• Characteristics and regulation of aspartate transport systems in rat ventral prostate epithelial cells

      Lao, Lixing; Franklin, Renty B.; Costello, Leslie (1992)
      A unique characteristic and function of rat ventral prostate, like human prostate, is the accumulation and secretion of high levels of citric acid. Aspartate is a proposed four-carbon precursor of citrate via transamination. Replenishment of endogenous aspartate requires continuous uptake of aspartate from circulation. This study was designed to identify aspartate transporters in isolated rat ventral prostate epithelial cells. The results indicated that two aspartate transporters, a high affinity (K{dollar}\sb{lcub}\rm m{rcub}{dollar} = 0.01 mM) and a low affinity transporter (K{dollar}\sb{lcub}\rm m{rcub}{dollar} = 0.8 mM) exist in these cells. Both transporters are Na{dollar}\sp+{dollar}-dependent and pH sensitive. The optimal pH for the high affinity transporter is about 7.5, whereas for the low affinity transporter the optimum is between 6.5 and 7.0. The high affinity transporter is also temperature dependent. Competitive inhibitory studies indicate that L-aspartate uptake by the high affinity transporter is inhibited by L-glutamate and D-aspartate, but not by L-alanine and L-lysine. The low affinity system is inhibited by D-aspartate, but not by L-glutamate or L-alanine. These different characteristics suggest that the high affinity and the low affinity transporters are two distinct systems. The high affinity aspartate transporter is sensitive to the Na{dollar}\sp+{dollar}-K{dollar}\sp+{dollar} ATPase inhibitor vanadate but less sensitive to ouabain. This suggests that an ouabain-insensitive Na{dollar}\sp+{dollar}-ATPase exists on the cell membrane. High affinity aspartate uptake is not dependent on K{dollar}\sp+{dollar}. However, a Na{dollar}\sp+{dollar}-H{dollar}\sp+{dollar} antiport might be involved. Aspartate uptake is stimulated by testosterone in vivo and in vitro. The in vitro effect is rapid and is inhibited by cycloheximide and actinomycin D. Prolactin also stimulated aspartate uptake independent of testosterone and is inhibited by cycloheximide. The high affinity aspartate transporter is subject to transstimulation by aspartate and citrate.
    • Testosterone regulates mitochondrial aspartate aminotransferase gene expression in rat ventral prostate

      Qian, Kaifeng; Franklin, Renty B.; Costello, Leslie (1991)
      One of the physiological functions of the rat ventral prostate, like human prostate, is to secrete an extraordinarily high quantity of citrate. This unique characteristic is under the influence of testicular androgen. The continuous secretion of citrate from prostate results in loss of a 6-carbon compound from the metabolic pool which must be replenished. Citrate is synthesized from the concentration of acetyl-CoA and oxaloacetate. Acetyl-CoA may come from various sources. However, recent studies indicated that the possible available source for oxaloacetate is the transamination of aspartate. Mitochondrial aspartate amino-transferase (mAAT) is an important enzyme which regulates citrate production in rat ventral prostate. Testosterone, as a major testicular androgen, stimulates citrate production and mAAT activity in a similar pattern. The hypothesis in this dissertation is that the ability of testosterone to increase citrate production and mAAT activity is the result of stimulation of mAAT gene expression. To verify this hypothesis, numerous in vivo and in vitro studies were utilized to analyze the steady-state level of mAAT mRNA in response to testosterone depletion and repletion. The rats were castrated, followed by testosterone or oil vehicle injection. Twenty-four (or forty-eight) hours later all animals were killed, prostates removed, RNA and nuclei isolated. In vitro studies were performed using primary cultured pig prostate cells. The quantity of mAAT mRNA was measured using northern hybridization. The rates of transcription and degradation of mAAT mRNA were determined by in vitro transcription assay and pulse chase labeling assay, respectively. Results show that castration caused a significant decrease in the content of mAAT mRNA and the transcription rate of the mAAT gene. However, testosterone administration reversed these hormonal depletion effects. Moreover, testosterone administration also prolonged the half life of mAAT mRNA. These results support the hypothesis that one of the major physiological functions of testosterone is to stimulate mAAT mRNA gene expression, which in turn enhances citrate production in rat ventral prostate. Furthermore, these results confirm that the increase in the steady-state level of mAAT mRNA in response to testosterone administration results from stimulation of mAAT gene transcription and inhibition of mAAT mRNA degradation.