, 1999 2, 51 (1), 25 30 25 Acta Physiologica Si nica L2Arg/ NO 1997212227 1998203230 3 3 (, 524023 ; 3, 100083) ( IRI) (NO), 15 min, 45 min, 30 ml KH 15 min, (LDH) NO2 - NOS L2 (L2Arg), IRI LDH 411, 514 1 ( P < 0101) NO 2-112 ( P < 0101) tnos inos cnos 4812 % 4312 % 5211 % ( P < 0101) NO 2 - inos, ( r = 017942, P < 0101) L2Arg IRI L2Arg (V max ) 48 % (, P < 0105) 2 (, P < 0101) ; Km 4714 %( P < 0105), Km L2Arg V max inos : IRI, L2Arg inos NO, L2Arg NO : ; ; ; L ; : Q463 ; R33113 (ischemia reperfusion injury, IRI), L2 / [1 (L2arginine/ nitric oxide, L2Arg/ NO) IRI ], [2, IRI, NO/ c GMP ] [3,4, NO IRI ] IRI NO, IRI L2Arg/ NO, IRI NO 1 111 IRI 150 250 g Wistar, (1 g/ kg) ip Langendorff, Krebs2Henselert ( KH) (95 % O 2-5 % CO 2 ), 37, 7185 kpa 15 min, : (1) ( n = 7) KH 45 min, 30 ml KH 15 min (2) 2 ( IR, n = 6), 45 min, NO - 2
26 51 30 ml 95 % O 2-5 % CO 2 KH 15 min (LDH) ( ) ( ) [5 ] ( ) [6 112 (nitric oxide synthase, NOS) ] 200 mg,,, 200 g, [ Tris2HCl 50 mmol/ L, p H 714, NADPH, CaCl 2, ( Ca 2 +, EGTA), A2Arg 5 mol/ L 3 H2A2Arg 01025 kbq/ ] 100 l, 37 30 min,, p H 810 Dowex A G50 W2X8 (Na + ), 4 1 h,, ( FJ 2115 ) 3 H 3 H NOS 3 H2 pmol Ca 2 + / NOS (tnos), Ca 2 + / NOS (in2 OS) NOS (cnos) 113 3 [7 H - A2Arg Durante ] 500 mg, 1 mm, 10, KH 1 ml,, 3 H - A2Arg, 0101 10 mmol/ L, 37 1 min, KH 3 32,, 012 ml, H 3 H - A2Arg, nmol/ (mg pr min) 114 Wistar L2[ 2,32 3 H ]2Arg Dupont N EN (2129 TBq/ mol), A2Arg Dowex A G 50W2X8 Sigma x s x,student s t test 2 45 min, 15 min LDH 411 514 1 ( P < 0101) 211 IRI 3 H- A2Arg A2Arg Eadie2Hofstee plot ( 1B) IR A2Arg ; (V max ) 48 %(, P < 0105) 2 ( 1 A2Arg Table 1 Effects of ischemia reperfusion injury on cardiac tissue L2arginine transport and Michaelis constant Group V max / nmol (mg pr min) - 1 Low affinity High affinity Low affinity Km / mol L - 1 High affinity Control IR x s x, n = 5. 1518 118 213 115 1405 347 49 27 2314 213 3 710 117 3 3 739 249 3 61 21 3 3 P < 0105, 3 P < 0101 vs control.
1 : L2Arg/ NO 27, P < 0101) Km 4714 %( P < 0105), Km ( 1, 1) 1 L Fig11 Effect of ischemia reperfusion injury on kinetics of cardiac L2arginine transport in rat 212 IRI NOS IRI NOS, tnos inos cnos 4812 % 4312 % 5211 %( 2) inos A2Arg V max, cnos V max ( 2) 2 NOS Table 2 Effects of ischemia reperfusion injury on nitrite content and nitric oxide syn2 t hase activity Group Control IR NO2 - / pmol mg - 1 w1w tnos NOS / pmol (mg pr min) - 1 inos cnos 5019 1213 3613 113 1716 311 1918 315 11219 2816 3 3 5318 414 3 3 2512 310 3 3 2816 612 3 3 3 3 P < 0101 vs control. 3 L Table 3 Correlation between L2arginine uptake or nitric oxide synthase activity and ni2 t rite content High affinity V max - NO - 2 017397 010145 Low affinity V max - NO - 2 019135 010002 cnos - NO - 2 015508 010989 inos - NO - 2 017942 010061 r P
28 51 2 L Fig12 Relationship between L2arginine transport and NOS activity 213 IRI NO IRI 112 ( P < 0101) ( 2) NO 2 - LDH, 019301, 018429 018724 ( P < 0101) NO2 - A2Arg inos, cnos ( 3) 3, 45 min, 15 min NO2 -, 30 ml KH 15 min NO2 -, LDH [2, NO ], NO NO, A2Arg, NOS A2Arg,,, A2Arg NOS A2Arg, (transporter), A2Arg A2Arg Na + 2 Y + (Na + 2independent system Y + [8 ), ] [7,9 11 ], A2Arg Na + Y + b o, +., A2Arg, Km (49 27) mol/ L (1 405 347) mol/ L A2Arg 50 100 mol/ L [9 ] IRI A2Arg A2Arg, A2Arg ( P < 0101) ; (V max ) ( P < 0101) ;
1 : L2Arg/ NO 29 Km ( P < 0105), IRI A2Arg A2Arg NOS, (cnos), (ecnos) (ncnos), Ca 2 + ; (inos), Ca 2 + cnos NO A2Arg, inos NO [9 A2Arg ] IRI, NOS,, IRI NOS, inos A2Arg V max ; cnos ; inos NO2 -, cnos NO2 - ; A2Arg V max NO2 - ( P < 0101),, IRI A2Arg inos NO, A2Arg NO Bogle [10 ], Durante [7,9 ], Green [11 ] [12 ], J 744, A2Arg NO, A2Arg L2Lysine L2ornithine A2Arg NO, A2Arg NO A2Arg, IRI [1 ] Yoshiki N. The role of nitric oxide in cardiac ischemia2reperfusion injury. J pn Circ J, 1997, 61 : 119 132. [ 2 ] Kosaka H, Komamura K, Minamino T et al. Plasma nitric oxide end products are increased in the ischemic canine heart. Biochem Biophys Res Com m un, 1995, 211 (2) : 370. [ 3 ] Li XS, Uriuda Y, Wang QD, et al. Role of L2arginine in preventing myocardial and endothelial injury fol2 lowing ischemia/ reperfusion in the rat isolated heart. Acta Physiol Scand, 1996, 156 : 37. [4 ] Naseem SA, Kontos MC, Rao PS, et al. Sustained inhibition of nitric oxide by N G 2nitro2L2arginine im2 proves myocardial function following ischemia/ reperfusion in isolated perfused rat heart. J Mol Cell Cardiol, 1995, 27 : 419. [ 5 ] Elz J S, Nayler WG. Quantification of calcium paradox in neonatal rat hearts. A m J Physiol, 1987, 253 : H1358. [6 ] Zhang XB ( ), Huang HL ( ), Zhang L Z ( ), et al. Measurement of nitric oxide syn2 thase activity and its applications. J Beijing Med U niv ( ), 1994, 26 (Suppl) : 173 176 (in Chinese with English abstract). [ 7 ] Durante W, Liao L, Iftikhar I, et al. Differential regulation of L2arginine transport and nitric oxide produc2 tion by vascular smooth muscle and endothelium. Cir Res, 1996, 78 : 1075. [ 8 ] Kim J W, Closs EI, Albritton LM,et al. Transport of cationic amino acids by the mouse ecotropic retrovirus receptor. N ature, 1991, 352 : 725 728. [9 ] Durante W, Lan L, Andrew IS. Differential regulation of L2arginine transport and inducible NOS in cul2 tured vascular smooth muscle cells. A m J Physiol, 1995, 268 : H1158 H1164. [ 10 ] Bogle R G, Baydoun AR, Pearson JD, et al. L2arginine transport is increased in macrophages generating ni2 tric oxide. J Biochem, 1992, 284 : 15 18. [11 ] Green B, Anthony J P. Wiley WB. Characterization of L2arginine transport by pulmonary artery endothelial cells. A m J Physiol, 1993, 264 : L351 L356.
30 Acta Physiologica Sinica Feb. 1999, 51 (1), 25 30 EFFECTS OF MYOCARD IAL ISCHEMIA REPERFUSION INJ URY ON L2ARGININE/ NITRIC OXIDE SYSTEM IN RAT HEART ZHEN G HU I2ZHEN, TAN G CHAO2SHU 3, SU J IA2L IN 3, WU TAO ( Depart ment of Physiology, Guangdong Medical College, Zhanjiang 524023 ; 3 Institute of Cardiovascular Research, Beijing Medical U niversity, Beijing 100083) ABSTRACT Ischemia reperf usion injury ( IRI) model of rat heart was prepared by preperf u2 sion for 15 min, then a suspension for 45 min and recycling reperfusion for 15 min wit h 30 ml KH buffer. The leakage of lactate dehydrogenase (LDH), protein, myo2 globine and nitrite (NO - 2 ) in t he circular perf usion fluid were measured. Myocardial nitric oxide synt hase (NOS) activity and L2arginine transport were observed. In t he IR group, the leakage of LDH, protein, myoglobine and NO - 2 were increased respec2 tively by 411, 514, 1 and 112 times ( P < 0101) and NOS (tnos, inos, cnos) activity by 4812 %, 4312 %and 5211 %, ( P < 0101, respectively) as compared with t he control group. L2arginine transport might be mediated by eit her high2 or low2 affinity transport system in cardiac tissue. In t he IR group, L2arginine transport in2 creased significantly with the V max being increased by 48 % and 2 times respectively for t he low2affinity and t he high2affinity transport as compared wit h control. Michaelis constant ( km) was decreased by 4714 % for low2affinity transport ( P < 0105), but not significantly changed for t he high2affinity transport. These results suggest t hat t he increase of nitric oxide generation might result f rom t he increased myocardial NOS activity and L2arginine transport during IRI. Key words : myocardium ; ischemia reperfusion injury ; nitric oxide ; L2arginine transport ; rat