Secretagogue response of goblet cells and columnar cells in
human colonic crypts
Dan R.
Halm and
Susan Troutman
Halm
Department of Physiology and Biophysics, Wright State University,
Dayton, Ohio 45435
 |
ABSTRACT |
Crypts of Lieberkühn were isolated from
human colon, and differential interference contrast microscopy
distinguished goblet and columnar cells. Activation with carbachol
(CCh, 100 µM) or histamine (10 µM) released contents from goblet
granules. Stimulation with prostaglandin
E2
(PGE2, 5 µM) or adenosine (10 µM) did not release goblet granules but caused the apical margin of
columnar cells to recede. Goblet volume was lost during stimulation
with CCh or histamine (~160 fl/cell), but not with
PGE2 or adenosine. Three-quarters
of goblet cells were responsive to CCh but released only 30% of goblet
volume. Half-time for goblet volume release was 3.7 min.
PGE2 stimulated a prolonged fluid
secretion that attained a rate of ~350 pl/min. Columnar cells lost
~50% of apical volume during maximal
PGE2 stimulation, with a half-time
of 3.3 min. In crypts from individuals with ulcerative colitis, goblet cells were hypersensitive to CCh for release of goblet volume. These
results support separate regulation for mucus secretions from goblet
cells and from columnar cells, with control mechanisms restricting
total release of mucus stores.
adenosine; cholinergic; histamine; inflammatory bowel disease; prostaglandin E2
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INTRODUCTION |
THE COLONIC EPITHELIUM of mammals secretes fluid and
mucus in response to various neurotransmitters and local mediators (9, 12, 13). Fluid secretion modifies luminal composition by increasing the
aqueous volume and by adjusting concentrations of electrolytes such as
K+ and
H+. Low rates of fluid secretion
could aid colonic motility and bacterial fermentation, whereas higher
rates seen in pathophysiological states would serve to flush bacteria
from the lumen. Mucus secretions contribute to the barrier function of
the epithelium by protecting the epithelial cells from abrasion and
also by slowing access of bacteria to those epithelial cells. Release
of mucus from goblet cells generally is separate from sustained fluid
secretion on the basis of sensitivity to specific secretagogues (35). A
mucous substance distinct from goblet cell mucin is released from crypt columnar cells during stimulation of fluid secretion (14, 16). Control
of secretion from goblet and columnar cells by separate secretagogues
permits alteration of the relative proportion of mucus types released
and the rate of fluid production.
Mucus secretion by goblet cells involves exocytotic release of granule
contents at the apical membrane, as indicated by ultrastructural studies (51). Movement of granules within the cell is a
microtubule-dependent process, and access of the granules to the apical
membrane is restricted by the actin cytoskeleton. Further advances in
examining dynamics of goblet cell secretion have been made using
video-enhanced differential interference contrast (DIC) microscopy of
living cells (4, 14, 20, 46, 52). Individual events of goblet granule
release were visualized as changes in light intensity, indicating a
rapid discharge (0.03-1.0 s) of mucus from granules (4, 20, 46).
The time course of stimulation with mucus secretagogues consists of a
rapid increase in the number of granule release events that reaches a
maximum in 1-3 min and then subsides to a much slower sustained
rate. Discharge of mucus from goblet cells in rabbit colonic crypts
occurs via a similar exocytotic process (14, 52), and columnar cells
also show stimulated emptying of contents from the apical pole of the
cell (14). The results presented in this study were obtained with DIC
microscopy of isolated human colonic crypts and indicate the presence
of two morphologically distinguishable types of cells: goblet and columnar cells. Distinct secretagogues stimulated release of mucuslike material from apically stored granules in each of these cell types.
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METHODS |
Specimens of human colon were obtained from surgical resection material
by a procedure approved by the Institutional Review Board (Ohio State
University and Wright State University). After release by the
pathologist, specimens were refrigerated in HEPES-buffered Ringer
solution until they were picked up and transported to the laboratory
(generally ~30 min). The epithelium was removed from underlying
muscle by blunt dissection and placed in ice-cold standard Ringer
solution. Colonic mucosal biopsies from cotton-top tamarins were
obtained during semiannual endoscopic screening of a colony maintained
by Dr. J. D. Wood at Ohio State University. This procedure was approved
by the Institutional Laboratory Animal Care and Use Committee at Ohio
State University. The tamarin biopsies were transported to the
laboratory in ice-cold HEPES-buffered Ringer solution within 20 min.
Human colonic crypts were isolated from resection material of 17 patients (9 women and 8 men, 15 Caucasians and 2 African-Americans; Ohio State University Hospitals) ranging in age from 35 to 69 yr.
Resections were roughly divided between ascending or transverse sites
(~55%) and descending or sigmoid sites (~45%); normal margins were released by the pathologist for experimental study. Resection had
been performed for cancer (12 of 17 patients), diverticulitis (2 of 17 patients), and inflammatory bowel disease (IBD, 3 of 17 patients). All
three patients with IBD were women: one had active ulcerative colitis,
one had nonactive colitis (resected for metastatic cancer), and one had
Crohn's disease. Information on patient medications was not available
for comparison with experimental results. Cotton-top tamarins are a
species of New World monkey that develops symptoms resembling
ulcerative colitis when in captivity (30). The tamarins supplying
colonic crypts for imaging (n = 4)
were assessed for extent of disease by histological measures, including
white blood cell infiltration: three had severe colitis, and one had
moderate colitis (personal communication, K. S. Tefend and J. D. Wood).
Differences among the human specimens were not readily discernible,
except for IBD tissue, which could be distinguished by using objective
measures presented in RESULTS. On the
basis of this general similarity and the pathologist's assessment of normal tissue margins, tissues from patients with cancer or
diverticulitis were grouped together as normal.
Individual colonic crypts were dissected from the colonic epithelium
with use of fine forceps on the stage of a dissecting microscope
(×40) in HEPES-buffered Ringer solution with 5% serum albumin
added. The standard mammalian Ringer solution contained (in mM) 145 Na+, 5 K+, 2 Ca2+, 1.2 Mg2+, 125 Cl
, 25 HCO
3, 4 POx
4, and 10 D-glucose. Solutions were
continually gassed with 95%
O2-5% CO2, which maintained solution pH
at 7.4. HEPES-buffered Ringer solution contained 10 mM HEPES with
HCO
3 replaced by
Cl
, and the pH was titrated
to 7.4.
Crypts were imaged in a chamber mounted on the stage of an inverted
microscope (Zeiss Axiovert); the bottom of the chamber was formed by a
no. 1 coverslip (14). Isolated crypts were held by glass pipettes or
adhered by a coat of polylysine to the chamber bottom coverslip. The
glass pipettes were made to accommodate the relatively short length of
colonic crypts and were manipulated with a system from Vestavia
Scientific (Vestavia Hills, AL). For luminal perfusion, the blind end
of the crypt was cut off with a sharpened needle before transfer to the
imaging chamber. Luminal perfusate was HEPES-buffered Ringer solution.
Luminal perfusion rate was estimated as 1-5 nl/min on the basis of
perfusion pressure, pipette dimensions, and previous measurements with
renal tubules (45). The bath was continuously perfused with standard
Ringer solution with use of a peristaltic pump, and effluent was
removed by suction tube; the solution was bubbled with 95%
O2-5%
CO2 and warmed by passage through
a heated water jacket to maintain bath temperature at 37°C. Bath
solution flow was 3 ml/min (~8 chamber vol/min).
Crypt images were formed with DIC optics: a ×40 oil immersion
(1.4 NA) lens and a nonimmersion (0.65 NA) condenser (6). A video
camera (model CCD300E, Videoscope International) was used to record
images to videotape (Sony) and to computer disk with use of the Image-1
system (Universal Imaging). A ×2 coupling lens (Diagnostic
Instruments) was used to fill the camera field.
Image analysis.
Morphometric measurements of recorded crypt images were performed with
Image-1 software. Quantification of volume was obtained with focus
adjusted to the midline of the crypt, because this plane of section
allowed ready measurement of crypt and lumen diameter as well as a
complete profile of the epithelium from base to apex. Crypt diameter
was taken at the base of epithelial cells on opposite sides of the
crypt (inside the pericryptal sheath); lumen diameter was taken at the
apexes of opposing cells. A consistent group of cells was monitored in
successive images by marking the boundaries between distinct,
identified cells near the lateral margins of the image frame. Diameters
were measured at nine evenly spaced points along the crypt segment
defined by the length markers. Segment length was determined at cell
apex, cell base, and midway along cell height for both epithelial
margins (6 values). Crypt (Vcr)
and luminal volumes (Vlu) (23)
were calculated on the basis of a piecewise cylindrical model with use
of average crypt diameter (D),
average lumen diameter (d), average
section length (L), and standard
deviations (
D and
d) of these values
(Eq. 1)
|
(1a)
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(1b)
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This
calculation assumes that cells seen in the plane of focus are
representative of all cells in that annulus of the crypt. Epithelial volume was calculated as the difference between crypt and
luminal volume. All volumes were normalized to epithelial volume
obtained in the initial control image for each experiment to
adjust for differences in defined segment lengths (cell number) between crypts. Generally, 300-400 crypt epithelial cells were represented by epithelial volume in each experiment. Repeated measures
on the same image indicated that reproducibility was ±0.1 µm for
diameter measurements and ±0.5 µm for length measurements. Propagation of these error estimates for volume calculations
(Eq. 1) gave an error estimate for
relative epithelial volume of ±0.6%.
Volume of the zone containing apical granules
(Vaz) was calculated (similar to
Eq. 1) by using the diameter of this
zone (Dg), measured from the base of goblet granule masses in cells on opposite sides of the lumen (Vaz = Vg
Vlu, where
Vg is goblet region volume); this
volume includes apexes of columnar cells, which often contain granules
(21, 40, 48). Normalization to control epithelial volume gave the
relative volume fraction for this apical zone. Error propagation
provides an error estimate for relative apical zone volume of ±3%.
Contribution of goblet granule masses to apical zone volume was
estimated by a point-counting technique (58). A square array of points
(2-µm image spacing) was overlaid (skewed from the crypt axis) on
crypt images, resulting in ~500 points over the apical zone. The
ratio of points within goblets to all points in the apical zone was the
fraction of volume contributed by goblets. This estimate requires two
corrections to reflect the actual volume distribution: section
thickness and crypt geometry. The highly refractile goblet granules
were readily recognized in images, but the contribution of goblet
granules is overestimated, because granules can be seen throughout the
depth of focus. The depth of focus expected for video imaging with this
lens-condenser combination, 0.7 µm (18), can be used to calculate a
correction of 0.88 (58). The other bias in the measurement
underestimates the contribution of goblets, because longitudinal crypt
midline sections were used, rather than random sections. Goblet
fractional areas measured by point counting were converted to volume
fractions (see APPENDIX) by use of
Eq. A5,
for each crypt, and
=
/4; the resulting correction factors ranged from 1.02 to 1.15.
Individual goblet granule masses were measured to monitor directly
volume changes in this apically located cell compartment. Volume of
goblets (Vgob) was calculated
from width (w) and height (h) of the granule mass by use of
Eq. 2, with the assumption of a
spheroidal shape
|
(2)
|
Eccentricity
[prolate (eprolate) and
oblate (eoblate)] in
goblet shape was calculated using Eq.
3 as a measure of distortions to this cellular
compartment
|
(3a)
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(3b)
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For
oblate goblets the ratio of width to height was inverted, and the
resulting eccentricity was assigned a negative value. These definitions
allow numerical comparisons of dissimilar goblets, with eccentricity
ranging from
1.0 to +1.0 as shape changes from short and wide
(oblate) to tall and narrow (prolate). Calculated goblet volumes
(Eq. 2) were corrected for
underestimation due to off-center optical sectioning of goblets within
the crypt midline images. Separate frequency histograms of raw width
and height measurements were deconvoluted to obtain an estimate of
actual mean width and height (58). Comparing these actual mean widths and heights with means of raw measured values gave a volume correction factor of 1.19 to adjust goblet volume calculations. Eccentricity values were not adjusted, because the distributions of width and height
produced similar underestimation, such that the ratio
(Eq. 3) was unaffected. Time courses
of goblet volume changes were obtained by measuring the same goblet
through a series of images taken during various stimulatory conditions.
In this analysis, only goblets that were clearly sectioned near the
center during the entire sequence were used. The average control volume
from these measurements was similar to the corrected volume obtained with the larger sample of all visible goblets.
Speed of refractile objects moving in the lumen was measured from video
recordings. Transit time between cursors (10- to 30-µm separation,
depending on rate of movement) was used to calculate speed. Size of
objects was obtained from width (orientation with lumen diameter) and
length. Object volume was calculated by assuming a spheroidal shape, as
for goblet granule masses (Eq. 2).
Values are means ± SE. Statistical comparisons were made using a
two-tailed Student's t-test for
paired comparisons, with significant difference accepted at
P < 0.05. For unpaired comparisons between groups, significant difference was accepted at
P < 0.05 from ANOVA with the method
of Newman and Keuls.
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RESULTS |
Isolated colonic crypts imaged with DIC microscopy (Fig.
1) exhibited the two
predominant cell types associated with this epithelium: columnar and
goblet cells (3, 14, 21, 40, 48). Focusing at the midline of the crypt
showed the full height of the epithelium, with apically located
granules above basally located nuclei. Surrounding the crypt epithelium
was a pericryptal sheath of myoepithelial cells (36, 44), which was
apparent as a fibrous layer with flattened nuclei. Goblet cells, in
particular, were distinct because of a densely packed cluster of highly
refractile granules stored in the apical pole, which had a
characteristic ovate profile. Generally, goblet cells were separated by
columnar cells having a more hourglass-shaped profile that arched over the neighboring goblet cells. Thus the luminal surface often was dominated by columnar apexes, even though goblet granules filled more
than one-half of the apical epithelial volume.

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Fig. 1.
Isolated colonic crypts. Differential interference contrast (DIC)
microscopy was used to image individual dissected colonic crypts. Focus
at midline of crypt tube provided a view of maximal crypt and lumen
diameters. A full epithelial profile from apex to base can be seen.
Pericryptal sheath is apparent at basal margin of crypt epithelium,
with oval nuclear profiles scattered along crypt (a few are marked by
*). Nuclei are apparent in basal pole of epithelial cells (a few are
marked by arrows), and goblet granule clusters in apical pole (a few
are marked by arrowheads). Columnar cell apexes generally appear as
triangular regions between goblet granule clusters. Crypt orifices are
to left. Scale bars, 20 µm.
A: central portion of a normal human
crypt shows goblet and columnar cells.
B: crypt from a patient with active
ulcerative colitis had a dilated lumen and an outside diameter (~110
µm) larger than most normal crypts. Epithelial height varied
considerably. Goblet granule clusters appeared narrower and shorter
than typical in normal crypts. C:
crypt from a patient with nonactive colitis appeared similar to normal
crypts, except lumen was relatively dilated and epithelial height
varied. D: crypt from a patient with
Crohn's colitis appeared normal. E:
crypt from a tamarin with severe colitis had goblet granule clusters
that were smaller than in tamarin with moderate colitis or normal human
crypts. Epithelial height varied considerably.
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Crypts also were obtained from colonic epithelium of patients with IBD
(ulcerative colitis and Crohn's disease; Fig. 1). During dissection
ulcerated regions of colon with active colitis had a fragile surface
epithelium and a sparse number of recognizable crypts (4 specimens with
active colitis were examined by dissection microscope; data not shown).
Those crypts present (Fig. 1B) were generally large in diameter (>100 µm). Goblet cells appeared less full of mucous granules than did normal crypts. Colonic epithelium from
patients with nonactive ulcerative colitis (Fig.
1C) or Crohn's disease (Fig.
1D) was indistinguishable on
dissection from normal epithelium in crypt density or crypt size.
Crypts from all IBD patients had more focal spots of short epithelial
height than did normal crypts. Colonic epithelium of tamarins had
densely packed crypts, even in specimens with severe colitis. As for
crypts from patients with IBD, the tamarin epithelium had focal spots of short cell height (Fig. 1E) and
relatively depleted granule stores in goblet cells. Results from
tamarin crypts are specifically indicated; all other results are for
human crypts.
Crypt dimensions were measured and assigned to three groups (Table
1): normal (see
METHODS), IBD, and tamarin. A
distinction also was made for crypts from the normal group that were
opened for luminal perfusion. Differences in size among these groups were not apparent statistically. The focal spots of short epithelial height seen in crypts from patients with IBD and in tamarin crypts were
not extensive enough to cause a decrease in average epithelial height;
however, the lumen diameter of perfused crypts and crypts from patients
with ulcerative colitis had a tendency to be larger.
Secretory stimulation.
Addition of secretagogues that stimulate mucus and fluid secretion
produced several changes in crypt geometry, as well as epithelial cell
morphology. The cholinergic agent carbachol (CCh) stimulated mucus
release from goblet cells, seen as shrinking of apical granule clusters
and as light flashes, presumably the result of refractile contents
exiting individual granules (4, 20, 46). In some cases, evanescent
plumes could be seen forming above the sites of flashes, which then
drifted away from the cell surface. Flashes ceased within a few seconds
of CCh removal. Concurrent with goblet granule release, crypt lumens
dilated (Fig. 2). Histamine also stimulated
mucus release from goblet cells, as indicated by numerous apical
flashes. Stimulation with prostaglandin
E2 (PGE2) or adenosine produced
fluid secretion and recession of columnar cell apexes without any
apparent release from goblet cells (Fig.
3). Occasional light flashes were seen at
columnar cell apexes, but these events were not consistently observed. Fluid flow toward the crypt orifice was visualized by the rapid movement of refractile particles along the lumen. The cholinergic response of tamarin crypts was dramatic, with nearly complete disgorging of mucous granules and large dilation of the lumen (Fig.
4). Large plumes of mucus stayed tethered
to the cell of origin (Fig. 4C).
Subsequent stimulation with adenosine (Fig. 4D) further shortened the epithelium
and produced rapid fluid flow along the lumen that eventually ripped
mucous plumes from goblet cells.

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Fig. 2.
Cholinergic stimulation. Goblet granule clusters (arrowheads) responded
to a cholinergic agonist. Individual granules are discernible within
some of these clusters. Crypt orifice is to
right. Scale bars, 10 µm.
A: midline DIC image from central
portion of a normal crypt in control condition.
B: crypt in
A 5 min after beginning of cholinergic
stimulation [100 µM carbachol (CCh)]. Lumen diameter
increased concurrent with loss of apically stored material from goblet
cells. Release of mucin granules produced craters in some goblet
cells.
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Fig. 3.
Prostaglandin E2
(PGE2) stimulation. Response of
columnar cells (arrowheads) to a fluid secretagogue. Crypt orifice is
to left. Scale bars, 10 µm.
A: midline DIC image from central
portion of a normal crypt in control condition (after return from
stimulation with 2 µM CCh). A large globule of goblet mucus (~10
µm diameter) can be seen in lumen (under 2nd downward arrowhead from
left), remaining from earlier
release event. B: crypt in
A 5 min after beginning of stimulation
with PGE2 (2 µM). Lumen diameter
increased through a recession of columnar apexes without any apparent
change in goblet cells.
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Fig. 4.
Secretory response of tamarin crypt. Hypersensitivity of goblet cells
to cholinergic stimulation was apparent in a crypt from a tamarin with
moderate colitis. Crypt orifice is to
left. Scale bars, 10 µm.
A: control goblet cells had apical
poles with moderately large granule clusters (arrowheads). Individual
granules were apparent within goblet clusters.
B: during stimulation (5 min) with CCh
(100 µM), almost all goblet clusters of granules were extruded into
lumen. Individual granules were still apparent within extruded luminal
mucous plumes. C: after removal of
CCh, further release of granule contents was not apparent, and lumen
remained full of goblet mucus. Lysis of extruded granules became
evident (10 min) from a uniform evanescent appearance of mucous plumes
above goblet cells. (Top center and
bottom right plumes in
B could not be clearly identified.)
D: subsequent stimulation of fluid
secretion by adenosine (10 µM) for 10 min pushed mucous plumes down
lumen toward crypt orifice, at left
~100 µm. (Only top right and
bottom center plumes in
B could still be identified.)
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Crypt shape changed during secretory stimulation through an increase in
lumen diameter and a small decrease in crypt diameter (Fig.
5). After sequential stimulation with these
two classes of secretagogues (goblet type and fluid type), the crypts
maintained an enlarged lumen diameter (Fig.
5D). The changes in crypt and lumen
diameters with each secretagogue (Fig. 5,
B and
C) are consistent with the decreased
epithelial volume expected from release of apically stored mucus.
Change toward a smaller crypt diameter and larger lumen diameter also
could have resulted from a simple crypt elongation caused by cells
becoming wider and shorter without losing volume. Including a
measurement of length to monitor a consistent group of crypt epithelial
cells would provide an unambiguous calculation of epithelial volume.




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Fig. 5.
Crypt dimensions. Lumen and crypt diameters of individual crypts are
shown. Dashed line, lumen diameter expected for case of epithelial
cells with zero height; dotted line, lumen diameter expected for case
of constant epithelial volume (with assumption of constant cell width),
fit to mean of normal control group.
A: crypt dimensions before any
stimulation. , Normal; , normal-perfused; , ulcerative colitis
(N, nonactive); , Crohn's disease; , tamarin.
B: changes in diameter induced by CCh
(10-min stimulation). Open symbols, control; filled symbols,
stimulated; circles, normal; squares, normal-perfused; triangles,
ulcerative colitis; inverted triangles, tamarin. Some control points
are return to control (~10 min) from another secretagogue.
C: changes in diameter induced (10-min
stimulation) by a fluid secretagogue,
PGE2 or adenosine (A); symbols as
in B. D: diameters after
return to control (10-15 min) from sequential stimulation by CCh
and a fluid secretagogue. Symbols as in
A.
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Secretagogue-induced changes in epithelial volume.
Epithelial volume of crypts was monitored during secretagogue
stimulation by picking a consistent segment of crypt length over which
to measure crypt diameter and lumen diameter. Segment length was
defined from the boundaries between identifiable cells positioned near
the edges of the recorded image fields. A representative experiment
from the central portion of a normal crypt is shown in Fig.
6. Volumes (Fig.
6B) were calculated from the
measured crypt dimensions (Fig. 6A;
see METHODS). Epithelial volume
decreased with CCh addition and did not return in the subsequent
control period. With addition of
PGE2, a decrease in epithelial
volume that was not recovered on return to control
conditions also occurred.


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Fig. 6.
Epithelial volume changes. A: a
representative time course of dimensions from central portion of a
normal crypt (~300 cells) during stimulation by CCh (100 µM) and
PGE2 (5 µM). Open symbols, equal
to prior measured value, indicate addition of CCh and
PGE2. Segment length represents
distance between distinct, identified cell boundaries positioned at
either margin of recorded image field. Epithelial height
(hep) was
calculated from crypt diameter (D)
and lumen diameter (d):
hep = (D d)/2. Error bars, SE.
B: crypt and lumen volumes calculated
using crypt and lumen diameters together with segment lengths (see
METHODS). Epithelial volume was
calculated as difference between crypt and lumen volumes.
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Relative changes in epithelial volume were measured in the central
portions of crypts during stimulation by CCh, histamine, PGE2, and adenosine (Fig.
7). Normal crypts had a sustained ~4% decrease in epithelial volume induced by CCh (Table
2). The only crypts that did not exhibit a
decrease in epithelial volume (Fig. 7A) were three perfused crypts with
dilated lumens that appeared relatively depleted of goblet granules
before stimulation. Tamarin crypts had the largest responses to CCh. As
a group, the responses to histamine showed no distinct change in
epithelial volume (Fig. 7B), even
though flashes indicative of granule release events were seen during
stimulation. Addition of PGE2
produced a sustained ~5% decrease in epithelial volume (Fig.
7C, Table 2), with only one perfused
normal crypt not showing a decline. Similarly, adenosine addition
resulted in a decrease in epithelial volume (Fig.
7D); a crypt from the patient with
nonactive ulcerative colitis was relatively unresponsive but lost
volume during subsequent PGE2 stimulation.




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Fig. 7.
Epithelial volume loss. Relative epithelial volume changes were
normalized to control value preceding stimulation. , Normal; ,
normal-perfused; , ulcerative colitis (N, nonactive); , Crohn's
disease; , tamarin. Last point generally was return to control (*).
A: CCh (maximal stimulation,
2-100 µM). B: histamine (10 µM). C:
PGE2 (2-5 µM).
D: adenosine (100 µM).
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The epithelial volume decreases that were sustained after return to
control (Fig. 7) are consistent with a volume loss due to release of
cellular material (presumably mucus) from apical stores (Figs.
2-4). Transient portions of epithelial volume responses may
reflect changes in cytoplasmic volume during secretion that recover
after removal of the stimulus. The portion of epithelial volume
contributed by apical granules in goblet and columnar cells (Vaz) was estimated for each
crypt from the radial extent of the apical goblets (see
METHODS);
Vaz ranged from 0.29 to 0.48 and averaged 0.35 ± 0.02. Loss of epithelial volume (Fig. 7) was
normalized to the individual measures of
Vaz to provide an indication of how much of the stored material was released (Table 2, Fig.
8). The total amount of
Vaz released after stimulation by
goblet- and fluid-type secretagogues was largest in those crypts from patients with ulcerative colitis and from tamarins (Fig.
9) but still did not exceed the measured
control apical stores.



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Fig. 8.
Relative volume loss of apical granule storage zone. Time courses in
Fig. 7 were normalized to apical zone volume fraction (~0.35) for
each crypt. , Normal; , normal-perfused; , ulcerative colitis
(N, nonactive); , Crohn's disease; , tamarin. Experiments
without clearly sustained volume decreases in Fig. 7 were not included.
* Return to control. A: CCh.
B:
PGE2.
C: adenosine.
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Fig. 9.
Depletion of apically stored materials. Proportion of epithelial volume
lost after sequential stimulation by CCh and a fluid secretagogue
(PGE2 or adenosine) is shown in
relation to measured initial contribution of apical zone to total
epithelial volume. , Normal; , normal-perfused; , ulcerative
colitis (N, nonactive); , Crohn's disease; , tamarin.
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The contribution of goblet granules to
Vaz was measured by point counting
in control crypt images (see
METHODS). For normal crypts the
goblet fraction was 0.65 ± 0.03 (n = 8). Crypts from patients with IBD had goblet fractions statistically
indistinguishable from crypts from normal patients (0.64 ± 0.04, n = 3). The CCh-induced loss of
Vaz (Table 2) can be transformed
by using the goblet fraction to estimate the percentage of goblet
granule volume released in normal crypts: ~30%. For crypts from the
two patients with ulcerative colitis, goblet volume release was
55-75%. Tamarin crypts released ~80% of goblet volume.
Similarly for PGE2-stimulated volume loss (Table 2), normal crypts released ~50% of nongoblet (columnar cell) Vaz. Release from
this columnar cell apical zone was 80-100% for crypts from
patients with ulcerative colitis and from tamarins.
Crypt diameter and length relationships may be controlled in part by
the contractile state of the pericryptal sheath (36, 44). Normal
crypts maintained length within 1% of control values during
stimulation with CCh (n = 6) and then
shortened by ~2% on return to control conditions. Perfused
crypts (n = 5) shortened by ~2%
during CCh stimulation and maintained that shortening on return to
control conditions. Crypts from patients and tamarins with ulcerative
colitis (2 humans and 2 tamarins) lengthened by ~4% during CCh
stimulation and then shortened to roughly control length on return to
the control condition. During PGE2
stimulation, normal crypts (perfused and nonperfused) maintained length
within 1% of control values (n = 7)
and then shortened by ~2% on return to control conditions. Crypts
from patients and tamarins with IBD (n = 3: active colitis, Crohn's disease, and tamarin) shortened by ~3%
during PGE2 stimulation and
maintained that shortening on return to control. Together with the
decreases in crypt diameter (Figs. 5 and 6), these changes in crypt
length suggest that the pericryptal sheath responds to
secretagogues and contributes to maintenance of crypt dimensions.
Luminal fluid flow.
Fluid flow along the lumen was apparent from movement of refractile
particles. Sustained movement of these objects in the lumen was seen
only during secretagogue stimulation. Presumably, the objects were made
up of released mucus and other cellular debris. In some crypts,
particles were small (~3 µm diameter) and seen infrequently,
whereas in other crypts the lumen was crowded with globular objects.
Stimulation by CCh of a crypt with globular objects already present in
the initial control condition (Fig. 10A)
produced a measurable particle flow toward the crypt orifice. Removal
of the stimulus stopped flow with a lag of ~1 min, although the
flashes indicative of granule release stopped within a few seconds.
PGE2 addition immediately produced
flow toward the crypt orifice. Movement was episodic, with particles
slowing down as larger globular objects squeezed past each other.
Dilation of the lumen alleviated this congestion and progressively led
to faster particle speeds. Addition of CCh together with
PGE2 led to reduced speed,
consistent with inhibition of fluid secretion. Particle speeds were
highest for smaller objects (Fig.
10B). All moving particles were
tracked during stimulation, but blurred streaks expected for very fast
particles were not observed. In crypts with relatively unobstructed
lumens, PGE2 produced particle speeds of ~10 µm/s throughout the period of stimulation, whereas CCh stimulation resulted in particle speeds comparable to
PGE2 for only 2-3 min, after
which movement slowed and became undetectable.


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Fig. 10.
Luminal fluid flow. Speeds of refractile particles moving in lumen of a
normal crypt (shown in Fig. 6) measured during stimulation with
secretagogues. A: time course of
particle speeds. Particle size was measured to compare cross-sectional
area with luminal cross-sectional area. , Particles with area <5%
that of lumen; , particles with area >20% that of lumen; ,
particles of intermediate cross section. Large particle monitored at
~25 min squeezed past several other objects at a relatively high
speed, propelled by full force of stream.
B: variation of particle speed with
particle volume during PGE2
stimulation. One large particle occluded lumen during speed measurement
(*).
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Generally, particles moved as discrete entities, with faster speeds
nearer the center of the lumen, as expected for laminar flow. Volume
flow through these crypt sections can be estimated from the speed of
the fastest particles and the cross-sectional area of the lumen. The
fastest speeds in Fig. 10 correspond to a crypt fluid flow of ~10
pl/min during CCh stimulation that increased to ~350 pl/min during
PGE2 stimulation (see
DISCUSSION); fluid flow decreased to
~210 pl/min after CCh addition with
PGE2. Retrograde flow consistent
with fluid absorption was not observed. For tamarin crypts, maximal
fluid flow with PGE2 or adenosine
stimulation tended to slowly push extruded goblet mucus globules along
the lumen walls rather than as free objects in the center of the lumen. The crypt from the patient with active colitis (Fig.
1B) formed a continuous filament
(~15 µm diameter) of presumptive goblet mucus in the center of the
lumen that was dragged toward the crypt orifice during
PGE2 stimulation. Particle
movement in the fluid surrounding the filament was comparable to that
in Fig. 10, but the speed of the filament was only ~0.5 µm/s.
Volume flow of mucus carried in the filament was ~5 pl/min, which
stopped at the end of PGE2 stimulation.
Stimulated release of cellular contents.
Loss of epithelial volume during stimulation by goblet- and fluid-type
secretagogues appeared to be restricted to specific cell types. Goblet
cells were the site of cholinergic volume loss. Release of mucin from
apical granules began at the luminal margin, occasionally progressing
to make deep craters within the apex of goblet cells, presumably by
fusion of individual pits (Fig. 11).
Columnar cell apexes continued to arch over goblet cells during CCh
stimulation, suggesting a lack of volume release from this cell type.
An en face view of the epithelium (Fig.
12) shows the craters centered within the
goblets and the spatial arrangement of goblet and columnar cells within
the epithelium. Craters generally did not persist, with goblet granule
clusters instead rearranging to form smaller spheroids.

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Fig. 11.
Cellular response to cholinergic stimulation. Addition of CCh
stimulated progressive release of mucin granules, producing apical
craters in goblet cells (arrowheads), without evidence of release from
columnar cells (bars with *). Scale bars, 5 µm. Three pairs of images
are shown for control and stimulated conditions
(A1 and B1,
A2 and B2, and
A3 and
B3).
A: control goblet cells had broad
clusters of granules. Columnar cells arched over goblet cells, such
that only goblet apexes were in contact with lumen.
B: during CCh stimulation (100 µM
for ~10 min), pits formed in apexes of goblets, with some becoming
deep invaginations into apical granule clusters.
C: goblet during CCh stimulation
(~10 min) shows an apical crater connected to lumen (apical margin is
along lower edge of goblet, with lumen below). Individual
~0.9-µm-diameter granules (arrowhead) also are visible. Scale bar,
2 µm.
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Fig. 12.
Goblet response to CCh. A deeper focal plane (below midline), along
crypt center line, provides an optical section through lower apical
region of crypt cells. This en face image plane shows goblet cells as
circular cross sections (arrowheads) and columnar cells as polygonal
shapes filling intervening spaces. Focus was ~16 µm above base of
cells along center line, which was determined from crypt width
(w) in these images compared with
crypt diameter
(Dcr) and with
assumption of a cylindrical crypt shape:
h = (Dcr/2)
{1 [1 (w/Dcr)2]1/2}.
Longitudinal axis of crypt runs from
bottom to
top of these images. Scale bars, 5 µm. A: control condition.
B: during CCh response (~5 min),
goblet granule clusters showed rearrangement and pitting. Deformation
of goblet cluster at top of field is readily apparent from convex edges
of cell.
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The response to CCh of a crypt from the patient with active ulcerative
colitis was consistent with hypersensitivity of goblet cells (Fig.
13). As with the crypt from a tamarin
with moderate colitis (Fig. 4), released mucus remained tethered to the
cells of origin, giving a cobblestone appearance to the apical surface. The crypts from tamarins with severe colitis had a similar appearance on CCh stimulation.

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Fig. 13.
Cholinergic response in ulcerative colitis. CCh addition stimulated
mucus release in a crypt from a patient with active ulcerative colitis
(Fig. 1B). Scale bars, 5 µm.
A: control goblet cells had relatively
depleted stores of granules. This crypt had been stimulated previously
by 5 µM PGE2.
B: during CCh stimulation (100 µM
for ~10 min) mucus was released and remained adherent to
epithelium.
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Stimulation of fluid secretion with
PGE2 or adenosine did not produce
any noticeable changes in granules of goblet cells. Although apical
vesicles or vacuoles were not generally discernible in columnar cells
(Figs. 11 and 14), fluid secretion was
accompanied by selective recession of columnar cell apical borders
(Fig. 14), consistent with loss of apically stored contents.

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Fig. 14.
Cellular response to PGE2
stimulation. PGE2 addition
produced recession of columnar cell apical margins (bars with *),
without evidence of release from goblet cells (arrowheads). Scale bars,
5 µm. Two pairs of images are shown for control and stimulated
conditions (A1 and
B1, and
A2 and
B2).
A: control columnar cells had narrow
profiles that fanned out near luminal margin. Lack of sharp contrast at
lumen edge suggests a lower density of cell contents than in
neighboring goblet granules. B: during
PGE2 stimulation (5 µM for ~10
min), luminal margins between goblet cells (presumptive columnar cells)
receded.
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Volume of stored mucus was calculated for individual goblet cells
(Table 3) by measuring the width and height
of the apical granule cluster, with the assumption of a spheroidal
shape (see METHODS). Goblet clusters
in crypts from patients with IBD were smaller than those in crypts from
normal patients. Eccentricity of the goblet granule cluster (ratio of
width to height, Eq. 3) provides an
indication of granule arrangement within the goblet cluster; goblet
granule clusters in perfused crypts and tamarin crypts were more nearly
spherical than those of normal crypts, suggesting less restrictive
packing. The relationship between control goblet volume and
eccentricity is shown in Fig. 15.
Distortion of large goblets into more prolate shapes suggests a
constraint on packing larger granule volumes into the tubular crypt
structure, with goblet crowding around the lumen.

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Fig. 15.
Size and shape of apical goblets. Goblet granule clusters were measured
for individual goblet cells in control conditions. Volume and
eccentricity (mean ± SE) were calculated for each crypt (number of
goblet cells measured ranged from 9 to 23/crypt). , Normal; ,
normal-perfused; , ulcerative colitis (N, nonactive); , Crohn's
disease; , tamarin. Positive eccentricities indicate a prolate
shape; negative values indicate an oblate shape (see
METHODS).
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Volume stored in individual goblets was measured before and after
maximal stimulation by CCh. The goblet volume released by human crypts
was ~25% of the control value (Fig.
16A).
Release at 2 and 10 µM CCh was >90% of the value at 100 µM,
indicating that the half-maximal stimulating concentration for CCh was
<0.2 µM. In response to histamine, ~30% of control goblet volume
was released in human and tamarin crypts (Fig.
16B). Addition of atropine, a
muscarinic antagonist, before and during histamine stimulation did not
alter the response (data not shown). Incomplete discharge of goblet
stores during maximal activation suggests stimulatory mechanisms
(cholinergic and histaminergic) that limit granule release. Goblets
from tamarin crypts, however, released nearly all the stored contents
with CCh stimulation (Fig. 16A) but
not with histamine stimulation (Fig.
16B), supporting a selective lack of
restrictive cholinergic regulation in these tamarins.


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Fig. 16.
Volume loss from apical goblets. Volume released from goblet granule
clusters is shown in relation to volume stored before stimulation (mean ± SE). Dashed line, total release.
A: volumes released with CCh
stimulation [100 or 2 µM (2) or 10 µM (10)]. ,
Normal; , normal-perfused; , ulcerative colitis (N, nonactive);
, Crohn's disease; , tamarin. Dotted line, 25% release. Some
crypts had been stimulated by histamine before stimulation by CCh (*).
B: volumes released with histamine
stimulation (10 µM). Symbols as in
A. Dotted line, 30% release.
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The two independent measures of CCh-induced goblet volume release
(Figs. 7A and Fig.
16A) were compared quantitatively by
using the goblet fraction of epithelial volume; Fig.
17 shows the goblet volume changes
obtained from these individual (goblet volume) and global (epithelial
volume) measures. Normal human and tamarin crypts had volume changes
close to the line of identity, supporting a conclusion that these two
measures of volume release represent the same cellular events. Crypts
from the two patients with ulcerative colitis deviated significantly
from the line of identity, suggesting that measurements of individual
goblet volumes underestimate total volume released in these crypts. The
larger response measured with epithelial volume changes could result
from a columnar cell contribution in ulcerative colitis, but a more
likely explanation is that smaller goblet clusters seen in ulcerative
colitis crypts (Table 3, Fig. 13) were underrepresented in measurements
of goblet volume, since these small clusters were excluded because of
difficulty in reliable tracking through the sequence of stimulation.

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Fig. 17.
Comparison of volume release measurements. Average relative volume
change of individual goblet granule clusters
(Vgob) in crypts produced by CCh
stimulation (mean ± SE) is shown in relation to relative volume
change of apical goblet zone [goblet fraction of epithelial
volume (Vep)]. ,
Normal; , normal-perfused; , ulcerative colitis (N, nonactive);
, tamarin. Dashed line, line of identity. Ulcerative colitis crypts
are significantly off line of identity; all other values are not
significantly different from line of identity.
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Responsiveness of individual goblet cells.
The distribution of CCh-induced fractional goblet volume release for
individual cells is shown in Fig.
18A. Two
distinct peaks occurred: one near zero volume release and one at higher
release. This bimodal distribution is consistent with a nonresponding
group of goblet cells dispersed among responding goblet cells.
Comparison of the fraction of goblet cells responding within a crypt to
the average initial goblet volume (Fig.
18B) indicated a greater proportion of nonresponding cells in crypts with larger goblet clusters. A similar
relation for histamine stimulation (Fig.
18C) suggested that the histamine
response was not related to goblet size. In those crypts exposed to
both CCh and histamine (3 crypts, 27 goblets), 43% of goblet cells
responded (defined as in Fig. 18) to both agents, but the
CCh-nonresponsive cells responded to histamine and the histamine-nonresponsive cells responded to CCh. Apparently all these
goblet cells were capable of stimulated release of granule contents.



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Fig. 18.
Responsiveness of apical goblets. A:
relative frequencies of CCh-induced fractional goblet volume release
for all goblet cells in normal crypts
(n = 74). Fitted curve is sum of 2 Gaussian distributions: peak at small fractional release contains 23%
of goblets with a mean of 0.04 ± 0.01 and peak at higher fractional
release contains 77% of goblets with a mean of 0.29 ± 0.02. B: fraction of CCh-responsive goblet
cells for each crypt relative to average control goblet volume (mean ± SE). , Normal; , normal-perfused; , ulcerative colitis
(N, nonactive); , Crohn's disease; , tamarin. Responding goblet
cells were defined as those with a volume release fraction 0.20.
C: fraction of histamine-responsive
goblet cells for each crypt relative to average control goblet volume
(mean ± SE). Symbols as in B.
Responding goblet cells were defined as those with a fractional goblet
volume release 0.20.
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The nonresponding goblet cells were not present in any apparent pattern
within the central portion of the crypts. In tamarin crypts, after CCh
stimulation had dramatically depleted central goblet cells of granules
(Fig. 4), those goblet cells in the most distal 25% of the crypt
(closest to the surface epithelium) retained large stores of granules
(data not shown).
The group of responding cells was used to provide a time course of the
responses to the goblet secretagogues CCh and histamine. A secretory
response from a representative crypt is shown in Fig. 19A.
Adenosine did not alter goblet volume, but subsequent additions of
histamine and CCh produced rapid losses of goblet volume. The shape of
goblets also changed on stimulation. Goblets became more spherical
during adenosine addition but returned to a prolate shape in control
conditions. An irreversible change toward spherical occurred with
subsequent CCh addition; histamine did not dramatically alter goblet
shape. Average time courses for CCh and histamine goblet volume
responses had similar kinetics and magnitude (Fig. 19B). The CCh response of goblet
cells measured from epithelial volume changes was indistinguishable
from the goblet volume measurement, further supporting that these
independent measures reflect the same cellular event. Goblets from
tamarin crypts had a similar half-time for goblet volume release, but
nearly all the contents were released. A time course for volume release
from the apical zone of columnar (nongoblet) cells indicates a
secretory response of comparable speed for fluid secretagogues (Fig.
19C).



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Fig. 19.
Stimulation of goblet cell and columnar cell release.
A: time course of changes in goblet
volume and eccentricity induced by adenosine (100 µM), histamine (10 µM), and CCh (100 µM) for responding goblet cells (defined as in
Fig. 18) within a single crypt (mean ± SE,
n = 10). Goblet volume was normalized
to initial control level. Positive eccentricities indicate a prolate
shape, and negative values indicate an oblate shape. Open symbols
indicate addition of agonist. B:
relative goblet volume (mean ± SE) for responding goblet cells
during CCh stimulation of normal crypts ( ; 6 crypts, 41 goblets).
Histamine stimulation was averaged from crypts in Fig.
16B ( ; 5 crypts, 32 goblets).
Average CCh response of goblet cells from tamarin crypts [ ; 2 crypts (1 severely affected and 1 moderately affected), 26 goblets] is also shown. Apical goblet zone volume changes during
CCh stimulation ( , 7 crypts) were obtained from epithelial volume by
using goblet volume fraction, including fraction of goblets responding
(0.77, from Fig. 18). Dashed line, exponential decline with a half-time
of 3.7 min, which fits all but earliest time point in response.
C: volume loss (mean ± SE) from
nongoblet (columnar cell) apical fraction of epithelial volume for
stimulation by fluid secretagogues
PGE2 and adenosine (8 crypts).
Half-time obtained from exponential fit was 3.3 min.
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Expansion of extruded goblet mucus was particularly apparent in the
crypt from the moderately affected tamarin (Fig. 4). For seven goblets
in this crypt that appeared to release all the mucin granules, a mucus
expansion ratio was calculated. Volume of the extruded mucus was
measured from width and height (Eq. 2) and compared with goblet volume before CCh
stimulation. The average expansion ratio was 2.1 ± 0.1 and was
half-maximal at ~5 min after onset. Particles moving in the lumen
(presumptive goblet mucus, Fig. 10) were often larger than expected for
the effluent of a single goblet cell (>200 fl: 335 fl average goblet
volume · 0.3 mean fractional release · 2-fold expansion ratio),
suggesting that these secretory plumes could coalesce.
Changes in goblet shape during secretagogue-stimulated release of
apically stored contents are consistent with a constraint on packing
within the crypt structure. Loss of goblet volume during CCh
stimulation was associated with a rounding of the goblet profile (Figs.
19A and
20A),
consistent with relief from deforming forces as volume in the apical
zone decreased. Stimulation with
PGE2 or adenosine led to similar
shape changes without loss of volume from goblets (Figs.
19A and
20B), consistent with lower
deforming forces due to volume release in neighboring columnar cells.
Return of goblet eccentricity after removal of fluid secretagogues may occur through compaction as crypt length shortens, presumably through
the action of the pericryptal sheath. The prolate shape of control
goblet clusters became more spherical with secretagogues through
changes in width and height (Table 4, Fig.
20C). Responding goblets became
shorter and narrower with CCh, whereas nonresponding goblets became
shorter but wider. During PGE2 or
adenosine stimulation, all goblets became shorter and wider. These
goblet shape changes indicate that cells neighboring goblet cells,
presumably columnar cells, lost volume on stimulation with
PGE2 or adenosine.



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Fig. 20.
Stimulated rearrangement of goblet volume.
A: goblet eccentricity in relation to
goblet volume (mean ± SE) during stimulation by CCh for responding
goblet cells (defined as in Fig. 18). Open symbols, control; filled
symbols, stimulated; circles, normal; triangles, ulcerative colitis;
diamonds, Crohn's disease; inverted triangles, tamarin. Some crypts
had been stimulated by histamine before stimulation by CCh (*).
B: goblet eccentricity (for all goblet
cells) in relation to goblet volume before (#), during, and after
stimulation by a fluid secretagogue,
PGE2 or adenosine. Symbols as in
A. C:
changes in goblet dimensions for CCh-responsive goblet cells of each
crypt ( , , ), for average of CCh-nonresponsive goblet cells
from all crypts ( , volume fraction released <0.10), and for all
goblet cells during fluid secretagogue stimulation of each crypt ( ,
, ).
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DISCUSSION |
A major task of the colonic epithelium as the most distal site along
the alimentary tract is to reabsorb fluid. In contrast to this
conservation function, the colonic epithelium also secretes fluid. This
fluid has a distinct electrolyte and macromolecular composition. The
largest macromolecule secreted is mucus (9), which serves to buffer
cells against abrasion by luminal contents and to create a microclimate
(unstirred layer) near the epithelial surface. Mucus released in crypts
of Lieberkühn is cleared into the colonic lumen by fluid
secretion. As shown in RESULTS, crypts from human colon release apically stored material (presumably mucus)
from goblet and columnar cells. Separate control of these mucus
secretions occurs through distinct types of secretagogues.
Epithelial cell types in colonic crypts.
The colonic epithelium is comprised of several cell types that form the
two major epithelial structures: the surface epithelium and the crypts
of Lieberkühn. Similar to other mammals (3, 14), human colonic
epithelium contains two predominant cell types: columnar and goblet
cells (21, 40, 48). Columnar cells have been distinguished further on
the basis of ultrastructural features associated with the degree of
differentiation. Enteroendocrine cells, which release signaling
molecules, generally make up <5% of the cells. Goblet cells are
distinguished by the large number of mucous granules in the apical
pole. Although in crypts these cells lack the characteristic narrow
basal pole seen in the surface epithelium, crypt goblet cells have a
rigid cytoskeletal arrangement that maintains the round profile of the
granule mass (43, 51). In living crypts, with use of DIC microscopy,
goblet cells can be recognized readily by the ovate shape of this
granule cluster because of the highly refractile contents of the
granules (Figs. 1-4 and 11-14). Columnar cells fill the
spaces between goblet cells; distinctions among these nongoblet cells
were not apparent. Deformation by the more rigid goblet cell neighbors
produced a narrow waist between basal and apical poles, giving columnar
cells an hourglass appearance. The apical poles of columnar cells fan
out over goblet cells, such that only the small apexes of goblet cells
contact the lumen. Whereas in rabbits and mice the apical poles of
columnar cells are filled with large vacuoles (3, 14), columnar cells in human colon have small apical vesicles (21, 40, 48). Thus goblet and
columnar cells have apically stored products positioned for release
into the lumen. The general epithelial appearance is of goblet ovals
pointing toward the lumen and fan-shaped deltas of columnar cells with
a wide luminal extent.
Mucus secretion by goblet and columnar cells.
Secretory control of columnar and goblet cells in colonic crypts is
distinct. Release of mucus from goblet cells is stimulated by
cholinergic agonists and by histamine (35); goblet cells do not release
mucus in response to vasoactive intestinal peptide (VIP) or cAMP and
theophylline (35), agents that stimulate fluid secretion (13).
Responsiveness was assessed in fixed specimens by the presence of
apical cavitation in goblet cells, a procedure that would miss modest
stimulation of granule release. A morphometric assessment of
cholinergic stimulation indicated that goblet cells that did not
cavitate, particularly in the surface epithelium and crypt base, did
show a decrease in mucous granules (37, 39). From these studies of
fixed colonic tissue, the strongest goblet secretagogues are
cholinergic agonists and histamine, but other agents are not
conclusively ruled out as minor secretagogues. In living human colonic
crypts, with use of morphometric assessment, it was found that CCh and
histamine stimulated goblet granule release (Figs. 11 and 16), but the
fluid secretagogues PGE2 and adenosine did not cause goblet granule release (Figs. 14, 19, and 20,
Table 4). Although these results are not comprehensive for all
potential agonists, goblet granule release was separable from sustained
fluid secretion.
Crypt columnar cells have vesicles in the apical pole that are
positioned to release contents into the lumen. Columnar cell vacuoles
of rabbit distal colonic crypts stain histochemically distinct from
goblet cell granules, and this vacuolar material was present in the
crypt lumen after PGE2 stimulation
(14). During PGE2 stimulation,
imaged with video microscopy in living crypts, the apical pole of
columnar cells became empty of refractile material, consistent with
exocytotic release (14). Release of material was also apparent in
guinea pig distal colonic crypts by use of electron microprobe analysis
(16). Columnar cell apical vacuoles had a characteristic composition of
high Ca2+ and sulfur with low
Na+ and
Cl
in control conditions
that changed to high Na+ and
Cl
with low
Ca2+ and sulfur during
PGE2 stimulation, consistent with
access of the vacuole interior to the extracellular space. Together
these observations clearly indicate a release of columnar cell material into the crypt lumen during stimulation by a fluid secretagogue. Intracellular ion concentrations of crypt columnar cells also changed
as expected for a Cl
secretory cell, with PGE2
stimulation producing an increase in Na+ (16) and a drop in
Cl
(15). Thus columnar
cells of colonic crypts respond to fluid secretagogues by actively
secreting Cl
and by
releasing a macromolecule into the lumen, indicating a cellular link
between fluid and mucus secretion. Living human colonic crypts
responded to PGE2 and adenosine by
secreting fluid (Figs. 4 and 10) and releasing apically stored material
from columnar cells (Figs. 3, 8, and 14). Cholinergic stimulation did
not alter columnar cells (Fig. 11), consistent with a segregation of
secretory responses to specific cell types.
Colonic epithelial cell lines.
Epithelial cell lines derived from colonic tumors have been used
extensively to study fluid and mucus secretion. Capacity for fluid
secretion generally is detected by measuring electrogenic Cl
secretion, which
produces the osmotically driven flow of fluid. Mucus secretion has been
measured by various methods that detect the presence of released
glycoproteins. The T84 cell line secretes Cl
in response to VIP,
prostaglandin E1
(PGE1) and CCh (5). Release of
mucus by T84 cells was stimulated with CCh,
PGE1, and VIP (28, 32); electron
micrographs showed that only ~5% of the cells contain large apical
granules, so that the mucus secretion may have emanated from a small
minority of the cells. Several subclones of the HT-29 cell line have
been developed (HT29.Cl16E, HT29.B6, and HT29.18N2) that have a
morphology similar to goblet cells (apically located granules).
Forskolin and CCh stimulate a short-circuit current consistent with
Cl
secretion in HT29.Cl16E
cells (33). With HT29.B6 cells, VIP and
PGE1 stimulate
Cl
secretion (25). The
HT29.Cl16E cell line releases mucus in response to CCh, neurotensin,
VIP, and ATP (1, 33). Mucus release from HT29.18N2 cells is stimulated
by a PGE2 derivative (38). The
ability of these cultured colonic epithelial cell lines to release
mucus in response to goblet secretagogues (CCh) and fluid secretagogues
(PGE2 and VIP) suggests that these
cell lines combine the traits of goblet and columnar cells, either within a single cell or in a culture of cells with mixed differentiation.
Fluid secretion.
Stimulation of human colonic crypts with goblet-type (CCh) or
fluid-type (PGE2) secretagogues
stimulated fluid flow toward the crypt orifice (Fig. 10), consistent
with active fluid secretion. This transepithelial fluid flow is driven
by the osmotic gradient developed by active
Cl
secretion; human colonic
epithelium can secrete fluid, as indicated by stimulated
Cl
secretion at distal and
transverse sites (47). Isolation of crypts from the interstitium may
alter the secretory osmotic gradient by removing any component of
interstitial hypotonicity that may normally develop. Fluid flow
stimulated by PGE2 was larger and more sustained than that stimulated by CCh (Fig. 10). In addition, significant flow toward the crypt orifice in the tamarin crypt was not
apparent with CCh but increased dramatically with adenosine (Fig. 4).
This difference in fluid secretory rates is consistent with
measurements of Cl
secretion, which indicate that cholinergic stimulation produces only a
transient increase in Cl
secretion compared with a sustained increase by fluid secretagogues such as PGE2 (5, 13). Reduction in
PGE2-stimulated fluid secretion
with CCh (Fig. 10) may reflect the inhibiting action of CCh on
Cl
secretion measured in
T84 cells (57). Thus the two types of mucus secretion (goblet cell
derived and columnar cell derived) are accompanied by different amounts
of fluid secretion.
Particle speeds (v) can be used to
calculate the Reynolds number (Re) for luminal fluid flow, which
provides a comparison of inertial and viscous forces acting in the
crypt lumen; high Re indicate dominance of inertia, and low Re indicate
a more viscous regimen (56). Lumen diameters can be used for the
characteristic length (l), and
estimates of fluid viscosity (
) and density (
) can be taken as
the values for water (Re =
lv/
). For a human crypt
secreting fluid as in Fig. 10, Re is 0.0004. Such a low Re clearly
indicates the dominance of viscous forces during crypt fluid secretion.
More realistic estimates of fluid viscosity would be higher, because of
secreted mucus, which would reduce Re further. The consequences for
crypt fluid flow are that turbulence does not occur and a laminar flow
condition develops immediately as flow starts.
An estimate of the fluid secretory rate for an entire crypt can be
calculated from the observed particle speeds and lumen dimensions.
Volume flow past a specific point is related to the cross-sectional
area of the lumen
(
d2/4) and the
velocity profile across the lumen (56). For laminar flow, total volume
flow (Jv) is
equal to one-half the product of cross-sectional area and speed at
lumen center (Jv = vmax
d2/8,
where vmax is
maximum velocity); the highest speeds in Fig. 10 should correspond to
the center of the velocity profile. Because the particle speeds were
obtained about halfway along the crypt, volume flow at the crypt
orifice would be twice as great (with the assumption of uniform
secretion over the entire crypt). The resulting total volume flow for
the crypt in Fig. 10 is ~350 pl/min during
PGE2 stimulation. Normalizing for
crypt length (Table 1) converts this fluid secretory rate to ~900
pl · min
1 · mm
1.
PGE2-stimulated fluid secretion
was ~40
pl · min
1 · mm
1
in isolated rabbit colonic crypts (24). In isolated perfused rat
colonic crypts (49), VIP reversed a fluid absorption of 350 pl · min
1 · mm
1
to a fluid secretion of 350 pl · min
1 · mm
1;
cholinergic stimulation reversed absorption to a fluid secretion of 330 pl · min
1 · mm
1. Interestingly, a
goblet secretagogue and a fluid secretagogue stimulated similar rates
of fluid flow with these rat colonic crypts, in contrast to the results
for human colonic crypts. Fluid absorption may be difficult to detect
in the present study, since the method relies on movement of refractile
particles that might be relatively scarce during absorption. In
addition, rat colonic crypts were thought to be absorptive because of
the absence of the pericryptal sheath (49); all dissected human and
tamarin crypts retained the pericryptal sheath (Fig. 1). Glands of the tracheal epithelium, which secrete fluid and mucus, can be stimulated to secrete fluid at much higher rates, ~10 nl/min (42, 53).
Secretory capacity for the human colon can be estimated from the output
of an individual crypt. Because crypt density in the human colonic
epithelium is ~25,000/cm2 (48),
the colonic mucosal surface area of ~4,000
cm2 indicates a total number of
crypts in the colon of ~108. The
total daily output of continuously stimulated maximal crypt fluid
secretion would be ~50 liters. Such a rate clearly could not be
sustained, but the ability to overwhelm the absorptive capacity of the
colon is apparent.
The hydrostatic pressure gradient necessary to push fluid toward the
crypt orifice can be obtained from the Hagen-Poiseuille equation
[
P = 128 l
Jv/(
d4),
where
P is pressure gradient] by using lumen dimensions, total crypt volume flow, and fluid viscosity (56). Viscosity is undoubtedly higher for the secreted fluid than for water because of the presence of
secreted mucus. Colonic mucus contains soluble and insoluble forms (2),
with the insoluble, or gel form, probably representing goblet granule
mucin. The less dense columnar cell material (14, 16) is more likely a
soluble form, which would be most pertinent for determining fluid
viscosity. Mucus viscosity depends on source, concentration, and shear
encountered (8, 22, 34) but could range from 10- to 1,000-fold that of
water. From this range of fluid viscosity, the expected pressure
difference would be 0.1-10 mmHg, consistent with a low
pressure-flow system.
Perfusion of the crypt lumen via pipettes mimics the state of fluid
secretion and allows introduction of compounds to the apical surface of
crypt epithelial cells. The measured perfusion rate was ~4 nl/min in
the study of fluid transport with perfused rat colonic crypts (49),
which is comparable to the estimated rate of 1-5 nl/min in this
study of human colonic crypts. These luminal perfusion rates are 3- to
15-fold faster than the secretory flow at crypt orifice produced with
PGE2 (Fig. 10), which may
contribute to the altered appearance (Figs. 5 and 15) and response
(Fig. 7) of perfused crypts compared with intact crypts. Higher
perfusion rates are produced by higher applied hydrostatic pressure,
which has been reported to be deleterious to rabbit colonic crypts
(26). Although luminal perfusion has the advantage of providing access to the apical membrane, nonphysiological fluid flow may lead to abnormal cellular responses.
Mucus secretory mechanism.
Mucus release by epithelial cells, and goblet cells in particular, has
been measured by several methods. These techniques can be separated
into two broad categories: those that detect mucus released (1, 28, 32,
38) and those that measure depletion of mucus from epithelial cells (4,
35, 37, 39, 46, 50, 52). Detection of mucus secreted from crypts is difficult, because collecting the mucus would require not only release
from the cell but clearance from the crypt lumen. Also, differences in
cellular responsiveness are difficult to determine from collection
methods. For these reasons, a morphological approach was chosen for the
present study. Mucus volume released was measured (see
METHODS) by two means: decreases in
epithelial volume and decreases in goblet volume. Epithelial volume
changes include goblet and columnar cell events, so that qualitative
observations are required to indicate which cell types produced a
particular response (Figs. 11 and 14). Goblet cell mucous granule
volume release could be measured directly, since the apical granules
formed a compact distinct grouping. Unfortunately, a similar measure
for columnar cells was not possible, because the extent of apical vesicles could not be distinguished consistently through the time course of secretory stimulation. Comparison of these two measures for
cholinergic responsiveness of goblet cells (Fig. 17) indicates that
epithelial volume can reliably report on the mucus volume released by
goblet cells, suggesting that the fluid secretagogue response measured
as epithelial volume change (Figs. 7,
C and D, and
19C) also reflects mucus release.
Goblet cell mucus secretion.
Similarity in the time courses for the CCh response of epithelial and
goblet volume (Fig. 19B) suggests
that cytoplasmic volume changes during this stimulation are minor and
limited to a small transient decrease during the first minute (seen as
excess volume loss in the epithelial volume trace compared with the
goblet volume trace). Histamine stimulation of goblet granule release
(Fig. 16) was not apparent in epithelial volume measurements (Fig.
7B), suggesting that some or all of
the crypt cells had cytoplasmic swelling that obscured the volume
released from mucous granules. Shape changes of goblet granule masses
during histamine stimulation also are consistent with swelling, because
goblet eccentricity stayed prolate during volume loss contrary to the
CCh response (Fig. 19A). Cytoplasmic
swelling would have compressed goblets laterally so that a more
spherical shape could not occur. Thus cholinergic and histaminergic
stimulation of goblet cells can be distinguished, in part, on the
apparent cytoplasmic swelling induced by histamine.
Goblet cells release mucus by an exocytotic mechanism in which mucous
granules fuse with the apical membrane (51). Maximal cholinergic
stimulation leads to deep cavitation of the goblet granule cluster by
compound exocytosis (50). This fusion of granules to other granules
that have already fused with the apical membrane produces craters in
the apex of goblet cells and results in loss of granule membrane as
successive fusions isolate some membrane fragments. Volume of the
granule pool in control conditions (Table 3) was similar to that in
rabbit distal colonic goblet cells (43); from the size of an individual
granule, ~0.4 fl (Fig. 11), the number of granules in a normal goblet
was ~850. Individual exocytotic events were visible in living crypts
as changes in light intensity of granules (flashes) that presumably result from a decrease in mucus refractility as expansive release from
granules occurs. These exocytotic flashes have been seen in other
goblet cells (4, 20, 52); the half-time for the process is ~0.2 s
(46). Supramaximal stimulation of human colonic goblet cells only
occasionally produced the deep cavitations seen in rabbit distal colon
(50). Instead, small craters were apparent at the apex of goblets
(Figs. 2 and 11). As the volume of goblet granule clusters decreased
(Figs. 16 and 19), the remaining granules rearranged to make goblets
shorter and narrower (Fig. 20). This more subtle response is similar to
goblet cells in colonic surface epithelium (39) and small intestinal
villi (37). A more dramatic goblet response was observed in a crypt
from the patient with active ulcerative colitis (Fig. 13) and in crypts
from tamarin colon (Figs. 4, 16, and 19). Although large quantities of
mucus were visibly released, cells appeared to rearrange behind the departing mucus by becoming shorter without a noticeable cavity. For
the tamarin with moderate colitis (Fig. 4), many of the granules appeared to be jettisoned intact as a group, presumably when more peripheral granules fused in a compound fashion to cut off connection with the cell. Within ~10 min of extrusion, the appearance of the
mucous plume became homogeneous, suggesting a lysis of these granules.
These results with human crypts support an involvement of compound
exocytosis in goblet release but suggest that remaining granules are
moved within the cell to alter cell shape.
Expansion of mucus results from electrostatic repulsion of multiple
anionic residues on these molecules (54). Packing of mucus into
granules is facilitated by charge screening with divalent cations such
as Ca2+ (55). Goblet granules in
the colonic epithelium contain high concentrations of
Ca2+ and
Mg2+ (16) that presumably serve
this purpose of condensing the filamentous mucous molecules. The
massive release from tamarin goblet cells (Fig. 4) indicated almost
twofold expansion for extruded mucus. Because this enlargement is much
smaller than the 600-fold expansion seen for slug goblet granules (55),
charge screening must be occurring in the crypt lumen to limit mucus
extension. Such a mechanism would have the advantage of not blocking
the crypt lumen, allowing easier passage of goblet mucus out of the crypt.
Observing living colonic crypts allowed individual goblet cells to be
followed through the course of stimulated granule release. Some goblet
cells were unresponsive to cholinergic stimulation, 23% (Fig.
18A), and tended to have larger
goblet volumes (Fig. 18B),
consistent with retention of granules. Apparently, unresponsiveness lasted long enough to fully pack the goblet zone. Responsive goblets, on average, released 29 ± 2% of mucus stores (none >70%, Fig. 18A). This incomplete release
suggests that an inhibitory control restricts granule emptying and can
block release in about one-fourth of the goblet cells.
Columnar cell mucus secretion.
The fluid secretagogues PGE2 and
adenosine produced decreases in epithelial volume that were sustained
after return to the control condition (Fig. 7,
C and
D), which suggests that the lost volume resulted from mucus release from columnar cells (14), as
visualized by the recession of the apical cell margin (Fig. 14).
Further support for this cellular specificity is the lack of goblet
volume decrease (Fig. 19A, Table 4). A
contribution of cytoplasmic volume change to the fluid secretagogue
response is more difficult to assess than with goblet secretagogues
because of the lack of a direct measure of apical vesicle volume. Crypt diameters have been used as an indicator of epithelial volume during
VIP stimulation of rat colonic crypts isolated without the pericryptal
sheath (7). Although changes in crypt diameter were altered by
furosemide, a blocker of
Na+-dependent
Cl
entry, interpretation of
these changes as indicative of epithelial volume is problematic without
measurements of lumen diameter and crypt length. Intracellular
Cl
concentration decreased
transiently during PGE2
stimulation in isolated rabbit colonic crypts, consistent with cell
shrinkage that returned to near the initial value in ~2 min (15). The membrane electrical potential difference of rabbit colonic crypt cells
also depolarized transiently during
PGE2 or adenosine stimulation, returning to near the initial value in 1-2 min (27). These results support an activation process for fluid secretagogues in which cytoplasmic volume decreases and returns to control levels; this response results from Cl
and K+ channels
opening followed by increasing Cl
influx, which supplies
continued Cl
secretion.
Therefore, the contribution of cytoplasmic changes to epithelial volume
would appear to be constrained to early time points, such that
sustained volume decreases in human colonic crypts (Figs. 7,
C and
D, and
19C) reflect release of apically stored mucus from columnar cells.
Columnar cells of colonic crypts release a material stored in apical
vesicles (vacuoles), but the mechanisms of release are much less
precisely known than for goblet cells. Stimulation by fluid
secretagogues in rabbit distal colon leads to release via opening of a
narrow connection between vacuole and crypt lumen (14). Viewed in these
crypts with DIC, initially the apical pole of the columnar cell becomes
empty (less refractile) and then collapses, such that goblet cells
appear closer together. Because in human crypts the apical vesicles
were small, it was not possible to view the emptying, but the collapse
of the apical zone was observed as a recession of the apical margin of
columnar cells, with goblet cells becoming closer together (Fig. 14).
The time course of the PGE2
response of human crypts (Fig. 19C)
indicates that volume release was essentially complete at 10 min of
stimulation, whereas fluid secretion continued. Apparently, fluid
secretion does not depend on constant mucus release, even though both
are initiated by the same agents.
IBD and secretory processes.
The IBD of ulcerative colitis and Crohn's disease lead to epithelial
damage and altered function (31, 41). Cell death may be the cause of
the focal points of short epithelial height seen in inflammatory crypts
(Fig. 1). In addition, the larger diameter of crypts from patients with
ulcerative colitis may reflect an early stage in cancer progression
(31, 41, 59). Whether epithelial changes are simply a consequence of
immune cell activity during inflammation or constitute part of the
cause for the pathology is uncertain.
One diagnostic feature is depletion of goblet cell mucus in ulcerative
colitis compared with normal mucosa or Crohn's disease, whereas
Crohn's disease mucosa contains only slightly less mucus than normal
mucosa (29). In colon from guinea pigs with experimental colitis, the
depletion of mucus was due to less mucus in goblet cells rather than a
decrease in goblet cell number (19). In addition to mucus depletion,
the composition of colonic mucus is altered in ulcerative colitis (41),
and some of the change may relate to the different mucus types in
goblet and columnar cells (11). In the colonic crypt goblet cells from
patients with ulcerative colitis and those from tamarins with severe
colitis, stores of mucus were smaller (Table 3, Fig. 15), consistent
with the interpretation that goblet cells are present in ulcerative colitis but are simply more difficult to identify because of reduced mucus content.
Mucus depletion may have resulted from chronic stimulation by
inflammatory mediators released in vivo, but the response to CCh in
vitro was larger than for normal crypts. Tamarin crypts released nearly
the complete apical supply of mucous granules (Figs. 4 and
16A). Both crypts from patients with
ulcerative colitis (active and nonactive disease) had indications of
hypersensitive goblet cells. Mucous plumes were apparent (Fig. 13), and
CCh-induced volume release was larger than in normal patients (Fig.
8A). The disparity between
CCh-induced epithelial volume changes and individual goblet changes may
result from preferential (or hypersensitive) release from goblets of
small size: small goblets would be difficult to monitor through the
time sequence of stimulation and would be underrepresented in direct
measures of goblet volume. Whereas all tamarin goblets were
hypersensitive (Figs. 16 and 18), the largest (and easiest to monitor)
goblets in human crypts were not hypersensitive, and some were
unresponsive. In contrast, tamarin and human ulcerative colitis goblet
cells did not appear to be hypersensitive to histamine, although an
increased sensitivity in cells with limited granule supplies cannot be
excluded. Thus goblet cells in ulcerative colitis are hypersensitive to
cholinergic stimulation, but those few that were the least responsive
would have retained more granules and have the largest granule stores.
Fluid secretion also is increased in ulcerative colitis (41), which may
be a direct consequence of elevation in the inflammatory mediator
PGE2 (17). Because isolated
ulcerative colitis crypts did not secrete fluid until stimulated, the
action of these fluid secretagogues was removed by the isolation of
crypts from the mucosa. The epithelial volume released in response to
PGE2 or adenosine (Fig. 8,
B and
C) also suggests a hypersensitivity
to these secretagogues in human and tamarin ulcerative colitis.
Hypersensitivity for release of goblet cell mucus and columnar cell
mucus would exacerbate hyperstimulation occurring through immune
responses. These results do not indicate whether epithelial cells
contribute to initiation of the inflammatory response, but the crypt
cells have an augmented mucus secretory response.
 |
APPENDIX |
A translation of relative area in crypt midline optical sections to
relative volume requires assumptions about crypt geometry. For random
sections, area measures will reflect volume (58). The rigid geometry of
crypts can be used to compensate for the nonrandom nature of midline
optical sectioning. Crypts can be modeled with roughly equal numbers of
goblet and columnar cells dispersed evenly in the epithelium (10). A
unit volume for the apical zone of the crypt can be defined as a
truncated pie piece (a bite removed from the tip to create the lumen)
with a single goblet in each; a series of these prismoid unit volumes
reconstruct the apical zone around the circumference of the lumen. The
goblet granule cluster can be approximated as a frustum of a right
circular cone. Volume within the pie piece, but not in the frustum,
would be the apical zone volume contributed by columnar cells. Volume of the frustum (Vfc,
Eq. A1) can be calculated from the
height (h) and two widths
(diameters), the smaller at the lumen
(wlu) and the
larger (w) at the base of the goblet
|
(A1)
|
Volume
of the unit apical zone (Vau,
Eq. A2) can be obtained from the
frustum measurements, since the frustum is inscribed within the unit
volume
|
(A2)
|
The
fraction of volume (fv)
contributed by goblets (Eq. A3) is
obtained by dividing Vfc by
Vau
|
(A3)
|
As
ranges from 0.0 to 1.0, fv
would increase from
/6 to
/4. The fraction of area
(fA) contributed in midline section by goblets (Eq. A4) is
obtained from the ratio of the frustum profile to the apical unit area
|
(A4)
|
The
term
is included to account for sectioning through the unit volume
at random distances into the frustum. The
fA obtained by point counting on
crypt midline optical sections can be converted to
fv by combining
Eqs. A3 and A4 to obtain Eq. A5
|
(A5)
|
Because
the lumen circumference is made up in increments of unit volumes, the
ratio
wlu/w
can be obtained from the measured diameters,
= d/Dg. As
approaches 1.0, the tubular crypt epithelium would transform into a
flat sheet, and the frustums would become cylinders. In this limit,
fv is equivalent to
fA and
=
/4. Sectioning
the crypt optically at the midline and measuring area along the crypt
segment is equivalent to sectioning unit volumes at various angles, by
rotating a unit around the axis defined by the center of the crypt.
Thus quasi-random sectioning of the unit volume is accomplished, since
each unit volume along the crypt segment has a random angle relative to
the optical sectioning plane. Equation A6 gives the average area
(Aau) from
sectioning the unit volumes
|
(A6)
|
The
angle subtended at the crypt axis by the wedge of the unit volume (pie
piece) is equal to 2
, where
= arcsin(2wcell/D). The ratio i/n gives the fractional
deviation of the unit volume from the midline. For goblet frustums,
Eq. A7 gives the average area from
sectioning
(Afc)
|
(A7)
|
Combining Eqs. A6 and A7 results in Eq. A4 and provides an explicit expression for
(Eq. A8) in the frustum model of
goblets
|
(A8)
|
In
the limit of many sectioning profiles (large
n),
fc is about
/4. The number
of profiles sampled in a typical crypt image was 20-30. For the
range of observed crypt diameters
(D) and cell widths,
fc is negligibly larger than
/4 (by a factor of 1.001-1.003).
 |
ACKNOWLEDGEMENTS |
We thank Karen S. Tefend and Dr. Jackie D. Wood for
providing tamarin biopsies and Dr. Donald R. DiBona for helpful
suggestions and support.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-39007.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. R. Halm,
Dept. of Physiology and Biophysics, Wright State University, 3640 Colonel Glenn Hwy., Dayton, OH 45435 (E-mail:
dan.halm{at}wright.edu).
Received 4 February 1999; accepted in final form 14 May 1999.
 |
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