[Truncated abstract] Steroid receptors belong to the superfamily of nuclear receptors, and include the androgen receptor (AR), estrogen receptors (ER[alpha] and ER[beta], glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and the progesterone receptors (PRA and PRB). Before binding ligand, the receptor undergoes biochemical and structural modifications through a series of interactions with molecular chaperones and cochaperones all within a receptor heterocomplex. The mature receptor complexes with the major chaperone Hsp90, the stabilising cochaperone p23, and one member of a group of cochaperones termed immunophilins. Steroid receptor-associated immunophilins include the cyclophilin, CyP40, two FK506-binding proteins, FKBP51 and FKBP52, and the protein phosphatase, PP5. Immunophilins are characterised by the presence of TPR domains which compete directly for the TPR-acceptor site within Hsp90. This leads to mutually exclusive, immunophilin-containing receptor complexes. While PP5 contains a C-terminal phosphatase domain, CyP40, FKBP51 and FKBP52 each contain an N-terminal peptidyl prolyl isomerase (PPIase) domain, which catalyses the cis/trans isomerisation of prolyl peptide bonds. FKBP52 has been demonstrated to potentiate the ligand-dependent activity of AR, GR and PR, but not ER[alpha]. Knowing that CyP40 is the preferred immunophilin associated with the ER[alpha] heterocomplex, it was hypothesised that this immunophilin plays a role in ER[alpha] function. ... As all mutants maintained this potentiating activity it was concluded that the five altered residues found within gpGR do not contribute to the altered interaction of FKBP52 and receptor. However, it cannot be discounted that FKBP51 is more competitive for gpGR. Immunophilins are hormonally regulated, with FKBP52 found to be essential for female fertility in mice. It was hypothesised that levels of immunophilins, associated with steroid receptors important in the menstrual cycle, would be regulated to reflect hormonal activity within cycling endometrium. Human pre-menopausal endometrial sections taken from different phases of the menstrual cycle were examined immunohistochemically for expression of CyP40, FKBP51, FKBP51 and PP5. Immunophilin levels peaked at the mid-secretory phase correlating with stromal decidualization, a process essential for eventual blastocyst implantation. The importance of immunophilins to steroid receptor action was therefore reinforced by the observation that immunophilins appear to be hormonally regulated in cycling pre-menopausal human endometrium. Further studies into the effects of immunophilin loss and knockdown on steroid receptor-mediated responses in specific mouse tissues, knockout-derived mouse embryo fibroblasts and cancer cell lines may contribute to our understanding of the receptor-selective and tissue-specific actions of the immunophilins. Elucidation of the mechanisms through which they modulate receptor function may provide opportunities for therapeutic intervention in steroid-related disease.

Extracellular adenosine 5′-triphosphate (ATP) is an agonist for the P2Z receptor of human leukaemic lymphocytes and opens a Ca ²⁺-selective ion channel, which also conducts Ba²⁺, Sr²⁺ and the small fluorescent dye, ethidium⁺. A wide range of receptor agonists, many of which raise cytosolic [Ca²⁺] activate phospholipase D (PLD). In the present study, it was shown that both ATP and 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) stimulated PLD activity in a concentration-dependent manner, and the inhibitory effects of suramin, oxidised ATP, extracellular Na⁺ and Mg²⁺ suggested that the effect of these agonists is mediated by P2Z receptors. The role of divalent cations in ATP-stimulated PLD activity was investigated. Several agonists (eg ATP, thapsigargin, ionomycin) stimulated a rise in cytosolic [Ca²⁺] in human lymphocytes, but only ATP and ionomycin stimulated PLD activity. When Ca²⁺ influx was prevented by EGTA, the majority of ATP-stimulated and all of ionomycin-stimulated PLD activity was inhibited. Preloading cells with the Ca²⁺ chelator, BAPTA, reduced cytosolic [Ca²⁺] and, paradoxically, ATP-stimulated PLD activity was potentiated. ATP-stimulated PLD activity was supported by both Ba²⁺ and Sr²⁺ when they were substituted for extracellular Ca²⁺. Furthermore, both ATP-stimulated PLD activity and ATP-stimulated ¹³³Ba²⁺ influx showed a linear dependence on extracellular [Ba²⁺]. Thus it was concluded that ATP stimulated PLD activity in direct proportion to the influx of divalent cations through the P2Z ion channel and this PLD activity was insensitive to changes in bulk cytosolic [Ca²⁺]. The calmodulin (Ca²⁺/CaM) inhibitor, trifluoperazine (TFP) inhibited ionomycin- and ATP-stimulated PLD activity and ATP-stimulated apoptosis, but had no effect on PLD activity already activated by ATP. However, TFP inhibited ATP-stimulated Ca²⁺, Ba²⁺ and ethidium⁺ fluxes, at concentrations below those which inhibit Ca²⁺/CaM, suggesting that TFP inhibits the P2Z receptor. Similarly, the isoquinolinesulphonamide, KN-62, a selective inhibitor of Ca²⁺/CaM-dependent protein kinase II (CaMKII), also prevented ATP-stimulated apoptosis, but had no effect on pre-activated PLD. In addition, KN-62, and an analogue, KN-04, which has no effect on CaMKII, potently inhibited ATP-stimulated Ba²⁺ influx (IC₅₀ 12.7 ± 1.5 and 17.3 ± 2.7 nM, respectively), ATP-stimulated ethidium⁺ uptake (IC₅₀ 13.1 ± 2.6 and 37.2 ± 8.9 nM, respectively), ATP-stimulated phospholipase D activity (50% inhibition 5.9 ± 1.2 and 9.7 ± 2.8 nM, respectively) and ATP-induced shedding of the surface adhesion molecule, L-selectin (IC₅₀ 31.5 ± 4.5 and 78.7 ± 10.8 nM, respectively). They did not inhibit phorbol ester- or ionomycin-stimulated PLD activity or phorbol ester-induced L-selectin shedding. Neither KN-62 nor KN-04 (both 500 nM) have any effect on UTP-stimulated Ca²⁺ transients in fura-2-loaded human neutrophils, a response which is mediated by the P2Y₂ receptor, neither did they inhibit ATP-stimulated contractile responses mediated by the P2X₁ receptor of guinea pig urinary bladder. Thus, KN-62 and KN-04 are almost equipotent as P2Z inhibitors with IC₅₀s in the nanomolar, indicating that their actions cannot be due to CaMKII inhibition, but rather that they are potent and direct inhibitors of the P2Z receptor. Extracellular ATP-induced shedding of L-selectin from lymphocytes into the medium is a Ca²⁺-independent response. L-selectin is either cleaved by a metalloproteinase or a PLD with specificity for glycosylphosphatidylinositol (GPI). The novel hydroxamic acid-based zinc chelator, Ro-31-9790 blocks ATP-induced L-selectin shedding, but was without effect on ATP-induced Ba²⁺ influx or ATP-stimulated PLD activity. Furthermore, another zinc chelator, 1,10-phenanthroline, an inhibitor of a GPI-PLD, potentiated rather than inhibited ATP-stimulated PLD activity, suggesting that ATP-induced L-selectin shedding and ATP-stimulated PLD activity are independent of each other. Although extracellular ATP is the natural ligand for the lymphocyte P2Z receptor, it is less potent than BzATP in stimulating Ba²⁺ influx. Concentration-response curves for BzATP- and ATP-stimulated ethidium⁺ influx gave EC₅₀s 15.4 ± 1.4 µM and 85.6 ± 8.8 µM, respectively. The maximal response to ATP was only 69.8 ± 1.9% of that for BzATP. Hill coefficients were 3.17 ± 0.24 and 2.09 ± 0.45 for BzATP and ATP respectively, suggesting greater positive cooperativity for BzATP than for ATP in opening the P2Z-operated ion channel. A rank order of agonist potency of BzATP > ATP = 2MeSATP > ATPγS was observed for agonist-stimulated ethidium⁺ influx, while maximal influxes followed a rank order of BzATP > ATP > 2MeSATP > ATPγS. When ATP (300 -1000 µM) was added simultaneously with 30 µM BzATP (EC₉₀), it reduced both ethidium⁺ and Ba²⁺ fluxes by 30 - 40% relative to values observed with BzATP alone. KN-62, previously shown to be a specific inhibitor of the lymphocyte P2Z receptor, was a less potent antagonist of BzATP-induced fluxes than ATP, when maximal concentrations of both agonists (50 and 500 µM respectively) were used. However, when BzATP (18 µM) was used at a concentration equiactive with a maximally effective ATP concentration, KN-62 showed the same inhibitory potency for both agonists. The ecto-ATPase antagonist, ARL-67156, inhibited both ATP- and BzATP-stimulated Ba²⁺ influx, suggesting that the lower efficacy of ATP compared with BzATP was not due to preferential hydrolysis of ATP. Thus, the natural ligand, ATP, is a partial agonist for the P2Z receptor while BzATP is a full agonist. Moreover the competitive studies show that only a single class of P2-receptor (P2Z class) is expressed on human leukaemic lymphocytes. Both ATP- and BzATP-stimulated PLD activity were significantly inhibited (P < 0.05) when cells were suspended in iso-osmotic choline Cl medium. Choline⁺ was found to be a permeant for the P2Z ion channel, since ATP induced a large uptake of [¹⁴C]choline⁺ (60 to 150 µmol/ml intracellular water) during a 5 min incubation, which remained in the cells for several hours, and ATP was used to load cells with these levels of choline⁺. Intracellular choline⁺ inhibited ATP-, BzATP-, PMA- and ionomycin-stimulated PLD activity. Brief exposure of lymphocytes to ATP increased the subsequent basal rate of ethidium⁺ uptake, and this was prevented by intracellular choline⁺. It is proposed that P2Z-mediated Ca²⁺ influx in lymphocytes activates PLD leading to significantly changes of the phospholipid composition of the plasma membrane, which subsequently produces a permeability lesion, which in turn contributes to cell death.

Graduation date: 1996

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 104-118).

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "December 2001." Typescript. Vita. Includes bibliographical references (leaves 228-272). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 123-159).

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains vi, 200 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains viii, 264 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy