INTRODUCTION

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PREGNANCY IS A UNIQUE PHYSIOLOGICAL state responsible for propagation of the species. Normal mammalian pregnancy is associated with increases in cardiac output and heart rate, a fall in systemic vascular resistance, and the redistribution of cardiac output (27). Uterine blood flow (UBF) increases from 3-5% of cardiac output to 20-25% at term, and it is the >30-fold rise in UBF that ensures normal fetal growth, development, and well-being (26, 27, 29). The rise in UBF occurs in three phases (34). The first is considered to be preimplantation and is due to vasodilation in all uterine tissues and endometrial neovascularization (25, 34). The second phase is associated with development and growth of the maternal and fetal placental circulations and, in the ovine species, is predominantly due to neovascularization and angiogenesis (26, 34, 44). The final phase occurs in the last third of gestation, during which time UBF rises exponentially, increasing three- to fourfold over 40 days (26, 27, 29, 34), and is essential for the increased delivery of oxygen and nutrients necessary for the parallel exponential increase in fetal size that occurs during this time (26, 29, 34). In morphometric studies, Teasdale (44) demonstrated this final rise in UBF in ovine pregnancy was predominantly due to progressive placental vasodilation, which is also likely to be true in women because there is no further anatomic development of the maternal placental circulation during the last trimester (26, 29). It also accounts for the marked redistribution of UBF previously observed (16, 28). The mechanisms responsible for uteroplacental vasodilation in the last third of pregnancy remain a mystery.

A growing body of evidence suggests that increases in vascular nitric oxide (NO) synthase (NOS) and thus NO may play an important role in the cardiovascular changes in pregnancy (41). Increases in uterine artery NOS may also play a pivotal role in the uterine vascular changes that normally occur in pregnancy (15, 41, 50), whereas local vascular prostaglandins do not appear to be involved (14, 19). For example, uterine cGMP synthesis increases 38-fold in pregnancy compared with the nonpregnant (30), and uterine artery endothelial NOS (eNOS) expression increases three- to fourfold (15, 50). The increases in uterine artery NOS expression may be due to increases in local placental estrogen synthesis because estradiol-17 (E₂) increases type III (eNOS) and type I (neuronal NOS) NOS in the uterine arteries of nonpregnant ewes (39, 46), which parallel increases in uterine cGMP production and UBF (30, 39). Moreover, E₂-induced increases in UBF are dose dependently inhibited by local infusions of N^G-nitro-L-arginine methyl ester (L-NAME), a nonspecific NOS antagonist (30, 47). However, in pregnant ewes, short-term L-NAME infusions decrease basal uteroplacental production of cGMP without altering UBF (30). We recently reported that systemic E₂ not only increases uterine artery NOS expression in nonpregnant ewes (39) but also increases the opening potential of large conductance calcium-dependent potassium channels (BK_Ca) in uterine artery myocytes (35). Furthermore, local infusions of tetraethylammonium chloride (TEA), a BK_Ca channel-selective antagonist at submillimolar concentrations (3, 21, 22), dose dependently inhibit E₂-mediated increases in UBF in nonpregnant ewes (35). Thus while NO contributes to E₂-induced vasodilation in nonpregnant ewes, BK_Ca channel activation may be the final mediator. No one has examined the role of BK_Ca channels in modulating basal UBF in pregnancy or the E₂-induced vasodilation previously observed in pregnant sheep (8, 33).

The purpose of the present investigation, therefore, was to determine in near-term pregnant sheep whether 1) BK_Ca channels are expressed in uterine artery myocytes, 2) local intra-arterial infusions of TEA alter basal UBF, and 3) TEA modifies the uterine vasodilation that follows systemic E₂ administration. These data would provide invaluable insights into the final cellular mechanisms responsible for the uterine vasodilation essential for normal fetal growth and well-being.

	METHODS

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Animal model. The animal model used in these experiments has previously been described (30). In brief, time-dated pregnant ewes (n = 5) of mixed Western breed at 118-125 days gestation (term = 145 days) were fasted overnight but allowed access to water. In the morning, animals were given atropine sulfate intramuscularly, and a percutaneous jugular venous catheter was placed for administration of preanesthetic pentobarbital sodium and ketamine hydrochloride. Animals were intubated and surgically prepared; isoflurane (Mallinckrodt Veterinary; Mundelein, IL) and oxygen were given via a rebreathing anesthesia machine. The gravid uterus was isolated through a midline abdominal incision, and an electromagnetic flow probe (inner diameter 6.0 or 7.0 mm; Carolina Medical; King, NC) was implanted on the main uterine artery of each uterine horn proximal to the first bifurcation. Polyvinyl catheters containing heparinized saline (100 U/ml) were implanted retrograde 2 cm into a distal branch of the uterine artery of each uterine horn for the local intra-arterial infusion of drugs. The abdomen was closed, and polyvinyl catheters were implanted via a groin incision into the femoral artery and vein to the level of the abdominal aorta and mid vena cava, respectively. Animals received antibiotics on the day of surgery and the next 2 days, as well as banamine (Schering-Plough Animal Health; Union, NJ) for pain. All animals were allowed 5 days for postoperative recovery before studies were initiated. These studies were approved by the Institutional Review Board for Animal Research at the University of Texas Southwestern Medical Center at Dallas.

Experimental protocols. Two protocols were used in these studies. In the first protocol, we determined whether TEA (Sigma; St. Louis, MO), a selective inhibitor of BK_Ca channels at submillimolar concentrations (22), infused directly into the uterine circulation would alter basal UBF at different times during the last third of gestation. Studies were performed in five pregnant ewes between 123 and 149 days gestation (term = 145 ± 5 days) using each uterine horn if the uterine artery catheters were patent and the flow probes were functional. On the day of study, a continuous infusion of TEA was initiated via one uterine artery catheter after a 30-min control period and maintained for 90 min. The arterial concentration of TEA was estimated from the rate of TEA infused (in µg/min) divided by the baseline measurement of UBF (in ml/min) (30). From preliminary studies, it has been determined that a continuous TEA infusion, resulting in an estimated uterine arterial concentration of 0.15 mM, decreased UBF significantly but did not exceed 50%. All subsequent studies were performed using this dose because greater decreases in UBF would potentially alter uterine oxygen delivery and thus fetal well-being (29), and we wanted to compare the magnitude of its effects at different gestational ages. Continuous recordings of UBF, mean arterial pressure (MAP), and heart rate were initiated 30 min before the infusion of TEA and maintained until 90 min after stopping the infusion. The study was repeated at 24-48 h, and each animal was studied up to three times at different gestational ages to determine if gestational age-dependent alterations in the responses occurred, resulting in 11 experiments.

Hemodynamic measurements. MAP in the lower abdominal aorta was monitored continuously via a femoral arterial catheter connected to a pressure transducer (type 4-327-0109, Bell and Howell; Pasadena, CA). Heart rate was determined from the phasic signal derived from the arterial pressure monitor. UBF was monitored continuously with square-wave electromagnetic flowmeters (model FM501, Carolina Medical). All measurements were continuously recorded on a six-channel pen recorder (model 3000, Gould; Cleveland, OH). Uteroplacental vascular resistance (UVR) was calculated from MAP (in mmHg) divided by UBF (in ml/min).

RT-PCR. At the termination of the above experiments, animals were euthanized with intravenous pentobarbital sodium (125 mg/kg), and samples of the third to fourth generation uterine artery were removed and placed in cold sterile physiological saline. Similar samples were obtained from nonpregnant ewes involved in other studies. With the use of sterile methods, the adventitia was removed with sharp dissection, the vessel was opened, and the endothelium was removed with a soft cotton swab as previously described (39). Arteries were placed in liquid nitrogen and stored at 80°C until assayed. At the time of assay, arteries were suspended in liquid nitrogen and ground to powder with a prechilled mortar and pestle. RNA was extracted using the guanidium thiocyanate-phenol-chloroform method (Trireagent, Sigma). Samples were then processed according to the reagent instructions, and the RNA was dissolved in diethyl pyrocarbonate-treated water and stored at 70°C. Optical density was measured to determine the RNA concentration. RNA (1 µg) was added to 11 µl of First Strand cDNA Synthesis reagent (Pharmacia) with random hexamers as primers in a final volume of 33 µl. Two microliters of this reverse transcript reaction were added for each PCR. The oligonucleotide primers used to amplify BK_Ca channel cDNA were based on the human sequence (45) and were (forward) 5'-CTACTGGGATGTTTCACTGGTGT-3' and (reverse) 5'-TGCTGTCATCAAACTGCATA-3', which yielded a product of 446 bp, consistent with that expected for human BK_Ca channels. Identity was confirmed with sequence analysis.

Statistical analysis. One-way ANOVA with multiple measures was used to determine whether significant differences occurred in responses of UBF and hemodynamic variables to treatment with either TEA infused locally or E₂ given systemically plus TEA at different gestational ages. When the effects of increasing gestational were shown to be nonsignificant (P > 0.05), one-way ANOVA with repeated measures was used to determine whether changes in UBF and hemodynamic measurements over time were significant. When significance was P < 0.05, a Student-Newman-Keuls test was then used to determine differences in responses between time periods for treatment with TEA alone (protocol 1) and TEA plus E₂ (protocol 2). Nonpaired and paired t-tests were employed where appropriate. Data are presented as means ± SE.

	RESULTS

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Effects of TEA on basal UBF. Although five animals were studied, two to three experiments were performed in each ewe at different gestational ages between 123 and 149 days gestation, resulting in 11 experiments. Because gestational age had no significant effect (P > 0.1) on uterine vascular or systemic hemodynamic responses to local infusions of TEA (all of which were calculated to achieve an arterial concentration of 0.15 mM), the data have been grouped for analysis. The basal hemodynamic data obtained before infusion of TEA are presented in Table 1 (protocol 1). These data are consistent with previously published values for pregnant ewes studied during this period of pregnancy (27). There was no significant effect of the local intra-arterial infusion of TEA on either MAP or heart rate (P  0.1, ANOVA) at any age. However, UBF in the treated uterine horn began to fall within 3-5 min after the local TEA infusion was started and achieved an apparent steady-state response by 45-60 min of infusion in each study (Fig. 1). During the period of the apparent steady-state response at 60 and 90 min of TEA infusion, UBF decreased (P < 0.0001, ANOVA) an average of 40 ± 8% and 55 ± 7% (Fig. 2), respectively. There was no significant difference in the magnitude of responses at this time. When the TEA infusion was stopped, UBF did not change significantly until 60- and 90-min postinfusion, increasing to an average of 335 ± 64 ml/min at 90-min post-TEA (Fig. 2). Of note, values remained 23 ± 7% less than that observed before TEA (P < 0.0001, ANOVA). There was no significant effect of TEA on UBF in the contralateral uterine horn at any gestational age (Fig. 2; P = 0.1, ANOVA). The fall in UBF was mirrored by a reciprocal rise in UVR (Fig. 3), which was >100% of baseline values and did not differ significantly after 30 min of TEA infusion (P > 0.05, ANOVA). While UVR began to fall soon after the local TEA infusion was stopped, the values remained significantly greater than control levels throughout the study period. As with UBF, contralateral UVR was unchanged at each gestational age studied (data not shown). All hemodynamic measurements returned to pre-TEA values within 24 h.

View this table: [in this window] [in a new window]   Table 1.   Basal hemodynamic data in near-term singleton pregnant sheep before study of TEA alone (protocol 1) and TEA plus E2 (protocol 2)

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Representative tracings obtained in a pregnant ewe at 135 days gestation demonstrating the effects of a local intra-arterial infusion of tetraethylammonium chloride (TEA; 0.15 mM) initiated via the left uterine artery catheter on mean arterial pressure, heart rate, and left and right uterine blood flow. Horizontal bar, duration of TEA infusion. bpm, Beats per minute.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   The effects of 90 min of continuous unilateral intra-arterial infusions of TEA (0.15 mM) on basal uteroplacental blood flow in pregnant ewes studied between 123 and 149 days gestation. Data are presented as means ± SE. Eleven studies were performed at different gestational ages as described in the text. a-d, Differences in blood flow between time periods in the treated uterine horn when analyzed by repeated-measures ANOVA, P < 0.0001. , Treated (Tx) animals; , untreated or control (non-Tx) animals.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   The effects of 90 min of continuous unilateral local intra-arterial infusions of TEA (0.15 mM) on basal uteroplacental vascular resistance in the treated uterine horn of pregnant ewes studied at different gestational ages between 123 and 149 days gestation. Data are presented as means ± SE. Eleven studies were performed at different gestational ages as described in the text. a and b, Differences in vascular resistance between time periods when analyzed by repeated-measures ANOVA, P = 0.0007.

Effects of TEA on E₂-induced vasodilation. Estrogen is a potent uterine vasodilator in pregnant ewes, increasing UBF 30-40% (29, 33). The mechanisms responsible for this vasodilation have not been studied. In nonpregnant sheep, TEA at submillimolar concentrations dose dependently decreases UBF responses to E₂, demonstrating that BK_Ca channels contribute to E₂-induced uterine vasodilation (35). Thus we investigated the effects of local intra-arterial TEA infusions on E₂-induced vasodilation in pregnant ewes during the third trimester. The effect of a systemic dose of E₂ (1 µg/kg) on UBF is illustrated in Fig. 4A. There is a 20- to 30-min delay, followed by a gradual rise in UBF that peaks and plateaus within 90-120 min. Eleven studies were performed in five ewes at different times between 123 and 149 days gestation. There was no evidence of a significant gestational age-mediated effect on the response to E₂ during the period of pregnancy studied (P > 0.1); therefore, the data have been grouped for analysis. Systemic E₂ administration alone had no effect on MAP (Table 2). However, 90 min after E₂ infusion, heart rate increased 10% (P < 0.0001) and UBF increased ~20% (P < 0.0001, ANOVA) in both uterine horns. This was paralleled by a reciprocal fall in UVR in both uterine horns (Table 2).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Representative tracings obtained in a pregnant ewe at 126 days gestation demonstrating the effects of a systemic dose of estradiol-17 (E2; 1 µg/kg) on left and right uteroplacental blood flow (A), and, on the next day, the inhibitory effects of a local intra-arterial infusion of TEA (0.15 mM) initiated via the right uterine artery catheter 30 min before systemic administration of E2 (B). Horizontal bar, duration of TEA infusion.

View this table: [in this window] [in a new window]   Table 2.   Systemic and uterine hemodynamic responses to systemic infusions of E2 (1 µg/kg) in near-term singleton pregnant sheep in the absence of local treatment with TEA

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View larger version (36K): [in this window] [in a new window]   Fig. 5.   Summary of the effects of E2 (1 µg/kg) on uteroplacental blood flow in near-term pregnant ewes in the absence of TEA (A) and in the presence of a 90-min continuous intra-arterial infusion of TEA (B; 0.15 mM) the following day. Data are presented as means ± SE. Thirteen studies were performed at different gestational ages as described in the text. a-c, Differences in uterine blood flow between time periods when analyzed by repeated-measures ANOVA.

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Summary of the effects of E2 (1 µg/kg) on uteroplacental vascular resistance in near-term pregnant ewes in the absence of TEA (A) and in the presence of a 90-min continuous intra-arterial infusion of TEA (B; 0.15 mM) on the following day. Data are presented as means ± SE. Thirteen studies were performed at different gestational ages as described in the text. a and b, Differences in uterine blood flow between time periods when analyzed by repeated-measures ANOVA.

Presence of the BK_Ca channel. Third and fourth generation uterine arteries were collected from four ewes at the completion of the studies, and the endothelium was removed to determine whether BK_Ca channels were expressed in the arterial smooth muscle. These samples included arteries from pregnant sheep at 107 and 145 days gestation and nonpregnant ewes exposed or not exposed to E₂ for 6 days as recently described (39). RT-PCR yielded a 446-bp fragment in vascular smooth muscle from all four animals (Fig. 7), which was highly homologous to the human sequences of the same molecule (45). The observations in the nonpregnant ewes were consistent with patch-clamp studies recently reported from this laboratory (35).

View larger version (58K): [in this window] [in a new window]   Fig. 7.   RT-PCR was performed on isolated total RNA prepared from denuded uterine arteries obtained from ewes at 107 (ewe 338) and 145 days (ewe 332) gestation and from nonpregnant E2-treated (ewe 308) and untreated (ewe 303) ewes. The housekeeping gene was 18S mRNA (488 bp). In all 4 samples, there was a single band at 446 bp, consistent with the human sequence for the large conductance Ca2+-dependent K+ (KCa) channel.

	DISCUSSION

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The pattern of the >30-fold rise in UBF occurring in ovine pregnancy is well described (26, 27, 29, 34), but the mechanisms involved remain unclear. Existing evidence suggests this exponential increase in UBF during the last two-thirds of pregnancy results from vascular growth and angiogenesis during placentation and subsequent vasodilation (26, 29, 34, 44). Furthermore, placentally derived estrogens may mediate uteroplacental vasodilation by activating existing NOS enzyme or increasing NOS expression in uterine artery endothelium and/or smooth muscle (8, 15, 30, 33, 39). We (35, 39) recently reported that E2 not only increases uterine artery NOS in the endothelium and smooth muscle but also enhances BK_Ca channel activation in uterine artery myocytes, and both NO and BK_Ca channels contribute to the E₂-induced uterine vasodilation. In the present investigation, we demonstrated the presence of BK_Ca channel mRNA in uterine artery myocytes from nonpregnant and pregnant ewes, confirming recent studies in the former using patch-clamp methods (35). Moreover, we provided new evidence that BK_Ca channels are responsible for maintaining a substantial proportion of the observed uteroplacental vasodilation normally seen at term and that BK_Ca channel activation contributes to the E₂-induced increases in UBF in pregnant sheep. These observations, therefore, provide the first molecular evidence of how UBF progressively increases during normal pregnancy and evidence, albeit indirect at present, that placental estrogens are involved.

Potassium channels regulate basal arterial tone and the myotropic responses to various agonists through the hyperpolarization of smooth muscle membranes, which inactivates Ca⁺² entry through potential-gated channels and results in vasorelaxation (for reviews, see Refs. 21 and 22). Because pregnancy is associated with a fall in systemic vascular resistance and attenuated systemic pressor responses to several vasoconstrictors (3, 12, 27, 28), it stands to reason that potassium channels may be the final mediator responsible for these alterations (18). At least four potassium channels are expressed in arterial vascular smooth muscle (22); however, only two have been studied in pregnancy: the ATP-sensitive potassium (K_ATP) channel and the BK_Ca channel (4, 11). In these studies it was evident that enhanced channel activity contributes to the systemic vascular changes associated with normal gestation, e.g., the fall in systemic and regional vascular resistances, and the attenuated pressor responses to several vasoconstrictors.

The uterine vascular bed also demonstrates a marked fall in vascular resistance and attenuated responses to several vasoconstrictors during pregnancy (12, 19, 27, 28). The decrease in basal UVR is not due to vasodilating prostaglandins, because intra-arterial and systemic indomethacin infusions do not alter basal UBF or UVR (14, 19). Furthermore, short-term intra-arterial infusions of L-NAME decrease the uterine synthesis of cGMP in pregnant ewes but do not significantly alter basal UBF or UVR, despite a greater than twofold increase in uterine artery NOS expression (15, 30). Although BK_Ca channels are expressed in uterine artery myocytes from nonpregnant ewes, they are not involved in maintaining basal uterine vascular tone (35). This was obtained from the observation that, in intact nonpregnant ewes, arterial concentrations of TEA of 1.0 mM, which are selective for blocking BK_Ca channels (3, 21, 22), do not alter basal UBF or UVR, findings consistent with patch-clamp studies (35) and studies in normotensive Wistar-Kyoto rats (38). We now report that TEA, at an arterial concentration of 0.15 mM, decreases basal UBF ~50% and increases UVR within minutes in near-term pregnant ewes. Furthermore, the response is similar throughout the last 3 wk of gestation and reversible within hours. This dose of TEA is well below the 50% inhibitory dose reported in vitro (22). Higher doses were not examined because we did not wish to disturb fetal well-being by further decreasing UBF and uterine oxygen delivery, which occurs when UBF falls >50% (29). Charybdotoxin and iberiotoxin, also BK_Ca channel-selective antagonists (22), were not used because of their cost and the potential for irreversible or prolonged binding. In human uterine arteries, TEA enhances in vitro vascular reactivity (10, 23); however, the concentrations used exceeded 1 mM, making channel selectivity unclear. In studies performed at a similar time in pregnant guinea pigs, i.e., 84% of gestation, systemic glibenclamide [a selective K_ATP antagonist (22)] infusions increased MAP and systemic vascular resistance but did not alter total UBF (11). Although the nonplacental portion of UBF fell modestly, placental blood flow was unaffected, suggesting that K_ATP channels are not expressed in the guinea pig placental vasculature and are not responsible for placental vasodilation. In nonpregnant ewes, glibenclamide does not alter basal or E₂-induced increases in UBF (unpublished results). Although the distribution of UBF was not measured in the present study, 85-90% of UBF in term ewes is placental (16, 28). Thus the maximum blood flow to nonplacental tissues in the treated uterine horn was 60-70 ml/min, yet TEA decreased unilateral UBF 200 ml/min. It is very likely, therefore, that the placental vascular bed was affected, providing the first evidence that BK_Ca channels play a prominent role in regulating basal placental vascular tone and may be responsible for the vasodilation and exponential rise in UBF in the last third of ovine gestation.

Although BK_Ca channel mRNA was observed in uterine artery myocytes by RT-PCR, it was of interest that expression may have actually decreased in the last third of pregnancy (Fig. 7). However, one must be cautious in making this conclusion from the preliminary data presented because it is known that uterine hypertrophy occurs in ovine pregnancy (1, 9) and we made no attempt to quantify BK_Ca channel mRNA in the present report. We also noted in these preliminary studies that E₂ had no obvious effect on BK_Ca channel mRNA in uterine artery smooth muscle from nonpregnant ewes. This, however, is not surprising because we (35) reported a 70-fold increase in BK_Ca channel activity within 30 min in myocytes studied with patch-clamp techniques, a time too short for channel upregulation. Thus it is possible that BK_Ca channel expression is quantitatively unchanged in pregnancy, but activity alone is increased. Studies to address this are presently underway.

Estrogen, a potent vasodilator in several vascular beds in humans and in other species including sheep (17, 29), has its greatest effect in reproductive tissues and, in particular, in the uterine vascular bed (29, 32, 33). Most studies of estrogen have used nonpregnant females. However, E₂ also increases UBF in pregnant sheep in a pattern resembling that in nonpregnant ewes (33). Furthermore, stimulated increases in endogenous placentally derived estrogens increase UBF in a pattern resembling that seen after exogenous E₂ treatment, suggesting that placental estrogens may regulate UBF during pregnancy (36, 37). However, the mechanism responsible for estrogen-induced vasodilation in pregnant animals has not been studied. We confirmed the effects of E₂ on UBF in the present study. As in nonpregnant ewes, UBF began to rise 30 min after E₂ infusion, suggesting a nongenomic mechanism may be involved. This is supported by the observation that actinomycin does not inhibit the effects of E₂ on UBF (24) and recent observations that E₂ increases eNOS activity in vitro within 2-5 min in a biphasic manner (40). In nonpregnant sheep, the acute rise in UBF parallels increases in uterine cGMP (30) and is associated with enhanced NOS activation or expression (39, 46, 47); this, however, has not been studied in pregnant animals. In nonpregnant ewes, BK_Ca channels in uterine artery myocytes are activated by E₂ independent of the endothelium and appear to interact with increasing NOS activity to contribute to and mediate E₂-induced vasodilation (35). We demonstrated that BK_Ca channels are expressed in uterine artery myocytes from pregnant ewes and that a TEA dose below the 50% inhibitory dose for E₂-induced vasodilation in nonpregnant ewes (35) will inhibit the UBF responses to E₂ in pregnant animals. The untreated uterine horn was minimally affected, and the systemic responses to E₂ were unchanged, with heart rate increasing 10% (13, 33). Because 0.15 mM TEA is selective for BK_Ca channels, and it is probable that smooth muscle exposure was even less, it is unlikely another potassium channel was inhibited (22). It is notable that the same local dose of TEA in nonpregnant ewes decreased E₂ responses ~20%; but, when infused with L-NAME, TEA resulted in complete inhibition (30, 35). Because increases in uterine cGMP follow E₂ exposure in pregnant and nonpregnant ewes (30), we propose that the acute rise in UBF in both groups is mediated, at least in part, by activating smooth muscle guanylyl cyclase by enhancing NOS activity and phosphorylating BK_Ca channels via a cGMP-dependent kinase (7, 35, 49). There also may be a direct effect of NO on the BK_Ca channel (2). Both hypotheses will need to be explored in future studies. Nonetheless, it is clear in pregnant ewes that additional BK_Ca channel activation follows E₂ exposure, and this contributes to E₂-induced rises in UBF. These observations also provide new insights into the potential role of the increase in placental estrogen synthesis on UBF that occurs with the onset of parturition (29).

In the present study, we provided new insights into the physiological role of BK_Ca channels in vascular smooth muscle. It was assumed previously that BK_Ca channels primarily regulated myogenic tone via a negative feedback mechanism (3, 21, 22), which required membrane depolarization induced by increases in intracellular Ca⁺² or transmural pressure derived from increases in arterial blood pressure. This caused BK_Ca channel hyperpolarization of smooth muscle membranes, inactivation of potential-gated Ca⁺² channels, vascular relaxation, and maintenance of blood flow. It is now obvious that estrogen activates BK_Ca channels in coronary and uterine myocytes in vivo and in vitro independent of elevations in myogenic tone (7, 35, 48, 49). We have provided the first evidence that BK_Ca channels contribute to both chronic and acute vasodilation in the uterine vascular bed in pregnancy. Furthermore, this may be independent of changes in potential-gated Ca⁺² channels, because these channels demonstrate decreased activity in uterine arteries from pregnant sows (42, 43). Alternatively, the inactivation of potential-gated Ca⁺² channels may be due to the negative feedback of hormonally activated BK_Ca channels (3, 22). Although our data suggest that placentally derived estrogens may regulate BK_Ca channel activity in placental vascular smooth muscle, it is possible that increases in UBF augment BK_Ca channel activation. This and other issues will be addressed in future studies as they impact our understanding of how placental blood flow is regulated in pregnancy and how new strategies may be developed for the treatment of pregnancies associated with fetal growth restriction due to abnormalities of UBF.