Analysis of glycoconjugate patterns of normal and hormone-induced dysplastic Noble rat prostates, and an androgen-independent Noble rat prostate tumor, by lectin histochemistry and protein blotting

BACKGROUND: Alteration of the expression of glycoconjugates is frequently observed in tumors. However, studies on the changes of cellular glycosylation in the early premalignant stage of prostate carcinogenesis are scarce. METHODS: The present study characterized and compared the glycoconjugates expressed in the dysplastic lateral prostate induced in Noble (Nb) rat by steroid hormones and a transplantable androgen-independent Nb rat prostatic carcinoma line (AIT) by lectin histochemistry and protein blotting. RESULTS: The results of lectin histochemistry show that the dysplastic prostatic epithelium elaborates altered patterns of glycosylation, which are distinct from the normal secretory epithelium. Some individual cells in the dysplastic epithelium were intensely labeled by the N-acetylgalactosamine (GalNAc)-specific (agglutins from Glycine max [SBA], Helix aspera [HAA], Helix pomatia [HPA], Vicia villosa [VVA], Erythrina cristigalli [ECA]) and complex-type oligosaccharide-specific (Phaseolus vulgaris agglutin [PHA-E]) lectins, indicating that these cells contained abundant GalNAc(alpha1,3)GalNAc/Gal and Gal(beta1,4)GlcNAc(alpha1,2)Man(alpha1,6) residues. These lectins also bound to some tumor cells in the AIT, suggesting that these sugar residues are common in some dysplastic and neoplastic prostatic cells. The study has also identified several lectins (agglutins from Griffonia simplicifolia [GS-I-B4], Arachis hypogaea [PNA], Ricinus communis [RCA-I], Maackia amurensis [MAA], Sambucus nigra [SNA]), which bound only to some AIT tumor cells but not to dysplastic epithelium, indicating that alpha/betaGal and sialic acid-containing glycoconjugates are expressed by neoplastic prostatic cells. The results of lectin blottings with Triticum vulgare agglutin [S-WGA] Ulex europaeus agglutin [UEA-I] and PHA-E have identified five major glycoprotein bands (of apparent molecular weights of 116, 79, 64, 61, and 57 kDa) in the microsomal fraction of testosterone plus 17beta-estradiol (T + E2)-treated lateral prostate. These lectin-reactive bands were not detected in the AIT extracts. In the AIT microsomal extract, two glycoprotein bands of molecular weights of 58 and 46 kDa were revealed by SBA and PNA. CONCLUSIONS: The present study shows that there is an increased expression of GalNAc(alpha1,3)GalNAc/Gal residues and triantennary complex-type oligosaccharides in the dysplastic epithelial cells as compared to normal secretory epithelial cells in rat lateral prostate. This altered expression of glycoconjugates revealed in the dysplastic epithelium indicates an aberrant glycosylation in the early premalignant stage of prostate carcinogenesis. The results also show that the AIT tumor cells are heterogeneous in their glycoconjugates and different from the dysplastic epithelial cells.     

Lectin-Binding Sites in Normal Human Testis/Lektinbindungsstellen normaler humaner Hoden

Nine fluorescein isothiocyanate (FITC)-labelled lectins have been used to investigate the distribution of glycoconjugates in unfixed frozen and Bouin-fixed sections of normal human testis. Interstitial cells and lamina propria of seminiferous tubuli were stained by PNA, HPA, RCA II, SBA, ConA, and WGA indicating an abundance of the following glycoconjugates: N-GlcNAc, N-GalNAc, Gal, and Man. The germinative cells were stained cytoplasmatically by ConA (Alpha-D-Man/-Glc). Sertoli cells showed the same pattern with ConA. Early spermatids fixed PNA and RCA II in the acrosomal region. Elongated spermatids fixed WGA on their acrosomes and fainty on the flagellae too indicating abundance of N-GlcNAc residues. The findings argue for differentiation-related modifications of lectin-binding sites on germinative cells and the usefulness of Bouin-fixed samples for lectin histochemistry.     

Lectin affinity of the seminiferous epithelium in healthy and cryptorchid post-pubertal boars

The present study describes the sugar content of the seminiferous epithelium, using lectin histochemistry, in healthy boars and in boars with unilateral and bilateral abdominal cryptorchidism. In healthy boars the apical cytoplasm of Sertoli cells exhibited abundant glucosyl (Con A and WGA lectins), galactosyl (HPA, DBA, SBA and PNA lectins), and fucosyl (AAA lectin) residues. Spermatogonia and spermatocytes contained abundant glucosyl (Con A and WGA lectins) and fucosyl (AAA lectin) residues. In spermatids, galactosyl (SBA and PNA lectins) and glucosyl (Con A and WGA lectins) residues increased progressively throughout spermiogenesis, and fucosyl (AAA lectin) residues decreased. As compared with healthy boars, the scrotal testis of unilateral cryptorchid boars showed decreased amounts of fucosyl (AAA lectin) and galactosyl (HPA and DBA lectins) residues on the Sertoli cell apical cytoplasm; spermatocytes exhibited higher content of glucosyl (Con A lectin) residues and spermatids showed altered nature of glucosyl (Con A and WGA lectins) and galactosyl (SBA and PNA lectins) complexes. In abdominal testes of unilateral and bilateral cryptorchid boars, immature Sertoli cells and spermatogonia showed decreased fucosyl (AAA lectin), and increased glucosyl (Con A and WGA lectins) and galactosyl (SBA and PNA lectins) contents. These results suggest that the seminiferous epithelium of healthy boars has polarized activity with the apical compartment implicated in germ cell-Sertoli cell adhesion and interaction, in transport of ions, substrates and fluids, and in acrosomal differentiation. In scrotal testes, unilateral abdominal cryptorchidism could lead to defective germ cell-Sertoli cell adhesion, impaired acrosomal differentiation and increased ionic transport in the apical compartment of the seminiferous epithelium. Unilateral and bilateral cryptorchidism could induce increased ionic transport and membrane permeability in the seminiferous epithelium of abdominal testes.     

Glycoconjugate Histochemistry of the Seminal Vesicles of the Japanese Miniature (Shiba) Goat

The present study localizes and characterizes complex glycoconjugates in the seminal vesicles of the Japanese Shiba goat, using several carbohydrate histochemical procedures, including lectin techniques at light and electron microscopic levels. Glandular epithelial cells and luminal secretions were shown to contain neutral and acidic glycoconjugates with various saccharide residues, such as α‐d ‐Man, α‐d ‐Glc, α‐l ‐Fuc, β‐d ‐Gal, GalNAc, GlcNAc, and NeuAc (sialic acid). The terminal oligosaccharide chains of sialoglycoconjugates present in the seminal vesicles were NeuAc α 2‐3Gal β 1‐3GalNAc and NeuAc α 2‐3Gal β 1‐4GlcNAc. In addition, in lysosomes of the glandular epithelial cells α‐d ‐Man, α‐d ‐Glc, GlcNAc and NeuAc (sialic acid) residues could be detected, the secretory vesicles contained α‐l ‐Fuc, and the endoplasmic reticulum exhibited α‐d ‐Man and α‐d ‐Glc residues. The complex glycoconjugates with various sugar residues found in the seminal vesicles of the goat may be involved in various fertilization‐related events.     

Capacitated and acrosome reacted spermatozoa of goat (Capra indicus): a fluorescence and electron microscopic study

Summary. Plasma membrane alterations accompanying in vitro capacitation and acrosome reaction of goat spermatozoa were studied using lectin labelling, scanning electron microscopy, and freeze-fracture methods. Fluorescein isothi-ocyanate linked lectins namely; Canavalia ensiformis (ConA), Maclura pomifera (MPA), Arachis hypogaea (PNA), Glycine max (SBA) and Triticum vulgaris (WGA) agglutinin were used to examine the distribution of surface carbohydrates during these two events. The head and the sperm tail reveal altered lectin labelling features after capacitation and acrosome reaction. After capacitation the surface coat components for MPA, SBA, and WGA are shed from the spermatozoon head. ConA receptors on the head are retained after capacitation but are partially shed in the acrosome reacted spermatozoa. SBA receptor sites appear on the sperm tail of the capacitated spermatozoa. Unusual morphological changes attending capacitation involve the sperm tail-end which develops a novel entity, which we have termed 'spatula'. The 'spatula' shows strong binding with ConA and WGA only. In the acrosome reacted spermatozoa the spatulated tail-end unwinds with a concomitant loss of lectin labelling. Highly ordered membrane particles, 'ladders' of the middle piece of the epididymal sperm tail, disappear and IMP clearings appear on the middle piece and in the spatulated ends of the capacitated spermatozoa. But in the acrosome reacted sperm IMPs reappear and are randomly disposed on the middle-piece and are clustered in small patches on the principal-piece. IMP free areas appear on the plasma membrane covering the acrosome and the outer acrosomal membrane (OAM) of the capacitated spermatozoa. The plasma membrane and OAM fuse at multiple foci and appear as acrosomal 'ghosts' which remain associated with the sperm head even after acrosome reaction.     

Lectins in diagnosis of bladder carcinoma.

With the purpose of studying changes in the expression of glycoconjugate structures in nonmalignant and cancerous lesions of urothelium the lectins ConA, TKA, PNA, DBA, STA, LFA, UEA, MPA, RCA, LCA, GSA1, SBA, GSA2, WGA, PHA and Lot were tested in formalin-fixed, paraffin-embedded tissue sections of (1) cold biopsies from normal urothelium and bladder cancer of different grades (G1-G3) in humans, (2) normal transitional epithelium and N-butyl-N(4-hydroxybutyl)nitrosamine (BBN)-induced bladder cancer in animal experiments (Wistar rat), and (3) human transitional cancer cell line HT 1376. In human urothelium TKA and SBA were positive markers demonstrating positive staining reactions in all tumor grades without binding to normal epithelium. They stained also the human transitional carcinoma cell line HT 1376 (G3). In Wistar rats DBA, ConA, LCA, SBA, GSA2 and WGA had a specific affinity to BBN-induced carcinoma. Findings of positive lectin marker in transitional cell cancer may offer progress in diagnostics and therapy.     

Localization of carbohydrate component in human synovial lining cells with fluorescent lectins and enzyme digestion

FITC-conjugated lectins, Con-A, DBA, GS-I, GS-II, PNA, MPA, RCA-I, SBA, UEA-I, WGA were used for demonstration of lectin bindings of human synovial lining cells, obtained from the patients with rheumatoid arthritis (RA), osteoarthritis (OA), aseptic necrosis (AN), and traumatic injury (TI). In the RA samples, GS-I binding to the cytoplasmic sites was predominantly noted and moderate SBA and MPA bindings were observed. However, PNA was not significant. In the OA samples, predominant binding was found in GS-I and SBA lectins, moderate binding in MPA and PNA. In the AN samples, binding was predominant in MPA, moderate in GS-I, SBA and PNA. After neuraminidase treatment the intensity of fluorescence increased significantly with PNA and moderately with SBA in the RA samples. These results suggested that the inflammatory lining cells produce galactose group and the content of neuraminic acids in the synovial membranes of the RA appears to be greater than in those of other diseases.     

Electrophoretic analysis of urinary glycoproteins in diabetic nephropathy using peroxidase-lectins

We examined the clinical usefulness determined by polyacrylamide gel electrophoresis, followed by reaction with peroxidase-coupled lectins using urinary glycoproteins for diabetic nephropathy in 20 patients with diabetes mellitus. Lectins used were Triticum vulgaris (WGA), Phaseolus vulgaris (PHA-E4), Dolichos biflorus (DBA), and Lens culinaris (LCA), which have high affinity for beta 1----4N-acetyl-D-glucosamine (GlcNAc beta 1----4GlcNAc), N-acetyl-D-galactosamine (GalNAc), alpha-galactosamine (alpha-GalNAc), and alpha-mannose (alpha-Man) residues, respectively. Electrophoretic patterns of urinary glycoproteins clearly showed the presence of lectin-reactive glycoproteins with molecular weights lower than that of albumin. The molecular weight of the main bands reacted with WGA, PHA-E4 or LCA were 50,000 and 38,000, and increased with the progress of diabetic nephropathy. WGA reacted strongly with many glycoproteins having a wide range of molecular weights. LCA and PHA-E4 reacted preferentially with glycoproteins of molecular weights glycoproteins of molecular weights lower than 50,000, but no reaction was observed by DBA. These results suggest that low molecular urinary glycoproteins have abundant carbohydrate residues such as GlcNAc beta 1----4GlcNAc, GalNAc, and alpha-Man. The excretion of low molecular weight glycoproteins with high affinities for some lectins suggests functional impairment in diabetic nephropathy.     

Tyrosine phosphorylation following lectin meiated endothelial cell stimulation

Abstract: Terminal alpha (1,3) galactosyl galactoside epitopes (α-gal) on membrane glycoproteins expressed by vascular endothelial cells represent the major xenoreactive antigens in pig to primate xenotransplantation. In other discordant xenotransplantation combinations, such as from guinea pig to rat, carbohydrate epitopes other than α-gal may be targeted by xenoreactive antibodies (XNA). We have shown that agonist binding to α-gal epitopes induces proinflammatory activation of porcine aortic endothelial cells (PAEC). Binding of α-gal epitopes by Bandeiraea simplicifolia isolectin B₄ results in both type I and type II PAEC activation. This includes the phosphorylation of tyrosine residue(s) of a protein with an apparent molecular weight of 130 kDa (p130). In order to investigate whether binding of other carbohydrate epitopes could induce a similar phosphorylation event, several lectins with different carbohydrate specificities were used to stimulate PAEC and human umbilical endothelial cells (HUVEC). In addition to BS-IB₄ binding to α-gal, lectins binding to sialic acid isolated from Sambucus nigra (SNA), Maackia amurensis (MAA), Wheat germ agglutinin (WGA), and lectin from jack bean (Concanavalin A, ConA), that binds to mannose residues within the core structure of N-glycosylated proteins all induced the phosphorylation of the p130 protein(s). Lectins with affinity to alpha bound N-acetylgalactosamine, Dolichos biflorus (DOB), and Sophora japonoca (SOJ) did not induce this phosphorylation event. A similar negative result was obtained with Ulex europaeus lectin I, which binds to fucose residues. Conclusively, endothelial cell activation can be observed upon binding of various lectins to the glycosylated moiety of surface glycoproteins. These carbohydrate epitopes against which XNA may exist in certain models might represent minor xenoantigens from porcine to primates or may comprise the major xenoepitopes in other discordant xenograft models. Binding of XNA and subsequently the elicited xenoreactive antibodies to carbohydrate epitopes may therefore contribute to xenograft rejection even in the absence of complement inactivation.     

胆汁成核活性蛋白中糖链的致石作用

目的 探讨糖链在胆石形成中的作用。方法 分别将5mg辣根过氧化酶（HRP）标记蔓陀罗凝集素（DSA）、麦胚凝集素（WGA）和伴刀豆凝集素（ConA），鉴定糖链结构与类型，并在透射电镜下观察亲和染色的模拟胆汁泡形态学变化；通过结晶生长试验检测胆汁泡蛋白（0.1g／L）促成核活性及其糖链水解、多肽降解产物的活性改变。结果 胆汁泡蛋白糖链为DSA强结合、ConA(一），属多天线复杂型聚糖。HRP－DSA标记的泡蛋白糖链，直接反映泡蛋白促进胆汁泡聚集、融合及胆固醇单水结晶析出的动态过程。胆汁泡蛋白具明显促成核活性，其It、Ig、Ic分别为0.57、1.52及1.63（P＜0.05）。糖苷酶切、肽链酶解后其成核活性均几乎丧失。结论 糖链可能参与成核效应蛋白的致石作用过程。     

Distribution of Lectin Binding in Rat Testis and Epididymis

Summary: Seven lectins (PNA, RCA I, SBA, Con A, WGA, UEA I, DBA) conjugated with rhodamine were employed to analyse the staining pattern of glycoproteins with varying sugar residues in the testis and epididymis of adult Wistar rats. Some lectins (UEA I, SBA, DBA) gave rather specific staining of the mature acrosome, while others (PNA, RCA I) showed affinity for the early stages of acrosome formation or had a wide affinity for germinal and non-germinal cells and structures (Con A, WGA). In the epididymis the sperm mass had a homogenous staining reaction with some lectins (PNA, RCA I, Con A, WGA, DBA) which also showed a rather strong reaction on the epithelial surface. It was concluded that this reaction is at least partially due to the secretory products synthetized by principal, apical, narrow and light cells of the epididymal epithelium. Some differences in the staining pattern of these cells were recorded indicating specialization of the cells for the production of distinct glycoproteins. The staining pattern of the interstitial and intertubular compartment of the testis and epididymis was also recorded. Zusammenfassung: Die Verteilung der Lektin-Bindung im Hoden und Nebenhoden der Ratte Sieben Lektine (PNA, RCA I, SBA, Con A, WGA, UEA I, DBA) wurden verbunden mit Rhodamin verwendet, um das Darstellungsmuster von Glykoproteinen mit verschiedenen Zuckerresten im Hoden und Nebenhoden erwachsener Wistar-Ratten zu analysieren. Einige Lektine (UEA I, SBA, DBA) gaben eine recht spezifische Darstellung des reifen Akrosoms, wohingegen andere (PNA, RCA I) eine Affinität zu den frühen Stadien der Akrosomformation zeigten oder eine breite Affinität zu Germinal- und Nichtgerminalzellen und -strukturen aufwiesen (Con A, WGA). Im Nebenhoden zeigte die Spermatozoenmasse ein homogenes Darstellungsmuster mit einigen Lektinen (PNA, RCA I, Con A, WGA, DBA), welche auf der epithelialen Oberfläche auch eine recht starke Reaktion aufwiesen. Es wurde daraus geschlossen, daβ diese Reaktion zumindest teilweise auf die sekretorischen Produkte, die in den Haupt-, Apikal-, Schmal- und Hellzellen des Nebenhodens synthetisiert werden, zurückzuführen ist. Einige Unterschiede im Darstellungsmuster dieser Zellen wurden aufgezeichnet, die die Spezialisierung der Zellen für die Produktion bestimmter Glykoproteine anzeigen. Das Darstellungsmuster des interstitiellen und intertubulären Anteils des Hodens und Nebenhodens wurde ebenfalls aufgezeichnet.     

Lectin Histochemical Investigations of the Distal Gut of Chicks with Special Emphasis on the Follicle‐associated Epithelium

Carbohydrates on epithelial cell surfaces play an important role as attachment sites for different microorganisms like bacteria, viruses and protozoa. To obtain more information about the distribution of carbohydrates on the luminal surface along the intestine, lectin histochemical studies on different gut segments of chicks of different age groups were carried out using a panel of 13 lectins with specificities for Man, Glc, Gal, GalNAc, GlcNAc or GlcNAc oligosaccharides and Sia. Furthermore, we tried to find out whether previously reported specificities of certain lectins for M cells (membranous or multifold cells) in the bursa of Fabricius (BF) can be observed also on M cells of the intestine. As a result we were able to demonstrate binding of all lectins employed in these studies in all investigated gut segments. In some cases, the application of the same lectin led to varying staining intensities of the same histological structures in different age‐groups (e.g. staining of the brush border with WGA, LEA, MAA or Conarva) or different gut segments (e.g. staining of goblet cells with CMA II, LEA and MPA). Hence, terminal carbohydrate residues of glycoconjugates on the intestinal epithelium vary depending on age and organ site. As glycoconjugates can act as attachment sites for microorganisms, these differences in the distribution of sugar residues may be one explanation for the site‐specificity of certain pathogens. Furthermore, the binding of lectins to the follicle‐associated epithelium (FAE) of the BF differs from that to the FAE of the intestine again stressing the site specificity of lectin binding. Thus, up to now no universal M‐cell marker along the chicken intestine exists.     

Characterizing the glycocalyx of poultry spermatozoa: I. Identification and distribution of carbohydrate residues using flow cytometry and epifluorescence microscopy

The aim of the present work was to use a battery of lectins to 1) delineate the carbohydrate content of sperm glycocalyx in the turkey and chicken using flow cytometry analysis, and 2) evaluate the distribution of existing sugars over the sperm plasma membrane surface with epifluorescent microscopy. Carbohydrate groups (corresponding lectins) that were investigated included galactose (GS-I, Jacalin, RCA-I, PNA), glucose and/or mannose (Con A, PSA, GNA), N-acetyl-glucosamine (GS-II, s-WGA, STA), N-acetyl-galactosamine (SBA, WFA), fucose (Lotus, UEA-I), sialic acid (LFA, LPA), and N-acetyl-lactosamine (ECA). Spermatozoa were assessed before and after treatment with neuraminidase to remove sialic acid. Mean fluorescence intensity (MnFI) was used as indicator of lectin binding for flow cytometry analysis. Nontreated spermatozoa from both species showed high MnFI when incubated with RCA-I, Con A, LFA, and LPA, as did chicken spermatozoa incubated with s-WGA. Neuraminidase treatment increased the MnFI for most lectins except LFA and LPA, as expected. Differences in MnFI between species included higher values for s-WGA and ECA in chicken spermatozoa and for WFA in turkey spermatozoa. Microscopy revealed segregation of some sugar residues into membrane-specific domains; however, the 2 staining techniques (cell suspension vs fixed preparation) differed in identifying lectin binding patterns, with fixed preparations yielding a high degree of nonspecific binding. We conclude that 1) the glycocalyx of turkey and chicken spermatozoa contains a diversity of carbohydrate groups, 2) these residues are extensively masked by sialic acid, 3) the glycocalyx composition is species-specific, and 4) some glycoconjugates appear to be segregated into membrane-specific domains. Characterization of the poultry sperm glycocalyx is the first step in identifying the physiological impact of semen storage on sperm function.     

Salivary Gland Basal Cell Adenocarcinoma: A Report of Cases in a Cat and Two Dogs

Basal cell adenocarcinoma of the salivary gland is described in a cat and two dogs; tumour tissue was characterized by cords and islands of epithelial cells with a distinct basal layer. The tumours were stained by various immunohistochemical methods. In addition to positive staining with cytokeratin 14 and pancytokeratin (CKs 5, 6, 8, 17 and 19), there was also staining with Jack bean agglutinin A (ConA) and soya bean agglutinin (SBA); this occurs in many other types of salivary gland tumours and is a feature of normal salivary gland acinar cells. In one dog there was also staining with SBA. This is the first report of this tumour in domestic animals; the immunohistochemical characteristics did not distinguish it from other salivary gland tumours.     

A study of oligosaccharide determinants expressed by prostatic glandular epithelium of the normal adult rat

Summary Normal prostates from Copenhagen/Fischer F1 hybrid rats were removed at 14 months of age. After routine formalin fixation and paraffin embedding, the expression of seven oligosaccharide structures by prostatic epithelial cells was assessed by an examination of lectin binding sites before and after neuraminidase digestion. Con-A bound to plasma membranes as well as the cytoplasm of all cells, thus confirming the presence of complex-type glycoconjugates. However, only two other oligosaccharides, apart from Con-A, were freely expressed on epithelial luminal plasma membranes. These were the Type I structure (Ga113GalNAc-) identified by PNA-binding and (GlcNAc14GlcNAc14-)_n identified by WGA. PNA, WGA, UEA-1 and SBA bound to the cytoplasm of almost all epithelial cells, although their intracellular distribution was not identical. DBF binding was not identified. ECG bound to only a very few cells and then only after digestion with neuraminidase when it was localised to the cytoplasm. Following removal of sialic acid groups by neuraminidase digestion, PNA-binding became more prominent, SBA-binding appeared localized to paranuclear intracellular vesicles and WGA binding sites were abolished. This study has now characterized the major oligosaccharide determinants expressed by rat normal prostatic epithelial cells and provides a baseline against which alterations occurring during ontogenesis and oncogenesis may be compared.Abbreviations Con-A Canavalia ensiformis - SBA Glycine max - DBF Dolichos biflorus - PNA Arachis hypogaea - ECG Erythrina cristagalli - WGA Triticum vulgaris - UEA-1 Ulex europaeus-1 This work was supported by a grant from the Hammersmith and Queen Charlotte's Special Health Authority     

Distribution of γ-Glutamyl Transpeptidase in Male Reproductive System of Rats and its Age-Related Changes

The present study is designed to investigate localization of gamma-glutamyl transpeptidase (gamma-GTP) in male reproductive organs and its age-related changes using Wistar rats aged 2 to 15 weeks. Histochemically, gamma-GTP activity was detected intensively in epithelial cells of epididymides and seminal vesicles and weakly in those of anterior prostates, but not in testes under the present conditions. Biochemically, the highest gamma-GTP activity was found in epididymal head portions. The order of the activity was epididymides (head greater than body greater than tail), seminal vesicles, prostates and testes. The activity increases with sexual maturation in epididymides and seminal vesicles, but not in prostates. Since gamma-GTP is an enzyme involved in the incorporation of amino acids, the present findings suggest that not only epididymides but also seminal vesicles possess uptake mechanisms for amino acids.     

Prostanoid transport by multidrug resistance protein 4 (MRP4/ABCC4) localized in tissues of the human urogenital tract

PURPOSE: The seminal vesicles are the major source of prostaglandins in seminal fluid. For prostanoid action on cell surfaces they must be released from synthesizing cells. MRP4/ABCC4 (multidrug resistance protein 4 adenosine triphosphate-binding cassette, subfamily C, member 4) is an adenosine triphosphate dependent export pump for organic anions that may mediate prostanoid transport across the plasma membranes. Therefore, we analyzed whether MRP4 is expressed in the seminal vesicles and other tissues of the human urogenital tract, whether MRP4 and prostanoid synthesizing enzymes are co-expressed in the same cell type and whether MRP4 functions as a prostanoid export pump. MATERIALS AND METHODS: The expression and localization of MRP4 and prostanoid synthesizing enzymes were investigated in several tissues of the male human urogenital tract by immunoblot and immunofluorescence analyses. Prostanoid transport was measured into inside-out membrane vesicles from cells expressing recombinant human MRP4. RESULTS: MRP4 and prostanoid synthesizing enzymes were co-expressed in the epithelial cells of human seminal vesicles. Moreover, MRP4 was localized in the plasma membrane of epithelial cells of the ureter, in the basolateral membrane of glandular epithelial cells of the prostate, and in smooth muscle cells of the bladder and corpus cavernosum. Transport studies established MRP4 as an efflux pump for prostaglandin E2 (Michaelis constant [Km] 3.5 muM), thromboxane B2 (Km 9.9 muM) and prostaglandin F2alpha (Km 12.6 muM). CONCLUSIONS: The co-expression of prostanoid synthesizing enzymes and MRP4 in epithelial cells of the human seminal vesicles and the function of MRP4 as a prostanoid efflux pump indicate that MRP4 mediates prostanoid transport from these cells, which are the main prostanoid synthesizing cells in the male urogenital tract.     

Vasopressin receptors in human seminal vesicles: identification, pharmacologic characterization, and comparison with the vasopressin receptors present in the human kidney

Because of the presence of a high density of vasopressin receptors in the epithelial cells of porcine seminal vesicles similar to the V2 vasopressin receptors of renal tubules, human seminal vesicles and kidney were investigated using quantitative binding and adenylate cyclase studies. Tissues were obtained at surgery from 17 patients with urologic diseases. A homogeneous class of vasopressin binding sites have been found in both seminal vesicles and renal medulla. However, the vasopressin receptors present in these tissues are different in terms of ligand specificity and adenylate cyclase activation. In seminal vesicles, the V1 vasopressin antagonist d(CH2)5 TyrMeAVP is 36-fold, more potent than the V2 agonist dVDAVP in displacing [3H]AVP binding, while in the medullopapillary portion of kidney dVDAVP is 24-fold, more selective than d(CH2)5 TyrMeAVP for the arginine vasopressin binding site. Furthermore, arginine vasopressin induces a dose-dependent increase in adenylate cyclase activity in renal membranes, while it was ineffective in seminal vesicle membranes. These results indicate that a very high affinity (0.2 nM), low capacity (14 fmoles/mg protein) class of vasopressin receptors is present in human seminal vesicles, having pharmacologic characteristics similar to the V1 subtype of vasopressin receptors. The presence of a high affinity (1.6 nM), high capacity (350 fmoles/mg protein) V2 subtype of vasopressin receptors in human renal membranes is also confirmed. The density of the vasopressin receptors present in human seminal vesicles is inversely correlated with patient age, consistent with a physiologic role for vasopressin in the regulation of accessory sex gland activity.     

Characteristic localization of carbohydrates in osteoclasts by lectin cytochemistry

Lectin cytochemistry was performed to clarify the process of glycosylation and the localization of glycocalyx in osteoclasts. Microslicer sections of decalcified rat tibiae were incubated in the presence of HRP-conjugated lectins (Con A, PNA, MPA, WGA, UEA-1). Lectin reactions in cell organelles revealed that glucose (Glc) and mannose (Man) are transferred to carbohydrate chains in nuclear envelopes, rough endoplasmic reticuli, and the cis and medial sides of the Golgi apparatus. N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and/or N-acetylneuraminic acic (NANA) residues are transferred, in turn, in the Golgi apparatus. Lectin reactions detected in lysosomal structures suggest that some sugar residues are incorporated into carbohydrate chains of hydrolytic enzymes, such as acid phosphatase and arylsulfatase. Others would be transported to plasma membranes as glycocalyx. PNA and MPA reactions were most evident on ruffled borders of osteoclasts. On the other hand, cement-line-like structures on bone surfaces displayed Con A, MPA, and WGA positive reactions. The following factors suggest that osteoclasts actively metabolize sugar: characteristic localization of glycocalyx in osteoclasts reflect the polarity of osteoclasts, and carbohydrate complexes in cementline-like structures seem to play an important role in the coupling phenomenon in bone tissue.     

Variable distribution of aminopeptidase A in male reproductive organs of mammals

Aminopeptidase A (AP-A) was analysed in the reproductive organs of the boar, bull, gerbil and man. High hydrolysis of alpha-L-glutamyl-beta-naphthylamide (GluNA) and alpha-L-aspartyl-beta-naphthylamide (AspNA) with activation by alkaline earth metals was detected in the ampulla, seminal vesicles, and seminal vesicle secretions of the bull and in the cauda epididymis of the boar and gerbil. In man, weak AP-A activity was found in all reproductive tissues. Histochemically, AP-A was localized in the epithelial cells of tissues having a high specific activity for the enzyme. AP-A was absent from human seminal fluid, whilst bovine seminal fluid had strong, and boar seminal fluid weaker, AP-A activity. Gel filtration of bull seminal vesicle secretions and seminal fluid, boar seminal fluid or an homogenate of boar and gerbil epididymal cauda and human epididymis and seminal vesicles on Sephacryl S-300 resulted in a major high-molecular-weight activity peak A at Ve/Vo = 1.17 and another low-molecular-weight peak B at Ve/Vo = 1.51 (man), 1.62 (boar, bull) or 1.75 (gerbil). This fractionation was not in all cases able to separate AP-A from aminopeptidase(s), which were active on L-alanine-beta-naphthylamide (AlaNA) but showed no activation by alkaline earth metals. Homogenates of bovine epididymis showed only the low-molecular-weight GluNA peak B, but two areas of activity for AlaNA hydrolysis. In bovine seminal vesicles and porcine epididymis, AP-A activity appeared to be linked with the functional maturity of these organs. The high-molecular-weight AP-A (peak A) appeared to be the predominant form in seminal fluid.