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Freeze-fracture and lead ion tracer evidence for a paracellular fluid secretory pathway in rat parotid glands.

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THE ANATOMICAL RECORD 208169-80 (1984)
Freeze-Fracture and Lead Ion Tracer Evidence for a
Paracellular Fluid Secretory Pathway in
Rat Parotid Glands
J.A.V. SIMSON AND H.L. BANK
Departments ofAnatomy (J.A.VS.) and Pathology (H.L.B.), Medical
University of South Carolina, Charleston, S C 29425
ABSTRACT
The morphology and permeability of tight junctions of the
three major epithelial constituents of rat parotid gland-acinus, intercalated
duct, and striated duct-have been examined ultrastructurally. Acinar and
intercalated duct junctions (including those surrounding intercellular canaliculi) averaged two to three sealing strands, whereas striated duct junctions had
five to eight sealing strands. When the permeability of the junctional complex
was probed by means of a recently devised lead ion tracer technique, acinar
junctions were found to be very permeable, intercalated duct junctions were
somewhat permeable, and striated duct junctions were essentially impermeable to the tracer. Thus, by both morphological and tracer-permeability criteria,
acinar tight junctions appear to be “leaky.” These data provide strong evidence
that, in rat parotid glands, a potential paracellular secretory pathway exists
in the acinar region for the transepithelial passage of fluid.
Saliva consists of a “carrier fluid” (composed ofwater and small inorganic ions) which
contains a variety of dissolved macromolecules. The carrier fluid is essential for the solubilization of secretory macromolecules and
for delivery of active secretory components to
their site of action. The intracellular synthesis and transfer of the macromolecular components of exocrine secretions have been
studied in detail (Castle et al., 1972; Palade,
1975);however, the routes and mechanisms of
transfer of water and ions (the “micromolecular components” of secretion) are less well
understood.
Certain diseases such as cystic fibrosis and
glaucoma apparently involve abnormalities
of water and ion transfer into or out of secretory fluid. In order to understand the morphological basis of secretory alterations in
these diseases, it is essential that the routes
and mechanisms of fluid and ion transfer be
elucidated. Rodent salivary glands are a n excellent model for dissecting the mechanisms
for secretion of fluid and macromolecules,
since P-adrenergic stimulation of these glands
elicits a predominantly macromolecular secretory response, whereas cholinergic and
a-adrenergic stimulation elicit a copious secretion which is low in macromolecules (see
Peterson, 1973; Young et al., 1979; Abe and
Dawes, 1980; Simson, 1982).
0 1984 ALAN R. LISS, INC.
The morphology of the three main epithelial segments of rodent parotid gland has been
previously described (Scott and Pease, 1959;
Rutberg, 1961; Simson, 1969a) and is diagramed in Figure l.The acinus is composed of
pyramidal cells containing abundant rough
endoplasmic reticulum and stored secretory
granules. These granules are rapidly discharged into the lumen in response to secretory stimuli such as isoproterenol (Simson,
1969b). The intercalated duct is a short segment of low cuboidal cells which often contain
a few apical granules that are not discharged
in response to isoproterenol. This segment is
surrounded by myoepithelial cells which are
presumed to contract during a secretory response (see Young and van Lennep, 1978; Emmelin, 1981). The morphology of the striated
duct is typical of the morphology of cells specialized for ion transport (see Berridge and
Oschman, 1972).Specifically, the cells of this
segment possess extensively invaginated and
interdigitated basolateral plasma membranes lined by numerous mitochondria. This
segment has been demonstrated to reabsorb
sodium from the luminal fluid (see Schneyer
et al., 1972).Salivary gland acinar cells do not
conform to the morphology expected of iontransporting epithelia. However, the primary
Received November 1, 1982; accepted September 2, 1983.
70
J.A.V. SIMSON AND H.L. BANK
Acinus
Intercalated Duct
Striated Duct
Fig. 1. Diagram of the three major epithelial cell types
in the salivary gland secretory unit. The acinus forms
the initial segment of the secretory lumen, which has
extensions between acinar cells known as intercellular
canaliculi (*I. Acinar cells possess abundant secretory
granules, while intercalated duct cells have few granules. The nuclei of striated duct cells are displaced api-
cally by the elaborate basal membrane infoldings, which
are lined by mitochondria. Occluding junctions (tight
junctions) are indicated by a thickening of the membrane line between cells; adhering junctions (zonulae
adherentes and desmosomes) are indicated by the “whiskered’ regions.
secretory product of these glands, including
the carrier fluid, is apparently elaborated in
the vicinity of the acinus-intercalated duct
junction (Martinez et al., 1966; Young, 1973;
Mangos et al., 1981).
Recent evidence has suggested that, in certain epithelia, a paracellular (extracellular)
pathway probably serves as a route for ion and
water flow (Fromter and Diamond, 1972;
Spring and Hope, 1978; Jansen et al., 1980;
Ernst et al., 1980). In the present study, we
have used both freeze-fracture and a lead-ion
tracer tracer techniques (Simson and Dom,
1983) to investigate junctional morphology
and permeability in the three main epithelial
segments of the rat parotid gland. Based upon
the number of tight junctional sealing strands
observed in freeze-fracture preparations, we
conclude that a potential paracellular secretory pathway exists between acinar cells and
may exist between intercalated duct cells, but
does not exist between cells of the striated
duct. In addition, lead tracer deposition patterns indicate that junctions between acinar
cells are highly permeable, junctions between
intercalated duct cells are less permeable, and
junctions between striated duct cells are essentially impermeable to this inorganic cationic tracer.
MATERIALS AND METHODS
Routine ultrastructural morphology was
obtained from parotid glands of perfusionfixed, anesthetized female rats (SpragueDawley) weighing about 200 gm. The primary fixative was 1%glutaraldehyde, 3%
paraformaldehyde in 0.1 M sodium phosphate, pH 7.0. After initial perfusion of the
fixative (8 minutes) via the aorta, glands
were removed and immersed in fresh fixative
for 23 hours, rinsed briefly (2 minutes) in
phosphate buffer, then transferred to 1%
OsO4 in 0.1 M collidine buffer for 1 hour.
Tissues were rinsed briefly in distilled water,
dehydrated in graded alcohols and propylene
oxide, infiltrated and embedded in epoxy
resin. Thin sections were stained with uranyl
acetate and lead citrate (UA-LC).
The lead ion tracer technique has been described in detail elsewhere (Simson and Dom,
1983). This method utilizes a soluble, inorganic cation as a n in vivo tracer. Since this
tracer is much smaller than the enzymes
(e.g., horseradish peroxidase or microperoxidase) usually used in in vivo tracer studies,
it rapidly distributes into compartments in
functional continuity with the vasculature
and the extracellular space. The technique
PARACELLULAR SECRETORY PATHWAY IN PAROTID
involves whole animal perfusion with a
blunted, 18-gauge needle via the ascending
aorta. The tracer sequence is designed to 1)
flush the cells from the vasculature and replace reactive anions (e.g., Cl-), with a 0.15
M sodium acetate preperfusion fluid 2)
briefly (1 minute) perfuse with a lead ioncontaining tracer fluid (0.02 M Pb acetate in
0.13 M sodium acetate); 3) perfuse the vasculature to remove the free tracer with a 0.15
M sodium acetate rinse fluid; and 4)fix the
tissue and simultaneously capture the lead
ion tracer, with 2% glutaraldehyde buffered
with 0.1 M sodium phosphate. All perfusion
solutions were between pH 7.0 and 7.4 and
were brought to room temperature before
use. Perfusion solutions were maintained a t
a constant height above the animal such that
the hydrostatic pressure at delivery was between 85-110 mm Hg (see Simson and Dom,
1983).After 5 minutes tissues were removed
and fixed for a n additional 55 minutes in the
same fixative as that used for perfusion. This
was followed by postfixation in phosphatebuffered osmium tetroxide. Dehydration, infiltration, and embedding were performed as
described above. Either en bloc or thin section staining with uranyl acetate was performed on some of the material.
To quantify permeability of junctions to the
lead tracer, all micrographs of material perfused with tracer for 1 minute were scored
for luminal deposits in acini, intercalated
ducts, and striated ducts. In acini, most lumina were intercellular canaliculi and hence,
bounded by only two junctions. In this segment, the numbers of tracer-containing and
empty lumina were counted and the percentage of lumina which contained tracer deposits was calculated. In intercalated ducts and
striated ducts, the number of tracer deposits
clearly on the luminal side of a junction (anywhere within the lumen) were counted and
expressed as a percentage of the total number of junctions surrounding each lumen.
Freezefracture studies were carried out on
tissues that had been taken from a second
series of animals fixed by perfusion with 1%
paraformaldehyde and 2% glutaraldehyde in
0.1 M phosphate buffer, pH 7.0, at room temperature. Tissues were fixed for 1hour, rinsed
for ?hhour in 0.1 M phosphate buffer and
gradually infiltrated in ascending concentrations of glycerol. Tissues were equilibrated
overnight in 30% glycerol a t 4"C,then cut or
teased into very small pieces, loaded in gold
hinges and quench-frozen in resolidifying
71
propane cooled by liquid nitrogen. The individual sample hinges were inserted into a
multiple sample holder under the surface of
liquid nitrogen and the holder transferred to
the precooled support stage of the freeze-etching unit. Freeze-etching was performed in a
modified Denton DFE-2 freeze-etch unit
mounted on a Varian ion pumping station.
The samples were fractured a t -125°C
5°C and immediately replicated (Bank and
Robertson, 1976). After replication, the samples were warmed to -3O"C, removed from
the vacuum system, and the adhering biological material digested in sodium hypochlorite. Following extensive washing, the
samples were recovered on 75-mesh Formvar-coated grids (Bank et al., 1978; Briggman et al., 1981).
Sections and replicas were viewed on either
a n Hitachi HS-9 or a JEOL 100 S electron
microscope operated a t 60 or 80 kV, photographed at magnifications ranging from
7,000 to 40,000, and photographically enlarged between two- and sixfold.
Well-focused,freeze-fracture micrographs of
junctions were used for measuring junctional
depth and for counting sealing strands (Table
1).A line was drawn perpendicular to the
luminal surface at the midpoint of the exposed junction. In very extensive junctions
(more than 3 pm in length parallel to the
luminal surface), two such lines were drawn,
each a third of the way from each end of the
junction. The distance between the luminal
strand (usually a t the luminal surface) and
the basal edge of the basalmost sealing
strand was measured with a n ocular micrometer graduated with a 0.1-mm scale. These
measurements were converted to pm (the
junctional depth) using a conversion factor
appropriate for the magnification of the micrograph. The number of sealing strands intersecting the perpendicular line (including
the most luminal and basal strands) was also
counted.
RESULTS
Normal Morphology
Junctional complexes between acinar cells
(Fig. 2 ) separated the lateral intercellular
space from the acinar lumen and from intercellular canaliculi. These canaliculi (see Fig.
1)are fingerlike extensions of the acinar lumen between acinar cells. Between acinar
cells, the region of close apposition of cell
membranes (tight junction or zonula occludens) was short and was usually accom-
72
J.A.V. SIMSON AND H.L. BANK
TABLE 1. Characterization of salivary gland junctions
Number of
sealing strandsa
Epithelial
segment
Depth (pmIa
X+O
X f O
Acinus
0.25 5 0.15
Intercalated duct
(N = 28)
0.21 0.05
Striated duct
(N = 14)
0.43 k 0.11
(N = 13)
2.5 f 0.8
(N = 28)
2.5
0.6
(N = 14)
6.0 5 1.6
(N = 13)
+
Percent of
junctions with
precipitatesb
63%
= 178)
9%
(N = 64)
5%
(N = 40)
(N
aN is the number of identifiable junctions analyzed. The means of the measurements on striated
duct junctions were significantly different (c< 0.01%confidence interval) from the means of acinar
and intercalated duct junctions in terms of both depth of junctzon and number of sealing strands.
There were no significant differences between acinar and intercalated duct cell junctions for either
arameter.
'The method used for evaluating permeable junctions is described in Materials and Methods. N is
either the total number of lumens (intercellular canaliculi) of acini or the total number of apical
junctions in the ductal segments.
panied by a single underlying desmosome.
Between intercalated duct cells, the region of
close apposition was also short (Fig. 3) and
numerous desmosomal attachments were
seen basal to the tight junctions. Between
the cells of the striated duct, a long region of
close apposition characterized apical tight
junctions (Fig. 4).Basal to the tight junction
was a well-developed zonula adherens (ZA),
with numerous underlying desmosomal attachments; a mat of cytoplasmic filaments
radiated from the ZA and desmosomes.
There was also considerable variability in
the depth of the junctions in this segment
(Table 1).
Because intercalated ducts were the shortest glandular segment, only five examples of
intercalated ducts with a total of 14 demonstrable junctions were unequivocally identified in freeze-fracture replicas. These
junctions were characterized by two parallel
sealing strands (Table 1) with intermediate
strands anastomosing at perpendicular angles rather than oblique angles as occurred
in acinar cell junctions (Fig. 11).DiscontinuLead Ion Tracer Technique
ities were not observed in the ridges of sealThe lead ion readily penetrated the junc- ing strands of these junctions, and there was
tions between acinar cells, gained access to less variability in the junctional depth than
most of acinar lumina, and was visible in was seen in acinar junctions (Table 1).
sections as luminal precipitates (Figs. 5, 6;
Junctions between striated duct cells were
Table 1). Tracer deposits were sometimes much more extensive than in the other two
present on the luminal side of junctional epithelial segments examined. These junccomplexes between intercalated duct cells tions consisted of five to eight sealing strands
(Fig. 7). However, these junctions appeared arranged in a bifurcating and anastomosing
to be much less permeable to the tracer than meshwork oriented roughly parallel to the
acinar junctions (Table 1).The lead tracer lumen (Fig. 12). The depth of the junction
rarely penetrated junctions between striated was about twice that of acinar and intercaduct cells (Fig. 8, Table 11, although it was lated duct junctions, and the sealing strands
present between the extensive basal and lat- were more tightly packed (Table 1).The depth
eral membrane infoldings (Fig. 9).
and number of sealing strands of striated
duct junctions were statistically significantly
Freeze Fracture
different (P < .01) from those parameters in
In junctions between acinar cells, few seal- junctions of either acini or intercalated ducts.
ing strands were present (Fig. 10, Table 1).
DISCUSSION
These sealing strands consisted of ridges (P
face) and grooves (E face) arranged in a loose
In this study, the permeability of junctions
meshwork with, usually, two strands paral- to an ionic tracer has been correlated with
lel t o the lumen and incomplete, auxillary the freeze-fracture morphology of tight juncstrands anastomosing at oblique angles. tions in three separate epithelial segments of
Ridges of sealing strands of acinar cell junc- rat parotid gland. Most junctions between
tions were often discontinuous (Fig. lob). acinar cells permitted ready passage of the
PARACELLULAR SECRETORY PATHWAY IN PAROTID
Fig. 2. Junctional region between two acinar cells.
The tight junction (arrow) is located subjacent to the
lumen (Lu) of an intercellular canaliculus. Two short
desmosomal junctions lie between the tight junction and
the intercellular space (ICS).Uranyl acetate, lead citrate
(UA-LC)-stainedthin section, ~ 5 6 , 0 0 0 .
Fig. 3. Junctional region between two intercalated
duct cells. The tight junction is indicated by the arrow.
Because of extensive desmosonal attachments between
73
these cells, the lumen (Lu) and intercellular space (ICS)
are more widely separated than in acini. UA-LC stain,
~56,000.
Fig. 4. Junctional region between two striated duct
cells illustrating the long tight junction (arrow) characteristic of this segment. The zonula adherens (ZA) and
its associated mat of filaments are cut tangentially; basal
to it lies a well-defined desmosome (D). UA-LC stained,
~56,000.
74
J.A.V. SIMSON AND H.L. BANK
Fig. 5. Lead tracer method, illustrating tracer (arrow)
in an acinar lumen Gu). The tracer has apparently penetrated a tight junction between adjacent acinar cells.
UA-stained thin section, ~40,000.
Fig. 6. Lead tracer method illustrating tracer in the
lumen (Lu) of a n intercellular canaliculus between two
acinar cells. Lead deposits are also present at the sites
of the tight junctions (arrows). En bloc UA-stained,
x21,OOO.
Fig. 7. Lead tracer method illustrating a rare tracer
deposit (arrow) in the lumen (Lu)of an intercalated duct.
Most of the lead deposits are trapped within the intercellular space (arrowhead) basal to the junctional complex.
Unstained, X30,OOO.
PARACELLULAR SECRETORY PATHWAY IN PAROTID
75
Fig. 8. Lead tracer method illustrating tracer deposits between striated duct cells (arrows) but not in the
lumen (Lu).Unstained, ~ 2 0 , 0 0 0 .
Fig. 9. The lead tracer method illustrating that tracer
readily enters the labyrinthine plasmalemmal folds of
striated ducts from the basal (B) extracellular space.
Unstained, ~ 2 0 , 0 0 0 .
lead tracer, and these junctions had few (two
to three) loosely-arranged, frequently discontinuous sealing strands, characteristic of
“leaky” tight junctions. In contrast, junctions between intercalated duct cells stopped
passage of most lead tracer ions and had two
to three well-ordered junctional sealing
strands with continuous apical and basal
strands. Most junctions between striated duct
cells were composed of several (five to eight)
closely packed sealing strands. These junctions almost totally impeded passage of the
lead tracer during the brief perfusion. Although acinar and intercalated duct junctions possessed approximately equal numbers
of sealing strands, the observed differences
76
J.A.V. SIMSON AND H.L. BANK
Fig. 10. Freeze-fracture images of acinar tight junctions adjacent to the lumen (Lu).Circled arrows indicate
direction of shadow. a) The sealing strands form ridges
on the P face (upper arrow) and grooves on the E face
(lower arrow). A large secretory granule (SG)identifies
this as an acinus. b) This micrograph illustrates the
variable and often discontinuous (arrows) nature of the
particle-studded ridges of acinar tight junction sealing
strands. An open sealing strand is indicated by the upper
arrow. ~60,000.
Fig. 11. Freeze-fracture images of intercalated duct
tight junctions parallel to the lumen (TAU). Circled arrows
indicate direction of shadow. The fracture face often
passes from one membrane face to the other (*) in the
junctional region of this segment. Note the continuity of
the apical and basal sealing strands, as well as the
regularity of cross strands between them. a) E face (* on
P face). b) P face (* on E face). x60.000.
PARACELLULAR SECRETORY PATHWAY IN PAROTID
77
Fig. 12. Freeze-fracture images of striated duct junctions illustrating the width and complexity of the tight
junctions (arrows) between these cells. Circled arrows
indicate direction of shadow. a) Tight junction illustrating E face grooves. b) A complex junction illustrating a
probable intersection between three cells. c) A junction
in which the fracture plane exposes both the E face
(apical, near arrow) and P face (more basal, near asterisk). ~60,000.
in the paracellular permeability of lead ion
tracer through the two types of junctions may
result from differences in the thickness and
continuity of apical and basal sealing strands
and the presence of intermediate cross
strands in the intercalated duct junctions.
Our data may underestimate the junctional permeability in the various segments
for two reasons: 1)in thin sections, the probability of encountering sparse precipitates is
less than one, and 2) we cannot be certain
that all tracer ions penetrating a junction
have been precipitated. It is possible that
lead ions which penetrated junctions in duct
regions were less readily precipitated than
those in the acinar intercellular canaliculi,
because of the larger fluid volume which may
have diluted the tracer in the ductal lumen.
The differential permeability to the tracer in
the various gland segments cannot be accounted for on the basis of differences in flow
rates, since the present studies were per-
78
J.A.V. SIMSON AND H.L. BANK
formed on nonsecreting glands, and there is
no basal (unstimulated) secretion in these
glands. Studies are currently under way to
determine the influence of secretogogues on
junctional morphology and tracer permeability of the three main epithelial segments.
Our results basically conform to the model
initially proposed by Claude and Goodenough (1973) and supported by others (Humbert et al., 1976) that a direct correlation
exists between the “tightness” (i.e., impermeability to water and ions) of tight junctions and the number of sealing strands
visualized by freeze fracture. However, it has
become clear that other factors besides number of sealing strands-for example, sealing
strand continuity and geometry-are also important in determining permeability characteristics (Claude, 1978;Briggman et al., 1981;
Cereijido et al., 1981). Claude (1978) has suggested that the proportion of “open strands”
in a junction more strongly influences junctional permeability than does the total number of sealing strands.
Our results are also in general agreement
with previous freeze-fractureand tracer studies of salivary glands. De Camilli et al. (1976)
illustrated, but did not describe or discuss,
the freeze-fracture morphology of junctions
of the three epithlial segments of rat parotid
gland. Their images conform to those obtained in the present study. Shimono et al.
(1980) examined junctional morphology as
well as lanthanum and ruthenium red impregnation in rat sublingual gland. Their
findings were similar t o those in the present
study, in that junctions of acini had less depth
and possessed fewer sealing strands than
those of striated ducts. However, their data
on intercalated duct junctions were intermediate between those of acini and striated
ducts, whereas, in the present study, intercalated duct junctions more closely resembled acinar junctions. This discrepancy could
reflect a real difference in the intercalated
ducts in the two glands (sublingual vs. parotid), or could result from sampling difficulties in obtaining identifiable fractures
through intercalated duct junctions. Garrett
et al. (1982) found a very slight but detectable passage of vascularly perfused horseradish peroxidase (MW - 40,000) tracer into
saliva in actively secreting dog salivary
glands, and provided cytochemical evidence
that this may occur via “leaky” tight junctions between acinar cells. Oliver and Hand
(1978) observed acinar junctional permeabil-
ity to luminally administered horseradish
peroxidase in isoproterenol-stimulated rat
parotid glands.
These results bear upon the question of the
route and mechanism of passage of ions and
water into the salivary secretory fluid. Two
classical models have been proposed to account for net transfer of fluid across an epithelium (Ogilvie et al., 1963; Diamond and
Bossert, 1967). Both models involve transcellular flow (across two cell membranes and
the intervening cytoplasm) of ions and water.
The model of Ogilvie et al. (1963) involves
differential permeabilities of basal and luminal membranes. This model is interesting
in that it requires little energy, but it has
been difficult to test in biological systems.
The Diamond and Bossert (1967) “standinggradient” model requires substantial energy
to drive the postulated ion pumps necessary
for ion movement across a membrane against
a concentration gradient. Recent evidence
from microprobe analysis (Gupta and Hall,
1979) not only lends credence to the standing-gradient model in certain epithelia, but
also suggests, at least in insect salivary
glands, a paracellular contribution to the
generation of isotonic secretions. In fact, it is
clear that, in some epithelia, a major route
for the flux of ions and small molecules is via
“leaky” tight junctions (Fromter and Diamond, 1972; Wright and Pietras, 1978; Cereijido et al., 1981). Additional evidence for a
route for passive transfer of small molecules
in salivary glands comes from earlier studies
by Martin and Burgen (19621, who found that
low molecular weight sugars can enter the
saliva passively and their concentration in
the saliva is inversely correlated with molecular size. Furthermore, permeability to these
sugars was increased by sympathetic
stimulation.
The present study was designed to determine if paracellular transport could occur in
the parotid gland by employing analysis of
both junctional morphology by freeze-fracture and junctional permeability to an ionic
tracer. Although results from either method
alone must be interpreted cautiously, we believe that, taken together, the data from the
two methods used in this study strongly support an interpretation of differential permeability in the three major epithelial segments
of the rat parotid gland. Based on the freezefracture and lead tracer evidence obtained,
we predict that a paracellular secretory pathway exists between parotid acinar cells. Such
PARACELLULAR SECRETORY PATHWAY IN PAROTID
a pathway would provide a low-resistance
route for the passage of water, inorganic ions,
and small organic molecules into the saliva.
Control of the fluid flux through this potential paracellular pathway (and hence control
of the rate of secretion) could be achieved in
a t least three ways. 1) Junctional sealing
strand organization could be altered by altering intracellular calcium concentration (Cereijido et al., 1981; Pinto da Silva and Kachar,
1982). 2) Blood flow or blood pressure could
be altered by modifying local intravascular
pressure (Shannon et al., 1974). 3) Osmotic
and hydrostatic pressure gradients across the
junction may be varied physiologically. Such
a model for a paracellular secretory mechanism has been proposed recently (Simson,
1982).
From our results it is clear that a potential
paracellular secretory pathway exists between parotid acinar cells. Further physiological studies will be required to determine
the contribution of paracellular fluid flux to
the total secretion and to investigate the postulated control mechanisms.
ACKNOWLEDGMENTS
We would like to express our sincere appreciation to Ms. Jan Condon for excellent technical assistance, to Ms. Marion Hinson for
skilled secretarial assistance, and to Dr. Patrick Moore for careful reading and helpful discussion of the manuscript. This work was supported in part by NIH grant RR05767 to the
College of Dental Medicine (J.A.V.S.) and NIH
grant AM 18115 (H.L.B.).
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