Browsing by Subject "Calcium channels"
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Item Application of parametric sensitivity analysis to calcium handling in cardiac myocytes(Texas Tech University, 2003-08) Smith, Charles NHeart failure disease (HF) kills 220,000 people in America every year. HF is characterized by the ineffective handling of calcium ions by the mechanisms present in cardiac myocytes resulting in decreased contractile force in the heart. This inefficiency leads to necrosis of the myocardium, cardiac hypertrophy, and eventual death of the patient. The mechanisms affected include various ion channels and active transport mechanisms. Numerous technologies and techniques have been developed recently to apply genetic and pharmacological therapies to these cells. Unfortunately, there are a large number of target parameters that can be manipulated in the myocytes. Mathematical models have been developed that accurately reproduce the calcium mechanisms. These models are reproducible and rapidly evaluated on desktop computers using current mathematical software. Sensitivity analysis is useful in determining the best targets for manipulation by external sources in the hopes of restoring the proper calcium ion handling by ion channels and calcium transporters. Sensitivity analysis uses matrix algebra and calculus to determine the normalized response of a control variable to a change in a parameter. More specifically, this technique calculates the responses of all control variables to changes in all of the parameters individually. This generates a matrix of values that can easily be analyzed. This method will save valuable time and resources that would have been spent on performing multiple experiments or simulations to determine the best targets for manipulation. The cytosolic calcium concentration is targeted as it is directly related to the contractile force of the heart. Reduction of complex models is also possible using the results of sensitivity analysis. Rota et al. describe a method of normalizing fluxes and sensitivities against their greatest magnitude as a characteristic for suggesting unimportant fluxes and parameters. Two models are examined: the Tang-Othmer (T-0) model and the Winslow, Rice, and Jafri (WRJ) model. Both of these models seek to describe the handling of calcium ions within cardiac myocytes; however the level of their complexities differs greatly. The T-O model contains much fewer state variables, parameters, and fluxes than the WRJ model. The greater incorporation of states and the related parameters and fluxes leads the WRJ model to yield a much greater complexity than that presented with the T-O model. This complexity is magnified upon formation of the sensitivity matrices for these models, where the T-O model yields a fifty-five element matrix and the WRJ model yields 1,881 elements in its sensitivity matrix. The T-O model is used to develop the application method whereas the WRJ model is used to determine the best targets for manipulation and to illustrate the efficacy of model reduction. Sensitivity analysis upon the T-O model yields the sodium-calcium exchanger parameters and the sarcolemmal leak coefficient as the optimal targets for manipulation to restore proper cytosolic calcium concentration. The best targets for genetic or pharmacological manipulation according to the WRJ model are the Na+ and Ca2+ background currents, the maximum current for the sodium-potassium pump, and the half saturation constants for the sodium-potassium pump. These parameters are not present in the T-O model and have greater sensitivity magnitudes than those that carry over into the WRJ model. Model reduction by the method of Rota et al. reveals that the Tang-Othmer model is irreducible in its present state. The WRJ model was found to have no reducibility with regards to the number of fluxes in the model. However the integration of some sensitivities was unattainable, and some of these parameters may be found to be removable upon further analysis of the model once these sensitivities are obtained. Further integration methods will be attempted, such as the use of a hard-coded implicit integrator. Sensitivity analysis also revealed a crossover phenomenon in both models. This phenomenon describes the change in sign of a sensitivity of a state variable to a parameter during the course of a heartbeat. When this occurs the desired effect of manipulating the parameter yields the opposite effect upon the state variable. This phenomenon may generate interesting side effects that require further study. The results of sensitivity analysis provide future direction for physical experiments. These experiments will both confirm the calculated sensitivities, and investigate their application to failing myocytes. The crossover phenomenon will provide interesting avenues of research into the side-effects of parameter manipulation based on the magnitude and location in the cycle of the crossover. Model reduction may play a key role in simplifying models for easier computation and analysis.Item Phospholipid headgroup superlattice modulation of cardiac calcium channel activity(Texas Tech University, 2000-08) Cannon, Brian L.The plasma membrane of cells consists of phospholipids, sterols, and proteins and maintains a steady composition through a regulatory mechanism that remains largely unknown. The interactions that occur among the membrane constituents are complex and are involved in compositional control. Among the proteins embedded is a class of transmembrane proteins called ion channels, specifically, the ryanodine receptors located in the sarcoplasmic reticulum of myocytes. These channels are responsible for the release of Ca ions, necessary for the activation of myofilaments to produce contractions. The receptors, through a process known as gating, undergo changes in conformation during the transition between open and closed states. Since proteins are surrounded by lipids and change conformations, the lipid-protein interactions must influence the protein state by hydrophobic coupling or on the headgroup level. By reconstituting the channels in planar lipid bilayers composed of two phospholipids with differing headgroup sizes, POPE and POPC and varying the ratio of the two lipids that form the bilayer, the channel functioned as sensors to determine how bilayer changes affect the channel. A peak in channel activity was observed over a narrow region of high PE:PC bilayers, indicating that lipid-protein interactions do have an important role in channel function. The results are interpreted as reflecting lateral organization by the lipids on the headgroup level in order to minimize the stress across the bilayer due to deformation, curvature, and packing, providing a favorable condition for the channel to enter its preferred conformation.Item Regulation of calcium stores in normal and diabetic endothelial cells(Texas Tech University, 2000-12) Sanka, Shankar ChittaranjanCytosolic Ca^^ ([Ca^^]*'^^) mediates many cellular ftinctions, e.g.. cell growth, motility, secretion, etc. In many cell types, ion transport processes appear to be dependent on metabolism of glucose for maximal activity. In certain cell types, a strict coupling between glycolysis and the acfivity of Endoplasmic Reticulum Ca^"-ATPases (SERCA). involved in regulating Ca^^ homeostasis, has been suggested. In diabetes, glucose homeostasis is altered. We hypothesize that Ca^^ homeostasis in microvascular endothelial cells from diabetic animals is altered due to a dysfunction of glycolysis coupling the activity of SERCA. We further hypothesize that endosomal/lysosomal (E/L) compartments exhibiting SERCA are involved in this dysfunction. Our data indicated that agonist stimulation (ATP, vasopressin, angiotensin-II)elicited [Ca^"]^^* increases (independent of extracellular Ca^^) that were larger in endothelial cells from diabetic than from normal animals. Simultaneous measurements of [Ca^^]'^^' and Ca^^ in E/L compartments ([Ca^^]^) using fluorescence spectroscopy, indicated that E/L compartments released Ca^^ following agonist-stimulation. The magnitude of the Ca'* release was significantly larger in microvascular endothelial cells from diabetic rats. SERCA inhibitors elicited Ca^^ releases from E/L compartments in both normal and diabetic models. The magnitude of the [Ca^^]^ release was however similar among normal and diabetic cells. Immunocytochemical experiments demonstrated that 60% of E/L compartments exhibited SERCA. These data indicate that (a) E/L compartments are important for Ca^^ homeostasis in microvascular endothelial cells from both normal and diabetic models; (b) Ca^^ regulation in E/L compartments is different in cells from a diabefic model, (c) the compartment involved in altered Ca'* homeostasis in diabetes is unknown.Item Taurine stimulation of calcium uptake in the retina: mechanism of action(Texas Tech University, 2003-05) Militante, Julius DTaurine 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.