Physiological and cytotoxic effects of Ca²⁺ ionophores on caco-2 paracellular permeability: Relationship of ⁴⁵ca²⁺ efflux to ⁵¹Cr release

The human intestinal cell line, Caco-2, and the Ca²⁺ ionophores, A23187 and ionomycin, were used to determine the interrelationships of ⁴⁵Ca²⁺ efflux, transepithelial electrical resistance (R_t), and [³H]-mannitol flux to changes in ^5lCr release and lactate dehydrogenase (LDH) activity. Treatment of Caco-2 monolayers with ionomycin at concentrations of between 0.25 and 2.50 umol/1 showed similar ⁴⁵Ca²⁺ efflux rate constants and coefficients. Analysis of the control and ionomycin-induced ⁴⁵Ca²⁺ efflux values showed the data to best fit a three Ca²⁺ compartmental model. All changes in Caco-2 R, and [³H]-mannitol flux were reversible with no significant increases in ^5lCr release with ionomycin concentrations of ≤ 2.5 µmol/1. Caco-2 monolayers treated with ionomycin at concentrations of between 5.0 and 50.0 umol/1 showed rapid non-exponential ⁴⁵Ca²⁺ effluxes with irreversible changes in R_t, [³H]-mannitol flux, and significant increases in ⁵¹Cr release. There was no change in media LDH activity using either ionomycin or A23187 at concentrations of up to 50 µmol/1 for 60 min. The results of our study show that: (1) disruption of Ca²⁺ homeostasis in Caco-2 cells will occur with the addition of Ca²⁺ ionophores at concentrations of > 2.50 µmol/1; (2) high concentrations (> 2.5 µmol/1) of ionomycin will cause non-exponential 45Ca²⁺ efflux rates with irreversible changes in transcellular R_t and ^l4C-mannitol flux, and (3) early signs of Ca²⁺ ionophore-induced damage can be detected by ^5lCr release from Caco-2 cells into the media and not by changes in LDH media activity.