Summary
A further analysis of the ultrastructural localization of a Mg++ dependant neutral ATPase in arteries (thoracic aorta and basilar artery) has been performed in light of recent findings concerning the use of differential fixation and pitfalls in the standard Wachstein-Meisel (W-M) technique. The localization of reaction product was documented following fixation in 5% and 10% formaldehyde and 5% glutaraldehyde, and following incubation in the standard W-M media with ATP, AMP, β-glycerophosphate as substrates. These results were compared to the localization using a modified W-M medium with ATP in which the lead ion concentration was reduced to 1.8 mM. Using the standard W-M procedure, formalin fixation gave a more intense but also a more diffuse (both intra- and extracellular) precipitate of reaction product than glutaraldehyde. The localization to cell structure remained the same in both cases, namely to the outer cell membrane, within its invaginations and in pinocytotic vesicles of both endothelial and smooth muscle cells. Following incubation in a medium with lower lead ion concentration, less extracellular precipitate was found and following glutaraldehyde fixation, very sparse precipitate of reaction product was localized to the cell membrane and its invaginations, often on the cytoplasmic side. The reduction of extracellular precipitate following pre-incubation in 5 mM cystein was believed to be due to inhibition of an unspecific alkaline phosphatase and phosphomonoesterase which had diffused out of the cell following fixation. Cysteine had no effect on the ATPase of the vascular wall. The significance of these results was discussed in light of previous studies on blood vessels and newer insights into this technique.
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References
Ahmed, Z., and I. L. Reis: The activation and inhibition of 5′ nuoleotidase. Biochem. J. 69, 386–387 (1958).
Cambell, W. G.: Localization of adenosine 5′ triphosphatase in vascular and cellular synovium of rabbits. Lab. Invest. 18, 304–316 (1968).
Hoff, H. F.: A comparison of the fine-structural localization of nucleoside phosphatase activity in large intracranial blood vessels and the thoracic aorta of rabbits. Histochemie 13, 183–191 (1968).
—, and J. Graf: An electron microscopy study of phosphatase activity in the endothelial cells of rabbit aorta. J. Histochem. Cytochem. 14, 719–724 (1966).
Lojda, Z., and Z. Zemplenyi: Histochemistry of some enzymes of the vascular wall in experimental rabbit atheromatosis. J. Atheroscler. Res. 1, 101–120 (1961).
Moses, H. L., and A. S. Rosenthal: Pitfalls in the use of lead ion for histochemical localization of nucleoside phosphatases. J. Histochem. Cytochem. 16, 530–539 (1969).
Novikoff, A. B., D. H. Hausman, and E. Podber: The localization of adenosine triphosphatase in liver: in situ staining and cell fractionation studies. J. Histochem. Cytochem. 6, 61–71 (1958).
Padykula, H. A., and E. Herman: Factors affecting the activity of adenosine triphosphatase and other phosphatase as measured by histochemical techniques. J. Histochem. Cytochem. 3, 161–195 (1955).
Rosenthal, A. S., H. L. Moses, D. L. Beaver, and S. S. Schuffman: Lead ion and phosphatase histochemistry. I. Nonenzymatic hydrolysis of nucleoside phosphatases by lead ion. J. Histochem. Cytochem. 14, 698–701 (1966).
Sabatini, D. D., K. G. Bensch, and R. I. Barknett: Preservation of ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19–58 (1963).
Santos-Buch, C. A.: Extrusion of ATPase activity from pinocytotic vesicles of abutting endothelium and smooth muscle to the internal elastic membrane of the major arterial circle of the iris of rabbits. Nature (Lond.) 211, 600–605 (1966).
Torack, R. M.: Adenosintriphosphatase activity in rat brain following differential fixation with formaldehyde, glutaraldehyde and hydroxyadipaldehyde. J. Histochem. Cytochem. 13, 191–205 (1965).
Wachstein, M., and E. Meisel: Histochemistry of hepatic phosphatases at a physiologic pH. With reference to the demonstration of bile canaliculi. Amer. J. clin. Path. 27, 11–23 (1957).
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Hoff, H.F. Further studies on the ultrastructural localization of a magnesium dependant neutral ATPase in arteries. Histochemie 18, 210–216 (1969). https://doi.org/10.1007/BF00306168
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DOI: https://doi.org/10.1007/BF00306168