Thesis (Ph. D.)--University of Chicago, Pritzker School of Medicine, Department of Molecular Genetics and Cell Biology, June 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, December 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, June 2003. / Includes bibliographical references. Also available on the Internet.

>Magister Scientiae - MSc / Cancer is defined as the abnormal growth of genetically mutated or perturbant cells. Nicotine is a known cancer promoter and an apoptotic suppressor. This alkaloid acts on the nicotinic acetylcholine receptors which affects the ubiquitin-proteasome protein degradation pathway and ultimately hinders apoptosis. The endoplasmic reticulum (ER) is an interconnecting organelle which synthesises proteins and its quality control processes ensures the proper protein folding, post-translational modifications and conformation of secretory and trans-membrane proteins. Studies demonstrated that the antibiotic, Tunicamycin (Tm) and the sesquiterpene lactone, Thapsigargin (Tg) causes ER stress and consequently cellular arrest. Tm interferes with N-glycosylation of newly synthesised proteins triggering the unfolded protein response, while Tg inhibits intracellular Ca2+ ATPases resulting in increased cytosolic Ca2+. Studies showed that these compounds have potential pro-apoptotic effects. The combinatorial effects of nicotine, Tm and Tg may produce antagonistic or synergistic effects and provide a therapeutic tool against breast cancer. The aim of the study was to determine the apoptotic effects of nicotine, Tm, and Tg on human breast carcinoma (MCF-7) at various time intervals and further to elucidate whether selected ratios of their combinations resulted in synergistic or antagonistic effects.

A prominent finding in the diabetic rat heart is a decrease in the rate at which the ventricular muscle can contract and relax. Since cardiac sarcoplasmic reticulum is thought to be intimately involved in muscle contraction and relaxation we studied the ability of diabetic rat cardiac sarcoplasmic reticulum to transport Ca²⁺ . Hearts were obtained from female Wistar rats 7, 30, 42, and 120 days after the induction of diabetes by a single i.v. injection of either alloxan (65 mg/kg) or streptozotocin (60 mg/kg). At all Ca²⁺ concentrations tested (0.2 μM-5.0 μM free Ca²⁺) cardiac sarcoplasmic reticulum obtained from 42 and 120 day diabetic rats showed a significant decrease in the rate of ATP-dependent tns-oxalate facilitated ²⁺ransport. This was accompanied by a decrease in Ca²⁺ -ATPase activity. The levels of long chain acylcarnitines associated with the microsomal sarcoplasmic reticulum preparation from 42 and 120 day diabetic rats were significantly higher than those present in sarcoplasmic reticulum from control rats. Palmitylcarnitine, the most abundant of the long chain acylcarnitines, in concentrations < 7 μM was found to be a potent time-dependent inhibitor of both Ca²⁺ transport and Ca²⁺ -ATPase in both control and diabetic rat sarcoplasmic reticulum preparations; inhibition of Ca²⁺ transport was found to be more marked in the control preparations. This would indicate that a degree of inhibition produced by the high endogenous levels of palmitylcarnitine may already be present in the diabetic rat heart preparations. Cardiac sarcoplasmic reticulum prepared from acutely diabetic rats (7 days) did not show any decrease in Ca²⁺ transport ability. Levels of long chain acylcarnitines associated with the microsomal preparation enriched in sarcoplasmic reticulum were also unchanged. Insulin treatment of diabetic rats could significantly increase the ability of cardiac sarcoplasmic reticulum to transport Ca²⁺, although at the time period obtested (30 days) the SR Ca²⁺ transport activity was only slightly depressed as compared to control. Insulin treatment also resulted in a slight, but non-significant, lowering of the levels of long chain acylcarnitines associated with the sarcoplasmic reticulum microsomal preparations. These findings suggest that the alteration in sarcoplasmic reticulum function in chronically diabetic rats may be due to the buildup of cellular long chain acylcarnitines which inhibit sarcoplasmic reticulum Ca²⁺ transport. The absence of any significant change in Ca²⁺ transport activity or levels of long chain acylcarnitines at 7 and 30 days suggests that the alterations in 42 and 120 day diabetic rats must be of gradual onset. Cardiac sarcoplasmic reticulum is known to be regulated by a number of factors, among them calmodulin, cAMP-dependent protein kinase, and K⁺. Since Ca²⁺ transport activity in cardiac sarcoplasmic reticulum from chronically diabetic rats is depressed, the role that these regulators play was investigated. Calmodulin (0.61 μM), cAMP (10 μM) plus cAMP-dependent protein kinase (0.2 mg/0.5 ml), and K⁺ (0-110 mM) all stimulated Ca transport in both control and streptozotocin-treated diabetic rats to the same degree. This suggests that the depression observed in sarcoplasmic reticulum function from diabetic rats is not due to altered regulation by these putative mediators of Ca²⁺ uptake. A number of studies suggest that carnitine administration may lower myocardial levels of long chain acylcarnitines in the diabetic rat. Therefore, D,L-carnitine (1 g/kg/day, orally) was administered to 120 day diabetic rats for a 30 day period. The elevated levels of long chain acylcarnitines normally seen in diabetic rats were significantly reduced in the diabetic rats administered carnitine. Carnitine administration, however, could not reverse the previously noted depression in diabetic rat heart function, as measured on an isolated working heart apparatus. In an effort to prevent the onset of the diabetic cardiomyopathy D,L-carnitine was administered (3 g/kg/day, orally) 3 days after the induction of diabetes for a 42 day period. As previously mentioned, sarcoplasmic reticulum Ca²⁺ transport activity was depressed in diabetic rats, as compared to control rats, at all free Ca²⁺ concentrations tested (0.1 μM-3.5 μM). Similarly, sarcoplasmic reticulum levels of long chain acylcarnitines were significantly elevated in these diabetic rats. The diabetic rats treated with carnitine did not show any depression in Ca²⁺ transport activity; long chain acylcarnitine levels were also similar to control. The carnitine-treated diabetic rats, however, showed no improvement in heart function compared to untreated-diabetic rats. These data suggest that although the long chain acylcarnitines are inhibiting cardiac sarcoplasmic reticulum function in chronically diabetic rats other factors must also be contributing to the depression in heart function. / Pharmaceutical Sciences, Faculty of / Graduate

The sarcoplasmic reticulum Ca²⁺ -pumping ATPase is the primary system responsible for the removal of calcium from the sarcoplasm during relaxation of skeletal and cardiac muscles. Since the rat heart SR is used frequently in our laboratory to study the Ca²⁺ -transport defects in disease states, the Ca²⁺ - ATPase activity of this system was characterized. Calmodulin (CaM) and cAMP-dependent protein kinase (cAMP-PK) are known to regulate the dog cardiac SR Ca²⁺ -pump. The effects of these regulators on the rat heart SR Ca²⁺ -pump were studied. Studies were also carried out to investigate the effects of Triton X-100 on SR Ca²⁺ -ATPase activity and the regulation of this activity by CaM. The rat heart SR Ca²⁺-ATPase was stimulated in a concentration-dependent manner by both Ca²⁺ and Mg²⁺ in the complete absence of the other cation. Magnesium produced a concentration-dependent increase in the basal ATPase activity without affecting the maximal ATPase activity. This appeared to result in a gradual disappearance of the Ca²⁺ dependency of the ATPase activity. Addition of 100µM CDTA (trans-1,2-diaminocyclo- hexane-N,N,N',N'-tetraacetic acid), in the absence of added magnesium, produced no effect on Ca²⁺ stimulation of ATPase activity. The results appear to indicate the presence of a low affinity non-specific divalent cation-stimulated ATPase. At a constant Mg: ATP ratio, ATP simulated the SR Ca²⁺-ATPase activity in a concentration-dependent manner. Double-reciprocal plots of the data suggest that the true substrate for rat heart SR Ca²⁺-ATPase may be ATP and not Mg.ATP. In the crude SR, CaM did not stimulate total or Ca²⁺-stimulated ATPase activity over a range of Ca²⁺ and Mg²⁺ concentrations. CaM also failed to stimulate membrane phosphorylation over a range of Mg²⁺ concentrations. Furthermore, CaM did not produce a significant effect on calcium transport into SR vesicles. The catalytic subunit of cAMP-dependent protein kinase was also ineffective in stimulating membrane phosphorylation and Ca²⁺ -ATPase activity. Two CaM antagonists, trifluperazine and compound 48/80, did not affect the rat heart SR ATPase activity. The ATPase activity in Triton-washed SR membranes appeared to be increased at low Triton concentrations. This effect was probably due to the removal of non-intrinsic proteins, leaky vesicles or altered membrane fluidity. At higher Triton X-100 concentrations, the ATPase activity was lost, probably due to loss of the phospholipid environment. When SR membranes phosphorylated under conditions similar to those used for the ATPase assay were analysed by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) followed by autoradiography, a single phosphorylated protein of 7,500-9,000 dalton was observed. This protein may represent the monomeric form of phospholamban. CaM, however, appeared to have no effect on the phosphorylation of this 7,500-9,000 dalton protein in either untreated or Tritan-washed SR membranes. It is speculated that the rat heart SR contains tightly bound CaM which cannot be removed by treatment with Triton X-100. / Pharmaceutical Sciences, Faculty of / Graduate

Elevated levels in blood serum (≥10μmol/L) of the amino acid homocysteine is strongly correlated with the incidence of heart failure (HF). We present evidence that the cyclic thioester, homocysteine thiolactone (HTL), a metabolic product of homocysteine, irreversibly modifies proteins that regulate the contractile process in cardiac muscle. Two proteins found in the sarcoplasmic reticulum (SR), the Ca2+ pump (SERCA2), and the ryanodine receptor (RyR2), are responsible for controlling the cytosolic Ca2+ concentration and hence the contractile state of the heart. While both improper Ca2+ handling and elevated homocysteine levels have been considered bio-markers in HF, a direct connection between the two has not previously been made. We show that HTL reacts with lysine residues on RyR2, generating a Nε-homocysteine-protein, which results in carbonyl formation and a change in the Ca2+ sensitivity of RyR2. This is a new molecular mechanism linking elevated levels of Homocysteine, improper Ca2+ handling and heart failure. This work was supported by NIH 1 R41 HL105063-01 to J. Abramson and R. Strongin.

The sarcoplasmic reticulum (SR) is an intracellular membrane system dedicated to the active regulation of cytosolic calcium in muscle. The opening of Ca²⁺ channels in the SR results in a rapid increase in the myoplasmic Ca²⁺ concentration and the initiation of contraction. Closure of these channels allows the SR to re-accumulate the released Ca²⁺ which results in muscle relaxation. While it is known that a muscle fiber is stimulated to contract by the depolarization of the sarcolemma, it is not understood how this signal is communicated to the SR. The focus of this dissertation is twofold. The first objective is to gain an understanding of the mechanism of Ca²⁺ release from the SR. To this end, three studies have been performed which indicate that Ca²⁺ release is mediated by an oxidation reaction. The second goal is to gain insight into the function of the Ca²⁺ release channel. This is addressed by a fourth study which characterizes the effect of the plant alkaloid, ryanodine on channel operation. The anthraquinones mitoxantrone , doxorubicin, daunorubicin, and rubidazone are shown to be potent stimulators of Ca²⁺ release from SR vesicles. Anthraquinoneinduced Ca²⁺ release is shown to be via a specific interaction with the Ca²⁺ release system of the SR. In addition, a strong interaction between anthraquinone and caffeine binding sites on the Ca²⁺ release channel is observed when monitoring Ca²⁺ fluxes across the SR. It is shown that Ca²⁺ release stimulated by anthraquinones is inhibited by preincubating the quinone with dithionite, a strong reducing agent. Spectrophotometric measurements show that the dithionite treated quinone is in a reduced state. Previous work in this lab has shown that the photooxidizing xanthene dye rose bengal stimulates rapid Ca²⁺ release from skeletal muscle SR vesicles. In this thesis, it is shown that following fusion of vesicles to a bilayer lipid membrane (BLM), Ca²⁺ channel activity is stimulated by nanomolar concentrations of rose bengal in the presence of a broad-spectrum light source. This stimulation is shown to be independent of the Ca²⁺ concentration but is inhibited by μM ruthenium red. The photooxidation of rose bengal is shown to not affect either the K+ or Cl- channels which are present in the SR. Exposure of the Ca²⁺ release channel to 500 nM rose bengal in the presence of light is shown to reverse the modification to the channel induced by μM ryanodine. This apparent displacement of bound ryanodine by nanomolar concentrations of rose bengal is directly observed upon measurement of [³H]ryanodine binding to TSR vesicles. Evidence is presented which suggests that Ca²⁺ release is mediated by singlet oxygen. Micromolar concentrations of the porphyrin meso-Tetra(4-N-methylpyridyl)porphine tetraiodide (TMPyP) is shown to induce the rapid release of Ca²⁺ from skeletal muscle SR vesicles. Porphyrin-induced Ca²⁺ release is stimulated by adenine nucleotides and μM Ca²⁺, and is inhibited by mM Mg²⁺ and μM ruthenium red. High-affinity [³H]ryanodine binding is also enhanced in the presence of the porphyrin. The presence of 1 mM Mg²⁺ in the assay medium sensitizes ryanodine binding to activation by ca²⁺. Porphyrin stimulated single channel activity is also sensitized to activation by Ca²⁺ in the presence of Mg²⁺. Reduction of the porphyrin by dithionite, a strong reducing agent, prior to exposure to the Ca²⁺ release channel inhibited the ability of TMPyP to stimulate Ca²⁺ release. These observations indicate that anthraquinones, rose bengal , and porphyrins induce a stimulation of the Ca²⁺ release protein from skeletal muscle SR by interacting with the ryanodine binding site. In addition, the mechanism of interaction for these compounds appears to be via an oxidation reaction. Nanomolar to micromolar concentrations of ryanodine are shown to alter the gating kinetics of the Ca²⁺ release channel from skeletal muscle SR fused with bilayer lipid membranes. In the presence of asymmetric CsCl, 5 to 40 nM concentrations of ryanodine are shown to activate the channel by increasing the open probability (P₀) without changing the conductance. Statistical analysis of gating kinetics reveal that the open and closed dwell times exhibit bi-exponential distributions that are significantly modified by nM ryanodine. The altered channel gating kinetics seen with low nM ryanodine is reversible and is shown to correlate with the binding kinetics of [³H]ryanodine with its highest affinity site under identical ionic conditions. Ryanodine concentrations between 20 and 50 nM are observed to induce occasional 1/2 conductance fluctuations while ryanodine concentrations greater than 50 nM stabilize the channel into a ½ conductance state which is not reversible. These results are shown to correlate with [³H]ryanodine binding to a second site having lower affinity than the first site. Ryanodine at concentrations greater than 70 μM from the 1/2 to a 1/4 conductance fluctuation , whereas ryanodine concentrations greater than 200 μM cause complete closure of the channel. The concentration of ryanodine required to stabilize either the 1/4 conductance transitions or channel closure do not directly correlate with the measured [³H]ryanodine equilibrium binding constants. However, these results can be explained by considering the association kinetics of ryanodine concentrations greater than 200 nM in the presence of 500 mM CsCl. These results indicate that ryanodine stabilizes four discrete states of the SR release channel and supports the existence of multiple interacting ryanodine binding sites on the channel protein.

Skeletal muscle contains an internal membrane system called the sarcoplasmic reticulum (SR) whose function is to regulate the Ca2+ concentration of the myoplasm. Ca2+ is transported into the SR from the myoplasm via a Ca2+ dependent ATPase thus lowering the myoplasmic Ca2+ concentration. Ca2+ exits from the SR via a Ca2+ releqse pathway resultingin the increase of myoplasmic Ca2+. Muscles contract when the myoplasmic Ca2+ concentration is > 5 uM and relax when the Ca2+ concentration is lowered below 1 uM. The Ca2+ dependent ATPase has been extensively studied but the Ca2+ release system is less well understood. SR vesicles release their internal Ca2+ when a reactive thiol group is oxidized (oxidation-induced Ca2+ release). It is shown in this dissertation that oxidation-induced Ca2+ release is stimulated by adenine nucleotides with an order of effectiveness of: ATP > AMP-PCP > cAMP > AMP > adenine. The stimulatory effect is not dependent upon phosphorylation of a protein because AMP-PCP, a nonhydrolyzable analogue of ATP, is almost as effective as ATP in stimulating oxidation-induced Ca2+ release. It is also shown in this dissertation that photooxidation of histidyl residues results in an increase Ca2+ permeability of the SR. Unlike oxidation-induced Ca2+ release, photooxidation-induced Ca2+ release is Mg2+ independent, not inhibited by ruthenium red and inhibited by adenine nucleotides. Covalent modification of histidyl residues with ethoxyformic anhydride results in the increased permeability of SR vesicles. Similar to photooxidation-induced Ca2+ efflux, EFA-induced Ca2+ efflux is Mg2+ independent and is inhibited by ATP. The AMP-PCP protection of SR proteins from modification with EFA is similar to non-competitive inhibition with a KI = 50 uM. The photooxidation effect is not on membrane lipids but on a protein component which may be an ion transport system, other than the Ca2+ release protein, altered in such a way that it now transports Ca2+.

No description available.