Figure 2.

Functional interaction of PMCA 4b and NOS-I. (A) Dose-dependent inhibition of NOS activity by PMCA 4b. Background cGMP production in untransfected HEK293 cells was set as one (first column), induced cGMP production is shown as fold increase of cGMP levels in relation to protein content of cell lysates. PMCA overexpression showed no effect on cGMP production (second column), whereas NOS-I expression resulted in an ∼60-fold increase in cGMP levels (third column). Increasing amounts of PMCA led to a dose-dependent reduction of NOS-I activity, insertion of the point mutation Asp⁶⁷²→Glu in the PMCA reduced inhibition dramatically (PMCAmut, grey columns). The ∼10-fold higher amounts of mutant PMCA necessary to inhibit NOS-I are in line with the fact that the mutation abolishes ∼90% of Ca²⁺ transport activity (Adamo et al., 1995). Transfection with pPMCAmut alone had no effect on cGMP production (column 12), treatment of NOS-I transfected cells with L-NAME (Lω-nitro-L-arginine methyl ester; 1 mM) resulted in the expected complete inhibition of cGMP production (penultimate column). Coexpression of the constitutively active mutant of PMCA 4b (“ct120”, last column) did not result in inhibition of NOS-I (n = 16, mean ±SEM, asterisk indicates P < 0.05 PMCA vs. PMCAmut). Representative Western blots demonstrated expression of relevant proteins: antibody JA3 showed expression of hPMCA4b and hPMCA4bmut, antibody 5F10, specific for a more NH₂-terminal epitope of PMCAs, demonstrated expression of PMCA4b(ct120), parallel to constant NOS-I expression in cotransfected cells. (B) Deletion of PDZ domain of NOS-I (ΔNOS-I) results in comparable NOS-I activity and complete loss of regulation by increasing amounts of wild-type hPMCA 4b (n = 16, mean ±SEM, changes in fold induction not significant). (C) NOS-III expression results in a highly increased production of cGMP, too, but this NO-dependent cGMP production was not inhibited by wild-type PMCA. The NOS inhibitor L-NAME (L-N) abolished cGMP production, proving the NOS-III–dependent cGMP production (n = 2 × 8, mean ± SEM, changes in fold induction not significant).