Summary
To show adenylate cyclase (AC) activity in rat calvaria, it is necessary first to decalcify the specimen. In hard tissues, several enzymes (adenosine triphosphatase (ATPase), alkaline phosphatase (APase), adenylate cyclase (AC) and perhaps pyrophosphatase (PPiase) are able to degrade adenosine triphosphate (ATP). The presence of sodium fluoride (NaF) in the incubation medium reduces the quantity of precipitate formed, compared to that observed using a NaF-free incubation medium. Levamisole, used under the same conditions, gives similar results. Possibly NaF inhibits pyrophosphohydrolase and/or phosphatases which mask the AC activity. Adenylylimidophosphate (AMP-PNP), which is a specific AC substrate, confirms the results obtained with ATP. AC activity is demonstrated cytochemically in the osteoblast and preosteoblast membranes, at the junction between two osteoblasts and along the cytoplasmic processes of the osteoblast which penetrate into the osteoid matrix. The osteocytes never show a precipitate, except those which present some osteoblastic features and then only on the membrane facing the osteogenic layer. An intracellular reaction is also evident and is discussed. Parathyroid hormone (PTH) does not reveal new sites of AC activity but increases the quantity of precipitate observed.
Similar content being viewed by others
References
Baykov AA, Bakuleva NP, Nazarova TI, Avaeva SM (1977a) Fluoride inhibition of inorganic pyrophosphatase. II. Isolation and characterization of a covalent intermediate between enzyme and entire substrate molecule. Biochim Biophys Acta 481:184–194
Baykov AA, Artjukov AA, Avaeva SM (1977b) Fluoride inhibition of inorganic pyrophosphatase. III. Dependence on the nature of substrate and metal ion cofactor. Biochim Biophys Acta 481:195–201
Baykov AA, Tam-Villoslado JJ, Avaeva SM (1979) Fluoride inhibition of inorganic pyrophosphatase. IV. Evidence for metal participation in the active center and a four-site model of metal effect on catalysis. Biochim Biophys Acta 569:228–238
Borgers M (1973) The cytochemical application of new potent inhibitors of alkaline phosphatases. J Histochem Cytochem 21:812–824
Chase LR, Fedak SA, Aurbach GD (1969) Activation of skeletal adenyl cyclase by parathyroid hormone in vitro. Endocrinology 84:761–768
Cheng H, Farquhar MG (1976) Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. II. Cytochemical localization within Golgi and ER membranes. J Cell Biol 70:671–684
Cutler LS (1975) Comments on the validity of the use of lead nitrate for the cytochemical study of adenylate cyclase. J Histochem Cytochem 23:786–787
Cutler LS, Rodan SB (1976) Biochemical and cytochemical studies on adenylate cyclase activity in the developing rat submandibular gland: differentiation of the acinar secretory compartment. J Embryol Exp Morphol 36:291–303
Cutler LS, Rodan G, Feinstein MB (1978) Cytochemical localization of adenylate cyclase and of calcium ion. Magnesium ion — activated ATPases in the dense tubular system of human blood platelets. Biochim Biophys Acta 542:357–371
Cutler LS, Christian CP (1980) Cytochemical localization of adenylate cyclase. J Histochem Cytochem 28:62–65
Davidovitch Z, Montgomery PC, Shanfeld JL (1977) Cellular localization and concentration of bone cyclic nucleotides in response to acute PTE administration. Calcif Tissue Res 24:81–91
Dietrich JW, Canalis EM, Maina DM, Raisz LG (1976) Hormonal control of bone collagen synthesis in vitro: effects of parathyroid hormone and calcitonin. Endocrinology 98:943–949
Doty SB (1980) Problems inherent in obtaining the alkaline phosphatase reaction. J Histochem Cytochem 28:66–68
Göthlin G, Ericsson JLE (1973a) Studies on the ultrastructural localization of adenosine triphosphatase activity in fracture callus. Histochemie 35:111–126
Göthlin G, Ericsson JLE (1973b) Fine structural localization of alkaline phosphatase in the fracture callus of the rat. Histochemie 36:225–236
Granström G, Linde A (1976) A comparison of ATP-degrading enzyme activities in rat incisor odontoblasts. J Histochem Cytochem 24:1026–1032
Granström G, Linde A (1977) ATP-ase activity in the odontoblastic layer of rat incisor. Determination with a radiochemical and a colorimetric method. Acta Odontol Scand 35:3–8
Guo MK, Messer HH (1978) A comparison of Ca2+-, Mg2+-ATPase and alkaline phosphatase activities of rat incisor pulp. Calcif Tissue Res 26:33–38
Howell SL, Whitfield M (1972) Cytochemical localization of adenyl cyclase activity in rat islets of Langerhans. J Histochem Cytochem 20:873–879
Jande SS, Robert P (1974) Cytochemical localization of parathyroid hormone activated adenylate cyclase in rat kidney. Histochemistry 40:323–327
Jones SJ, Ness AR (1977) A study of the arrangement of osteoblasts of rat calvarium cultured in medium with, or without, added parathyroid extract. J Cell Sci 25:247–263
Jones SJ, Boyde A (1978) Scanning electron microscopy of bone cells in culture. In: Copp DH, Talmage RV (eds) Endocrinology of calcium metabolism. Proc 6th parathyroid conf. Excerpta Medica, Amsterdam Oxford, pp 97–104
Kempen HJM, de Pont JJHHM, Bonting SL, Stadhouders AM (1978) The cytochemical localization of adenylate cyclase: fact or artifact? J Histochem Cytochem 26:298–312
Korhonen LK, Hämäläinen M, Kaivosoja M (1977) Inorganic pyrophosphatase activity distinct from alkaline phosphatase in rat bone. Clin Orthop 128:332–339
Krstic R (1977) Ultracytochemical localization and comparison of adenyl cyclase activities in pineal bodies of wistar rats and mongolian gerbils. Histochemistry 53:249–255
Kvinnsland S (1979) Adenylate cyclase cytochemistry: a methodological evaluation. Histochem J 11:669–684
Lemay A, Jarett L (1975) Pitfalls in the use of lead nitrate for the histochemical demonstration of adenylate cyclase activity. J Cell Biol 65:39–50
Linde A, Magnusson BC (1975) Inhibition studies of alkaline phosphatases in hard tissue-forming cells. J Histochem Cytochem 23:342–347
Linde A, Granström G (1978) Odontoblast alkaline phosphatases and Ca2 + transport. J Biol Buccale 6:293–308
Luben RA, Wong GI, Cohn DV (1976) Biochemical characterization with parathormone and calcitonin of isolated bone cells: provisional identification of osteoclasts and osteoblasts. Endocrinology 99:526–534
Magnusson BC, Linde A (1974) Alkaline phosphatase, 5′-nucleotidase and ATPase activity in the molar region of the mouse. Histochemistry 42:221–232
Mato M, Uchiyama Y (1978) Studies on the relationship between uptake of biogenic amines in leucocytes and their adenylate cyclase. Acta Histochem Cytochem 11:64–74
Moses HL, Rosenthal AS (1968) Pitfalls in the use of lead ion for histochemical localization of nucleoside phosphatases. J Histochem Cytochem 16:530–639
Parfitt AM (1976) The actions of PTH on bone: relation to bone remodelling and turnover, calcium homeostasis, and metabolic bone disease. Part III of IV parts: PTH and osteoblasts, the relationship between bone turnover and bone loss, and the state of the bones in primary hyperparathyroidism. Metabolism 25:1033–1069
Peck WA, Klahr S (1979) Cyclic nucleotides in bone and mineral metabolism. In: Greengard P, Robison GA (eds) Advances in cyclic nucleotide research, vol 11. Raven Press, New York, pp 89–30
Peck WA, Burks JK, Wilkins J, Rodan SB, Rodan GA (1977) Evidence for preferential effects of parathyroid hormone, calcitonin and adenosine on bone and periosteum. Endocrinology 100:1357–1364
Raible DG, Cutler LS, Rodan GA (1978) Localization of adenylate cyclase in skeletal muscle sarcoplasmic reticulum and its relation to calcium accumulation. FEBS Lett 85:149–152
Ramp WK (1975) Cellular control of calcium movements in bone. Interrelationships of the bone membrane, parathyroid hormone and alkaline phosphatase. Clin Orthop 106:311–322
Reik L, Petzold GL, Higgins JA, Greengard P, Barnett RJ (1970) Hormone-sensitive adenyl cyclase cytochemistry localization in rat liver. Science 168:382–384
Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212
Rodbell M, Birnbaumer L, Pohl SL, Krans HMJ (1971) The glucagon sensitive adenyl cyclase system in plasma membranes of rat liver. V. An obligatory role of guanyl nucleotides in glucagon action. J Biol Chem 246:1877–1882
Schulze W, Krause EG, Wollenberger A (1972) Cytochemical demonstration and localization of adenyl cyclase in skeletal and cardiac muscle. In: Greengard P, Paoletti R, Robison GA (eds) Advances in cyclic nucleotide research, Vol 1. Raven Press, New York, pp 249–260
Schulze W, Hinterberger U, Wollenberger A, Krause E-G, Janiszewski E (1977) Problems of the cytochemical demonstration of adenylate cyclase. Acta Histochem Cytochem 10:371–378
Severson AR (1971) Histochemical demonstration of nucleoside triphosphate hydrolysis in the mouse dentition. Acta Histochem Cytochem 40:86–97
Severson AR, Tonna EA, Paulec M (1967) Histochemical demonstration of adenosine triphosphatase in osteoblasts. J Histochem Cytochem 15:550–552
Smith DM, Johnston CC (1974) Hormonal responsiveness of adenylate cyclase activity from separated bone cells. Endocrinology 95:130–139
Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct Res 26:31–43
Spierto F, Rogler JC, Parker HE (1969) The effect of magnesium and fluoride on bone pyrophosphatase activity. Proc Soc Exp Biol Med 132:568–570
Thomas ML, Ramp WK (1978) Effects of parathyroid hormone on bone alkaline phosphatase and ATPase activities in vitro. In: Copp DH, Talmage RV (eds) Endocrinology of calcium metabolism. Proceedings 6th Parathyroid conference. Excerpta Medica, Amsterdam Oxford, p 377
Tsukahara S, Maezawa N (1978) Cytochemical localization of adenyl cyclase in the rabbit ciliary body. Exp Eye Res 26:99–106
Wadskov S, Søndergaard J, Kobayasi T (1977) Electron microscopic cytochemistry for demonstration of sodium fluoride sensitive adenyl cyclase in normal human epidermis. Acta Derm-Venereol 57:1–5
Walzer C (1978) Fluoride and adenylate cyclase activity in bone tissue studied by electron microscopy. In: Courvoisier B, Donath A, Baud CA (eds) Fluoride and bone. 2nd symposium CEMO. Hans Huber, Bern Stuttgart, pp 22–26
Walzer C, Schönenberger N (1979) Ultrastructure and cytochemistry of the yolk syncytial layer in the alevin of trout (Salmo fario trutta L.) after hatching. I. The vitellolysis zone. Cell Tissue Res 196:59–73
Warshawsky H, Moore G (1967) A technique for the fixation and decalcification of rat incisors for electron microscopy. J Histochem Cytochem 15:542–549
Wong GL, Cohn DV (1974) Separation of parathyroid hormone and calcitonin-sensitive cells from non-responsive bone cells. Nature [New Biol] 252:713–715
Wong GL, Cohn DV (1975) Target cells in bone for parathormone and calcitonin are different: enrichment for each cell type by sequential digestion of mouse calvaria and selective adhesion to polymeric surfaces. Proc Natl Acad Sci USA 72:3167–3171
Yoshiki S, Umeda T, Kurahashi Y (1972) An effective reactivation of alkaline phosphatase in hard tissue completely decalcified for light and electron microscopy. Histochemie 29:296–304
Yount RG, Babcock D, Ballantyne W, Ojala D (1971a) Adenylylimidophosphate, an adenosine triphosphate analog containing a PNP linkage. Biochemistry 10:2484–2489
Yount RG, Ojala D, Babcock D (1971b) Interaction of PNP and PCP analogs of adenosine triphosphate with heavy meromyosin, myosin and actinomyosin. Biochemistry 10:2490–2496
Zull JE, Krug S, Abel D, Caplan AI (1978) Development of parathyroid hormone- and calcitonin-activated adenylate cyclases in embryonic chicken limb and in cultured cells from embryonic chicken limb. Proc Natl Acad Sci USA 75:3871–3875
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Walzer, C. An attempt at localizing adenylate cyclase in rat calvaria. Influence of sodium fluoride and parathyroid hormone. Histochemistry 68, 281–296 (1980). https://doi.org/10.1007/BF00493257
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00493257