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Oleic Acid Inhibits the KATP Channel Subunit Kir6.1 and the KATP Current in Human Umbilical Artery Smooth Muscle Cells
BASIC INVESTIGATION
Oleic Acid Inhibits the KATP Channel Subunit Kir6.1 and the KATP Current in Human Umbilical Artery Smooth Muscle Cells Xiao-Jun Bai, MD, Hong-Yan Tian, PhD, Ting-Zhong Wang, PhD, Yuan Du, MD, Yu-Tao Xi, MD, Yue Wu, MD, Jie Gao, MD and Ai-Qun Ma, PhD
Abstract: Background: The objective of the present study was to determine the effect of various concentrations of oleic acid (OA) on KATP channel expression and the potential relationship to exogenous nitrogen monoxide and protein kinase C levels. Methods: Human umbilical artery smooth muscle cells (HUASMCs), between the 7th and 10th passages, were divided into control group, OA group (final OA concentration of 0, 50, 100 or 200 mmol/L), nitric oxide (NO) intervention group, protein kinase C inhibitor group or GF-109203X (GFX) intervention group. Western immunoblotting was used to detect the protein expression of the KATP channel subunit Kir6.1. Also, quantitative real-time polymerase chain reaction analysis to determine Kir6.1 messenger RNA levels and whole-cell patch clamping to measure KATP currents were performed. Results: The results suggested that OA inhibited Kir6.1 protein and messenger RNA expression in HUASMCs. Under a high concentration of potassium (140 mmol/L), 100 mmol/L OA significantly reduced ATP-sensitive potassium current density, whereas a low extracellular concentration of potassium (5.4 mmol/L) did not influence KATP density. Pretreatment with either exogenous NO or GFX weakened the OA-induced inhibition of KATP in HUASMCs. Conclusions: The study demonstrated that OA inhibited Kir6.1, a KATP channel subunit, in HUASMCs, and indirectly inhibited the KATP current. In addition, the results indicated that NO and/or GFX partially reversed OA inhibition in HUASMCs. Key Indexing Terms: Vascular smooth muscle cell; Oleic acid; KATP channel; Nitric oxide; PKC depressor. [Am J Med Sci 2013;346 (3):204–210.]
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ardiac and cerebrovascular diseases are the leading cause of death in China. Hypertension is a primary risk factor associated with these diseases. Recent studies have reported that increased free fatty acid (FFA) concentrations contribute to hypertension via multiple pathways. These pathways include alpha1-adrenergic stimulation, endothelial cell dysfunction, increased oxidative stress and stimulation of vascular cell growth.1–3 Resistance from medium-sized arteries and arterioles in the periphery plays a pivotal role in maintaining blood pressure. Furthermore, ion channels, such as the K+, Ca2+, Na+ and Cl2 channels, are active in stabilizing angiostasis. K+ channels are highly responsible for regulating the resting potential of the vascular smooth muscle (VSM) membrane and determining vascular tone and cavosurface. Alterations in K+ channel From the Department of Cardiovascular Medicine, the First Affiliated Hospital of the Xi’an Jiaotong University School of Medicine, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an, China. Submitted April 06, 2012; accepted in revised form July 25, 2012. The authors have no financial or other conflicts of interest to disclose. Correspondence: Ai-Qun Ma, PhD, Department of Cardiovascular Medicine, the First Affiliated Hospital of the Xi’an Jiaotong University School of Medicine, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, No. 277 West Yanta Road, Xi’an, Shaanxi 710061, China (E-mail: [email protected]).
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expression and dysfunction of vascular smooth muscle cells (VSMCs) affect K+ outflow and membrane potential level, thus changing vascular tone.4 Our previous study confirmed that ATP-sensitive potassium channels (ATP-sensitive K+ channels, KATP) regulated VSM tension and blood pressure by maintaining the resting potential of the VSMC membrane. The bridging role between myocyte electrical and metabolic activity has drawn much attention regarding metabolism and hypertension.5–8 The molecular structure of the KATP channels is composed of 4 Kir6.3 subunits and 4 sulfonylurea receptors (SUR) subunits. The KATP channels found in VSMCs are mainly composed of the SUR2B/Kir6.1 subtype. The Kir6.1 and SUR2B channels have several properties similar to vascular KATP channels, such as pharmacological properties and sensitivity to nucleotide diphosphates.9,10 Northern blot and real-time polymerase chain reaction (RT-PCR) evidence also suggests that the combination of Kir6.1 and SUR2B is likely to be a KATP channel isoform in VSM.11,12 Mouse knockout models targeting the Kir6.1 and SUR2B genes emphasize the critical role of KATP in the cardiovascular system.13 Many studies have shown that one of the most representative FFAs in the blood circulation is oleic acid (OA), which is able to reduce ATP sensitivity of the pancreatic b-cell KATP channel,14 and inhibit the cardiac KATP channel.15 Studies have shown that OA impairs endothelial cell function, causing a decrease in nitric oxide (NO), and in turn dilating blood vessels via activation of the KATP channel of vascular endothelial cells.16,17 Another study revealed that OA could activate protein kinase C (PKC) in VSMCs, thereby enhancing the inhibition of the KATP channel.18,19 However, the mechanisms underlying KATP channel function and OA-induced high blood pressure remain unclear. The purpose of the present study was to investigate the underlying mechanisms by which OA inhibits the KATP channel to regulate blood pressure, and examine the mechanisms related to the reduction of NO synthesis and activation of the PKC pathway.
MATERIALS AND METHODS Human Umbilical Artery Smooth Muscle Cell Preparation Upon recovery, cryopreserved human umbilical artery smooth muscle cell (HUASMCs; Sciencell Company, San Diego, CA) were grown in a cell culture flask and plated with Smooth Muscle Cell Medium (Sciencell) supplemented with 2% fetal bovine serum (Gibco Company, New York, NY) and penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO2 and 95% air. The medium was changed every 1 to 2 days, and cells were characterized as smooth muscle cells (SMCs) by morphological criteria and the expression of smooth muscle a-actin. The cells were grown until 70% to 80% confluence, trypsinized using 0.25% trypsin every 4 to 6 days and passed in proportion of 1:2 or 1:3. Cells in the 7th to 10th passage of the logarithmic growth phase were employed for
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subsequent experiments. At approximately 90% confluence, cells were cultivated in serum-free SMCM medium for 12 hours, allowing cultured cells to be quiescent before experiments. Our experimental groups were divided as follows: control group (without intervention and OA), OA group (medium containing final OA concentrations of 0, 50, 100 and 200 mmol/ L), NO intervention group (cells were first cultured with medium supplemented with a final diethylenetriamine-NO concentration of 20 mmol/L for 30 minutes and then 100 mmol/L OA was added) and GF-109203X (GFX) intervention group (cells were cultured with a final GFX concentration of 100 nmol/L for 1 hour and then 100 mmol/L OA was added). The duration for all treatments was 24 hours, and the treatment time for each group was 24 hours. HUASMCs were harvested using trypsinization at the end of the experiment. Preparation of the Fatty Acid-Bovine Serum Albumin Complex OA (75.3 mL; Sigma, St. Louis, MO) was added to 58 mL of 0.1 mM NaOH, and the solution was heated to 95°C for 5 minutes. Next, 50 mL of this solution was added to 950 mL of 10% bovine serum albumin (BSA) solution and incubated for 30 minutes at 55°C. The final solution contained a 5 mM OA/10% BSA stock solution. Addition of 10, 20 and 40 mL/mL of the OA/10% BSA stock solution to SMCM media corresponded to the final experimental concentrations of 50, 100 and 200 mmol/L OA, respectively. The BSA-bound OA stock solution was then cooled to room temperature and sterile filtered. Western Immunoblotting Cell proteins were extracted using a RIPA protein lysate buffer (Biomiga, San Diego, CA). The protein concentration was measured using the Bradford method. Cell proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene fluoride membrane at a constant current of 350 mA for 90 minutes at 4°C in a solution containing 25 mM Tris, 193 mM glycine and 10% methanol. The membrane was incubated at room temperature first with Tris-buffered saline containing 5% nonfat dried milk and 0.1% Tween 20, and then with the anti-Kir6.1 antibody (Alomone, Jerusalem, Israel) at a working dilution of 1:200. The immune complexes were detected with an IRDye 800CWlabeled anti-rabbit IgG secondary antibody at a working dilution of 1:3000. Lastly, enhanced chemiluminescene reagent was applied and the membrane underwent X-ray film exposure, developing and fixing. The strip was scanned and analyzed using densitometric analyses. HUASMC Kir6.1 Messenger RNA Expression by Quantitative RT-PCR Total cellular RNA was extracted according to the manufacturer’s instructions. Reverse transcription was conducted according to the steps described in the reverse transcription kit (TaKaRa, Kyoto, Japan). The Kir6.1 sequence was as follows: upstream, 5’-GAT CAT CTG CCA CGT GAT TGA-3’ and downstream, 5’-GCA ATG TAG GAG GTT CGT GCT-3’. The sequence for b-actin was as follows: upstream, 5’-GCA AAG ACC TGT ACG CCA ACA-3’ and downstream, 5’-ACA CGG AGT ACT TGC GCT CAG-3’. Quantitative RT-PCR amplification was performed using a TaKaRa SYBR RT-PCR kit under the following process: pre-denaturation for 2 minutes at 95°C, denaturation for 15 seconds at 95°C and annealing for 30 seconds at 60°C, at a total of 50 cycles. Fluorescence data were acquired during the annealing/extension step. Threshold Ó 2012 Lippincott Williams & Wilkins
cycles were measured at the end of each extension step using the second-derivative method offered by the iCycler IQ optical system software (version 3.0a; Bio-Rad Laboratories, Hercules, CA). Kir6.1 messenger RNA (mRNA) levels were quantified using the relative standard curve method, and each gene amplification in triplicate underwent the same conditions. Electrophysiological Recording of KATP Channel Activity Whole-cell patch-clamp techniques were used to record KATP channel currents. Briefly, cells were placed in a Petri dish mounted on the stage of an inverted phase-contrast microscope (Olympus IX71; Tokyo, Japan). Currents were recorded using an Axopatch-700B patch clamp amplifier (Axon Instruments Inc, Foster City, CA), controlled by a Digidata 1314 interface and a pClamp software package (version 9.2; Axon Instruments Inc). Membrane currents were filtered at 1 kHz using an 8-pole Bessel filter and stored on a computer for offline analyses. At the beginning of each experiment, the junction potential between the pipette solution and the bath solution was electronically adjusted to zero. No leakage subtraction was applied to the original recordings, and all cells with visible changes in leakage currents during the course of study were excluded from further analyses. The pipette resistances were 2 to 4 MV. The pipette solution contained (in mM) KCl 140, CaCl2 1, MgCl2 1, HEPES 10 and ethylene glycol tetraacetic acid 10. The pH was adjusted to 7.3 with KOH. Seals were made in 5.4 mM K+ extracellular solution containing (in mM) NaCl 135, KCl 5.4, CaCl2 1, MgCl2 1.2, HEPES 10 and glucose 5. The solution pH was adjusted to 7.3 with NaOH, but most recordings were made under symmetrical conditions of 140 mM K+ using an extracellular solution of the same composition as described previously, except that KCl was 140 mM and NaCl was omitted. The experimental bath (volume 2 mL) was perfused continuously with the extracellular solution at a rate of 2 mL/min. All experiments were performed at room temperature (20–23°C). Statistical Analyses The software SPSS 13.0 was used for statistical analyses. Data are expressed as mean 6 standard error of the mean. The mean comparisons were performed with a single factor analysis of variance. a , 0.05 was considered statistically significant.
RESULTS Effect of Various Concentrations of OA on the Protein Expression of Kir6.1 Three concentrations (50, 100 and 200 mmol/L) of OA had a negative effect on the protein expression of Kir6.1. During Western immunoblot analyses, Kir6.1 protein expression in the OA intervention group was significantly decreased, as compared with the control group (P , 0.05), following OA administration for 24 hours (Figure 1). As the OA concentration increased, the protein expression gradually decreased (P , 0.05). Effect of NO Pretreatment on Kir6.1 Protein Expression After OA Intervention HUASMCs were pretreated with 20 mmol/L NO for 30 minutes, followed by 100 mmol/L OA administration, and then incubated for 24 hours. Kir6.1 protein expression significantly decreased (P , 0.05 in the NO intervention plus OA group; 100 mmol/L) in comparison with the control group. In contrast, the Kir6.1 protein expression in the NO intervention
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FIGURE 1. Effect of 3 concentrations of oleic acid (OA) on the expression of Kir6.1. Western immunoblot analyses showed that 3 concentrations (50, 100 and 200 mmol/L) of OA inhibited protein expression of Kir6.1. There was a significant difference in the KATP channel subunit Kir6.1 protein expression between the OA intervention group and the control group (P , 0.05). As OA concentration increased, protein expression gradually decreased (P , 0.05).
group increased compared with that found in the OA group (100 mmol/L; P , 0.05; Figure 2).
(100 mmol/L), but without statistical significance (P . 0.05; Figure 5).
Effect of GFX Pretreatment on Kir6.1 Protein Expression After OA Intervention HUASMCs were pretreated with 100 mmol/L of GFX for 1 hour, followed by 100 mmol/L OA administration, and then incubated for 24 hours. Western immunoblot analyses confirmed that the Kir6.1 protein expression levels decreased (P , 0.05) in the GFX intervention plus OA group (100 mmol/L) compared with the control group. The Kir6.1 protein expression was higher in the GFX intervention group than in the OA group (100 mmol/L; P , 0.05; Figure 3).
Effect of GFX Pretreatment on Kir6.1 mRNA Expression After OA Intervention HUASMCs were pretreated with 100 mmol/L GFX for 1 hour, followed by 100 mmol/L OA administration, and then incubated for 24 hours. Quantitative RT-PCR revealed that Kir6.1 mRNA expression levels decreased (P , 0.05) in the GFX intervention plus OA group (100 mmol/L), as compared with the control group. The Kir6.1 mRNA expression was higher in the GFX intervention group than in the OA group (100 mmol/L; P , 0.05; Figure 6), but the mRNA expression did not reach the level of the control group.
Effect of Different Concentrations of OA on Kir6.1 mRNA Expression Various concentrations (50, 100 and 200 mmol/L) of OA were administered to HUASMCs for 24 hours. Kir6.1 mRNA was significantly lower in the OA treatment group than in the control group (P , 0.05; Figure 4). Effect of NO Pretreatment on the Expression of Kir6.1 mRNA After OA Intervention HUASMCs were pretreated with 20 mmol/L NO for 30 minutes, followed by 100 mmol/L OA administration, and then incubated for 24 hours. RT-PCR analysis indicated that Kir6.1 mRNA expression levels decreased (P , 0.05) in the NO intervention plus OA group (100 mmol/L) compared with the control group. The Kir6.1 mRNA expression was higher in the NO intervention group than in the OA group
Recordings of Cultured HUASMC KATP Currents Induced by OA and GFX Using the Gap-Free Mode The gap-free mode was employed to record HUASMC KATP currents for the 3 groups (control group, OA group and GFX intervention group). The sampling time was set for 100 mseconds, and the patch camp voltage was maintained at 0 mV. In the control group (Figure 7A), the KATP current was weaker than the OA group and the GFX intervention group. We used a high potassium concentration (140 mmol/L) and perfused a potassium channel-specific opener, pinacidil, for 10 minutes, elevating the channel current to the largest opening range with a 2300 pA current density. Because hypokalemia (5.4 mmol/L) perfusion was able to wash out the current, the current most likely came from a KATP channel. When 100 mmol/L OA was applied for 24 hours, the cell KATP channel was significantly inhibited (Figure 7B), with a maximum KATP current of
FIGURE 2. Effect of nitric oxide (NO) pretreatment on HUASMC Kir6.1 protein expression under oleic acid (OA) intervention. Western immunoblot analyses showed that pretreatment with NO followed by OA decreased Kir6.1 protein expression levels (P , 0.05) in comparison with the control group. In contrast, Kir6.1 protein expression in the NO intervention group increased compared with the OA group (P , 0.05).
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FIGURE 3. Effect of GF109203X (GFX) pretreatment on HUASMC Kir6.1 protein expression after oleic acid (OA) intervention. Western immunoblot analyses showed that pretreatment with GFX followed by OA decreased Kir6.1 protein expression levels in the GFX intervention plus OA group (100 mmol/L), as compared with the control group (P , 0.05). Kir6.1 protein expression was higher in the GFX intervention group than in the OA group (100 mmol/L; P , 0.05).
approximately 2150 pA. HUASMCs were pretreated with 100 mmol/L GFX for 1 hour, followed by 100 mmol/L OA administration, and then incubated for 24 hours. This treatment moderated the inhibition of OA on the KATP channel (Figure 7C), with a maximum KATP current density of more than 2200 pA. Recordings of Cultured HUASMC KATP Currents Induced by OA and GFX Using the Episodic Stimulation Mode To further elucidate the effect of OA and GFX on KATP channels, we employed the episodic stimulation mode to record HUASMC KATP currents in the 3 groups (control group, OA group and GFX intervention group). To eliminate the discrepancy of the current from cell size, we used current density as the measuring unit for recording the current. Current density was calculated as the measured current divided by the measured cell membrane capacitance. Analysis and the comparison of results concluded that under low extracellular potassium (5.4 mmol/L), current densities recorded were as follows: the control group averaged 1.87 6 0.54 pA/pF, the 100 mmol/L OA group averaged
FIGURE 4. Effect of different concentrations of oleic acid (OA) on the expression of Kir6.1 messenger RNA (mRNA) in HUASMCs. Various concentrations (50, 100 and 200 mmol/L) of OA were administered to HUASMCs for 24 hours, and quantitative real-time polymerase chain reaction analysis indicated that Kir6.1 mRNA in the OA treatment group was significantly lower than in the control group (P , 0.05). Ó 2012 Lippincott Williams & Wilkins
1.68 6 0.93 pA/pF and the GFX intervention group averaged 1.97 6 0.39 pA/pF. There was no significant difference among the 3 groups (P . 0.05). Under a high potassium (140 mmol/L) perfusion, the average current density was 4.34 6 0.38 pA/pF in the control group and 2.99 6 0.42 pA/pF in the 100 mmol/L OA group (P , 0.05). The current density in the GFX intervention group was 3.81 6 0.49 pA/pF, which was significantly different (P , 0.05) compared with the aforementioned 2 groups. After perfusion with pinacidil, the average current density of the control group was 30.05 6 4.74 pA/pF and the density in the 100 mmol/L OA group was 20.13 6 1.76 pA/pF (P , 0.01). The current density in the GFX intervention group was 23.91 6 1.97 pA/ pF, which was significantly different compared with the aforementioned groups (P , 0.01; Figure 8).
DISCUSSION
Bulow et al20 first discovered the relationship between FFA and blood pressure in 1990. Grekin et al21 later
FIGURE 5. Effect of nitric oxide (NO) pretreatment on the expression of messenger RNA (mRNA) in HUASMCs after oleic acid (OA) intervention. Real-time polymerase chain reaction analysis indicated that pretreatment with NO followed by OA decreased Kir6.1 mRNA expression levels (P , 0.05) in the NO intervention plus OA group (100 mmol/L) when compared with the control group. Kir6.1 mRNA expression was higher in the NO intervention group than in the OA group (100 mmol/L); however, this was not statistically significant (P . 0.05).
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FIGURE 6. Effect of GF-109203X (GFX) pretreatment on HUASMC Kir6.1 messenger RNA (mRNA) expression after oleic acid (OA) intervention. Quantitative real-time polymerase chain reaction analysis revealed that pretreatment with GFX followed by 100 mmol/L OA caused Kir6.1 mRNA expression levels to decrease (P , 0.05) in the GFX intervention plus OA group (100 mmol/L) when compared with the control group. Kir6.1 mRNA expression was higher in the GFX intervention group than in the OA group (100 mmol/L; P , 0.05).
demonstrated that Sprague-Dawley rats exhibited an instant increase in blood pressure following administration of a sodium oleate solution via either the portal or femoral vein. In addition, there are a number of clinical trials revealing that acute FFA increases in blood can generate an elevation in blood pressure.22–26 K+ channels play an important role in the regulation of membrane resting potentials in VSMCs, but little is known regarding the effect of OA on the expression and function of VSMC KATP channels. In this study, Western immunoblot and quantitative RT-PCR analyses revealed that OA attenuated Kir6.1 protein
and mRNA expression of HUASMCs. In addition, as the concentrations of OA increased, the inhibition became prominently enhanced in a dose-dependent manner. Pretreatment of either exogenous NO or GFX (PKC inhibitor) in cultured HUASMCs abrogated the inhibition of Kir6.1 mRNA and protein expression induced by OA. This study indicated that OA may directly affect the KATP channel gene and protein. The contribution of OA to KATP channels was determined in our study. First, exogenous DETA-NO was applied to the culture medium, relieving the suppression of OA on Kir6.1 mRNA and protein expression. DETA-NO is used extensively to represent exogenous NO27 and consistently releases NO at certain concentrations in the culture medium. NO is one of the most widely used vasodilators in vivo and is synthesized and secreted by the endothelial cells. Our previous study showed that HUASMCs could synthesize NO.28 Miyoshi et al29 showed that the NO/cGMP pathway could be activated by opening KATP channels in VSMCs. Other studies also showed that NO could activate the KATP channels in ventricular myocytes.30 We discovered that OA inhibited the synthesis and release of NO in endothelial cells (unpublished data, Xiao-Jun Bai, 2008). This study not only revealed that NO could act directly on the KATP channel but also implied that OA might have affected KATP channel function and influenced the state of relaxation and contraction of VSMCs. Furthermore, before OA was administered, the pretreatment of GFX weakened OA inhibition of KATP channels in VSMCs. GFX15 is a new PKC inhibitor with a strong inhibitive potency. The PKC pathway is involved in the regulation of VSMCs in the presence of allergens. In the VSM cells of the rabbit mesenteric artery, vasoactive substances, such as norepinephrine, serotonin, neuropeptide Y and histamine, can suppress KATP current activated by pinacidil. However, application of phospholipase C inhibitors and PKC inhibitors can cause a reversal, suggesting that these vasoconstrictor substances possibly activate phospholipase C, yielding diacylglycerol to activate the PKC pathway. Our findings further indicated that OA might have downregulated the gene expression of KATP and reduced its protein expression via the activated PKC pathway. Furthermore, results from our patch-clamp
FIGURE 7. Whole-cell patch clamp KATP current. (A) High potassium and pinacidil perfusion, (B) oleic acid (OA) intervention and (C) pretreatment with GF-109203X (GFX) and OA intervention. Under normal circumstances, the KATP current was weaker in the OA group and the GFX intervention group (A). OA of 100 mmol/L significantly inhibited cellular KATP channel (B). Pretreatment with 100 mmol/L GFX moderated the inhibition of OA on the KATP channel (C).
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FIGURE 8. The current recorded by the episodic stimulation mode. (A) Cell current at low potassium, (B) high potassium, (C) pinacidil perfusion and (D) comparison of current among the different groups. *P , 0.05 versus control group; **P , 0.01 versus control group; #P , 0.05 versus oleic acid (OA) group; ##P , 0.01 versus OA group. Under low extracellular potassium, there was no significant difference in current densities among the 3 groups (P . 0.05; A). Under a high potassium (140 mmol/L) perfusion, the current density in the GF-109203X (GFX) intervention group was significantly different (P , 0.05) compared with the control and OA groups (B). After perfusion with pinacidil, current density in the GFX intervention group was significantly different compared with the aforementioned groups (P , 0.01; C). PKCI, protein kinase C inhibitor.
studies indicated that either the administration of OA alone or in combination with a PKC inhibitor significantly blocked KATP current in HUASMCs. Presumably, OA may have an effect on gene subunits and protein expression of KATP channels. In this study, whole-cell patch clamping revealed that the application of OA markedly inhibited the KATP channel current of cultured HUASMCs. This inhibition was weaker under low concentrations of extracellular potassium because of fewer open KATP channels. The inhibitory effect of OA became predominant after perfusion with high potassium or the specific KATP channel opener, pinacidil, was employed. Pretreatment of the PKC inhibitor, GFX partially recovered the KATP current after OA treatment. This confirmed the effect of OA on the KATP channel, and also implied that OA works via multiple pathways, rather than just the PKC pathway. In addition to the gap-free mode, the episodic stimulation mode was employed to directly probe the open state of the KATP channels. The results showed that OA significantly inhibited the KATP channel current and GFX could partially reverse this effect, consistent with that in the literature.31 Most researchers now believe that OA and other lipids with an anionic charge have a direct inhibition on KATP channels. KATP channels serve as a bridge in extensive tissues across cellular metabolism and membrane excitability. As an important energy substrate, OA can regulate KATP channels in a variety of tissues. In the nervous system, OA can activate the hypothalamic KATP channels to repress glucose outcome in the liver and regulate blood glucose concentration.32 For pancreatic Ó 2012 Lippincott Williams & Wilkins
b cells, an elevated concentration of OA can activate its KATP channel, resulting in cell membrane hyperpolarization, which inactivates voltage-dependent Ca2+ channels, decreases Ca2+ influx and leads to reduced insulin secretion. For myocardial cells,33 coenzyme A derived by OA esterification can open the KATP channel, whereas direct OA inhibits the KATP channel. When myocardial ischemia occurs, coenzyme A, the direct substrate to b-oxidation, first accumulates to promote the opening of KATP channels and shortens the action potential. This causes the Ca2+ influx to decrease and myocardial contractility and oxygen consumption drop. Five minutes after myocardial ischemia occurs, OA begins to accumulate, inhibiting the KATP channels and delaying the action potential during heart muscle ischemia. There are multiple pathways by which OA inhibits KATP channels. First, OA directly targets the lipid bilayer structure of the cell membrane, forming lipid holes to exert channel-like activity, and thus interfering with the opening of the KATP channel.34 Second, OA activates certain cell signaling pathways, such as the PKC pathway, to inhibit the expression and function of the KATP channels. Third, OA enters into the cytoplasm through the cell membrane with the assistance of fatty acyl coenzyme A synthetase to transfer into olein acyl coenzyme A, stimulating the opening of the KATP channel.35 In this study, we speculated that OA in the culture medium has sufficient time to enter the cytoplasm and is converted to olein acyl coenzyme A to activate the KATP channel, which
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partially counteracts the inhibitory effect of OA on the KATP channel. Using the whole-cell patch-clamp technique, we observed that OA significantly inhibited KATP current in VSMCs, indicating that OA not only interferes with KATP channel mRNA and protein expression but also directly inhibits its function. PKC inhibitors partially reversed this inhibition, indicating that the PKC pathway may be involved.
16. Sobey CG. Potassium channel function in vascular disease. Arterioscler Thromb Vasc Biol 2001;21:28–38. 17. Waldron GJ, Cole WC. Activation of vascular smooth muscle K+ channels by endothelium-derived relaxing factors. Clin Exp Pharmacol Physiol 1999;26:180–4. 18. Davda RK, Stepniakowski KT, Lu G, et al. Oleic acid inhibits endothelial nitric oxide synthase by a protein kinase C-independent mechanism. Hypertension 1995;26:764–70.
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7. Yokoshiki H, Sunagawa M, Seki T, et al. ATP-sensitive K+ channels in pancreatic, cardiac, and vascular smooth muscle cells. Am J Physiol 1998;274:C25–37. 8. Brayden JE. Functional roles of KATP channels in vascular smooth muscle. Clin Exp Pharmacol Physiol 2002;29:312–6. 9. Yamada M, Isomoto S, Matsumoto S, et al. Sulphonylurea receptor 2B and Kir6.1 form a sulphonylurea-sensitive but ATP-insensitive K+ channel. J Physiol 1997;499(pt 3):715–20. 10. Satoh E, Yamada M, Kondo C, et al. Intracellular nucleotide-mediated gating of SUR/Kir6.0 complex potassium channels expressed in a mammalian cell line and its modification by pinacidil. J Physiol 1998;511 (pt 3):663–74. 11. Inagaki N, Tsuura Y, Namba N, et al. Cloning and functional characterization of a novel ATP-sensitive potassium channel ubiquitously expressed in rat tissues, including pancreatic islets, pituitary, skeletal muscle, and heart. J Biol Chem 1995;270:5691–4. 12. Isomoto S, Kondo C, Yamada M, et al. A novel sulfonylurea receptor forms with BIR (Kir6.2) a smooth muscle type ATP-sensitive K+ channel. J Biol Chem 1996;271:24321–4. 13. Chutkow WA, Pu J, Wheeler MT, et al. Episodic coronary artery vasospasm and hypertension develop in the absence of Sur2 K(ATP) channels. J Clin Invest 2002;110:203–8. 14. Branstrom R, Corkey BE, Berggren PO, et al. Evidence for a unique long chain acyl-CoA ester binding site on the ATP-regulated potassium channel in mouse pancreatic beta cells. J Biol Chem 1997;272:17390–4. 15. Blackshear PJ. Approaches to the study of protein kinase C involvement in signal transduction. Am J Med Sci 1988;296:231–40.
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26. Lopes HF, Martin KL, Nashar K, et al. DASH diet lowers blood pressure and lipid-induced oxidative stress in obesity. Hypertension 2003;41:422–30. 27. Hrabie JA, Klose JR, Wink DA, et al. New nitric oxide-releasing zwitterions derived from polyamines. J Org Chem 1993;58:1472–6. 28. Balligand JL, Kelly RA, Marsden PA, et al. Control of cardiac muscle cell function by an endogenous nitric oxide signaling system. Proc Natl Acad Sci U S A 1993;90:347–51. 29. Miyoshi H, Nakaya Y, Moritoki H. Nonendothelial-derived nitric oxide activates the ATP-sensitive K+ channel of vascular smooth muscle cells. FEBS Lett 1994;345:47–9. 30. Han J, Kim N, Kim E, et al. Modulation of ATP-sensitive potassium channels by cGMP-dependent protein kinase in rabbit ventricular myocytes. J Biol Chem 2001;276:22140–7. 31. Tucker SJ, Baukrowitz T. How highly charged anionic lipids bind and regulate ion channels. J Gen Physiol 2008;131:431–8. 32. Obici S, Feng Z, Morgan K, et al. Central administration of oleic acid inhibits glucose production and food intake. Diabetes 2002;51:271–5. 33. Liu GX, Hanley PJ, Ray J, et al. Long-chain acyl-coenzyme A esters and fatty acids directly link metabolism to K(ATP) channels in the heart. Circ Res 2001;88:918–24. 34. Woodbury DJ. Pure lipid vesicles can induce channel-like conductances in planar bilayers. J Membr Biol 1989;109:145–50. 35. Kim D, Duff RA. Regulation of K+ channels in cardiac myocytes by free fatty acids. Circ Res 1990;67:1040–6.
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Oleic Acid Inhibits the KATP Channel Subunit Kir6.1 and the KATP Current in Human Umbilical Artery Smooth Muscle Cells Xiao-Jun Bai, MD, Hong-Yan Tian, PhD, Ting-Zhong Wang, PhD, Yuan Du, MD, Yu-Tao Xi, MD, Yue Wu, MD, Jie Gao, MD and Ai-Qun Ma, PhD
Abstract: Background: The objective of the present study was to determine the effect of various concentrations of oleic acid (OA) on KATP channel expression and the potential relationship to exogenous nitrogen monoxide and protein kinase C levels. Methods: Human umbilical artery smooth muscle cells (HUASMCs), between the 7th and 10th passages, were divided into control group, OA group (final OA concentration of 0, 50, 100 or 200 mmol/L), nitric oxide (NO) intervention group, protein kinase C inhibitor group or GF-109203X (GFX) intervention group. Western immunoblotting was used to detect the protein expression of the KATP channel subunit Kir6.1. Also, quantitative real-time polymerase chain reaction analysis to determine Kir6.1 messenger RNA levels and whole-cell patch clamping to measure KATP currents were performed. Results: The results suggested that OA inhibited Kir6.1 protein and messenger RNA expression in HUASMCs. Under a high concentration of potassium (140 mmol/L), 100 mmol/L OA significantly reduced ATP-sensitive potassium current density, whereas a low extracellular concentration of potassium (5.4 mmol/L) did not influence KATP density. Pretreatment with either exogenous NO or GFX weakened the OA-induced inhibition of KATP in HUASMCs. Conclusions: The study demonstrated that OA inhibited Kir6.1, a KATP channel subunit, in HUASMCs, and indirectly inhibited the KATP current. In addition, the results indicated that NO and/or GFX partially reversed OA inhibition in HUASMCs. Key Indexing Terms: Vascular smooth muscle cell; Oleic acid; KATP channel; Nitric oxide; PKC depressor. [Am J Med Sci 2013;346 (3):204–210.]
C
ardiac and cerebrovascular diseases are the leading cause of death in China. Hypertension is a primary risk factor associated with these diseases. Recent studies have reported that increased free fatty acid (FFA) concentrations contribute to hypertension via multiple pathways. These pathways include alpha1-adrenergic stimulation, endothelial cell dysfunction, increased oxidative stress and stimulation of vascular cell growth.1–3 Resistance from medium-sized arteries and arterioles in the periphery plays a pivotal role in maintaining blood pressure. Furthermore, ion channels, such as the K+, Ca2+, Na+ and Cl2 channels, are active in stabilizing angiostasis. K+ channels are highly responsible for regulating the resting potential of the vascular smooth muscle (VSM) membrane and determining vascular tone and cavosurface. Alterations in K+ channel From the Department of Cardiovascular Medicine, the First Affiliated Hospital of the Xi’an Jiaotong University School of Medicine, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an, China. Submitted April 06, 2012; accepted in revised form July 25, 2012. The authors have no financial or other conflicts of interest to disclose. Correspondence: Ai-Qun Ma, PhD, Department of Cardiovascular Medicine, the First Affiliated Hospital of the Xi’an Jiaotong University School of Medicine, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, No. 277 West Yanta Road, Xi’an, Shaanxi 710061, China (E-mail: [email protected]).
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expression and dysfunction of vascular smooth muscle cells (VSMCs) affect K+ outflow and membrane potential level, thus changing vascular tone.4 Our previous study confirmed that ATP-sensitive potassium channels (ATP-sensitive K+ channels, KATP) regulated VSM tension and blood pressure by maintaining the resting potential of the VSMC membrane. The bridging role between myocyte electrical and metabolic activity has drawn much attention regarding metabolism and hypertension.5–8 The molecular structure of the KATP channels is composed of 4 Kir6.3 subunits and 4 sulfonylurea receptors (SUR) subunits. The KATP channels found in VSMCs are mainly composed of the SUR2B/Kir6.1 subtype. The Kir6.1 and SUR2B channels have several properties similar to vascular KATP channels, such as pharmacological properties and sensitivity to nucleotide diphosphates.9,10 Northern blot and real-time polymerase chain reaction (RT-PCR) evidence also suggests that the combination of Kir6.1 and SUR2B is likely to be a KATP channel isoform in VSM.11,12 Mouse knockout models targeting the Kir6.1 and SUR2B genes emphasize the critical role of KATP in the cardiovascular system.13 Many studies have shown that one of the most representative FFAs in the blood circulation is oleic acid (OA), which is able to reduce ATP sensitivity of the pancreatic b-cell KATP channel,14 and inhibit the cardiac KATP channel.15 Studies have shown that OA impairs endothelial cell function, causing a decrease in nitric oxide (NO), and in turn dilating blood vessels via activation of the KATP channel of vascular endothelial cells.16,17 Another study revealed that OA could activate protein kinase C (PKC) in VSMCs, thereby enhancing the inhibition of the KATP channel.18,19 However, the mechanisms underlying KATP channel function and OA-induced high blood pressure remain unclear. The purpose of the present study was to investigate the underlying mechanisms by which OA inhibits the KATP channel to regulate blood pressure, and examine the mechanisms related to the reduction of NO synthesis and activation of the PKC pathway.
MATERIALS AND METHODS Human Umbilical Artery Smooth Muscle Cell Preparation Upon recovery, cryopreserved human umbilical artery smooth muscle cell (HUASMCs; Sciencell Company, San Diego, CA) were grown in a cell culture flask and plated with Smooth Muscle Cell Medium (Sciencell) supplemented with 2% fetal bovine serum (Gibco Company, New York, NY) and penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO2 and 95% air. The medium was changed every 1 to 2 days, and cells were characterized as smooth muscle cells (SMCs) by morphological criteria and the expression of smooth muscle a-actin. The cells were grown until 70% to 80% confluence, trypsinized using 0.25% trypsin every 4 to 6 days and passed in proportion of 1:2 or 1:3. Cells in the 7th to 10th passage of the logarithmic growth phase were employed for
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subsequent experiments. At approximately 90% confluence, cells were cultivated in serum-free SMCM medium for 12 hours, allowing cultured cells to be quiescent before experiments. Our experimental groups were divided as follows: control group (without intervention and OA), OA group (medium containing final OA concentrations of 0, 50, 100 and 200 mmol/ L), NO intervention group (cells were first cultured with medium supplemented with a final diethylenetriamine-NO concentration of 20 mmol/L for 30 minutes and then 100 mmol/L OA was added) and GF-109203X (GFX) intervention group (cells were cultured with a final GFX concentration of 100 nmol/L for 1 hour and then 100 mmol/L OA was added). The duration for all treatments was 24 hours, and the treatment time for each group was 24 hours. HUASMCs were harvested using trypsinization at the end of the experiment. Preparation of the Fatty Acid-Bovine Serum Albumin Complex OA (75.3 mL; Sigma, St. Louis, MO) was added to 58 mL of 0.1 mM NaOH, and the solution was heated to 95°C for 5 minutes. Next, 50 mL of this solution was added to 950 mL of 10% bovine serum albumin (BSA) solution and incubated for 30 minutes at 55°C. The final solution contained a 5 mM OA/10% BSA stock solution. Addition of 10, 20 and 40 mL/mL of the OA/10% BSA stock solution to SMCM media corresponded to the final experimental concentrations of 50, 100 and 200 mmol/L OA, respectively. The BSA-bound OA stock solution was then cooled to room temperature and sterile filtered. Western Immunoblotting Cell proteins were extracted using a RIPA protein lysate buffer (Biomiga, San Diego, CA). The protein concentration was measured using the Bradford method. Cell proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene fluoride membrane at a constant current of 350 mA for 90 minutes at 4°C in a solution containing 25 mM Tris, 193 mM glycine and 10% methanol. The membrane was incubated at room temperature first with Tris-buffered saline containing 5% nonfat dried milk and 0.1% Tween 20, and then with the anti-Kir6.1 antibody (Alomone, Jerusalem, Israel) at a working dilution of 1:200. The immune complexes were detected with an IRDye 800CWlabeled anti-rabbit IgG secondary antibody at a working dilution of 1:3000. Lastly, enhanced chemiluminescene reagent was applied and the membrane underwent X-ray film exposure, developing and fixing. The strip was scanned and analyzed using densitometric analyses. HUASMC Kir6.1 Messenger RNA Expression by Quantitative RT-PCR Total cellular RNA was extracted according to the manufacturer’s instructions. Reverse transcription was conducted according to the steps described in the reverse transcription kit (TaKaRa, Kyoto, Japan). The Kir6.1 sequence was as follows: upstream, 5’-GAT CAT CTG CCA CGT GAT TGA-3’ and downstream, 5’-GCA ATG TAG GAG GTT CGT GCT-3’. The sequence for b-actin was as follows: upstream, 5’-GCA AAG ACC TGT ACG CCA ACA-3’ and downstream, 5’-ACA CGG AGT ACT TGC GCT CAG-3’. Quantitative RT-PCR amplification was performed using a TaKaRa SYBR RT-PCR kit under the following process: pre-denaturation for 2 minutes at 95°C, denaturation for 15 seconds at 95°C and annealing for 30 seconds at 60°C, at a total of 50 cycles. Fluorescence data were acquired during the annealing/extension step. Threshold Ó 2012 Lippincott Williams & Wilkins
cycles were measured at the end of each extension step using the second-derivative method offered by the iCycler IQ optical system software (version 3.0a; Bio-Rad Laboratories, Hercules, CA). Kir6.1 messenger RNA (mRNA) levels were quantified using the relative standard curve method, and each gene amplification in triplicate underwent the same conditions. Electrophysiological Recording of KATP Channel Activity Whole-cell patch-clamp techniques were used to record KATP channel currents. Briefly, cells were placed in a Petri dish mounted on the stage of an inverted phase-contrast microscope (Olympus IX71; Tokyo, Japan). Currents were recorded using an Axopatch-700B patch clamp amplifier (Axon Instruments Inc, Foster City, CA), controlled by a Digidata 1314 interface and a pClamp software package (version 9.2; Axon Instruments Inc). Membrane currents were filtered at 1 kHz using an 8-pole Bessel filter and stored on a computer for offline analyses. At the beginning of each experiment, the junction potential between the pipette solution and the bath solution was electronically adjusted to zero. No leakage subtraction was applied to the original recordings, and all cells with visible changes in leakage currents during the course of study were excluded from further analyses. The pipette resistances were 2 to 4 MV. The pipette solution contained (in mM) KCl 140, CaCl2 1, MgCl2 1, HEPES 10 and ethylene glycol tetraacetic acid 10. The pH was adjusted to 7.3 with KOH. Seals were made in 5.4 mM K+ extracellular solution containing (in mM) NaCl 135, KCl 5.4, CaCl2 1, MgCl2 1.2, HEPES 10 and glucose 5. The solution pH was adjusted to 7.3 with NaOH, but most recordings were made under symmetrical conditions of 140 mM K+ using an extracellular solution of the same composition as described previously, except that KCl was 140 mM and NaCl was omitted. The experimental bath (volume 2 mL) was perfused continuously with the extracellular solution at a rate of 2 mL/min. All experiments were performed at room temperature (20–23°C). Statistical Analyses The software SPSS 13.0 was used for statistical analyses. Data are expressed as mean 6 standard error of the mean. The mean comparisons were performed with a single factor analysis of variance. a , 0.05 was considered statistically significant.
RESULTS Effect of Various Concentrations of OA on the Protein Expression of Kir6.1 Three concentrations (50, 100 and 200 mmol/L) of OA had a negative effect on the protein expression of Kir6.1. During Western immunoblot analyses, Kir6.1 protein expression in the OA intervention group was significantly decreased, as compared with the control group (P , 0.05), following OA administration for 24 hours (Figure 1). As the OA concentration increased, the protein expression gradually decreased (P , 0.05). Effect of NO Pretreatment on Kir6.1 Protein Expression After OA Intervention HUASMCs were pretreated with 20 mmol/L NO for 30 minutes, followed by 100 mmol/L OA administration, and then incubated for 24 hours. Kir6.1 protein expression significantly decreased (P , 0.05 in the NO intervention plus OA group; 100 mmol/L) in comparison with the control group. In contrast, the Kir6.1 protein expression in the NO intervention
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FIGURE 1. Effect of 3 concentrations of oleic acid (OA) on the expression of Kir6.1. Western immunoblot analyses showed that 3 concentrations (50, 100 and 200 mmol/L) of OA inhibited protein expression of Kir6.1. There was a significant difference in the KATP channel subunit Kir6.1 protein expression between the OA intervention group and the control group (P , 0.05). As OA concentration increased, protein expression gradually decreased (P , 0.05).
group increased compared with that found in the OA group (100 mmol/L; P , 0.05; Figure 2).
(100 mmol/L), but without statistical significance (P . 0.05; Figure 5).
Effect of GFX Pretreatment on Kir6.1 Protein Expression After OA Intervention HUASMCs were pretreated with 100 mmol/L of GFX for 1 hour, followed by 100 mmol/L OA administration, and then incubated for 24 hours. Western immunoblot analyses confirmed that the Kir6.1 protein expression levels decreased (P , 0.05) in the GFX intervention plus OA group (100 mmol/L) compared with the control group. The Kir6.1 protein expression was higher in the GFX intervention group than in the OA group (100 mmol/L; P , 0.05; Figure 3).
Effect of GFX Pretreatment on Kir6.1 mRNA Expression After OA Intervention HUASMCs were pretreated with 100 mmol/L GFX for 1 hour, followed by 100 mmol/L OA administration, and then incubated for 24 hours. Quantitative RT-PCR revealed that Kir6.1 mRNA expression levels decreased (P , 0.05) in the GFX intervention plus OA group (100 mmol/L), as compared with the control group. The Kir6.1 mRNA expression was higher in the GFX intervention group than in the OA group (100 mmol/L; P , 0.05; Figure 6), but the mRNA expression did not reach the level of the control group.
Effect of Different Concentrations of OA on Kir6.1 mRNA Expression Various concentrations (50, 100 and 200 mmol/L) of OA were administered to HUASMCs for 24 hours. Kir6.1 mRNA was significantly lower in the OA treatment group than in the control group (P , 0.05; Figure 4). Effect of NO Pretreatment on the Expression of Kir6.1 mRNA After OA Intervention HUASMCs were pretreated with 20 mmol/L NO for 30 minutes, followed by 100 mmol/L OA administration, and then incubated for 24 hours. RT-PCR analysis indicated that Kir6.1 mRNA expression levels decreased (P , 0.05) in the NO intervention plus OA group (100 mmol/L) compared with the control group. The Kir6.1 mRNA expression was higher in the NO intervention group than in the OA group
Recordings of Cultured HUASMC KATP Currents Induced by OA and GFX Using the Gap-Free Mode The gap-free mode was employed to record HUASMC KATP currents for the 3 groups (control group, OA group and GFX intervention group). The sampling time was set for 100 mseconds, and the patch camp voltage was maintained at 0 mV. In the control group (Figure 7A), the KATP current was weaker than the OA group and the GFX intervention group. We used a high potassium concentration (140 mmol/L) and perfused a potassium channel-specific opener, pinacidil, for 10 minutes, elevating the channel current to the largest opening range with a 2300 pA current density. Because hypokalemia (5.4 mmol/L) perfusion was able to wash out the current, the current most likely came from a KATP channel. When 100 mmol/L OA was applied for 24 hours, the cell KATP channel was significantly inhibited (Figure 7B), with a maximum KATP current of
FIGURE 2. Effect of nitric oxide (NO) pretreatment on HUASMC Kir6.1 protein expression under oleic acid (OA) intervention. Western immunoblot analyses showed that pretreatment with NO followed by OA decreased Kir6.1 protein expression levels (P , 0.05) in comparison with the control group. In contrast, Kir6.1 protein expression in the NO intervention group increased compared with the OA group (P , 0.05).
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FIGURE 3. Effect of GF109203X (GFX) pretreatment on HUASMC Kir6.1 protein expression after oleic acid (OA) intervention. Western immunoblot analyses showed that pretreatment with GFX followed by OA decreased Kir6.1 protein expression levels in the GFX intervention plus OA group (100 mmol/L), as compared with the control group (P , 0.05). Kir6.1 protein expression was higher in the GFX intervention group than in the OA group (100 mmol/L; P , 0.05).
approximately 2150 pA. HUASMCs were pretreated with 100 mmol/L GFX for 1 hour, followed by 100 mmol/L OA administration, and then incubated for 24 hours. This treatment moderated the inhibition of OA on the KATP channel (Figure 7C), with a maximum KATP current density of more than 2200 pA. Recordings of Cultured HUASMC KATP Currents Induced by OA and GFX Using the Episodic Stimulation Mode To further elucidate the effect of OA and GFX on KATP channels, we employed the episodic stimulation mode to record HUASMC KATP currents in the 3 groups (control group, OA group and GFX intervention group). To eliminate the discrepancy of the current from cell size, we used current density as the measuring unit for recording the current. Current density was calculated as the measured current divided by the measured cell membrane capacitance. Analysis and the comparison of results concluded that under low extracellular potassium (5.4 mmol/L), current densities recorded were as follows: the control group averaged 1.87 6 0.54 pA/pF, the 100 mmol/L OA group averaged
FIGURE 4. Effect of different concentrations of oleic acid (OA) on the expression of Kir6.1 messenger RNA (mRNA) in HUASMCs. Various concentrations (50, 100 and 200 mmol/L) of OA were administered to HUASMCs for 24 hours, and quantitative real-time polymerase chain reaction analysis indicated that Kir6.1 mRNA in the OA treatment group was significantly lower than in the control group (P , 0.05). Ó 2012 Lippincott Williams & Wilkins
1.68 6 0.93 pA/pF and the GFX intervention group averaged 1.97 6 0.39 pA/pF. There was no significant difference among the 3 groups (P . 0.05). Under a high potassium (140 mmol/L) perfusion, the average current density was 4.34 6 0.38 pA/pF in the control group and 2.99 6 0.42 pA/pF in the 100 mmol/L OA group (P , 0.05). The current density in the GFX intervention group was 3.81 6 0.49 pA/pF, which was significantly different (P , 0.05) compared with the aforementioned 2 groups. After perfusion with pinacidil, the average current density of the control group was 30.05 6 4.74 pA/pF and the density in the 100 mmol/L OA group was 20.13 6 1.76 pA/pF (P , 0.01). The current density in the GFX intervention group was 23.91 6 1.97 pA/ pF, which was significantly different compared with the aforementioned groups (P , 0.01; Figure 8).
DISCUSSION
Bulow et al20 first discovered the relationship between FFA and blood pressure in 1990. Grekin et al21 later
FIGURE 5. Effect of nitric oxide (NO) pretreatment on the expression of messenger RNA (mRNA) in HUASMCs after oleic acid (OA) intervention. Real-time polymerase chain reaction analysis indicated that pretreatment with NO followed by OA decreased Kir6.1 mRNA expression levels (P , 0.05) in the NO intervention plus OA group (100 mmol/L) when compared with the control group. Kir6.1 mRNA expression was higher in the NO intervention group than in the OA group (100 mmol/L); however, this was not statistically significant (P . 0.05).
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FIGURE 6. Effect of GF-109203X (GFX) pretreatment on HUASMC Kir6.1 messenger RNA (mRNA) expression after oleic acid (OA) intervention. Quantitative real-time polymerase chain reaction analysis revealed that pretreatment with GFX followed by 100 mmol/L OA caused Kir6.1 mRNA expression levels to decrease (P , 0.05) in the GFX intervention plus OA group (100 mmol/L) when compared with the control group. Kir6.1 mRNA expression was higher in the GFX intervention group than in the OA group (100 mmol/L; P , 0.05).
demonstrated that Sprague-Dawley rats exhibited an instant increase in blood pressure following administration of a sodium oleate solution via either the portal or femoral vein. In addition, there are a number of clinical trials revealing that acute FFA increases in blood can generate an elevation in blood pressure.22–26 K+ channels play an important role in the regulation of membrane resting potentials in VSMCs, but little is known regarding the effect of OA on the expression and function of VSMC KATP channels. In this study, Western immunoblot and quantitative RT-PCR analyses revealed that OA attenuated Kir6.1 protein
and mRNA expression of HUASMCs. In addition, as the concentrations of OA increased, the inhibition became prominently enhanced in a dose-dependent manner. Pretreatment of either exogenous NO or GFX (PKC inhibitor) in cultured HUASMCs abrogated the inhibition of Kir6.1 mRNA and protein expression induced by OA. This study indicated that OA may directly affect the KATP channel gene and protein. The contribution of OA to KATP channels was determined in our study. First, exogenous DETA-NO was applied to the culture medium, relieving the suppression of OA on Kir6.1 mRNA and protein expression. DETA-NO is used extensively to represent exogenous NO27 and consistently releases NO at certain concentrations in the culture medium. NO is one of the most widely used vasodilators in vivo and is synthesized and secreted by the endothelial cells. Our previous study showed that HUASMCs could synthesize NO.28 Miyoshi et al29 showed that the NO/cGMP pathway could be activated by opening KATP channels in VSMCs. Other studies also showed that NO could activate the KATP channels in ventricular myocytes.30 We discovered that OA inhibited the synthesis and release of NO in endothelial cells (unpublished data, Xiao-Jun Bai, 2008). This study not only revealed that NO could act directly on the KATP channel but also implied that OA might have affected KATP channel function and influenced the state of relaxation and contraction of VSMCs. Furthermore, before OA was administered, the pretreatment of GFX weakened OA inhibition of KATP channels in VSMCs. GFX15 is a new PKC inhibitor with a strong inhibitive potency. The PKC pathway is involved in the regulation of VSMCs in the presence of allergens. In the VSM cells of the rabbit mesenteric artery, vasoactive substances, such as norepinephrine, serotonin, neuropeptide Y and histamine, can suppress KATP current activated by pinacidil. However, application of phospholipase C inhibitors and PKC inhibitors can cause a reversal, suggesting that these vasoconstrictor substances possibly activate phospholipase C, yielding diacylglycerol to activate the PKC pathway. Our findings further indicated that OA might have downregulated the gene expression of KATP and reduced its protein expression via the activated PKC pathway. Furthermore, results from our patch-clamp
FIGURE 7. Whole-cell patch clamp KATP current. (A) High potassium and pinacidil perfusion, (B) oleic acid (OA) intervention and (C) pretreatment with GF-109203X (GFX) and OA intervention. Under normal circumstances, the KATP current was weaker in the OA group and the GFX intervention group (A). OA of 100 mmol/L significantly inhibited cellular KATP channel (B). Pretreatment with 100 mmol/L GFX moderated the inhibition of OA on the KATP channel (C).
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FIGURE 8. The current recorded by the episodic stimulation mode. (A) Cell current at low potassium, (B) high potassium, (C) pinacidil perfusion and (D) comparison of current among the different groups. *P , 0.05 versus control group; **P , 0.01 versus control group; #P , 0.05 versus oleic acid (OA) group; ##P , 0.01 versus OA group. Under low extracellular potassium, there was no significant difference in current densities among the 3 groups (P . 0.05; A). Under a high potassium (140 mmol/L) perfusion, the current density in the GF-109203X (GFX) intervention group was significantly different (P , 0.05) compared with the control and OA groups (B). After perfusion with pinacidil, current density in the GFX intervention group was significantly different compared with the aforementioned groups (P , 0.01; C). PKCI, protein kinase C inhibitor.
studies indicated that either the administration of OA alone or in combination with a PKC inhibitor significantly blocked KATP current in HUASMCs. Presumably, OA may have an effect on gene subunits and protein expression of KATP channels. In this study, whole-cell patch clamping revealed that the application of OA markedly inhibited the KATP channel current of cultured HUASMCs. This inhibition was weaker under low concentrations of extracellular potassium because of fewer open KATP channels. The inhibitory effect of OA became predominant after perfusion with high potassium or the specific KATP channel opener, pinacidil, was employed. Pretreatment of the PKC inhibitor, GFX partially recovered the KATP current after OA treatment. This confirmed the effect of OA on the KATP channel, and also implied that OA works via multiple pathways, rather than just the PKC pathway. In addition to the gap-free mode, the episodic stimulation mode was employed to directly probe the open state of the KATP channels. The results showed that OA significantly inhibited the KATP channel current and GFX could partially reverse this effect, consistent with that in the literature.31 Most researchers now believe that OA and other lipids with an anionic charge have a direct inhibition on KATP channels. KATP channels serve as a bridge in extensive tissues across cellular metabolism and membrane excitability. As an important energy substrate, OA can regulate KATP channels in a variety of tissues. In the nervous system, OA can activate the hypothalamic KATP channels to repress glucose outcome in the liver and regulate blood glucose concentration.32 For pancreatic Ó 2012 Lippincott Williams & Wilkins
b cells, an elevated concentration of OA can activate its KATP channel, resulting in cell membrane hyperpolarization, which inactivates voltage-dependent Ca2+ channels, decreases Ca2+ influx and leads to reduced insulin secretion. For myocardial cells,33 coenzyme A derived by OA esterification can open the KATP channel, whereas direct OA inhibits the KATP channel. When myocardial ischemia occurs, coenzyme A, the direct substrate to b-oxidation, first accumulates to promote the opening of KATP channels and shortens the action potential. This causes the Ca2+ influx to decrease and myocardial contractility and oxygen consumption drop. Five minutes after myocardial ischemia occurs, OA begins to accumulate, inhibiting the KATP channels and delaying the action potential during heart muscle ischemia. There are multiple pathways by which OA inhibits KATP channels. First, OA directly targets the lipid bilayer structure of the cell membrane, forming lipid holes to exert channel-like activity, and thus interfering with the opening of the KATP channel.34 Second, OA activates certain cell signaling pathways, such as the PKC pathway, to inhibit the expression and function of the KATP channels. Third, OA enters into the cytoplasm through the cell membrane with the assistance of fatty acyl coenzyme A synthetase to transfer into olein acyl coenzyme A, stimulating the opening of the KATP channel.35 In this study, we speculated that OA in the culture medium has sufficient time to enter the cytoplasm and is converted to olein acyl coenzyme A to activate the KATP channel, which
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partially counteracts the inhibitory effect of OA on the KATP channel. Using the whole-cell patch-clamp technique, we observed that OA significantly inhibited KATP current in VSMCs, indicating that OA not only interferes with KATP channel mRNA and protein expression but also directly inhibits its function. PKC inhibitors partially reversed this inhibition, indicating that the PKC pathway may be involved.
16. Sobey CG. Potassium channel function in vascular disease. Arterioscler Thromb Vasc Biol 2001;21:28–38. 17. Waldron GJ, Cole WC. Activation of vascular smooth muscle K+ channels by endothelium-derived relaxing factors. Clin Exp Pharmacol Physiol 1999;26:180–4. 18. Davda RK, Stepniakowski KT, Lu G, et al. Oleic acid inhibits endothelial nitric oxide synthase by a protein kinase C-independent mechanism. Hypertension 1995;26:764–70.
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Volume 346, Number 3, September 2013