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Academic Unit of Child Health (P.B., C.P.S.), University of Manchester, St. Mary’s Hospital, Manchester M13 0JH, United Kingdom; and School of Animal and Microbial Sciences (N.J.B., R.J.W., P.J.L., N.M.P.), University of Reading, Reading RG6 6AJ, United Kingdom
Address all correspondence and requests for reprints to: Paul Brownbill, Academic Unit of Child Health, University of Manchester, St. Mary’s Hospital, Hathersage Road, Manchester M13 0JH, United Kingdom. E-mail: paul.brownbill{at}man.ac.uk.
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Introduction |
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The purpose of this study was to test the hypothesis that NKB is vasoactive in the fetal placental vasculature. Using the in vitro dually perfused human placental cotyledon, we measured endogenous basal NKB production and determined the effect of exogenous NKB on fetal arterial hydrostatic pressure (FAHP). The effect of NKB was measured under control conditions and after preconstriction of the fetal vessels with the thromboxane A2 mimetic, U-46619. RT-PCR and selective tachykinin receptor antagonists were used to elucidate the receptors involved in NKB action. Finally, we investigated whether NKB action was mediated through NO or prostacyclin release.
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Materials and Methods |
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Aliquots of 0.1 µM to 0.1 mM NKB (Bachem AG, Bubendorf, Switzerland) were prepared in a 50 µg/ml poly arginine, proline, threonine (1:1:1, Sigma-Aldrich Co., Poole, UK), 100 mM NaHCO3, 54 mM NaCl diluent. This acted as a carrier to reduce binding of the tachykinin to plastic and perfusion tubing. Aliquots were stored at -20 C. Then, 1 mM U-46619 thromboxane A2 mimetic (Calbiochem, Nottingham, UK) stock was prepared in 100% ethanol and stored at -20 C. This was diluted to 2.5 x 10-7 M with PBS before experimentation and kept on ice. L-732,138 was used as a specific NK1 receptor antagonist (13, 14). A 0.1 M L-732,138 (Sigma-Aldrich) stock was prepared in 100% ethyl acetate and stored at -20 C. This was serially diluted by 1:100 with ethyl acetate and by 1:40 with PBS to 2.5 x 10-5 M (2.5% vol/vol ethyl acetate) before experimentation and kept on ice. The specific NK2 receptor antagonist (S)-(-)-N-[[4-(4-acetylamino-4-phenyl) piperidin-1-yl]-2-(3,4-dichloro-phenyl)-butyl]-N-methyl-benzamide succinate (SR48968, a kind gift from Sanofi-Synthelabo, Paris, France) was prepared to 1 mM in 100% ethyl acetate and stored at -20 C. This was serially diluted to 2.5 x 10-5 M with PBS before experimentation and kept on ice. Nitro-L-arginine methyl ester hydrochloride (L-NAME; Sigma-Aldrich) was stored desiccated at -20 C, prepared to 10 mM with PBS before experimentation, and then stored on ice. Indomethacin (160 mM; Sigma-Aldrich) stock was prepared in ethyl acetate daily and stored on ice. This was serially diluted by 1:40 with PBS to 4 mM (2.5% vol/vol ethyl acetate) before experimentation and kept on ice.
Earle’s bicarbonate buffer containing 402 µM L-arginine (Fluka, Poole, UK), 5.6 mM glucose, 0.5 mM dextran 70 (average molecular mass, 60–90 kDa; Sigma-Aldrich), 0.017 mM BSA (Sigma-Aldrich), and 5,000 IU/liter heparin (sodium mucous: Multiparin, CP Pharmaceuticals, Wrexham, UK) was used as the perfusate, equilibrated with 95% O2/5% CO2 to pH 7.4.
Perfusion
The method of perfusion was described by Schneider et al. (15), as adapted in our laboratory (16, 17). Term placentas were obtained within 30 min from normal delivery or cesarean section. Because these placentas were used anonymously, ethical approval was not required, as stipulated by UK Department of Health guidelines current at the time of this study. Cotyledons were dually perfused in vitro (6.0 and 14.0 ml/min through the fetal and maternal circulatory systems, respectively) in a humidified cabinet maintained at 37 C. When fetal venous outflow was less than 95% of fetal arterial inflow at the commencement of experimentation, preparations were rejected from study. This criterion was reduced to 80% for preparations not used in solvent transfer evaluation. FAHP and maternal arterial hydrostatic pressure (MAHP) were continually recorded with a chart recorder (Lectromed Multitrace 2, Lectromed Ltd., Letchworth, UK) via two pressure transducers (SensoNor, Horten, Norway) linked to the perfusate tubing.
Investigation of potential constrictor effects of NKB
In two preparations, successive 0.5-ml boluses of the carrier alone and then incremental NKB doses (up to 0.1 mM) were administered to the fetal-side circulation via the fetal arterial perfusate tubing, and FAHP and MAHP were recorded.
Investigation of potential dilator effects of NKB
Five cotyledons were preconstricted with an appropriate dose of the thromboxane mimetic U-46619 to elevate baseline FAHP, administered by constant infusion into the fetal perfusate line, using a syringe pump (Precidor model, Infors AG, Basel, Switzerland). U-46619 infusion was maintained throughout the rest of the experiment. An elevated steady state FAHP baseline was achieved and maintained throughout the experiment (interexperimental variation, U-46619 perfusate concentration, 2.0–2.8 nM; steady state FAHP, 60.8–114 kPa). Preparations not achieving and maintaining a steady elevated baseline FAHP were rejected from the study. Successive 0.5-ml boluses of the carrier alone and then seven incremental NKB doses (0.1–5 µM) were administered to the fetal-side circulation. FAHP and MAHP were recorded throughout the experiment. Response recovery times between administering bolus doses of NKB were 10 min at concentrations less than 1 µM and 20 min at concentrations of 1–5 µM. FAHP in response to the carrier or each dose of NKB was standardized against its respective previous steady state value and expressed as a percentage change in FAHP. Effects of NKB were compared with the effect of the carrier.
Estimation of endogenous NKB secretion into the fetal placental circulation
Sample collection. In six cotyledons, 1-min samples were taken from the fetal venous cannula at 10-min intervals for 1 h. Collection was performed from the commencement of fetal-side perfusion. Samples were centrifuged at 4 C at 1100 x g for 10 min to remove blood cells. The supernatant was recovered, snap-frozen in liquid nitrogen, and stored at -80 C before NKB assay.
Extraction of NKB from plasma. NKB standards were prepared in modified Earle’s bicarbonate buffer, described earlier. The standards contained 1280, 640, 320, 160, and 80 pg/ml NKB. Each 1-ml sample or plasma standard was acidified by addition of 220 µl 1 M HCl containing 0.21 M glycine. They were then diluted to 10 ml with 0.9% saline and subjected to centrifugation at 3000 x g for 20 min to ensure complete clarity. Sep-Pak C18 1CC cartridges (Peninsula Laboratories Europe, St. Helens, Merseyside, UK) were primed before use by perfusion with 2 ml of the following solutions: 1) water containing 0.1% trifluoroacetic acid and 0.1% Polypep gelatin hydrolysate (Sigma-Aldrich; 2) water containing 0.1% trifluoroacetic acid; 3) water containing 80% (vol/vol) acetonitrile; and 4) water containing 0.1% trifluoroacetic acid. After loading, cartridges were washed with 1 ml 0.1 M HCl containing 0.02 M glycine, followed by 1 ml 0.1% trifluoroacetic acid in water. Additional washes with 1 ml 0.1% trifluoroacetic acid in water containing 10% and 20% acetonitrile were followed by elution with 1 ml 0.1% trifluoroacetic acid in a mixture of 50% water and acetonitrile. Eluted fractions were reduced to dryness under vacuum after adding 1 mg mannitol and 100 µg Polypep.
Measurement of NKB in plasma extracts. Plasma extracts were reconstituted in 500 µl of buffer supplied as part of a commercial NKB RIA kit RIK 7357 (Peninsula Laboratories, Inc., Belmont, CA) to which had been added 0.2% Igepal CA-630 nonionic detergent (Sigma-Aldrich). Subsamples of 25 µl were taken from extracted and nonextracted standards and mixed with 75 µl of the above buffer. Standards were prepared in buffer containing Igepal, to which 200 µg/ml Polypep had been added. Anti-NKB antibody solution (100 µl) was added to all assay tubes except blanks, and the assay was conducted as described in the General Protocol for RIA Kit instructions. Assays were performed in duplicate, and results were corrected by reference to extracted standards.
Estimation of exogenous NKB concentration reaching the cotyledon
The concentration of NKB in the bolus administered may be diminished by dilution within the tubing and bubble-trap, binding to the perfusate tubing, and breakdown, leading to an overestimation of the effective dose. To assess this, a 5-µM NKB bolus was administered to the fetal perfusion line, as described above, and sequential 30-sec sampling followed at the open-ended fetal arterial cannula (time, 1–4 min; n = 6). Samples were processed and assayed for NKB as above.
Effects of NKB on fetal vessel solvent loss
At the point of maximal response in FAHP to NKB, 1-min samples of fetal venous perfusate were collected into tared bijoux. Similar collections of perfusate were made before NKB bolus administration during the steady state periods. The difference between fetal arterial perfusion rate and fetal venous outflow gave values relating to the rate of perfusate loss from the fetal placental vasculature in response to each NKB dose for each experiment. These values were then expressed as a percentage change in the rate of fetal-side solvent loss compared with their respective pre-NKB steady states. Because we were considering water movement along a hydrostatic pressure gradient, we calculated the difference in fetal and MAHP (FMAHP) at the midpoints of fetal venous perfusate sampling. Linear regression analysis was used to investigate whether there was an association between changes in FMAHP differential and changes in fetal-side solvent loss from fetal placental vessels.
Identification of tachykinin receptors in the placenta mediating vasodilation
This was investigated in two ways:
Molecular study—RNA preparation. Complete normal placentas were obtained, carefully dissected from the chorionic plate, and pooled. RNA was extracted immediately by using an acid guanidium thiocyanate-phenol-chloroform extraction method (18). Every 100 mg of tissue was homogenized in 1 ml of Tri Reagent (Sigma-Aldrich), and the mixture was left to stand at room temperature for 5 min. The mixture was spun at 7,500 x g (4 C) for 1 min to remove excess cellular debris, and the supernatant was collected. The supernatant was mixed with 0.2 ml of chloroform and vortexed vigorously for 15 sec before being left on ice for 5 min and centrifuged at 12,000 x g (4 C) for 10 min. The upper aqueous phase was transferred to a fresh tube, and the RNA was precipitated with isopropanol (0.5 ml) and collected by centrifuging at 12,000 x g (4 C) for 10 min.
The supernatant was removed, and the pellet was washed with 75% (vol/vol) ethanol by vortexing and centrifugation at 7500 x g (4 C) for 5 min. The pellet was briefly dried under vacuum for 10 min and dissolved in nuclease-free water. All total RNA samples were treated with DNase I to remove any contaminating DNA and re-purified as above. The final amount and purity of total RNA was determined as previously described (19).
A total of 1.5 µg of each purified total RNA was subjected to first strand cDNA synthesis using the SMART RACE amplification kit (CLONTECH Laboratories, Inc. Basingstoke, UK) to produce 3' SMART RACE cDNA as previously described (20). RT-PCR was used to determine mRNA expression of the tachykinin NK1, NK2, and NK3 receptors in the placenta and human umbilical vein endothelial cell (HUVEC) cells. Gene-specific primers were designed to amplify the human NK1 (accession no. M84425; forward, 5'-GGCCATGAGCTCCACCATGTACAACCCC; reverse, 5'-GCATGAAGGGAGGCAGGTCAAAGGCA GTGG); human NK2 (accession no. M57414; forward, 5'-TGCTGGTGG TGC TGACGTTTGCCATCTGCT; reverse, 5'-CTGTTGACTCTCGTGGAGAGGGAGGTCGT); and human NK3 (accession no. M89473; forward, 5'-GGCTGGCAATGAGCTCAACCATGTACAATCCCA; reverse, 5'-GGTGAGCTTATGAAACTTGAAGTGGCGGAGGCA) tachykinin receptors. PCR amplification was performed at 95 C for 30 sec and 68 C for 1 min for 35 cycles using each set of gene-specific primers by following the manufacturer’s protocol (CLONTECH Laboratories, Inc.). Reactions were performed as previously described (20). Fetal brain from a human total RNA master panel (CLONTECH Laboratories, Inc.; catalog no. K4005-1) was used as a positive control tissue for NK3 tachykinin receptor expression. Human ß-actin was used as a housekeeping gene to assess loading efficiency for human placenta, HUVEC, and fetal brain samples.
Functional study. The effects of NK1 and NK2 receptor antagonists (L-732,138 and SR48968, respectively) on the vasodilatory response to NKB in the in vitro perfused cotyledon were investigated separately (n = 4, both blockers): Each preparation was preconstricted with U-46619, as above (interexperimental variation, U-46619 perfusate concentration, 2.2–3.0 nM and 2.2–2.5 nM; primary steady state FAHP, 78.1–115.8 kPa and 75.5–121.7 kPa, for L-732,138 and SR48968, respectively). Then, 2.5 x 10-7 M L-732,138 or 2.5 x 10-7 M SR48968 (including 0.025% vol/vol ethyl acetate) was continually delivered to the fetal-side vasculature using a second syringe pump 30 min before the administration of 1-, 2.5-, and 5-µM NKB boluses, as described above. These doses of antagonists are those shown to be maximally effective in other tissues, or in receptor-ligand dissociation studies (21, 22, 23). For both the L-732,138 and SR48968 investigations, FAHP values were calculated from the chart recordings at comparative points to the primary study on NKB effects, as described above. Controls for these antagonist studies were performed by repeating the protocol, but with the administration of 0.025% ethyl acetate alone (n = 2) to the fetal vasculature using the second syringe pump.
Role of NO and/or prostacyclin in mediating the NKB effect
Three placental cotyledons were dually perfused and preconstricted with U-46619, as above, to achieve an initial elevated FAHP baseline less than 76 kPa (inter-experimental variation, U-46619 perfusate concentration, 1.4–3.3 nM; steady state FAHP, 60.4–68.6 kPa). On achieving steady state, the effects of a 0.5-ml carrier bolus and a 0.5-ml 5-µM NKB bolus were sequentially investigated, as described above. The fetal placental vasculature was then subjected to an infusion of 100 µM of the NO synthase blocker L-NAME using a second syringe pump into the fetal arterial perfusate line. This infusion was maintained throughout the remaining course of the experiment. After attainment of a new steady state in FAHP and at least 20 min after L-NAME administration, another 0.5-ml 5-µM NKB bolus was administered to the fetal arterial perfusate tubing. After the ensuing response and the reestablishment of steady state FAHP, the fetal reservoir was switched to one containing an elevated concentration of L-arginine (3 mM), but otherwise identical to that described above. After attainment of steady state FAHP, the fetal vasculature was again challenged with a 0.5-ml 5-µM NKB bolus, as above. Maximal response to the 5-µM NKB doses was calculated and compared with their previous steady state values. The steady state FAHP value resulting from the effect of L-NAME was compared with its previous steady state FAHP value. Finally, the effect of 5 µM NKB in the presence and absence of L-NAME was compared.
Five additional cotyledons were dually perfused and pretreated with U-46619 (inter-experimental variation, U-46619 perfusate concentration, 2.0–2.8 nM; steady state FAHP, 66.8–128.2 kPa), and the effect of the carrier and 5 µM NKB was established, as above. A total of 40 µM of the prostacyclin synthesis blocker, indomethacin, was then delivered to the fetal placental vasculature, using the second syringe pump, infusing into the fetal perfusate line (perfusate concentration of ethyl acetate was 0.025%). This infusion was maintained. After 30 min of indomethacin treatment, the fetal vasculature was again challenged with a 0.5-ml 5-µM NKB bolus. Maximal responses to the 5-µM NKB doses were calculated and compared with their previous steady state values. The steady state FAHP value resulting from the effect of indomethacin was also calculated and compared with its previous steady state FAHP value. The effect of 5 µM NKB in the presence and absence of indomethacin was compared.
In a further study, the combined effect of L-NAME and indomethacin on the dilatory response of NKB was investigated, following the same procedure as for single inhibitor-administered studies above (interexperimental variation, U-46619 perfusate concentration, 2.0–2.5 nM; primary steady state FAHP, 50.7–82.9 kPa; n = 4). Then, 100 µM L-NAME and 40 µM indomethacin were administered as a cocktail from the second syringe pump. Similar calculations and analyses were made as for the single inhibitor-administered studies above.
Statistical analysis
Data from dose response experiments, effects of NKB with and without L-732,138, SR48968, L-NAME, and indomethacin, and all steady state data were expressed as mean ± SEM, with n representing the number of placentas studied. For the NKB dose response investigation, ANOVA was used to investigate the possibility of a relationship between FAHP and NKB dosage, and a Dunnett post hoc test was used to assess whether individual doses might have significantly altered the FAHP differential from the effect of the carrier. In investigations into the effects of L-732,138 and SR48968 on the dose response to NKB, ANOVA was used for each dose, with a Bonferroni post hoc test to compare the effect of NKB doses alone with the effect on addition of each blocker. In L-NAME and indomethacin single- and multiple-administered studies, paired t tests were used to compare the effects of the 5 µM NKB bolus with and without the inhibitor. In the investigation into the relationship between changes in FAHP and fetomaternal solvent transfer, linear regression analysis was used on merged data from all NKB dose response experiments. In all cases, a significant effect was reported when the P value was less than 0.05.
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Results |
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NKB alone, i.e. without preconstriction, did not have any effect on FAHP, even at the very high doses used (0.1 mM, data not shown). In the U46619 preconstricted cotyledon, NKB caused a significant dose-dependent decrease in FAHP (ANOVA, P < 0.0001; n = 5). The 2.5- and 5-µM NKB doses decreased the FAHP to 77.8 ± 2.3% and 74.9 ± 4.5% of previous steady state values (mean ± SEM; Fig. 1
). These highest doses significantly relaxed the vessels, compared with the effect of the carrier alone (P < 0.01, both doses; Fig. 1). No tachyphylaxis was observed in the resting FAHP after the application of successive boluses of NKB.
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Detectable levels of NKB were found in fetal venous perfusate samples in five of six cotyledons investigated (mean = 57.7 ± 36.0 pM at 60 min; n = 6; Fig. 2). Endogenous basal NKB output was quite steady over the collection period (Fig. 2).
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After upstream administration of a 0.5-ml 5-µM bolus of NKB, before the fetal-side pump, the measured peak concentration of the tachykinin at the open arterial end was 1.89 ± 0.54 nM in the 2.0- to 2.5-min sampling period (n = 6). A similar trial with a bromophenol blue bolus resulted in a similar elution profile, but a concentration reduction factor of only 36.2 (data not shown). Hence, it is estimated that tubing binding and breakdown of the peptide accounts for a 73.1-fold reduction in concentration of NKB in our system.
Effects of NKB on fetal vessel solvent loss
There was a weak, but significant positive linear correlation between changes in water loss from fetal vessels and changes in FMAHP differential during the dilatory responses to NKB (Fig. 3). MAHP remained constant throughout the course of the experiments (data not shown).
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Molecular evidence.
After 35 cycles of RT-PCR, the NK1 tachykinin receptor appeared to predominate with some NK2 tachykinin receptor expression (Fig. 4a). No expression of the NK3 receptor subtype could be found in term placental mRNA (Fig. 4a). Furthermore, using this technique, we found NK1 and NK2, but not NK3 receptor subtype expression in the HUVEC line (Fig. 4b). Control reactions performed with total RNA and with no cDNA template all produced clean tracks with no visible bands (data not shown). Expression levels for human ß-actin were found to be even for all samples studied (Fig. 4c).
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]. The NKB vasodilation was not significantly different in the presence or absence of the selective NK2 receptor blocker, SR48968 (n = 4; Fig. 5). There was no effect of 0.025% ethyl acetate on the NKB dilatory response at any of the doses investigated (data not shown).
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As expected, L-NAME significantly elevated the FAHP in the presence of U-46619 to a new steady state level (177.6 ± 29.6% of previous steady state, mean ± SEM; n = 3; Fig. 6). However, L-NAME had no significant effect on the vasodilatory potential of NKB, expressed as a percentage change in FAHP (68.4 ± 5.9% without L-NAME; 68.5 ± 6.9% with L-NAME; n = 3; Fig. 6). Subsequent elevation of the fetal reservoir L-arginine concentration to 3 mM did not alter this finding (post 3 mM L-arginine, 72.2 ± 3.4%; n = 3).
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). However, in the presence of indomethacin, NKB caused a further vasodilation, similar in magnitude from its new steady state level to that observed in the absence of indomethacin (70.2 ± 6.2% with indomethacin and 66.8 ± 1.5% without indomethacin; n = 5).
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). There was no significant difference in the vasodilatory potential of NKB, expressed as a percentage change in FAHP (73.9 ± 1.1% and 70.8 ± 6.4%, with and without the inhibitor cocktail, respectively; n = 4; Fig. 8).
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Discussion |
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The finding of a correlation in FMAHP differential and fetomaternal solvent transfer during the dilatory responses to collective NKB doses infers that this agonist reduces the fetomaternal transfer of water in the capillaries at the exchange barrier. This must result from a reduction in the hydrostatic pressure gradient between the fetal capillary bed lumen, the presumed site of water exchange (24, 25, 26), and the maternal intervillous space. MAHP was unaffected by NKB, so changes in FMAHP are a sole consequence of altered FAHP. We have previously shown indirect evidence that U-46619 induces fetomaternal solvent transfer by predominantly constricting fetal placental vasculature downstream of the capillaries (27). Hence, it is likely that NKB counteracts this effect by predominantly vasodilating the venous end of the fetal vascular bed.
The vasodilatory effect of NKB appears to be solely mediated through its interaction with the NK1 tachykinin receptor subtype, because there was a largely diminished response in the presence of the NK1 receptor antagonist L-732,138 and no effect with the NK2 receptor antagonist SR48968. These data contrast markedly with findings in a recent report on NKB-induced lung edema that appeared to be mediated via a tachykinin receptor-independent mechanism (28). These data are consistent with the RT-PCR results, showing the predominant expression of NK1, some expression of NK2, and absent expression of NK3 receptor mRNA in the whole placental homogenate after 35 cycles of PCR. A similar pattern of expression of tachykinin receptor subtypes in the HUVEC cell line suggests a similar role of NKB in the umbilical vein, supporting the notion of a uniform dilatory action of NKB throughout the fetoplacental circulation, downstream of its site of synthesis. Additionally, tachykinin receptor expression in the HUVEC line supports the possibility of NKB mediating its effect through the endothelium and poses the possibility of an endocrine role for this tachykinin in the umbilical vein. There is a suggestion from the NK2 receptor antagonist data (Fig. 5
) that the 1-µM NKB dose elicits a minor concurrent constrictor effect, mediated through the NK2 receptor, which is not apparent at higher NKB doses, perhaps due to a more predominant vasodilation via NK1. This is conceivable given the relative affinity of the tachykinin receptors for NKB (NK2 > NK1). However, the effect is statistically insignificant for the number of experiments performed, and further investigation would be required to evaluate this suggestion.
In some other vascular beds, vasodilatory effects of NKB are mediated through the release of NO or prostacyclin (29, 30, 31). However, the vasodilatory response of the fetal placental vasculature was not mediated through NO synthesis, because L-NAME failed to inhibit the NKB effect. Evidently, the administered L-NAME dose was sufficient to significantly raise the fetal placental vascular tone in response to U-46619, as expected from previous studies (32, 33). Similarly, prostacyclin does not appear to mediate the NKB effect, because this was not altered by indomethacin. The observed reduction in FAHP by indomethacin is in keeping with the work of others (34). Similarly, a cocktail of L-NAME and indomethacin failed to inhibit the NKB dilatory response, so mediation of this response is possible through a NO- and prostacyclin-independent mechanism. It remains undetermined whether other endothelial-derived signaling factors might influence smooth muscle relaxation in response to NKB. Therefore, the data suggest that the vasodilatory effect of NKB in the fetal placental circulation is predominantly due to direct activation of the NK1 tachykinin receptor, perhaps mediating the release of the putative endothelial-derived hyperpolarizing factor.
Tachykinin family members are prone to different levels of biodegradation by various endogenous placental peptidases, including neutral endopeptidase 24.11, angiotensin I-converting enzyme, bestatin-sensitive aminopeptidases, and amastatin-sensitive aminopeptidases (35, 36, 37, 38, 39, 40, 41, 42, 43). There is indirect evidence for specific patterns of tachykinin catabolism governed by the localized expression of these enzymes within human placenta tissue (44, 45, 46, 47, 48, 49, 50, 51). Taken together, the literature suggests that fetally derived substance P may survive passage through the fetal placental vasculature, where it could influence tone. However, its potential for survival after passage through the maternofetal transplacental route would be very low. Conversely, NKB is likely to have a high capacity for survival along the transplacental axis, suggesting the possibility of a paracrine role in the regulation of fetal placental vascular tone after its synthesis at the syncytiotrophoblast (5).
In conclusion, NKB may act as a paracrine regulator of fetal placental vascular tone in the human placenta. In the placental bed, the overall action of NKB is to cause vasodilation that is mediated predominantly through the tachykinin NK1 receptor, while the vasoconstrictor tachykinin NK3 receptor is absent or expressed at extremely low levels (20). Increased syncytiotrophoblast production in preeclampsia could mean that NKB has a significant local role in this condition, vasodilating the fetoplacental circulation in compensation for the reduced blood flow through the uterine circulation. This has been suggested previously, in regard to other vasoactive agents (52). Further work investigating fetal serum levels of NKB and its potential to affect tone in placentas from pregnancies affected by preeclampsia would help to clarify this question.
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Acknowledgments |
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Footnotes |
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Abbreviations: FAHP, Fetal arterial hydrostatic pressure; FMAHP, fetal and MAHP; HUVEC, human umbilical vein endothelial cell; L-NAME, nitro-L-arginine methyl ester hydrochloride; MAHP, maternal arterial hydrostatic pressure; NK, neurokinin; NO, nitric oxide.
Received November 6, 2002.
Accepted February 5, 2003.
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-fetoprotein transfer in the perfused human placental cotyledon from uncomplicated pregnancy. J Clin Invest 96:2220–2226
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; n = 5; mean ± SEM). The x-axis is shown as a log scale, representing administered bolus NKB concentration. Pressure data are expressed as a percentage change from respective prebolus steady state values. Significance of NKB doses were tested against the effect of the carrier alone (
; n = 5; mean ± SEM) using an ANOVA (P < 0.0001), followed by Dunnett’s multiple comparison post hoc test (**, P < 0.01 for 2.5 and 5 µM).
