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Molecular structure of phospholamban deduced from chemical and cDNA analyses
j Mol Cell Cardiol 19 (Supplement I) (1987)
34
P L A S M A LEVELS OF A T R I A L N A T R I U R E T I C P O L Y P E P T I D E IN PATIENTS WITH CONGESTIVE HEART FAILURE. T. Tanahashi, K. Ogawa, T. Ito, M. H a s h i m o t o , A. Ishida, T. Satake. The 2nd Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan. We have measured plasma concentration of immunoreactive human atrial natriuretic polypeptide (h-ANP) and cyclic GMP by using radioimmunoassay as well as hemodynamic indices in p a t i e n t s w i t h c o n g e s t i v e h e a r t failure (CHF) and other heart diseases. P l a s m a h - A N P was s i g n i f i c a n t l y r e l a t e d to the NYHA f u n c t i o n a l classes, p u l m o n a r y artery (PA) pressure, pulmonary capillary wedge pressure (PCWP), and plasma cyclic GMP level. And high levels of h-ANP have been observed in patients with paroxysmal atrial fibrillation. Plasma h-ANP was highest in coronary sinus and decreased in the order of aorta (Ao), PA, right a t r i u m (RA), and i n f e r i o r vena cava (IVC). The d i f f e r e n c e of p l a s m a c o n c e n t r a t i o n of h - A N P b e t w e e n PA and IVC was greater in patients with higher RA pressure, and that between An and PA was greater in patients with higher PCWP. These findings suggest that, although the main source of plasma hANP is the molecules released via coronary sinus, h-ANP is released into each portion of the heart according to hemodynamics of each patient.
35
THE EFFECT OF RAPID COOLING CONTRACTURE ON THE RECOVERY FROM ANOXIA-INDUCED IMPAIRMENT OF CONTRACTILE ACTIVITY IN RAT VENTRICULAR MUSCLE. T. Okada. Department of Physiology, Juntendo University School of Medicine, Tokyo, Japan. The effect of rapid cooling contracture (RCC) on the recovery from anoxic contraeture and on the regain of tension development on reoxygenatien were examined in rat right ventricuar muscle. Muscles were made anoxic by superfusing N 2saturated, glucose-free Krebs solution at 34~ A contracture developed slowly by the anoxia and reached near the peak twitch tension level 60 min after the induction of anoxia. With reoxygenation at this point, the muscle did not show complete recovery f~om the contracture with the active tension development beeing less than 30% of the control level at 60 min after the reoxygenation. On the other hand, when the muscle was cooled to 4~ for about i0 sec (RCC) just before the reoxygenation, the recovery from anoxic contracture was significantly accelerated and almost complete. At the same time, RCC improved the regain of tension development i.e., active tension development was 50-70% of the control at 60 min after the reoxygenation. RCC similarly accelerated the recovery from contracture induced by Ca overload, but showed no e f f e c t o n the recovery from CN--induced contracture. Thus, it was suggested that RCC facilitates the recovery of mitochondrial ATP synthesis by removing excess intracellular Ca ions and it can produce the faster recovery from anoxia-induced impairment of contractile activity.
36
M O L E C U L A R S T R U C T U R E O F P H O S P H O L A M B A N D E D U C E D F R O M C H E M I C A L AND eDNA ANALYSES. M. Kadoma, J. Fujii, Y. K i m u r a , Y. K i j i m a , M. Tada. D i v i s i o n of Cardiology, First D e p a r t m e n t of M e d i c i n e and P a t h o p h y s i o l o g y , Osaka University School of Medicine, Osaka 553, Japan. Amino acid sequence of p h o s p h o l a m b a n (PN) f r o m SR e l u c i d a t e d unique characteristics. Sequence analyses of PN, cleaved by BrCN and TPCK-trypsin, showed 45 a m i n o acid r e s i d u e s s t a r t i n g w i t h NCC-acetylated Met. C o m p l e t e p r i m a r y s t r u c t u r e including the remaining C-terminal residues was clarified by cloning and sequencing eDNA encoding PN. These results showed that a phospholamban monomer consists of 52 amino acid residues with a calculated M W of 6,080. The majority of this molecule is p r e d i c t e d to e x h i b i t ~ - h e l i c a l structure. H y d r o p a t h y p r o f i l e i n d i c a t e d that PN monomer is an amphipathic molecule composed of two domains; domain I with hydrophilic c y t o p l a s m i c p o r t i o n at N - t e r m i n a l r e g i o n ( p o s i t i o n s 1-30) and d o m a i n If w i t h hydrophobic transmembrane portion at C-terminal. Domain I is further subdivided into two portions, domains IA a n d IB, at a r o u n d P r o 21. D o m a i n IA c o n t a i n s p h o s p h o r y l a t a b l e sites (Ser 16 and Thr 17), w h i c h a l l o w a l t e r a t i o n s in charge distribution, whereas domain IB serves to maintain pentameric assembly of PN monomers by intermolecular hydrogen bonding between Gln and Asn residues, stabilizing molecular assembly of PN holoprotein. It is of prime importance to define which moiety of PN should exert a direct regulatory influence on a moiety of Ca pump ATPase.
S.34
34
P L A S M A LEVELS OF A T R I A L N A T R I U R E T I C P O L Y P E P T I D E IN PATIENTS WITH CONGESTIVE HEART FAILURE. T. Tanahashi, K. Ogawa, T. Ito, M. H a s h i m o t o , A. Ishida, T. Satake. The 2nd Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan. We have measured plasma concentration of immunoreactive human atrial natriuretic polypeptide (h-ANP) and cyclic GMP by using radioimmunoassay as well as hemodynamic indices in p a t i e n t s w i t h c o n g e s t i v e h e a r t failure (CHF) and other heart diseases. P l a s m a h - A N P was s i g n i f i c a n t l y r e l a t e d to the NYHA f u n c t i o n a l classes, p u l m o n a r y artery (PA) pressure, pulmonary capillary wedge pressure (PCWP), and plasma cyclic GMP level. And high levels of h-ANP have been observed in patients with paroxysmal atrial fibrillation. Plasma h-ANP was highest in coronary sinus and decreased in the order of aorta (Ao), PA, right a t r i u m (RA), and i n f e r i o r vena cava (IVC). The d i f f e r e n c e of p l a s m a c o n c e n t r a t i o n of h - A N P b e t w e e n PA and IVC was greater in patients with higher RA pressure, and that between An and PA was greater in patients with higher PCWP. These findings suggest that, although the main source of plasma hANP is the molecules released via coronary sinus, h-ANP is released into each portion of the heart according to hemodynamics of each patient.
35
THE EFFECT OF RAPID COOLING CONTRACTURE ON THE RECOVERY FROM ANOXIA-INDUCED IMPAIRMENT OF CONTRACTILE ACTIVITY IN RAT VENTRICULAR MUSCLE. T. Okada. Department of Physiology, Juntendo University School of Medicine, Tokyo, Japan. The effect of rapid cooling contracture (RCC) on the recovery from anoxic contraeture and on the regain of tension development on reoxygenatien were examined in rat right ventricuar muscle. Muscles were made anoxic by superfusing N 2saturated, glucose-free Krebs solution at 34~ A contracture developed slowly by the anoxia and reached near the peak twitch tension level 60 min after the induction of anoxia. With reoxygenation at this point, the muscle did not show complete recovery f~om the contracture with the active tension development beeing less than 30% of the control level at 60 min after the reoxygenation. On the other hand, when the muscle was cooled to 4~ for about i0 sec (RCC) just before the reoxygenation, the recovery from anoxic contracture was significantly accelerated and almost complete. At the same time, RCC improved the regain of tension development i.e., active tension development was 50-70% of the control at 60 min after the reoxygenation. RCC similarly accelerated the recovery from contracture induced by Ca overload, but showed no e f f e c t o n the recovery from CN--induced contracture. Thus, it was suggested that RCC facilitates the recovery of mitochondrial ATP synthesis by removing excess intracellular Ca ions and it can produce the faster recovery from anoxia-induced impairment of contractile activity.
36
M O L E C U L A R S T R U C T U R E O F P H O S P H O L A M B A N D E D U C E D F R O M C H E M I C A L AND eDNA ANALYSES. M. Kadoma, J. Fujii, Y. K i m u r a , Y. K i j i m a , M. Tada. D i v i s i o n of Cardiology, First D e p a r t m e n t of M e d i c i n e and P a t h o p h y s i o l o g y , Osaka University School of Medicine, Osaka 553, Japan. Amino acid sequence of p h o s p h o l a m b a n (PN) f r o m SR e l u c i d a t e d unique characteristics. Sequence analyses of PN, cleaved by BrCN and TPCK-trypsin, showed 45 a m i n o acid r e s i d u e s s t a r t i n g w i t h NCC-acetylated Met. C o m p l e t e p r i m a r y s t r u c t u r e including the remaining C-terminal residues was clarified by cloning and sequencing eDNA encoding PN. These results showed that a phospholamban monomer consists of 52 amino acid residues with a calculated M W of 6,080. The majority of this molecule is p r e d i c t e d to e x h i b i t ~ - h e l i c a l structure. H y d r o p a t h y p r o f i l e i n d i c a t e d that PN monomer is an amphipathic molecule composed of two domains; domain I with hydrophilic c y t o p l a s m i c p o r t i o n at N - t e r m i n a l r e g i o n ( p o s i t i o n s 1-30) and d o m a i n If w i t h hydrophobic transmembrane portion at C-terminal. Domain I is further subdivided into two portions, domains IA a n d IB, at a r o u n d P r o 21. D o m a i n IA c o n t a i n s p h o s p h o r y l a t a b l e sites (Ser 16 and Thr 17), w h i c h a l l o w a l t e r a t i o n s in charge distribution, whereas domain IB serves to maintain pentameric assembly of PN monomers by intermolecular hydrogen bonding between Gln and Asn residues, stabilizing molecular assembly of PN holoprotein. It is of prime importance to define which moiety of PN should exert a direct regulatory influence on a moiety of Ca pump ATPase.
S.34