Relaxation of the Aorta During Hypoxia Is Impaired in Chronically Hypertensive Rats
Abstract We investigated mechanisms by which hypoxia produces relaxation of the aorta and tested the hypothesis that these mechanisms are altered during chronic hypertension. Tension of thoracic aortae from normotensive Wistar-Kyoto (WKY) rats and stroke-prone spontaneously hypertensive rats (SHRSP) was measured in an organ bath under control conditions and at two levels of hypoxia. In WKY rats, mild and severe hypoxia produced relaxation of the aortae (precontracted with phenylephrine) by 33±4% and 82±3%, respectively (mean±SEM). Removal of endothelium or administration of NG-nitro-l-arginine (10−4 mol/L), an inhibitor of nitric oxide synthase, abolished relaxation of the aortae in response to mild hypoxia but did not affect relaxation during severe hypoxia. Glibenclamide (10−6 mol/L), an inhibitor of potassium channels, attenuated relaxation of the aortae during mild and severe hypoxia by 49±16% and 74±4%, respectively. In SHRSP, mild hypoxia produced little relaxation of the aortae (3±4%, P<.05 compared with WKY). Indomethacin did not increase relaxation to mild hypoxia in SHRSP, which suggests that a cyclooxygenase-derived contracting factor does not contribute to impaired relaxation. Severe hypoxia relaxed the aortae by 86±4% in SHRSP, and glibenclamide inhibited this response by 60±9%. These findings suggest that relaxation of the aorta in response to mild hypoxia in WKY rats is mediated primarily by endothelium-derived relaxing factor, and the response to mild hypoxia is markedly impaired in SHRSP. In contrast, relaxation during severe hypoxia is mediated, in large part, by activation of glibenclamide-sensitive potassium channels, and the response to severe hypoxia is preserved in SHRSP.
- potassium channels
- endothelium-derived relaxing factor
- rats, inbred WKY
- rats, inbred SHR
- nitro compounds
Hypoxia produces marked relaxation of blood vessels in vitro and in vivo.1 2 The precise mechanism by which hypoxia produces vasorelaxation is unclear. Relaxation of coronary arteries and cerebral arterioles in response to hypoxia appears to be mediated in part by activation of glibenclamide-sensitive potassium channels.3 4 Although some studies suggest that endothelium-derived relaxing factor (EDRF) mediates vasorelaxation during hypoxia,5 6 other studies do not support this hypothesis.7 8 9 The first goal of the present study was to determine whether either activation of glibenclamide-sensitive potassium channels or production of EDRF mediates hypoxia-induced relaxation of the thoracic aorta. We measured relaxation of the aortae from normotensive Wistar-Kyoto (WKY) rats in response to two levels of hypoxia and examined effects of glibenclamide (an inhibitor of potassium channels) and an inhibitor of nitric oxide synthase on hypoxia-induced relaxation.
Vasodilator responses to endothelium-dependent agonists are impaired in stroke-prone spontaneously hypertensive rats (SHRSP).10 We have shown that dilatation of the basilar artery in response to submaximal concentrations of aprikalim, a direct activator of ATP-sensitive potassium channels, is reduced in SHRSP.11 We anticipated that if either EDRF or activation of potassium channels were involved, hypoxia-induced vasorelaxation may be impaired in SHRSP. Thus, the second goal of the present study was to determine whether relaxation of the aorta in response to hypoxia is impaired in SHRSP.
Preparation of Vascular Rings
Male WKY rats (6 to 8 months old, n=24) and age-matched SHRSP (n=22) were used in this study. Blood pressure was measured by the tail-cuff method in unanesthetized animals. Systolic blood pressure was 221±16 mm Hg in SHRSP and 158±7 mm Hg in WKY (n=4, P<.05). Rats were anesthetized with pentobarbital (50 mg/kg IP), and the thoracic aorta of each rat was removed immediately and placed in Krebs’ buffer with the following composition (mmol/L): NaCl 118.3, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, and glucose 12. Loose connective tissue in the adventitia was removed and the vessel was cut into rings (3 to 4 mm in length).
Aortic rings were suspended in an organ bath containing 20 mL Krebs’ solution maintained at 37°C and bubbled with a mixture of 95% O2 and 5% CO2. High partial pressures of oxygen are routinely used for control conditions in studies of the aorta in vitro to provide adequate delivery of oxygen throughout the vessel wall.12 The rings were connected to a force transducer to measure isometric tension. Resting tension was increased stepwise to reach the final tension of 1.0 g, and the rings were allowed to equilibrate for at least 30 minutes. Phenylephrine (10−9 to 10−5 mol/L) was applied in a cumulative manner, and the concentration of phenylephrine that caused a half-maximal response was used to precontract the rings.
After precontraction with phenylephrine, hypoxia was produced by changing the bubbling gas from the control mixture (95% O2 and 5% CO2) to a hypoxic mixture, and changes in tension were measured. The gas mixture for mild hypoxia contained 9% O2, 5% CO2, and 86% N2, and the mixture for severe hypoxia contained 95% N2 and 5% CO2. After hypoxia-induced relaxation reached a steady state, an aliquot of the buffer was collected for measurement of pH, Po2, and Pco2. During mild and severe hypoxia, Po2 in the buffer decreased from 345±26 mm Hg to 93±3 and 48±3 mm Hg, respectively. pH and Pco2 were not altered during hypoxia.
We examined the effects of NG-nitro-l-arginine (L-NNA), an inhibitor of nitric oxide synthase,13 and glibenclamide, an inhibitor of ATP-sensitive potassium channels,8 14 on relaxation of the aortae during hypoxia. L-NNA at a concentration of 10−4 mol/L was applied 30 minutes before and during exposure to hypoxia. This concentration of L-NNA abolished acetylcholine-induced relaxation of the aortae without affecting vasorelaxation in response to sodium nitroprusside in both strains.
Glibenclamide (10−6 mol/L) was applied 10 minutes before and during exposure to hypoxia. Because glibenclamide was dissolved in dimethyl sulfoxide (DMSO), control experiments were performed in the presence of DMSO vehicle (0.1%). Glibenclamide produced almost complete inhibition of relaxation of the aortae in response to aprikalim, a selective activator of ATP-sensitive potassium channels,14 without affecting sodium nitroprusside–induced vasorelaxation in both strains. In an additional set of experiments (n=5), we also examined the effects of charybdotoxin (5×10−8 mol/L), a selective inhibitor of calcium-activated potassium channels,15 on responses to hypoxia.
An endothelium-derived contracting factor (EDCF), which can be inhibited by indomethacin, is released in response to some stimuli in SHRSP.16 In an additional set of experiments (n=4), we attempted to examine responses to hypoxia in the presence of indomethacin (10−5 mol/L). In the presence of indomethacin, phenylephrine failed to produce sustained contraction of the thoracic aortae from SHRSP. Thus, we could not examine responses to hypoxia in SHRSP after indomethacin. We therefore used KCl (40 mmol/L) to produce submaximal contraction of the aortae in additional experiments. Responses to hypoxia were examined in the absence and presence of indomethacin (10−5 mol/L).
We also tested the effects of removal of endothelium on vasorelaxation during hypoxia. To denude endothelium, we removed the intimal surface by gentle rolling with forceps. Denudation abolished acetylcholine-induced relaxation of the rings without affecting vasorelaxation in response to sodium nitroprusside (data not shown).
All data are expressed as mean±SEM. Student’s t test was used to compare absolute values, and the Mann-Whitney U test was used to compare percent changes. P<.05 was considered to be statistically significant.
Hypoxia-Induced Vasorelaxation in WKY Rats
Mild hypoxia produced relaxation of the aortae from normotensive WKY rats by 41±6% (Fig 1⇓). Severe hypoxia produced transient slight contraction of the aortae that was followed by relaxation, which reached a maximum of 86±5% within 15 minutes after the onset of severe hypoxia (Fig 1⇓).
L-NNA (10−4 mol/L) almost abolished relaxation of the aortae in response to mild hypoxia. In contrast, L-NNA did not affect vasorelaxation produced by severe hypoxia (P>.05) (Fig 1⇑). L-NNA also did not alter relaxation of the aortae in response to nitroprusside: the aortae relaxed by 27±6% and 31±7% in response to 3×10−9 mol/L nitroprusside and 80±3% and 85±3% in response to 3×10−8 mol/L nitroprusside in the absence and presence of L-NNA, respectively. Removal of endothelium also abolished relaxation of the aortae during mild hypoxia, but did not attenuate severe hypoxia-induced vasorelaxation (P>.05) (Fig 1⇑).
Glibenclamide (10−6 mol/L) inhibited relaxation in response to mild and severe hypoxia by 49±16% and 74±4%, respectively (P<.05, Fig 2⇓). Glibenclamide did not alter relaxation in response to nitroprusside. Nitroprusside (10−8 and 10−7 mol/L) relaxed the aortae by 58±2% and 100±0%, respectively. In the presence of glibenclamide, 10−8 and 10−7 mol/L nitroprusside produced 50±2% and 98±2% relaxation, respectively.
In other experiments we tested the effects of charybdotoxin, an inhibitor of calcium-activated potassium channels, on hypoxia-induced vasorelaxation. Charybdotoxin (5×10−8 mol/L), in contrast to glibenclamide, did not affect hypoxia-induced vasorelaxation. Mild and severe hypoxia relaxed the aortae by 36±4% and 84±4% in the absence and 29±6% and 73±6% in the presence of charybdotoxin (n=5), respectively.
Hypoxia-Induced Vasorelaxation in SHRSP
Mild hypoxia did not produce significant relaxation of the aortae from SHRSP (3±4%). This response was profoundly impaired compared with responses of aortae from WKY rats (P<.05). In contrast, severe hypoxia relaxed the aortae by 86±4% in SHRSP (Fig 3⇓), a response similar to that observed in WKY (Fig 2⇑). Glibenclamide (10−6 mol/L) did not significantly affect response of the aortae from SHRSP to mild hypoxia, and attenuated vasorelaxation in response to severe hypoxia by 60±9% (P<.05, Fig 3⇓). Nitroprusside produced marked relaxation of the aortae from SHRSP that was not affected by glibenclamide (data not shown). Charybdotoxin (5×10−8 mol/L) did not significantly affect the response of the aortae from SHRSP to mild and severe hypoxia. Mild and severe hypoxia relaxed the aortae by 3±2% and 89±2% in the absence and 2±2% and 92±2% in the presence of charybdotoxin (n=5), respectively.
Mild hypoxia failed to produce relaxation of the aortae from SHRSP after indomethacin; they contracted by 13±2%. Indomethacin did not alter the response to severe hypoxia in aortae from SHRSP, which relaxed by 74±4%.
There are two major new findings in the present study. First, in WKY, relaxation of the aorta in response to mild hypoxia is mediated primarily by EDRF. Relaxation of the aorta to severe hypoxia is mediated, in large part, by activation of glibenclamide-sensitive potassium channels. Second, relaxation of the aorta in SHRSP in response to mild hypoxia is profoundly impaired. In contrast, relaxation of the aorta in response to severe hypoxia is not impaired in SHRSP. Vasorelaxation during severe hypoxia is mediated by activation of glibenclamide-sensitive potassium channels in SHRSP as well as in WKY.
Hypoxia-Induced Vasorelaxation in WKY Rats
Hypoxia produced relaxation of the aortae from WKY rats. Severe hypoxia produced significantly greater responses than mild hypoxia. Thus, mechanisms that mediate relaxation of the aorta during hypoxia are dependent on the level of Po2.
NG-nitro-l-arginine, an inhibitor of nitric oxide synthase, or removal of endothelium abolished relaxation of the aortae from WKY rats during mild hypoxia. These results suggest that endothelium and production of EDRF mediate relaxation of the aorta in WKY rats during mild hypoxia. Glibenclamide also produced some inhibition, suggesting that activation of potassium channels may also be involved in relaxation of the aorta during mild hypoxia.
It is not clear whether mild hypoxia increases production of EDRF or prolongs its activity. Vascular endothelium appears to contain ATP-sensitive potassium channels,17 and hyperpolarization produced by activation of potassium channels in endothelium may activate calcium influx through receptor-operated cation channels18 and thereby release EDRF. Alternatively, reduction in formation of oxygen radicals (which inactivate EDRF) during mild hypoxia may also contribute to enhanced activity of EDRF in isolated vessels.
Glibenclamide produced marked inhibition of relaxation of the aorta during severe hypoxia. The results suggest that relaxation of the aorta in response to severe hypoxia is mediated, in large part, by activation of glibenclamide-sensitive potassium channels. Because glibenclamide did not completely inhibit vasorelaxation during severe hypoxia, other factors may also contribute to relaxation in response to severe hypoxia. In several experiments, we tried a higher concentration of glibenclamide (10−5 mol/L) but found that contraction to phenylephrine was impaired, probably because a higher concentration of vehicle (1% DMSO) was needed.
The concentration of glibenclamide used in this study is generally considered to be relatively specific for ATP-sensitive potassium channels.19 It has been suggested that higher concentrations of glibenclamide (10−5 mol/L) may modulate the activity of large conductance calcium-activated potassium channels as well as ATP-sensitive potassium channels in the rabbit aorta.16 Our findings with glibenclamide at a concentration of 10−6 mol/L suggest that relaxation of the aorta may be mediated by activation of ATP-sensitive potassium channels. We cannot exclude the possibility, however, that calcium-activated potassium channels play some role.
We also tested the effects of charybdotoxin, an inhibitor of calcium-activated potassium channels,15 on hypoxia-induced relaxation of the aorta. Charybdotoxin had no effect on relaxation of the aortae during hypoxia, suggesting that calcium-activated potassium channels do not mediate this response. The concentration of charybdotoxin used in this study (50 nmol/L) was selected on the basis of previous reports in which 1 to 50 nmol/L of the toxin inhibited relaxation of large arteries in vitro.15 20 21 However, other studies suggest that the efficacy of charybdotoxin may be reduced in intact preparations (compared with its effects in patch-clamp studies) examined in the presence of physiological levels of extracellular calcium.22 23 Thus, we cannot completely exclude the possibility that stimulation of calcium-activated potassium channels may contribute to vasorelaxation during hypoxia.
Hypoxia-Induced Vasorelaxation in SHRSP
Mild hypoxia failed to relax the aortae from SHRSP. Endothelium-dependent relaxation of the aorta is reduced during chronic hypertension.10 24 Thus, impaired responses of the aorta to mild hypoxia may be due to impairment of endothelium-dependent relaxation in SHRSP.
Impairment of endothelium-dependent relaxation in SHRSP in response to some stimuli appears to be due to formation of an EDCF that can be inhibited by indomethacin.24 We considered the possibility that formation of an EDCF contributes to the impaired relaxation of the aorta in response to mild hypoxia in SHRSP. We found that in the presence of indomethacin, mild hypoxia had little effect in the KCl-contracted aortae of SHRSP. These data suggest that a cyclooxygenase-derived EDCF may not contribute to impaired relaxation in response to mild hypoxia in SHRSP.
Severe hypoxia produced similar relaxation of the aortae from WKY rats and SHRSP, and glibenclamide inhibited vasorelaxation during severe hypoxia. Thus, activation of glibenclamide-sensitive potassium channels is the primary mechanism by which severe hypoxia produced vasorelaxation, and the response is preserved in SHRSP. Dilatation in response to submaximal, but not maximal, activation of glibenclamide-sensitive potassium channels is impaired in SHRSP.11 Therefore, we speculate that relaxation of the aorta during severe hypoxia may be mediated by maximal activation of glibenclamide-sensitive potassium channels, and thus is not impaired in SHRSP.
In conclusion, relaxation of the aorta in response to mild hypoxia is mediated primarily by EDRF, and the response to severe hypoxia is mediated, in large part, by activation of glibenclamide-sensitive potassium channels. Vasorelaxation in response to mild hypoxia is markedly impaired in SHRSP, probably because endothelium-dependent relaxation is impaired. Vasorelaxation in response to severe hypoxia is not impaired in SHRSP, probably because maximal activation of glibenclamide-sensitive potassium channels by hypoxia is not impaired in these rats.
This study was supported by National Institutes of Health grants HL-16066, NS-24621, AG-10269, HL-14388, and HL-38901; by research funds from the Veterans Administration; and by a Grant-in-Aid from the American Heart Association (92015170). F.M. Faraci is an Established Investigator of the American Heart Association.
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