Am J Physiol Heart Circ Physiol 307: H1093–H1102, 2014. First published August 15, 2014; doi:10.1152/ajpheart.00240.2013.

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Type 2 diabetes: increased expression and contribution of IKCa channels to vasodilation in small mesenteric arteries of ZDF rats Christian Schach,* Markus Resch,* Peter M. Schmid, Guenter A. Riegger, and Dierk H. Endemann Department of Cardiology, University Hospital Regensburg, Regensburg, Germany Submitted 15 March 2013; accepted in final form 6 August 2014

Schach C, Resch M, Schmid PM, Riegger GA, Endemann DH. Type 2 diabetes: increased expression and contribution of IKCa channels to vasodilation in small mesenteric arteries of ZDF rats. Am J Physiol Heart Circ Physiol 307: H1093–H1102, 2014. First published August 15, 2014; doi:10.1152/ajpheart.00240.2013.—Impaired endothelial function, which is dysregulated in diabetes, also precedes hypertension. We hypothesized that in Type 2 diabetes, the impaired endothelium-dependent relaxation is due to a loss of endotheliumderived hyperpolarization (EDH) that is regulated by impaired ion channel function. Zucker diabetic fatty (ZDF), Zucker heterozygote, and homozygote lean control rats were used as the experimental models in our study. Third-order mesenteric arteries were dissected and mounted on a pressure myograph; mRNA was quantified by RT-PCR and channel proteins by Western blotting. Under nitric oxide (NO) synthase and cyclooxygenase inhibition, endothelial stimulation with ACh fully relaxes control but not diabetic arteries. In contrast, when small-conductance calcium-activated potassium (KCa) channels and intermediate- and large-conductance KCa (I/BKCa) are inhibited with apamin and charybdotoxin, NO is able to compensate for ACh-induced relaxation in control but not in diabetic vessels. After replacement of charybdotoxin with 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; IKCa inhibitor), ACh-induced relaxation in diabetic animals is attenuated. Specific inhibition with TRAM-34 or charybdotoxin attenuates ACh relaxation in diabetes. Stimulation with 1-ethyl-2-benzimidazolinone (IKCa activator) shows a reduced relaxation in diabetes. Activation of BKCa with 1,3-dihydro1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)- 2 Hbenzimidazol-2-one NS619 leads to similar relaxations of control and diabetic arteries. RT-PCR and Western blot analysis demonstrate elevated mRNA and protein expression levels of IKCa in diabetes. Our results suggest that the compensatory effect of NO and EDHassociated, endothelium-dependent relaxation is reduced in ZDF rats. Specific blockade of IKCa with TRAM-34 reduces NO and EDH-type relaxation in diabetic rats, indicating an elevated contribution of IKCa in diabetic small mesenteric artery relaxation. This finding correlates with increased IKCa mRNA and protein expression in this vessel. diabetes; calcium-activated potassium channel; vasodilation; endothelium; nitric oxide; endothelium-derived hyperpolarization; mesenteric artery CARDIOVASCULAR DISEASE (CVD) is the major cause of mortality in diabetic patients (1). Up to 75% of CVD in diabetes may be attributable to hypertension, with arterial hypertension approximately twice as frequent in individuals with diabetes. Control

* C. Schach and M. Resch contributed equally to this project. Address for reprint requests and other correspondence: C. Schach, Klinik und Poliklinik für Innere Medizin II, Franz-Josef-Strauss-Allee 11, Univ. of Regensburg, 93042 Regensburg, Germany (e-mail: [email protected]). http://www.ajpheart.org

of blood pressure, therefore, provides an important therapeutic goal (43, 46). Diabetes and hypertension frequently correspond, and endothelial dysfunction is thought to be the initial step in CVD development (7). With the lining of all blood vessels, the endothelium modulates smooth muscle tone and vasorelaxation under healthy conditions. It has been established that endothelial function in diseases, such as diabetes and hypertension, is impaired in animal models and in humans as well (17). Cardiovascular complications develop by deficient function of nitric oxide (NO), following enhanced oxidative stress in diabetes (36). The loss of NO regulation is thought to lead to hypertension by arterial stiffening and chronic activation of the sympathetic nervous system (23, 25). The expected compensatory role for endothelium-derived hyperpolarization (EDH) for a loss of NO regulation is not observed uniformly, and indeed, a reduced function of EDH has been suggested to play an important role in the genesis of endothelial dysfunction in diabetes. Dysregulated, EDH-mediated responses were also observed in Type 1 diabetes. Mesenteric arteries of streptozotocin-induced Type 1 diabetic rats had an enhanced EDH-mediated relaxation (41), whereas other observations show an impaired EDH-dependent response in that vasculature (20, 49). In mice, streptozotocin-induced Type 1 diabetes leads to diminished EDH relaxation with a compensatory increase in the NO response (18, 32). In both studies, isometric force of mesenteric arteries was measured. Type 2 diabetic mice show an attenuated, EDH-mediated vasodilation in coronary arterioles (38), whereas in small mesenteric arteries (SMA), its contribution to endothelium-dependent vasodilation is unaltered or even elevated (37). In humans, NO- and EDH-mediated vasorelaxation is reduced in Types 1 and 2 diabetes patients (3). Otsuka Long-Evans Tokushima fatty (OLETF) Type 2 diabetic rats exhibited an impaired EDHtype relaxation measuring isometric force (30, 31). In an electrophysiological study, using a combination of Zucker diabetic fatty (ZDF) rat SMA and bovine aortic endothelial cells (ECs), an impaired EDH response was observed due to a reduced function of small-conductance calcium-activated potassium channels (SKCa) (8), and diminished EDH relaxation in ZDF rat mesenteric arteries could be restored via pharmacological activation of SKCa and intermediate-conductance KCa (IKCa) (6). These observations were generated under different conditions and until now, there appears to be an absence of investigations demonstrating a compensatory role for KCa function and their expression levels and role in NO- and EDH-mediated relaxation using pressure myography in Type 2 diabetic rats. Furthermore, it is not known if the commonly used control

0363-6135/14 Copyright © 2014 the American Physiological Society

H1093

H1094

ROLE OF KCa IN TYPE 2 DIABETIC SMA

animal for ZDF rats, the Zucker heterozygote lean (ZL) rat, has reduced vasofunction due to its heterogeneity in a leptin receptor defect compared with the Zucker homozygote lean control (Z⫹/⫹) rat, which is negative for a leptin receptor defect. To gain additional insight, the molecular approaches, RT-PCR and Western blot, were used to examine and compare KCa expression levels among these animal models. As SKCa [isoform three of SKCa (SK3), KCa2.3, KCNN3], IKCa (IK1, KCa3.1, KCNN4), as well as large-conductance KCa (BKCa; BK, KCa1.1, KCNMA1) have been observed in EDH-mediated responses (6, 8, 33), we determined mRNA and protein expression levels of these channels. METHODS

Animals. Male ZDF rats (homozygote for leptin receptor defect, fa/fa) and their respective heterozygote (ZL, fa/⫹) and homozygote (Z⫹/⫹, ⫹/⫹) control rats were purchased from Charles River Laboratories (Brussels, Belgium) at the age of 10 wk. Rats had unlimited access to the diabetogenic diet Purina 5008 (energy rich, containing 26.8% protein, 16.7% fat, and 56.4% carbohydrates). The animals were housed on a 12/12-h light/dark cycle with constant temperature (22–23°C), with access to food and tap water ad libitum. Body weight, blood pressure, and fasting blood glucose concentrations were measured (BPrecorder No. 8005; W⫹W electronic AG, Basel, Switzerland; ACCU-CHEK sensor; Roche, Mannheim, Germany). At the age of 18 wk, animals were weighted; systolic blood pressure was measured using an automated tail-cuff, inflator-pulse detection system (CODA 2 multichannel, computerized; emka Technologies, Paris, France); and fasting (6 h) blood glucose level was assessed. Afterwards, rats were euthanized by decapitation. Blood samples were collected in tubes, and tibia length (TL) was obtained. All animal care and experimental procedures followed German law as well as the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health, and were approved by the Institutional Review Board at the University of Regensburg. Drugs and preparation. 4-[(1R)-2-Amino-1-hydroxyethyl]benzene1,2-diol [noradrenaline (NA)] and sodium pentacyanonitrosylferrate(II) [sodium nitroprusside (SNP)] were purchased from Fluka (Neu-Ulm, Germany); 2-acetoxy-N,N,N-trimethylethanaminium (ACh), 1,3dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)2 H-benzimidazol-2-one (NS1619), N␻-nitro-L-arginine methyl ester (LNAME), and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM34) were purchased from Sigma (Taufkirchen, Germany); 1-ethyl-2benzimidazolinone (1-EBIO), [2-(2,6-dichlorophenylamino)phenyl13C6]acetic acid sodium salt heminonahydrate (diclofenac), apamin, and charybdotoxin were purchased from Axxora (Loerrach, Germany). Primers for RT-PCR were purchased from Biomers (Ulm, Germany). ACh, NA, SNP, L-NAME, apamin, and charybdotoxin were dissolved in water; 1-EBIO and NS1619 in ethanol; and diclofenac, as well as TRAM-34, in DMSO. Preparation of mesenteric arteries. Following euthanasia, the mesenteric arcade was isolated and instantly bathed in cold Krebs solution (consisting of NaCl 118, KH2PO4 1.18, KCl 4.7, MgSO4 1.18, CaCl2 2.5, D-glucose 5.5, NaHCO2 25, and EDTA 0.026 mM and adjusted to pH 7.4). For myograph experiments a third-order artery (200 –300 ␮m in diameter), with a length of 3– 4 mm, was dissected from surrounding adipose tissue. Afterwards, the vessel was mounted onto two glass cannulas in an organ chamber filled with Krebs solution (pH 7.4) and secured with a suture on each side. The preparation was then transferred to a myograph (111P; Danish Myo Technology, Aarhus, Denmark), the organ chamber perfused with warmed Krebs solution (adjusted to pH 7.4 with continuous bubbling of an air/CO2 mixture 95/5%), and the intraluminal pressure set to 45 mmHg with a servocontrolled pump (Watson-Marlow, Nordrhein-Westfalen, Germany). Vessels now were equilibrated for 40 min under constant intraluminal

pressure in the superfused organ chamber (6 ml vol, 2 ml/min perfusion speed). Cross-sectional diameter was quantified at three different points along the vessel wall with the help of a calibrated video system (Danish Myo Technology). Myograph protocols. After stabilization and equilibration, arteries were stimulated with NA (10 ␮M). Endothelial function was examined by superfusion with ACh (1 ␮M). All vessels with intact endothelium used for analysis produced an ACh-induced relaxation of ⬎70%. For some experiments, preparation with denuded endothelia was used. Transient air insufflation was used to eliminate the endothelium functionally, and successful denudation was assumed when the ACh-induced relaxation was ⬍20%. Relative relaxation (R) was calculated by the following formula in all experiments: R ⫽ (Dx ⫺ Dc)/(Dr ⫺ Dc), where Dx is the actual measured diameter at a given concentration of ACh, SNP, 1-EBIO (primarily activates IKCa and with lower affinity also SKCa), or NS1619 (activates BKCa); Dc is the diameter when contracted with 10 ␮M NA; and Dr is the resting diameter after the 40-min equilibration period. Each value for Dx, Dc, or Dr represents the average from three measured diameters. After NA precontraction (10 ␮M) and testing endothelium-dependent relaxation with ACh (1 ␮M), agonists were washed out for 30 min. To evaluate the relaxation of SMA, cumulative concentration-response curves to the endothelium-dependent relaxant ACh (1 nM– 0.1 mM), endothelium-independent relaxant SNP (0.1 nM–100 ␮M), as well as 1-EBIO (1 nM–100 ␮M) and NS1619 (1–100 ␮M) were examined after precontraction with NA (10 ␮M). Additionally, ACh-induced responses were conducted after 20 min incubation with given combinations of L-NAME (100 ␮M), a nonselective NO synthase (NOS) inhibitor; diclofenac (10 ␮M), a nonselective cyclooxygenase (COX) inhibitor; apamin (0.1 ␮M), a blocker of SKCa; charybdotoxin (0.1 ␮M), a blocker of IKCa and BKCa; as well as voltage-gated K⫹ channels (Kv) and TRAM-34 (0.1 ␮M), a selective IKCa inhibitor. These KCa inhibitors block the respective channels selectively at specific concentrations. When vessels were treated with the combination of L-NAME, diclofenac, and apamin plus charybdotoxin, at first, a concentration-response curve to ACh was generated after incubation with L-NAME and then again after a 30-min washout period, with the combination of L-NAME plus diclofenac with a 30-min washout afterwards. In experiments with isolated application of TRAM-34, charybdotoxin, and apamin, these inhibitors were applied in respective order with a wash-out period of 30 min afterwards. For experiments studying the effect of applying individual channel inhibitors (apamin, charybdotoxin, or TRAM-34; see Fig. 3) and the effect of combined channel inhibition (apamin plus charybdotoxin or apamin plus TRAM-34) in the absence and presence of NOS and COX inhibition (by L-NAME and diclofenac), a second set of rats was used (n ⫽ 6 for each group). Quantitative RT-PCR. mRNA of KCa proteins (SKCa3, IKCa, and BKCa) was measured in the first- through third-order branches of

Table 1. Physiological data n Body weight, g Tibia length, mm Body weight/tibia length, g/mm Blood glucose, mg/dl Systolic blood pressure, mmHg Heart rate, beats/min Lumen diameter, ␮m

Z⫹/⫹

ZL

ZDF

6 372 ⫾ 4.2 45.0 ⫾ 0.4a

6 375.9 ⫾ 8.4 44.7 ⫾ 0.3a

6 370.7 ⫾ 5.8 42.8 ⫾ 0.4

8.3 ⫾ 1.1 90.4 ⫾ 2.2a

8.4 ⫾ 1.9 86.7 ⫾ 1.9a

P

0.76 0.02

8.9 ⫾ 1.6 0.08 375.3 ⫾ 13.1 ⬍0.01

112.1 ⫾ 4.1 109.9 ⫾ 4.3 103.2 ⫾ 6.3 586.7 ⫾ 19.7a 622.1 ⫾ 24.9a 493.7 ⫾ 17.1 281.6 ⫾ 9.0 276.7 ⫾ 8.7 265.4 ⫾ 9.1

0.47 0.01 0.91

Physiological data and lumen diameter of studied 3rd-order mesenteric resistance arteries from Zucker homozygote lean control (Z⫹/⫹), Zucker heterozyote lean control (ZL), and Zucker diabetic fatty (ZDF) rats. aP ⬍ 0.05 vs. ZDF (1-way ANOVA).

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00240.2013 • www.ajpheart.org

H1095

ROLE OF KCa IN TYPE 2 DIABETIC SMA 0

*

*

Relaxation (%)

25

* *#

*#

50

75

Z+/+ ZL ZDF

100 -9

-8

-7 SNP

-6

-5

-4

[ACh] (logM) Fig. 1. Cumulative dose-response curves of small mesenteric arteries (SMA) from Zucker homozygote lean control (Z⫹/⫹), Zucker heterozygote lean control (ZL), and Zucker diabetic fatty (ZDF) rats to increasing concentrations of ACh (1 nM– 0.1 mM) after precontraction with noradrenaline (NA; 10 ␮M); n ⫽ 6 experiments. Results are shown as means ⫾ SE. *P ⬍ 0.05 vs. Z⫹/⫹; #P ⬍ 0.05 vs. ZL, 2-way repeated-measures ANOVA.

SMA. After homogenization of frozen tissue sections (Peqlab, Erlangen, Germany), total mRNA was extracted following the manufacturer’s directions, with additional DNase digestion to remove all traces of genomic DNA. Total RNA was reverse transcribed into complementary DNA (cDNA), according to standard protocols, as described previously (4). In brief, cDNA probes were synthesized in 20 ␮l reaction volume containing 1 ␮g total RNA, 0.5 ␮g oligo(dT) primer (Sigma-Aldrich, Munich, Germany), 40 U RNasin (Promega, Mannheim, Germany), 0.5 mM dNTP (Amersham, Freiburg, Germany), 4 ␮l 5⫻ transcription buffer, and 200 U Moloney murine leukemia virus RT, and no template controls were performed. Real-time quantitative RT-PCR was performed on an ABI PRISM 7900 detection system (Applied Biosystems, Darmstadt, Germany) using TaqMan Gene Expression Assays for the genes KCNN33, KCNN44, and KCNMA1, together with TaqMan Gene Expression Master Mix (Applied Biosystems). ␤-Actin was used as a reference gene. All water controls and single-stranded RT controls were negative. Data were analyzed using SDS version 2.2.2 software (Applied Biosystems). Western blots. Western blots were performed from the first-, second-, and third-order branches of superior mesenteric arteries after carefully dissecting the vessels from surrounding tissue. Two animals from the same treatment group were pooled and considered as n ⫽ 1. The arteries were homogenized in 300 ␮l lysis buffer (100 mM NaCl, 10 mM Tris, 2 mM EDTA, 0.5% wt/vol Na deoxycholate, 1% vol/vol

Triton X-100, pH 7.4, plus a protease inhibitor cocktail) on ice. Then, 20% SDS (15 ␮l) was added and incubated for 10 min. Centrifugation at 800 g for 5 min at 4°C and with 12,000 g for 20 min at 4°C of the supernatant was used to generate aliquots. The total protein concentration was quantified using the two-dimensional Quant-iT protein assay kit (GE Healthcare, Solingen, Germany), and equal amounts of protein homogenate were subjected to SDS-PAGE (12.5% SDS; Precast; Bio-Rad Laboratories, Hercules, CA) and run for 1.5 h at 35 mA, 100 V (Mini-PROTEAN; Bio-Rad Laboratories), before being transferred (Trans-Blot Turbo; Bio-Rad Laboratories) to a polyvinylidene fluoride membrane. After 10 min at 1.5 A and 25 V, the membrane was blocked with 5% BSA in Tris-buffered saline-Tween 20 (TBS-T) for 1 h. The primary antibodies against SKCa3 APC-025 (lot AN-09), IKCa APC-064 (AN-04), and BKCa-␣ APC-021 (AN-12), all 1:500 rabbit polyclonal anti-rat antibodies (Alomone Labs, Jerusalem, Israel), were incubated at 4°C overnight and then washed four times in TBS-T before being incubated for 1 h with the secondary antibody (1:10,000 anti-rabbit horseradish peroxidase; Cell Signaling Technology, Danvers, MA). Afterwards, the membrane was washed four times in TBS-T and once in TBS and then developed using an enhanced chemiluminescence kit (Clarity Western ECL substrate kit; Bio-Rad Laboratories). The relative amount of protein was quantified by densitometry (ChemiDoc; Bio-Rad Laboratories) and expressed as the ratio of loading control (Image Lab 5.0; Bio-Rad Laboratories). Antibody specificity was assessed by incubation of the antibody with its cognate peptide to block its specific binding site. The peptide was mixed with the antibody (1:1 ratio/wt) and incubated for 1 h at 37°C and afterwards, overnight at 4°C. Further handling of this blocked antibody for Western blotting was as described above. As molecular-weight markers, peqGOLD Protein-Marker IV (M1; Peqlab) and Precision Plus Protein WesternC (M2; Bio-Rad Laboratories) were used. Statistical analysis. All values are given as means ⫾ SE. Concentration-response curves from isolated rat mesenteric arteries were computer fitted using nonlinear regression (Prism version 6.0; GraphPad Software, San Diego, CA) to calculate the sensitivity of each agonist and presented as pD2 values (pD2 ⫽ ⫺log[EC50]). Maximum relaxation (Rmax) to ACh, 1-EBIO, or NS1619 was measured as a percentage of NA-induced precontraction. Rmax and pD2 values were compared by one-way ANOVA with post hoc analysis using Bonferroni’s test or Student’s unpaired t-test. Further statistical analyses were carried out using Student’s t-test and one- and two-way repeatedmeasures ANOVA as appropriate. Post hoc testing was performed using Fisher’s least significant difference test. P ⬍ 0.05 was considered significant. RESULTS

Animal characteristics. Morphological characteristics as well as blood values are shown in Table 1. Fasting blood glucose levels were higher, and heart rate was significantly lower in ZDF rats compared with ZL or Z⫹/⫹ rats. Blood

Table 2. Role of EDH-type relaxation on Rmax and pD2 values in small mesenteric arteries Rmax, %, n ⫽ 6

ACh CTL ⫹L ⫹L⫹D ⫹L⫹D⫹A⫹C

pD2, n ⫽ 6

Z⫹/⫹

ZL

ZDF

Z⫹/⫹

ZL

ZDF

100.1 ⫾ 3.4a 108.3 ⫾ 7.4a 103.3 ⫾ 5.4a,c 3.7 ⫾ 1.8b

96.6 ⫾ 1.9a 91.1 ⫾ 16.2a 70.6 ⫾ 17.4a 12.9 ⫾ 9.1b

62.5 ⫾ 6.1 21.6 ⫾ 7.1b 16.3 ⫾ 4.4b ⫺2.5 ⫾ 2.8b

6.7 ⫾ 0.18a 6.5 ⫾ 0.14 6.5 ⫾ 0.15 n.d.

6.5 ⫾ 0.17 6.1 ⫾ 0.22 6.2 ⫾ 0.25 n.d.

6.2 ⫾ 0.22 n.d. n.d. n.d.

Parameters measured in small mesenteric arteries from 18-wk-old Z⫹/⫹, ZL, and ZDF rats. EDH, endothelium-derived hyperpolarization. Sensitivity (pD2) and maximal relaxation (Rmax) to ACh in the absence [control (CTL)] or presence of N␻-nitro-L-arginine methyl ester [L-NAME (L); 100 ␮M]; [2-(2,6dichlorophenylamino)phenyl13C6]acetic acid sodium salt heminonahydrate [diclofenac (D); 10 ␮M]; apamin (A; 0.1 ␮M); and charybdotoxin (C; 0.1 ␮M). n.d., not determined. Values are means ⫾ SE. aP ⬍ 0.05 vs. ZDF; bP ⬍ 0.05 vs. corresponding CTL; cP ⬍ 0.05 vs. ZL (1-way ANOVA). AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00240.2013 • www.ajpheart.org

H1096

ROLE OF KCa IN TYPE 2 DIABETIC SMA

A Relaxation (%)

0

*

*

*

* # § * #§ * # §

25

Z+/+

50 75 CTL + L-NAME + L-NAMA+Diclo + L-NAME+Diclo+Apa+Chtx

100

-9

-8

-7

-6

-5

-4

[ACh] (logM)

B

* # § *# § *# §

Relaxation (%)

0

NA-induced contraction. Cumulative doses of NA were applied to the vessel after an equilibration period of 40 min. ZDF and control rats contracted without a significant difference, neither in maximal contraction (Cmax) nor in NA sensitivity, to ⬃70% of the initial diameter (Cmax ZDF 72.1 ⫾ 1.5% vs. control 70.4 ⫾ 1.2%, P ⫽ not significant), indicating neither a loss of contractile ability nor a hyper-responsiveness to catecholamines. ACh-induced relaxation in ZDF rats vs. control rats. The application of increasing ACh concentrations to the arteries of homozygote healthy control rats (Z⫹/⫹) produced a complete relaxation at high concentrations, whereas vessels from ZDF rats reached only 58% of Rmax. At nanomolar ACh concentrations, vessels of Z⫹/⫹ rats are already partly dilated, which is significantly different from the behavior of arteries from ZDF rats. Compared with Z⫹/⫹ rats, ACh-induced relaxations in

25

Z+/+

ZL

50

Relaxation (%)

75 100 -9

-8

-7

-6

-5

-4

[ACh] (logM)

Relaxation (%)

C

*

*

0

A

0 25 50 75 CTL +Apa +Chtx +TRAM34

100

*

-11

25

-10

-9

-8

-7

-6

-5

-4

[ACh] (logM) ZDF

50 #§ #§

75

ZDF

#§

B

0

-9

-8

-7

-6

-5

-4

[ACh] (logM) Fig. 2. Cumulative dose-response curves of SMA isolated from (A) Z⫹/⫹, (B) ZL, and (C) ZDF rats to ACh. Arteries were exposed to cumulative doses of ACh (1 nM– 0.1 mM) without incubation [control (CTL)] and after incubation with N␻-nitro-L-arginine methyl ester (L-NAME; 100 ␮M), L-NAME plus diclofenac (Diclo; 10 ␮M), and the combination of apamin (Apa) plus charybdotoxin (Chtx; each 0.1 ␮M) in the presence of L-NAME (100 ␮M) and Diclo (10 ␮M). Arteries were precontracted with NA (10 ␮M); n ⫽ 6 experiments. Results are shown as means ⫾ SE. *P ⬍ 0.05 vs. CTL; #P ⬍ 0.05 vs. L-NAME; §P ⬍ 0.05 vs. L-NAME ⫹ Diclo, 2-way repeated-measures ANOVA.

pressure was not significantly different in ZDF rats compared with the control groups. Body weight showed no difference among the three groups (Table 1); however, TL was lower in ZDF rats compared with the control groups, resulting in a trend to (P ⫽ 0.08) elevated relative body weight of ZDF rats (body weight/TL; Table 1).

Relaxation (%)

100 25

#§

50

#§ #§

75

100 -11 -10

-9

-8

-7

-6

-5

-4

[ACh] (logM) Fig. 3. Cumulative dose-response curves to ACh (0.01 nM– 0.1 mM) in the absence (CTL) and presence of Apa, Chtx, or 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), respectively (each 0.1 ␮M) in Z⫹/⫹ (A) and ZDF (B) rats. Relaxations to ACh were induced after stable precontraction with NA; n ⫽ 6 experiments. Results are shown as means ⫾ SE. #P ⬍ 0.05 vs. TRAM-34; §P ⬍ 0.05 vs. Chtx, 2-way repeated-measures ANOVA.

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00240.2013 • www.ajpheart.org

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ROLE OF KCa IN TYPE 2 DIABETIC SMA

ZL-derived arteries are slightly less sensitive to ACh without statistical significance (Fig. 1 and Table 2). Endothelium-independent relaxation. Endothelium-independent relaxation was assessed by a dose-response curve to SNP (0.01–100 ␮M). SMA of ZDF, as well control rats, increased diameter without difference, although complete relaxation was not achieved by high concentrations of SNP (data not shown). Selective blockade of ACh-induced relaxation. Stimulation with ACh produced a full relaxation in the nondiabetic control groups, Z⫹/⫹ and ZL, whereas in diabetic ZDF animals, ACh-induced relaxation was reduced significantly. Inhibition of endothelial NOS (eNOS) with L-NAME (100 ␮M) significantly reduced relaxation at low doses of ACh in Z⫹/⫹ animals (Fig. 2 and Table 2). Further addition of diclofenac (10 ␮M) did not alter the dose-response curve to ACh significantly. Additional blockade of KCa by apamin plus charybdotoxin (each 0.1 ␮M) led to a significant suppression of the AChinduced vasorelaxation in the Z⫹/⫹ rat mesenteric artery, indicating a substantial contribution of EDH-type relaxation. In nondiabetic heterozygote (fa/⫹) ZL rats, without NOS and COX inhibition, the endothelium-dependent stimulation with ACh led to a complete relaxation of SMA (Fig. 2A and Table 2). In the presence of NOS and COX inhibition, ACh-induced relaxation was not altered significantly, whereas further application of the toxin combination apamin plus charybdotoxin abolished ACh-induced vasorelaxation in ZL animals (Fig. 2B and Table 2). In ZDF rats, ACh-induced relaxation of SMA was decreased significantly, and additive inhibition of NOS further reduced the ACh-induced relaxation in a significant way (Fig. 2C and Table 2). As in the two control groups, COX inhibition did not significantly alter the ACh-induced relaxation. Under NOS and COX blockade and inhibition of SKCa and I/BKCa by apamin and charybdotoxin, ACh was not able to relax mesenteric arteries from ZDF rats. Blockade of KCa. In another set of rats, we tested the influence of potassium channels by selective application of apamin, charybdotoxin, and TRAM-34 (block at specific concentrations SKCa, IKCa/BKCa/KV, and IKCa, respectively, each 0.1 ␮M), comparing ZDF with Z⫹/⫹ rats. When applied separately, the channel blockers did not affect ACh-induced relaxation in Z⫹/⫹ rats, whereas there was an inhibition in ZDF rats (Fig. 3 and Table 3). In the latter, isolated inhibition of SKCa with apamin did not significantly reduce ACh-induced relaxation. Blockade of IKCa (with TRAM-34) or IKCa, BKCa, and KV (with charybdotoxin) led to a significant reduction in ACh-induced relaxation compared with the control group in diabetic animals (Fig. 3B). Combined application of apamin and charybdotoxin decreased ACh-induced relaxation in Z⫹/⫹ animals in a nonsignificant manner, whereas ACh-induced relaxation of the ZDF SMA was reduced significantly (Fig. 4, A and B, and Table 3). Additional blockade of eNOS and COX in Z⫹/⫹ resulted in an abolished ACh-induced relaxation. After substitution of charybdotoxin by TRAM-34, the AChinduced relaxation was not reduced significantly in Z⫹/⫹ rats. However, the substitution of charybdotoxin with TRAM-34 on this combination showed no difference in ZDF animals (Fig. 4, C and D, and Table 3). Activation of KCa. The stimulation of mesenteric arteries with cumulative concentrations of 1-EBIO, which activates IKCa more specifically than SKCa, showed a significant divergence in relaxations between Z⫹/⫹ and ZDF animals at 1, 10,

and 100 ␮M. However, Rmax in ZDF was significantly lower in ZDF than in Z⫹/⫹ animals (32.7 ⫾ 6.4% vs. 15.8 ⫾ 5.2%; Fig. 5A), whereas control experiments with denuded arteries showed no difference between the groups (Fig. 5B). The dose-response curve to the BKCa activator NS1619 was significantly different for Z⫹/⫹ vs. ZDF at 10 ␮M vs. both groups treated with iberiotoxin at 5, 10, and 50 ␮M and vs. the iberiotoxin-treated ZDF group at 100 ␮M, with no significant difference of Rmax between Z⫹/⫹ and ZDF (Rmax Z⫹/⫹ 49.2 ⫾ 8.1% vs. ZDF 38.6 ⫾ 9.4%; Fig. 5C). Denudation, however, led to an altered iberiotoxin sensitivity of NS1619-induced relaxation. Relaxation in denuded, iberiotoxin-treated Z⫹/⫹ and ZDF vessels was reduced significantly vs. denuded Z⫹/⫹ at 10 –100 ␮M NS1619, and denuded ZDF vessels relaxed more than those treated with iberiotoxin at 100 ␮M (Fig. 5D). Quantitative RT-PCR studies of KCa. Analyses of mesenteric arteries of ZDF and control rats did not reveal a significant difference in mRNA levels of BKCa (Fig. 6). In ZDF, expression of SK3 was elevated compared with Z⫹/⫹, and expression of IKCa (IK1) was significantly augmented compared with Z⫹/⫹ and ZL (Fig. 6). Western blot analysis of KCa. SKCa and BKCa protein expression levels were not altered significantly in ZDF mesenteric arteries (Figs. 7, A and D), whereas expression of IKCa monomeric proteins (appearing at 50 kDa), as well as channel complexes (appearing at high molecular weight, ⬎200 kDa), which were described as the functional homotetrameric channel (11), was upregulated by diabetes compared with Z⫹/⫹ mesenteric arteries (Fig. 7, B and F). DISCUSSION

In SMA, EDH-type relaxation entirely supersedes the loss of NO (Fig. 2, A and B) and vice versa (Fig. 4, A and C). In ZDF, the loss of NO blockage of the EDH pathway with SKCa and IKCa inhibitors could not be overridden by other relaxing mechanisms after stimulation with ACh (Fig. 4, B and D). Inhibition of IKCa with charybdotoxin or with TRAM-34 Table 3. ACh-induced Rmax and pD2 under control condition and potassium channel inhibition Rmax, %, n ⫽ 6 Z⫹/⫹

ACh CTL 91.3 ⫾ 5.9 ⫹A 103.6 ⫾ 6.8 ⫹C 98.9 ⫾ 17.8 ⫹T 87.4 ⫾ 8.2 ⫹A⫹C 64.6 ⫾ 17.1d ⫹A⫹T 72.4 ⫾ 19.6 ⫹L⫹D⫹A⫹C 8.7 ⫾ 2.6d ⫹ L ⫹ D ⫹ A ⫹ T 55.6 ⫾ 21.4e

ZDF

62.2 ⫾ 5.7a 37.4 ⫾ 11.0a 17.3 ⫾ 11.4b,c 25.2 ⫾ 7.1a,c 10.3 ⫾ 3.4b,d 10.8 ⫾ 2.0b,d 7.4 ⫾ 2.4d 12.1 ⫾ 9.6d

pD2, n ⫽ 6 Z⫹/⫹

ZDF

6.3 ⫾ 0.25 6.0 ⫾ 0.23 6.4 ⫾ 0.15 n.d. 6.2 ⫾ 0.14 n.d. 6.2 ⫾ 0.11 n.d. c 5.8 ⫾ 0.29 n.d. 6.3 ⫾ 0.22 n.d. n.d. n.d. 6.0 ⫾ 0.28 n.d.

Parameters measured in small mesenteric arteries from 18-wk-old Z⫹/⫹, ZL, and ZDF rats. pD2 and Rmax to ACh in the absence (CTL) or presence of apamin (0.1 ␮M), charybdotoxin (0.1 ␮M), 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole [TRAM-34 (T); 0.1 ␮M], apamin plus charybdotoxin, apamin plus TRAM-34, L-NAME (100 ␮M) plus diclofenac (10 ␮␮M) plus apamin plus charybdotoxin, or L-NAME plus diclofenac plus apamin plus TRAM-34. Values are means ⫾ SE. aP ⬍ 0.01 vs. Z⫹/⫹; bP ⬍ 0.05 vs. Z⫹/⫹ (Student’s t-test); cP ⬍ 0.05 vs. corresponding CTL; dP ⬍ 0.01 vs. corresponding CTL; eP ⬍ 0.01 vs. corresponding ⫹ L-NAME ⫹ diclofenac ⫹ apamin ⫹ charybdotoxin (1-way ANOVA).

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ROLE OF KCa IN TYPE 2 DIABETIC SMA

Z++

ZDF

A

B *

Relaxation (%)

0

§ 25

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50

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75 CTL + Apa+Chtx + L-NAME+Diclo+Apa+Chtx

-10

-9

-8

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-6

*

0

*# *#

25

100

C

-5

-4

§

-10

-9

-8

-7

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-6

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-4

*

*

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75 CTL + Apa+TRAM34 + L-NAME+Diclo+Apa+TRAM34

-10

-9

-8

-7

-6

-5

-4

§

§

100 -10

[ACh] (logM)

resulted in a significant reduction of ACh-induced relaxation in ZDF but not in control SMA (Fig. 3). Given that mRNA and protein expression levels of IKCa are elevated in the animal model (Figs. 6 and 7), the IKCa, therefore, appears to maintain an important role in endothelium-dependent, ACh-induced relaxation of diabetic SMA. Since it also has been shown in a variety of studies (26, 34, 37) that EDH is able to compensate for decreased availability of the NO pathway, the hypothesis suggesting that EDH-type relaxation is an important compensatory, vasodilatory mechanism is not unanimously accepted, as it has been shown that EDH-type relaxation is severely impaired in diabetes and hypertension (20, 31, 47). Oxidative stress plays an early role leading to endothelial dysfunction and cardiovascular complications in diabetes (36). Hyperglycemia activates reduced NADP oxidases and leads to uncoupling of the homodimer eNOS to form superoxide (12, 24). Elevated formation of reactive oxygen species (ROS) is thought to be a key event in the genesis of endothelial dysfunction (35, 50), and ROS leads to enhanced oxidative stress, especially when antioxidant systems are impaired. One protein involved in this regulation is

§

[ACh] (logM)

0

100

*

*

100

[ACh] (logM)

Relaxation (%)

Fig. 4. Cumulative concentration-response curves to ACh (0.01 nM– 0.1 mM) before and after incubation with the combination of Apa and Chtx (each 0.1 ␮M) in the absence and presence of L-NAME (100 ␮M) and Diclo (10 ␮M) in Z⫹/⫹ rats (A) and ZDF rats (B). Chtx was replaced by TRAM-34 (0.1 ␮M) under otherwise uniform experimental procedure in Z⫹/⫹ (C) and ZDF rats (D); n ⫽ 6 experiments. Results are shown as means ⫾ SE. *P ⬍ 0.01 vs. CTL; #P ⬍ 0.05 vs. Apa ⫹ Chtx; §P ⬍ 0.01 vs. CTL, 2-way repeatedmeasures ANOVA.

#

-9

-8

-7

-6

-5

-4

[ACh] (logM)

heme oxygenase, which is responsible for maintaining normal metabolic cellular functions, and when absent, oxidative stress and chronic inflammation markedly increase (2, 21). Metabolites of the heme-containing cytochrome P450 epoxygenases, the epoxyeicosatrienoic (EET) acids, lead to an increased activity of heme oxygenase in rat mesenteric microvessels and to an attenuation of ROS levels, thereby protecting vessels against oxidative stress (27, 28, 39). EET acids can act as an EDH factor (EDHF) via autocrine activation of transient receptor potential channels in ECs with a consecutive opening of SKCa and IKCa to amplify endothelial signals (9), and they also possess a capability to act without involvement of the endothelial S/IKCa system by activating BKCa (33). As BKCa are sensitive to charybdotoxin, and ACh-induced relaxation in ZDF was markedly reduced by this toxin, a contribution of EET acids cannot be ruled out. However, selective inhibition of IKCa with TRAM-34 led to a comparable reduction of AChinduced relaxation, suggesting no relevant contribution of BKCa for ACh-induced vasorelaxation in this vessel. A mechanism of ECs to hyperpolarize the cell membrane of smooth muscle cells (SMCs) is mediated by electrical commu-

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ROLE OF KCa IN TYPE 2 DIABETIC SMA

Fig. 5. Concentration-response curves of SMA precontracted with NA (10 ␮M) to increasing concentrations of 1-ethyl-2-benzimidazolinone (1-EBIO; 1 nM–100 ␮M) or 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS1619; 1–100 ␮M) with endothelium (A and C) and without endothelium (⫺E; B and D) in the absence and presence of TRAM-34 (0.1 ␮M) or iberiotoxin (Ibtx; 0.1 ␮M), respectively, isolated from Z⫹/⫹ and ZDF rats; n ⫽ 5–7 experiments. Results are shown as means ⫾ SE. *P ⬍ 0.05 vs. ZDF. A: §P ⬍ 0.05 vs. Z⫹/⫹ ⫹ TRAM-34 and vs. ZDF ⫹ TRAM34. C: §P ⬍ 0.05 vs. Z⫹/⫹ ⫹ Ibtx and vs. ZDF ⫹ Ibtx; #P ⬍ 0.05 vs. ZDF ⫹ Ibtx. D: §P ⬍ 0.05 vs. Z⫹/⫹ ⫺E ⫹ Ibtx and vs. ZDF ⫺E ⫹ Ibtx; #P ⬍ 0.05 vs. ZDF ⫺E ⫹ Ibtx, 2-way repeated-measures ANOVA.

nication via myoendothelial gap junctions (MEGJs), spreading the signal from the luminal cell layer into the muscular layer. In the arterial mesenteric bed, it has been shown that MEGJs quantitatively increase with the decline of vessel diameter (40).

Fig. 6. Comparison of mRNA levels of isoform 3 of small (SKCa3)-, intermediate (IKCa)-, and large (BKCa)-conductance calcium-activated potassium channels in mesenteric arteries of Z⫹/⫹, ZL, and ZDF rats; n ⫽ 6. Results are shown as means ⫾ SE. *P ⬍ 0.05, 1-way ANOVA.

A prerequisite for this pathway of SMC hyperpolarization is EC hyperpolarization, which is conducted via activation of SKCa and IKCa in the membrane of ECs. In the present study, inhibition of EC hyperpolarization by apamin plus TRAM-34 led to a reduction of ACh-induced relaxation in diabetic vessels (Fig. 4D). An elongation or amplification of the electrical signal by gap junctions could be suppressed, so reduced gap junctional communication in the studied vessel may additionally occur, as observed in other studies (19, 29), and could act independently of SK/IKCa signaling, as proposed by Chadha et al. (11). Our results show a compensation of endothelium-dependent relaxation when potassium channels are inhibited in control animals but a loss of compensation in the diabetic model. Whereas products of COX do not have a role for endotheliumdependent vasorelaxation in control and diabetic animals in this study, NO-induced relaxation might be diminished because of reduced NO bioavailability. In ZDF animals, AChinduced relaxation is already severely affected by the combination of apamin and charybdotoxin, suggesting the NO system cannot compensate or alternatively, that EDH-type relaxation is predominant in the endothelium-dependent relaxation in that vessel under diabetic conditions. ROS play an important role in modulating NO levels, but next to a reduced bioavailability, the reason for the reduced NO-induced relaxation in diabetes can be upstream of NO production. eNOS is

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ROLE OF KCa IN TYPE 2 DIABETIC SMA

A

C

B IKCa

SKCa3

D IKCa +p

BKCa

250 170

150

130

kDa 100 70 55 40

M1 Z+/+ ZDF M2

M1 Z+/+ ZDF M2

M1 Z+/+ ZDF M2

M1 Z+/+ ZDF

-Actin

F

90 kDa

0.8

0.6

0.4

0.2

250 kDa

*

0.8

0.6

* 0.4

0.2

0.0

0.0

Z+/+

ZDF

Z+/+

ZDF

G

50 kDa

0.8

Densitometric ratio (BKCa/ -Actin)

250 kDa

Densitometric ratio (IKCa/ -Actin)

Densitometric ratio (SKCa3/ -Actin)

E

pZ+/+ pZDF

0.6

0.4

0.2

0.0

Z+/+

ZDF

Fig. 7. Representative Western blots of mesenteric artery revealing expression of SKCa3 (A), IKCa (B), and BKCa (D) expression. Monomeric forms appear at 90 kDa for SKCa3, 50 kDa for IKCa, and 110 kDa for BKCa. Channel protein complexes appear at 250 kDa. Specificity of anti-IKCa was characterized with its antigenic peptide (⫹p; C). Lanes M1 and M2, molecular-weight markers. Summary of Western blot data with histograms showing normalized values to actin protein levels for SKCa3 (E), IKCa (F), and BKCa (G). Unfilled rectangles represent values for the high molecular-weight region at 250 kDa; pZ⫹/⫹ and pZDF, preincubation with their control peptide; n ⫽ 3– 4 experiments. Results are shown as means ⫾ SE. *P ⬍ 0.05 vs. Z⫹/⫹ for the same molecular weight, using the Student’s unpaired t-test.

a Ca2⫹-dependent enzyme (10), so a reduction in endothelial Ca2⫹ concentration could result in reduced enzyme activation and consecutively reduced vasodilation. Furthermore, dampened activation of IKCa or SKCa and attenuated hyperpolarization of EC with decreased driving force for capacitative Ca2⫹ entry through store-operated channels are results of initial attenuation of agonist-induced Ca2⫹ elevation in ECs. It has been shown that Ca2⫹ release from endothelial stores is reduced with no significant alienation in store-operated calcium entry in Type 1 diabetic mice (16). Of course, a reduced NO bioavailability, as well as an altered upstream signaling, can occur coincidentally and lead to reduced vasodilation in diabetes. In our study, 1-EBIO-induced relaxation is impaired in diabetic arteries, similar to studies using isometric tension in SMA of Type 2 diabetic OLETF and Type 1 diabetic rats (31,

49). IKCa mRNA and protein are quantitatively upregulated in ZDF SMA, suggesting a reduced function of IKCa in diabetes, although besides monomeric channel protein, also channel complexes are upregulated. As SKCa and IKCa have been shown to be expressed exclusively in ECs of rat mesenteric artery (13, 14), our results go in line with other observations, stating an impaired function of the endothelial S/IKCa dilator system in cardiovascular diseases (6, 8). The observed increase in IKCa protein expression in diabetes could be part of a compensatory, regulatory mechanism in mesenteric ECs of ZDF rats. Theoretically, the channel protein could be upregulated in SMCs, as observed in studies of vascular beds other than rat mesenteric artery, and also cultured cells (22, 45). Although the proportion of increased IKCa expression cannot be attributed precisely to EC or SMC, as whole vessel tissue was used in this study, following literature, we suppose an

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00240.2013 • www.ajpheart.org

ROLE OF KCa IN TYPE 2 DIABETIC SMA

upregulation in EC rather than SMC. As IKCa activation via 1-EBIO stimulation results in reduced vasorelaxation in diabetic SMA, the observed mRNA and protein upregulation of IKCa most likely reflects non- or dysfunctional protein, which leads to a reduced relaxation in ZDF mesenteric arteries when endothelium-dependently stimulated with ACh. BKCa, which are expressed exclusively in SMCs (15, 48), possibly act as a target of endothelium-derived relaxing factors. In this study EC activation is already reduced, so a lessened downstream activation of BKCa could be expected if EDHF would target this channel. NS1619-induced relaxation showed no difference in ZDF animals, suggesting no impairment of this pathway by diabetes. Study limitations. Due to technical limitations of our laboratory, we were not able to provide electrophysiological data to demonstrate EDH of SMCs; instead, we use the term EDHtype relaxation. As we used whole mesenteric arteries, thirdorder branch for myograph experiments, and first- through third-order branches for mRNA and protein quantification, we cannot distinguish between effects on EC or SMC with certainty. However, there are robust data stating that in intact rat mesenteric arteries, BKCa are expressed exclusively in SMC (15, 48) and S/IKCa in EC (13, 14). The accuracy of the Western blot analysis depends on antibody specificity. The antibodies used in this study to detect SKCa3 and BKCa are well characterized (5, 42, 44). The antibody APC-064 against IKCa is less well characterized; therefore, we determined specificity by preincubation of the antibody with the antigenic peptide, as shown in our data in Fig. 7. We nevertheless note that differences may arise with different antibody batches. Conclusion. In the present study, we examined the role of endothelium-derived relaxation in Type 2 diabetes with focus on KCa. We introduced a previously uninvestigated control group, the Z⫹/⫹ rat, which has no allele for the leptin receptor defect. To the best of our knowledge, we present, for the first time, molecular data of the relevant KCa, i.e., SKCa3, IKCa, and BKCa, in ZDF, as well as Z⫹/⫹ SMA, and show functional data of the Z⫹/⫹ mesenteric vessels in this study. NO-associated and EDH-type relaxation can compensate for each other in ACh-induced relaxation of mesenteric arteries in control but not in Type 2 diabetic ZDF rats. Particularly, the NOS- and COX-independent relaxation is sensitive to the combination of apamin and TRAM-34 in our diabetes model. Although relaxation after IKCa activation is reduced in diabetes, mRNA and protein levels were elevated in mesenteric arteries of diabetic animals. These observations, when taken together with the shown reduction of ACh-induced relaxation by selective IKCa inhibition, suggest an important role for IKCa in diabetic SMA as a target for innovative treatments of diabetic microvascular complications. ACKNOWLEDGMENTS We thank Prof. Dr. Warth for offering expertise and critically reviewing the data and manuscript and Gabriela Piertrzyk for excellent technical help. GRANTS Support for this work was provided by a grant from the Deutsche Forschungsgemeinschaft (EN 472/4-1; to D. H. Endemann). DISCLOSURES The authors declare that there is no competing interest.

H1101

AUTHOR CONTRIBUTIONS Author contributions: C.S., M.R., and D.H.E. conception and design of research; C.S. and P.M.S. performed experiments; C.S., M.R., and D.H.E. analyzed data; C.S., M.R., P.M.S., G.A.R., and D.H.E. interpreted results of experiments; C.S. prepared figures; C.S. and M.R. drafted manuscript; C.S. and D.H.E. edited and revised manuscript; C.S., M.R., P.M.S., G.A.R., and D.H.E. approved final version of manuscript. REFERENCES 1. National High Blood Pressure Education Program Working Group report on hypertension in diabetes. Hypertension 23: 145–158; discussion 159 – 160, 1994. 2. Abraham NG, Kappas A. Pharmacological and clinical aspects of heme oxygenase. Pharmacol Rev 60: 79 –127, 2008. 3. Angulo J, Cuevas P, Fernandez A, Gabancho S, Allona A, MartinMorales A, Moncada I, Videla S, Saenz de Tejada I. Diabetes impairs endothelium-dependent relaxation of human penile vascular tissues mediated by NO and EDHF. Biochem Biophys Res Commun 312: 1202–1208, 2003. 4. Bergler T, Stoelcker B, Jeblick R, Reinhold SW, Wolf K, Riegger GA, Kramer BK. High osmolality induces the kidney-specific chloride channel CLC-K1 by a serum and glucocorticoid-inducible kinase 1 MAPK pathway. Kidney Int 74: 1170 –1177, 2008. 5. Berrout J, Mamenko M, Zaika OL, Chen L, Zang W, Pochynyuk O, O’Neil RG. Emerging role of the calcium-activated, small conductance, SK3 K⫹ channel in distal tubule function: regulation by TRPV4. PLoS One 9: e95149, 2014. 6. Brondum E, Kold-Petersen H, Simonsen U, Aalkjaer C. NS309 restores EDHF-type relaxation in mesenteric small arteries from type 2 diabetic ZDF rats. Br J Pharmacol 159: 154 –165, 2010. 7. Brunner H, Cockcroft JR, Deanfield J, Donald A, Ferrannini E, Halcox J, Kiowski W, Luscher TF, Mancia G, Natali A, Oliver JJ, Pessina AC, Rizzoni D, Rossi GP, Salvetti A, Spieker LE, Taddei S, Webb DJ, Working Group on Endothelins and Endothelial Factors of the European Society of Hypertension. Endothelial function and dysfunction. Part II: association with cardiovascular risk factors and diseases. A statement by the Working Group on Endothelins and Endothelial Factors of the European Society of Hypertension. J Hypertens 23: 233– 246, 2005. 8. Burnham MP, Johnson IT, Weston AH. Impaired small-conductance Ca2⫹-activated K⫹ channel-dependent EDHF responses in Type II diabetic ZDF rats. Br J Pharmacol 148: 434 –441, 2006. 9. Busse R, Edwards G, Feletou M, Fleming I, Vanhoutte PM, Weston AH. EDHF: bringing the concepts together. Trends Pharmacol Sci 23: 374 –380, 2002. 10. Busse R, Mulsch A. Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. FEBS Lett 265: 133–136, 1990. 11. Chadha PS, Haddock RE, Howitt L, Morris MJ, Murphy TV, Grayson TH, Sandow SL. Obesity up-regulates intermediate conductance calcium-activated potassium channels and myoendothelial gap junctions to maintain endothelial vasodilator function. J Pharmacol Exp Ther 335: 284 –293, 2010. 12. Ding H, Aljofan M, Triggle CR. Oxidative stress and increased eNOS and NADPH oxidase expression in mouse microvessel endothelial cells. J Cell Physiol 212: 682–689, 2007. 13. Doughty JM, Plane F, Langton PD. Charybdotoxin and apamin block EDHF in rat mesenteric artery if selectively applied to the endothelium. Am J Physiol Heart Circ Physiol 276: H1107–H1112, 1999. 14. Edwards G, Dora KA, Gardener MJ, Garland CJ, Weston AH. K⫹ is an endothelium-derived hyperpolarizing factor in rat arteries. Nature 396: 269 –272, 1998. 15. Ellis A, Goto K, Chaston DJ, Brackenbury TD, Meaney KR, Falck JR, Wojcikiewicz RJ, Hill CE. Enalapril treatment alters the contribution of epoxyeicosatrienoic acids but not gap junctions to endothelium-derived hyperpolarizing factor activity in mesenteric arteries of spontaneously hypertensive rats. J Pharmacol Exp Ther 330: 413–422, 2009. 16. Estrada IA, Donthamsetty R, Debski P, Zhou MH, Zhang SL, Yuan JX, Han W, Makino A. STIM1 restores coronary endothelial function in Type 1 diabetic mice. Circ Res 111: 1166 –1175, 2012. 17. Feletou M, Vanhoutte PM. Endothelium-dependent hyperpolarizations: past beliefs and present facts. Ann Med 39: 495–516, 2007. 18. Fitzgerald SM, Kemp-Harper BK, Parkington HC, Head GA, Evans RG. Endothelial dysfunction and arterial pressure regulation during early

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19.

20.

21.

22.

23. 24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

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