Formal analysis of electrogenic sodium, potassium, chloride and bicarbonate transport in mouse colon epithelium

Abstract

1. The mammalian colonic epithelium carries out a number of different transporting activities simultaneously, of which more than one is increased following activation with a single agonist. These separate activities can be quantified by solving a set of equations describing these activities, provided some of the dependent variables can be eliminated. Using variations in the experimental conditions, blocking drugs and comparing wild type tissues with those from transgenic animals this has been achieved for electrogenic ion transporting activity of the mouse colon. 2. Basal activity and that following activation with forskolin was measured by short circuit current in isolated mouse colonic epithelia from normal and cystic fibrosis (CF) mice. 3. Using amiloride it is shown that CF colons show increased electrogenic sodium absorption compared to wild type tissues. CF mice had elevated plasma aldosterone, which may be responsible for part or all of the increased sodium absorbtion in CF colons. 4. The derived values for electrogenic chloride secretion and for electrogenic potassium secretion were increased by 13 and 3 fold respectively by forskolin, compared to basal state values for these processes. 5. The loop diuretic, frusemide, completely inhibited electrogenic potassium secretion, but apparently only partially inhibited electrogenic chloride secretion. However, use of bicarbonate-free solutions and acetazolamide reduced the frusemide-resistant current, suggesting that electrogenic bicarbonate secretion accounts for the frusemide-resistant current. 6. It is argued that the use of tissues from transgenic animals is an important adjunct to pharmacological analysis, especially where effects in tissues result in the activation of more than one sort of response.

Figure 1

The effects of sequential administration of amiloride (100 μM, apical) and frusemide (1 mM, basolateral), with (a) or without (b) forskolin (10 μM, both sides) given between, in wild type and CF colonic epithelia. Specimen SCC traces for epithelia of 20 mm² are shown. Note when forskolin is not added (b) the changes caused by frusemide are small. In this situation frusemide increased or decreased the SCC in wild type resulting in only a small mean change, whereas in CF epithelia an overall significant increase in current was found (see Figure 4a).

Figure 2

Cumulative data from 22 wild type colonic epithelia and 42 CF epithelia. Shown are the values (means±s.e.mean) for the basal SCC (a) and changes in SCC caused by the sequential addition of amiloride (100 μM, apical) (b), forskolin (10 μM, both sides) (c) and frusemide (1 mM, basolateral) (d), all as μA cm⁻². P values are shown and were obtained with Student's t-test, comparing wild type with CF preparations.

Figure 3

The total data from Figure 2 presented as actual SCC values. The mean values of the currents and their standard errors are given. Peak values have been used and no allowance made for any decline in the response during 10 min before the next drug was added.

Figure 4

(a) Effects of frusemide (1 mM, basolateral) addition to wild type and CF colons, without addition of forskolin and in the presence of amiloride. (b) Effects on SCC by the sequential addition of amiloride (100 μM, apical), forskolin (10 μM, both sides) and frusemide (1 mM, basolateral) in 12 wild type colonic epithelia bathed in HCO⁻₃-free medium. Means±s.e.mean are given in both (a) and (b).

Figure 5

SCC records from two separate colonic epithelia, one bathed in KHS (a) and the other in HCO⁻₃-free KHS (b) Each was subject to the sequential addition of amiloride (100 μM, apically), forskolin (10 μM, both sides), frusemide (1 mM, basolaterally) and acetazolamide (100 μM, both sides). Both tissues had an area of 20 mm².