I. S. Longmuir

Intracellular measurement of oxygen by quenching of fluorescence of pyrenebutyric acid.

Abstract We have examined pyrenebutyric acid as a possible fluorescent probe to determine intracellular concentrations of oxygen. Isolated rat liver cells rapidly take up pyrenebutyric acid, and the fluorescence of the cells is quenched by oxygen to the same extent as is free pyrenebutyric acid. Therefore, this technique appears to be applicable to the physiological range of oxygen concentrations.

Intracellular oxygen measurements of mouse liver cells using quantitative fluorescence video microscopy

Our currently developed fluorescence video microscope can measure fluorescence intensities with an error of +/- 1.5% of full scale in 65536 different positions of a microscope field. With a video frame freeze acquisition time of 33 ms, time-dependent changes of this order of time or slower can be followed. Using cells which have absorbed pyrene-1-butyrate to an intracellular concentration of 0.05 to 1 mM, the changes in fluorescence intensity with oxygen concentration are easily measured. The spatial resolution for data collection is 0.5 micron when a 54X objective is used. The individual Stern-Volmer quenching constants of each individual pixel were measured for agar slices and mouse liver cells treated with pyrenebutyric acid. The distribution of quenching constants for agar follows a normal curve about a mean value of 16 . 10(-4) torr-1. The data for mouse liver cells gave a non-normal distribution of quenching constants with a mean value of 18 . 10(-4) torr-1. The greater spread of the data from cells is interpreted as evidence for a real biological variation in the solubility coefficient of oxygen in different locations within the cell. In all the cells examined, this distribution has been observed to be non-random and appears to be associated with specific cell structures.

Nonclassical respiratory activity of tissue slices

Abstract The relationship between respiration rate and ambient oxygen tension has been measured for fresh liver, kidney, heart, and brain slices. Statistical procedures are developed for discriminating between the kinetics predicted by the classical diffusion model and those of the Michaelis-Menten equation suggested in earlier papers. In all tissue slices the Michaelis-Menten equation tended to fit better than the classical model. The differences in fit were most pronounced in liver slices and smallest for kidney and brain slices. The classical model is clearly unsatisfactory for liver and heart tissue. Several explanations are discussed including the hypothesis that a significant part of the oxygen transport is by carrier. The possible nature of such a carrier is considered.

A hypothetical tissue oxygen carrier

Abstract The relation between the respiration rate of fresh liver, kidney, or heart slices and the ambient partial pressure of oxygen follow “saturation” or Michaelis-Menten kinetics rather than those predicted if passive diffusion is the mechanism of tissue oxygen transport. This suggests that the transport of oxygen in those tissues is facilitated by a “carrier” mechanism. In the case of brain and artificial tissue slices, however, the kinetic pattern is different, and passive diffusion adequately describes oxygen transport in this tissue.

Evidence for nonclassical respiratory activity from oxygen gradient measurements in tissue slices

Abstract Direct measurement of steady-state oxygen concentration gradients using oxygen-sensitive recessed microelectrodes (2- to 4-μm tip size) were obtained in brain and liver slices. Experimental observations were normalized to examine the deviation from a classical passive diffusion model with concentration-independent (zero-order) chemical reaction kinetics. Discrepancies were noted between gradients measured during inward or outward electrode movement, which can be attributed to tissue compression effects. Gradients obtained during outward electrode movement were therefore judged to be more accurate than gradients measured during inward movement. Concentration gradients from both tissue types demonstrated significant departures from the gradient predicted by classical respiratory activity. A tendency towards a greater departure from the classical model was observed for gradients obtained in liver. The shape of the observed normalized gradients indicates that oxygen diffuses further into the tissue than predicted by the classical model. Nonclassical models of respiratory activity must be formulated to account for the experimental results from brain and liver tissues observed in this study.

The Induction of Cytochrome P-450 by Hypoxia

A number of apparently unrelated observations have led us to look for daily fluctuations in the level of liver cytochrome P-450. We have observed that there seems to be a rather wide variation in P-450 concentrations in different animals of the same sex, age, and species. In addition the Chouteau phenomenon is pronounced in men after midnight, whereas the same stress has no effect in the afternoon. The third observation is that the response to certain drugs is greater in man after midnight than during the day, whereas in nocturnal species the same phenomenon is seen twelve hours out of phase. Since cytochrome P-450 appears to play an important role in both tissue oxygen transport and drug inactivation, these latter effects could be associated with a diurnal variation in the concentration of this pigment.

CEREBRAL MICROCIRCULATORY CHANGES DURING EXPOSURE TO HYPOXIA

Changes in the red blood cell content of the cat cerebral cortical vessels were monitored using reflecting light during a decrease in PIO2. The following observations were made: 1) the capillary bed red blood cell content increased during hypoxia prior to the arteriole response at an arterial oxygen tension of 52.0 mmHg; 2) at arterial oxygen tensions below 52 mmHg the red blood cell content increased in all vessels simultaneously; 3) as the PIO2 decreased, the increase in red blood cell content occurred earlier in all vessels. Capillaries, therefore, can respond to hypoxia independently of the arteriole in cat cerebral cortical tissue.

The Intracellular Heterogeneity of Oxygen Concentrations as Measured by Ultraviolet Television Microscopy of PBA Fluorescence Quenching by Oxygen

If oxygen moves through the cell along special channels (Longmuir, 1977) and the mean solubility coefficient for oxygen in liver cells is rather low as Zander (1976) has shown, then there should be considerable intracellular heterogeneity of oxygen concentration even in a non-respiring cell with uniform PO2 throughout. Since the Polarographie electrode measures PO2 (Longmuir and Allen, 1960), this heterogeneity cannot be detected by this method. However, the fluorescence of pyrenebutyric acid (PBA) is quenched in proportion to the concentration of oxygen in a volume of a few hundred Augstrom’s radius around each photofluor molecule (Longmuir and Knopp, 1976). Thus, this method, with a theoretical geometric resolution of 0.2 μm appears to have this capability.

The role of facilitated diffusion of oxygen in tissue hypoxia

The evidence that cytochrome P-450 can act as a tissue oxygen carrier is outlined, and a new experimental approach to the measurement of the fraction of oxygen carried this way is described. This fraction is increased when cytochrome P-450 concentration is increased, which occurs on exposure of the experimental animal to hypoxia. This appears to be a new mechanism of tissue acclimation to hypoxia.

The Measurement of the Fraction of Oxygen Carried by Facilitated Diffusion

Evidence has been presented that the cytochrome P-450 in the endoplasmic reticulum can facilitate the diffusion by oxygen through tissue (Longmuir, 1970). This hypothesis is based on the observation that the respiration rate of liver slices as a function of ambient oxygen tension more closely fits a model equation based on purely passive diffusion. However, the fit is not perfect at higher oxygen tensions, and it seems highly improbable that there can be no passive diffusion proceeding concomitantly. The respiration rate of a slice in which there is only passive diffusion would rise as the square root of the ambient oxygen tension, whereas if transport is purely by carrier, the respiration rate would show “saturation” kinetics. That is, the rate of rise would fall off rapidly, becoming asymptotic to a maximum rate. Thus it seems certain that both passive and facilitated diffusion are proceeding simultaneously. That this is the case can be shown by the application of specific inhibitors to tissue slices.