Prostacyclin Synthesis and Prostacyclin Receptor Expression in the Porcine Myometrium: Prostacyclin Potential to Regulate Fatty Acid Transporters, Cytokines and Contractility-Related Factors

Abstract

Although prostacyclin (PGI2) has been well described as a regulator of smooth muscle activity, limited data are available concerning its role in the myometrium of pigs. The present research aimed to examine profiles of PGI2 synthase (PTGIS) and PGI2 receptor (PTGIR) expression and 6-keto PGF1α (a PGI2 metabolite) concentrations in the myometrium of gilts throughout the estrous cycle and during early pregnancy using qPCR, Western blot, and/or ELISA methods. Furthermore, myometrial explants were exposed to iloprost (a stable PGI2 analog) to investigate the effect of PGI2 on the mRNA expression of factors engaged in smooth muscle contraction, nutrient transport, prostaglandin synthesis and action, and inflammatory response. PTGIS mRNA expression was greater in cyclic than in pregnant gilts on days 11-12 after estrus and was accompanied by greater concentrations of 6-keto PGF1α detected in cyclic than in pregnant animals on days 11-20. Iloprost stimulated fatty acid transporters and contractility-related calponin 1 and caldesmon 1 mRNA expression and decreased interleukin 1β and tumor necrosis factor transcript abundance. The obtained results indicate a physiologically relevant role of PGI2 during the estrous cycle in the porcine myometrium with its importance for regulating the expression of contractility-, nutrient transport- and inflammatory response-related factors.

Keywords: contractility-related factors; cytokines; myometrium; nutrient transporters; pig; pregnancy; prostacyclin; prostacyclin receptor; prostaglandin receptors; the estrous cycle.

Figure 1

Expression of prostaglandin I2 (PGI2; prostacyclin) synthase (PTGIS) mRNA (A) and protein (B) and concentrations of 6-keto PGF1α (a PGI2 metabolite; C) in the myometrium of cyclic and early pregnant gilts. Values from real-time PCR for PTGIS were normalized to geometric averaging of GAPDH and HPRT1 gene expression. Values from densitometric analyses of PTGIS protein were normalized to ACTB. Representative blots are presented (c—cyclic, p—pregnant; original western blots are included in Figure S1). Data are expressed as the mean ± SEM (n = 5–7). Bars marked with various letters (ab—for cyclic gilts; xy—for pregnant gilts) are different among groups. Asterisks specify differences between cyclic and pregnant animals on particular days after estrus (* p < 0.05; *** p < 0.001).

Figure 2

Prostaglandin I2 receptor (PTGIR) mRNA (A) and protein (B) expression in the myometrium of cyclic and early pregnant gilts. Values from real-time PCR for PTGIR were normalized to geometric averaging of GAPDH and HPRT1 gene expression. Values from densitometric analyses of PTGIR protein were normalized to GAPDH. Representative blots are presented (c—cyclic, p—pregnant; original western blots are included in Figure S1). Data are expressed as the mean ± SEM (n = 5–7). Bars marked with various letters are different among the group of pregnant females. Asterisk specifies the difference between cyclic and pregnant animals on days 19–20 after estrus (* p < 0.05).

Figure 3

Effect of prostaglandin I2 (PGI2; prostacyclin) analog, iloprost, on the mRNA expression of contractility-related genes (panel A; GJA1, OXTR, CNN1, and CALD1) and genes encoding transporters (panel B) of glucose (SCL5A1), amino acids (SLC38A1), and fatty acids (CD36 and SLC27A4) in the myometrium. Myometrial explants were incubated for 8 h without (control) or with 1 µM of iloprost. Values from real-time PCR for each studied mRNA transcript were normalized to geometric averaging of GAPDH and HPRT1 mRNA expression. Data are expressed as the mean ± SEM (n = 6). Asterisk specifies the difference compared with the control value (* p < 0.05).

Figure 4

Effect of prostaglandin I2 (PGI2; prostacyclin) analog, iloprost, on the mRNA expression of genes involved in PG synthesis (panel A; PTGS2) and action (panel A; PTGER2, PTGER4, and PTGFR) and genes involved in the inflammatory response (panel B; TNF, IL1B, CXCL8, and NFKB1) in the myometrium. Myometrial explants were incubated for 8 h without (control) or with 1 µM of iloprost. Values from real-time PCR for each studied mRNA transcript were normalized to geometric averaging of GAPDH and HPRT1 mRNA expression. Data are expressed as the mean ± SEM (n = 6). Asterisks specify the difference compared with the control value (* p < 0.05; ** p < 0.01).