The interaction of Na and H ions with the extracellular and intracellular sites of the Na+/H + exchanger of the osteosarcoma cell line UMR-106 was investigated. Na ions interact with a single, saturable extracellular transport site. H + and amiloride appear to compete with Na + for binding to this site. The apparent affinity for extracellular Na § (Na~) and amiloride was independent of intracellular H + (Hi+), Nai +, or an outwardly directed H + gradient. The interaction of H + with the intracellular face of the exchanger had a sigmoidal characteristic with a Hill coefficient of ~2. The apparent affinity for Hi+ was independent of Na~ between 25 and 140 mM. The apparent affinity for Hi+, but not the number of intracellular sites, increased with the increase in the outwardly directed H § gradient across the membrane. Nai+/Ho + exchange (reverse mode) is an electroneutral process with a Na+/H + stoichiometry of 1. The dependence of Na~+/H 2 exchange on Na~ + was sigmoidal, with a Hill coefficient of 2.16. Nai + competes with Hi+ for binding to at least the transport site. The apparent affinity for Nai + decreased with the increase in the outwardly directed H § gradient. High H 2 inhibited exchange activity in the reverse mode. We conclude that intracellular Na § and H § can activate the exchanger. The exchanger has two separate and asymmetric extracellular and intracellular transport sites. The relative apparent affinities of the internal transport site for Na + and H + are determined by the direction and magnitude of the H § gradient across the membrane. Kinetic characterization of the exchanger suggests that Na+/H § exchange is compatible with a simultaneous transport model, although a ping-pong transport model could not be excluded.

Measurements of cytosolic pH (pHi), S6C1 fluxes and free cytosolic Ca ~+ concentration ([Ca2+]i) were performed in the clonal osteosarcoma cell line UMR-106 to characterize the kinetic properties of CI-/HCO~ (OH-) exchange and its regulation by pHi and [Ca2+]i. Suspending cells in C1--free medium resulted in rapid cytosolic alkalinization from phi 7.05 to ~7.42. Subsequently, the cytosol acidified to pHi 7.31. Extracellular HCO~ increased the rate and extent of cytosolic alkalinization and prevented the secondary acidification. Suspending alkalinized and Cl--depleted cells in O--containing solutions resulted in cytosolic acidification. All these pHi changes were inhibited by 4',4',-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) and H~DIDS, and were not affected by manipulation of the membrane potential. The pattern of extracellular CI-dependency of the exchange process suggests that C1-ions interact with a single saturable external site and HCO~ (OH-) compete with CI-for binding to this site. The dependencies of both net anion exchange and CI-self-exchange fluxes on phi did not follow simple saturation kinetics. These findings suggest that the anion exchanger is regulated by intracellular HCO~ (OH-).A rise in [Ca~+]i, whether induced by stimulation of protein kinase C-activated Ca 2+ channels, Ca 2+ ionophore, or depolarization of the plasma membrane, resulted in cytosolic acidification with subsequent recovery from acidification. The Ca2+-activated acidification required the presence of Cl-in the medium, could be blocked by DIDS, and H2DIDS and was independent of the membrane potential. The subsequent recovery from acidification was absolutely dependent on the initial acidification, required the presence of Na + in the medium, and was blocked by amiloride. Activation of protein kinase C without a change in [Ca~+]i did not activate the Na+/H + exchanger in UMR-106 cells. These findings indicate that an increase in [Ca~+]~ was sufficient to activate the CI-/HCO~ exchanger, which results in the acidification of the cytosol. The accumulated H + in the cytosol activated the Na+/H + exchanger. Kinetic analysis of the anion exchange showed that at saturating intracellular OH-, a [Ca2+]i increase did not modify the properties of the extracellular site. A rise in [Ca~+]i increased the apparent affinity for intracellular OH-(or HCO;) of both net anion and Cl-self exchange. These results indicate that [Ca2+]i modifies the interaction of intraceilular OH-(or HCO~-) with the proposed regulatory site of the anion exchanger in UMR-106 cells.

A wide spectrum of prostaglandins (PG) stimulate both the production of cyclic AMP and an increase in free cytosolic Ca2+ concentration [( Ca2+]i) in the osteogenic osteosarcoma cell line, UMR-106-01, which has characteristics compatible with osteoblasts. Using PG-stimulated determinations of the second messengers cyclic AMP and [Ca2+]i, a method for classification of PG receptors is presented. UMR-106-01 cells demonstrate three subclasses of PG receptors. One receptor interacts with PGF2 alpha, PGD2, and thromboxane B2 (TxB2) to increase [Ca2+]i. A second receptor binds PGE2, PGE1, PGI2, PGA2 and 6-oxo-PGF1 alpha to increase [Ca2+]i by stimulation of a second separate phospholipase C pool. A third receptor accepts PGE2, PGE1, PGA2, PGI2 and to a lesser extent PGF2 alpha, PGD2 and TxB2 to increase cyclic AMP. Such a classification system may be applicable to other cells responding to multiple PGs by inducing changes in cellular second messengers.

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