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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.
Thus, although by ultrastructural criteria, the striated
and excretory ducts of the rat parotid gland appear to be
mature at age 15 days, both functionally and histochemically, there are further developmental changes until about
age 30 days.
ACKNOWLEDGMENTS
The authors thank Rodney L. McNutt for technical
assistance and Dr. Ruth B. Field for critically reviewing
the manuscript.
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