Tibor Barka

HISTOCHEMICAL METHODS FOR ACID PHOSPHATASE USING HEXAZONIUM PARAROSANILIN AS COUPLER

pimatase activity is largely vitiated by the insperfections of the available histochenmical methods. The sources of error its the lead sulfide nmetimod weme appreciated by Gomori, but this ha.s usot discouraged the uncritical use of this technique in the area of “appbied” histochenmsistmy. ‘[he azo (lye nmethods have suffered fronms two deficiencies : a sui)strate that would yield a hydrolysis iroduct (If low soiubilitv and Imigh substantivity, amsd a capture reagent tisat would couple rapidly ins acid nmedia to productaum ussoluble anmsorphous azo dye. TIme pisos mhate esters of various usapimthob AS derivatives, as developed by Burstone (9), gave imimroved localizatious its freeze-dried material because of the increased l)roteius affinity aumci cxtrenumeby low’ solubibity of the naphtlmobic isycirolysis product. On the other sicica major refineummeust in diazomuiunms coupling tec-immmique followed time introduction of hexazonium b)ararosausilin by Davis and Ornstein (13). ‘[he use of freslsbv diazotizecl pararosanilin w-itim a-msaphthyb pisosphate its a sinmultaneous coupling azo dye method for acid phosphatase (4) resulted in significant improveumment because of the nmaumy desirable characteristics of the final azo dye (1, 2). Several logical msmodificatiomss that conmbimse time advantages of time naphthol AS sui)strates and the isexazonium pararosanibin coupler have givo-us reproducible artifact-free bocabizatioum of acid

Induced cell proliferation: The effect of isoproterenol

Abstract Isoproterenol (IPR) in pharmacologic doses produces enlargement of the salivary glands in rat and mouse. Alterations in weight, DNA-content and incorporation of 3H-thymidine in the submaxillary gland of rat were studied using autoradiographic and chemical techniques during a four-week course of IPR treatment. Within 2 days 80 to 100 mg of IPR markedly stimulated the incorporation of 3H-thymidine into the nuclei of acinar cells of the submaxillary gland and into the nuclei of hepatocytes. Continuous administration of the drug produced lesser peaks of labeling by the 7th and 21st day. The percentage of labeled interstitial and duct cells was the highest after treatment for seven days. The weight of the submaxillary gland significantly increased after the administration of IPR for 4 days and reached its maximum 3.5 times normal, after 2 weeks. The amount of DNA per gland was also significantly higher after 4 days of IPR treatment increasing further up to 14 days. The incorporation of 3H-thymidine into DNA per gland was severalfold above control level from the second day on with a 1255 per cent peak after injections of IPR for 4 days. The specific activity of DNA rose to 8 times the control after only 2 days of IPR treatment. These alterations quickly regressed after withdrawal of the drug. Studies on the turnover rate of DNA indicated reutilization of DNA or its catabolites by the cells of submaxillary gland during IPR treatment. Dichloroisoproterenol caused no alteration of the weight, DNA-content and incorporation of thymidine in the submaxillary gland and it partly prevented the effect of IPR.

Stimulation of DNA synthesis by isoproterenol in the salivary gland

Abstract A single dose of isoproterenol greatly stimulated the incorporation of tritiated thymidine in the submaxillary gland of rat. The maximum of incorporation occurred 24 hr after the administration of the drug. The activities of soluble thymidine kinase and DNA polymerase also rose severalfold of the control level with sharp rises at about 18 hr. The activities of both enzymes returned to resting level in about 48 hr. Actinomycin D, given prior to the administration of isoproterenol or 12 hr after it, inhibited the incorporation of thymidine. It also prevented, partly, the rise in thymidine kinase and DNA polymerase to occur. Under these circumstances there was a good correlation between the incorporation of thymidine and the activities of the two enzymes. The results suggested that the cell proliferation in the salivary gland induced by isoproterenol may be regulated by a number of factors but the triggering mechanism probably acts on a genetic level.

STUDIES OF ACID PHOSPHATASE. I. ELECTROPHORETIC SEPARATION OF ACID PHOSPHATASES OF RAT LIVER ON POLYACRYLAMIDE GELS

The technique of Polyacrylamide gel electrophoresis for acid phosphatases is described. This permits the separation of the soluble acid phosphatases of rat liver into three fractions.

Localization of peroxidase activity in the developing submandibular gland of normal and isoproterenol-treated rats.

The localization of peroxidase activity was studied in the developing submandibular gland of normal and isoproterenol-treated rats using a diaminobenzidine-H2O2 method. During postnatal development peroxidase activity was localized in the proacinar and acinar cells. The proacinar cells were characterized by the presence of polymorphic secretory granules that gave a strong peroxidase reaction, particularly in the glands of 1-day-old rats. In addition to the secretory granules, enzyme activity was demonstrated in the rough endoplasmic reticulum and nuclear envelope. The terminal tubule cells and duct cells were devoid of peroxidase activity. The secretory granules in the fully developed acinar cells revealed little or no enzyme activity. Isoproterenol stimulated the secretion of the peroxidase-positive granules from the proacinar cells. The stimulation was followed by a reaccumulation of peroxidase-positive secretory material. During this process enzyme activity was demonstrable in the Golgi complex. Isoproterenol had no effect on the terminal tubule cells. A less effective depletion of the secretory granules from the proacinar cells was seen after pilocarpine administration. Chronic administration of isoproterenol to 5-day-old rats led to an acceleration of the differentiation of acinar cells and to a hypertrophy of the gland, without significant change in the localization of peroxidase activity.

Beta-adrenergic stimulation of c-fos gene expression in the mouse submandibular gland.

Isoproterenol (IPR), a beta-adrenergic agonist, induces division of acinar cells in the parotid and submandibular glands of adult rodents and produces hyperplastic and hypertrophic enlargements of these organs. We analyzed the effects of IPR on thymidine incorporation, c-fos mRNA levels, and the immunocytochemical localization of c-fos protein in the submandibular glands of adult and of 5- and 14-day-old mice. In the glands of untreated mice c-fos transcripts were not detectable. In all experimental groups, administration of IPR led to a rapid, transient increase in the c-fos mRNA level. Propranolol blocked the IPR effect, while treatment with IPR and cycloheximide led to superinduction. We observed no correlation between the effect of IPR on cell replication or organ growth and stimulation of c-fos expression, and conclude that the latter is the result of beta-adrenergic receptor-IPR interaction. The c-fos protein was localized immunocytochemically in both the cytoplasm and the nuclei of acinar cells and in the nuclei of duct cells.

Electron microscopic alterations of submaxillary gland produced by isoproterenol

Electron microscopic alterations of submaxillary gland of rat were studied 1–24 hours after a single dose of isoproterenol. This sympathomimetic amine stimulates excretion of saliva as well as DNA synthetic and mitotic activity of acinar cells and in chronic experiment produces hypertrophy and hyperplasia of salivary glands. Within 4 hours and following a complete depletion of secretory material, the rough-surfaced endoplasmic reticulum and Golgi apparatus became markedly hypertrophied and ribosomes and polysomes increased in number. Nuclei were swollen and rounded with prominent, enlarged nucleoli. The prominent nucleolus-associated chromatin penetrated into the nucleolus, which revealed well developed nucleolonema. Nuclear and nucleolar dense particles increased significantly. The secretory material was restored in 12–18 hours, and acinar cell structure returned to a resting stage in 24 hours. Actinomycin D given simultaneously with isoproterenol modified the structural response of the gland and produced highly condensed nucleoli. Attempt was made to correlate the morphologic alterations with the physiological and biochemical information available.

ELECTRON HISTOCHEMICAL LOCALIZATION OF ACID PHOSPHATASE ACTIVITY IN THE SMALL INTESTINE OF MOUSE

In formalin-fixed or glutaraldehyde-fixed frozen sections of mouse jejunum acid phosphatase activity is localized electron histochemically to a group of cytoplasmic bodies. These bodies are structurally heterogeneous, sometimes with internal membranous components. The common position for these structures is below the terminal web. Following oil-feeding the number of enzyme-carrying granules is decreased. Enzyme activity occasionally surrounds lipid droplets.

CELLULAR LOCALIZATION OF ACID PHOSPHATASE ACTIVITY

A hsistochcussical survevt of the distribution of acici l)hosPhsnttnm se activity reveals an alnsost universnol occurrence of this cnzvnne in various cell types. However both the intensity and the cytologic localization of the enzynse staining reaction show coussiderabhc variat ion in different locations. An ammahvsis of the relation of cell structures, tlenssoumstrated i:iy acid phosphatase staining, to the lvsosomuues of 1)e I)uve (in Subcelliilar Particles, Anss. Phvsioh. Soc., Washington, I). C., 1959), led to the folloss-ing conclusions: 1) With acid

Immunocytochemical localization of renin in the submandibular gland of the mouse.

Renin was localized in the submandibular gland of the adult mouse at light and electron microscopic levels by the unlabeled antibody enzyme method of Sternberger. At the light microscopic level, renin was confined to the granular convoluted tubule (GCT) segment of the gland with considerable variation among GCT cells in intensity of staining. Some GCT cells failed to stain for renin. The pattern of staining was the same in the gland of male and female mice, but in the glands of females GCT segments were smaller and less numerous. At the electron microscopic level, staining for renin was also confined to the GCT cells, and was localized exclusively to the secretory granules. The intensity of staining of the secretory granules within a given GCT cell varied; some cells contained only minimally reactive or negative secretory granules. All other organelles within the GCT cell, except condensing vacuoles, failed to stain.