Response of sigmoïd epithelium to secretory stimuli in adults with constipation

Approximately 12-27% of the population may suffer from functional constipation
which seems to further increase with age. It is recognized by infrequent, hard
stools, incomplete evacuation of stools and the involuntary loss of faeces in
the underwear. It is a debilitating condition that is often associated with
abdominal pain. To date however, treatment modalities are mainly based on
empiricism and have little evidence of long-term efficacy. This is mainly due
to the lack of knowledge on the pathophysiology which is most likely
multifactorial and can result from behavioural factors, motility abnormalities
or disturbances in fluid and / or electrolyte balance.
With respect to possible disturbances in fluid and / or electrolyte balance it
should be noted that secretion of water and electrolytes by the
gastrointestinal tract are central to its physiological functions. Factors that
increase net water absorption (as opposed to secretion) will lead to harder
stools and possibly constipation. The predominant electrolyte that drives
intestinal fluid secretion is chloride, and its secretion is mediated by
chloride channels in the apical membrane of the enterocyte. The importance of
intestinal chloride secretion is emphasized by the congenital disorder cystic
fibrosis that is accompanied by severe intestinal obstruction. In cystic
fibrosis constipation is caused by a mutation in one of the two Chloride
channels that is important for intestinal chloride secretion, namely the cystic
fibrosis transmembrane conductance regulator (CFTR). The other chloride channel
that may be involved in non-cystic-fibrosis-related constipation is the
eneterocyte-expressed ClC-2 channel located at the tight junction of the apical
membrane.
The possible relevance of the ClC-2 ion-channel became apparent from
preliminary studies on the chloride-channel activating compound Lubiprostone
(SPI-0211). Its use showed a statistically significant increase in bowel
movement frequency in constipated adults; from a range of 2.8*3.5 bowel
movements/week in the placebo group to a range of 5.1*5.7 in the SPI-0211
treated group (P < .002). The main drug-associated side effect was nausea, but
only a few patients withdrew because of this and no patients developed
dehydrating diarrhoea. At this moment already 4 studies have been performed in
constipated adults using this compound with positive results increasing
frequency of bowel movements or accelerating colonic transit time. In a recent
investigation, a human enterocyte cell line as well as ClC-2 and CFTR
transfected human embryonic kidney cells were used to investigate the possible
mechanism of action of SPI-0211. It was shown that the most probable target of
this drug is not CFTR but the ClC-2 channel instead. However, only very few
studies on the functional role of ClC-2 exist and functional investigations on
intact human tissues have never been performed. Furthermore, there is a
complete lack of publications on in situ expression levels of these
chloride-channels in intestinal tissue of patients suffering from functional
constipation.

To investigate a) the functional role of chloride secretion in constipated
adults (Ussing chamber studies) and b) inventory possible aberrancies in in
situ ClC-2/ CFTR expression levels by immuno-histochemistry staining
techniques.

Experiments:

Ussing Chambers
Functional ex vivo investigations on electrolyte transport can be performed in
miniaturized Ussing chambers; forcepal endoscopic biopsies are mounted between
the chamber halves where buffers circulate separately on the luminal and
serosal sides. The spontaneous potential difference generated by the tissue is
negated by injecting a current, known as the short-circuit current (Isc).
Pharmacological agents added to either side of the chamber may alter active ion
transport, which is shown by a change in Isc. A change of current indicates a
change in chloride secretion.

In situ immunofluorescence investigations
The expression patterns of ClC-2 and CFTR in rectal mucosa of adults with
functional constipation will be compared with controls. For this staining we
will use the same biopsy that is present in the Ussing chamber. After
completing the Ussing chamber experiments one biopsy will be used for
immunohistochemistry staining.

The burden is that patients will undergo a sigmoidoscopy. This means that
patients undergo such a procedure lasting for a total of 10 minutes.
The only benefit for those patients is that when we find a disturbed chloride
secretion in those suffering from constipation, we will have a grounded reason
for using Lubiprostone in our clinic in the future.

Case reports of perforation after sigmoidoscopy are described. However this
risk is not high, approximately 0.5 pro mille.