r e s u l t i n e l e c t r o l y t e changes s i m i l a r t o t h o s e found f o l l o w i n g exogenous hormone a d m i n i s t r a t i o n . In o t h e r words, does t h e p h y s i o l o g i c a l r e l e a s e o f c a l c i t o n i n have any r e a l e f f e c t on e x c r e t i o n o f s u c h i o n s as Na, Ca, P, and Mg v i a t h e k i d n e y i n t h e normal a n i m a l ? Numerous s t u d i e s have been performed w h i c h d e m o n s t r a t e t h a t t h e i n f u s i o n o f Ca s a l t s does r e s u l t i n d r a m a t i c changes i n t h e e x c r e t i o n o f c e r t a i n u r i n a r y e l e c t r o l y t e s both i n man ( 5 1 , 85, 86, 53, 87) and i n a n i m a l s ( 4 9 , 50, 88, 52, 5 4 ) . The u r i n a r y changes t h a t a r e found f o l l o w i n g Ca i n f u s i o n i n c l u d e an i n c r e a s e i n u r i n e volume, and an i n c r e a s e i n Na, Ca, P, and Mg e x c r e t i o n . These changes have n o t been c o n s i d e r e d i n r e l a t i o n t o changes t h a t would o c c u r i n c i r c u l a t i n g l e v e l s o f c a l c i t o n i n f o l l o w i n g an induced h y p e r c a l c e m i a . C a r e e t a_l_. (47) and D e f t o s e t a l . ( 4 8 ) have shown t h a t a p r o p o r t i o n a l r e l a t i o n s h i p e x i s t s between t h e degree o f h y p e r c a l c e m i a and t h e r a t e o f s e c r e t i o n o f c a l c i t o n i n . S i n c e t h e u r i n a r y changes t h a t o c c u r f o l l o w i n g Ca i n f u s i o n i n i n t a c t a n i m a l s a r e v e r y s i m i l a r t o t h o s e found f o l l o w i n g exogenous c a l c i t o n i n a d m i n i s t r a t i o n , i t i s p o s s i b l e t h a t c a l c i t o n i n i s p a r t i c i p a t i n g i n such changes. 103 C a l c i u m i n f u s i o n i n i n t a c t , c o n s c i o u s sheep produced a l t e r a t i o n s in plasma Ca and P l e v e l s t h a t would be e x p e c t e d i f a c o n c u r r e n t i n c r e a s e i n endogenous c a l c i t o n i n had o c c u r r e d . T h a t i s , a h y p e r c a l c e m i a was found w h i c h was r a p i d l y c o r r e c t e d f o l l o w i n g c e s s a t i o n o f t h e Ca i n f u s i o n , and a hypophosphatemia s i m i l a r t o t h a t found f o l l o w i n g exogenous hormone a d m i n i s t r a t i o n was a l s o o b s e r v e d . Plasma Mg l e v e l s were a l s o found t o d e c r e a s e . The u r i n e changes i n t h e i n t a c t sheep were c o n s i s t e n t w i t h f i n d i n g s of o t h e r s f o l l o w i n g Ca i n f u s i o n i n man o r a n i m a l s . These changes i n c l u d e d an i n c r e a s e d volume o f u r i n e and an i n c r e a s e i n t h e e x c r e t i o n o f Na, C a , P, and Mg. The removal o f t h e c a I c i t o n i n - c o n t a i n i n g "C" c e l l s o f t h e sheep by t h y r o i d e c t o m y r e s u l t e d i n changes i n plasma and u r i n a r y e l e c t r o l y t e s i n r e s p o n s e t o Ca i n f u s i o n w h i c h were d i f f e r e n t from t h o s e found i n t h e i n t a c t sheep. The impairment w h i c h was o b s e r v e d i n t h e c o n t r o l o f plasma Ca l e v e l s f o l l o w i n g t h e h y p e r c a I c e m i c s t r e s s i n t h e TX sheep was s i m i l a r t o t h a t o b s e r v e d by Sanderson e t a j _ . i n I960 (89) i n dogs. However, t h e s e a u t h o r s f a i l e d t o r e a l i z e t h e s i g n i f i c a n c e o f t h e i r f i n d i n g s . I t remained f o r Copp e t a l _ . i n 1961 (90) t o d e m o n s t r a t e t h e p r e s e n c e o f a h y p o c a l c e m i c f a c t o r i n t h e t h y r o - p a r a t h y r o i d g l a n d complex wh i c h was r e l e a s e d i n t h e p r e s e n c e o f h y p e r c a l c e m i a and c o u l d a c c o u n t f o r 1he improved c o n t r o l o f h y p e r c a l c e m i a i n a n i m a l s w i t h i n t a c t g l a n d s . These f i n d i n g s have been c o n f i r m e d by many w o r k e r s and l n s k e e p and Kenny (83) have a l s o o b s e r v e d an i m p a i r e d c o n t r o l o f h y p e r c a l c e m i a i n TX sheep f o l l o w i n g an i . p . Ca g l u c o n a t e l o a d . The r i s e i n plasma P which o c c u r r e d f o l l o w i n g t h e s t a r t o f t h e Ca i n f u s i o n i n t h e TX sheep, i n c o n t r a s t t o t h e f a l l found i n t h e i n t a c t g r o u p , s u g g e s t e d t h a t endogenous r e l e a s e o f c a l c i t o n i n c o u l d a l s o a f f e c t 104 plasma P l e v e l s i n a s i m i l a r way t o t h a t found f o l l o w i n g exogenous hormone t r e a t m e n t . Kennedy and TaImage (57) a l s o f ound a r i s e i n plasma P l e v e l s i n TX r a t s f o l l o w i n g an i . p . Ca l o a d . TaI mage e t a I.(91) s u g g e s t t h a t c a l c i t o n i n c a u s e s a g e n e r a l movement o f P i n t o s o f t t i s s u e s as w e l l as bone, i n a d d i t i o n t o s t o p p i n g t h e movement o f t h i s i o n from bone. They a t t r i b u t e t h e r i s e i n plasma P i n t h e TX a n i m a l s f o l l o w i n g a Ca s t r e s s t o an i m p a i r e d phosphate t r a n s p o r t i n t o c e l l s s e c o n d a r y t o Ca a c c u m u l a t i o n on c e l l membranes. They s u g g e s t c a l c i t o n i n c a n c o u n t e r - a c t t h e i n h i b i t i o n o f phosphate t r a n s p o r t i n t o c e l l s and t h u s r e d u c e plasma P l e v e l s ( 7 4 ) . A f a l l i n plasma Mg l e v e l s i n t h e TX sheep was o b s e r v e d w h i c h was not s i g n i f i c a n t l y d i f f e r e n t from t h a t found i n t h e i n t a c t sheep. S i n c e t h i s f a l l was s i m i l a r , t h e i n v o l v e m e n t o f endogenous c a l c i t o n i n c o u l d not be a s s o c i a t e d w i t h t h e s e o b s e r v a t i o n s . The d e c r e a s e i n plasma Mg i n b o t h t h e i n t a c t and t h e TX g r o u p s c o u l d be a s s o c i a t e d w i t h t h e d e c r e a s e i n PTH a c t i v i t y f o l l o w i n g t h e h y p e r c a l c e m i a , and t h u s t h e d e c r e a s e d movement o f t h i s i o n from bone. However, when c a l c i t o n i n was g i v e n t o t h e TX sheep, a s i g n i f i c a n t l y g r e a t e r f a l l i n plasma Mg was seen w h i c h s u g g e s t s a p o s s i b l e hypomagnesemic e f f e c t o f t h i s hormone. In c o n t r a s t t o t h e i n c r e a s e d volume o f u r i n e t h a t was found f o l l o w i n g t h e s t a r t o f t h e Ca i n f u s i o n i n t h e i n t a c t sheep, a s i g n i f i c a n t and m a i n t a i n e d d e c r e a s e i n u r i n e volume was found i n t h e TX a n i m a l s . Accompanying t h i s d e c r e a s e i n u r i n e volume, s i g n i f i c a n t l y s m a l l e r i n c r e a s e s i n o s m o l a l o u t p u t were a l s o o b s e r v e d i n t h e TX g r o u p . 105 In t h e TX sheep, changes i n V were reduced as would be e x p e c t e d from t h e d e c r e a s e d o s m o l a l o u t p u t which was o b s e r v e d i n t h i s g r o u p . In a d d i t i o n , d e c r e a s e d changes i n UpV and V were a l s o s e e n . The s m a l l i n c r e a s e i n V i n t h e TX sheep as compared t o t h a t found i n t h e i n t a c t a n i m a l s o c c u r r e d d e s p i t e t h e f a c t t h a t t h e mean o u t p u t o f Ca was g r e a t e r i n t h e TX g r o u p . T h i s i n c r e a s e d LL. V would be e x p e c t e d s i n c e plasma Ca l e v e l s r e a c h e d a h i g h e r a b s o l u t e l e v e l i n t h e TX group and were m a i n t a i n e d f o r a l o n g e r p e r i o d o f t i m e . Thus, a d i s s o c i a t i o n o f u r i n a r y Ca and Na was a p p a r e n t i n t h e TX sheep. In f a c t , two o f t h e TX sheep showed no i n c r e a s e i n V when a l a r g e i n c r e a s e i n LL. V o c c u r r e d . T h i s s u g g e s t s t h a t c a l c i t o n i n may p l a y a r o l e i n m a i n t a i n i n g a p a r a l l e l i s m i n t h e r e n a l c o n t r o l o f t h e s e two i o n s . P h o s p h a t e o u t p u t s i n t h e sheep were v e r y v a r i a b l e , n o t o n l y i n r e s t i n g c o n t r o l v a l u e s between t h e d i f f e r e n t a n i m a l s , b u t a l s o i n t h e a b s o l u t e changes o b s e r v e d f o l l o w i n g t h e Ca s t r e s s . V a r i a t i o n s i n U^V were a l s o o b s e r v e d i n a g i v e n a n i m a l from day t o day. Thus, i t was i m p o s s i b l e t o pool r e s u l t s from any one s e r i e s o f e x p e r i m e n t s . Never- t h e l e s s , a n a l y s i s o f t h e d a t a from i n d i v i d u a l a n i m a l s i n d i c a t e d t h a t whereas LL. V i n t h e i n t a c t and TX sheep appeared t o r e f l e c t t h e plasma Ca c h a n g e s , t h i s d i d n o t a p p l y t o changes i n UpV i n r e l a t i o n t o t h e plasma P changes t h a t were s e e n . In t h e i n t a c t sheep, d e s p i t e a f a l l i n g plasma P, t h e u r i n a r y P e x c r e t i o n was i n c r e a s e d i n each o f t h e f o u r a n i m a l s o b s e r v e d . S i n c e t h e sheep were h y p e r c a I c e m i c a t t h i s t i m e , PTH-induced p h o s p h a t u r i a i s n o t l i k e l y t o be t h e c a u s e o f t h i s f i n d i n g s i n c e PTH s e c r e t i o n r a t e s d i m i n i s h w i t h r i s i n g plasma Ca l e v e l s as has been shown by Sherwood e t a I . ( 9 2 ) . I t i s more l i k e l y t h a t a d e c r e a s e d 106 r e a b s o r p t i o n o f P had o c c u r r e d i n t h e k i d n e y s i n c e a d e c r e a s e d f i l t e r e d l o a d o f P was i n d i c a t e d . A p h o s p h a t u r i c r e s p o n s e has been found by a number of w o r k e r s f o l l o w i n g a d m i n i s t r a t i o n o f c a l c i t o n i n ( I I , 14, 30, 16), and t h e s e e f f e c t s a r e a l s o found where PTH i n t e r v e n t i o n c a n be r u l e d o u t (12 , 13, 31, 15). I t i s p o s s i b l e t h a t induced i n c r e a s e s i n endogenous c a l c i t o n i n l e v e l s c o u l d c a u s e a p h o s p h a t u r i c e f f e c t s i m i l a r t o t h a t seen f o l l o w i n g exogenous hormone t r e a t m e n t . When salmon c a l c i t o n i n was g i v e n t o t h e TX sheep, t h e p a t t e r n o f b o t h plasma and u r i n a r y changes f o l l o w i n g t h e Ca i n f u s i o n was r e s t o r e d i n p a r t t o one s i m i l a r t o t h a t f ound i n t h e i n t a c t sheep. Plasma Ca l e v e l s were l i m i t e d i n t h e i r a b s o l u t e r i s e and t h e r e t u r n t o b a s e - l i n e l e v e l s was a c c e l e r a t e d . Plasma P l e v e l s i m m e d i a t e l y f e l l f o l l o w i n g t h e s t a r t o f t h e Ca i n f u s i o n a n d , i n a d d i t i o n , t h e r e was a g r e a t e r f a l l i n plasma Mg t h a n t h a t f ound i n e i t h e r t h e i n t a c t o r TX sheep. I n c r e a s e s i n u r i n e volume and t h e e x c r e t i o n o f Na, P, and Mg were a l s o o b s e r v e d i n t h e TX sheep when c a l c i t o n i n was a d m i n i s t e r e d d u r i n g and f o l l o w i n g t h e Ca i n f u s i o n . A d e c r e a s e i n t h e e x c r e t i o n o f Ca was a l s o f ound w h i c h appeared t o be s e c o n d a r y t o t h e c a l c i t o n i n - induced changes i n plasma Ca l e v e l s . The changes i n plasma Ca and u r i n a r y e x c r e t i o n o f Ca when c a l c i t o n i n was a d m i n i s t e r e d t o t h e TX group were s i m i l a r t o t h o s e found i n t h e i n t a c t sheep. The a l t e r a t i o n s i n both plasma and u r i n a r y r e s p o n s e s i n t h e TX sheep as compared t o t h o s e found i n t h e i n t a c t (endogenous c a l c i t o n i n ) o r TX+CT (exogenous c a l c i t o n i n ) a n i m a l s s u g g e s t t h a t t h e r e l e a s e o f c a l c i t o n i n i n r e s p o n s e t o a Ca s t r e s s a f f e c t s plasma Ca, P, and p o s s i b l y Mg, as w e l l a s u r i n a r y volume and t h e e x c r e t i o n o f Na, C a , P, and Mg. 107 I I I . EXTRACELLULAR VOLUME EXPANSION The " s t r a i n on volume h o m e o s t a s i s " (93) which i s b r o u g h t a b o u t f o l l o w i n g c a l c i t o n i n a d m i n i s t r a t i o n s u g g e s t s t h a t t h i s hormone may p l a y a s i g n i f i c a n t r o l e i n t h e c o n t r o l o f body Na. S t u d i e s i n both man and animals f o l l o w i n g exogenous hormone a d m i n i s t r a t i o n have shown t h a t a c o n s i d e r a b l e l o s s o f Na and volume o c c u r s a s a r e s u l t o f t h e t r e a t m e n t . However, an impairment i n c o n t r o l o f body volume i n man o r a n i m a l s w i t h o u t t h e c a I c i t o n i n - c o n t a i n i n g "C" c e l l s has n o t been d e m o n s t r a t e d . I t i s d i f f i c u l t t o c o r r e l a t e t h e a c t i v i t i e s o f t h i s hormone i n r e l a t i o n t o Na c o n t r o l w i t h o u t such e v i d e n c e . A. Volume E x p a n s i o n i n R a t s The volume e x p a n s i o n s t u d i e s i n r a t s i m p l i c a t e d c a l c i t o n i n and t h e p a r a t h y r o i d g l a n d s i n Na h o m e o s t a s i s . T h i s was n o t i m m e d i a t e l y a p p a r e n t , however, s i n c e a n a t r i u r e t i c and d i u r e t i c r e s p o n s e f o l l o w i n g s a l i n e l o a d i n g o c c u r r e d i n TPTX r a t s s i m i l a r i n m a g n i t u d e t o t h a t found i n t h e i n t a c t a n i m a l s ( F i g 16). O n l y when c a l c i t o n i n was a b s e n t i n t h e s e a n i m a l s (TX r a t s ) was an i n h i b i t i o n o f t h e normal n a t r i u r e t i c r e s p o n s e e v i d e n t f o l l o w i n g volume e x p a n s i o n . The d i f f e r e n c e i n r e s p o n s e between t h e TPTX and TX r a t s s u g g e s t e d t h a t t h e p r e s e n c e o f t h e t r a n s p l a n t e d p a r a - t h y r o i d t i s s u e m i g h t be r e s p o n s i b l e f o r t h e d e l a y e d n a t r i u r e s i s . To s u p p o r t t h i s s u g g e s t i o n i t was h y p o t h e s i z e d t h a t any p o s s i b l e a n t i n a t r i u r e t i c a c t i v i t y e x h i b i t e d by p a r a t h y r o i d t i s s u e i n t h e i n t a c t r a t s (where an immediate n a t r i u r e s i s was found) c o u l d be a n t a g o n i z e d by t h e a c t i o n o f c a l c i t o n i n r e l e a s e d i n r e s p o n s e t o volume e x p a n s i o n . 108 The p o s s i b i l i t y t h a t t h e p r e s e n c e o f t h e p a r a t h y r o i d t i s s u e was r e s p o n s i b l e f o r t h e a n t i n a t r i u r e t i c r e s p o n s e i n t h e TX r a t s was s t r e n g t h e n e d by t h e f i n d i n g t h a t exogenous a d m i n i s t r a t i o n o f PTE t o TPTX r a t s r e s u l t e d i n a s i m i l a r i n h i b i t i o n o f Na o u t p u t f o l l o w i n g a s a l i n e l oad ( F i g 17). In t h e TPTX r a t s , t h e i n c r e a s e s i n C a , P, and Mg o u t p u t s d u r i n g t h e f i r s t t h r e e h o urs f o l l o w i n g t h e s a l i n e l o a d were n o t s i g n i f i c a n t , i n c o n t r a s t t o t h e s i g n i f i c a n t i n c r e a s e s i n t h e e x c r e t i o n o f t h e s e i o n s i n t h e i n t a c t r a t s ( T a b l e X I V ) . However, t h e volume and Na i n c r e a s e s were n o t a p p r e c i a b l y d i f f e r e n t between t h e i n t a c t and TPTX g r o u p s . I t i s p o s s i b l e t h a t t h e u r i n a r y changes i n t h e TPTX r a t s i n r e s p o n s e t o t h e s a l i n e l o a d were s e c o n d a r y t o an i n c r e a s e i n GFR. I t was found t h a t a 23? i n c r e a s e i n U C r e a + V o c c u r r e d i n t h e s e a n i m a l s i n t h e f i r s t t h r e e hours f o l l o w i n g t h e s a l i n e l o a d ( T a b l e X I V ) a s compared t o an 8% i n c r e a s e i n t h e i n t a c t r a t s . The a b s e n c e o f f u n c t i o n i n g p a r a t h y r o i d t i s s u e may a l s o have been a f a c t o r . The f i n d i n g t h a t a n a t r i u r e s i s o c c u r s i n i n t a c t r a t s i n r e s p o n s e t o a s a l i n e load and i s i n h i b i t e d i n t h o s e a n i m a l s w h i c h a r e " C " - c e l I d e f i c i e n t (TX r a t s ) s u g g e s t s t h a t c a l c i t o n i n may be r e l e a s e d i n r e s p o n s e t o volume e x p a n s i o n . In t h e s e s t u d i e s where i s o t o n i c s a l i n e was used t o c a u s e t h e e x t r a c e l l u l a r volume e x p a n s i o n , one m i g h t have e x p e c t e d e o c t l y t h e o p p o s i t e t o o c c u r . A d i l u t i o n o f t h e e x t r a c e l l u l a r Ca c o n c e n t r a t i o n by t h e s a l i n e load would be e x p e c t e d t o d e c r e a s e t h e r e l e a s e o f c a l c i t o n i n and enhance p a r a t h y r o i d s e c r e t i o n . However, t h e u r i n a r y changes t h a t o c c u r f o l l o w i n g volume e x p a n s i o n w i t h s a l i n e ( 4 4 ) , 109 even when GFR i s reduced (53), are not c o n s i s t e n t with the known u r i n a r y changes t h a t occur f o l l o w i n g PTH i n t e r v e n t i o n . The only renal response found t h a t i s c o n s i s t e n t with the PTH a c t i v i t y i s a phosphaturia, but a phosphaturic response i s a l s o observed with c a l c i t o n i n . The changes t h a t are found f o l l o w i n g volume expansion in i n t a c t animals are c o n s i s t e n t with the renal responses observed in animals f o l l o w i n g the a d m i n i s t r a t i o n of exogenous c a l c i t o n i n . The f a c t t h a t these changes d i d not occur in thyroidectomized r a t s lends support to t h i s hypothesis. B. PTH and Renal E l e c t r o l y t e Changes In 1929, A l b r i g h t and E l l s w o r t h (27) f i r s t noted t h a t a decrease in u r i n a r y e x c r e t i o n of Ca occur r e d , in a d d i t i o n t o an increase in phosphate e x c r e t i o n , f o l l o w i n g the a d m i n i s t r a t i o n of PTE i n a hypopara- t h y r o i d p a t i e n t . The decrease in Ca e x c r e t i o n continued u n t i l the serum Ca le v e l rose t o a " c r i t i c a l v a l u e " of 8.5 mg%, above which p o i n t u r i n a r y Ca increased. Talbot et a l . ( 2 l ) in r e - e v a l u a t i n g t h i s data along with t h e i r own f i n d i n g s suggested t h a t PTH a c t s "to boost the lowered serum Ca c o n c e n t r a t i o n towards normal, in part by i n c r e a s i n g the e f f i c i e n c y of Ca reabs o r p t i o n by the renal t u b u l e s " . Talmage and K r a i n t z (5) observed an increase i n Ca e x c r e t i o n in r a t s f o l l o w i n g acute parathyroidectomy which was reversed by the a d m i n i s t r a t i o n of PTE. Since serum Ca l e v e l s were f a l l i n g a t t h i s time, they concluded t h a t PTH has a d i r e c t e f f e c t on the renal t u b u l e s , enhancing Ca r e a b s o r p t i o n . S i m i l a r f i n d i n g s were observed by I 10 Kleeman e+ aj_. (22) in man and dogs. They s+a+ed t h a t "the e x c r e t i o n of Ca i s a f u n c t i o n of the f i l t e r e d load of i o n i c and complexed Ca and the homeostatic f a c t o r s r e g u l a t i n g a c t i v e t u b u l a r r e a b s o r p t i o n . " One of the f a c t o r s involved in homeostatic r e g u l a t i o n of t u b u l a r r e a b s o r p t i o n i s PTH which they found enhanced Ca r e a b s o r p t i o n . In hyperparathyroid p a t i e n t s , f o r example, they found t h a t the C_ was decreased, and i n hypoparathyroid p a t i e n t s or in PTX dogs, d e s p i t e a decreased f i l t e r e d load of Ca, was increased. Massry et a I.(25) a l s o found an increased in TPTX dogs. Stop-flow s t u d i e s by Widrow and Levinsky i n 1962 (23) i n d i c a t e d t h a t the s i t e of a c t i o n of PTH on Ca r e a b s o r p t i o n was in the d i s t a l t u b u l e . Microperfus ion and micropuncture s t u d i e s by F r i c k et a l . i n I965((94) s u b s t a n t i a t e d these f i n d i n g s by demonstrating t h a t PTH does not a f f e c t Ca r e a b s o r p t i o n p r o x i m a l l y . Therefore, the changes in r e a b s o r p t i o n of t h i s ion modified by PTH must occur a t a more d i s t a l s i t e . Changes i n Mg r e a b s o r p t i o n have a l s o been found in c o n j u n c t i o n with changes in Ca r e a b s o r p t i o n f o l l o w i n g PTH a d m i n i s t r a t i o n . In 1963, Maclntyre et a I.(24), using p u r i f i e d PTH or L i l l y PTE, observed decreases in both U^V and U^V. Studies in hypoparathyroid man by G i l l e t a I . (77) and Haas et aj_. (30) a l s o showed t h a t decreases in u r i n a r y Ca and Mg occurred f o l l o w i n g PTE. G i l l et a I.(77) found t h a t as plasma Ca and Mg rose f o l l o w i n g PTE, the i n i t i a l decrease in u r i n a r y Ca and Mg was reversed. They suggested t h a t the increased f i l t e r e d load of these ions masked any change in r e a b s o r p t i v e c a p a c i t y of the renal t u b u l e c e l l s . Peacock e+ a_l_. (95) a l s o s t a t e d t h a t PTH promotes t u b u l a r r e a b s o r p t i o n of Ca. When c a l c i u r i a i s evident i n hyperparathyroidism, i t I 11 i s s e c o n d a r y t o t h e i n c r e a s e d f i l t e r e d l o a d o f Ca. The p h o s p h a t u r i c a c t i o n o f PTH has been r e c o g n i z e d f o r many y e a r s . In a r e c e n t m i c r o p u n c t u r e s t u d y i n dogs by G o l d b e r g e t a I.(76);, t h e d e c r e a s e i n phosphate r e a b s o r p t i o n t h a t o c c u r r e d f o l l o w i n g PTH a d m i n i s t r a t i o n was found t o o c c u r i n t h e p r o x i m a l t u b u l e . They found t h a t t h e r e j e c t e d p h o s p h a t e from t h i s s i t e was t h e n e x c r e t e d i n t h e u r i n e w i t h l i t t l e f u r t h e r a l t e r a t i o n . These a u t h o r s a l s o found t h a t t h e d e c r e a s e i n p h o s p h a t e r e a b s o r p t i o n a t t h e p r o x i m a l t u b u l e was accompanied by a p a r a l l e l d e c r e a s e i n Na r e a b s o r p t i o n a t t h i s same s i t e . However, a n a t r i u r e s i s was n o t f o u n d t o accompany t h e p h o s p h a t u r i a , and t h i s t h e y a t t r i b u t e d t o a r e a b s o r p t i o n o f t h i s i o n i n t h e d i s t a l n ephron. They s u g g e s t e d t h a t PTH v i a i t s m e d i a t o r cAMP may be i n i t i a l l y a c t i n g on Na r e a b s o r p t i o n and t h a t t h e i n h i b i t i o n o f p r o x i m a l p h o s p h a t e r e a b s o r p t i o n i s a c o n sequence o f t h i s e v e n t . However, t h i s h y p o t h e s i s does n o t i n d i c a t e what f a c t o r ( s ) may be i n v o l v e d i n t h e i n c r e a s e d r e a b s o r p t i o n o f Na t h a t o c c u r r e d i n a more d i s t a l s i t e so t h a t no n e t i n c r e a s e i n Na e x c r e t i o n was o b s e r v e d . These f i n d i n g s by G o l d b e r g e t a 1.(76) c o u l d be i n t e r p r e t e d somewhat d i f f e r e n t l y s i n c e t h e y do not c o n f i r m t h a t PTH was a c t i n g d i r e c t l y on Na r e a b s o r p t i o n a t t h e p r o x i m a l t u b u l e s i t e . A d e c r e a s e d Na r e a b s o r p t i o n was s i m p l y o b s e r v e d i n c o n j u n c t i o n w i t h a d e c r e a s e d phosphate r e a b s o r p t i o n . I t i s p o s s i b l e t h a t PTH's a c t i o n on Na r e a b s o r p t i o n was a t a more d i s t a l s i t e (where PTH has been found t o i n c r e a s e t h e r e a b s o r p t i o n o f Ca ( 2 3 ) ) , e n h a n c i n g t h e r e a b s o r p t i o n o f Na, c o u n t e r a c t i n g t h e i n c r e a s e d d e l i v e r y o f Na t o t h i s r e g i o n so t h a t t h e e x p e c t e d n a t r i u r e s i s d i d not o c c u r . I 12 In many o f t h e s t u d i e s where an i n c r e a s e i n Na e x c r e t i o n i s o b s e r v e d f o l l o w i n g PTH a d m i n i s t r a t i o n , f a c t o r s s e c o n d a r y t o PTH a c t i o n have not a l w a y s been c o n s i d e r e d . F o r example, (a) i s t h e n a t r i u r e s i s which i s sometimes o b s e r v e d a r e s u l t o f r e n a l hemodynamic changes ( 9 6 ) ; (b) i s i t s e c o n d a r y t o an i n c r e a s e i n plasma Na wh i c h may f o l l o w PTH a c t i v i t y ( 7 ) ; ( c ) i s t h e n a t r i u r e s i s s e c o n d a r y t o t h e r e l e a s e o f c a l c i t o n i n i n r e s p o n s e t o PTH-induced h y p e r c a l c e m i a ? Such f a c t o r s may be p e r t i n e n t i n i n t e r p r e t i n g t h e Na changes i n t h e u r i n e f o l l o w i n g PTH i n t e r v e n t i o n . If one i s t o a c c e p t t h a t t h e immediate e f f e c t o f PTH i s t o d e c r e a s e u r i n a r y Ca e x c r e t i o n b e f o r e any changes i n plasma Ca o c c u r which may o b s c u r e t h e e f f e c t , a c l o s e r e x a m i n a t i o n o f some o f t h e s t u d i e s f o l l o w i n g PTH i n t e r v e n t i o n r e l a t i n g t h e s i m u l t a n e o u s changes i n Na and Ca m i g h t c l a r i f y t h e pr o b l e m . In s t u d i e s on TPTX dogs, M a s s r y e t a I.(52) found t h a t i n r e s p o n s e t o PTE, I L V f e l l on Day I o f t r e a t m e n t d e s p i t e a r i s i n g serum Ca. ua I n c r e a s e s i n U., V were m i n i m a l a t t h i s t i m e even when serum Na was Na e l e v a t e d . On Day 2 o f t r e a t m e n t , UQ gV was i n c r e a s e d o n l y when t h e dog was h y p e r c a I c e m i c . u N a V o n Day 2 was reduced o r m o d e r a t e l y e l e v a t e d d e s p i t e a l a r g e i n c r e a s e i n serum Na c o n c e n t r a t i o n . In a s i m i l a r s t u d y i n man, Wi I Is e t a_l_. (53) found t h a t on Day I t r e a t m e n t w i t h PTE, I L V i n c r e a s e d w i t h no measured change i n serum Ca. ua U^gV was a l s o i n c r e a s e d a t t h e same t i m e but was accompanied by an i n c r e a s e i n serum Na. On Days 2 and 3, I L V was s t i l l e l e v a t e d b u t serum Ca l e v e l s I 13 were a l s o e l e v a t e d a t t h i s t i m e . V d e c r e a s e d d u r i n g Days 2 and 3 and was not s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l v a l u e s when normal serum Na l e v e l s were f o u n d . In a more r e c e n t s t u d y by P a u n i e r e t a I.(26) i n man, Na and Ca showed s i m i l a r changes i n t h e i r e x c r e t o r y p a t t e r n s f o l l o w i n g PTE i n f u s i o n . The r a t i o s o f Na and Ca o u t p u t s t o c o n t r o l Na and Ca o u t p u t s showed a s i m i l a r i n c r e a s e i n t h e f i r s t hour f o l l o w i n g PTE, and i n t h e second hour showed s i m i l a r d e c r e a s e s . T h a t i s , when Ca e x c r e t i o n was d e c r e a s e d , a s i m i l a r d e c r e a s e i n Na e x c r e t i o n v/as f o u n d . R e s u l t s f r o m such s t u d i e s i n a n i m a l s and man do not c o n v i n c i n g l y d e m o n s t r a t e t h a t exogenous a d m i n i s t r a t i o n o f PTH c a u s e s a p r i m a r y n a t r i u r e t i c r e s p o n s e . In T a b l e X V I I , t h e changes i n t h e e x c r e t i o n o f Ca, P, and Mg i n TPTX r a t s f o l l o w i n g a d m i n i s t r a t i o n o f PTE a r e c o n s i s t e n t w i t h t h e f i n d i n g s of o t h e r s . P a r a t h y r o i d hormone by i n c r e a s i n g t h e r e a b s o r p t i o n o f Ca and Mg and d e c r e a s i n g t h e r e a b s o r p t i o n o f P c a u s e s an i n i t i a l d e c r e a s e i n t h e e x c r e t i o n o f Ca and Mg and a p h o s p h a t u r i c r e s p o n s e . Accompanying t h e u r i n a r y C a , P, and Mg c h a n g e s , a d e c r e a s e i n Na e x c r e t i o n and u r i n e volume was o b s e r v e d w i t h i n c r e a s i n g d o s e s o f hormone. As i s seen i n F i g . 19, U^ gV was l i n e a r l y r e l a t e d t o t h e l o g - d o s e o f PTE. P r e v i o u s l o a d i n g w i t h s a l i n e d i d not a l t e r t h e r e s p o n s e s o f r a t s t o e i t h e r T C A - p r e c i p i t a t e d PTH o r PTE, as i s seen i n T a b l e X V I . An a n t i n a t r i u r e s i s was o b s e r v e d w i t h b o t h hormone p r e p a r a t i o n s , a l t h o u g h a g r e a t e r and more p r o l o n g e d a n t i n a t r i u r e t i c a c t i v i t y was o b s e r v e d w i t h t h e L i l l y PTE. The i n h i b i t i o n o f Ca and Mg e x c r e t i o n was a l s o more marked w i t h PTE. I 14 In T a b l e XV, t h e a n t i n a t r i u r e t i c r e s p o n s e t o L i l l y PTE i n non-loaded and s a I i n e - I o a d e d TPTX r a t s e x h i b i t e d d u r i n g t h e f i r s t t h r e e h o u r s f o l l o w i n g t r e a t m e n t was f o l l o w e d by an " e s c a p e " whereby a n a t r i u r e s i s and a d i u r e s i s were o b s e r v e d . Thus t h e a n t i n a t r i u r e t i c a c t i v i t y t h a t was found f o l l o w i n g PTE was a t r a n s i e n t i n h i b i t i o n w h i c h s u g g e s t e d t h a t d u r i n g t h i s t i m e p e r i o d , PTE, i n a d d i t i o n t o i n c r e a s i n g t h e r e a b s o r p t i o n o f Ca and Mg from t h e k i d n e y t u b u l e s , a l s o i n c r e a s e d Na r e a b s o r p t i o n . The p o s s i b i l i t y t h a t d e c r e a s e s i n GFR accompanied t h e s e changes c a n n o t be o v e r l o o k e d , a l t h o u g h d e c r e a s e s i n L l ^ V were no t a l w a y s a s s o c i a t e d w i t h d e c r e a s e s i n ̂ Qrea^. V. In s t u d i e s by L e v i t t e t a j _ . (78) i n man and monkey, t h e i n f u s i o n o f a C a - c h e l a t i n g a g e n t w h i c h lowered plasma Ca l e v e l s r e s u l t e d i n s i m i l a r e l e c t r o l y t e changes t o t h o s e o b s e r v e d i n t h e TPTX r a t s f o l l o w i n g PTE t r e a t m e n t ( T a b l e X V I I ) . T h a t i s , immediate d e c r e a s e s i n U.. V and U~ V ' Na Ca were found t o o c c u r i n a d d i t i o n t o a c o n c u r r e n t p h o s p h a t u r i a w h i c h p e r s i s t e d even a f t e r t h e c h e l a t i n g i n f u s i o n was s t o p p e d . The a u t h o r s s u g g e s t e d t h a t t h e p h o s p h a t u r i a was s e c o n d a r y t o s t i m u l a t e d r e l e a s e o f PTH i n r e s p o n s e t o h y p o c a l c e m i a but d i d n o t comment on t h e c a u s e o f t h e o t h e r u r i n a r y changes t h a t o c c u r r e d a t t h e same t i m e . I t i s e q u a l l y p o s s i b l e t h a t r e l e a s e o f endogenous PTH c o u l d have c a u s e d t h e d e c r e a s e found i n t h e u r i n a r y e x c r e t i o n o f Na and Ca, a s w e l l as b e i n g r e s p o n s i b l e f o r t h e p h o s p h a t u r i a . H i g h l y p u r i f i e d PTH d i d not c a u s e a s i g n i f i c a n t d e c r e a s e i n U V when g i v e n i n graded doses t o TPTX r a t s ( T a b l e X V I I I ) . However, I 15 s i g n i f i c a n t i n c r e a s e s i n U^V were o b s e r v e d i n t h e 10 and 100 U/100 g dose range which s u g g e s t e d t h e p o s s i b i l i t y t h a t an i n c r e a s e d r e a b s o r p t i o n o f Na i n exchange f o r K was o c c u r r i n g . I t i s p o s s i b l e t h a t PTH may a l t e r t h e r e a b s o r p t i o n s i t e f o r Na from t h e p r o x i m a l t o t h e d i s t a l t u b u l e :thus r e d u c i n g exchange f o r hydrogen i o n ( a t a p r o x i m a l s i t e ) i n p r e f e r e n c e f o r exchange f o r K a t a more d i s t a l s i t e . G o l d b e r g e t a I.(76) found t h a t a d e c r e a s e d r e a b s o r p t i o n o f Na o c c u r r e d i n t h e p r o x i m a l t u b u l e f o l l o w i n g PTH a d m i n i s t r a t i o n . S i n c e t h i s i s t h e major s i t e f o r Na-hydrogen exchange, an i n h i b i t i o n o f t h i s mechanism a s p o s t u l a t e d by HelIman e t aj _ . (97) i s not u n r e a s o n a b l e . The r e l a t i v e e f f e c t s o f t h e d i f f e r e n t PTH p r e p a r a t i o n s on n e t Na e x c r e t i o n c o u l d be a t t r i b u t e d t o changes i n GFR. In some s t u d i e s w i t h PTE, f o r example, a d e c r e a s e d e x c r e t i o n o f endogenous c r e a t i n i n e was o b s e r v e d when u r i n a r y Na was d e c r e a s e d . The r e a s o n f o r t h i s a p p a r e n t d e c r e a s e i n GFR i n some o f t h e s e s t u d i e s c a n n o t be a c c o u n t e d f o r . The f a c t t h a t l l . , V was n o t i n c r e a s e d i n s a I i n e - I o a d e d TX r a t s ( w i t h Na f u n c t i o n i n g p a r a t h y r o i d t r a n s p l a n t s ) as compared t o TPTX r a t s s u g g e s t s t h a t PTH c a n compensate f o r a d e c r e a s e d p r o x i m a l r e a b s o r p t i o n o f Na vh i c h has been shown t o o c c u r f o l l o w i n g volume e x p a n s i o n (76) by i n c r e a s i n g t h e r e a b s o r p t i o n o f t h i s i o n a t a more d i s t a l s i t e . The i n c r e a s e d e x c r e t i o n o f K which was o b s e r v e d i n t h e s t u d i e s w i t h h i g h l y p u r i f i e d PTH ( T a b l e X V I I I ) s u b s t a n t i a t e d t h e c o n c e p t t h a t PTH may enhance d i s t a l t u b u l a r r e a b s o r p t i o n o f Na. I 16 C. Volume E x p a n s i o n i n Sheep I . Plasma changes The i n i t i a l d i l u t i o n o f t h e plasma volume by a f a c t o r of 23$* as a r e s u l t o f t h e d e x t r a n i n f u s i o n r e s u l t e d i n d e c r e a s e s i n t h e c o n c e n t r a t i o n s o f t o t a l C a , P, and Mg i n t h e plasma. S i n c e a d i l u t i o n o f s a l i n e o n l y c a u s e s s m a l l i n c r e a s e s i n t h e u I t r a f i I t r a b I e f r a c t i o n o f Ca and Mg a c c o r d i n g t o B l y t h e e t a j _ . ( 4 4 ) , i t i s l i k e l y t h a t i n t h e s e s t u d i e s t h e u I t r a f i I t r a b I e c o n c e n t r a t i o n s of Ca, Mg, and P ( w h i c h i s " b t a l l y u I t r a f i I t r a b I e) were a l s o d e c r e a s e d . In t h e i n t a c t sheep ( F i g s . 20 and 2 1 ) , t h e s e plasma e l e c t r o l y t e c hanges were m a i n t a i n e d t h r o u g h o u t t h e e x p e r i m e n t a l p e r i o d . In t h e TX a n i m a l s , a s i m i l a r d i l u t i o n e f f e c t was o b s e r v e d . However, i n t h i s group plasma Ca l e v e l s s l o w l y r e t u r n e d t o normal i n t h e t h r e e - h o u r p e r i o d f o l l o w i n g t h e d e x t r a n l o a d . The o b s e r v a t i o n t h a t a c a l c i u r i a was found i n t h e i n t a c t group w h i c h d i d n o t o c c u r i n t h e TX group s u g g e s t s t h a t t h e r e n a l h a n d l i n g o f t h i s i o n c o n t r i b u t e d i n p a r t t o t h e plasma changes f o u n d . If c a l c i t o n i n was r e l e a s e d i n r e s p o n s e t o volume e x p a n s i o n i n t h e i n t a c t sheep, plasma Ca l e v e l s would be e x p e c t e d t o be u n a l t e r e d *Based on t h e a s s u m p t i o n t h a t i n sheep plasma volume = 3.4$ t o t a l body w e i g h t (98). 117 or even decreased f u r t h e r f o l l o w i n g the i n i t i a l d i l u t i o n with the dextran load. C a l c i t o n i n by depressing bone r e s o r p t i o n and by inducing a c a l c i u r i a could cause such plasma changes as were observed. If PTH was released in response to the hypocalcemia in the TX animals, : unapposed by c a l c i t o n i n , the observed changes in the plasma and u r i n e would a l s o be expected. I t i s not p o s s i b l e to determine without knowledge of the c i r c u l a t i n g l e v e l s of these two hormones, c a l c i t o n i n and PTH, what a c t u a l mechanism may have been o p e r a t i n g , i f indeed these hormones were involved a t a l l . - However, the observed changes in plasma and u r i n a r y Ca in the i n t a c t and TX sheep d i d suggest t h a t c a l c i t o n i n might be i n v o l v e d . This was s u b s t a n t i a t e d by the f i n d i n g t h a t when the TX sheep were given c a l c i t o n i n , a p r o g r e s s i v e and sustained f a l l i n plasma Ca occurred. In a d d i t i o n , a c a l c i u r i a was observed i n s p i t e of the low plasma Ca l e v e l . 2. U r i ne Changes The i n f u s i o n of a s a l i n e load with the a d d i t i o n of the macro- molecule dextran increases the c o l l o i d osmotic pressure of the plasma so t h a t the increased volume i s maintained. Dextran (Gentran 75) i s a glucose polymer with an average molecular weight of 75,000 and i s only slowly degraded t o glucose which can then be e l i m i n a t e d from the v a s c u l a r space. As a r e s u l t , i n f u s i o n of a s a l i n e load with the a d d i t i o n of dextran increases the v a s c u l a r volume f o r a longer period of time than t h a t of s a l i n e alone. N i z e t (99) found t h a t the i n f u s i o n of dextran f o l l o w i n g a s a l i n e load depressed the increase in Na and volume e x c r e t i o n t h a t occurred with s a l i n e alone. This he a t t r i b u t e d t o a measured increase in plasma c o l l o i d osmotic pressure which may have been I 18 r e s p o n s i b l e f o r a simultaneous decrease in GFR. However, N i z e t could not r u l e out haemodynamic i n t r a - r e n a l changes as a l s o being involved i n the response. Howards et a I.(100) in micropuncture s t u d i e s in the dog found t h a t dextran decreased Na re a b s o r p t i o n by the proximal t u b u l e but net Na e x c r e t i o n was s t i l l depressed. Therefore, i t i s p o s s i b l e t h a t the depression of Na e x c r e t i o n f o l l o w i n g dextran i n f u s i o n as compared to s a l i n e expansion alone can be a t t r i b u t e d t o haemodynamic changes w i t h i n the kidney enhancing r e a b s o r p t i o n of Na in s i t e s d i s t a l from the proximal t u b u l e . I t would appear t h a t dextran i s simply modifying the changes found i n response t o s a l i n e loading and o f f e r s some advantages in such s t u d i e s by compensating f o r the d i l u t i o n of plasma p r o t e i n s and, thereby, reducing the degree of e l e c t r o l y t e loss which could be secondary t o a s h i f t of the " S t a r l i n g e q u i l i b r i u m " in the kidney. If dextran a f f e c t s the abs o l u t e changes t h a t are found i n response t o volume expansion, i t does not a l t e r the d i r e c t i o n of e l e c t r o l y t e changes t h a t normally occurs f o l l o w i n g s a l i n e loading in normal animals. Increases in u r i n a r y volume, Na, Ca, P, and Mg e x c r e t i o n were found in the i n t a c t sheep f o l l o w i n g the dextran load. The n a t r i u r e t i c and d i u r e t i c responses which occurred in the i n t a c t sheep were a l s o found in the TX sheep. Only when salmon c a l c i t o n i n was given to t h i s l a t t e r group was an a l t e r e d response found. At t h i s time, exogenous c a l c i t o n i n a d m i n i s t r a t i o n doubled the increase in Na and volume e x c r e t i o n f o l l o w i n g dextran i n f u s i o n . The most s i g n i f i c a n t d i f f e r e n c e s t h a t occurred in the u r i n e of these three groups of sheep were those in r e l a t i o n t o Ca e x c r e t i o n . Whereas the changes in U M V in the i n t a c t and TX sheep were of a s i m i l a r I 19 magni+ude, a c a l c i u r i a d i d not accompany t h e n a + r i u r e s i s i n t h e TX sheep a s was found i n t h e i n t a c t a n i m a l s . In f a c t , U"Q V a c t u a l l y d e c r e a s e d i n t h e s e a n i m a l s f o l l o w i n g t h e d e x t r a n l o a d , and c l e a r a n c e v a l u e s as shown i n T a b l e XXII i n d i c a t e d no s i g n i f i c a n t c hange. C a l c i t o n i n a d m i n i s t r a t i o n t o t h e TX sheep was f o l l o w e d by a c a l c i u r i a and an i n c r e a s e d C'Ca' a l t h o u g h t h e s e changes were not as g r e a t a s t h o s e found i n t h e i n t a c t a n i m a l s . T h i s c o u l d be e x p l a i n e d by t h e g r e a t e r f a l l i n plasma Ca l e v e l s w h i c h o c c u r r e d i n t h e TX sheep t r e a t e d w i t h salmon c a l c i t o n i n . B l y t h e e t aj_. (44) i n s t u d i e s i n dogs f o l l o w i n g volume e x p a n s i o n w i t h s a l i n e o b s e r v e d i n c r e a s e s i n U., V and I L V and Na Ca i n c o n s i s t e n t changes i n U KV, UpV, and V when GFR was reduced by i n f l a t i o n o f a b a l l o o n i n t h e a o r t a . D e s p i t e t h e d e c r e a s e i n GFR, t h e f r a c t i o n o f t h e f i l t e r e d l o a d o f a l l t h e s e i o n s e x c r e t e d i n t h e u r i n e was i n c r e a s e d . In r e l a t i n g Ca and Na, B I y t h e e t a j _ . s u g g e s t e d t h a t d e c r e a s e d r e a b s o r p t i o n o f e i t h e r Ca o r Na m i g h t c o n s e q u e n t l y a f f e c t t h e r e a b s o r p t i o n o f t h e o t h e r . B u t , w h a t e v e r t h e mechanism, an i n c r e a s e d e x c r e t i o n o f Ca i s an " i n t e g r a l p a r t " o f t h e k i d n e y s r e s p o n s e t o volume e x p a n s i o n . A n t o n i o u e t a_l_. (45) found t h a t a p r o g r e s s i v e i n c r e a s e i n t h e c l e a r a n c e s o f Na, C a , and Mg o c c u r r e d i n dogs i n r e s p o n s e t o s a l i n e l o a d i n g . S i m i l a r changes were p r e v i o u s l y o b s e r v e d by WaIser (41) and D u a r t e and Watson ( 4 3 ) . S i n c e t h e s e i o n changes o c c u r i r r e s p e c t i v e o f changes i n GFR, m i n e r a l o c o r t i c o i d o r ADH a c t i v i t y ( 4 4 ) , i t would a p p e a r t h a t t h e y a r e caused by some o t h e r f a c t o r ( s ) a f f e c t i n g t h e i r r e a b s o r p t i o n as t h e y p a s s t h r o u g h t h e nephron. 120 The d i s s o c i a t i o n between Ca and Na e x c r e t i o n i n t h e TX sheep was a t v a r i a n c e w i t h t h e many o b s e r v a t i o n s t h a t d e m o n s t r a t e t h e p a r a l l e l r e l a t i o n s h i p o f t h e s e two i o n s i n t h e i r e x c r e t i o n by t h e k i d n e y . T h i s d i s s o c i a t i o n c o u l d be a t t r i b u t e d t o t h e l a c k o f c a l c i t o n i n s i n c e i t was o n l y f ound i n t h y r o i d e c t o m i z e d sheep. When c a l c i t o n i n was p r e s e n t , e i t h e r e n d o g e n o u s l y o r e x o g e n o u s l y , b o t h e x c r e t i o n and c l e a r a n c e o f Ca were found t o i n c r e a s e f o l l o w i n g t h e d e x t r a n l o a d when Na e x c r e t i o n was i n c r e a s e d . The e x c r e t i o n o f Mg was a l s o reduced i n t h e TX sheep a s compared t o t h e i n t a c t and TX+CT a n i m a l s . Plasma changes c o u l d n ot a c c o u n t f o r t h e e x c r e t o r y changes i n t h i s i o n . M a s s r y e t aj _ . (25) found t h a t PTE d e c r e a s e d t h e p e r c e n t o f f i l t e r e d Mg e x c r e t e d w h i c h s u p p o r t s t h e c o n c e p t t h a t PTH c a n enhance t u b u l a r r e a b s o r p t i o n o f Mg. The changes t h a t were o b s e r v e d i n t h e e x c r e t i o n o f Ca and Mg i n t h e TX sheep c o u l d a I so be m o d i f i e d by t h e i n t e r v e n t i o n o f PTH unopposed by c a l c i t o n i n i n t h e s e a n i m a l s . If t h i s argument i s c o n t i n u e d i n r e l a t i o n t o P e x c r e t i o n , t h e r e s u l t s s h o u l d i n d i c a t e s i m i l a r p a t t e r n s o f P e x c r e t i o n i n t h e t h r e e g r o u p s o f a n i m a l s s i n c e both c a l c i t o n i n and PTH c a n c a u s e a p h o s p h a t u r i a . Both i n c r e a s e s and d e c r e a s e s o r no change a t a 11 i n P e x c r e t i o n were o b s e r v e d ( T a b l e XXI 11). C a l c i t o n i n a p p e a r e d t o have no d i r e c t i n f l u e n c e on K e x c r e t i o n i n t h e s e s t u d i e s . In p r e l i m i n a r y c o n t r o l s t u d i e s i n sheep where no d e x t r a n i n f u s i o n was g i v e n , U^V was found t o d e c r e a s e o v e r a p e r i o d o f 4-5 ho u r s o f u r i n e c o l l e c t i o n i n a way s i m i l a r t o t h a t o b s e r v e d i n t h e d e x t r a n i n f u s i o n s t u d i e s . T h i s i s p r o b a b l y a r e f l e c t i o n o f d i e t a r y i n p u t 121 s i n c e t h e a n i m a l s were s t a r v e d . I t c o u l d a l s o be r e l a t e d t o t h e d i u r n a l p a t t e r n o f a l d o s t e r o n e s e c r e t i o n . O n l y when t r a n s i e n t i n c r e a s e s i n UQrea_|_ V were o b s e r v e d f o l l o w i n g t h e d e x t r a n load were s m a l l i n c r e a s e s i n U^V o b s e r v e d . These changes a r e p r o b a b l y d i r e c t l y r e l a t e d t o t r a n s i e n t i n c r e a s e s i n GFR. E v i d e n c e i s not c o n c l u s i v e t h a t c a l c i t o n i n has any d i r e c t e f f e c t on K m e t a b o l i s m . P o t a s s i u m i s n o t a b o n e - s e e k i n g m i n e r a l and a l l o f t h e K i n bone i s r e a d i l y e x c h a n g e a b l e . Changes i n K e x c r e t i o n , a p a r t from changes i n d i e t a r y i n p u t , a r e r e l a t e d t o the movement o f Na and t h e c o n c e n t r a t i o n o f t h i s i o n i n t h e d i s t a l p o r t i o n o f t h e nephron where K i s s e c r e t e d . I n c r e a s e d c o n c e n t r a t i o n s o f Na i n t h e d i s t a l nephron can i n c r e a s e t h e p a s s i v e movement o f K i n r e s p o n s e t o t h e e s t a b l i s h m e n t o f a more f a v o u r a b l e e l e c t r o - c h e m i c a l g r a d i e n t (75). The u r i n a r y e l e c t r o l y t e changes i n t h e TX sheep i n r e s p o n s e t o volume e x p a n s i o n w i t h d e x t r a n i n f u s i o n showed c e r t a i n s i m i l a r i t i e s t o t h e changes found i n t h e TX r a t s i n r e s p o n s e t o volume e x p a n s i o n w i t h an i . p . s a l i n e l o a d . B o t h a n i m a l s showed an im p a i r m e n t i n t h e c a l c i u r e t i c and m a g n e s u r i c r e s p o n s e t h a t n o r m a l l y f o l l o w s volume e x p a n s i o n . In t h e TX r a t s , a p h o s p h a t u r i a was n o t o b s e r v e d and i n t h e TX sheep t h i s was t h e f i n d i n g i n 4/6 of t h e s t u d i e s p e rformed i n t h e s e a n i m a l s . Whereas t h e r a t s showed a h i g h l y s i g n i f i c a n t i m p a i r m e n t i n t h e n a t r i u r e t i c and d i u r e t i c r e s p o n s e , t h i s was not o b s e r v e d i n t h e sheep. T h i s d i s p a r i t y i n t h e Na and volume r e s p o n s e s c o u l d be a r e s u l t o f t h e d i f f e r e n t methods used f o r volume e x p a n s i o n i n t h e two a n i m a l s , o r t h e n a g a i n i t may be a s p e c i e s d i f f e r e n c e . N e v e r t h e l e s s , i t was a p p a r e n t i n b o t h t h e r a t and t h e sheep s t u d i e s t h a t a l t e r a t i o n s i n t h e normal e x c r e t i o n o f c e r t a i n body e l e c t r o l y t e s f o l l o w i n g volume e x p a n s i o n o c c u r when c a l c i t o n i n i s no t p r e s e n t . T h i s s u g g e s t s t h a t t h i s hormone may p l a y some r o l e i n t h e e l e c t r o l y t e changes t h a t occur f o l l o w i n g e x t r a c e l l u l a r volume expansion. 123 SUMMARY AND CONCLUSIONS I. Exogenous C a I c i+on i n Admi n i s t r a t i o n 1. The a d m i n i s t r a t i o n o f salmon c a l c i t o n i n lowered t h e c o n c e n t r a t i o n o f c e r t a i n e l e c t r o l y t e s i n t h e plasma. These i n c l u d e d , i n a d d i t i o n t o Ca, i n o r g a n i c P and Mg. D e c r e a s e s i n t h e c o n c e n t r a t i o n s o f t h e s e i o n s f o l l o w i n g c a l c i t o n i n t r e a t m e n t were o b s e r v e d i n t h e r a t and t h e sheep. The p i a s m a - l o w e r i n g e f f e c t o f c a l c i t o n i n on C a , P, and Mg i s a t t r i b u t e d t o t h e d e c r e a s e i n bone r e s o r p t i o n which o c c u r s f o l l o w i n g a d m i n i s t r a t i o n o f t h e hormone. However, c o n c u r r e n t changes i n t h e r e n a l e x c r e t i o n o f t h e s e i o n s c o u l d a l s o c o n t r i b u t e t o t h e n e t p i a s m a - l o w e r i n g e f f e c t . I t has a l s o been s u g g e s t e d t h a t c a l c i t o n i n c a n enhance t h e movement o f P i n t o c e l l s (91). 2. C a l c i t o n i n a p p e a r s t o d e c r e a s e t h e r e a b s o r p t i o n o f C a , P, and Mg from t h e k i d n e y t u b u l e so t h a t a c a l c i u r i a , p h o s p h a t u r i a , and magnesuria a r e found f o l l o w i n g hormone t r e a t m e n t . The ma g n i t u d e of t h e u r i n a r y changes may be dependent on t h e plasma changes t h a t o c c u r . Where c a l c i t o n i n i s c a u s i n g d e c r e a s e s i n t h e plasma c o n c e n t r a t i o n s o f Ca, P, and Mg, s e c o n d a r y t o i t s a c t i o n on bone, t h e s u b s e q u e n t d e c r e a s e i n f i l t e r e d load o f t h e s e i o n s c o u l d m o d i f y o r even o b s c u r e t h e e f f e c t s o f a d e c r e a s e i n t u b u l a r r e a b s o r p t i o n a t t h e r e n a l s i t e . 124 3. Accompanying t h e changes i n t h e r e n a l e x c r e t i o n o f C a , P, and Mg f o l l o w i n g c a l c i t o n i n t r e a t m e n t , a d i u r e t i c and n a t r i u r e t i c e f f e c t was a l s o o b s e r v e d . C a l c i t o n i n a l s o c a u s e d an i n c r e a s e i n Na e x c r e t i o n i n r a t s p r e - t r e a t e d w i t h l a r g e doses o f t h e s y n t h e t i c m i n e r a l o c o r t i c o i d , DOCA, whi c h d i d not d i f f e r from t h e i n c r e a s e i n Na e x c r e t i o n f ound i n non-DOCA t r e a t e d r a t s . T h i s s u g g e s t s t h a t t h e n a t r i u r e t i c e f f e c t o f t h i s hormone i s i n d e p e n d e n t o f changes i n m i n e r a l o c o r t i c o i d a c t i v i t y . 4. S i n c e i n c r e a s e s i n t h e e x c r e t i o n of endogenous c r e a t i n i n e were not c o n s i s t e n t l y found t o accompany t h e n a t r i u r e t i c and d i u r e t i c r e s p o n s e s , i t i s u n l i k e l y t h a t i n c r e a s e s i n GFR were p r i m a r i l y r e s p o n s i b l e f o r t h e u r i n a r y e l e c t r o l y t e changes f o u n d . 5. The r e n a l r e s p o n s e s o b s e r v e d f o l l o w i n g c a l c i t o n i n t r e a t m e n t were no t s e c o n d a r y t o s t i m u l a t i o n o f t h e p a r a t h y r o i d g l a n d s . F o l l o w i n g c a l c i t o n i n a d m i n i s t r a t i o n , TPTX r a t s ( w i t h o u t p a r a t h y r o i d t i s s u e ) showed s i m i l a r e l e c t r o l y t e changes t o t h o s e found i n i n t a c t r a t s . S i n c e t h e n a t r i u r e t i c r e s p o n s e i n t h e TPTX r a t s f o l l o w i n g c a l c i t o n i n t r e a t m e n t was c o n s i s t e n t l y g r e a t e r i n r e s p o n s e t o a g i v e n dose o f hormone t h a n t h a t found i n r a t s w i t h i n t a c t p a r a t h y r o i d t i s s u e ( i n t a c t and TX r a t s ) , i t would a p p e a r t h a t t h e p r e s e n c e o f p a r a t h y r o i d t i s s u e can a l t e r t h e m a gnitude of t h e n a t r i u r e t i c r e s p o n s e . 6. The u r i n a r y e l e c t r o l y t e changes t h a t o c c u r r e d i m m e d i a t e l y f o l l o w i n g t h e a d m i n i s t r a t i o n o f c a l c i t o n i n were g r e a t e r i n t h e young r a t a s compared t o t h e o l d e r a n i m a l . 125 I I . C a l c i u m I n f u s i o n S t u d i e s (Endogenous C a I c i t o n i n R e l e a s e ) 1. Plasma Ca l e v e l s i n i n t a c t sheep were r a p i d l y r e s t o r e d t o normal v a l u e s f o l l o w i n g t h e c o m p l e t i o n o f a one-hour Ca i n f u s i o n . Plasma P l e v e l s showed an immediate f a l l f o l l o w i n g t h e s t a r t o f t h e Ca i n f u s i o n . These a f f e c t s on plasma Ca and P a r e a t t r i b u t e d t o an i n c r e a s e d endogenous r e l e a s e o f c a l c i t o n i n w h i c h i s known t o o c c u r f o l l o w i n g a h y p e r c a I c e m i c s t r e s s induced by t h e i n f u s i o n o f Ca s a l t s . T h i s i s s u b s t a n t i a t e d by t h e o b s e r v a t i o n t h a t i n t h e TX s h e e p , an impairment i n t h e c o n t r o l o f plasma Ca was o b s e r v e d , and plasma P l e v e l s were found t o r i s e d u r i n g and f o l l o w i n g t h e Ca i n f u s i o n . When salmon c a l c i t o n i n was a d m i n i s t e r e d t o t h e TX sheep d u r i n g and f o l l o w i n g t h e Ca i n f u s i o n , t h e changes i n plasma Ca and P were s i m i l a r t o t h o s e found i n i n t a c t sheep. 2. An i n c r e a s e d endogenous r e l e a s e o f c a I c i t o n i n may be r e s p o n s i b l e f o r some o f t h e changes i n u r i n a r y e l e c t r o l y t e e x c r e t i o n t h a t o c c u r r e d f o l l o w i n g Ca i n f u s i o n i n i n t a c t sheep. These changes i n c l u d e d an i n c r e a s e i n Na, P, and Mg, i n a d d i t i o n t o an i n c r e a s e i n u r i n e volume. T h i s was i n d i c a t e d by t h e o b s e r v a t i o n t h a t t h e changes i n Na e x c r e t i o n were s i g n i f i c a n t l y reduced i n t h e TX sheep when l a r g e i n c r e a s e s i n Ca e x c r e t i o n were f o u n d . S m a l l e r changes i n t h e e x c r e t i o n o f P and Mg i n t h e TX sheep were a l s o o b s e r v e d , i n a d d i t i o n t o a d e c r e a s e i n u r i n e volume. I t would a p p e a r t h a t a d i s s o c i a t i o n i n t h e r e n a l e x c r e t i o n o f Ca and Na c a n o c c u r i n sheep when c a l c i t o n i n i s n o t p r e s e n t . 126 I I I . C a I c i t o n i n and Volume H o m e o s t a s i s 1. An impairment i n t h e normal e x c r e t i o n o f c e r t a i n body e l e c t r o - l y t e s was o b s e r v e d i n r e s p o n s e t o volume e x p a n s i o n i n r a t s and sheep when t h e c a I c i t o n i n - c o n t a i n i n g "C" c e l l s were removed. In i n t a c t a n i m a l s , volume e x p a n s i o n i s f o l l o w e d by i n c r e a s e s i n u r i n e volume and t h e e x c r e t i o n o f Na, K, C a , P, and Mg. T h y r o i d e c t o m i z e d r a t s showed no s i g n i f i c a n t i n c r e a s e s i n any o f t h e s e p a r a m e t e r s i n t h e f i r s t t h r e e hours f o l l o w i n g volume e x p a n s i o n . In t h e TX sheep, both t h e C^ g and t h e e x c r e t i o n o f Ca were u n a l t e r e d by volume e x p a n s i o n and o n l y s m a l l i n c r e a s e s i n t h e e x c r e t i o n o f Mg were o b s e r v e d as compared t o t h e i n t a c t sheep. 2. The f i n d i n g o f a m a i n t a i n e d r e d u c t i o n i n plasma Ca l e v e l s f o l l o w i n g d e x t r a n i n f u s i o n i n t h e i n t a c t sheep, w h i c h was n o t o b s e r v e d i n t h e TX sheep, s u g g e s t e d f u r t h e r t h a t t h e r e l e a s e o f c a l c i t o n i n may be i n v o l v e d i n t h e r e s p o n s e t o volume e x p a n s i o n . 3. The p r e s e n c e o f f u n c t i o n i n g p a r a t h y r o i d t i s s u e i n a d d i t i o n t o l a c k o f c a l c i t o n i n was i m p l i c a t e d i n t h e i n h i b i t i o n o f normal e l e c t r o l y t e e x c r e t i o n i n TX r a t s f o l l o w i n g volume e x p a n s i o n . Exogenous a d m i n i s t r a t i o n of PTE t o TPTX r a t s p r e v e n t e d t h e n a t r i u r e t i c and d i u r e t i c r e s p o n s e s w h i c h were found i n t h e s e r a t s f o l l o w i n g volume e x p a n s i o n . I t i s p o s t u l a t e d t h a t PTH may enhance t h e r e a b s o r p t i o n o f Na i n a d d i t i o n t o t h a t of Ca and Mg from t h e r e n a l t u b u l e . I t i s f u r t h e r p o s t u l a t e d t h a t f o l l o w i n g volume e x p a n s i o n , endogenous r e l e a s e o f c a l c i t o n i n ( i n a d d i t i o n t o d e c r e a s i n g p r o x i m a l r e a b s o r p t i o n o f Na, Ca, P, and Mg) may a n t a g o n i z e t h e a c t i o n of PTH a t a more d i s t a l 127 s i t e . Thus, an i n c r e a s e d u r i n e volume and e l e c t r o l y t e e x c r e t i o n i s o b s e r v e d . The e v i d e n c e from t h e volume e x p a n s i o n s t u d i e s i n r a t s and sheep i m p l i c a t e d c a l c i t o n i n and PTH i n t h e e l e c t r o l y t e changes t h a t were f o u n d . However, t h i s e v i d e n c e was i n d i r e c t . The d e f i n i t i v e e x p e r i m e n t would be a c o n c u r r e n t e v a l u a t i o n by means of r e l i a b l e a s s a y methods o f t h e c i r c u l a t i n g l e v e l s o f b o t h c a l c i t o n i n and PTH i n r e s p o n s e t o volume e x p a n s i o n o r volume d e p l e t i o n . T h i s a p p r o a c h would c l a r i f y t o what e x t e n t t h e two hormones a r e i n v o l v e d i n volume c o n t r o l . S t o p - f l o w and m i c r o p u n c t u r e s t u d i e s s h o u l d e n a b l e a d e t e r m i n a t i o n o f t h e s i t e o f a c t i o n of c a l c i t o n i n a t t h e t u b u l a r l e v e l . Such t e c h n i q u e s would a l s o c l a r i f y t o what e x t e n t PTH i s a f f e c t i n g t u b u l a r r e a b s o r p t i o n o f Na a t v a r i o u s s i t e s i n t h e n e p h r o n , and m i g h t a l s o e s t a b l i s h whether t h e a c t i o n o f e i t h e r hormone c a n a n t a g o n i z e t h e a c t i o n o f t h e o t h e r a t t h e r e n a l s i t e . 128 BIBLIOGRAPHY 1. B e l a n g e r , L.F. O s t e o l y s i s : An o u t l o o k on i t s mechanism and c a u s a t i o n . j_n The P a r a t h y r o i d Gl and s . E d i t e d by P. J . G a i l l a r d , R. V. Talmage, and A. M. Budy. U n i v e r s i t y o f C h i c a g o P r e s s , C h i c a g o , 1965. pp. 137-143. 2. Sherwood, L. M., Herrman, I . , and B a s s e t t , C. A. P a r a t h y r o i d hormone s e c r e t i o n i n v i t r o : r e g u l a t i o n by c a l c i u m and magnesium i o n s . N a t u r e 225, 1056-1058, 1970. 3. Heaton, F. W. 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