title: 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.
abstract: 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 ionomycin (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 (1-2 h) 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 kinase signalling pathways which can be both activated and down-regulated by PMA.
authors.affiliation: Molecular Devices Corporation, Menlo Park, California 94025.
authors.forename: H G
authors.initials: HG
authors.lastname: Wada
authors.forename: S R
authors.initials: SR
authors.lastname: Indelicato
authors.forename: L
authors.initials: L
authors.lastname: Meyer
authors.forename: T
authors.initials: T
authors.lastname: Kitamura
authors.forename: A
authors.initials: A
authors.lastname: Miyajima
authors.forename: G
authors.initials: G
authors.lastname: Kirk
authors.forename: V C
authors.initials: VC
authors.lastname: Muir
authors.forename: J W
authors.initials: JW
authors.lastname: Parce
mesh_terms: D016227:Benzoquinones; D001853:Bone Marrow / Q000378:metabolism*; D001854:Bone Marrow Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002455:Cell Division; D002460:Cell Line; D004988:Ethers, Cyclic / Q000494:pharmacology; D019833:Genistein; D005947:Glucose / Q000378:metabolism*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015759:Ionomycin / Q000494:pharmacology; D007529:Isoflavones / Q000494:pharmacology; D007700:Kinetics; D047029:Lactams, Macrocyclic; D009281:Naphthalenes; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D011083:Polycyclic Compounds / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction; D017923:Sodium-Hydrogen Exchangers; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D016227:Benzoquinones; D002352:Carrier Proteins; D004988:Ethers, Cyclic; D007529:Isoflavones; D047029:Lactams, Macrocyclic; D009281:Naphthalenes; D011083:Polycyclic Compounds; D011809:Quinones; D017923:Sodium-Hydrogen Exchangers; D019319:Okadaic Acid; D017828:Rifabutin; D015759:Ionomycin; C020754:herbimycin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; C058819:calphostin C; D005947:Glucose; D013755:Tetradecanoylphorbol Acetate