FIGURE 9.

Tadalafil induction of AMPK and inhibition of high glucose-induced laminin γ1 increment requires guanylyl cyclase activation. A, cyclic GMP (cGMP) content was measured by a commercial kit. B, podocytes were preincubated with or without ODQ for 30 min followed by incubation with or without tadalafil (TAD) for 8 h. Cell lysates were immunoblotted with antibody against the indicated proteins. C, cells were preincubated with or without ODQ for 30 min followed by incubation with or without tadalafil for 1 h. Cell lysates were immunoblotted with antibodies against the indicated proteins. D, under conditions described in C, hydrogen sulfide generation was measured as described in Fig. 6A. E, cells were preincubated with ODQ for 30 min followed by incubation with tadalafil and glucose as described in Fig. 1C. Cell lysates were immunoblotted with antibody against laminin γ1 (Lamγ1) antibody and actin. F, 8-bromo-cyclic GMP (Br-cGMP) was added to the cells for the indicated duration, and immunoblotting was done with antibodies against CSE and actin. G, 8-bromo-cyclic GMP was added to the cells for the indicated duration, and immunoblotting was done with antibodies against phospho-AMPK (P-AMPK) and AMPK. In A–G, composite data from three to six experiments are shown in graphs; error bars represent S.E.