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| Author(s): |
Diogo Coutinho Soriano
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Faculdade de Engenharia Elétrica e de Computação |
| Defense date: | 2007-06-20 |
| Examining board members: |
José Wilson Magalhães Bassani;
Antonio Carlos Guimarães de Almeida;
Eduardo Tavares Costa;
Vera Lúcia da Silveira Nantes Button
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| Advisor: | Rosana Almada Bassani; José Wilson Magalhães Bassani |
| Abstract | |
The goal of this work was to develop an instrument for simultaneous measurement of cell shortening and cytosolic Ca2+ concentration ([Ca2+]i), by using the fluorescent Ca2+ indicator fluo-3. The instrument was applied to study the uptake of cytosolic Ca2+ by the sarcoplasmic reticulum (RS) Ca2+- ATPase (SERCA, the main transporter responsible for cell relaxation in intact isolated rat ventricular myocytes). For this purpose, the development of a specific experimental protocol was required. In this protocol, the main competitor of SERCA by cytosolic Ca2+, the Na+/Ca2+ exchanger (NCX), was disabled by removal of extracellular Ca2+ and Na+. In this situation, it may be assumed that the other Ca2+ transporters (sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uniporter) are too slow to compete with SERCA. SR Ca2+ release was induced by short, rapid caffeine pulses, and the amplitude (?[Ca2+]i) and time for 50% [Ca2+]i decline (t1/2) of the resulting [Ca2+]I transients were measured in the absence and in the presence of drugs that affect the rate of SR Ca2+ uptake by SERCA, namely the beta-adrenergic receptor agonist isoproterenol (ISO), which causes increase in SERCA activity, and 2,5-di-(tert-butyl) hydroquinone (tBQ), which inhibits the enzyme. ISO caused significant reduction of t1/2 (p< 0,01), without any significant change in ?[Ca2+]i (p> 0,05). In case of tBQ, significant increase of t1/2 (p< 0,01) and reduction of ?[Ca2+]I (p< 0,05) were observed. A theoretical model (Tang & Othmer, 1994) was adapted for mathematical description of the experimental model proposed. Simulation of the effects of the drugs, in which SERCA kinetic parameters were changed according to data obtained from literature, were reasonably successful at reproducing the cytosolic [Ca2+]i kinetics observed experimentally (AU) | |