Inhibitory Effect of High Concentration of Glucose on Relaxations to Activation of ATP-Sensitive K+ Channels in Human Omental Artery

Inhibitory Effect of High Concentration of Glucose onRelaxations to Activation of ATP-Sensitive K� Channels in

Human Omental ArteryHiroyuki Kinoshita, Toshiharu Azma, Katsutoshi Nakahata, Hiroshi Iranami, Yoshiki Kimoto,

Mayuko Dojo, Osafumi Yuge, Yoshio Hatano

Objective—The present study was designed to examine in the human omental artery whether high concentrations ofD-glucose inhibit the activity of ATP-sensitive K� channels in the vascular smooth muscle and whether this inhibitoryeffect is mediated by the production of superoxide.

Methods and Results—Human omental arteries without endothelium were suspended for isometric force recording.Changes in membrane potentials were recorded and production of superoxide was evaluated. Glibenclamide abolishedvasorelaxation and hyperpolarization in response to levcromakalim. D-glucose (10 to 20 mmol/L) but not L-glucose(20 mmol/L) reduced these vasorelaxation and hyperpolarization. Tiron and diphenyleneiodonium, but not catalase,restored vasorelaxation and hyperpolarization in response to levcromakalim in arteries treated with D-glucose.Calphostin C and Go6976 simultaneously recovered these vasorelaxation and hyperpolarization in arteries treated withD-glucose. Phorbol 12-myristate 13 acetate (PMA) inhibited the vasorelaxation and hyperpolarization, which arerecovered by calphostin C as well as Go6976. D-glucose and PMA, but not L-glucose, significantly increased superoxideproduction from the arteries, whereas such increased production was reversed by Tiron.

Conclusions—These results suggest that in the human visceral artery, acute hyperglycemia modulates vasodilationmediated by ATP-sensitive K� channels via the production of superoxide possibly mediated by the activation of proteinkinase C. (Arterioscler Thromb Vasc Biol. 2004;24:2290-2295.)

Key Words: ATP-sensitive K� channels � high glucose � human artery � protein kinase C � superoxide

Increasing evidence suggests that ATP-sensitive K� chan-nels play important roles in physiological and pathophys-

iological vasodilation.1 Previous studies on the diabetic ani-mal models suggest that hyperglycemia impairs the activityof ATP-sensitive K� channels in the vascular smooth musclecells.2,3 Although a recent study on coronary arterioles fromthe diabetic patients has documented the reduction of vasore-laxation mediated by ATP-sensitive K� channels,4 the acuteeffect of high glucose on the activity of K� channels has notbeen studied in the human blood vessels.

Studies using several diabetic animal models indicate thatsuperoxide reduces the activity of ATP-sensitive K� channelsin the vascular smooth muscle cells.5 However, the evidenceshowing that hyperglycemia-induced formation of reactiveoxygen species modulates the activity of ATP-sensitive K�

channels is scarce. Recent studies on the rat as well as therabbit demonstrated that protein kinase C activation inhibitsATP-sensitive K� channels expressed on vascular smoothmuscle cells.6,7 In animal models, hyperglycemia is report-

edly capable of increasing the activity of protein kinase C,whereas this has not been well-documented in the humanvasculature.8 In addition, it is unclear whether in the humanblood vessels the activation of protein kinase C via acuteexposure of high glucose may induce increased production ofsuperoxide, resulting in the inhibitory effect on the functionof K� channels.

Therefore, the present study was designed to examine inthe human omental artery, whether high concentrations ofD-glucose inhibit the activity of ATP-sensitive K� channels,and whether this inhibitory effect is mediated by the produc-tion of superoxide via the activation of protein kinase C.

MethodsThe institutional research committee approved this study and thewritten informed consent was obtained from each patient enrolled inthis study. The part of human greater omentum was obtained frompatients scheduled for the elective gastric surgery, and all of enrolledpatients (40 patients, aged 40 to 75 years) were without heart diseaseand coronary risk factors, including diabetes mellitus, hypertension,

Original received August 8, 2004; final version accepted September 28, 2004.From the Department of Anesthesia (H.K., K.N., H.I.), Japanese Red Cross Society, Wakayama Medical Center, and the Department of Anesthesiology

(Y.K., M.D., Y.H.), Wakayama Medical University, Wakayama; and the Department of Anesthesia (T.A.), Hiroshima General Hospital, and theDepartment of Anesthesiology (O.Y.), Hiroshima University School of Medicine, Hiroshima, Japan.

Correspondence to Hiroyuki Kinoshita, MD, PhD, Department of Anesthesiology, Wakayama Medical University, 811-1 Kimiidera, Wakayama641-0012, Japan. E-mail [email protected]

© 2004 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000148006.78179.c7

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and hypercholesterolemia. Immediately after the resection, thegreater omentum was put in ice-cold modified Krebs-Ringer bicar-bonate solution (control solution, pH 7.4).9 All experiments wereperformed in the presence of D-glucose (11 mmol/L) in thecontrol condition.

Organ Chamber ExperimentsEach omental artery (2-mm ring, 0.5 to 1.0 mm in diameter) withoutendothelium was connected to an isometric force transducer. Weremoved endothelium to avoid the involvement of endothelium-derived factors in this study. Optimal tension was achieved at �1.0g. During contraction in response to a prostaglandinH2/thromboxane receptor agonist U46619 (10�7 mol/L),concentration-response curves to levcromakalim (a generous giftfrom GlaxoSmithKline PLC, Greenford, United Kingdom) or dilti-azem were obtained in the absence or in the presence of gliben-clamide, D-glucose, L-glucose, or phorbol 12-myristate 13-acetate(PMA) in combination with calphostin C, Go 6976, Tiron, diphe-nyleneiodonium (DPI), catalase, genistein, or PD98059, which wereadded 60 minutes or 15 minutes (for PMA) before the contraction toU46619. The relaxations were expressed as a percentage of themaximal relaxation to papaverine (3�10�4 mol/L).9

Electrophysiological ExperimentsArterial rings were longitudinally cut and fixed on the bottom of anexperimental chamber. The arteries were continuously perfused withcontrol solution (37°C) bubbled with 95% O2–5% CO2 gas mixture.A glass microelectrode (tip resistance 40 to 80 mol/L�) filled with3 mol/L KCl and held by a micromanipulator (Narishige, Tokyo,Japan), was inserted into a smooth muscle cell from the intimal sideof the vessel.10,11 The electrical signal was amplified using arecording amplifier (Electro 705; World Precision Instruments Inc).The membrane potential was continuously monitored and recordedon a chart recorder (SS-250F-1; SENKONIC Inc). The validity of asuccessful impalement was assessed by a sudden negative shift,followed by a stable negative voltage for �2 minutes.10,11 Changesin membrane potentials produced by levcromakalim (10�5 mol/L)were continuously recorded. D-glucose, L-glucose, glibenclamide,calphostin C, Go 6976, Tiron, or DPI was applied 60 minutes beforemembrane potential recordings.

Chemiluminescence Detection of SuperoxideSuperoxide yielded from human omental arteries without endotheli-um was detected by using a luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-�]pyrazin-3-one (MCLA;Tokyo Kasei Kogyo, Tokyo, Japan), as a chemiluminescenceprobe.12 Pieces of the omental artery, dissected 1-cm-long, openedlongitudinally to expose the internal surface, were put into dishes ofa 96-well multititer plate of 40 �mol/L MCLA-containing Krebs–HEPES (70 �L). This multiplate was then promptly transferred to adark room to perform the following steps under red safe lights. A70-�L aliquot of drugs dissolved in Krebs–HEPES–MCLA bufferwas added to each well containing vessel segments. The plate wasthen covered with a blue light-sensitive roentgen film, especiallyequipped for chemiluminescence detection of immunoblotting ex-periments (Hyperfilm ECL; Amersham Biosciences). After a 15-minute exposure to vessel segments, the film was developed using anautomatic X-ray film processor. The film was then scanned and theoptical density of each well was evaluated by using an imageprocessing ImageJver 1.30 (Research Services Branch, NationalInstitute of Mental Health). The intensity of chemiluminescencefrom each well was expressed as percentage, assuming the opticaldensity of autoradiography from the buffer of 0% and that fromvessels loaded with 20 mmol/L D-glucose to be 100%.

Statistical AnalysisThe data are expressed as means�SD, and n refers to the number ofpatients from whom the omental artery was taken. Statistical analysiswas performed using repeated measures analysis of variance, fol-

lowed by Scheffe F test for multiple comparisons. Differences wereconsidered to be statistically significant at P�0.05.

ResultsOrgan Chamber ExperimentsDuring submaximal contraction to U46619 (10�7 mol/L), aselective ATP-sensitive K� channel opener, levcromakalim(10�8 to 10�5 mol/L) induced concentration-dependent relax-ation in the human omental artery without endotheliumtreated with L-glucose (20 mmol/L) (please see http://atvb.ahajournals.org.). A selective ATP-sensitive K� channel an-tagonist, glibenclamide, completely abolished this vasorelax-ation, whereas it did not affect the basal tone of the omentalartery.

D-glucose (10 to 20 mmol/L) concentration-dependentlyreduced vasorelaxation induced by levcromakalim (Figure 1),whereas D-glucose (20 mmol/L) did not affect vasorelaxationproduced by a voltage-dependent Ca2� channel antagonistdiltiazem (10�7 to 10�4 mol/L).

A superoxide scavenger, Tiron (10 mmol/L), and aNAD(P)H oxidase inhibitor, DPI (10�6 mol/L), restoredvasorelaxation in response to levcromakalim in the omentalarteries treated with D-glucose (20 mmol/L), whereas ahydrogen peroxide scavenger catalase (1200 U/mL) did notalter the inhibitory effect of D-glucose (20 mmol/L) (Figure2a). These inhibitors themselves did not affect the vasorelax-ation produced by levcromakalim. Protein kinase C inhibi-tors, calphostin C (3�10�7 mol/L), and Go 6976 (3�10�7

mol/L) restored vasorelaxation in response to levcromakalimin the omental arteries treated with D-glucose (20 mmol/L)(Figure 2b), whereas these inhibitors themselves did not alterthe vasorelaxation produced by levcromakalim. Phorbor 12-myristate 13-acetate ester, PMA (10�7 mol/L), impairedvasorelaxation in response to levcromakalim in the arteriestreated with L-glucose (20 mmol/L), which is completelyrecovered by calphostin C (3�10�7 mol/L) or Go 6976(3�10�7 mol/L) (Figure 2c). A nonselective tyrosine kinaseinhibitor genistein (10�6 mol/L) and a mitogen-activatedprotein kinase inhibitor PD98059 (10�5 mol/L) did not affectthe inhibitory effect induced by D-glucose (20 mmol/L)

Figure 1. Concentration-response curves to levcromakalim inthe absence or in the presence of L-glucose or D-glucose,obtained in the human artery without endothelium. *Differencebetween rings treated with L-glucose and rings treated withD-glucose is statistically significant (P�0.05).

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(Figure 2d). In each Figure, maximal relaxations in responseto papaverine (3�10�4 mol/L) were not different betweengroups.

Electrophysiological ExperimentsLevcromakalim (10�5 mol/L) produced hyperpolarization ofsmooth muscle cells of the human mental artery treated withL-glucose (20 mmol/L), which is abolished by glibenclamide(5�10�6 mol/L), and this hyperpolarization was reduced bythe treatment with D-glucose (20 mmol/L) (Figure 3). Tiron(10 mmol/L), DPI (10�6 mol/L), calphostin C (3�10�7

mol/L), and Go 6976 (3�10�7 mol/L) restored hyperpolar-ization in response to levcromakalim in the omental arteriestreated with D-glucose (20 mmol/L) (Figures 4 and 5).Resting membrane potentials did not differ among the groupsstudied (see legends for Figures 3, 4, and 5).

Chemiluminescence Detection of SuperoxideThe intensity of MCLA-dependent chemiluminescence fromhuman omental arteries loaded with D-glucose or PMA wassignificantly higher than those loaded with L-glucose (Figure6). A superoxide scavenger, Tiron, remarkably decreased thechemiluminescence from vessels loaded with D-glucose (Fig-ure 6).

DiscussionIn the human omental artery, glibenclamide abolished vasore-laxation as well as hyperpolarization in response to levcro-makalim.13,14 However, glibenclamide did not affect the basaltension as well as membrane potential of the omental artery,indicating that ATP-sensitive K� channels may not play a rolein the resting tone of visceral circulation in humans. Incontrast to this finding, a recent human study has documentedthat direct administration of glibenclamide to the largecoronary artery provokes reduction of resting vessel diameter,

suggesting that these channels may modulate resting tone ofthe large coronary artery in humans.15 We cannot rule out thepossible involvement of the regional difference in the mod-ulator role of ATP-sensitive K� channels in the humanvascular tone.

In the current study, addition of 10 or 20 mmol/LD-glucose, but not 20 mmol/L L-glucose, reduced vasorelax-ation and hyperpolarization mediated by ATP-sensitive K�

channels, whereas 20 mmol/L D-glucose did not alter vasore-laxation induced by a voltage-dependent Ca2� channel antag-onist. These results suggest that in the human visceral artery,acute exposure of high concentration D-glucose (�378

Figure 3. Changes in membrane potential of smooth musclecells induced by levcromakalim (10�5 mol/L) in the humanomental artery. Levcromakalim-induced hyperpolarization is sig-nificantly reduced by glibenclamide plus L-glucose or D-glucose(*P�0.05). Resting membrane potentials did not differ amongthe groups studied (L-glucose [20 mmol/L]�37.6�2.6 mV;L-glucose [20 mmol/L] plus glibenclamide [5�10�6 mol/L]�42.6�4.3 mV; D-glucose [20 mmol/L]�40.8�4.8 mV).

Figure 2. a, Concentration-responsecurves to levcromakalim in the absenceor in the presence of Tiron, catalase, orDPI obtained in the human artery withoutendothelium treated with D-glucose.*Difference between rings treated withD-glucose and rings treated withD-glucose in combination with Tiron orDPI is statistically significant (P�0.05). b,Concentration-response curves tolevcromakalim in the absence or in thepresence of calphostin C or Go 6976obtained in the human artery withoutendothelium treated with D-glucose.*Difference between rings treated withD-glucose and rings treated withD-glucose in combination with calphos-tin C or Go 6976 is statistically signifi-cant (P�0.05). c, Concentration-response curves to levcromakalim in theabsence or in the presence of phorbol12-myristate 13-acetate (PMA) in combi-nation with calphostin C or Go 6976,obtained in the porcine coronary arterywithout endothelium treated withL-glucose. *Differences between rings

treated with L-glucose plus PMA and rings treated with L-glucose or rings treated with L-glucose plus PMA in combination with cal-phostin C or Go 6976 are statistically significant (P�0.05). d, Concentration-response curves to levcromakalim in the absence or in thepresence of PD98059 or genistein obtained in the human artery without endothelium treated with D-glucose.

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mg/dL) inhibits the activity of ATP-sensitive K� channels invascular smooth muscle cells in an osmolarity-independentfashion, in which the effect is relatively selective on K�

channels. Our results with high glucose appear to be consis-tent with a recent study on diabetic patients, demonstratingthe reduction of vasorelaxation induced by hypoxia viaATP-sensitive K� channels in the human coronary arteriole.4

Similarly, high glucose impaired the preconditioning effectstoward the ischemic in the canine heart, indicating theinhibitory effect of high glucose on the activity of mitochon-drial ATP-sensitive K� channels in animals.16 These resultsalso suggest that acute exposure of high glucose may simul-taneously modulate the activity of different subtypes ofATP-sensitive K� channels expressed on blood vessels aswell as mitochondria.17,18

In animals, incubation of vascular smooth muscle cellswith high glucose reportedly increases intracellular levels ofdiacylglycerol, subsequently leading to protein kinase Cactivation.19 In the human omental artery, calphostin C andGo 6976 restored vasorelaxation as well as hyperpolarizationin response to levcromakalim in the arteries treated withD-glucose, although the restoration induced by Go 6976 wasrather augmented. Mutual targets of the protein kinase Cisozyme for calphostin C and Go 6976 are reportedly�-isozymes and �- isozymes.20,21 Therefore, it is speculatedthat protein kinase C �-isozymes and �- isozymes maycontribute to the inhibitory effect induced by acute exposureof high glucose on the activity of ATP-sensitive K� channelsin the human visceral arterial smooth muscle cells. Thisconclusion is also supported by the notion from previousstudies, demonstrating the activation of protein kinase C �-isozymes induced by the high concentrations of glucose.8 Inthe current study, PMA impairs vasorelaxation as well ashyperpolarization in response to levcromakalim, which iscompletely recovered by calphostin C as well as Go 6976. In

addition, neither calphostin C nor Go 6976 affected vasore-laxation to levcromakalim in arteries treated with L-glucose.These results simultaneously support the conclusion thatprotein kinase C activation may selectively play a role in theinhibitory effects induced by acute exposure to high glucoseon the activity of ATP-sensitive K� channels. Our results arein agreement with previous animal studies, showing that theprotein kinase C activation inhibits vasorelaxation as well ascurrents via ATP-sensitive K� channels.6,7,22–26

The ATP-sensitive K� channel is a complex of 2 proteins:the sulfonylurea receptor (SUR) and the pore forming sub-unit, which belongs to the inward rectifier K� channel (Kir)family.17 Because the SUR of ATP-sensitive K� channel is aprimary target of the channel openers, the action of D-glucoseon some components of SUR may play a role in theseinhibitory effects.27 Protein kinase C activation seems to becapable of modulating the limited subtype of ATP-sensitiveK� channel expressed in the vascular smooth muscle cells(SUR 2B � Kir6.1).28,29 Importantly, the activity of thechannel subtypes produced by SUR 2B and Kir6.2 was notaltered by the kinase, suggesting the crucial role of Kir6.1compartment of ATP-sensitive K� channels in the modulatoreffect of protein kinase C.29 Therefore, it is also possible thatD-glucose may modulate Kir6.1 compartment of ATP-sensitiveK� channels, leading to the inhibition of vasorelaxation medi-ated by these channels in the human omental artery.

Previous studies demonstrated that protein tyrosine phosphor-ylation as well as augmented activity of mitogen-activatedprotein kinase modulate the activity of ATP-sensitive K� chan-nels.30–34 However, in the current study, neither genistein norPD98059 affected the effects produced by high glucose. There-fore, it is unlikely that tyrosine and mitogen-activated proteinkinases mediate the inhibitory effect of high glucose exposure onATP-sensitive K� channels in the human artery.35–37

Figure 4. Changes in membrane potential of smooth musclecells induced by levcromakalim (10�5 mol/L), in the humanomental artery treated with D-glucose. Levcromakalim-inducedhyperpolarization is significantly recovered by calphostin C orGo 6976, respectively (*P�0.05). Resting membrane potentialsdid not differ among the groups studied (D-glucose [20 mmol/L]�43.8�4.6 mV; D-glucose [20 mmol/L] plus calphostin C[3�10�7 mol/L]�42.4�3.8 mV; D-glucose [20 mmol/L] plus Go6976 [3�10�7 mol/L]45.6�2.4 mV, respectively).

Figure 5. Changes in membrane potential of smooth musclecells induced by levcromakalim (10�5 mol/L) in the humanomental artery treated with D-glucose. Levcromakalim-inducedhyperpolarization is significantly recovered by Tiron or DPI,respectively (*P�0.05). Resting membrane potentials did notdiffer among the groups studied (D-glucose [20 mmol/L]�44.2�1.5 mV; D-glucose [20 mmol/L] plus DPI[10�6 mol/L]�45.0�1.6 mV; D-glucose [20 mmol/L] plus Tiron[10 mmol/L]�42.8�1.9 mV, respectively).

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Acute exposure toward high glucose produced increasedlevels of superoxide in smooth muscle cells of the humanomental artery. Our results are in agreement with previousstudies on human blood vessels, showing that high concen-trations of glucose can induce augmentation of production ofsuperoxide in the endothelial cells.38 In the human omentalartery, protein kinase C activation by a phorbor ester, similarto high glucose, increased superoxide production in thevascular smooth muscle cells, indicating that activation of thekinase contributes to the augmented production of superoxideby acute exposure to high glucose in the human visceralartery. Our results are certainly concurrent with previousstudies demonstrating an important role of protein kinase Cactivation in the increased production of superoxide in theblood vessels.39–41

Studies using several diabetic animal models indicate thatsuperoxide reduces the activity of ATP-sensitive K� channelsin the vascular smooth muscle cells.5 Our results with acuteexposure to high glucose have demonstrated the evidencesupporting the inhibitory effects of superoxide produced byhigh glucose on the activity of ATP-sensitive K� channels. Asimilar inhibitory effect of superoxide on K� channels wasreported in the study on the rat coronary artery, documentingthe inhibitory effect of high glucose on voltage-dependent K�

channel currents.42 In the current study, the inhibitory effectsof high glucose on vasorelaxation as well as hyperpolariza-tion mediated by ATP-sensitive K� channels were abolishedby Tiron or DPI, suggesting that the increased production ofsuperoxide seen in arteries treated with high glucose may bemediated by NAD(P)H oxidase in the smooth muscle cells,which has been shown to be activated by protein kinaseC.41,43 However, further studies are warranted in this aspect,because the activity of NAD(P)H oxidase was not evaluatedin our study.

This is the first study examining the acute effects of highglucose on the activity of ATP-sensitive K� channels in thehuman blood vessel. Results in the current study suggest that inthe human visceral artery, acute hyperglycemia modulates va-sodilation mediated by ATP-sensitive K� channels via theproduction of superoxide possibly mediated by the activation ofprotein kinase C. It appears that in the visceral circulation,

similar to the cerebral one, acidosis corresponding with ischemiaactivates ATP-sensitive K� channels, resulting in visceral arte-rial dilation.44,45 Therefore, it is speculated that even short-termacute exposure to high glucose reduces the beneficial effect viaATP-sensitive K� channels, which may play important roles inregulation of human visceral circulation during diverse patho-physiological situations. In addition, it is not clinically rare toadminister vasodilators, such as nicorandil, which act via ATP-sensitive K� channels, to diabetic patients. Our results indicatethat in that occasion, one might have to consider the possibilitythat these vasodilator compounds acting on ATP-sensitive K�

channels cannot afford to produce their effects to supportappropriate visceral blood flow.46

AcknowledgmentsThis work was supported in part by grants-in-aid 16390458 (H.K.),16659426 (H.K.), and 13470327 (Y.H.) for Scientific Research fromthe Ministry of Education, Science, Sports, and Culture of Japan,Tokyo, and 11–7 for Medical Research from Wakayama prefecture,Wakayama, Japan (H.K.). This work was presented in part at theannual meeting of the American Society of Anesthesiologists, SanFrancisco, Calif, October 11 to 15, 2003.

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Kinoshita et al Effects of High Glucose on the Human Artery 2295

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Mayuko Dojo, Osafumi Yuge and Yoshio HatanoHiroyuki Kinoshita, Toshiharu Azma, Katsutoshi Nakahata, Hiroshi Iranami, Yoshiki Kimoto,

Channels in Human Omental Artery+ATP-Sensitive KInhibitory Effect of High Concentration of Glucose on Relaxations to Activation of

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hang

e in

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sion

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Levcromakalim (log mol/L)

L-glucose (20 mmol/L) + Glibenclamide (5 × 10-6 mol/L)

L-glucose (20 mmol/L)

**

**

*

n=5

*

Fig. I. Concentration-response curves to levcromakalim in the absence or in the presence of glibenclamide, obtained in the humanomental artery without endothelium treated with L-glucose. *Difference between control rings and rings treated with glibenclamide is statistically significant (P< 0.05). Data are expressed as percent of maximal vasorelaxation induced by papaverine (3×10-4 mol/L;100% = 2.09 ± 1.03 g [n = 5] and 2.00 ± 1.13 g [n = 5] for rings treated with L-glucose (20 mmol/L) or L-glucose plusglibenclamide [5 × 10-6 mol/L], respectively [NS]).

% C

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Diltiazem (log mol/L)

L-glucose (20 mmol/L)D-glucose (20 mmol/L)

n=5

Fig. II. Concentration-response curves to levcromakalim or diltiazem in the absence or in the presence of L-glucose or D-glucose,obtained in the human artery without endothelium. ∗Difference between rings treated with L-glucose and rings treated with D-glucoseis statistically significant (P< 0.05). Data are expressed as percent of maximal vasorelaxation induced by papaverine (3×10-4 mol/L;100% = 2.94 ± 1.01 g [n = 5] and 2.98 ± 0.84 g [n = 5] for rings treated with L-glucose [20 mmol/L] or D-glucose [20 mmol/L],respectively [NS]).

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Levcromakalim (log mol/L)

L-glucose (20 mmol/L) + DPI (10 -6 mol/L)

L-glucose (20 mmol/L) + Tiron (10 mmol/L)

L-glucose (20 mmol/L))

n = 5

Fig. III. Vasorelaxation in response to levcromakalim in the absence or in the presence of Tiron or DPI obtained

in the human artery without endothelium treated with L-glucose. Data are expressed as percent of maximal vasorelaxation

induced by papaverine (3×10-4 mol/L; 100% = 1.96 ± 0.84 g [n = 5], 2.44 ± 0.43 g [n = 5] and 1.68 ± 0.49 g [n = 5] for rings

treated with L-glucose [20 mmol/L], L-glucose [20 mmol/L] plus Tiron [10 mmol/L] or

L-glucose [20 mmol/L] plus DPI [10-6 mol/L], respectively [NS].

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Levcromakalim (log mol/L)

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L-glucose (20 mmol/L) + Calphostin C (3 × 10-7 mol/L)

L-glucose (20 mmol/L)

n = 5

Fig. IV. Vasorelaxation in response to levcromakalim in the absence or in the presence of calphostin C or Gö 6976

obtained in the human artery without endothelium treated with L-glucose. Data are expressed as percent of maximal vasorelaxation

induced by papaverine (3×10-4 mol/L; 100% = 2.42 ± 0.37 g [n = 5], 2.18 ± 0.79 g [n = 5] and 2.77 ± 0.29 g [n = 5] for rings

treated with L-glucose [20 mmol/L], L-glucose [20 mmol/L] plus calphostin C [3 × 10-7 mol/L] or

L-glucose [20 mmol/L] plus Gö 6976 [3 × 10-7 mol/L], respectively [NS].

A B C D

Fig. V. A representative autoradiograph of human omental arteries incubated with a chemiluminescence probe, MCLA (40 µM). Vessel segments were loaded with: (A) L-glucose (20 mmol/L); (B) D-glucose (20 mmol/L); (C) D-glucose (20 mmol/L) plus Tiron (10 mM); and (D) L-glucose (20 mmol/L) plus PMA (10-7 mol/L).

Supplemental figure legends

Fig. 1. Data are expressed as percent of maximal vasorelaxation induced by papaverine (3×10-4 mol/L; 100% = 2.66 ± 0.96 g [n = 5], 2.71 ± 0.55 g [n = 5] and 2.71 ± 1.11 g [n = 5] for rings treated with L-glucose [20 mmol/L], L-glucose [10 mmol/L] plus D-glucose [10 mmol/L] or D-glucose [20 mmol/L] in vasorelaxation to levcromakalim, respectively [NS]).

Fig. 2. (a) Data are expressed as percent of maximal vasorelaxation induced by papaverine (3×10-4 mol/L; 100% = 2.38 ± 0.51 g [n = 6],2.65 ± 1.26 g [n = 6] and 2.55 ± 1.05 g [n = 6] for rings treated with D-glucose [20 mmol/L], D-glucose [20 mmol/L] plus Tiron [10 mmol/L]or D-glucose [20 mmol/L] plus catalase [1200 U/ml], respectively [left, NS], 100% = 1.95 ± 1.26 g [n = 6] and 1.38 ± 0.51 g [n = 6] for ringstreated with D-glucose [20 mmol/L] or D-glucose [20 mmol/L] plus DPI [10-6 mol/L], respectively [right, NS]). (b) Data are expressed aspercent of maximal vasorelaxation induced by papaverine (3×10-4 mol/L; 100% = 2.52 ± 0.56 g [n = 7] and 2.71 ± 0.49 g [n = 7] for ringstreated with D-glucose [20 mmol/L] or D-glucose [20 mmol/L] plus calphostin C [3 × 10-7 mol/L], respectively [left, NS], 100% = 3.13 ±

1.27 g [n = 6] and 3.06 ± 1.53 g [n = 6] for rings treated with D-glucose [20 mmol/L] or D-glucose [20 mmol/L] plus Gö 6976 [3 × 10-7 mol/L],respectively [right, NS]). (c) Data are expressed as percent of maximal vasorelaxation induced by papaverine (3×10-4 mol/L;100% = 2.38 ± 1.02 g [n = 6], 1.99 ± 0.73 g [n = 6] and 2.18 ± 0.71 g [n = 6] for rings treated with L-glucose [20 mmol/L], L-glucose [20 mmol/L] plus PMA [10-7 mol/L] or L-glucose [20 mmol/L] plus PMA [10-7 mol/L] in combination with calphostin C [3 × 10-7 mol/L], respectively [left, NS], 100% = 2.02 ± 0.92 g [n = 5], 2.04 ± 0.64 g [n = 5] and 2.39 ± 1.49 g [n = 5] for rings treated with L-glucose [20 mmol/L], L-glucose [20 mmol/L] plus PMA [10-7 mol/L], L-glucose [20 mmol/L] plus PMA [10-7 mol/L] in combination withGö 6976 [3 × 10-7 mol/L], respectively [right, NS]). (d) Data are expressed as percent of maximal vasorelaxation induced bypapaverine (3×10-4 mol/L; 100% = 2.54 ± 1.13 g [n = 5], 2.67 ± 1.23 g [n = 5] and 2.62 ± 0.96 g [n = 5] for rings treated withD-glucose [20 mmol/L], D-glucose [20 mmol/L] plus PD 98059 [10-5 mmol/L] or D-glucose [20 mmol/L] plus genistein [10-6 mol/L], respectively [NS].