Previous research has demonstrated that the protein containing copper, ceruloplasmin, plays an important role as antioxidant in the extracellular space (1,2).
Bioscience Reports 5, 473-476 (1985) Printed in Great Britain
473
Inhibition by c e r u l o p l a s m i n of the c a r d i a c sarcolemmal adrenochrome formation
Carlo GUARNIERI and Carlo VENTURA Istituto Chimica Biologica, Centro Studi e Ricerche del Metabolismo Cardiaco, via Irnerio 48, 40126 Bologna, Italy (Received 23 April 1985)
The a d d i t i o n of c e r u l o p l a s m i n to bovine c a r d i a c s a r c o l e m m a l vesicles supplemented with NADPH was able to reduce the formation of adrenochrome from adrenaline. This inhibitory e f f e c t appears at 2.5 IJM ceruloplasmin and it is almost complete at the level of 20 IJM. P r e v i o u s r e s e a r c h has demonstrated that the protein containing copper, ceruloplasmin, plays an important role as antioxidant in the e x t r a c e l l u l a r space ( 1 , 2 ) . The mechanisms proposed include the s activity of ceruloplasmin which, maintaining the iron in the Fe3+ state~ prevents the Fe 2+ stimulated lipid peroxidation process (3). In addition, ceruloplasmin can prevent copper ions from stimulating lipid peroxidation (4). Ceruloplasmin is also able to scavenge the superoxide radicals 02" by a noncatalytic reaction, less efficient than those produced by the enzyme superoxide dismutase (5). Among the various tissues~ the heart muscle seems to be particularly damaged by active oxygen metabolites generated in the extracellular space, for example by the activated neutrophils (6)7 or by the interaction of sarcolemma with catecholamines (7). Considering that the heart muscle contains receptors for ceruloplasmin (g), the purpose of this study is to examine the possibility that ceruloplasmin may prevent the formation of adrenochrome induced in the cardiac sarcolemma by the superoxide radicals. Materials and Methods Ceruloplasmin (type VI human), catalase, superoxide dismutase and bovine albumin were from Sigma Chem. Corp. All other reagents were of the highest quality available form Merck. Bovine cardiac sarcolemma was prepared by the method of Reeves and Sutko (9), modified as suggested by Lamers and Stinis (10). A f t e r i s o l a t i o n , the vesicles were suspended in !60 mM KCI and 20 mM 3-(N-morpholine)-propane sulfonic acid, pH 7.t~ at a protein concentration of about 1.5-2 mg/ml and stored frozen in small aliquots at -S0~ The p r e p a r a t i o n was highly enriched in sarcolemmal m e m b r a n e s , as was revealed by the high specific activity of the m a r k e r e n z y m e s ouabain s e n s i t i v e Na+/K+-ATPase (65.8 t~mol/mg p r o t . h ) and 5 ' - n u c l e o t i d a s e (28.9 tJmol/mg p r o t . h ) ( 1 1 ) . The formation of adrenochrome was monitored at 480 nm at 25~ in a
474
GUARNIERI
& VENTURA
double-beam Perkin Elmer s p e c t r o p h o t o m e t e r , model 559, in a total vol. of 1.0 ml. The assay mixture consisted of 50 mM Tris/HCl, pH 7.5, 1 mM EDTA and 30 pg of sarcolemmal protein. Adrenaline was added at 1 mM final concentration. The concent rat i on of adrenochrome was determined using F ~ 0 = 4.02 x l03 M-1 cm-1. Protein concentration was determined by the method of Bradford (12) using bovine albumin protein as standard. Results
Table 1 shows that the formation of adrenochrome from adrenaline by b o v i n e cardiac sarcolemmal vesicles stimulated by NADPH was m a r k e d l y i n h i b i t e d by s u p e r o x i d e d i s m u t a s e , while the reduction e x e r t e d by catalase and mannitol was less evident. The production of sarcolemmal adrenochrome was also inhibited by ceruloplasmin, while the addition of Cu2+ at the same level which is present in i0 #M ceruloplasmin did stimulate almost 5 times the rate of adrenochrome formation. Moreover, the heat denaturated ceruloplasmin lost its ability to reduce the conversion of adrenaline to adrenochrome. Fig. 1 shows t h a t t h e i n h i b i t i o n of a d r e n o c h r o m e f o r m a t i o n by ceruloptasmin followed a dose dependent response. Already at concentrations ranging f r o m 5-7 pM c e r u l o p l a s m i n r e d u c e d t he r a t e of a d r e n o c h r o m e formation by 50%, while at concentrations above 20 lJM the reaction was almost completely blocked. Discussion
The present study indicates t hat ceruloplasmin is able to reduce the formation of adrenochrome by cardiac sarcolemmal vesicles supplemented with NADPH. The inhibitory e f f e c t requires the molecular integrity of the protein because its denaturation does not modify the r ate of conversion of adrenaline to adrenochrome. The experi m ent
Table i. Effect of ceruloplasmin and other oxygen radical scavengers on the rate of formation of adrenochrome by bovine cardiac sarcolemmal membranes NADPH was added to the reaction mixture immediately after the addition of adrenaline. All the other agents were added s e p a r a t e l y before the addition of NADPH (final concentration indicated in parenthesis). The values are means +- SEM of 3 preparations (triplicate measurements in each heart). Incubation system Sarcolemmal vesicles/NADPH (0.4 mM) superoxide dismutase (2 ~M) catalase (2 ~M) mannitol (5 mM) ceruloplasmin (i0 ~M) ceruloplasmin denatured copper (0.02 ~g)
nmol/min.mg prot 96 4 - + 0 . 8 123+_0.5 702+0.4 108 4 + 0.8 328+0.3 115 7 +- 0.9 569 9 +- 2.2
CERULOPLASMIN
AND A D R E N O C H R O M E
FORMATION
475
100 0
E c
80
-5 E .- 60 9
E ID
E 2 20 0 121 r
