Fenamates: A novel class of reversible gap junction blockers

The effect of fenamates on gap junctional intercellular communication was i nvestigated in monolayers of normal rat kidney (NRK) fibroblasts and of SKH ep1 cells overexpressing the gap junction protein connexin43 (Cx43). Using two different methods to study gap junctional intercellular communication, single electrode voltage-clamp step response measurements and dye microinje ction, we show that fenamates are reversible blockers of Cx43-mediated inte rcellular communication. After adding fenamates to a confluent monolayer of electrically coupled NRK fibroblasts, the voltage step-induced capacitive current transient changed from a transient characteristic for charging mult iple coupled cell capacitances to one characteristic for a single cell in i solation. The capacitance of completely uncoupled cells was 19.7 +/- 1.0 pF (mean +/- S.E.M.; n = 11). Junctional conductance between the patched cell and the surrounding cells in the monolayer changed from >140.7 +/- 9.6 nS (mean +/- S.E.M.; n = 14) to <1.4 :+/- 0.4 nS (mean :+/- S.E.M.; n = 11) af ter uncoupling. Electrical coupling could be restored to >51.8 +/- 4.2 nS ( mean +/- S.E.M.; n = 11) by washout of the fenamates. Voltage-clamp step re sponse measurements showed that the potency of fenamates in inhibiting elec trical coupling decreases in the order meclofenamic acid > niflumic acid > flufenamic acid. The half-maximal concentration determined by dye-coupling experiments was 25 and 40 muM for meclofenamic acid and flufenamic acid, re spectively. Inhibition of gap junctional communication by fenamates did not involve changes in intracellular calcium or pH, and was unrelated to prote in kinase C activity or an inhibition of cyclooxygenase activity. Voltage-c lamp step response measurements in confluent monolayers of SKHep1 cells tha t had been stably transfected with Cx43 revealed that fenamates are potent blockers of Cx43-mediated intercellular communication. In conclusion, fenam ates represent a novel class of reversible gap junction blockers that can b e used to study the role of Cx43-mediated gap junctional intercellular comm unication in biological processes.