Abstract
Activated neutrophils undergo a large burst of metabolic acid generation, yet maintain their cytosolic pH (pHi) within physiological limits. To analyze the underlying regulatory mechanisms, pHi was measured fluorimetrically in suspensions of human neutrophils. In acid loaded but otherwise unstimulated cells, pHi recovered rapidly via Na+/H+ exchange. Upon Na+ removal, recovery from an imposed acid load was negligible. Phorbol ester activation of acidified cells induced a rapid recovery of pHi partly due to a Zn(2+)-sensitive H(+)-conductive pathway. A third component of the regulatory response was apparent in Na(+)-free media containing Zn2+. Acid extrusion through this alternate pathway was voltage sensitive and capable of translocating H+ equivalents against their electrochemical gradient. This active H+ transport was inhibited by N-ethylmaleimide, by N,N'-dicyclohexylcarbodiimide and by nanomolar doses of bafilomycins A1 or B1, suggesting the involvement of vacuolar (V)-type H+ pumps. Cytosolic alkalinization was accompanied by extracellular acidification, indicative of translocation of H+ equivalents across the surface membrane and consistent with the sensitivity of the alkalinization to changes in plasma membrane potential. The activity of the V-type H+ pumps was virtually undetectable in resting cells, becoming apparent only after treatment with phorbol esters or other, chemically unrelated agonists of protein kinase C. These H+ pumps are likely to play a role in pHi homeostasis during the metabolic burst that accompanies neutrophil activation during infection and inflammation.
Highlights
Activated neutrophils undergoa large burstof met- potent microbicidal agents, essential for host defense against abolic acid generation, yet maintain their cytosopliHc infection
To analyze the un- The one-electron reduction of molecular oxygen to 0; is derlying regulatory mechanisms, pHi was measured effected by the NADPH oxidase, a membrane-bound multifluorimetrically in suspensions of human neutrophils. subunit complex [1,2]
Cytosolic alkalinization was accompanied by ification is unmasked extracellular acidification, indicative of translocation when neutrophils are stimulated in media free ofNa’ or in of H+ equivalents across the surface membrane and the presence of amiloride, a potent inhibitor of the antiport
Summary
Exist inmacrophages [10,11,12], which are phagocytic cells closely related to neutrophils. In order topreclude any contribution toH' (equivalent) extrusion by the anion exchangers, all the experiments demycin and other blockers of V-type ATPases. This behavior scribed in this work were performed in the nominalabsence differs from that reportfeodr unstimulated neutrophilsw, hich of HCO;. H' pumping across the plasmalemma isnegligible in resting peny1)amino amiloride (1p ~ (n)ot shown),or by omission of cells, even after acid loading,but becomes markedly activated extracellular Na+ (Fig. lA, lower truce). Together, these reupon stimulation of PKC at both normal and acidic pH,. TO sultsconfirmearlierreportsthatthesteep pHi recovery our knowledge, this is the first report directly linking activa- observed in Na' solution is mediated largely by the Na+/H' tion of V-type ATPases with stimulatioonf a protein kinase. antiport [4].It is noteworthy that, in medium void ofNa', pH
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