Patch clamp and calcium studies on human colonic mucosal cells

Abstract: Background: The main function of the large intestine is to fine-tune electrolyte and water balance. This is achieved through the consorted action of ion channels, transporters and pumps. Failures of this system can lead to diarrhoea. Many gastrointestinal disorders have diarrhoea as the main symptom but the underlying mechanisms are not always clear. Most studies of electrolyte and water flow across the mucosa have been done on different animals, but since there is a marked interspecies variation, care has to be taken when data are being evaluated. To increase our knowledge of the normal and pathologically changed colonic mucosa and to be able to find better drugs to treat diseases, more studies have to be done on human tissue. Methods: Patch clamp and Ca++ measurement studies were made on a colonic tumour cell line (HT29) in paper I-II and on human colonic crypts isolated from biopsies obtained at colonoscopy in paper III-IV. Results and conclusion: The results from paper I show that HT29 cells respond to low K' with a depolarization. This effect is believed to be caused by an increase in the relative ratio of Na+ to K+ permeability (PNa+/PK+). When K+ is removed from the extracellular solution, this will lead to an inflow of Na+ leading to a depolarization. An additional depolarizing effect is caused by an inhibition of the Na+/K+-ATPase when K+ is lowered. This kind of depolarization has not been reported in epithelial cells before and could be important to salt and water balance. In paper II, HT29 cells are exposed to carbachol (CCh), an agonist that increases intracellular Ca++. Both membrane potential as well as intracellular Ca++ levels started to oscillate in response to CCh. The underlying mechanism is an activation of Ca++-activated Cl- and K+ currents that depolarize and hyperpolarize the cell respectively. Oscillations in intracellular Ca++ are a means by which the cell can modulate the response to Ca++-increasing agents. In paper III and IV, human colonic crypts are isolated (non-enzymatically) from mucosal biopsies. A frequently found channel in the basolateral membrane of the colonocytes, is a K+ channel called KCNN4. It has a conductance of 28 pS, is activated by increased levels of intracellular Ca++ and is blocked by Ba++. In paper III, the inwardly rectifying property of KCNN4 is shown not to be caused by Mg++ or polyamines, two factors that in many other channels cause rectification. Inward rectification means that the channel conducts ions more readily at negative potentials. In colonocytes, inward rectification in a K+ channel could facilitate Cl- secretion. In paper IV, it is shown that KCNN4 has a role in cellular volume regulation. The colonocytes are constantly being exposed to an environment with varying concentrations of osmolytes. This will cause the cells to either swell (in a hypotonic environment) or shrink (in a hypertonic environment). This is the first study of the effects of cell swelling in human colonocytes.