Cystic Fibrosis research background
Research into the mechanisms of small intestinal secretion
For many years it was not well understood which compartment of the small intestinal
epithelium was responsible for the secretion of salt and water that is essential for the
proper digestion and absorption of nutrients after a meal. This question was made
crucial by the clinical importance of lethal diseases of intestinal secretion
such as diarrhoea and cystic fibrosis.
The first stage of the research was to devise a low temperature method
to isolate populations of small intestinal crypts that could be clearly identified by
biochemical assay for the specific marker enzymes of the villus and crypt regions,
alkaline phosphatase and thymidine kinase respectively. Having obtained a viable
preparation of small intestinal crypts, membrane potential and volume measurements
were made to (a) demonstrate that the small intestinal crypt compartment secreted KCl
and water; (b) that the crypt probably mediates a tonic secretion of KCl, as the membrane potential of
unstimulated crypts is determined by both potassium and chloride channel conductances;
(c) that acetylcholine (ACh), a neurotransmitter released from the myenteric plexus of the intestine,
stimulates a calcium-dependent increase in the rate of fluid and electrolyte (salt) secretion by upregulating the
potassium channel conductance alone, implying the existence of a constitutive Cl- pathway in the apical membrane
(i.e. facing the gut lumen) and that; (d) VIP, another myenteric plexus neurotransmitter,
augments fluid and electrolyte secretion through an increase in cAMP levels which, directly or indirectly,
activates chloride and probably also potassium channels. Further VIP and ACh were shown to act synergistically
in activating a chloride conductance.
Perforated-patch and single-channel modes of the "patch-clamp" technique were used to
characterise the role of the various ion channels in crypt fluid and electrolyte secretion.
As only the serosal, or basolateral membrane, of the intact isolated crypts was accessible,
this obligated a study of the K+ channels of the small intestinal crypt. In addition to a Ca2+-dependent
inwardly-rectifying K+ channel, the serosal membrane also contains a cation channel
and a very small K+ channel as two of the channels that were responsive to ACh.
Further a K+ channel that was too small to observe as unitary events, and which later was suggested to be the
MinK channel, was discovered, which probably plays a role in the regulation of the crypt membrane potential.
The cAMP-responsive K+ channel was not identified, but might well be MinK.
Measurements of crypt cell volume changes in response to changes in external osmolarity and secretagogues were performed by John O'Brien.
These suggested that the crypt basolateral membrane contains a Na-K-2Cl cotransporter which maintains Cl- and K+ fluxes
into the crypt in response to cell shrinkage, induced either by ACh or VIP, or by high external osmotic loads.
Radioimmunoassays were performed to identify the neurotransmitter or hormone that stimulates intestinal secretion via
the production of cGMP and also to determine whether Angiotensin II might stimulate an increase in cAMP levels. Contrary to
expectations, the hormone Atrial Natriuretic Peptide (ANP) did not increase cGMP levels as the STa heat-stable enterotoxin does.
Soon afterwards, guanylin was identified as the natural ligand for the STa-'hijacked' receptor.
In other collaborations the crypt isolation method was used to examine changes in the expression
of Angiotensin II receptor protein and mRNA along the rat jejunal
crypt-villus axis. Radioimmunoassay and membrane conductance
measurements were performed to elucidate the actions of angiotensin II on the
rat small intestinal crypt. In addition a truncated form of the CFTR gene product (deltaF508) was
expressed in Hela cells using a vaccinia/lipofectin transfection system (courtesy of the group of Professor C.F.Higgins), and gene function was
evaluated using the conventional whole-cell patch-clamp recording technique. Due to difficulties with the transfection system, only a fraction of cells were found to
express the mutant CFTR chloride channel.
