1 Molecular Medicine Unit, Somatostatin is a powerful inhibitor of
intestinal Cl
chloride secretion; G proteins
CHLORIDE SECRETORY DIARRHEA, whether infective,
inflammatory, or neurohumoral in origin, remains a common and costly
clinical problem that often responds poorly to conventional
antidiarrheal drugs. The dominant transport process, electrogenic
Cl Somatostatin is a tetradecapeptide normally present in intestinal
mucosa and has long been recognized as a potent antisecretory peptide
capable of inhibiting all forms of
Ca2+- and cyclic
nucleotide-mediated Cl Isolation of Colonic Crypts
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
secretion. We
used patch-clamp recording techniques to investigate the effects of
somatostatin on low-conductance (23-pS)
K+ channels in the basolateral
membrane of human colonic crypts, which are an important component of
the Cl
secretory process.
Somatostatin (2 µM) elicited a >80% decrease in
"spontaneous" K+ channel
activity in cell-attached patches in nonstimulated crypts (50%
inhibition =~8 min), which was voltage-independent and
was prevented by pretreating crypts for 18 h with pertussis toxin (200 ng/ml), implicating a G protein-dependent mechanism. In crypts stimulated with 100-200 µM dibutyryl cAMP, 2 µM somatostatin
and its synthetic analog octreotide (2 µM) both produced similar
degrees of K+ channel inhibition
to that seen in nonstimulated crypts, which was also present under
low-Cl
(5 mM) conditions.
In addition, 2 µM somatostatin abolished the increase in
K+ channel activity stimulated by
2 µM thapsigargin but had no effect on the thapsigargin-stimulated
rise in intracellular Ca2+. These
results indicate that somatostatin peptides inhibit 23-pS basolateral
K+ channels in human colonic crypt
cells via a G protein-dependent mechanism, which may result in loss of
the channel's inherent Ca2+ sensitivity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
secretion through apical
Cl
channels, occurs
primarily in small intestinal and colonic crypts and is triggered by
soluble mediators acting through
Ca2+-dependent and/or
cAMP-dependent intracellular signaling pathways (5, 18). In the
currently proposed model of electrogenic Cl
secretion in intestinal
epithelia, in which an increase in apical Cl
conductance is the
initial step in the secretory process, a subsequent increase in
basolateral K+ conductance
promotes cell hyperpolarization and recycling of K+ taken up via basolateral
Na+-K+-2Cl
cotransport and
Na+-K+-ATPase.
This maintains the electrochemical gradient required for sustained
apical Cl
exit (1). As a
result, maneuvers leading to a decrease in basolateral
K+ conductance have a profound
inhibitory effect on electrogenic Cl
secretion. Serosal
application of Ba2+ to polarized
monolayers of the T84 human colonic epithelial cell line markedly
inhibits basolateral
86Rb+
(a proxy for K+) efflux and
cAMP-stimulated Cl
secretion by blocking a basolateral
K+ conductance (27, 29).
Inhibition of basolateral K+
conductance has also been implicated in the mechanism of action of
other pharmacological agents with antisecretory properties in vitro
(25, 34). Patch-clamp studies have confirmed that Ba2+-sensitive 23-pS
K+ channels are present in
abundance in the basolateral membrane of native human colonic crypt
cells (35).
secretion (10, 22, 41). This peptide exerts its antisecretory effects
via G protein-coupled receptors on the basolateral membrane of the
epithelium (39, 41), which in turn interact in a complex way with the
Cl
secretory process.
Although somatostatin lowers intracellular cAMP concentrations in
epithelial cells via G protein-dependent inhibition of adenylate
cyclase (38), it has additional antisecretory effects distal to the
intracellular second messenger production/activation cascades (41).
Somatostatin and octreotide, its more widely used synthetic analog, may
therefore act at a fundamental regulatory site in the
Cl
secretory process. The
aim of this study was to investigate whether basolateral
K+ channels are a possible target
for the antisecretory effects of these peptides.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Measurement of K+ Channel Activity
Patch-clamp recordings were made from basolateral membranes of cells in the middle third of intact crypts using cell-attached and excised inside out configurations (19, 35). The bath solution routinely contained (in mM) 140 Na+, 4.5 K+, 1.2 Ca2+, 1.2 Mg2+, 149 ClBecause of the difficulty experienced in maintaining high-resistance membrane seals throughout the long experimental protocols (up to 40 min), some experiments on individual crypts from each patient had to be aborted. This invariably resulted in only one complete experimental protocol being completed in crypts from a single patient. The number of patches used for each protocol therefore is equal to the number of patients from whom crypts were obtained.
Effect of Somatostatin Peptides on Intracellular cAMP
Crypts were resuspended in Krebs bicarbonate buffer solution containing (in mM) 146 Na+, 4.2 K+, 1.2 Ca2+, 1.2 Mg2+, 126 ClMeasurement of Intracellular Ca2+ Concentration
Intact human colonic crypts were isolated as previously described and prepared for fluorescence imaging by incubating in NaCl Ringer solution containing 200 µM dibutyryl cAMP and 5 µM fura 2-AM for 15 min at room temperature, followed by repeated washing (3 times) with fresh NaCl Ringer solution. The 200 µM dibutyryl cAMP was present during all subsequent manipulations of the crypts to maintain similar conditions to those used in the patch-clamp studies. Two or three drops of crypt suspension were placed on a polyethyleneimine-coated glass coverslip mounted in a small chamber on the stage of a Nikon Diaphot inverted microscope, which facilitated continuous perfusion (2 ml/min) of the fura 2-loaded crypts. To reduce possible loss of dye from the crypts, measurements of intracellular Ca2+ concentration ([Ca2+]i) were started immediately after a 5- to 10-min equilibration period, during which time the cells were perfused continuously with NaCl Ringer solution. Cells were epi-illuminated alternately at 340 nm and 380 nm, and the emitted light above 520 nm was captured through a ×40 1.3 numerical aperture objective by an extended ISIS-M video camera (Photonic Science, Robertsbridge, UK) and digitized using a PT50 frame grabber (Perceptics, Knoxville, TN). Consecutive frames obtained during 340 nm and 380 nm excitation were analyzed pixel by pixel using Ionvision software (Improvision, Coventry, UK) to give ratio images every 5 s. [Ca2+]i was calculated with reference to a calibration curve relating nanomolar concentrations of Ca2+ to the 340/380 ratio using sigmoid curve-fitting software. The calibration curve was constructed using commercially available Ca2+-EGTA buffer solutions yielding known free Ca2+ concentrations in the range 0-39.8 µM (Molecular Probes, Eugene, OR) and containing 50 µM fura 2.Statistical Analyses
Data are expressed as means ± SE. Differences between two means were compared using paired or unpaired Student's t-test as appropriate. ![]() |
RESULTS |
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Patch-Clamp Studies
Studies in the absence of dibutyryl cAMP.
To evaluate the effect of somatostatin on basolateral
K+ channel activity in the absence
of a Cl secretory agonist,
a series of experiments was performed using human colonic crypts not
previously exposed to dibutyryl cAMP. Under these conditions,
"spontaneous" low-conductance
K+ channel activity was identified
in 10 out of 34 cell-attached basolateral membrane patches in the
middle third of crypts studied on the day of isolation. In excised
inside out patches, current recordings over a range of holding voltages
(Fig.
1A)
provided a current-voltage relationship (Fig.
1B) that confirmed the high K+-to-Na+
permeability ratio (49 ± 6:1) and low unitary conductance (24 ± 3 pS; n = 8 patches) of the channel.
Addition of 2 µM somatostatin markedly decreased the
Po of
spontaneously active
K+ channels in six out of six
cell-attached patches (Fig.
2A).
Figure 2B shows the full time course
of three of these experiments (in which recordings were continued
during a washout period) in which 2 µM somatostatin produced
reversible inhibition of K+
channel activity,
Po decreasing
from 0.41 ± 0.04 to 0.07 ± 0.06 (P < 0.01). A 50% inhibition
occurred after ~8 min, with maximal inhibition at ~20 min after the
addition of somatostatin.
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Studies in the presence of dibutyryl cAMP.
We then went on to study the relation between intracellular cAMP,
electrogenic Cl
secretion,1 and the ability of
somatostatin to inhibit the 23-pS basolateral K+ channel. Somatostatin inhibits
adenylate cyclase (see Effect of Somatostatin Peptides on
Intracellular cAMP) and reduces intracellular cAMP concentrations
in many cell types, including intestinal epithelial cells (23, 41). We
therefore performed a further series of experiments to evaluate the
inhibitory effect of somatostatin on 23-pS
K+ channel activity using human
colonic crypts pretreated for 30 min with 100 µM dibutyryl cAMP to
maintain high intracellular cAMP levels. Somatostatin inhibited
K+ channel activity in dibutyryl
cAMP-pretreated crypts to a similar degree
(Po decreasing
from 0.45 ± 0.03 to 0.19 ± 0.05, n = 9 patches, P < 0.01) and over the same time
course as that seen in crypts not pretreated with dibutyryl cAMP.
Pretreatment with a higher concentration (200 µM) of dibutyryl cAMP
also failed to attenuate the inhibitory effect of somatostatin on
K+ channel activity (data not
shown). Octreotide (2 µM), a long-acting synthetic somatostatin
analog used in the treatment of secretory diarrhea, also markedly
reduced K+ channel activity in
crypts pretreated with dibutyryl cAMP
(Po decreasing
from 0.47 ± 0.03 to 0.18 ± 0.04, n = 5 patches,
P < 0.05).
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Studies in the presence of thapsigargin.
In addition to the evidence that somatostatin inhibits intestinal
Cl secretion independently
of changes in intracellular cAMP concentration, this peptide has been
shown to prevent Cl
secretion stimulated by
Ca2+-dependent secretagogues in a
human colonocyte line, apparently by acting at a site distal to the
process(es) involved in raising [Ca2+]i
(41). Because the 23-pS basolateral
K+ channels in human colonic
crypts are Ca2+ sensitive (26,
35), we explored the possibility that somatostatin might alter this
channel characteristic. As shown in Fig. 5,
addition of 2 µM thapsigargin in the absence of somatostatin produced
a twofold increase in K+ channel
activity after 10 min in cell-attached patches
(n = 3 patches,
P < 0.01). In contrast, when crypts
were pretreated with somatostatin for 20 min, subsequent addition of
thapsigargin had no effect on K+
channel activity (n = 4 patches). The
ability of somatostatin to block
Ca2+ activation of this channel
appeared to be independent of intracellular [Ca2+]i
per se, because thapsigargin elicited similar sustained increases in
[Ca2+]i
in both the absence (from 207 ± 24 to 321 ± 31 nM,
n = 6 crypts) and in the presence
(from 201 ± 12 to 304 ± 25 nM,
n = 6 crypts) of somatostatin (Fig.
6). These data suggest that the
antisecretory effect of somatostatin may reflect, at least in part, a
crucial loss of the K+ channel's
ability to respond to
[Ca2+]i.
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Effect of Somatostatin Peptides on Intracellular cAMP
With the use of crypt preparations from four patients, the intracellular cAMP level was 15.1 pM/mg cell protein under basal conditions, rising to 332 pM/mg cell protein after the addition of 2 µM forskolin to stimulate adenylate cyclase. Whereas the prior addition of 2 µM somatostatin or 2 µM octreotide had no effect on the basal intracellular cAMP level, both somatostatin peptides produced modest decreases in intracellular cAMP [to 88.7 ± 2.8% (P < 0.005) and 83.7 ± 2.7% (P < 0.005), respectively, of the maximal level] in the forskolin-treated crypts. These data indicate that the ability of somatostatin and octreotide to decrease spontaneous basolateral K+ channel activity does not reflect inhibiton of adenylate cyclase. In addition, although we used exogenous dibutyryl cAMP rather than forskolin to activate electrogenic Cl ![]() |
DISCUSSION |
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In the accepted model of intestinal
Cl secretion, activation of
basolateral K+ channels plays a
key role in maintaining the driving force for Cl
secretion by promoting
cell hyperpolarization and the recycling of
K+ taken up across the basolateral
membrane via the
Na+-K+-2Cl
cotransporter and
Na+-K+-ATPase
(1). Studies based on transepithelial electrical measurements suggest
the presence of two pharmacologically distinct basolateral K+ conductances in cultured
monolayers of T84 human colonic adenocarcinoma cells (4, 9). One is
activated during cAMP-stimulated electrogenic Cl
secretion and is
profoundly inhibited by serosal
Ba2+, whereas the other is
stimulated by Ca2+-mediated
Cl
secretory agonists and
is Ba2+ insensitive. With the use
of patch-clamp recording techniques, we have also identified two
distinct types of basolateral K+
channel in human colonic crypt cells. One is the 23-pS
K+ channel we describe here, which
is activated by exogenous cAMP and carbachol (a
Ca2+-mediated secretory agonist)
and is readily inhibited by Ba2+
(35). The other is a 138-pS K+
channel, which is also Ba2+
sensitive but is unaffected by cAMP and carbachol (26). It is obviously
difficult to make a direct comparison between single-channel data
obtained from native human colonic crypt cells and transepithelial data
obtained from a malignantly transformed colonic epithelial cell line.
It therefore remains unclear whether the inwardly rectifying 23-pS
basolateral K+ channel we have
identified in human colonic crypts equates with either of the
basolateral K+ conductances
described in T84 cells. However, the 23-pS
K+ channel may correspond to the
Ca2+-activated inwardly rectifying
K+ channel of intermediate
conductance identified in single (nonpolarized) T84 cells (7). The
apparent responsiveness of 23-pS
K+ channels to dibutyryl cAMP in
human colonic crypt cells (35) may therefore reflect basal
[Ca2+]i
being higher in these cells (207 ± 24 nM; see Fig. 6) than in T84
cells (117 ± 7 nM; see Ref. 9), leading to a complex interplay
between cAMP- and Ca2+-mediated
signaling pathways and secondary activation of basolateral K+ channels during cAMP-stimulated
Cl
secretion. Indeed, a
rise in basal
[Ca2+]i
during the crypt isolation procedure may account for the significant levels of spontaneous 23-pS K+
channel activity that we saw in some crypts even in the absence of
dibutyryl cAMP.
Although we have shown that 23-pS
K+ channels predominate in the
basolateral membrane of native human colonic crypt cells (26), they
differ widely from basolateral K+
channels in intestinal epithelia in other species. Rat small intestinal
enterocytes possess Ca2+- and
voltage-dependent 250-pS basolateral
K+ channels (30), but their
physiological role is unclear. The basolateral membrane of cells at the
base of rat distal colonic crypts is rich in
Ca2+- and voltage-dependent 12-pS
K+ channels that may help to
maintain cell membrane voltage (3), has prostaglandin
E2-stimulated 27- to 39-pS
nonselective cation channels that may be involved in active
K+ secretion (36), and contains an
infrequent 187-pS K+ channel with
no known function (3). The 12-pS
K+ channel in rat colon may
correspond to the Ca2+-sensitive
inwardly rectifying 10- to 20-pS basolateral
K+ channel that is activated
during carbachol-induced (that is, Ca2+-mediated)
Cl secretion and
inactivated during cAMP-induced
Cl
secretion (2, 17, 32).
Thus despite the evidence that basolateral
K+ channels are an important
component of the generally proposed model of intestinal
Cl
secretion (1), the
precise nature of these channels and their mode(s) of regulation are
likely to show appreciable species-to-species variability.
Studies over many years have shown that somatostatin and its related
peptides are extremely effective inhibitors of intestinal Cl secretion stimulated by
all types of secretagogue, including those acting through cAMP- and
Ca2+-dependent processes (12, 22,
41). This has been the rationale for the clinical use of long-acting
somatostatin analogs (such as octreotide) in the treatment of
persistent Cl
secretory
diarrhea (15). Octreotide therapy is now widely used with varying
degrees of success to treat refractory diarrheas, such as those
associated with carcinoid syndrome as well as high-output ileostomies
(6, 42). Although previous studies in several species have alluded to
an inhibitory interaction between somatostatin peptides and enterocyte
K+ channels, the cellular
mechanisms by which they exert such a comprehensive inhibition of the
Cl
secretory process remain
unclear. Inhibition of adenylate cyclase, as observed in the
HT-29-cl.19A colonic cell line (41) and forskolin-treated human colonic
crypts in the present study, suggests that direct interference with the
production of intracellular second messengers is likely to be one
component of somatostatin's antisecretory action. However, these
peptides are also effective against agents that activate secretion
distal to second messenger production. In the same study using
HT-29-cl.19A cells, Cl
secretion activated by the
Ca2+-mediated agonist carbachol
was markedly inhibited by somatostatin via a pertussis toxin-dependent
process without affecting the agonist-stimulated rise in
[Ca2+]i
(41). Similarly, somatostatin peptides inhibited dibutyryl cAMP-activated Cl
secretion
in HT-29-c1.29A cells and rat colon (39, 41). In rat colonic crypts,
somatostatin decreased carbachol-activated whole-cell
K+ currents, although the type of
K+ channel involved and its
membrane location were not defined (11). Furthermore, studies in guinea
pig enterocytes showed that activation of G proteins by the
intracellular application of GTP
S inhibited whole cell
K+ currents (14), but no attempt
was made to identify the physiological regulator of this process.
Despite the implication from such studies that somatostatin receptors
couple to a fundamental regulatory site in the
Cl
secretory process, which
lies downstream to the production of intracellular second messengers,
the nature and location of this site have been unclear.
In the present study, we show that somatostatin and octreotide
reversibly inhibit spontaneous basolateral
K+ channel activity via a
mechanism that involves pertussis toxin-sensitive inhibitory G
protein(s). Moreover, their inhibitory effect is independent of raised
intracellular cAMP levels and
Cl secretion per se.
Indeed, based on the studies with thapsigargin (Figs. 5 and 6), one
possible interpretation of our data is that somatostatin- and
octreotide-induced inhibition of the 23-pS
K+ channel reflects loss of the
channel's inherent Ca2+
sensitivity. An alternative interpretation, that somatostatin peptides
cause a loss of the channel's sensitivity to cAMP, is possible but is
not supported by our finding that somatostatin and octreotide both
inhibited spontaneous K+ channel
activity in crypts not pretreated with dibutyryl cAMP. In any event,
the inhibitory effect appears to be restricted to the 23-pS
K+ channel because somatostatin
had no effect on the 138-pS K+
channel, which is also located in the basolateral membrane of human
crypt cells (Fig. 3A and Ref. 26).
The precise mechanism through which somatostatin peptides inhibit 23-pS basolateral K+ channels in human colonic crypts is unknown and will be an important goal of future studies. One possibility to be considered is that the proposed loss of K+ channel sensitivity to Ca2+ may reflect a change in the phosphorylation state of the channel protein, which is an important determinant of the Ca2+ sensitivity of intestinal K+ channels (21). Furthermore, in rat pituitary tumor cells, somatostatin induces dephosphorylation of Ca2+-activated K+ channels via activation of arachidonic acid release and metabolism (13). In this respect it is interesting that arachidonic acid has recently been shown to directly inhibit Ca2+-dependent K+ channels in the T84 human colonic cell line (8).
The selective blockade of basolateral
K+ channels as a means of
controlling epithelial Cl
secretion has been explored by several groups. At the simplest level,
the nonspecific K+ channel blocker
Ba2+ inhibits 23-pS basolateral
K+ channels in human colonic
crypts (35) and causes almost complete cessation of
Cl
secretion when added to
the basolateral surface of colonic cell monolayers (27, 29). Recently,
novel K+ channel blockers have
been shown to inhibit cAMP-activated
K+ conductances and
Cl
secretion in rabbit
colon (25). The antifungal antibiotic clotrimazole has also been shown
to prevent fluid and electrolyte secretion in rabbit and mouse
intestine triggered by Ca2+- and
cAMP-mediated agonists via specific inhibition of basolateral K+ conductances (34). Furthermore,
levamisole (and other phenylimidazothiazoles) inhibits
Ca2+- and cAMP-activated
Cl
secretion in T84 cell
monolayers and isolated human distal colon, apparently by blocking
basolateral K+ channels (31). Our
finding that somatostatin, one of the most potent and comprehensive
inhibitors of intestinal secretion, has a marked inhibitory effect on
23-pS basolateral K+ channels in
human colonic crypts highlights this component of the intestinal
Cl
secretory process as the
starting point for new antidiarrheal strategies. To this end, we should
emphasize that somatostatin peptides elicit their diverse cellular
actions by activating a family of G protein-coupled somatostatin
receptors (SSTR) (33), several of which are expressed in
colonic epithelia (24, 39, 40). Studies using receptor-selective
agonists in rat colon indicate that the antisecretory actions of
somatostatin in this epithelium are mediated by a specific receptor,
SSTR2 (28, 39). Identification of
the SSTR subtypes linked to basolateral
K+ channels in human small
intestinal and colonic epithelia and a better understanding of the
signaling processes involved may provide a basis for the development of
new antisecretory drugs with greater potency and specificity.
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ACKNOWLEDGEMENTS |
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This study was supported by grants from the Sir Jules Thorn Charitable Trust, Medical Research Council, Wellcome Trust, Royal Society, and the North West Regional Health Authority.
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FOOTNOTES |
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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.
1
Dibutyryl cAMP-stimulated
Cl secretion was studied in
confluent monolayers of the human colonic HT-29-cl.19A cell line
mounted between Ussing chambers containing Krebs bicarbonate solution (38). Increases in short-circuit current
(Isc) reflected
dibutyryl cAMP-stimulated electrogenic
Cl
secretion (41). For
experiments under low-Cl
(5 mM) conditions, monolayers were bathed with a solution in which 121 mM
Cl
was replaced with
equimolar gluconate.
Isc was monitored
at 5-min intervals before and after the basolateral addition of 200 µM dibutyryl cAMP. Under
high-Cl
conditions,
dibutyryl cAMP elicited a peak rise in
Isc of 20.3 ± 2.2 µA/cm2 after 30-35 min
(n = 5). In contrast, under
low-Cl
conditions dibutyryl
cAMP stimulated only a small rise in
Isc of 2.4 ± 0.5 µA/cm2
(n = 5). These data confirm that
dibutyryl cAMP-stimulated electrogenic Cl
secretion by human
colonocytes is substantially reduced (by 88%, P < 0.001) under
low-Cl
conditions.
2
The aim of this study was to establish the
principle that somatostatin peptides influence basolateral
K+ channels in
Cl secretory epithelia. We
therefore used both somatostatin and octreotide at a concentration of 2 µM, because the addition of 5 µM somatostatin to the basolateral
bathing solution has been shown to inhibit prostaglandin
E2-induced electrogenic
Cl
secretion by 90% in
HT-29-cl.19A cell monolayers (41).
Address for reprint requests and other correspondence: G. I. Sandle, Molecular Medicine Unit, St. James's Univ. Hospital, Beckett St., Leeds LS9 7TF, UK (E-mail: g.i.sandle{at}leeds.ac.uk).
Received 16 June 1998; accepted in final form 2 August 1999.
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