Measurement of intracellular chloride ion concentration in ICC in situ and in explant culture.

BACKGROUND

Chloride channels are proposed to play a central role in the electrical pacemaking mechanism of interstitial cells of Cajal (ICC). A key unknown factor in the consideration of this role is the chloride equilibrium potential (E(Cl)), as determined by the relative concentrations of intra- (Cl(-)) and extracellular (Cl(-)) chloride ions.

METHODS

To calculate the E(Cl) of ICC, Cl(-) was measured with the fluorescent chloride indicator N-(6-methoxyquinolyl) acetoethyl ester (MQAE). Pacemaker ICC in explant cultures or in situ, i.e. ICC associated with the myenteric plexus of the small intestine, were loaded with MQAE and fluorescence was measured by laser scanning confocal microscopy. The dye fluorescence was then calibrated against known Cl(-) by treating the explants or in situ preparations with chloride ionophore and varying bath chloride concentrations.

KEY RESULTS

In explants, ICC Cl(-) was measured as 13 mmol L(-1) with Cl(-) of 100 mmol L(-1), giving an E(Cl) of -52 mV [corrected]. With Cl(-) at 166 mmol L(-1), Cl(-) was 26 mmol L(-1), giving an E(Cl) of -47 mV[corrected]. In situ, ICC Cl(-) was measured as 26 mmol L(-1) with Cl(-) of 130 mmol L(-1), giving an E(Cl) of -41 mV [corrected]. Importantly ICC compensate for changes in extracellular chloride by changing Cl(-) and thus maintain E(Cl). In ICC explant clusters, Cl(-) was seen to fluctuate, possibly evoked by rhythmic changes in intracellular calcium.

CONCLUSIONS & INFERENCES: The intracellular chloride concentration in ICC fluctuates to keep its equilibrium potential constant. The identification of E(Cl) as positive to the resting membrane potential of ICC indicates that opening of chloride channels will depolarize ICC.