Browsing by Subject "Calcium -- physiology"
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Item Inhibition of virus replication by cocaine: alterations in interferon production and calcium regulation(Texas Tech University, 1999-12) Grattendick, Kenneth JohnPrevious studies have demonstrated cocaine to have a myriad of effects on the immune system. Depending on which component of the immune system is studied and the methods used, cocaine has been shown to possess both stimulatory and inhibitory effects. Cocaine has also been associated with an increase in the risk of developing infectious diseases, although a direct correlation has yet to be determined. The studies presented in this dissertation describe the effects of cocaine on host resistance to viral infections. Cocaine was found to possess antiviral properties in macrophages (Mø). L929 cells, and Madin Darby canine kidney (MDCK) cells in vitro. This effect was dose-dependent in all of these cells with 100 µg/ml exhibiting the maximal inhibition. A similar dose-dependent inhibition was observed in M0 exposed to norcocaine, a metabolite of cocaine. However, other metabolites did not show any effects on virus replication. Cocaine induced a time-dependent increase in the antiviral activity of Mǿ that was not reproduced in L929 or MDCK cells. On average, the antiviral effect of cocaine required approximately 24—48 hr to appear, indicating that the effect was not due to a direct interaction of cocaine and virus. Cocaine's inhibition of virus replication could be reversed by the addition of either antibodies to interferons (IFN) or calcium ionophores. An indication that the effect of cocaine was due to the secretion of an antiviral protein was found in experiments showing that the antiviral effect could be transferred from cells incubated with cocaine to unstimulated cells. This antiviral product was found to accumulate over time in the media and could be neutralized by the addition of anti-IFN. In studies to determine the direct effects of cocaine on IFN production, cocaine was found to induce a 2-3-fold increase in IFN secretion in both L929 cells and M0, with similar increases in IFN transcripts. Cocaine also demonstrated the ability to inhibit cell proliferation, an effect attributed to the production of IFN. Experiments were also conducted to determine if the antiviral effects of cocaine observed in vitro could be demonstrated in an animal model. C57B1/6 mice were infected with Influenzavirus A and given daily i.p. injections of 10 mg/kg cocaine or saline. Cocaine-injected mice were visibly less sick than control animals and had 50% less virus in their lungs, as determined by hemagglutination. This reduction in virus load was consistent with the previous in vitro experiments with cocaine. Because the addition of calcium ionophores had been demonstrated to reverse the antiviral effect of/« vitro cocaine exposure, studies were conducted to determine the effects of cocaine on intracellular calcium (Ca^2+) regulation. Mø incubated with 100 µg/ml cocaine for 48-72 hr demonstrated a 41% increase in steady-state Ca^2+ concentrations. This effect was observed when cells were assayed either in the presence or absence of extracellular Ca^2+ indicating an alteration in calcium regulation that was localized to either the cytoplasm or intracellular membranes. Studies on the mobilization of Ca^2+ showed an increase m ATP-induced Ca^2+ transients when Mø were incubated with cocaine. Calcium ATPase inhibitors reduced the calcium increases in cocaine-treated Mø, further supporting the hypothesis that cocaine was increasing calcium mobilization. This result also indicated that the mechanism by which cocaine altered intracellular Ca levels was most likely localized to the endoplasmic reticulum Ca^2+ -ATPases. In summary, cocaine was found to inhibit virus replication by increasing IFN production and altering cytoplasmic Ca^2+ levels.Item Intracellular calcium and pacemaker activity in the sinoatrial node(Texas Tech University, 1997-05) Li, JinThe importance of the sinoatrial (S-A) node as the pacemaker of the heart is well known; however, little is known about calcium homeostasis and excitation-contraction coupling in the pacemaker cells. Thus, the major objectives of this dissertation research were: (1) to quantitatively measure intracellular calcium (Cai^2+) in isolated pacemaker cells; (2) to examine the relationship between Cai^2+ and electrical activity; and (3) to measure Ca^2+ release from the sarcoplasmic reticulum (SR) and determine its effect on pacemaker activity. We used the fluorescent Ca^2+ indicator, Indo-1, to assess the changes in Cai^2+ and perforated-patch whole-cell recordings to monitor the electrical activity of single, cultured pacemaker cells that had been isolated from the rabbit S-A node. Indo-1 fluorescence ratios were calibrated both in vitro and in vivo. The contributions to action potential-induced Cai^2+ transients from Ca^2+ entry through voltage-gated channels and Ca^2+ release from the SR were demonstrated. The results indicate the existence of an internal Ca^2+ store, the SR, and that action potential-induced Cai^2+ transients are produced mainly by Ca^2+ entry through voltage-gated Ca^2+ channels, rather than by Ca^2+ release from the SR. Simultaneous recordings of membrane potentials and the Indo-1 fluorescence ratio showed that automaticity can be modified by isoproterenol and caffeine, agents that modulate Cai^2+. Changes in beat rate were correlated with changes in Cai^2+; however, Cai^2+ transients were not essential for cells to generate spontaneous firing. Using the perforated-patch voltage-clamp technique, we studied the effects of ryanodine and thapsigargin, inhibitors of SR Ca^2+ release, on pacemaker activity, hyperpolarization-activated inward current, time-independent inward current, L- and T-type Ca ^2+ currents, and inward Na^+-Ca^2+ exchange current. Both ryanodine and thapsigargin slowed pacemaker activity significantly, in part by reducing inward Na^+-Ca^2 exchange current; ryanodine also had a direct inhibitory effect on T-type Ca^2+ current. In summary, rabbit S-A node pacemaker cells display a complex set of positive and negative feedback mechanisms that control intracellular Ca^2+ and pacemaker activity.