p38 Mitogen-activated protein kinase (MAPK) increases arginase activity and contributes to endothelial dysfunction in corpora cavernosa from angiotensin-II-treated mice

Abstract

Introduction: Angiotensin II (AngII) activates p38 mitogen-activated protein kinase (MAPK) and elevates arginase activity in endothelial cells. Upregulation of arginase activity has been implicated in endothelial dysfunction by reducing nitric oxide (NO) bioavailability. However, signaling pathways activated by AngII in the penis are largely unknown.

Aim: We hypothesized that activation of p38 MAPK increases arginase activity and thus impairs penile vascular function in AngII-treated mice.

Methods: Male C57BL/6 mice were implanted with osmotic minipumps containing saline or AngII (42 µg/kg/h) for 14 days and cotreated with p38 MAPK inhibitor, SB 203580 (5 µg/kg/day), beginning 2 days before minipump implantation. Systolic blood pressure (SBP) was measured. Corpus cavernosum (CC) tissue was used for vascular functional studies and protein expression levels of p38 MAPK, arginase and constitutive NO synthase (NOS), and arginase activity.

Main outcome measures: Arginase expression and activity; expression of phospho-p38 MAPK, endothelial NOS (eNOS) and neuronal NOS proteins; endothelium-dependent and nitrergic nerve-mediated relaxations were determined in CC from control and AngII-infused mice.

Results: AngII increased SBP (22%) and increased CC arginase activity and expression (∼twofold), and phosphorylated P38 MAPK levels (30%) over control. Treatment with SB 203580 prevented these effects. Endothelium-dependent NO-mediated relaxation to acetylcholine was significantly reduced by AngII and this effect was prevented by SB 203580 (P < 0.01). AngII (2 weeks) did not alter nitrergic function. However, SB 203580 significantly increased nitrergic relaxation in both control and AngII tissue at lower frequencies. Maximum contractile responses for phenylephrine and electrical field stimulation were increased by AngII (56% and 171%, respectively) and attenuated by SB 203580 treatment. AngII treatment also decreased eNOS phosphorylation at Ser-1177 compared to control. Treatment with SB 203580 prevented all these changes.

Conclusion: p38 MAPK inhibition corrects penile arginase activity and protects against erectile dysfunction caused by AngII.

Figure 1. SB 203580 ameliorates AngII-induced endothelial dysfunction

Endothelium-dependent NO-mediated relaxation to acetylcholine (ACh, 10⁻⁹ to 10⁻⁵ M, panel A) in cavernosal segments from control (CTL, open circle), AngII (closed circle), AngII+SB 203580 (closed square) and CTL+SB 203580 (open square) mice. Treatment with SB 203580 significantly prevented endothelial dysfunction in AngII mice. Panel B, Nitrergic relaxation induced by electrical field stimulation (EFS). Nitrergic relaxation induced by EFS (1–32 Hz) in cavernosal strips from CTL (open bars), AngII (grey bars), CTL+SB 203580 (solid bars) and AngII+SB 203580 (hatched bars). Data were calculated relative to the maximal changes from the contraction produced by phenylephrine (PE, 10⁻⁵ M) in each tissue, which was taken as 100%. Data represent the means ± S.E.M. of 5–7 experiments. *P < 0.05 compared with CTL mice; ^†P < 0.05 and ^††P < 0.01, compared with the respective control group; ^#P < 0.05 compared with AngII+SB 203580. No differences were observed in CTL vs SB 203580 tissues.

Figure 2. SB 203580 treatment prevents AngII-induced augmented corporal contractile responses

Contractile responses upon stimulation of α-₁-adrenergic receptor phenylephrine (PE, 10⁻⁹ to 10⁻⁴ M, panel A) or adrenergic nerves (electric field stimulation, EFS 1–64 Hz) in cavernosal segments from control (CTL), AngII, CTL+SB 203580 and AngII+SB 203580 mice. Frequency-response curves elicited by EFS were performed in the absence (panel B) or in the presence of L-NAME (10⁻⁴ M) and atropine (10⁻⁶ M, panel C). Data were calculated relative to the maximal changes from the contraction produced by KCl (80 mM), which was taken as 100%. Data represent the mean ± S.E.M. of 6 animals. *P < 0.05; **P < 0.01, and ***P < 0.001 compared with CTL mice; ^#P < 0.05, compared to AngII mice.

Figure 3. Treatment with SB 203580 significantly attenuates AngII-induced increased corporal arginase activity

Arginase activity in cavernosal tissues from CTL (open bar), AngII (grey bar), CTL+SB 203580 (closed bar) and AngII+SB 203580 (hatched bar) mice was determined by the conversion of L-arginine to urea and L-ornithine. AngII mice markedly increased arginase activity. Treatment with SB 203580 significantly attenuated arginase activity in AngII mice. Data are expressed as % of control. Data represents the mean ± S.E.M. of 5 experiments. *P < 0.05, compared to CTL mice; ^#P < 0.05, compared to AngII mice.

Figure 4. AngII increases corporal expression of arginase II

Western blot analysis of arginase II expression in cavernosal tissues of CTL (open bar), AngII (grey bar), AngII+SB 203580 (hatched bar) and CTL+SB 203580 mice (closed bar). SB 203580 treatment suppressed arginase II expression in CC from AngII mice. A representative blot is shown in the top panel. Results were quantified by densitometry. Protein expression of arginase II was normalized to α-actin levels and expressed as % of control. Data represent the mean ± S.E.M. of 6 experiments (each group). **P < 0.01, compared to CTL mice; ^#P <0.05, compared to AngII mice.

Figure 5. AngII increases corporal p38 MAPK activity

Western blot analysis of phophorylated (P)-p38 MAPK and total p38 MAPK expression in cavernosal tissue of CTL (open bar), AngII (grey bar) and AngII+SB 203580 mice (hatched bar). SB 203580 treatment significantly attenuated increased P-p38 MAPK expression in AngII mice. Representative blot of P-p38 MAPK and total p38 MAPK is shown in the top panel. P-p38 MAPK and total p38 MAPK were normalized by α-actin levels and expressed as % of control. A summarized bar graph shows that increased P-p38 MAPK was detected in tissues from AngII mice when normalized to total p38 MAPK (N = 5, each group).*P < 0.01, compared to CTL mice; ^#P < 0.05, compared to AngII mice.

Figure 6. SB 203580 prevents decreased corporal expression of eNOS-Ser1177

Western blot analysis of total eNOS, phosphorylated eNOS in the regulatory site at Ser-1177, at Thr-495 (panel A) and nNOS expression (panel B) in cavernosal tissues of CTL (open bar), AngII (grey bar) and AngII+SB 203580 (hatched bar). Representative blot is shown in the top of each panel. Protein expression of constitutive NOS were normalized by α-actin levels and expressed as % of control. A summarized bar graph shows phosphorylated eNOS at Ser-1177 was decreased in tissues from AngII mice when normalized to total eNOS (N = 5, each group) **P < 0.01, compared to CTL mice.