Masando Hayashi

THE CYTOLOGIC DEMONSTRATION OF β-GLUCURONIDASE EMPLOYING NAPHTHOL AS-BI GLUCURONIDE AND HEXAZONIUM PARAROSANILIN; A PRELIMINARY REPORT

The preparation of a new substrate for β-glucuronidase, naphthol AS-BI β-d-glucuronide, has been described. The potassium salt of naphthol AS-BI and acetobromomethyl glucuronate were reacted in ethanol. Unreacted anilide was removed and deacetylation and deesterification were carried out with barium hydroxide. The barium salt of naphthol AS-BI glucuronide was separated and was then converted to free acid. The compound has an elemental analysis which agrees with the theoretical one. It has also been possible to devise a satisfactory cytochemical technique for the in situ demonstration of β-glucuronidase employing sodium salt of naphthol AS-BI glucuronide and hexazonium pararosanilin. The optimal staining reaction was obtained with 0.25 mM substrate and 1.8 mM diazo reagent at pH 5.2 in 20 to 30 minutes at 37°C for rat liver and kidney. The brilliant red dye was visualized at the site of enzyme activity mostly as discrete granules. A brief discussion concerned the evaluation of the present method as a cytochemical technique for the demonstration of β-glucuronidase.

HISTOCHEMICAL DEMONSTRATION OF N-ACETYL-β-GLUCOSAMINIDASE EMPLOYING NAPHTHOL AS-BI N-ACETYL-β-GLUCOSAMINIDE AS SUBSTRATE

The N-acetyl-β-glucosaminide of naphthol AS-BI (7-bromo-3-hydroxy-2-naphth-o-anisidide) was obtained by reacting the anisidide with acetochloroglucosamine in aqueous alkaline acetone. After removal of O-acetyl groups with methanolic ammonia and recrystallization from aqueous methanol, the compound may be used as a substrate for N-acetyl-β-glucosaminidase. Formol-calcium fixed frozen sections were incubated with 0.5 mM substrate in the presence of hexazonium pararosanilin or fast garnet GBC at pH 5.2. Staining appeared in the cytoplasm mostly as discrete granules at the presumed site of enzyme activity and was inhibited in the presence of N-acetylglucosaminolactone. In the rat kidney a number of spherical granules which stained for the enzyme were observed in the proximal portion of the proximal convoluted tubules. In the liver staining was recognized as small granules localized at the pericanalicular cytoplasm of parenchymal hepatic cells. The general cytological localization in these tissues was quite similar to that of other lysosomal enzymes. The limitations of the technique were briefly discussed.

AN IMPROVED METHOD OF FIXATION FOR FORMALIN-SENSITIVE ENZYMES WITH SPECIAL REFERENCE TO MYOSIN ADENOSINE TRIPHOSPHATASE

Studies have been made on the effect of fixatives containing substrate and other additives on the histochemical staining reaction for myosin adenosine triphosphatase activity in the leg muscle, and α-glycerophosphate and succinate dehydrogenase activities in the kidney of the rat. Myosin adenosine triphosphatase was well preserved in the presence of adenosine triphosphate in fixative prepared from methanol-free formaldehyde and buffered to pH 7.2 with cacodylate. Addition of sucrose was found to increase the enzyme preservation. Similar protection of the enzyme activity was afforded by other polyphosphates, particularly sodium pyrophosphate and, to less degree, thiamine pyrophosphate, adenosine diphosphate and uridine triphosphate. α-Glycerophosphate dehydrogenase was also preserved to greater degree by a similar fixative containing α-glycerophosphate than by substrate-free fixative. Succinate dehydrogenase was not significantly preserved in succinate-containing fixative. On the other hand, sucrose in the fixative increased the preservation of succinate dehydrogenase but had no appreciable effect on α-glycerophosphate dehydrogenase.

DISTRIBUTION OF β-GLUCURONIDASE ACTIVITY IN RAT TISSUES EMPLOYING THE NAPHTHOL AS-BI GLUCURONIDE HEXAZONIUM PARAROSANILIN METHOD

The histochemical distribution of β-glucuronidase activity in rat tissues as demonstrated by the naphthol AS-BI glucuronide and hexazonium pararosanilin method has been described. The present method has demonstrated a close correlation between the amount of stain derived from the β-glucuronidase reaction and the reported enzyme activity by homogenate assay in every tissue examined. Tissues known to be enzyme-rich by homogenate assay showed a rapid and intense staining in a great number of cells. These tissues were the preputial gland, spleen, liver, epididymis, ovary, lymph node, thymus, kidney and thyroid. In enzyme-poor tissues, the staining was negative or limited to a small number of specific cells. These tissues were the brain, spinal cord, eye, testis, and muscle. Cells responsible for the intense staining in all tissues examined were those of the macrophage system, parenchymal cells of liver and kidney and several types of mucous membrane cells. In each cell the major staining appeared as discrete and, most often, spherical granules in the cytoplasm, and no staining was observed in the nucleus, Size, number and location of the granules was quite variable depending on the type of cells. Large granules were especially abundant in macrophages. The possible significance of this enzyme distribution is discussed briefly.

COMPARATIVE HISTOCHEMICAL LOCALIZATION OF LYSOSOMAL ENZYMES IN RAT TISSUES

Localization of acid phosphatase, β-glucuronidase and N-acetyl-β-glucosaminidase has been studied in various tissues of the rat with comparable simultaneous coupling azo dye techniques employing naphthol AS-BI compounds as substrates and hexazonium pararosanilin as a capture reagent. These three lysosomal hydrolases were widely distributed in tissues and were generally demonstrated as discrete granules in certain portions of the cytoplasm. Granules stained for each enzyme were localized at identical sites in most, but not all, tissues in which they were present. However, the intensity or number of granules stained for each of the enzymes was found to differ in varying degree among tissues. Acid phosphatase appeared to be more widespread in tissues than the two glycosidases. Distribution of the glycosidases was also found to differ considerably. Cells of the macrophage system, for example, displayed high β-glucuronidase activity, while N-acetyl-β-glucosaminidase was much less active in these cells. The latter enzyme was more active in the epithelium of the mucous membrane. Furthermore, in several tissues, the three enzymes exhibited qualitatively different localization. In these sites, one or two of the enzymes were not or were only faintly demonstrable while the other showed an intense reaction. These findings suggest that a functional differentiation among lysosomes in various tissues may exist.

The Distribution of Abcc6 in Normal Mouse Tissues Suggests Multiple Functions for this ABC Transporter

We have studied the tissue distribution of Abcc6, a member of the ABC transmembrane transporter subfamily C, in normal C57BL/6 mice. RNase protection assays revealed that although almost all tissues studied contained detectable levels of the mRNA encoding Abcc6, the highest levels of Abcc6 mRNA were found in the liver. In situ hybridization (ISH) demonstrated abundant Abcc6 mRNA in epithelial cells from a variety of tissues, including hepatic parenchymal cells, bile duct epithelia, kidney proximal tubules, mucosa and gland cells of the stomach, intestine, and colon, squamous epithelium of the tongue, corneal epithelium of the eye, keratinocytes of the skin, and tracheal and bronchial epithelium. Furthermore, we detected Abcc6 mRNA in arterial endothelial cells, smooth muscle cells of the aorta and myocardium, in circulating leukocytes, lymphocytes in the thymus and lymph nodes, and in neurons of the brain, spinal cord, and the specialized neurons of the retina. Immunohistochemical analysis using a polyclonal Abcc6 rabbit antibody confirmed the tissue distribution of Abcc6 suggested by our ISH studies and revealed the cellular localization of Abcc6 in the basolateral plasma membrane in the epithelial cells of proximal convoluted tubules in the kidney. Although the function of Abcc6 is unknown, mutations in the human ABCC6 gene result in a heritable disorder of connective tissue called pseudoxanthoma elasticum (PXE). Our results demonstrating the presence of Abcc6 in epithelial and endothelial cells in a variety of tissues, including those tissues affected in PXE patients, suggest a possible role for Abcc6 in the normal assembly of extracellular matrix components. However, the presence of Abcc6 in neurons and leukocytes, two cell populations not associated with connective tissue, also suggests a more complex multifunctional role for Abcc6.

Analysis of ABCC6 (MRP6) in normal human tissues

To determine the tissue distribution of the ABC transporter ABCC6 in normal human tissues, we analyzed tissue arrays for the presence of ABCC6 mRNA by in situ hybridization and ABCC6 protein by immunohistochemistry using the polyclonal antibody HB-6. We detected ABCC6 mRNA and protein in various epithelial cells of exocrine and endocrine tissues, such as acinar cells in the pancreas, mucosal cells of the intestine and follicular epithelial cells of the thyroid. We obtained a very strong immunostaining for enteroendocrine G cells in the stomach. In addition, ABCC6 mRNA and protein were present in most neurons of the brain, in alveolar macrophages in the lungs and lymphocytes in the lymph node. Immunohistochemisty using the monoclonal antibody M6II-31 confirmed the widespread tissue distribution of ABCC6. The physiological substrate(s) of ABCC6 are yet unknown, but we suggest that ABCC6 fulfills multiple functions in different tissues. The strong immunostaining for ABCC6 in G cells suggests that it plays an important role in these endocrine cells.

Transcriptional regulation of type X collagen during chondrocyte maturation

The 18-day embryonic chick sternum expresses type X collagen only in the cephalic, presumptive mineralization zone which consists primarily of hypertrophic chondrocytes. The smaller chondrocytes of the caudal, permanent cartilaginous zone do not express this protein. Run-off transcriptions of nuclei isolated directly from cephalic and caudal 18-day embryonic sterna demonstrate that type X collagen is specifically transcribed by cells of the cephalic zone. In addition, type X mRNA is localized to the cephalic zone as detected by in situ hybridization. The results suggest that the cell-specific expression of the type X collagen gene is due entirely to transcriptional regulation.

Müllerian inhibiting substance blocks epidermal growth factor receptor phosphorylation in fetal rat lung membranes

Neonatal males develop respiratory distress syndrome more frequently than females for unknown reasons. The fetal testis secretes testosterone and müllerian inhibiting substance (MIS); MIS has been shown to inhibit fetal lung maturation in vitro and in vivo and to block phosphorylation of epidermal growth factor (EGF) receptors in A431 cells. We hypothesized that MIS would also inhibit membrane phosphorylation of EGF receptors in fetal lung, and that ultrastructural study of MIS-exposed lung might complement the biochemical data by assessing the effect of MIS on tissue morphology. Lung membranes were prepared from 19.5-day fetal rats and phosphorylation assays performed with 3 to 4 micrograms of membrane protein, with or without EGF (26 nmol/L), 0.025 mCi AT32P (0.136 mumol/L), and either recombinant human MIS (rhMIS, 30 pmol) from media of Chinese hamster ovary (CHO) cells, rhMIS dialysis buffer, or wild-type CHO media. The 170,000 molecular weight EGF receptor, visualized by autoradiography of polyacrylamide gels, was phosphorylated in both female and male membranes. rhMIS, when added to EGF-stimulated membranes, caused significant inhibition of EGF receptor phosphorylation (females: 32.42% +/- 11.5%; males: 32.3% +/- 19.1%, P less than 0.001; rhMIS-treated v EGF-stimulated state, P = NS, male v female, Cerenkov counting). Electron microscopy (EM) of rhMIS-exposed lung showed decreased lamellar bodies (LB) in both male alveolar spaces and female parenchyma, and, unexpectedly, increased numbers in female alveoli. Immunoabsorption experiments using coincubation of rhMIS with anti-rhMIS IgG polyclonal antibodies or equiprotein normal IgG demonstrated MIS antibody-specific reversal of rhMIS activity in membrane phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative differences in goblet cells in the tracheal epithelium of male and female rats.

The cause of higher morbidity rates among men than women from diseases of the respiratory tract may be complex and involve several factors, one of which may be structural differences within airways. In an attempt to determine whether such differences exist, 13 male rats and 45 female rats (15 each in proestrous, estrous, and diestrous) were evaluated. Tracheas were fixed in situ by injection of a mixture of formaldehyde and glutaraldehyde. Longitudinal sections cut from paraffin blocks were stained and combined alcian blue (pH 2.5) and periodic acid-Schiff stain to count goblet cells, or with Weigerts hematoxylin and eosin stain to determine epithelial thickness. In the normal rat trachea, cells containing periodic acid-Schiff-positive granules at their apices constituted a major population of goblet cells. The number of these goblet cells present in the trachea was greater in female than in male rats at each stage of the estrous cycle, with the values in the diestrous females closest to the value in the males. Among females, estrous and proestrous rats contained significantly more of these goblet cells than did diestrous animals. The tracheal epithelium of male rats was significantly thicker than that of female rats. These studies demonstrate that the tracheal epithelium differs in male and female rats and varies in the female rat as a function of hormonal cycles. These results raise the question of whether similar differences might be important in the pathogenesis of human disease, and they deserve further clarification in man.