THE ANATOMICAL RECORD 256:72–77 (1999) Enzyme Histochemical Localization of Na⫹,K⫹-ATPase and NADH-DE in the Developing Rat Parotid Gland 1Oral FREDERICK D. PEAGLER1,2 AND ROBERT S. REDMAN1,3* Pathology Research Laboratory, Department of Veterans Affairs Medical Center, Washington, District of Columbia 2Department of Histopathology, College of Dentistry, Howard University, Washington, District of Columbia 3Department of Oral Pathology, Baltimore College of Dental Surgery, University of Maryland, Baltimore, Maryland ABSTRACT Information on ductal differentiation in the developing rat parotid gland is sparse. Striated and excretory ducts are rich in a number of enzymes related to ion movement. The objective of this investigation was to delineate histochemically the chronology of two of these, ouabain-sensitive Na⫹,K⫹-ATPase and NADH-DE, in the developing rat parotid gland. Parotid glands were excised from rats at representative ages from 20 days in utero to 42 days. Enzyme histochemistry was performed on air-dried frozen sections. For Na⫹,K⫹-ATPase, some sections also were fixed in phosphate-buffered formalin. Ouabain blocked Na⫹,K⫹-ATPase activity, and neither enzyme reacted without substrate. Weak Na⫹,K⫹-ATPase reactions were initially seen in unfixed sections at 1 day, and increased steadily to the adult pattern of strong (concentrated basolaterally) in striated ducts and excretory ducts, respectively, and weak to modest (diffuse) in acini and intercalated ducts at 28 days. In fixed sections, localization was sharper but the reaction was somewhat reduced. NADH-DE was modest in terminal buds and ducts before birth, then progressively changed to the adult pattern of weak in acini and intercalated ducts and strong (concentrated basally and luminally) in striated and excretory ducts at 28 days. As demonstrated by enzyme histochemistry of Na⫹,K⫹-ATPase and NADHDE, differentiation of rat parotid striated ducts and excretory ducts occurs mainly between birth and 28 days. Anat Rec 256:72–77, 1999. Published 1999 Wiley-Liss, Inc.† Key words: development; differentiation; histochemistry; Na⫹,K⫹-activated adenosine triphosphatase; nicotinamide adenine dinucleotide (reduced form)-dependent enzyme(s); parotid gland; rat; salivary gland Considerable work has been done on acinar and myoepithelial differentiation in the developing rat parotid gland, but there have been only a few reports on ductal differentiation (Redman and Sreebny, 1971; Taga and Sesso, 1979; Bordeianu et al., 1983; Redman, 1987; Ogawa et al., 1998; Peagler et al., 1998). This investigation is part of a systematic study of ductal differentiation in this gland. Enzyme histochemistry was chosen as the investigative tool because it can provide morphologic and functional biochemical observations simultaneously. Published 1999 WILEY-LISS, INC. †This article is a U.S. Government work and, as such, remains in the domain of the United States of America. RATIONALE One of the major functions of the larger ducts of the rat parotid gland is the transfer of ions to and from the Grant support: Department of Veterans Affairs. *Correspondence to: Dr. Robert S. Redman, Oral Pathology Research Laboratory (151-I), Department of Veterans Affairs Medical Center, 50 Irving Street, NW, Washington, DC 20422. Received 21 December 1998; Accepted 1 June 1999 73 ATPASE AND NADH-DE IN DEVELOPING RAT PAROTID primary fluid (Mangos et al., 1973). This requires large amounts of energy, and the structure of the striated and excretory ducts reflects this by having large numbers of elongated mitochondria concentrated along complex basolateral membrane infoldings (Hand, 1987). Two enzymes were chosen for this phase of the investigation because their location in these large ducts indicates that they are involved in the transfer of ions either directly or by making available the required energy. Ouabain-sensitive, sodium/ potassium adenosine triphosphatase (Na⫹,K⫹- ATPase) has been localized to the basolateral membranes (Speight and Chisholm, 1984; Iwano et al., 1987; Winston et al., 1988), while nicotinamide adenine dinucleotide-dependent enzymes (NADH-DE) have been localized mainly in the mitochondrial and luminal membranes (Bordeianu et al., 1983), of the rat parotid gland. Both were localized more intensely in the striated and excretory ducts than in other parenchymal structures. Retrograde injection of ouabain into the duct system has been shown to greatly decrease the reabsorption of both potassium and sodium ions in the rat parotid gland (Mangos and Braun, 1966), testifying to the functional importance of the ATPase. NAD is reduced to NADH by a number of dehydrogenases, and the NADH is oxidized by cytochrome c or b5 (and by tetrazolium salts) in the presence of a specific reductase, NADH diaphorase (Pearse, 1972; Troyer, 1980). A number of dehydrogenases, e.g., alcohol, glutamate, malate and mitochondrial isocitrate, are NAD-linked or dependent (Troyer, 1980). These are heavily involved in the utilization of the energy required for the transport of ions. The chronology of the localization and reaction intensity of these enzymes thus can provide an overview of an important aspect of the functional development of these ducts. MATERIALS AND METHODS Experimental Animals Sprague-Dawley rats (Rattus norvegicus albinus) were obtained at precise pre- and postnatal ages from a breeding colony (breeders purchased viral pathogen-free from Charles River) maintained by Dr. William D. Ball at Howard University. Representative ages were selected for analysis, as follows: 20 days in utero (i.u.) and 0 (newborn), 1, 7, 14, 21, 28 and 42 days. At all ages, one or more animals of each sex were obtained from each of two litters. In order to obtain sufficient tissue, glands were pooled by litter at 1 day or younger and by sex at 7 days. Animals were killed by cervical section immediately prior to dissection. The experimental protocol was approved by the Research and Development Committee of this Department of Veterans Affairs Medical Center. Enzyme Histochemistry Parotid glands were removed and cleaned of extraneous tissues. Samples were frozen in a tube of 4-methyl butane (isopentane) immersed in liquid nitrogen, sectioned at 8 µm in a cryostat, thaw-mounted on 5% gelatin-coated glass slides, and air-dried for 20 min. Na⫹,K⫹-ATPase histochemistry was performed by the method of Mayahara et al. (1980), with a few modifications, as follows. Some sections were fixed for 20 min in 5% formalin, 0.1 M NaH2PO4, 0.075 M sucrose, 0.001 M CaCl2, and 10% dimethylsulfoxide (DMSO) for 20 min prior to incubation, followed by three washes in 0.162 M glycine-KOH buffer, pH 9.0, with .0075 M sucrose and 10% TABLE 1. Histochemical assessment of Naⴙ,Kⴙ-activated adenosine triphosphatase activity in developing rat parotid gland* Ducts Age (days) Acini Intercalated Striated Excretory 0 1 7 14 21 21 (F) 28 28 (F) 42 42 (F) 0 1 1 1 1 1 2 1 1 1 0 1 1 1 1 1 2 1 2 1 0 1 3B 3B 4B 3B 5B 4B 5B 4B 0 1 3B 4B 4B 3B 5B 4B 5B 4B *Scores ranged from 0 (none) to 5 (heaviest stain), and represent the consensus of two observers. Letter F after age indicates formalin fixed sections; all others were air-dried only. Localization within structures was diffuse, except mostly basolateral where score is followed by the letter B. DMSO. The incubation medium consisted of 0.25 M glycineKOH buffer, pH 9.0, 0.002 M MgSO4, 25% DMSO, 0.025 M levamisole, 0.005 M p-nitrophenylphosphate, and 0.004 M Pb3(C6H5O7)2. Incubation was for 20 min. The time was reduced to 15 min at 21 days and 10 min at 28 and 42 days with the unfixed sections to avoid obscuring the localization. Control sections were pre-incubated in 0.0125 M ouabain (3-[(6-deoxy-␣-L-mannopyrosyl)oxyl]-1,5,11␣,14,19pentahydroxycard-20(22)-enolide), 0.25 M glycine-KOH buffer, pH 9.0, and 0.23 M sucrose, and had 0.0125 M ouabain added to the incubation medium, or were incubated without p-nitrophenylphosphate. Alternate sections were counterstained with hematoxylin. NADH-DE histochemistry utilized Nitro-Blue Tetrazolium (NBT) as the chromogen and NADH as the substrate, using the method described by Shapiro (1967). Control sections were incubated without NADH. Sections then were dehydrated sequentially with ethanol and xylenes and coverslipped with Permount. The ouabain blocked Na⫹,K⫹-ATPase activity, and neither enzyme visibly reacted without substrate. Intensity of stains was judged on a scale of 0 (none) to 5 (maximum) for each locality, and final scores were assigned by consensus. RESULTS The results of the Na⫹,K⫹-ATPase histochemistry are summarized in Table 1 and illustrated in Figure 1. No reaction was seen in unfixed sections until 1 day, when it was weak in both ducts and acini. Between 7 and 28 days, it increased steadily to strong (concentrated basolaterally) in striated ducts and excretory ducts, respectively, and modest (diffuse) in acini and intercalated ducts. In fixed sections, localization was sharper but scores were about one point lower. The results of the NADH-DE histochemistry are presented in Table 2 and illustrated in Figure 2. Reaction was modest in terminal buds and ducts before birth. Between birth and 28 days, it decreased to weak in acini and intercalated ducts and increased steadily to strong (concentrated basally and luminally) in striated and excretory ducts. 74 PEAGLER ET AL. Fig. 1. Photomicrographs of histochemically localized Na⫹,K⫹activated adenosine triphosphatase activity (dark reaction product) in rat parotid glands at ages 1 day (A), 7 days (B), 21 days (C–E), and 28 days (F). All sections except A and C were fixed in phosphate-buffered formalin. Incubation time was 15 min in C and 20 min in all others. Light, diffuse reactions occurred in all elements at 1 day. The progressively heavier and more basally concentrated reaction product in the striated and excretory ducts increasingly demarcated these structures from the acini and intercalated ducts to 28 days, with very little change thereafter. The basolateral localization of reaction product in the striated ducts in D is more easily appreciated at higher magnification in E. a, acini; e, excretory ducts; i, intercalated ducts; s, striated ducts; t, terminal buds. Magnification A–D, F, ⫻130; E, ⫻310. ATPASE AND NADH-DE IN DEVELOPING RAT PAROTID Fig. 2. Photomicrographs of histochemically localized nicotinamide adenine dinucleotide (reduced form)-dependent enzyme(s) activity (blue reaction product) in rat parotid glands at ages 20 days in utero (A, B), 0 days (newborn) (C), 14 days (D), 28 days (E), and 42 days (F). Modest, diffuse reactions have occurred in all elements prior to birth. Reaction product is somewhat less intense in the acini and progressively heavier in 75 the striated and excretory ducts with increasing postnatal age to 28 days. The basal and luminal concentration of reaction product in the striated and excretory ducts of the older rats is more easily visualized with the higher magnification of E. a, acini; e, excretory ducts; i, intercalated ducts; s, striated ducts; t, terminal buds. Magnification A, ⫻27; B–D, F, ⫻110; E, ⫻225. 76 PEAGLER ET AL. TABLE 2. Histochemical assessment of nicotinamide adenine dinucleotide (reduced form)-dependent enzyme(s) activity in developing rat parotid gland* Ducts Age (days) Acini 20 i.u. 0 7 14 21 28 42 2B 2B 1B, 2L 1B, 1L 1B 1B 1B Intercalated Striated Excretory 2B, 2L 2B, 3L 3B, 3L 3B, 3L 4B, 5L 4B, 5L 2B, 2L 2B, 2L 2B, 3L 3B, 4L 4B, 4L 5B, 5L 5B, 5L 2B, 2L 1B, 2L 1B, 2L 2B, 2L 2B, 2L 1B, 2L 1B, 2L *Scores ranged from 0 (none) to 5 (heaviest stain), and represent the consensus of two observers. I.u. ⫽ in utero. Letters following scores refer to localization within structures: B, basal; L, luminal. Intercalated and striated ducts were not reliably distinguished in these preparations at 20 days i.u.; one score is given for both. The histochemical reactions of both enzymes thus reached the adult pattern by 28 days. DISCUSSION Comparisons The distribution of reaction product with both of these enzymes at ages 28 and 42 days in this study is similar to those reported by others in adult rat parotid glands (Bordeianu et al., 1983; Speight and Chisholm, 1984; Iwano et al., 1987; Winston et al., 1988). At the ultrastructural level, the localization of Na⫹,K⫹-ATPase is almost exclusively to the folds and interdigitations of the basolateral membranes of serous acini and striated and excretory ducts with both enzyme- and immunocytochemistry. It also is localized to the plasmalemma of the intercalated ducts immediately subjacent to myoepithelial processes (Speight and Chisholm, 1984). The concentration of reaction product per unit of membrane appears to be equal among acini and ducts (Speight and Chisholm, 1984; Iwano et al., 1987). However, the much greater infolding and interdigitation of these membranes in the striated and excretory ducts spreads the enzyme over a proportionally much greater area of these ducts than in the acini and intercalated ducts. This difference in distribution appears to account for much of the stronger reactions in these ducts at the light microscopic level. It seems difficult to reconcile the basolateral localization of Na⫹,K⫹-ATPase in salivary glands with one aspect of their function: the supposed direction of ion transport. Thus, Na⫹ should either enter or exit at the bases of all of these cells, not enter the acini and exit the striated ducts. One explanation that has been suggested is that a buildup of Na⫹ in the intercellular spaces, which in rat parotid acini but not ducts are adjacent to secretory canaliculi (Hand, 1987), may facilitate water and ion transport into the lumen (Winston et al., 1988). In the present study, NADH-DE activity differed from that of Na⫹,K⫹-ATPase in two important respects. 1) In the older rats, it was moderate instead of weak in the acini and intercalated ducts, and in the striated and excretory ducts, it was localized to the luminal membranes and basal cytoplasm, rather than the basolateral membranes. 2) It was already moderate in both terminal buds and ducts prior to birth, while that of Na⫹,K⫹-ATPase was imperceptible in all parenchymal elements until one day after birth. The histochemical reactions reported here indicate that both enzymes are useful as markers of ductal differentiation in the developing rat parotid gland. However, Na⫹,K⫹ATPase may be somewhat superior in this regard because it has weaker reactions in the terminal buds, acini and intercalated ducts during the perinatal period. Correlation with Other Reports on Ductal Differentiation in Rat Parotid Gland In general, the changes in the histochemical reactions of the two enzymes presented here are consistent with the chronology of ductal differentiation in the developing rat parotid gland in previous reports. Bordeianu et al. (1983) described an increase in histochemical reaction of NADH-DE in the larger ducts of the developing rat parotid gland between birth and 30 days, similar to that in this study. However, their reactions were considerably weaker among the acini and small ducts during the perinatal period. Similarly, carbonic anhydrase increases in both activity and amounts of enzyme in the striated and excretory ducts during the same period (Ogawa et al., 1998; Peagler et al., 1998). Ultrastructurally, the striated ducts are recognizable by 5 days after birth and mature by 15 days, in terms of cell size, the extent of infolding of basolateral membranes, and the accumulation of large mitochondria (Taga and Sesso, 1979). Functionally, although the transductal fluxes of Na⫹ and K⫹ are much greater in the immature (18 days) rat, the concentrations of these ions in the saliva in a range of matched flow rates are not similar to those of the adult until age 32 days (Schneyer and Schneyer, 1961; Schneyer and Hall, 1968; Mangos, 1978). As discussed previously (Peagler et al., 1998), there are insufficient data, e.g., on fluxes of other ions such as HCO3⫺ and Cl⫺, to satisfactorily explain why the capacity of these ducts to transport ions seems to diminish between 18 and 32 days. 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