Journal of Histochemistry & Cytochemistry

YAMASHINA, SHOHEI; BARKA, TIBOR

doi: 10.1177/20.11.855pmid: 4118196

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.

HOWELL, S. L.; WHITFIELD, MARGARET

doi: 10.1177/20.11.873pmid: 4118197

A cytochemical method has been used to investigate the localization of adenyl cyclase activity in A and B cells of isolated rat islets of Langerhans. Adenosine triphosphate was initially utilized as substrate, the pyrophosphate liberated being precipitated by lead ions at its site of production. The specificity of the method was increased by the use of adenylyl-imidodiphosphate as an alternative substrate; this adenosine triphosphate analogue was not hydrolyzed by adenosine triphosphatase but provided an effective substrate for adenyl cyclase. Adenyl cyclase activity, which was found to retain its glucagon and fluoride sensitivity in glutaraldehyde-fixed tissue, was found exclusively and almost uniformly in the plasma membranes of A and B cells. Storage granule membrane, incorporated into the plasma membrane during secretion of the granule content by exocytosis, appeared to be devoid of adenyl cyclase activity.

ACKERMAN, G. ADOLPH; CLARK, MICHAEL A.

doi: 10.1177/20.11.880pmid: 4118198

Normal human blood and bone marrow cells exposed to the pyroantimonate reaction exhibit a selective binding of pyroantimonate to sites associated with the plasmalemma of neutrophilic leukocytes, erythrocytes and certain of their precursors. Chemical distinctions exist between the plasmalemma of different types of blood and bone marrow cells, and major changes in the cationic binding ability of neutrophilic and erythrocytic cells can be correlated with different phases in their differentiation. Plasmalemmal pyroantimonate-osmium-reactive cation is bound with glycoprotein in the erythrocytic elements and to both phospholipid and glycoprotein moieties in neutrophilic cells. The cellular distribution, membrane localization and degree of membrane reactivity obtained with the pyroantimonate-osmium reaction is distinct from that obtained with Thorotrast and ruthenium red.

MARKWALD, ROGER R.; SMITH, WILLIAM N. ADAMS

doi: 10.1177/20.11.896pmid: 4118199

Mucosubstances (MS) were examined in 10½-14½-day embryonic rat hearts utilizing nonaqueous fixatives or formaldehyde vapor-fixed frozen sections hydrated in concentrated solutions of cetylpyridinium chloride. Ribonuclease-resistant, polyanionic sites were limited to the extracellular cardiac jelly, endocardium and fibroblastic cells (cushion tissue) associated with the endocardium. The cardiac jelly and endocardium of day 10½ embryos principally contained a hyaluronic acid-like carboxylated mucosubstance whose alcianophilia at pH 2.5 was abolished by hyaluronidase but was resistant to NaOH extraction and neuraminidase and trypsin digestion. A critical electrolyte concentration of 0.2 M MgCl2 abolished alcianophilia. On days 13½-14½ carboxylated MS were restricted to cushion tissue and partially resisted mild methylation. Sulfated MS were limited to primitive endocardial cells which gave origin to cushion tissue. Dye deposits of aldehyde fuchsin, high iron diamine or Alcian Blue (pH 1.0) were localized on cell surfaces and such staining was prevented by strong (60°C) methylation. Hyaluronidase sensitivity of sulfated MS decreased with gestation. The critical electrolyte concentration varied from 0.5-0.7 M MgCl2 on days 11½-12½ to 0.8-0.9 M MgCl2 after day 12½. The sulfated MS of endocardial cells were preceded by a transitory accumulation of diastase-resistant, periodic acid-Schiff-positive material. Possible roles of MS in normal and abnormal cardiac septation processes are discussed.

HÅKANSON, R.; LARSSON, L.-I.; NOBIN, A.; SUNDLER, F.

doi: 10.1177/20.11.908pmid: 4639929

In all species studied certain endocrine cells of the adenohypophysis exhibited intense fluorescence after treatment with formaldehyde-ozone but only weak fluorescence after treatment with formaldehyde alone. This is characteristic of both tryptamine and peptides with NH2-terminal tryptophan in protein droplet models. Excitation and emission maxima of the fluorophore in the pituitary were very similar to those of authentic tryptamine or tryptophyl peptide fluorophores. However, chemical analysis failed to demonstrate tryptamine in extracts of the pituitary, whereas large amounts of peptides with NH2-terminal tryptophan could be isolated and identified by fluorometry and thin layer chromatography. It is therefore suggested that the formaldehyde-ozone-induced fluorescence in the endocrine cells reflects the presence of tryptophyl peptides rather than tryptamine.

WILKINSON, DAVID I.; GLICK, DAVID

doi: 10.1177/20.11.917pmid: 4629587

In an attempt to clarify the question of whether histidine is stored in the mast cell for coversion to histamine or whether the rate of conversion is rapid enough to prevent accumulation of histidine so that the rate-limiting step is the histidine uptake, it was found that no histidine was demonstrable in rat peritoneal mast cells by either quantitative analysis or paper chromatographic detection. Microadaptation of Hassall's method, based on conversion of l-histidine by histidase to urocanic acid and measurement of the latter by its absorbance at 277 nm, was made to permit determination of histidine in nanogram amounts in the presence of histamine. This adaptation was found reliable when compared with the o-phthalaldehyde method in estimation of l-histidine in serum and in insulin hydrolysate, and then it was applied to analysis of mast cells before and after l-histidine uptake in vitro. The adaptation should be generally useful in microanalysis of l-histidine in histologically and cytologically defined samples.

ORENBERG, ELAINE K.; GLICK, DAVID

doi: 10.1177/20.11.923pmid: 4344838

Adaptations of both the enzymatic radioisotope displacement and the luminescence methods for measurement of adenosine-3',5'-monophosphate in picomole amounts in sub-milligram samples (e.g., fresh-frozen microtome sections of tissue or 10 µl homogenate) were made and applied to establishing the quantitative histologic distribution of adenosine-3',5'-monophosphate in the adrenals of untreated and adrenocorticotropic hormone-treated rats. A sharp peak in concentration of 24 pmoles/mg wet weight (1.3 pmoles/µg protein-nitrogen) was observed in the reticular zone of the untreated rats and the values fell off rapidly in the medulla to a minimum of less than one-third the peak value, and in the fasciculata and glomerulosa to less than one-half. The adrenocorticotropic hormone injections caused a second peak to appear in the outer fasciculata with a height comparable to that in the reticularis, but the concentrations in all zones were elevated. These two peaks were about 30 pmoles/mg wet weight (2 pmoles/µg protein-nitrogen), and the concentrations also fell to a minimum in the medulla, about one-third the peak values, and to relatively low values in inner fasciculata and glomerulosa, almost one-half the peak value.

LILLIE, R. D.; DONALDSON, P. T.

doi: 10.1177/20.11.929pmid: 4118200

The xanthoproteic reaction is accomplished with only 20% HNO3 with glacial acetic acid (HAc) as the solvent; in water 40% HNO3 is required. Tyrosine and 3-mononitrotyrosin readily azo-couple in alkaline solution with fresh p-nitrodiazobenzene in vitro; 3,5-dinitrotyrosine does not. In vitro p-nitrodiazobenzene at pH 8.5 does not couple with histidine, tryptophan or tyrosine after overnight nitration in tetranitromethane (TNM)-pyridine-0.1 N HCl 1:20:40. Histochemical nitration of tissue adequate to prevent the p-diazobenzenesulfonic acid, pH 1-0.02% azure A sequence reaction of hair medulla and arterial elastin of man, dog and rodents can be achieved by 40% HNO3 in glacial acetic acid-acetic anhydride (Ac2O) mixtures. Acetic anhydride should be 10% only, to restrict evolution of brown oxides of nitrogen and prevent undue section losses. Exposures of 4 hr at 3°C to 4:5:1 HNO3:HAc:Ac2O are effective and well tolerated. Nitration is also effectively accomplished by 6-hr 25°C exposures to mixtures of 1% tetranitromethane-pyridine and two volumes of water (pH 8.2) or 0.1 N HCl (pH 6.6). Even 10% TNM in dry pyridine and 2.5% in 16.3% pyridine alcohol (6 hr), 3°C exposures gave only partial tyrosine blockades. The pyridine-water TNM mixtures also prevented the Morel-Sisley tyrosine reaction and, with greater exposures, the postcoupled benzylidene indole reaction of tryptophan as well. Sites reactive to diazobenzenesulfonic acid-azure A after the best nitrations are probably assignable largely to histidine, though the presence of some unblocked tryptophan, purines and other reactive substances must be considered.

LAGUNOFF, DAVID

doi: 10.1177/20.11.938pmid: 4118201

Ruthenium red, at a concentration of 0.005%, stains extracellular but not intracellular mast cell granules. This vital stain can be used to demonstrate that after degranulation with polymyxin B sulfate many granules retained within the domain of the cell have been extruded into the extracellular space. This interpretation is confirmed by electron microscopy. Indirect evidence indicates that some extruded granules may be recovered by the cell and sequestered in an intracellular space not accessible to ruthenium red.

BRODY, M. J.; HÅKANSON, R.; OWMAN, C.; SUNDLER, F.

doi: 10.1177/20.11.945pmid: 4639930