Effects and mechanisms of action of sildenafil citrate in human chorionic arteries

Abstract

Objectives: Sildenafil citrate, a specific phosphodiesterase-5 inhibitor, is increasingly used for pulmonary hypertension in pregnancy. Sildenafil is also emerging as a potential candidate for the treatment of intra-uterine growth retardation and for premature labor. Its effects in the feto-placental circulation are not known. Our objectives were to determine whether phosphodiesterase-5 is present in the human feto-placental circulation, and to characterize the effects and mechanisms of action of sildenafil citrate in this circulation.

Study design: Ex vivo human chorionic plate arterial rings were used in all experiments. The presence of phosphodiesterase-5 in the feto-placental circulation was determined by western blotting and immunohistochemical staining. In a subsequent series of pharmacologic studies, the effects of sildenafil citrate in pre-constricted chorionic plate arterial rings were determined. Additional studies examined the role of cGMP and nitric oxide in mediating the effects of sildenafil.

Results: Phosphodiesterase-5 mRNA and protein was demonstrated in human chorionic plate arteries. Immunohistochemistry demonstrated phosphodiesterase-5 within the arterial muscle layer. Sildenafil citrate produced dose dependent vasodilatation at concentrations at and greater than 10 nM. Both the direct cGMP inhibitor methylene blue and the cGMP-dependent protein kinase inhibitor Rp-8-Br-PET-cGMPS significantly attenuated the vasodilation produced by sildenafil citrate. Inhibition of NO production with L-NAME did not attenuate the vasodilator effects of sildenafil. In contrast, sildenafil citrate significantly enhanced the vasodilation produced by the NO donor sodium nitroprusside.

Conclusion: Phosphodiesterase-5 is present in the feto-placental circulation. Sildenafil citrate vasodilates the feto-placental circulation via a cGMP dependent mechanism involving increased responsiveness to NO.

Figure 1

Schematic representation of the ex vivo incubation model system used in the isolated vessel experiments.

Figure 2

Panel A: Demonstration of PDE-5 mRNA in chorionic plate arterial rings. cDNA was prepared from RNA extracted from chorionic plate arterial rings, and amplified using specific primers for 400 bp regions of the indicated genes. PCR products were electrophoresed on 1% agarose. Panel B: Demonstration of PDE-5 protein in chorionic plate arterial rings. Human chorionic plate arterial rings homogenates, representing various amounts of protein, were examined by SDS-PAGE Western blot for the presence of PDE-5.

Figure 3

Immunohistochemical demonstration of the localization of PDE-5 protein in chorionic plate arterial rings. Haematoxylin and eosin staining of a paraffin embedded 15 μm section from a chorionic plate arterial ring demonstrates the structure of the ring (Panel A). Subsequent images, enlarged from the section of the wall of this ring indicated by the rectangle, demonstrate nuclear staining with DAPI (Panel B), staining with PDE-5 primary and phycoerythrin labeled secondary (Sigma) antibodies (Panel C), and the superimposition of panels B and C to demonstrate the cytoplasmic location of PDE-5 (Panel D).

Figure 4

Panel A: Cumulative concentration-response curve for sildenafil citrate from 10^-10 M to 10^-3 M compared to control (vehicle) at a submaximal U46619 induced contraction. *P < 0.05 compared to baseline. † P < 0.05 compared to control. Panel B: Cumulative concentration-response curve for sildenafil citrate from 10^-7 M to 10^-3 M compared to vehicle at a submaximal U46619 induced contraction, in the presence and absence of methylene blue. *P < 0.05 compared to baseline. † P < 0.05 compared to sildenafil citrate.

Figure 5

Panel A: Cumulative concentration-response curve for sildenafil citrate from 10^-7 M to 10^-3 M compared to vehicle at a submaximal U46619 induced contraction, in the presence and absence of cGMPS (3 × 10^-6 M). Panel B: Cumulative concentration-response curve for sildenafil citrate from 10^-7 M to 10^-3 M compared to vehicle at a submaximal U46619 induced contraction, in the presence and absence of cGMPS (3 × 10^-5 M). *P < 0.05 compared to baseline. † P < 0.05 compared to sildenafil citrate.

Figure 6

Panel A: Cumulative concentration-response curve for sildenafil citrate from 10^-7 M to 10^-3 M compared to vehicle at a submaximal U46619 induced contraction, in the presence and absence of L-NAME. *P < 0.05 compared to baseline. Panel B: Histogram of concentration dependent relaxation for sodium nitroprusside in the presence of increasing concentrations of sildenafil citrate. †P < 0.05 compared to control.