| Popis: |
Taurine is a free amino acid found in millimolar concentrations inside most animal cells, and the retina appears to possess the greatest amount of taurine compared to the other cell types. Taurine modulates calcium uptake in retinal tissue, suggesting that the physiologic function of taurine may be related to calcium. Taurine is known to stimulate calcium uptake in the presence of low calcium levels (-10-500 iM) in the presence of ATP, and the mechanism behind this effect of taurine was studied. Much is unknown about this specific effect of taurine. What is the site of action of taurine? What is the nature of the calcium uptake? Most importantly, is this particular type of calcium uptake and in turn the effects of taurine, physiologically relevant? Truly, the main interest of this thesis is the possible physiologic relevance of the stimulation produced by taurine. The specific questions that were addressed relate to the nature of the ATPdependence of stimulation by taurine, the site of action of taurine, and the nature of the calcium uptake that taurine increases. Chelerythrine (CHT) is a potent protein kinase C (PKC) and ATPase inhibitor that has been previously shown to inhibit taurine-related effects, specifically in vitro CHT treatment produced an increased in the phosphorylation of proteins that taurine specifically inhibited. The discovery of the possible interaction between taurine and CHT suggested the use of CHT as a possible pharmacological tool in the study of the effects of taurine. Experiments using CHT were conducted that tested the hypothesis that taurine modulates ATPase activity in the retina. CHT inhibited the stimulatory effects of taurine on retinal calcium uptake, and it was used to help define the mechanism of action of taurine. Among other effects, CHT inhibits ATPase activity in the retina, and because the stimulatory effects of taurine are ATP-dependent, the data suggested that ATPase activity may be involved in taurine stimulation of calcium uptake. Thus, the role of ATPase activity in the mechanism of action of taurine was studied. Taurine had no direct effect on ATPase activity and so the involvement of ATPase activity was discounted. CHT also inhibited taurine uptake, suggesting another mechanism by which CHT may antagonize the stimulatory effects of taurine on calcium uptake. Taurine uptake was studied relative to its stimulatory effects. The kientics of taurine uptake were determined in both whole retinal homogenate and in isolated rod outer segments (ROS). In the whole retina, two uptake components were defined, one of low-affinity and the other of high-affinity. In contrast, only one uptake system of high-affinity was observed in ROS. Another series of experiments were conducted to address the second hypothesis that the inhibidon of taurine uptake abolishes or attenuates the stimulatory effects of taurine on calcium uptake. An analogue of taurine, guanidinoethane sulfonic acid (GES) was found to effectively inhibit taurine uptake. Inhibidon of taurine uptake with GES surprisingly did not produce any effects, eliminating taurine uptake as a necessary event behind taurine-dependent stimulation of calcium uptake. The data suggested that taurine binds to the membrane to produce its effects. The nature of the calcium uptake was a logical succeeding question to the stimulatory effects of taurine. Reference literature described the modulatory effects of taurine on ion channels in the heart and in the skeletal muscle, which suggested the possible involvement of calcium channel activation in the mechanism of action of taurine. Experiments were conducted to test the third hypothesis that specific inhibition of calcium channels abolishes or attenuates the stimulatory effects of taurine. The effects of taurine were antagonized by cation channel blockers, specifically by pharmacologic blockers of cGMP-gated channels. The data strongly suggested that taurine exerts a stimulatory effect on this channel to increase calcium uptake. The mechanism behind this effect on ion channels is unknown. Lastly, experiments were conducted to test the fourth hypothesis that taurine does not affect calcium binding to rednal membranes. Taurine is known to modulate calcium binding to sarcolemmal membranes and so the stimulatory effects of taurine may include the stimulation of calcium binding. The involvement of calcium binding was ruled out with the use of binding experiments and calcium ionophore treatments, suggesting that the increase in calcium uptake induced by taurine is solely due to increased flux through calcium channels. The mechanism of taurine, thus, can be summarized: Taurine binds to membranes to modulate the activation of calcium channels and increase calcium uptake, a process which does not involve ATPase activity, taurine uptake or calcium binding. |
