A major problem in understanding the regulation of cellular metabolism is the heterogeneity of tissue structure, not only with regard to the cellular composition, but also within the individual cells. An ingenious step was the development of a method for measuring oxygen consumption of single cells, though without spacial resolution around the cell itself2. In this study, a solitary amphibian rod photoreceptor was used: these cells come from the retina, a tissue with an especially high metabolic rate3, reflecting primarily the contribution of the photoreceptors4. Previous measuremnts of retinal oxygen consumption have suggested that the metabolic activity of the vertebrate retina varies with light intensity5, but neuromodulators, such as dopamine, may also substantially alter the metabolic activity of retinal neurons6,7. With the one exception2, these studies have been conducted using whole retinas, composed of a variety of cell types. Consequently it has been almost impossible to determine oxygen consumption of different categories of cell. Oxygen-consumption experiments performed on isolated identifiable retinal cells, with good spatial resolution, would provide new information on the control and modulation of metabolic activity in photoreceptors and provide an approach applicable to numerous other varieties of cells. The purpose of the present study was to measure oxygen consumption by isolated photoreceptors where there is a high degree of structural polarization, with improved spatial resolution. This involved the use of a new technique incorporating polarographic oxygen-selective microelectrodes in a self-referencing, non-invasive modality (serp-electrode)8. In the present experiment, oxygen -selective electrodes with final tip diameters of 1-4 mm were prepared according to the method of Linsenmeier and Yancey9 giving an expected spatial resolution in the region of mm2. Isolated photoreceptors from the all-rod retina of the skate (Raja erinacea or R. ocellata) were prepared according to an enzymatic dissociation protocol described in detail by Malchow et al.10. The cells used in the present experiments possessed outer and inner segments but no obvious synaptic terminals. All of the recordings reported here were from light-adapted photoreceptors continuously bathed in bright white light. Vertebrate photoreceptors are highly polarized in structure, with most of the mitochondria located in a specific portion of the inner segment called the ellipsoid. The rest of the cell is largely devoid of mitochondria. This distinctly compartmentalized distribution should result in oxygen concentrations being lowest at the ellipsoid; i.e., the oxygen consumption is high in this area, but very low near the tip of the outer segment. Indeed, when electrodes are situated next to the ellipsoid, we obtain differential readings indicative of a higher rate of oxygen consumption in that region. In eight cells examined, the average differential current measured next to the cell is 138.8fA+41fA. The conversion of these currents to an oxygen flux can be calculated by theformula: Flux (mmol cm-2 s-1) = -D[(DA.S) Dr-1] where D is the diffusion constant for oxygen (2.5 . 10-5 cm2 s-1), DA (picoAmps) is the recorded current differential, S is the slope of the electrode (mmol ml-1 picoAmps-1), and Dr the distance between the measured points in cm. Applying this equation to our data, we find that the average amount of oxygen flux into the ellipsoid is 0.015+0.004mmol cm-1s-1. Current readings obtained at the tip of the outer segment of the same eight cells are indistinguishable from readings at background, indicating no detectable oxygen uptake by the cells in this region. Our data demonstrate that polarographic, self-referencing, oxygen-selective electrodes can be used to monitor oxygen consumption from isolated retinal photoreceptors and can do so with a spatial resolution that reveals single cell inhomogenieties in the consumption of oxygen. We have shown significant differences in the oxygen concentration profile around cells, probably reflecting similar profiles within. We anticipate that these differences will have important physiological implications. 1. Malchow, R. P., Patel, L. and Smith, P. J. S. 1997. Biol. Bull. In press. 2. Poitry, S., M. Tsacopoulos, A. Fein, and M. C. Cornwall. 1996. J. Gen. Physiol. 108: 75-87. 3. Graymore, C. 1969. Pp. 601-645 in The Eye, 2nd Ed., vol.1, Dawson, H., ed. Academic Press:New York. 4. Ames III, A., Y-Y. Li, E. C. Heher, and C. R. Kimble. 1992. J. Neurosci. 12: 840-853. 5. Linsenmeier, R. A. 1986. J. Gen Physiol. 88: 521-542. 6. Medrano, C. J., and D. A. Fox. 1995. Exp. Eye Res. 61: 273-284. 7. Shulman, L. M., and D. A. Fox. 1996. Proc. Natl. Acad. Sci. U. S. A. 93: 8034-8039. 8. Smith, P. J. S., R. H. Sanger, and L. F. Jaffe. 1994. Meth. Cell Biol. 40: 115-134. 9. Linsenmeier, R. A., and C. M. Yancey. 1987. J. Appl. Physiol. 63: 2554-2557. 10. Malchow, R. P., H. Qian, H. Ripps, and J. E. Dowling. 1990. J. Gen. Physiol. 95 177-198.
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