Endothelium-derived prostanoids reduce 5-hydroxytryptamine-induced contraction in the human uterine artery

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1 Human Reproduction vol.13 no.7 pp , 1998 Endothelium-derived prostanoids reduce 5-hydroxytryptamine-induced contraction in the human uterine artery Caroline Karlsson 1, Gunilla Bodelsson 1, Mikael Bodelsson 2 and Martin Stjernquist 1,3 1 Department of Obstetrics and Gynaecology, University Hospital, S Malmö and 2 Department of Anaesthesiology and Intensive Care, University Hospital, S Lund, Sweden 3 To whom correspondence should be addressed The contribution of endothelium-linked mechanisms to the contraction induced by 5-hydroxytryptamine (5-HT) was investigated in the isolated human uterine artery. 5-HT contracted the uterine artery concentration-dependently. Removal of the endothelium or treatment with the cyclooxygenase inhibitor indomethacin potentiated the contractile response to 5-HT. The nitric oxide synthase inhibitor L-N G -monomethyl-arginine (L-NMMA) did not influence the contraction induced by 5-HT. Indomethacin did not affect the response to 5-HT in endothelium-denuded vessels. The 5-HT 1 receptor agonist 5-carboxyamidotryptamine (5-CT) did not relax precontracted arteries. Removal of the endothelium did not change the response to 5-HT in the presence of the 5-HT 1B/D receptor antagonist GR and the 5-HT 1A and 5-HT 1B receptor antagonist pindolol. The 5-HT 1B receptor antagonist SB did not affect the contraction induced by 5-HT. The results indicate that the 5-HT-induced contraction in the human uterine artery is accompanied by the release of an endothelium-derived relaxing factor (EDRF). This EDRF seems to be a prostanoid, probably prostacyclin (PGI 2 ). The endotheliumlinked mechanism seems to be mediated via a 5-HT 1 receptor, but it is not possible to further classify the receptor subtype by the information obtained in this study. Key words: EDRF/endothelium/5-hydroxytryptamine/prostanoids/uterine artery Introduction The vascular supply of the female internal genital organs has fundamental importance for several reproductive functions such as the development of the endometrium, implantation of the ovum and fetal growth. The uterine arteries are responsible for the major part of the blood flow to the uterus, and an adequate uterine circulation has been shown to be mandatory for the implantation of the ovum (Tekay et al., 1996). 5-Hydroxytryptamine (5-HT), which is released during platelet activation (Stoltz, 1985), has been suggested to play a role in the pathogenesis of pre-eclampsia (Tamura et al., 1990; Poulsen et al., 1993; Taniguchi, 1994; Taniguchi et al., 1994). The vascular endothelium releases different endothelium- derived relaxing factors (EDRF) and endothelium-derived contracting factors (EDCF; Furchgott and Zawadski, 1980; Furchgott, 1983; Ignarro, 1989; Lüscher et al., 1992). Endothelial cell injury with impaired synthesis of vasodilators and/ or increased production of vasoconstrictors has been proposed as one possible pathophysiological mechanism behind the increased vasopressor sensitivity in pre-eclampsia (Roberts et al., 1989). In isolated uterine arteries with intact endothelium, 5-HT contracts the circular smooth muscle predominantly via 5-HT 2 receptors with a minor contribution of 5-HT 1 -like receptors (Karlsson et al., 1997). In vessels without endothelium the contractile response to 5-HT seems to be mediated by receptors only of the 5-HT 2 subtype (Fontes Ribeiro et al., 1991). This difference may be due to different pharmacological approaches or to the state of the endothelium. The objective of the present study was to investigate the contribution of endothelium-linked mechanisms to the contraction induced by 5-HT in the isolated human uterine artery. Materials and methods Human material Preparations were obtained from 34 women (mean age 49 years, range 29 77) undergoing abdominal hysterectomy for benign gynaecological diseases (fibromyoma, endometriosis, adenomyosis, menometrorrhagia). Immediately after removal of the uterus, segments of the ascending branches of the uterine arteries were excised and placed in cold (4 C) modified Krebs Ringer solution (composition: see below) and immediately transported to the laboratory. In-vitro pharmacology The vessel specimens were dissected free from adjacent veins and connective tissue. Within h they were cut into cylindrical segments with a length of ~5 mm, and mounted in organ baths (volume 4 ml) on two metal hooks, one which was fixed to a Grass FT03C force displacement transducer and one which could be moved in order to adjust the smooth muscle tension. The mechanical activity was measured isometrically and recorded on a Grass model 7D polygraph. The organ bath contained a modified Krebs Ringer solution of the following composition (in mm): NaCl, 118; KCl, 4.7; CaCl 2 2H 2 O, 2.0; MgSO 4 7H 2 O, 1.2; NaHCO 3, 24.8; KH 2 PO 4, 1.2; glucose, 5.6. A gas mixture consisting of 88.5% O 2 and 11.5% CO 2 was used for continuous aeration, giving a ph of The temperature of the fluid was thermostatically maintained at 37 C (Stjernquist and Owman, 1985). The vessels were stretched to an initial tension of 40 mm (Stjernquist and Owman, 1990). The Krebs Ringer solution was changed repeatedly at least every 30 min. After an equilibration period of 1 h the circular smooth muscle was European Society for Human Reproduction and Embryology 1947

2 C.Karlsson et al. contracted by exchanging the bathing fluid for one containing 126 mm KCl and no NaCl (KCl Krebs Ringer). The resulting contraction was used as a contraction reference for each segment. Four organ baths were run in parallel. In a first series of experiments the endothelium was removed in two of the vessel segments by gentle injection through the lumen of the O 2 /CO 2 gas mixture with a 0.3 mm cannula for 5 min (Mikkelsen et al., 1988, Bodelsson and Stjernquist, 1994). Two vessel segments were left intact and used as controls. The absence or presence of the endothelium was tested by confirming that substance P ( M), after precontraction with KCl Krebs Ringer, failed to relax the gas treated segments but relaxed the untreated segments (Bodelsson and Stjernquist, 1992). After thorough wash-out, the contraction in response to KCl Krebs Ringer was registered again. Following washout 5-HT was added cumulatively (10 8 to 10 4 M). In a second series of experiments two vessel segments from each patient were incubated with indomethacin ( M), a cyclooxygenase inhibitor, or with L-N G -monomethyl-arginine (L- NMMA, 10 4 M), a nitric oxide synthase inhibitor. After 20 min the Krebs Ringer solution was changed and the inhibitors added again after which 5-HT was supplied cumulatively (10 8 to 10 4 M). Two other segments were used as controls and were only exposed to 5- HT. In a separate series of experiments the effect of indomethacin on 5-HT-induced contraction was investigated in endotheliumdenuded vessels. In a third series of experiments, aiming to investigate any 5-HT 1 receptor-mediated relaxation, the endothelium was removed from two of the vessel segments and two vessel segments were left with intact endothelium. All four vessel segments were then precontracted with prostaglandin F 2α (PGF 2α,10 6 M). When a stable contraction had developed the 5-HT 1 receptor agonist 5-carboxyamidotryptamine (5- CT) was added cumulatively (10 12 to 10 4 M). In a fourth series of experiments two vessel segments from each patient were preincubated with the 5-HT 1B antagonist, SB (10 7 M). Two vessel segments were left untreated as controls. After 20 min the Krebs Ringer solution was changed and SB added again, after which 5-HT was supplied cumulatively (10 8 to 10 4 M). In a fifth series of experiments, the endothelium was removed from two segments as described above. After subsequent testing with substance P, the 5-HT 1B/D receptor antagonist GR ( M) and the 5-HT 1A and 5-HT 1B receptor antagonist pindolol ( M) were added to the organ baths. After 20 min the Krebs Ringer solution was changed and the antagonists added again, after which 5-HT was supplied cumulatively (10 8 to 10 4 M). Calculations and statistics The contractile responses are expressed as a percentage of the contraction induced by KCl Krebs Ringer. In the experiments involving destruction of the endothelium, the second contraction elicited by KCl Krebs Ringer was used as the reference. The mean and the standard error of the mean for the contractile response from experiments on arteries obtained from several patients were calculated. Possible significant interactions between agonists and the effects of different treatments were evaluated using analysis of variance (ANOVA) for repeated measurements (Ludbroock, 1994). P 0.05 was chosen as the level of significance. Drugs 5-Hydroxytryptamine creatinine sulphate; L-NMMA; substance P (Sigma, St Louis, MO, USA); sodium indomethacin (Confortid ; Dumex, Copenhagen, Denmark); 5-CT (Research Biochemicals Incorporated, MA, USA); PGF 2α (Dinoprost ; Upjohn, Kalamazoo, MI, USA); GR (Glaxo Wellcome, Stevenage, UK); SB Figure 1. Concentration response curves for 5-hydroxytryptamine (5-HT) in vessels with and without endothelium. Values are means SEM (n 6). Removal of the endothelium increased the contraction induced by 5-HT. Repeated measurements ANOVA revealed a significant (P 0.001) interaction between 5-HT and the factor presence or absence of endothelium. (Smith Kline Beecham Pharmaceuticals, Harlow, UK); pindolol (Visken, Sandoz, Täby, Sweden). All substances were dissolved in 0.9% NaCl except for indomethacin, of which 50 mg was dissolved in a buffer consisting of 27.1 mg NaH 2 PO 4 and 12 mg NaOH in 10 ml distilled water, and SB , which was dissolved in dimethylsulphoxide. Addition of indomethacin to the organ baths to achieve M did not affect the ph of the Krebs-Ringer solution. Ethics The study was approved by the Ethics Committee of the Faculty of Medicine, University of Lund. Results 5-HT contracted the smooth muscle in the human uterine artery in a concentration-dependent manner. The contraction was more pronounced in vessel segments without endothelium, compared to segments with intact endothelium (Figure 1). Repeated measurements ANOVA revealed a significant (P 0.001) interaction between 5-HT and the factor presence or absence of endothelium. Indomethacin enhanced the contraction induced by 5-HT in segments with intact endothelium (Figure 2) and ANOVA yielded a significant (P 0.001) interaction between 5-HT and the factor presence or absence of indomethacin. In endothelium-denuded segments indomethacin did not affect the contraction in response to 5-HT (Figure 3). L-NMMA did not influence the 5-HT-induced contraction (n 7, data not shown). 5-CT did not relax any of the precontracted segments but induced a further contraction which was similar in vessels with and without endothelium (n 7, data not shown). In the presence of GR and pindolol no differences in the response to 5-HT were encountered between intact and endothelium-denuded vessels (Figure 4). SB did not affect the contraction induced by 5-HT (n 5, data not shown).

3 Endothelium and 5-HT in uterine artery Figure 2. Concentration response curves for 5-hydroxytryptamine (5-HT) in segments with intact endothelium in absence or presence of indomethacin ( M). Values are means SEM (n 8). Indomethacin increased the contraction induced by 5-HT. Repeated measurements ANOVA revealed a significant (P 0.001) interaction between 5-HT and the factor presence or absence of indomethacin. Figure 3. Concentration response curves for 5-hydroxytryptamine (5-HT) in vessels without endothelium, in the absence or presence of indomethacin ( M). Values are means SEM (n 5). Indomethacin did not affect the contraction induced by 5-HT (not significant). Discussion 5-HT induced a concentration-dependent contractile response in the isolated human uterine artery. This is consistent with earlier studies performed on both intact (Karlsson et al., 1997) and endothelium-free (Fontes Ribeiro et al., 1991) human uterine arteries. The 5-HT-induced contraction was greater in endothelium-denuded segments compared to intact ones. It indicates that some endothelium-linked mechanism reduces the contractile response to 5-HT. A similar observation was made by Cocks and Angus (1983) when studying coronary arteries. However, the present finding of an inhibitory effect of the endothelium on the contraction in response to 5-HT in the human uterine artery contrasts to earlier studies performed with other vasoconstrictors on the same vascular preparation. Thus, the contractions induced by noradrenaline (Stjernquist Figure 4. Concentration response curves for 5-hydroxytryptamine (5-HT) in uterine arteries in the presence of GR ( M) and Pindolol ( M). Values are means SEM (n 5). No differences in the contractile response between intact and endothelium-denuded vessels were encountered. and Owman, 1990), prostaglandin F 2α (Kimura et al., 1995; Grbovic et al., 1996), oxytocin (Jovanovic et al., 1997) and arginine vasopressin (Jovanovic et al., 1995c) are not affected by removal of the endothelium. One explanation for the present finding of an inhibitory effect of the endothelium could be that it releases an EDRF. This factor could counteract the contraction induced by 5-HT. Acetylcholine relaxes the human uterine artery only in the presence of an intact endothelium (Bodelsson and Stjernquist, 1992; Jovanovic et al., 1994a). This relaxation is inhibited by methylene blue and L-NMMA, indicating that it is mediated via endothelial release of nitric oxide (Jovanovic et al., 1994b). In the present study L-NMMA did not affect the contraction induced by 5-HT, indicating that nitric oxide is not involved in the endothelial modulation of the 5-HT-induced contraction. Prostacyclin (PGI 2 ) is another vasorelaxatory substance that can be released from the endothelium (Moncada and Vane, 1979). The PGI 2 analogue, beroprost, relaxes the human uterine artery in vitro (Kimura et al., 1995). Indomethacin does not affect the endothelium-dependent relaxation induced by acetylcholine, indicating that prostanoid synthesis is not involved in the cholinergic relaxation (Jovanovic et al., 1994b). However, in this study it was found that indomethacin increased the 5-HT-induced contraction similar to the effect of removal of the endothelium. When the endothelium was absent, indomethacin did not affect the 5-HT-induced contraction. These results suggest that prostanoids, presumably PGI 2, synthesized by the endothelium, reduce the contraction in response to 5- HT in the human uterine artery. Non-endothelial prostanoid production in the human uterine artery has previously been demonstrated (Kimura et al., 1995; Grbovic et al., 1996). The present study is thus, to our knowledge, the first indication of endothelial prostanoid production in this preparation. Apparently there exist differences in the endothelial contribution to vascular tone in the uterine artery between species, and pregnancy is also a factor of importance. The isolated uterine arteries from pregnant guinea-pigs display an endo- 1949

4 C.Karlsson et al. thelium-independent contractile response to vasopressin (Jovanovic et al., 1995b) in accordance with the results from the non-pregnant human uterine artery (Jovanovic et al., 1995c). On the other hand removal of the endothelium from the pregnant guinea-pig uterine artery results in an augmented contractile response to noradrenaline, an effect that does not seem to involve nitric oxide or PGI 2 (Jovanovic et al., 1995a). The contractile response to PGF 2α of the pregnant guinea-pig artery is augmented by endothelial removal, an effect that is also encountered after treatment with indomethacin (Grbovic and Jovanovic, 1996). The next step was to determine if 5-HT receptor agonists could actively generate endothelium-dependent 5-HT 1 receptor-mediated relaxation in the human uterine artery as previously described in other vessels (Cohen et al., 1983; Schoeffter and Hoyer, 1990; Bodelsson et al., 1993). The 5-HT 1 receptor agonist, 5-CT, did not relax precontracted segments, neither in the presence or in the absence of the endothelium. These findings do not support the presence of any endothelial 5- HT 1 receptors mediating vasorelaxation. However, the human uterine artery contains a small population of contractionmediating 5-HT 1 -like receptors (Karlsson et al., 1997). In fact, 5-CT induced a contraction that might be due to an activation of these receptors. Such a 5-HT 1 -like receptor mediated contraction could possibly mask a potential endothelium-dependent relaxation. In another experiment to try to disclose any endotheliumdependent 5-HT 1 receptor-mediated relaxation, attempts were made to block 5HT 1B/D receptors with GR and 5-HT 1A and 5-HT 1B receptors with pindolol. In the presence of these antagonists the contraction induced by 5-HT can be assumed to be mediated by 5-HT 2 receptors solely. In addition any 5- HT 1 receptor-mediated relaxation would be blocked. In this situation the difference in response to 5-HT between intact and endothelium-denuded vessels disappeared. These findings might indicate that the inhibitory effect of the endothelium on the response to 5-HT is mediated via a receptor belonging to the 5-HT 1 receptor subgroup. However, the results do not permit any further subclassification of this suggested endothelial 5-HT 1 -like receptor. The 5-HT 1B receptor antagonist SB had no effect on the 5-HT-induced contraction, indicating the absence of receptors of that subtype. Another possibility is that the uterine artery, as with many other blood vessels, has a basal production and secretion of prostacyclin from the endothelium with a relaxing effect on the vascular smooth muscle (Lamping et al., 1985; Lüscher and Vanhoutte, 1986). If so, removing the endothelium might eliminate this secretion, resulting in a potentiating effect on the 5-HT-induced contraction. Endothelial removal does not, however, affect the response to several other vasoconstrictors (Stjernquist and Owman, 1990; Jovanovic et al., 1995c; Kimura et al., 1995; Grbovic et al., 1996). These findings contradict the concept of a receptor-independent mechanism and a nonstimulated basal release of PGI 2. In conclusion, removal of the endothelium potentiates the contraction induced by 5-HT in the isolated human uterine artery. This effect seems to be due to the elimination of an inhibitory prostanoid released from the endothelium, presum ably PGI 2. This endothelium-linked mechanism appears to be mediated via a receptor belonging to the 5-HT 1 subgroup. Further subclassification cannot be made on the basis of the present results. It might be speculated that loss of such an endotheliumdependent serotonergic vasorelaxatory mechanism could be involved in the pathogenesis of pre-eclampsia. Endothelial damage has been proposed as a main feature of pre-eclampsia (Roberts, 1989) and increased vascular response to 5-HT has been demonstrated in vessels from pre-eclamptic patients (Johnston et al., 1987). Further research will hopefully elucidate this problem. Acknowledgements Supported by grants from the faculty of Medicine, University of Lund, the Swedish Medical Research Council (Grant No ) and the Foundations of the Health Authorities of Malmö, Sweden. References Bodelsson, G. and Stjernquist, M. (1992) Smooth muscle dilatation in the human uterine artery induced by substance P, vasoactive intestinal polypeptide, calcitonin gene-related peptide and atrial natriuretic peptide; relation to endothelium-derived relaxing substances. Hum. Reprod., 7, Bodelsson, G. and Stjernquist, M. (1994) Endothelium-dependent relaxation to substance P in human umbilical artery is mediated via prostanoid synthesis. Hum. Reprod., 9, Bodelsson, M., Törnebrandt, K. and Arneklo-Nobin, B. 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