Ultracytochemistry of ouabain-sensitive K+-dependent p-nitrophenyl phosphatase in rat incisor enamel organ.код для вставкиСкачать
THE ANATOMICAL RECORD 216~1-9(1986) Ultracytochemistry of Ouabain-Sensitive K+-Dependent p-Nitrophenyl Phosphatase in Rat Incisor Enamel Organ PHILIAS R. GARANT AND TAKAHISA SASAKI Department of Oral Biology and Pathology, School of Dental Medicine, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY 11794-8700 ABSTRACT Sprague-Dawley strain rats of 4-5 weeks old were perfusion-fixed with either a mixture containing 0.1 or 0.25%glutaraldehyde and 2%formaldehyde, or a 2% formaldehyde in 0.1 M sodium cacodylate buffer for 10 minutes. Nondecalcified 30-50-pm sections of the enamel organ taken from lower incisors were then processed for ultracytochemical demonstration of ouabain-sensitive, K -dependent, p-nitrophenyl phosphatase, by use of the one-step lead method, representing the second dephosphorylative step of Na+-K+-ATPase.Throughout the secretory, transition, and maturation stages of amelogenesis, the enzymatic activity was demonstrated along the cytoplasmic side of the plasma membranes of the stratum intermedium and the papillary layer cells, especially along their numerous microvilli. The plasma membranes forming gap junctions and desmosomes were free of reaction or showed slight focal precipitates of reaction products. The stellate reticulum and the outer enamel epithelium exhibited either a weak reaction or were reaction negative. Secretory ameloblasts showed a weak trace-like reaction along the basal and lateral cell surfaces; however, the latter surfaces were sometimes completely free of reaction. Tomes’ processes were usually reaction negative. Ameloblasts in the transition and maturation stages were devoid of enzymatic activity, except for a slight reaction along the plasma membranes of the basal cell surfaces of transition ameloblasts facing the papillary layer. The enzymatic activity described above was completely dependent on the presence of potassium and substrate in the incubation media and was almost completely inhibited by an addition of 10 mM ouabain to the incubation media, + Enamel maturation involves the removal of protein and water from young enamel concomitant with crystal growth to form the hardest substance in the body (Deakins, 1942). It has been demonstrated that a large portion of enamel matrix proteins undergo rapid breakdown and removal after secretion (Seyer and Glimcher, 1977; Glimcher et al., 1977; Robinson et al., 1981; Robinson et al., 1982; Nanci et al., 1985). The removal of matrix breakdown products and water is thought to be under the control of secretory and maturation ameloblasts and the overlying papillary layer cells (Reith, 1970; Reith and Cotty, 1967; Kallenbach, 1966, 1968; Garant and Nalbandian, 1968; Garant, 1972; Josephsen and Fejerskov, 1977; Sasaki, 1984a,b;Nanci et al., 1985). Most investigators of enamel organ structure agree that during enamel maturation the papillary layer and maturation ameloblasts take on the appearance of a transport epithelium (Kallenbach, 1966, 1968; Garant and Nalbandian, 1968; Garant, 1972; Reith, 1970). This suggests that the movement of water and solutes out of the enamel may not be due solely to the permeability and porosity of the enamel organ and the enamel, but also to an active cellular process. No clearly defined hypothesis of how the enamel organ might function as a transporting epithelium capable of 0 1986 ALAN R.LISS, INC. effecting water and solute movement out of the enamel has as of yet been put forth. In this report, we demonstrate the ultrastructural localization of Naf-K+ATPase on the plasma membranes of the stratum intermedium and the papillary layer cells, and we propose that local osmotic gradients created in the enamel organ might provide a driving force to move water and solutes out of the maturing enamel. MATERIALS AND METHODS Sprague-Dawley strain rats, 4-5 weeks old, were anesthetized by an intraperitoneal injection of sodium pentobarbital and fixed by transcardiac perfusion with one of the following fixatives for 10 minutes at room temperature: (1) a mixture containing 2% formaldehyde and 0.25% glutaraldehyde in a 0.1 M sodium cacodylate buffer (pH 7.4); (2) a mixture containing 2% formaldehyde and 0.1%glutaraldehyde in the same buffer; and (3) 2% formaldehyde in the same buffer. After fixation, the fixative was immediately washed out by perfusion with 0.1 M sodium cacodylate buffer for 5 minutes, then the lower incisors were dissected out of the jaws and Received January 8,1986; accepted April 15, 1986. 2 P.R. GARANT AND T. SASAKI ULTRACYTOCHEMISTRY OF Na+-K+-ATPaseIN RAT INCISOR ENAMEL ORGAN placed in a 0.01 M sodium cacodylate buffer containing 0.25 M sucrose and 10% dimethylsulfoxide (DMSO; Sigma Chemical Company), pH 7.4,overnight. DMSO is a selective activator of the K+-stimulated phosphatase and shifts its pH optimum to 9.0 (Albers and Koval, 1972).Nonfrozen sections of the enamel organ of 30-50pm thickness were prepared using a vibratome (Oxford Vibratome Sectioning System) without any decalcification of the tissues. For ultracytochemical demonstration of the enzyme, the sections were incubated for 10 min at room temperature (about ZZOC)in a medium (Mayahara et al., 1980) consisting of 250 mM glycine-KOH buffer, pH 9.0,10 mM p-nitrophenyl phosphate (magnesium salt; Sigma Chemical Company), 25% DMSO, 4.0 mM lead citrate (dissolved in 50 mM KOH), and 2.5 mM levamisole. Final pH was adjusted to 8.8 to 9.0 using 10 M KOH. To ensure substrate specificity, ouabain sensitivity, and K+ dependency, the sections were incubated in one of the following media: (1) standard medium described above lacking substrate (p-nitrophenyl phosphate); (2) standard medium containing 10 mM ouabain; and (3) standard medium, in which K + was replaced by Naf (glycine-KOH buffer was replaced by glycine-NaOH buffer). I n these experiments, we used freshly prepared solutions for incubation, and all incubation media were clear of any precipitates. After incubation, the sections were rinsed with 0.1 M sodium cacodylate buffer (pH 7.4) several times and postfixed with 1.5% potassium ferrocyanide-reduced 1% osmium tetroxide for 30 minutes a t 4°C. They were then block-stained with 1% uranyl acetate in 10% ethanol for 30 minutes at room temperature, dehydrated through a graded ethanol series and propylene oxide, and embedded in Poly Bed 812.To avoid a dislocation of reaction products, tissue processing from fixation to pre-embedding in a 1:l mixture of propylene oxide and Poly Bed 812 were carried out within 2 days. Thin sections were cut using a diamond knife on a n LKB ultratome 111and, without any additional staining, were examined with a JEOL lOOB electron microscope at 60 kV. Fig. 1 . The proximal portion of secretory ameloblasts (Ab) and the stratum intermedium cells (SI). Reaction products of lead phosphates are seen along the plasma membranes of these cells. Inset figure shows a higher magnification of microvilli of the stratum intermedium cells. Precipitates of reaction products are visible along the cytoplasmic side of the plasma membranes but not in the extracellular spaces. The plasma membranes forming gap junctions (GJt and desniosome (D) are reaction negative or show a slight focal reaction. Mt, mitochondria. x 17,000;inset, ~ 4 5 , 6 0 0 . Fig. 2. The outer enamel epithelium (OEE) and adjacent fibroblast F b ) showing no enzymatic reaction. EM, extracellular matrix of surrounding connective tissue. ~ 2 2 , 8 0 0 . Fig. 3. The supranuclear region of secretory ameloblasts. Very slight reaction products are seen along the plasma membranes (indicated by arrows). The Golgi membranes (Go) and secretion granules (SG) are free of reaction. ~ 3 4 , 2 0 0 . Fig. 4. The Tomes process (TP) of secretory ameloblast showing no enzymatic reaction. x 34,200. Fig. 5. The proximal parts of the Tomes processes (TP). The enamel matrix of the interrod growth region contains reaction precipitates (indicated by arrows), but the cytoplasmic side of the plasma membranes is reaction negative. ~28,500. 3 RESULTS Secretory Stage In the enamel organ at the stage of enamel secretion, a n enzymatic reaction was observed mainly along the cytoplasmic side of the plasma membranes of the stratum intermedium cells, especially along their microvilli. Reaction precipitates of lead phosphates were usually adjacent to the plasma membranes or within the subsurface cytoplasm, but were not observed in deeper cytoplasm nor in the extracellular spaces. Gap junctions were usually free of reaction, while desmosomal membranes occasionally showed slight reaction precipitates (Fig. 1). Enzymatic activity along the plasma membranes became weaker towards the stellate reticulum and the outer enamel epithelium, which seldom showed a positive reaction (Fig. 2).Secretory ameloblasts exhibited a weak reaction in the plasma membranes of the basal cell surfaces facing the stratum intermedium cells, and less frequently along their lateral cell surfaces. Cytoplasmic organelles and secretion granules were always reaction negative (Fig. 3). In Tomes’ processes, both the plasma membranes and cytoplasmic inclusions showed no enzymatic reaction. Adjacent newly secreted matrix of rod- and interrod-growth regions sometimes contained scattered reaction precipitates; but the plasma membranes themselves were free of reaction (Figs. 4,5). TransitionStage Between Enamel Secretion and Maturation At the transition stage, the enamel organ consisted of transition ameloblasts and the papillary layer cells. Enzymatic reaction was observed mainly along the cytoplasmic side of the plasma membranes of the papillary layer cells. The plasma membranes forming gap junctions and desmosomes between papillary layer cells and between papillary layer cells and ameloblasts were usually reaction negative. Cytoplasmic matrix and cell organelles such as mitochondria, Golgi membranes, and endoplasmic reticulum were free of reaction (Fig. 6). The papillary layer cells at this stage sometimes contained large phagosomes containing the remnants of various cell organelles; these were also reaction negative (Fig. 7).The transition ameloblasts showed no enzymatic reaction along their plasma membranes, except at the basal cell surfaces facing the papillary layer cells (Figs. 63). Although cell organelles and secretion granules of transition ameloblasts were always free of enzymatic reaction, some membrane-bounded bodies resembling phagosomes contained a considerable amount of reaction product (Fig. 9). Maturation Stage At the stage of enamel maturation, the enamel organ consisted of ruffle-ended and smooth-ended ameloblasts and the papillary layer cells. Many fenestrated capillaries were situated between the epithelial ridges of the papillary layer. A most intense enzymatic activity was found along the cytoplasmic side of the plasma membranes of papillary layer cells, especially along their numerous microvilli (Fig. 10). Gap junctions and desmosomes were reaction negative. Cytoplasmic matrix and cell organelles of papillary layer cells showed little or no enzymatic reaction except in regions subadjacent to the plasma membrane (Fig. 10). Both ruffle-ended and smooth-ended ameloblasts showed no enzymatic reac- 4 P.R. GARANT AND T. SASAKI ULTRACYTOCHEMISTRY OF Na+-K+-ATPaseIN RAT INCISOR ENAMEL ORGAN tion in their cell organelles and along their plasma membranes (Figs. 11,12>.The endothelial plasma membranes of capillaries in the papillary layer were usually reaction negative (Fig. 13, inset). We found little enhancement or reduction of enzymatic activity and no change in enzyme localization among the three different fixatives employed in this study (Figs. 13,141. Control Experiments When p-nitrophenyl phosphate was omitted from the incubation medium, no precipitated reaction products were observed in any cell organelles or plasma membranes of papillary layer cells and other enamel organ cells (Figs. 15,16). Substitution of K+ with Na+ and addition of 10 mM ouabain into the incubation media produced a strong inhibition of the enzymatic reaction (Figs. 17,18). Only slight focal precipitates of lead phosphates were still observed in the subsurface cytoplasm of papillary cells when K+ was substituted for Na+ (Fig. 17). DISCUSSION Biochemically, Mornstad (1978) demonstrated the presence of a ouabain-sensitive, K+-stimulated, p-nitrophenyl phosphatase in the homogenates of the rat incisor enamel organ. This hydrolytic reaction represents the second step of ATP dephosphorylation by Na+-K+ATPase (Skou, 1974; Ernst, 1975). In Mornstad's experiment, only 30% of the enzyme activity was inhibited by ouabain. Furthermore, because of the use of tissue homogenates, it was impossible to associate the enzyme activity with any defined cytological structures. In 1980, Mayahara et al. developed a one-step lead method for the electron microscopic localization of ouabain-sensitive, K f -dependent, p-nitrophenyl phosphatase: i.e., the equivalent of Naf-Kf-ATPase. This is the method we have used to localize Na+-K+-ATPaseat the ultrastructural level in the enamel organ. The present observations demonstrate the site of Na K+-ATPase, and presumably that of a sodium pump, throughout the stratum intermedium and the papillary layer cells, and although somewhat weaker, along the basal surfaces of secretory and transition ameloblasts. Localization of reaction products at the cytoplasmic side of the plasma membranes indicates the internal release of the hydrolyzed phosphates by Na+-K+-ATPaseand, by extrapolation, the transport of sodium across the + Fig. 6.The proximal portion of transition ameloblast (Ab) and adjacent papillary cells (PC). Reaction precipitates are visible along the plasma membranes of these cells excepting gap junctions (GJ) and desmosomes 0).~28,500. Fig. 7. Large electron-dense phagosome in a papillary cell, which contains no reaction products. X 17,000. Fig. 8.The distal portion of transition ameloblasts in the regions close to the maturation ameloblast layer and to the secretory ameloblast layer (inset). Transition ameloblasts in both regions show no enzymatic activity. x 17,000; inset, ~22,800. Fig. 9. The supranuclear cytoplasm of transition ameloblasts. Membrane-bounded lysosomal bodies contain reaction products in their matrix. Inset figure shows a higher magnification view of a lysosomal body containing reaction products and many vesicular and tubular structures. Go, the Golgi membranes. ~28,500;inset, ~57,000. 5 membrane to the extracellular milieu (Ernst, 1975; Mayahara et al., 1980; Ueno, et al., 1984). Considerable evidence has been accumulated in various kinds of epithelial cells that a Na+ pump, namely Na+-K+-ATPase (E.C. 188.8.131.52.), functions in reabsorption, exchange, and active transport of solutes across the plasma membranes CBonting, 1970; Blaustein, 1974; Leuenberger and Novikoff, 1974; Ernst, 1975; Hoshi et al., 1976; Berman et al., 1977; Mills and DiBona, 1977, 1978; Ueno et al., 1984). Water movement is thought to be a secondary consequence of active sodium transport across the plasma membranes (Curren and MacIntoch, 1962; Diamond and Bossert, 1967; Mills and DiBona, 1980). Most epithelia that engage in transport of water and solutes through the active maintenance of Nat pump-generated osmotic gradients possess the barrier of zonulae occludentes across the distal (i.e., luminal) ends of the cells (Claude and Goodenough, 1973). The intercellular barrier at this location may be total or partial (Machen et al., 1972; Martinez-Palomo and Erlij, 1975). In epithelia that are involved in isosmotic reabsorption of water and solutes such as the colon and the gall bladder, the barrier is not fully sealed (Machen et al., 1972). In these leaky epithelia, tracers such as lanthanum penetrate through the zonula occludens junctions, and the intercellular space is a pathway of lowered electrical resistance (Machen et al., 1972). The tall columnar cells that make up these epithelia possess Na+K'-ATPase along their basolateral surfaces (i.e., opposite the end with the zonula occludens junction), and thus are thought to be able to pump Na+ into the basolateral intercellular spaces creating a local osmotic gradient (DiBona and Mills, 1979). Despite the fact that the luminal zonulae occludentes are leaky, gradients are believed to be maintained because of the complexity of the basolateral intercellular space and the amplification of the plasma membrane that borders this space (Welling et al., 1978; Welling and Welling, 1976). In fact, the leakiness of the zonula occludens may account for the high volume of water that moves across these epithelia (Machen et al., 1972). As Na+ is pumped across the basolateral plasma membrane, the intracellular level of sodium is replenished by passive entry of Na+ across the luminal surface of the cell. The structure of the enamel organ possesses some severe problems to a conceptualization of how it might function as a transport epithelium. First of all, the enamel organ is made up of several cell layers. Ameloblasts border onto the enamel and the stratum intermedium, and papillary layers are located between the ameloblasts and the blood supply to the enamel organ (Kallenbach, 1967;Garant and Nalbandian, 1968;Reith, 1970; Garant, 1972). To our knowledge, only frog skin epithelium possessed Na+-K+-ATPase at the inwardfacing membranes, different from other nonstratified epithelia (Mills and DiBona, 1977). A second difficulty is presented by the fact that not all ameloblasts have distal zonulae occludentes. Secretory ameloblasts have two sets of tight junctions at both the proximal and distal ends of cell bodies, and only the latter one is considered to be a zonula occludens (Warshawsky, 1978; Sasaki et al., 1982). The proximal tight junction has been shown to be permeable to horseradish peroxidase (Kallenbach, 1980b; Sasaki, 1984a).Maturation ameloblasts exist in cohorts of ruffle-ended @A) and smooth-ended (SA) cell types along the surface of the 6 P.R. GARANT AND T.SASAKI ULTRACYTOCHEMISTRY OF Na+-K+-ATPaseIN RAT INCISOR ENAMEL ORGAN enamel (Josephsen and Fejerskov, 1977; Reith and Boyde, 1979). A well developed zonula occludens junction, made up of about ten rows of tight junctional strands, is present on the distal end of RA and on the proximal end of SA (Sasaki and Garant, in press). On the other hand, a permeable intercellular space is present at the proximal end of the RA and the distal end of the SA (Josephsen and Fejerskov, 1977; Kallenbach, 1980s; Sasaki et al., 1983; Sasaki and Garant, in press). Because of the different location of the zonulae occludentes in RA and SA, an open pathway is believed to exist from the enamel space to the papillary layer. This pathway is an indirect one from the enamel beneath the SA, proceeding along the lateral intercellular spaces to the region of RA, where access to the papillary layer intercellular spaces is gained through the open proximal borders of the RA (Kallenbach, 1980a). A bidirectional exchange of substances is thought to occur along this pathway. Intravenously injected tracers such as horseradish peroxidase have been shown to gain access to the enamel at the location of the SAs (Kallenbach, 1980a; Takano and Ozawa, 1980; Sasaki et al., 1983). The concentration of Na+-K+-ATPaseon the plasma membranes of the cells of stratum intermedium and papillary layer suggests that if an osmotic gradient is established in the enamel organ, it is within the intercellular spaces of the stratum intermedium and papillary layers. Thus, it would seem that the positioning of a zonulae occludentes in the secretory and maturation ameloblasts at either the distal or the proximal ends of these cells would work equally well in establishing a barrier between the stratum intermedium and papillary layers and the enamel. If these zonulae occludentes are leaky, water would be drawn across the junctions from the enamel to the stratum intermedium and the papillary layers. Kallenbach (1980a) and Sasaki (1984) documented that, at least in early secretion zone, the distal zonula occludens of secretory ameloblast is permeable to molecules as large as those of horseradish peroxidase (molecular weight 43,000). Movement of water from the 7 enamel might drag matrix breakdown products out of the enamel toward the secretory and maturation ameloblasts. Cytochemical analysis of rat incisor enamel indicated that a relatively steep rise of mineral content was observed when it was expressed in terms of tissue volume, suggesting the replacement of matrix by water (Hiller et al., 1975). They also described that the subsequent increase in Ca and P per volume of enamel reflected the replacement of water as further mineral was acquired. In an earlier chemical study of developing pig molar enamel, Deakins (1942) demonstrated that Ca influx into the enamel was compensated chiefly by a loss of water, which had been preceded by a loss of organic matrix. Accordingly, water movement across the enamel organ probably mediated by Na+-K+-ATPase may be a necessary process for the replacement of matrix and, eventually, water during progressive enamel mineralization. We visualize a net flow of water across the enamel toward the papillary layer and vasculature. Such a flow would not preclude diffusion of substances from ameloblasts into the enamel, nor from the pulpal blood vessels into the enamel. In other transport epithelia, such as small intestine, renal proximal tubule, and gall bladder, sodium enters the cells passively at the Iuminal surfaces (Blaustein, 1974;Hoshi et al., 1976; DiBona and Mills, 1979).IfNa+ were to enter the secretory and maturation ameloblasts passively at the distal (i.e., next to the enamel) surfaces, it could move down a concentration gradient across the extensive gap junctions between the ameloblasts and the cells of stratum intermedium and papillary layers. The colloid osmotic pressure of serum within the extensive, closely juxtaposed, and fenestrated capillaries could serve as a sink to remove water and small molecular weight substances from the intercellular spaces of the papillary layer (Garant and Gillespie, 1969; Sasaki et al., 1984; Sasaki and Garant, in press). ACKNOWLEDGMENTS We wish to thank Mrs. Kris Vandenberg for her typing of the manuscript. This study was supported by grant DE 05586 to Dr. P.R. Garant from the National Institute of Dental Research. Fig. 10. The papillary layer cells in the transition stage showing reaction precipitates along the cytoplasmic side of the plasma memLITERATURE CITED branes. The plasma membranes forming gap junctions (GJ) and desmosomes (D) contain few reaction products. Inset higher magnification Albers, R.W., and G.J. Koval (1972) Na+-K+-activated adenosine triphosphatase. VII. Concurrent inhibition of Na+-K+-adenosinetrifigure shows a localization of reaction precipitates in the microvilli of phosphatase and activation of K+-nitrophenylphosphatase papillary cells. x 17,000; inset, ~ 4 5 , 6 0 0 . activities. J. Biol. Chem., 2473088-3092. Fig. 11. The proximal (inset) and distal portions of ruffle-ended mat- Berman, A.L., A.M. Azimora, and F.G. Gribakin (1977) Localization of Na+, K+-ATPase and Ca++-activatedMg+ +-dependentATPase in uration ameloblasts (Ab) having the ruffled borders (RB) and adjacent retinal rods. Vision Res., 17527-536. papillary cells (PC). Enzymatic reaction is seen along the plasma membranes of papillary cells’ microvilli and of the ameloblast’s basal Blaustein, M.P. (1974) The interrelationship between sodium and calcium fluxes across cell membranes. Rev. Physiol. Biochem. Pharcell structures. GJ, gap junctions. x 17,000. macol., 70:33-82. Fig. 12. The proximal (inset) and distal portions of smooth-ended Bonting, S.L. (1970) Sodium-potassiumactivated adenosinetriphosphatase and cation transport. In: Membrane and Ion Transport, Val. 1. maturation ameloblasts (Ab), which lack both the ruffled border and E.E. Bittar, ed. New York, Wiley-Interscience, pp. 257-368. the distal junctional complexes, and adjacent papillary cells (PC). Enzymatic reaction is seen prominently in the papillary cells and weakly Claude, P., and D.A. Goodenough (1973) Fracture faces of zonulae occludentes from “tight” and “leaky” epithelia. J. Cell Biol., in the basal cell surface of ameloblast. Neither lateral nor distal cell 58390400. surfaces of ameloblasts show enzymatic reaction. X 17,000. Curran, P.F., and J.R. Macintosh (1962) A model system for biological water transport. Nature, 193:347-348. Fig. 13. The papillary cells fixed with 2% formaldehyde. Reaction precipitates are seen intensely along the cytoplasmic side of the plasma Deakins, M. (1942) Changes in the ash, water and organic contents of pig enamel during calcification. J. Dent. Res., 21:429-435. membranes. Inset shows a reaction-negative fenestrated portion of Diamond, J.M., and W.H. Bossert (1967) Standing-gradient osmotic capillary endothelium. X 28,500. flow: A mechanism for coupling of water and solute transport in epithelia. J. Gen. Physiol., 50:2061-2083. Fig. 14. The ruffled border zone of maturation ameloblast fixed with 2% formaldehyde, whose plasma membranes show no enzymatic reac- DiBona, D.R., and J.W. Mills (1979) Distribution of Na+-pumpsites in transporting epithelia. Fed. Roc.,38134-143. tion. ~ 2 8 , 5 0 0 . 8 P.R. GARANT AND T. SASAKI ULTRACYTOCHEMISTRY OF Na+-K+-ATPaseIN RAT INCISOR ENAMEL ORGAN Ernst, S.A. (1975) Transport ATPase cytochemistry: Ultrastructural localization of potassium-dependent and potassium-independent phosphatase activities in rat kidney cortex. J. Cell Biol., 66586608. Garant, P.R., and J. 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