Endothelin-1 regulates cardiac sympathetic innervation in the rodent heart by controlling nerve growth factor expression
J. Clin. Invest. Masaki Ieda, et al. 113:876 doi:10.1172/JCI19480 [Go to this article.]

Figure 2
The ET-1/ET_A receptor augments NGF expression via Giβγ, PKC, EGFR, ERK, p38MAPK, and AP-1 and C/EBPδ elements. (A) Preincubation of cardiomyocytes with either PTX or H89. NGF mRNA expression was determined 2 hours after ET-1 stimulation. (B) Cardiomyocytes were pretreated with LacZ or βARK-ct to inhibit the function of Giβγ, and stimulated with ET-1. βARK-ct attenuated ET-1–induced NGF expression, but not BNP. (C and D) Stimulation with PMA (a PKC activator) for 2 hours augmented NGF expression. In contrast, pretreatment with chelerythrine (che; a PKC inhibitor) for 30 minutes or PMA for 24 hours inhibited ET-1–induced NGF expression. (E and F) Pretreatment with PD98059 (PD; an MAPK inhibitor), AG1478 (AG; an EGFR inhibitor), SB203580 (SB; a p38MAPK inhibitor), or PP2 (an Src family inhibitor), but not with wortmannin (WM; a PI3K inhibitor) or KN62 (a calmodulin kinase II/Iv inhibitor) attenuated ET-1–induced NGF mRNA expression. BNP was affected only with PD98059 pretreatment. (G) The results of the densitometry of four separate experiments are shown. *P < 0.001 vs. control; **P < 0.01 vs. ET-1 alone. NS, not significant vs. ET-1 alone. (H and I) Cardiomyocytes were pretreated with DN-ERK or DN-p38MAPK. (J and K) Identification of ET-1–responsive elements in the NGF promoter using luciferase assay. Black bars, control; white bars, ET-1 stimulation (n = 4). (L) Specific negative regulatory plasmid of the EGFR (533delEGFR) or the Src family (Csk) inhibited NGF transcription (n = 4). *P < 0.001, **P < 0.01, ^#P < 0.05 vs. relative control. NS, not significant.