One of the critical functions of the osteoblast is matrix mineralization. To accomplish this task, the osteoblast must be able to influence the pH and ion current of the fluid in the demarcation zone. It is likely that the osteoblasts are capable of generating and maintaining gradients of HCO3-, Cl-, Na+, K+ and Ca2+ between the plasma and bone extracellular fluid of the demarcation zone. These cells also undergo marked shape changes during hormonal stimulation. Maintenance of the ionic gradients, in particular Ca2+ and HCO3-, are essential for bone remodeling. Establishing vectorial transport and transcellular ionic gradients require polarized localization of ion transport pathways. The nature of the ionic pathways and their localization in the osteoblasts are not known. It is also not known whether the shape changes observed in response to calcitropic hormones are also accompanied by volume changes. We will use the osteosarcoma cell line UMR 106, and primary cultures of osteoblast-like cells from long bone to: a) identify and then kinetically characterize the ionic pathways which participate in cytosolic pH regulation by the osteoblast. These ionic pathways include Cl- dependnt HCO3- secretion (Cl-/HCO3-), Na+/H+ exchanger K+ and Cl- conductances nd KCl or 3NaKCl2 cotransporters. This will be achieved by measurements of pHi and (Ca2+)i with the pH and Ca2+ sensitive dyes BCECF and Fura 2, respectively, and net, undirectional and exchange transport of 36 Cl-, 22Na, and 86Rb.; b) localize these ionic pathways to the membrane facing the BECF or the membrane facing plasma. UMR-106 and normal rat osteoblast-like cells will be grown on collagen coated filters and placed in chamber. The ionic transporters facing the collagen matrix or the medium will be identified. In parallel, the ionic transport characteristics of vesicles prepared from different portions of the osteoblast plasma membrane will be studied. These ionic pathways will be examined within the framework of cell volume regulation by these cells. Finally, the effect of hormones such as PTH2+ Vitamin D, prostoglandins, epidermal growth factor, and second messengers such as Ca2+, cAMP, and protein Kinase C activation, on these ionic pathways will be studied as outlined above.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR039245-04
Application #
3159244
Study Section
General Medicine B Study Section (GMB)
Project Start
1988-12-01
Project End
1992-09-30
Budget Start
1990-09-01
Budget End
1992-09-30
Support Year
4
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
Green, J (1994) Cytosolic pH regulation in osteoblasts. Miner Electrolyte Metab 20:16-30
Green, J (1994) The physicochemical structure of bone: cellular and noncellular elements. Miner Electrolyte Metab 20:7-15
Zhang, B X; Zhao, H; Loessberg, P A et al. (1992) Regulation of agonist-evoked [Ca2+]i oscillation by intracellular Ca2+ and Ba2+ in AR42J cells. Am J Physiol 262:C1125-33
Muallem, S (1992) The ins and outs of Ca2+ in exocrine cells. Adv Second Messenger Phosphoprotein Res 26:351-68
Green, J; Kleeman, C R (1992) Role of calcium and cAMP messenger systems in intracellular pH regulation of osteoblastic cells. Am J Physiol 262:C111-21
Merritt, B S; Yamaguchi, D T; Green, J et al. (1992) Dissociation between parathyroid hormone-stimulated cAMP and calcium increase in UMR-106-01 cells. J Cell Physiol 152:520-8
Muallem, S; Zhang, B X; Loessberg, P A et al. (1992) Simultaneous recording of cell volume changes and intracellular pH or Ca2+ concentration in single osteosarcoma cells UMR-106-01. J Biol Chem 267:17658-64
Star, R A; Zhang, B X; Loessberg, P A et al. (1992) Regulatory volume decrease in the presence of HCO3- by single osteosarcoma cells UMR-106-01. J Biol Chem 267:17665-9
Khademazad, M; Zhang, B X; Loessberg, P et al. (1991) Regulation of cell volume by the osteosarcoma cell line UMR-106-01. Am J Physiol 261:C441-7
Loessberg, P A; Zhao, H; Muallem, S (1991) Synchronized oscillation of Ca2+ entry and Ca2+ release in agonist-stimulated AR42J cells. J Biol Chem 266:1363-6

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