Date of Award

Spring 1995

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Rick H Cote

Abstract

Cyclic GMP and calcium are known to be messengers involved with visual transduction in vertebrate rod photoreceptor outer segments (ROS). However, it has been difficult to study the interrelationship of the calcium and cGMP signaling pathways using biochemical methods. An electropermeabilized rod outer segment (EP-ROS) preparation has been developed to examine these problems.

Square-wave electric field pulses were used to generate permanent disruptions in the plasma membrane of ROS. This treatment also permeabilized the disk membranes within the organelle. EP-ROS retain the characteristic morphology of osmotically intact ROS at the light microscopic level, but are permeable to low molecular weight compounds such as didansyl cysteine and GTP. Because EP-ROS are permeable to calcium buffers it is possible to exogenously control the intracellular free calcium concentration.

EP-ROS exhibit similar cGMP responses to flash illumination as do osmotically intact outer segments retaining the ellipsoid portion of the inner segment (OSIS). Reducing the intracellular free calcium concentration from 250 nM to 50 nM delays the recovery of the light-induced cGMP decrease. This delay is contrary to predictions based on the activity of GC and rhodopsin kinase at low calcium concentrations. The intensity-response relationship for EP-ROS is also similar to that of osmotically intact OSIS, but the stoichiometry of cGMP hydrolyzed per R*/ROS is ten-fold lower in EP-ROS. Reducing the free intracellular calcium concentration of EP-ROS from 100 nM to approximately 0 nM results in a two-fold increase in stoichiometry, consistent with the two-fold increase in free cGMP at low calcium concentrations.

Visual transduction requires energy in the form of ATP and GTP. The source of the energy required for transduction has been examined using EP-ROS. GTP is synthesized mainly by glycolysis; however transphosphorylation of GDP by nucleoside diphosphate kinase (NDPK) may also serve to buffer GTP levels. ATP is synthesized by glycolysis, but creatine kinase and adenylate kinase also provide alternative pathways for ATP synthesis.

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