The Prostate 67:1211^1218 (2007) Effect of Metabolic Inhibitors on ATP and Citrate Content in PC3 Prostate Cancer Cells B.K. Matheson,* J.L. Adams, J. Zou, R. Patel, and R.B. Franklin Department of Biomedical Sciences,University of Maryland Dental School, Baltimore, Maryland BACKGROUND. In normal prostate epithelial cells low m-aconitase activity decreases citrate oxidation leading to citrate accumulation. In prostate cancer cells m-aconitase activity is increased and citrate content is lower. The effect of inhibition of m-aconitase on ATP production by prostate cancer cells (PC3) is not known nor is the contribution of glycolysis versus respiration. METHODS. ATP content of PC3 cells as affected by inhibition of m-aconitase (fluoroacetate (FA), zinc), inhibition of glycolysis (2DxG), or respiration (DNP, oligomycin) was determined. The ability to maintain ATP using glucose or glutamine as sole substrate was also determined. Intermediates including ATP, lactate, glucose, and glutamine were assayed in neutralized perchloric acid (PCA) cell extracts, virgin, and conditioned medium by enzymatic fluorometry. RESULTS. Data show that inhibition of m-aconitase, glycolysis, or respiration alone did not decrease ATP content. Inhibition of both glycolysis and respiration were required to decrease ATP content. PC3 cells were able to produce ATP with either glucose or glutamine as sole substrate. Though FA clearly inhibited m-aconitase there was no evidence that zinc had a similar effect. CONCLUSION. PC3 cells can support ATP production when m-aconitase is inhibited by using glycolysis or oxidation of substrate (e.g., glutamine) entering the TCA cycle distal to citrate. Prostate 67: 1211 – 1218, 2007. # 2007 Wiley-Liss, Inc. KEY WORDS: citrate; ATP; m-aconitase; fluoroacetate; oligomycin INTRODUCTION Citrate, produced in the normal prostate epithelial cell, is not further metabolized in the TCA cycle but accumulates for secretion into prostatic fluid [1,2,3]. The metabolic adaptation which supports this secre- tory function is the inhibitory effect of high intracellular zinc on m-aconitase [4,5]. High intracellular zinc and citrate content are peculiar to prostate epithelial cells. Both the zinc transporter (ZIP1) and m-aconitase found in prostate are regulated at genetic and functional levels by hormones including testosterone and prolac- tin [6,7,8]. Citrate is produced from acetyl CoA (ACoA) and oxaloacetate (OAA). In the prostate, as in other cells, the source of ACoA is glucose via glycolysis [9,10]. However, in prostate the OAA is not supplied by metabolism via the TCA cycle or from glycolysis but is derived by transamination of aspartate which is taken up actively from plasma using a specific high-affinity- aspartate transporter of the EAAT family [11,12]. Activity and expression of prostate m-aspartate amino- transferase and the aspartate transporter is stimulated by prolactin and testosterone [12,13]. Low oxidation of citrate by normal prostate has been proposed to limit ATP production by the TCA cycle [14]. Costello et al. [2] have shown in suspensions of rat ventral prostate cells that glutamate, a possible alter- native fuel, is used to produce a-ketoglutarate which Grant sponsor: NIH/NIDCR Training program; Grant number: T32 DE07309; Grant sponsor: Department of Biomedical Science, Uni- versity of Maryland Dental School, Baltimore, Maryland, USA; Grant sponsor: NIH Grant; Grant number: CA79903. *Correspondence to: B.K. Matheson, Department of Biomedical Sciences, University of Maryland Dental School 650 West Baltimore Street, Baltimore, MD 21201. E-mail: bmatheson@umaryland.edu Received 1 December 2006; Accepted 24 April 2007 DOI 10.1002/pros.20617 Published online 24 May 2007 in Wiley InterScience (www.interscience.wiley.com). ß 2007 Wiley-Liss, Inc.