Heterogeneity of apical glycoconjugates in kidney collecting ducts: Further studies using simultaneous detection of lectin binding sites and immunocytochemical detection of key transport enzymes

Summary In the search for a functional role for the polarized glycoconjugates of rat collecting duct epithelial cells, the relation between binding of various lectins and expression of cellular transport enzyme profile of the cells was studied. For this purpose, principal and intercalated cells of rat kidney collecting duct were identified by morphological criteria and by their immunocytochemically determined content of (Na⁺+K⁺)-ATPase and carbonic anhydrase (CA II), respectively. VariousN-acetylgalactosamine-specific lectins such as those fromHelix pomatia andMaclura pomifera revealed heterogeneity among both principal and intercalated cells, whereas -N-acetylgalactosa nine-specific lectin fromDolichos biflorus andVicia villosa bound preferentially to principal cells. Still another lectin fromArachis hypogaea reacted with most collecting duct cells in the cortex and outer medulla, but only with a subpopulation of cells in the inner medulla. Interestingly, some lectins reacted exclusively with the apical aspect of the collecting duct epithelial cells, whereas others revealed both an apical and basolateral distribution of lectin reactive glycoconjugates. The results thus show subtle differences in the glycocalyx structure of principal and intercalated cells and differences in the intracellular polarization of glycoconjugates of these cells. Thus, lectins may be useful tools in the study of the molecular mechanisms which establish and maintain the polarized functions of principal and intercalated cells.     

Immunocytochemistry of ion transport mediators in the genital tract of female rodents

With immunocytochemistry, we have determined distribution of sodium, potassium-adenosine triphosphatase (Na+, K+-ATPase) and of three isoenzymes of carbonic anhydrase (CA) and have shown absence of the chloride channel, Band 3 protein, in the genital tract of female rodents. Staining for Na+,K+-ATPase was strongest in the ampullary oviduct and uterine glands in the mouse. In the mouse and rat ovary, immunostaining evidenced CA I, II, and III in theca interna cells where the enzyme could affect the pH of follicular fluid. The zona pellucida of the ovary and cytoplasmic foci in follicular granulosa cells stained for content of only CA I in mouse ovary, suggesting synthesis of a zona pellucida component by granulosa cells. CA II in mouse oviductal epithelium increased from the negative infundibulum to the variably positive ampulla and isthmus to the uniformly positive interstitial segment. The content of CA III varied inversely to that of CA II. The prevalence of CA II-positive cells apparently corresponded with that of nonciliated cells, whereas abundance of CA III-positive cells concurred with that of ciliated cells in regions of the mouse oviduct. The rat oviduct lacked CA II but, like that of the mouse, showed CA III in the proximal region. The staining for CA II in surface epithelium exceeded the reactivity in glandular epithelium in the mouse uterus, except during estrus. In contrast, rat uterus evidenced CA II in glandular but not surface epithelium. These results testify to possible significance of various ion transport mechanisms for biologic activities of diverse cells in the female genital tract.     

Relationship between concentrations of immunoreactive insulin-like growth factor-I in follicular fluid and various biochemical markers of differentiation in bovine antral follicles

Three experiments were conducted to determine the relationship between concentrations of insulin-like growth factor-I (IGF-I) in ovarian follicular fluid and various biochemical markers of follicular differentiation in bovine follicles. In Experiment I, ovaries were removed on Days 7, 14, 28, 42, or 56 after parturition from a total of 21 cows. In Experiment II, ovaries of 31 cows were removed between Days 20 and 30 postpartum after 48 or 96 h of either saline (0.9% NaCl, 5 ml) or luteinizing hormone-releasing hormone (LHRH, 500 ng/5 ml saline) injections given every 2 h via jugular cannulae. In Experiment III, ovaries of six cows were removed 48-50 h after a 35-mg injection of prostaglandin F2 alpha during the midluteal phase of an estrous cycle. In Experiments I and II, all follicles greater than or equal to 8.0 mm in diameter were removed from each ovary (n = 33 and 46, respectively). In Experiment III, fluid from all follicles greater than 4 mm in diameter were removed individually (n = 10), and fluid from follicles 1-4 mm in diameter were pooled for each cow. Follicles for each experiment were further categorized as either estrogen-active (E-A, concentration of estradiol greater than progesterone in follicular fluid) or estrogen-inactive (E-I, concentration of progesterone greater than estradiol in follicular fluid). Measurements of immunoreactive IGF-I (i-IGF-I) were made after separating IGFs from their binding proteins with an acid-ethanol extraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunolocalization of band 3 protein in normal and cystic fibrosis skin

Current evidence indicates that the defect in cystic fibrosis (CF) involves chloride transport in various epithelial cells. The sweat gland, one site of altered chloride transport in CF, was examined immunocytochemically for localization of a chloride-channel membrane protein, designated band 3 protein. Immunoreactivity was observed in sweat duct cell membranes of both normal and CF samples, whereas secretory coil regions were entirely unreactive. No difference was observed in the pattern or intensity of immunoreactivity between the two groups at the light microscopic (LM) level of resolution.     

Lectin histochemistry of complex carbohydrates in human cervix

Summary Complex carbohydrates in the human cervix were studied histochemically using lectins, conjugated to horseradish peroxidase and correlated procedures. Stratified squamous epithelium of the exocervix and columnar epithelium of the endocervix in some, but not all specimens showed staining for terminal -N-acetyl-d-galactosamine, -d-galactose, -d-galactose and -l-fucose. the staining for -N-acetylgalactosamine and -galactose, the terminal sugars in blood group A and B antigens respectively, corresponded to a large extent with ABO blood type. One exception was the lack of staining for terminal -N-acetylgalactosamine in endocervical secretions in three of nine blood type A patients. A second exception was the staining for terminal -galactose in endocervical secretions in about half of blood type O and A specimens. The type and amount of glycoprotein formed by endocervical columnar cells differed according to location in superficial compared with deep portions of the glands and according to location at the junction with exocervix compared with the more internal regions. Staining of endothelial cells for blood group A and B antigens was confined to subjects of blood type A and B respectively, although three of nine type A specimens showed no lectin reactivity for group, A antigen. Endothelial cells evidenced affinity forUlex europeus I agglutinin demonstrative of fucose in all specimens. Mast cells disclosed lectin affinity consistent with the presence of terminal or internal mannose orN-acetylglucosamine residues. Two blood type O specimens were examined with conjugated lectins at the ultrastructural level. Secretory granules stained for content of terminal -galactose, -galactose and fucose. These results support and concur with biochemical studies of complex carbohydrates in human cervical tissues. They reveal, in addition, the location of the blood group antigens in the human exocervix and endocervix and the marked heterogeneity among endocervical columnar cells in glycoprotein production.     

Histochemical evaluation of secretory glycoproteins in human salivary glands with lectin-horseradish peroxidase conjugates

Paraffin sections of submandibular, sublingual, minor salivary, and parotid glands from ten human autopsy cases were stained with a battery of ten lectins conjugated to horseradish peroxidase. Variable affinity for one or another lectin between mucous cells in a gland evidenced cellular heterogeneity in mucin production. Mucous cells of a given type of gland varied among individuals, but for a single individual appeared markedly but not completely similar from one type of salivary gland to another. The individual variation related, in part, to the ABO blood group and secretor status of the individual. For mucous cells in secretors of blood group A and B all antigens stained strongly for the presence of terminal alpha-N-acetylgalactosamine or alpha-galactose, respectively. Mucous cells in AB secretors contained both antigens, whereas those of O (H) secretors lacked both. Mucous cells of three presumed nonsecretors, two of whom were immature infants and possibly too young to produce ABO antigen, failed to stain. Mucous cells in glands from the presumed nonsecretors, however, revealed a staining pattern consistent with the presence of Lea antigen. Mucous cells of nonsecretors stained with Lotus tetragonolobus agglutinin but not with Ulex europeus I agglutinin, whereas mucous cells of ABO secretors stained with both lectins. This difference in lectin binding indicated that sites reactive only with Lotus tetragonolobus agglutinin contain 1----4 linked fucosyl residues and sites stained by both lectins contain fucose linked 1----2 to the oligosaccharide. Staining of mucous cells of nonsecretors with Pisum sativum agglutinin indicate that either the lectin binds to internal N-acetylglucosamine of Lea substance or the mucous cells contain an N-glycosidic glycoprotein of the type thought to bind this lectin. Serous cells stained less strongly than mucous cells and differed in lectin affinities from one type of gland to another in an individual. Staining of serous cells of a given gland varied markedly among different subjects. This individual variability did not relate to blood group as terminal sugars demonstrative of A or B blood group antigens were not detected in any serous cells. Serous cells in the submandibular glands from the two immature infants were unreactive with all lectin conjugates. Secretions in parotid and submandibular serous cells generally contained a higher content of fucose than those in sublingual serous cells, which contained higher levels of a terminal galactose-sialic acid dimer. Some but not other cells of striated and interlobular ducts of submandibular glands of one subject stained for alpha-N-acetylgalactosamine.     

Lectin cytochemical evaluation of somatosensory neurons and their peripheral and central processes in rat and man

Summary Paraffin sections of the trigeminal nerve root of the rat, and human spinal nerve root and trigeminal ganglion were stained with a battery of lectin-horseradish peroxidase conjugates to localize and characterize glycoconjugate (GC) in situ. In the rat the myelin sheath of the peripheral segment contained GC with sialic acid most probably linked to the penultinate disaccharide galactose(1 4)-N-acetylglucosamine (Gal(1 )-GlcNAc), and complex type N-glycosidic side chains. The myelin sheath in the central segment differed in containing little if any of the GC named above and in containing GC with terminal -Gal linked to N-acetylgalactosamine (GalNAc), terminal GalNAc and fucose. Schwann cells stained for GC with GlcNAc or mannose whereas oligodendroglia stained for GC with the terminal disaccharide Gal-(1 3)-GalNAc and N-glycosidic side chains, especially in presumed Golgi zones, but also in processes continued as the outer myelin sheath. The human myelin sheath in the central segment differed from that of the rat in not staining with lectins specific for fucose and terminal GalNAc. Sialic acid and terminal -Gal were seen in the human central segment but these sugars appeared to bind to astroglial structures rather than to the myelin sheath as in the rat. Astrocytes in both rat and man were stained by two fucose-binding lectins. Several lectins revealed affinity for GC in the neurilemmal sheath, and staining of this structure was stronger in the human specimens. Neurons in the human trigeminal ganglion ranged from unstained to strongly positive for fucoconjugate in cytoplasmic bodies and plasmalemma. Positive ganglion cells gave rise to unmyelinated fibers which also stained for fucoconjugate. Remak fibers and their extensions into the substantia gelatinosa of the human spinal cord stained strongly for content of fucose.The stronger lectin affinity for N-glycosidic core sugars in the peripheral as compared with the central segment suggests that lectins localize P_o protein in peripheral myelin. The reactivity for several sugars in the central segment can possibly be attributed to myelin-associated glycoprotein (MAG) of central myelin, but lectin staining for GalNAc shows in addition a biochemically unrecognized GC with O-glycosidic linked oligosaccharides in myelin. The lectin cytochemistry indicates that the 170 K Dalton glycoprotein with PNA affinity obtained from rat sciatic nerves occurs in nodes of Ranvier.This research was supported by NIH Grants AM-10956, HL-29775 and United Health and Medical Research Foundation of South Carolina, Inc. Grant No. 79     

ULTRASTRUCTURAL LOCALIZATION OF DIALYZED IRON-REACTIVE MUCOSUBSTANCE IN RABBIT HETEROPHILS, BASOPHILS, AND EOSINOPHILS

For ultrastructural localization of acid mucosubstances in rabbit granulocytes, bone marrow and buffy coat specimens were fixed with formalin, glutaraldehyde, or osmium tetroxide, sectioned at 40 µ, and stained with the Rinehart and Abul-Haj solution of dialyzed iron (DI). Heterophils revealed DI staining on the outer surface of the plasma membrane, in the Golgi complex involved in primary granulogenesis, and in primary granules. The intragranular distribution of DI-stained material varied at different stages in the maturation of primary granules. Immature granules of heterophils fixed by any of the three methods contained a peripheral concentric band of DI-positive material; however, fully mature primary granules possessed a core of DI-reactive material in heterophils fixed with osmium tetroxide, but they contained little or no staining in heterophils fixed with formalin or glutaraldehyde. Secondary granules of rabbit heterophils failed to stain with DI. Tertiary granules, observed only in late heterophils, contained distinct DI-positive particles. Basophil granules exhibited intensely DI-stained material distributed in an orderly pattern throughout the granule. In eosinophils, DI staining was localized in the Golgi complex and in the rims of a few immature cytoplasmic granules.     

Human placental cathepsin B1. Isolation and some physical properties

A reproducible procedure for the isolation, from human placenta, of a cathepsin B1 in a homogeneous state, demonstrated by electrophoretic, ultracentrifugal and enzymic criteria, was carried out. The pH optimum was near pH5.5. The placental enzyme catalysed the release of acid-soluble u.v.-dense products from haemoglobin and myoglobin. It was inhibited by heavy metals and several compounds which react with the thiol groups. The optimum temperature was between 37° and 42°C. The molecular weight of the enzyme was calculated to be 24250.