Molecular origin of plasma membrane citrate transporter in human prostate epithelial cells

The prostate is a highly specialized mammalian organ that produces and releases large amounts of citrate. However, the citrate release mechanism is not known. Here, we present the results of molecular cloning of a citrate transporter from human normal prostate epithelial PNT2‐C2 cells shown previously to express such a mechanism. By using rapid amplification of cDNA ends PCR, we determined that the prostatic carrier is an isoform of the mitochondrial transporter SLC25A1 with a different first exon. We confirmed the functionality of the clone by expressing it in human embryonic kidney cells and performing radiotracer experiments and whole‐cell patch‐clamp recordings. By using short interfering RNAs targeting different parts of the sequence, we confirmed that the cloned protein is the main prostatic transporter responsible for citrate release. We also produced a specific antibody and localized the cloned transporter protein to the plasma membrane of the cells. By using the same antibody, we have shown that the cloned transporter is expressed in non‐malignant human tissues.     

Human prostate stromal cells stimulate increased PSA production in DHEA-treated prostate cancer epithelial cells

Dehydroepiandrosterone (DHEA) is commonly used as a dietary supplement and may affect prostate pathophysiology when metabolized to androgens and/or estrogens. Human prostate LAPC-4 cancer cells with a wild type androgen receptor (AR) were treated with DHEA, androgens dihydrotestosterone (DHT), T, or R1881), and E(2) and assayed for prostate specific antigen (PSA) protein and gene expression. In LAPC-4 monocultures, DHEA and E(2) induced little or no increase in PSA protein or mRNA expression compared to androgen-treated cells. When prostate cancer-associated (6S) stromal cells were added in coculture, DHEA stimulated LAPC-4 cell PSA protein secretion to levels approaching induction by DHT. Also, DHEA induced 15-fold more PSA mRNA in LAPC-4 cocultures than in monocultures. LAPC-4 proliferation was increased 2-3-fold when cocultured with 6S stromal cells regardless of hormone treatment. DHEA-treated 6S stromal cells exhibited a dose- and time-dependent increase in T secretion, demonstrating stromal cell metabolism of DHEA to T. Coculture with non-cancerous stroma did not induce LAPC-4 PSA production, suggesting a differential modulation of DHEA effect in a cancer-associated prostate stromal environment. This coculture model provides a research approach to reveal detailed endocrine, intracrine, and paracrine signaling between stromal and epithelial cells that regulate tissue homeostasis within the prostate, and the role of the tumor microenvironment in cancer progression.     

Receptors for vasoactive intestinal peptide on isolated epithelial cells of rat ventral prostate

Receptors for porcine vasoactive intestinal peptide have been characterized in isolated epithelial cells of rat ventral prostate. The interaction of ¹²⁵I-labelled VIP with cells was rapid, reversible, specific, saturable and dependent on temperature. Degradation of peptide and receptors was minimized at 15°C. At apparent equilibrium, the binding of ¹²⁵I-labelled peptide was competitively inhibited by native VIP in the 1·10⁻¹⁰−10⁻⁷ M range concentration. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a K_d = 4.0 nM and a low binding capacity (0.12 pmol VIP/mg cell protein), and a low-affinity class with a K_d = 17.8 nM and a high binding capacity (1.6 pmol VIP/mg cell protein). Chicken VIP and porcine secretin exhibited a 7-fold higher and a 7-fold lower affinity than porcine VIP for binding sites, respectively. Glucagon, Leu-enkephalin, Met-enkephalin and somatostatin were ineffective. The presence of high-affinity receptors for VIP together with previous reports on the occurrence of VIP-containing neurones innervating the male genitourinary tract strongly suggest that this peptide may be important in the physiological regulation of the functions of prostatic epithelium.     

Regulation of migration of primary prostate epithelial cells by secreted factors from prostate stromal cells

Stromal-epithelial interactions, which regulate the migration of prostate epithelial cells, play an important role in prostate development, prostatic hyperplasia, and prostate cancer. The objective of this study was to determine how the prostate stroma stimulates the migration of primary prostate epithelial cells (PECs). In the Boyden chamber assay, PEC migration was strongly induced by the conditioned medium of primary prostate stromal cells (PSC-CM). Stimulation of PEC migration depended on the concerted action of adhesion and motility factors in the PSC-CM. Immobilized proteins from PSC-CM mediated adhesion, spreading, and head-to-tail polarization of PECs. Migration induced by immobilized PSC-CM proteins was significantly increased by hepatocyte growth factor/scatter factor (HGF/SF). Inhibition of P13-kinase or Src-family kinases, but not MEK or PLCchi, abolished migration in the Boyden chamber assay. Consistent with their concerted activity in migration assays, the combination of adhesion and motility factors was required for efficient activation of the P13-kinase/Akt pathway. HGF/SF in the PSC-CM was the principal stimulator of the P13-kinase/Akt pathway and an important mediator of PSC-CM-induced PEC migration. In conclusion, our data show that the migration of primary PECs is regulated by the P13-kinase and Src-family kinase signaling pathways and that the activation of the P13-kinase pathway requires adhesion and motility factors from the prostate stroma.     

Prostaglandin production by type II alveolar epithelial cells

Prostaglandin production was studied in fetal and adult type II alveolar epithelial cells. Two culture systems were employed, fetal rat lung organotypic cultures consisting of fetal type II cells and monolayer cultures of adult lung type II cells. Dexamethasone, thyroxine, prolactin and insulin, hormones which influence lung development, each reduced the production of prostaglandin E and F_α by the organotypic cultures. The fetal cultures produced relatively large quantities of prostaglandin E and F_α and smaller quantities of 6-keto-prosta-glandin F_1α and thromboxane B₂. However, prostaglandin E₂ production was predominant. In contrast, the adult type II cells in monolayer culture produced predominantly prostacyclin (6-keto-prostaglandin F_1a) along with smaller quantities of prostaglandin E₂ and F_2α. The type II cells were relatively unresponsive to prostaglandins. Exogenously added prostaglandin E₂ had no effect on cell growth, and only a minimal effect on cyclic AMP levels in the monolayer cultures.     

Cyclic AMP-stimulating effect of vasoactive intestinal peptide in isolated epithelial cells of rat ventral prostate

Vasoactive intestinal peptide (VIP) has been shown to increase cyclic AMP content in isolated epithelial cells of rat ventral prostate. The stimulatory effect of VIP was dependent on time and temperature and was potentiated by a phosphodiesterase inhibitor. At 15°C, the response occurred in the 1·10⁻¹⁰−10⁻⁷ M range of VIP concentrations. Half-maximal stimulation of cellular cyclic AMP was obtained at 1.4 nM and maximal stimulation (3-fold basal level) at about 100 nM VIP. Chicken VIP and porcine secretin were agonists of porcine VIP but exhibited a 2-times higher and a 170-times lower potency, respectively. A high concentration (1·10⁻⁶ M) of glucagon, somatostatin, neurotensin, substance P, Met-enkephalin or Leu-enkephalin did not modify cAMP levels. The finding of a VIP-stimulated cAMP system in rat prostatic epithelial cells together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, as well as the presence of VIP-containing neurones innervating the male genitourinary tract, strongly suggest that VIP may be involved in prostatic growth regulation and function.     

Cyclic AMP-stimulating effect of vasoactive intestinal peptide in isolated epithelial cells of rat ventral prostate

Vasoactive intestinal peptide (VIP) has been shown to increase cyclic AMP content in isolated epithelial cells of rat ventral prostate. The stimulatory effect of VIP was dependent on time and temperature and was potentiated by a phosphodiesterase inhibitor. At 15 degrees C, the response occurred in the 1 X 10(-10)-10(-7)M range of VIP concentrations. Half-maximal stimulation of cellular cyclic AMP was obtained at 1.4 nM and maximal stimulation (3-fold basal level) at about 100 nM VIP. Chicken VIP and porcine secretin were agonists of porcine VIP but exhibited a 2-times higher and a 170-times lower potency, respectively. A high concentration (1 X 10(-6)M) of glucagon, somatostatin, neurotensin, substance P, Met-enkephalin or Leu-enkephalin did not modify cAMP levels. The finding of a VIP-stimulated cAMP system in rat prostatic epithelial cells together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, as well as the presence of VIP-containing neurones innervating the male genitourinary tract, strongly suggest that VIP may be involved in prostatic growth regulation and function.     

Prostaglandin production by type II alveolar epithelial cells.

Prostaglandin production was studied in fetal and adult type II alveolar epithelial cells. Two culture systems were employed, fetal rat lung organotypic cultures consisting of fetal type II cells and monolayer cultures of adult lung type II cells. Dexamethasone, thyroxine, prolactin and insulin, hormones which influence lung development, each reduced the production of prostaglandin E and F alpha by the organotypic cultures. The fetal cultures produced relatively large quantities of prostaglandin E and F alpha and smaller quantities of 6-keto-prostaglandin F1 alpha and thromboxane B2. However, prostaglandin E2 production was predominant. In contrast, the adult type II cells in monolayer culture produced predominantly prostacyclin (6-keto-prostaglandin F1 alpha) along with smaller quantities of prostaglandin E2 and F2 alpha. The type II cells were relatively unresponsive to prostaglandins. Exogenously added prostaglandin E, had no effect on cell growth, and only a minimal effect on cyclic AMP levels in the monolayer cultures.     

Characteristics of amino acid accumulation by isolated intestinal epithelial cells

Summary Accumulation of neutral amino acids by isolated chick epithelial cells has been studied and the results discussed in terms of the ion gradient model, and a model invoking a direct input of metabolic energy. The cells establish four- to eightfold concentration gradients of amino acids at an extracellular concentration of 1mm. The accumulation is sodium-dependent, inhibited by high extracellular potassium concentrations, and is sensitive to a variety of metabolic inhibitors. Also, amino acid uptake is depressed by actively transported sugars, and certain other amino acids, and is stimulated by phloridzin.Cells equilibrated with valine and loaded with 30 to 40mm intracellular sodium begin immediately to actively accumulate valine when suddenly introduced to media containing 20mm sodium. The cells establish a threefold gradient of amino acid during the interval when intracellular sodium is higher than extracellular sodium.Amino acid accumulation and²²Na efflux were monitored simultaneously in cells treated with phloridzin. While phloridzin causes a 30% stimulation of amino acid uptake, no variation in the rate of²²Na efflux or the steady-state level of²²Na maintained by the cells can be detected. Similarly, either 2.5mm glucose or 2.5mm 3-O-methylglucose cause approximately a 50% inhibition of 1mm valine uptake, but no detectable change in steady-state cellular²²Na content. Several aspects of the data seem inconsistent with concepts embodied in the ion gradient hypothesis, and it is suggested that a directly energized transport mechanism can better accommodate all of the data.     

Uridine uptake by isolated intestinal epithelial cells of guinea pig

Uptake of uridine was studied in isolated intestinal epithelial cells of guinea pig. Uptake was not severely influenced by metabolism. Free uridine was accumulated within cells 13-fold. Uptake was saturable with an apparent Km value of 46 microM and a Vmax of 0.9 nmol/mg protein per min. Uracil inhibited uptake only slightly; adenosine, guanosine and cytosine inhibited strongly. Antimycin A and ouabain inhibited almost 90%. If the extracellular Na+ concentration was decreased to 5 mM, the rate of uptake decreased 6.5-fold. The stimulatory effect of Na+ was related to the transmembraneous Na+-gradient. Cells from jejunum transported about 30% faster than cells from ileum. In conclusion, isolated enterocytes of guinea pig posses an active transport system for uridine.     

Uridine uptake by isolated intestinal epithelial cells of guinea pig

Uptake of uridine was studied in isolated intestinal epithelial cells of guinea pig. Uptake was not severely influenced by metabolism. Free uridine was accumulated within cells 13-fold. Uptake was saturable with an apparent K_m value of 46 μM and a V_max of 0.9 nmol/mg protein per min. Uracil inhibited uptake only slightly; adenosine, guanosine and cytosine inhibited strongly. Antimycin A and ouabain inhibited almost 90%. If the extracellular Na⁺ concentration was decreased to 5 mM, the rate of uptake decreased 6.5-fold. The stimulatory effect of Na⁺ was related to the transmembraneous Na⁺-gradient. Cells from jejunum transported about 30% faster than cells from ileum. In conclusion, isolated enterocytes of guinea pig posses an active transport system for uridine.     

Kinetics of glycylsarcosine transport by isolated chicken intestinal epithelial cells

Kinetics of Glycylsarcosine (Gly-Sar) uptake by isolated chicken enterocytes was studied by measuring its intracellular concentration, and by discriminating between the saturable and the diffusive components of the total uptake. The diffusive component was greater at pH 6.0 than at pH 7.4, and the J max was also increased by lowering external pH, whereas the Km remained in the same order of magnitude. Carnosine competitively inhibits Gly-Sar uptake, indicating that both share a common transport system.     

Characterization of insulin receptors in isolated epithelial cells of rat ventral prostate: effect of fasting

Insulin receptors have been characterized in rat prostatic epithelial cells by using [125I]insulin and a variety of physicochemical conditions. The binding data at equilibrium (2 h at 15 degrees C) could be interpreted in terms of two populations of insulin receptors: a class of receptors with high affinity (Kd = 2.16 nM) and low binding capacity (28.0 fmol mg-1 protein), and another class of receptors with low affinity (Kd = 0.29 microM) and high binding capacity (1.43 pmol mg-1 protein). Proinsulin exhibited a 63-fold lower affinity than insulin for binding sites whereas unrelated peptides were ineffective. The specific binding of insulin increased by about 50 per cent after 96 h of fasting; this increase could be explained by an increase of both the number of the high affinity-low capacity sites and the affinity of the low affinity-high capacity sites. These results together with previous studies on insulin action at the prostatic level strongly suggest that insulin may exert a physiological role on the prostatic epithelium.     

The effect of testosterone on citrate synthesis and citrate oxidation and a proposed mechanism for regulation of net citrate production in prostate

Citrate oxidation by rat ventral prostate was reduced by castration and increased by testosterone administration. Similarly, the mitochondrial aconitase activity was decreased by castration; whereas cytosol aconitase was unaffected. The rate of citrate oxidation is extremely low in prostate. Castration also decreased mitochondrial aspartate aminotransferase activity while having no effect on the cytosol isoenzyme. Testosterone markedly stimulated the net production of citrate from aspartate plus glutamate by prostate mitochondria. These studies support the proposal that aspartate is a major source of oxalacetate for citrate production, and that a "glutamate-aspartate-citrate" pathway may be functional in prostate mitochondria. In addition, testosterone can regulate citrate production by a specific effect on mitochondrial aspartate aminotransferase activity. Testosterone also regulates the flux of citrate through the Krebs cycle, but this represents only a small proportion of the citrate accumulated. These conditions would be consistent with the function of prostate epithelium in accumulating and secreting citrate.     

Clomiphene citrate alters surface ultrastructure of uterine luminal epithelial cells.

Scanning electron microscopy was used to evaluate the uterine luminal epithelium from ovariectomized rats treated with a single minimal physiological dose of clomiphene citrate, oestradiol-17 beta or progesterone. It was found that clomiphene treatment produced some ultrastructural surface features which were similar to those seen with both oestrogen and progesterone treatment, but in addition it produced features unique to clomiphene treatment.     

Inhibition of androgen and oestrogen production by clomiphene citrate in avian theca cells

Isolated theca cells (2 X 10(5)/ml) were pre-incubated for 1 h in the presence or absence of clomiphene citrate (10(-12)-10(-4) M). Ovine LH (50 ng/ml) was added and cells were incubated for an additional 3 h. A 50% inhibition of LH-stimulated androstenedione and oestrogen production was obtained with doses of 10(-8) M and 2 X 10(-7) M clomiphene, respectively. Furthermore, the effect of clomiphene on LH-stimulated androstenedione production was reversed by washing clomiphene from the cells before stimulation with LH. In subsequent experiments, the effects of clomiphene on C17-20-lyase and aromatase activities were examined. Conversion of [3H]17-hydroxyprogesterone to androstenedione was inhibited by 50% when theca cells were pretreated with 10(-5) M-clomiphene. In addition, conversion of testosterone to oestrogen by theca cells was inhibited in a dose-dependent manner by clomiphene, with 50% inhibition occurring at a dose of 5 X 10(-6) M. The results show that clomiphene treatment in vitro inhibits androgen and oestrogen production in theca cells by inhibitory effects on the activities of C17-20-lyase and aromatase. In addition to the widely-accepted effects of clomiphene on the hypothalamic-pituitary axis, the present findings add further support to the suggestion that clomiphene exerts direct effects on ovarian steroidogenesis.     

2-Deoxyglucose transport by intestinal epithelial cells isolated from the chick

Summary Characteristics of 2-deoxyglucose uptake (2DG) by intestinal epithelial cells isolated from chickens were evaluated as a means of discriminating between the concentrative transport system for monosaccharides, associated with the mucosal brush border, and other possible routes of monosaccharide entry. 2DG was chosen as it is not a substrate for the mucosal transport system. The deoxysugar enters via a saturable pathway which is not Na⁺-dependent, is not inhibited by K⁺, does not accumulate solute against a concentration gradient; exhibits a high sensitivity to inhibition by phloretin; is relatively insensitive to phlorizin inhibition; and has low affinity [but high capacity relative to Na⁺-dependent mucosal transport of 3-O-methylglucose (3-OMG) and other monosaccharides]. These characteristics confirm those established in an earlier report for Na⁺-independent uptake of 3-OMG. Complications encountered in the use of 2DG as a test sugar include significant rates of metabolic conversion to an anionic form which presumably is a phosphorylated species. Methods for distinguishing between transport and subsequent metabolism are described. Inhibition of 2DG entry by several other sugars is described and inhibitory constants (K's) given for each.     

Prostaglandin E2 output by isolated rat gastric parietal cells and non-parietal epithelial cells

Prostaglandins have acid antisecretory and cytoprotective effects in gastric mucosa when given exogenously. This study's purpose was to isolate preparations of parietal and non-parietal cells from rat stomachs and to compare prostaglandin output by these cells. Gastric epithelial cells were isolated from rat stomachs using pronase. Cells from different incubation times were collected separately and enriched by discontinuous Percoll gradient. Cell types were identified by hematoxylin and eosin stain, succinic dehydrogenase activity (parietal cells), periodic acid Schiff staining (mucous cells), Bowie staining (chief cells) and electron microscopy. Prostaglandin E2 activity was measured by radio-immunoassay. Parietal cells were purified to over 90% while the non-parietal preparation contained 67% chief cells and over 31% mucous cells. By electron-microscopy, cell integrity was seen to be maintained. The parietal cell enriched fraction contained two and one-half times the amount of prostaglandin E2 that the non-parietal chief cell enriched fraction did, p less than 0.01. These results raise the question as to whether output of PGE2 by parietal cells could play a role in modulating gastric acid secretion directly by parietal cells as well as in protecting the deeper layers of gastric mucosa against damaging agents in-vivo.