Abstract
The effect of fenamates on gap junctional intercellular communication was investigated in monolayers of normal rat kidney (NRK) fibroblasts and of SKHep1 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 microinjection, we show that fenamates are reversible blockers of Cx43-mediated intercellular 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 multiple coupled cell capacitances to one characteristic for a single cell in isolation. 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) after 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 response measurements showed that the potency of fenamates in inhibiting electrical coupling decreases in the order meclofenamic acid > niflumic acid > flufenamic acid. The half-maximal concentration determined by dye-coupling experiments was 25 and 40 μM for meclofenamic acid and flufenamic acid, respectively. Inhibition of gap junctional communication by fenamates did not involve changes in intracellular calcium or pH, and was unrelated to protein kinase C activity or an inhibition of cyclooxygenase activity. Voltage-clamp step response measurements in confluent monolayers of SKHep1 cells that had been stably transfected with Cx43 revealed that fenamates are potent blockers of Cx43-mediated intercellular communication. In conclusion, fenamates represent a novel class of reversible gap junction blockers that can be used to study the role of Cx43-mediated gap junctional intercellular communication in biological processes.
Footnotes
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↵1 Current address: Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, The Netherlands.
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↵2 Current address: Institute of Environmental Studies, Department of Chemistry, Toxicology and Ecology, Free University of Amsterdam, The Netherlands.
- Abbreviations:
- NRK
- normal rat kidney
- GJIC
- gap junctional intercellular communication
- Cx43
- connexin43
- PKC
- protein kinase C
- DMEM
- Dulbecco's modified Eagle's medium
- MFA
- meclofenamic acid
- FFA
- flufenamic acid
- TPA
- 12-O-tetradecanoylphorbol-13-acetate
- Received January 31, 2001.
- Accepted May 8, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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