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Malpighian tubules of A. dorsalis mosquito larvae : general characteristics and mechanism of magnesium… Ng, Karen Karpui 1985

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MALPIGHIAN TUBULES OF A. DORSALIS MOSQUITO LARVAE: GENERAL CHARACTERISTICS AND MECHANISM OF MAGNESIUM TRANSPORT by KAREN K. NG B . S c , The U n i v e r s i t y of B r i t i s h Columbia, 1982 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA JULY 1985 © Karen K. Ng, 1985 to In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of /7s ffrOlt) Cw/ The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 i i ABSTRACT M a l p i g h i a n t u b u l e s of A. d o r s a l i s mosquito l a r v a e , s t u d i e d i n v i t r o , a c t i v e l y t r a n s p o r t e d magnesium a t h i g h r a t e s a g a i n s t c o n c e n t r a t i o n g r a d i e n t s as l a r g e as 1 6 - f o l d and t r a n s e p i t h e l i a l p o t e n t i a l g r a d i e n t s of a p p r o x i m a t e l y -l5mV. F l u i d s e c r e t i o n r a t e s , d e t e r m i n e d over 90 minute p e r i o d s , i n the presence and absence of cAMP, i n d i c a t e d t h a t A. d o r s a l i s t u b u l e s were v i a b l e and had s e c r e t i o n r a t e s of the same magnitude as those r e p o r t e d f o r A. t a e n i o r h y n c h u s t u b u l e s . Having c h a r a c t e r i z e d the i n v i t r o p r e p a r a t i o n of M a l p i g h i a n t u b u l e s , the main h y p o t h e s i s t h a t M g 2 + t r a n s p o r t i s d r i v e n p r e d o m i n a t e l y by c o u n t e r t r a n s p o r t w i t h Na + was t e s t e d . T h i s h y p o t h e s i s was not s u p p o r t e d by k i n e t i c , N a - s u b s t i t u t i o n , or i n h i b i t o r s t u d i e s . K i n e t i c and Bumetanide s t u d i e s suggest b a c k f l u x of K d r i v e s J ^ g ' however, t h i s was not c o n s i s t e n t l y found i n o t h e r s t u d i e s . i i i TABLE OF CONTENTS Page LIST OF TABLES i v LIST OF FIGURES v LIST OF ABBREVIATIONS v i i i ACKNOWLEDGEMENTS i x INTRODUCTION 1 MATERIALS AND METHODS 15 RESULTS 23 S u r v i v a l of Aedes d o r s a l i s i n Waters of High Mg 2 + Content 23 V i a b i l i t y and C h a r a c t e r i s t i c s of In V i t r o M a l p i g h i a n Tubule P r e p a r a t i o n 26 I n f l u e n c e of E x t e r n a l M g 2 + L e v e l s on F l u i d and Ion S e c r e t i o n Rates 43 E f f e c t of N a - f r e e S a l i n e s on Mg 2 + S e c r e t i o n 68 C h o l i n e S u b s t i t u t i o n f o r Na 68 K + S u b s t i t u t i o n f o r Na 76 I n h i b i t o r S t u d i e s 83 A m i l o r i d e 83 Bumetanide 91 DISCUSSION 99 C h a r a c t e r i s t i c s and V i a b i l i t y of M a l p i g h i a n T u b u l e s 99 P r o p e r t i e s of Mg T r a n s p o r t 103 REFERENCES 112 APPENDIX A: I n f l u e n c e of cAMP on C o n c e n t r a t i o n s of e l e c t r o l y t e s i n the S e c r e t e d F l u i d 120 APPENDIX B: I o n i c f l u x ( J . ) and I o n i c C o m p o s i t i o n of io n S e c r e t e d F l u i d 122 i v LIST OF TABLES Table Page 1. C o m p o s i t i o n of 50% Seawater 15 2. C o m p o s i t i o n of E x p e r i m e n t a l S o l u t i o n s 17 3. Pe r c e n t a g e s u r v i v a l of l a r v a e hatched and r e a r e d i n 50% seawater a f t e r they were t r a n s f e r r e d a t day 2 t o t e s t s o l u t i o n s of d i f f e r e n t M g 2 + and Na* c o n t e n t .24 4. Pe r c e n t a g e s u r v i v a l of l a r v a e hatched i n d i s t i l l e d water and r e a r e d i n 50% seawater a f t e r they were t r a n s f e r r e d at day 5 t o s o l u t i o n s c o n t a i n i n g d i f f e r e n t amounts of Mg 2 + 24 5. T r a n s e p i t h e l i a l e l e c t r i c a l g r a d i e n t s a c r o s s A. d o r s a l i s M a l p i g h i a n t u b u l e s 27 6. C o n c e n t r a t i o n of e l e c t r o l y t e s i n s e c r e t e d f l u i d 39 7. F l u x of e l e c t r o l y t e s from A. d o r s a l i s M a l p i g h i a n t u b u l e s 40 V LIST OF FIGURES F i g u r e Page 1. S c h e m a t i c i l l u s t r a t i o n of the i n s e c t e x c r e t o r y system 2 2. E l e c t r i c p o t e n t i a l a c r o s s an i n s e c t e p i t h e l i u m 13 3. The e f f e c t of bath cAMP on s e c r e t i o n r a t e s of A. d o r s a l i s M a l p i g h i a n t u b u l e s 28 4. S e c r e t i o n r a t e s of i n d i v i d u a l M a l p i g h i a n t u b u l e s of A. d o r s a l i s r e a r e d i n 126mM MgSO„ seawater and s t i m u l a t e d w i t h 2mM cAMP 30 5. The e f f e c t of cAMP (1mM t o the b a t h i n g medium) on c o n c e n t r a t i o n s of e l e c t r o l y t e s s e c r e t e d by A. d o r s a l i s M a l p i g h i a n t u b u l e s 33 6. The e f f e c t of cAMP (imM t o the b a t h i n g medium) on s e c r e t i o n r a t e s of e l e c t r o l y t e s by A. d o r s a l i s M a l p i g h i a n t u b u l e s 36 7. R a t e s of f l u i d s e c r e t i o n from A. d o r s a l i s M a l p i g h i a n t u b u l e s v e r s u s c o n c e n t r a t i o n of Mg i n e x t e r n a l s a l i n e s o l u t i o n 44 8. C o n c e n t r a t i o n s of Mg s e c r e t e d v e r s u s e x t e r n a l c o n c e n t r a t i o n s of b a t h Mg i n A. d o r s a l i s M a l p i g h i a n t u b u l e s . L a r v a e r e a r e d i n 126mM MgSO a medium 46 9. R a t i o of S e c r e t e d / E x t e r n a l c o n c e n t r a t i o n s of Mg v e r s u s e x t e r n a l c o n c e n t r a t i o n s of Mg 48 10. F l u x of Mg from A. d o r s a l i s M a l p i g h i a n t u b u l e s w i t h i n c r e a s i n g e x t e r n a l Mg c o n c e n t r a t i o n . 52 1.1. A d o u b l e - r e c i p r o c a l ( Lineweaver-Burk) p l o t of j*Mg v e r s u s e x t e r n a l c o n c e n t r a t i o n of Mg (n = 52) 54 12.1. C o n c e n t r a t i o n s of Mg and K i n the s e c r e t e d f l u i d v e r s u s e x t e r n a l c o n c e n t r a t i o n of Mg. ... 56 v i 12.2. S e c r e t i o n r a t e s of Mg and K v e r s u s e x t e r n a l c o n c e n t r a t i o n of Mg 58 13.1. C o n c e n t r a t i o n s of Mg and Na i n the s e c r e t e d f l u i d v e r s u s e x t e r n a l c o n c e n t r a t i o n of Mg. ... 60 13.2. S e c r e t i o n r a t e s of Mg and Na v e r s u s e x t e r n a l c o n c e n t r a t i o n of Mg 62 14.1. C o n c e n t r a t i o n s of Mg and CI v e r s u s e x t e r n a l c o n c e n t r a t i o n of Mg 64 14.2. S e c r e t i o n r a t e s of Mg and CI v e r s u s e x t e r n a l c o n c e n t r a t i o n of Mg 66 15. Rates of f l u i d s e c r e t i o n by A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n N a - s u b s t i t u t e d w i t h c h o l i n e s a l i n e s o l u t i o n 70 16. C o n c e n t r a t i o n of i o n s s e c r e t e d by A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n N a - s u b s t i t u t e d w i t h c h o l i n e s o l u t i o n 72 17. S e c r e t i o n r a t e s of i o n s ( J . ) of A. d o r s a l i s I on M a l p i g h i a n t u b u l e s bathed i n N a - s u b s t i t u t e d w i t h c h o l i n e s a l i n e s o l u t i o n 74 18. Rates of f l u i d s e c r e t i o n of A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n N a - s u b s t i t u t e d w i t h K s a l i n e s o l u t i o n 77 19. C o n c e n t r a t i o n of i o n s s e c r e t e d by A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n c o n t r o l and " N a - f r e e " s a l i n e 79 20. S e c r e t i o n r a t e s of i o n s ( J . ) of A. d o r s a l i s ion M a l p i g h i a n t u b u l e s bathed i n c o n t r o l and " N a - f r e e " s a l i n e 81 21. F l u i d s e c r e t i o n r a t e s of A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n s a l i n e w i t h and w i t h o u t 1mM a m i l o r i d e ( i n the presence of 1mM CAMP) 85 22. C o n c e n t r a t i o n of i o n s s e c r e t e d by A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n s a l i n e w i t h and w i t h o u t 1mM a m i l o r i d e ( i n the p r e s ence of 1mM cAMP) 87 v i i 23. S e c r e t i o n r a t e s of i o n s ( J . ) of A. d o r s a l i s i o n :  M a l p i g h i a n t u b u l e s bathed i n s a l i n e w i t h and w i t h o u t 1mM a m i l o r i d e ( i n the presence of 1mM cAMP) 89 24. Rates of f l u i d s e c r e t i o n of A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n s a l i n e w i t h and w i t h o u t 5X10~ 5M bumetanide ( i n the presence of 1mM cAMP) 93 25. C o n c e n t r a t i o n of i o n s s e c r e t e d by A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n s a l i n e w i t h and w i t h o u t 5X10~ 5M bumetanide ( i n the presence of 1mM cAMP) 95 26. S e c r e t i o n r a t e s of i o n s ( J . ) of A. d o r s a l i s I on M a l p i g h i a n t u b u l e s bathed i n s a l i n e w i t h and w i t h o u t 5X10" 5M bumetanide ( i n the presence of 1mM cAMP) 97 v i i i LIST OF ABBREVIATIONS mM m i l l i m o l e s per l i t e r uM micromoles per l i t e r M moles per l i t e r ml m i l l i l i t e r n l n a n o l i t e r mm m i l l i m e t e r mV m i l l i v o l t h hour mOsm m i l l i o s m o l e pmol/min picomole per minute p l / m i n p i c o l i t e r per minute mEquiv/1 mi 1 1 i e q u i v a l e n t per l i t e r U/P r a t i o of u r i n e - t o - p l a s m a Sec/Ext r a t i o of s e c r e t e d f l u i d t o e x t e r n a l f l u i d J . f l u x of i o n io n J v f l u x of f l u i d V t t r a n s e p i t h e l i a l p o t e n t i a l Km M i c h a e l i s c o n s t a n t Vmax maximum v e l o c i t y c y c l i c 3 5 ' - a d e n o s i n e monophosphate cAMP PAH para-amino h i p p u r a t e cTAL c o r t i c a l t h i c k a s c e n d i n g l i m b HEPES N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 e t h a n e s u l f o n i c a c i d (a weak a c i d b u f f e r i n g compound) i x ACKNOWLEDGEMENTS I would l i k e t o thank my r e s e a r c h s u p e r v i s o r , Dr. J.E. P h i l l i p s f o r h i s a s s i s t a n c e , g u i d a n c e , and p a t i e n c e w i t h me throughout the cour s e of my work. I am i n d e b t e d t o Dr. Gary Quamme f o r u s e f u l d i s c u s s i o n s , f o r u s e f u l comments c o n c e r n i n g the f i n a l form of t h i s m a n u s c r i p t and f o r e l e c t r o n m i c r o p r o b e a n a l y s i s . I would l i k e t o thank C h r i s t i n e M c C a f f r e y and Maggie P i r a n i a n f o r e l e c t r o n m i c r o p r o b e a n a l y s e s . I am g r a t e f u l t o Drs. Dave R a n d a l l and Reuben Kaufmann f o r t h e i r u s e f u l comments r e g a r d i n g the form of t h i s t h e s i s . I would l i k e t o thank Joan M a r t i n , Brent and Joyce Thomson, and R i c h L e c h l e i t n e r f o r t h e i r a s s i s t a n c e and f o r c r e a t i n g a good atmosphere t o work i n . I am i n d e b t e d t o Dr. Laur a Kramer f o r p r o v i d i n g A. d o r s a l i s eggs i n time of need! I would l i k e t o thank Junko Kazumi, Jeyne Eng, Romilda Ang, L o u i s Tsang, Kathy D a l i n g h a u s , Ken and Kim f o r t h e i r support and u s e f u l d i s c u s s i o n s o u t s i d e of the l a b thro u g h o u t the cours e of t h i s work. I am d e e p l y g r a t e f u l t o M i c h a e l and Dorothy R a c i c h f o r t h e i r encouragement and t h e i r s u p port a t a l l t i m e s . I would l i k e t o thank Bruce M c G i l l i v r a y f o r h i s p a t i e n c e and p u n c t u a l i t y i n p r e p a r i n g t h i s t h e s i s . 1 INTRODUCTION Mo s q u i t o l a r v a e , Aedes d o r s a l i s a r e commonly found i n s a l i n e ponds of d i v e r s e i o n i c c o m p o s i t i o n i n the Great b a s i n of western N o r t h A m e r i c a . F o u r t h - i n s t a r stage of Aedes d o r s a l i s l a r v a e have a s u r v i v a l r a t e near 100% i n 200mM magnesium s u l p h a t e medium ( B r a d l e y and Sheplay, 1982). These l a r v a e d r i n k the e x t e r n a l s a l i n e media a t a r a t e e q u a l t o 130% of t h e i r body weight per day (Str a n g e et a l , 1982). I t f o l l o w s t h a t i n o r d e r t o s u r v i v e i n such M g - r i c h w a t e r s , t h e s e mosquito l a r v a e must e l i m i n a t e e x c e s s Mg v i a the e x c r e t o r y system, i . e . M a l p i g h i a n t u b u l e s or rectum, a t h i g h r a t e s . I t i s u n l i k e l y t h a t hemolymph Mg 2 + c o n c e n t r a t i o n s i n Aedes d o r s a l i s v a r y s u b s t a n t i a l l y under d i f f e r e n t e x t e r n a l c o n d i t i o n s , because hemolymph c o n c e n t r a t i o n s of t h i s c a t i o n a r e m a i n t a i n e d a t low l e v e l s (l.5-4mM Mg 2 +) i n a c l o s e l y r e l a t e d s p e c i e s , Aedes c a m p e s t r i s , when exposed t o a wide range of e x t e r n a l magnesium l e v e l s (4-95mM);(Kiceniuk and P h i l l i p s , 1975). Strange(1982) measured magnesium l e v e l s of 3.8mM i n hemolymph of Aedes d o r s a l i s r e a r e d i n medium c o n t a i n i n g 0.5mM magnesium. As e x p l a i n e d l a t e r , the e x c r e t o r y system of A. d o r s a l i s o f f e r s an unusual o p p o r t u n i t y t o i n v e s t i g a t e the n a t u r e of magnesium t r a n s p o r t a c r o s s i n s e c t e p i t h e l i a and inde e d e p i t h e l i a i n g e n e r a l . I n s e c t e x c r e t o r y systems work on the same b a s i c p r i n c i p l e s as o t h e r r e n a l organs (see F i g . 1 ). M a l p i g h i a n F i g u r e 1 : Schematic i l l u s t r a t i o n of the i n s e c t e x c r e t o r y system. 2 MIDGUT MALPIGHIAN T U B U L E S ANTERIOR J HINDGUT V "~z~rS *> i hypo-or isosmotic RECTUM Selective secretion of small solutes /J£^3 Diffusion of Hr small solutes KCI.Na* Fluid secretion Some KCI and H z O reabsorption ANUS. Bulk of water, ion and metabolite strongly hyperosmotic or hyposmotic excreta 3 t u b u l e s of i n s e c t s a r e commonly found a t t a c h e d t o the a l i m e n t a r y c a n a l d e l i n e a t i n g the s e p a r a t i o n of the midgut and the h i n d g u t . A. d o r s a l i s l a r v a e have 5 M a l p i g h i a n t u b u l e s which l i e f r e e i n t h e hemocoel. They are a p p r o x i m a t e l y 2.8mm i n l e n g t h and 0.15mm i n d i a m e t e r , and c o n s i s t of l a r g e p r i n c i p a l c e l l s ( p r o b a b l y the p r i m a r y s i t e of s a l t and water s e c r e t i o n ) and s m a l l e r s t e l l a t e c e l l s of unknown f u n c t i o n which make up l e s s than 10% of the t o t a l t i s s u e mass ( B r a d l e y e t a l , 1983). I t has been p o s t u l a t e d t h a t p r i n c i p a l c e l l s w i t h t h e i r abundant m i t o c h o n d r i a might s e c r e t e a N a - r i c h f l u i d w h i l e s t e l l a t e c e l l s r e a b s o r b Na + and s e c r e t e K + ( B e r r i d g e et a l . , 1969) but t h e r e i s no d i r e c t e v i d e n c e f o r t h i s s u g g e s t i o n . H i s t o l o g i c a l l y , the M a l p i g h i a n t u b u l e s c o n s i s t of a si m p l e t u b u l a r e p i t h e l i u m . The hemolymph s i d e of the e p i t h e l i u m i s covered by a b a s a l l a m e l l a whose p r o b a b l e f u n c t i o n i s t o f i l t e r out p a r t i c l e s and c e l l s so as t o prev e n t c l o g g i n g of i n f o l d s of the b a s a l c e l l u l a r membrane. The h i g h degree of f o l d i n g of the b a s a l plasma membrane i n c r e a s e s s u r f a c e a r e a and i n c r e a s e s the number of t r a n s p o r t p r o t e i n s which can be i n s e r t e d t o maximize s o l u t e - w a t e r c o u p l i n g d u r i n g f l u i d t r a n s p o r t ( B e r r i d g e and Oschman, 1969). M i t o c h o n d r i a a r e found i n c l o s e p r o x i m i t y t o the b a s a l membrane i n f o l d s ( B r a d l e y , 1983). M i t o c h o n d r i a a r e a l s o found near the a p i c a l s u r f a c e which i s e x t e n s i v e l y c o v e r e d by m i c r o v i l l i . However, m i c r o v i l l i of the i n s e c t 4 a r e d i f f e r e n t from m i c r o v i l l i of o t h e r a n i m a l s i n t h a t they commonly c o n t a i n smooth endoplasmic r e t i c u l u m , rough endoplasmic r e t i c u l u m , and m i t o c h o n d r i a . S e p t a t e j u n c t i o n s , the major j u n c t i o n s between M a l p i g h i a n t u b u l e c e l l s , a r e found on the l a t e r a l membranes near the a p i c a l s u r f a c e (Lane and Skaer, 1980). Below the s e p t a t e j u n c t i o n s a r e gap j u n c t i o n s , a common s t r u c t u r e i n i n s e c t M a l p i g h i a n t u b u l e s . I n t e r c e l l u l a r c l e f t s occupy v e r y l i t t l e s u r f a c e a r e a of the M a l p i g h i a n t u b u l e because i n d i v i d u a l c e l l s of the t u b u l e a r e l a r g e and o f t e n extend h a l f w a y around each t u b u l e . I n t r a c e l l u l a r s t o r a g e g r a n u l e s , p o s s i b l y f o r s t o r a g e of e x c r e t o r y p r o d u c t s , a re p r e s e n t i n the p r i m a r y c e l l s of A. t a e n i o r h y n c h u s . These i n t r a c e l l u l a r c r y s t a l s c o n t a i n c a l c i u m , magnesium, and phosphate ( p r o b a b l y PO u 2~; Ry e r s e , 1979). In t h i s t h e s i s , the mechanism of magnesium t r a n s p o r t a c r o s s M a l p i g h i a n t u b u l e s of A. d o r s a l i s was i n v e s t i g a t e d . In a d d i t i o n , g e n e r a l c h a r a c t e r i s t i c s of i o n i c and f l u i d s e c r e t i o n i n the M a l p i g h i a n t u b u l e s of t h i s s p e c i e s were examined. B e f o r e c o n s i d e r i n g Mg t r a n s p o r t i n t h i s e p i t h e l i a , a g e n e r a l review of Mg t r a n s f e r and c o n t r o l i n a n i m a l s i s i n o r d e r . C o n s i d e r i n g the i m p o r t a n t r o l e of Mg 2 + i n many p h y s i o l o g i c a l p r o c e s s e s , i n p a r t i c u l a r neuromuscular e v e n t s , 5 rema r k a b l y l i t t l e i s known about the mechanisms of magnesium t r a n s p o r t a c r o s s b i o l o g i c a l membranes, e s p e c i a l l y e p i t h e l i a i n v o l v e d i n r e g u l a t i n g b l o o d magnesium l e v e l s i n most a n i m a l s . Some s p e c i f i c p r o c e s s e s t h a t a re s t r o n g l y i n f l u e n c e d by magnesium l e v e l s i n c l u d e : n u c l e i c a c i d and p r o t e i n s y n t h e s i s ( L i n d a h l e t a l , 1966), r e a c t i o n s w i t h p y r u v a t e k i n a s e ( S l o a n et a l , 1976; M i l d v a n et a l , 1976), c r e a t i n e k i n a s e (Cohn, 1963), e n o l a s e (Winstead e t a l , 1965), phosphoglucomutase (Ray e t a l , 1973), a l k a l i n e phosphatase ( V a l l e e e t a l , 1970), a d e n y l a t e c y c l a s e s ( A l v a r e z e t a l , 1977), a d e n o s i n e t r i p h o s p h a t a s e s ( R a c k e r , 1977), b r a i n h e x o k i n a s e ( B a c h e l a r d , 1970), p y r u v a t e dehydrogenase (Hucho, 1974), c r e a t i n e phosphokinase (Saks e t a l , 1975), a s p a r t a t e t r a n s c a r b a m o y l a s e ( C h r i s t o p h e r s o n e t a l , 1977), second messenger events d u r i n g i n s u l i n s t i m u l a t i o n ( L o s t r o h e t a l , 1974), and muscle c o n t r a c t i o n (Stephenson et a l , 1977; S o l a r o e t a l , 1976). R e g u l a t i o n and t r a n s p o r t of magnesium i s best u n d e r s t o o d i n s i n g l e b a c t e r i a and i n v e r t e b r a t e c e l l s , i . e . f o r t r a n s f e r a c r o s s s i n g l e plasma membranes r a t h e r than e p i t h e l i a . Lusk and Kennedy (1969) showed t h a t a t r a n s p o r t system s p e c i f i c f o r magnesium e x i s t s i n E. c o l i . T h i s t r a n s p o r t system i s i n h i b i t e d by a z i d e , d i n i t r o p h e n o l , or p o t a s s i u m c y a n i d e i n d i c a t i n g an energy r e q u i r i n g p r o c e s s . However, i t i s u n c l e a r whether i t i n v o l v e s a p r i m a r y or secondary t r a n s p o r t p r o c e s s . When temperature i s lowered or 6 when c e l l s a r e t r e a t e d w i t h a s u l f h y d r y l p o i s o n such as N - e t h y l m a l e i m i d e , magnesium uptake d e c r e a s e s . C o b a l t a l s o appears t o be t r a n s p o r t e d i n t o E. c o l i by the same t r a n s p o r t system; however, t h i s system has a h i g h e r a f f i n i t y f o r magnesium. A Na-Mg exchanger found i n some i n v e r t e b r a t e c e l l s w i l l be reviewed i n d e t a i l l a t e r . E x p e r i m e n t s on mammalian systems have p r o v i d e d l i t t l e i n f o r m a t i o n on magnesium t r a n s p o r t a c r o s s e p i t h e l i a . However, f a c t o r s t h a t might i n f l u e n c e magnesium e x c r e t i o n i n the k i d n e y have been i n v e s t i g a t e d (Quamme e t a l , 1982). These f a c t o r s i n c l u d e e f f e c t s of volume e x p a n s i o n , d i u r e t i c s , h y p e r c a l c e m i a , and a number of c o n d i t i o n s such as a c i d o s i s , phosphate d e p l e t i o n , and c h r o n i c a l c o h o l i n g e s t i o n . The p o s s i b i l i t y of hormonal c o n t r o l of r e n a l magnesium t r a n s f e r has a l s o been i n v e s t i g a t e d (Quamme, 1982). E x p e r i m e n t s performed on the p r o x i m a l t u b u l e suggest p o s s i b l e a c t i v e t r a n s p o r t of Mg 2 + from the lumen which i s dependent on sodium and water t r a n s p o r t (Wong et a l , 1981). The n a t u r e of magnesium f l u x i n the deep d e s c e n d i n g l i m b i s more c o n t r o v e r s i a l . T h i s i s p r o b a b l y due t o s p e c i e s d i f f e r e n c e s ( D eRouffignac e t a l , 1973); d i f f u s i o n a l f l u x of magnesium from i n t e r s t i t i u m i n t o the lumen may account f o r a l l o b s e r v a t i o n s . The a s c e n d i n g l i m b of the l o o p of Henle appears t o be the major s i t e f o r magnesium r e a b s o r p t i o n ; 50-60% of f i l t e r e d magnesium i s r e a b s o r b e d between the 7 p r o x i m a l and e a r l y d i s t a l t u b u l e ( M o r e l et a l , 1973, 1969). There appears t o be some i n t e r a c t i o n between c a l c i u m and magnesium t r a n s p o r t i n t h i s segment (Quamme, 1981; Mussny et a l , 1973). Furosemide i n h i b i t s t r a n s p o r t of sodium, c a l c i u m , and magnesium i n t h i s p o r t i o n of the t u b u l e and t h e r e i s g r e a t e r i n h i b i t i o n of a b s o l u t e magnesium and c a l c i u m t r a n s p o r t as compared t o sodium t r a n s p o r t (Quamme,1978). In a d d i t i o n , c a l c i t o n i n , i n the presence of low plasma l e v e l s of c a l c i u m , and p a r a t h y r o i d hormone both i n c r e a s e magnesium r e a b s o r p t i o n i n the l o o p of Henle (Quamme, 1980). The d i s t a l c o n v o l u t e d t u b u l e and c o l l e c t i n g d u c t both r e a b s o r b a p p r o x i m a t e l y 2-5% and 1-3% r e s p e c t i v e l y of f i l t e r e d magnesium (Quamme, 1980; B r u n e t t e , 1978). S i n c e i t i s d i f f i c u l t t o e l u c i d a t e mechanisms of magnesium t r a n s p o r t a c r o s s r e n a l t u b u l e s , i t may be more r e w a r d i n g t o study e p i t h e l i a known t o t r a n s p o r t magnesium a t h i g h r a t e s and a g a i n s t l a r g e e l e c t r o c h e m i c a l d i f f e r e n c e s . The M a l p i g h i a n t u b u l e s and r e c t a of mosquito l a r v a e which i n h a b i t magnesium and sodium s u l p h a t e l a k e s appear i d e a l f o r t h i s purpose. These e p i t h e l i a have c e l l s of u n u s u a l l y l a r g e s i z e and s i m p l e s t r u c t u r e which can p r o b a b l y be im p a l e d w i t h m i c r o e l e c t r o d e s . S t u d i e s of M a l p i g h i a n t u b u l e s i n n i n e s p e c i e s of i n s e c t s i n d i c a t e lumen p o s i t i v e p o t e n t i a l s w i t h r e s p e c t t o the hemocoel; the range of p o t e n t i a l r e a d i n g s i s between 10 and 30 mV. The c e l l i n t e r i o r of the 4 s p e c i e s s t u d i e d i s e l e c t r i c a l l y n e g a t i v e t o both lumen and hemocoel 8 ( M a d d r e l l , 1 9 7 1 ; Ramsey, 1953; see F i g u r e 2 ) . C o n c e n t r a t i o n s of K + i n the s e c r e t i o n a r e n o r m a l l y 3-30 tim e s h i g h e r than those i n the hemolymph and do not change much when b a t h i n g Na +/K + i s v a r i e d i n v i t r o over a wide range ( M a d d r e l l , 1971). C l e a r l y , K + t r a n s p o r t a t the a p i c a l membrane i s the predominate i o n t r a n s p o r t p r o c e s s i n most i n s e c t t u b u l e s . Of p a r t i c u l a r i n t e r e s t , Aedes l a r v a e t u b u l e s c o n t i n u e t o s e c r e t e f l u i d c o n t a i n i n g h i g h c o n c e n t r a t i o n s of p o t a s s i u m f o r some time when l a r v a e a r e bathed i n a pure NaCl s o l u t i o n (Ramsay, 1953). In a l l ca s e s t o d a t e , M a l p i g h i a n t u b u l e f l u i d i s e i t h e r i s o s m o t i c 'or s l i g h t l y h y p e r o s m o t i c ( P h i l l i p s , 1981). U n l i k e p l a n t f e e d e r s , the. b l o o d - s u c k i n g s p e c i e s Rhodnius p r o l i x u s must e l i m i n a t e e x c e s s NaCl r a t h e r than K + a f t e r f e e d i n g , and f l u i d s e c r e t i o n i s d r i v e n by bo t h Na + and K + t r a n s p o r t w i t h r e c o v e r y of K + i n a lower t u b u l e segment ( M a d d r e l l , 1 9 6 9 , 1 9 7 7 ) . E x p e r i m e n t s on M a l p i g h i a n t u b u l e s of t h i s s p e c i e s suggest t r a n s p o r t of Na + and K + by a common pump s i t u a t e d on the a p i c a l plasma membrane ( M a d d r e l l , 1977,1978). E n t r y of K + (from the e x t e r n a l s o l u t i o n t o c e l l i n t e r i o r ) may p o s s i b l y i n v o l v e exchange f o r Na + by a Na +/K +-ATPase which i s o u a b a i n - i n s e n s i t i v e (Anstee et a l , 1979; B e r r i d g e , 1 9 6 8 ) . T h e r e f o r e , i t i s p o s s i b l e t h a t some Na + may be r e c y c l e d from the t u b u l e lumen t o the c e l l i n t e r i o r because both the e l e c t r i c a l and c o n c e n t r a t i o n g r a d i e n t s a c r o s s both the a p i c a l and b a s a l membrane f a v o u r 9 p a s s i v e e n t r y of Na + i n t o the c e l l . The c o n c e n t r a t i o n of Na + i n the c e l l i s p r o b a b l y m a i n t a i n e d a t low l e v e l s by a Na +/K +-ATPase on the b a s o l a t e r a l membrane. There i s e v i d e n c e f o r c o n t r o l of f l u i d s e c r e t i o n i n M a l p i g h i a n t u b u l e s i n 20 s p e c i e s of i n s e c t s ( M a d d r e l l , 1 9 8 1 ) by a f a m i l y of d i u r e t i c hormones ( r e v i e w e d by P h i l l i p s , 1981). D i u r e t i c hormone i s thought t o i n c r e a s e t u b u l a r s e c r e t i o n by r a i s i n g i n t r a c e l l u l a r l e v e l s of cAMP. In support of t h i s view, e x t e r n a l l y a p p l i e d cAMP mimics the a c t i o n of d i u r e t i c hormone i n 8 s p e c i e s . F u r t h e r m o r e , d i u r e t i c hormone s t i m u l a t i o n of t u b u l e s i n Rhodnius i n c r e a s e s t i s s u e l e v e l s of cAMP (Anstee and Bowler, 1979) and causes a t r a n s i e n t r e v e r s a l of the t r a n s m u r a l e l e c t r o p o t e n t i a l d i f f e r e n c e ( M a d d r e l l , 1980). As a r e s u l t of a c c e l e r a t e d s a l t and hence f l u i d s e c r e t i o n , o t h e r s u b s t a n c e s i n the s e c r e t e d f l u i d a re d i l u t e d i n the lumen of the t u b u l e . T h i s i n c r e a s e s the f a v o u r a b l e c o n c e n t r a t i o n g r a d i e n t f o r s u b s t a n c e s t h a t e n t e r p a s s i v e l y and reduces o p p o s i n g c o n c e n t r a t i o n d i f f e r e n c e s c r e a t e d by a c t i v e t r a n s p o r t ( t h u s r e d u c i n g back ' d i f f u s i o n of t r a n s p o r t e d s u b s t a n c e s from the lumen). In both c a s e s , s u b s t a n c e s are t h e r e b y removed from the hemolymph a t a h i g h e r r a t e a f t e r s t i m u l a t i o n of KC1 t r a n s p o r t by cAMP. P h i l l i p s et a l (1974; 1975) demonstrated t h a t a c t i v e t r a n s p o r t of magnesium can occur a g a i n s t b o t h l a r g e e l e c t r i c a l and c o n c e n t r a t i o n g r a d i e n t s i n M a l p i g h i a n t u b u l e s 10 of A. c a m p e s t r i s . Larvae c a p a b l e of s u r v i v i n g i n medium of lOOmM magnesium a c t i v e l y t r a n s p o r t e d magnesium a g a i n s t a t e n f o l d c o n c e n t r a t i o n g r a d i e n t and an e l e c t r i c a l p o t e n t i a l d i f f e r e n c e of +l5mV. Magnesium t r a n s p o r t approaches s a t u r a t i o n when the b a t h i n g medium c o n t a i n s 5-6mM Mg 2 + and h a l f maximal r a t e i s reached a t 2.5mM. The r a t e of t r a n s p o r t of magnesium i s not a f f e c t e d by changes i n the r a t e of f l u i d s e c r e t i o n which suggests t h a t b a c k f l u x of t h i s c a t i o n i s n e g l i g i b l e . When M a l p i g h i a n t u b u l e s were exposed t o a r t i f i c i a l hemolymph c o n t a i n i n g 2mM Mg 2 + and e i t h e r 4mM or 76mM K +, t u b u l e s s e c r e t e d Mg 2 + a t average r a t e s of 6.72 pmol/min and 6.95 pmol/min r e s p e c t i v e l y . The t u b u l e s i n K + - r i c h medium s e c r e t e d f l u i d c o n t a i n i n g 8.2mM Mg 2 + a t an average r a t e of 852 p l / m i n , whereas t u b u l e s i n K + - p o o r medium s e c r e t e d f l u i d c o n t a i n i n g 25.5mM Mg 2 + a t an average r a t e of 260 p l / m i n . T h e r e f o r e , a t h i g h f l u i d s e c r e t i o n r a t e s , the M g 2 + c o n c e n t r a t i o n i n the t u b u l a r f l u i d i s lower ( P h i l l i p s and M a d d r e l l , 1974). The major h y p o t h e s i s t e s t e d i n t h i s t h e s i s r e s t s on ev i d e n c e for. a Na +-Mg 2 + exchange mechanism i n g i a n t s q u i d axon (Baker e t a l , 1972) and s i n g l e c r u s t a c e a n muscle f i b e r s ( A s h l e y e t a l , 1972), which e x t r u d e s M g 2 + a c t i v e l y from t h e s e c e l l s . Cyanide and low temperatures both reduce t h i s magnesium e f f l u x , i n d i c a t i n g the presence of a m e t a b o l i c a l l y dependent pump. Ouabain does not i n h i b i t M g 2 + t r a n s p o r t i n e i t h e r s q u i d axon or c r u s t a c e a n f i b e r s , s u g g e s t i n g t h a t 11 Na +/K +-ATPase i s not d i r e c t l y and i m m e d i a t e l y i n v o l v e d . P a l a t y (1974) has demonstrated t h a t an o u t w a r d l y d i r e c t e d Mg 2 + f l u x i n r a t v a s c u l a r smooth muscle u t i l i z e s the energy a v a i l a b l e i n the spontaneous i n f l u x of sodium. In a d d i t i o n , many o t h e r c e l l u l a r t r a n s p o r t mechanisms a r e known t o i n v o l v e c o t r a n s p o r t w i t h , or exchange f o r , e x t e r n a l sodium. In a l l t h e s e cases low i n t r a c e l l u l a r l e v e l s of sodium a r e m a i n t a i n e d by Na +/K +-ATPase i n the plasma membrane ( H a r r i s o n e t a l , 1980). The inward d i r e c t e d b a c k f l u x of Na + i s used t o d r i v e secondary t r a n s p o r t of many s u b s t a n c e s : eg. a b s o r p t i o n of D-hexose (Busse et a l , l 9 7 2 ) (Crane, 1977), d i and t r i c a r b o x y l i c a c i d s (Kippen et a l , 1978), b i l e a c i d s ( W i l s o n e t a l , 1980), (Lucke e t a l 1978), L - a s c o r b a t e ( S i l l i p r a n d i e t a l , 1979), L - l a c t a t e (Barac e t a l , 1980), (Hildmann e t a l , 1980), d i p e p t i d e s ( S i g r i s t - N e l s o n , 1975), L-aminoacids ( B u r c k h a r d t e t a l , 1980),(Fromter, 1979), ( H o p f e r , 1 9 7 7 a ) , i n o r g a n i c s u l f a t e (Lucke et a l , 1979), i n o r g a n i c phosphate (Murer e t a l , 1980), (Berner e t a l , 1976), c h l o r i d e ( E v e l o f f e t a l , 1980), c a l c i u m (Gmaj e t a l , 1979), (Hildmann e t a l , 1980), s e c r e t i o n of c h l o r i d e ( E v e l o f f e t a l , 1978), and H + s e c r e t i o n (Murer e t a l , 1977). Based on t h i s w i d e s p r e a d o c c u r r e n c e of sodium-coupled t r a n s p o r t p r o c e s s e s , i n c l u d i n g t h a t of magnesium t r a n s p o r t i n some s i n g l e c e l l s , i t seemed p o s s i b l e t h a t magnesium s e c r e t i o n by mosquito M a l p i g h i a n t u b u l e s i s a l s o d r i v e n by a sodium exchange p r o c e s s l o c a t e d a t the 1 2 mucosal b o r d e r . I t i s u n l i k e l y t h a t magnesium c o u l d c r o s s the a p i c a l membrane by d i f f u s i o n s i n c e the c e l l i n t e r i o r would have t o c o n t a i n g r e a t e r than 200mM Mg 2 +- t o e x p l a i n s e c r e t i o n of t h i s c a t i o n (20mM i n lumen) a g a i n s t an op p o s i n g a p i c a l p o t e n t i a l , as low as 29mV (see F i g . 2 ) . I f M a l p i g h i a n t u b u l e s of A. d o r s a l i s l a r v a e have t r a n s e p i t h e l i a l p o t e n t i a l p r o f i l e s s i m i l a r t o those of o t h e r i n s e c t s d e s c r i b e d above, then d o w n h i l l movement of Na + a c r o s s the a p i c a l membrane i n t o the c e l l c o u l d d r i v e a c t i v e e x t r u s i o n of Mg 2 + from c e l l t o lumen. However, g i v e n t h a t Na + l e v e l s a r e o f t e n low i n the s e c r e t i o n s of i n s e c t t u b u l e s , i n c l u d i n g A. d o r s a l i s ( P h i l l i p s , 1981), a l t e r n a t e mechanisms may have e v o l v e d i n these e p i t h e l i a , e.g. exchange of c e l l u l a r magnesium f o r l u m i n a l K +, H +, or C a 2 + . A l t e r n a t e l y , magnesium may be c o - t r a n s p o r t e d w i t h s u l p h a t e or b i c a r b o n a t e . P h i l l i p s and M a d d r e l l found t h a t t u b u l e s of A. t a e n i o r h y n c h u s (1978) and A. c a m p e s t r i s ( M a d d r e l l , S . H . P . et a l , 1975) a c t i v e l y s e c r e t e s u l p h a t e but they d i d not c o n s i d e r the p o s s i b i l i t y of c o - t r a n s p o r t w i t h magnesium. I n d u c t i o n of s u l p h a t e t r a n s p o r t was shown by M a d d r e l l and P h i l l i p s (1978) i n M a l p i g h i a n t u b u l e s of A. t a e n i o r h y n c h u s . They found t h a t f o u r t h stage l a r v a e were a b l e t o r e g u l a t e hemolymph s u l p h a t e l e v e l s a t low v a l u e s (<lOmM) even when e x t e r n a l l e v e l s were as h i g h as 89mM. Larvae exposed t o 33.3mM s u l p h a t e were a b l e t o s e c r e t e t h i s a n i o n a t a r a t e n e a r l y f o u r t i m e s f a s t e r than i n s e c t s r e a r e d i n 13 F i g u r e 2: E l e c t r i c p o t e n t i a l a c r o s s an i n s e c t e p i t h e l i u m . 1 4 s u l p h a t e - f r e e water. C o n c e i v a b l y the Mg 2 + t r a n s p o r t system i n A. d o r s a l i s M a l p i g h i a n t u b u l e s i s a l s o i n d u c i b l e . T h i s p o s s i b i l i t y was i n v e s t i g a t e d i n the p r e s e n t s t u d y . A re m a i n i n g p o s s i b i l i t y i s t h a t Mg-ATPase e x i s t s i n A. d o r s a l i s M a l p i g h i a n t u b u l e s ; however, such a t r a n s p o r t ATPase has not been r e p o r t e d t o dat e i n any e p i t h e l i a or s i n g l e c e l l . To d etermine whether magnesium s e c r e t i o n i n A. d o r s a l i s M a l p i g h i a n t u b u l e s o c c u r s by a Na +-Mg 2 + exchange mechanism, the f o l l o w i n g s t u d i e s were performed. M a l p i g h i a n t u b u l e s were bathed i n v a r y i n g c o n c e n t r a t i o n s of magnesium (0-4mM) and s e c r e t e d d r o p l e t s were a n a l y z e d t o determine the r e l a t i o n s h i p between M g 2 + and Na + (as w e l l as w i t h HPO a", S O f l 2 " , C I " , and K + ) . Second, s e c r e t e d d r o p l e t s from M a l p i g h i a n t u b u l e s b a t h e d i n sodium-free s a l i n e s were a n a l y z e d f o r any c o r r e l a t i o n between r e s i d u a l Na + and Mg 2 + c o n t e n t . These s t u d i e s a l s o y i e l d e d i n f o r m a t i o n c o n c e r n i n g the r e l a t i o n s h i p of o t h e r s e c r e t o r y p r o c e s s e s (e.g. HPO„~, S0„ 2-, C I " , K + ) w i t h M g 2 + . T h i r d , the e f f e c t s of i n h i b i t o r s of t r a n s e p i t h e l i a l sodium t r a n s p o r t ( e . g . a m i l o r i d e , bumetanide) on Mg 2 + s e c r e t i o n were s t u d i e d . 1 5 MATERIALS AND METHODS  Aedes d o r s a l i s eggs were k i n d l y p r o v i d e d by Dr. L a u r a Kramer, Department of B i o m e d i c a l and E n v i r o n m e n t a l H e a l t h S c i e n c e s , S c h o o l of P u b l i c H e a l t h , B e r k e l e y , C a l i f o r n i a . Eggs were hatched as p r e v i o u s l y d e s c r i b e d ( B r a d l e y , 1976) and l a r v a e were r e a r e d a t 28°C i n 50% seawater. C o m p o s i t i o n of seawater i s shown i n T a b l e 1. T a b l e 1: C o m p o s i t i o n of 50% Seawater Ion Concentration(mM) Na + 229.43 K + 4.89 M g 2 + 26.30 C a 2 + 7.54 CI" 272.57 HC0 3- 1.25 Br" 0.38 SO a 2" 13.90 F o u r t h i n s t a r l a r v a e were used i n a l l e x p e r i m e n t s because they s u r v i v e i n h i g h e r magnesium wat e r s compared t o e a r l i e r s t a g e s ( B r a d l e y and S h e p l a y , 1982). Larvae were f e d d a i l y w i t h d r y f i s h food ( T e t r a m i n S t a p l e Food; D4520 M e l l e 1,W.Germany) and the r e a r i n g media was a e r a t e d g e n t l y and changed p e r i o d i c a l l y t o p r e v e n t s t a g n a t i o n . S u r v i v a l s t u d i e s were performed by t r a n s f e r r i n g f o u r t h i n s t a r l a r v a e , f i v e t o s i x days a f t e r b e i n g hatched i n 50% seawater, t o s o l u t i o n s c o n t a i n i n g 50% seawater p l u s 16 one of the f o l l o w i n g c o m b i n a t i o n of s a l t s : (a) OmM MgSO«+500mM NaCl ( a b b r e v i a t e d 0/500), (b)50/450, (C)100/400. A f t e r 24 h o u r s , the number of dead and s u r v i v i n g a n i m a l s was det e r m i n e d , as d e s c r i b e d by B r a d l e y ( 1 9 8 2 ) . Based on these s u r v i v a l s t u d i e s , i n a l l subsequent e x p e r i m e n t s l a r v a e were t r a n s f e r r e d t o lOOmM MgSOa/400mM NaCl medium f i v e t o s i x days a f t e r b e i n g h a t c h e d ; e x p e r i m e n t s were performed on l a r v a e b e f o r e and a f t e r t r a n s f e r t o lOOmM MgSO„/400mM NaCl medium. P h y s i o l o g i c a l s a l i n e s used t o bathe i s o l a t e d M a l p i g h i a n t u b u l e s were s i m i l a r i n c o m p o s i t i o n t o s a l i n e s o l u t i o n s used by Strange (1982) and were based on measured i o n i c , o s m o t i c , and o r g a n i c s o l u t e c o n c e n t r a t i o n s of n a t u r a l hemolymph (Table 2 ) . To study k i n e t i c s of M g 2 + t r a n s p o r t , M g C l 2 c o n c e n t r a t i o n s of t h i s p h y s i o l o g i c a l s a l i n e .were a d j u s t e d between 0.5 and 4 mM. In sodi u m - f r e e s a l i n e s , Na* was r e p l a c e d by K + or c h o l i n e and a l l o t h e r components were the same. The f i n a l c o n c e n t r a t i o n of K + was 15.OmM i n c o n t r o l s a l i n e as compared t o 75.4mM i n the K + - s u b s t i t u t e d s o d i u m - f r e e s a l i n e . The " s o d i u m - f r e e " s a l i n e c o n t a i n e d a p p r o x i m a t e l y 1mM Na, as measured by e l e c t r o n microprobe a n a l y s i s (Cameca MBX). T h i s c o n c e n t r a t i o n of Na found i n "s o d i u m - f r e e " s a l i n e may be due t o sodium from r e a g e n t s or may be due t o background i n t e r f e r e n c e of o t h e r i o n s as measured by t h e e l e c t r o n m i c r o p r o b e . As the source of Na was not d e t e r m i n e d , t h i s v a l u e of Na (1mM) found i n "Na-T a b l e 2: Composition of Experimental S a l i n e s S a l i n e # Na< Mg ! Ca 2 H C O 3 - CI" SO 1 Cycl Succ. C i t r a t e Hepes Amil. Burnet. cAMP pH osmol i ) i i ) i i i ) i v ) v) v i ) v i i ) v i i i ) i x ) x) x i ) x i i ) x i i i ) x i v) 60.4 15 13 15 60.4 75 15 4.0 7.9 41.3 5.0 10.3 7.4 2.4 2.0 4.0 1 .0 4.0 62.4 G2 . 4 20.6 73 . 5 5.0 8 . 1 3.9 7 . 2 1 .0 1 .0 2.0 0.05 195 200 200 200 200 200 2 10 220 240 300 220 210 220 220 C y c l . Am i 1 . Burnet. = cyclamate = am i1 or i de = bumetanide ( A l l e x p e r i m e n t a l s a l i n e s c o n t a i n 10mM g l u c o s e , 4mM glutamine, 20mM p r o l i n e , 5mM a l a n i n e , and 3mM g l y c i n e ) 18 f r e e " s a l i n e was not s u b t r a c t e d from a l l Na v a l u e s . Because of s o l u b i l i t y problems, s u l p h a t e , s u c c i n a t e , c i t r a t e , and cy c l a m a t e were o m i t t e d from the s a l i n e s c o n t a i n i n g the i n h i b i t o r a m i l o r i d e (imM). The c o n t r o l s a l i n e i n t h i s c a s e was the same as the e x p e r i m e n t a l s a l i n e , but w i t h o u t a m i l o r i d e . A m i l o r i d e was a g i f t from W.D. D o r i a n , Merck Merck F r o s s t L a b o r a t o r i e s ( D o r v a l , Quebec). Bumetanide was a g i f t from Hoffman-La Roche L t d . ( E t o b i c o k e , O n t a r i o ) . The i n v i t r o p r e p a r a t i o n of M a l p i g h i a n t u b u l e s from A. d o r s a l i s f o l l o w s the proce d u r e d e s c r i b e d by Ramsey (1953) and P h i l l i p s and M a d d r e l l (1974). A p a i r of f o r c e p s was used t o h o l d the head of a l a r v a on a g l a s s d i s h . The c u t i c l e of the p o s t e r i o r r e g i o n (from midgut t o a n a l p a p i l l a e ) was c u t and f r e e d from the r e s t of the l a r v a by g r a s p i n g the t r a c h e a ( s i p h o n ) w i t h another p a i r of f o r c e p s and p u l l i n g . The 5 M a l p i g h i a n t u b u l e s came f r e e and were s e p a r a t e d from each o t h e r and from the mid-gut w i t h g l a s s n e e d l e s made from c a p i l l a r y t u b e s ( F i s h e r , P i t t s b u r g h , P a . ) which were p u l l e d t o o b t a i n f i n e t i p s u s i n g a v e r t i c a l g l a s s e l e c t r o d e p u l l e r ( M o d e l 700C, D a v i d Kopf i n s t r u m e n t s , Tujunga, C a l i f o r n i a ) and the t i p s s e a l e d . A p p r o x i m a t e l y 8 t u b u l e s were p l a c e d i n 0.1 ml d r o p l e t of p h y s i o l o g i c a l s a l i n e immersed under h y d r a t e d l i g h t p a r a f f i n o i K s p e c i f i c gravity=0.845-0.875 a t 25°C, MCB r e a g e n t s ) i n a g l a s s d i s h h a l f - f i l l e d w i t h S y l g a r d ( M i d l a n d , M i c h i g a n ) . The c u t ends of t u b u l e s were p u l l e d out of the s a l i n e d r o p i n t o the o i l and wrapped around f i n e g l a s s pegs s t u c k i n t o the S y l g a r d 19 f l o o r of the d i s h . Pegs were p l a c e d 0.8mm from the b a t h i n g drop t o pr e v e n t b a c k f l u x of s e c r e t e d f l u i d i n t o the b a t h i n g drop. S e c r e t i o n r a t e s of f l u i d and e l e c t r o l y t e s were det e r m i n e d over a p e r i o d of time as d e s c r i b e d by P h i l l i p s and M a d d r e l K 1 974) . F l u i d s e c r e t i o n r a t e s were d e t e r m i n e d by measuring the d i a m e t e r of the s e c r e t e d d r o p l e t s a t 30,60, and 90 minutes u s i n g an e y e - p i e c e micrometer and a W i l d d i s s e c t i n g m i c r o s c o p e , (Heerbrugg, S w i t z e r l a n d ) and then d e t e r m i n i n g the volume of f l u i d s e c r e t e d by the e q u a t i o n , V=4/3 i r r 3 . The a c c u r a c y of t h i s method has been r e p e a t e d l y c o n f i r m e d f o r drops of l e s s than 0.4mm d i a m e t e r ( P h i l l i p s and M a d d r e l l , 1 974) . A f t e r the volumes were d e t e r m i n e d , s e c r e t e d d r o p l e t s were.drawn up i n c a p i l l a r y t u b e s ( F i s h e r , P i t t s b u r g h , P a . ) p r e v i o u s l y r i n s e d w i t h n i t r i c a c i d and f i l l e d w i t h h y d r a t e d p a r a f f i n o i l c o n t a i n i n g Sudan B l a c k B (MCB R e a g e n t s ) . Each f l u i d sample was a n a l y z e d f o r c o n c e n t r a t i o n s of Mg, Ca, K, Na, C I , t o t a l S and t o t a l P, u s i n g a Cameca MBX e l e c t r o n m i c r o p r o b e . Methods a r e d e s c r i b e d by M o r e l and R o i n e l (1969) and R o i n e l (1975). T h i s method of element a n a l y s i s i s s u i t a b l e f o r d e t e r m i n i n g c o n c e n t r a t i o n s of elements i n n a n o l i t e r samples t o an a c c u r a c y of 2% f o r a c o n c e n t r a t i o n of 1mM ( R o i n e l . e t a l . , 1982). Samples of e x t e r n a l s a l i n e 20 b a t h i n g t u b u l e s were a l s o a n a l y z e d t o c o n f i r m the a c c u r a c y of the p i p e t t i n g procedure and of probe a n a l y s i s . In a d d i t i o n , d r o p l e t s of d i s t i l l e d water were a n a l y z e d t o determine background. At 30, 60, and 90 m i n u t e s , the r a t e of s e c r e t i o n of v a r i o u s e l e c t r o l y t e s was e s t i m a t e d from the volume and the r e s p e c t i v e c o n c e n t r a t i o n of the s e c r e t e d d r o p l e t s . Osmotic c o n c e n t r a t i o n s of n a n o l i t e r f l u i d samples were measured w i t h a ' C l i f t o n T e c h n i c a l P h y s i c s ' n a n o l i t e r osmometer ( H a r t f o r d , N.Y.) and l a r g e r samples w i t h an osmometer from Advanced I n s t r u m e n t s I n c . (Newton H i g h l a n d s , Mass.). NaCl s o l u t i o n s (100, 200, 300mOsm) were used as s t a n d a r d s f o r the C l i f t o n T e c h n i c a l P h y s i c s osmometer. S t u d i e s w i t h i a C - i n u l i n were performed i n i t i a l l y t o c o n f i r m the i n t e g r i t y of d i s s e c t e d t u b u l e s and t o i n d i c a t e whether any leakage o c c u r r e d a l o n g the t u b u l e s u r f a c e i n the o i l between b a t h i n g and s e c r e t e d d r o p l e t s . T h i s t r a c e r was added at h i g h s p e c i f i c a c t i v i t i e s t o the b a t h i n g s a l i n e . D r o p l e t s ( a p p r o x i m a t e l y 24.0 n l ) of s e c r e t e d f l u i d were c o l l e c t e d w i t h time and p l a c e d i n p l a s t i c v i a l s c o n t a i n i n g 1.0ul n o n - r a d i o a c t i v e s a l i n e s o l u t i o n and 3.33 ml of ACS I I aqueous c o u n t i n g s c i n t i l l a t e f l u i d (Amersham C o r p o r a t i o n , O a k v i l l e , O n t a r i o ) . Samples of the l a b e l l e d b a t h i n g s a l i n e were s i m i l a r l y p r e p a r e d . The 1 " C - a c t i v i t y of t h e s e samples were measured by l i q u i d s c i n t i l l a t i o n counting(Beckmann 9000 s c i n t i l l a t i o n c o u n t e r ) w i t h a p p r o p r i a t e c o r r e c t i o n s f o r 21 background r a d i a t i o n and quenching. These s t u d i e s on A. d o r s a l i s t u b u l e s y i e l d e d a U/P r a t i o of 0.0181±0.004 1 f o r 1 4 C - i n u l i n (n=9). T h i s low v a l u e proves t h a t t u b u l e s were not s i g n i f i c a n t l y damaged d u r i n g d i s s e c t i o n and t h a t no m i x i n g o c c u r s e x t e r n a l l y between b a t h i n g and s e c r e t e d d r o p s . U/P r a t i o s f o r i n u l i n (of 0.007-0.05) are commonly r e p o r t e d f o r o t h e r i n s e c t t u b u l e s ( M a d d r e l l , 1974). I f t h e r e , i s a major l e a k i n the t u b u l e s U/P c o u l d approach 1; i f a compound i s a c t i v e l y net s e c r e t e d , U/P c o u l d be g r e a t e r than 1 . The t r a n s e p i t h e l i a l p o t e n t i a l d i f f e r e n c e f o r the t u b u l e s bathed i n s a l i n e i v was r e c o r d e d u s i n g f i n e g l a s s m i c r o e l e c t r o d e s f i l l e d w i t h 3M KC1, as d e s c r i b e d by R.C. Thomas (1978). C a p i l l a r y tubing. ( F r e d e r i c k Haer and Co., B r u n s w i c k , Maine, U.S.A.) was used t o make t h e s e g l a s s e l e c t r o d e s . These were i n s e r t e d i n m i c r o e l e c t r o d e h o l d e r s ( t y p e EH-3FS) from WPI (New Haven, CT.) which c o n t a i n e d i n t e r n a l Ag-AgCl h a l f c e l l s . The two m i c r o e l e c t r o d e s , were i n s e r t e d w i t h the a i d of m i c r o m a n i p u l a t o r s ( K a n e t s u , MB-W, Tokyo, Japan) i n t o the b a t h i n g and s e c r e t e d d r o p l e t s r e s p e c t i v e l y , and were connected t o a h i g h impedance v o l t m e t e r ( K e i t h l e y Model 602, C l e v e l a n d , O h i o ) . The t r a n s e p i t h e l i a l p o t e n t i a l d i f f e r e n c e (P.D.) was r e c o r d e d as d e s c r i b e d by P h i l l i p s and M a d d r e l l (1974). The asymmetry p o t e n t i a l was determined w i t h both s a l t b r i d g e s i n the b a t h i n g d r o p l e t and was s u b t r a c t e d from t r a n s e p i t h e l i a l r e a d i n g s . 22 Phenol r e d (MCB) was used as pH i n d i c a t o r t o d e t e r m i n e pH of the b a t h i n g s a l i n e w i t h time as w e l l as the s e c r e t e d d r o p l e t s from t u b u l e s . A p p r o x i m a t e l y 4.10x10" 6 ml of phenol r e d was mixed w i t h an e q u a l volume of e i t h e r s t a n d a r d b u f f e r (pH=7.0-7.5) or t e s t s o l u t i o n ( b a t h i n g drop or s e c r e t e d d r o p l e t ) . These drops were immersed i n a d i s h c o n t a i n i n g h y d r a t e d p a r a f f i n o i l . . The pH of t h e s e t e s t s o l u t i o n s were d e t e r m i n e d by v i s u a l l y comparing the c o l o u r of phenol r e d i n unknown s o l u t i o n s w i t h s t a n d a r d b u f f e r s of known pH. 23 RESULTS S u r v i v a l of Aedes d o r s a l i s i n Waters of High Mg 2" Content E x p e r i m e n t s s i m i l a r t o those conducted by B r a d l e y e t a l . ( l 9 8 2 ) on A. d o r s a l i s l a r v a e were r e p e a t e d t o determine the upper s u r v i v a l l i m i t t o e x t e r n a l Mg 2 + and t o see i f a Mg 2 + t o l e r a n c e s t r a i n c o u l d be s e l e c t e d by e x p o s i n g s u c c e s s i v e g e n e r a t i o n s t o h i g h Mg 2 + w a t e r s . Such l a r v a e would be e x p e c t e d t o have w e l l d e v e l o p e d Mg t r a n s p o r t mechanisms i n t h e i r e x c r e t o r y systems. Eggs were hatched and l a r v a e were r e a r e d f o r 2 days i n 50% seawater c o n t a i n i n g 26mM MgSO„ and % s u r v i v a l was then d e t e r m i n e d 1 and 2 days ( i . e . 3 r d i n s t a r ) a f t e r t r a n s f e r t o t e s t s o l u t i o n s of v a r y i n g Na + and Mg 2 + s o l u t i o n (Table 3, Exp. 1 ) . C l o s e l y r e l a t e d s p e c i e s of s a l i n e - w a t e r mosquito l a r v a e r e a c h new st e a d y l e v e l s of hemolymph i o n s w i t h i n 1-2 days of t r a n s f e r t o new s a l i n e s ( K i c e n i u k and P h i l l i p s , 1974); t h e r e f o r e , t h i s time p e r i o d was judged a p p r o p r i a t e i n the c u r r e n t s t u d y . T h i r d i n s t a r l a r v a e s u r v i v e d w e l l the f i r s t day i n 126mM M g 2 + but m o r t a l i t y exceeded 50% a f t e r 1 day and was n e a r l y 100% a f t e r 1 day a t 226mM Mg 2 + (Table 3, Exp. 1). On t h i s b a s i s , 126mM Mg 2 + was s u b s e q u e n t l y used f o r r o u t i n e r e a r i n g of l a r v a e . In c o n t r a s t , B r a d l e y r e p o r t s s u r v i v a l r a t e s of 85% and 15% f o r t h i r d i n s t a r l a r v a e of t h i s s p e c i e s i n 226mM and 326mM Mg 2 + r e s p e c t i v e l y . He a l s o r e p o r t s good s u r v i v a l (80-100%) f o r l a r v a e h a t c h e d i n 26 t o 24 T a b l e 3: Percentage s u r v i v a l of l a r v a e hatched i n d i s t i l l e d water and r e a r e d i n 50% seawater a f t e r they were t r a n s f e r r e d a t day 2 t o t e s t s o l u t i o n s of d i f f e r e n t M g 2 + and Na + c o n t e n t . Two s e p a r a t e e x p e r i m e n t s were performed. ( n = o r i g i n a l number of l a r v a e i n each experiment) CONCENTRATION %SURVIVAL AFTER TRANSFER n OF TEST SOLUTION 1 Day 2 Days (mM) Na Mg Exp.1 Exp.2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 729 26 95 98 90 98 1 32 1 12 629 1 26 91 92 27 79 215 1 00 579 1 70 48 - 0 - 92 529 226 0.01 0 - - 1 56 429 326 0 0 - - 1 54 329 426 0 0 - - 1 42 229 526 0 0 — — 1 49 Ta b l e 4: Per c e n t a g e s u r v i v a l of l a r v a e h a t c h e d i n d i s t i l l e d water and r e a r e d i n 50% seawater a f t e r they were t r a n s f e r r e d a t day 5 t o s o l u t i o n s c o n t a i n i n g d i f f e r e n t amounts of M g 2 + . ( n = o r i g i n a l number of l a r v a e i n each e x p e r i m e n t ) . CONCENTRATION %SURVIVAL n of TEST SOLUTION (mM) AFTER TRANSFER Na Mg 1 Day 2Days 729 26 96 92 50 679 76 92 88 50 629 126 92 80 50 25 226mM Mg 2 + s o l u t i o n whereas n e g l i g i b l e s u r v i v a l f o r l a r v a e h a t c h e d i n 126mM Mg 2 + was o b s e r v e d i n p r e s e n t study ( d a t a not shown). A c c o r d i n g l y , l a r v a e were hatched i n d i s t i l l e d water and t r a n s f e r r e d t o s o l u t i o n c o n t a i n i n g 26mM Mg 2 + and Mg 2 + was i n c r e a s e d t o 126mM a t the t h i r d i n s t a r . A f t e r a p p r o x i m a t e l y 6 g e n e r a t i o n s ( 3 , months), the t o l e r a n c e of l a r v a e t o e x t e r n a l M g 2 + was r e t e s t e d . In t h i s case l a r v a e were t r a n s f e r r e d t o h i g h Mg 2 + t e s t s o l u t i o n s a f t e r e i t h e r 2 days ( i . e . T a b l e 3, Exp. 2) or 5 days ( 4 t h i n s t a r , Table 4) because of the s u g g e s t i o n ( B r a d l e y et a l . , 1982) t h a t t o l e r a n c e i n c r e a s e s w i t h d e v e l o p m e n t a l s t a g e . As shown i n T a b l e s 3 and 4, 20% m o r t a l i t y of 3rd and 4 t h i n s t a r l a r v a e o c c u r r e d a f t e r 2 days i n 26 to 126mM Mg 2 + w a t e r s and 100% m o r t a l i t y a t l70mM M g 2 + , s u g g e s t i n g t h a t t h e r e was a s m a l l but not a d r a m a t i c i n c r e a s e i n M g 2 + t o l e r a n c e as a r e s u l t of re p e a t e d s e l e c t i o n . As compared t o l a r v a e r e a r e d a t low Mg 2 + c o n c e n t r a t i o n , t h o s e exposed t o 126mM MgSO„ medium were s m a l l e r i n s i z e . The l e s s e r t o l e r a n c e of l a r v a e t o Mg 2 + i n our e x p e r i m e n t s compared t o those of B r a d l e y et a l . (1982) may r e f l e c t the f a c t t h a t our c o l o n y had p r e v i o u s l y been r e a r e d i n h i g h H C 0 3 _ / l o w Mg 2 + w a t e r s by-Strange (1982) f o r s e v e r a l y e a r s . 26 V i a b i l i t y and C h a r a c t e r i z a t i o n of In V i t r o  M a l p i g h i a n Tubule P r e p a r a t i o n The v i a b i l i t y of i n v i t r o M a l p i g h i a n t u b u l e s was a s s e s s e d by f o l l o w i n g the m e t a b o l i c a l l y dependent f l u i d s e c r e t i o n r a t e ( J v ) w i t h time and i t s response t o the common s t i m u l a n t of i n s e c t t u b u l e s , cAMP. The c o n t r o l b a t h i n g s a l i n e ( i v , T a b l e 2) resembled l a r v a l hemolymph and i s known t o s u s t a i n i o n t r a n s p o r t a t steady r a t e s f o r many hours ( S t r a n g e , 1982). U n s t i m u l a t e d t u b u l e s s e c r e t e d a t an average r a t e of 0.30±0.06 nl/min-mm over the f i r s t 1.5h. ( F i g . 3) and j * v d e c l i n e d from 0.54 to 0.20 nl/min-mm d u r i n g t h i s t i m e , which compares f a v o u r a b l y w i t h r e s u l t s f o r o t h e r i n s e c t M a l p i g h i a n t u b u l e p r e p a r a t i o n s ; e g . , r a t e s of s e c r e t i o n by t u b u l e s of fe d A. t a e n i o r h y n c h u s l a r v a e were found t o be 1.95±0.16 nl / m i n (n=l0) d u r i n g the f i r s t 5 minutes a f t e r i s o l a t i o n , f a l l i n g t o 0.98±0.06 n l / m i n (n=27) a f t e r 15 min. and t o 0.41+0.05 n l / m i n (n=l0) d u r i n g the subsequent 15 min. ( M a d d r e l l e t a l . , 1978). In two ex p e r i m e n t s a t d i f f e r e n t t imes of the year ( F i g . 3 ) , cAMP s t i m u l a t e d the average J v by about 6 - f o l d t o 1.96±0.23 (1mM cAMP) and 1.6±0.19 (2mM cAMP) nl/min-mm r e s p e c t i v e l y over the f i r s t 1.5h. S t i m u l a t e d J y d e c l i n e d by about 30% over t h i s p e r i o d which i s a s m a l l d e c r e a s e compared t o p r e p a r a t i o n s of o t h e r i n s e c t t u b u l e s . These r e s u l t s demonstrate the c a p a c i t y of mosquito t u b u l e c e l l s t o respond t o the m e t a b o l i c demands of the 27 g r e a t l y i n c r e a s e d i o n t r a n s p o r t r a t e s which d r i v e s t i m u l a t e d J . An i n d i c a t i o n of the range and time c o u r s e of J v f o r i n d i v i d u a l c A M P - s t i m u l a t e d (2mM) t u b u l e s i s shown i n F i g . 4. W h i l e the range i n J v i s q u i t e l a r g e , the d e c l i n e w i t h time i s r e a s o n a b l y c o n s i s t e n t . The t r a n s - e p i t h e l i a l p o t e n t i a l d i f f e r e n c e of 8 M a l p i g h i a n t u b u l e s i s o l a t e d i n a b a t h i n g s o l u t i o n c o n t a i n i n g 2mM Mg 2 + (15mM K +, 60mM N a + , 4mM C a 2 + , 41mM C I " , 5mM S0„ 2', 8mM HC0 3") v a r i e d w i d e l y but averaged -15mV at 90 minutes (lumen n e g a t i v e w i t h r e s p e c t t o b a t h i n g s o l u t i o n ) . V a r i a t i o n s w i t h time f o r i n d i v i d u a l t u b u l e s were l e s s than d i f f e r e n c e s between t u b u l e s (Table 5 ) . Tab l e 5: P o t e n t i a l r e a d i n g s a c r o s s t u b u l e s . (mV) A. d o r s a l i s M a l p i g h i a n Time(min.) 30 60 90 -15 -19 -17 -14.5 -1 1 -20 -28 -12 -24 + 5 + 8 + 4 + 8 + 2 + 4 28 F i g u r e 3. The e f f e c t of cAMP on s e c r e t i o n r a t e s of A. d o r s a l i s M a l p i g h i a n t u b u l e s when added t o the b a t h i n g drop. L a r v a e were r e a r e d i n seawater c o n t a i n i n g 126mM MgSO„. V e r t i c a l l i n e s a t t a c h e d t o the b a r s r e p r e s e n t ±S.E. of the mean. £ E I C > 3.0 2.5 2.0 1.5 1.0 0.5r-0 . 0 ^ 0 20 + 2mM cAMP o ImM cAMP M w i t h o u t r. A M P 60 80 100 Time (min,) -X) 30 F i g u r e 4. S e c r e t i o n r a t e s of i n d i v i d u a l M a l p i g h i a n t u b u l e s of A. d o r s a l i s r e a r e d i n 126mM MgSOft seawater and s t i m u l a t e d w i t h 2mM cAMP (n=9). (WUI-UJUI/tU) AP 32 F i g u r e 5 shows the average e l e c t r o l y t e c o m p o s i t i o n of the s e c r e t i o n from u n s t i m u l a t e d and cA M P - s t i m u l a t e d t u b u l e s over the f i r s t 1 . 5h i n i n i t i a l e x p e r i m e n t s . L i k e many o t h e r i n s e c t c A M P - s t i m u l a t e d t u b u l e s i n c l u d i n g A. c a m p e s t r i s and A. t a e n i o r h y n c h u s ( P h i l l i p s and M a d d r e l l , 1 9 7 4 ) ; ( M a d d r e l l and P h i l l i p s , 1978) those of A. d o r s a l i s l a r v a e s e c r e t e a KC1(68mM)-rich f l u i d (U) as compared t o the b a t h i n g s a l i n e (P) which c o n t a i n e d 15mM K + and 40mM C I " . C l e a r l y , s e c r e t i o n of thes e i o n s o c c u r s a g a i n s t c o n c e n t r a t i o n d i f f e r e n c e s (U/P r a t i o of 4.5 f o r K + and 1.7 f o r C l " ) . S i n c e the t r a n s e p i t h e l i a l p o t e n t i a l ( V t ) ranges between -20 t o +8mV (mean =-15mV,sign r e f e r s t o lumen); (T a b l e 5) f o r d i f f e r e n t t u b u l e s , c l e a r l y K + i s a c t i v e l y s e c r e t e d a g a i n s t an average net e l e c t r o c h e m i c a l d i f f e r e n c e of -38mV. C l " may a l s o be s e c r e t e d by a c t i v e t r a n s p o r t a g a i n s t a net e l e c t r o c h e m i c a l d i f f e r e n c e of -8.54 mV, as p r e v i o u s l y r e p o r t e d f o r t u b u l e s of a c l o s e l y r e l a t e d l a r v a , ( A . c a m p e s t r i s , P h i l l i p s and M a d d r e l l , 1 9 7 4 ) . C y c l i c AMP s t i m u l a t i o n s i g n i f i c a n t l y i n c r e a s e d K + but not C l " c o n c e n t r a t i o n i n the s e c r e t i o n (P=0.05). As f o r many i n s e c t t u b u l e s , Na + l e v e l s i n the s e c r e t i o n (16 t o 25mM) a r e low r e l a t i v e t o the b a t h i n g s a l i n e (60mM, U/P r a t i o of 0.3) and are not changed s i g n i f i c a n t l y by cAMP s t i m u l a t i o n . Thus Na + p r o b a b l y d i f f u s e s p a s s i v e l y i n t o the t u b u l a r f l u i d due t o a c t i v e KC1 s e c r e t i o n . The same may be t r u e f o r C a 2 + s i n c e i t s U/P r a t i o i s l e s s than one. 33 F i g u r e 5. The e f f e c t (of a d d i n g 1mM cAMP t o the b a t h i n g medium) on c o n c e n t r a t i o n s of i o n s s e c r e t e d by A. d o r s a l i s M a l p i g h i a n t u b u l e s . Mean i s average of v a l u e s a t 60' and 90'.. V e r t i c a l l i n e s a t t a c h e d t o ba r s r e p r e s e n t ±S.E. of the mean ( n = l 0 ) . * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e . C o n c e n t r a t i o n (mM) ro o o m o o o o H H rt i-> rt IE c rt 35 As p r e v i o u s l y r e p o r t e d f o r A. c a m p e s t r i s l a r v a e from (Na+Mg)SO« l a k e s , A. d o r s a l i s t u b u l e s i n s a l i n e c o n t a i n i n g 2mM Mg s e c r e t e M g 2 + (32mM) a g a i n s t a U/P of 1 6 - f o l d . L e v e l s of M g 2 + i n the t u b u l a r s e c r e t i o n f a l l t o 8mM a f t e r s t i m u l a t i o n , not because M g 2 + s e c r e t i o n i s reduced (see F i g . 6 ) , but because t h i s p r o c e s s i s independent of j * v and enhanced K C l - d r i v e n j " v d i l u t e s Mg i n the t u b u l e lumen. T h i s was p r e v i o u s l y observed f o r Mg 2 + s e c r e t i o n by t u b u l e s of A. c a m p e s t r i s l a r v a e by P h i l l i p s and M a d d r e l l (1974). G i v e n the low t r a n s e p i t h e l i a l p o t e n t i a l a c r o s s A. d o r s a l i s t u b u l e s ( v a r y i n g from +8mV t o -20mV f o r d i f f e r e n t t u b u l e s ) , M g 2 + s e c r e t i o n must i n v o l v e a c t i v e t r a n s p o r t assuming t h a t , as f o r A. c a m p e s t r i s , t h i s c a t i o n does not e x i s t as a p r e c i p i t a t e i n the s e c r e t i o n (see P h i l l i p s and M a d d r e l l , 1974). As g e n e r a l l y o b s e r v e d f o r o t h e r i n s e c t t u b u l e s ( P h i l l i p s , 1981) A. d o r s a l i s t u b u l e s s e c r e t e phosphate a g a i n s t a l a r g e c o n c e n t r a t i o n d i f f e r e n c e ( 5 - f o l d ) and t h i s i s not s i g n i f i c a n t l y changed by cAMP s t i m u l a t i o n . L i k e w i s e , t o t a l s u l p h u r , presumably as S0« 2~, i s s e c r e t e d a g a i n s t a d i f f e r e n c e of n e a r l y 2 - f o l d by u n s t i m u l a t e d t u b u l e s , i n agreement w i t h p r e v i o u s o b s e r v a t i o n s f o r A. c a m p e s t r i s ( P h i l l i p s and M a d d r e l l , 1975) and A. t a e n i o r h y n c h u s t u b u l e s ( M a d d r e l l and P h i l l i p s , 1 9 7 8 ) . C y c l i c AMP causes l e v e l s of SO, 2" t o f a l l i n the s e c r e t e d f l u i d because of d i l u t i o n but 36 F i g u r e 6. The e f f e c t (of a d d i n g 1mM cAMP t o the b a t h i n g medium) on s e c r e t i o n r a t e s of i o n s by A. d o r s a l i s M a l p i g h i a n t u b u l e s . Mean i s average of v a l u e s at 60' and 90'. V e r t i c a l l i n e s a t t a c h e d t o bars r e p r e s e n t ±S.E. of the mean ( n = l 0 ) . * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e . without CAMP e l Mg Na Ca Cl Ions 38 s t i m u l a t i o n a c t u a l l y i n c r e a s e s net S O i , 2 " s e c r e t i o n r a t e s (see F i g . 6) . Rates of i o n s e c r e t i o n ( J i o n ) a r e a f u n c t i o n of both J v ( F i g . 3) and i o n l e v e l s i n the s e c r e t i o n ( F i g . 5 ) . The p o o l e d v a l u e s f o r t u b u l e s d e t e r m i n e d a t 1.0 and 1.5h. a r e shown i n F i g . 6. As e x p e c t e d , i f KC1 t r a n s p o r t l a r g e l y d r i v e s f l u i d s e c r e t i o n , cAMP causes 1 1 - f o l d and 7 - f o l d i n c r e a s e r e s p e c t i v e l y (P<0.05) i n j " K and Jc±r compared t o an i n c r e a s e i n j ~ v of 6 - f o l d ( F i g . 3 ) . S t i m u l a t i o n a l s o causes s m a l l e r but s i g n i f i c a n t i n c r e a s e s i n Na + ( 4 - f o l d ; P<0.05), C a 2 + ( 3 - f o l d ; P<0.05), s u l p h u r ( 2 . 5 - f o l d ; P<0.05), and P ( 6 . 5 - f o l d ; P<0.05) s e c r e t i o n r a t e s . No s i g n i f i c a n t change i n M g 2 + s e c r e t i o n r a t e i s apparent a f t e r a d d i t i o n of cAMP. A comparison of t o t a l e q u i v a l e n t s of c a t i o n s ( M g 2 + , C a 2 + , N a + , K +) and a n i o n s ( C I " , S as SO,,2', P as H 2PO„-) i n d i c a t e s no a n i o n d e f i c i t i n the s e c r e t i o n . In a l l exp e r i m e n t s i n T a b l e s 6 and 7, the average d i f f e r e n c e between t o t a l a n i o n s and t o t a l c a t i o n s ranged between 0 and 10 mEquiv.L" 1. T h i s suggest t h a t HC0 3~ and o r g a n i c a n i o n s ar e not major components i n the s e c r e t i o n . Moreover, s i g n i f i c a n t a l k a l i n i z a t i o n of s e c r e t i o n due t o HC0 3~ or OH" s e c r e t i o n was not o b s e r v e d . The f l u i d s e c r e t e d by A. d o r s a l i s M a l p i g h i a n t u b u l e s (204±1.38 mosm, (n= l 9 ) ) i s not s i g n i f i c a n t l y d i f f e r e n t (P=0.05) than the b a t h i n g s o l u t i o n (198.8±1.83 mosm, (n=6)). L i k e most o t h e r i n s e c t M a l p i g h i a n t u b u l e s , the c o m p o s i t i o n T a b l e 6 C o n c e n t r a t i o n of Ions i n S e c r e t e d F l u i d (mM) and J (nl/min-mm) C o n c e n t r a t i o n of Mg i n A r t i f i c i a l Mg ! + Ca2* K + Na* CI" P S u y Hemol ymph(mM) c s/n c s/n c s/n c s/n c s/n c Sx/n c s-Jn J s-Jn a . 0.0 b.9.1 0.4 3.3 0.9 52.8 4.1 43.1 11.6 47.5 3.0 4.2 1.8 36.4 7.4 0.28 0.22 a.6.5 0.6 3.3 0.7 50.75.8 36.2 8.2 55.2 1.4 3.9 0.7 27.52.7 0.40 0.11 0.5 b.8.2 1.0 2.3 0.2 67.7 3.8 25.9 3.4 63.0 2.5 4.9 0.7 18.6 0.5 0.54 0.11 a.8.4 0.7 3.1 0.8 44.5 3.7 45.1 2.7 64.5 2.9 4.3 1.7 25.5 5.3 0.37 0.07 1.0 b.15.0 1.7 3.0 0.5 52.8 3.6 36.6 4.4 62.2 3.6 5.2 0.9 27.9 2.7 0.41 0.06 a.22.0 2.9 5.7 1.2 34.7 3.0 40.6 5.9 63.9 1.2 5.4 1.0 39.3 2.9 0.43 0.09 2.0 b.32.0 3.3 2.7 0.4 35.5 1.9 26.0 4.8 56.6 1.1 4.6 2.2 35.2 1.8 0.35 0.07 a.27.9 3.0 6.7 1.2 43.4 3.6 28.1 4.8 87.9 3.3 3.7 0.9 30.3 3.1 0.47 0.06 4.0 b.30.3 4.1 5.7 1.0 37.3 6.3 33.7 4.5 75.9 1.4 3.6 1.2 35.2 3.5 0.44 0.06 a= l a r v a e r e a r e d i n 26mM MgS0« s o l u t i o n . b = l a r v a e r e a r e d i n 126mM MgSOa s o l u t i o n . Table 7: Flux of Ions from A. d o r s a l i s M a l p i g h i a n t u b u l e s (pmole/min-mm) and <Jv (nl/min-mm) C o n c e n t r a t i o n of Mg i n A r t i f i c i a l Mg ! + C a ! * K + Na* C1 P S u y Hemolymph(mM) u s^n J s/n u sjn J sjn J s,/n J s,/n J s/n J s/n a. 0.0 b.3.62 0.9 1.1 0.2 21.8 6.3 16.0 4.2 19.5 5.2 1.4 0.3 ' 13.8 3.8 0.28 0.22 a.2.8 0.9 1.5 0.6 21.2 6.2 15.5 4.7 23.6 6.9 1.6 0.6 11.5 3.2 0.40 0.11 0.5 b.3.7 0.6 1.0 0.1 33.6 7.3 11.6 2.1 30.4 5.7 2.2 0.3 9.0 1.7 0.54 0.11 a.2.5 0.4 0.8 0.1 14.5 3.4 13.9 2.2 20.3 3.8 1.5 0.6 7.4 1.1 0.37 0.07 1.0 b.5.4 1.0 1.1 0.2 18.5 2.7 10.9 2.2 21.9 3.3 1.9 0.6 9.9 1.7 0.41 0.06 a.6.8 1.7 1.8 0.6 10.1 1.7 11.6 2.5 19.1 3.6 1.5 0.4 12.1 2.8 0.43 0.09 2.0 b.9.3 2.4 0.9 0.3 11.43.8 7.73.0 17.45.2 0.9 0.2 11.23.6 0.35 0.07 a. 10.1 1.5 2.6 0.6 16.2 2.6 9.5 2.3 33.0 4.7 1.3 0.4 10.6 1.3 0.47 0.06 4.0 b.11.5 3.8 2.2 0.6 15.3 5.0 12.1 2.0 29.1 5.5 1.1 0.2 13.7 2.7 0.44 0.06 a=l a r v a e r e a r e d i n 26mM MgSOa s o l u t i o n . b = l a r v a e r e a r e d i n 126mM MgS0« s o l u t i o n . 41 of s e c r e t e d f l u i d i s i s o s m o t i c t o the b a t h i n g s o l u t i o n . In a d d i t i o n , no s i g n i f i c a n t d i f f e r e n c e s were found between o s m o l a r i t y of f l u i d s e c r e t e d by t u b u l e s bathed i n e i t h e r 0.5, 1, 2, or 4mM Mg s a l i n e s o l u t i o n and between l a r v a e r e a r e d i n e i t h e r 26mM or 126mM Mg 2 + medium (P=0.05). A comparison of pH of b a t h i n g d r o p l e t s ( s a l i n e i v , Tab l e 2) b e f o r e and a f t e r the 90 minute e x p e r i m e n t a l p e r i o d i n d i c a t e d a change i n pH of 0.2 u n i t s (n=4) from 7.2 t o 7.4 r e s p e c t i v e l y . S e c r e t e d d r o p l e t s had an average pH of 7.24±0.07 (n=5) a t the end of the 90 minute e x p e r i m e n t a l p e r i o d . T h e r e f o r e , pH of b a t h i n g and s e c r e t e d d r o p l e t s were not s i g n i f i c a n t l y d i f f e r e n t (P=0.05). Another i n d i c a t o r of t u b u l e v i a b i l i t y i s the c o n s t a n c y i n the c o m p o s i t i o n of the s e c r e t i o n w i t h t i m e , s i n c e a d e t e r i o r a t i o n i n c e l l u l a r m etabolism s h o u l d reduce a c t i v e s e c r e t i o n of i o n s as opposed t o p a s s i v e d i f f u s i o n and th e r e b y change i o n i c r a t i o s . S i g n i f i c a n t changes i n i o n c o n c e n t r a t i o n i n the s e c r e t i o n d i d not oc c u r between 0.5, 1.0, and 1.5h. a f t e r the s t a r t of experiments ( d a t a i n Appendix A, F i g . 5 f o r u n s t i m u l a t e d and s t i m u l a t e d t u b u l e s ) . The 30% d e c l i n e i n s t i m u l a t e d J y over 1.5h., which c l e a r l y r e f l e c t s KC1 s e c r e t i o n r a t e , may i n d i c a t e e i t h e r some d e t e r i o r a t i o n i n t i s s u e v i a b i l i t y or a l t e r n a t e l y a r e g u l a t o r y decrease i n cAMP s t i m u l a t i o n of t r a n s p o r t p r o c e s s e s w i t h t i m e , or b o t h . 42 Having c h a r a c t e r i z e d s e c r e t i o n by i n v i t r o t u b u l e s of A. d o r s a l i s and h a v i n g c o n f i r m e d M g 2 + s e c r e t i o n a g a i n s t l a r g e c o n c e n t r a t i o n d i f f e r e n c e s i n t h i s s p e c i e s , the k i n e t i c s of Mg 2 + s e c r e t i o n was next i n v e s t i g a t e d t o d e c i d e on a s u i t a b l e c o n c e n t r a t i o n of t h i s c a t i o n t o be used i n a l a t e r s t u d i e s of Na-dependency. S i n c e the Mg 2 + g r a d i e n t s d e v e l o p e d a c r o s s the t u b u l e w a l l a r e much l a r g e r i n u n s t i m u l a t e d t u b u l e s ( F i g . 5) and s i n c e i s not changed by cAMP s t i m u l a t i o n ( F i g . 6 ) , subsequent experiments were conducted on u n s t i m u l a t e d t u b u l e s . 43 I n f l u e n c e of E x t e r n a l Mg 2* L e v e l s on F l u i d and Ion S e c r e t i o n  Rates U n s t i m u l a t e d t u b u l e s were bathed i n s a l i n e s c o n t a i n i n g 0, 0.5, 1.0, 2.0, or 4.0 mM Mg 2 + but o t h e r w i s e i d e n t i c a l i n c o m p o s i t i o n , and s e c r e t i o n was measured. S i n c e S O „ 2secretion i n A. c a m p e s t r i s i s i n c r e a s e d by exposure of l a r v a e t o h i g h e r e x t e r n a l l e v e l s of t h i s a n i o n f o r a, day ( P h i l l i p s and M a d d r e l l , 1 9 7 5 ) , a s i m i l a r s t i m u l a t i o n of t u b u l a r M g 2 + s e c r e t i o n might be a n t i c i p a t e d . A c c o r d i n g l y , t u b u l e s were compared from A. d o r s a l i s l a r v a e r e a r e d t o t a l l y i n 26mM Mg 2 + and o t h e r s exposed f o r 1 t o 2 days t o 126mM Mg 2 + j u s t b e f o r e use. As shown i n F i g . 7, J v i s not s i g n i f i c a n t l y a f f e c t e d by Mg 2 + c o n c e n t r a t i o n s (0-4mM) i n the s a l i n e b a t h i n g t u b u l e s or i n the wate r s (26 and 126mM) i n which l a r v a e were r e a r e d . The i o n i c c o m p o s i t i o n and s e c r e t i o n r a t e s at 1.0 and 1.5h (pooled) a r e g i v e n i n T a b l e s 6 and 7. As shown i n F i g . 8, t u b u l e s i n n o m i n a l l y Mg-free s a l i n e (measured c o n c e n t r a t i o n of 0.07mM Mg) c o n t i n u e t o s e c r e t e f l u i d c o n t a i n i n g 9mM Mg, presumably from the c e l l u l a r c o n t e n t s . M g 2 + l e v e l s i n the s e c r e t i o n r e a c h a maximum of about 30mM when the b a t h i n g s a l i n e c o n t a i n s 2-4mM M g 2 + . As shown i n F i g . 9, the Mg 2 + r a t i o d e v e l o p e d a c r o s s the t u b u l e w a l l d e c l i n e s , as exp e c t e d f o r a s a t u r a b l e t r a n s p o r t p r o c e s s , as the e x t e r n a l l e v e l of t h i s c a t i o n i n c r e a s e s but the t r e n d i s not marked over the l i m i t e d range of c o n c e n t r a t i o n s t e s t e d . 44 F i g u r e 7. Rates of f l u i d s e c r e t i o n of s e c r e t e d d r o p l e t s from A. d o r s a l i s M a l p i g h i a n t u b u l e s v e r s u s c o n c e n t r a t i o n of Mg i n e x t e r n a l s a l i n e s o l u t i o n . Mean i s average of v a l u e s a t 60' and a t 90'. V e r t i c a l l i n e s a t t a c h e d t o p o i n t s i n d i c a t e ±S.E. of the mean (n=52). in a z Ol z z e z (0 6 CVl to (VI o * z E ni u c o CJ 4 J X HI CD a to a a CM o a (lUUI-U-tUl/tU) A P 46 F i g u r e 8.- C o n c e n t r a t i o n s of Mg s e c r e t e d v e r s u s e x t e r n a l c o n c e n t r a t i o n s of Mg b a t h i n g A. d o r s a l i s M a l p i g h i a n t u b u l e s . Larvae were r e a r e d i n 126mM MgSO„ medium. Mean i s average of v a l u e s a t 60' and a t 90'. V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean (n=27). Conc'n of Mg Secreted (mM) 48 F i g u r e 9. R a t i o of Sec/Ext c o n c e n t r a t i o n s of Mg v e r s u s e x t e r n a l ( e x t . ) c o n c e n t r a t i o n s of Mg. Mean i s average of v a l u e s a t 60' and at 90'. V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean (n=52). 50 Two mM Mg i s the lowest c o n c e n t r a t i o n a t which J., i s Mg markedly i n c r e a s e d over the b a s e l i n e l e v e l (OmM e x t e r n a l Mg) and a t which the U/P r a t i o i s s t i l l v e r y l a r g e (10-15). T h e r e f o r e , 2mM Mg 2 + i n the s a l i n e was s e l e c t e d f o r l a t e r e x p e r i m e n t s on Na-dependency. R e a r i n g l a r v a e i n 126 as opposed t o 26 mM Mg had a s l i g h t but not d r a m a t i c e f f e c t on JMg ^ F^-9' 1 u ) a n Q l t u b u l a r Mg c o n c e n t r a t i o n ( F i g . 8 ) . Only the d i f f e r e n c e a t 1mM Mg i s s i g n i f i c a n t (P<0.05). Perhaps i f l a r v a e were r e a r e d i n Mg-free w a t e r s , a more s i g n i f i c a n t e f f e c t on j" Mg might be demonstrated as was the case f o r JgQ 2- i - n A. t a e n i o r h y n c h u s t u b u l e s ( P h i l l i p s and M a d d r e l l , 1 9 7 8 ) . E x p r e s s e d as mEquiv.L" 1, the l e v e l s of s e c r e t e d M g 2 + ( 6 0 ) from t u b u l e s bathed i n 2 and 4 mM Mg 2 + a c t u a l l y exceed th o s e of both K + (36) and Na + (26-34) and indee d almost e q u a l s these two monovalent i o n s ( T a b l e s 6 and 7 ) . The d a t a s u g g e s t s t h a t as l e v e l s of Mg 2 + i n the s e c r e t i o n r i s e , those of K + d e c l i n e whereas Na + does not change ( F i g s . 12.1, 13.1). T o t a l E q u i v a l e n t s of a l l . i o n s (assuming monovalent P, d i v a l e n t S as SO a 2~) are h i g h e r a t 4mM Mg (293 mEquiv. L " 1 ) than a t 0.5mM Mg (220 mEquiv. L" 1) s u g g e s t i n g an i n c r e a s e i n o s m o l a r i t y ( T a b l e 6 ) . I n c r e a s e d Mg l e v e l s seem t o be a s s o c i a t e d w i t h i n c r e a s e d C I" l e v e l s (compare 0 and 0.5mM Mg v s . 4mM Mg i n T a b l e 6 and 7) r a t h e r than o t h e r a n i o n s (eg. SO„ 2~). In a d d i t i o n , F i g u r e 14 i n d i c a t e s g e n e r a l i n c r e a s e s i n CI w i t h Mg a t 4mM Mg (compared t o 51 OmM Mg) a l t h o u g h v a l u e s f l u c t u a t e i n between the s e c o n c e n t r a t i o n s . However, t h e r e i s enough v a r i a b i l i t y between d i f f e r e n t e x p e r i m e n t s ( T a b l e 6 and 7) t o e x c l u d e any r i g o r o u s statement at t h i s time t h a t i n c r e a s e d Mg s e c r e t i o n s i g n i f i c a n t l y a l t e r s l e v e l s of o t h e r i o n s i n the s e c r e t i o n ( T a b l e s 6 and 7 ) . I f the i n c r e a s e i n J M ^ above b a s e l i n e i n F i g . 1 0 i s c o n s i d e r e d , then s e c r e t i o n appears t o be a s a t u r a b l e p r o c e s s as e x p e c t e d f o r c a r r i e r mediated a c t i v e t r a n s p o r t w i t h a V of 14.94±2.89 pmoles/min-mm and a K of 1.57±0.16 max r m mM Mg 2 + ( f o r l a r v a e r e a r e d i n 126mM MgSO<,). These v a l u e s a r e s i m i l a r t o those r e p o r t e d by P h i l l i p s and M a d d r e l l (1974) f o r J M g i n t u b u l e s of A. c a m p e s t r i s . For l a r v a e r e a r e d i n 26mM Mg medium, V was 9.17±8.16 pmole/min-mm 3 max r w h i l e K m was 1.40±0.77 ( F i g . 11). 52 F i g u r e 10. of A. d o r s a l i s M a l p i g h i a n t u b u l e s w i t h i n c r e a s i n g e x t e r n a l Mg c o n c e n t r a t i o n s . V e r t i c a l b ars a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean (n=52). * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e . 126mM Mg 54 F i g u r e 11. A d o u b l e - r e c i p r o c a l (Lineweaver-Burk) p l o t of J M v e r s u s e x t e r n a l c o n c e n t r a t i o n of Mg (n=52). 56 F i g u r e 12.1. C o n c e n t r a t i o n s of Mg and K i n the s e c r e t e d f l u i d v e r s u s e x t e r n a l c o n c e n t r a t i o n of Mg. Mean i s average of v a l u e s a t 60' and 90'. V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean (n=27). C o n c e n t r a t i o n (mEquiv./l) 58 F i g u r e 12.2. S e c r e t i o n r a t e s of Mg and K v e r s u s e x t e r n a l c o n c e n t r a t i o n of Mg. Mean i s average of v a l u e s a t 60' and 90'. V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean (n=27). 60 F i g u r e 13.1. C o n c e n t r a t i o n s of Mg and Na i n the s e c r e t e d f l u i d v e r s u s e x t e r n a l c o n c e n t r a t i o n of Mg. Mean i s average of v a l u e s at 60' and 90'. V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean (n=27). Concentration (mEquiv./I) 62 F i g u r e 13.2. S e c r e t i o n r a t e s of Mg and Na v e r s u s e x t e r n a l c o n c e n t r a t i o n of Mg. Mean i s average of v a l u e s at 60' and 90'. V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean (n=27). 64 F i g u r e 14.1. C o n c e n t r a t i o n s of Mg and C l v e r s u s e x t e r n a l c o n c e n t r a t i o n of Mg. Mean i s average of v a l u e s a t 60' and 90'. V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean (n=27) . Concentration (mEquiv./I) 66 F i g u r e 14.2. S e c r e t i o n r a t e s of Mg and CI v e r s u s e x t e r n a l c o n c e n t r a t i o n of Mg. Mean i s average of v a l u e s a t 60' and 90'. V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean (n=27) . 68 E f f e c t of N a - f r e e S a l i n e s on Mq 2 + S e c r e t i o n S i n c e KC1 s e c r e t i o n d r i v e s J v i n many i n s e c t t u b u l e s , i t seemed f e a s i b l e t o r e p l a c e Na + i n the b a t h i n g s a l i n e so as t o reduce l u m i n a l Na + t o t r a c e l e v e l s . I f Mg s e c r e t i o n does occur by an a p i c a l Na +/Mg 2 + exchanger, then such a t r e a t m e n t s h o u l d a b o l i s h J w . Mg a. C h o l i n e s u b s t i t u t i o n f o r Na When Na was r e p l a c e d by c h o l i n e J v dropped markedly from 0.53 t o 0.18 nl/min-mm between 30 and 60 min. ( F i g . 15) i n d i c a t i n g t h a t e i t h e r c h o l i n e or absence of Na + does i n f l u e n c e s e c r e t i o n r a t e s compared t o c o n t r o l s ( j " v of c o n t r o l t u b u l e s d e c r e a s e d from 0.54 t o 0.30 nl/min-mm between 30 and 60 m i n u t e s ) . However, i o n i c c o m p o s i t i o n of the s a l i n e d i d not change over t h i s p e r i o d ( F i g . 16) e x cept f o r P and S (P<0.05). Mg c o n c e n t r a t i o n i n c r e a s e d from 19 t o 28 mM ( F i g . 16) and J" Mg changed from 10 t o 5 pmoles/min-mm ( F i g . 1 7 ) between 30 and 90 m i n u t e s , a l t h o u g h not s i g n i f i c a n t l y . Thus Mg s e c r e t i o n c o n t i n u e d a g a i n s t a U/P r a t i o of 10 t o 14 ( i . e . o n l y s l i g h t l y l e s s than c o n t r o l s . , F i g . 7,at 2mM e x t . Mg) when average Na i n the s e c r e t i o n was reduced t o 2mM from 30 t o 40 mM i n c o n t r o l s ( T a b l e 6 ) . J V T Na was o n l y 10% of J M g ( F i g . 17). As seen i n F i g . 17, a t 30 m i n u t e s , J M g , J^' ^ c i a n c ^ ^P a r e s i m i l a r o r g r e a t e r than 69 c o n t r o l v a l u e s ( i . e . Na i n s a l i n e ; T a b l e 7 ) . J g was reduced by 50% c o n c u r r e n t w i t h decrease (of 50%) i n S i n s a l i n e . In a d d i t i o n , j " N a was v e r y low (10% of c o n t r o l v a l u e s , T a b l e 7) i n n o m i n a l l y N a - f r e e s a l i n e . J C a was not d e c r e a s e d i n N a - f r e e s a l i n e , a t 30 minutes ( F i g . 17) a l t h o u g h i t s b a t h i n g c o n c e n t r a t i o n was 25% of c o n t r o l . 70 F i g u r e 15. Rates of f l u i d s e c r e t i o n of A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n N a - s u b s t i t u t e d w i t h c h o l i n e s a l i n e s o l u t i o n . V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean (n=6). a d o o o (UlU-UCUI/IU) AP 72 F i g u r e 16. C o n c e n t r a t i o n s of i o n s s e c r e t e d by A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n N a - s u b s t i t u t e d w i t h c h o l i n e s a l i n e s o l u t i o n . V e r t i c a l l i n e s a t t a c h e d t o bars r e p r e s e n t ±S.E. of the mean (n=6). * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e . c c E E o a to cn 4 J ro 4 J ro H f r-H a o o o co o a (WW) uoi^ej^uasuoo 74 F i g u r e 17. S e c r e t i o n r a t e s of i o n s ( J . ) of A. d o r s a l i s 3 I on M a l p i g h i a n t u b u l e s bathed i n N a - s u b s t i t u t e d w i t h c h o l i n e s a l i n e s o l u t i o n . V e r t i c a l l i n e s a t t a c h e d t o bars r e p r e s e n t ±S.E. of the mean (n=6). * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e . f c E o a o to a in o a co a 2 o (ujui-uTui/atouid) uoxr 76 b) K* s u b s t i t u t i o n f o r Na" When Na was r e p l a c e d w i t h K, j " v d e c r e a s e d from 1.1 t o 0.47 nl/min-mm between 30 and 90 min., s i m i l a r t o c o n t r o l v a l u e s of 1.32 and 0.64 nl/min-mm r e s p e c t i v e l y ( F i g . 1 8 ) . T h i s i n d i c a t e s t h a t s u b s t i t u t i o n of K f o r Na does not s i g n i f i c a n t l y a l t e r the s e c r e t i o n r a t e over 90 min.(P=0.05). A n a l y s i s of i o n i c c o m p o s i t i o n of s e c r e t e d f l u i d from t u b u l e s bathed i n 'Na-free' s a l i n e ( a t 60 and 90 min.) i n d i c a t e t h a t c o n c e n t r a t i o n s of S,K,Ca,and C l are 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 u b u l e s i n c o n t r o l s a l i n e (P>0.05). However, c o n c e n t r a t i o n s of Mg and P a r e s i g n i f i c a n t l y i n c r e a s e d i n s e c r e t e d f l u i d of t u b u l e s i n n o m i n a l l y N a - f r e e s a l i n e (imM). Na c o n c e n t r a t i o n i n the s e c r e t e d f l u i d d e c r e a s e d t o 2mM from 15mM i n c o n t r o l t u b u l e s w h i l e Mg s e c r e t i o n i n c r e a s e d t o 23mM from 6mM i n c o n t r o l t u b u l e s ( F i g . 1 9 ) . In a d d i t i o n , J N d e c r e a s e d t o 0.8 pmole/min-mm from c o n t r o l v a l u e of 10 pmole/min-mm, w h i l e J M g i n c r e a s e d t o 11 pmole/min-mm (from average c o n t r o l v a l u e of 4 pmole/min-mm). J p a l s o i n c r e a s e d by 4 - f o l d a f t e r Na-replacement whereas J C a d i d not change. J , J _ , , and J _ a l l d e c r e a s e d s i g n i f i c a n t l y (P<0.05; F i g . 2 0 ) . 77 F i g u r e 18. Rates of f l u i d s e c r e t i o n of A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n N a - s u b s t i t u t e d w i t h K s a l i n e s o l u t i o n . L a r v a e were r e a r e d i n 126mM MgSO„ s o l u t i o n . V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean (n=9). 79 F i g u r e 19. C o n c e n t r a t i o n s of i o n s s e c r e t e d by A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n c o n t r o l and "N a - f r e e " s a l i n e . L a r v a e were r e a r e d i n 126mM MgSO„ s o l u t i o n . Mean i s average a t 60' and a t 90'. V e r t i c a l l i n e s a t t a c h e d t o ba r s r e p r e s e n t ±S.E. of the mean (n=9). * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e . C o n c e n t r a t i o n (mM) ru o o o GD O a o O H £ 3 3 H tN\\\\\\\\\\^^^^ 3 n • 3 rt--J a Z i oi rr tn D -* • 81 F i g u r e 20. S e c r e t i o n r a t e s of i o n s ( J - ) of A. d o r s a l i s J i o n M a l p i g h i a n t u b u l e s bathed i n c o n t r o l and "Na - f r e e " s a l i n e . L a r v a e were r e a r e d i n l26mM MgSOi, s o l u t i o n . Mean i s average of v a l u e s a t 60' and at 90'. V e r t i c a l l i n e s a t t a c h e d t o ba r s r e p r e s e n t ±S.E. of the mean (n=9). * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e . 83 I n h i b i t o r S t u d i e s The i n f l u e n c e s of known i n h i b i t o r s of Na + t r a n s f e r mechanisms i n b i o l o g i c a l membranes on t u b u l a r s e c r e t i o n was i n v e s t i g a t e d as another means t o e x p l o r e Na c o u p l i n g of J. Mg' J and o t h e r J . v i o n s a) A m i l o r i d e A m i l o r i d e i n h i b i t s Na c h a n n e l s (uM range) and Na +-H + exchangers (mM range) i n v a r i o u s e p i t h e l i a (Gee, 1976). T h e r e f o r e , t h i s agent might decrease e n t r y of Na + i n t o t u b u l e c e l l s and hence the lumen, thereby reduce l u m i n a l Na a v a i l a b l e f o r a p i c a l exchange f o r c e l l u l a r Mg, and c o n s e q u e n t l y reduce J M i f a Na-Mg exchanger i s i n v o l v e d . By b l o c k i n g b a s o l a t e r a l Na + c h a n n e l s , a m i l o r i d e might a l s o c o n c e i v a b l y s t o p b a s a l Na r e c y c l i n g v i a a Na/K ATPase and t h e r e b y i n d i r e c t l y i n h i b i t K C l - d r i v e n t u b u l a r s e c r e t i o n i f K e n t r y o c c u r s i n exchange f o r c e l l u l a r Na. T h i s i d e a has not p r e v i o u s l y been t e s t e d i n any K C l - s e c r e t i n g i n s e c t t u b u l e . As shown i n F i g . 21, 1mM a m i l o r i d e does reduce K C l - d r i v e n f l u i d s e c r e t i o n by 80% as p r e d i c t e d by the b a s a l N a - r e c y c l i n g h y p o t h e s i s . Moreover as shown i n F i g . 22, K + l e v e l s i n the s e c r e t i o n i s reduced 50% (P<0.05) w h i l e N a + , C a 2 + , C I " , and S c o n c e n t r a t i o n s are unchanged by t h i s i n h i b i t o r . Remarkably, c o n c e n t r a t i o n of M g 2 + i n c r e a s e s by 240% (P<0.05) and phosphorus by 200% (P<0.05; F i g . 2 2 ) . T h i s can be e x p l a i n e d i f J*Mg and J p do not d e c rease s u b s t a n t i a l l y 84 or s i g n i f i c a n t l y when a m i l o r i d e i n h i b i t s J K by 90% (P<0.05), J N A by 81% (P<0.05), J C L by 80% (P<0.05), J C q by 66% (P<0.05),and J G by 44% (P<0.05; F i g . 2 3 ) . 85 F i g u r e 21. F l u i d s e c r e t i o n r a t e s of A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n s a l i n e w i t h and w i t h o u t 1mM a m i l o r i d e ( i n the presence of 1mM cAMP). L a r v a e were r e a r e d i n l26mM MgSOft s o l u t i o n . V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean (n=lO). * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e . 87 F i g u r e 22. C o n c e n t r a t i o n s of i o n s s e c r e t e d by A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n s a l i n e w i t h and w i t h o u t 1mM a m i l o r i d e ( i n the presence of 1mM cAMP). Larvae were r e a r e d i n 126mM MgSO„ s o l u t i o n . Mean i s average of v a l u e s a t 60' and a t 90'. V e r t i c a l l i n e s a t t a c h e d t o b a r s r e p r e s e n t ±S.E. of the mean ( n = l 0 ) . * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e . C o n c e n t r a t i o n ( m M ) ro o .u o cn o m o o ro o o o ~ i \\\\\\\^ 3 H 3 3 s rt U O • C a o z 01 H-3 rr M - IT O T a 89 F i g u r e 23. S e c r e t i o n r a t e s of i o n s ( J . ) of A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n s a l i n e w i t h and w i t h o u t 1mM a m i l o r i d e ( i n the presence of 1mM cAMP). Larvae were r e a r e d i n l26mM MgSOfl s o l u t i o n . Mean i s average of v a l u e s a t 60' and a t 90'. V e r t i c a l l i n e s a t t a c h e d t o ba r s r e p r e s e n t +S.E. of the mean ( n = l 0 ) . * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e . 8 0 -7 0 -6 0 -5 0 -4 0 -3 0 -2 0 -1 0 -0 — la m. rti * fo 'A. W' 1 . f i r with amiloride without nmllnrlriR Mg Na K Ca CI Ions 91 b) Bumetanide There i s e v i d e n c e i n Rhodnius M a l p i g h i a n t u b u l e s ( M a d d r e l l , 1969) t h a t C l " e n t e r s c e l l s by a NaCl or (Na,K,2Cl) c o t r a n s p o r t mechanism as commonly found i n v a r i o u s v e r t e b r a t e e p i t h e l i a and which i s i n h i b i t e d by such d i u r e t i c s as furosemide and bumetanide ( S c h l a t t e r et a l . , 1 9 8 3 ) . A d d i t i o n of bumetanide, a t 5X10~ 5M t o b a t h i n g s o l u t i o n , reduced s e c r e t i o n r a t e s i g n i f i c a n t l y (P=0.05) from 0.72±0.12 ( c o n t r o l tubules,n=4) t o 0.41±0.06 nl/min-mm (n=4) at 90 minutes ( F i g . 2 4 ) . A n a l y s i s of average i o n i c c o n c e n t r a t i o n s of s e c r e t e d f l u i d a t 60 and 90 minutes ( F i g . 25) i n d i c a t e s i g n i f i c a n t (P<0.05) d e c r e a s e s i n Mg (of lOmEquiv), and K (of 16mEquiv) and P (of 4mEquiv). C o n c e n t r a t i o n of Na i n the s e c r e t e d f l u i d i n c r e a s e d s i g n i f i c a n t l y from c o n t r o l v a l u e of 26mM t o 47mM (P<0.05). C o n c e n t r a t i o n s of Ca and C l remain unchanged from c o n t r o l v a l u e s ( F i g . 2 5 ) . C o n c e n t r a t i o n of S i n s e c r e t e d f l u i d was a p p r o x i m a t e l y 2 . 5 - f o l d g r e a t e r than c o n t r o l v a l u e of 2.0mM. j " N a and j * C a a r e not s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l v a l u e s w h i l e s i g n i f i c a n t d e c r e a s e s i n J M g , Jci' a n < ^ J P ar e e v i d e n t . j \ , d e c r e a s e d 60% from i t s c o n t r o l v a l u e , Mg ' w h i l e J " k and d e c r e a s e d 45% and 34% from t h e i r c o n t r o l v a l u e s , r e s p e c t i v e l y (P=0.05; F i g . 2 6 ) . T h i s experiment s h o u l d be r e p e a t e d u s i n g h i g h e r c o n c e n t r a t i o n s of bumetanide. S i n c e J„ was not d e c r e a s e d Na 92 by t h i s t r e a t m e n t whereas J" K and were, these r e s u l t s do not suggest the p r e s e n c e of a b a s a l K, Na, CI c o t r a n s p o r t e r u n l e s s a b a s a l Na r e c y c l i n g o c c u r s which i s independent of t r a n s e p i t h e l i a l net Na f l u x . R e g a r d l e s s , i n t h i s experiment the r e d u c t i o n i n J.. i s a s s o c i a t e d w i t h i n c r e a s e d r a t h e r Mg than d e c r e a s e d l u m i n a l Na which i s not the e x p e c t e d r e s u l t i f and a p i c a l Na/Mg exc h a n g e . i s i n v o l v e d . 93 F i g u r e 24. Rates of f l u i d s e c r e t i o n of A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n s a l i n e w i t h and w i t h o u t 5X10" 5M bumetanide ( i n the presence of 1mM cAMP). Larvae were r e a r e d i n l26mM MgSO„ s o l u t i o n . V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean (n=8). * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e . B B I C •rt E \ c > 2.2 -2.0 -1.8 -1.6 -1.4 -1.2 -1.0 -0.B -0.6 -0.4 -0.2 -O.O4L 20 with Bumetanide without Bumetanide 60 BO 100 Time (min.) 95 F i g u r e 25. C o n c e n t r a t i o n s of i o n s s e c r e t e d by A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n s a l i n e w i t h and w i t h o u t 5X10 _ 5M bumetanide ( i n the presence of 1mM cAMP). L a r v a e were r e a r e d i n 126mM MgSO„ s o l u t i o n . Mean i s average of v a l u e s a t 60' and at 90'. V e r t i c a l l i n e s a t t a c h e d t o b a r s r e p r e s e n t ±S.E. of the mean (n=8). * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e . C o n c e n t r a t i o n (mM) ro cn o CD o o o ro .h. o o i r i r 3 N \ \ \ \ \ \ \ \ \ \ \ ^ ± H • 3 3 z CD M c n 3 3 CD O rt C oi n 3 a CD 97 F i g u r e 26. S e c r e t i o n r a t e s of i o n s ( J . ) of A. d o r s a l i s 3 i on M a l p i g h i a n t u b u l e s bathed i n s a l i n e w i t h and w i t h o u t 5X10 _ 5M bumetanide ( i n the presence of 1mM cAMP). L a r v a e were r e a r e d i n 126mM MgSO„ s o l u t i o n . Mean i s average of v a l u e s a t 60' and at 90'. V e r t i c a l l i n e s a t t a c h e d t o ba r s r e p r e s e n t ±S.E. of the mean (n=8). * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e . 99 DISCUSSION C h a r a c t e r i z a t i o n and V i a b i l i t y of M a l p i g h i a n T u b u l e s The e x p e r i m e n t s d e s c r i b e d i n t h i s t h e s i s c l e a r l y show t h a t i s o l a t e d M a l p i g h i a n t u b u l e s of A. d o r s a l i s can t r a n s p o r t magnesium a c t i v e l y a t h i g h r a t e s a g a i n s t a c o n s i d e 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 . C o n c e n t r a t i o n g r a d i e n t s as l a r g e as 1 6 - f o l d were d e v e l o p e d and the average t r a n s e p i t h e l i a l p o t e n t i a l d i f f e r e n c e was -l5mV ( c a l c u l a t e d e q u i l i b r i u m v a l u e of -35mV f o r Mg) when t u b u l e s were bathed i n s a l i n e c o n t a i n i n g 2mM Mg ( i v , T a b l e 2 ) . Moreover, the lumen i s a c t u a l l y p o s i t i v e i n some t u b u l e s , as r e p o r t e d f o r A. c a m p e s t r i s ( M a d d r e l l and P h i l l i p s , 1974). F l u i d s e c r e t i o n r a t e s , d e t e r m i n e d over 90 minute p e r i o d s , i n the presence and absence of cAMP, i n d i c a t e d t h a t A. d o r s a l i s t u b u l e s used i n t h i s study were v i a b l e and had s e c r e t i o n r a t e s of the same magnitude as those r e p o r t e d f o r A. t a e n i o r h y n c h u s t u b u l e s ( M a d d r e l l and P h i l l i p s , 1978). R a p i d hormonal s t i m u l a t i o n of s e c r e t o r y p r o c e s s e s commonly i n v o l v e s i n c r e a s e s i n i n t r a c e l l u l a r c o n c e n t r a t i o n s of cAMP (B e r r i d g e , 1 9 7 5 ) and t h i s n u c l e o t i d e s t i m u l a t e s f l u i d s e c r e t i o n of a l l i n s e c t M a l p i g h i a n t u b u l e s s t u d i e d t o date ( M a d d r e l l et a l . , 1971). Moreover, t u b u l e s from f e d A. t a e n i o r h y n c h u s l a r v a e s e c r e t e f l u i d a t h i g h e r r a t e s (1.95±0.16 nl/ m i n - m i n , n=l0) t h a n t u b u l e s from u n f e d l a r v a e (0.26±0.04 nl/min-min, n=22) s u g g e s t i n g hormonal s t i m u l a t i o n 1 00 d u r i n g f e e d i n g i n s i t u ; s e c r e t i o n r a t e s of t u b u l e s from f e d l a r v a e a r e comparable t o t u b u l e s bathed i n s a l i n e c o n t a i n i n g 5 - h y d r o x y t r y p t a m i n e which mimics the a c t i o n s of d i u r e t i c hormone i n s e v e r a l i n s e c t s . Moreover, homogenates of the b r a i n and the t h o r a c i c g a n g l i a of A. t a e n i o r h y n c h u s s t i m u l a t e t u b u l a r s e c r e t i o n , s u g g e s t i n g they a r e the source of a d i u r e t i c neurohormone which i s r e l e a s e d a f t e r l a r v a e f e e d ( M a d d r e l l and P h i l l i p s , 1978). To e x c l u d e d i f f e r e n c e s between s e c r e t i o n r a t e s of f e d and unfed l a r v a e , l a r v a e used i n a l l e x p e r i m e n t s i n the p r e s e n t study were f e d an hour p r i o r t o e x p e r i m e n t s . A l t h o u g h e v i d e n c e f o r a neurohormone c a u s i n g changes i n t i s s u e cAMP (and hence i n c r e a s e f l u i d and i o n s e c r e t i o n r a t e s ) was not t e s t e d i n t h i s s t u d y , the a d d i t i o n of 1mM cAMP d i d s i g n i f i c a n t l y i n c r e a s e J R , J N a , J C 1 ' J " C a , Jp,and Jg . E x p e r i m e n t s on Rhodnius suggest t h a t cAMP a c t s f i r s t on an e l e c t r o g e n i c c a t i o n pump i n the a p i c a l membrane and l a t e r on the u p h i l l t r a n s p o r t of C l ~ a c r o s s the b a s a l plasma membrane ( M a d d r e l l , 1 9 7 1 ) . T h i s t h e o r y agrees w i t h the p r e s e n t o b s e r v a t i o n of i n c r e a s e d J _ , , J„, and J X T C l r\ N a a f t e r s t i m u l a t i n g A. d o r s a l i s t u b u l e s . M a d d r e l l ( 1 9 7 7 ) has h y p o t h e s i z e d t h a t Na + and K + t r a n s p o r t i n i n s e c t M a l p i g h i a n t u b u l e s o c c u r s by a common a p i c a l pump and the predominant i o n t r a n s p o r t e d depends on r e l a t i v e i n t r a c e l l u l a r l e v e l s . As most i n s e c t s s e c r e t e f l u i d w i t h h i g h K + - t o - N a + r a t i o s , J R s h o u l d i n c r e a s e more than J N a a f t e r s t i m u l a t i o n so t h a t t r a n s p o r t of KC1 would be 101 p r i m a r i l y r e s p o n s i b l e f o r d r i v i n g f l u i d s e c r e t i o n ( P h i l l i p s , 1981). The p r e s e n t r e s u l t s agree w i t h t h i s p a t t e r n i n t h a t J„ and J _ , i n c r e a s e d more t h a t J„ ( F i g . 6 ) . Of i n t e r e s t i s K c i Na 3 the i n c r e a s e d J g a f t e r a d d i t i o n of 1mM cAMP. T h i s f i n d i n g i s s i m i l a r t o t h a t of M a d d r e l l and P h i l l i p s (1978) f o r A. t a e n i o r h y c h u s t u b u l e s which show i n c r e a s e d r a t e of SO,,2' t r a n s p o r t as r a t e of f l u i d s e c r e t i o n i s i n c r e a s e d . T h i s i n d i c a t e s the p o s s i b i l i t y t h a t the i n c r e a s e d f l u i d s e c r e t i o n r a t e reduces the c o n c e n t r a t i o n d i f f e r e n c e d r i v i n g back d i f f u s i o n of S from lumen t o the b a t h i n g s o l u t i o n ; t h i s has been h y p o t h e s i z e d f o r o t h e r t u b u l e s ( P h i l l i p s , 1 9 8 1 ) . S i m i l a r l y J p and J " C a are s i g n i f i c a n t l y i n c r e a s e d i n the p r e s e n c e of 1mM cAMP i n the b a t h i n g medium, s u g g e s t i n g t h a t at low r a t e s of f l u i d s e c r e t i o n , the c o n c e n t r a t i o n s of P and C a 2 + r i s e ( i n the s e c r e t e d f l u i d ) so t h a t net d i f f u s i o n of these i o n s through the t u b u l e w a l l i n t o the b a t h i n g s o l u t i o n i s d e c r e a s e d , t h e r e b y r e d u c i n g the r a t e of e l i m i n a t i o n of P and C a 2 + . Changes i n i n A. d o r s a l i s t u b u l e s i s n e g l i g i b l e a f t e r a d d i t i o n of 1mM cAMP t o the b a t h i n g medium which i s s u g g e s t i v e t h a t back f l u x ( i . e . p e r m e a b i l i t y ) i s n o r m a l l y low. As mentioned p r e v i o u s l y , d e t e r i o r a t i o n of f l u i d s e c r e t i o n r a t e s from 30 t o 90 minutes ( F i g . 3) i n d i c a t e s reduced v i a b i l i t y of A. d o r s a l i s M a l p i g h i a n t u b u l e s or d e c r e a s e d s t i m u l a t i o n . I t has been p o s t u l a t e d t h a t 5 - H y d r o x y t r y p t a m i n e , a p o t e n t s t i m u l a t o r of s e c r e t i o n 1 02 through m e d i a t i o n of cAMP, i s broken down or e l i m i n a t e d by Rhodnius t u b u l e s a f t e r 30 minutes when added a t c o n c e n t r a t i o n s of 10~ 7 M t o the b a t h i n g d r o p l e t . I t i s known t h a t M a l p i g h i a n t u b u l e s break down the d i u r e t i c hormone ( M a d d r e l l , 1 9 6 4 ; P i l c h e r , 1969). P o s s i b l y , the r e d u c t i o n of f l u i d s e c r e t i o n r a t e a t 90 minutes may be due t o the f a c t t h a t e x t e r n a l l y a p p l i e d cAMP i s broken down, s i m i l a r l y t o 5-Hydroxytryptamine. The d e t e r i o r a t i o n of s e c r e t i o n r a t e a t 90 minutes i n t u b u l e s not exposed t o cAMP may be due t o e l i m i n a t i o n of DH p r e s e n t on membrane s i t e s a t the b e g i n n i n g of the expe r i m e n t . Lack of a c o n s i s t e n t d e c l i n e i n t r a n s e p i t h e l i a l p o t e n t i a l r e a d i n g s w i t h time ( T a b l e 5) a l s o i n d i c a t e d t h a t t u b u l e s were v i a b l e over the 90 minute e x p e r i m e n t a l p e r i o d . E l e c t r o p o t e n t i a l measurements a t 90 minutes d i d not d e v i a t e more than 6mV f o r any t u b u l e from measurements taken a t 30 min u t e s . As observ e d by P h i l l i p s and M a d d r e l l (1974) on A. c a m p e s t r i s t u b u l e s , A. d o r s a l i s t u b u l e s had a t r a n s - w a l l p o t e n t i a l which was on average lumen n e g a t i v e but v a r i a b l e . However, as the medium b a t h i n g A. c a m p e s t r i s t u b u l e s was a l t e r e d t o a K - r i c h C l - p o o r one, t h e t u b u l e s ' lumina became more p o s i t i v e w i t h r e s p e c t t o the b a t h . The a b i l i t y of A. c a m p e s t r i s t u b u l e s t o t r a n s p o r t Mg a g a i n s t a l a r g e c o n c e n t r a t i o n g r a d i e n t and a g a i n s t an e l e c t r i c a l g r a d i e n t i n d i c a t e s t h a t an a c t i v e t r a n s p o r t mechanism i s i n v o l v e d . I t would be of i n t e r e s t t o p e r f o r m s i m i l a r a l t e r a t i o n s i n 103 KC1 l e v e l s i n the medium b a t h i n g A. d o r s a l i s t u b u l e s t o s u b s t a n t i a t e t h a t t r a n s p o r t o c c u r s a g a i n s t both a c o n c e n t r a t i o n as w e l l as an e l e c t r i c a l g r a d i e n t s i m u l t a n e o u s l y , a l t h o u g h i t a p p a r e n t l y d i d i n thos e few t u b u l e s w i t h lumen p o s i t i v e p o t e n t i a l s . P r o p e r t i e s of Mg T r a n s p o r t Having c h a r a c t e r i z e d the i n v i t r o p r e p a r a t i o n of M a l p i g h i a n t u b u l e s , i n c l u d i n g t h e i r c a p a c i t y t o t r a n s p o r t Mg, I t e s t e d my main h y p o t h e s i s t h a t M g 2 + t r a n s p o r t i s d r i v e n p r e d o m i n a n t l y by c o u n t e r t r a n s p o r t w i t h N a + . T h i s h y p o t h e s i s was not su p p o r t e d by k i n e t i c , N a - s u b s t i t u t i o n , or i n h i b i t o r s t u d i e s . The, d a t a i n Table 7 suggest t h a t the i n c r e a s e d J M g which o c c u r s when s a l i n e Mg l e v e l s a r e r a i s e d c o u l d not oc c u r by a 2Na +/Mg 2 + n e u t r a l , or a 1 N a + / l M g 2 + e l e c t r o g e n i c exchanger at the l u m i n a l b o r d e r . J N g i s 12 t o 20 pmole/min-mm when i s low (eg. b a s e l i n e 3.7 a t 0 or 0.5 mM e x t e r n a l Mg 2 + i n the s a l i n e ; T a b l e 7 ) . R a i s i n g s a l i n e Mg t o 4mM ( s e r i e s 6, T a b l e 7) causes an i n c r e a s e i n JMg °^ 8pmole/min-mm or 16 pEquiv./min-mm but a t most J N a may d e c l i n e by 4 pEquiv./min-mm ( F i g . 13.2). However, the maximum p o s s i b l e d e c l i n e i n of 11 pEquiv./min-mm (see 0.5 mM, s e r i e s b t o 4mM i n T a b l e 7; F i g . 12.2) would be s u f f i c i e n t f o r a 2K +/Mg 2 + n e u t r a l exchanger. A l t h o u g h c o r r e s p o n d i n g d e c r e a s e s i n J R does not occur w i t h i n c r e a s e s i n a t each c o n c e n t r a t i o n of Mg t e s t e d i n the e x t e r n a l 1 04 s o l u t i o n ( F i g . 12.2), g e n e r a l t r e n d s a re e v i d e n t over a 90 minute p e r i o d ( i . e . as J„ d e c r e a s e s , J„ i n c r e a s e s ) . At Mg K each c o n c e n t r a t i o n of e x t e r n a l Mg, K c o n c e n t r a t i o n d e c r e a s e s as Mg c o n c e n t r a t i o n i n c r e a s e s i n the s e c r e t i o n ( F i g . 12.1). An i n v e r s e r e l a t i o n s h i p , however, does not e x i s t between Na and Mg c o n c e n t r a t i o n s a t a l l c o n c e n t r a t i o n s of e x t e r n a l Mg t e s t e d ( F i g . 13.1). In a d d i t i o n , d e c r e a s e s i n Na (of 10 mEquiv./L) i s not s u f f i c i e n t t o e x p l a i n i n c r e a s e i n Mg (of 40mEquiv./L) over the range of e x t e r n a l Mg t e s t e d . Thus da t a i n t h i s e x p e r i m e n t a l s e r i e s s u g g e s t s t h a t a Na/Mg 2 + c o u n t e r t r a n s p o r t p r o c e s s i s u n l i k e l y i n A. d o r s a l i s t u b u l e s because the ex p e c t e d i n c r e a s e d r e c o v e r y of l u m i n a l Na + from the b a s e l i n e s e c r e t i o n t o b a l a n c e i n c r e a s e J w i s not Mg ob s e r v e d . However, t h e r e i s a d r i v i n g f o r c e f o r back f l u x of l u m i n a l . K + ( h i g h ) t o the hemocoel s i d e (low K + ) and l u m i n a l K + may d e c l i n e enough t o account f o r obser v e d i n c r e a s e s i n J., . However, t h e r e i s no c o n s i s t e n t Mg r e l a t i o n s h i p between and or l u m i n a l K l e v e l s i n o t h e r t y p e s of e x p e r i m e n t s . The o n l y o t h e r e v i d e n c e s u g g e s t i n g b a c k f l u x of K d r i v e s J"Mg i s shown i n the bumetanide study ( F i g . 26) where and J R both decrease s i g n i f i c a n t l y and t o the same magnitude ( 2 - f o l d ) i n t u b u l e s t r e a t e d w i t h bumetanide. Other s t u d i e s do not suggest t h a t b a c k f l u x of K d r i v e s J w . When t u b u l e s were t r e a t e d w i t h a m i l o r i d e , J„ Mg K 105 d e c r e a s e d s i g n i f i c a n t l y whereas d i d not decrease s u b s t a n t i a l l y or s i g n i f i c a n t l y . In N a - s u b s t i t u t e d w i t h c h o l i n e s t u d y , J R and J M ^ d i d not decrease s i g n i f i c a n t l y ; t h e r e f o r e , no c o n c l u s i o n s can be made about the e f f e c t of de c r e a s e d J R on J" Mg. In the presence of cAMP i n the b a t h i n g s o l u t i o n , J K i n c r e a s e d w h i l e no s i g n i f i c a n t changes i n J M ^ was n o t e d . F u r t h e r m o r e , i n N a - s u b s t i t u t e d w i t h K s t u d y , J K d e c r e a s e d s i g n i f i c a n t l y and J M g i n c r e a s e d s i g n i f i c a n t l y s u g g e s t i n g t h a t a K/Mg exchange mechanism i s u n l i k e l y s i n c e J" M i n c r e a s e d r a t h e r than d e c r e a s e d a l o n g w i t h J R . To o b t a i n a more a c c u r a t e c o r r e l a t i o n of i o n f l u x e s w i t h Mg f l u x , the k i n e t i c s of Mg would have t o be "studied f u r t h e r a t more c o n c e n t r a t i o n s t o o b t a i n an a c c u r a t e k i n e t i c s p l o t . When Na was s u b s t i t u t e d w i t h c h o l i n e , J X T was ' Na reduced t o 101 or c o n t r o l v a l u e s (Table 7) w h i l e J w , Jv, Mg K J c ^ , and Jp were a l l s i m i l a r or g r e a t e r than c o n t r o l v a l u e s ( T a b l e 7 ) . T h i s does not support the h y p o t h e s i s of a Na/Mg exchange mechanism s i n c e d e c r e a s e s i n would be ex p e c t e d w i t h c o r r e s p o n d i n g d e c r e a s e s i n l u m i n a l Na l e v e l s and ^ N a • Any d e t e r i o r a t i o n of t u b u l e s over the 60 minute p e r i o d d i d not a f f e c t r a t e of f l u x e s s i g n i f i c a n t l y , except f o r Ca 1 06 ( F i g . 17). In a d d i t i o n , the c o n c e n t r a t i o n of i o n s (except P and S) i n the s e c r e t e d f l u i d d i d not change s i g n i f i c a n t l y over the e x p e r i m e n t a l p e r i o d ( F i g . 16). However, t o o b t a i n a more s i g n i f i c a n t c o r r e l a t i o n between i o n s , a p r o p e r c o n t r o l would have t o be used i n t h i s experiment w i t h c o n c e n t r a t i o n s of s o l u t e s s i m i l a r t o e x p e r i m e n t a l s a l i n e e x c ept f o r Na; i n a d d i t i o n cAMP can be added t o keep r a t e s of f l u i d s e c r e t i o n c o n s t a n t over a l o n g e r p e r i o d of t i m e . When Na was s u b s t i t u t e d w i t h K, f l u i d s e c r e t i o n r a t e s d i d not a l t e r s i g n i f i c a n t l y , i n d i c a t i n g s i m i l a r v i a b i l i t y as c o n t r o l t u b u l e s ( F i g . 18). When Na c o n c e n t r a t i o n i n the s e c r e t e d f l u i d was d e c r e a s e d t o 2mM from l5mM, Mg l e v e l s i n the s e c r e t i o n . i n c r e a s e d s i g n i f i c a n t l y t o 23mM from 6mM ( F i g . 19). In a d d i t i o n , as J N a d e c r e a s e d s i g n i f i c a n t l y by 1 2 - f o l d , i n c r e a s e d s i g n i f i c a n t l y by 3 - f o l d . T h i s s t r o n g l y s u g g e s t s t h a t a Na/Mg exchange mechanism i s u n l i k e l y s i n c e Mg i n c r e a s e s r a t h e r than d e c r e a s e s i n response t o d e c r e a s e d J * N a . I t i s i n t e r e s t i n g t h a t J R and i n Na s u b s t i t u t e d w i t h K s t u d y a r e l e s s t h a t J" K and J c ^ i n c o n t r o l study a l t h o u g h h i g h e r c o n c e n t r a t i o n s of K and CI are found i n the b a t h i n g s o l u t i o n of the former. Perhaps Na was not p r e s e n t i n h i g h enough c o n c e n t r a t i o n t o s t i m u l a t e KC1 t r a n s p o r t ( i . e . 2mM Na i n the b a t h i n g s a l i n e i s lower than the Kffl r e q u i r e d t o s t i m u l a t e KC1 t r a n s p o r t ) . I t has been shown i n p r e v i o u s e x p e r i m e n t s 1 07 t h a t Na i s r e q u i r e d ( i n the b a t h i n g medium) t o d r i v e K t r a n s p o r t when c o n c e n t r a t i o n of K i s low i n the b a t h i n g medium ( P h i l l i p s , 1981). Perhaps a s i m i l a r mechanism e x i s t s f o r A. d o r s a l i s M a l p i g h i a n t u b u l e s and Na i s r e q u i r e d i n the b a t h i n g medium. t o s t i m u l a t e KC1 t r a n s p o r t even when c o n c e n t r a t i o n of K i s h i g h i n the medium. To f u r t h e r study i n v o l v e m e n t of R + i n Mg 2 + t r a n s p o r t , K + c o n c e n t r a t i o n s s h o u l d be v a r i e d perhaps by u s i n g PAH t r a n s p o r t t o d r i v e f l u i d s e c r e t i o n t o study the c o r r e l a t i o n between J R and J* Mg-When a m i l o r i d e (imM) i s added t o the b a t h i n g s o l u t i o n , j " N a i s i n h i b i t e d by 81%, J R by 90%, J c l by 80%, JCa ky r Jp by 44%, J v by 80% whereas J M g and J g a r e not de c r e a s e d s u b s t a n t i a l l y or s i g n i f i c a n t l y ( F i g . 21, 2 3 ) . I t has been p r e v i o u s l y shown t h a t a m i l o r i d e i n h i b i t s J v of t u b u l e s of the t s e t s e f l y which l i k e o t h e r b l o o d f e e d i n g i n s e c t s , s e c r e t e s N a C l - r i c h r a t h e r than K C l - r i c h f l u i d . However, even KC1 s e c r e t i n g t u b u l e s o f t e n a r e s t i m u l a t e d by low l e v e l s of Na when e x t e r n a l K + i s low ( P h i l l i p s , 1981). One e x p l a n a t i o n of t h i s e f f e c t i s t h a t K + i s pumped i n t o t u b u l e c e l l s from the hemolymph i n exchange f o r c e l l u l a r Na + by a t y p i c a l Na +/K +-ATPase: Na + then l e a k s down a l a r g e e l e c t r o c h e m i c a l g r a d i e n t from lumen t o c e l l ( i . e . i s r e c y c l e d ) . In t h i s way Na t h a t i s r e c y c l e d can be exchanged f o r Mg. However, Mg s e c r e t i o n i s seen a g a i n t o be independent of changes i n J N a and i n t h i s case a l s o J R . 108 Furosemide, bumetanide, and p i r e t a n i d e a r e known t o i n h i b i t Na:K:2Cl t r a n s p o r t p r o c e s s i n e r y t h r o c y t e s ( P a l f r e y e t a l . , 1980), E h r l i c h a s c i t e s c e l l (Geek et a l . , 1 9 8 1 ) , shark r e c t a l g l a n d ( P a l f r e y e t a l . , 1 9 7 9 ) , and c o r t i c a l t h i c k a s c e n d i n g l i m b of r a b b i t ( S c h l a t t e r e t a l . , 1983). The c o n c e n t r a t i o n s of furosemide and bumetanide f o r h a l f - m a x i m a l i n h i b i t i o n of a c t i v e t r a n s p o r t i n r a b b i t c o r t i c a l t h i c k a s c e n d i n g l i m b was 5x10"" M and 2x10" 7 from the lumen and b a t h r e s p e c t i v e l y . In the shark r e c t a l g l a n d these i n h i b i t o r s a r e l e s s e f f i c i e n t than they a r e i n the cTAL segment by a f a c t o r of about 100. In a d d i t i o n , i t i s known t h a t f u r o s e m i d e , a t 10" 3 M, causes i n h i b i t i o n of g l y c o l y s i s and Na/K-ATPase ( K l a h r e t a l . , 1 9 7 1 ) so h i g h e r l e v e l s of t h e s e ' d i u r e t i c s ' may not g i v e m e a n i n g f u l i n f o r m a t i o n . A l t h o u g h t h e s e a c t i o n s have not been i n v e s t i g a t e d f o r bumetanide, t h i s p o s s i b i l i t y remains. On t h i s b a s i s , a c o n c e n t r a t i o n of 5x10" 5 M bumetanide was chosen f o r t h i s s t u d y . At the c o n c e n t r a t i o n of bumetanide used, J N a was not s i g n i f i c a n t l y d e c r e a s e d (P=0.05) a l t h o u g h J M g , J R , J c i ' an<^ J p were s i g n i f i c a n t l y d e c r e a s e d (P=0.05); ( F i g . 26). T h i s a g a i n s u g g e s t s Na independence of Mg f l u x . I t has been proposed i n Rhodnius t u b u l e s ( M a d d r e l l , 1 9 6 9 ) t h a t Na + d r i v e s C I " e n t r y i n t o c e l l s a c r o s s the b a s o l a t e r a l membrane by a c o t r a n s p o r t mechanism and Na + i s then pumped out a c r o s s the b a s o l a t e r a l membrane i n exchange f o r K +. In t h i s way Na + i s r e c y c l e d and may s t i m u l a t e C I " and K + s e c r e t i o n ( P h i l l i p s , 109 1981) and a l s o M g 2 + s e c r e t i o n . S c h l a t t e r e t a l . (1983) have proposed a s i m i l a r mechanism f o r (Na:K:2Cl t r a n s p o r t ) a c r o s s the TAL of r a b b i t t u b u l e s . R e s u l t s from these v e r t e b r a t e s t u d i e s i n d i c a t e t h a t furosemide causes a g r e a t e r i n h i b i t i o n of magnesium and c a l c i u m t r a n s p o r t over t h a t of- sodium (Quamme,1978). T h e r e f o r e , a Na-independent component i s s u g g e s t e d a g a i n . A h i g h e r c o n c e n t r a t i o n of bumetanide s h o u l d be t e s t e d t o see i f i t might i n h i b i t J N a s i n c e a t low c o n c e n t r a t i o n s I found o n l y i n h i b i t i o n of J„, J„,, and J w J K C l Mg ( F i g . 2 1 ) . T h e r e f o r e , the a c t i o n of bume'tanide found i n t h i s s t u d y i s d i f f i c u l t , t o i n t e r p r e t e i n terms of past s t u d i e s mentioned above. The d e c rease i n may be e x p l a i n e d by a d e c r e a s e i n J R ; p o s s i b l y b a c k f l u x of K from the s e c r e t e d f l u i d i n t o the c e l l d r i v e s Mg s e c r e t i o n . T h e r e f o r e , i f J„ i s d e c r e a s e d and l u m i n a l K c o n c e n t r a t i o n i s l o w e r , l e s s K i s a v a i l a b l e t o d i f f u s e from the s e c r e t e d f l u i d i n t o the c e l l t o d r i v e Mg s e c r e t i o n and t h e r e f o r e J w Mg would d e c r e a s e . I t seems u n l i k e l y t h a t t r a n s p o r t of an a n i o n ( b e s i d e s C l " ) accompanies Mg 2 + because i n c o n s i s t e n t c o r r e l a t i o n s between P, S, and Mg are o b s e r v e d i n k i n e t i c s t u d i e s (Table 7 ) . O r g a n i c a n i o n s and HC0 3" a l s o are p r o b a b l y not major components i n the s e c r e t i o n because the d i f f e r e n c e between t o t a l c a t i o n s and a n i o n s i n the s e c r e t e d 110 f l u i d d i d not v a r y more than 1OmM i n a l l e x p e r i m e n t s , e x c e p t c o n t r o l s used i n a m i l o r i d e and bumetanide e x p e r i m e n t s . The amount of bound as opposed t o f r e e Mg 2 + i n the s e c r e t i o n i s a l s o u n c e r t a i n . To dete r m i n e the amount of " f r e e " Mg, M g - s e l e c t i v e m i c r o e l e c t r o d e s would be r e q u i r e d t o d e t e r m i n e the a c t i v i t y of Mg. However, the p o s s i b i l i t y of an i n t r a c e l l u l a r p o o l of bound M g 2 + which can be m o b i l i z e d t o f r e e Mg 2 + i s p o s s i b l e . Indeed, the f a c t t h a t t u b u l e s bathed i n s a l i n e c o n t a i n i n g OmM Mg 2 + c o n t i n u e d t o s e c r e t e s u b s t a n t i a l amounts of t h i s c a t i o n suggests t h a t such a p o o l e x i s t s ( F i g . 10). P r e v i o u s s t u d i e s ( S c a r p a , 1974) i n d i c a t e t h a t M g 2 + as w e l l as C a 2 + e x i s t l a r g e l y as "bound" f r a c t i o n s i n c o n c r e t i o n s found i n M a l p i g h i a n t u b u l e c e l l s . T h i s , would a l t e r the a c t u a l v a l u e of Mg found i n s e c r e t e d f l u i d . An i n d u c t i o n of Mg t r a n s p o r t i n A. d o r s a l i s t u b u l e s was not c o n v i n c i n g l y shown i n t h i s study because Mg t r a n s p o r t o n l y i n c r e a s e d s i g n i f i c a n t l y over c o n t r o l s i n t u b u l e s from l a r v a e r e a r e d i n 126mM Mg-waters when bathed i n  v i t r o i n 1mM Mg s a l i n e . P o s s i b l y the 26mM Mg i n the c o n t r o l s a l i n e was s u f f i c i e n t t o a c t i v a t e t u b u l a r Mg s e c r e t i o n . T h e r e f o r e , comparisons between l a r v a e r e a r e d i n Mg-free and 126mM Mg _waters might g i v e a c l e a r e r i n d i c a t i o n of i n d u c t i o n s i m i l a r t o t h a t found f o r SO f t 2 _ t r a n s p o r t i n A. t a e n i o r h y n c h u s . T h i s would be of i n t e r e s t as a c a r r i e r - m e d i a t e d mechanism i s suggested by k i n e t i c s of Mg t r a n s p o r t ( F i g . 10). J M i n c r e a s e s as the e x t e r n a l 111 c o n c e n t r a t i o n of Mg i n c r e a s e s , and J M ^ approaches a maximum at 4mM Mg ( e x t e r n a l ) f o r l a r v a e r e a r e d i n 26 and 126mM Mg w a t e r s . For l a r v a e r e a r e d i n 126mM Mg medium, V was 3 max 14.92±2.89 pmole/min-mm and K was 1.57±0.16. For l a r v a e r m r e a r e d i n 26mM Mg medium, V was 9.17±8.16 pmole/min-mm ^ ' max ^ ' w h i l e K was 1.40±0.77. R e g r e s s i o n a n a l y s i s i n d i c a t e V m max and K m of l a r v a e r e a r e d i n 26mM MgSO« medium a r e 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 o s e of l a r v a e r e a r e d i n 126mM MgSOfl medium. These r e s u l t s suggest t h a t magnesium t r a n s p o r t i s not induced a c r o s s M a l p i g h i a n t u b u l e s of A. d o r s a l i s l a r v a e when t r a n s f e r r e d from 26mM t o 126mM MgSO„ medium. However, o n l y 4 s e t s of v a l u e s were compared. I f more e x t e r n a l Mg c o n c e n t r a t i o n s were t e s t e d f o r l a r v a e r e a r e d i n low and h i g h MgSO« media, more d a t a p o i n t s would be g e n e r a t e d ; t h i s would g i v e more a c c u r a t e c o n c l u s i o n s of d i f f e r e n c e s (of V and K ) between l a r v a e r e a r e d i n 26mM max m MgSO a and 126mM MgSO„ media. T h i s t h e s i s i n d i c a t e s t h a t Mg t r a n s p o r t i n A. d o r s a l i s t u b u l e s i s a c t i v e , c a r r i e r mediated and l a r g e l y Na-independent. There i s no c o n s i s t e n t e v i d e n c e f o r Mg s e c r e t i o n by a p i c a l exchange f o r l u m i n a l K +. 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Med. 98:741-749. 1 20 APPENDIX A INFLUENCE OF CAMP ON CONCENTRATIONS OF IONS IN THE SECRETED FLUID Appendix A: Concentrations of Ions in Secreted Fluid (mM) T ime (min.) 30' i)30 i i ) 8 5112 82 + 2 12 94 Ca* + 2 . 39±0.39 2. 14±0.63 42 .62±2. 10 73.71+7.69 Na + 20.25±4.04 19.59±2.75 Cl " 64.5711.44 66.79+8.55 2.19+0.98 5.0912.09 34.00±1.91 14.7410.85 60' )34 . i )7. 26 + 3 . 7713 . 73 51 2.4310.37 1.5110.44 36.7611.58 70.22+6.95 23.1615.74 17.6412.79 59.7910.55 68.7617.47 4.3411.90 4.01+2.34 34.9811.68 14.78+0.86 90' i )29 1 i )8 7912 0613 76 93 2.98+0.36 1.7210.38 34.29+ 2.30 64.13110.72 28.9213.88 22.0616.22 53.3311 .59 65.9917.91 5.00+2.53 4.6512.69 35.35+1.95 16.7211.49 i=unstimu1ated tubules ii=stimu1ated tubules(with 1mM cAMP) 1 22 APPENDIX B JIONS AND CONCENTRATIONS OF IONS IN SECRETED FLUID 123 F i g u r e B.1 Rates of f l u i d s e c r e t i o n by A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n OmM Mg, OmM cAMP s o l u t i o n . L a r v a e were r e a r e d i n 126mM MgS04 s o l u t i o n . V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean (n=4). 125 F i g u r e B.2 C o n c e n t r a t i o n s of i o n s s e c r e t e d by A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n OmM Mg, OmM cAMP s o l u t i o n . L a r v a e were r e a r e d i n 126mM MgS04 s o l u t i o n . Mean i s average of v a l u e s a t 60' and at 90'. V e r t i c a l l i n e s a t t a c h e d t o b a r s r e p r e s e n t ±S.E. of the mean (n=4). Concentration (mM) ro o o o o cn o x 2 01 n 01 o cn in °IZI 1 27 F i g u r e B.3 S e c r e t i o n r a t e s of i o n s ( J . ) of A. d o r s a l i s 1 on M a l p i g h i a n t u b u l e s bathed i n OmM Mg, OmM cAMP s o l u t i o n . L a r v a e were r e a r e d i n 126mM MgS04 s o l u t i o n . Mean i s average of v a l u e s a t 60' and a t 90'. V e r t i c a l l i n e s a t t a c h e d t o ba r s r e p r e s e n t ±S.E. of the mean (n=4). a I2<Z H to c a ID a z O) 3: a ro in cu o cu in in (ujui-uiiu/aiouid) uoip 1 29 F i g u r e B.4 Rates of f l u i d s e c r e t i o n by A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n 0.5mM Mg, OmM cAMP s o l u t i o n . V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean ( n = l 0 ) . 131 F i g u r e B.5 C o n c e n t r a t i o n s of i o n s s e c r e t e d by A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n 0.5mM Mg, OmM cAMP s o l u t i o n . Mean i s average of v a l u e s a t 60' and at 90'. V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean ( n = l 0 ) . * r e p r e s e n t s s i g n i f i c a n t d i f f e r e n c e . Concentration (mM) ro o it o cn o 2 H J CD O 51 3 H ro cn 3 2 1 33 F i g u r e B.6 S e c r e t i o n r a t e s of i o n ( J . ) of A. d o r s a l i s 3 I on M a l p i g h i a n t u b u l e s bathed i n 0.5mM Mg, OmM cAMP s o l u t i o n . Mean i s average of v a l u e s a t 60' and at 90'. V e r t i c a l l i n e s a t t a c h e d t o ba r s r e p r e s e n t ±S.E. of the mean (n=!0). X E (0 CU H - H G C o 10 o o ( U l U I - U X U I / B I O U I C J ) U O t p 1 35 F i g u r e B.7 F l u i d s e c r e t i o n r a t e s of A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n 1mM Mg, OmM cAMP s o l u t i o n . V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean (n=13). /3b (VI o o d CD a ID a ( U I U I — U T U i / I U ) A P 1 37 F i g u r e B.8 C o n c e n t r a t i o n s of i o n s s e c r e t e d by A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n 1mM Mg, OmM cAMP s o l u t i o n . Mean i s average of v a l u e s a t 60' and at 90'. V e r t i c a l l i n e s a t t a c h e d t o b a r s r e p r e s e n t ±S.E. of the mean (n=13). * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e . E to (VI ^ \ \ \ \ \ \ \ ^ rfl c I I I I I I L o r» a to o in o a tn a CM 2 o (Wui) uoi^ej^uasuoQ 139 F i g u r e B.9 S e c r e t i o n r a t e s of i o n ( J . ) of A. d o r s a l i s 3 1 on M a l p i g h i a n t u b u l e s bathed i n 1mM Mg, OmM cAMP s o l u t i o n . Mean i s average of v a l u e s a t 60' and at 90'. V e r t i c a l l i n e s a t t a c h e d t o b a r s r e p r e s e n t ±S.E. of the mean (n=13). * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e . Hi 6 l I I 1 1 L o in o CO CM OJ Q i  o in o m o (uiui-uTui/atQuid) u o i p 141 F i g u r e B.10 F l u i d s e c r e t i o n r a t e s of A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n 2mM Mg, OmM cAMP s o l u t i o n . V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ' ±S.E. of the mean ( n = 1 l ) . 1 43 F i g u r e B.11 C o n c e n t r a t i o n s of i o n s s e c r e t e d by A . d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n 2mM Mg, OmM cAMP s o l u t i o n . Mean i s average of v a l u e s a t 60' and at 90'. V e r t i c a l l i n e s a t t a c h e d t o b a r s r e p r e s e n t ±S.E. of the mean ( n = 1 l ) . C o n c e n t r a t i o n (mM) ro o o -j r u & n oi M o o o o o 1 1 1 1 1 \\\\\\\\\\\\\\^^H 3 3 3< 3 1 45 F i g u r e B.12 S e c r e t i o n r a t e s of i o n ( J . ) of A. d o r s a l i s 3 i o n M a l p i g h i a n t u b u l e s bathed i n 2mM Mg, OmM cAMP s o l u t i o n . Mean i s average of v a l u e s a t 60' and at 90'. V e r t i c a l l i n e s a t t a c h e d t o ba r s r e p r e s e n t ±S.E. of the mean ( n = 1 l ) . z z E IO OJ r - r ^ N \ \ \ \ \ ^ r — ^ ^ ^ ^ ^ ^ ^ i 1 H I I i L in OJ o Ol in in (umi-u;iu/atou/d) uotr* 1 47 F i g u r e B.13 F l u i d s e c r e t i o n r a t e s of A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n 4mM Mg, OmM cAMP s o l u t i o n . V e r t i c a l l i n e s a t t a c h e d t o p o i n t s r e p r e s e n t ±S.E. of the mean (n=14). 1 49 F i g u r e B.14 C o n c e n t r a t i o n s of i o n s s e c r e t e d by A. d o r s a l i s M a l p i g h i a n t u b u l e s bathed i n 4mM Mg, OmM cAMP s o l u t i o n . Mean i s average of v a l u e s a t 60' and at 90'. V e r t i c a l l i n e s a t t a c h e d t o bars r e p r e s e n t ±S.E. of the mean (n=14). * r e p r e s e n t s s i g n i f i c a n t d i f f e r e n c e . Concentration (mM) ru a o cn o CD O r-H I 1 5 H 3 3 N \ \ \ \ \ \ \ \ ^ f H o o 1 ro cn 151 F i g u r e B.15 S e c r e t i o n r a t e s of i o n ( J . ) of A. d o r s a l i s 3 i o n M a l p i g h i a n t u b u l e s bathed i n 4mM Mg, OmM cAMP s o l u t i o n . Mean i s average of v a l u e s a t 60' and at 90'. V e r t i c a l l i n e s a t t a c h e d t o ba r s r e p r e s e n t ±S.E. of the mean (n=14). C 1 - f H I-BZ: o a m o cu (uiaj-u;ui/etoujd) uo\p 

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