Angiotensin II-triggered p44/42 mitogen-activated protein kinase mediates sympathetic excitation in heart failure rats

Abstract

Angiotensin II (Ang II), acting via angiotensin type 1 receptors in the brain, activates the sympathetic nervous system in heart failure (HF). We reported recently that Ang II stimulates mitogen-activated protein kinase (MAPK) to upregulate brain angiotensin type 1 receptors in HF rats. In this study we tested the hypothesis that Ang II-activated MAPK signaling pathways contribute to sympathetic excitation in HF. Intracerebroventricular administration of PD98059 and UO126, 2 selective p44/42 MAPK inhibitors, induced significant decreases in mean arterial pressure, heart rate, and renal sympathetic nerve activity in HF rats, but had no effect on these variables in sham-operated rats. Pretreatment with losartan attenuated the effects of PD98059. Intracerebroventricular administration of the p38 MAPK inhibitor SB203580 and the c-Jun N-terminal kinase inhibitor SP600125 had no effect on mean arterial pressure, heart rate, or renal sympathetic nerve activity in HF. The phosphatidylinositol 3-kinase inhibitor LY294002 induced a small decrease in mean arterial pressure and heart rate but no change in renal sympathetic nerve activity. Immunofluorescent staining demonstrated increased p44/42 MAPK activity in neurons of the paraventricular nucleus of the hypothalamus of HF rats, colocalized with Fra-like activity (indicating chronic neuronal excitation). Intracerebroventricular PD98059 and UO126 reduced Fra-like activity in the paraventricular nucleus of the hypothalamus neurons in HF rats. In confirmatory acute studies, intracerebroventricular Ang II increased mean arterial pressure, heart rate, and renal sympathetic nerve activity in baroreceptor-denervated rats and Fra-like immunoreactivity in the paraventricular nucleus of the hypothalamus of neurally intact rats. Central administration of PD98059 markedly reduced these responses. These data demonstrate that intracellular p44/42 MAPK activity contributes to Ang II-induced neuronal excitation in the paraventricular nucleus of the hypothalamus and augmented sympathetic nerve activity in rats with HF.

Figure 1

Original tracings illustrating the effects of ICV p44/42 MAPK inhibitor PD98059 on AP, HR and RSNA in SHAM rats (A), HF rats (B) and HF rats pretreated with the angiotensin type-1 receptor antagonist losartan (C). (D), grouped data showing the changes from baseline in MAP, HR and RSNA elicited by ICV PD98059 in SHAM and HF rats. * P<0.05 compared with baseline; † P<0.05 compared with SHAM or losartan pretreated HF rats. AP: arterial pressure; MAP: mean arterial pressure; HR: heart rate; RSNA: renal sympathetic nerve activity (integrated voltage).

Figure 2

Original tracings illustrating the effects of ICV p44/42 MAPK inhibitor UO126 on AP, HR and RSNA in SHAM (A) and HF (B) rats. (C), grouped data showing the changes from baseline in MAP, HR and RSNA elicited by ICV UO126 in SHAM and HF rats. * P<0.05 compared with baseline; †P<0.05 compared with SHAM.

Figure 3

Original tracings showing AP, HR and RSNA in HF rats treated with ICV VEH (A), JNK inhibitor SP600125 (B), p38 MAPK inhibitor SB203580 (C) and PI₃-kinase inhibitor LY294002 (D). (E), grouped data showing the changes from baseline in MAP, HR and RSNA elicited by ICV VEH, SP600125, SB203580 and LY294002 in HF rats. * P<0.05 compared with baseline; † P<0.05 compared with VEH treated HF rats.

Figure 4

Immunohistochemical analysis of Fra-LI immunoreactivity in the PVN of VEH-treated SHAM rats, and of HF rats treated with ICV VEH, PD98059, UO126, SP600125, SB203580, and LY294002. (A) Representative sections of PVN from animals undergoing each treatment protocol (3^rd ventricle to the left). Scale bar: 0.3 mm. (B) Grouped data showing numbers of Fra-LI positive neurons counted in ventrolateral parvocellular (PVN-vlp), dorsal parvocellular (PVN-dp) and posterior magnocellular (PVN-pm) subdivisions of PVN. Values are expressed as means ± SEM. * P<0.05, HF vs. SHAM; † P<0.05 Drug vs. VEH in HF rats.

Figure 5

(A) Immunofluorescent images of the PVN, triple-labeled for Fra-LI (red), phosphorylated p44/42 MAPK (green) and nucleus (blue). Left panels, low power views from a SHAM (top) and a HF (bottom) rat, showing full expanse of PVN (unilateral, 3^rd ventricle to the left). Right panels, high power views taken from the ventrolateral PVN of the same SHAM (top) and HF (bottom) rat, in the regions indicated by the yellow rectangles in left panels. Pink to purple appearance indicates the merge of red Fra-LI positive neurons with the blue nuclear marker. (B) Grouped data showing numbers of PVN neurons positive for phosphorylated p44/42 MAPK (left) and for both p44/42 MAPK and Fra-LI-immunoreactivity (right) counted in ventrolateral parvocellular (PVN-vlp), dorsal parvocellular (PVN-dp) and posterior magnocellular (PVN-pm) subdivisions of PVN in SHAM and HF rats. Values are expressed as means ± SEM. * P<0.05, HF vs. SHAM; † P< 0.05, PVN-vlp, PVN-pm vs. PVN-dp in SHAM or HF. (C) Correlation between the numbers of p44/42 MAPK active neurons and Fra-LI positive neurons in the PVN in SHAM and HF rats. The regression analysis included p44/42 MAPK active neurons and Fra-LI positive neurons in PVN-dp, PVN-vlp and PVN-pm from both SHAM and HF rats.

Figure 6

Representative tracings showing the effects of ICV angiotensin II (ANG II) on AP, HR and RSNA in baroreceptor-denervated rats (A) and in baroreceptor-denervated rats pretreated with 1-hour ICV p44/42 MAPK inhibitor PD98059 (B). (C), grouped data demonstrating the changes from baseline in MAP, HR and RSNA elicited by ICV ANG II in baroreceptor-denervated rats before and after the ICV administration of PD98059. * P<0.05 compared with baseline; † P<0.05 compared with ICV ANG II alone in baroreceptor-denervated rats.

Figure 7

Confocal immunofluorescent images showing the double-labeled Fra-LI (red), nucleus (blue) in PVN (unilateral, 3^rd ventricle to the left) in the rats treated with ICV administration of VEH (A), ANG II (B) and combined ANG II and PD98059 (C) for 1 hour. (D) Grouped data showing the numbers of Fra-LI positive neurons counted in ventrolateral parvocellular (PVN-vlp), dorsal parvocellular (PVN-dp) and magnocellular (PVN-pm) subdivisions of PVN in the rats treated with ICV VEH, ANG II, or combined ANG II and PD98059. Values are expressed as means ± SEM. * P<0.05, compared with VEH; † P<0.05 compared with ICV ANG II.