Abstract

Neutrophils are terminally differentiated cells with a lifespan of hours to days, terminated by programmed cell death, or apoptosis. In culture, apoptosis can be delayed by the addition of G-CSF or other growth factors or inflammatory mediators. We present preliminary data that neutrophils possess an acid endonuclease that is responsible for the internucleosomal cleavage seen in apoptosis. We have partially purified the endonuclease and show it has an apparent molecular weight of 35 kDa, a pH optimum of 5.5, and a threshold for activity of pH 6.8. In addition, we show that apoptotic neutrophils acidify to a pH value as low as pH 6.0 after G-CSF deprivation, and that pH homeostasis is dependent upon activity of the Na+/H+ exchanger (NHE) and the vacuolar-type proton ATPase. We propose to purify and clone the acid endonuclease and to examine the regulation of the NHE.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08AI001345-03
Application #
2390112
Study Section
Cancer Institutional Fellowship Review Committee (CT)
Project Start
1995-04-01
Project End
1998-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
3
Fiscal Year
1997
Total Cost
Indirect Cost

  Institution

Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037

  Publications

Adachi, S; Gottlieb, R A; Babior, B M (1998) Lack of release of cytochrome C from mitochondria into cytosol early in the course of Fas-mediated apoptosis of Jurkat cells. J Biol Chem 273:19892-4
Gottlieb, R A; Babior, B M (1997) Regulation of Fas-mediated apoptosis. Curr Top Cell Regul 35:69-105
Niessen, H; Meisenholder, G W; Li, H L et al. (1997) Granulocyte colony-stimulating factor upregulates the vacuolar proton ATPase in human neutrophils. Blood 90:4598-601
Adachi, S; Cross, A R; Babior, B M et al. (1997) Bcl-2 and the outer mitochondrial membrane in the inactivation of cytochrome c during Fas-mediated apoptosis. J Biol Chem 272:21878-82
Krippner, A; Matsuno-Yagi, A; Gottlieb, R A et al. (1996) Loss of function of cytochrome c in Jurkat cells undergoing fas-mediated apoptosis. J Biol Chem 271:21629-36
Gottlieb, R A; Dosanjh, A (1996) Mutant cystic fibrosis transmembrane conductance regulator inhibits acidification and apoptosis in C127 cells: possible relevance to cystic fibrosis. Proc Natl Acad Sci U S A 93:3587-91
Gottlieb, R A; Nordberg, J; Skowronski, E et al. (1996) Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification. Proc Natl Acad Sci U S A 93:654-8
Gottlieb, R A; Gruol, D L; Zhu, J Y et al. (1996) Preconditioning rabbit cardiomyocytes: role of pH, vacuolar proton ATPase, and apoptosis. J Clin Invest 97:2391-8
Meisenholder, G W; Martin, S J; Green, D R et al. (1996) Events in apoptosis. Acidification is downstream of protease activation and BCL-2 protection. J Biol Chem 271:16260-2