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Demonstration of anionic sites in rat myocardium and isolated cardiac myocytes with polyethylenimine
j Mol Cell Cardiol 18 (Supplement 2) (1986) 7ADRENERGIC MECHANISMS IN THE CALCIUM PARADOX? A.N. @ksendal, P. Jynge. Department of Pharmacology and Toxicology, Faculty of Medicine, University of Trondheim, Norway. The aim of the present study was to establish the potential role of the adrenergic system in the calcium paradox (CaPa). Two injur~ levels (minimal and total CaPa) caused by different volumes (5 and 45 ml) of CaZ+-free perfusion (5 min) prior to calcium repletion (15 min) were examined in the normothermic isolated rat heart model. The presence of the ~-blocker propranolol (5 9 10 -6 M) or the el-blocker prazosin (10-7 M) in the perfusion media (i0 min prior to, during and 5 min following Ca2+-free perfusion) had no effect upon tissue injury in any of the two injury models (total and minimal) of the CaPa. Supplementation of noradrenaline (10-8 M) i0 min prior to calcium depletion depressed the myocardial function (LV-contracture, LVDP decrease) and metabolism (CP 45% and ATP 65% of control). However, this supplementation of noradrenaline significantly reduced the myocardial tissue injury in the minimal CaPa, as assessed by CK-release and initial contracture during repletion, and final values of CP and Ca2+ content (whole tissue). The presence of propranolol or prazosin combined with noradrenaline did not induce any further changes. It is concluded that the release of endogenous catecholamines may not be an important factor contributing to myocardial injury in the CaPa. The noradrenaline-induced reduction in the minimal CaPa may be related to a decrease in cellular energy prior to calcium repletion or to an increase in cellular calcium prior to calcium depletion.
8DEMONSTRATION OF ANIONIC SITES IN RAT MYOCARDIUM AND ISOLATED CARDIAC MYOCYTES WITH POLYETHYLENIMINE. P. Kirchner, Jutta Schaper, Max-Planck-Institute, Bad Nauheim, FRG Knowledge of distribution and localisation of anionic sites (AS) on cell surfaces of cardiac tissue and isolated myocytes (ICM) is increasingly important for receptor related studies and also reflects the binding properties of cell membranes (M). We analysed AS in vivo in myocardium and in vitro on ICM Using electron microscopy (EM) after labeling with polyethylenimine (PEI), a protein precipitant with strong cationic character. Isolated rat hearts were perfused by the Langendorff method with modified Krebs-Ringer-Hepes buffer containing 0.05% PEI and perfusion fixed. ICM were obtained by enzymatic digestion (0.06% collagenase) of isolated, Langendorff perfused hearts and incubated in buffered PEI. EM revealed regular deposition of PEI particles roughly 20nm in diameter in the lamina densa of the basement M of myocytes and vascular endothelium in vivo. Average spacing was 70nm. PEI was present in the surface coat, in T-tubules, sarcoplasmic reticulum and intercalated discs. On ICM a regular deposition was absent. Diffuse and inhomogeneous PEI deposits occurred on the sarcolemma and in ICM with sarcolemmal defects PEI was deposited intracellularly. Conclusion: a regular distribution pattern of AS can be morphologically demonstrated in normal rat myocardium. The structure of cell surface M and distribution of AS is, however, strongly altered during the isolation process of ICM. This may have implications for further studies involving structures of surface M and receptors in ICM.
9 DISTRIBUTION
OF
8-HEXOSAMINIDASE
V. M a r j o m ~ k i a n d A. Jyv~skyl~, Finland.
Salminen.
A AND B ISOENZYMES
Department
of C e l l
IN M Y O C A R D I U M .
Biology,
University
of
8-Hexosaminidase (Hex), a l y s o s o m a l a c i d h y d r o l a s e , o c c u r s in t w o i s o e n z y m i c f o r m s A a n d B in m a m m a l i a n t i s s u e s . H e x A c a n be d e n a t u r e d b y h e a t i n g at 5 0 ~ C (pH 4.5) for 2 hr. T h e p e r c e n t a g e of H e x A v a r i e d b e t w e e n 34.2 a n d 36.5 a n d c o r r e s p o n d i n g l y t h a t of H e x B b e t w e e n 63.5 a n d 65.8 in the r i g h t a n d l e f t ( s u b e p i - a n d s u b e n d o m y o c a r d i a l layers) rat v e n t r i c l e s . In a t r i a the r a t i o w a s 46.8 a n d 53.2 p e r c e n t b e t w e e n H e x A a n d H e x B. In the h e a r t s of n e w b o r n m i c e t h e p e r c e n t a g e of H e x A w a s 52.6 a n d s t a y e d at a p p r o x 45 % u p to s e n e s c e n c e ( p < 0.05). A f r a c t i o n a t i o n s t u d y s h o w e d t h a t t h e P e r c e n t a g e s of H e x A a n d H e x B w e r e n e a r l y e q u a l (Hex A r a n g e d b e t w e e n 37.8 a n d 47.7 %) in s a r c o l e m m a l , s a r c o p l a s mic reticulum and mitochondrial fractions, although the total activity of B - H e x w a s s i g n i f i c a n t l y s m a l l e r in m i t o c h o n d r i a l f r a c t i o n . T h e d i s t r i b u t i o n of 8 - H e x to free, r e l e a s a b l e (by T r i t o n X - 1 0 0 ) a n d m e m b r a n e b o u n d f r a c t i o n s s h o w e d t h a t H e x A v a l u e w a s l o w e s t in f r e e f r a c t i o n a n d h i g h e s t in r e l e a s a b l e f r a c t i o n , p o s s i b l y o r i g i n a t i n g in l y s o s o m e s .
8DEMONSTRATION OF ANIONIC SITES IN RAT MYOCARDIUM AND ISOLATED CARDIAC MYOCYTES WITH POLYETHYLENIMINE. P. Kirchner, Jutta Schaper, Max-Planck-Institute, Bad Nauheim, FRG Knowledge of distribution and localisation of anionic sites (AS) on cell surfaces of cardiac tissue and isolated myocytes (ICM) is increasingly important for receptor related studies and also reflects the binding properties of cell membranes (M). We analysed AS in vivo in myocardium and in vitro on ICM Using electron microscopy (EM) after labeling with polyethylenimine (PEI), a protein precipitant with strong cationic character. Isolated rat hearts were perfused by the Langendorff method with modified Krebs-Ringer-Hepes buffer containing 0.05% PEI and perfusion fixed. ICM were obtained by enzymatic digestion (0.06% collagenase) of isolated, Langendorff perfused hearts and incubated in buffered PEI. EM revealed regular deposition of PEI particles roughly 20nm in diameter in the lamina densa of the basement M of myocytes and vascular endothelium in vivo. Average spacing was 70nm. PEI was present in the surface coat, in T-tubules, sarcoplasmic reticulum and intercalated discs. On ICM a regular deposition was absent. Diffuse and inhomogeneous PEI deposits occurred on the sarcolemma and in ICM with sarcolemmal defects PEI was deposited intracellularly. Conclusion: a regular distribution pattern of AS can be morphologically demonstrated in normal rat myocardium. The structure of cell surface M and distribution of AS is, however, strongly altered during the isolation process of ICM. This may have implications for further studies involving structures of surface M and receptors in ICM.
9 DISTRIBUTION
OF
8-HEXOSAMINIDASE
V. M a r j o m ~ k i a n d A. Jyv~skyl~, Finland.
Salminen.
A AND B ISOENZYMES
Department
of C e l l
IN M Y O C A R D I U M .
Biology,
University
of
8-Hexosaminidase (Hex), a l y s o s o m a l a c i d h y d r o l a s e , o c c u r s in t w o i s o e n z y m i c f o r m s A a n d B in m a m m a l i a n t i s s u e s . H e x A c a n be d e n a t u r e d b y h e a t i n g at 5 0 ~ C (pH 4.5) for 2 hr. T h e p e r c e n t a g e of H e x A v a r i e d b e t w e e n 34.2 a n d 36.5 a n d c o r r e s p o n d i n g l y t h a t of H e x B b e t w e e n 63.5 a n d 65.8 in the r i g h t a n d l e f t ( s u b e p i - a n d s u b e n d o m y o c a r d i a l layers) rat v e n t r i c l e s . In a t r i a the r a t i o w a s 46.8 a n d 53.2 p e r c e n t b e t w e e n H e x A a n d H e x B. In the h e a r t s of n e w b o r n m i c e t h e p e r c e n t a g e of H e x A w a s 52.6 a n d s t a y e d at a p p r o x 45 % u p to s e n e s c e n c e ( p < 0.05). A f r a c t i o n a t i o n s t u d y s h o w e d t h a t t h e P e r c e n t a g e s of H e x A a n d H e x B w e r e n e a r l y e q u a l (Hex A r a n g e d b e t w e e n 37.8 a n d 47.7 %) in s a r c o l e m m a l , s a r c o p l a s mic reticulum and mitochondrial fractions, although the total activity of B - H e x w a s s i g n i f i c a n t l y s m a l l e r in m i t o c h o n d r i a l f r a c t i o n . T h e d i s t r i b u t i o n of 8 - H e x to free, r e l e a s a b l e (by T r i t o n X - 1 0 0 ) a n d m e m b r a n e b o u n d f r a c t i o n s s h o w e d t h a t H e x A v a l u e w a s l o w e s t in f r e e f r a c t i o n a n d h i g h e s t in r e l e a s a b l e f r a c t i o n , p o s s i b l y o r i g i n a t i n g in l y s o s o m e s .