Augmented EDHF signaling in rat uteroplacental vasculature ... · pregnant (LP) rats. Changes in SMC membrane potential of pressur-ized arteries from LP rats were assessed using glass

Augmented EDHF signaling in rat uteroplacental vasculature duringlate pregnancy

N. I. Gokina, O. Y. Kuzina, and A. M. VanceDepartment of Obstetrics, Gynecology, and Reproductive Sciences, College of Medicine, University of Vermont,Burlington, Vermont

Submitted 4 March 2010; accepted in final form 27 August 2010

Gokina NI, Kuzina OY, Vance AM. Augmented EDHF signalingin rat uteroplacental vasculature during late pregnancy. Am J PhysiolHeart Circ Physiol 299: H1642–H1652, 2010. First published Sep-tember 3, 2010; doi:10.1152/ajpheart.00227.2010.—A successfulpregnancy outcome relies on extensive maternal cardiovascular adap-tation, including enhanced uteroplacental vasodilator mechanisms.The objective of the present study was to determine the contributionof the endothelium-derived hyperpolarizing factor (EDHF) signalingin pregnancy-enhanced uterine vasodilation, to define the role ofCa2�-activated K� channels in mediating EDHF effects, and toexplore the impact of endothelial Ca2� signaling in pregnancy-specific upregulation of EDHF. Fura 2-based measurements of smoothmuscle cell (SMC) and endothelial cell cytosolic Ca2� concentration([Ca2�]i) were performed simultaneously with measurements of thediameter of uterine radial arteries from nonpregnant (NP) and latepregnant (LP) rats. Changes in SMC membrane potential of pressur-ized arteries from LP rats were assessed using glass microelectrodes.After blockade of nitric oxide and prostacyclin production, a cumu-lative application of ACh induced rapid and effective dilatation ofuterine vessels from both NP and LP rats. This vasodilation wasassociated with SMC hyperpolarization and SMC [Ca2�]i reductionand was abolished by a high-K� solution, demonstrating that NG-nitro-L-arginine (L-NNA)- and indomethacin-resistant responses areattributable to EDHF. Pregnancy significantly potentiates EDHF-mediated vasodilation in part due to enhanced endothelial Ca2�

signaling. L-NNA- and indomethacin-resistant responses were insen-sitive to iberiotoxin but abolished by a combined treatment withapamin and charybdotoxin, supporting the key role of small- andintermediate-conductance K� channels in mediating EDHF signalingin the maternal uterine resistance vasculature.

acetylcholine; smooth muscle cell hyperpolarization; endothelial cal-cium signaling; calcium-activated potassium channels

SUCCESSFUL PREGNANCY OUTCOME relies on extensive maternalcardiovascular adaptation, including dramatic increase inuteroplacental blood flow. Current studies implicate growthand remodeling of the maternal uterine vasculature and en-hanced uterine vasodilation as major underlying mechanisms(1, 3, 36, 37, 43, 44). Pregnancy is associated with a markedchange in uterine endothelial function, resulting in increasedbasal and stimulated release of nitric oxide (NO) and prosta-cyclin (PGI2). In large conductive uterine arteries of animalsand humans, these two autocoids mostly mediate endothelium-dependent dilatation (3, 50). Recent studies indicate that asignificant part of agonist-induced vasodilation of more distalhuman myometrial arteries or smaller rat uterine radial arteriesis resistant to inhibitors of nitric oxide synthase (NOS) and

cyclooxygenase (COX), implicating the role of endothelium-derived hyperpolarizing factor (EDHF) (6, 28).

Extensive in vivo and in vitro studies during the last decadeconvincingly demonstrate an essential role of EDHF in regu-lating the tone of small-resistance arteries and arterioles in avariety of vascular beds (5, 8, 13, 16, 22, 27, 30, 48). Thenature of EDHF remains the matter of substantial controversy,and EDHF is no longer considered as a single endothelium-derived factor. Several diffusible substances or even the mech-anism of electrical coupling between endothelial cells (ECs)and smooth muscle cells (SMCs) were proposed for the role ofEDHF in different vascular beds. These multiple mechanismscan work separately or in combination depending on the typeof EC stimulation as well as the origin of blood vessels (5, 8,9, 16, 22). An elevation of endothelial cytosolic Ca2� concen-tration ([Ca2�]i), a key event in response to stimulation of ECsby numerous neurohumoral mediators or mechanical forces, iscritically involved in EDHF-evoked vasodilation (5, 8, 22, 34,41). This [Ca2�]i rise in endothelial cells results in activationof multiple Ca2�-dependent pathways mediating EDHF. Forexample, Ca2�-induced activation of arachidonic acid cancause formation of epoxyeicosatrienoic acids (EETs), a diffus-ible mediator of EDHF-induced vasodilation in coronary, re-nal, and skeletal circulations. One of the established mecha-nisms of EET-induced vasodilation is activation of large-conductance Ca2�-activated K� channels (BKCa) of vascularSMCs, resulting in their hyperpolarization and relaxation (5, 8,9, 16). In the majority of resistance arteries and arterioles,endothelial [Ca2�]i rise activates Ca2�-dependent small(SKCa)- and intermediate (IKCa)-conductance K� channels (5,8, 16, 22, 27, 34, 41). Subsequent efflux of K� from ECsthrough SKCa and IKCa channels can hyperpolarize and relaxunderlying vascular SMCs through activation of inward-recti-fier K� channels, Na�-K� pump, or both (15). Recentlydemonstrated colocalization of IKCa channels and myoendo-thelial gap junctions provides a structural basis for such mech-anism (33, 46). Hyperpolarization of ECs due to activation ofSKCa and IKCa channels can also electrotonically spread toneighboring SMCs through myoendothelial gap junctions, re-sulting in SMC hyperpolarization and relaxation. Contributionof other proposed mediators, such as hydrogen peroxide andC-type natriuretic peptide, in EDHF-related mechanisms iscurrently under investigation (5, 8, 16).

In the course of rodent and human pregnancy, maternalpreplacental spiral arteries are transformed into large dilatedvessels with a loss of their constrictor function. More proximalradial uterine arteries become a major site regulating uteropla-cental vascular resistance and, as a consequence, fetal growthand development (4, 21, 37, 43, 44). In spite of the acknowl-edged importance of these vessels in the control of uteropla-

Address for reprint requests and other correspondence: N. I. Gokina, Dept.of Obstetrics, Gynecology, and Reproductive Sciences, College of Medicine,The Univ. of Vermont, Burlington, VT 05405 (e-mail: [email protected]).

Am J Physiol Heart Circ Physiol 299: H1642–H1652, 2010.First published September 3, 2010; doi:10.1152/ajpheart.00227.2010.

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cental blood flow, the nature of EDHF and its role in theregulation of uteroplacental vascular tone remains unknown. Inthe present study, we hypothesized that EDHF importantly con-tributes to pregnancy-specific enhancement of vasodilation in thematernal uterine circulation because of augmented EC Ca2�

signaling and activation of SKCa and IKCa channels. Therefore,the purpose of this study was to: 1) characterize the contributionof EDHF to ACh-induced uterine vasodilation; 2) define the effectof pregnancy on EDHF-mediated responses; 3) explore the role ofEC [Ca2�]i in pregnancy-induced modulation of EDHF; and4) study the role of BKCa, SKCa, and IKCa channels in EDHF-induced responses of uterine vessels.

METHODS

Animals and preparation of arteries. All experiments were con-ducted in accordance with the National Institutes of Health Guide forthe Care and Use of Laboratory Animals (NIH Publications No.85–23, Revised 1996), and the experimental protocols were approvedby the Institutional Animal Care and Use Committee of the Universityof Vermont.

Virgin cycling nonpregnant (NP, n � 50) or late pregnant (19–20day, LP, n � 66) female Sprague-Dawley rats of 14–16 wk old wereused for this study. The estrous cycle of NP rats was determined byexamination of vaginal smears on the day of experimentation. In thecurrent study, we used 11 rats in proestrous, 23 rats in metestrous, and16 rats in diestrous stage of the estrous cycle. Animals were anesthe-tized by an intraperitoneal injection of Nembutal (50 mg/kg) andkilled by decapitation. The abdominal wall was transected, and theentire uterus and uterine vasculature was rapidly removed and pinnedin a dissecting dish filled with aerated cold physiological salt solution(PSS; see Solutions and drugs for composition). Second-order uterineradial arteries were identified within the mesometrial arcade anddissected free of connective tissue. Only radial arteries feeding theplacenta (uteroplacental arteries) were dissected from LP rats. Arterialsegments were cannulated from both ends in the arteriograph andcontinuously superfused at 3 ml/min with aerated (10% O2-5%CO2-85% N2) PSS at 37°C. To minimize mechanical stimulation ofECs and SMCs within the arterial wall during the equilibration period,cannulated arteries were initially pressurized to 10 mmHg using theservo pressure system (Living System Instrumentation, Burlington,VT). All experiments were performed at 50 mmHg and under nointraluminal flow conditions. In contrast to uterine radial arteries fromNP rats, uteroplacental arteries from LP animals can develop vaso-constriction (myogenic tone) in response to elevations of pressureexceeding 50 mmHg (52). Therefore, to avoid development of myo-genic tone and to equalize experimental conditions for arteries of NPand LP rats, they were pressurized to a similar level: 50 mmHg. Bloodpressure measured in vivo in rat distal uteroplacental arteries justbefore entering the placenta was �14 mmHg (37). Uteroplacentalarteries are located in the middle section of the mesometrial vascula-ture between the main uterine artery and the placenta. Therefore,physiological levels of pressure experienced by these vessels in vivoshould approximate 50–70 mmHg.

Selective loading of endothelial or SMCs with fura 2 and measure-ment of intracellular [Ca2�]i. Detailed description of the procedurefor selective loading of ECs or SMCs of uterine arteries with theCa2�-sensitive dye fura 2 was previously published (20, 52). Briefly,heat-polished glass cannulas were used in all experiments to preventaccidental damage of the endothelial layer during the cannulationprocedure and to avoid diffusion of fura 2 to the SMC layer. ECs wereloaded with fura 2 at room temperature by intraluminal perfusion ofpressurized arteries with fura 2-AM-containing solution (5 �M) for 5min followed by 10 min of washout with regular PSS. A similarprotocol was used in our previous study where preferential loading ofECs with fura 2 was confirmed by nearly complete disappearance of

fluorescent signal after arterial denudation (20). SMC loading withfura 2 was performed by extraluminal incubation of arteries in fura2-AM (5 �M) solution at room temperature in the dark for 60 min.Fura 2-loaded arteries were washed two to three times and thencontinuously superfused with aerated PSS at 37°C. Preferential load-ing of SMCs over ECs with this protocol can be confirmed by lack of[Ca2�]i responses to 3 �M ACh in uterine arteries depolarized withhigh (35–45 mM)-K� solution (see Fig. 4B). It has been shown that,in pressurized arterioles, ACh-induced increase in EC [Ca2�]i was notmodulated by high-K� depolarization (10). Similar experimentalprotocols were previously used for selective loading of ECs or SMCsin microvessels (7, 35, 53).

Ratiometric measurements of fura 2 fluorescence from ECs orSMCs were performed using a photomultiplier system (IonOptix,Milton, MA). Experimental ratios were corrected for backgroundfluorescence taken from each artery before loading with fura 2.Background-corrected ratios of 510 nm emission were obtained at asampling rate of 5 Hz from arteries alternately excited at 340 and 380nm. The arterial lumen diameter was simultaneously monitored usingthe SoftEdge Acquisition Subsystem (IonOptix). All experimentalprotocols were started following an additional 15-min equilibrationperiod at 10 mmHg to allow intracellular deesterification of fura2-AM.

Measurements of membrane potential from SMCs of pressurizeduterine arteries. For intracellular measurement of SMC membranepotential (MP) from pressurized arteries, we used short arterial seg-ments (400–500 �m) that were carefully cleaned of any residualconnective tissue. Lumen diameter and MP changes were simulta-neously recorded from arteries pressurized at 50 mmHg. Each glassmicroelectrode was positioned on the top of the arterial segment, andimpalement was made from the adventitial surface of the artery byadvancing the electrode down the long axis of SMCs. Such position ofthe glass microelectrode and some flexibility of the microelectrode tipallowed for better maintenance of the impalement during movementsof the vessel wall. All MP measurements were performed on utero-placental arteries of LP rats. Small diameters and stiff vessel walls ofradial uterine arteries of NP rats did not allow continuous recordingsof SMC MP during ACh application. For measurement of MP, weused microelectrodes filled with 0.5 M KCl having tip resistances of110–150 M�; an Ag-AgCl pellet was used as an indifferent electrode.A microelectrode was connected to a motorized micromanipulator(World Precision Instruments), and MP was recorded using a high-input impedance amplifier Electro 705 (World Precision Instruments).Changes in MP and arterial diameter were simultaneously displayedand recorded on a desktop computer using a data acquisition program(IonOptix). The following criteria were used for acceptance of MPrecordings: 1) abrupt negative change in voltage upon impalement ofthe cells; 2) a sharp return to zero voltage following withdrawal of amicroelectrode tip; 3) tip potential of �7 mV; and 4) unchangedresistance of microelectrodes after impalement. A stable MP record-ing for at least 1 min was accepted for data collection.

Protocols for studying EDHF-mediated responses of uterinearteries. After equilibration and loading of cells with fura 2, arterieswere incubated with 200 �M NG-nitro-L-arginine (L-NNA, NOSinhibitor) and 10 �M indomethacin (COX inhibitor) to abolish theproduction of NO and PGI2, respectively. A number of publishedobservations demonstrated a nearly complete inhibition of agonist-induced endothelial NO production in the presence of 100–200 �MNOS inhibitors [L-NNA or NG-monomethyl-L-arginine (L-NMMA)]measured with the NO-sensitive dye DAF-2 (14, 31, 57). The arterialdiameters and levels of EC or SMC [Ca2�]i were recorded during 5min at 10 mmHg followed by an elevation of intraluminal pressure to50 mmHg.

After 20 min of treatment of vessels with L-NNA and indometha-cin, phenylephrine (PE) was added in increasing concentrations (1–3doses) to produce a constriction of 50–70% of the initial diameter.Following stabilization of vasoconstriction, ACh was applied in in-

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creasing concentrations. For each artery, dose-dependent effects ofACh were studied only one time. A combination of papaverine (100�M, a phosphodiesterase inhibitor) and diltiazem (10 �M, a Ca2�

channel blocker) was added at the end of each experiment to obtainthe diameter under maximally dilated conditions. ACh-induced vaso-dilation was expressed as the percentage of maximal vasodilation inresponse to papaverine and diltiazem (Dmax). Additional experimentswere performed to determine the effects of 3 �M ACh in arteriestreated with L-NNA and indomethacin and preconstricted by 50 –70% of the initial diameters with moderate (35– 45 mM) concen-trations of K�.

To test the role of SMC BKCa channels in EDHF-mediated re-sponses, L-NNA- and indomethacin-treated uterine arteries were pre-constricted with PE by �20–30% of their initial diameters. Subse-quent extraluminal application of iberiotoxin (IBTX), a specific in-hibitor of BKCa channels, resulted in an additional constriction thatstabilized within 5 min. Final levels of preconstriction by combinedtreatment with PE and IBTX were 60–70% of the initial diameters.ACh was then tested in concentrations of 0.03, 0.1, and 10 �M. In aseparate set of experiments, we studied ACh-induced responses aftertreatment of the vessels with a combination of 100 nM apamin and 50nM charybdotoxin (CTX) or 100 nM apamin and 100 nM IBTX. Alltoxins were delivered intraluminally by producing a transient flow of30–50 �l/min for 5–8 min. PE was then applied in increasingconcentrations to preconstrict arteries, and a combination of apaminand CTX (or IBTX) was added extraluminally with a superfusionsolution for an additional 5–10 min. ACh was applied to PE-precon-

stricted vessels in the presence of apamin and CTX (or IBTX) in30–40 min after cessation of intraluminal flow.

In our electrophysiological experiments, each artery was pressur-ized to 50 mmHg in the presence of L-NNA and indomethacin, andtwo to three concentrations of PE were added to preconstrict arteries.After stabilization of the PE-induced constriction, microelectrodeimpalement of SMC was made, and MP was recorded for 2–3 min.Simultaneous changes in SMC MP and the arterial diameter inresponse to application of 0.1 ACh were then recorded during the next3–5 min. A combination of papaverine and diltiazem was added at theend of each experiment to maximally dilate the artery.

Solutions and drugs. The PSS contained (in mM): 119 NaCl, 4.7KCl, 24.0 NaHCO3, 1.2 KH2PO4, 1.6 CaCl2, 1.2 MgSO4, 0.023EDTA, and 11.0 glucose, pH � 7.4. For the fura 2 calibrationprocedure, we used a solution of the following composition: 140 mMKCl, 20 mM NaCl, 5 mM HEPES, 5 mM EGTA, 1 mM MgCl2, 5 �Mnigericin, and 10 �M ionomycin, pH � 7.1.

The majority of chemicals was purchased from Sigma Chemical(St. Louis, MO) with the exception of ionomycin and nigericin, whichwere obtained from Calbiochem (La Jolla, CA). Fura 2-AM andpluronic acid were purchased from Invitrogen (Carlsbad, CA). Fura2-AM was dissolved in dehydrated DMSO as a 1 mM stock solution,frozen in small aliquots, and used within 1 wk of preparation. L-NNA,PE, ACh, and papaverine were dissolved in deionized water on theexperimental day. Diltiazem and indomethacin were prepared as 10mM stock solutions in deionized water and alcohol, respectively, andkept refrigerated until use. Ionomycin and nigericin were dissolved in

Fig. 1. Relative contribution of endothelium-derived hyperpo-larizing factor (EDHF) to initial (I) and sustained (S) compo-nents of ACh-induced uterine vasodilation. A and B: represen-tative tracings showing changes in lumen diameter of thecontrol uteroplacental artery (A) and the artery treated withNG-nitro-L-arginine (L-NNA) and indomethacin (B) in responseto application of 1 �M ACh. Solid lines indicate the exposureof arteries to tested compounds. Dotted lines show maximallydilated diameters of the arteries in the presence of 100 �Mpapaverine and 10 �M diltiazem. C and D: summary graphsshowing no difference in initial dilatation to 1 �M ACh incontrol arteries of nonpregnant (NP) and late pregnant (LP) ratsand arteries treated with L-NNA and indomethacin. Dmax,maximal vasodilation in response to papaverine and diltiazem.E and F: summary graphs demonstrating the effects of L-NNAand indomethacin on sustained vasodilation induced by ACh inarteries from NP and LP rats. Initial and sustained componentswere calculated at �3 and 10 min of ACh application and areexpressed as Dmax. *Significantly different at P � 0.05 (un-paired Student’s t-test); n, no. of arteries tested.

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methanol (10 mM) and kept at �20°C. Stock solutions of IBTX,apamin, and CTX were prepared in deionized water and stored at�20°C until use.

Calculations and statistical analysis. EC or SMC [Ca2�]i wascalculated using the following equation (23): [Ca2�]i � Kd�(R �Rmin)/(Rmax � R), where Kd is the dissociation constant, R is anexperimentally measured ratio (340/380 nm) of fluorescence intensi-ties, Rmin is a ratio in the absence of [Ca2�]i, Rmax is a ratio atCa2�-saturated fura 2 conditions, and � is a ratio of the fluorescenceintensities at 380 nm excitation wavelength at Rmin and Rmax. Rmin,Rmax, and � were determined by an in situ calibration procedure fromthe arteries treated with the ionophores ionomycin (10 �M) andnigericin (5 �M) to increase cell membrane permeability to Ca2� andminimize Ca2� extrusion mechanisms through Na�/Ca2� exchange,respectively (56). Calibration was performed for two separate sets ofvessels loaded intraluminally (endothelial cell loading, n � 9) orextraluminally (SMC loading, n � 8) with fura 2. These values werethen pooled and used to convert the ratio values into a [Ca2�]i. The Kd

(the dissociation constant for fura 2) was 282 nM, as determined by insitu titration of Ca2� in fura 2-loaded small arteries (29). Arterialdiameter and pressure and ratio values were simultaneously recordedusing an IonOptix data acquisition program and imported into Sig-maPlot and SigmaStat programs for graphical representation, calcu-lations, and statistical analysis. In view of significant oscillatoryactivity of uterine arteries, all measurements were made by averagingrecords of arterial diameters or SMC [Ca2�]i during 15–20 s. Data areexpressed as means SE, where each n is the number of arterialsegments studied. One or two arteries from the same animal were usedon each experimental day with one vessel per animal used for aparticular protocol. A paired or unpaired Student’s t-test or two-wayrepeated-measures ANOVA was used to determine the significance ofdifferences between sets of data, with P � 0.05 considered significant.The concentration of ACh required to produce half-maximal vasodi-lation, EC50, was determined for each tested artery using standardcurve analysis from data imported into a SigmaPlot program.

RESULTS

Temporal characterization of uterine vasodilation beforeand after inhibition of NOS and COX. Late pregnancy wasassociated with a significant increase in passive lumendiameters of radial uterine arteries at 50 mmHg that aver-aged 195.8 5.7 �m (n � 63) vs. 120.3 4.0 �m (n � 53)in the nonpregnant state.

Under control conditions, an application of 1 �M AChresulted in a rapid and complete vasodilation of arteries pre-constricted with PE (Fig. 1A). This response remained stableuntil washout of ACh. Similar experiments were performedafter blockade of NO and PGI2 production with L-NNA andindomethacin. Initial lumen diameters of arteries in the pres-ence of these inhibitors were comparable to passive lumendiameters of the same arteries treated with papaverine anddiltiazem (NP rats: 119.9 4.2 vs. 120.3 4.0 �m, n � 53;LP rats: 193.3 5.8 vs. 195.8 5.7 �m, n � 63). These dataindicate that arteries of both NP and LP rats pressurized at 50mmHg develop no significant spontaneous tone in the presenceof L-NNA and indomethacin. To test vasodilatory effects ofACh in these and other experiments, arteries were precon-stricted with PE by 58.4 1.8% (NP, n � 38) and 58.7 2.0% (LP, n � 49) of their initial diameters. The concentra-tions of PE used to preconstrict vessels from NP rats (0.70 0.10 �M) were significantly higher than those used for arteriesof LP rats (0.26 0.04 �M). Application of 1 �M AChinduced vasodilation that reached the maximal value within 3min and was followed by a partial restoration of the constric-tion (Fig. 1B). Initial maximal responses were similar in controland L-NNA- and indomethacin-treated arteries (Fig. 1, C and D).Sustained vasodilation at the end of a 10-min period of ACh

Fig. 2. Pregnancy enhances L-NNA- and in-domethacin-resistant vasodilation of utero-placental arteries. A and B: representativechanges in lumen diameters of pressurizeduterine arteries from NP and LP rats in re-sponse to cumulative application of ACh inincreasing concentrations. Arteries weretreated with L-NNA and indomethacin andpreconstricted with phenylephrine (PE) be-fore testing ACh. Dotted lines indicate thediameters of maximally dilated arteries ob-tained at the end of each experiment bytreating vessels with a combination of papav-erine and diltiazem. Solid horizontal linesdepict the time of exposure of arteries totested compounds. C: summary graphs dem-onstrating the degree of initial vasodilationas a function of ACh concentrations in arter-ies from NP and LP rats. ACh-induced va-sodilation is expressed as Dmax. *Signifi-cantly different at P � 0.05 (2-way repeated-measures ANOVA). D: bar graph showingsignificant decrease in the concentration ofACh required for half-maximal dilatation(EC50) of uterine arteries in late gestation.*Significantly different at P � 0.05 (un-paired Student’s t-test); n, no. of arteriestested.




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administration was significantly diminished from 93.5 3.2 and92.0 4.2% to 62.2 11.8 and 58.0 9.4% in arteries of NPand LP rats, respectively (Fig. 1, F and E). Time for reachingtwo-thirds of maximal vasodilation in treated arteries from NP(23.1 5.3 s, n � 7) and LP (19.7 3.4 s, n � 8) rats wasnot significantly different from NP (20.8 3.7 s, n � 6) andLP (15.8 2.2 s, n � 5) controls.

Pregnancy enhances L-NNA- and indomethacin-resistant va-sodilation of uteroplacental arteries. The effect of pregnancyon EDHF-mediated responses of uterine arteries was charac-terized next. Figure 2 shows representative changes in thediameters of arteries from NP (A) and LP (B) rats in responseto cumulative application of ACh. Vessels were pretreated withL-NNA and indomethacin and preconstricted with PE. Thethreshold concentration of ACh producing a minimal responsewas lower for LP arteries (0.03 �M) compared with NPcontrols (0.1 �M). In both types of vessels, maximal vasodi-lation was induced by ACh at 10 �M (Fig. 2C). The EC50 wassignificantly decreased at near-term pregnancy from 0.264 0.04 �M (n � 8, NP rats) to 0.122 0.03 �M (n � 8, LP rats;Fig. 2D).

EDHF-mediated uterine vasodilation is associated with amarked reduction in SMC [Ca2�]i. We next determinedwhether EDHF-mediated reduction in SMC [Ca2�]i was dif-ferent in arteries of NP and LP rats. Changes in SMC [Ca2�]i

and the diameter of uteroplacental artery from a LP rat inresponse to threshold, intermediate, and maximally effectiveconcentrations of ACh are shown in Fig. 3A. An application of

PE resulted in a marked elevation of SMC [Ca2�]i withsuperimposed [Ca2�]i oscillations that were associated withsynchronous oscillations in the arterial diameter. PE-inducedoscillations in SMC [Ca2�]i and the diameter were smaller andless frequent in arteries of NP rats. The averaged levels ofPE-induced SMC [Ca2�]i and the frequency of oscillationswere significantly reduced or abolished by ACh with a result-ant concentration-dependent arterial dilatation. As evidentfrom summary graphs, both EDHF-mediated [Ca2�]i responsesand vasodilatation to ACh were significantly enhanced bypregnancy (Fig. 3, B and C).

Abolition of EDHF-mediated responses of uterine arteriesdepolarized with high-K� solution. We next studied the effectof high-K� depolarization on EDHF-mediated responses ofuterine arteries to 3 �M ACh, the concentration that producednear-maximal dilatation of arteries from both NP and LP rats.As shown in Fig. 4A, in the artery from a LP rat preconstrictedwith PE in the presence of L-NNA and indomethacin, 3 �MACh induced a marked reduction in SMC [Ca2�]i associatedwith vasodilation. In contrast, in arteries treated with 35–45mM K�, application of ACh resulted in no significant reduc-tion in [Ca2�]i or vasodilation (Fig. 4B). Similar data wereobtained in our experiments using vessels from NP rats (Fig. 4,C and D). These findings demonstrate that depolarization ofSMCs with high-K� prevents ACh-induced reduction in[Ca2�]i and vasodilation, suggesting that these responses aremediated by hyperpolarization of SMCs.

Fig. 3. Increased EDHF-mediated uterine vasodilation in lategestation is associated with enhanced smooth muscle cell(SMC) cytosolic Ca2� concentration ([Ca2�]i) response.A: representative changes in SMC [Ca2�]i and the lumendiameter of the artery from a LP rat to cumulative applicationof ACh. The artery was pretreated with L-NNA and indometh-acin and preconstricted with PE before testing ACh. B andC: bar graphs showing the reduction in SMC [Ca2�]i andvasodilation induced by ACh in arteries of NP and LP rats.Vasodilation is expressed as Dmax. *Significantly different atP � 0.05 (2-way repeated-measures ANOVA); n, no. of arter-ies tested.




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L-NNA- and indomethacin-resistant uteroplacental vasodi-lation is associated with SMC hyperpolarization. Direct evi-dence that ACh can hyperpolarize SMCs after blockade of NOand PGI2 production was obtained by microelectrode measure-ments of MP from SMCs in the wall of uteroplacental arteriesfrom LP rats pressurized at 50 mmHg. Resting MP of SMCswas �48.4 1.5 mV (n � 6). Application of PE resulted inmarked SMC depolarization to �26.3 0.7 mV that wasassociated with vasoconstriction (data not shown). Adminis-tration of ACh in the concentration 0.1 �M hyperpolarizedSMCs to 38.7 1.5 mV and resulted in a considerablevasodilation. Representative recordings of simultaneous changesin MP and in the arterial diameter in response to ACh are shownin Fig. 5A. ACh-induced partial hyperpolarization of PE-depolarized SMCs resulted in the appearance of oscillations inMP that were followed by transient arterial constrictions. Thesefindings are summarized in Fig. 5, B and C.

Pregnancy-increased EDHF-mediated vasodilation is asso-ciated with enhanced endothelial [Ca2�]i responses. In ourprevious study, we demonstrated that ACh-induced uterinevasodilation is preceded by a significant rise in EC [Ca2�]i

(20). Similar responses were obtained in uterine arteries afterblockade of NO and PGI2 production (data not shown). Theonset of vasodilation was delayed from the onset of [Ca2�]i

elevation in response to 10 �M ACh by 4.8 0.4 and 5.7 0.8 s in arteries of NP (n � 5) and LP (n � 6) rats, respectively.

Recent studies indicate that Ca2� and/or inositol trisphos-phate (IP3), elevated in response to agonist stimulation ofSMCs, can diffuse to ECs through myoendothelial gap junc-tions and modulate Ca2� signaling in endothelial cells (26, 32).To minimize a possible contribution of this mechanism toendothelial Ca2� signaling, we studied ACh-induced endothe-lial [Ca2�]i responses in vessels that were not preconstrictedwith PE. In the presence of L-NNA and indomethacin, basallevels of EC [Ca2�]i measured at 50 mmHg were significantlyhigher in uterine arteries of LP animals (123 7 nM, n � 21)

compared with NP controls (81 5 nM, n � 20; Fig. 6, A, B,and C). Results of these studies, shown in Fig. 6, D and E,indicate that, in the absence of SMC stimulation with PE,ACh-induced EC [Ca2�]i responses were significantly en-hanced in late gestation.

Blockade of SKCa and IKCa channels abolishes EDHF-mediated uterine vasodilation. Ca2�-activated K� channels arecommonly implicated in EDHF-mediated vasodilation. Wefirst tested the contribution of SMC BKCa channels to EDHF inuterine arteries by studying the effects of ACh in the presenceof extraluminal IBTX. As evident from the graphs in Fig. 7,EDHF-mediated SMC [Ca2�]i (Fig. 7A) and dilator responses(Fig. 7B) of uterine arteries in the presence of IBTX were notdifferent from the control responses shown in Fig. 3. Re-sponses of arteries from LP rats were significantly enhancedcompared with those of vessels from NP controls.

To evaluate the role of endothelial BKCa, SKCa, and IKCa

channels in EDHF-mediated uterine vasodilation, we testedACh-induced changes in SMC [Ca2�]i and arterial diameterafter blockade of these channels with a combination of apaminand CTX (or apamin and IBTX). To ensure effective inhibitionof endothelial K� channels, all toxins were delivered to thearteries intra- and extraluminally. Summary graphs in Fig. 8demonstrate an abolition of both SMC [Ca2�]i (Fig. 8, A andC) and dilator (Fig. 8, B and D) responses to ACh of uterinearteries treated with apamin and CTX. At the same time, thevasodilation induced by 10 �M ACh was preserved aftercombined treatment of arteries with apamin and IBTX (NPrats: 84 5%, n � 5; LP rats: 81 4%, n � 5).

DISCUSSION

This study aimed to explore the role of EDHF in endothelium-dependent vasodilation of maternal uterine resistance arteries andto define the effect of pregnancy on EDHF-mediated vascularresponses with a specific focus on endothelial cell Ca2� signaling.

Fig. 4. Inhibition of EDHF-mediated responsesof uterine arteries by high-K� solution. A: repre-sentative tracings showing changes in SMC[Ca2�]i and lumen diameter of the uteropla-cental artery from a LP rat in response toapplication of 3 �M ACh. B: ACh at thesame concentration failed to induce changesin SMC [Ca2�]i and the diameter of theartery preconstricted with 40 mM of K�.L-NNA (200 �M) and indomethacin (10�M) were present throughout all experi-ments. C and D: bar graphs summarizingACh-induced SMC [Ca2�]i and dilator re-sponses of uterine arteries from NP and LPrats preconstricted with PE or high-K� solu-tion. Vasodilation is expressed as Dmax. Nos.in parentheses indicate the no. of tested ar-teries. *Significantly different at P � 0.05(unpaired Student’s t-test).




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The main findings of this study are: 1) ACh can effectively dilateuterine resistance arteries after blockade of NO and PGI2

production; 2) L-NNA- and indomethacin-resistant vasodila-tion to ACh is abolished by high-K� solution and is associatedwith SMC hyperpolarization and a reduction in SMC [Ca2�]i;3) late pregnancy increases EDHF-mediated vasodilation inpart due to enhanced endothelial Ca2� signaling; 4) EDHF-dependent responses are insensitive to IBTX; and 5) combinedtreatment of uterine arteries with apamin and CTX abolishesNO- and PGI2-resistant responses to ACh, supporting the keyrole of SKCa and IKCa channels in mediating EDHF effects.

The importance of EDHF in the control of regional vascularresistance is well established (16); however, the exact natureand role of EDHF in regulating maternal uterine blood flowremains unclear. The present study utilizing small-resistance-size radial uterine arteries from both NP and LP rats demon-strates that ACh can induce rapid and effective dilatation ofthese vessels after blockade of NO and PGI2 production. Thisvasodilation was associated with SMC [Ca2�]i reduction, SMChyperpolarization, and was abolished by a high-K� solution,suggesting that L-NNA- and indomethacin-resistant responsesare attributable to EDHF.

Prevalence of EDHF in the endothelium-dependent controlof small uterine artery tone. Previous studies performed onlarge (main or arcuate) uterine arteries of rodents demonstratedonly small and transient endothelium-dependent uterine vaso-dilation to ACh after inhibition of the production of NO andPGI2 (12, 40, 51). In experiments using uterine arteries fromboth nonpregnant and pregnant women, NOS inhibition re-sulted in a complete abolition of ACh-induced dilatation (39).These data are consistent with a relatively minor contributionof EDHF to endothelium-dependent dilatation of large uterinearteries. However, smaller myometrial arteries from nonpreg-nant or late pregnant women can be markedly dilated withbradykinin or placental growth factor in the presence of NOSand COX inhibitors (28, 42). EDHF also importantly contrib-utes to ACh-induced dilatation of radial uterine arteries fromovariectomized NP rats (6). Collectively, previous observa-tions and our current findings indicate that contribution ofEDHF to the control of maternal uterine vascular tone in-creases in more distal uterine vasculature. The inverse relation-ship between arterial size and magnitude of EDHF-mediateddilatation was previously established in mesenteric, cerebral,and rabbit ear circulations (2, 5, 24, 49).

The exact mechanism(s) underlying an increased role ofEDHF in endothelium-dependent vasodilation of smaller uter-ine arteries remains unknown. It has been demonstrated thatthe augmented contribution of EDHF to the control of vasculartone in more distally located mesenteric arteries correlates wellwith a higher incidence of myoendothelial gap junctions (47).An increased density of myoendothelial gap junctions mayresult in more effective electrotonic spreading of agonist-induced hyperpolarization from ECs to neighboring SMCs. Itis also conceivable that electrical spreading of hyperpolariza-tion from ECs to SMCs might be more effective in the wall ofsmaller arteries and arterioles with one to two layers of SMCscompared with large conductive vessels with five to six layersof SMCs.

It is generally accepted that endothelial SKCa and IKCa

channels play a pivotal role in EDHF effects in the majority ofmicrocirculatory vascular beds (5, 8, 16, 22, 27, 34). In thisregard, relative expression of SKCa and IKCa channels wassignificantly higher in fourth- compared with first-order mes-enteric arteries (24). Therefore, both increased density ofmyoendothelial gap junctions and/or upregulation in SKCa andIKCa channel gene expression may be responsible for aug-mented EDHF signaling in more distal resistance uterine vas-culature.

Late gestation enhances the contribution of EDHF to thecontrol of uteroplacental vascular tone. In this study, wedemonstrated for the first time that both EDHF-mediated uter-ine vasodilation and associated reduction in SMC [Ca2�]i weresignificantly enhanced in late pregnancy. These findings con-vincingly establish the importance of EDHF in pregnancy-specific upregulation of vasodilatory mechanisms in the ma-ternal uteroplacental circulation. Late gestation was also asso-ciated with an increased functional role of EDHF in themesenteric arteries (17). The previous observation and ourcurrent findings suggest that a common mechanism might beresponsible for pregnancy-induced upregulation of EDHF indifferent vascular beds. The role of estrogen in enhancement ofendothelium-dependent vasodilation is well documented inhuman and animal studies (1, 25, 48). Estrogen effects are

Fig. 5. L-NNA- and indomethacin-resistant uteroplacental vasodilation iscaused by SMC hyperpolarization. A: representative tracings showing simul-taneous changes in SMC membrane potential and lumen diameter of the arteryfrom a LP rat in response to application of 0.1 �M ACh. The artery waspressurized to 50 mmHg and depolarized with PE in the presence of L-NNAand indomethacin before testing ACh. Note a close association betweenmembrane hyperpolarization and vasodilation in response to ACh. Depolariz-ing oscillations in membrane potential were followed by transient arterialconstrictions. B: bar graphs summarizing the effect of PE and ACh on SMCmembrane potential. RP, resting membrane potential measured from SMCs ofpressurized (50 mmHg) arteries before application of PE. C: bar graph showingdilatation of the same vessels in response to 0.1 ACh, which is expressed as thepercentage of maximal responses induced by papaverine and diltiazem. *Sig-nificantly different at P � 0.05 (paired Student’s t-test); n, no. of arteriestested.




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especially prominent in the reproductive vasculature, andmarked estrogen-induced sensitization of the uterine artery toACh was first demonstrated by Bell (1). The high estrogenstate of pregnancy increases endothelial NOS activity in themain uterine artery of the guinea pig (54). Several recentstudies suggest that estrogen can also regulate vascular tonethrough modulation of EDHF-mediated vascular responses(25, 45, 48). We recently demonstrated that EDHF-mediatedresponses of uterine arteries were significantly potentiated bysupplementation of ovariectomized rats with estrogen (6).Taken together, these data strongly suggest an important roleof estrogen in pregnancy-induced upregulation of EDHF inuterine and systemic circulations.

Increased endothelial Ca2� signaling contributed to preg-nancy-enhanced EDHF. Although the exact nature of EDHFremains the matter of controversy, it is generally accepted thatelevation of intracellular [Ca2�]i in response to chemical ormechanical stimulation of endothelial cells is an initial criticalstep in EDHF-induced vasodilation (5, 8, 16, 22, 34, 41, 46). Inour previous study, we found that chelation of endothelial[Ca2�]i with BAPTA abolished ACh-induced [Ca2�]i rise andassociated vasodilation of rat radial uterine arteries, indicatingthat Ca2� is a key mediator for production of endothelium-derived factors, including EDHF (20). In the current study,ACh-induced EC [Ca2�]i elevation was followed by EDHF-mediated vasodilation with a time delay of only �5 s. In this

Fig. 6. Late gestation results in increased basal levels ofcytoplasmic concentration of Ca2� in endothelial cells (EC[Ca2�]i) and in augmented EC [Ca2�]i responses to ACh. A andB: representative changes in EC [Ca2�]i in response to increas-ing concentrations of ACh in pressurized uterine arteries froma NP rat (A) and a LP rat (B) that were not preconstricted withPE. The arteries were treated with 200 �M L-NNA and 10 �Mindomethacin. Solid lines indicate time of exposure of arteriesto ACh, while dotted lines show basal levels of EC [Ca2�]i.Transient (maximal) and sustained (3 min after ACh applica-tion) components of the response are shown in A. C–E: sum-mary graphs showing a significant increase in basal levels ofEC [Ca2�]i (C) and in transient (D) and sustained (E) EC[Ca2�]i responses to ACh of uterine arteries from LP ratscompared with NP controls. *Significantly different at P � 0.05(2-way repeated-measures ANOVA); n, no. of arteries tested.

Fig. 7. Iberiotoxin failed to inhibit EDHF-mediated changes inSMC [Ca2�]i and dilatation induced by ACh. A and B: graphsshowing reduction in SMC [Ca2�]i and vasodilation induced byACh in arteries treated with L-NNA and indomethacin andpreconstricted with 100 nM iberiotoxin and PE. *Significantlydifferent at P � 0.05 (2-way repeated-measures ANOVA); n,no. of arteries tested.




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regard, it has been reported that onset of endothelial NOproduction determined with the NO-sensitive dye DAF-2 sig-nificantly delayed by 20–60 s from the onset of agonist-induced elevation in EC [Ca2�]i (14, 31, 57). These datasuggest that initial uterine vasodilation in response to theCa2�-mobilizing agonist ACh is mostly mediated by EDHF.This conclusion is also supported by the fact that the magnitudeand time for reaching two-thirds of initial ACh-induced dila-tation in the presence of L-NNA and indomethacin was notdifferent from those of control untreated vessels. Sustainedcomponent, however, was significantly diminished, demon-strating that NO and PGI2, in addition to EDHF, contribute tosustained uterine vasodilation in the nonpregnant and pregnantstates.

In uterine vessels treated with L-NNA and indomethacin,both basal levels of EC [Ca2�]i and ACh-stimulated [Ca2�]i

responses were significantly enhanced in late pregnancythrough as-yet-unidentified mechanisms. The levels of cytoso-lic [Ca2�]i in ECs are finely regulated through Ca2� releasefrom internal stores and Ca2� influx into cells as well as bycellular Ca2� extrusion and sequestration mechanisms. Bind-ing of ACh to endothelial muscarinic receptors results inactivation of phospholipase C with subsequent Ca2� releasefrom internal stores and Ca2� influx through Ca2� permeablechannels (41); both of these processes may be modulated inlate pregnancy. In view of the absence of voltage-gated Ca2�

channels in endothelium of the majority of blood vessels, Ca2�

entry in cells is determined by Ca2� electrochemical potential.In addition to a strong Ca2� concentration gradient, membranehyperpolarization is another driving force for endothelial Ca2�

influx (41). Therefore, pregnancy may affect endothelial Ca2�

signaling through modulation of the expression and/or functionof ion channels that regulate MP.

Finally, close anatomical and functional communicationsbetween ECs and SMCs through myoendothelial gap junctionsare convincingly demonstrated in recent publications (8, 13,16, 26, 32, 33, 46). Previous studies indicate that Ca2� and/or

IP3 can diffuse from SMCs through gap junctions and subse-quently modulate Ca2� signaling and Ca2�-dependent mech-anisms in endothelial cells (26, 32, 33). We found that pres-sure-induced elevation of SMC [Ca2�]i in small uterine arteriesis significantly enhanced in late pregnancy (52). This observa-tion raises the possibility that IP3/Ca2� diffusion from SMCsmay contribute to a pregnancy-induced increase in basal levelsof EC [Ca2�]i and also modulate Ca2� responses to ACh inpressurized uterine vessels. Earlier, we did not find significantchanges in basal EC [Ca2�]i during development of myogenictone in uteroplacental arteries (20). Although our data do notexclude the role of SMCs in regulation of Ca2� signaling inadjacent endothelial cells, this mechanism is unlikely respon-sible for the higher basal levels of [Ca2�]i in endothelial cellsof uterine vessels in LP compared with NP rats.

In our experiments, PE application was associated withSMC [Ca2�]i elevation and vasoconstriction of uterine radialarteries from LP and NP rats. We also noted a remarkable[Ca2�]i oscillatory activity that was more frequent in vessels ofLP rats compared with NP controls. Previously, we observedsimilar [Ca2�]i oscillations and associated uteroplacental va-somotions in response to elevation of intracellular pressure.Pressure-induced vasomotions were preceded by oscillations inSMC MP that were most likely responsible for [Ca2�]i tran-sients (52). The origin of [Ca2�]i and vessel wall rhythmicactivity in response to PE in the current study, as well asmechanisms underlying its augmentation in late pregnancy,still remain unknown and deserves further investigation.

A key role of endothelial SKCa-IKCa channels in EDHF-mediated uterine vasodilation. In the current experiments, weobserved a close relationship between EC [Ca2�]i rise andEDHF-mediated vasodilation. Elevation of EC [Ca2�]i resultsin activation of several Ca2�-dependent intracellular mecha-nisms accompanied by activation of the EDHF system. It isgenerally accepted that endothelial SKCa and IKCa channelsplay a pivotal role in EDHF effects in the majority of micro-circulatory vascular beds. In the present study, ACh-induced

Fig. 8. Abolition of EDHF-mediated responsesof uterine arteries by charybdotoxin (CTX) andapamin (AP). A–D: bar graphs demonstratinginhibition of ACh-induced SMC [Ca2�]i anddilator responses by a combined treatment ofarteries with 50 nM CTX and 100 nM AP. Bothtoxins were applied intra- and extraluminally.Arteries were preconstricted with PE before test-ing ACh. L-NNA (200 �M) and indomethacin(10 �M) were present throughout the wholeexperiment. ACh-induced vasodilation is ex-pressed as Dmax. Nos. in parentheses indicate theno. of tested arteries. *Significantly different atP � 0.05 (2-way repeated-measures ANOVA).




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responses were abolished by a combined treatment withapamin and CTX (but not with apamin and IBTX), demon-strating a critical involvement of SKCa and IKCa channels inEDHF-mediated vasodilation of rat uterine arteries as well.Similar data were published for small human myometrialarteries (18). From our study, we concluded that increased EC[Ca2�]i responses followed by activation of SKCa and IKCa

channels is an important mechanism of enhanced EDHF-mediated uterine vasodilation during pregnancy. This, how-ever, does not rule out a contribution of other mechanisms.[Ca2�]i elevation in ECs resulted in generation of arachidonicacid and formation of a number of diffusible molecules pro-posed for the role of EDHF (8, 16). So far, the most convincingevidence was obtained on the role of EETs as EDHF incoronary and renal arteries. In these vessels, EETs can hyper-polarize SMCs through activation of SMC BKCa channels (9).In our experiments, IBTX, a highly potent and selective inhib-itor of these channels, did not modify L-NNA- and indometha-cin-resistant uterine vasodilation, indicating no role for BKCa

channels in EDHF. We cannot, however, exclude an involve-ment of EETs in paracrine modulation of endothelial SKCa andIKCa channel function of uterine vessels. As was reportedrecently, EETs can promote Ca2� influx in endothelial cellsthrough Ca2�-permeable channels or directly activate endothe-lial K� channels (38, 55). Finally, expression of SKCa and IKCa

channels and/or connexins may also be increased during lategestation, adding more complexity to the potential mechanismscontributing to augmented EDHF signaling in the maternaluterine circulation.

The role of EDHF in the control of regional blood flow aswell as in systemic blood pressure regulation is confirmed bynumerous current studies. A number of cardiovascular dis-eases, including hypertension and diabetes, are accompaniedby endothelial dysfunction, which in part may be due to defectsin the EDHF system (5, 11, 22, 30). Recently, it has beenshown that the development of preeclampsia in pregnantwomen is associated with compromised EDHF function ofsmall myometrial arteries (28). We have found that EDHF-mediated uterine vasodilation is impaired in the rat model ofdiabetic pregnancy (19). Further studies on the mechanismscontributing to an impaired EDHF system are essential in thesearch for pharmacological strategies to improve maternaluteroplacental circulation in pregnancies complicated by hy-pertension, preeclampsia, or diabetes.

In conclusion, we demonstrated a prominent role of EDHFin ACh-induced vasodilation of small-resistance uterine arter-ies and established the importance of EDHF in pregnancy-specific upregulation of vasodilatory mechanisms in the ma-ternal uteroplacental circulation. Increased EC [Ca2�]i signal-ing and concomitant activation of SKCa and IKCa channelsimportantly contribute to the augmented role of EDHF in theregulation of maternal uteroplacental blood flow in late gesta-tion.

ACKNOWLEDGMENTS

We thank Tara Goecks and Ashley Zucker for excellent technical assistanceand Rita Lemire for help with editing of the manuscript.

GRANTS

This study was supported by National Heart, Lung, and Blood InstituteGrants HL-067250 and HL-088245.

DISCLOSURES

No conflicts of interest are declared by the authors.

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Augmented EDHF signaling in rat uteroplacental vasculature ... · pregnant (LP) rats. Changes in SMC membrane potential of pressur-ized arteries from LP rats were assessed using glass