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J Orlowski

Publications and source records attributed to J Orlowski.

At least 73 records · Page 4Linked to original sources
PubMed1993

Primary structure and functional expression of a novel gastrointestinal isoform of the rat Na/H exchanger.

A cDNA encoding a new isoform of the rat Na/H exchanger (NHE) family has been identified by cross-hybridization with an NHE-1 cDNA probe. The 3.9-kilobase stomach cDNA encodes a protein of 813 amino acids with an M(r) of 91,296. The amino acid sequence of the protein, termed NHE-2, is 42% identical to NHE-1, 36% identical to NHE-3, and 57% identical to NHE-4. Na/H exchanger-deficient Chinese hamster ovary cells that were transfected with an expression construct containing the complete coding sequence of this cDNA exhibit amiloride-sensitive, H+-dependent 22Na+ influx, demonstrating that the protein it encodes is a functional Na/H exchanger. Northern hybridization analyses show that the corresponding mRNA is expressed predominantly in small intestine, colon, and stomach, with much lower levels present in skeletal muscle, kidney, brain, testis, uterus, heart, and lung. This suggests that NHE-2 plays an important role in gastrointestinal physiology and functions in many other organ systems as well.

Amino Acid Sequence↗
PubMed1993

Pregnancy in women with end-stage renal disease: treatment of anemia and premature labor.

There is little experience with the use of various therapies in the end-stage renal disease patient who becomes pregnant. Erythropoietin for the treatment of anemia has become part of the standard treatment regimen of dialysis patients, but experience with its use in pregnancy is limited. We report five cases of its use in dialysis patients during pregnancy. We found no evidence that it crossed the placenta or that it made blood pressure control more difficult. We found that patients required a higher dose of erythropoietin to maintain hematocrit levels than they had before pregnancy. Another therapy involves the treatment for premature labor, which is the most common cause of pregnancy loss in dialysis patients. Two of our patients were successfully treated with indomethacin for premature labor. Both drugs are useful tools in the management of pregnant dialysis patients.

Abnormalities, Drug-Induced↗
PubMed1993

Na(+)-K(+)-ATPase gene expression in rat intestine and Caco-2 cells: response to thyroid hormone.

Expression of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) gene family in rat intestinal epithelial cells was examined using RNA blot hybridization analyses. Rat intestinal epithelial cells express only the alpha 1- and beta 1-subunit mRNAs. A gradient in expression of alpha 1- and beta 1-subunit mRNA was seen along the villus-crypt unit in both jejunum and ileum, i.e., villus tip >> crypt cells. Regional differences in expression were observed along the intestine. alpha 1- and beta 1-subunit mRNA abundance was similar in jejunum, ileum, and colon while enzymatic activity was highest in the jejunum and lowest in the ileum. Administration of thyroid hormone to thyroidectomized rats increased the expression of alpha 1- and beta 1-subunit mRNAs in jejunum but not in colon. Hypothyroidism had no effect on subunit mRNA expression. The human intestinal cell line Caco-2 was also studied. These cells also expressed only the alpha 1- and beta 1-isoform mRNAs and demonstrated a developmental profile in both mRNA and enzymatic activity. Furthermore, in Caco-2 cells both alpha 1- and beta 1-mRNAs and Na(+)-K(+)-ATPase enzymatic activity were stimulated by thyroid hormone. Caco-2 cells transfected with 5' flanking regions of the human Na(+)-K(+)-ATPase beta 1-gene linked to the chloramphenicol acetyltransferase (CAT) reporter gene responded to 3,5,3'-triiodothyronine (T3) treatment with increased expression of CAT activity. This suggests that the 5' flanking region of the beta 1-gene contains a thyroid hormone response element and that T3 upregulation occurs at the transcriptional level.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Molecular cloning of putative members of the Na/H exchanger gene family. cDNA cloning, deduced amino acid sequence, and mRNA tissue expression of the rat Na/H exchanger NHE-1 and two structurally related proteins.

Biochemical and pharmacological data support the existence of multiple forms of the Na/H exchanger (NHE). Two isoforms, termed NHE-1 and NHE-2, have recently been isolated from rabbit ileal villus epithelial cells (Tse, C. M., Ma, A. I., Yang, V. W., Watson, A. J. M., Levine, S., Montrose, M. H., Potter, J., Sardet, C., Pouysségur, J., and Donowitz, M. (1991) EMBO J. 10, 1957-1967; Tse, C. M., Watson, A. J. M., Ma, A. I., Pouysségur, J., and Donowitz, M. (1991) Gastroenterology 100, A258). To identify additional molecular forms of the exchanger, rat brain, heart, kidney, stomach, and spleen cDNA libraries were screened for their presence using an NHE-1 cDNA probe under low stringency hybridization conditions. cDNAs encoding rat NHE-1 and two structurally related proteins, designated NHE-3 and NHE-4, have been isolated. Based on the deduced amino acid sequences, NHE-1, -3, and -4 are similar in size, having relative molecular masses of 91,506, 92,997, and 81,427, respectively. Overall, the proteins exhibit approximately 40% amino acid identity to each other and have similar hydropathy profiles, suggesting that they have the same transmembrane organization. The predicted N-terminal transmembrane regions of the three proteins, which span between 453 and 503 amino acids, exhibit the highest degree of identity (45-49%). In contrast, the C-terminal cytoplasmic regions, which span between 247 and 378 amino acids, exhibit very low amino acid identity (24-31%). Tissue distribution studies reveal that the NHE-1 mRNA is present at varying levels in all tissues examined, whereas NHE-3 and NHE-4 mRNAs exhibit a more limited distribution. NHE-3 mRNA is expressed at high levels in colon and small intestine, with significant levels also present in kidney and stomach. NHE-4 mRNA is most abundant in stomach, followed by intermediate levels in small intestine and colon and lesser amounts in kidney, brain, uterus, and skeletal muscle. These data suggest that the molecular basis for the functional diversity of the Na/H exchanger in mammals is based, at least in part, on expression of multiple members of a gene family.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and developmental expression of the rat cardiac-specific isoform of troponin I.

Troponin I is the subunit of the troponin complex in striated muscle which inhibits actomyosin ATPase activity. We have isolated a full-length cDNA clone for rat cardiac troponin I and determined its nucleic acid sequence. The amino acid sequence deduced from this clone shows 88%-92% similarity with previously reported amino acid sequences for rabbit (Wilkinson and Grand, 1978) and bovine (Leszyk et al.) cardiac troponin I. Examination of cardiac troponin I mRNA abundance during development revealed a 15-fold induction in its expression in the adult heart compared to that in embryonic (14 day) heart muscle. Furthermore, expression of cardiac troponin I mRNA was restricted to heart muscle and was not detected in skeletal muscle at any developmental stage.

Amino Acid Sequence↗
PubMed1991

Apical polarity of Na,K-ATPase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton.

In striking contrast to most other transporting epithelia (e.g., urinary or digestive systems), where Na,K-ATPase is expressed basolaterally, the retinal pigment epithelium (RPE) cells display Na,K-ATPase pumps on the apical membrane. We report here studies aimed to identify the mechanisms underlying this polarity "reversal" of the RPE Na,K-ATPase. By immunofluorescence on thin frozen sections, both alpha and beta subunits were localized on the apical surface of both freshly isolated rat RPE monolayers and RPE monolayers grown in culture. The polarity of the RPE cell is not completely reversed, however, since aminopeptidase, an apically located protein in kidney epithelia, was also found on the apical surface of RPE cells. We used subunit- and isoform-specific cDNA probes to determine that RPE Na,K-ATPase has the same isoform (alpha 1) as the one found in kidney. Ankyrin and fodrin, proteins of the basolateral membrane cytoskeleton of kidney epithelial cells known to be associated with the Na,K-ATPase (Nelson, W. J., and R. W. Hammerton. 1989. J. Cell Biol. 110:349-357) also displayed a reversed apical localization in RPE and were intimately associated to Na,K-ATPase, as revealed by cross-linking experiments. These results indicate that an entire membrane-cytoskeleton complex is assembled with opposite polarity in RPE cells. We discuss our observations in the context of current knowledge on protein sorting mechanisms in epithelial cells.

Animals↗
PubMed1991

Epithelial-stromal interactions in the regulation of rat ventral prostate function: identification and characterization of pathways for androgen metabolism in isolated cell types.

Androgen metabolism plays a significant role in the androgen regulation of prostate cell function. In this report the various pathways for androgen metabolism in primary cultures of rat ventral prostate epithelial and stromal cells were identified and characterized by in vitro whole cell assays, using HPLC. Confluent cultures of both cell types were incubated with supraphysiological concentrations (50 nM) of tritiated androgens (testosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha(and 3 beta), 17 beta-diols, and delta 4-androstene-3,17-dione), and the metabolites were analyzed at several time points over a 24-h period. The metabolism studies indicated that 5 alpha-reductase activity, the oxidative reactions of 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid oxidoreductases, and the reductive reaction of 3 beta-hydroxysteroid oxidoreductase were expressed at significantly higher levels in epithelial cells compared to stromal cells. The reductive reactions of 3 alpha- and 17 beta-hydroxysteroid oxidoreductases were similar in both cell types. In contrast, stromal cells exhibited substantially higher levels of 6 alpha/7 alpha-hydroxylase activity. In addition, stromal cells were capable of metabolizing 5 alpha-dihydrotestosterone directly to a new unidentified polar androgen metabolite (HO5 alpha-DHT). Overall, epithelial cells were approximately 29 times more capable than stromal cells of forming the biologically active androgen 5 alpha-dihydrotestosterone. Conversely, stromal cells were more capable of forming biologically inactive polar androgen metabolites.

17-Hydroxysteroid Dehydrogenases↗
PubMed1990

Thyroid and glucocorticoid hormones regulate the expression of multiple Na,K-ATPase genes in cultured neonatal rat cardiac myocytes.

The Na,K-ATPase alpha isoform (alpha 1, alpha 2, and alpha 3) and beta subunit genes exhibit a complex pattern of expression during heart development. To identify possible molecular signals that regulate the differential expression of these genes, isolated neonatal rat myocardial and non-myocardial cells were cultured in chemically defined medium and the responses of the multiple Na,K-ATPase subunit mRNAs to various hormones were tested. Myocardiocytes in control cultures express primarily alpha 1 and beta mRNAs. Triiodothyronine (T3) induced the expression of alpha 2, alpha 3, and beta mRNAs without influencing alpha 1 mRNA levels. Dexamethasone (DEX) treatment similarly induced alpha 2 mRNA levels, but the abundance of the other subunit transcripts remained unaltered. T3 and DEX together caused increases in alpha 2 and beta mRNA, increments similar to that observed with T3 alone. However, DEX specifically repressed the induction of alpha 3 mRNA by T3. Both hormones stimulated corresponding changes in the sarcolemma concentration of these Na,K-ATPase isozymes. Addition of norepinephrine to the cultures had little appreciable effect on expression of the alpha isoform and beta mRNAs. Although characterized less extensively, control cultures of non-myocardiocytes expressed alpha 1, alpha 3, and beta mRNAs, of which only the beta mRNA was stimulated by T3. These data indicate that thyroid and glucocorticoid hormones differentially regulate the expression of multiple alpha isoform and beta subunit mRNAs of Na,K-ATPase in cardiocytes in vitro and, therefore, may also be important physiological modulators in vivo.

Animals↗
PubMed1990

Molecular genetics of Na,K-ATPase.

Researchers in the past few years have successfully used molecular-genetic approaches to determine the primary structures of several P-type ATPases. The amino-acid sequences of distinct members of this class of ion-transport ATPases (Na,K-, H,K-, and Ca-ATPases) have been deduced by cDNA cloning and sequencing. The Na,K-ATPase belongs to a multiple gene family, the principal diversity apparently resulting from distinct catalytic alpha isoforms. Computer analyses of the hydrophobicity and potential secondary structure of the alpha subunits and primary sequence comparisons with homologs from various species as well as other P-type ATPases have identified common structural features. This has provided the molecular foundation for the design of models and hypotheses aimed at understanding the relationship between structure and function. Development of a hypothetical transmembrane organization for the alpha subunit and application of site-specific mutagenesis techniques have allowed significant progress to be made toward identifying amino acids involved in cardiac glycoside resistance and possibly binding. However, the complex structural and functional features of this protein indicate that extensive research is necessary before a clear understanding of the molecular basis of active cation transport is achieved. This is complicated further by the paucity of information regarding the structural and functional contributions of the beta subunit. Until such information is obtained, the proposed model and functional hypotheses should be considered judiciously. Considerable progress also has been made in characterizing the regulatory complexity involved in expression of multiple alpha-isoform and beta-subunit genes in various tissues and cells during development and in response to hormones and cations. The regulatory mechanisms appear to function at several molecular levels, involving transcriptional, posttranscriptional, translational, and posttranslational processes in a tissue- or cell-specific manner. However, much research is needed to precisely define the contributions of each of these mechanisms. Recent isolation of the genes for these subunits provides the framework for future advances in this area. Continued application of biochemical, biophysical, and molecular genetic techniques is required to provide a detailed understanding of the mechanisms involved in cation transport of this biologically and pharmacologically important enzyme.

Amino Acid Sequence↗
PubMed1990

Differential androgen modulation of acid phosphatase isozymes in primary cultures of rat ventral prostate epithelial and stromal cells.

The influence of androgen on prostate differentiated cell function was investigated using primary cultures of rat ventral prostate epithelial and stromal cells developed from sexually immature animals (21 days of age). As a biochemical marker of androgen action, total acid phosphatase activity, which comprises both the secretory and lysosomal isoforms, was measured. Testosterone increased total acid phosphatase activity approximately 2-fold in epithelial cell cultures. This increase occurred only after the cessation of cell proliferation (i.e. upon reaching a confluent monolayer). In contrast, stromal cells showed no significant change in total acid phosphatase activity in response to androgen. Polyacrylamide gel isoelectric focusing of total acid phosphatase activity from epithelial and stromal cell extracts revealed that secretory acid phosphatase activity was localized exclusively in the epithelial cells while lysosomal acid phosphatase activity was present in both cell types. Furthermore, the androgen-induced increases in epithelial total acid phosphatase activity were found to result from increases in the secretory isoform.

Acid Phosphatase↗
PubMed1989

Developmental regulation of expression of the alpha 1 and alpha 2 subunits mRNAs of the voltage-dependent calcium channel in a differentiating myogenic cell line.

The voltage-dependent calcium channel (VDCC) in skeletal muscle probably plays a key role in transducing membrane charge movement to the calcium release channel. We report here that the expression of VDCC alpha 1 and alpha 2 mRNAs is developmentally regulated in differentiating C2C12 myogenic cells. The alpha 1 mRNA is not detectable in the myoblast form of C2C12 cells while its expression is induced 20-fold in differentiated myotubes. In contrast, the alpha 2 mRNA is weakly expressed in myoblasts but is also induced upon myogenic differentiation.

Animals↗
PubMed1988

Differential expression of the Na,K-ATPase alpha 1 and alpha 2 subunit genes in a murine myogenic cell line. Induction of the alpha 2 isozyme during myocyte differentiation.

Expression of Na,K-ATPase catalytic alpha isoform (alpha 1, alpha 2, and alpha 3) and beta subunit genes in rodent muscle was investigated using the murine C2C12 myogenic cell line. RNA blot analyses of myoblasts revealed expression primarily of the alpha 1 mRNA and low levels of alpha 2 mRNA. Fusion of the proliferating myoblasts to form myotubes was accompanied by an approximate 12-fold induction of the alpha 2 mRNA. In contrast, expression of alpha 1 mRNA remained constant throughout myogenesis. The alpha 3 mRNA was not detected in either myoblasts or myotubes. The beta mRNA abundance also increased 2-3-fold during myotube formation. In rodent tissues, low and high affinity cardiac glycoside (e.g. ouabain) receptors have been shown to be associated with the Na,K-ATPase catalytic alpha 1 and alpha 2 isoform subunits, respectively. The existence of these two functional classes of Na,K-ATPase in myoblasts and myotubes correlated with the biphasic ouabain inhibition of Na,K-ATPase activity. Confluent myoblasts expressed primarily the alpha 1 isozyme (IC50 = 3.6 X 10(-5) M; 95% of total activity) and lesser amounts of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 5% of total activity). In contrast, the myotubes showed significant levels of the alpha 1 isozyme (IC50 = 4.0 X 10(-5) M; 68% of total activity) and, in addition, showed a 6-fold increase in the relative levels of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 32% of total activity). To quantitate further the expression of the high affinity, ouabain-sensitive alpha 2 isozyme, a whole cell [3H]ouabain-binding assay was used. Results revealed that myotubes have an approximately 6-fold greater concentration of [3H]ouabain-binding sites than myoblasts with an apparent dissociation constant (Kd) of 1.4 X 10(-7) M. The results indicate that muscle cells can express multiple isozymes of Na,K-ATPase and that expression of the alpha 2 isozyme is developmentally regulated during myogenesis.

Animals↗
PubMed1988

Tissue-specific and developmental regulation of rat Na,K-ATPase catalytic alpha isoform and beta subunit mRNAs.

The developmental expression of the multiple isozymes of Na,K-ATPase in rat brain, heart, lung, kidney, and skeletal muscle from fetal (14 days gestation) to adult (55 days) was investigated at the molecular level with cDNA probes specific for the multiple catalytic alpha isoform (alpha 1, alpha 2, alpha 3) and beta subunit mRNAs. Northern and RNA slot blot analyses revealed that these mRNAs are regulated in a tissue-specific manner. The multiple alpha isoform and beta subunit mRNAs appear to be regulated coordinately during ontogenesis with maximum expression occurring between 15 and 25 days of age for brain, heart, kidney, and skeletal muscle, whereas peak expression in lung was observed between 2 and 4 days of neonatal life. Brain tissue showed between 10- and 17-fold increases in the levels of expression for the three individual alpha isoform mRNAs. The alpha 3 mRNA was found to be the predominant alpha isoform transcript in fetal as well as adult brain. Examination of heart tissue showed alpha 1 mRNA to be the major catalytic subunit during development. However, a developmentally regulated transition in alpha 2 and alpha 3 mRNA expression was observed in heart between 7 and 14 days after birth. The alpha 3 mRNA was expressed primarily in fetal and neonatal heart tissue, while alpha 2 mRNA was expressed in juvenile and adult tissue. In kidney and lung, alpha 1 mRNA was the predominant alpha isoform transcript showing temporary increases in expression of 2- and 4-fold, respectively, during development. In contrast to the other tissues, muscle expressed predominantly alpha 2 mRNA following birth, the levels increasing approximately 89-fold during myogenesis. Thus, each tissue examined exhibits a distinct pattern of expression for the Na,K-ATPase catalytic alpha isoforms during ontogenesis.

Aging↗
PubMed1988

Effect of a 4-methyl-4-aza steroid on androgen metabolism by rat ventral prostate epithelial and stromal cell cultures: selective inhibition of 5 alpha-reductase activity.

The effect of a potent steroid metabolic inhibitor, 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (DMAA), on androgen metabolism was investigated in primary monolayer cultures of rat ventral prostate epithelial and stromal cells. Using testosterone (T) as substrate, 5 alpha-reductase (5 alpha-R) activity in both cell types was inhibited by greater than 98% at an inhibitor concentration of 1000 nM. The concentrations required to produce a 50% inhibition (IC50) were 7.4 and 9.0 nM for epithelial and stromal cells, respectively. To examine the specificity of this compound, its effect on other steroid-metabolic enzymes was examined. DMAA at a concentration of 1,000 nM had no effect on 3 alpha-hydroxysteroid oxidase (3 alpha-HSORox), 3-ketosteroid reductase (3 alpha-HSORred), and 6/7-hydroxylase (6/7-HSH) activities in both cell types; 17 beta-hydroxysteroid oxidase (17 beta-HSORox) activity, located primarily in epithelial cells, also was not influenced by DMAA. In contrast, epithelial 3 beta-hydroxysteroid oxidase (3 beta-HSORox) and 3-ketosteroid reductase (3 beta-HSORred) activities were inhibited by 65% (P less than .001) and 58% (P greater than .05), respectively, albeit the latter result was not statistically significant. Stromal 3 beta-HSORox and 3 beta-HSORred activities were negligible; hence the effect of the inhibitor of these enzymes could not be assessed. In conclusion, DMAA is a relatively selective and potent inhibitor of 5 alpha-R activity in primary cultures of rat ventral prostate epithelial and stromal cells and should be a useful compound for antagonizing androgen-mediated actions in the prostate and other androgen target tissues.

5-alpha Reductase Inhibitors↗
PubMed1988

Estrogen metabolism by primary cultures of rat ventral prostate epithelial and stromal cells.

Estrogen metabolism was examined in primary cultures of rat ventral prostate epithelial and stromal cells developed from young (approximately 3 weeks old) animals. Supraphysiologic concentrations (50 nM) of tritium-labelled estradiol (E2) and estrone (E1) were incubated separately with each cell type and the metabolites formed were measured at selected time points over a 24 h period. The metabolites were analyzed using high performance liquid chromatography. Epithelial cells exhibited an equal capability to interconvert E2 and E1 thus demonstrating the presence of similar oxidative and reductive activities for 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) [0.45 and 0.40 pmol/3 h/microgram DNA respectively]. In contrast, stromal cells showed a 6-fold lower rate of oxidation of E2 to E1 (0.08 pmol/3 h/microgram DNA) but exhibited an approx 5-fold higher rate of reduction of E1 to E2 (1.81 pmol/3 h/microgram DNA). Estriol (E3) formation from either substrate was not detected in the two cell types. The results demonstrate that rat ventral prostate epithelial cells have similar capabilities to form or remove biologically active E2. In contrast, prostate stromal cells exhibited a preferential capability to form and possibly maintain high levels of biologically active E2. These findings are discussed with reference to the actions of estrogens on prostate epithelial-stromal cellular interactions.

17-Hydroxysteroid Dehydrogenases↗
PubMed1988

Androgen metabolism and regulation of rat ventral prostate growth and acid phosphatase during sexual maturation.

Androgen metabolism and the regulation of rat ventral prostate cell proliferation and secretory function were examined during sexual maturation. Changes in acid phosphatase (AP) characteristics were measured as a marker of androgen-dependent prostatic secretory function. In immature (21-day-old) rats, total AP activity per cell was low (14.2 +/- 1.3 mol p-nitrophenol phosphate hydrolysed/h per mg DNA); it increased threefold as the weight, protein and DNA contents of the prostate increased to adult (65-day) levels. This corresponded with significant (P less than 0.001) increases in the staining intensities of three of the four bands of secretory AP on isoelectric focusing gels. The extent of inhibition of AP by tartrate decreased at the same time. Secretory AP is known to be relatively tartrate-resistant. The changes in AP activity occurred after prostatic 5 alpha-dihydrotestosterone (5 alpha-DHT) levels increased from 4.6 +/- 0.7 pmol/mg DNA (21 days) to reach a peak of 17.6 +/- 2.3 pmol/mg DNA at 58 days. Prostatic 5 alpha-DHT concentrations were always higher than testosterone levels. Prostatic 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-Adiol) levels were lower than 5 alpha-DHT levels except on day 58 when levels peaked dramatically at 26.2 +/- 5.5 pmol/mg DNA. Changes in prostatic 5 alpha-DHT and 3 alpha-Adiol levels corresponded with changes in 5 alpha-reductase and 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) activities. The oxidative reaction of 3 alpha-HSOR was approximately fourfold higher than the reductive reaction, indicating a preference for the formation of 5 alpha-DHT. The plasma levels of testosterone, 5 alpha-DHT and 3 alpha-Adiol cannot account for their respective prostatic levels, indicating the importance of the steroid-metabolizing enzymes in regulating intracellular androgen levels. Changes in the AP characteristics could be correlated with the androgen status of the prostate.

Acid Phosphatase↗
PubMed1986

Androgen metabolism and actions in rat ventral prostate epithelial and stromal cell cultures.

The rat ventral prostate requires androgens for normal development, growth, and function. To investigate the relationship between androgen metabolism and its effects in the prostate and to examine differences between the epithelial and stromal cells, we have established a system of primary cell cultures of immature rat ventral prostate cells. Cultures of both cell types after reaching confluency (6-7 days) actively metabolized 3H-labelled testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT), 5 alpha-androstane-3 alpha,17 beta-diol, and 5 alpha-androstane-3 beta,17 beta-diol. The epithelial cells actively reduced T to 5 alpha-DHT and formed significant amounts of 5 alpha-androstane-3,17-dione from T, 5 alpha-DHT, and 5 alpha-androstane-3 alpha,17 beta-diol. All substrates were converted to significant amounts of C19O3 metabolites. The stromal cells also metabolized all substrates, but very little 5 alpha-androstane-3,17-dione was formed. The metabolism studies indicate that both cell types have delta 4-5 alpha-reductase, 3 alpha- and 3 beta-hydroxysteroid oxidoreductase and hydroxylase activities. The epithelial cells have significant 17 beta-hydroxysteroid oxidoreductase activity. The epithelial cells cultures grown in the presence of T have higher acid phosphatase (AP) contents (demonstrated histochemically and by biochemical assay). Tartrate inhibition studies indicate that the epithelial cells grown in the presence of T are making secretory AP. Stromal cell AP is not influenced by T. The results indicate that the cultured cells maintain differentiated prostatic functions: ability to metabolize androgens and, in the case of the epithelial cells, synthesize secretory AP.

Acid Phosphatase↗