Critical oxygen tension measurements in striated muscle in vivo and in vitro

Abstract

The coupling of oxygen delivery to oxygen consumption is thought to occur as the result of an increase in the production of vasodilating substances which diffuse from the tissue and act on the smooth muscle of arterioles, resulting in an increase in regional blood flow (metabolic regulation). A majority of the hypotheses regarding the nature of metabolic regulation of blood flow focus on the role of cytochrome oxidase and oxidative phosphorylation. The oxygen tension (PO₂) at which the rate of cytochrome oxidase is one-half maximal (K(m)) has been determined in isolated mitochondria but has not been determined in vivo and some evidence suggests that it may be higher than in vitro. In these experiments we determine the PO₂ at which NADH fluorescence began to increase in vivo and in vitro in response to oxygen limitation (critical PO₂). These changes in fluorescence are assumed to reflect oxygen limitation of oxidative phosphorylation and can be compared to the cytochrome oxidase K(m) values. This assumption was tested using imaging techniques on single striated myocytes to determine whether the change in NADH fluorescence that occurs with oxygen removal was associated with mitochondrial dense regions. Our findings support the use of NADH fluorescence to monitor oxidative phosphorylation. Using this technique the critical PO₂ was measured at tissue sites in post-capillary venular regions and in 20 micron diameter post-capillary venules. The critical PO₂ in the tissue was 2.4 mmHg and was higher in the venule (7.7 mmHg), consistent with predicted diffusion gradients for oxygen from vessel to tissue. The difference between the extracellular tissue PO₂ and the of Km cytochrome oxidase for oxygen (.05-0.05 mmHg) is consistent with theorized gradients existing between cell membrane and mitochondria and does not necessarily indicate a higher critical PO₂ in vivo. Using techniques similar to those used in vivo we measured the critical PO₂ from single isolated striated cells and determined it was significantly different from the critical PO₂ measured in vivo. This difference is likely due to the differences between in vivo and in vitro measurement conditions rather than real differences in the critical PO₂.