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Specific small interfering RNA (siRNA) constructs were used to test for the functional relation of subunits α, β, and γ of the epithelial Na + channel (ENaC) to the hypertonicity-induced cation channel (HICC) in confluent rat hepatocytes. In current-clamp recordings, hypertonic stress (300 → 400 mosM) increased membrane conductance from 75.4 ± 9.4 to 91.1 ± 11.2 pS ( p < 0.001). The effect was completely blocked by 100 μM amiloride and reduced to 46, 30, and 45% of the control value by anti-α-, anti-β-, and anti-γ-rENaC siRNA, respectively. Scanning acoustic microscopy revealed an initial shrinkage of cells from 6.98 ± 0.45 to 6.03 ± 0.43 pl within 2 min. This passive response was then followed by a regulatory volume increase (RVI) by 0.42 ± 0.05 pl ( p < 0.001). With anti-α-, anti-β-, and anti-γ-rENaC siRNA, the volume response was reduced to 31, 31, and 36% of the reference level, respectively. It is concluded that all three subunits of the ENaC are functionally related to RVI and HICC activation in rat hepatocytes.

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