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1Department of Physiology and 2Cardiovascular Research Center, Medical College of Wisconsin, and 3Clement Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin
Submitted 26 December 2006 ; accepted in final form 28 September 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-hydroxylase metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE), and generation of superoxide in the hypoxia-evoked activation of the KCa channel current in rat cerebral arterial muscle cells (CAMCs) and cerebral vasodilation. Patch-clamp analysis of K+ channel current identified a voltage- and Ca2+-dependent 238 ± 21-pS unitary K+ currents that are inhibitable by tetraethylammonium (TEA, 1 mM) or iberiotoxin (100 nM). Hypoxia (<2% O2) reversibly enhanced the open-state probability (NPo) of the 238-pS unitary KCa current in cell-attached patches. This effect of hypoxia was not observed on unitary KCa currents recorded from either excised inside-out or outside-out membrane patches. Inhibition of CYP 4A -hydroxylase activity increased the NPo of KCa single-channel current. Hypoxia reduced the basal endogenous level of 20-HETE by 47 ± 3% as well as catalytic formation of 20-HETE in cerebral arterial muscle homogenates as determined by liquid chromatography-mass spectrometry analysis. The concentration of authentic 20-HETE was reduced when incubated with the superoxide donor KO2. Exogenous 20-HETE (100 nM) attenuated the hypoxia-induced activation of the KCa current in CAMCs. Hypoxia did not augment the increase in NPo of KCa channel current induced by suicide inhibition of endogenous CYP 4A -hydroxylase activity with 17-octadecynoic acid. In pressure (80 mmHg)-constricted cerebral arterial segments, hypoxia induced dilation that was partly attenuated by 20-HETE or by the KCa channel blocker TEA. Exposure to hypoxia caused the generation of intracellular superoxide as evidenced by intense staining of arterial muscle with the fluorescent probe hydroethidine, by quantitation using fluorescent HPLC analysis, and by attenuation of the hypoxia-induced activation of the KCa channel current by superoxide dismutation. These results suggest that the exposure of CAMCs to hypoxia results in the generation of superoxide and reduction in endogenous level of 20-HETE that may account for the hypoxia-induced activation of arterial KCa channel currents and cerebral vasodilation.
cytochrome P-450 4A -hydroxylase; superoxide; vasodilation; patch-clamp recording
Although the signaling mechanism has not been completely resolved, the hypoxia-induced change in electrical activity of cerebral, pulmonary, or coronary arterial muscle cells is hyperpolarization that results upon activation of Ca2+-activated K+ (KCa) channel currents and/or the ATP-sensitive K+ channel (KATP) and accounts for the associated cerebral vasodilation (2, 3, 7–9, 11, 34, 35). Hypoxia can also induce suppression of K+ currents in some tissues, resulting in depolarization and vasoconstriction (2, 28, 29). Unlike the KATP channel that is regulated by the metabolic state of the cell under hypoxic condition, it is not known whether the cerebral arterial KCa channel interaction with hypoxia involves changes in the level of endogenous oxidative products in the arterial smooth muscle cell itself. On the other hand, possible cellular event that could be assumed to link the effect of hypoxia and indirect activation of arterial KCa channel currents could be the occurrence of a transient change in intracellular pH or intracellular Ca2+ concentration ([Ca2+]i) that could accompany the hypoxic stimulus. However, such a possibility appears unlikely since no change in [Ca2+]i or intracellular pH levels has been previously observed in cerebral arterial muscle cells (CAMCs) of the cat exposed to a similar grade of hypoxia (11). In contrast, a recent study reported hypoxia-induced inhibition of transient KCa channel current in cerebral arterial muscle that was suggested to be related to reduced apparent Ca2+ sensitivity and KCa channel uncoupling to Ca2+ sparks and proposed to oppose hypoxic cerebrovascular dilation (44). Given that the dominant effect of hypoxia in the cerebral circulation is vasodilation and an increase in CBF, the physiological implication of such an inhibitory influence of hypoxia on transient KCa channel activity in cerebral arterial muscle, in the face of an unchanged global Ca2+ (44), is interesting and deserves further investigation.
The KCa channel is ubiquitous in CAMC membranes and plays a critical role in regulating the reactivity of the cerebral arteries to a variety of physical and physiological stimuli (5, 11, 30, 40). Both cat and rat cerebral and renal arterial muscle cells express the enzymes of the cytochrome P-450 4A (CYP 4A) -hydroxylase gene family that catalyze the formation of 20-HETE from arachidonic acid (AA) (13, 14, 19, 46). 20-HETE is a potent endogenous inhibitor of the KCa channel current in CAMCs that primarily acts by stimulating protein kinase C activity to induce phosphorylation of the KCa channel 
-subunit, resulting in the inhibition of the activities of the channel and membrane depolarization followed by cerebral vasoconstriction (19, 22). 20-HETE can also enhance the openings of L-type Ca2+ channel and cause an increase in [Ca2+]i to induce vasoconstriction (14). A recent report relates the contractile effect of 20-HETE to the activation of Rho-kinase phosphorylation of myosin light chain phosphatase and sensitization of the contractile apparatus to Ca2+ in small porcine coronary arterial muscle (31).
Although the CAMCs are known to express the CYP 4A -hydroxylase protein and produce 20-HETE, its role in the hypoxia-induced activation of cerebral arterial KCa channel current and cerebral microvascular vasodilation is not known. The present studies investigated the interaction between hypoxia and native KCa channel currents. We also examined whether a cytosolic mediator is obligatory to transduce the effect of hypoxia on KCa channel current in rat CAMCs using different modalities of the patch-clamp, ion channel current recording technique (12, 15, 18). These studies investigated whether hypoxia per se alters the level of 20-HETE in rat CAMCs and that an exogenous application of synthetic 20-HETE influences the ability of hypoxia to modulate the activities of KCa channel currents and diameter of pressurized cerebral arteries. Furthermore, in an attempt to examine whether exposure to hypoxia is associated with the generation of the reactive O2 species (ROS) superoxide in cerebral arterial muscle that could contribute to the activation of KCa channel current (42), we quantitated the production of superoxide in CAMCs during exposure to hypoxia using the fluorescent probe hydroethidine (HE) and the recently developed fluorescent HPLC assay method (43).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The animal protocols used in this study were approved by the Medical College of Wisconsin Animal Care and Use Committee. Adult, 10–12-wk-old male Sprague-Dawley rats (Harlan, Indianapolis, IN) were anesthetized with pentobarbital sodium (65 mg/kg ip, Anpro Pharmaceutical, Acradia, CA), and middle cerebral arteries were carefully dissected after removing the brain. The dissected rat middle cerebral arterial segments were first placed in a vial containing 1 ml solution of bovine serum albumin (0.5 mg/ml) in low-Ca2+ dissociation solution composed of (in mM) 134 NaCl, 5.2 KCl, 1.2 MgSO4·7H2O, 1.18 KH2PO4, 0.05 CaCl2, 24 NaHCO3, 11 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) with pH adjusted to 7.4, for 10 min at room temperature (25°C). The cerebral arterial segments were then transferred to another vial and incubated for 20 min in a 1-ml solution of dithiothreitol (0.5 mg/ml; Sigma, St. Louis, MO) and papain (60 U/ml; Worthington, Freehold, NJ) in low-Ca2+ dissociation solution and placed in a water-jacketed beaker at 37°C. After a 20-min incubation, the supernatant layer was removed and replaced with 0.5 ml dissociation solution containing collagenase type II (240 U/ml; Worthington), elastase (15 U/ml), and trypsin inhibitor (0.1 mg/ml) in low-Ca2+ dissociation solution and incubated at 37°C and pH 7.4. Supernatant fractions were then collected at 5-min intervals and diluted to 1 ml with fresh low-Ca2+ dissociation solution. The procedure was repeated by incubating the remaining microvascular tissue with fresh enzyme containing low-Ca2+ dissociation solution. A complete dispersion of the cerebral arterial segments to single smooth muscle cells was usually attained within 30–40 min following incubation with the dissociation solution. The fractions which contained single cerebral arterial smooth muscle cells were pooled and kept at 4°C. Patch-clamp recordings of K+ channel currents were performed within 6 h.
Changing of PO2 in Solutions Bathing Isolated Cells and Membrane Patches.
The freshly isolated rat middle CAMCs were placed and allowed to adhere to the bottom of a 1-ml suffusion chamber. The chamber was perfused at 7 ml/min by gravity flow from a reservoir containing solutions aerated with 21% O2 (
150 mmHg). A second reservoir was aerated with N2 to bring the O2 content of solution below 2% (14 mmHg). Following 2–6 min of recording time in the cell-attached, inside-out, outside-out, or whole cell mode, the gravity flow was switched to the N2-aerated reservoir, and after 6 min, another 2–6-min recording period was carried out. Control measurements were made when O2 content was 21%, and experimental measurements were made when O2 content was <2%. Bath O2 content was measured with a Clark-type electrode (Microelectrodes, New Brunswick, NH). Switching from the control to the N2-aerated reservoir produced a rapid and reproducible change in suffusion chamber O2 content as previously described (11). We have previously reported that there were no detectable changes in intracellular pH or Ca2+ during the 3–10-min superfusion of the cells with either 21% or <2% O2 bath solution (11). The effects of the KCa channel blocker tetraethylammonium (TEA, 1 mM) or the CYP 4A -hydroxylase AA metabolite 20-HETE (100 nM or 1 µM) on the hypoxia-induced activation of rat cerebral arterial KCa channel currents or cerebral arterial dilation were investigated by adding directly to the hypoxic solution that continuously superfusing the patch-clamped arterial muscle cells or the pressurized arterial segment.
Single-Channel K+ Current Recordings.
Single-channel K+ currents were recorded at room temperature from cell-attached and excised outside-out and inside-out membrane patches of freshly isolated rat middle CAMCs using the patch-clamp technique as previously described (12, 15, 18). Briefly, recording pipettes were fabricated from borosilicate glass pulled on a two-stage micropipette puller (PC-84) and heat-polished under a microscope (Narishige MF-83 heat polisher). The recording pipettes were mounted on a three-way hydraulic micromanipulator (Narishige, Tokyo, Japan) for placement of the tips on the cell membrane. High-resistance seals (>l g
) were established by applying a slight suction between fire-polished pipette tips (3-l0 m) and cell membranes. The offset potentials between pipette and bath solution were corrected with an offset circuit before each experiment. Pipette potential was clamped, and single-channel currents were recorded under normoxic or hypoxic condition through a List EPC-7 patch-clamp amplifier (List Biological, Campbell, CA). The amplifier output was low-pass filtered at 1 kHz with an eight-pole Bessel filter (Frequency Devices, Haverhill, MA). Current signals were digitized at a sampling rate of 2.5 kHz. Single-channel currents were analyzed using a pClamp software package (pClamp versions 5.5 and 6.04; Axon Instruments, Foster City, CA) to determine mean current amplitudes and open-state probability (NPo). The mean NPo was expressed as NPo = I/i, where I is the time-averaged current, N is the number of open channels per patch determined following maximal activation of the single-channel current by increasing bath Ca2+ to 1 µM, i is the amplitude of the unitary current, and Po is the probability of a channel being open (1). Slope conductance was determined by fitting the unitary current-voltage relation using least-square linear regression.
Patch-Clamp Solutions.
Pipette solutions for both cell-attached and excised inside-out patches contained (in mM) 145 KCl, 1.8 CaCl2, 1.1 MgCl2, and 5 HEPES, with the final pH adjusted to 7.2 with KOH. During recording from cell-attached patches and excised inside-out patches, the bath solution was composed of (in mM) 145 KCl, 1.8 CaCl2, 1.1 MgCl2, 5 HEPES, and 5 ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), with pH adjusted to 7.2 with KOH, to allow for the optimum control of membrane potential of the cell. This resulted in a calculated final bath [Ca2+] of 10–7 M (16). In some studies, cell-attached patches of CAMCs were bathed in physiological K+- and Ca2+-containing bath solution, and single-channel KCa currents were recorded at a resting or physiological membrane potential to determine the effects of exposure to hypoxia. The bath was contained in a volume of 1 ml that was continually exchanged with fresh normoxic (21% O2, 146 mmHg) or hypoxic (<2% O2, 14 mmHg) solution at a rate of 7 ml/min by gravitational flow. To study the sensitivity to changes in [Ca2+]i of the single-channel KCa currents recorded from inside-out membrane patches of rat middle CAMCs, the different [Ca2+]i values were calculated using a computer program (16). Single-channel KCa currents from excised outside-out membrane patches were recorded using normal physiological salt solution (PSS) bath and a pipette solution containing 150 mM KCl, 3 mM HEPES, and 10–7 M CaCl2, at pH 7.2 (1, 12). The selectivity of the channel identified in rat CAMCs for K+ over Na+ was examined by replacing the extracellular K+ with equimolar concentration of Na+.
Isolated Rat Middle Cerebral Arterial Segment Studies.
Rat middle cerebral arterial segments isolated from adult Sprague-Dawley rats (8–10 mm in length and 200–250 µm outer diameter) were placed in a perfusion chamber, cannulated at both ends with glass micropipettes, and secured in place with 8-0 polyethylene suture (Ethicon, Somerville, NJ) using a stereo microscope. Side branches of the arteries were tied off with 10-0 polyethylene suture. The arterial segments were perfused and superfused with PSS aerated with a 21% O2-5% CO2 gas mixture (balance N2) and were maintained at 37°C and pH 7.4. A bolus of air was passed through the lumen to cause damage to the endothelium of the arterial segments. The inflow cannula was connected in series with a volume reservoir and a pressure transducer (Gould Instruments Division, Cleveland, OH) to allow for a continuous monitoring of transmural pressure. The internal diameter of the arteries was measured using a videomicroscopy system composed of a television camera and a videomicrometer, as previously described (20). After an equilibration period of 15 min at 20 mmHg and the taking of control diameter measurements at this pressure, the cannulated arteries were then pressurized to 80 mmHg and maintained at this pressure throughout the course of the experiment. Following an additional 15 min of equilibration, the vessels were constricted further with 10 µM serotonin, and the relaxation response to 1 µM acetylcholine was determined in an attempt to judge the integrity of the endothelium. Arterial segments that constricted to serotonin and failed to dilate in response to acetylcholine were studied. Following repeated washout and a further 30-min equilibration of the pressurized (80 mmHg) arterial segments in PSS aerated with 21% O2-5% CO2 gas (balance N2) mixture (normoxic solution), diameter measurements were taken. The PO2 of the superfusate and luminal perfusate was then lowered by aerating the respective PSS reservoirs with 2% O2-5% CO2 (balance N2) gas (hypoxic solution), and changes in internal diameter of the arterial segments were redetermined. The PO2 of the superfusate and the perfusate was then increased to the normoxic level by aerating the reservoirs with 21% O2-5% CO2 (balance N2) gas mixture (normoxic solution), and arterial segments were equilibrated until their diameters returned to the control level. The equilibrated arterial segments were then treated with the KCa channel blocker TEA (1 mM) or 20-HETE (1 µM) or the superoxide dismutase mimic Tempol (1 mM) by adding to the reservoirs containing the perfusing or superfusing solutions, and changes in diameter were determined. The PO2 of the perfusing or superfusing solution was reduced to hypoxic level (2% O2) by aerating the reservoirs with 2% O2-5% CO2-93% N2 gas in the continued presence of the KCa channel blocker 1 mM TEA or 1 µM 20-HETE or the superoxide dismutase mimic Tempol (1 mM). Under these conditions, changes in the internal diameter of the arterial segments were measured every 1 min for 5 min to examine whether the hypoxia-induced cerebral arterial dilation is attenuated by blocking the arterial KCa channel current and is inhibitable by the addition of either exogenous 20-HETE or by superoxide dismutation with Tempol. At the end of each set of the studies, the arterial segments were repeatedly washed with normoxic solution and allowed to recover for 30 min, and measurements of the diameter responses of the arterial preparations to the applied stimuli were found not to be different from the respective controls.
Liquid Chromatography-Mass Spectrometry Analysis.
Isolated brain vessels of Sprague-Dawley rats were washed three times with 3 ml of HEPES buffer composed of (in mM) 10 HEPES, 149 NaCl, 5 KCl, 1.8 CaCl2, 1 MgCl2, and 5.5 glucose (pH 7.4). HEPES buffer (4 ml) was added to the vessels segments that were then exposed to normoxic (21% O2) or hypoxic condition (<2% O2) at 37°C for 10 min. The normoxic or hypoxic vessels were then transferred to a centrifuge tube and quickly frozen in liquid nitrogen followed by homogenization. A 50-µl aliquot of homogenate was used for protein determination using the Bio-Rad protein assay method. The remaining homogenates of the normoxic or hypoxic samples were then separately subjected to solid-phase extraction after an addition of ethanol to a final concentration of 25% followed by an addition of 5 µl of the internal standards deuterated [2H8]epoxyeicosatrienoic acids, [2H8]dihydroeicosatrienoic acids, and 20-[2H2]HETE. The normoxic or hypoxic samples were mixed, sonicated, and centrifuged at 1,500 rpm for 5 min. The supernatant was applied to the C18 Bond Elut SPE columns that had been preconditioned with 5 ml of ethanol and 15 ml of water. The columns were washed with 20 ml of water and allowed to run dry. The eicosanoids were then eluted from the column with 5 ml of ethyl acetate. The ethyl acetate layer was separated from the aqueous layer and transferred to a clean tube. The aqueous layer was then extracted twice with 1 ml of ethyl acetate. The ethyl acetate extracts of the normoxic or hypoxic samples were separately combined and dried under a stream of nitrogen gas. The dried samples were then reconstituted in 20 µl of acetonitrile and analyzed by liquid chromatography-mass spectrometry (LC-MS) according to a previously published procedure (26). In additional studies we also examined the effect of hypoxia on the formation of 20-HETE in cerebral arterial homogenates prepared as previously described (19), incubated at 37°C for 30 min with nonradiolabeled AA (20 µM), the cofactor NADPH (2 mM), and the internal standards, in the presence or absence of the superoxide dismutase mimic Tempol (1 mM) or polyethylene glycol, covalently linked to superoxide dismutase (PEG-SOD, 50 U/ml), under normoxic or hypoxic conditions. At the end of the incubation period, the homogenate reaction mixture was extracted twice with 3 ml of diethyl ether, and the pooled diethyl ether extract was dried under a stream of nitrogen, reconstituted in 20 µl of acetonitrile, and subjected to LC-MS analysis. In further studies, we also examined the effect of the superoxide donor (K+ peroxide) KO2 on the stability of standard 20-HETE in aqueous solution. Authentic 20-HETE, obtained from Biomol, was incubated with 10 µM KO2 or with the vehicle DMSO alone or with 10 µM KO2 in the presence of the superoxide dismutase mimic Tempol (1 mM) at 37°C for 10 min. At the end of the incubation period, the samples were separately extracted with two volumes of diethyl ether, dried under N2 gas, and the extract products were analyzed by LC/MS.
Assessment of Intracellular Superoxide Generation in Isolated Cerebral Arterial Muscle Exposed to Hypoxia
Fluorescent detection.
Freshly dissociated rat CAMCs or isolated cerebral arterial segments of 100 µm diameter were allowed to adhere on coverslips in 1 ml Dulbecco's phosphate buffered saline (D-PBS, Gibco) and placed in a chamber at 37°C connected to tubing of low-O2 permeability supplying a continuous inflow of normoxic (21% O2 ) or hypoxic (<2% O2) gas. The humidity of the chamber was maintained by placing a culture dish containing 10 ml of D-PBS in the chambers. The isolated arterial muscle cells or arterial segments were first treated with the cell permeable fluorescence probe HE (10 µM; Molecular Probes), which is preferentially oxidized by superoxide to form the fluorescent product 2-hydroxyethidium (2-OH-E+) (43), and its specificity for superoxide over other ROS has been confirmed previously (43), and then exposed to normoxia or hypoxia for 10 min with or without pretreatment with the superoxide dismutase mimic 4-hydroxy-2,2,6,6-tetramethyl piperidine 1-oxyl (Tempol, 1 mM). Fluorescence was monitored using Nikon E-600 fluorescent microscope equipped with tetramethyl rhodamine iso-thiocyanate (TRITC) filters with an excitation at 510 nm and an emission at 590 nm.
Measurement of intracellular superoxide generation by HPLC. Freshly dissociated CAMCs were first treated in dark conditions with 10 µM HE, prepared in D-PBS, and then incubated in normoxic (21% O2) or hypoxic (<2% O2) chambers at 37°C for 10 min. The medium was then removed by centrifugation at 5,000 g for 5 min, and the CAMCs were washed twice with D-PBS in dark. The CAMCs were then lysed in 0.25 ml lyses buffer (0.1% Triton X-100 in D-PBS, pH 7.4), and 50 µl of the cell lysate were used to determine the cell protein level. The remaining lysate solution was mixed with 0.5 ml of n-butanol and extracted in dark conditions by vortexing for 1 min followed by centrifugation at 10, 000 g for 10 min at 24°C. The n-butanol phase was separated and evaporated under a stream of nitrogen gas. The dried samples were taken up by adding 0.1 ml of HPLC-grade water. The HE, E+, and superoxide oxidation product of HE 2-OH-E+ were separated on an HPLC system equipped with fluorescence and UV detectors. The mobile phase was H2O/CH3CN (acetonitrile). The stationary phase was a C18 reverse-phase column (Partisil ODS-3 250 x 4.5 mm, Alltech Association, Deerfield, IL). Sample (50 µl) was injected into the HPLC system (HP 1100, Agilent Technologies, Palo Alto, CA) with a C18 column (250 x 4.5 min) equilibrated with 10% CH3CN in 10% trifluoroacetic acid. HE, E+, and 2-OH-E+ were separated by a linear increase in CH3CN concentration from 10% to 70% in 46 min at a flow rate of 0.5 ml/min. The elution was monitored for hydroethidium and its oxidation products by a variable UV detector at 210 and 350 nm and a fluorescence detector with excitation and emission at 510 and 595 nm. The HPLC peak area for each experiment was normalized to the protein concentration as previously described (43).
Drugs and Chemicals
All chemicals used are of analytical grade and were obtained from Sigma. 20-HETE and 17-octadecynoic acid (17-ODYA) were obtained from Biomol (Plymouth, PA), and HE, from Invitrogen (Carlsbad, CA).
Statistical Analysis
Data are expressed as means ± SE. Differences between mean values were assessed using a Student's t-test or analysis of variance (ANOVA) for multiple comparisons followed by Duncan's new multiple range tests. P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The single-channel K+ current recorded from excised inside-out membrane patches of rat CAMCs displayed changes in unitary current amplitude and random opening when measured at different patch potentials. The current-voltage relationship determined over the voltage range of –60 and +60 mV in 20-mV increments using symmetrical KCl (145 mM) solution containing 0.1 µM Ca2+ revealed a unitary conductance of 238 ± 21 pS (Fig. 1A,a). The sensitivity to changes in [Ca2+]i of the 238-pS K+ channel was determined by varying the concentration of free Ca2+ in the external solution bathing the cytoplasmic surface of excised inside-out membrane patches of CAMCs during recording at a patch potential of +40 mV. The increase in [Ca2+]i from 0.1 to 0.3 µM significantly increased the mean NPo of the 238-pS K+ channel current from 0.016 ± 0.003 to 0.076 ± 0.0065 [n = 4–18, P < 0.05, Fig. 1A,b]. External application of low concentration of TEA (1 mM, Fig. 1A,c,I) or iberiotoxin (100 nM, Fig. 1A,c,II), known blockers of the KCa channel, reversibly inhibited the openings of the 238-pS single-channel K+ current recorded from outside-out membrane patches. This 238-pS K+ channel current recorded from inside-out patches of rat CAMCs was highly selective for K+ since a reduction of the concentration of K+ in the solution bathing the cytoplasmic surface of the inside-out membrane patches from 145 to 40 mM, by equimolar replacement with Na+, significantly reduced the unitary current amplitude of the single-channel K+ current recorded at different potentials and shifted the reversal potential for K+ from near 0 to 32.4 mV (equilibrium potential for K+ = 33.5), indicative of a high selectivity of this channel for K+. These findings indicate that the 238-pS single-channel K+ current recorded from rat CAMC membranes displays sensitivity to changes in [Ca2+]i, selectivity for K+, and pharmacological properties similar to the KCa single-channel currents previously identified in rat cerebral arterial muscle and other tissues (5, 25, 40).
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We examined single-channel KCa current activity in cell-attached and in excised inside-out membrane patches at a patch potential of +40 mV and cytosolic Ca2+ concentration of 10–7 M using symmetrical KCl (145 mM) solution. An inside-out patch excision from a cell-attached patch was accompanied by a rise in the NPo (from 0.0037 ± 0.001 to 0.017 ± 0.008, n = 6 for each group, P < 0.05, Fig. 1B,a and B,b, left) of the 238-pS KCa channel current without a detectable change in the amplitude of the single-channel current. As depicted in the Fig. 1B,a, top, and B,b, left, inhibition of the CYP 4A -hydroxylase activity in intact isolated CAMCs by the suicide substrate inhibitor 17-ODYA (5 µM) evoked a marked increase in the NPo of the 238-pS single-channel KCa current (from 0.0038 ± 0.001 to 0.0167 ± 0.0018; n = 7 for each group) when recorded from cell-attached patches under conditions of symmetrical KCl (145 mM) solution at a patch potential of +40 mV. The observed increase in NPo following CYP 4A -hydroxylase inhibition was closely similar to the rise in NPo observed following the excision of an inside-out patch (Fig. 1B,b, right, white bars). Furthermore, as depicted in this figure, application of 17-ODYA at the concentration used above to inhibit the CYP 4A -hydroxylase activity in intact cells did not influence the NPo of the KCa channel current in excised inside-out patches (NPo, before 0.0172 ± 0.0018 and after 0.0186 ± 0.0013, n = 7, P > 0.05), suggesting that the rise in NPo following treatment of the cell-attached patches with 17-ODYA was due to inhibition of CYP 4A -hydroxylase catalyzed 20-HETE formation in the arterial muscle cell, which appears to function as an endogenous inhibitor of the KCa channel in these CAMCs.
Effect of Hypoxia on Single-Channel KCa Currents in Rat CAMCs
Previous studies from our laboratory have demonstrated that hypoxia or reduced O2 availability induces indirect but reversible activation of single-channel KCa current in cat CAMCs (11). In the present study we investigated the effects of exposure to hypoxia on the activities of the 238-pS single-channel KCa current recorded from cell-attached patches, excised inside-out or outside-out membrane patches of rat CAMCs. Exposure to hypoxia induced a significant increase in the openings and NPo of single-channel KCa currents in cell-attached patches of cerebral arterial muscles during recording at a resting membrane potential (pipette potential, 0 mV) using normal PSS in the bath (Fig. 2, n = 5 cells). In an additional set of experiments, symmetrical KCl (145 mM) solution was used to optimally control the membrane potential and to record single-channel KCa currents from either cell-attached or inside-out patches under normoxic (21% O2) or hypoxic (<2% O2) conditions. In the cell-attached patches bathed in symmetrical KCl solution and held at a patch potential of +40 mV, hypoxia (<2% O2) increased the mean NPo of the 238-pS single-channel KCa current from 0.0035 ± 0.001 to 0.0127 ± 0.0036 (P < 0.05, n = 18; Fig. 3A, a and b). This hypoxia-induced increase in the NPo of the 238-pS KCa channel current returned to the control level when the bath solution superfusing the cell-attached patches was switched to the one aerated with 21% O2 (Fig. 3A, a and b). To determine whether this effect of hypoxia on cerebral arterial KCa single-channel currents is direct or requires a cytosolic mediator(s), we examined the effect of hypoxia on single-channel KCa current activity in excised inside-out or outside-out membrane patches of rat CAMCs. In contrast to the response of the cell-attached patches, exposure to hypoxia (<2% O2) had no detectable effect on the openings of single-channel KCa currents recorded from either inside-out or outside-out membrane patches [Fig. 3, B,I and B,II]. Thus, in the inside-out configuration, the mean NPo of the single-channel KCa current recorded at +40 mV averaged 0.0174 ± 0.005 under normoxic condition and 0.0182 ± 0.0046 during superfusion with hypoxic bath solution (<2% O2) (P > 0.05; n = 7, Fig. 3B,I, a and b). Similarly, the hypoxic stimulus also had no significant effect on the NPo of single-channel KCa currents recorded in outside-out membrane patches as depicted in Fig. 3B,II, a and b (mean NPo, 0.170 ± 0.025 under normoxia and 0.176 ± 0.034 under hypoxia, P > 0.05, n = 3). Taken together, these results obtained from cell-free excised membrane patches of rat CAMCs suggest that hypoxia cannot directly activate KCa channel currents in rat CAMCs but appears to require cytosolic mediatory factor(s).
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We examined the effect of incubation of cerebral arterial muscle of the rat under hypoxic condition (<2% O2 at 37°C for 10 min) on the level of endogenous 20-HETE. The level of 20-HETE in cerebral arterial muscle was determined using LC-MS analysis. Figure 4A depicts the amounts of 20-HETE following a 10-min incubation of cerebral arterial muscle under normoxic (21% O2) or hypoxic (<2% O2) conditions. As depicted in Fig. 4A, the level of 20-HETE averaged 1.18 ± 0.05 pg/mg and 0.58 ± 0.025 pg/mg protein following a 10-min incubation of the cerebral arterial muscle under normoxia and hypoxia, respectively [P < 0.01, n = 4]. These data indicated that subacute hypoxia markedly reduces the level of endogenous 20-HETE in cerebral arterial muscle. In additional studies we also examined the effect of hypoxia on the formation of 20-HETE in cerebral arterial homogenates incubated with nonlabeled or cold AA and the cofactor NADPH in the presence or absence of the superoxide dismutase mimic Tempol (1 mM) or PEG-SOD (50 U/ml) under normoxic or hypoxic conditions. As shown in Fig. 4B, the cerebral arterial homogenates incubated with cold AA produced a significant amount of 20-HETE under normoxia (Fig. 4B, left) that was significantly reduced under hypoxic condition (Fig. 4B, right) but could not be reversed by pretreatment with the two different superoxide scavengers Tempol or PEG-SOD. These later findings may suggest that mechanisms or conditions other than the generated superoxide during hypoxia might contribute to the reduction in 20-HETE levels observed in cerebral arterial homogenate assay reactions under a hypoxic condition that awaits exploration in future studies. In an attempt to examine the effect of exogenous superoxide on 20-HETE, we also performed additional studies in which authentic 20-HETE was incubated with the superoxide donor KO2 in the presence or absence of the superoxide dismutase mimic Tempol (1 mM) for 10 min and analyzed the extract of the reaction product by LC/MS. As depicted in Fig. 4C, superoxide reduced the concentration of authentic 20-HETE by a factor of three, and this effect was partly reversible in the presence of the superoxide dismutase Tempol (1 mM).
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-Hydroxylase Activity in the Hypoxia-Evoked Activation of KCa Channel Current in Rat CAMCs
The expression of different isoforms of the CYP 4A -hydroxylase has been previously identified in the CAMCs of the cat and rat (13, 14, 19). Furthermore, the formation of 20-HETE by the catalytic action of CYP 4A -hydroxylase on AA in cerebral arterial muscle requires the presence of molecular O2 and the cofactor NADPH (14). Under a normoxic condition, application of exogenous 20-HETE causes protein kinase C-dependent cerebral constriction and inhibition of openings of KCa channels (22). In the present study we investigated the possibility that reduced levels of endogenous 20-HETE during hypoxia might contribute to the hypoxia-induced activation of KCa channel currents in rat CAMCs. We found that an application of exogenous 20-HETE (100 nM) to a hypoxic solution continuously superfusing patch-clamped rat CAMCs significantly reduced hypoxia-induced increases in mean NPo of single-channel KCa currents from 0.012 ± 0.00126 to 0.0036 ± 0.0006 (P < 0.05, n = 9, Fig. 5A, a and b) when recorded using symmetrical 145 mM KCl bath solution. On the other hand, inhibition of endogenous formation of 20-HETE in CAMCs by preincubation with the suicide substrate inhibitor of CYP 4A -hydroxylase, 17-ODYA (5 µM), for 15 min, did not augment further the hypoxia-induced increase in mean NPo of the 238-pS single-channel KCa current (NPo, 0.012 ± 0.0026 before and 0.0115 ± 0.0021 after treatment with 17-ODYA, P > 0.05, n = 5, Fig. 5B, a and b).
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We sought to determine whether the hypoxia-induced activation of KCa channel current in rat CAMCs was partly mediated through the generation of the ROS superoxide. Under a normoxic condition, the treatment of CAMCs with 1 mM of the superoxide dismutase mimic Tempol (41) for 10 min did not change the single-channel KCa current activity compared with that of untreated controls. However, Tempol pretreatment significantly abrogated the ability of hypoxia to activate single-channel KCa currents. Thus, before treatment with 1 mM Tempol, the NPo values of the single-channel KCa current were 0.0053 ± 0.00034 under normoxia and 0.0195 ± 0.0015 under hypoxia (P < 0.05). However, after treatment with 1 mM Tempol, the NPo averaged 0.0056 ± 0.00034 during normoxia and 0.0057 ± 0.00042 during hypoxia [Fig. 6, top; n = 5 for each group; P < 0.05 (hypoxia + Tempol)]. These findings suggest that superoxide generation during hypoxia contributes to the hypoxia-induced activation of KCa channel currents in rat CAMCs. The generation of superoxide in CAMCs exposed to hypoxia was further confirmed by the use of the fluorescent probe HE (10 µM, 10 min). As shown in Fig. 6, bottom, a 10-min exposure to hypoxia of freshly dissociated rat CAMCs (Fig. 6, bottom, A) or rat isolated cerebral arterial segments (Fig. 6, bottom, B), pretreated with HE (10 µM) produced intense red fluorescence staining compared with that observed under the normoxic condition and was markedly reduced in isolated arterial muscle cells or arterial segments pretreated with the superoxide dismutase mimic Tempol (1 mM). These findings revealed an intracellular production of superoxide in isolated arterial muscle cells or arterial segments during exposure to hypoxic condition. The observed high intensity of the HE fluorescent signal, indicative of an increased generation of superoxide in CAMCs during exposure to hypoxia, was further quantitated using the recently developed HPLC fluorescence assay method (43). Figure 7A depicts profiles of the HPLC chromatogram of standard HE, E+, and 2-OH-E+. Figure 7B summarizes the concentrations of 2-OH-E+, the fluorescent oxidation product between HE and superoxide, per milligram protein generated in response to exposure of isolated arterial muscle cells to 10 min normoxia or hypoxia, following pretreatment with 10 µM HE for 10 min, before and after inhibition of CYP 4A -hydroxylase activity with the suicide substrate inhibitor 17-ODYA (5 µM). As clearly shown in Fig. 7, hypoxia induced increased amounts of 2-OH-E+ that was significantly reduced in arterial muscle cells in which CYP 4A -hydroxylase activity has been inhibited with 17-ODYA (n = 4, P < 0.05 for each group). In contrast, inhibition of CYP 4A -hydroxylase activity had no effect on the intracellular level of superoxide under normoxic condition. These findings suggest that CYP 4A -hydroxylase activity might contribute to the increased intracellular generation of superoxide during exposure of the CAMCs to hypoxia.
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The effects of normoxia or hypoxia on the diameter of pressure (80 mmHg)-constricted rat cerebral arterial segments was examined in the absence and presence of the KCa channel blocker TEA (1 mM) or the CYP 4A -hydroxylase metabolite of AA, 20-HETE (1 µM). Under a normoxic condition (21% O2), the internal diameter of the cannulated cerebral arterial segments averaged 211 ± 6 µM at rest, 205 ± 5 µm at 20 mmHg, and 164 ± 12 µm at 80 mmHg (n = 5, P < 05). Exposure to hypoxia (2% O2) reversed the pressure (80 mmHg)-induced reduction in diameter observed under normoxia and dilated the cerebral arterial segment by 33 ± 3% that returned to the control level upon restoring to normoxic condition (21% O2, not shown). This hypoxia-induced cerebral vasodilation was not significantly reversed by pretreatment with Tempol. The pressure (80 mmHg)-induced constriction response observed under the normoxic condition was markedly enhanced in the presence of TEA (1 mM), a relatively selective inhibitor of the KCa channel at this concentration (23), or exogenous 20-HETE (1 µM) (Fig. 8). Replacement of the normoxic solution containing TEA or 20-HETE and bathing the arterial segments with a hypoxic solution that also contained TEA or 20-HETE failed to evoke a dilation or an increase in diameter of the cerebral arterial segments (Fig. 8, n = 5 to 6 vessels/each group).
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We found that hypoxia indirectly enhances the NPo of the 238-pS single-channel KCa current recorded in rat CAMCs. Our finding that hypoxia increases the NPo of single-channel KCa currents in cell-attached patches when recorded at a resting membrane potential may have physiological relevance in that it could be one of the ionic mechanism by which hypoxia increases cerebral arterial diameter in vivo to increase CBF. Exposure to hypoxia also caused significant enhancement of the magnitude of the whole cell KCa current, the macroscopic equivalent of the 238-pS unitary KCa channel current, which was markedly attenuated by an application of 1 mM external TEA (data not shown). The fact that the stimulatory effect of hypoxia on KCa single-channel currents was not evident during recording from either excised inside-out or outside-out membrane patches suggests that this native KCa channel current in CAMCs is not by itself directly responsive to the hypoxic stimulus and that it probably requires an intracellular mediator. It, therefore, appears that the observed response of the KCa channel current in rat CAMCs to hypoxia is a secondary response mediated by a cytosolic factor(s), the concentration of which in rat CAMCs is regulated by reduced PO2. Indeed, the CYP 4A -hydroxylase-catalyzed formation of the potent cerebral arterial vasoconstrictor 20-HETE from AA in arterial muscle cells is dependent on the presence of molecular O2 (13, 14, 19), and, as demonstrated in the present study, its endogenous level decreases during O2 deprivation (Fig. 4A). Since 20-HETE functions as a potent arterial constrictor enabled by its inhibitory action on arterial KCa channel current under normoxic conditions (13, 19, 22, 46), it is conceivable that its deficient state during hypoxia could lead to increased openings of the KCa channel current-initiated cerebral vasodilation.
The present findings that exogenously applied 20-HETE attenuated the hypoxia-induced activation of the KCa channel current and that the failure of the hypoxic stimulus to further enhance the increased KCa channel current activity elicited by suicide substrate inhibition of CYP 4A -hydroxylase (Fig. 5, A and B) suggest that hypoxia-related reduction of endogenous 20-HETE level and/or a reduced efficiency of an inhibitory signaling pathway activated by 20-HETE (22, 33) may contribute to the hypoxia-induced activation of the KCa channel currents, which could lead to hyperpolarization and relaxation of rat cerebral arterial muscle. The hypoxia-induced cerebral vasodilation could also result from a reduction in the activities of voltage-gated L-type channel current, the openings of which in CAMCs is enhanced by 20-HETE and inactivated by membrane hyperpolarization (14). However, the effect of hypoxia on L-type Ca2+ channel current does not appear clear, since hypoxia was reported to reduce L-type Ca2+ channel currents in rabbit cerebral, femoral, celiac, and main pulmonary arterial muscle cells (10), whereas it had no effect on hamster cremaster muscle cells (6), thus prompting a requirement for further studies to define the functional role of L-type Ca2+ channel in the hypoxia-induced reduction of cerebral arterial tone.
An alternative possibility that could be raised to explain the mechanisms mediating hypoxia-induced activation of KCa channel currents is the generation of ROS, specifically superoxide, the release of which has been proposed to increase during hypoxia (37, 38). Indeed, it has been previously reported that brief hypoxia causes a release of superoxide that was specifically implicated in preconditioning of cardiacmyocytes (38). To determine whether such a possibility prevails in our studies of the effect of hypoxia on arterial KCa channel current activity, we also examined the generation of superoxide during exposure of CAMCs or cerebral arterial segments to acute hypoxia using 1) the fluorescence probe HE that reacts with superoxide to form a red fluorescent oxidation product (43), 2) the recently developed HPLC/fluorescence assay that uses HE as a probe (43), and 3) the cell permeable superoxide dismutase mimic Tempol (1 mM) (41). The results of these studies revealed that cerebral arterial muscle exposed to hypoxia generates superoxide as determined by the increased intensity of a red fluorescent HE-staining signal and quantitation of the amounts of the oxidation product formed from the reaction between HE and superoxide. These observations were further justified pharmacologically, since pretreatment of CAMCs with the cell permeable superoxide dismutase mimic Tempol (1 mM, Fig. 6) reduced the generation of superoxide in cerebral arterial muscle and attenuated the capacity of hypoxia to activate cerebral arterial KCa single-channel currents. The diminution of KCa channel activity in the presence of the superoxide dismutase mimic Tempol appears to suggest that it is the generated superoxide rather than its dismutation product or other types of ROS that may contribute to the hypoxia-induced increased KCa channel activity. Taken together, the present findings suggest that hypoxia is associated with the generation of superoxide in CAMCs. Although the physiological significance of the increased superoxide production in cerebral arterial muscle during exposure to hypoxia has yet to be established, the generated superoxide itself may contribute to the hypoxia-induced activation of cerebral arterial KCa channel currents (42), resulting in hyperpolarization, vasodilation, and an increase in CBF that could promote nutrient and O2 delivery to the brain during oxidative stress.
The source of superoxide in the CAMCs under conditions of hypoxia is, however, not known. It is possible that it could originate from the mitochondria, the site that has been previously identified and confirmed to generate or release superoxide in cardiomyocytes exposed to a hypoxic insult (38). Despite the fact that the role of mitochondria as a source of superoxide generation during hypoxia is currently debated, it has been argued that hypoxia by either limiting the availability of the substrate O2 at the end of the electron transport chain or by inhibiting the activity of one of the complexes within the electron transport chain could accelerate the rate of electrons leak leading to an increase of superoxide production (37). Such a possibility could be considered to explain possible sources of the generated superoxide following exposure of CAMCs to hypoxia but awaits rigorous investigation.
Another intriguing finding of the present study is the reduction in 20-HETE level in rat CAMCs following exposure to hypoxia. Given that 20-HETE is an endogenous inhibitor of the KCa channel (Refs. 19, 22, and 46 and the present study), the reduction in the endogenous level of 20-HETE under hypoxic condition could lead to increased openings of the KCa channel current due to the removal of the endogenous inhibitory influence imposed by 20-HETE itself. The reduced level of 20-HETE under hypoxic condition might be achieved through the action of superoxide generated during hypoxia, similar, if not identical, to a decreased amount of 20-HETE detected during an analysis of arachidonate CYP 4A -hydroxylase activity in the presence of the superoxide donor KO2 or the superoxide-generating system xanthine/xanthine oxidase or the reduction of the concentration of authentic 20-HETE following incubation with the superoxide donor KO2 (Ref. 21 and Fig. 4). The present finding that suicide inhibition of CYP 4A -hydroxylase attenuates superoxide formation appears to indicate that either the CYP 4A -hydroxylase itself (39) or its catalytic product 20-HETE (17, 32) could generate superoxide, which could in turn impose a negative impact on the activity of CYP 4A -hydroxylase to produce 20-HETE or on the level of preformed 20-HETE (Ref. 21 and Fig. 4), leading to events favorable for increased openings of arterial KCa channel currents. This possibility could also be raised to explain the inability of antioxidant pretreatment to restore the fall in CYP 4A -hydroxylase-derived 20-HETE production observed under hypoxic condition (Fig. 4C), and the inability of Tempol to completely reverse the hypoxia-induced increase in cerebral arterial diameter.
Under physiological level of PO2, optimum concentration of endogenous 20-HETE is achieved by the catalytic action of CYP 4A -hydroxylase on the substrate AA that operates to maintain a depolarized membrane potential and developed myogenic tone necessary for efficient autoregulation of CBF (13, 14). The recently reported hypoxia-induced shift in the autoregulation curve of CBF to higher blood flow levels in vivo in the rat (42) could be assumed to result from vasodilation evoked by an increased generation of superoxide or a decline in the 20-HETE level occurring under hypoxic condition. Based on the present findings, we propose the emergence of a possible hypoxia-signaling mechanism comprising of increased superoxide generation and reduced 20-HETE level (Fig. 9), which might become operational under hypoxic condition to result in activation of KCa channel currents that could initiate hyperpolarization and CAMC relaxation.
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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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). Reduction of extracellular K+ concentration from 145 to 40 mM by equimolar replacement with Na+ on the cytoplasmic surface of inside-out patches reduced unitary current amplitude of the 238 pS and shifted the current-voltage relationship curve to the right indicative of a high K+ selectivity of the channel for K+ (
). A,b: elevation of the intracellular Ca2+ concentration from 0.1 to 0.3 µM on the cytoplasmic surface of excised inside-out membrane patches significantly increased the openings of the 238-pS single-channel K+ current recorded at a patch potential of +40 mV. A,c: representative single-channel K+ current tracings recorded from excised outside-out membrane patches of cerebral arterial muscle cells at patch potential of about +10 mV in the absence (control) and in the presence of the Ca2+-activated K+ (KCa) channel blocker tetraethylammonium (TEA, 1 mM; I) or iberiotoxin (IBX, 100 nM; II). Application of TEA or IBX to the bath reversibly blocked the openings of the 238-pS single-channel K+ current. B, a and b: effects of patch excision and inhibition of cytochrome P-450 4A (CYP 4A) 
