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.
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