Geoffrey I. Sandle

Effect of glucocorticoids on rectal transport in normal subjects and patients with ulcerative colitis.

The acute effects of single pharmacological doses of glucocorticoid hormones on net electrolyte and water transport and electrical potential difference (pd) in the rectum was studied in control subjects and in patients with either active or inactive ulcerative colitis, using a dialysis technique. Compared with 17 control subjects, nine patients with active ulcerative colitis exhibited marked decreases in net sodium absorption and rectal pd, while these transport parameters were normal in six patients with inactive ulcerative colitis. Intravenous administration of hydrocortisone hemisuccinate (100 mg) resulted five hours later in significant and quantitatively similar increases in net sodium and water absorption and pd in nine control subjects, seven patients with active ulcerative colitis, and six patients with inactive ulcerative colitis. Intravenous administration of methylprednisolone phosphate (40 mg) to eight control subjects produced increases in net sodium and water absorption and pd five hours later, which did not differ significantly from those produced by hydrocortisone; methylprednisolone induced similar changes in two patients with active ulcerative colitis. These results indicate that single pharmacological doses of glucocorticoids stimulate acute increases in rectal sodium and water absorption in control subjects and in patients with acute ulcerative colitis. The ability of systemically administered glucocorticoids to reduce diarrhoea in ulcerative colitis may therefore be related to direct effects on distal colonic sodium and water transport, as well as to their better known anti-inflammatory action.

Enhanced large intestinal potassium permeability in end-stage renal disease.

The capacity of the colon for potassium (K+) secretion increases in end‐stage renal disease (ESRD), to the extent that it makes a substantial contribution to K+ homeostasis. This colonic K+ adaptive response may reflect enhanced active K+ secretion, and be associated with an increase in apical membrane K+ permeability. In this study, this hypothesis was tested in patients with normal renal function or ESRD, by evaluating the effect of barium ions (a K+ channel inhibitor) on rectal K+ secretion using a rectal dialysis technique, and the expression of high conductance (BK) K+ channel protein in colonic mucosa by immunohistochemistry. Under basal conditions, rectal K+ secretion was almost threefold greater (p < 0.02) in ESRD patients (n = 8) than in patients with normal renal function (n = 10). Intraluminal barium (5 mmol/l) decreased K+ secretion in the ESRD patients by 45% (p < 0.05), but had no effect on K+ transport in patients with normal renal function. Immunostaining using a specific antibody to the BK channel α‐subunit revealed greater (p < 0.001) levels of BK channel protein expression in surface colonocytes and crypt cells in ESRD patients (n = 9) than in patients with normal renal function (n = 9), in whom low levels of expression were mainly restricted to surface colonocytes. In conclusion, these results suggest that enhanced colonic K+ secretion in ESRD involves an increase in the apical K+ permeability of the large intestinal epithelium, which most likely reflects increased expression of apical BK channels. Copyright

Altered cryptal expression of luminal potassium (BK) channels in ulcerative colitis.

Decreased sodium (Na+), chloride (Cl−), and water absorption, and increased potassium (K+) secretion, contribute to the pathogenesis of diarrhoea in ulcerative colitis. The cellular abnormalities underlying decreased Na+ and Cl− absorption are becoming clearer, but the mechanism of increased K+ secretion is unknown. Human colon is normally a K+ secretory epithelium, making it likely that K+ channels are expressed in the luminal (apical) membrane. Based on the assumption that these K+ channels resembled the high conductance luminal K+ (BK) channels previously identified in rat colon, we used molecular and patch clamp recording techniques to evaluate BK channel expression in normal and inflamed human colon, and the distribution and characteristics of these channels in normal colon. In normal colon, BK channel α‐subunit protein was immunolocalized to surface cells and upper crypt cells. By contrast, in ulcerative colitis, although BK channel α‐subunit protein expression was unchanged in surface cells, it extended along the entire crypt irrespective of whether the disease was active or quiescent. BK channel α‐subunit protein and mRNA expression (evaluated by western blotting and real‐time PCR, respectively) were similar in the normal ascending and sigmoid colon. Of the four possible β‐subunits (β1–4), the β1‐ and β3‐subunits were dominant. Voltage‐dependent, barium‐inhibitable, luminal K+ channels with a unitary conductance of 214 pS were identified at low abundance in the luminal membrane of surface cells around the openings of sigmoid colonic crypts. We conclude that increased faecal K+ losses in ulcerative colitis, and possibly other diseases associated with altered colonic K+ transport, may reflect wider expression of luminal BK channels along the crypt axis. Copyright

Non-genomic regulation of intermediate conductance potassium channels by aldosterone in human colonic crypt cells

Background: Aldosterone has a rapid, non-genomic, inhibitory effect on macroscopic basolateral K+ conductance in the human colon, reducing its capacity for Cl− secretion. The molecular identity of the K+ channels constituting this aldosterone inhibitable K+ conductance is unclear. Aim: To characterise the K+ channel inhibited by aldosterone present in the basolateral membrane of human colonic crypt cells. Methods: Crypts were isolated from biopsies of healthy sigmoid colon obtained during colonoscopy. The effect of aldosterone on basolateral K+ channels, and the possible involvement of Na+:H+ exchange, were studied by patch clamp techniques. Total RNA from isolated crypts was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using primers specific to intermediate conductance K+ channels (KCNN4) previously identified in other human tissues. Results: In cell attached patches, 1 nmol/l aldosterone significantly decreased the activity of intermediate conductance (27 pS) K+ channels by 31%, 53%, and 54% after 1, 5 and 10, minutes, respectively. Increasing aldosterone concentration to 10 nmol/l produced a further 56% decrease in channel activity after five minutes. Aldosterone 1–10 nmol/l had no effect on channel activity in the presence of 20 μmol/l ethylisopropylamiloride, an inhibitor of Na+:H+ exchange. RT-PCR identified KCNN4 mRNA, which is likely to encode the 27 pS K+ channel inhibited by aldosterone. Conclusion: Intermediate conductance K+ channels (KCNN4) present in the basolateral membranes of human colonic crypt cells are a target for the non-genomic inhibitory effect of aldosterone, which involves stimulation of Na+:H+ exchange, thereby reducing the capacity of the colon for Cl− secretion.

Potential role of reduced basolateral potassium (IKCa3.1) channel expression in the pathogenesis of diarrhoea in ulcerative colitis.

Diarrhoea in ulcerative colitis (UC) mainly reflects impaired colonic Na+ and water absorption. Colonocyte membrane potential, an important determinant of electrogenic Na+ absorption, is reduced in UC. Colonocyte potential is principally determined by basolateral IK (KCa3.1) channel activity. To determine whether reduced Na+ absorption in UC might be associated with decreased IK channel expression and activity, we used molecular and patch clamp recording techniques to evaluate IK channels in colon from control patients and patients with active UC. In control patients, immunolabelling revealed basolateral IK channels distributed uniformly along the surface‐crypt axis, with substantially decreased immunolabelling in patients with active UC, although IK mRNA levels measured by quantitative PCR were similar in both groups. Patch clamp analysis indicated that cell conductance was dominated by basolateral IK channels in control patients, but channel abundance and overall activity were reduced by 53% (p = 0.03) and 61% (p = 0.04), respectively, in patients with active UC. These changes resulted in a 75% (p = 0.003) decrease in the estimated basolateral membrane K+ conductance in UC patients compared with controls. Levels of IK channel immunolabelling and activity in UC patients in clinical remission were similar to those in control patients. We conclude that a substantial decrease in basolateral IK channel expression and activity in active UC most likely explains the epithelial cell depolarization observed in this disease, and decreases the electrical driving force for electrogenic Na+ transport, thereby impairing Na+ absorption (and as a consequence, Cl− and water absorption) across the inflamed mucosa. Copyright

Over-expression of colonic K + channels associated with severe potassium secretory diarrhoea after haemorrhagic shock

A 67-year-old woman with end-stage renal disease (ESRD) was referred with chronic diarrhoea, severe hypokalaemia and recurrent colonic pseudo-obstructions following haemorrhagic shock. The cause of secretory diarrhoea was uncertain, but an ileostomy identified the colon as the source of the watery diarrhoea and potassium (K(+)) losses, and symptoms only resolved after colectomy. Immunohistochemistry of the colon revealed over-expression of high conductance K(+) (BK) channel protein in surface colonocytes and crypt cells compared with controls and other patients with ESRD. We hypothesize that colonic ischaemia during haemorrhagic shock led to increased BK channel expression and thus enhanced colonic K(+) and water secretion, resulting in severe hypokalaemia and colonic pseudo-obstruction.

Regulation of colonic apical potassium (BK) channels by cAMP and somatostatin

High-conductance apical K+ (BK) channels are present in surface colonocytes of mammalian (including human) colon. Their location makes them well fitted to contribute to the excessive intestinal K+ losses often associated with infective diarrhea. Since many channel proteins are regulated by phosphorylation, we evaluated the roles of protein kinase A (PKA) and phosphatases in the modulation of apical BK channel activity in surface colonocytes from rat distal colon using patch-clamp techniques, having first increased channel abundance by chronic dietary K+ enrichment. We found that PKA activation using 50 μmol/l forskolin and 5 mmol/l 3-isobutyl-1-methylxanthine stimulated BK channels in cell-attached patches and the catalytic subunit of PKA (200 U/ml) had a similar effect in excised inside-out patches. The antidiarrheal peptide somatostatin (SOM; 2 μmol/l) had a G protein-dependent inhibitory effect on BK channels in cell-attached patches, which was unaffected by pretreatment with 10 μmol/l okadaic acid (an inhibitor of protein phosphatase type 1 and type 2A) but completely prevented by pretreatment with 100 μmol/l Na+ orthovanadate and 10 μmol/l BpV (inhibitors of phosphoprotein tyrosine phosphatase). SOM also inhibited apical BK channels in surface colonocytes in human distal colon. We conclude that cAMP-dependent PKA activates apical BK channels and may enhance colonic K+ losses in some cases of secretory diarrhea. SOM inhibits apical BK channels through a phosphoprotein tyrosine phosphatase-dependent mechanism, which could form the basis of new antidiarrheal strategies.

Evidence that two distinct crypt cell types secrete chloride and potassium in human colon

Background Human colon may secrete substantial amounts of water secondary to chloride (Cl−) and/or potassium (K+) secretion in a variety of diarrhoeal diseases. Ion secretion occurs via Cl− and K+ channels, which are generally assumed to be co-located in the colonocyte apical membrane, although their exact cellular sites remain unclear. Objective To investigate the location of apical Cl− (CFTR) and apical K+ (large conductance; BK) channels within human colonic epithelium. Design Whole-cell patch clamp recordings were obtained from intact human colonic crypts. Specific blockers of K+ channels and CFTR identified different types of K+ channel and CFTR under resting conditions and after stimulating intracellular cAMP with forskolin. The BK channel β3-subunit was localised by immunostaining. Results Two types of crypt cells were identified. One (73% of cells) had whole-cell currents dominated by intermediate conductance (IK) K+ channels under resting conditions, which developed large CFTR-mediated currents in response to increasing intracellular cAMP. The other (27% of cells) had resting currents dominated by BK channels inhibited by the BK channel blocker penitrem A, but insensitive to both forskolin and the IK channel blocker clotrimazole. Immunostaining showed co-localisation of the BK channel β3-subunit and the goblet cell marker, MUC2. Conclusions In human colon, Cl− secretion originates from the dominant population of colonocytes expressing apical CFTR, whereas K+ secretion is derived from a smaller population of goblet cells expressing apical BK channels. These findings provide new insights into the pathophysiology of secretory diarrhoea and should be taken into account during the development of anti-diarrhoeal drugs.

Enhanced K(+) secretion in dextran sulfate-induced colitis reflects upregulation of large conductance apical K(+) channels (BK; Kcnma1).

Defective colonic Na(+) and Cl(-) absorption is a feature of active ulcerative colitis (UC), but little is known about changes in colonic K(+) transport. We therefore investigated colonic K(+) transport in a rat model of dextran sulfate-induced colitis. Colitis was induced in rat distal colon using 5% dextran sulfate sodium (DSS). Short-circuit current (Isc, indicating electrogenic ion transport) and (86)Rb (K(+) surrogate) fluxes were measured in colonic mucosa mounted in Ussing chambers under voltage-clamp conditions in the presence of mucosal orthovanadate (a P-type ATPase inhibitor). Serum aldosterone was measured by immunoassay. Control animals exhibited zero net K(+) flux. By contrast, DSS-treated animals exhibited active K(+) secretion, which was inhibited by 98, 76, and 22% by Ba(2+) (nonspecific K(+) channel blocker), iberiotoxin (IbTX; BK channel blocker), and TRAM-34 (IK channel blocker), respectively. Apical BK channel α-subunit mRNA abundance and protein expression, and serum aldosterone levels in DSS-treated animals, were enhanced 6-, 3-, and 6-fold respectively, compared with controls. Increasing intracellular Ca(2+) with carbachol (CCH), or intracellular cAMP with forskolin (FSK), stimulated both active Cl(-) secretion and active K(+) secretion in controls but had no or little effect in DSS-treated animals. In DSS-induced colitis, active K(+) secretion involves upregulation of apical BK channel expression, which may be aldosterone-dependent, whereas Cl(-) secretion is diminished. Since similar ion transport abnormalities occur in patients with UC, diarrhea in this disease may reflect increased colonic K(+) secretion (rather than increased Cl(-) secretion), as well as defective Na(+) and Cl(-) absorption.

Infective and inflammatory diarrhoea: mechanisms and opportunities for novel therapies.

There have been significant advances in unravelling the cellular mechanisms of diarrhoea in common gut infections and colonic inflammation, as well as in the identification of targets for potential antidiarrhoeal drugs. Infective diarrhoea reflects activation of electrogenic Cl⁻ secretion, inhibition of electroneutral NaCl absorption and in some cases, downregulation of tight junctional proteins and increased apoptosis. In colonic inflammation, diarrhoea mainly reflects impairment of colonic Na⁺ and Cl⁻ absorption by inflammatory cytokines, leading to decreased water absorption. Stimulation of endogenous opiate-dependent pathways, manipulation of epithelial ion (Na⁺, K⁺ and Cl⁻) channels and suppression of proinflammatory cytokine production by a variety of drugs and novel molecules, offer opportunities to move evaluation of these potential antisecretory and anti-inflammatory agents from the laboratory into clinical trials.