Endothelin-induced constriction of the ductus venosus in fetal sheep: developmental aspects and possible interaction with vasodilatory prostaglandin

Abstract

1. The ductus venosus is actively regulated in the fetus, but questions remain on the presence of a functional sphincter at its inlet. Using fetal sheep (0.6-0.7 gestation onwards), we have examined the morphology of the vessel and have also determined whether endothelin-1 (ET-1) qualifies as a natural constrictor being modulated by prostaglandins (PGs). 2. Masson's staining and alpha-actin immunohistochemistry showed a muscular, sphincter-like formation at the ductus inlet and a muscle layer within the wall of the vessel proper. This muscle cell component increased with age. 3. ET-1 contracted dose-dependently isolated sphincter and extrasphincter preparations of the ductus from term fetus. This ET-1 effect also occurred in the premature, but its threshold was higher. 4. BQ123 (1 microm) caused a rightward shift in the ET-1 dose-response curve, while indomethacin at a threshold concentration (28 nm) tended to have an opposite effect. 5. Big ET-1 also contracted the ductus sphincter but differed from ET-1 for its lesser potency and inhibition by phosphoramidon (50 microm). 6. The ductus sphincter (term and preterm) and extrasphincter (term) released 6-keto-PGF(1alpha) (hence PGI(2)) and, to a lesser degree, PGE(2) at rest and their release increased dose-dependently upon ET-1 treatment. Both basal and stimulated release was curtailed by endothelium removal. 7. BQ123 and phosphoramidon reduced slightly the contraction of ductus sphincter to indomethacin (2.8 microm). 8. We conclude that the ductus contains a contractile mechanism in the sphincter and extrasphincter regions. ET-1 lends itself to a role in the generation of contractile tone and its action may be modulated by prostaglandins.

Figure 1

Fetal lamb at term. Longitudinal section of the ductus venosus showing the inlet region, with a prominent sphincter-like formation, and a segment of the vessel proper. (a) Masson's trichrome staining. (b) α-Actin immunostaining. Note the accumulation of muscle at the site of the inlet and the muscle layer beneath the endothelium along the vessel. Bar 100 μm for both panels.

Figure 2

Fetal lamb at 0.7 gestation. Longitudinal section of the ductus venosus showing the inlet region with a sphincter-like formation and a segment of the vessel. (a) Masson's trichrome staining. (b) α-Actin immunostaining. The muscle component was less developed than in the term preparation but, otherwise, it was similarly arranged. Bar 100 μm.

Figure 3

Concentration–response curve to ET-1 in the ductus venosus from fetal lamb. (a) Intact ductus sphincter at different gestation ages. The ED₅₀ for the contraction is 0.86±0.49 (n=3), 1.84±0.37 (n=5), and 1.9±0.54 nm (n=5), respectively at term, 119–125 days, and 104–108 days. Dose–response curves for the two preterm groups are significantly different (P<0.001) from the curve at term, but do not reach a significant difference among themselves. (b) Intact ductus sphincter at term; contraction during treatment with BQ123 1 μM (n=3–4, except for the 0.1 μM dose where n=2). (c) Endothelium-denuded ductus sphincter at term; contraction before and during the treatment with BQ123 1 μM (n=9 and 3, respectively). (d) Intact ring preparation from the extrasphincter part of term ductus. The ED₅₀ for the contraction is 1.2±0.3 nM (n=3).

Figure 4

Ductus sphincter from term fetal lamb. Representative responses to ET-1, (a) before and (b) during treatment with BQ123, and to (c) big ET-1. Note that tracings (a) and (c) are taken from the same experiment. Calibration, 0.1 g weight=0.98 mN.

Figure 5

Concentration–response curve to big ET-1 in the ductus sphincter from term fetal lamb. (a) Intact vs endothelium-denuded vessel (n=3–11). Note that big ET-1 had no effect on one endothelium-denuded preparation (see text) and this result was not included in the computation. (b) and (c) Effect of big ET-1 on intact (n=4–7) and endothelium-denuded (n=3) preparations before and during treatment with phosphoramidon 50 μm (Phos) (P<0.01 for control vs treatment in panels (b) and (c)). The same treatment had no effect on the ET-1 contraction in both the intact (242±47 and 299±57 mg at 1 nM before and during treatment; n=5) and the endothelium-denuded (231 and 237 mg at 3.5 nm before and during treatment; n=1) preparations. Note that different preparations were used in panels (a) vs (b) and (c).

Figure 6

Fetal lamb at term. Prostaglandin release from the ductus venosus sphincter through a period of 4 h. (a) 6-keto-PGF_1α. (b) PGE₂ (n=4–5 for both compounds). With either prostaglandin, differences between individual samples are not significant. In this and the following figures, the two compounds were measured in the same sample and reference weight applies to the wet weight of the tissue (see Methods).

Figure 7

Fetal lamb at term. Effect of ET-1 on prostaglandin release from the intact vs endothelium-denuded ductus venosus sphincter. (a) 6-keto-PGF_1α. (b) PGE₂ (n=3 for both compounds and conditions). P<0.001 intact vs endothelium-free for both panels.

Figure 8

Fetal lambs at term. Effect of ONO-11113 on prostaglandin release from the ductus venosus sphincter. (a) 6-keto-PGF_1α, (b) PGE₂ (n=4–5 for both compounds). Contractile responses to ONO-11113 are 387±46, 647±75, and 821±133 mg, respectively with 1, 7, and 100 nM.

Figure 9

Fetal lamb before term. Effect of ET-1 on prostaglandin release from the ductus venosus sphincter at 119–125 days vs 104–108 days gestation. (a) 6-keto-PGF_1α (n=4–5). (b) PGE₂ (n=3). Differences between the two age groups do not reach significance. Note that release of 6-keto-PGF_1α at rest is 1147±383 and 686±229 pg 100 mg⁻¹ min⁻¹, respectively, for the older and the younger age group, while the equivalent values for PGE₂ are 63±27 and 64±52 pg 100 mg⁻¹ min⁻¹. With either compound, basal output in the premature does not differ significantly from that seen at term.

Figure 10

Ductus sphincter from term lamb. Effect of (a) BQ123 1 μM (n=7) and (b) phosphoramidon 50 μM (Phos; n=4 and 9, respectively, for the intact and endothelium-denuded vessel) on the contractile response to indomethacin 2.8 μM. Indomethacin was tested sequentially and normal Krebs was passed through the bath between applications until the contraction had fully subsided (see Methods). Where indicated, BQ123 or phosphoramidon was added to the perfusion fluid 10 min prior to starting the indomethacin test and the treatment was continued throughout the period of exposure to indomethacin. In each panel, the same tissues were studied in the absence or presence of BQ123 (or phosphoramidon). Note that in panel (b) the difference between control and treated endothelium-denuded preparations reaches significance (P<0.05) by paired t-test.