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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
Halm, Dan R., and Susan Troutman Halm. Secreta- indicated by ultrastructural studies (51). Movement of
gogue response of goblet cells and columnar cells in human granules within the cell is a microtubule-dependent
colonic crypts. Am. J. Physiol. 277 (Cell Physiol. 46): C501– process, and access of the granules to the apical mem-
C522, 1999.—Crypts of Lieberkühn were isolated from hu- brane is restricted by the actin cytoskeleton. Further
man colon, and differential interference contrast microscopy advances in examining dynamics of goblet cell secretion
distinguished goblet and columnar cells. Activation with
carbachol (CCh, 100 µM) or histamine (10 µM) released
have been made using video-enhanced differential inter-
contents from goblet granules. Stimulation with prostaglan- ference contrast (DIC) microscopy of living cells (4, 14,
din E2 (PGE2, 5 µM) or adenosine (10 µM) did not release 20, 46, 52). Individual events of goblet granule release
goblet granules but caused the apical margin of columnar were visualized as changes in light intensity, indicating
cells to recede. Goblet volume was lost during stimulation a rapid discharge (0.03–1.0 s) of mucus from granules
with CCh or histamine (⬃160 fl/cell), but not with PGE2 or (4, 20, 46). The time course of stimulation with mucus
adenosine. Three-quarters of goblet cells were responsive to secretagogues consists of a rapid increase in the num-
CCh but released only 30% of goblet volume. Half-time for ber of granule release events that reaches a maximum
goblet volume release was 3.7 min. PGE2 stimulated a in 1–3 min and then subsides to a much slower
prolonged fluid secretion that attained a rate of ⬃350 pl/min. sustained rate. Discharge of mucus from goblet cells in
Columnar cells lost ⬃50% of apical volume during maximal rabbit colonic crypts occurs via a similar exocytotic
PGE2 stimulation, with a half-time of 3.3 min. In crypts from
process (14, 52), and columnar cells also show stimu-
individuals with ulcerative colitis, goblet cells were hypersen-
sitive to CCh for release of goblet volume. These results lated emptying of contents from the apical pole of the
support separate regulation for mucus secretions from goblet cell (14). The results presented in this study were
cells and from columnar cells, with control mechanisms obtained with DIC microscopy of isolated human co-
restricting total release of mucus stores. lonic crypts and indicate the presence of two morphologi-
cally distinguishable types of cells: goblet and colum-
adenosine; cholinergic; histamine; inflammatory bowel dis-
ease; prostaglandin E2
nar cells. Distinct secretagogues stimulated release of
mucuslike material from apically stored granules in
each of these cell types.
METHODS
THE COLONIC EPITHELIUM of mammals secretes fluid and
mucus in response to various neurotransmitters and Specimens of human colon were obtained from surgical
local mediators (9, 12, 13). Fluid secretion modifies resection material by a procedure approved by the Institu-
luminal composition by increasing the aqueous volume tional Review Board (Ohio State University and Wright State
University). After release by the pathologist, specimens were
and by adjusting concentrations of electrolytes such as
refrigerated in HEPES-buffered Ringer solution until they
K⫹ and H⫹. Low rates of fluid secretion could aid colonic were picked up and transported to the laboratory (generally
motility and bacterial fermentation, whereas higher ⬃30 min). The epithelium was removed from underlying
rates seen in pathophysiological states would serve to muscle by blunt dissection and placed in ice-cold standard
flush bacteria from the lumen. Mucus secretions contrib- Ringer solution. Colonic mucosal biopsies from cotton-top
ute to the barrier function of the epithelium by protect- tamarins were obtained during semiannual endoscopic screen-
ing the epithelial cells from abrasion and also by ing of a colony maintained by Dr. J. D. Wood at Ohio State
slowing access of bacteria to those epithelial cells. University. This procedure was approved by the Institutional
Release of mucus from goblet cells generally is separate Laboratory Animal Care and Use Committee at Ohio State
from sustained fluid secretion on the basis of sensitivity University. The tamarin biopsies were transported to the
laboratory in ice-cold HEPES-buffered Ringer solution within
to specific secretagogues (35). A mucous substance
20 min.
distinct from goblet cell mucin is released from crypt Human colonic crypts were isolated from resection mate-
columnar cells during stimulation of fluid secretion (14, rial of 17 patients (9 women and 8 men, 15 Caucasians and 2
16). Control of secretion from goblet and columnar cells African-Americans; Ohio State University Hospitals) ranging
by separate secretagogues permits alteration of the in age from 35 to 69 yr. Resections were roughly divided
relative proportion of mucus types released and the between ascending or transverse sites (⬃55%) and descend-
rate of fluid production. ing or sigmoid sites (⬃45%); normal margins were released by
Mucus secretion by goblet cells involves exocytotic the pathologist for experimental study. Resection had been
release of granule contents at the apical membrane, as 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
The costs of publication of this article were defrayed in part by the active ulcerative colitis, one had nonactive colitis (resected for
payment of page charges. The article must therefore be hereby metastatic cancer), and one had Crohn’s disease. Information
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 on patient medications was not available for comparison with
solely to indicate this fact. experimental results. Cotton-top tamarins are a species of
0363-6143/99 $5.00 Copyright r 1999 the American Physiological Society C501
Downloaded from journals.physiology.org/journal/ajpcell (176.076.226.016) on April 4, 2024.

C502 RESPONSE OF GOBLET AND COLUMNAR CELLS IN COLONIC CRYPTS
New World monkey that develops symptoms resembling (L), and standard deviations (␴D and ␴d ) of these values
ulcerative colitis when in captivity (30). The tamarins supply- (Eq. 1)
ing colonic crypts for imaging (n ⫽ 4) were assessed for extent
of disease by histological measures, including white blood cell ␲
Vcr ⫽ L 1D2 ⫹ ␴2D2 (1a)
infiltration: three had severe colitis, and one had moderate 4
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 distin- Vlu ⫽ L 1d2 ⫹ ␴d2 2 (1b)
4
guished 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 This calculation assumes that cells seen in the plane of focus
with cancer or diverticulitis were grouped together as normal. are representative of all cells in that annulus of the crypt. Epi-
Individual colonic crypts were dissected from the colonic thelial volume was calculated as the difference between crypt
epithelium with use of fine forceps on the stage of a dissecting and luminal volume. All volumes were normalized to epithe-
microscope (⫻40) in HEPES-buffered Ringer solution with lial volume obtained in the initial control image for each
5% serum albumin added. The standard mammalian Ringer experiment to adjust for differences in defined segment lengths
solution contained (in mM) 145 Na⫹, 5 K⫹, 2 Ca2⫹, 1.2 Mg2⫹, (cell number) between crypts. Generally, 300–400 crypt epi-
125 Cl⫺, 25 HCO3⫺, 4 PO4x⫺, and 10 D-glucose. Solutions were thelial cells were represented by epithelial volume in each
continually gassed with 95% O2-5% CO2, which maintained experiment. Repeated measures on the same image indicated
solution pH at 7.4. HEPES-buffered Ringer solution con- that reproducibility was ⫾0.1 µm for diameter measurements
tained 10 mM HEPES with HCO3⫺ replaced by Cl⫺, and the and ⫾0.5 µm for length measurements. Propagation of these
pH was titrated to 7.4. error estimates for volume calculations (Eq. 1) gave an error
Crypts were imaged in a chamber mounted on the stage of estimate for relative epithelial volume of ⫾0.6%.
an inverted microscope (Zeiss Axiovert); the bottom of the Volume of the zone containing apical granules (Vaz ) was
chamber was formed by a no. 1 coverslip (14). Isolated crypts calculated (similar to Eq. 1) by using the diameter of this zone
were held by glass pipettes or adhered by a coat of polylysine (Dg ), measured from the base of goblet granule masses in cells
to the chamber bottom coverslip. The glass pipettes were on opposite sides of the lumen (Vaz ⫽ Vg ⫺ Vlu, where Vg is
made to accommodate the relatively short length of colonic goblet region volume); this volume includes apexes of colum-
crypts and were manipulated with a system from Vestavia nar cells, which often contain granules (21, 40, 48). Normaliza-
Scientific (Vestavia Hills, AL). For luminal perfusion, the tion to control epithelial volume gave the relative volume
blind end of the crypt was cut off with a sharpened needle fraction for this apical zone. Error propagation provides an
before transfer to the imaging chamber. Luminal perfusate error estimate for relative apical zone volume of ⫾3%.
was HEPES-buffered Ringer solution. Luminal perfusion Contribution of goblet granule masses to apical zone volume
rate was estimated as 1–5 nl/min on the basis of perfusion was estimated by a point-counting technique (58). A square
pressure, pipette dimensions, and previous measurements array of points (2-µm image spacing) was overlaid (skewed
with renal tubules (45). The bath was continuously perfused from the crypt axis) on crypt images, resulting in ⬃500 points
with standard Ringer solution with use of a peristaltic pump, over the apical zone. The ratio of points within goblets to all
and effluent was removed by suction tube; the solution was points in the apical zone was the fraction of volume contrib-
bubbled with 95% O2-5% CO2 and warmed by passage through uted by goblets. This estimate requires two corrections to
a heated water jacket to maintain bath temperature at 37°C. reflect the actual volume distribution: section thickness and
Bath solution flow was 3 ml/min (⬃8 chamber vol/min). crypt geometry. The highly refractile goblet granules were
Crypt images were formed with DIC optics: a ⫻40 oil readily recognized in images, but the contribution of goblet
immersion (1.4 NA) lens and a nonimmersion (0.65 NA) granules is overestimated, because granules can be seen
condenser (6). A video camera (model CCD300E, Videoscope throughout the depth of focus. The depth of focus expected for
International) was used to record images to videotape (Sony) video imaging with this lens-condenser combination, 0.7 µm
and to computer disk with use of the Image-1 system (Univer- (18), can be used to calculate a correction of 0.88 (58). The
sal Imaging). A ⫻2 coupling lens (Diagnostic Instruments) other bias in the measurement underestimates the contribu-
was used to fill the camera field. tion of goblets, because longitudinal crypt midline sections
Image analysis. Morphometric measurements of recorded were used, rather than random sections. Goblet fractional areas
crypt images were performed with Image-1 software. Quanti- measured by point counting were converted to volume fractions
fication of volume was obtained with focus adjusted to the (see APPENDIX ) by use of Eq. A5, ␣ for each crypt, and ␥ ⫽ ␲/4;
midline of the crypt, because this plane of section allowed the resulting correction factors ranged from 1.02 to 1.15.
ready measurement of crypt and lumen diameter as well as a Individual goblet granule masses were measured to moni-
complete profile of the epithelium from base to apex. Crypt tor directly volume changes in this apically located cell
diameter was taken at the base of epithelial cells on opposite compartment. Volume of goblets (Vgob ) was calculated from
sides of the crypt (inside the pericryptal sheath); lumen width (w) and height (h) of the granule mass by use of Eq. 2,
diameter was taken at the apexes of opposing cells. A consis- with the assumption of a spheroidal shape
tent group of cells was monitored in successive images by ␲
marking the boundaries between distinct, identified cells Vgob ⫽ w2h (2)
near the lateral margins of the image frame. Diameters were 6
measured at nine evenly spaced points along the crypt
Eccentricity [prolate (eprolate ) and oblate (eoblate )] in goblet
segment defined by the length markers. Segment length was
shape was calculated using Eq. 3 as a measure of distortions
determined at cell apex, cell base, and midway along cell
to this cellular compartment
height for both epithelial margins (6 values). Crypt (Vcr ) and
luminal volumes (Vlu ) (23) were calculated on the basis of a eprolate ⫽ 31 ⫺ 1w/h2241/2 (3a)
piecewise cylindrical model with use of average crypt diam-
eter (D), average lumen diameter (d), average section length eoblate ⫽ ⫺31 ⫺ 1h/w2 4 2 1/2
(3b)

RESPONSE OF GOBLET AND COLUMNAR CELLS IN COLONIC CRYPTS C503
For oblate goblets the ratio of width to height was inverted, Goblet cells appeared less full of mucous granules than
and the resulting eccentricity was assigned a negative value. did normal crypts. Colonic epithelium from patients
These definitions allow numerical comparisons of dissimilar with nonactive ulcerative colitis (Fig. 1C) or Crohn’s
goblets, with eccentricity ranging from ⫺1.0 to ⫹1.0 as shape disease (Fig. 1D) was indistinguishable on dissection
changes from short and wide (oblate) to tall and narrow
(prolate). Calculated goblet volumes (Eq. 2) were corrected for
from normal epithelium in crypt density or crypt size.
underestimation due to off-center optical sectioning of goblets Crypts from all IBD patients had more focal spots of
within the crypt midline images. Separate frequency histo- short epithelial height than did normal crypts. Colonic
grams of raw width and height measurements were deconvo- epithelium of tamarins had densely packed crypts,
luted to obtain an estimate of actual mean width and height even in specimens with severe colitis. As for crypts from
(58). Comparing these actual mean widths and heights with patients with IBD, the tamarin epithelium had focal
means of raw measured values gave a volume correction spots of short cell height (Fig. 1E) and relatively
factor of 1.19 to adjust goblet volume calculations. Eccentric- depleted granule stores in goblet cells. Results from
ity values were not adjusted, because the distributions of tamarin crypts are specifically indicated; all other
width and height produced similar underestimation, such
results are for human crypts.
that the ratio (Eq. 3) was unaffected. Time courses of goblet
volume changes were obtained by measuring the same goblet Crypt dimensions were measured and assigned to
through a series of images taken during various stimulatory three groups (Table 1): normal (see METHODS ), IBD, and
conditions. In this analysis, only goblets that were clearly tamarin. A distinction also was made for crypts from
sectioned near the center during the entire sequence were the normal group that were opened for luminal perfu-
used. The average control volume from these measurements sion. Differences in size among these groups were not
was similar to the corrected volume obtained with the larger apparent statistically. The focal spots of short epithelial
sample of all visible goblets. height seen in crypts from patients with IBD and in
Speed of refractile objects moving in the lumen was mea- tamarin crypts were not extensive enough to cause a
sured from video recordings. Transit time between cursors
decrease in average epithelial height; however, the
(10- to 30-µm separation, depending on rate of movement)
was used to calculate speed. Size of objects was obtained from lumen diameter of perfused crypts and crypts from
width (orientation with lumen diameter) and length. Object patients with ulcerative colitis had a tendency to be
volume was calculated by assuming a spheroidal shape, as for larger.
goblet granule masses (Eq. 2). Secretory stimulation. Addition of secretagogues that
Values are means ⫾ SE. Statistical comparisons were stimulate mucus and fluid secretion produced several
made using a two-tailed Student’s t-test for paired compari- changes in crypt geometry, as well as epithelial cell
sons, with significant difference accepted at P ⬍ 0.05. For morphology. The cholinergic agent carbachol (CCh)
unpaired comparisons between groups, significant difference stimulated mucus release from goblet cells, seen as
was accepted at P ⬍ 0.05 from ANOVA with the method of
shrinking of apical granule clusters and as light flashes,
Newman and Keuls.
presumably the result of refractile contents exiting
individual granules (4, 20, 46). In some cases, evanes-
RESULTS
cent plumes could be seen forming above the sites of
Isolated colonic crypts imaged with DIC microscopy flashes, which then drifted away from the cell surface.
(Fig. 1) exhibited the two predominant cell types associ- Flashes ceased within a few seconds of CCh removal.
ated with this epithelium: columnar and goblet cells (3, Concurrent with goblet granule release, crypt lumens
14, 21, 40, 48). Focusing at the midline of the crypt dilated (Fig. 2). Histamine also stimulated mucus
showed the full height of the epithelium, with apically release from goblet cells, as indicated by numerous
located granules above basally located nuclei. Surround- apical flashes. Stimulation with prostaglandin E2
ing the crypt epithelium was a pericryptal sheath of (PGE2 ) or adenosine produced fluid secretion and reces-
myoepithelial cells (36, 44), which was apparent as a sion of columnar cell apexes without any apparent
fibrous layer with flattened nuclei. Goblet cells, in release from goblet cells (Fig. 3). Occasional light
particular, were distinct because of a densely packed flashes were seen at columnar cell apexes, but these
cluster of highly refractile granules stored in the apical events were not consistently observed. Fluid flow to-
pole, which had a characteristic ovate profile. Gener- ward the crypt orifice was visualized by the rapid
ally, goblet cells were separated by columnar cells movement of refractile particles along the lumen. The
having a more hourglass-shaped profile that arched cholinergic response of tamarin crypts was dramatic,
over the neighboring goblet cells. Thus the luminal with nearly complete disgorging of mucous granules
surface often was dominated by columnar apexes, even and large dilation of the lumen (Fig. 4). Large plumes of
though goblet granules filled more than one-half of the mucus stayed tethered to the cell of origin (Fig. 4C).
apical epithelial volume. Subsequent stimulation with adenosine (Fig. 4D) fur-
Crypts also were obtained from colonic epithelium of ther shortened the epithelium and produced rapid fluid
patients with IBD (ulcerative colitis and Crohn’s dis- flow along the lumen that eventually ripped mucous
ease; Fig. 1). During dissection ulcerated regions of plumes from goblet cells.
colon with active colitis had a fragile surface epithe- Crypt shape changed during secretory stimulation
lium and a sparse number of recognizable crypts (4 through an increase in lumen diameter and a small
specimens with active colitis were examined by dissec- decrease in crypt diameter (Fig. 5). After sequential
tion microscope; data not shown). Those crypts present stimulation with these two classes of secretagogues
(Fig. 1B) were generally large in diameter (⬎100 µm). (goblet type and fluid type), the crypts maintained an

Fig. 1. Isolated colonic crypts. Differen-

tial interference contrast (DIC) micros-
copy was used to image individual dis-
sected colonic crypts. Focus at midline
of crypt tube provided a view of maxi-
mal 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 arrow-
heads). 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 mod-
erate colitis or normal human crypts.
Epithelial height varied considerably.
enlarged lumen diameter (Fig. 5D). The changes in from a simple crypt elongation caused by cells becom-
crypt and lumen diameters with each secretagogue ing wider and shorter without losing volume. Including
(Fig. 5, B and C) are consistent with the decreased a measurement of length to monitor a consistent group
epithelial volume expected from release of apically of crypt epithelial cells would provide an unambiguous
stored mucus. Change toward a smaller crypt diameter calculation of epithelial volume.
and larger lumen diameter also could have resulted Secretagogue-induced changes in epithelial volume.
Epithelial volume of crypts was monitored during
Table 1. Crypt dimensions secretagogue stimulation by picking a consistent seg-
ment of crypt length over which to measure crypt
Crypt Lumen Epithelial Crypt diameter and lumen diameter. Segment length was
Diameter, Diameter, Height, Length,
µm µm µm µm defined from the boundaries between identifiable cells
positioned near the edges of the recorded image fields. A
Normal 73.5⫾3.4 (8) 16.2⫾2.8 (8) 28.7⫾1.5 (8) 433⫾25 (6)
Normal (per-
representative experiment from the central portion of a
fused) 80.6⫾4.4 (6) 26.2⫾4.3 (6) 27.2⫾2.7 (6) normal crypt is shown in Fig. 6. Volumes (Fig. 6B) were
IBD 87.1⫾11.2 (3) 25.4⫾6.3 (3) 30.8⫾3.9 (3) 399⫾75 (2) calculated from the measured crypt dimensions (Fig.
Tamarin 67.6⫾3.2 (4) 17.4⫾3.2 (4) 25.1⫾2.6 (4) 383⫾36 (4) 6A; see METHODS ). Epithelial volume decreased with
Values are means ⫾ SE of number of observations in parentheses. CCh addition and did not return in the subsequent
IBD, inflammatory bowel disease. control period. With addition of PGE2, a decrease in

(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.
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 (presum-
ably 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
Fig. 2. Cholinergic stimulation. Goblet granule clusters (arrow-

heads) 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 diam-
eter increased concurrent with loss of apically stored material from
goblet cells. Release of mucin granules produced craters in some
goblet cells.
epithelial volume that was not recovered on return to

control conditions also occurred.
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 Fig. 3. Prostaglandin E2 (PGE2 ) stimulation. Response of columnar
cells (arrowheads) to a fluid secretagogue. Crypt orifice is to left. Scale
appeared relatively depleted of goblet granules before bars, 10 µm. A: midline DIC image from central portion of a normal
stimulation. Tamarin crypts had the largest responses crypt in control condition (after return from stimulation with 2 µM
to CCh. As a group, the responses to histamine showed CCh). A large globule of goblet mucus (⬃10 µm diameter) can be seen
no distinct change in epithelial volume (Fig. 7B), even in lumen (under 2nd downward arrowhead from left), remaining from
earlier release event. B: crypt in A 5 min after beginning of
though flashes indicative of granule release events stimulation with PGE2 (2 µM). Lumen diameter increased through a
were seen during stimulation. Addition of PGE2 pro- recession of columnar apexes without any apparent change in goblet
duced a sustained ⬃5% decrease in epithelial volume cells.

Fig. 4. Secretory response of tamarin

crypt. Hypersensitivity of goblet cells to
cholinergic stimulation was apparent
in a crypt from a tamarin with moder-
ate colitis. Crypt orifice is to left. Scale
bars, 10 µm. A: control goblet cells had
apical poles with moderately large gran-
ule 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 mu-
cous plumes. C: after removal of CCh,
further release of granule contents was
not apparent, and lumen remained full
of goblet mucus. Lysis of extruded gran-
ules became evident (10 min) from a
uniform evanescent appearance of mu-
cous plumes above goblet cells. (Top
center and bottom right plumes in B
could not be clearly identified.) D: sub-
sequent 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.)
crypt from the radial extent of the apical goblets (see control conditions. Perfused crypts (n ⫽ 5) shortened by
METHODS ); Vaz ranged from 0.29 to 0.48 and averaged ⬃2% during CCh stimulation and maintained that
0.35 ⫾ 0.02. Loss of epithelial volume (Fig. 7) was shortening on return to control conditions. Crypts from
normalized to the individual measures of Vaz to provide patients and tamarins with ulcerative colitis (2 hu-
an indication of how much of the stored material was mans and 2 tamarins) lengthened by ⬃4% during CCh
released (Table 2, Fig. 8). The total amount of Vaz stimulation and then shortened to roughly control
released after stimulation by goblet- and fluid-type length on return to the control condition. During PGE2
secretagogues was largest in those crypts from patients stimulation, normal crypts (perfused and nonperfused)
with ulcerative colitis and from tamarins (Fig. 9) but maintained length within 1% of control values (n ⫽ 7)
still did not exceed the measured control apical stores. and then shortened by ⬃2% on return to control
The contribution of goblet granules to Vaz was mea- conditions. Crypts from patients and tamarins with
sured by point counting in control crypt images (see IBD (n ⫽ 3: active colitis, Crohn’s disease, and tamarin)
METHODS ). For normal crypts the goblet fraction was shortened by ⬃3% during PGE2 stimulation and main-
0.65 ⫾ 0.03 (n ⫽ 8). Crypts from patients with IBD had tained that shortening on return to control. Together
goblet fractions statistically indistinguishable from with the decreases in crypt diameter (Figs. 5 and 6),
crypts from normal patients (0.64 ⫾ 0.04, n ⫽ 3). The these changes in crypt length suggest that the peri-
CCh-induced loss of Vaz (Table 2) can be transformed by cryptal sheath responds to secretagogues and contrib-
using the goblet fraction to estimate the percentage of utes to maintenance of crypt dimensions.
goblet granule volume released in normal crypts: ⬃30%. Luminal fluid flow. Fluid flow along the lumen was
For crypts from the two patients with ulcerative colitis, apparent from movement of refractile particles. Sus-
goblet volume release was 55–75%. Tamarin crypts tained movement of these objects in the lumen was
released ⬃80% of goblet volume. Similarly for PGE2- seen only during secretagogue stimulation. Presum-
stimulated volume loss (Table 2), normal crypts re- ably, the objects were made up of released mucus and
leased ⬃50% of nongoblet (columnar cell) Vaz. Release other cellular debris. In some crypts, particles were
from this columnar cell apical zone was 80–100% for small (⬃3 µm diameter) and seen infrequently, whereas
crypts from patients with ulcerative colitis and from in other crypts the lumen was crowded with globular
tamarins. objects. Stimulation by CCh of a crypt with globular
Crypt diameter and length relationships may be objects already present in the initial control condition
controlled in part by the contractile state of the peri- (Fig. 10A) produced a measurable particle flow toward
cryptal sheath (36, 44). Normal crypts maintained the crypt orifice. Removal of the stimulus stopped flow
length within 1% of control values during stimulation with with a lag of ⬃1 min, although the flashes indicative of
CCh (n ⫽ 6) and then shortened by ⬃2% on return to granule release stopped within a few seconds. PGE2

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. r, Normal; s, normal-perfused; l, ulcerative colitis (N, nonactive); p, Crohn’s disease; m,
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.
addition immediately produced flow toward the crypt crypt sections can be estimated from the speed of the
orifice. Movement was episodic, with particles slowing fastest particles and the cross-sectional area of the
down as larger globular objects squeezed past each lumen. The fastest speeds in Fig. 10 correspond to a
other. Dilation of the lumen alleviated this congestion crypt fluid flow of ⬃10 pl/min during CCh stimulation
and progressively led to faster particle speeds. Addition that increased to ⬃350 pl/min during PGE2 stimulation
of CCh together with PGE2 led to reduced speed, (see DISCUSSION ); fluid flow decreased to ⬃210 pl/min
consistent with inhibition of fluid secretion. Particle after CCh addition with PGE2. Retrograde flow consis-
speeds were highest for smaller objects (Fig. 10B). All tent with fluid absorption was not observed. For tama-
moving particles were tracked during stimulation, but rin crypts, maximal fluid flow with PGE2 or adenosine
blurred streaks expected for very fast particles were stimulation tended to slowly push extruded goblet
not observed. In crypts with relatively unobstructed mucus globules along the lumen walls rather than as
lumens, PGE2 produced particle speeds of ⬃10 µm/s free objects in the center of the lumen. The crypt from
throughout the period of stimulation, whereas CCh the patient with active colitis (Fig. 1B) formed a
stimulation resulted in particle speeds comparable to continuous filament (⬃15 µm diameter) of presumptive
PGE2 for only 2–3 min, after which movement slowed goblet mucus in the center of the lumen that was
and became undetectable. dragged toward the crypt orifice during PGE2 stimula-
Generally, particles moved as discrete entities, with tion. Particle movement in the fluid surrounding the
faster speeds nearer the center of the lumen, as ex- filament was comparable to that in Fig. 10, but the
pected for laminar flow. Volume flow through these speed of the filament was only ⬃0.5 µm/s. Volume flow

goblet granule clusters instead rearranging to form

smaller spheroids.
The response to CCh of a crypt from the patient with
active ulcerative colitis was consistent with hypersensi-
tivity 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 appear-
ance on CCh stimulation.
Stimulation of fluid secretion with PGE2 or adeno-
sine 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 reces-
sion of columnar cell apical borders (Fig. 14), consistent
with loss of apically stored contents.
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 assump-
tion of a spheroidal shape (see METHODS ). Goblet clus-
ters 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 vol-
ume 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.
Fig. 6. Epithelial volume changes. A: a representative time course of Volume stored in individual goblets was measured
dimensions from central portion of a normal crypt (⬃300 cells) during before and after maximal stimulation by CCh. The
stimulation by CCh (100 µM) and PGE2 (5 µM). Open symbols, equal
to prior measured value, indicate addition of CCh and PGE2. Seg-
goblet volume released by human crypts was ⬃25% of
ment length represents distance between distinct, identified cell the control value (Fig. 16A). Release at 2 and 10 µM
boundaries positioned at either margin of recorded image field. CCh was ⬎90% of the value at 100 µM, indicating that
Epithelial height (hep ) was calculated from crypt diameter (D) and the half-maximal stimulating concentration for CCh
lumen diameter (d): hep ⫽ (D ⫺ d)/2. Error bars, SE. B: crypt and
lumen volumes calculated using crypt and lumen diameters together
was ⬍0.2 µM. In response to histamine, ⬃30% of
with segment lengths (see METHODS ). Epithelial volume was calcu- control goblet volume was released in human and
lated as difference between crypt and lumen volumes. tamarin crypts (Fig. 16B). Addition of atropine, a
muscarinic antagonist, before and during histamine
stimulation did not alter the response (data not shown).
of mucus carried in the filament was ⬃5 pl/min, which Incomplete discharge of goblet stores during maximal
stopped at the end of PGE2 stimulation. activation suggests stimulatory mechanisms (choliner-
Stimulated release of cellular contents. Loss of epithe- gic and histaminergic) that limit granule release. Gob-
lial volume during stimulation by goblet- and fluid-type lets from tamarin crypts, however, released nearly all
secretagogues appeared to be restricted to specific cell the stored contents with CCh stimulation (Fig. 16A)
types. Goblet cells were the site of cholinergic volume but not with histamine stimulation (Fig. 16B), support-
loss. Release of mucin from apical granules began at ing a selective lack of restrictive cholinergic regulation
the luminal margin, occasionally progressing to make in these tamarins.
deep craters within the apex of goblet cells, presumably The two independent measures of CCh-induced gob-
by fusion of individual pits (Fig. 11). Columnar cell let volume release (Figs. 7A and Fig. 16A) were com-
apexes continued to arch over goblet cells during CCh pared quantitatively by using the goblet fraction of
stimulation, suggesting a lack of volume release from epithelial volume; Fig. 17 shows the goblet volume
this cell type. An en face view of the epithelium (Fig. 12) changes obtained from these individual (goblet volume)
shows the craters centered within the goblets and the and global (epithelial volume) measures. Normal hu-
spatial arrangement of goblet and columnar cells within man and tamarin crypts had volume changes close to
the epithelium. Craters generally did not persist, with the line of identity, supporting a conclusion that these

Fig. 7. Epithelial volume loss. Relative epithelial volume changes were normalized to control value preceding
stimulation. r, Normal; s, normal-perfused; l, ulcerative colitis (N, nonactive); p, Crohn’s disease; m, 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).
two measures of volume release represent the same ulcerative colitis crypts (Table 3, Fig. 13) were underrep-
cellular events. Crypts from the two patients with resented in measurements of goblet volume, since these
ulcerative colitis deviated significantly from the line of small clusters were excluded because of difficulty in
identity, suggesting that measurements of individual reliable tracking through the sequence of stimulation.
goblet volumes underestimate total volume released in Responsiveness of individual goblet cells. The distri-
these crypts. The larger response measured with epithe- bution of CCh-induced fractional goblet volume release
lial volume changes could result from a columnar cell for individual cells is shown in Fig. 18A. Two distinct
contribution in ulcerative colitis, but a more likely peaks occurred: one near zero volume release and one
explanation is that smaller goblet clusters seen in at higher release. This bimodal distribution is consis-
tent with a nonresponding group of goblet cells dis-
Table 2. Relative volume response to secretagogues persed among responding goblet cells. Comparison of
the fraction of goblet cells responding within a crypt to
Cell-Specific the average initial goblet volume (Fig. 18B) indicated a
Epithelial Apical Zone Apical Zone
greater proportion of nonresponding cells in crypts
CCh ⫺4.2 ⫾ 1.4‡ (11) ⫺19.0 ⫾ 3.7‡ (8) ⫺29.2 ⫾ 6.5*‡ (8) with larger goblet clusters. A similar relation for hista-
PGE2 ⫺4.7 ⫾ 1.8‡ (7) ⫺17.7 ⫾ 4.6‡ (6) ⫺50.6 ⫾ 13.1†‡ (6) mine stimulation (Fig. 18C) suggested that the hista-
Values are means ⫾ SE expressed as percentage; number of crypts mine response was not related to goblet size. In those
is in parentheses. Change in relative volume was calculated as crypts exposed to both CCh and histamine (3 crypts, 27
difference between control period after stimulation and control period goblets), 43% of goblet cells responded (defined as in
preceding stimulation. Carbachol (CCh) was added at 2–100 µM
(maximum stimulation). Prostaglandin E2 (PGE2 ) was added at 2–5
Fig. 18) to both agents, but the CCh-nonresponsive
µM (maximal stimulation). * Goblet. † Columnar. ‡ Significantly differ- cells responded to histamine and the histamine-
ent from zero, P ⱕ 0.05. nonresponsive cells responded to CCh. Apparently all

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. r, Normal; s, normal-perfused; l, ulcerative
colitis (N, nonactive); p, Crohn’s disease; m, tamarin. Experiments
without clearly sustained volume decreases in Fig. 7 were not
included. * Return to control. A: CCh. B: PGE2. C: adenosine.
these goblet cells were capable of stimulated release of crypts. In tamarin crypts, after CCh stimulation had
granule contents. dramatically depleted central goblet cells of granules
The nonresponding goblet cells were not present in (Fig. 4), those goblet cells in the most distal 25% of the
any apparent pattern within the central portion 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 repre-
sentative crypt is shown in Fig. 19A. Adenosine did not
alter goblet volume, but subsequent additions of hista-
mine and CCh produced rapid losses of goblet volume.
The shape of goblets also changed on stimulation.
Goblets became more spherical during adenosine addi-
tion but returned to a prolate shape in control condi-
tions. An irreversible change toward spherical occurred
with subsequent CCh addition; histamine did not dra-
matically alter goblet shape. Average time courses for
CCh and histamine goblet volume responses had simi-
lar kinetics and magnitude (Fig. 19B). The CCh re-
sponse of goblet cells measured from epithelial volume
changes was indistinguishable from the goblet volume
measurement, further supporting that these indepen-
Fig. 9. Depletion of apically stored materials. Proportion of epithelial dent measures reflect the same cellular event. Goblets
volume lost after sequential stimulation by CCh and a fluid secreta- from tamarin crypts had a similar half-time for goblet
gogue (PGE2 or adenosine) is shown in relation to measured initial
contribution of apical zone to total epithelial volume. r, Normal; s,
volume release, but nearly all the contents were re-
normal-perfused; l, ulcerative colitis (N, nonactive); p, Crohn’s leased. A time course for volume release from the apical
disease; m, tamarin. zone of columnar (nongoblet) cells indicates a secretory

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. Stimula-
tion 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 secreta-
gogues may occur through compaction as crypt length
shortens, presumably through the action of the peri-
cryptal 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 stimula-
tion, all goblets became shorter and wider. These goblet
shape changes indicate that cells neighboring goblet
cells, presumably columnar cells, lost volume on stimu-
lation with PGE2 or adenosine.
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)
Fig. 10. Luminal fluid flow. Speeds of refractile particles moving in near the epithelial surface. Mucus released in crypts of
lumen of a normal crypt (shown in Fig. 6) measured during stimula- Lieberkühn is cleared into the colonic lumen by fluid
tion with secretagogues. A: time course of particle speeds. Particle secretion. As shown in RESULTS, crypts from human
size was measured to compare cross-sectional area with luminal colon release apically stored material (presumably mu-
cross-sectional area. r, Particles with area ⬍5% that of lumen; j,
particles with area ⬎20% that of lumen; s, particles of intermediate
cus) from goblet and columnar cells. Separate control of
cross section. Large particle monitored at ⬃25 min squeezed past these mucus secretions occurs through distinct types of
several other objects at a relatively high speed, propelled by full force secretagogues.
of stream. B: variation of particle speed with particle volume during Epithelial cell types in colonic crypts. The colonic
PGE2 stimulation. One large particle occluded lumen during speed epithelium is comprised of several cell types that form
measurement (*).
the two major epithelial structures: the surface epithe-
lium and the crypts of Lieberkühn. Similar to other
response of comparable speed for fluid secretagogues mammals (3, 14), human colonic epithelium contains
(Fig. 19C). two predominant cell types: columnar and goblet cells
Expansion of extruded goblet mucus was particularly (21, 40, 48). Columnar cells have been distinguished
apparent in the crypt from the moderately affected further on the basis of ultrastructural features associ-
tamarin (Fig. 4). For seven goblets in this crypt that ated with the degree of differentiation. Enteroendo-
appeared to release all the mucin granules, a mucus crine cells, which release signaling molecules, gener-
expansion ratio was calculated. Volume of the extruded ally make up ⬍5% of the cells. Goblet cells are
mucus was measured from width and height (Eq. 2) distinguished by the large number of mucous granules
and compared with goblet volume before CCh stimula- in the apical pole. Although in crypts these cells lack
tion. The average expansion ratio was 2.1 ⫾ 0.1 and the characteristic narrow basal pole seen in the surface
was half-maximal at ⬃5 min after onset. Particles epithelium, crypt goblet cells have a rigid cytoskeletal
moving in the lumen (presumptive goblet mucus, Fig. arrangement that maintains the round profile of the
10) were often larger than expected for the effluent of a granule mass (43, 51). In living crypts, with use of DIC
single goblet cell (⬎200 fl: 335 fl average goblet volume · microscopy, goblet cells can be recognized readily by the
0.3 mean fractional release · 2-fold expansion ratio), ovate shape of this granule cluster because of the
suggesting that these secretory plumes could coalesce. highly refractile contents of the granules (Figs. 1–4 and
Changes in goblet shape during secretagogue-stimu- 11–14). Columnar cells fill the spaces between goblet
lated release of apically stored contents are consistent cells; distinctions among these nongoblet cells were not

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.
apparent. Deformation by the more rigid goblet cell lumen and fan-shaped deltas of columnar cells with a
neighbors produced a narrow waist between basal and wide luminal extent.
apical poles, giving columnar cells an hourglass appear- Mucus secretion by goblet and columnar cells. Secre-
ance. The apical poles of columnar cells fan out over tory control of columnar and goblet cells in colonic
goblet cells, such that only the small apexes of goblet crypts is distinct. Release of mucus from goblet cells is
cells contact the lumen. Whereas in rabbits and mice stimulated by cholinergic agonists and by histamine
the apical poles of columnar cells are filled with large (35); goblet cells do not release mucus in response to
vacuoles (3, 14), columnar cells in human colon have vasoactive intestinal peptide (VIP) or cAMP and theoph-
small apical vesicles (21, 40, 48). Thus goblet and ylline (35), agents that stimulate fluid secretion (13).
columnar cells have apically stored products positioned Responsiveness was assessed in fixed specimens by the
for release into the lumen. The general epithelial presence of apical cavitation in goblet cells, a procedure
appearance is of goblet ovals pointing toward the that would miss modest stimulation of granule release.

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 diam-
eter (Dcr ) and with assumption of a cylindri-
cal crypt shape: h ⫽ (Dcr /2) 51 ⫺ [1 ⫺
(w/Dcr )2]½6. 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 pit-
ting. Deformation of goblet cluster at top of
field is readily apparent from convex edges
of cell.
A morphometric assessment of cholinergic stimulation secretagogues. In living human colonic crypts, with use
indicated that goblet cells that did not cavitate, particu- of morphometric assessment, it was found that CCh
larly in the surface epithelium and crypt base, did show and histamine stimulated goblet granule release (Figs.
a decrease in mucous granules (37, 39). From these 11 and 16), but the fluid secretagogues PGE2 and
studies of fixed colonic tissue, the strongest goblet adenosine did not cause goblet granule release (Figs.
secretagogues are cholinergic agonists and histamine, 14, 19, and 20, Table 4). Although these results are not
but other agents are not conclusively ruled out as minor 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 stimu-
lation, 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 obser-
vations 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 secreta-
gogues by actively secreting Cl⫺ and by releasing a
Fig. 13. Cholinergic response in ulcerative colitis. CCh addition macromolecule into the lumen, indicating a cellular
stimulated mucus release in a crypt from a patient with active link between fluid and mucus secretion. Living human
ulcerative colitis (Fig. 1B). Scale bars, 5 µm. A: control goblet cells colonic crypts responded to PGE2 and adenosine by
had relatively depleted stores of granules. This crypt had been
stimulated previously by 5 µM PGE2. B: during CCh stimulation (100
secreting fluid (Figs. 4 and 10) and releasing apically
µM for ⬃10 min) mucus was released and remained adherent to stored material from columnar cells (Figs. 3, 8, and 14).
epithelium. Cholinergic stimulation did not alter columnar cells

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.
(Fig. 11), consistent with a segregation of secretory study fluid and mucus secretion. Capacity for fluid
responses to specific cell types. secretion generally is detected by measuring electro-
Colonic epithelial cell lines. Epithelial cell lines de- genic Cl⫺ secretion, which produces the osmotically
rived from colonic tumors have been used extensively to driven flow of fluid. Mucus secretion has been mea-
sured by various methods that detect the presence of
Table 3. Mucous goblet released glycoproteins. The T84 cell line secretes Cl⫺ in
response to VIP, prostaglandin E1 (PGE1 ) and CCh (5).
Volume, fl Eccentricity N (n) Release of mucus by T84 cells was stimulated with
Normal 335 ⫾ 27 ⫹0.74 ⫾ 0.03 8 (111) CCh, PGE1, and VIP (28, 32); electron micrographs
Normal (perfused) 310 ⫾ 37 ⫹0.45 ⫾ 0.11* 6 (95) showed that only ⬃5% of the cells contain large apical
IBD 230 ⫾ 27* ⫹0.70 ⫾ 0.02 3 (55) granules, so that the mucus secretion may have ema-
Tamarin (severe) 137 ⫾ 12* ⫹0.22 ⫾ 0.15* 3 (43) nated from a small minority of the cells. Several
Values are means ⫾ SE; N, number of crypts; n, number of subclones of the HT-29 cell line have been developed
individual goblets. * Significantly different from normal, P ⱕ 0.05. (HT29.Cl16E, HT29.B6, and HT29.18N2) that have a

types of mucus secretion (goblet cell derived and colum-

nar 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 characteris-
tic 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 secre-
tion. More realistic estimates of fluid viscosity would be
higher, because of secreted mucus, which would reduce
Fig. 15. Size and shape of apical goblets. Goblet granule clusters
Re further. The consequences for crypt fluid flow are
were measured for individual goblet cells in control conditions. that turbulence does not occur and a laminar flow
Volume and eccentricity (mean ⫾ SE) were calculated for each crypt condition develops immediately as flow starts.
(number of goblet cells measured ranged from 9 to 23/crypt). r, An estimate of the fluid secretory rate for an entire
Normal; s, normal-perfused; l, ulcerative colitis (N, nonactive); p, crypt can be calculated from the observed particle
Crohn’s disease; m, tamarin. Positive eccentricities indicate a prolate
shape; negative values indicate an oblate shape (see METHODS ).
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 stimu-
lated 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 secre-
tagogues (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 ) secreta-
gogues 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 compo-
nent 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
Fig. 16. Volume loss from apical goblets. Volume released from goblet
measurements of Cl⫺ secretion, which indicate that granule clusters is shown in relation to volume stored before stimula-
cholinergic stimulation produces only a transient in- tion (mean ⫾ SE). Dashed line, total release. A: volumes released
crease in Cl⫺ secretion compared with a sustained with CCh stimulation [100 or 2 µM (2) or 10 µM (10)]. r, Normal; s,
increase by fluid secretagogues such as PGE2 (5, 13). normal-perfused; l, ulcerative colitis (N, nonactive); p, Crohn’s
disease; m, tamarin. Dotted line, 25% release. Some crypts had been
Reduction in PGE2-stimulated fluid secretion with CCh stimulated by histamine before stimulation by CCh (*). B: volumes
(Fig. 10) may reflect the inhibiting action of CCh on Cl⫺ released with histamine stimulation (10 µM). Symbols as in A. Dotted
secretion measured in T84 cells (57). Thus the two line, 30% release.

one-half the product of cross-sectional area and speed

at lumen center (Jv ⫽ vmax␲d2/8, where vmax is maxi-
mum 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 secre-
tion 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 secre-
tion of 350 pl · min⫺1 · mm⫺1; cholinergic stimulation
Fig. 17. Comparison of volume release measurements. Average reversed absorption to a fluid secretion of 330 pl · min⫺1 ·
relative volume change of individual goblet granule clusters (Vgob ) in mm⫺1. Interestingly, a goblet secretagogue and a fluid
crypts produced by CCh stimulation (mean ⫾ SE) is shown in relation secretagogue stimulated similar rates of fluid flow with
to relative volume change of apical goblet zone [goblet fraction of
epithelial volume (Vep )]. r, Normal; s, normal-perfused; l, ulcer- these rat colonic crypts, in contrast to the results for
ative colitis (N, nonactive); m, tamarin. Dashed line, line of identity. human colonic crypts. Fluid absorption may be difficult
Ulcerative colitis crypts are significantly off line of identity; all other to detect in the present study, since the method relies
values are not significantly different from line of identity. on movement of refractile particles that might be
relatively scarce during absorption. In addition, rat
speeds and lumen dimensions. Volume flow past a colonic crypts were thought to be absorptive because of
specific point is related to the cross-sectional area of the the absence of the pericryptal sheath (49); all dissected
lumen (␲d2/4) and the velocity profile across the lumen human and tamarin crypts retained the pericryptal
(56). For laminar flow, total volume flow (Jv ) is equal to sheath (Fig. 1). Glands of the tracheal epithelium,
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). r,
Normal; s, normal-perfused; l, ulcerative colitis (N, nonactive); p, Crohn’s disease;
m, tamarin. Responding goblet cells were defined as those with a volume release
fraction ⱖ0.20. 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 ⱖ0.20.

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 (r; 6 crypts,
41 goblets). Histamine stimulation was averaged from crypts in Fig. 16B (p; 5 crypts,
32 goblets). Average CCh response of goblet cells from tamarin crypts [m; 2 crypts (1
severely affected and 1 moderately affected), 26 goblets] is also shown. Apical goblet
zone volume changes during CCh stimulation (s, 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 exponen-
tial fit was 3.3 min.
which secrete fluid and mucus, can be stimulated to 0.1–10 mmHg, consistent with a low pressure-flow
secrete fluid at much higher rates, ⬃10 nl/min (42, 53). system.
Secretory capacity for the human colon can be esti- Perfusion of the crypt lumen via pipettes mimics the
mated from the output of an individual crypt. Because state of fluid secretion and allows introduction of
crypt density in the human colonic epithelium is compounds to the apical surface of crypt epithelial cells.
⬃25,000/cm2 (48), the colonic mucosal surface area of The measured perfusion rate was ⬃4 nl/min in the
⬃4,000 cm2 indicates a total number of crypts in the study of fluid transport with perfused rat colonic crypts
colon of ⬃108. The total daily output of continuously (49), which is comparable to the estimated rate of 1–5
stimulated maximal crypt fluid secretion would be ⬃50 nl/min in this study of human colonic crypts. These
liters. Such a rate clearly could not be sustained, but luminal perfusion rates are 3- to 15-fold faster than the
the ability to overwhelm the absorptive capacity of the secretory flow at crypt orifice produced with PGE2 (Fig.
colon is apparent. 10), which may contribute to the altered appearance
The hydrostatic pressure gradient necessary to push (Figs. 5 and 15) and response (Fig. 7) of perfused crypts
fluid toward the crypt orifice can be obtained from the compared with intact crypts. Higher perfusion rates
Hagen-Poiseuille equation [⌬P ⫽ 128 l␩Jv/(␲d4 ), where are produced by higher applied hydrostatic pressure,
⌬P is pressure gradient] by using lumen dimensions, which has been reported to be deleterious to rabbit
total crypt volume flow, and fluid viscosity (56). Viscos- colonic crypts (26). Although luminal perfusion has the
ity is undoubtedly higher for the secreted fluid than for advantage of providing access to the apical membrane,
water because of the presence of secreted mucus. nonphysiological fluid flow may lead to abnormal cellu-
Colonic mucus contains soluble and insoluble forms (2), lar responses.
with the insoluble, or gel form, probably representing Mucus secretory mechanism. Mucus release by epithe-
goblet granule mucin. The less dense columnar cell lial cells, and goblet cells in particular, has been
material (14, 16) is more likely a soluble form, which measured by several methods. These techniques can be
would be most pertinent for determining fluid viscosity. separated into two broad categories: those that detect
Mucus viscosity depends on source, concentration, and mucus released (1, 28, 32, 38) and those that measure
shear encountered (8, 22, 34) but could range from 10- depletion of mucus from epithelial cells (4, 35, 37, 39,
to 1,000-fold that of water. From this range of fluid 46, 50, 52). Detection of mucus secreted from crypts is
viscosity, the expected pressure difference would be difficult, because collecting the mucus would require

Fig. 20. Stimulated rearrangement of goblet volume. A: goblet eccentric-

ity 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 adeno-
sine. Symbols as in A. C: changes in goblet dimensions for CCh-
responsive goblet cells of each crypt (r, l, m), for average of CCh-
nonresponsive goblet cells from all crypts (j, volume fraction released
⬍0.10), and for all goblet cells during fluid secretagogue stimulation of
each crypt (s, q, o).
not only release from the cell but clearance from the vesicles could not be distinguished consistently through
crypt lumen. Also, differences in cellular responsive- the time course of secretory stimulation. Comparison of
ness are difficult to determine from collection methods. these two measures for cholinergic responsiveness of
For these reasons, a morphological approach was cho- goblet cells (Fig. 17) indicates that epithelial volume
sen for the present study. Mucus volume released was can reliably report on the mucus volume released by
measured (see METHODS ) by two means: decreases in goblet cells, suggesting that the fluid secretagogue
epithelial volume and decreases in goblet volume. response measured as epithelial volume change (Figs.
Epithelial volume changes include goblet and columnar 7, C and D, and 19C) also reflects mucus release.
cell events, so that qualitative observations are re- Goblet cell mucus secretion. Similarity in the time
quired to indicate which cell types produced a particu- courses for the CCh response of epithelial and goblet
lar response (Figs. 11 and 14). Goblet cell mucous volume (Fig. 19B) suggests that cytoplasmic volume
granule volume release could be measured directly, changes during this stimulation are minor and limited
since the apical granules formed a compact distinct to a small transient decrease during the first minute
grouping. Unfortunately, a similar measure for colum- (seen as excess volume loss in the epithelial volume
nar cells was not possible, because the extent of apical trace compared with the goblet volume trace). Hista-
Table 4. Changes in size and shape of mucous goblet

⌬ Volume, fl ⌬ Eccentricity ⌬ Width, µm ⌬ Height, µm N (n)
CCh
High responders ⫺158 ⫾ 8* ⫺0.29 ⫾ 0.11* ⫺0.62 ⫾ 0.23* ⫺3.00 ⫾ 0.56* 6 (47)
Nonresponders ⫺6 ⫾ 7 ⫺0.11 ⫾ 0.03* ⫹0.39 ⫾ 0.15* ⫺0.91 ⫾ 0.25* 6 (21)
Fluid secretagogues
All goblets ⫹1 ⫾ 9 ⫺0.17 ⫾ 0.06* ⫹0.43 ⫾ 0.05* ⫺0.85 ⫾ 0.23* 5 (66)
Values are means ⫾ SE; N, number of crypts; n, number of individual goblets. ⌬ values were calculated from stimulated condition compared
with prior control condition. High responders released ⱖ20% of goblet volume (see Fig. 18); nonresponders released ⬍10% of goblet volume.
* Significantly different from zero, P ⱕ 0.05.

mine stimulation of goblet granule release (Fig. 16) was charge screening with divalent cations such as Ca2⫹
not apparent in epithelial volume measurements (Fig. (55). Goblet granules in the colonic epithelium contain
7B), suggesting that some or all of the crypt cells had high concentrations of Ca2⫹ and Mg2⫹ (16) that presum-
cytoplasmic swelling that obscured the volume released ably serve this purpose of condensing the filamentous
from mucous granules. Shape changes of goblet granule mucous molecules. The massive release from tamarin
masses during histamine stimulation also are consis- goblet cells (Fig. 4) indicated almost twofold expansion
tent with swelling, because goblet eccentricity stayed for extruded mucus. Because this enlargement is much
prolate during volume loss contrary to the CCh re- smaller than the 600-fold expansion seen for slug
sponse (Fig. 19A). Cytoplasmic swelling would have goblet granules (55), charge screening must be occur-
compressed goblets laterally so that a more spherical ring in the crypt lumen to limit mucus extension. Such
shape could not occur. Thus cholinergic and histaminer- a mechanism would have the advantage of not blocking
gic stimulation of goblet cells can be distinguished, in the crypt lumen, allowing easier passage of goblet
part, on the apparent cytoplasmic swelling induced by mucus out of the crypt.
histamine. Observing living colonic crypts allowed individual
Goblet cells release mucus by an exocytotic mecha- goblet cells to be followed through the course of stimu-
nism in which mucous granules fuse with the apical lated granule release. Some goblet cells were unrespon-
membrane (51). Maximal cholinergic stimulation leads sive to cholinergic stimulation, 23% (Fig. 18A), and
to deep cavitation of the goblet granule cluster by tended to have larger goblet volumes (Fig. 18B), consis-
compound exocytosis (50). This fusion of granules to tent with retention of granules. Apparently, unrespon-
other granules that have already fused with the apical siveness lasted long enough to fully pack the goblet
membrane produces craters in the apex of goblet cells zone. Responsive goblets, on average, released 29 ⫾ 2%
and results in loss of granule membrane as successive of mucus stores (none ⬎70%, Fig. 18A). This incomplete
fusions isolate some membrane fragments. Volume of release suggests that an inhibitory control restricts
the granule pool in control conditions (Table 3) was granule emptying and can block release in about one-
similar to that in rabbit distal colonic goblet cells (43); fourth of the goblet cells.
from the size of an individual granule, ⬃0.4 fl (Fig. 11), Columnar cell mucus secretion. The fluid secreta-
the number of granules in a normal goblet was ⬃850.
gogues PGE2 and adenosine produced decreases in
Individual exocytotic events were visible in living crypts
epithelial volume that were sustained after return to
as changes in light intensity of granules (flashes) that
the control condition (Fig. 7, C and D), which suggests
presumably result from a decrease in mucus refractility
that the lost volume resulted from mucus release from
as expansive release from granules occurs. These exocy-
columnar cells (14), as visualized by the recession of the
totic flashes have been seen in other goblet cells (4, 20,
52); the half-time for the process is ⬃0.2 s (46). Supra- apical cell margin (Fig. 14). Further support for this
maximal stimulation of human colonic goblet cells only cellular specificity is the lack of goblet volume decrease
occasionally produced the deep cavitations seen in (Fig. 19A, Table 4). A contribution of cytoplasmic vol-
rabbit distal colon (50). Instead, small craters were ume change to the fluid secretagogue response is more
apparent at the apex of goblets (Figs. 2 and 11). As the difficult to assess than with goblet secretagogues be-
volume of goblet granule clusters decreased (Figs. 16 cause of the lack of a direct measure of apical vesicle
and 19), the remaining granules rearranged to make volume. Crypt diameters have been used as an indica-
goblets shorter and narrower (Fig. 20). This more tor of epithelial volume during VIP stimulation of rat
subtle response is similar to goblet cells in colonic colonic crypts isolated without the pericryptal sheath
surface epithelium (39) and small intestinal villi (37). A (7). Although changes in crypt diameter were altered by
more dramatic goblet response was observed in a crypt furosemide, a blocker of Na⫹-dependent Cl⫺ entry,
from the patient with active ulcerative colitis (Fig. 13) interpretation of these changes as indicative of epithe-
and in crypts from tamarin colon (Figs. 4, 16, and 19). lial volume is problematic without measurements of
Although large quantities of mucus were visibly re- lumen diameter and crypt length. Intracellular Cl⫺
leased, cells appeared to rearrange behind the depart- concentration decreased transiently during PGE2 stimu-
ing mucus by becoming shorter without a noticeable lation in isolated rabbit colonic crypts, consistent with
cavity. For the tamarin with moderate colitis (Fig. 4), cell shrinkage that returned to near the initial value in
many of the granules appeared to be jettisoned intact ⬃2 min (15). The membrane electrical potential differ-
as a group, presumably when more peripheral granules ence of rabbit colonic crypt cells also depolarized tran-
fused in a compound fashion to cut off connection with siently during PGE2 or adenosine stimulation, return-
the cell. Within ⬃10 min of extrusion, the appearance ing to near the initial value in 1–2 min (27). These
of the mucous plume became homogeneous, suggesting results support an activation process for fluid secreta-
a lysis of these granules. These results with human gogues in which cytoplasmic volume decreases and
crypts support an involvement of compound exocytosis returns to control levels; this response results from Cl⫺
in goblet release but suggest that remaining granules and K⫹ channels opening followed by increasing Cl⫺
are moved within the cell to alter cell shape. influx, which supplies continued Cl⫺ secretion. There-
Expansion of mucus results from electrostatic repul- fore, the contribution of cytoplasmic changes to epithe-
sion of multiple anionic residues on these molecules lial volume would appear to be constrained to early
(54). Packing of mucus into granules is facilitated by time points, such that sustained volume decreases in

human colonic crypts (Figs. 7, C and D, and 19C) reflect (or hypersensitive) release from goblets of small size:
release of apically stored mucus from columnar cells. small goblets would be difficult to monitor through the
Columnar cells of colonic crypts release a material time sequence of stimulation and would be underrepre-
stored in apical vesicles (vacuoles), but the mechanisms sented in direct measures of goblet volume. Whereas all
of release are much less precisely known than for goblet tamarin goblets were hypersensitive (Figs. 16 and 18),
cells. Stimulation by fluid secretagogues in rabbit the largest (and easiest to monitor) goblets in human
distal colon leads to release via opening of a narrow crypts were not hypersensitive, and some were unre-
connection between vacuole and crypt lumen (14). sponsive. In contrast, tamarin and human ulcerative
Viewed in these crypts with DIC, initially the apical colitis goblet cells did not appear to be hypersensitive to
pole of the columnar cell becomes empty (less refractile) histamine, although an increased sensitivity in cells
and then collapses, such that goblet cells appear closer with limited granule supplies cannot be excluded. Thus
together. Because in human crypts the apical vesicles goblet cells in ulcerative colitis are hypersensitive to
were small, it was not possible to view the emptying, cholinergic stimulation, but those few that were the
but the collapse of the apical zone was observed as a least responsive would have retained more granules
recession of the apical margin of columnar cells, with and have the largest granule stores. Fluid secretion
goblet cells becoming closer together (Fig. 14). The time also is increased in ulcerative colitis (41), which may be
course of the PGE2 response of human crypts (Fig. 19C) a direct consequence of elevation in the inflammatory
indicates that volume release was essentially complete mediator PGE2 (17). Because isolated ulcerative colitis
at 10 min of stimulation, whereas fluid secretion contin- crypts did not secrete fluid until stimulated, the action
ued. Apparently, fluid secretion does not depend on of these fluid secretagogues was removed by the isola-
constant mucus release, even though both are initiated tion of crypts from the mucosa. The epithelial volume
by the same agents. released in response to PGE2 or adenosine (Fig. 8, B
IBD and secretory processes. The IBD of ulcerative and C) also suggests a hypersensitivity to these secreta-
colitis and Crohn’s disease lead to epithelial damage gogues in human and tamarin ulcerative colitis. Hyper-
and altered function (31, 41). Cell death may be the sensitivity for release of goblet cell mucus and colum-
cause of the focal points of short epithelial height seen nar cell mucus would exacerbate hyperstimulation
in inflammatory crypts (Fig. 1). In addition, the larger occurring through immune responses. These results do
diameter of crypts from patients with ulcerative colitis not indicate whether epithelial cells contribute to initia-
may reflect an early stage in cancer progression (31, 41, tion of the inflammatory response, but the crypt cells
59). Whether epithelial changes are simply a conse- have an augmented mucus secretory response.
quence of immune cell activity during inflammation or
constitute part of the cause for the pathology is uncer- APPENDIX
tain.
One diagnostic feature is depletion of goblet cell A translation of relative area in crypt midline optical
mucus in ulcerative colitis compared with normal mu- sections to relative volume requires assumptions about crypt
cosa or Crohn’s disease, whereas Crohn’s disease mu- geometry. For random sections, area measures will reflect
cosa contains only slightly less mucus than normal volume (58). The rigid geometry of crypts can be used to
mucosa (29). In colon from guinea pigs with experimen- compensate for the nonrandom nature of midline optical
tal colitis, the depletion of mucus was due to less mucus sectioning. Crypts can be modeled with roughly equal num-
bers of goblet and columnar cells dispersed evenly in the
in goblet cells rather than a decrease in goblet cell epithelium (10). A unit volume for the apical zone of the crypt
number (19). In addition to mucus depletion, the compo- can be defined as a truncated pie piece (a bite removed from
sition of colonic mucus is altered in ulcerative colitis the tip to create the lumen) with a single goblet in each; a
(41), and some of the change may relate to the different series of these prismoid unit volumes reconstruct the apical
mucus types in goblet and columnar cells (11). In the zone around the circumference of the lumen. The goblet
colonic crypt goblet cells from patients with ulcerative granule cluster can be approximated as a frustum of a right
colitis and those from tamarins with severe colitis, circular cone. Volume within the pie piece, but not in the
stores of mucus were smaller (Table 3, Fig. 15), consis- frustum, would be the apical zone volume contributed by
tent with the interpretation that goblet cells are pre- columnar cells. Volume of the frustum (Vfc, Eq. A1) can be
sent in ulcerative colitis but are simply more difficult to calculated from the height (h) and two widths (diameters),
the smaller at the lumen (wlu ) and the larger (w) at the base of
identify because of reduced mucus content. the goblet
Mucus depletion may have resulted from chronic
stimulation by inflammatory mediators released in ␲
vivo, but the response to CCh in vitro was larger than Vfc ⫽ h 1w2 ⫹ wwlu ⫹ wlu
2
2 (A1)
12
for normal crypts. Tamarin crypts released nearly the
complete apical supply of mucous granules (Figs. 4 and Volume of the unit apical zone (Vau, Eq. A2) can be obtained
16A). Both crypts from patients with ulcerative colitis from the frustum measurements, since the frustum is in-
(active and nonactive disease) had indications of hyper- scribed within the unit volume
sensitive goblet cells. Mucous plumes were apparent 1
(Fig. 13), and CCh-induced volume release was larger Vau ⫽ h 1w2 ⫹ wwlu2 (A2)
2
than in normal patients (Fig. 8A). The disparity be-
tween CCh-induced epithelial volume changes and The fraction of volume (fv ) contributed by goblets (Eq. A3) is
individual goblet changes may result from preferential obtained by dividing Vfc by Vau

1 2
␲ 1 ⫹ ␣ ⫹ ␣2 wlu Address for reprint requests and other correspondence: D. R.
fv ⫽ , ␣⫽ (A3) Halm, Dept. of Physiology and Biophysics, Wright State University,
6 1⫹␣ w 3640 Colonel Glenn Hwy., Dayton, OH 45435 (E-mail:
dan.halm@wright.edu).
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 Received 4 February 1999; accepted in final form 14 May 1999.
goblets (Eq. A4) is obtained from the ratio of the frustum
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Secretagogue response of goblet cells and

columnar cells in human colonic crypts

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Fig. 1. Isolated colonic crypts. Differen-

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Fig. 2. Cholinergic stimulation. Goblet granule clusters (arrow-

epithelial volume that was not recovered on return to

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Fig. 4. Secretory response of tamarin

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goblet granule clusters instead rearranging to form

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Fig. 8. Relative volume loss of apical granule storage zone. Time

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with a constraint on packing within the crypt structure.

A major task of the colonic epithelium as the most

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Fig. 12. Goblet response to CCh. A deeper

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types of mucus secretion (goblet cell derived and colum-

morphology similar to goblet cells (apically located

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one-half the product of cross-sectional area and speed

Fig. 18. Responsiveness of apical goblets. A: relative frequencies of CCh-induced

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Fig. 20. Stimulated rearrangement of goblet volume. A: goblet eccentric-

Table 4. Changes in size and shape of mucous goblet

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