Combined biochemistry and histocytochemistry as a tool to investigate Ecto‐ATPase in the cardiac muscle

Ecto‐ATPase (ecto‐adenosine triphosphatase), a key enzyme of cardiac metabolism, is responsible for modulation of the concentration of extracellular nucleotides in the heart. We present methodology consisting of the combined use of biochemical and histocytochemical techniques to study its properties. Using samples from essentially the same preparation, we applied biochemistry and histocytochemistry to determine biochemical characteristics of ecto‐ATPase and an in situ localization of its reactivity. Our results indicated that detected enzyme resists fixation, depends on divalent ions, and hydrolyzes ATP, but not AMP or ADP‐β‐S. Reaction product of the enzyme activity was found confined to the extracellular surface of the plasma membrane of cardiac myocytes and endothelial cells due to the corresponding orientation of the enzyme active sites. Experiments using an inhibitor justified specificity of the reaction. When used together with molecular biological and immunocytochemical techniques, the present methodological approach should be capable of yielding important information about the actual ability of ecto‐ATPase to operate. Microsc. Res. Tech. 58:427–431, 2002. © 2002 Wiley‐Liss, Inc.

[1]  G. Vassort Adenosine 5'-triphosphate: a P2-purinergic agonist in the myocardium. , 2001, Physiological reviews.

[2]  B S Khakh,et al.  International union of pharmacology. XXIV. Current status of the nomenclature and properties of P2X receptors and their subunits. , 2001, Pharmacological reviews.

[3]  R. Evans,et al.  Comparison of P2X receptors in rat mesenteric, basilar and septal (coronary) arteries. , 2000, Journal of the autonomic nervous system.

[4]  Toshihiro Kobayashi,et al.  Ecto-ATPase activity in the rat cardiac muscle: biochemical characteristics and histocytochemical localization , 1999, Cell and Tissue Research.

[5]  A. Knowles,et al.  Ectonucleotidases of avian gizzard smooth muscle and liver plasma membranes: a comparative study. , 1999, Archives of biochemistry and biophysics.

[6]  G Burnstock,et al.  Receptors for purines and pyrimidines. , 1998, Pharmacological reviews.

[7]  T. M. Smith,et al.  Immunological detection of ECTO‐atpase in chicken and rat tissues: Characterization, distribution, and a cautionary note , 1998, Biochemistry and molecular biology international.

[8]  Toshihiro Kobayashi,et al.  Diethyl Pyrocarbonate Is an Inhibitor of Cardiac, Intestinal and Renal Ecto-ATPase , 1997 .

[9]  A. Marcus,et al.  The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39. , 1997, The Journal of clinical investigation.

[10]  D. Candinas,et al.  Loss of ATP Diphosphohydrolase Activity with Endothelial Cell Activation , 1997, The Journal of experimental medicine.

[11]  H. Zimmermann BIOCHEMISTRY, LOCALIZATION AND FUNCTIONAL ROLES OF ECTO-NUCLEOTIDASES IN THE NERVOUS SYSTEM , 1996, Progress in Neurobiology.

[12]  W. Stühmer,et al.  Molecular cloning and functional expression of a novel rat heart P2X purinoceptor , 1996, FEBS letters.

[13]  L. Plesner Ecto-ATPases: identities and functions. , 1995, International review of cytology.

[14]  H. Zimmermann Signalling via ATP in the nervous system , 1994, Trends in Neurosciences.

[15]  G. Dubyak,et al.  Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. , 1993, The American journal of physiology.

[16]  W. Frederiks,et al.  Cerium methods for light and electron microscopical histochemistry , 1993, Journal of microscopy.

[17]  A. Megías,et al.  Transverse tubule Mg(2+)-ATPase of skeletal muscle. Evidence for extracellular orientation of the chicken and rabbit enzymes. , 1991, The Journal of biological chemistry.

[18]  J. Robinson,et al.  Localization of cerium-based reaction products by scanning laser reflectance confocal microscopy. , 1990, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[19]  H. Seguchi,et al.  Cerium-based cytochemical method for detection of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity at physiological pH. , 1987, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[20]  C. Hulstaert,et al.  Decreased ATPase activity in adriamycin nephrosis is independent of proteinuria. , 1987, Kidney international.

[21]  J. Robinson Improved localization of intracellular sites of phosphatases using cerium and cell permeabilization. , 1985, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[22]  K. Gable,et al.  Characterization of the membrane bound Mg2+-ATPase of rat skeletal muscle. , 1983, Biochimica et biophysica acta.

[23]  G. Burnstock,et al.  Studies on the stereoselectivity of the P2‐purinoceptor , 1983, British journal of pharmacology.

[24]  J. Williamson,et al.  Release of macromolecular markers (enzymes) from liposomes treated with antibody and complement. An attempt at correlation with electron microscopic observations. , 1973, Biochimica et biophysica acta.

[25]  H. Belle Kinetics and inhibition of alkaline phosphatases from canine tissues. , 1972 .

[26]  S. Karasaki Subcellular localization of surface adenosine triphosphatase activity in preneoplastic liver parenchyma. , 1972, Cancer research.

[27]  Theodor Bücher Über ein phosphatübertragendes gärungsferment , 1947 .