Bone resorption involves osteoclast-mediated acidification via a vacuolar type H+-ATPase (V-ATPase) found in lysosomes and at the ruffled border membrane. V-ATPases are proton pumps that include the a3 subunit, one of four isoforms (a1-a4) in mammals. The a3 isoform is enriched in osteoclasts where it is essential for bone resorption. Over 50% of humans with osteopetrosis have mutations in the a3 subunit and a3 mutations in mouse also result in osteopetrosis. A mouse founder with an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This mouse bears a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit resulting in the replacement of a highly conserved amino acid, arginine 740, with serine (R740S). The heterozygous mice (+/R740S) exhibit high bone density but otherwise have a normal appearance, size and weight. Osteoblast parameters are unaffected whereas osteoclast number and marker expression are increased along with a decreased number of apoptotic osteoclasts. V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport along with wild type levels of ATP hydrolysis, indicating that the R740S mutation uncouples ATP hydrolysis from proton transport. The mutation however has no effect on ruffled border formation or polarization of +/R740S osteoclasts. Mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis than +/R740S mice and die by postnatal day 14. Similarly to the mouse models that lack the a3 subunit (oc/oc and Tcirg1-/-) R740S/R740S osteoclasts do not polarize and lack ruffled border membranes. However R740S/R740S osteoclasts exhibit unique phenotypic traits, including increased apoptosis and defective early stage autophagy. Intracellular and extracellular acidification is absent in R740S/R740S osteoclasts, providing evidence for a requirement for lysosomal acidification for cytoplasmic distribution of key osteoclast enzymes such as TRAP and other important osteoclast phenotypic traits. This work provides evidence that the a3 subunit of V-ATPases and the proton pumping function of a3-containing V-ATPases play a major role in osteoclast survival, maturation and function.

Bone resorption involves osteoclast-mediated acidification via a vacuolar type H+-ATPase (V-ATPase) found in lysosomes and at the ruffled border membrane. V-ATPases are proton pumps that include the a3 subunit, one of four isoforms (a1-a4) in mammals. The a3 isoform is enriched in osteoclasts where it is essential for bone resorption. Over 50% of humans with osteopetrosis have mutations in the a3 subunit and a3 mutations in mouse also result in osteopetrosis. A mouse founder with an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This mouse bears a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit resulting in the replacement of a highly conserved amino acid, arginine 740, with serine (R740S). The heterozygous mice (+/R740S) exhibit high bone density but otherwise have a normal appearance, size and weight. Osteoblast parameters are unaffected whereas osteoclast number and marker expression are increased along with a decreased number of apoptotic osteoclasts. V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport along with wild type levels of ATP hydrolysis, indicating that the R740S mutation uncouples ATP hydrolysis from proton transport. The mutation however has no effect on ruffled border formation or polarization of +/R740S osteoclasts. Mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis than +/R740S mice and die by postnatal day 14. Similarly to the mouse models that lack the a3 subunit (oc/oc and Tcirg1-/-) R740S/R740S osteoclasts do not polarize and lack ruffled border membranes. However R740S/R740S osteoclasts exhibit unique phenotypic traits, including increased apoptosis and defective early stage autophagy. Intracellular and extracellular acidification is absent in R740S/R740S osteoclasts, providing evidence for a requirement for lysosomal acidification for cytoplasmic distribution of key osteoclast enzymes such as TRAP and other important osteoclast phenotypic traits. This work provides evidence that the a3 subunit of V-ATPases and the proton pumping function of a3-containing V-ATPases play a major role in osteoclast survival, maturation and function.

KATP channels couple cell metabolic status to the membrane excitability by sensing the cytoplasmic ATP/ADP ratio. Present studies examined how conserved motifs (Walker A (WA), signature sequence (L), and Walker B (WB)) within each NBF of SUR2A bind to Syn-1A to affect its actions on cardiac KATP channels. In vitro binding experiments illustrated that Syn-1A binds cardiac SUR2A at WA and L of NBF-1 and WA, L, and WB of NBF-2. Electrophysiology experiments on stably expressing SUR2A/Kir6.2 cell-lines showed that only L and WB of NBF-1 and all three NBF-2 motifs could abrogate the inhibitory effect of Syn-1A on SUR2A/KATP channels. These results lead me to hypothesize that more independent motif in NBF-2 can bind and abrogate Syn-1A’s inhibition than NBF-1 on SUR2A/KATP channels. A corollary postulate is that Syn-1A acts as a scaffold to secure the NBF-1 and -2 in dimer conformation required for SUR2A to modulate Kir6.2 gating.

KATP channels couple cell metabolic status to the membrane excitability by sensing the cytoplasmic ATP/ADP ratio. Present studies examined how conserved motifs (Walker A (WA), signature sequence (L), and Walker B (WB)) within each NBF of SUR2A bind to Syn-1A to affect its actions on cardiac KATP channels. In vitro binding experiments illustrated that Syn-1A binds cardiac SUR2A at WA and L of NBF-1 and WA, L, and WB of NBF-2. Electrophysiology experiments on stably expressing SUR2A/Kir6.2 cell-lines showed that only L and WB of NBF-1 and all three NBF-2 motifs could abrogate the inhibitory effect of Syn-1A on SUR2A/KATP channels. These results lead me to hypothesize that more independent motif in NBF-2 can bind and abrogate Syn-1A’s inhibition than NBF-1 on SUR2A/KATP channels. A corollary postulate is that Syn-1A acts as a scaffold to secure the NBF-1 and -2 in dimer conformation required for SUR2A to modulate Kir6.2 gating.

Mitochondria are the main sites for adenosine triphosphate (ATP) generation within most cells. Structural and functional alterations of mitochondria due to genetic abnormalities of mitochondria can cause respiratory chain dysfunction. In this study, the important role of mitochondria in energy metabolism was determined by comparing the effect of mitochondrial DNA (mtDNA) mutations on growth patterns and oxidative phosphorylation (OXPHOS) enzyme activities of six isolated clones (B5, B12, D4, D9, E1 and E8); as well as the effect of ATP supplement to culture using the slowest growing clone. The isolated clones had shown distinct growth pattern and morphology. The difference in proliferation rates among the clones was ascertained by the doubling times (B5=26.4h. B12=43.2h. D4=25.7h. D9=33.6h. E1=26.9h and E8=28.8h). The clone's slow growth rate was likely the result of mitochondrial mutations in the 16S rRNA gene, ND1, ND4, ND6 and COX III. Five heteroplasmic mutations were found in clone B12 (G2480T, C2513G, A2520T, C9527T and C14263G), one heteroplasmic mutation in clone D9 (A4137G) and one homoplasmic mutation in clone D4 (C11496). The mutations in clone B12 appeared to be deleterious to the cell by disrupting mitochondrial OXPHOS activities and reducing energy output. Additionally, extracellular ATP supplement to OXPHOS deficient clone B12 facilitated cell growth and enhances the gene expression. Increased expression of mtDNA-encoded respiratory chain complexes observed in clone B12 compared to clone D4 may reflect mitochondrial genomic adaptation to perturbations in cellular energy requirements. The stimulation of mitochondrial biogenesis may be a cellular response in compensation for defects in OXPHOS associated with mtDNA mutations. My data support the hypothesis that the variability in functional manifestations of mtDNA is attributed to the nature of the mutation, number of mutation and the gene specifically affected. These results will help to further our understanding of the relationship between mitochondrial mutation and cellular function.

Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X7 receptor. These responses include opening of a cation selective channel/pore which allows entry of the fluorescent dye, ethidium+ and activation of a membrane metalloprotease which sheds the adhesion molecule L-selectin. In this thesis, the surface expression of P2X7 receptors was measured in normal leucocytes, platelets and B-CLL lymphocytes and compared with their functional responses. Monocytes showed 4-5 fold greater expression of P2X7 than B-, T- and NK- lymphocytes, while P2X7 expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed surface P2X7 at about the same level as for B-lymphocytes from normal subjects. P2X7 function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes. However, the ATP-induced uptake of ethidium into the malignant B-lymphocytes in 3 patients was low or absent. The lack of P2X7 function in these B-lymphocytes was confirmed by the failure of ATP to induce Ba2+ uptake into their lymphocytes. This lack of function of the P2X7 receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule which directs the recirculation of lymphocytes from blood into the lymph node. To study a possible genetic basis of non-functional P2X7 receptor, we sequenced DNA coding for the carboxyl terminal tail of P2X7. In 33 of 130 normal subjects a heterozygous nucleotide substitution (1513A--C) was found while 3 subject carried the homozygous substitution which codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this 496Glu--Ala polymorphism demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the 496Glu--Ala homozygote subject expressed non-functional receptor while heterozygotes showed P2X7 function which was half that of wild type P2X7. Results of transfection experiments showed the mutant P2X7 receptor was non-functional when expressed at low receptor density but regained function at a high receptor density. This density-dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma which upregulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with wild type. The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor. Apart from the 496Glu--Ala polymorphism, three other single nucleotide polymorphisms, 155His--Tyr, 348Ala--Thr and 568Ile--Asn were also found in the P2X7 receptor. The site directed mutant cDNA were generated for all 3 polymorphisms and transfected into HEK293 cells to study the impact of these polymorphisms on P2X7 function. Results suggested that Ile568 is important for P2X7 protein trafficking to cell surface. Further study of these two loss-of-function polymorphisms (496Glu--Ala and 568Ile--Asn) may help better understanding of the functional domains in the P2X7 receptor and its role in CLL, lymphoma and infectious diseases. Conclusions: 1.P2X7 receptor is expressed in human leukocytes, including lymphocytes, natural killer cells as well as monocytes, on both surface and intracellular locations. 2.Both the expression and function of P2X7 are highly variable between in human individuals. Non-functional P2X7 receptors are found in some subjects, including both normal subjects and CLL patients, and are often associated with defects in ATP-induced cytotoxicity and L-selectin shedding. 3.Two single nucleotide polymorphisms (SNPs), 496Glu--Ala and 568Ile--Asn, are found at low frequency in the human population and lead to the loss-of-function of P2X7. Both permeabllity function and the downstream effects mediated by P2X7 are affected by these two SNPs. The mechanisms for the loss-of-function differs between the two polymorphisms.

Master of Science / In vivo many forms of glia utilise both intercellular and extracellular pathways in the form of IP3 permeable gap junctions and cytoplasmic ATP diffusion to produce calcium waves. We introduce a model of ATP and Ca2+ waves in clusters of glial cells in which both pathways are included. Through demonstrations of its capacity to replicate the results of existing theoretical models of individual pathways and to simulate experimental observations of retinal glia the validity of the model is confirmed. Characteristics of the waves resulting from the inclusion of both pathways are identified and described.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 4 uppsatser.

Thesis (M.Sc.). / Written for the Dept. of Experimental Medicine. Title from title page of PDF (viewed 2008/12/07). Includes bibliographical references.