Introduction

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Corticotropin-releasing factor (CRF), a 41-amino acid peptide, is produced by the hypothalamus (Rivier et al., 1982). It acts on the anterior pituitary to release adrenocorticotropin, which, in turn, stimulates cortisol production by the adrenal glands (Rivier et al., 1982). Central administration of CRF in rats has been shown to elevate blood pressure and heart rate (Fisher et al., 1983). In contrast, peripherally administered CRF lowers blood pressure (Gardiner et al., 1988). In addition to control of cortisol levels in the body, CRF may play a direct role in regulation of blood pressure.

In humans, the levels of CRF are normally undetectable in the nonpregnant state, increase exponentially during pregnancy, particularly during the final weeks, and then decline in the immediate postpartum period (Campbell et al., 1987). The placenta is the main source of this increased production of CRF during pregnancy (Riley et al., 1991). CRF levels are higher in pregnancies complicated with pre-eclampsia as compared with uncomplicated pregnancies (Riley et al., 1991). Pregnancy is associated with various cardiovascular changes such as increased blood volume and cardiac output and decreased blood pressure and peripheral vascular resistance (Poston et al., 1995). The decrease in peripheral vascular resistance has been attributed to decreased responsiveness to vasopressor agents and increased production of vasorelaxants. We and others have shown that CRF is a potent vasorelaxant (Lei et al., 1993; Clifton et al., 1995; Jain et al., 1997, 1998). Therefore, CRF may have a role in the modulation of the peripheral vascular resistance during pregnancy, especially of the uteroplacental vascular bed.

The mechanism of the vasodilatory effect of CRF is not well characterized. Vasorelaxants are known to act on the vascular endothelium to cause the release of relaxant factors such as prostacyclin (Moncada and Vane, 1979), nitric oxide (NO) (Furchgott, 1993), and endothelium-derived hyperpolarizing factor (EDHF) (Vanhoutte, 1996), which, in turn, diffuse to the vascular smooth muscle and cause relaxation. Vasorelaxants can also act directly on the vascular smooth muscle causing an increase in the intracellular cAMP/cGMP levels (Little et al., 1984) or opening of the membrane K⁺ channels (Brayden and Nelson, 1992), thereby causing relaxation. The vascular endothelium as well as smooth muscle has CRF-binding sites (Dashwood et al., 1987). We have shown previously that the relaxant effect of CRF in rat aorta is predominantly endothelium-dependent and mediated by the NO-cGMP pathway (Jain et al., 1997). Our results agree with those of Clifton et al. (1995), who demonstrated that NO and cGMP are involved in the vasodilatory effect of CRF in the human fetoplacental circulation. However, Lei et al. (1993) showed that in the rat mesenteric artery, vasodilation by CRF is endothelium-independent. Hence, the mechanism of action of CRF remains a subject of controversy.

Because the uterine vasculature may be an important target for CRF during pregnancy, the objective of this study was to examine the mechanism of action of CRF on the uterine artery of pregnant rats. We hypothesized that CRF causes relaxation of the uterine artery by predominantly endothelium-dependent mechanisms.

	Materials and Methods

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Animals. Timed-pregnant Sprague-Dawley rats (on day 18 of gestation) were obtained from Charles River Laboratories (Wilmington, MA). They were housed separately in temperature- and humidity-controlled quarters with constant light/dark cycles of 12 h:12 h and were provided with food and water ad libitum. The pregnant rats used in these studies have a gestation of 22 days, with day 1 as the day the sperm plug was observed. The animals were sacrificed by CO₂ inhalation. Each experimental group consisted of five to eight rats. All procedures were approved by the Animal Care and Use Committee of the University of Texas Medical Branch.

Drugs and Solutions. The drugs used in the experiments were acetylcholine hydrochloride, CRF, -helical corticotropin-releasing factor 9-41 (CRF-A), N-nitro-L-arginine methyl ester (L-NAME), norepinephrine bitartrate (NE), phentolamine hydrochloride, sodium nitroprusside, thiopental sodium, indomethacin, and tetrabutyl ammonium chloride (TBA) were purchased from Sigma (St. Louis, MO); 6-anilino-5,8-quinolinedione (AQD, LY-83,583; Alexis, San Diego); cicaprost (ZK-96,480) was pruchased from Schering AG (Berlin, Germany); and glibenclamide, pinacidil, and 9-tetrahydro-2-furanyl-9H-purin-6-amine (TFPA, SQ-22,536) were purchased from Research Biochemicals International (Natick, MA). Stock solutions of all of the drugs were prepared in deionized water with the exception of indomethacin (10² mol), which was prepared in a 150-mmol solution of NaHCO₃ (pH 8.3), and of AQD (10¹ mol), glibenclamide (10¹ mol), and pinacidil (10¹ mol), which were dissolved in dimethyl sulfoxide. Stock solutions for NE were freshly prepared for each experiment. The composition of physiological salt solution was as follows: NaCl, 115 mM; KCl, 5 mM; NaH₂PO₄, 1.2 mM; NaHCO₃, 25 mM; MgCl₂, 1.2 mM; CaCl₂, 2.5 mM; EDTA, 0.026 mM; and glucose, 11mM. The depolarizing solution (high-K⁺ physiological salt solution) was made by replacing NaCl with equimolar KCl; the final K⁺ concentration in that solution was 120 mM.

In Vitro Experiments. Two-millimeter segments of the uterine artery (o.d. 300-400 µm) were mounted in the jaws of a wire myograph (model 410A; J.P. Trading I/S, Aarhus, Denmark) over 25-µm tungsten wires. The preparations were bathed in physiological salt solution maintained at 37°C, pH ~7.4. A mixture of 95% O₂ and 5% CO₂ was bubbled continuously through the solution. The vessels were given a preload based on the length-tension curve for each vessel. Myosight software (J.P. Trading I/S) was used for estimating the circumference that each vessel would have had under a transmural pressure of 100 mm Hg in situ, and the circumference of the preparation was adjusted to 90% of the estimated circumference (Mulvany and Halpern, 1977). The vessels were equilibrated for 1 h. Then, two successive stimulations of 15-min duration were given with high-K⁺ physiological salt solution, separated by a 15-min equilibration in physiological salt solution. The endothelium was removed in some vessels by rubbing their luminal surface with a human hair (Osol et al., 1989). The presence or absence of endothelium in the preparations was confirmed by contracting with NE (10⁶ mol) and eliciting a relaxation with acetylcholine (10⁶ mol). After washing and rest, the preparations with or without endothelium were contracted with NE (10⁶ mol) and the relaxant responses to cumulative concentrations of CRF (10¹⁰ to 10⁶ mol) were studied. The force was recorded by an isometric force transducer and analyzed using Windaq data acquisition and playback software (DataQ Instruments, Inc., Akron, OH).

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Data Analysis. Data are expressed as mean ± S.E., and n represents the number of rats used in each experiment. The effect of CRF on the uterine artery was quantified as the percentage of relaxation of the preexisting tone in preparations contracted with NE or depolarizing solution. Concentration-response curves were generated based on responses to cumulative concentrations of CRF. The median effective dose (ED₅₀) values for CRF (concentration of CRF producing 50% of the maximal relaxation) were calculated. Area under the dose-response curves was calculated and expressed in arbitrary units. For statistical analysis, Student's t test or one-way analysis of variance followed by Newman-Keuls Multiple Comparisons Test was used as appropriate, and p < .05 was considered to be statistically significant.

	Results

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CRF relaxed the uterine artery of day 18 pregnant rats in a concentration-dependent manner (Fig. 1). Blockade of the CRF receptor by CRF-A abolished relaxation of the uterine artery by CRF (Fig. 2). The area under the concentration-response curve was significantly decreased (control, 195.15 ± 7.15; CRF-A, 58.05 ± 11.93, p < .001).

View larger version (5K): [in this window] [in a new window]   Fig. 1.   Representative tracing of relaxation responses of the uterine artery of day 18 pregnant female rats to cumulative concentrations of CRF. Isometric tension measured from the uterine artery in vitro is shown. The uterine artery with intact endothelium was precontracted with NE (106 mol). The concentrations of CRF are expressed in log mol units.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effect of CRF antagonist (CRF-A) on responses of the uterine artery of day 18 pregnant rats to cumulative concentrations of CRF. The vessels with intact endothelium were preincubated with CRF-A (106 mol) for 15 min and contracted with NE (106 mol). The 100% contraction induced by NE was 1.82 ± 0.14 g and 1.91 ± 0.11 g in the absence and presence of CRF-A, respectively. Each point represents mean ± S.E., n = 5. Student's t test was used; control versus CRF-A: **p < .01; ***p < .001.

Relaxation responses of the uterine artery to CRF were significantly inhibited by removal of the vascular endothelium (Fig. 3). The ED₅₀ value as well as the area under the concentration-response curve were significantly decreased by removal of endothelium (ED₅₀: control, 8.32 ± 0.1; deendothelized, 7.83 ± 0.09, p < .01; area under the curve: control, 189.26 ± 8.29; deendothelized, 84.44 ± 13.75, p < .001). In addition, relaxation of the uterine artery by CRF was decreased in the presence of inhibitor of NO synthase (L-NAME, Fig. 4A) or guanylate cyclase (AQD, Fig. 4B). Both the ED₅₀ value and the area under the curve were significantly decreased by L-NAME (ED₅₀: control, 8.09 ± 0.18; L-NAME, 7.40 ± 0.16, p < .05; area under the curve: control, 165.35 ± 19.40; L-NAME, 41.13 ± 13.36, p < .001), whereas only the decrease in the area under the curve was significant in the case of AQD (ED₅₀: control, 7.90 ± 0.18; AQD, 7.47 ± 0.20, p > .05; area under the curve: control, 142.26 ± 17.83; AQD, 45.45 ± 2.15, p < .001).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of removal of the vascular endothelium on responses of the uterine artery of day 18 pregnant rats to cumulative concentrations of CRF. Endothelium-intact preparations that relaxed less than 50% with acetylcholine (106 mol) and endothelium-denuded preparations that relaxed with acetylcholine were discarded. The preparations were precontracted with NE (106 mol). The 100% contraction induced by NE was 1.95 ± 0.15 g and 2.14 ± 0.15 g in preparations with or without endothelium, respectively. Each point represents mean ± S.E., n = 6. Student's t test was used; control versus deendothelized: *p < .05, **p < .01, ***p < .001.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Effect of inhibition of NO synthase (L-NAME, A) or guanylate cyclase (AQD, B) on responses of the uterine artery of day 18 pregnant rats to cumulative concentrations of CRF. Uterine arteries with intact endothelium were preincubated with L-NAME (104 mol) or AQD (105 mol) for 30 min and contracted with NE (106 mol). The 100% contraction induced by NE was 1.92 ± 0.09 g and 2.17 ± 0.16 g in the absence and presence of L-NAME (n = 6) and 1.87 ± 0.14 g and 2.06 ± 0.18 g in the absence and presence of AQD (n = 5), respectively. Each point represents mean ± S.E. Student's t test was used; control versus L-NAME/AQD: *p < .05, **p < .01, ***p < .001.

When depolarizing solution (120 mmol of K⁺) was used for contracting the uterine artery, relaxation by CRF was decreased as compared with uterine artery contracted with NE (Fig. 5). In addition, in preparations contracted with NE, preincubation with thiopental or miconazole (inhibitors of cytochrome P-450) significantly decreased relaxation responses to CRF (Fig. 5). The ED₅₀ values were not significantly decreased by 120 mmol of K⁺, thiopental, or miconazole, but the areas under the curves were decreased significantly (Table 1). Inhibition of cyclo-oxygenase by indomethacin did not affect the relaxation responses to CRF in the uterine artery (Fig. 6). The ED₅₀ value or the area under the curve was not significantly different from controls (ED₅₀: control, 7.73 ± 0.07; indomethacin, 7.63 ± 0.04, p > .05; area under the curve: control, 129.74 ± 13.54; indomethacin, 129.42 ± 10.26, p > .05).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effect of membrane depolarization (120 mmol of K+) or inhibition of cytochrome P-450 (thiopental/miconazole) on responses of the uterine artery of day 18 pregnant rats to cumulative concentrations of CRF. The vessels with intact endothelium were contracted with depolarizing solution (120 mmol of K+) or preincubated with thiopental (3 × 104 mol) or miconazole (3 × 105 mol) for 30 min and contracted with NE (106 mol). The 100% contraction induced by NE was 1.95 ± 0.09 g, by depolarizing solution was 1.41 ± 0.08 g, by NE/thiopental was 1.88 ± 0.08 g, and by NE/miconazole was 1.91 ± 0.06. Each point represents mean ± S.E., n = 16, 8, 8, and 8 for the four groups, respectively. One-way analysis of variance followed by Newman-Keuls Multiple Comparisons Test was used; NE versus 120 mmol of K+, NE/thiopental, and NE/miconazole: *p < .05.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effect of inhibition of cyclo-oxygenase (indomethacin) on responses of the uterine artery of day 18 pregnant rats to cumulative concentrations of CRF. Uterine arteries with intact endothelium were preincubated with indomethacin (105 mol) for 45 min and contracted with NE (106 mol). The 100% contraction induced by NE was 1.98 ± 0.10 g and 2.00 ± 0.13 g in the absence or presence of indomethacin, respectively. Each point represents mean ± S.E., n = 8. Student's t test was used; control versus indomethacin: p > .05.

In vessels denuded of endothelium, the relaxation was not decreased by inhibition of adenylate cyclase with TFPA, inhibition of adenosine triphosphate-sensitive K⁺ channels with glibenclamide, or nonspecific inhibition of K⁺ channels with TBA (Fig. 7). The ED₅₀ values and the areas under the curves were not significantly different (Table 2). In addition, selective activation of voltage-gated Ca⁺⁺ channels by depolarizing solution (120 mmol of K⁺, in presence of phentolamine) or inhibition of voltage-gated Ca⁺⁺ channels with nitrendipine did not alter the relaxation responses to CRF (Fig. 8). The ED₅₀ values and the areas under the curves were not significantly different (ED₅₀: NE, 6.80 ± 0.16; 120 mmol of K⁺, 6.86 ± 0.14; NE/nitrendipine, 7.23 ± 0.20, p > .05; area under the curve: NE, 34.60 ± 13.15; 120 mmol of K⁺, 27.86 ± 6.90; NE/nitrendipine, 39.60 ± 11.49, p > .05).

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Effect of inhibition of adenylate cyclase (TFPA), adenosine triphosphate-dependent K+ channels (glibenclamide), or nonspecific inhibition of K+ channels (TBA) on responses of deendothelized uterine artery of day 18 pregnant rats to cumulative concentrations of CRF. The vessels were preincubated with TFPA (2 × 104 mol, 30 min), glibenclamide (105 mol, 30 min), or TBA (103 mol, 15 min) and contracted with NE (106 mol). The 100% contraction induced by NE was 1.89 ± 0.12 g, 2.15 ± 0.14 g, 2.00 ± 0.12 g, and 2.03 ± 0.13 g in control, TFPA, glibenclamide, and TBA groups, respectively. Each point represents mean ± S.E., n = 8. One-way analysis of variance was used; p > .05.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Effect of selective activation of voltage-gated Ca++ channels (120 mmol of K+) or inhibition of voltage-gated Ca++ channels (nitrendipine) on responses of deendothelized uterine artery of day 18 pregnant rats to cumulative concentrations of CRF. The vessels were preincubated with phentolamine (105 mol) and contracted with depolarizing solution (120 mmol of K+) or preincubated with nitrendipine (106 mol) for 30 min and contracted with NE (106 mol). The 100% contraction induced by NE was 2.02 ± 0.12 g, by depolarizing solution was 1.76 ± 0.14 g, and by NE/nitrendipine was 1.13 ± 0.17. Each point represents mean ± S.E., n = 6. One-way analysis of variance was used; p > .05.

	Discussion

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CRF is a potent vasodilator that has been shown to cause hypotension when administered i.v. in rats (Gardiner et al., 1988). We have shown previously that CRF, when administered chronically in pregnant rats, causes a decrease in blood pressure (Jain et al., 1998). We also have shown that CRF is a relaxant of the rat aorta in vitro and this relaxant effect is endothelium-dependent and mediated by the NO-cGMP pathway (Jain et al., 1997). The objective of the present study was to characterize the effects of CRF on rat uterine artery during pregnancy. Our study demonstrates that CRF is a potent relaxant of the uterine artery of pregnant rats. Vasorelaxation by CRF is a specific, receptor-operated effect. This effect is predominantly endothelium-dependent and mediated by the NO-cGMP pathway as well as EDHF but not prostacyclin. There is also a smaller endothelium-independent component of vasorelaxation by CRF. Neither cAMP, K⁺ channels, nor Ca⁺⁺ channels are involved in endothelium-independent relaxation by CRF.

CRF has binding sites on the vascular endothelium as well as smooth muscle (Dashwood et al., 1987). Two subtypes of the CRF receptor have been identified in humans and rats: CRF₁ and CRF₂ (Grigoriadis et al., 1996). CRF₂ has two splice variants, i.e., CRF₂ and CRF₂. The results from a previous study suggest that the CRF receptor in the vasculature may be type 2 (Rohde et al., 1996). Our studies on the CRF receptor indicate that the isoform expressed in the vasculature is CRF₂ (unpublished observations). In this study, CRF receptor antagonist, CRF-A, abolished the relaxant effect of CRF. Hence, our studies support the conclusion that vasorelaxant effect of CRF is mediated by the receptor CRF₂.

Vascular endothelium is known to be important in the regulation of vascular smooth muscle tone through various contracting (endothelin, angiotensin II) and relaxing factors (NO, prostacyclin, EDHF) (Rubanyi, 1993). In the present study, we show that removal of endothelium abolishes a major component of relaxation of the uterine artery by CRF, especially at lower (and more physiological) concentrations of CRF. This supports the conclusion that vasorelaxant effect of CRF is endothelium-dependent. Release of endothelial relaxing factors is mediated by an increase in free cytoplasmic Ca⁺⁺ levels in the endothelial cells (Furchgott, 1983). This increase is effected by various mechanisms such as influx of extracellular Ca⁺⁺, Na⁺-Ca⁺⁺ exchange, and liberation of Ca⁺⁺ from intracellular stores (Singer and Peach, 1982; Winquist et al., 1985; Luckhoff and Busse, 1986). CRF has been shown to cause an increase in intracellular calcium through receptor-operated Ca⁺⁺ channels (Kiang, 1994). Hence, CRF may enhance the production of endothelial relaxing factors by increasing intracellular Ca⁺⁺ in the endothelium. Our studies on localization of the CRF receptor by immunohistochemistry show that the CRF receptor is present predominantly in the endothelium and, to a lesser extent, in the vascular smooth muscle (unpublished observations) and therefore support this conclusion.

Endothelium-derived relaxing factor or NO is generated in the vascular endothelium from L-arginine by a Ca⁺⁺-dependent NO synthase (Furchgott, 1993; Wu, 1995). NO activates the soluble guanylate cyclase of the vascular smooth muscle and increases cGMP, which causes relaxation of the smooth muscle (Holzmann, 1982). In the present study, blockade of the NO synthase with L-NAME or of soluble guanylate cyclase with AQD caused a significant inhibition of the relaxant effect of CRF. L-NAME is a prodrug that lacks NO synthase blocking activity and is rapidly hydrolyzed in biological tissues to its active form, i.e., N-nitro-L-arginine (Pfeiffer et al., 1996). L-NAME is also known to possess other effects such as inhibition of muscarinic receptors (Pfeiffer et al., 1996). Similarly, AQD, an inhibitor of soluble guanylate cyclase, can potentiate intracellular release of NO (Kawada et al., 1994). However, in spite of potential nonspecific actions of these agents, the similarity of the effects of de-endothelization, L-NAME, and AQD support this role of endothelium, NO, and cGMP in vasorelaxation by CRF. It may be noted that NO may cause relaxation of smooth muscle by mechanisms other than stimulation of guanylate cyclase (Barany, 1996). Prostacyclin, the first endothelial relaxing factor to be identified, is produced by the cyclo-oxygenase enzyme (Moncada and Vane, 1979). However, inhibition of cyclo-oxygenase with indomethacin failed to have any effect on the vasorelaxation by CRF. This indicates that prostacyclin may not be an important factor that mediates endothelium-dependent relaxation by CRF.

A recently described endothelial factor, EDHF, has been shown to cause relaxation of the smooth muscle by activation of Ca⁺⁺-activated K⁺ channels (Vanhoutte, 1996). At least one class of EDHFs (epoxyeicosatrienoic acids) has been shown to be produced by cytochrome P-450 (Lischke et al., 1995; Campbell et al., 1996). Because EDHF acts by causing hyperpolarization of the membrane potential, use of depolarizing solution for the contraction of vessels in vitro eliminates this effect of EDHF. Precontraction of the uterine artery with depolarizing solution (120 mmol of K⁺) as well as inhibition of cytochrome P-450 with thiopental or miconazole significantly inhibited the relaxation responses to CRF. This supports the role of a cytochrome P-450-dependent EDHF (possibly epoxyeicosatrienoic acids) in vasorelaxation by CRF.

Removal of endothelium did not completely abolish the relaxation of uterine artery by CRF. Thus, CRF may also cause vasorelaxation by acting directly on the vascular smooth muscle. Vascular smooth muscle is known to relax in response to cAMP (Little et al., 1984). The central effects of CRF are mediated by the activation of adenylate cyclase and an intracellular increase in cAMP (Battaglia et al., 1987). Vasorelaxants may also activate the various membrane K⁺ channels (e.g., adenosine triphosphate-sensitive K⁺ channels, Ca⁺⁺-activated K⁺ channels, and voltage-dependent K⁺channels) and relax the smooth muscle by membrane hyperpolarization (Brayden and Nelson, 1992; Barany, 1996). Inhibition of adenylate cyclase by TFPA (a specific adenylate cyclase inhibitor) (Lippe and Ardizzone, 1991) or blockade of adenosine triphosphate-sensitive K⁺ channels (with glibenclamide) or nonspecific inhibition of K⁺ channels (with TBA) failed to inhibit the direct relaxant effect of CRF in deendothelized uterine artery. Hence, neither K⁺ channels nor cAMP may mediate the direct relaxant effect of CRF on the vascular smooth muscle. In addition, selective activation of voltage-gated Ca⁺⁺ channels by depolarizing solution in the presence of -adrenoceptor blocker phentolamine as well as inhibition of voltage-gated Ca⁺⁺ channels by nitrendipine did not decrease relaxation responses to CRF, indicating that Ca⁺⁺ channels may not be directly involved in this effect.

Our data confirm the results from our previous study (Jain et al., 1997) as well as those of Clifton et al. (1995) which show that the relaxant effect of CRF is endothelium-dependent and mediated by the NO-cGMP system. However, our results are only in partial agreement with Lei et al. (1993), who showed that the response to CRF in small mesenteric arteries from male Wistar rats (precontracted with arginine vasopressin) is endothelium-independent. Because CRF exhibits regional differences in its effects (Gardiner et al., 1988), it is possible that the mechanism characterized in our study is different from the one examined in the previous report. Differences in the agonist used to precontract the vessel and the gender and strain of rats used are other factors that may account for the observed differences.

This study shows that CRF is a potent relaxant of the pregnant rat uterine artery. The relaxation responses to CRF in the rat uterine artery as well as aorta are decreased at the term of gestation (Jain et al., 1997, 1998). The nonpregnant rat uterine artery is also relaxed by CRF (unpublished data). In addition, CRF receptor expression in the rat uterine artery is decreased at the end of pregnancy. Thus, this mechanism of relaxation is actively regulated during pregnancy. In humans, CRF production is increased exponentially from low-picomolar concentrations in the nonpregnant state to ~2 nmol toward the end of pregnancy (Campbell et al., 1987). The placenta is the main source for CRF in pregnant women as opposed to the nonpregnant state, when the hypothalamus produces very low amounts of this peptide (Riley et al., 1991). However, the circulating levels of CRF levels were not increased during pregnancy in rat (Jain et al., 1998). Nonetheless, infusion of the CRF antagonist in pregnant rats caused a decrease in blood pressure, supporting the presence of a vasoactive level of CRF bioactivity during pregnancy in rats (Jain et al., 1998). Various other peptides are known to act on the CRF receptor, e.g., urocortin, urotensin I, sauvagine, and epidermal growth factor (Brown et al., 1982; Polk et al., 1987; Vaughan et al., 1995). Urocortin, which is produced by the placenta (Petraglia et al., 1996), has been shown to be more active on the CRF receptor type 2 (Vaughan et al., 1995), suggesting that it may be the true high-affinity ligand for this receptor isoform. Therefore, it would be reasonable to assume that not only CRF but also other CRF-related peptides that may be increased during pregnancy may act on the CRF receptors and cause vasorelaxation in the uteroplacental bed. The increase in CRF levels in pregnant women is even greater in pregnancies complicated by pre-eclampsia (Campbell et al., 1987). Because pre-eclampsia is associated with uteroplacental hypoperfusion (Sibai, 1996), the abnormal increase in CRF in these conditions may represent a compensatory response of the placenta to underperfusion.

In summary, the present study shows that CRF is a potent vasorelaxant that relaxes the uterine artery predominantly through endothelium-dependent but additionally through endothelium-independent mechanisms. This effect may be important in modulating uteroplacental blood flow during pregnancy.