Beta microseminoprotein is not a prostate-specific protein. Its identification in mucous glands and secretions.

Beta microseminoprotein (beta inhibin, PSP94), an unglycosylated protein of 94 amino acids with unknown function, is one of the predominating proteins in the secretion of the human prostate gland. In this work the authors have demonstrated that the expression of beta microseminoprotein is not restricted to the prostate and that the protein has a previously unrecognized widespread occurrence in the human body. According to radioimmunoassay, beta microseminoprotein immunoreactivity is present in many nonprostatic body fluids. The highest concentrations were found in secretions from the respiratory tract; in tracheobronchial fluid sometimes even at concentrations comparable to that in seminal plasma (about 1 g/l). Intermediate concentrations were found in gastric juice and some samples of secretion from the uterine cervix, whereas tears, saliva, pancreatic juice, bile, and mucus from the colon had low concentrations. According to gel chromatography, the molecular size of the beta microseminoprotein immunoreactivity present in tracheal fluid, gastric juice, and secretion from the uterine cervix did not differ from that of beta microseminoprotein in seminal plasma. The beta microseminoprotein immunoreactive component present in gastric juice had the same amino-terminal amino acid sequence as prostatic beta microseminoprotein (14 residues identified in material purified from gastric juice), providing further evidence for chemical identity of a nonprostatic beta microseminoprotein with the prostatic protein. Immunohistochemical staining with affinity-purified antibodies demonstrated the presence of beta microseminoprotein in many tissues, including the goblet cells in the tracheobronchial epithelium, tracheobronchial submucosal glands, certain mucosal cells in the antrum of the stomach, some glands of Brunner in the duodenum, and in parts of the mucosa of the colon. At least in the respiratory tract, the staining was localized to mucus-containing cells. beta microseminoprotein immunoreactivity also was localized to the cilia of the ciliated epithelium in the respiratory tract, the fallopian tubes, and the Gartner ducts of the uterine cervix. The pattern of tissue distribution of beta microseminoprotein found in this work indicates a connection of beta microseminoprotein with mucous secretions.

Distribution of acid stable trypsin inhibitor immunoreactivity in normal and malignant human tissues.

The distribution and localization of acid stable trypsin inhibitor (ASTI) in normal and malignant human tissues from various organs were examined using immunohistochemical techniques that used goat antibody raised against highly purified ASTI from human urine. Tissues were assessed as positive only when they were stained by both the biotin-avidin-peroxidase complex system and biotin-streptavidin-beta-galactosidase complex system, and the staining was abolished by absorption with purified ASTI. Under normal conditions, ASTI immunoreactivity was observed in only a few organs. Positive tissues for ASTI immunoreactivity included the kidney proximal tubules, glial cells of the cerebrum, fibrillar structures of the lamina propria of the stomach and colon, and bronchial epithelial cells. No ASTI immunoreactivity was observed in the cardiovascular system, reproductive system, or other tissues examined. As is not the case for normal tissues, ASTI immunoreactivity was found to be widely distributed in malignant tumors. Staining was observed in the extracellular space, i.e., in the stroma of the tumor and in connective tissues around the tumor invasion, whereas no ASTI immunoreactivity was detected in the malignant cells. Considering the identity of the first 36 NH2-terminal residues of ASTI purified from plasma or urine with a recently reported endothelial cell growth factor, the present findings suggest that ASTI could play an important role, not limited to its function as a protease inhibitor, in the invasive growth of malignant neoplasms.

[Experimental studies on sympathetic innervation of lower urinary tract. Quantitation of nerve terminals and urethral pressure response after hypogastric denervation].

On 6 female mongrel dogs (denervated group), bilateral hypogastric nerves were cut distally to the inferior mesenteric ganglion. Five dogs were kept intact as a control group. After 2-5 months, urethral pressure response to continuous noradrenaline infusion (0.1 microgram/kg/min) was monitored. The urethral pressure rose significantly after noradrenaline loading in each group, however there was no significant difference in the degree of the response between the denervated group and control group. Subsequently, the bladder and the urethra were extirpated to determine the intrinsic noradrenaline content. The tissue noradrenaline concentration was highest in the posterior urethra, intermediate in the bladder base and lowest in the bladder dome. Although these values tended to be lower in the denervated group than in the control group, no significant difference was obtained between the groups in each portion. These results suggest that the majority of sympathetic components which consists in the hypogastric nerve may involve short adrenergic neurons. Thus, chronic hypogastric denervation alone does not induce sympathetic denervation supersensitivity. Simultaneous decentralization of the pelvic nerve may be necessary for inducing sympathetic denervation supersensitivity.

Distribution of rabbit mucosal glycoprotein throughout urinary tract.

The mucin layer covering the transitional epithelium of the bladder is thought to be an anti-adherence substance for bacteria. We describe the use of an immunoperoxidase staining technique to demonstrate the presence of glycoprotein lining the urothelium of both the upper and lower urinary tracts of the rabbit. Antisera against this glycoprotein (GP1) were raised in Swiss-Webster mice. The genitourinary tracts of male and female NZW rabbits were removed and sequentially treated with mouse anti-GP1 sera, biotin-labelled anti-mouse IgG, and an avidin-biotinylated horseradish peroxidase complex. The results demonstrated that an antigenically similar (or identical) glycoprotein covers the distal renal tubules and urothelium of the renal pelvis, ureters, bladder, and urethra, suggesting that it may function as an antibacterial defense mechanism throughout the urinary tract.

Characterization of autonomic receptors in neonatal urinary tract smooth muscle.

In the 1-day-old rabbit there are a large number of alpha-2- (labeled with [3H]yohimbine) than alpha-1- (labeled with [3H]prazosin) adrenergic receptors in the various parts of urinary tract smooth muscle, but the alpha-1 receptors are functionally more responsive to agonists than are the alpha-2 receptors. Unlike the findings in the adult rabbit, the neonatal bladder dome is functionally more sensitive to alpha-adrenergic agonists than the neonatal bladder base. The rank order of beta-adrenergic receptor densities (labeled with [3H]dihydroalprenolol) in various urinary tract smooth muscles correlates with the magnitude of the relaxant response to isoproterenol. There is a high number of functional muscarinic receptors (labeled with [3H]quinuclidinyl benzylate) in the neonatal urinary tract. The data demonstrate the existence of functional autonomic receptors in neonatal urinary tract smooth muscle.

Occurrence and distribution of glycoconjugates in human tissues as detected by the Erythrina cristagalli lectin.

We applied a horseradish peroxidase-Erythrina cristagalli agglutinin (HRP-ECA) conjugate for histochemical staining of tissue sections from various formalin-fixed, paraffin-embedded human tissue specimens. The HRP-ECA conjugate showed broad reactivity, but there was a distinct distribution of native (not masked by sialic acid) and sialic acid-masked ECA binding sites in the various organs. Free ECA binding sites could be detected on red blood cells, lymphocytes of thymus, tonsil, lymph node, and in mucous substances of different organs. Independent of blood group type, the vascular endothelium exhibited strong ECA reactivity. Free ECA binding sites occurred in the cytoplasm of Kupffer's cells in liver, in histiocytic cells of thymus, lymph node, tonsil, and in bone marrow. Podocytes of kidney glomerulus, syncytiotrophoblasts of placenta, megakaryocytes in bone marrow, myelin sheath of nerve, medullary thymocytes, and hepatocytes, as well as islet cells of pancreas, contained only sialic acid-capped ECA binding sites. Inhibiting studies with galactose, lactose, and N-acetyl-lactosamine, as well as other sugars, revealed that this lectin is specific for galactosyl residues. In comparison to galactose and lactose, N-acetyl-lactosamine exhibited the highest inhibitory activity on lectin binding, supporting the concept that this lectin is most reactive with N-acetyl-lactosamine-type (type 2 chain) glycoconjugates.

Progesterone receptors in the female lower urinary tract.

When female estrogenized rabbits were injected i.v. with 3H-progesterone, the tritium concentration determined after one hour was about two to three times higher in urethra, urinary bladder and vagina than in the heart. High affinity progesterone receptors (KD = 1-2 nM) could be demonstrated in both cytoplasmic and nuclear fractions prepared from estrogenized rabbit urethra, bladder and vagina. The cytosolic receptor concentration in both urethra and bladder was about half of that in the vagina. The concentration of nuclear receptors in urethra was not significantly different from that in the vagina, but in the bladder the concentration was only about one fourth of that in the vagina or urethra. The mean KD of cytosolic receptors from bladder was significantly higher than the corresponding values in urethra and vagina. Progesterone binding sites in the bladder had a broader hormonal specificity than those in the urethra or vagina. The present demonstration of specific progesterone receptors in the female urethra might provide a possible link between estrogen progesterone interaction and the appearance of urinary incontinence during pregnancy in women.

Immunoanatomic distribution of cytostructural and tissue-associated antigens in the human urinary tract.

The main objective of the present study is to define the expression and/or modulation of antigenic phenotypes in cells of the normal human kidney and urothelium according to cell type. Fourteen antibodies detecting differentiation and structural antigens expressed in the human urinary tract have been used to define the immunoanatomic distribution of these antigenic systems. They include urinary tract antigens (Tamm-Horsfall glycoprotein and prostate-specific antigen), tissue-associated antigens (epithelial membrane antigen, Factor VIII antigen, and Protein S-100), and cytoskeletal antigens of the intermediate filament classes (cytokeratins, vimentin, desmin, glial fibrillary acidic protein, and neurofilaments. Immunofluorescence and immunoperoxidase analyses performed on normal human fetal and adult tissue sections have demonstrated that these antigens are expressed by different cell types and domains of the nephron. Studies correlating normal fetal and adult tissues reveal that some of the antigens appear at distinct stages of maturation, representing early and late antigenic expression events. These antibodies offer a wide range of potential applications that include studies of embryogenesis of the human urinary tract and immunopathologic analyses of neoplastic and nonneoplastic diseases of the human kidney and urothelium.

Calcitonin gene-related peptide immunoreactivity in afferent neurons supplying the urinary tract: combined retrograde tracing and immunohistochemistry.

The innervation of rat and guinea pig urinary tract was examined using immunohistochemistry, radioimmunoassay and True Blue retrograde tracing techniques and was further assessed following both surgical and chemical denervation experiments. Substantial amounts of calcitonin gene-related peptide-like immunoreactivity (range 20-150 pmol/g) were detected in tissue extracts and localised to nerve fibres distributed throughout the urinary tract of both species, these being concentrated in the ureter and base of the bladder. In the guinea pig, the number and distribution pattern of calcitonin gene-related peptide-like immunoreactive nerves appeared to be identical to that of substance P-containing nerves, whereas in the rat the former predominated. Seven days after injection of the fluorescent dye True Blue into tissues of the urinary tract, retrogradely labelled cells were found in the dorsal root ganglia. These cells had a segmental distribution pattern which was specific for each of the injection sites. Thus, after injection of True Blue into the left kidney hilum a single group of labelled cells were found in the ipsilateral T10-L2 dorsal root ganglia. In contrast, injection into the left ureter produced labelled cells in two separate groups of ipsilateral ganglia (T11-L3 and L6-S1). Injection into the wall of the bladder and upper urethra resulted in bilateral labelling, with most labelled cells occurring in L6 and S1 ganglia. Approximately 90% of labelled cells in T10-L3 dorsal root ganglia displayed calcitonin gene-related peptide-like immunoreactivity, but only 60% of retrogradely labelled bladder neurons in L6-S1 ganglia were immunoreactive for this peptide. Adult guinea pigs and neonatal rats injected systemically with capsaicin subsequently exhibited a marked reduction both in the amount of calcitonin gene-related peptide immunostaining and the concentration of immunoreactive material in the urinary tract, dorsal root ganglia and spinal cord. In rats treated neonatally with capsaicin, there was a significant reduction in the number of retrogradely labelled cells and a hypertrophy of the bladder. Sectioning of the pelvic and hypogastric nerves in the rat also resulted in a depletion of calcitonin gene-related peptide-like immunoreactive nerves in the bladder, whereas chemical sympathectomy appeared to have no effect. The results indicate that calcitonin gene-related peptide immunoreactivity occurs in a major proportion of afferent neurons supplying the urinary tract of the rat and guinea pig.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunohistochemical distribution of S-100 antigen in the urinary system of man and rat.

The immunohistochemical distribution of S-100, a protein originally isolated from the brain, has been investigated at the light and electron microscopic levels in rat and man urinary systems. In both species the antigen essentially exhibited the same location, restricted, with different degrees of staining, to certain cells in the kidney, i.e. collecting tubules, thin limbs of Henle's loop and renal papillae.

Calcitonin gene-related peptide (CGRP) in the female rat urogenital tract.

CGRP-immunoreactivity was found throughout the female rat urogenital tract by specific radioimmunoassay, and shown to be present in nerve fibres by immunocytochemistry. The highest concentrations of CGRP-like immunoreactivity were found in the urinary tract, with lower levels in regions of the genitalia. Chromatographic analysis of bladder and vaginal extracts on Sephadex G-50 columns and HPLC revealed at least three CGRP-immunoreactive peaks. The major peak emerged in the same position as synthetic rat CGRP. CGRP nerve fibres were associated mainly with blood vessels, non-vascular smooth muscle, squamous epithelium and uterine and cervical glands, and were particularly abundant in the ureter and bladder. CGRP-immunoreactivity was depleted by neonatal treatment with capsaicin and after surgical section of pelvic and/or hypogastric nerves. Immunocytochemistry demonstrated that depletion occurred predominantly in the mucosal layer of the urogenital tract. These findings indicate a sensory function for most of the CGRP-immunoreactive nerves in the rat urogenital tract.

Immunohistochemical localization of smooth muscle myosin in normal human tissues.

Immunohistochemical localization of smooth muscle myosin, an immunologically distinct contractile protein, was achieved using rabbit anti-human uterine smooth muscle myosin antibodies. In immunodiffusion studies and in cryostat sections, these antibodies were highly specific and reacted with smooth muscle myosin but not with platelet, skeletal muscle, or cardiac muscle myosin. To evaluate comprehensively the structural profile of smooth muscle elements in normal human tissues, an indirect immunoperoxidase technique (peroxidase-antiperoxidase) was applied to a wide variety of specimens. Parallel studies comparing cryostat sections with fixed (10% formalin, B5, Bouin's, or Zenker's solution) paraffin-embedded tissues revealed optimal immunoreactivity, sensitivity, and specificity of staining for smooth muscle myosin using frozen tissues. Strong immunoreactivity was present in muscular tissues such as blood vessels and the muscularis of gastrointestinal and genitourinary tracts. Distinct delineation of smooth muscle elements, including individual smooth muscle cells, and their specific patterns of alignment and organization, were observed, e.g., cells comprising the muscularis mucosae and extending into the lamina propria of the gastrointestinal tract, and myoepithelial cells of skin, exocrine glands, and breast. This method provides excellent morphologic preservation and readily permits unambiguous identification of individual cells containing smooth muscle myosin.

Dermorphin-like immunoreactivity in guinea pig and rat stomach.

Dermorphin is an opioid peptide containing one D-amino acid isolated from amphibian skin. We examined the presence of dermorphin-like immunoreactivity (DMP-IR) in mammalian brain and tissues using an antiserum developed in our own laboratory which works at a dilution of 1:120,000 for Radioimmunoassay (RIA) and can detect 2.1 pg of dermorphin. We were unable to find DMP-IR in guinea pig, rat and toad brain and rat and toad spinal cord either extracted with methanol or with HCl. The guinea pig and rat stomach contained significant amounts of DMP-IR (228.9 pg/mg protein and 97.5 pg/mg protein respectively) when extracted with HCl but not with methanol. The DMP-IR we detected in the stomach is not dermorphin per se. A single peak of DMP-IR was found on Sephadex G-25 gel filtration. When re-chromatographed on reverse phase HPLC, this peak of DMP-IR gave rise to three peaks of DMP-IR.

Repeated application of first-layer antiserum improves immunofluorescence staining: a modification of the indirect immunofluorescence staining procedure.

Repeated application of the first-layer antiserum in the indirect immunofluorescence technique considerably improves the immunostaining. The modified method reveals more antigenic sites and increases the contrast between specific and background stainings, particularly where sparsely distributed antigenic areas are to be investigated. The effect of this novel immunostaining procedure is compared with that of the routine procedure and of other modifications. Possible mechanisms for the improving effect are discussed. A procedure of combined modifications is recommended for exploration of non-abundant antigens or for achieving a high-quality photograph.

The secretory immunoglobulin system in urothelial neoplasia.

The role of the secretory immunoglobulin system in urinary tract neoplasia has been investigated by staining formalin-fixed, paraffin-wax embedded sections of normal and abnormal urinary tract epithelium for secretory component (SC), IgA and J (joining) chain. Normal mucosa contains IgA with J chain and SC as a thin superficial layer on surface umbrella cells and in small amounts in the cytoplasm of intermediate cells. Inflamed mucosa is infiltrated by many IgA and J chain positive plasma cells in the lamina propria and there is IgA, SC and J chain in the cytoplasm of the second layer of the epithelium as much as in surface umbrella cells. Epithelium with moderate to severe dysplasia and invasive transitional cell carcinoma is almost always negative, containing only occasional cells with cytoplasmic IgA, J chain and SC, whilst surface staining is entirely absent.

Autoradiographic demonstration of binding sites for oestradiol and dihydrotestosterone in the urinary tract of male and female baboons.

By light microscopic autoradiography, the kidneys, ureters and urinary bladder of male and female baboons were examined in an effort to define which cells in these three organs were targets for oestradiol (E2) and dihydrotestosterone (DHT). In the parenchyma of the kidney, there was no specific uptake of either 3H-E2 or 3H-DHT, whereas the cortical and medullary connective tissue cells sequestered only 3H-E2. The latter hormone was also found in cells comprising the tunica intima and tunica media of the renal, interlobar and arcuate arteries. The two radiolabelled steroids were concentrated in connective tissue cells of the lamina propria of the ureter and bladder and in the tissue adjacent to smooth muscle fasciculi. Only 3H-DHT was retained by the smooth muscle in these two organs. These observations indicate that specific steroid binding sites are present in the urinary system of the baboon. Their role in the physiology of the kidney, ureter and urinary bladder remains at this point unclear.