Time-dependent effect of orchidectomy on vascular nitric oxide and thromboxane A2 release. Functional implications to control cell proliferation through activation of the epidermal growth factor receptor

Abstract

This study analyzes whether the release of nitric oxide (NO) and thromboxane A2 (TXA2) depends on the time lapsed since gonadal function is lost, and their correlation with the proliferation of vascular smooth muscle cells (VSMC) mediated by the epidermal growth factor receptor (EGFR). For this purpose, aortic and mesenteric artery segments from control and 6-weeks or 5-months orchidectomized rats were used to measure NO and TXA2 release. The results showed that the basal and acetylcholine (ACh)-induced NO release were decreased 6 weeks post-orchidectomy both in aorta and mesenteric artery, but were recovered 5 months thereafter up to levels similar to those found in arteries from control rats. The basal and ACh-induced TXA2 release increased in aorta and mesenteric artery 6 weeks post-orchidectomy, and was maintained at high levels 5 months thereafter. Since we previously observed that orchidectomy, which decreased testosterone level, enlarged the muscular layer of mesenteric arteries, the effect of testosterone on VSMC proliferation was analyzed. The results showed that treatment of cultured VSMC with testosterone downregulated mitogenic signaling pathways initiated by the ligand-dependent activation of the EGFR. In contrast, the EGFR pathways were constitutively active in mesenteric arteries of long-term orchidectomized rats. Thus, the exposure of mesenteric arteries from control rats to epidermal growth factor (EGF) induced the activation of EGFR signaling pathways. However, the addition of EGF to arteries from orchidectomized rats failed to induce a further activation of these pathways. In conclusion, this study shows that the release of NO depends on the time lapsed since the gonadal function is lost, while the release of TXA2 is already increased after short periods post-orchidectomy. The alterations in these signaling molecules could contribute to the constitutive activation of the EGFR and its downstream signaling pathways after long period post-orchidectomy enhancing the proliferation of the vascular muscular layer.

Figure 1. Time-dependent effect of orchidectomy on the release of NO from rat aorta and mesenteric artery.

The plots present the basal- and ACh-induced NO release in aorta (A) and mesenteric (B) segments from control, 6 weeks and 5 months post-orchidectomized rats. Results (mean ± SEM) are expressed as arbitrary units (AU)/mg of tissue. * p<0.05, compared with its respective basal condition; # p<0.05 compared with basal NO release in control animals; + p<0.05, ⁺⁺ p<0.001 compared with ACh-induced NO release in control animals. The number of animals used were: control, 6; 6 week post-orchidectomy, 4; 5 months post-orchidectomy, 7.

Figure 2. Time-dependent effect of orchidectomy on the release of TXA₂ from rat aorta and mesenteric artery.

The plots present the basal- and ACh-induced TXA₂ release in aorta (A) and mesenteric (B) segments from control, 6 weeks and 5 months post-orchidectomized rats. Results (mean ± SEM) are expressed as pg/mL/mg tissue. Number of animals: 4–7. * p<0.05, ** p<0.001 compared with its respective basal condition; # p<0.01 compared with basal TXA₂ release in control animals; + p<0.01 compared with ACh-induced TXA₂ release in control animals. The number of animals used were: control, 6; 6 week post-orchidectomy, 4; 5 months post-orchidectomy, 7.

Figure 3. Effect of testosterone treatment on ligand-dependent activation of the EGFR and downstream signaling pathways in smooth muscle vascular cells.

Serum starved SMVC were incubated with 10% (w/v) trichloroacetic acid. The samples were processed by Western blots using the indicated antibodies to determine the phosphorylation levels of the EGFR, p115, Akt, and ERK1/2, and the total levels of EGFR Akt, ERK1/2 and GAPDH as described in Materials and Methods. The total EGFR level decreased after EGF stimulation due to the expected proteolytic processing of the receptor after ligand-dependent internalization. The total Akt, ERK1/2 and GAPDH levels were used as loading controls. The intensity of the bands was measured densitometrically and the signal of the different phosphoproteins was corrected using appropriate loading controls. The photograph (A) shows typical Western blots of the proteins. The top and bottom arrows point to the phosphorylated EGFR and p115, respectively. The plots (B–E) present the mean ± SEM phosphorylation of the EGFR (n = 4) (B), p115 (n = 5) (C), Akt (n = 6) (D), and ERK1/2 (n = 6) (E) from a set of experiments similar to those shown in A.

Figure 4. Effect of orchidectomy on the ligand-dependent activation of the EGFR and downstream signaling pathways in the mesenteric artery.

Segments of the superior mesenteric artery from control and 5 months post-orchidectomized rats were incubated in the absence (−) and presence (+) of 10 nM EGF during 2 min, and the reaction was thereafter arrested freezing the samples in liquid nitrogen as described in Materials and Methods. The frozen samples were stored and thereafter homogenized and processed by Western blots using the indicated antibodies. The photograph shows the phosphorylation levels of the EGFR, p115, Akt and ERK1/2, and the total levels of Akt and ERK1/2 and segments of PVDF membranes stained with Fast Green used as additional loading control. The top and bottom arrows point to the phosphorylated EGFR and p115, respectively. The figure shows a typical experiment from control (n = 4) and orchidectomized (n = 4) rats.