Prolactin specifically increases pyruvate dehydrogenase Elα in rat lateral prostate epithelial cells

Prolactin is an important regulator of prostate citrate production. In rats this regulatory effect of prolactin is specific for lateral prostate, and has no effect on either ventral or dorsal prostate. The mechanisms by which prolactin regulates prostate citrate production have not been elucidated. Two key regulatory enzymes involved in citrate synthesis by prostate epithelial cells are mitochondrial aspartate aminotransferase (mAAT) which provides oxalacetate, and PDH E1α (pyruvate dehydrogenase) which provides acetyl CoA for citrate synthesis. Our previous studies demonstrated that prolactin regulates mAAT. However, an increase in citrate synthesis would require an increase in both oxalacetate and acetyl CoA. Therefore, we investigated the possibility that prolactin might also regulate PDH E1α in LP epithelial cells. The present studies demonstrate that prolactin administration (1 mg/rat) to rats resulted in an increased level of E1α in LP epithelial cells within 6 hr, but had no effect on the E1α level of VP epithelial cells. In vitro studies demonstrated that exposure of freshly prepared LP epithelial cells to prolactin (0.1–1.0 μg/ml) resulted in increased levels of E1α. Prolactin had no effect on either VP or DP epithelial cells. The stimulatory effect of prolactin on E1α was inhibited by actinomycin and cycloheximide, thereby indicating that prolactin stimulated the biosynthesis of E1α. The studies reveal that prolactin specifically stimulates E1α levels of LP epithelial cells, whereas testosterone specifically stimulates E1α levels of VP epithelial cells. At this time, we propose that the effects of prolactin and testosterone involve increased expression of the E1α gene of LP and VP epithelial cells, respectively.     

Concepts of citrate production and secretion by prostate: 2. Hormonal relationships in normal and neoplastic prostate

A unique and major function of prostate secretory epithelial cells is to synthesize, accumulate, and secrete extraordinarily high levels of citrate. This function is regulated by testosterone and by prolactin. Concepts of the mechanisms of hormonal regulation are presented. The relationship of testosterone and prolactin to the origin and homologies of different prostate cell lines is described. The metabolic differentiation of citrate and non-citrate producing prostate secretory epithelial cells is discussed. Concepts of the pathogenesis of prostatic neoplasms are presented based on hormonal, metabolic, and homologous relationships associated with citrate production. Characterization of normal and neoplastic secretory epithelial cells by their citrate function is emphasized. The urgency and necessity for research relating to all aspects of prostate citrate production in normal and pathological prostate are emphasized.     

Prolactin and testosterone regulation of mitochondrial zinc in prostate epithelial cells

The prostate gland of many animals accumulates extremely high levels of zinc and citrate. Evidence currently exists in support of a concept that zinc might be an important regulator of m-aconitase and citrate oxidation of prostate epithelial cells. No information concerning the mitochondrial levels of zinc has been available. The roles of testosterone and prolactin in the regulation of zinc have not been established. In this report, we determined the levels of tissue and mitochondrial zinc of rat lateral prostate (LP), ventral prostate (VP), dorsal prostate (DP), liver, and kidney. The results demonstrate that the mitochondrial zinc levels of the prostate cells were higher than levels of nonprostatic cells. The LP contained severalfold higher zinc levels than DP and VP. The effects of testosterone and prolactin in vivo and in vitro on the zinc levels were also determined. Both hormones significantly increased cellular and mitochondrial zinc levels of LP cells; decreased the zinc levels of VP cells; and had no effect on the zinc levels of DP, liver, or kidney cells. These effects are direct and physiological effects of the hormones on the targeted prostate epithelial cells. The hormonal effects on mitochondrial zinc of LP and VP epithelial cells correlate perfectly with their effects on citrate oxidation. The results support the concept that mitochondrial zinc is an inhibitor of m-aconitase and citrate oxidation; and that prolactin and testosterone regulation of mitochondrial zinc provides a mechanism for their regulation of citrate oxidation in citrate-producing prostate epithelial cells. Prostate 30:26–32, 1997 © 1997 Wiley-Liss, Inc.     

Novel role of zinc in the regulation of prostate citrate metabolism and its implications in prostate cancer

The prostate gland of humans and many other animals has the major function of accumulating and secreting extraordinarily high levels of citrate. This specialized metabolic process of “net citrate production” is the result of unique metabolic capabilities of the secretory epithelial cells. Most importantly, in prostate cancer (Pca) the capability for net citrate production is lost. In addition to citrate, the normal and BPH (benign prostatic hyperplasia) prostate also accumulates the highest levels of zinc in the body. As with citrate, in Pca the ability for high zinc accumulation is diminished. These and other correlations between zinc and citrate in the prostate have been indicative of an important role of zinc in the regulation of citrate metabolism in normal and malignant prostate epithelial cells. The link between zinc and citrate metabolism has now been established. The intramitochondrial accumulation of high zinc levels inhibits mitochondrial (m-) aconitase activity, which inhibits citrate oxidation. This essentially truncates the Krebs cycle and markedly decreases the cellular energy (ATP) production normally coupled to citrate oxidation. It is also clear that zinc accumulation in citrate-producing prostate epithelial cells is regulated by testosterone and by prolactin. These relationships form the basis for a new concept of the role of zinc and citrate-related energy metabolism in prostate malignancy. The inability of malignant prostate cells to accumulate high zinc levels results in increased citrate oxidation and the coupled ATP production essential for the progression of malignancy. The concept offers new approaches to the treatment of Pca. Prostate 35:285–296, 1998. © 1998 Wiley-Liss, Inc.     

Effect of prolactin on the prostate

Normal and benign prostate hyperplasia (BPH) prostate is characterized by the presence of extraordinarily high levels of citrate. Presumably, this results from the inability of the prostate epithelial cells to oxidize citrate due to a limiting mitochondrial (m-) aconitase. In contrast, prostate carcinoma (CA) is not characterized by high citrate levels. Malignant prostate epithelial cells apparently undergo a metabolic transformation from citrate-producing to citrate-oxidizing cells. A consequence of citrate production in normal and BPH cells is an inefficient and low level of ATP production. It is proposed that the process of malignancy necessitates an energy production that cannot be provided by citrate-producing cells. Consequently, the transformation of prostate epithelial cells to citrate-oxidizing cells which increases the energy production capability is essential to the process of malignancy and metastasis. The metabolic transformation likely occurs as a premalignant or early malignant stage. This bioenergetic theory of prostate malignancy, if correct, will provide new approaches to the diagnosis and treatment of CA. © 1994 Wiley-Liss, Inc.     

Androgen-dependent regulation of medium and long chain fatty acids uptake in prostate cancer

BACKGROUND: Epidemiological and experimental studies suggest that both fatty acids and androgens have a role in the development and progression of prostate cancer (PC). Plasma membrane fatty acid binding protein (FABP(pm)) is a transporter of medium and long chain fatty acids (MCFA and LCFA) across the plasma membrane, and is identical to the mitochondrial protein aspartate aminotransferase (mAAT) that is regulated by testosterone only in prostate epithelial cells, a site where PC initially develops. We therefore hypothesized that FABP(pm) is also regulated by androgens. METHODS: We examined the effect of a synthetic androgen, R1881, and that of androgen receptor (AR) blocker, bicalutamide, on the expression of FABP(pm) and mAAT and on the uptake of fatty acids in the androgen-sensitive LNCaP, androgen responsive 22rv1 and androgen-independent CL1 human PC cells. This was done using immunofluorescence and confocal microscopy, Western blot, flow cytometry, and (3)H-oleate uptake studies. RESULTS: Androgen supplementation increased the cellular and surface expression of FABP(pm) and mAAT and increased the uptake of fluorescently labeled MCFA and LCFA and that of (3)H-oleate only in PC cells that express the AR. Bicalutamide inhibited this phenomenon. CONCLUSIONS: The uptake of MCFA and LCFA into PC cells is androgen regulated as well as the expression of FABP(pm) and mAAT.     

Prostate epithelial cells utilize glucose and aspartate as the carbon sources for net citrate production

The prostate gland (human, rat ventral prostate) has the major function of accumulating and secreting extremely high levels of citrate. This function requires unique and specialized metabolic pathways associated with prostate secretory epithelial cells by which exogenous substrates must be utilized as the six-carbon sources of citrate. Recent studies demonstrated that aspartate can serve as the four-carbon source of oxalacetate for citrate synthesis. Identification of the two-carbon source of acetyl CoA (AcCoA) had not been established. The present study investigated the probability that exogenous glucose, via pyruvate oxidation, is a physiological source of AcCoA for net citrate production by isolated epithelial cells from rat ventral prostate. Under adequate oxygenation, 5 mM glucose in the presence of aspartate plus glutamate markedly stimulated citrate production. Exogenous and endogenous pyruvate also stimulated net citrate production. We propose that glucose via aerobic glycolysis and pyruvate oxidation provides AcCoA, which condenses with oxalacetate obtained from aspartate transamination for citrate synthesis. Prostate epithelial cells do not readily oxidize citrate, which permits accumulation and secretion of the synthesized citrate.     

Concepts of citrate production and secretion by prostate. 1. Metabolic relationships

Accumulation and secretion of extraordinarily high levels of citrate are principal functions of the prostate gland of humans and other animals. To achieve this, prostate secretory cells must possess unique metabolic relationships which distinguish them from virtually all other cells. Furthermore, citrate metabolism is markedly altered in benign prostatic hyperplasia (BPH) and in prostatic carcinoma (CA). This review assimilates existing information and presents current concepts related to 1) the pathway of metabolism associated with net citrate production, 2) the involvement of transporting mechanisms associated with citrate secretion, 3) energy implications of citrate production, 4) altered metabolic relationships in BPH and CA, and 5) the importance of citrate relationships as biochemical markers for characterizing prostate secretory epithelial cells. It is hoped that this review will bring attention to the importance and urgency of elucidating and understanding the metabolic relationships associated with citrate production by normal and neoplastic prostate epithelial cells. Research in these areas has been severely neglected despite the fact that the combined incidence of BPH and CA constitutes the most prevalent neoplastic disease among men.     

Ionic and pharmacologic characteristics of epithelial cells in a semi-intact preparation of the rat ventral prostate gland

BACKGROUND: The essential ionic and pharmacologic characteristics of epithelial cells within the ducts of the prostate gland are not well known. METHODS: Experiments were carried out on segments of ventral prostate glands from adult male rats. By using sharp microelectrodes, intracellular epithelial cell and transepithelial (lumen) potentials were recorded in response to ionic substitution and application of ion channel blockers, hormones, and other pharmacologic agents related to prostatic function. RESULTS: Membrane permeabilities to K(+), Na(+), and Cl(-) were found to account for approximately 43% of the resting membrane potential, whereas some 39% was likely to be metabolic in origin. The membrane potential also responded to adrenaline, acetylcholine, insulin, prolactin, testosterone, nerve growth factor, and nitric oxide. The lumen potential was found to be particularly sensitive to citrate, prolactin, and testosterone. CONCLUSION: It was concluded that the basal membrane potential of prostatic epithelial cells is associated with a relatively high Na(+):K(+) permeability ratio and metabolic dependence. The hormonal and pharmacologic sensitivity observed is consistent with the functional characteristics of the prostate gland.     

Announcement

Histologic specimens of ventral and dorsolateral prostate from ACI male rats of varying ages (3, 6, 12, 18, 24, and 29–32 months) were examined to identify changes associated with the development of adenocarcinoma. The same specimens were examined immunohistochemically for their distribution of prolactin binding sites. At 3 months of age ventral lobes of these ACI rats exhibited normal histology and prolactin binding patterns. At 6 and 12 months of age most ventral prostate alveoli showed epithelial cell atrophy, along with occasional acini containing normal-looking epithelial cells. Prolactin binding activity was present in most epithelial cells at 6 months of age but disappeared from atrophied cells at 12 months of age. At 18 months of age epithelial atrophy was more advanced and devoid of prolactin binding activity while zones of epithelial cell proliferation were expanded containing populations of normal-looking cells, and cells that were either hyperplastic or dysplastic (abnormal looking), all of which were capable of binding prolactin. Older rats, 24–32 months of age, also showed prostatic adenocarcinoma in ventral prostate and these cancerous cells bound prolactin. A continuum was frequently seen between areas of normal epithelial cells, hyperplasia, dysplasia, and adenocarcinoma of ventral prostate. Morphology and prolactin binding patterns in dorsal and lateral prostates of old ACI rats were similar to young adult rats and did not contain adenocarcinoma. In conclusion, age-dependent atrophy of the ventral prostate gland of ACI rats occurs early in life and precedes adenocarcinoma. Precancerous dysplastic changes appear to arise from normal and hyperplastic epithelial cells that are capable of binding prolactin rather than those that undergo precocious atrophy and lose their prolactin binding activity. Although atrophic epithelial cells do not appear to be progenitors of prostatic cancer, the possible relationship between precocious senescence (atrophy) and adenocarcinoma in ventral prostates of ACI rats is discussed.     

High-affinity L-aspartate transporter in prostate epithelial cells that is regulated by testosterone

The prostate gland produces and secretes extraordinarily high levels of citrate. Studies with rat ventral prostate (VP) have demonstrated that aspartate can serve as a four-carbon source of oxalacetate in the synthesis of citrate. To achieve this, prostate secretory epithelial cells must contain a transport system for the active uptake of aspartate from circulation. The present studies with VP epithelial cells confirm the existence of a Na⁺ -dependent high-affinity L-aspartate transporter. The transporter has an optimal pH ≈ 7.5 and is temperature dependent. It appears to be an anionic amino acid transporter capable of transporting L-glutamate but not basic or neutral amino acids. The transporter is inhibited by ATPase inhibitors, thereby indicating its dependency on a Na⁺ gradient. The characteristics of the high-affinity L-aspartate transporter are consistent with its operation at the basilar membrane for the transport of circulating aspartate into the cell. Castration (24 hr) resulted in a significant decrease- in the ability of VP epithelial cells to transport L-aspartate. The administration of testosterone to castrated rats completely restored L-aspartate transport. In addition, in vitro testosterone addition (10^?8 M for 30 min) to isolated prostate epithelial cells markedly increased L-aspartate transport. Both cycloheximide and actinomycin inhibited the testosterone effect. The studies reveal that testosterone is a regulator of this Na⁺ -dependent high-affinity L-aspartate transporter. The mechanism of this testosterone effect appears to involve both RNA and protein synthesis. We now have a model system to elucidate this novel effect of testosterone. © 1993 Wiley-Liss, Inc.     

Effect of metabolic inhibitors on ATP and citrate content in PC3 prostate cancer cells

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.     

Regional variations in the testicular dependence of prolactin binding and its possible relationship to castration-induced involution in rat prostate gland

Prolactin binding sites of ventral, lateral, and dorsal lobes of rat prostate were examined immunohistochemically 1, 2, 4, and 8 days after castration or sham operation. In sham-operated rats each lobe exhibited a distinct pattern of intracellular and intraluminal prolactin binding. A loss in prolactin binding from epithelial cells of ventral prostate, which was visualized postcastration, was quantitated with the aid of an image analyzer and then statistically evaluated. The proportion of ventral prostate epithelial cells devoid of prolactin binding increased from approximately 13% in sham-operated rats to approximately 29% 1 day after castration, and reached a peak level of about 71% 4 days postcastration. No loss of prolactin binding was evident in either lateral or dorsal prostate up to 8 days postcastration. Direct measurement of epithelial cell heights and subsequent statistical evaluation revealed similar regional differences in the rates and extent of prostate involution. Eight days after castration ventral prostate epithelial cell heights decreased by 56% whereas lateral and dorsal lobe epithelial cells heights decreased about 25% and 14%, respectively. The apparent relationship between testicular dependence of prostatic prolactin binding and castration-induced prostatic involution are discussed in terms of possible regional variations in the prolactin-androgen interplay in prostate.     

Biochemical and immunohistochemical characterization of prolactin binding in rat ventral,lateral, and dorsal prostate lobes

A radioligand assay and the soluble peroxidase-antiperoxidase (PAP) technique were used to compare ovine and rat prolactin as ligands for the demonstration of prolactin binding sites in rat ventral, lateral, and dorsal prostate lobes. Biochemical techniques were effective in demonstrating prolactin binding sites in ventral prostate with both ovine and rat prolactin; however, binding sites could not be characterized in lateral or dorsal lobes with either ligand. Using the PAP technique, rat prolactin consistently produced a discrete, dense reaction product over binding sites in the Golgi zone of the ventral prostate; ovine prolactin produced a more dispersed pattern of staining throughout the entire supranuclear region of the cells. In ventral prostate, most epithelial cells were immunoreactive when either ligand was used. However, lateral prostate contained fewer densely immunoreactive cells than did ventral prostate, and rat prolactin was more effective in demonstrating these binding sites than was ovine prolactin. Dorsal prostate epithelium was highly immunoreactive for binding of both ligands; however, there was considerably more staining in this lobe that remained after absorption of the antibody with prolactin. These data suggest that the inability to demonstrate prolactin binding sites in the lateral lobe using radioligand assay may have been due to the fewer numbers of binding sites that were diluted below the sensitivity of the assay when a tissue homogenate preparation was used. Noncompetitive binding in the dorsal lobe, as seen in the radioligand assay, coincided with immunocytochemical findings of relatively high nonabsorbable background staining.     

Endogenous and exogenous citrate transport and release in prostatic preparations: semi-polarized two-dimensional cultures of human PNT2-C2 cells and isolated tubules and segments of rat prostate

BACKGROUND: Electrophysiological characterization of normal human prostate epithelial cells showed exogenous trivalent citrate transport (release) to be K(+)-dependent. METHODS: (1) Ussing chamber recordings of short circuit current (SCC) were used to study citrate transport in the same (PNT2-C2) cell line grown on micro-pore filters as a monolayer. (2) Release of endogenous citrate from confluent cultures and tubules and segments of rat prostate was measured using a fluorescence technique. (3) Enzyme-spectrophotometry was employed to detect citrate release from segments of rat prostate. RESULTS: Citrate transport across the PNT2-C2 monolayer was asymmetrical, consistent with release into the lumen-side. Fluorescence and/or enzyme-spectrophotometric measurements showed that time-dependent citrate release (endogeneous and preabsorbed) occurred from rat prostate (tubules and segments), but not kidney or lung. The release was dependent on extracellular K(+) but not Na(+). CONCLUSIONS: Citrate release from prostatic cells and tissues (rat and human) was K(+)-dependent, consistent with the previous electrophysiological data.     

Hormonal effects on zinc concentration and morphology of rat lateral prostate gland

Hypophysectomized rats were injected with prolactin and/or testosterone, and luteinizing hormone (LH) and prolactin and/or follicle-stimulating hormone (FSH). Tissue from the lateral prostate was processed for spectrophotometric determination of zinc and for electron microscopy. Changes in zinc concentration, epithelial height, and morphological characteristics were used to assess the effect of hormonal replacement on functional activity. Although testosterone administration resulted in full restoration of all parameters, there was no evidence of a synergistic effect of prolactin and testosterone. Prolactin alone decreased the zinc concentration and increased the epithelial height compared with control animals, and there was evidence of epithelial hyperplasia. Luteinizing hormone, alone or combined with prolactin or prolactin and FSH, resulted in a marked increase of epithelial height and restoration of morphological features found in intact animals; however, only a slight increase of zinc concentration over control values was elicited. Combined LH and FSH administration reduced epithelial height and zinc concentration compared with the other LH regimes, and there was evidence of cellular degeneration.     

Characterization of rat ventral prostatic epithelial cells in collagen gel culture

The characterization of rat ventral prostatic epithelial cells grown in collagen gel culture was undertaken to determine the usefulness of this culture technique for the study of prostatic growth and differentiation in vitro. The results of these studies demonstrate that embedding prostatic epithelial cells in a matrix of collagen gel permitted rapid proliferation and maintenance of the cells for a prolonged period of time in a state that showed remarkable structural resemblance and physiological responsiveness to that found in vivo. Three-dimensional outgrowths from dissociated single epithelial cells and/or small aggregates of epithelial cells were obtained which resembled structures actually found within the prostate gland, as determined by histological and visual examination of the cellular growths present within the gels. In addition, the responsiveness of the cells in the collagen gel cultures to various hormonal additives, including insulin, testosterone, prolactin, and estradiol correlated closely to the manner in which these cells have been shown to respond in vivo. The general characteristics of growth of the cells in the collagen gel cultures, the effect of various sera and different pH levels upon growth, and the ability of the cells to produce acid phosphatase were also investigated.     

Short-term primary culture of rat prostate tumor epithelial cells on reconstituted basement membrane as a useful in vitro model to assess drug action on prostatic cancer: efficacy of the thiazolidinedione derivative CGP 19984

A model system has been developed that permits short-term culture of rat R3327 prostate adenocarcinoma epithelial cells on a reconstituted basement membrane. Growth of prostate tumor cells under these conditions resulted in an enriched epithelial cell population that exhibited an eightfold increase in cell number in 10 days. This model system was used to test the efficacy of the thiazolidinedione derivative CGP 19984, a drug that inhibits luteinizing hormone secretion in vivo. At concentrations ranging between 1 and 25 micrograms/ml, CGP 19984 inhibited growth of the prostate tumor epithelial cells in a dose-dependent manner. The results thus demonstrate a direct effect of CGP 19984, which complements its indirect antitumor action in vivo, and suggest that this drug might be an effective agent for treatment of prostatic cancer. Moreover, growth of prostate tumor epithelial cells on a reconstituted basement membrane provides a useful system for in vitro testing of drugs for prostate cancer.