Adenosine triphosphate hydrolysis in rat dental tissues

Histochemistry 47, 303 314 (1976) Histochemistry �9 by Springer-Verlag 1976

Adenosine Triphosphate Hydrolysis in Rat Dental Tissues A Histochemical Study to Differentiate the Enzymes Involved

Hfikan M6rnstad and Bengt SundstrSm Department of Oral Histopathology, School of Dentistry, Carl Gustav V/ig 34, University of Lurid, Malm6, Sweden

Summary. The purpose of this study was to try to differentiate histochemically between the various enzymes which may catalyze the hydrolysis of ATP in developing rat dental tissues. Freeze cut and freeze dried sections of molar and incisor teeth were incubated in lead capture-based media at pH 5.0, 7.2 or 9.4 with one of the following substrates:/~-glycerophosphate, AMP, ADP, ATP, AMP-PNP and tetrasodium pyrophosphate. To establish the enzymatic nature of the hydrolysis parallel sections were incubated after prior fixation in either formaldehyde or glutaraldehyde.

By comparing the enzymatic stainings obtained with the various substrates and at the different pH:s, it was concluded that ATP can be visibly hydrolyzed in rat dental tissues by alkaline phosphatase (stratum intermedium, apical part of maturation ameloblasts, basal part of all ameloblasts, odontoblasts and subodontoblastic layer), specific ATPase (apical and basal parts of secretory ameloblasts) and ATP pyrophosphatase and/or adenylate cyclase (stratum intermedium, odontoblasts). Acid phosphatase, specific ADPase, 5'-nucleotidase, inorganic pyrophosphatase, 3': 5'-cyclic-AMP-phosphodiesterase and adenylate kinase on the other hand, seem not to be engaged in the ATP hydrolysis to such a degree as to complicate the interpretation of the histochemical staining. The alkaline phosphatase part of the ATP hydrolysis appeared to be rather insensitive to aldehyde fixation, while the hydrolysis effected by specific ATPase and ATP pyrophosphatase and/or adenylate cyclase was extinguished after fixation with formaldehyde for 4 h or glutaraldehyde for 10 rain.

Introduction

Adenosine triphosphate (ATP) can be dephosphorylated by several enzymes, and this makes enzyme histochemical studies with this substrate especially diffi- cult. Up to now the following enzymes have been reported to be able to dephos- phorylate ATP : alkaline phosphatase (E.C.3.1.3.1), acid phosphatase (E.C.3.1.3.2), 5'-nucleotidase (E.C.3.1.3.5), adenosine diphosphatase (E.C.3.6.1.6), adenosine triphosphatase (Mg, Ca and Na-K-dependent) (E.C.3.6.1.3), adenosine triphosphate pyrophosphatase (E.C.3.6.1.8) and adeny-

304 H. M6rnstad and B. Sundstr6m

late cyclase (E.C.4.6.1.1) (Commission on Biochemical Nomenclature, 1973; Novikoff et al., 1952; Padykula and Herman, 1955; Freiman and Kaplan, 1960). The interpretation of histochemical results may also be complicated by a possible further dephosphorylation of the initial reaction products ADP, AMP, and PPI by adenosine diphosphatase (E.C.3.6.1.6), 51-nucleotidase (E.C.3.1.3.5) and inorganic pyrophosphatase (E.C.3.6.1.1) respectively (Goldfischer, Essner and Novikoff, 1964). The reaction product cAMP may be converted to AMP by the action of 3': 5'-cyclic-AMP phosphodiesterase (E.C.3.1.4.17). Still another complication may be present due to transphosphorylations by adenylate kinase (E.C.2.7.4.3) (Novikoff et al., 1952; Freiman and Kaplan, 1960).

Several methods have been applied histochemically to differentiate between dephosphorylating enzyme activities. The most frequently used method is to compare the histochemical staining patterns obtained with specific substrates. When cross-over reactions occur, i.e. when several specific substrates are found to be hydrolyzed at the same histological site, the use of inhibitors, which extinguish one or more well defined enzyme activities, has been adhered to.

The purpose of this study was to localize histochemically the hydrolysis of ATP in rat dental tissues and to try to distinguish between the enzymes involved. Experiments were made with histochemical media based on lead-capture at acid, neutral and alkaline pH:s and with a range of substrates. The influence of formaldehyde and glutaraldehyde fixation was studied in order to establish the enzymatic nature of the histochemical reactions, and to see whether these fixations could be used for the differentiation of the various enzymes.

Material and Methods Nine-day-old Sprague-Dawley rats were anesthetized by ethyl ether inhalation and then decapitated. The heads were mounted on a chuck with an aqueous solution of carboxymethylcellulose and frozen in a mixture of dry ice and hexane ( - 7 0 ~ C). They were then sectioned sagitally at 20 gm through the upper incisor and/or molar tooth levels and the sections taken up on adhesive tape (Ullberg, 1954~ 1958; Hammarstr6m and Hasselgren, 1974). The sections were subsequently freeze-dried at - 2 0 ~ C and stored at this temperature until used within two weeks.

Every second serial section was fixed in freshly prepared, ice cold solutions of formaldehyde or glutaraldehyde. The fixatives consisted of 2.5 per cent formaldehyde or glutaraldehyde in an 85 mM cacodylate-HC1 buffer pH 7.2, containing 3 per cent glucose. The formaldehyde was prepared by the depolymerization of paraformaldehyde, and the glutaraldehyde (Merck, Darmstadt, W. Germany) was charcoal-shaken and filtered before use. Fixation time was varied from 10 min to 24 h. The unfixed sections were stored in the fixative buffer for corresponding periods of time. After fixation all sections were washed for 30 rain in three changes of an 80 mM tris-maleate buffer, pH 7.2, containing 8 per cent glucose. Sections to be incubated at acid or alkaline pH were subsequently transferred to the same buffer at pH 5.0 or 9.4 respectively.

Unfixed and fixed sections were incubated together in one of the following solutions: (a) 80 mM tris-maleate buffer, pH 5.0, containing 8 per cent glucose, 3 mM lead nitrate,

3 mM magnesium chloride and 3 mM substrate (this solution was prepared by the drop-wise addition of an 0.2 per cent aqueous solution of lead nitrate to the otherwise complete medium).

(b) 80 mM tris-maleate buffer, pH 7.2, containing 8 per cent glucose, 3 mM lead nitrate, 3 mM magnesium chloride and 3 mM substrate (modified from Reik et al., 1970).

(c) 80 mM tris-maleate buffer, pH 9.4, containing 8 per cent glucose, 3 mM lead citrate, 3 mM magnesium chloride and 3 mM substrate (prepared according to Sundstr6m and MSrnstad, 1975).

The following substrates were used: ATP, ADP, AMP (all as sodium salts; obtained from Sigma Co., St. Louis, Mo., USA), sodium-fi-glycerophosphate, tetrasodium pyrophosphate (BDH, Poole, England) and 5'-adenylyl imidodiphosphate (AMP-PNP, sodium salt; ICN Pharmaceuticals Inc., Irvine, Calif., USA). In media containing ATP, NaCI (120 mM) and KC1 (30 mM) were

Adenosine Triphosphate Hydrolysis in Dental Tissue 305

sometimes added for the demonstration of Na-K-dependent ATPase. In other such media CaC12 was substituted for MgC12 in order to demonstrate Ca-activated ATPase. Control media were prepared as the experimental media except that substrates were omitted.

Semi-quantitative evaluation of the staining was made on sections incubated for 30 min at room temperature. For optimal recording of the localization of the reaction products, shorter incubation times were sometimes used. After incubation, the sections were washed in several changes of an 8e mM tris-maleate buffer, pH 5.0, 7.2 or 9.4 respectively, containing 8 per cent glucose, and then in deionized water, before treatment with 1 per cent ammonium sulphide for 1 rain. After repeated washing in deionized water, the sections were mounted in glycerol-gelatin.

The nomenclature proposed by Reith (1970) was used when describing the various functional stages in the enamel organ. Secretory and maturation ameloblasts were distinguished by their heights. Differentiation between transitional and preabsorptive ameloblasts was not possible. Although the stratum intermedium does not persist as a distinct layer after the transitional period, the name was anyhow used to designate the cells most proximal to the maturation ameloblasts.

Results

All incubation media, except the sodium pyrophosphate containing medium at pH 7.2, were free from precipitates during the incubation period. The reaction products were generally distinctly localized. The only exception was, that the

Table 1.

Incubation media

p H C a p t u r e S u b s t r a t e

L e a d ~ - g l y c e r o p h o s

5 . 0 n i t r a t e A T P

~-glycerophos ++

A M P ++

A D P L e a d

7.2 nitrate ATP

A M P ' P N P

N a P P i

~ - g l y c e r o p h o s +++

A M P ++

A D P ++

L e a d A T P ++ 9.4 citr at e

A M P . P N P (+)

N a P P i

D e n t a l h a r d t i s s u e f o r m i n g c e l l s

S t r a t u m O d o n t o - S u b o d o n t c intermed i A m e l o b l a s t s blasts layer

I P r e s e c r S e e r Transi- I Mat i tional rati ) and p r e a b - sorptive

Earlyllate basla p Ibasla~ bas ap bas p

+

+ ++ ++ ++ ++ + ++

+ (++) + + + + + +

(+)

(+) (++)

++ +++ +++ I +++ H-+ + +++

+ (++) ++ ++ ++ ++ + ++

+ ++ (+) ++ ++ ++ + ++

+ ++ (+++) ++ (+) ++ ++ (+) + ++

(+)

(4-) ( + + ~ )

(+++) t (+++)

I C a p i l - S t r i a t e d l a r i e s m u s c l e s i n t e e t

r(+)

(+) (+++) (++) i (+++)

+

+

Early stratum intermedium denotes that part of stratum intermedium which overlies presecretory, secretory, transitional, preabsorptive, and early maturation ameloblasts. Late stratum intermedium denotes that part of stratum intermedium which overlies late maturation ameloblasts bas=basal part of the ameloblast: between the nucleus and the stratum intermedium, ap=apical part of the ameloblast: between the nucleus and the enamel. + denotes weak, + + moderate, + + + strong and + inconsistent histochemical staining. ( ) denotes enzymatic activity extinguished after fixation with formaldehyde for 4 h or glutaraldehyde for i0 min


Fig. 1. Incubation with ATP, pH 9.4 for 20 min. Arrows mark the junctions between, from the left, presecretory, secretory, transitional and preabsorptive, and maturation ameloblasts. Note staining in secretory ameloblasts, x 20

Fig. 2. Incubation with fl-glycerophosphate, pH 9.4 for 10 min. Arrows as in Figure 1. Note absence of staining in secretory ameloblasts, x 20

Fig. 3, Incubation with AMP-PNP, pH 9.4 for 30 min. Arrows as in Figure 1. Note absence of staining in ameloblasts, x 20

Fig. 4. Incubation with fl-glycerophosphate, pH 9.4 for 20 min. Note the double lines in the stratum intermedium-ameloblast junctional area (arrows). Am ameloblasts, x 100

Fig. 5. Incubation with ATP, pH 9.4 for 20 min. Dotted lines mark the junctions between, from the bottom, presecretory, secretory, transitional and preabsorptive, and maturation ameloblasts. x 30

Fig. 6. Incubation with ATP, pH 9.4 for 10 min showing strong staining in the secretory ameloblasts (between arrows). E enamel, D dentine, x 100


Fig. 7. Incubation with AMP, pH 9.4 for 20 min. Dotted lines as in Figure 5. x 30

Fig. 8. Incubation with ATP, pH 9.4 for 30 rain after the treatment of the section for 10rain With glutaraldehyde. Only structures which also are stained with fi-glycerophosphate are seen. Dotted lines as in Figure 5. x 30

Fig. 9. Incubation with ATP, pH 7.2 for 30 rain. Dotted lines as in Figure 5. • 30

Fig, 10. Incubation with ADP, pH 7.2 for 30 rain. Dotted lines as in Figure 5. x 30

Fig. 11. Incubat ion with AMP, pH 7.2 for 30 rain. Dotted lines as in Figure 5. x 30

Fig, 12. Incubation with ATP, pH 7.2 for 30 rain after the treatment of the section for 10 min with glutaraldehyde. No enzymatic staining. Dotted lines as in Figure 5. x 30


staining obtained with the sodium-fi-glycerophosphate medium at pH 5.0 was rather diffuse. Nuclear staining was consistently slight and substrate-free media showed staining of calcified structures only.

The results of the various incubations are summarized in Table 1 and illus- trated in Figures 1-12.

Exchange of Ca 2 + for Mg 2+ , or the addition of Na + and K + to the Mg-ATP- media did not change the staining pattern.

Aldehyde fixation gave an overall grey-brownish tint to the specimens, and always affected the intensity of the histochemical staining. The staining observed with sodium-fl-glycerophosphate at pH 7.2 and 9.4, and the staining of the stratum intermedium, the basal part of all ameloblasts, the apical part of the maturation ameloblasts and the subodontoblastic layer with AMP, ADP and ATP, were only slightly reduced, even after fixation for 24 h. Additional stainings observed with AMP, ADP and ATP were completely extinguished after fixation with formaldehyde for 4 h or with glutaraldehyde for 10 min. Sections incubated with the sodium-fi-glycerophosphate medium at pH 5.0 showed no apparent loss of enzymatic activity following fixation, whereas when using ATP at this pH blood vessel staining was extinguished. No staining was observed with AMP- PNP after fixation.

Discussion

The structural localization of adenosine triphosphate hydrolysis demonstrated in this study differs in one particular respect from what has been reported by most earlier authors (Burstone, 1960; Heyden and From, 1969, 1970; Heyden, 1969, 1970a, b; Severson, 1968, 1971; Magnusson, Heyden and Arwill, 1974; Magnusson, 1974; Magnusson and Linde, 1974). We have thus found ATP hydrolysis in the apical part of secretory ameloblasts. It is uncertain whether this localization was observed by Pourtois (1962a, b, 1963). However, differen- cies in techniques and stages of ameloblast function investigated may explain this discrepancy. The fact, that the addition of Na + and K § at incubations with ATP did not change the staining pattern should be expected, as the Na +- K+-dependent ATPase is inhibited by lead (Tormey, 1966; Jacobsen and Jor- gensen, 1969). The problem of differentiating Mg 2+- and Ca2+-activated ATPases in non-decalcified sections of dental tissues has been discussed earlier by Magnusson, Heyden and Arwill (1974) and Magnusson (1974).

In order to approach a qualitative interpretation of the observed staining reactions, i.e. in our study to determine which enzymes are responsible for the ATP hydrolysis, we have primarily chosen to use several different substrates. Before evaluating the similarities and dissimilarities between the stainings obtained with these substrates and ATP, each individual staining pattern will be briefly discussed.

The localization of sodium-fi-glycerophosphate hydrolysis at neutral and alkaline pH:s, interpreted as the activity of alkaline phosphatase (E.C.3.1.3.1), is the same as has been reported by several other authors using a variety of histo- and cytochemical techniques (see e.g. Burstone, 1960; Leonard and


Provenza, 1973; Magnusson, Heyden and Arwill, 1974). The staining in the stratum intermedium-ameloblast junctional area in our sections sometimes appeared as two distinct lines, and we have tentatively ascribed the apical one of these lines to the basal part of the ameloblasts. This would be in accordance with the electron microscopic finding of Leonard and Provenza (1973).

Our observation at acid pH, that fi-glycerophosphate hydrolysis could be seen in the apical part of the secretory ameloblasts and in odontoblasts is in accordance with the results of e.g. Kurahashi et al. (1967), using the Gomori (1956) lead capture method and Hammarstr6m et al. (1971), using a simul- taneous coupling azo dye method. The diffuse staining observed at acid pH is probably caused by interaction between the lead capture and released phosphate from the mineralized tissues.

AMP and ADP-hydrolysis appeared at the same sites as shown earlier by Magnusson, Heyden and Arwill (1974) using a calcium capture technique.

The hydrolysis of AMP-PNP is considered specific for adenylate cyclase (Rodbell et al., 1971), and the substrate has been utilized cytochemically to demonstrate adenylate cyclase activity as distinct from other ATPases in a variety of tissues (Howell and Whitfield, 1972; Jande and Robert, 1974; M6rnstad and Sundstr6m, 1974). By raising the pH from 7.2 to 9.4, it was now possible to demonstrate hydrolysis of this substrate also at the light microscopical level. In the enamel organ, the localization of the staining was in agreement with that earlier reported by us cytochemically at pH 7.2 (M6rnstad and Sundstr6m, 1974). However, the reliability of this technique has recently been questioned by Lemay and Jarett (1975) on the grounds, that adenylate cyclase is totally inhibited by concentrations of lead higher than 0.5 mM. It may then be, that the observed staining reflect adenosine triphosphate pyrophosphatase. The difference in enzymatic activity on ATP or ATP-PNP by adenylate cyclase and adenosine triphosphate pyrophosphatase is that the former enzyme, after the release of inorganic pyrophosphate, cyclisizes the residual AMP to cAMP, whereas the latter enzyme does not. Histochemically these two enzymes can not be distinguished, because it is the relased phosphate (PP~ or PNP) which-trough lead capture-underlies the staining.

The absence of inorganic pyrophosphate hydrolysis light microscopically in dental tissues is in agreement with the study by Magnusson, Heyden and Arwill (1974). These results are, nevertheless, surprising because the presence of inorganic pyrophosphate hydrolysis has been demonstrated biochemically in dental tissues (W61tgens, Bonting and Bijvoet, 1970; Fred6n, Linde and Magnusson, 1975), and the responsible enzyme has been held to be identical with alkaline phosphatase. Larsson (1974), in an apparent agreement with the biochemical data, demonstrated inorganic pyrophosphate hydrolysis in developing dental tissues at the cytochemical level.

When comparing the staining patterns obtained with ATP and fi-glycerophosphate at pH 9.4, it is obvious, that alkaline phosphatase (E.C.3.1.3.1) is responsible for a substantial part of the ATP hydrolysis in the stratum intermedium, the basal part of all ameloblasts, the apical part of maturation ameloblasts, odontoblasts and the subodontoblastic layer. If this pattern is substracted from the ATP-pattern, only the activity in the apical part of the secretory ameloblasts


is left in the dental tissues to be ascribed to a specific ATPase (E.C.3.6.1.3). This conclusion is naturally limited by the fact, that histochemical enzyme studies rely upon the appearance of visible staining obtained under defined experimental conditions. All cells are provided with ATPase activities in e.g. mitochondria and plasma membranes, and what is histochemically demonstrated primarily reflects strong, and not total, enzyme activity.

Incubations with ATP at pH 7.2 again showed hydrolysis in the apical part of the secretory ameloblasts. In addition the basal part of secretory ameloblasts was also stained. The absence of staining in the stratum intermedium and the subodontoblastic layer under these conditions makes it probable that the ATP hydrolysis in the basal part of the ameloblasts is due to a specific ATPase and not to alkaline phosphatase.

Specific ATPase with alkaline pH optima may be present in structures, which also show alkaline phosphatase activity. Many such cross-over reactions are connected with the limited resolution of the light microscope, and electron microscopical studies will probably help to separate enzyme activities on the basis of dissimilar subcellular localization. Ultimate problems will be encoun- tered in those structures which possess more than one enzyme activity, with similar pH optimum, at the same subcellular site. To solve this problem at the light microscopical level many authors (Severson, 1968, 1971 ; Heyden and From, 1969, 1970; Magnusson, Heyden and Arwill, 1974; Magnusson and Linde, 1974) have applied special tests and inhibitors to exclude alkaline phosphatase activity. In summary these authors have reported specific ATPase activity in the cells of stratum intermedium, the basal part of secretory ameloblasts and in the odontoblasts. These locations harbour numerous mitochondria, and the recorded ATPase activities may represent mitochondrial ATPase.

As acid phosphatase activity is present in the apical part of secretory ameloblasts, ATP hydrolysis in this place at higher pH:s might be thought of as representing tail activity of acid phosphatase (Novikoff, 1958). Absence of ATP hydrolysis at acid pH, however, indicates a very low interference of this enzyme on the ATP-hydrolysis even at acid pH, and any tail activity at pH: s above neutrality should therefore be insignificant when lead-methods are used.

As stated in the introduction, ATP hydrolysis may be directly catalyzed by 5'-nucleotidase and ADPase. Such reactions, however, seem not to interfere with the interpretation of ATP hydrolysis in dental tissues. Specific 5'-nucleotidase activity, as distinct from nonspecific hydrolysis of AMP by alkaline phosphatase, was recorded in the cytoplasm of presecretory ameloblasts, the stellate reticulum and the outer enamel epithelium, i.e. in structures showing no ATP hydrolysis. Specific ADPase activity was recorded only in the apical cytoplasm of the secretory ameloblasts, i.e. in this case in a structure which also shows ATP hydrolysis. The ADP hydrolysis is, however, much weaker than the ATP hydrolysis, and it therefore appears as if ADPase in this local is not responsible for the substantial part of the ATP hydrolysis. Substrate specific ADPase activity may be expected to be present in the well developed endoplasmatic reticulum of the secretory ameloblasts.

AMP-PNP hydrolysis occurred in the stratum intermedium and the odontoblasts, and part of the ATP hydrolysis in these structures may thus be catalyzed


by ATP pyrophosphatase and/or adenylate cyclase. The absence of AMP-PNP hydrolysis in the basal and apical parts of secretory ameloblasts, excludes the possibility that the ATP hydrolysis in these structures is caused by ATP pyrophosphate and/or adenylate cyclase.

When ATP is used as substrate, reaction products may be found at sites even when these sites lack ATPase activity (Goldfischer, Essner and Novikoff, 1964). This can occur when other structures in the tissue (e.g. blood vessels and striated muscles) hydrolyze ATP to liberate sufficient ADP or AMP, and when the site in question has the proper ADPase or 5'-nucleotidase activity. It follows from the discussion of 5'-nucleotidase and ADPase above that this complication does not apply to the interpretation of ATP hydrolysis in dental tissues.

The influence on the recorded stainings by the further hydrolysis of the reaction product PPi can be disregarded, as the intentional use of 3 mM inorganic pyrophosphate as a primary substrate did not cause any staining. Absence of specific 5'-nucleotidase staining after incubations with ATP or AMP-PNP demonstrates, that the action of 3':5'-cyclic-AMP-phosphodiesterase is of no significance for the evaluation of the ATP hydrolysis. Also, the absence of specific AMP and/or ATP staining after incubation with ADP rules out interference by adenylate kinase.

Alkaline phosphatase is generally considered rather insensitive to aldehyde fixation (e.g. Danielli, 1958; Sabatini, Bensch and Barrnett, 1963; Pearse, 1968 ; Geyer, 1969). This enzyme has also often been demonstrated on fixed, ethanol- dehydrated and paraffin-embedded specimens. This study has shown, that alkaline phosphatase in dental tissues also is very resistant to fixation. As regards nucleoside phosphatases, these have been reported to show a variable inhibition by aldehyde fixation in other tissues (Sabatini, Bensch and Barrnett, 1963; Torack, 1965; Guth and Samaha, 1969; Ernst and Philpott, 1970; Koenig and Vial, 1973). In dental tissues, specific nucleoside phosphatase appear very sensitive to fixation. Treatment with these common fixatives thus seems to be a way to display alkaline phosphatase activity in dental tissues uncontaminated by specific nucleotidase phosphatase activities. Treatments with -SH compounds (Heyden, 1969, 1970a, b; Heyden and From, 1969, 1970), levamisole and R 8231 (Magnusson and Linde, 1974) or EDTA (Severson, 1968, 197I), on the other hand, retain specific nucleoside phosphatase activities and extinguish alkaline phosphatase activity. A combination of fixation and treatment with these latter chemicals would then inhibit all ATP hydrolysis.

As with the nucleoside phosphatases, ATP pyrophosphatase and/or adenylate cyclase were readily inhibited by fixation. Acid phosphatase, in contrast to the study by Hanker et al. (1972), was apparently unaffected by fixation. This discrepancy may be explained by the fact that the staining obtained with the lead method showed severe diffusion artifacts.

Lead capture-based media for the histochemical demonstration of ATP and AMP-PNP-hydrolyzing enzymes have been criticized on the grounds that lead ions can give rise to a non-enzymatic hydrolysis of these substrates (c.f. Ernst, 1972; Lemay and Jarett, 1975). This criticism has especially been applied to the lead medium described by Wachstein and Meisel (1957), which contains


a low substrate-lead ratio and a high concentration of magnesium. Our ATP- media are close to that proposed by Jacobsen and Jorgensen (1969) in which non-enzymatic, lead catalyzed ATP-hydrolysis is greatly reduced. Furthermore, absence of specific nucleoside phosphatase activity after fixation would generally be interpreted to favour an enzymatic nature of this activity in unfixed sections.

In conclusion, it appears that ATP can be visibly hydrolyzed in dental tissues by alkaline phosphatase, specific ATPase and ATP pyrophosphatase and/or adenylate cyclase. Acid phosphatase, 5'-nuc.leotidase, specific ADPase, inorganic pyrophosphatase, 3':5'-cyclic-AMP-phosphodiesterase and adenylate kinase are not engaged in the ATP hydrolysis to such a degree as to complicate the interpretation of the histochemical staining.

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Received December 9, 1975

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Adenosine triphosphate hydrolysis in rat dental tissues