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Am J Physiol Heart Circ Physiol 294: H272-H284, 2008. First published November 2, 2007; doi:10.1152/ajpheart.00216.2007
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Decreased function of voltage-gated potassium channels contributes to augmented myogenic tone of uterine arteries in late pregnancy

Vsevolod Telezhkin, Tara Goecks, Adrian D. Bonev, George Osol, and Natalia I. Gokina

Departments of Obstetrics and Gynecology, and Pharmacology, University of Vermont, College of Medicine, Burlington, Vermont

Submitted 19 February 2007 ; accepted in final form 28 October 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Increased pressure-induced (myogenic) tone in small uteroplacental arteries from late pregnant (LP) rats has been previously observed. In this study, we hypothesized that this response may result from a diminished activity of vascular smooth muscle cell (SMC) voltage-gated delayed-rectifier K+ (Kv) channels, leading to membrane depolarization, augmented Ca2+ influx, and vasoconstriction (tone). Elevation of intraluminal pressure from 10 to 60 and 100 mmHg resulted in a marked, diltiazem-sensitive rise in SMC cytosolic Ca2+ concentration ([Ca2+]i) associated with a vasoconstriction of uteroplacental arteries of LP rats. In contrast, these changes were significantly diminished in uterine arteries from nonpregnant (NP) rats. Gestational augmentation of pressure-induced Ca2+ influx through L-type Ca2+ channels was associated with an enhanced SMC depolarization, the appearance of electrical and [Ca2+]i oscillatory activities, and vasomotion. Exposure of vessels from NP animals to 4-aminopyridine, which inhibits the activity of Kv channels, mimicked the effects of pregnancy by increasing pressure-induced depolarization, elevation of [Ca2+]i, and development of myogenic tone. Furthermore, currents through Kv channels were significantly reduced in myocytes dissociated from arteries of LP rats compared with those of NP controls. Based on these results, we conclude that decreased Kv channel activity contributes importantly to enhanced pressure-induced depolarization, Ca2+ entry, and increase in myogenic tone present in uteroplacental arteries from LP rats.

membrane potential; intracellular calcium; patch clamp; potassium currents; 4-aminopyridine

ADEQUATE DELIVERY OF OXYGEN AND NUTRITION is critical for normal fetal growth and development and is ensured by a remarkable increase in uteroplacental blood flow due to structural and functional adaptations of the maternal uterine vasculature (33, 38, 53). In humans and animals with hemochorial placentas, maternal spiral uterine arteries undergo transformation to large funnel-shaped vessels caused by trophoblast invasion (7, 41, 55). This results in destruction and/or dedifferentiation of smooth muscle cells (SMCs) and loss of vasoconstriction (7, 11, 16, 42). Uteroplacental radial arteries that connect larger main and arcuate uterine vessels to structurally and functionally modified spiral arteries become the primary site regulating uterine vascular resistance in late gestation.

Normal pregnancy is characterized by enhanced endothelium-dependent vasodilation of uterine arteries in response to chemical or mechanical stimulation, which is, in turn, determined by increased expression and activity of key endothelial enzymes (endothelial nitric oxide synthase, cyclooxygenase), as well as an upregulation of Ca2+ signaling in endothelial cells (5, 15, 28, 44, 52, 56, 64). These important changes occur in parallel with augmented responses of these vessels to some vasoconstrictors and to intraluminal pressure (12, 14, 35, 48, 59, 61). The balance between augmented vasodilator and vasoconstrictor mechanisms determines uterine arterial tone and, consequently, blood flow to the placenta and fetus.

Pressure-induced myogenic constriction represents an essential property of resistance-size arteries contributing to autoregulation of regional blood flow and maintenance of capillary hydrostatic pressure (10, 22, 23, 30, 46). Pressure-induced vasoconstriction has been described in uterine arteries from pregnant animals and women (9, 15, 16, 28, 48, 61). The effect of pregnancy on myogenic behavior of uterine arteries varies significantly, depending on their anatomic location and the type of placentation. Several studies from different laboratories, including our own, have demonstrated that pressure-induced vasoconstriction (myogenic tone) of small radial arteries of the rat is significantly enhanced in late gestation (15, 48, 61).

The causes and underlying mechanisms of augmented uterine artery myogenic tone in late pregnancy remain largely unknown. The contractile state of SMCs is a primary determinant of vascular diameter and is regulated by changes in the cytosolic concentrations of Ca2+ ([Ca2+]i), as well as in the Ca2+ sensitivity of the contractile process (22, 29, 57, 60). Ca2+ influx, a major mechanism for increasing [Ca2+]i, occurs through multiple Ca2+ channels. Voltage-gated Ca2+ channels represent a key pathway for Ca2+ entry into vascular SMCs and are under the control of membrane potential.

Voltage-gated delayed-rectifier K+ (Kv) channels are important regulators of smooth muscle membrane potential and excitability and are involved in the control of myogenic behavior of small resistance arteries and arterioles in different vascular beds (1, 10, 23, 27, 31, 43). Activation of these channels provides a negative feedback pathway regulating the degree of pressure-induced depolarization and, hence, myogenic tone (1, 10, 23, 29, 43).

In this study, we hypothesized that late gestation results in a diminished activity of Kv channels, leading to augmented SMC depolarization, Ca2+ influx, and myogenic tone of uteroplacental arteries. The aims of the present study were to 1) characterize changes in vessel diameter and in SMC [Ca2+]i levels in response to pressure elevation in arteries from late pregnant (LP) and nonpregnant (NP) rats, 2) study the role of membrane potential in pregnancy-induced potentiation of myogenic tone, and 3) assess the role of Kv channels in pregnancy-specific modulation of pressure-induced myogenic tone by testing the effects of 4-aminopyridine (4-AP) and characterizing ion currents through Kv channels.

Several methodological approaches were combined to provide direct evidence that decreased activity of arterial smooth muscle Kv channels, with a resultant increase in pressure-induced depolarization and Ca2+ influx, importantly contributes to pregnancy-specific enhancement of myogenic tone in small uteroplacental arteries of the rat.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and preparation of arteries. All experiments were conducted in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publications no. 85-23, revised 1996), and the experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Vermont.

Virgin and cycling NP and LP (19–20 day) female Sprague-Dawley rats 14–16 wk old (n = 73) were anesthetized by an intraperitoneal injection of Nembutal (50 mg/kg) and killed by decapitation. The abdominal wall was transected, and the entire uterus and uterine vasculature were rapidly removed and pinned in a dissecting dish filled with aerated cold physiological salt solution (PSS; see Solutions and drugs for composition). First- or second-order uterine radial arteries were identified within the mesometrial arcade and dissected free of connective tissue. Only uteroplacental arteries (radial arteries feeding the placenta) were dissected from the vasculature of pregnant uteri. Arterial segments were cannulated from both ends in the arteriograph and continuously superfused at 3 ml/min with aerated (10% O2-5% CO2-85% N2) PSS at 37°C. Initial intraluminal pressure was set at 10 mmHg with a servo pressure system (Living System Instrumentation, Burlington, VT). All experiments were performed under no intraluminal flow conditions.

In a separate set of experiments, the endothelium was removed by infusing air into the arterial lumen for 5–6 min, followed by gentle and brief (5 s) perfusion with regular PSS before pressurization of the artery. The effectiveness of this denudation procedure was confirmed by the absence of a dilatory response to ACh (3 µM).

Measurement of SMC [Ca2+]i in pressurized arteries. After an equilibration period of 20 min at 37°C at 10 mmHg and measurement of background fluorescence, SMCs within the wall of arteries pressurized at 10 mmHg were loaded with 5 µM fura 2. An arterial segment was incubated extraluminally in fura 2-AM loading solution at room temperature in the dark for 45–60 min under a no-perfusion condition. Fura 2-loaded arteries were then continuously superfused at 3 ml/min with aerated PSS at 37°C. Ratiometric measurements of fura 2 fluorescence were performed with a photomultiplier system (IonOptix, Milton, MA). Background-corrected ratios of 510-nm emission were obtained at a sampling rate of 5 Hz from arteries alternately excited at 340 and 380 nm. Lumen diameter was simultaneously monitored with the SoftEdge Acquisition Subsystem (IonOptix). All experimental protocols were started after an additional 15- to 20-min equilibration period at 10 mmHg to allow intracellular deesterification of fura 2-AM.

After equilibration, the levels of smooth muscle [Ca2+]i and arterial diameters were recorded for 5 min at 10 mmHg, and intraluminal pressure was then elevated to 60 and 100 mmHg. Changes in arterial diameter and in [Ca2+]i were recorded until stabilization of myogenic constriction (typically 10 min for each level of pressure). Papaverine (100 µM) and diltiazem (10 µM) were applied at the end of each experiment, and the arterial diameter was recorded at 10, 60, and 100 mmHg from a maximally dilated artery. The degree of myogenic tone was expressed as a percentage of the reduction in maximal arterial diameter at a given level of intraluminal pressure.

Measurement of membrane potential. In a separate set of experiments, simultaneous changes in diameter and membrane potential were recorded from arteries at different levels of intraluminal pressure. For measurement of membrane potential, we used glass microelectrodes filled with 0.5 M KCl and with tip resistances of 110–150 M{Omega}; an Ag-AgCl pellet was used as an indifferent electrode. Microelectrode impalements of SMCs were made from the adventitial surface of the arterial segments. A microelectrode was connected to a motorized micromanipulator (World Precision Instruments, Sarasota, FL), and membrane potential was recorded with a high-input impedance amplifier Electro 705 (World Precision Instruments). Changes in membrane potential and arterial diameter were displayed and recorded on a desktop computer using a data-acquisition program (IonOptix). The following criteria were used for acceptance of membrane potential recordings: 1) abrupt negative change in voltage after impalement of the cells, 2) a sharp return to zero voltage after withdrawal of the microelectrode tip, and 3) tip potential of <7 mV.

Whole cell patch-clamp experiments. Arteries from NP or LP rats were cleaned of connective tissue and placed in low-Ca2+ dissociation media (see Solutions and drugs for composition) containing papain (1 mg/ml) and BSA (0.7 mg/ml); samples were kept refrigerated for 20–30 min. 1,4-DTT (0.8 mg/ml) was then added, and arteries were incubated for an additional 12 min at 37°C. Digested tissue was washed out and gently triturated with a fire-polished glass pipette. A suspension of single SMCs was refrigerated until use (typically 6–8 h). Outward K+ currents were recorded from single SMCs at room temperature with a perforated or conventional whole cell patch-clamp technique. Recording electrodes with resistances of 2–4 M{Omega} were pulled from borosilicate glass and backfilled with a pipette solution of appropriate composition (see Solutions and drugs for composition). Kv currents were recorded from cells on an Axopatch 200B amplifier, filtered at 5 kHz using a low-pass Bessel filter, and digitized at 10 kHz (Digidata 1322A, Axon Instruments). pCLAMP-9 software (Axon Instruments) was used for data registration and analysis.

Solutions and drugs. PSS contained (in mM) 119 NaCl, 4.7 KCl, 24.0 NaHCO3, 1.2 KH2PO4, 1.6 CaCl2, 1.2 MgSO4, 0.023 EDTA, and 11.0 glucose (pH 7.4). For the fura 2 calibration procedure, we used a solution of the following composition (in mM): 140 KCl, 20 NaCl, 5 HEPES, 5 EGTA, and 1 MgCl2, as well as 5 µM nigericin and 10 µM ionomycin (pH 7.1). The solution for enzymatic dissociation of SMCs contained (in mM) 110 NaCl, 10 NaHCO3, 5 KCl, 0.5 NaH2PO4, 0.5 KH2PO4, 2 MgCl2, 0.16 CaCl2, 10 HEPES, and 10 glucose (pH 7.0). The bath solution for patch-clamp experiments was of the following composition (in mM): 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose; the pH was adjusted to 7.4 with 5 M NaOH solution. For perforated patch-clamp experiments, patch pipettes were filled with 110 mM potassium aspartate, 30 mM KCl, 10 mM NaCl, 10 mM HEPES, 1 mM MgCl2, and 250 µg/ml amphothericin (pH 7.2). For experiments with a conventional patch-clamp configuration, the pipette solution contained (in mM) 110 KCl, 30 KOH, 10 HEPES, 10 EGTA, 1 MgCl2, 3 Na2ATP, and 0.5 Na2GTP (pH 7.2).

All chemicals were purchased from Sigma (St. Louis, MO) with the exception of ionomycin and nigericin, which were obtained from Calbiochem (La Jolla, CA). Fura 2-AM and pluronic acid were purchased from Invitrogen (Carlsbad, CA). Fura 2-AM was dissolved in dehydrated DMSO as a 1 mM stock solution, frozen in small aliquots, and used within 1 wk of preparation. Papaverine was dissolved in deionized water and used the same day. Diltiazem was prepared as a 10 mM stock solution in deionized water and kept refrigerated until use (2–3 wk). Ionomycin and nigericin were prepared as 10 mM stock solutions in methanol and kept at –20°C until use. 4-AP was dissolved in deionized water and used after the pH was adjusted to 7.4. A stock solution of iberiotoxin was made with deionized water and was stored at –20°C until use.

Calculations and statistical analysis. SMC [Ca2+]i was calculated with the following equation (17): [Ca2+]i = Kdβ(R – Rmin)/(Rmax – R), where R is experimentally measured ratio (340/380 nm) of fluorescence intensities, Rmin is a ratio in the absence of [Ca2+]i, Rmax is a ratio at Ca2+-saturated fura 2 conditions, and β is a ratio of the fluorescence intensities at 380-nm excitation wavelength at Rmin and Rmax. Rmin, Rmax, and β were determined by an in situ calibration procedure from the arteries treated with nigericin (5 µM) and ionomycin (10 µM). Calibration was performed from the vessels loaded extraluminally with fura 2 (n = 4). These values were then pooled and used to convert the ratio values into [Ca2+]i. The Kd (the dissociation constant for fura 2) was 282 nM, as determined by in situ titration of Ca2+ in fura 2-loaded small arteries (25). Arterial diameter, pressure, and ratio values were simultaneously recorded with an IonOptix data-acquisition program and imported into SigmaPlot and SigmaStat programs for graphical representation, calculations, and statistical analysis. Data are expressed as means ± SE, where each n = number of arterial segments studied. One to two arteries from the same animal were used on each experimental day. Only one vessel per animal was used for a particular protocol. Diltiazem was tested in nine arteries from six rats. One to three cells from each rat were used for a specific protocol in patch-clamp studies. A paired or unpaired Student's t-test or two-way repeated-measures ANOVA was used to determine the significance of differences between sets of data, with P < 0.05 considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Pregnancy-enhanced myogenic tone is associated with an increase in SMC [Ca2+]i. In uterine radial arteries from NP rats, a stepwise elevation of intraluminal pressure from 10 to 60 and to 100 mmHg did not lead to a marked elevation of [Ca2+]i or vasoconstriction (elevation in [Ca2+]i above basal levels was 7 ± 3 and 16 ± 6 nM at 60 and 100 mmHg, respectively; Fig. 1, A, C, and D). In contrast, uteroplacental arteries from LP rats constricted robustly to pressure elevation (25 ± 3% and 32 ± 5% at 60 and 100 mmHg, respectively). This myogenic tone was preceded by a significant increase in [Ca2+]i consisting of slow [Ca2+]i rise with superimposed fast spikelike oscillations in [Ca2+]i. The latter were clearly associated with rhythmic vasoconstrictions or vasomotion. The average increment in [Ca2+]i above the basal level was 112 ± 15 and 168 ± 25 nM at 60 and 100 mmHg, respectively (Fig. 1, B–D).


Figure 1
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  		Fig. 1. Pregnancy-induced enhancement of myogenic tone in uteroplacental arteries is associated with augmented smooth muscle cytosolic Ca2+ ([Ca2+]i) responses to pressure elevation. A and B: representative tracings showing the effects of stepwise elevation in intraluminal pressure from 10 to 60 and 100 mmHg on smooth muscle [Ca2+]i and diameters of arteries from nonpregnant (NP; A) and late pregnant (LP; B) rats. C: summary graph demonstrating potentiation of pressure-induced [Ca2+]i responses in arteries of LP rats compared with NP controls. D: graph summarizing effect of pregnancy on myogenic tone of intact uterine arteries in response to elevations in intraluminal pressure from 10 to 60 and 100 mmHg. E and F: summary graphs showing [Ca2+]i (E) and constrictor (F) responses to pressure elevation of endothelium-denuded arteries from NP and LP rats. Myogenic tone is expressed as a percentage of the reduction in the arterial diameter under completely dilated conditions, measured in the presence of papaverine and diltiazem. Numbers in parentheses (n) indicate the number of arteries tested. *Significantly different from NP controls at P < 0.05.

 
Arteries from NP rats did not develop a significant myogenic tone after endothelial denudation. Similar to intact arteries, denuded vessels showed minimal changes in SMC [Ca2+]i in response to pressure elevation from 10 to 60 mmHg (11 ± 5 nM) and then to 100 mmHg (22 ± 8 nM, n = 5; Fig. 1E). Pressure-induced constriction was 2 ± 1% and 6 ± 2% at 60 and 100 mmHg, respectively (n = 10; Fig. 1F). Endothelium-denuded arteries from LP rats developed a marked myogenic tone of 21 ± 4% and 29 ± 6% in response to pressure elevation from 10 to 60 and then to 100 mmHg (n = 10). These constrictor responses were associated with significant increases in SMC [Ca2+]i above basal levels of 97 ± 32 and 245 ± 31 nM at 60 and 100 mmHg, respectively (n = 6; Fig. 1, E and F). Pressure-induced constrictor and [Ca2+]i responses of denuded arteries were not significantly different from those of intact vessels from both NP and LP rats. Therefore, enhancement of myogenic tone of uterine arteries in late pregnancy is due to specific changes in the properties of SMCs.

Both pressure-induced [Ca2+]i oscillatory activity and sustained [Ca2+]i elevation in arteries of LP rats were effectively abolished by diltiazem, an inhibitor of L-type Ca2+ channels (Fig. 2, A and B). Treatment with diltiazem also abolished the myogenic tone developed in response to pressure elevation (Fig. 2C).


Figure 2
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  		Fig. 2. Diltiazem, a specific inhibitor of voltage-gated L-type Ca2+ channels, abolishes pressure-induced [Ca2+]i response and myogenic tone of uteroplacental arteries. Nine arteries obtained from 6 rats were tested for this protocol. A: representative tracings showing inhibition of pressure-induced smooth muscle [Ca2+]i elevation and vasoconstriction by 10 µM diltiazem. B: summary graph demonstrating the effect of diltiazem on the levels of smooth muscle [Ca2+]i in response to pressure elevation from 10 to 60 mmHg. *Significantly different from the [Ca2+]i levels at 10 mmHg. C: bar graph summarizing the effect of diltiazem on vasoconstriction (myogenic tone) induced by pressure elevation from 10 to 60 mmHg. Myogenic tone is expressed as a percentage of the reduction in the diameters of the arteries maximally dilated with a combination of papaverine and diltiazem (Dmax). *Significantly different from myogenic tone before application of diltiazem at P < 0.05.

 
Pressure-induced SMC membrane depolarization is augmented in late pregnancy. We next examined the hypothesis that the pregnancy-specific enhancement of pressure-induced Ca2+ influx is due to augmented SMC depolarization. This aim was assessed by direct measurements of SMC membrane potential in arteries from NP and LP rats subjected to different levels of intraluminal pressure. Resting membrane potential measured from SMCs of NP arteries (n = 8) at 10 mmHg was slightly more negative than membrane potential from LP controls (n = 8), but the difference did not reach significance (–55 ± 1 vs. –53 ± 1 mV).

An elevation of intraluminal pressure from 10 to 60 mmHg resulted in a passive vasodilation of arteries from NP rats associated with a small but significant SMC depolarization from –55 ± 1 to –49 ± 1 mV (P < 0.05, n = 5; Fig. 3, A, C–E). In contrast, arteries from LP rats developed myogenic tone of 30 ± 4% associated with a marked depolarization from –53 ± 1 to –37 ± 1 mV (n = 8; Fig. 3, B–E).


Figure 3
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  		Fig. 3. Pregnancy-augmented myogenic tone is associated with increased smooth muscle cell depolarization in response to elevation of intraluminal pressure. A and B: representative microelectrode recordings of membrane potential from smooth muscle cells in the wall of the same artery of an NP rat (A) and an LP rat (B) before and after elevation of intraluminal pressure from 10 to 60 mmHg; note an appearance of slow waves of depolarization and action potential generation in the artery of LP rat after pressure elevation. C and D: bar graphs showing the levels of smooth muscle membrane potential in the arteries of NP and LP rats pressurized to 10 (C) and to 60 (D) mmHg. E: summary graph demonstrating an augmentation of myogenic tone in association with enhanced depolarization of uterine arteries in LP animals (shown in D). Numbers in parentheses indicate the number of arteries tested. *Significantly different from NP controls at P < 0.05.

 
Membrane potential of SMCs in LP arteries pressurized to 60–100 mmHg was unstable; generations of slow waves of depolarization with superimposed bursts of action potentials of 10–30 mV were frequently observed. The levels of membrane potential at 60 mmHg (–37 ± 1 mV, n = 8) were significantly different from those at 100 mmHg (–32 ± 1 mV, n = 5; P < 0.05). Continuous measurements of changes in membrane potential and arterial diameter obtained in some arteries demonstrated that stepwise elevations or reductions in intraluminal pressure were clearly associated with membrane depolarization and repolarization, respectively (Fig. 4).


Figure 4
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  		Fig. 4. Pressure-induced smooth muscle depolarization as a function of intraluminal pressure. Representative traces demonstrate simultaneous changes in smooth muscle membrane potential, in frequency of action potentials and in arterial diameter in response to stepwise changes in intraluminal pressure.

 
The duration of slow waves in membrane potential and [Ca2+]i and the frequency of action potentials and [Ca2+]i spikes within each wave were all observed to be a function of intraluminal pressure (Fig. 5, A–D). A causal relationship between electrical events and [Ca2+]i signaling in SMCs of uteroplacental arteries was evidenced by a similar duration of slow waves (12 ± 1 and 12 ± 2 s at 60 mmHg and 26 ± 7 and 30 ± 5 s at 100 mmHg for membrane potential and [Ca2+]i, respectively) and frequency of spikes (26 ± 4 and 26 ± 3 s at 60 mmHg and 62 ± 11 and 49 ± 5 spikes/min at 100 mmHg for action potentials and [Ca2+]i, respectively; Fig. 5, E and F).


Figure 5
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  		Fig. 5. Similarity in the pattern of electrical and [Ca2+]i activity and arterial vasomotion at the same level of intraluminal pressure. A and B: representative tracings showing changes in membrane potential and smooth muscle cell [Ca2+]i and associated vasomotion of arteries from LP rats at 60 mmHg. C and D: representative tracings of electrical activity and smooth muscle cell [Ca2+]i changes and associated vasoconstriction at 100 mmHg. E: summary graph showing a significant increase in the duration of waves in membrane potential (MP) and in [Ca2+]i in response to pressure elevation from 60 to 100 mmHg. There was no significant difference in the duration of the membrane potential waves vs. duration of [Ca2+]i waves at the same level of intraluminal pressure. F: bar graph demonstrating increased frequency of action potentials (APs) and [Ca2+]i spikes at 100 vs. 60 mmHg. The frequency of action potentials was not different from that of [Ca2+]i spikes at the same level of intraluminal pressure. Numbers in parentheses indicate the number of arteries tested. *Significantly different at P < 0.05.

 
4-AP enhances myogenic tone in arteries from NP and LP rats. We next tested the hypothesis that reduced activity of Kv channels contributes to enhanced pressure-induced SMC depolarization and uteroplacental myogenic tone in late pregnancy. We first studied the effect of 4-AP, a widely used specific inhibitor of voltage-gated Kv channels, on the ability of arteries from NP rats to develop myogenic tone.

Application of 5 mM 4-AP to arteries of NP rats pressurized at 10 mmHg did not alter SMC [Ca2+]i or arterial diameter. In the presence of 4-AP, subsequent elevation of pressure from 10 to 60 and to 100 mmHg led to the development of myogenic tone of 45 ± 9% and 57 ± 4% and significant [Ca2+]i elevations from 88 ± 20 to 162 ± 26 nM and 203 ± 16 nM, respectively. In addition, 4-AP also induced a marked [Ca2+]i oscillatory activity and vasomotion similar to results observed in arteries of LP rats (Fig. 6, A–D). Similar treatment of arteries from LP rats with 5 mM 4-AP resulted in slight enhancement of [Ca2+]i and constrictor responses to elevation of intraluminal pressure (Fig. 6, E and F). These results suggest that elevation of pressure in NP rats leads to a substantial activation of Kv channels, which counteracts the depolarizing influence of pressure and hence an elevation in [Ca2+]i and vasoconstriction.


Figure 6
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  		Fig. 6. 4-Aminopyridine (4-AP) enhances pressure-induced smooth muscle [Ca2+]i responses and myogenic tone of uterine arteries from NP and LP rats. A and B: representative tracings showing [Ca2+]i and diameter responses to pressure elevation before (A) and after (B) application of 5 mM 4-AP. C: bar graph summarizing changes in smooth muscle [Ca2+]i in response to pressure elevation from 10 to 60 and then to 100 mmHg under control conditions and in the presence of 4-AP. D: summary graph demonstrating a marked augmentation of myogenic tone in arteries from NP rats treated with 4-AP. E and F: bar graphs summarizing the effects of 4-AP on [Ca2+]i responses and myogenic tone induced by pressure elevation from 10 to 60 and then to 100 mmHg in uteroplacental arteries from LP rats. Myogenic tone is expressed as a percentage of the reduction in the maximal diameter of arteries dilated in response to 100 µM papaverine and 10 µM diltiazem. Numbers in parentheses indicate the number of arteries tested. *Significantly different from controls at P < 0.05.

 
To test this prediction, SMC membrane potential was measured from the same pressurized artery before and after application of 5 mM 4-AP (Fig. 7, A and B). The SMCs were significantly more depolarized in the presence of 4-AP (–34 ± 1 vs. –48 ± 1 mV, n = 3; P < 0.05). Generation of action potentials with amplitudes of 15–20 mV was observed in all 4-AP-treated arteries. Marked pressure-induced depolarization in the presence of 4-AP was also associated with the development of considerable myogenic tone (54 ± 5%; Fig. 7, C and D). These data suggest that a decrease in Kv channel activity is an important mechanism in mediating the pregnancy-induced augmentation of myogenic tone in rat uteroplacental arteries.


Figure 7
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  		Fig. 7. 4-AP-induced myogenic tone in uterine arteries of NP rats is associated with marked smooth muscle depolarization. A and B: representative tracings showing membrane potential recordings from the same artery before (A) and after (B) application of 4-AP. Membrane depolarization and generation of action potentials were associated with development of myogenic tone. The 15-min time break was the period used to obtain intracellular impalement and stable reading of the membrane potential. C: summary bar graph showing augmentation in pressure-induced depolarization in arteries treated with 4-AP. D: summary bar graph demonstrating 4-AP-induced increase in myogenic tone of arteries from NP rats. Myogenic tone is expressed as a percentage of the reduction in the maximal diameter of arteries dilated in response to 100 µM papaverine and 10 µM diltiazem. Numbers in parentheses indicate the number of arteries tested. *Significantly different from control at P < 0.05.

 
Pregnancy-specific inhibition of currents through Kv channels. Direct evidence for the decreased function of Kv channels in late pregnancy was obtained from a comparative study of currents through Kv channels recorded from single SMCs freshly dissociated from arteries of NP and LP rats. SMCs from LP rats had significantly larger cell capacitance (19.0 ± 0.5 pF, n = 65) than SMCs from NP rats (15.9 ± 0.4 pF, n = 54). The increased capacitance of SMCs most likely reflects a significant enlargement of SMCs during gestation (8) and correlates well with the increase in passive diameters of uterine radial arteries in late pregnancy from 124 ± 10 to 201 ± 18 µm measured at 60 mmHg (P < 0.05) in the present study.

Outward K+ currents from myocytes of NP and LP rats were recorded in perforated patch-clamp configuration in response to the voltage steps from –80 to +80 mV (holding potential of –60 mV) in 10-mV increments and with a duration of 500 ms (Fig. 8A). Outward currents were characterized by large fluctuations at positive levels of membrane potentials and were significantly reduced in myocytes from LP rats (14 ± 2 pA/pF, n = 6) compared with NP controls (24 ± 3 pA/pF, n = 7) at +80 mV (Fig. 8B).


Figure 8
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  		Fig. 8. Pregnancy-induced suppression of Kv currents in smooth muscle cells freshly dissociated from uterine radial arteries. A: representative families of outward K+ currents induced by 500-ms voltage steps from holding potential of –60 mV recorded from myocytes of uterine arteries of NP and LP rats using perforated patch-clamp technique. B: graph summarizing the effect of pregnancy on the current density of outward K+ currents. Seven myocytes from 6 NP rats and 6 myocytes from 5 LP rats were used for these experiments. C and D: families of currents through voltage-dependent delayed-rectifier K+ (Kv) channels recorded from myocytes of NP and LP rats before and after treatment with 4-AP. Glyb, glybenclamide; IbTX, iberiotoxin. E: graph showing a decrease in Kv currents in myocytes of LP rats compared with NP controls. F and G: graphs summarizing the inhibitory effect of 4-AP on Kv currents of uterine vascular myocytes from NP (F) and LP (G) rats. Kv currents were obtained from 16 myocytes of 9 NP rats and 17 myocytes of 8 LP rats. *Significantly different from NP controls at P < 0.05.

 
Ion currents through Kv channels of myocytes from NP and LP rats were characterized and next compared with the use of the whole cell patch-clamp technique (Fig. 8, A and B). K+ currents through ATP-sensitive K+ (KATP) and large-conductance calcium-activated K+ (BK) channels were minimized by the inclusion of 5 mM ATP and 10 mM EGTA in pipette solution and by adding 100 nM iberiotoxin and 10 µM glybenclamide, specific inhibitors of BK and KATP channels, respectively, to external solutions. Kv currents were recorded in response to the voltage steps from –80 to +80 mV (holding potential of –60 mV) in 10-mV increments; they were rapidly activating and showed very little inactivation over 500 ms (Fig. 8C). Kv currents of SMCs from both NP and LP animals could be detected at membrane potentials above –40 mV. Current density was significantly reduced in myocytes of LP rats compared with NP controls [9.3 ± 0.8 pA/pF (n = 17) vs. 16.7 ± 2.2 pA/pF (n = 16), at +80 mV; Fig. 8D]. Application of 4-AP (5 mM) resulted in a significant decrease in Kv current amplitude in myocytes from both NP and LP rats (Fig. 8, E and F).

We next studied the effect of pregnancy on the voltage dependence of steady-state activation and inactivation of Kv currents by means of standard double-pulse protocols. Steady-state activation was assessed from peak tail currents in response to testing pulses (500 ms) to –20 mV after preconditioning pulses (300 ms) from –60 to +30 mV. Representative families of currents recorded with this protocol from myocytes of NP and LP rats are shown in Fig. 9A. A summary graph of peak tail currents as a function of the voltage is shown in Fig. 9B. For both NP and LP myocytes, tail current amplitude reached maximal value at voltages above +20 mV. Consistent with data shown in Fig. 8, Kv currents recorded with this protocol were also significantly reduced in myocytes from LP rats (0.7 ± 0.1 pA/pF, n = 6) compared with NP controls (2.0 ± 0.5 pA/pF, n = 8) at +30 mV.


Figure 9
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  		Fig. 9. Voltage dependence of steady-state activation and inactivation of Kv currents in uterine vascular myocytes. A: representative families of currents in response to the first conditioning pulse (300 ms) from –60 to +30 mV and deactivating tail currents in response to the second testing pulse (500 ms) on return to –20 mV from myocytes of NP and LP rats. B: graph showing pregnancy-induced reduction in Kv tail currents from uterine vascular myocytes. C: representative families of Kv currents recorded in response to a test pulse of the double-pulse voltage protocol to study a steady-state inactivation in myocytes of NP and LP rats. The first voltage pulse of 10 s from –100 to +20 mV was followed by the second test voltage pulse (shown) of 50 ms from –60 to +60 mV. D: plots of steady-state activation (circles) and steady-state inactivation (rectangles) from single smooth muscle cells of NP (open symbols) and LP (solid symbols) rats. Solid (NP) or dotted (LP) lines are fits to the Boltzmann distribution. For the activation protocol, 8 myocytes from 4 NP rats and 6 myocytes from 5 LP rats were used. For the inactivation protocol, 8 myocytes from 7 NP rats and 11 myocytes from 8 LP rats were used. *Significantly different from NP controls at P < 0.05.

 
The effect of pregnancy on steady-state inactivation of Kv channels was assessed by means of standard double-pulse protocol (Fig. 9, C and D). Preconditioning 10-s voltage steps from –100 to +20 mV were applied in 10-mV increments from holding potential of –60 mV. These steps were followed by a test voltage step of 50 ms to +60 mV. Families of currents in response to a test voltage step recorded from myocytes of NP and LP rats are shown in Fig. 9C. Currents from activation and inactivation protocols were normalized by the maximal current amplitude and were fitted with a Boltzmann distribution function (Fig. 9D). Inactivation of Kv currents was incomplete in myocytes from both NP and LP rats. The slopes of inactivation and voltages for half-maximal inactivation were not different between two groups of myocytes: 12.3 ± 1.6 vs. 11.3 ± 1.0 and –24.7 ± 2.1 vs. –28.1 ± 1.3 mV for NP (n = 8) and LP (n = 11) rats, respectively. The voltages for half-maximal activation were similar for myocytes of NP and LP rats [0.2 ± 0.5 mV (n = 8) vs. –2.6 ± 0.8 mV (n = 6), respectively]. The slopes of activation were not significantly different between the two groups: 7.9 ± 0.7 vs. 6.3 ± 0.4 for NP and LP myocytes, respectively.


   DISCUSSION
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 ABSTRACT
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This study provides the first evidence that a pregnancy-specific decrease in Kv channel function contributes to enhanced myogenic behavior of small uteroplacental arteries. This conclusion is based on the following observations: 1) pregnancy-induced enhancement of myogenic tone was linked to augmented SMC [Ca2+]i responses to pressure elevation, 2) increased Ca2+ influx through L-type Ca2+ channels was associated with an enhanced pressure-induced SMC depolarization, 3) the excitability of uteroplacental SMCs was increased in late pregnancy and manifested by the appearance of electrical and [Ca2+]i oscillatory activity and vasomotion, 4) exposure to 4-AP mimicked the effects of pregnancy by increasing pressure-induced depolarization and the [Ca2+]i response in vessels from NP animals and resulting in the development of myogenic tone, and 5) currents through Kv channels were significantly diminished in myocytes of arteries from LP rats compared with those of NP controls.

The present study confirmed previously published findings that uterine arteries supplying hemochorial placentas develop a sustained myogenic tone in response to pressure elevation (9, 16, 28, 48, 61). The significant enhancement of myogenic tone shown in the present and previous studies most likely reflects functional adaptive changes that are important for the regulation of uteroplacental vascular resistance in late pregnancy. An enhanced myogenic tone of uterine arteries in late pregnancy can increase the vasodilator reserve necessary for a rapid adjustment of uteroplacental blood flow when needed.

Another essential role of arterial myogenic tone is the protection of downstream arterioles and capillaries from high levels of intraluminal pressure. As previously shown by Moll and Künzel (41) in rodents, the maternal compartment of a hemochorial placenta operates under relatively low levels of pressure (15–20 mmHg), which is important for an adequate maternal-fetal exchange. It is very likely that the development of myogenic tone in late pregnancy is one of the adaptive mechanisms that protects maternal-placental perfusion from high levels of blood pressure. This mechanism probably is not important in animals with an epitheliochorial type of placenta such as sheep, where the maternal microvascular system is intact and major resistance to placental blood flow is located inside the placenta (40). In this regard, it was recently demonstrated that myogenic tone of uterine arteries of sheep was significantly reduced in late pregnancy (63).

The present study demonstrates that pregnancy-specific development of myogenic tone in uteroplacental arteries is due to an augmented SMC [Ca2+]i response. A typical pressure-induced [Ca2+]i response consists of a sustained [Ca2+]i elevation with superimposed Ca2+ oscillations followed by vasoconstriction. Both pressure-induced [Ca2+]i and constrictor responses were abolished by diltiazem, implicating Ca2+ influx through L-type Ca2+ channels as the primary pathway for Ca2+ entry being responsible for the development of myogenic tone. Direct measurement of SMC membrane potential indicates that the major cause of this increased Ca2+ influx during pregnancy is an augmented pressure-induced depolarization. Continuous records of changes in membrane potential and arterial diameter clearly demonstrate that the extent of membrane depolarization depends on the level of intraluminal pressure. In addition, SMCs show remarkable regenerative oscillations in membrane potential. The results of this study show that the frequency of action potentials is determined by the degree of membrane depolarization, which is a function of intraluminal pressure. The similarities in the frequency and pattern of action potentials, in the spiking oscillations in [Ca2+]i, and in arterial vasomotion at the same level of intraluminal pressure indicate that these three events are closely related. These observations suggest that generation of action potentials is the mechanism for triggering the oscillations in [Ca2+]i, which in turn results in the subsequent development of rhythmic vasoconstrictions. Partial summation of action potential-induced individual vasoconstrictions forms larger periodic constrictions, which are evidenced as vasomotion. The origin of electrical and [Ca2+]i rhythmic activity in pressurized uteroplacental arteries still remains unclear. Generation of action potentials was preceded by slow waves of depolarization of 1–2 mV that might, in turn, be triggered by slow Ca2+ waves of intracellular origin. Alternatively, periodic activation of Ca2+ and K+ channels in response to pressure-induced depolarization might be the major underlying mechanism (18, 37). The physiological role of vasomotion in the maternal uteroplacental circulation is not clear. However, similar intermittent functioning of monkey uteroplacental arteries was demonstrated in earlier studies using radioangiography (36, 54). Spontaneous vasomotion might be responsible for the blood spurts characterizing blood entry into the intervillous space of the hemochorial placenta of humans and animals (38, 53).

Pressure-induced membrane depolarization as an underlying mechanism of myogenic tone was demonstrated in a number of pressurized arterial preparations (6, 19, 20, 26, 47, 62). It is well known that the extent of pressure-induced depolarization is in part under control of K+ channels (6, 10, 43, 60). The increased excitability and augmented depolarization in response to pressure elevation found in the present study strongly suggest that late pregnancy might result in decreased activity of Kv channels. Similar to previous data from cerebral (26) and mesenteric (50) arteries, we found that, in uterine arteries of NP rats, 4-AP can potentiate the development of myogenic tone and, in this way, can mimic the effect of pregnancy. 4-AP-induced myogenic tone was associated with membrane depolarization and elevations in [Ca2+]i that were remarkably similar to those of pressurized arteries from LP rats (Figs. 6 and 7). 4-AP application also resulted in the generation of action potentials and Ca2+ spikes in arteries of NP rats. These data suggest that decreased activity of Kv channels contributes to pregnancy-specific augmentation of myogenic tone and an increased excitability of uterine vascular SMCs.

Direct evidence to support this idea was obtained from Kv current measurements under different experimental protocols. Outward K+ currents recorded from vascular myocytes using perforated patch-clamp technique were significantly reduced in late pregnancy (Fig. 8, A and B). In experiments minimizing the contribution of BK and KATP channels, outward Kv currents were significantly decreased in myocytes of LP vessels compared with NP controls (Fig. 8, C and E). Voltage dependence of steady-state activation and inactivation of Kv channels was essentially similar for myocytes of both NP and LP rats and was comparable to results reported for small resistance arteries from different circulations (21, 32). There was a significant window current between –40 and +10 mV, implicating Kv channels in the control of uterine SMC membrane potential. These findings indicate that the decrease in the activity of Kv channels induced by pregnancy is not caused by changes in their activation and inactivation properties.

We found that the tail currents from uterine vascular myocytes were significantly reduced in pregnancy even at high positive voltages at which open probability of Kv channels is reaching the maximum (Fig. 9, A and B). Kv current amplitude is proportional to the number of the functional channels and the open probability of the individual channels (43). Therefore, these data suggest that the pregnancy-induced decrease in Kv currents is due to a reduction in the number of the functional Kv channels rather than a change in their open probability. Together, our data strongly support the idea that the reduction in the activity of Kv channels is an essential cause of enhanced pressure-induced depolarization of uteroplacental arteries in late pregnancy.

Recently, it has been shown that heteromultimeric Kv1.2 to Kv1.5 channels underlie 4-AP-sensitive Kv currents in myocytes of the rabbit portal vein (24). 4-AP-sensitive Kv1 channels also contribute to myogenic control of small mesenteric arteries (50). Here, we demonstrated that 4-AP can potently inhibit the Kv currents of uterine vascular myocytes, especially at negative levels of membrane potentials (Fig. 8). These data suggest that decreased activity of 4-AP-sensitive Kv1 channels contributes to enhanced pressure-induced depolarization in uterine arteries during late gestation.

A significant part of the outward current of uterine vascular myocytes was insensitive to 4-AP, suggesting a role of other types of Kv channels in its production. The molecular composition of SMC Kv channels in rat uteroplacental arteries remains unknown and is most likely heterogeneous. The Kv channel superfamily represents a large group of channels consisting of 12 members that are expressed in a vessel-specific manner (10, 27). A recent study using SMCs from the main uterine artery of nonpregnant women demonstrated a great diversity in the expression pattern of Kv {alpha}-subunits in freshly dissociated SMCs; expression and distribution of Kv {alpha}-subunits were significantly modulated in proliferating SMCs in culture, suggesting a link between Kv channels expression and phenotypic remodeling (39).

The mechanisms that underlie the pregnancy-induced reduction in Kv currents were not defined in the present study and might be due to suppression of channel function, modulation of channel expression, or of channel trafficking (27, 45, 65). The remarkable growth of the uterine vasculature in late pregnancy is well documented and is due to hypertrophy and hyperplasia of SMCs (2, 49, 54). It is well known that cellular growth is associated with augmented Ca2+ cell signaling that, in part, is due to depolarization of SMCs (3, 4, 34, 51). Reductions in Kv channel function and expression during vascular SMC growth resulting in a more depolarized state were demonstrated in a number of studies (34, 51). It is well documented that pregnancy is characterized by a dramatic rise in the levels of circulating estrogen, which are, in part, responsible for some of the cardiovascular adaptations during gestation (33, 38, 53). Elevated estrogen provides an essential link between cell hypertrophy and downregulation of K+ channels in the heart and the myometrium in late pregnancy (13, 58). Whether a similar estrogen-dependent mechanism operates in uteroplacental arteries remains unknown and deserves further investigation.

In conclusion, the present data demonstrate that uteroplacental artery myogenic tone is significantly increased in late pregnancy and that this phenomenon is associated with enhanced pressure-induced depolarization, Ca2+ signaling, and cellular excitability. A decrease in Kv channel activity is an important mechanism mediating the pregnancy-induced augmentation of myogenic tone in rat uteroplacental arteries. However, in contrast to the reduction in Kv currents during pathological conditions like hypertension or diabetes mellitus, pregnancy-specific modulation of Kv channel function is likely one of the normal physiological mechanisms underlying the remarkable adaptation of the maternal uteroplacental circulation in late gestation.


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This study was supported by National Heart, Lung, and Blood Institute Grants HL-67250, HL-73895, and HL-44455.


   ACKNOWLEDGMENTS
 
We thank Dr. Mark Nelson for valuable comments on this manuscript and Olga Kuzina for skilled technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. I. Gokina, Dept. of Obstetrics and Gynecology, The Univ. of Vermont, College of Medicine, Burlington, VT 05405 (e-mail: natalia.gokina{at}uvm.edu)

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


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294/1/H272    most recent
00216.2007v1
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