GM-CSF triggers a rapid, glucose dependent extracellular acidification by TF-1 cells: Evidence for sodium/proton antiporter and PKC mediated activation of acid production

The extracellular acidification rate of the human bone marrow cell line, TF‐1, increases rapidly in response to a bolus of recombinant granulocyte‐macrophage colony stimulating factor (GM‐CSF). Extracellular acidification rates were measured using a silicon microphysiometer. This instrument contains micro‐flow chambers equipped with potentiometric sensors to monitor pH. The cells are immobilized in a fibrin clot sandwiched between two porous polycarbonate membranes. The membranes are part of a disposable plastic “cell capsule” that fits into the microphysiometer flow chamber. The GM‐CSF activated acidification burst is dose dependent and can be neutralized by pretreating the cytokine with anti‐GM‐CSF antibody. The acidification burst can be resolved kinetically into at least two components. A rapid component of the burst is due to activation of the sodium/proton antiporter as evidenced by its elimination in sodium‐free medium and in the presence of amiloride. A slower component of the GM‐CSF response is a consequence of increased glycolytic metabolism as demonstrated by its dependence on D‐glucose as a medium nutrient. Okadaic acid (a phospho‐serine/threonine phosphatase inhibitor), phorbol 12‐myristate 13‐acetate (PMA, a protein kinase C (PKC) activator), and ionmycin (a calcium ionophore) all produce metabolic bursts in TF‐1 cells similar to the GM‐CSF response. Pretreatment of TF‐1 cells with PMA for 18 h resulted in loss of the GM‐CSF acidification response. Although this treatment is reported to destroy protein kinase activity, we demonstrate here that it also down‐regulates expression of high‐affinity GM‐CSF receptors on the surface of TF‐1 cells. In addition, GM‐CSF driven TF‐1 cell proliferation was decreased after the 18 h PMA treatment. Short‐term treatment with PMA (1–2h) again resulted in loss of the GM‐CSF acidification response, but without a decrease in expression of high‐affinity GM‐CSF receptors. Evidence for involvement of PKC in GM‐CSF signal transduction was obtained using calphostin C, a specific inhibitor of PKC, which inhibited the GM‐CSF metabolic burst at a subtoxic concentration. Genistein and herbimycin A, tyrosine kinase inhibitors, both inhibited the GM‐CSF response of TF‐1 cells, but only at levels high enough to also inhibit stimulation by PMA. These results indicate that GM‐CSF activated extracellular acidification of TF‐1 cells is caused by increases in sodium/proton antiporter activity and glycolysis, through protein kinas