Fetal to maternal blood flow matching in the placenta, necessary for optimal fetal blood oxygenation, may occur via hypoxic fetoplacental vasoconstriction (HFPV). We hypothesized that HFPV is mediated by K+ channel inhibition in fetoplacental vascular smooth muscle, as occurs in several other O2-sensitive tissues. With the use of an isolated human placental cotyledon perfused at a constant flow rate, we found that hypoxia reversibly increased perfusion pressure by >20%. HFPV was unaffected by cyclooxygenase or nitric oxide synthase inhibition. HFPV and 4-aminopyridine, an inhibitor of voltage-dependent K+(Kv) channels, increased pressure in a nonadditive manner, suggesting they act via a common mechanism. Iberiotoxin, a large conductance Ca2+-sensitive K+(BKCa) channel inhibitor, had little effect on normoxic pressure. Immunoblotting and RT-PCR showed expression of several putative O2-sensitive K+ channels in peripheral fetoplacental vessels. In patch-clamp experiments with smooth muscle cells isolated from peripheral fetoplacental arteries, hypoxia reversibly inhibited Kv but not BKCa or ATP-dependent currents. We conclude that human fetoplacental vessels constrict in response to hypoxia. This response is largely mediated by hypoxic inhibition of Kv channels in the smooth muscle of small fetoplacental arteries.
- fetal growth retardation
- Kv1.5 channel
tissue hypoxia is a physiologically important regulator of vascular tone. In most organs, hypoxia causes local vasodilatation. This response increases blood flow to the affected organ and thus promotes restoration of tissue oxygenation. In the lung, which has low O2 consumption and serves as an oxygenator for the organism, alveolar hypoxia causes local vasoconstriction. This hypoxic pulmonary vasoconstriction reduces perfusion of poorly ventilated alveoli, diverting blood flow toward better ventilated areas. In this manner, the systemic blood oxygenation is optimized.
In the fetus, the organ responsible for blood oxygenation is the placenta. The maternal blood flow to the placenta, and therefore the supply of O2 to the organ, is not spatially homogeneous (33). Therefore, a mechanism for matching fetal to maternal placental blood flow would be advantageous. Although little studied, hypoxic vasoconstriction of fetal vessels in placental regions receiving poor maternal blood supply could serve this function. We hypothesize that such hypoxic fetoplacental vasoconstriction (HFPV) would redirect the flow of fetal blood from insufficiently oxygenated areas of the placenta toward regions receiving better maternal perfusion. The Po 2 in the fetal arterial blood would thus be optimized.
Despite the potential importance of HFPV, there has been minimal experimental investigation of this phenomenon (8, 15). The goal of the present study was to evaluate the mechanism of HFPV in humans. In the other known O2-sensitive tissues, such as pulmonary vessels, carotid body glomus cells, neuroepithelial bodies, and the ductus arteriosus, a role for O2-sensitive K+ channels has been identified (21, 39). In general, changes in Po 2 inhibit one or more K+ channels, thereby causing membrane depolarization, Ca2+ channel activation, Ca2+ influx, and vasoconstriction. By analogy, we hypothesized that O2-sensitive K+ channels are involved in the mechanism of HFPV.
All experiments were performed on tissue from healthy human placentas delivered at or near term vaginally or (less frequently) via an elective Cesarean section. The preparation of the isolated cotyledon for perfusion experiments started immediately after the delivery. For all other experiments (isolated vascular rings, patch clamp, RT-PCR, and immunoblotting), a sample of placental tissue was placed into an ice-cold physiological saline solution and stored for <1 h before isolation of fetoplacental vessels.
All reagents were purchased from Sigma (St. Louis, MO, or Prague, Czech Republic) unless stated otherwise. Drug doses were based on preliminary experiments.
Isolated perfused human placental cotyledon.
An intact cotyledon that was not damaged (as judged by visual inspection) was chosen for perfusion (14, 15, 36). The arterial and venous branches supplying the selected cotyledon were injected with heparin (2,500 IU each; Léciva, Prague, Czech Republic) and cannulated within 10 min of the end of the third stage of parturition.
The fetoplacental vasculature of the cotyledon was perfused with 4% dextran in Earle's balanced salt solution (Sigma) at 38°C. The perfusate was oxygenated by bubbling with a 40% O2-5% CO2-55% N2 gas mixture. Perfusion was initiated within 10 min of the delivery of the placenta. The venous outflow was discarded until it was visibly blood free (usually ∼30 min) and then it was recirculated. The initial flow rate of 2 ml/min was gradually increased to 7–8 ml/min and then kept constant. The measured changes in perfusion pressure therefore directly reflected changes in vascular resistance.
All visible vessels crossing the boundaries of the selected cotyledon were ligated. A piece of placental tissue containing the perfused cotyledon plus a ∼2-cm margin was excised and placed, maternal side down, on a wire mesh atop a funnel-shaped reservoir. The preparation was then covered with a heated (38°C) glass lid with a central opening. Through this hole, three 20-gauge needles were inserted into the lacunae of the cotyledon, thereby allowing perfusion of the maternal side of the cotyledon with a separate pool of heated and oxygenated Earle's/dextran solution (38°C, equilibrated with 40% O2-5% CO2-55% N2). To resemble the in vivo conditions, the flow to the maternal side of the isolated cotyledon was kept 2–3 times higher than the flow to the fetal side. The perfusate of the maternal side left the placenta through the remnants of the spiral arteries at the bottom of the preparation, passed through the wire mesh into the funnel, and after 1 h of perfusion, once visibly free of blood, was recirculated.
To elicit a hypoxic vasoconstrictor response, the gas mixture bubbling both perfusates (maternal and fetal) was changed to 95% N2-5% CO2 until the fetoplacental perfusion pressure stabilized (10–20 min). Repeated hypoxic challenges were separated by reoxygenation (40% O2) intervals of at least 20 min. To confirm that hypoxia rather than fluctuations in CO2 was the stimulus for HFPV, a supplementary experiment was performed with three perfused cotyledons challenged repeatedly with moderate hypercapnia (10% CO2) while the oxygenation was kept constant (40% O2).
The perfused cotyledon was used in four separate experiments. The first experiment (n = 6 preparations) was designed to test the possible modulation of HFPV by endogenous prostanoids, nitric oxide (NO), and preexisting vascular tone. The role of prostaglandins was addressed by the addition of a cyclooxygenase inhibitor (20 μM sodium meclofenamate) to the perfusate after a control hypoxic response. The hypoxic responses before and after meclofenamate were compared. Similarly, the role of NO in the modulation of HFPV was tested using an inhibitor of NO biosynthesis,N ω-nitro-l-arginine methyl ester (l-NAME; 50 μM).
To test whether the magnitude of HFPV depends on the level of preexisting vascular tone, the responses to hypoxia were compared before and after angiotensin II injection (0.2 μg bolus into fetoplacental inflow cannula). To determine the degree of active tone in the fetoplacental circuit under resting conditions, we assessed the response to sodium nitroprusside (3 doses each of 120 μg). No significant difference in perfusion pressure occurred between the second and third bolus, indicating that a maximal vasodilator dose had been reached. Perfusion pressure after the maximal dose of sodium nitroprusside compared with the value at the start of perfusion was taken as a measure of the active basal tone.
The second (n = 6) and third (n = 7) series of experiments investigated, respectively, the roles of voltage-dependent K+ channels (Kv) and large conductance Ca2+-sensitive K+(BKCa) channels in the mechanism of HFPV by measuring the effects of their inhibitors on the hypoxic responses of the perfused cotyledon. 4-Aminopyridine (4-AP; 5 mM) and iberiotoxin (100 nM) were used to block Kv and BKCa channels, respectively. A supplementary experiment with four preparations tested the effect of 4-AP on nonhypoxic vasoconstriction (induced by 0.2 μg angiotensin II).
The fourth experiment was a time control with four perfused cotyledons challenged repeatedly with hypoxia without any other intervention.
Fetoplacental vascular rings in tissue bath.
To assess the localization of hypoxic vasoconstriction in the placenta, we studied rings cut from the large arteries and veins running on the placental surface (∼1 mm resting diameter). Although we were able to isolate small arteries for measurement of mRNA and protein (see below), we could not measure tension in isolated peripheral arteries due to their small internal dimensions and fragility. However, we reasoned that if larger conduit vessels failed to constrict, the net change in vascular resistance during hypoxia in the isolated cotyledon model should reflect the contribution of the small arteries.
Placental vascular rings were studied in ring baths, as previously described for other vessels (2). Optimal resting tension was found to be ∼1,100 mg. Changes in tension in response to hypoxia were recorded. Rings were studied in Krebs solution composed of (in mM) 22.6 NaHCO3, 119 NaCl, 50 sucrose, 4.7 KCl, 1.17 MgSO4, 1.18 KH2PO4, 5.5d-glucose, and 3.2 CaCl2 at 37°C during normoxia (21% O2-5% CO2-55% N2, pH 7.4, and 120 mmHg Po 2) or hypoxia (O2 content of the bubbling gas mixture reduced to 2.5% for ∼40 min, pH 7.4, Po 2 ∼40 mmHg).
Isolation of fetoplacental vessels.
Isolation of small, peripheral fetoplacental arteries for detection of K+ channel protein (by immunoblotting) and mRNA (by RT-PCR) and for electrophysiology was initiated by making a cut into the placenta along a large fetoplacental artery running on the fetal surface of the organ. This exposed small branches of the artery diving into the placental tissue (diameter ∼500 μm and less). Segments (∼5 mm long) of up to 10 of these arteries were dissected free from the surrounding trophoblast and connective tissue under a dissecting microscope and placed into ice-cold Hanks' balanced salt solution composed of (in mM) 140 NaCl, 4.2 KCl, 1.2 KH2PO4, 0.5 MgCl2, 10 HEPES, and 0.1 EGTA, pH 7.4. The arteries were either enzymatically digested and cells dispersed for patch clamping or homogenized for RT-PCR and immunoblotting. For comparison, samples of large conduit arteries running on the surface of the placenta (∼1 mm diameter) were also taken and processed identically.
After the adventitia were carefully removed under a dissecting microscope, the fetoplacental arteries were opened longitudinally, cut in small pieces, and placed in Ca2+-free Hanks' solution for 20 min. They were then transferred for 15 min to cold (4°C) Hanks' solution without EGTA that contained (in mg/ml) 1.0 papain, 0.75 DTT, 0.8 collagenase, and 0.8 BSA. The incubation then continued for 10 min at 37°C. The arteries were then transferred to ice-cold Hanks' solution supplemented with glucose (1 mg/ml) and dispersed by a gentle trituration with the use of a Pasteur pipette. Cells were transferred to a perfusion chamber on the stage of an inverted microscope for patch-clamp studies and left to attach to the bottom of the chamber for 10–15 min.
Whole cell patch-clamp recordings were performed as previously described (40). Micropipettes (resistance 1–5 MΩ) were filled with a solution (pH 7.2) containing (in mM) 140 KCl, 1.0 MgCl2, 10 HEPES, 5 EGTA, and 10 glucose. The chamber containing the cells was slowly perfused (2 ml/min) in a nonrecirculating manner with a solution that contained (in mM) 145 NaCl, 5.4 KCl, 1.0 MgCl2, 1.5 CaCl2, 10 HEPES, and 10 glucose. This solution was bubbled in its reservoir with a gas mixture of 21% O2-5% CO2-74% N2, resulting in Po 2 of ∼140 and pH of 7.4. The current-voltage relationship was measured by increasing the membrane potential in 20 mV steps (0.1 Hz, 200 ms each) from a holding potential of −70 mV. Currents were filtered at 1 kHz and sampled at 2 or 4 kHz. Current density was expressed by dividing the whole cell K+current (I K) by cell capacitance.
The effect of hypoxia was studied by switching the gas mixture bubbling the external solution to one containing 5% CO2-95% N2, which resulted in a Po 2 of ∼40 mmHg, without a change in pH. The contribution of several types of K+ channels to I K was assessed by using preferential inhibitors: iberiotoxin (100 nM, a highly specific inhibitor of BKCa channels), glyburide (10 μM, an inhibitor of ATP-sensitive K channels), BaCl (50 μM, an inhibitor of inward rectifying K+ channels), and 4-AP (5 mM).
Total RNA was isolated from homogenized peripheral fetoplacental vessels using an RNeasy mini Kit (Qiagen; Missisauga, ON, Canada). RNA (2 μg) was reverse transcribed using Qiagen Omniscript reverse transcriptase, as previously described (3). For K+ channels previously implicated in O2sensitivity (2, 4, 9, 16, 30), PCR primers were designed based on human cloned sequences from GeneBank (Table1). The number of cycles chosen for each primer was within the linear region of the amplification curve. PCR products were sequenced and confirmed to be identical to the intended amplified sequence for each primer set. A no-RT control was used for each primer (shown only for β-actin) to ensure the absence of DNA contamination.
Immunoblotting was performed as previously described (3, 4,22). Antibodies against putative O2-sensitive K+ channels (Alomone; Jerusalem, Israel) were detected with secondary antibodies (Pierce; Rockford, IL) and enhanced chemiluminescence (Amersham; Buckinghamshire, UK). The specificity of each primary antibody for the intended antigen was confirmed in competition experiments in which incubation with an excess of the relevant antigen neutralized the antibody.
The immunobloting and PCR data are qualitative. Hypoxia- and pharmacological K+ channel blocker-induced changes in current-voltage curves were evaluated using ANOVA for repeated measures. All other data were analyzed using a pairedt-test. P < 0.05 was considered significant. The results are reported as means ± SE. Sample sizes are noted in the figure legends.
Hypoxia invariably caused significant fetoplacental vasoconstriction. The time course of the hypoxic response is illustrated in Fig. 1. Vasoconstriction started within 5 min after the switch to hypoxia and reached a plateau within 10–20 min. The response was reproducible with repetition in the same preparation (not shown). The HFPV was ∼80% reversible within minutes of reoxygenation. The average magnitude of control hypoxic responses from all perfusion experiments (n = 36) was 23 ± 3% of baseline perfusion pressure (from 38 ± 2 to 46 ± 2 mmHg; P < 0.0001) with a fetoplacental effluent Po 2 decrease from 122 ± 8 to 61 ± 2 mmHg (P < 0.0001). The pH was 7.32 ± 0.02 before and 7.28 ± 0.02 during hypoxia (P = 0.051). Pco 2 was 45.9 ± 1.4 mmHg before and 48.6 ± 1.7 mmHg during hypoxia (P = 0.07). In individual preparations, HFPV was similar regardless of whether pH and Pco 2slightly increased, decreased, or were unchanged during hypoxia.
The possible role of pH/Pco 2 changes was further examined in a small supplementary experiment with hypercapnic challenges. In this particular group, eucapnic hypoxia increased fetoplacental perfusion pressure by 29 ± 1%. Hypercapnia (CO2 increased from 5 to 10%) with unchanged oxygenation significantly increased effluent Pco 2 (from 52 ± 3 to 70 ± 2 mmHg, P < 0.05,n = 3) and reduced pH (from 7.24 ± 0.03 to 7.07 ± 0.04, P < 0.01), but had no effect on perfusion pressure (27.3 ± 1.5 mmHg before and 27.9 ± 1.6 mmHg during hypercapnia, P = 0.09).
Meclofenamate, a cyclooxygenase inhibitor, had no effect on baseline perfusion pressure (29.1 ± 0.9 mmHg before and 29.6 ± 1.1 mmHg after meclofenamate) or the magnitude of HFPV (Fig.2). l-NAME, an inhibitor of NO synthase, increased the baseline perfusion pressure from 31 ± 1 to 36 ± 2 mmHg (P = 0.02, n = 6). However, the magnitude of HFPV was not significantly affected (Fig.2). Thus, although NO appears to contribute to maintaining low tone in the fetoplacental vasculature, it does not have a major role in HFPV. Furthermore, the data with NO synthase inhibition show that elevating vascular wall tension does not enhance HFPV. This conclusion is further supported by the observation that an additional elevation of perfusion pressure by angiotensin II to 47 ± 2 mmHg (a plateau achieved after a transient peak to 61 ± 4 mmHg) does not increase the subsequent hypoxic response (Fig. 2). The initial basal tone at the start of the perfusion was minimal, as judged from the negligible vasodilatation that occurred in response to a high dose of sodium nitroprusside. The baseline perfusion pressure in this series was 27 ± 2 mmHg at the beginning of the perfusion and 24 ± 3 mmHg after sodium nitroprusside (P = 0.2,n = 6).
To assess the localization of HFPV, the response to hypoxia was measured in isolated fetoplacental vessels mounted in an organ bath. Large conduit fetoplacental arteries responded to hypoxia by relaxation: their tension decreased from 1,065 ± 35 mg in normoxia to 789 ± 106 mg in hypoxia (P < 0.05,n = 9). Large veins exhibited a similar tendency. In seven venous rings, tension was 1,101 ± 68 mg in normoxia and 960 ± 39 mg in hypoxia (P = 0.17). Although we could not measure tone in the small peripheral vessels, the existence of hypoxic vasoconstriction in the whole cotyledon and its absence in the large fetoplacental vessels implicate fetoplacental microvessels as the site of HFPV.
RT-PCR analysis of K+ channels implicated in O2 sensing (2, 4, 16, 30) showed that homogenates from peripheral human fetoplacental arteries contain mRNA for Kv1.5, Kv2.1, and BKCa, but not Kv1.2 (Fig.3). All primers (except Kv3.1b) detected the relevant channel mRNA in human brain homogenate (Fig. 3), which was used as a standard because of its known abundant expression of virtually all Kv channel types. Immunoblotting confirmed the presence of proteins for the putative O2-sensitive K+ channels, where the mRNA was detected by RT-PCR. Kv3.1b protein was also detected, although we did not have a primer that detected the relevant mRNA under our experimental conditions (Fig.4).
Initial patch-clamp measurements using smooth muscle cells isolated from a mixture of large and small fetoplacental vessels showed that within minutes of being switched to hypoxia (Po 2 ∼ 40 mmHg), I Kdropped by ∼30%, and this response was fully reversible on reoxygenation (Fig. 5). Because the results with isolated vascular rings suggested a differential involvement of small and large fetoplacental vessels (see above), cells from these two sources were separated in subsequent patch-clamp experiments.
Smooth muscle cells isolated from small peripheral arteries had significantly higher I K density than cells from large conduit arteries (Fig. 6). The combination of iberiotoxin, glyburide, and barium (to inhibit K+ channels other than Kv) diminishedI K to a similar degree in both cell populations, whereas 4-AP caused significant I K reduction only in cells from small arteries (Fig. 6). Iberiotoxin alone had similar effects as it had in combination with glyburide and barium, and glyburide alone did not affect I K in these cells (data not shown). The conclusion that the two smooth muscle cell populations have similar non-Kv current and cells from small arteries differ from those from large vessels by also having a significant Kv current was confirmed by subtraction analysis (Fig. 7).
In the presence of iberiotoxin, glyburide, and barium, hypoxia significantly reduced I K in cells from small, but not large, arteries (Fig. 6, E and F). In the presence of 4-AP, hypoxia did not cause any furtherI K inhibition in any cell population (Fig. 6,A and D). Subtraction analysis revealed that the hypoxia-sensitive portion of I K was considerably greater in cells from peripheral than from conduit arteries, where it was minimal (Fig. 7). Importantly, subtraction current-voltage relationships were almost superimposable for hypoxia and 4-AP (Fig. 7), implying that both hypoxia and 4-AP act on the same channel(s).
The functional relevance of the hypoxic reduction ofI K in fetoplacental vascular smooth muscle was studied in isolated perfused cotyledon. The Kv channel blocker 4-AP (5 mM) mimicked the vasoconstrictor effect of hypoxia. In this series of six experiments, adding 4-AP (5 mM) into the perfusate caused a rise in perfusion pressure from 41 ± 4 to 52 ± 6 mmHg (Fig. 8, A andB). This increase was similar in magnitude to that elicited by hypoxia (Fig. 8, A and B). When the preparation was challenged with hypoxia in the presence of 4-AP, the fetoplacental perfusion pressure did not rise above the level to which it had already been elevated by 4-AP (Fig. 8, A andB).
To confirm that the inhibitory effect of 4-AP was selective for the hypoxic response, a small supplementary experiment was performed with a similar protocol except that after reaching the plateau of the 4-AP vasoconstriction (from 35 ± 4 to 44 ± 5 mmHg, P < 0.05), angiotensin II was given instead of the hypoxic challenge. Unlike hypoxia, angiotensin II caused a large vasoconstriction in the fetoplacental vascular bed already constricted by 4-AP (to a peak of 66 ± 6 mmHg, P < 0.01,n = 4). This shows that the abolition of HFPV by 4-AP was not due to achieving maximal vasoconstriction with 4-AP.
In a separate series of seven experiments, iberiotoxin, a selective BKCa inhibitor, had no significant effect on normoxic perfusion pressure (48 ± 4 mmHg before and 53 ± 5 mmHg after iberiotoxin, P = 0.126), although there was a small tendency for an increase in three of the preparations (Fig. 8,C and D). In the presence of iberiotoxin, HFPV was reduced by ∼30% (Fig. 8, C and D).
This study clearly demonstrates a marked vasoconstriction of the human fetoplacental vessels in response to acute hypoxia. We show that hypoxic inhibition of Kv channels present in the fetoplacental vascular smooth muscle underlies this phenomenon. HFPV is most likely localized in small fetoplacental vessels, as implicated by the absence of hypoxic constriction in the larger fetoplacental vessels and because the hypoxic response of K+ channels in smooth muscle cells from small arteries is considerably greater than that in large arteries.
Although the existence of HFPV has been frequently assumed (23,32), it has not previously been well experimentally documented. Its existence has been inferred from the fetoplacental vasoconstriction elicited by a mechanical restriction of the perfusion of the uterus (37). However, factors other than hypoxia could act as stimuli in this situation, e.g., reduced supply of nutrients or impaired removal of fetal metabolites. The first direct indication that hypoxia per se causes fetoplacental vasoconstriction was in a study by Howard et al. (15). In their experiments on perfused human cotyledons, the increases in perfusion pressure with hypoxia were small (∼10% above baseline). In our study, the hypoxic responses were more than twice as large. This demonstration of a functionally relevant magnitude of HFPV is in agreement with a recent study by Byrne et al. (8).
Because vascular reactivity in many organs is modulated by endothelial production of vasodilatory prostaglandins, we hypothesized that HFPV might be increased by cyclooxygenase inhibition. That proved not to be the case (Fig. 2), suggesting that endogenous production of prostaglandins does not modulate HFPV in humans. Our experiments do not distinguish whether the reason for this finding is that cyclooxygenase activity is not altered by hypoxia or that the human HFPV is insensitive to changes in prostaglandin levels. Our data are consistent with those of King et al. (19), who showed no effect of cyclooxygenase inhibition on baseline perfusion pressure and on vasoconstrictor reactivity to the thromboxane analog U-46619 in perfused lobules of human placenta.
Another potent modulator of reactivity in many vascular beds is endothelial NO. NO synthase is richly expressed in the fetoplacental vasculature (25, 26). Therefore, we addressed the possibility that endogenous NO modulates HFPV in a similar manner as it alters reactivity to numerous pharmacological vasoconstrictors (19). In agreement with several previous studies (8,11, 19, 24), we found NO synthase inhibition byl-NAME to cause vasoconstriction during normoxia (confirming effectiveness of the dose). The hypoxic vasoconstriction, however, was unaltered (Fig. 2). Our interpretation of this finding is that in the human placenta there is a basal, “tonic” production of NO (the inhibition of which increases vascular tone), which is not altered during acute exposure to hypoxia.
Our finding of HFPV unaltered by NO synthase inhibition contradicts the study of Byrne et al. (8), where l-NAME abolished hypoxic reactivity in perfused placental cotyledon. This discrepancy may be related to a considerably higher l-NAME dose (0.3 mM) or to the lower Po 2 (∼40 mmHg) achieved during the hypoxic challenges in the study of Byrne et al. (8). High doses of NO synthase blockers may be associated with nonspecific effects (7, 31, 38), possibly contributing to unexpected results. The l-NAME dose used in our study has been shown to be sufficient to inhibit NO synthase (12).
In general, lowering Po 2 is expected to inhibit NO biosynthesis, because O2 is a substrate for NO synthase. However, because of various cellular compensatory mechanisms, the drop in Po 2 needed to cause a significant inhibition of NO synthase appears quite large (13, 35). It is thus possible that the Po 2 achieved by Byrne et al. (8) was low enough to inhibit NO synthesis, whereas Po 2 in our experiments was not. Although the degree of hypoxia reported by Byrne et al. (8) is closer to that expected in vivo, our finding of a normal HFPV during NO synthase inhibition refutes their conclusion that reduction of NO synthesis is the basis of HFPV. This shows that other mechanisms must be in action, and we turned to the possible role of K+channels.
In several O2-sensing tissues, hypoxic inhibition of K+ channels (particularly homo- or heterotetramers containing Kv1.2, Kv1.5, and Kv2.1 α-subunits, and possibly Kv3.1b) is thought to contribute to the mechanism of hypoxic sensing (2,4, 16, 30). BKCa channels are also implicated in O2 sensing in fetal pulmonary vessels (9). We found that these K+ channels, except for Kv1.2, are expressed in peripheral fetoplacental vessels (Figs. 3and 4). The presence of a 4-AP and voltage-sensitive Kvcurrent in the patch-clamp studies is consistent with a contribution of Kv1.5 and Kv2.1 to the I K ensemble. The ability of iberiotoxin to reduce I K shows that BKCa channels also contribute to I K. However, the portion of the current that is inhibited by hypoxia is sensitive to 4-AP but not iberiotoxin. Importantly, we demonstrated the specificity of all RT-PCR products and antibodies and furthermore showed that these probes effectively detected channel mRNA and protein in the human brain, a rich source of K+ channels.
The fetoplacental vascular smooth muscle cells display rapid and reversible hypoxic inhibition of I K (Figs. 5 and7). Because K+ channels are central to the regulation of membrane potential in vascular smooth muscle (28), their inhibition is expected to cause membrane depolarization that, in turn, activates the voltage-gated Ca2+ channels and thereby causes Ca2+ influx. The resulting increase in intracellular Ca2+ concentration is a known stimulus for activation of the contractile apparatus. This proposed chain of events remains to be tested in human fetoplacental arterial smooth muscle cells. The mechanism whereby hypoxia inhibits K+ channels also has yet to be found. This may be a direct effect of hypoxia on the channel or alternatively act through an O2 sensor mechanism (for review, see Ref. 5).
Our data with K+ channel inhibitors in perfused placenta show that the electrophysiologically documented inhibition of fetoplacental vascular smooth muscle K+ channels by low Po 2 does indeed underlie the vasoconstrictor response of the placenta to hypoxia. Importantly, Kvchannels are much more important in this respect than BKCachannels. A dose of 4-AP that is selective for Kv channel inhibition (27, 28) mimics and blocks HFPV, suggesting that 4-AP and hypoxia act by the same mechanism. By contrast, iberiotoxin at a dose highly selective for BKCa channel inhibition (27, 28) has a minimal effect on fetoplacental perfusion pressure and only partially reduces HFPV. In light of our electrophysiological finding of small hypoxic sensitivity of BKCa-dominated I K in smooth muscle cells from large arteries, contrasted with large hypoxic reactivity of Kv-dominated I K of cells from small arteries, these data are consistent with an interpretation that a major portion of HFPV is mediated by Kv closure in small vessels. Hypoxic inhibition of BKCa channels (also in large vessels) plays a minor role.
The absence of hypoxic contraction in isolated rings of large conduit fetoplacental arteries suggests that HFPV is localized in small vessels. An alternative explanation could be that placental parenchyma responds to hypoxia by releasing constrictor factor(s) that subsequently increase placental tone. HFPV thus would be lost once the arteries are isolated, regardless of their size. Certain, although probably not crucial, involvement of surrounding tissue in O2 sensing related to vascular regulation has been documented in the lung (1, 10, 18), which is analogous to the placenta by also responding to hypoxia with vasoconstriction. However, our patch-clamp data show that hypoxia-sensitive K+ current was much higher in the cells from small arteries, whereas it was minimal in cells from large vessels (Figs. 6and 7). Because of the demonstration that the hypoxic K+channel participates in HFPV (see above), these data strongly support the idea that small arteries are much more important in HFPV than the large ones.
A methodological issue that requires a brief comment is that of baseline perfusion pressures in isolated cotyledons. Because the preparations differed considerably in the size of the perfused placental tissue while the perfusion flow rate was always similar, the values of baseline perfusion pressure varied greatly (range 20–61 mmHg). In many vascular beds, the vasoconstrictor reactivity is affected by the level of pretone, typically in the sense of greater vasoconstriction if the baseline tension is higher. In the present study, however, we found no correlation between the baseline perfusion pressure and HFPV magnitude within our range of pressures (analysis not shown). The average baseline perfusion pressure (38 ± 2 mmHg,n = 36) was close to the value reported in vivo (41).
One methodological limitation of this study that has not been yet mentioned is that of Po 2 during the hypoxic challenges. Because of the O2 permeability of tubing in perfusion systems and of the placental tissue itself, we were not able to lower effluent Po 2 to <33 mmHg. In fact, the average Po 2 during the hypoxic challenges was ∼60 mmHg (effluent) in perfusion experiments and ∼40 mmHg (bath) in patch-clamp studies. Although the drop in Po 2 from baseline was considerable, these values are still higher than the normal Po 2 in the fetoplacental circulation in vivo (∼30 mmHg) (29). On the basis of an analogy with other O2-sensing tissues, it is unlikely that HFPV is fundamentally different at lower Po 2; however, the exact characteristics of HFPV in vivo remain to be determined.
We speculate that HFPV is pathophysiologically important in humans. In fact, despite the limited published information on HFPV to date, it is often cited as a principal factor in the pathogenesis of intrauterine growth retardation (IUGR) (23, 32), a major cause of neonatal mortality and morbidity affecting 3% of newborns (17,20, 34). Newborns with IUGR, especially when combined with prematurity, are at increased risk for extreme respiratory distress syndrome (20), severe bronchopulmonary dysplasia (17), and adverse neurodevelopmental outcome (34). Because of that risk, management of premature newborns with IUGR remains one of the main challenges in neonatology. Of those who survive the neonatal period, 8–10% do not catch up postnatally in growth. In addition, the long-term consequences of IUGR include cardiovascular disease, obesity, and non-insulin-dependent diabetes mellitus (6).
How does HFPV relate to IUGR? We speculate that if HFPV affects most of the placenta (due to maternal hypoxia or uteroplacental dysfunction), it increases the total fetoplacental hemodynamic resistance and consequently impairs the perfusion of the fetal side of the placenta (23, 32). Reduced fetoplacental blood flow, in turn, is thought to impair fetal growth (23, 32). Understanding the mechanism of HFPV thus might ultimately facilitate new treatments to prevent or minimize IUGR.
The authors thank Dr. Jan Stulc for help with the perfused cotyledon model, Dr. Jan Simak and Stacy Freeman for help in arranging the supply of the placentas, Ross Waite for assistance with immunoblotting, and Dr. Bernard Thébaud for a critical review of the study. We also thank the obstetrical staff of the Institute for the Care of Mother and Child (Prague, Czech Republic) for help in obtaining fresh placental tissue, as well as the mothers whose placentas were used.
This work was supported by a grant from the Hospital Foundation at the University of Alberta and North Atlantic Treaty Organization Science Program Collaborative Research Grant LST-CLG 975202. V. Hampl is supported by Czech Ministry of Health Grant 4538 and Czech Ministry of Education Research Project 111300002. J. Bı́bová is supported by the Grant Agency of the Charles University Grant 55/2001/c/2.LF. S. L. Archer and E. D. Michelakis are supported by the Heart and Stroke Foundation of Canada, the Alberta Heritage Foundation for Biomedical Research, the Canadian Institutes of Health Research, the Canadian Foundation for Innovation, and the University of Alberta Hospital Foundation.
Address for reprint requests and other correspondence: V. Hampl, Dept. of Physiology, Charles Univ., Second Medical School, 15006 Prague 5, Czech Republic (E-mail:).
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.
August 22, 2002;10.1152/ajpheart.01033.2001
- Copyright © 2002 the American Physiological Society