In vivo studies of metabolism and visual phototransduction of the isolated toad retina by phosphorus-31 nuclear magnetic resonance spectroscopy
Apte, Dipali V.
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Permalink
https://hdl.handle.net/2142/21623
Description
Title
In vivo studies of metabolism and visual phototransduction of the isolated toad retina by phosphorus-31 nuclear magnetic resonance spectroscopy
Author(s)
Apte, Dipali V.
Issue Date
1992
Doctoral Committee Chair(s)
Ebrey, Thomas G.
Department of Study
Biophysics and Computational Biology
Discipline
Biophysics
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Neuroscience
Biology, Animal Physiology
Biophysics, General
Language
eng
Abstract
Retinal physiology and metabolism, active areas of inquiry twenty years ago, have been relatively inactive recently. During the last twenty years, however, the development of nuclear magnetic resonance spectroscopy as a tool to study tissue metabolism, has changed the nature of the field of physiology. In this thesis I demonstrate the use of this technique to study several aspects of retinal metabolism and phototransduction. General considerations and the potential of the technique for studying the retina is presented in Chapter 3. With the improvements in the method of maintaining retinae (Chapter 4) I could conduct longer experiments than my preliminary studies. Thus, I measured the concentrations of phosphorus-metabolites to see if these change with light adaptation (Chapter 5) and if cGMP could be observed in vivo (Chapter 6).
A summary of the main results in these chapters are as follows: (1) The intracellular pH of dark-adapted retinae at 4$\sp\circ$C is about 7.3. At least 70% of nucleotide triphosphates (NTP) is bound to magnesium. As in the brain, phosphocreatine functions as an ATP buffer in the retinae. (2) At 20$\sp\circ$C, superfusion maintains retina(e) for at least 8 hours without adversely affecting normal electrophysiology, biochemistry, and histology if the solutions prepared for superfusion adequately buffer protons and contain glucose. (3) The metabolic energy-requirement of the retina decreases in light as was demonstrated by the increased level of phosphocreatine and no change in the level of NTP. The cause of this is likely due to the decreased entry of sodium ions and hence a reduction in the ATP-requirement. The concentrations of NTP and PCr in the dark are 1.5 mM and 0.7 mM, respectively and in light are 1.5 mM and 1.0 mM, respectively. (4) A peak that is observed only in the presence of the phosphodiesterase inhibitor, IBMX, in in vivo spectra of retinae is identified as cGMP. Most of the cGMP in the dark-adapted retinae is NMR-invisible, most likely due to binding to phosphodiesterase, and it is the free cGMP that increases in the presence of IBMX.
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