• 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.
    • Interactions of immune and neuroendocrine systems in response to the superantigen staphylococcal enterotoxin B

      Weng, Ching-Feng; Franklin, Renty B.; Tseng, Jeenan (1994)
      The Immune and neuroendocrine systems interact with each other through cytokines and hormones. Staphylococcal enterotoxin B (SEB) is a powerful inducer of cytokines. The present studies were conducted to investigate the effect of SEB on the interaction between the immune and neuroendocrine systems. In monkeys challenged with SEB, efforts were made to investigate the interrelationships among the circulating cytokines, lymphoid cells, ACTH, and glucocorticoids (GCs). It was found that in dying monkeys there was an increase of cytokines (TNF, IL-2, IL-6, and IFN-gamma) that paralleled the appearance of ACTH and GCs and various lymphocyte subpopulations. To further study the complicated interactions among cytokines, lymphoid cells, and GCs, lymphoid cell cultures were established and stimulated with SEB, SEB plus dexamethasone (DEX), or SEB plus cytokines. DEX, if added after SEB to lymphoid cell cultures, generally suppressed SEB-induced mitosis. However, if DEX was added simultaneously with SEB, it enhanced SEB-induced mitosis of spleen cell cultures but not of lymphocytes from other lymphoid tissues. The enhancement appeared to be regulated primarily by IL-1 and to a lesser extent by IL-6. The significance of DEX-enhanced SEB-induced mitosis was further studied in mice challenged with SEB, or SEB plus DEX. Spleen cells from these mice were stimulated in culture with SEB, SEB plus DEX, and SEB plus cytokines. It was found that spleen cells from mice challenged with SEB or SEB plus DEX became tolerant to SEB-induced mitosis in culture. Because there were increases in the total number of splenic T cells and of Vbeta8 T cells, which are normally SEB-reactive, this tolerance appeared to be due to anergy rather than cell depletion. The anergic state of these cells could not be overcome by adding DEX and various cytokines to the lymphoid cell cultures. In conclusion, SEB may affects the interaction of the immune and neuroendocrine systems through the production of large amounts of cytokines that act on the neuroendocrine system which, in turn, releases ACTH and then GCs which subsequently regulate the immune system. The increased levels of cytokines and GCs may result in an over-stimulation of the nervous and immune systems resulting in pathophysiological changes of various organs and organ systems. A profound effect of GCs and SEB on the immune system is the development of anergy, which may confer resistance to subsequent SEB insult.
    • Studies on prolactin regulation of mitochondrial aspartate aminotransferase gene expression and protein kinase C activity and expression in prostate cells

      Gorski, Ewa; Franklin, Renty B. (1996)
      Citrate accumulation and secretion are physiological functions of the normal prostate gland in most species. Prolactin (PRL) stimulates citrate accumulation in the rat lateral prostate (LP) and pig prostate by increasing the expression of mitochondrial aspartate aminotransferase (mAAT), the enzyme that catalyzes aspartate transamination. In the present studies, we established the role of PRL and the phorbol ester 12-O-tetra-decanoylphorbol 13-acetate (TPA) in the regulation of mAAT in LNCaP and PC-3 cells. We isolated total RNA from the cells and hybridize it with a mAAT cDNA. Next, we assayed protein kinase C (PKC) activity and expression in PRL and TPA treated prostate cells. We, then determined the effect of PRL and TPA on gene transcription using a mAAT-chloramphenicol acetyltransferase (CAT) reporter gene construct, transiently transfected into PC-3 cells. A fragment of the 5{dollar}\sp\prime{dollar} gene region containing putative TPA response elements (TRE) was cloned into the pCAT-Promoter vector from Promega. The reporter vector was introduced into the cells by the calcium phosphate-DNA co-precipitation method. The cells were then incubated for 48 h with or without PRL or TPA.;The results indicated that PRL and TPA increased the mAAT mRNA level 2-4 fold. Upon PRL and TPA treatment the level of PKC activity in rat LP, and LNCaP and PC-3 cells increased 20-60% and 40-207%, respectively. In addition, long term PRL and TPA treatment resulted in increased PKC expression (10-60% and 10-240%, respectively) in the LP cytosol fraction. Furthermore, the results showed that PRL at a concentration of {dollar}1\times10\sp{lcub}-9{rcub}{dollar} M and TPA (0.1 {dollar}\mu{dollar}g/ml) treatment increased CAT activity in PC-3 cells. These results suggest that PRL regulates mAAT at the transcriptional level. Moreover, these effects of PRL on mAAT mRNA and PKC activity were eliminated by PKC down-regulation. Since both PRL and TPA induced PKC activity and since the effects of PRL and TPA were eliminated by PKC down-regulation, we postulate that the PRL effect on mAAT is mediated via the diacylglycerol (DAG)-PKC signal transduction pathway in rat LP and prostate cancer (CA) cells.
    • 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.