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Use of 2-deoxyglucose-6-phosphate as an indicator to determine intracellular pH in the perfused guinea-pig heart by 31P NMR spectroscopy
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M E C H A N I S M S C O N T R O L L I N G Ca 2+ D I S T R I B U T I O N IN HEART. G.J. B A R ~ I T T and A.M. LEE. D e p a r t m e n t of Clinical Biochemistry, Flinders U n i v e r s i t y School of Medicine, F l i n d e r s Medical Centre, Bedford Park, South A u s t r a l i a 5042. Key elements w h i c h control the c o n c e n t r a t i o n of free Ca 2+ in the m y o p l a s m in cardiac muscle cells are the stores of Ca 2+ on the g l y c o c a l y x and in the sarcop l a s m i c reticulum, and the a c t i v i t y of the t r a n s p o r t e r s w h i c h move Ca 2+ across the m e m b r a n e s of the s a r c o l e m m a and s a r c o p l a s m i c reticulum. In o r d e r to determine the relative c o n t r i b u t i o n made by each of these e l e m e n t s e x p e r i m e n t s w e r e p e r f o r m e d w i t h i s o l a t e d cardiac myocytes u s i n g 45Ca2+ e x c h a n g e techniques. At 1.3 m M Ca 2+ e l e c t r i c a l s t i m u l a t i o n i n c r e a s e d by 20% the a m o u n t of 45Ca2+ e x c h a n g e d in the p e r i o d 0.25-3 min following the addition of 45Ca2+ to i s o l a t e d myocytes. For b e a t i n g myocytes the a m o u n t of 4 5 C a 2 + e x c h a n g e d was r e d u c e d by 15 and 80% in the presence of i0 ~M verapamil and 1 mM La 3+, respectively. I n c u b a t i o n of myocytes w i t h the a c e t o x y m e t h y l esters of the i n t r a c e l l u l a r Ca 2+ chelating agents BAPTA (BAPTA/AM) or Quin 2 led to an i n h i b i t i o n of contraction. At 0.5 m M Ca 2+ h a l f - m a x i m a l i n h i b i t i o n was given by 200 DM BAPTA/AM. The i n h i b i t o r y e f f e c t of BAPTA was almost completely r e v e r s e d by i n c r e a s i n g the concentration of Ca 2+ from 0.5 to 5.0 mM. T r e a t m e n t of cells with B A P T A / A M caused a small reduction in the a m o u n t of 45Ca2+ exchanged. It is c o n c l u d e d that B A P T A prevents the transient increase in free Ca 2+ in the m y o p l a s m w h i c h follows d e p o l a r i s a t i o n of the sarcolemma. The results indicate that this pool of Ca 2+ represents a small p r o p o r t i o n of the rapidly e x c h a n g e a b l e Ca 2+ a s s o c i a t e d w i t h the myocytes. {Supported by a grant from the National Heart F o u n d a t i o n of Australia.)
USE OF 2 - D E O X Y G L U C O S E - 6 - P H O S P H A T E AS AN I N D I C A T O R TO DETERMINE I N T R A C E L L U L A R pH IN THE P E R F U S E D G U I N E A - P I G HEART BY 31p N M R SPECTROSCOPY. W i l l i a m M. Brooks and Roger J. Willis, School of Science, Griffith University, Nathan, 4111, Australia. The 31p NMR spectrum of freshly p r e p a r e d g u i n e a - p i g heart p e r f u s e d in the Langend o r f f mode at 37~ differs from the spectrum o b t a i n e d from rat heart under similar conditions in that no inorganic p h o s p h a t e peak is present. This makes the d e t e r m i n a t i o n of i n t r a c e l l u l a r pH from the chemical shift of the inorganic p h o s p h a t e peak impossible. By incorporating 2 - d e o x y g l u c o s e - 6 - p h o s p h a t e into the heart to act as an alternative pH indicator, i n t r a c e l l u l a r pH was d e t e r m i n e d to be 6.98. In c o n t r a s t to the rat, 2 deoxyglucose was not taken up and p h o s p h o r y l a t e d by g u i n e a - p i g heart w h e n glucose was p r e s e n t in the p e r f u s i o n medium. Uptake and p h o s p h o r y l a t i o n did p r o c e e d if glucose was o m i t t e d from the m e d i u m but a large inorganic p h o s p h a t e p e a k was p r e s e n t in the intial s p e c t r u m p r i o r to 2-deoxyglucose addition. W i t h p y r u v a t e as sole energy source, an inorganic p h o s p h a t e p e a k was absent and 2-deoxyglucose was taken up and phosphorylated. During a 12 min p e r i o d of global ischaemia at 200C, when an i n o r g a n i c p h o s p h a t e p e a k was visible together with p r e v i o u s l y i n c o r p o r a t e d 2 - d e o x y g l u c o s e - 6 - p h o s p h a t e , a close correlation was found b e t w e e n the pH estimated from the chemical shift of inorganic phosphate and 2 - d e o x y g l u c o s e - 6 - p h o s p h a t e over the pH range 6.9 - 6.3.
CARDIAC PURKINJE CELLS IN CULTURE E. CANALE, J.H. CAMPBELL2~ G.R. CAMPBELL1, 1ANATOMYDEPARTMENT, UNIVERSITY OF MELBOURNE, PARKVILLE, 5052 2BAKER MEDICAL R E S E A R C H INSTITUTE, COMMERCIAL ROAD, PRAHRAN, 3131 AUSTRALIA. V o l t a g e clamp studies on Purkinje cells using single cell p r e p a r a t i o n s are limited by the relatively short time the cells can be m a i n t a i n e d in vitro. Similar studies on long term c u l t u r e d cells should be p r a c t i c a b l e and offer the a d v a n t a g e of both a c o n t r o l l e d e n v i r o n m e n t for e x p e r i m e n t a t i o n and an extended v i a b i l i t y for the tissue. This paper describes one method for the i s o l a t i o n and growth of cardiac Purkinje cells in culture. Hearts were obtained from young rabbits, pigs or fetal lambs. The false tendons were d i s s e c t e d free in Hanks' b a l a n c e d salt solution, and p l a c e d in serum-free E a g l e ' s Minima] Essential Medium with Hanks' salts c o n t a i n i n g 5 mg/ml c o l l a g e n a s e (Worthington CLS &196) at 37~ Enzymatic d i g e s t i o n was a l t e r n a t e d b e t w e e n collagenase and elastase, (i mg/ml, type III N~ Sigma) at ZTOC. A f t e r dispersal, the cell suspension was c e n t r i f u g e d and the pellet r e s u s p e n d e d in m e d i u m with 10% fetal calf serum, p r o - p l a t e d onto a plastic culture dish ( s t e r i l i n ) , and left at 57~ for Z0 min. The Purkinje cell e n r i c h e d s u s p e n s i o n was then p l a t e d onto c o l l a g e n - c o a t e d culture dishes. In culture Purkinje cells are s p o n t a n e o u s l y contractile, u s u a l l y for 4 days but up to 7 days in some cases. C o n c o m i t a n t with the loss of c o n t r a c t i l i t y there is a gradual loss of o r g a n i s a t i o n of the m y o f i b r i l s in the cells. However the cells remain viable in culture for at least two weeks, being the longest time studied.
M E C H A N I S M S C O N T R O L L I N G Ca 2+ D I S T R I B U T I O N IN HEART. G.J. B A R ~ I T T and A.M. LEE. D e p a r t m e n t of Clinical Biochemistry, Flinders U n i v e r s i t y School of Medicine, F l i n d e r s Medical Centre, Bedford Park, South A u s t r a l i a 5042. Key elements w h i c h control the c o n c e n t r a t i o n of free Ca 2+ in the m y o p l a s m in cardiac muscle cells are the stores of Ca 2+ on the g l y c o c a l y x and in the sarcop l a s m i c reticulum, and the a c t i v i t y of the t r a n s p o r t e r s w h i c h move Ca 2+ across the m e m b r a n e s of the s a r c o l e m m a and s a r c o p l a s m i c reticulum. In o r d e r to determine the relative c o n t r i b u t i o n made by each of these e l e m e n t s e x p e r i m e n t s w e r e p e r f o r m e d w i t h i s o l a t e d cardiac myocytes u s i n g 45Ca2+ e x c h a n g e techniques. At 1.3 m M Ca 2+ e l e c t r i c a l s t i m u l a t i o n i n c r e a s e d by 20% the a m o u n t of 45Ca2+ e x c h a n g e d in the p e r i o d 0.25-3 min following the addition of 45Ca2+ to i s o l a t e d myocytes. For b e a t i n g myocytes the a m o u n t of 4 5 C a 2 + e x c h a n g e d was r e d u c e d by 15 and 80% in the presence of i0 ~M verapamil and 1 mM La 3+, respectively. I n c u b a t i o n of myocytes w i t h the a c e t o x y m e t h y l esters of the i n t r a c e l l u l a r Ca 2+ chelating agents BAPTA (BAPTA/AM) or Quin 2 led to an i n h i b i t i o n of contraction. At 0.5 m M Ca 2+ h a l f - m a x i m a l i n h i b i t i o n was given by 200 DM BAPTA/AM. The i n h i b i t o r y e f f e c t of BAPTA was almost completely r e v e r s e d by i n c r e a s i n g the concentration of Ca 2+ from 0.5 to 5.0 mM. T r e a t m e n t of cells with B A P T A / A M caused a small reduction in the a m o u n t of 45Ca2+ exchanged. It is c o n c l u d e d that B A P T A prevents the transient increase in free Ca 2+ in the m y o p l a s m w h i c h follows d e p o l a r i s a t i o n of the sarcolemma. The results indicate that this pool of Ca 2+ represents a small p r o p o r t i o n of the rapidly e x c h a n g e a b l e Ca 2+ a s s o c i a t e d w i t h the myocytes. {Supported by a grant from the National Heart F o u n d a t i o n of Australia.)
USE OF 2 - D E O X Y G L U C O S E - 6 - P H O S P H A T E AS AN I N D I C A T O R TO DETERMINE I N T R A C E L L U L A R pH IN THE P E R F U S E D G U I N E A - P I G HEART BY 31p N M R SPECTROSCOPY. W i l l i a m M. Brooks and Roger J. Willis, School of Science, Griffith University, Nathan, 4111, Australia. The 31p NMR spectrum of freshly p r e p a r e d g u i n e a - p i g heart p e r f u s e d in the Langend o r f f mode at 37~ differs from the spectrum o b t a i n e d from rat heart under similar conditions in that no inorganic p h o s p h a t e peak is present. This makes the d e t e r m i n a t i o n of i n t r a c e l l u l a r pH from the chemical shift of the inorganic p h o s p h a t e peak impossible. By incorporating 2 - d e o x y g l u c o s e - 6 - p h o s p h a t e into the heart to act as an alternative pH indicator, i n t r a c e l l u l a r pH was d e t e r m i n e d to be 6.98. In c o n t r a s t to the rat, 2 deoxyglucose was not taken up and p h o s p h o r y l a t e d by g u i n e a - p i g heart w h e n glucose was p r e s e n t in the p e r f u s i o n medium. Uptake and p h o s p h o r y l a t i o n did p r o c e e d if glucose was o m i t t e d from the m e d i u m but a large inorganic p h o s p h a t e p e a k was p r e s e n t in the intial s p e c t r u m p r i o r to 2-deoxyglucose addition. W i t h p y r u v a t e as sole energy source, an inorganic p h o s p h a t e p e a k was absent and 2-deoxyglucose was taken up and phosphorylated. During a 12 min p e r i o d of global ischaemia at 200C, when an i n o r g a n i c p h o s p h a t e p e a k was visible together with p r e v i o u s l y i n c o r p o r a t e d 2 - d e o x y g l u c o s e - 6 - p h o s p h a t e , a close correlation was found b e t w e e n the pH estimated from the chemical shift of inorganic phosphate and 2 - d e o x y g l u c o s e - 6 - p h o s p h a t e over the pH range 6.9 - 6.3.
CARDIAC PURKINJE CELLS IN CULTURE E. CANALE, J.H. CAMPBELL2~ G.R. CAMPBELL1, 1ANATOMYDEPARTMENT, UNIVERSITY OF MELBOURNE, PARKVILLE, 5052 2BAKER MEDICAL R E S E A R C H INSTITUTE, COMMERCIAL ROAD, PRAHRAN, 3131 AUSTRALIA. V o l t a g e clamp studies on Purkinje cells using single cell p r e p a r a t i o n s are limited by the relatively short time the cells can be m a i n t a i n e d in vitro. Similar studies on long term c u l t u r e d cells should be p r a c t i c a b l e and offer the a d v a n t a g e of both a c o n t r o l l e d e n v i r o n m e n t for e x p e r i m e n t a t i o n and an extended v i a b i l i t y for the tissue. This paper describes one method for the i s o l a t i o n and growth of cardiac Purkinje cells in culture. Hearts were obtained from young rabbits, pigs or fetal lambs. The false tendons were d i s s e c t e d free in Hanks' b a l a n c e d salt solution, and p l a c e d in serum-free E a g l e ' s Minima] Essential Medium with Hanks' salts c o n t a i n i n g 5 mg/ml c o l l a g e n a s e (Worthington CLS &196) at 37~ Enzymatic d i g e s t i o n was a l t e r n a t e d b e t w e e n collagenase and elastase, (i mg/ml, type III N~ Sigma) at ZTOC. A f t e r dispersal, the cell suspension was c e n t r i f u g e d and the pellet r e s u s p e n d e d in m e d i u m with 10% fetal calf serum, p r o - p l a t e d onto a plastic culture dish ( s t e r i l i n ) , and left at 57~ for Z0 min. The Purkinje cell e n r i c h e d s u s p e n s i o n was then p l a t e d onto c o l l a g e n - c o a t e d culture dishes. In culture Purkinje cells are s p o n t a n e o u s l y contractile, u s u a l l y for 4 days but up to 7 days in some cases. C o n c o m i t a n t with the loss of c o n t r a c t i l i t y there is a gradual loss of o r g a n i s a t i o n of the m y o f i b r i l s in the cells. However the cells remain viable in culture for at least two weeks, being the longest time studied.