Direct determination of adp in hypoxic porcine carotid artery using 31p nmr

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Abstract
31‐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 m̈moles/g wet wt.) and creatine (2.04 ± 0.14 m̈mol/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 m̈mol/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 × 108 M–1. It was used to compute free ADP levels (m̈mol/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 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.