Histochemical and elemental localization of calcium in the granular cell subapical granules of the amphibian urinary bladder epithelium.

THE ANATOMICAL RECORD 218:229-236 (1987)
Histochemical and Elemental Localization of
Calcium in the Granular Cell Subapical Granules of
the Amphibian Urinary Bladder Epithelium
WALTER L. DAVIS, RUTH GWENDOLYN JONES, H.K. HAGLER, GENE R. FARMER,
AND DAVID B.P. GOODMAN
Department of Anatomy, Baylor College of Dentistry, Dallas, TX 75246 (WLD., R.G.J.,
G.RX), Department of Pathology, The University of Texas Health Science Center,
Southwestern Medical School, Dallas, TX 75212 (H.K.H.),Department of Pathology and
Laboratory Medicine, The University of Pennsylvania Medical School, Philadelphia, PA
19104 @. B. P. G.)
ABSTRACT
The ultrahistochemical analysis of apical granules in the epithelial
cells, i.e., granular cells, of the amphibian urinary bladder using the N,N-naphthaloylhydroxylamine procedure identified the presence of calcium in these structures.
Subsequent analytical microscopy employing fresh-frozen ultrathin cryosections
for X-ray microanalysis of the granules further confirmed the above histochemical
findings. In addition to calcium, elemental analysis indicated the presence of magnesium, phosphorus, sulfur, silicon, potassium, and chlorine either within or in close
proximity to the granules. The possibility that these granules function as subcellular
compartments for the uptake and storage of calcium ions, in a way similar to
mitochondria, and thus function in intracellular calcium homeostasis, is discussed.
Additionally, a role for this cation in the secretion of granular glycoproteins, i.e.,
stimulus-secretion coupling, is hypothesized.
The amphibian urinary bladder has been used exclusively as a simple in vitro model system for the mammalian distal nephron (Leaf, 1965). The peptide hormone
vasopressin (antidiuretic hormone or ADH) produces two
well-established physiological effects on the toad urinary bladder: 1)the hormone induces a n increase in the
permeability of the tissue to both water and low molecular weight solutes (urea, etc.) allowing for the bulk
movement of fluid (water) down a mucosal to a serosal
osmotic gradient, and 2) ADH stimulates active transepithelial sodium transport (Leaf, 1965,1967).
The mucosal epithelium is a transitional epithelium
and has been the subject of numerous fine structural
studies (Peachey and Rasmussen, 1961; Choi, 1961;
Davis et al., 1974). It is this epithelium that represents
the major permeability barrier to water (Leaf, 1967;
Parisi and Piccinni, 1973). The epithelium is comprised
of four cell types: 1)granular cells, 2) mitochondria-rich
(MR) cells, 3) goblet cells, and 4) basal cells (Peachey and
Rasmussen, 1961). Only the basal cells lack a luminal
face. Of these four cells, the granular cells are the most
numerous, comprising approximately 90-95% of the epithelial cell population (Peachey and Rasmussen, 1961;
DiBona et al., 1969a,b,; Davis et al., 1974). The next
most abundant cell of the mucosal epithelium is the MRcell (10-15%) (Peachey and Rasmussen, 1961; DiBona et
al., 1969a,b; Davis et al., 1974). The granular cells are
believed to be the major site of vasopressin action in this
tissue (DiBona et al., 1969a,b; Davis et al., 1974).
Granular cells, as their name implies, are characterized by the presence of numerous membrane delimited,
electron dense granules, which are located often in clusters or “stacks” immediately subadjacent to the luminal
0 1987 ALAN R. LISS, INC.
(apical) microvillated border (Choi, 1963; Masur et al.,
1972; Gronowicz et al., 1980). Both ultrastructural and
cytochemical studies have indicated that these granules
contain complex carbohydrates (glycoconjugates, glycoproteins), which represent precursor molecules that are
secreted by exocytosis apically to become components of
the PAS-positive, luminal membrane associated glycocalyx (Choi, 1963; Masur et al., 1972; Pisam and Ripoche, 1973; Strum and Ekblad, 1977; Gronowicz et al.,
1980; Masur et al., 1986).
All of these observations prompted our present investigation of the ionic composition of the apical granules,
particularly the calcium content of these structures since
this cation is known to be involved in a variety of cellular secretory processes (Rasmussen and Goodman, 1977).
MATERIALS AND METHODS
Large female toads (Bufo marinus) were obtained from
National Reagents Inc., Bridgeport, CT, and maintained
at room temperature on a bed of moistened peat. The
urinary hemibladders were excised from doubly pithed
animals and placed immediately in Ling-Ringer’s phosphate buffer of the following composition (mM): 1) NaC1,
92.7, 2) KC1, 2.5, 3) CaC12, 1.0, 4) MgS04, 1.2, 5) NaHC03, 7.8, 6) NaH2P04, 1.8, 7) Na2HP04, 1.2, pH 7.47.6; osmolarity, 200-220 mosmolkg H2O (Ling, 1962).
The hemibladders were then gently stretched and
mounted, mucosal surface up, while under buffer to tefReceived September 3, 1986; accepted March 2, 1987.
Address reprint requests to Dr. Walter L. Davis, Baylor College of
Dentistry, Dallas, TX 75246.
230
W.L. DAVIS ET AL.
lon screens. After mounting, the buffer was replenished
with fresh solution. The mounted hemibladders were
maintained in plastic dishes containing 25 ml of fullstrength buffer. The solution was continuously aerated
during this preincubation period. Buffer was changed
each 30 min. For this study, hemibladders from 6 toads
were used for each phase of the project. Thus, matched
hemibladders from 6 toads were used for the histochemical study (see below) and another 6 were used for elemental analysis (see below). Continuing, one block of
embedded tissue from each hemibladder (12 blocks), or
one block of fresh-frozen tissue from each hemibladder
(12 blocks) were sectioned. Two grids from each sample
were evaluated, photographed, andor analyzed.
For the selective ultrahistochemical localization of cellular calcium, the procedure of Zeichmeister (1979) was
employed. This technique uses the sodium salt of N,Nnaphthaloylhydroxylamine (1,8-CloH6CON(ONa)CO),
NHA, to localize subcellular calcium in the form of the
electron dense precipitates of the NHA-calcium salt. Potassium pyroantimonate and potassium hydroxyantimonate procedures have been used extensively for the
ultrastructural localization of calcium ions (Simson and
Spicer, 1975). However, the specificity of this technique
has been criticized (Klein et al., 1972). A much more
specific ultrahistochemical technique for the localization of calcium, employing NHA, was utilized in the
present study. The NHA method appears to have a
greater affinity, a greater sensitivity, a greater selectivity, and the potential to detect sites with tightly bound
calcium (Zeichmeister, 1979; Slocum and Wang, 1982)
than the previously employed antimonate procedures,
which are not specific, selective reagents since sodium
and magnesium can also participate in the reaction.
NHA has been used to detect calcium in microanalytical
chemistry as well a s microscopic demonstration of this
cation (Voight, 1957). NHA is characterized by its sensitivity for calcium, which is approximately 100 times
higher than other detection reagents currently employed in ultrahistochemistry, i.e., potassium pyroantimonate, potassium oxalate, and murexide (Zeichmeister,
1979).
For this procedure, the physiologic buffer covering the
mounted hemibladders was decanted and replaced with
freshly prepared, cacodylate-buffered (0.05) M, pH 7.2)
2.5% glutaraldehyde. Tissues were incubated in this
prefixation medium for 30-60 min at 4°C. After this
primary fixation step, the tissues were washed in cacodylate buffer prior to incubation in 0.01 mM NHA
(Sigma Chemical Co., St. Louis, MO) for 5 min a t RT. A
secondary fixation was then carried out for 60 min a t
4°C in the previously mentioned glutaraldehyde. After
several washes in cacodylate buffer, the tissues were
postfixed in 1.0% osmium tetroxide buffered with cacodylate (0.05 M, pH 7.2) for 60 min. After osmication, the
tissues were washed several times in cacodylate buffer.
While in the last buffer wash, the hemibladders were
removed from their frames and diced into longitudinal
strips approximately 2 mm in width. The tissues were
then dehydrated and subsequently placed into a 1:l
mixture of propylene 0xide:Durcupan (Fluka AG, Buchs
SG, Switzerland) for 120 min. This was followed by pure
Durcupan (14 hr) and finally by embedment in Durcupan (12-24 hr) at 37OC-45"C. Ultrathin sections were
cut with freshly prepared glass knives, mounted to cop-
per grids, and examined unstained in a Philips 300
transmission electron microscope (Philips Electronics
Inc., Mount Vernon, NY). Subsequently, sections were
doubly stained with uranyl acetate and lead citrate and
photographed. For the microanalysis of the reaction
products, dark gold sections were prepared, mounted to
copper grids, and subsequently carbon coated prior to
elemental analysis (see below).
For the preparation of fresh-frozen cryosections for
analytical X-ray microanalysis, the techniques of Hagler et al. (1983, 1984) were employed. Hemibladders
were removed from the buffer wash still attached to
their screens and placed directly into dissection dishes
under liquid nitrogen (LN). While under LN, the hemibladders were sectioned into strips with LN-cooled razor
blades. A single specimen was then mounted in a specimen holder and subsequently transferred and mounted
in the specimen arm of a DuPont-Sorvall FS-1000 cryostem (DuPont Instruments, Sorvall Operations, Newtown, CT) attachment for the MT-5000 ultramicrotome.
The specimen and knife temperatures were maintained
a t - 120°C or colder in order to prevent the freeze drying
of sections within the cryosystem. Dark gold sections
(1,000-1,200 A in thickness) were cut on 45" glass knives
using a clearance angle of 6". Sections were picked up
on 50 mesh, formvar-carbon coated copper grids. The
grids were then transferred to a holding block within
the cryosystem. While in the holding block, the sections
were "sandwiched" beneath a formvar film. The cold
stage was then used to transfer the frozen-hydrated ultrathin sections to the electron microscope. Due to the
lack of recognizeable morphology in the hydrated state,
all sections were freeze-dried prior to X-ray microanalysis. This was accomplished in the electron microscope
by "warming" the cold stage to -100°C. Usually, after
30 min the sections were dry as judged by a stable
continuum and the recognition of fine structural detail.
For analytical electron microscopy, a JEOL lOOC
TEMSCAN (JEOL, U.S.A., Medford, MA) equipped with
a 32 mm2, 158 eV resolution, lithium-drifted, silicon,
energy dispersive X-ray detector was used. The X-ray
spectra were collected and analyzed by a Tracor-Northern TN-2000 multichannel analyzer. The microscope was
operated in the STEM mode a t 80 keV, 50 microamps
emission current, 30" specimen tilt, with a beam diameter of 20 nm. X-ray spectra were collected over the
energy range of 0-20 keV for 50 sec a t a resolution of 20
evlchannel. The selected-area raster mode was used to
collect spectra from each sample. Ten granules were
analyzed per sample for 20 sec each and their spectra
summed. The latter were then subjected to a multipleleast squares fitting routine using reference spectra obtained by the analysis of single crystal specimens. The
analysis of spectra were performed with the DEC PDP
11/05 computer-based Tracor-Northern NS 880 unit.
RESULTS
In routinely fixed and prepared ultrathin sections of
the amphibian urinary bladder for transmission electron microscopy, the granular cells of the epithelium are
characterized by the presence of numerous subapical
dense granules (Fig. 1). These structures are round to
ovoid (ellipsoid) in shape and are located immediately
subadjacent to the lumina1 plasmalemma. A few morphologically similar granules were occasionally ob-
TOAD BLADDER GRANULAR CELL
231
Fig. 1. Transmission electron micrograph (TEM) of the mucosal epi- dense, ovoid secretory granules (arrows). Several of these granules
thelium from a hemibladder fixed in cacodylate-buffered paraformal- seem to possess eccentric densities (arrowheads). A few of the granules
dehyde, osmicated, and subsequently embedded in low viscosity resin. appear to be in the process of exocytosis (e). L, bladder lumen; M,
Section double-stained with uranyl acetate and lead citrate. Several microvilli; g, glycocalyx; N, nuclei. The morphology of the epithelial
granular cells are seen (G). Note the numerous subapical, electron cells is identical when the tissues are fixed in glutaraldehyde. X11,500.
served in mitochondria-rich cells. Fine structural
observations showed the granular cell granules to be
membrane delimited with varying degrees of electron
density. A few showed a n eccentric electron dense structure or core. Some granular membranes were in close
proximity to the luminal cell membrane remniscent of
ongoing exocytosis. In tangential sections, the ovoidshaped granules were organized into clusters or
aggregates.
Cytochemically, a n electron dense reaction product
was seen within the granules in tissues exposed to the
sodium salt of NHA (Fig. 2). Apical and basolateral cell
membranes, mitochondria, nuclei, and nucleoli also
showed evidence of a positive reaction. Direct evidence
for the presence of calcium, as calcium N,N-naphthaloylhydroxylamine, in the electron dense reaction product seen within the subapical granules of these cells was
confirmed by X-ray microanalysis of semithin sections
(Fig. 3). This X-ray spectrum represents a summed analysis of 10 separate granules, each analyzed for 20 sec.
The latter procedure was performed a total of 10 times.
The identifiable peaks shown in this spectrum are: 1)
silicon (Si-K,), 2) phosphorus (P-K,), 3) cholorine (Cl-Ka),
4) calcium (Ca-K,, Kb), 5) copper (Cu-K,, Kb), and 6)
osmium (OS-K,). The presence of osmium, chlorine, and
copper is the direct result of the preparative techniques
utilized for the tissues. Thus, osmium is from the postosmication step, chlorine is from the embedding medium,
and copper is from the grid. From this the granules
appear to contain silicon, calcium, and phosphorus.
Using STEM, freeze-dried cryosections photographed
at - 100°C showed good preservation and retention of
the fine structure of the hemibladder epithelium (Fig.
4). Large areas showing minimal ice damage were seen.
Under these conditions, the granules appeared a s ovoid
to elongate structures of varying density (Figs. 4, 5). Xray microanalysis of the granules in freeze-dried sections showed the presence of the following ions: 1)magnesium (Mg-K,), 2) silicon (Si-Ka), 3) phosphorus (P-K,),
4) sulfur (S-K,), 5 ) chlorine (Cl-K,), potassium (K-K,), 6)
calcium (Ca-K,, Kb), and 7) copper (not shown) (Fig. 6).
Generally the more dense granules contained considerably more calcium and phosphorus than did the light,
less dense granules. The granules were analyzed as described above, i.e., 10 granules were analyzed for 20 sec
each and their spectra summed. This procedure was
repeated 10 times. All granules analyzed showed the
presence of calcium. Quantitative analyses, using the
Hall peak-to-continuum approach (Hall et al., 1973), indicate that the granules contain calcium in a concentration of (mM/Kg dry weight f S.D.) 10.1 f 8.9 (N = 10).
These quantitative results reflect a random sampling of
both light and dark granules.
DISCUSSION
The granular cells of the amphibian urinary bladder
mucosal epithelial cells represent the most numerous
cell type of this tissue (Peachey and Rasmussen, 1961;
DiBona et al., 1969a,b,; Davis et al., 1974). Ultrastructural studies have shown the granular cells to be characterized by a n “inverted pyramid” morphology with a
narrow basal aspect and a n expansive, microvillated
luminal surface (Peachey and Rasmussen, 1961; Choi,
1963; DiBona et al., 1969a,b; Davis et al., 1974). These,
W.L. DAVIS ET AL.
232
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Fig. 2. TEM of a granular cell from a hemibladder reacted by the
NHA-method for the histochemical localization of calcium. Section
stained with uranyl acetate only. An electron dense reaction product
is seen along the lumina1 microvillated border and within the subadjacent secretory granules. These precipitates contain calcium (see below). X42,500.
Fig. 3.X-ray microanalysis of secretory granules from a hemibladder
incubated by the NHA-method for the localization of cell calcium. This
spectrum shows the presence of: 1) magnesium (Mg), 2) silicon (Si), 3)
phosphorus (PI, 4) chlorine (Cl), 5) calcium (Ca), 6) copper (Cu), 7)
osmium (OS). C1 is from the embedding medium; Cu is from the copper
grid; OS is from the postosmication step following primary fixation.
Summed spectrum from the analysis of 10 granules.
TOAD BLADDER GRANULAR CELL
233
Fig. 4. Low magnification TEM of a freeze-dried section of the hemibladder epithelium. The morphology
of the two granular cells (G)shown is well preserved with excellent retention of fine structure. Minimal
ice damage is apparent. L, bladder lumen; MV, microvilli; N, nucleus; Arrows, secretory granules.
x21,000.
plus additional morphological studies, have all described the presence of regular aggregates of membrane
delimited, ovoid to ellipsoid-shaped, electron dense granules immediately subadjacent to the apical plasmalemma of the granular cells (Masur et al., 1971, 1972,
1986). Continuities between the granule membrane and
the surface cell membrane, perhaps indicative of granular secretion or exocytosis, have been described (Peachey
and Rasmussen, 1961; Masur et al., 1972; Gronowicz et
al., 1980). Despite the numerous ultrastructural studies
on the granular cells, the precise nature and function of
the subapical granules is only now beginning to be
appreciated.
Early electron microscopic studies on the bladder mucosal cells described a similarity in the morphology,
texture, etc., of the granule contents to that of the lu-
234
W.L. DAVIS ET AL.
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Fig. 5. High magnification STEM from a freeze-dried cryosection
prepared as in Figure 4. Sample photographed and analyzed at -100°C.
A cluster of secretory granules is seen (1-4). Note the difference in
density between individual secretory granules. Generally, the denser
the granule, the greater the calcium concentration. X144,OOO.
Fig. 6. X-ray spectrum from the subapical secretory granules shown
in Figure, 5. The granules contain: 1)Mg, 2) Si, 3) P, 4) sulfur (S), 5) C1,
6) potassium, K, 7 ) Ca. All analyses were performed at the higher
magnification where it was possible to thoroughly analyze a single
granule. The presence of K, the intracellular ion, is indicative that the
cells were adequately preserved and not disrupted by rapid freezing
and the subsequent freeze-drying.
TOAD BLADDER GRANULAR CELL
mina1 fibrous glycocalyx (Choi, 1963). This observation
was subsequently supported by both light microscopic
and transmission electron microscopic histochemical
procedures, which showed both the granules and the
glycocalyx to be: 1) PAS-positive (Masur et al., 19721,
and 2) silver methenamine positive using the procedure
of Rambourg (1967), a technique performed on ultrathin
sections (Pisam and Ripoche, 1976; Gronowicz, 1980).
Such information indicates that both the subapical granules as well a s the glycocalyx are rich in carbohydrate
moieties.
The source of the glycocalyx associated with the surface membrane of the granular cells has been investigated. Histochemical and autoradiographic studies
employing tritiated-fucose (a precursor of glycoproteins)
have both indicated that the carbohydrate-rich glycocalyx is derived from the subapical granules via secretion
or exocytosis (Masur et al., 1972; Pisam and Ripoche,
1976; Strum and Ekblad, 1977).Variations in the nature
of these surface glycoconjugates can be further ascertained by even more specific light and electron microscopic histochemical procedures (Spicer et al., 1981). In
the present study, X-ray microanalysis of freeze-dried
sections confirms the presence of complex carbohydrates
and glycoconjugates. Thus, the sulfur peak (Fig. 6) probably identifies sulfated glycosaminoglycans (mucopolysaccharides) and even more complex proteoglycans
within the granular matrix. It is conceivable that at
least a portion of these glycoconjugates may indicate the
presence of a glycoprotein, perhaps a calcium-binding
glycoprotein with a high affinity for this cation. Such
proteins have been identified in mitochondria (Carafoli,
1980). This may help to explain the qualitative analytical data reported here, which shows the granules to
contain considerable calcium. This is supported by quantitative data as well (see below). Granular calcium may
be associated with a calcium-binding protein located
within the granular matrix. Thus, these granules may
play a role in intracellular calcium homeostasis similar
to the well-established ion transport capabilities of mitochondria, smooth endoplasmic reticulum, etc. (Rasmussen, 1966, 1970; Rasmussen and Goodman, 1977;
Carafoli, 1980). In the amphibian urinary bladder, ADH
induces a change in the cytosolic calcium ion concentration (Thorn and Schwartz, 1965; Schwartz and Walter,
1969; Rasmussen, 1970; Cuthbert and Wong, 1974; Berridge, 1975). The granular cells of the bladder epithelium are believed to be the primary site of action for
ADH (DiBona et al., 1969a,b). Our quantitative elemental analyses indicate that the granules contain approximately 10.1 f 8.9 m W g dry weight + S.D., N = 10.
In comparison, the concentration of calcium in normal
cardiac myocyte mitochondria, a s determined by X-ray
microanalysis of similarly prepared freeze-dried tissue
sections analyzed at -1OO"C, was determined to be 10.5
& 9.2 ( m W g dry weight calcium k S.D., N = 10)
(Hagler et al., 1984). Quantitatively, the calcium content
for these very different organelles appears to be similar.
Additionally, the ionic profile for the secretory granules
is comparable to that of normal cardiac myocyte mitochondria (Hagler et al., 1984).
The presence of calcium in association with cytoplasmic secretory granules is not a new observation.
This relationship has been repeatedly demonstrated in
many diverse systems: 1)cortical (secretory) granules of
235
unfertilized sea urchin eggs (Cardasis et al., 1976)., 2)
chromaffin cells of the rat adrenal medulla (Ravazzola,
1976), 3) in endocrine cells of the rat pars distalis (Schecter, 1976),4)in the zymogen granules of the guinea pig
exocrine pancreas (Clemente and Meldolesi, 1975), and
5) in the secretory granules of rat odontoblasts (Reith,
1976). The majority of these studies have utilized cation
localization with osmium pyroantimonate coupled with
selective calcium chelation with EGTA and/or microprobe analysis.
It has been firmly established that calcium plays a n
essential role in the secretory process (Rasmussen, 1970;
Rasmussen and Goodman, 1977). With regard to cell
secretory mechanisms, this cation appears to be involved in one or more of the following ways: 1)calcium
may be involved in the packaging of secretory materials
(Clemente and Meldolesi, 1969), 2) calcium appears to
be involved in regulating plasma membrane permeability, including those changes in membrane permeability
that occur after the stimulation of secretion (Rasmussen,
1970; Dean and Matthews, 1970); the latter may be
associated with the known lumina1 membrane permeability changes that take place in the toad urinary bladder following exposure to ADH, and 3) calcium seems to
be involved in the mechanism of stimulus-secretion coupling (Douglas and Rubin, 1961; Rasmussen, 1970; Rasmussen and Goodman, 1977). Since the pioneering work
of Douglas on the adrenal medulla (Douglas and Rubin,
1961; Douglas, 1966), it was generally assumed that the
calcium involved in excitation-secretion coupling was of
extracellular origin (Robberecht and Christophe, 1971).
However, recent kinetic studies have determined that
the calcium involved in this phenomenon is derived
from a n intracellular calcium pool (Case and Clausen,
1973; Matthews et al., 1973). Thus, the mechanism involved in stimulus-secretion coupling is similar to that
established for excitation-contraction coupling in skeletal muscle with regard to the intracellular source for
calcium ions (Ebashi and Endo, 1968).
In the amphibian urinary bladder, the calcium associated with the subapical granules may function in the
exocytosis of glycocalyx components via a mechanism
not unlike calcium-dependent excitation-secretion coupling. Several reports have adduced that ADH stimulates the exocytosis of these granules (Masur et al., 1972;
Gronowicz et al., 1980). In addition to calcium, magnesium was also identified in the subapical granules by Xray microanalysis (Fig. 6). Like calcium, magnesium
may also be involved in cellular secretion methods
(Douglas and Rubin, 1961; Douglas, 1968).
The phosphorus (phosphate) seen in the subapical
granules is probably involved in the mechanism of calcium uptake by the secretory granules. A similar mechanism is present in mitochondria (Rasmussen, 1970;
Rasmussen and Goodman, 1977).
Also present in the granular matrix was silicon. Although the precise function of this ion cannot be explained at the present time, silicon has been identified
in all toad tissues thus far analyzed. These include urinary bladder, intestine, skeletal muscle, kidney, and
liver (Davis, unpublished observations). The presence of
silicon may simply reflect the habitat in which these
animals normally live, i.e., swamps.
There is little doubt that the various precipitation
techniques for the ultrahistochemical localization of cal-
236
W.L. DAVIS ET AL
cium ions present potential problems and artifacts. The
initial precipitation of electron dense calcium salts no
doubt serve as potential nucleation sites for additional
calcium epitaxis. The latter probably involves the artifactual translocation of this cation from other sources.
In the study reported here, the NHA-technique is more
than likely inducing the translocation of calcium from
extragranular sources. This can effectively explain the
increase in calcium seen in the NHA spectrum when the
latter is compared to the calcium in the spectrum obtained from freeze-dried sections. Despite this, histochemical procedures have proven themselves to be
valuable in assessing the distribution of cations within
cells and tissues.
ACKNOWLEDGMENTS
We thank Rhonda B. Davis for excellent manuscript
preparation. This research was supported in part by a
grant from Baylor College of Dentistry, Dallas, TX, and
by grants from Mr. and Mrs. Thomas Bedford and by
the General Dynamics Corporation of Fort Worth, TX,
to W.L. Davis.
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