Journal of Histochemistry & Cytochemistry

PELLETIER, GEORGES; NOVIKOFF, ALEX B.

doi: 10.1177/20.1.1pmid: 4333663

All five known secretory cell types of the rat anterior pituitary gland display nucleoside diphosphatase (NDPase) activity throughout the endoplasmic reticulum (ER), including the nuclear envelope but not the specialized region of ER at the inner aspect of the Golgi apparatus known as GERL. The functions of the ER diphosphatase are currently unknown. However, speculations concerning its association with glucuronyl transferase may focus on the metabolic roles of the ER in pituitary cells other than those directly related to secretory protein transport. The gonadotrophs have been studied for thiamine pyrophosphatase and acid phosphatase activities as well as NDPase activity. The results suggest that the secretory granules of gonadotrophs arise from GERL and not from the inner element of the Golgi apparatus. The innermost Golgi element of this cell type shows NDPase and thiamine pyrophosphatase activities and appears to be composed, in part at least, of anastomosing tubules. Nucleoside phosphatase activity is also present at the surfaces of all five secretory cell types and between the cells and adjacent blood capillaries.

ERNST, STEPHEN A.

doi: 10.1177/20.1.13pmid: 4333662

The optimal kinetic parameters for the K-dependent, ouabain-sensitive hydrolysis of p-nitrophenyl phosphate by K-nitrophenyl phosphatase, under conditions closely approximating those employed for cytochemistry, were determined in the avian salt gland as a necessary prerequisite for the ultrastructural localization of the enzyme. The enzyme was characterized in 50-µ cryostat sections of paraformaldehyde-fixed tissue, incubated at room temperature in a medium containing 5 mM nitrophenyl phosphate, 10 mM MgCl2, 20 mM SrCl2 and 100 mM Tris-HCl buffer (pH 9.0), either with or without 10 mM KCl. For comparison, parallel assays were conducted in the absence of Sr, the heavy metal salt used to precipitate phosphate for cytochemistry. Enzymatic activity was determined by measuring spectrophotometrically the amount of nitrophenol hydrolyzed. In this system, Sr acts as a pure noncompetitive inhibitor of the enzyme, causing 50% inhibition at 3 mM and 87% at 20 mM. The Km for the enzyme is 4.5 mM. Sr (20 mM) causes an 8-fold reduction in the apparent affinity of the enzyme for Mg but has little effect on K affinity. The sensitivity of the enzyme to ouabain is decreased 50-fold in the presence of 20 mM Sr. The relationship of this enzymatic activity to Na-K-activated adenosine triphosphatase and the application of this defined medium to transport adenosine triphosphatase cytochemistry are discussed.

ERNST, STEPHEN A.

doi: 10.1177/20.1.23pmid: 4333664

A cytochemical procedure is described for the ultrastructural localization of K-dependent, ouabain-sensitive nitrophenyl phosphatase activity in avian salt gland. Cryostat sections (50 µ) of paraformaldehyde-fixed tissue were incubated in a kinetically defined medium containing: 5 mM p-nitrophenyl phosphate, 10 mM MgCl2, 10 mM KCl, 100 mM Tris-HCl buffer (pH 8.5 or 9.0) and 20 mM SrCl2 to precipitate hydrolyzed phosphate. After incubation at room temperature, the sections were treated with Pb(NO3)2 to convert SrPi to PbPi precipitates for visualization in the electron microscope. Reaction product was localized on the cytoplasmic side of the secretory cell lateral and basal plasma membranes. Little, if any, reaction product was associated with the apical surfaces of the secretory cells or with endothelial surfaces of capillaries. Appropriate control experiments indicated that deposition of reaction product was dependent on Mg and K and was sensitive to ouabain. Furthermore, nonenzymatic hydrolysis of nitrophenyl phosphate did not occur in the medium, and deposition of artifactually produced precipitates did not resemble deposition of enzymatically produced precipitates. The relationship of this localization to transport adenosine triphosphatase cytochemistry is discussed, and the physiologic implications of the localization for tracing the route of active Na transport in the salt gland are considered.

BERG, GEORGE G.; LYON, DOROTHEA; CAMPBELL, MARILYN

doi: 10.1177/20.1.39pmid: 4110187

A very rapid localization of nucleoside triphosphate phosphohydrolase activity is given by incubation mixtures containing 2 M chlorides of monovalent cations. An optimal procedure is to fix overnight in cold formaldehyde-calcium, store in gum-sucrose, freeze-section and incubate 4 min or less at 30°C in a solution of 3 mM Na2 adenosine triphosphate (ATP), 2 M KCl, 3 mM MgCl2, and 1 mM Pb(NO3)2 in 24 mM Tris buffer adjusted to pH 7.2 with HCl. In rat intestine, for example, a strong, selective stain was developed in myenteric plexus, blood vessels, lamina propria mucosae and smooth muscle, while paired preparations without 2 M KCl were pale or unstained. The reaction was specific to nucleoside triphosphates and required Mg++. Acceleration by 2 M chloride was also found in the adenosine triphosphate phosphohydrolase reaction given by a single protein fraction (thin disc) in acrylamide gel electropherograms. The histochemical reaction for ATP phosphohydrolase produced residues of lead in formaldehyde-fixed sections, with two-thirds of lead captured as a precipitate of products of enzyme reaction and one-third bound directly to tissue. Only the enzyme-generated precipitate gave an anatomically and chemically selective and time-dependent pattern of staining, while direct binding gave a diffuse and time-independent background stain enhanced by omitting the substrate. Less than 1% of precipitate originated from nonenzymatic breakdown of ATP. Conditions of precipitation were studied. Reaction products were captured by lead as mixed crystals of nucleotides and phosphate. Addition of chloride salts decreased the concentration of lead required to saturate the reaction mixture, and this effect probably accounted for the enhanced histochemical reaction.

BJÖRKLUND, ANDERS; EHINGER, BERNDT; FALCK, BENGT

doi: 10.1177/20.1.56pmid: 4550857

Treatment with HCl induces changes in the excitation spectra of the formaldehyde condensation products of dopamine and noradrenaline that permit the distinction of these two catecholamines on the cellular level. Using histochemical models and tissue sections, these spectral shifts and changes in the ratio of the two main excitation peaks at about 320 and 370 nm have been measured. Acidification of the dopamine fluorophore rapidly leads to the conversion of the quinoidal dihydroisoquinoline fluorophore into its tautomeric nonquinoidal form. The process probably goes to completion within a few seconds following treatment with HCl. The fluorophore remains stable during prolonged acid treatment. The conversion of the noradrenaline fluorophore into its nonquinoidal state is equally rapid; however, the subsequent dehydration to the fully aromatic form is a slower process, and it is apparently not complete even after several minutes, when the rising over-all tissue fluorescence makes measurement increasingly difficult. The results, however, indicate that this characteristic fluorophore is formed from the noradrenaline fluorophore in a high degree also in tissue sections. In the present report, the various states of the catecholamine fluorophores are characterized in more detail, and the evaluation of excitation spectra for the cellular identification of dopamine and noradrenaline, alone or in mixtures, is discussed. A detailed description of the practical performance of the microspectrofluorometric differentiation procedure is given.

KLEIN, RICHARD L.; YEN, SHYUE-SHONG; THURESON-KLEIN, ÅSA

doi: 10.1177/20.1.65pmid: 4621708

The histochemical method employing potassium pyroantimonate in conjunction with electron microscopy has been investigated using carefully controlled preparation techniques and very sensitive atomic absorption analysis of cations. A critique on the reliability and limitations of the method based on test tube and in vitro experiments is given. The method is sensitive to Ca++, Mg++ and Na+ at the 10–6, 10–5 and <10–2M levels, respectively. Under defined conditions a linear ~l:l ratio of cation present to cation precipitated occurs above these levels. Approximate solubility products have been estimated. Under the test conditions, K+ does not precipitate as a pyroantimonate salt, and neither K+ nor OsO4 influences cation precipitaton at physiologic concentrations. Unbuffered, Tris-HCl-buffered and weakly buffered NaHCO3 media at pH 7.2-7.8 give statistically similar results with Na+ precipitation. The pyroantimonate ion can compete with chelators, ethylenedinitrilotetraacetic acid and ethylene glycol bis-N, N'-tetraacetic acid, for divalent cations when employed simultaneously. These chelators effectively remove Ca++ but not Mg++ from embryonic myocardium, and their effects on Na+ and K+ balance are not marked if employed for relatively short periods. Electron micrographic examples of cation precipitates are given in support of certain findings. A brief discussion of the significance of pyroantimonate grain size, the discrepancy between the ratio of intra- and extracellular precipitates and guidelines for the use of the method are included.

VACCA, LINDA; LILLIE, R. D.

doi: 10.1177/20.1.79pmid: 4110188

doi: 10.1177/20.1.85pmid: N/A

In the article "Subpopulations of Blood Lymphocytes Demonstrated by Quantitative Cytochemistry," by Filiberto Giacomelli, Joseph Wiener, Joseph B. Kruskal, Joanna V. Pomeranz and Alden V. Loud, which appeared in the July issue of the Journal (volume 19, no. 7, pp. 426-433, 1971), the formula in the footnote of Table IV on page 432 should read:[See equation in the PDF file]