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Am J Physiol Regul Integr Comp Physiol 281: R261-R268, 2001;
0363-6119/01 $5.00
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Vol. 281, Issue 1, R261-R268, July 2001

Effect of in vivo fetal infusion of dexamethasone at 0.75 GA on fetal ovine resistance artery responses to ET-1

Cheryl C. Docherty1, Judit Kalmar-Nagy1, Marc Engelen1, Steven V. Koenen1, Mark Nijland1, Rhoda E. Kuc2, Anthony P. Davenport2, and Peter W. Nathanielsz1

1 Laboratory for Pregnancy and Newborn Research, Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; and 2 Clinical Pharmacology Unit, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 2QQ, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

At 110-111 days gestation, instrumented fetal sheep were administered saline or dexamethasone (2.2 µg · kg-1 · h-1 iv) for 48 h. Measurement of fetal blood pressure showed a greater increase in dexamethasone-treated (n = 6) compared with control (n = 5) fetuses (7.3 ± 2.3 vs. 0.6 ± 2.3 mmHg, P < 0.05). Fetuses were delivered by cesarean section, and the femoral muscle and brain were obtained under halothane anesthesia. Femoral and middle cerebral arteries (~320-µm internal diameter) were evaluated using wire myography. Sensitivity to KCl (2.5-125 mM) and the magnitude of the maximal vasoconstriction to 125 mM K+ were similar in femoral and middle cerebral arteries from dexamethasone-treated vs. control fetuses. Acetylcholine-induced vasorelaxation was similar in femoral arteries from control and dexamethasone-treated fetuses. Middle cerebral arteries did not relax to acetylcholine. Sensitivity to endothelin-1 (ET-1; 0.1 pM-0.1 µM) and magnitude of the ET-1-induced vasoconstriction were greater in femoral arteries from dexamethasone-treated vs. control fetuses (P < 0.05). Autoradiographical studies with receptor-specific ligands demonstrated increased ETA-receptor binding, the principal receptor subtype, in femoral muscle vessels (P < 0.001) but decreased ETA-receptor binding in middle cerebral arteries (P < 0.01) from dexamethasone-treated compared with control fetuses. Relatively little ETB-receptor binding was evident in all tissues examined. We conclude that hyperreactivity to ET-1, due to increased ETA-receptor binding, may be involved in the dexamethasone-induced increase in peripheral vascular resistance in fetal sheep in vivo.

glucocorticoids; blood pressure; fetal sheep


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GLUCOCORTICOIDS REGULATE IMPORTANT functions that prepare the fetus for metabolic adaptations essential for extrauterine life, in particular, fetal lung maturation (4). Treatment of women in preterm labor with antenatal glucocorticoids lessens the incidence of neonatal morbidity (8). The National Institutes of Health recently advised routine administration of antenatal betamethasone or dexamethasone (DM) for 48 h to all pregnant women at risk of premature delivery before 32 wk of gestation (30a).

Direct infusion of the synthetic glucocorticoids, DM and betamethasone, to the fetal sheep at 125 days gestation (~0.8 gestation) increases fetal blood pressure (13). A rise in blood pressure could be manifested via an increase in cardiac output and/or an increase in total peripheral resistance. The fetal heart operates near the upper limit of its function curve and therefore has a limited capacity to increase its cardiac output (42). Therefore, any change in blood pressure in the fetus is likely to result from an increase in total peripheral resistance. In support of this view, we previously reported that betamethasone-induced fetal hypertension in sheep is associated with increased fetal femoral vascular resistance (13). Furthermore, isolated femoral resistance arteries from fetuses exposed to betamethasone exhibit altered vascular responsiveness (1).

The mechanisms by which glucocorticoids alter vascular responsiveness in specific fetal vascular beds are unknown but may involve a disturbance in the balance of vasodilator- and/or vasoconstrictor-mediated effects. Endothelin-1 (ET-1), produced by endothelial (44) and vascular smooth muscle (34) cells, is a potent vasoconstrictor peptide that probably plays an important role in the maintenance of basal vasomotor tone (16). ET-1 may contribute to fetal hemodynamic changes (19, 31). Glucocorticoids increased the pre-pro-ET-1 mRNA levels in vascular smooth muscle cells (25) and isolated rat aorta (33). Cortisol and/or DM have also been shown to selectively induce ET-1 release (20) and to potentiate ET-1-induced inositol trisphosphate (IP3) production (35) in vascular smooth muscle cells.

We hypothesized that antenatal glucocorticoids increase peripheral vascular resistance in the ovine fetus by augmenting vasoconstrictor responses to ET-1. We therefore examined vasoconstrictor responses to ET-1 in isolated fetal ovine femoral and middle cerebral resistance arteries, following exposure of the fetal sheep to a DM infusion for 48 h at 110-111 days gestational age (dGA; 0.75 gestation). Autoradiographic techniques using receptor subtype-specific ligands were used to quantify the density of ETA and ETB receptors in the vessels and surrounding tissue. Because alteration in vascular responsiveness with DM exposure may reflect changes in the endothelium, we also examined responses of preconstricted arteries to the endothelium-dependent relaxatory agent ACh. We chose these specific fetal vascular beds because the cerebral circulation is "protected" at times of oxygen deficiency and undergoes vasodilation and increased flow (7). In contrast, the femoral bed is unprotected, acting as a reservoir of peripheral vascular resistance to maintain perfusion pressure in protected vascular beds. We chose a stage of gestation to approximate the period of gestation at which antenatal glucocorticoids are used therapeutically in human pregnancy.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Rambouillet-Columbia crossbred ewes (Ovis aries) carrying a single fetus of known gestational age were studied. Animals had free access to food and water except for the 24 h before surgery. The experimental protocol was approved by the Institutional Animal Care and Use Committee at Cornell University. All facilities were approved by the American Association for the Accreditation of Laboratory Animal Care.

Surgical preparation. Surgery was performed at 104-105 dGA as previously described (30). The ewes were pretreated with 1 g of ketamine intramuscularly and 1 mg of glycopyrrolate intramuscularly and induced with 4% halothane for 4 min before intubation. In brief, both ewes and fetuses were instrumented with polyvinyl catheters inserted into the carotid artery and jugular vein. A catheter was also placed in the amniotic cavity. All animals were allowed 5 days postoperative recovery. During this time, the ewes received a daily dose of 1 g of ampicillin sodium intramuscularly (Polycillin-N, Bristol Laboratories, Syracuse, NY) and 500 mg of ampicillin sodium into the amniotic sac. Phenylbutazone (EQUAPHEN, Pharma Cerricalas, Shirley, NY) was administered twice daily (0.5 g orally) for 3 days postsurgery to provide analgesia.

DM treatment and tissue collection. At 110-111 dGA, fetuses received an infusion of either saline (n = 5) or DM (AZIUM, Schering, NY) (2.2 µg · kg-1 · h-1; n = 6) intravenously for 48 h. After 48 h of infusion, fetuses were removed during cesarean section (112-113 dGA), and femoral muscle from the left side and the brain were obtained under halothane general anesthesia and collected in ice-cold physiological salt solution (PSS) for wire myography studies. Another section of the same tissue was embedded in tissue-freezing medium (Triangle Biomedical Sciences, Durham, NC), immersed in ice-cold isopentane, snap-frozen in liquid nitrogen, and stored at -80°C. Fetuses and ewes were then euthanized by exsanguination under halothane general anesthesia.

Wire myography. Second-order middle cerebral arteries and femoral arteries were immediately dissected from the collected tissue. Isolated arteries (~2 mm in length) were mounted on two 40-µm tungsten wires in a small vessel wire myograph as previously described (27). It was not always possible to dissect out enough vessels from each animal to study all the agonists in each animal. This accounts for the variation in the number of vessels examined in the different portions of the study. Vessels were equilibrated in PSS at 37°C and continuously aerated with 95% O2-5% CO2 (pH 7.4) for 30 min. Vessel dimensions were then normalized as previously described (27). Briefly, arteries were stretched in a stepwise manner, and the internal circumference and corresponding wall tension as a result of each stretch were calculated by a Myodaq program (J. P. Trading) and plotted to produce a resting wall tension-internal circumference curve for that particular artery. Intersection of the curve with a 100-mmHg isobar line determined the point on the fitted exponential curve corresponding to the internal circumference that the artery would have attained in situ when relaxed and exposed to a transmural pressure of 13.3 kPa (100 mmHg), as determined by the Laplace relationship; termed L100 (27). Preliminary studies conducted by us on fetal ovine middle cerebral and femoral vessels showed that active tension development is maximal in ovine fetal resistance arteries when set to 0.9 L100 (normalization value obtained from interpolation of wall tension-internal circumference curve). Thus experiments described in this paper were performed at 0.9 L100. There was no significant difference in the calculated internal diameter of arteries between control and DM-treated groups for femoral arteries [control: 314.1 ± 39.6 µm, means ± SE (n = 4) vs. DM: 232.6 ± 34.2 µm (n = 5)]. However, middle cerebral arteries from DM-treated fetuses (355.2 ± 18.6 µm, n = 4) were smaller than those from age-matched control fetuses (444.2 ± 26.2 µm, n = 4, P < 0.05).

After several washes and a further 30-min equilibration, vessel responsiveness was tested by stimulation with NEK [5 µM norepinephrine (NE) in 125 mM potassium-substituted PSS (KPSS)], KPSS alone, 5 µM NE alone, and a second exposure to NEK. Cumulative concentration-response curves (CRC) were performed to KCl (2-125 mM) and ET-1 (10 pM-0.3 µM). The relaxing effects of the cumulative addition of ACh (1 nM-10 µM) on femoral arteries were assessed after preconstriction with 5 µM NE. Because NE-induced vasoconstriction was not maintained in middle cerebral arteries, 50 mM KCl were used to constrict these arteries. Additions were performed after a plateau had been attained to the preceding concentration.

Receptor autoradiography. ETA receptors were visualized in duplicate cryostat sections chosen at random following incubation with 125I-PD-151242 using quantitative autoradiography, using methods developed for human vessels (23, 32). 125I-PD-151242 has previously been characterized in vascular smooth muscle having subnanomolar affinity for the ETA receptor but micromolar affinity at ETB (10, 11). At the concentration used, 125I-PD-151242 would occupy <0.03% of the ETB subtype and the ligand would not detect any ETB receptors present on endothelial cells. At present, there is no general agreement as to the diameter of a resistance vessel; the term has been applied to vessels ranging in diameter from <2 mm (40) to <500 µm (26). The diameter of ~100 vessels analyzed by autoradiography ranged from 45 to 450 µm. It is likely that vessels of varying diameter along the vascular tree would contribute in some degree to vascular resistance.

Briefly, 10-µm thick cryostat-cut sections of femoral muscle and frontal cortex were mounted on Poly-Prep slides (Sigma, St. Louis, MO) and stored at -80°C until they were processed. Slides were preincubated in HEPES buffer (50 mM HEPES containing 5 mM MgCl2 and 0.3% wt/vol BSA, pH 7.4) for 30 min at room temperature (23°C). To visualize the ET-receptor subtypes present in these tissues, the wash buffer was replaced with HEPES buffer containing 0.1 nM 125I-ET-1 (to label both receptor subtypes), 0.1 nM 125I-PD-151242 (10) (to label ETA receptors), or 0.3 nM 125I-BQ3020 (24) (to identify ETB receptors). Nonspecific binding was defined by the inclusion of 1 µM of the corresponding unlabeled peptide. After a 2-h incubation period, the slides were washed in ice-cold Tris buffer (50 mM, pH 7.4) and apposed to radiation-sensitive film (Hyperfilm Bmax, Amersham Pharmacia Biotech, Buckinghamshire, UK) for 5 days. Autoradiograms were analyzed by measuring the diffuse-integrated optical density using a computer-assisted image-analysis system (Quantimet 970, Leica, Milton Keynes, UK) equipped with a shading corrector (with the correction performed in real time before each scan). The shading corrector compensated for any variation in illumination so that optical densities were measured accurately throughout the autoradiogram. The white level (100% transmission) was set, and the scanner dark current (the current flowing in the scanner in the absence of a signal) that would otherwise contribute to the gray image was eliminated.

The density of ET receptors within the vessels present throughout the tissue sections was measured by digitizing each autoradiographic image into an array of 630,000 image points each with a gray value in the range 0-255. A cursor was used to draw around each vessel wall displayed on a high-resolution monitor; the computer isolated each area defined by these marks and measured the integrated optical density. When all measurements had been made for a particular section, the threshold for detecting the autoradiogram was increased to produce a template that was used to align the autoradiographic image of an adjacent section used to define the nonspecific binding. The second image was digitally subtracted from the first to measure the amount of specific binding. The resulting optical densities were converted to the amount of specifically bound radioligand in attomoles per millimeter squared by interpolation from the standard curve. Corrections were made for decay of the radioligand from the time at which these measurements were made to the midpoint between apposing and developing the film.

A standard curve was constructed for each film by coexposing calibrated 125I standards with each film and generating an appropriate curve. The autoradiographic image of each standard was detected, and the cursor was used to draw around and isolate the image. The integrated optical density for each standard together with the area was determined. The radioactivity of each standard, measured by gamma counting (corrected for the efficiency of the counter), was divided by the area to calculate radioactivity in dpm per millimeter squared. For each standard curve, the image analyzer generated a natural log plot of optical density vs. radioactivity that gave a linear relationship (see Ref. 9 for a discussion of the statistical analysis of the data). Tissue sections were exposed to produce optical density values falling within the linear range (0.1 to 0.8) that is below the optical density of one, at which point radiation-sensitive films saturate and cannot record further increases in tissue radioactivity. The interassay coefficient of variation was <3% (9).

Drugs and solutions. PSS was of the following composition (in mM): 119 NaCl, 4.7 KCl, 1 KH2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, 0.9 MgSO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 2.5 CaCl2, 11.1 glucose, 2.5 NaHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and 0.021 EDTA. KPSS was prepared by equimolar substitution of NaCl by KCl in PSS. Chemicals for PSS, NE, bitartrate salt, and ACh bromide were purchased from Sigma. ET-1 was obtained from Bachem (Torrance, CA).

Data analysis. Tension is expressed as milliNewton per millimeter artery length or as a percentage of the maximal response to K+. Relaxation is expressed as a percentage of the preinduced tension. CRC were constructed by fitting data to the logistic sigmoid equation (log agonist concentration vs. effect), using the GraphPad Prism program (GraphPad Software, San Diego, CA). Sensitivity (pEC50) to the agonists is expressed as the negative log of the effective molar concentration of the agonist required to elicit 50% of the maximum response. Magnitude of responses and pEC50 values were compared between groups by Student's unpaired t-test. Data from receptor autoradiography studies were analyzed by computer-assisted image analysis (Quantimet 970, Leica). The autoradiographic image of nonspecific binding was digitally subtracted from the total to calculate specific binding densities in attomoles per millimeter squared by interpolation from a standard curve. Differences were considered statistically significant at P < 0.05. All results are presented as means ± SE, and n refers to the number of animals studied.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Blood pressure measurements. Measurement of fetal blood pressure following the 48-h in vivo protocol showed a significantly greater increase in the DM-treated compared with the saline-treated control fetuses [7.3 ± 2.3 (n = 6) vs. 0.6 ± 2.3 mmHg (n = 5), P < 0.05], compared with pretreatment values for each group. Data on fetal arterial blood gases and fetal cardiovascular function before and after 46 h of infusion in both groups are given in Table 1.

                              
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  		Table 1.   BL fetal blood gases 2 h before administration of vehicle (n = 5) or Dex (n = 5) and after 46 h of Inf

K+-induced vasoconstriction. Sensitivity to KCl-induced vasoconstriction was similar in femoral (Fig. 1A) and middle cerebral (Fig. 1B) arteries from control and DM-treated fetuses (pEC50 ~1.45 and ~1.8 for femoral and middle cerebral arteries, respectively). The magnitude of the maximal vasoconstriction evoked by 125 mM K+ was also similar in femoral (0.49 ± 0.15 vs. 0.83 ± 0.14 mN/mm) and middle cerebral (1.3 ± 0.28 vs. 0.9 ± 0.1 mN/mm) arteries from control and DM-treated fetuses.


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  		Fig. 1.   KCl-induced vasoconstriction (as % of own maximum response) in femoral (A) and middle cerebral (B) resistance arteries from saline-treated control (open circle ) and dexamethasone (DM)-treated () fetal sheep (means ± SE, n = 4-6).

ACh-induced response. Sensitivity to ACh was similar in femoral arteries from control (pEC50: 6.99 ± 0.14) and DM-treated fetuses (pEC50: 7.3 ± 0.37). Arteries from both groups relaxed to near baseline tension (Fig. 2A). Preconstricted middle cerebral arteries did not relax to ACh. Indeed, increasing concentrations of ACh tended to augment the preinduced tone, and this was particularly notable in arteries from DM-treated fetuses (Fig. 2B).


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  		Fig. 2.   Response to acetylcholine (ACh) (as % of preinduced tone) in femoral (A) and middle cerebral (B) resistance arteries from saline-treated control (open circle ) and DM-treated () fetal sheep (means ± SE, n = 4-6).

ET-1-induced vasoconstriction. ET-1 sensitivity was significantly greater in femoral arteries from DM-treated fetal sheep compared with controls (pEC50: 8.17 ± 0.03 vs. 7.83 ± 0.14, P < 0.05) (Fig. 3). In middle cerebral arteries from DM-treated fetuses, a pronounced tachyphylaxis was noted to ET-1 concentrations greater than 10 nM. Because a plateau in the ET-1-induced vasoconstriction was not obtained in the middle cerebral arteries in this group, it was not possible to calculate a pEC50 value. pEC50 for ET-1 in middle cerebral arteries from control fetuses was calculated as 8.34 ± 0.02 (n = 4). The contraction elicited by ET-1 (expressed as mN/mm) was augmented in femoral arteries from DM-treated compared with control fetuses (Fig. 3A). In middle cerebral arteries from control fetuses, the maximum vasoconstriction was attained at 0.1 µM ET-1 (1.51 ± 0.29 mN/mm). However, a comparatively smaller maximum response was evoked by 0.1 nM ET-1 in middle cerebral arteries from DM-treated fetuses (0.95 ± 0.06 mN/mm); tachyphylaxis occurred with higher concentrations (Fig. 3B). Tachyphylaxis was not seen in femoral arteries in the ET-1 concentration range examined.


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  		Fig. 3.   Endothelin-1 (ET-1)-induced vasoconstriction (mN/mm) in femoral (A) and middle cerebral (B) resistance arteries from saline-treated control (open circle ) and DM-treated () fetal sheep (means ± SE, n = 4-6, *P < 0.05 control vs. DM).

Distribution of ET-receptor subtypes. 125I-PD-151242 identified a significantly higher density of ETA in small femoral muscle vessels from DM-treated compared with control fetuses, but no effect of DM was noted in the muscle tissue surrounding the femoral arteries (Figs. 4 and 5, C and D). In contrast, DM treatment was associated with a decrease in ETA receptors in middle cerebral artery branches (Figs. 4 and 5, A and B). In sections from both vascular beds, in marked difference to the intense binding visualized with 125I-PD-151202, relatively little ETB-receptor density could be detected within the vessel walls using 125I-BQ3020, and there was no difference between experimental groups (results not shown).


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  		Fig. 4.   Measurements of optical density for specific binding of 125I-PD-151242 (amol/mm2) in cerebral arteries, vessels located in femoral muscle, and femoral muscle tissue from saline-treated control (open bars) and DM-treated (filled bars) fetal sheep (means ± SE, n = 4-5). **P < 0.01; ***P < 0.001.



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  		Fig. 5.   Computer-generated, color-coded images showing binding of the ETA-selective ligand 125I-PD-151242 to sections of frontal cortex from control vehicle-treated fetus (A) and DM-treated fetus (B) and to femoral muscle from control vehicle-treated fetus (C) and DM-treated fetus (D). The autoradiographic image of nonspecific binding was digitally subtracted from the total to produce the image of specific binding. Density of binding in amol/mm2 was coded according to the scale bar by interpolation from a standard curve. Highest levels of binding are shown in red, with black below the level of detection.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we demonstrated that ET-1-induced in vitro vasoconstriction of fetal ovine femoral resistance arteries was increased following in vivo administration of DM directly to the fetus for 48 h at 110-111 dGA. As in previous studies, we chose to administer DM directly to the fetus so that we could have complete control over fetal exposure without confounds due to maternal and placental metabolism (13). We have previously demonstrated that a dose of 10 µg/h in fetuses weighing ~3.32 kg produced concentration of fetal plasma DM of 8 ± 1 ng/ml. These plasma levels are in the same range as human fetal plasma levels obtained at cesarean section within 24 h following maternal glucocorticoid administration (22). The stimulatory effect of prenatal DM exposure on femoral vascular reactivity was manifested by a decrease in the concentration of ET-1 required to elicit 50% of the maximal vasoconstriction and an augmented maximal response. In accordance with the functional results, autoradiography studies demonstrated an increase in ETA receptor-ligand binding number in small resistance-sized femoral vessels from DM-treated fetuses. Hence, DM-induced increase in ET-1 responsiveness appears to be, at least in part, manifested by an increase in ETA-receptor binding.

To date, relatively few studies have examined the effect of in vivo glucocorticoid administration on intact artery ET-1 responsiveness in either fetal or adult vessels. In contrast to our own findings, reactivity to ET-1 was not altered in isolated aortic and carotid conduit arteries from rabbits treated with DM in vivo (37). Discrepancies in results may be related to differences in species, developmental age, vessel size, and vascular preparation.

Middle cerebral arteries from DM-treated sheep showed a pronounced tachyphylaxis to ET-1 concentrations greater than 10 nM. However, in cerebral arteries from control fetuses, the maximal vasoconstriction was not attained until 0.1 µM ET-1. Compared with the femoral vascular bed, a downregulation of ETA-receptor binding was shown in middle cerebral artery branches from treated fetuses, indicating vascular bed specificity in DM-induced changes. The greater susceptibility of middle cerebral arteries from DM-exposed compared with control fetuses to ET-1-induced tachyphylaxis may be related to the comparatively smaller number of vasoconstrictor ETA receptors in the former; therefore, tachyphylaxis occurs with relatively lower concentrations of ET-1 compared with control arteries. However, the aforementioned explanation is entirely speculative, and this interesting phenomenon warrants further investigation.

Several studies indicate that glucocorticoids alter ETA receptors. Glucocorticoids were shown to induce upregulation of ETA receptor mRNA in human osteoblastic cells (5). However, in A7r5 vascular smooth muscle cells, although DM increased pre-pro-ET-1 mRNA abundance, it decreased ETA receptor mRNA abundance and reduced the number of maximal ET-1-binding sites without altering their binding affinity. This suggests that in in vitro culture conditions, DM downregulates the expression of this receptor subtype, possibly in part by the autocrine production of ET-1 (21). Similarly, RU-28362, a pure glucocorticoid agonist, caused a decrease in ETA-receptor number but not affinity in vascular smooth muscle cells in vitro from spontaneously hypertensive adult rats (33). Likewise, using a rat vascular smooth muscle cell line (A-10) that displays high-density and high-affinity ETA receptors, pretreatment with DM was shown to reduce ETA receptor mRNA and the number of ET receptors by 50-60% without changing their affinity. Furthermore, this downregulation of ET receptors was accompanied by an attenuated response to ET-1 in DM-pretreated cells (29). Similar findings have been reported in endothelial cells. For example, in human cerebromicrovascular endothelial cells that express high-affinity ETA receptors in culture, pretreatment with DM decreased the number of ET-1-binding sites without changing the binding affinity (39).

Importantly, the majority of previous studies examining the interaction of glucocorticoids and ET-1 has used isolated or cultured vascular-derived cells. However, phenotypic changes in ET-receptor subtypes have been demonstrated in cultured vascular smooth muscle cells (36). For example, ETA receptor was shown to be predominant in early-passage cells and ETB receptors in late-passage cells, whereas only ETA receptor mRNA was expressed in intact media of rat aorta (14). Therefore, attempting to relate the previous findings in cultured vascular cells to those we have obtained in isolated intact arteries becomes complex. Furthermore, of considerable importance is the fact that we examined fetal arteries, which are functionally distinct from adult arteries. Because antenatal glucocorticoid treatment of women in premature labor is now almost universally practiced, it is essential to study effects both on the fetus and in vivo.

Our findings suggest that glucocorticoid treatment may increase peripheral vascular resistance via an increase in ET-1 responsiveness resulting from an increase in ETA-receptor binding. However, this does not exclude other mechanisms that may contribute to such functional changes. For example, augmentation of ET-1 responses by glucocorticoids may be related to relative inhibition of vasodilatory responses and hence increased vasoconstriction. This is indicated by the finding that potentiation of ET-1-induced IP3 production in rat vascular smooth muscle cells by DM was partially due to inhibition of prostaglandin synthesis (35). Moreover, ET-1-induced release of the vasodilators PGI2 (12) and PGE2 (41) functionally antagonized the direct vasoconstrictor effect of ET-1. In addition, several studies suggest that production of the potent vasodilator PGI2 by the vasculature is decreased by glucocorticoids (3, 17). Moreover, evidence exists for the interaction of the endothelial-derived agents nitric oxide (NO) and ET-1 (6, 18). Accordingly, we used the endothelial-dependent agent ACh to examine the role of NO in arterial responsiveness. However, a similar response to ACh was noted in femoral arteries from both groups, suggesting that a reduction in baseline and/or agonist-induced NO is not involved in increased ET-1 responsiveness. In contrast to femoral arteries, second-order middle cerebral arteries from control and DM-treated fetuses did not relax to ACh. Similarly, examination of endothelium-dependent relaxations in isolated arteries from adult rabbits and dogs demonstrated a reduced effect of ACh in cerebral compared with extracerebral (e.g., femoral) arteries (28). Hayashi et al. (15) reported that isolated cerebral artery strips from premature and newborn baboons showed a marked contractile response to ACh, whereas arteries from adult baboons showed little response.

ETB receptors present on the endothelium can mediate vasodilation through the NO pathway (43), although the resolution of the macroautoradiography used in this study was not high enough to visualize receptors expressed by this single layer of cells. Smooth muscle cells within the vessel wall can also express ETB receptors, but the ratio varies between species and vascular beds. In humans, a relatively consistent pattern has emerged; the density of ETB receptors is less than 15% of the ETA subtype (11). Autoradiographic examination of femoral and cerebral vascular beds in our studies detected little presence of ETB receptors in these arteries, confirming that ETA receptors are the predominant subtypes in these vessels. Furthermore, preliminary studies in isolated femoral and middle cerebral arteries showed negligible responses to ET-3. Therefore, the markedly greater potency of ET-1 compared with ET-3 in these arteries provides further evidence for the predominance of ETA-receptor subtype. This pattern is comparable to the human vasculature and suggests that the fetal sheep is a relevant animal model to elucidate further the action of glucocorticoids on the ET system.

Our findings suggest that the nature of the increased ET-1-induced responsiveness in arteries from DM-treated fetuses is vascular bed specific. Tissue specificity of glucocorticoid-mediated effects has previously been reported. For example, DM was shown to increase ET-1 mRNA before downregulation of ETB receptor mRNA level at the hypothalamus and cerebellum but not other areas of the rat brain (38). Thus the influence of glucocorticoids on ET-1 responsiveness of cerebral arteries may well be differential depending on their anatomic location. The tachyphylaxis we noted in DM-exposed middle cerebral arteries may represent a tissue-specific protective mechanism to ensure maintained and adequate cerebral blood flow, particularly at this crucial time of development.

In summary, the findings of our study provide further evidence for the interaction of glucocorticoids and the potent vasoconstrictor ET-1. Our results indicate that the DM-induced increase in peripheral vascular resistance, which we have previously demonstrated in fetal sheep (13), may involve increased ET-1 responsiveness in femoral arteries. The mechanism of this increased ET-1-induced vasoconstriction may, at least in part, be due to an increase in ETA-receptor binding. Therefore, hyperreactivity to vasoconstrictor agonists, such as ET-1, may be involved in glucocorticoid-induced hypertension in fetal sheep.


    ACKNOWLEDGEMENTS

This research was supported by grants from Wellcome Trust, UK and National Institutes of Health HD-21350. A. P. Davenport acknowledges grant support from the British Heart Foundation and Royal Society, UK. C. C. Docherty was supported by a Wellcome Trust Travelling Research Fellowship, UK. S. V. Koenen was supported by the Ter Meulen Funds, Royal Netherlands Academy of Arts and Sciences.


    FOOTNOTES

Address for reprint requests and other correspondence: P. W. Nathanielsz, Laboratory for Pregnancy and Newborn Research, Dept. of Biomedical Sciences, College of Veterinary Medicine, Cornell Univ., Ithaca, NY 14853 (E-mail: pwn1{at}cornell.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 18 July 2000; accepted in final form 6 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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