Direct determination of ADP in hypoxic porcine carotid artery using 31P NMR.
31P-NMR spectroscopy was performed on vascular smooth muscle (VSM; porcine carotid artery) superfused with a substrate-free high K(+)-PSS. Scans were collected before (control), during (hypoxia), and after (post-control) hypoxia, and chemical measurements of ATP (0.070 +/- 0.13 mumoles/g wet wt.) and creatine (2.04 +/- 0.14 mumol/g wet wt.) were made. During hypoxia, well-defined beta-ADP signals were consistently resolved. Their areas indicated that after 30, 60, and 90 min of hypoxia, free ADP was 0.05 +/- 0.01, 0.09 +/- 0.01, and 0.12 +/- 0.01 mumol/g wet wt., respectively. The apparent tissue equilibrium constant (Kck) for creatine kinase (CK) was calculated using 90 min hypoxic data and was 7.6 +/- 0.6 x 10(8) M-1. It was used to compute free ADP levels (mumol/g wet wt.) for control (0.028 +/- 0.002) and post-control (0.23 +/- 0.003) periods, since ADP signals could not be directly detected, and for the 30 and 60 min hypoxic periods (0.05 +/- 0.01 and 0.08 +/- 0.01, respectively). The Kck-dependent ADP values for the 30 and 60 min hypoxic periods periods were the same as the ADP values determined directly from the beta-ADP peak areas, suggesting that the CK reaction is in equilibrium in smooth muscle. These data show that 31P-NMR provides a means of directly measuring free ADP in hypoxic smooth muscle and a more accurate means of computing free ADP levels in normoxic VSM through the use of an in situ tissue Kck vs an assumed or in vitro Kck.
Fisher
M J
MJ
Department of Physiology, Michigan State University, East Lansing 48824.
Dillon
P F
PF
D000244:Adenosine Diphosphate / Q000032:analysis*; D000818:Animals; D002339:Carotid Arteries; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009131:Muscle, Smooth, Vascular / Q000032:analysis*; D010100:Oxygen / Q000502:physiology*; D013552:Swine
D000244:Adenosine Diphosphate; D010100:Oxygen