Fig. 7.

cGMP-dependent inhibition of NHE3 activity in Caco-2/bbe cells expressing cGK II is NHERF2 dependent. A: infection of cGK II-expressing Caco-2/bbe cells with small interfering RNA (siRNA) constructs directed against NHERF2 dramatically reduced NHERF2 protein expression. Infection with siRNA construct 1 or construct 2 strongly reduced NHERF2 expression (75%), and the coinfection of both these constructs produced a knockdown of >85%. GAPDH was used as a loading control. B: in cells with NHERF-2 knocked down (Adeno-NHERF2, siRNA constructs 1 + 2, infected at same time as Adeno-HA-NHE3), there was no longer 8-pCPT-cGMP-induced inhibition of NHE3 activity, while in simultaneously studied scrambled siRNA control cells 8-pCPT-cGMP inhibited NHE3 activity (paired t-tests; n = 3 for each). C: NHE3 activities (initial rates) were measured in Caco-2/bbe cells in which NHERF1 or NHERF2 was knocked down with lentivirus-shRNA and lentivirus-GFP-shRNA was used as a negative control. NHERF1-KD cells expressing cGK II showed reduced NHE3 activity when treated with 100 μM 8-pCPT-cGMP, but NHERF2-KD cells expressing cGK II had no effect when treated with cGMP. P values indicate effect of cGMP (paired t-tests; n = 3).