Twenty-Four-Hour Exposure to Altered Blood Flow Modifies Endothelial Ca -Activated K Channels in Rat Mesenteric Arteries

We tested the hypothesis that changes in arterial blood flow modify the function of endothelial Ca -activated K channels [calcium-activated K channel (KCa), small-conductance calciumactivated K channel (SK3), and intermediate calcium-activated K channel (IK1)] before arterial structural remodeling. In rats, mesenteric arteries were exposed to increased [ 90%, high flow (HF)] or reduced blood flow [ 90%, low flow (LF)] and analyzed 24 h later. There were no detectable changes in arterial structure or in expression level of endothelial nitric-oxide synthase, SK3, or IK1. Arterial relaxing responses to acetylcholine and 3-oxime-6,7dichlore-1H-indole-2,3-dione (NS309; activator of SK3 and IK1) were measured in the absence and presence of endothelium, NO, and prostanoid blockers, and 6,12,19,20,25,26-hexahydro-5,27:13,18: 21,24-trietheno-11,7-metheno-7H-dibenzo [b,n] [1,5,12,16]tetraazacyclotricosine-5,13-diium dibromide (UCL 1684; inhibitor of SK3) or 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM34; inhibitor of IK1). In LF arteries, endothelium-dependent relaxation was markedly reduced, due to a reduction in the endothelium-derived hyperpolarizing factor (EDHF) response. In HF arteries, the balance between the NO/prostanoid versus EDHF response was unaltered. However, the contribution of IK1 to the EDHF response was enhanced, as indicated by a larger effect of TRAM-34 and a larger residual NS309-induced relaxation in the presence of UCL 1684. Reduction of blood flow selectively blunts EDHF relaxation in resistance arteries through inhibition of the function of KCa channels. An increase in blood flow leads to a more prominent role of IK1 channels in this relaxation. The endothelial smalland intermediate-conductance calcium-activated K channels (KCa2.3 or SK3 and KCa3.1 or IK1, respectively) have been proposed to initiate endothelium-derived hyperpolarizing factor (EDHF) signaling in resistance-sized arteries (Edwards et al., 1998; Burnham et al., 2002; Crane et al., 2003; Eichler et al., 2003; Hilgers et al., 2006). These calcium-activated K channel (KCa) channels can be activated by pharmacological stimuli to mediate K efflux and result in endothelial hyperpolarization with subsequent smooth muscle hyperpolarization, leading to closure of voltage-activated Ca channels and relaxation. KCa channels and other mechanosensitive cation channels, such as endothelial transient receptor potential V4 channels, can play a crucial role in acute endothelium-dependent vasodilator responses to elevated shear stress (Olesen et al., 1988; Köhler et al., 2006). Indeed, pharmacological blockade of KCa channels blunts shear stress-induced vasodilatations in isolated small arteries (Popp et al., 1998), suggesting release of EDHF and/or spread of an electrical current via activation of KCa channels. Whether the expression of KCa channels is modulated by in vivo shear stress alterations remains elusive. Given their distinct spatial location in the endothelial layer (SK3 at endothelial cell borders and IK1 at sites of This work was supported by the European Vascular Genomics Network European Community’s 6th Framework Programme [Grant LSHM-CT-2003503254]. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.161448. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: KCa, calcium-activated K channel; SK3, small-conductance calcium-activated K channel; IK1, intermediate calciumactivated K channel; EDHF, endothelium-derived hyperpolarizing factor; ACh, acetylcholine; NS309, 3-oxime-6,7-dichlore-1H-indole-2,3-dione; LF, low flow; HF, high flow; NF, normal flow; MA, mesenteric artery(ies); KRB, Krebs-Ringer buffer; NE, norepinephrine; PHE, phenylephrine; INDO, indomethacin; L-NAME, N -nitro-L-arginine methyl ester; ODQ, 1H-[1,2,4]oxadiazole[4,3a]quinoxaline-1-one; UCL 1684, 6,12,19,20,25,26-hexahydro-5,27:13,18:21,24-trietheno-11,7-metheno-7H-dibenzo [b,n] [1,5,12,16]tetraazacyclotricosine-5,13-diium dibromide; TRAM-34, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; eNOS, endothelial nitric-oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMSO, dimethyl sulfoxide; pEC50, sensitivity; Emax, maximal effect. 0022-3565/10/3331-210–217$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 333, No. 1 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 161448/3566154 JPET 333:210–217, 2010 Printed in U.S.A. 210 http://jpet.aspetjournals.org/content/suppl/2009/12/29/jpet.109.161448.DC1 Supplemental material to this article can be found at: at A PE T Jornals on A ril 9, 2017 jpet.asjournals.org D ow nladed from myoendothelial gap junctions; Sandow et al., 2006; Dora et al., 2008), it may be expected that the expression and function of KCa channels is under differential control by the frictional force of blood flow. Hence, we investigated whether subchronic (24-h) alterations in shear stress modulate expression and function of SK3 and IK1 channels in rat mesenteric arteries. This time point of 24 h after ligation reflects a transition from acute flow-mediated responses to structural flow-induced remodeling processes. Previously, relationships were proposed between acute vasomotor responses and chronic arterial structural responses to altered blood flow (De Mey et al., 2005). Understanding the molecular events that occur in response to early changes in arterial blood flow might provide us with more insight into the structural arterial changes in response to chronic alterations in blood flow. The function of each KCa channel was analyzed by recording relaxing responses to acetylcholine (ACh) and the KCa channel activator NS309 during inhibition of NO synthases, cyclooxygenases, and soluble guanylate cyclase. We used a surgical ligation method in which first-order mesenteric arteries were either exposed to low flow (LF; 90%), high flow (HF; 90%), or normal flow (NF) in vivo (Pourageaud and De Mey, 1997; Buus et al., 2001). After several days, this intervention resulted in adaptive changes of the structural arterial diameter and wall mass (Unthank et al., 1996; Buus et al., 2001; Tuttle et al., 2001). After 1 day, LF, HF, and NF first-order mesenteric arteries were isolated to study EDHF responses to ACh and NS309 in the absence and presence of pharmacological inhibitors of KCa channel subtypes and protein expression levels of SK3 and IK1 channels using Western blotting techniques and immunohistochemistry. Materials and Methods Animals. In total, 32 male Wistar Kyoto rats of 12 weeks of age were obtained from Charles River (Maastricht, The Netherlands). All animals were caged separately and had free access to standard food (SRMA-1210; Hope Farms, Woerden, The Netherlands) and tap water. Experimental protocols were performed in accordance with institutional guidelines and were approved by the Ethics Committee on Experimental Animal Welfare of the Maastricht University. Ligation Model. Small mesenteric arteries (MA) were exposed to altered blood flow via a surgical ligation method as described previously (Pourageaud and De Mey, 1997). In brief, in anesthetized (isoflurane; Abbott Laboratories Ltd., Maidenhead, UK) rats, laparotomy was performed and the mesentery was spread out on gauze tissue wetted with sterile saline solution. Local blood flow was lowered (LF) by distal ligation of three alternate second-order MA branches. The MA running between these had compensatory higher blood flows (HF). We previously observed in Wistar Kyoto rats that the blood flow averages 10 and 200% in LF and HF compared with second-order MA outside of the surgical area (NF) (Pourageaud and De Mey, 1997; Buus et al., 2001). Animals received buprenorphine (0.05 mg/kg s.c. Temgesic; Schering Plough, Utrecht, The Netherlands) as an analgetic before and after surgical intervention. Pressure Myograph Experiments. Twenty-four hours after surgery, rats were killed by CO2 inhalation, and the mesentery was removed and placed in cold (4°C) Krebs-Ringer buffer (KRB) with the following composition: 118.5 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25.0 mM NaHCO3, and 5.5 mM glucose. From each experimental rat, one segment of first-order MA (4–5 mm in length) that had been exposed to LF, one segment to HF, and one segment to NF were carefully dissected. In an organ chamber, segments were cannulated at both ends on two glass micropipettes (outer diameter, 200 m), tied with nylon knots, and incubated in 10 ml of calcium-free physiological salt solution of the following composition: 144 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 14.9 mM HEPES, and 5.5 mM glucose, pH 7.4. The organ chamber was placed on the stage of an inverted microscope (Nikon, Tokyo, Japan) equipped with a black-and-white video camera (Stemmer, Puchheim, Germany). An arteriograph system (Living Systems Instruments, Burlington, VT) analyzed the signal obtained from the video image and continuously determined the wall thickness and internal diameter, while intraluminal pressure was controlled (Halpern et al., 1984). Internal diameter and wall thickness were recorded using PowerLab and Chart 5 software (ADInstruments Ltd., Chalgrove, Oxfordshire, UK). Passive pressure-diameter relations were constructed as described previously (Hilgers et al., 2004). Wire-Myograph Experiments. From each experimental rat, segments of first-order MA (2 mm in length) that had been exposed to LF, HF, or NF in vivo (for 1 day) were carefully dissected and mounted in a wire-myograph (Danish Myotech, Aarhus, Denmark) for the recording of isometric force development (Hilgers et al., 2006). Segments were incubated for 0.5 h in KRB that was continuously aerated with 95% O2, 5% CO2 and maintained at 37°C. Each experiment started by progressively stretching the arterial segment to the diameter at which the largest contractile response to 10 M norepinephrine (NE) could be obtained (optimal diameter). To exclude any vasodilator influences of sensory motor nerves (De Mey et al., 2008), arteries were exposed to 1 M capsaicin (during 20 min). During contraction with a single concentration of phenylephrine (PHE; 20 M), relaxing responses to the endothelium-dependent muscarinic vasodilator ACh (0.001–10 M) were recorded in the absence of any inhibitors (control). After washing and a 30-min rest period, arterial segments were again contracted with 20 M PHE. When a stable contraction was achieved, relaxing responses to the KCa channel activator NS309 (0.1–12.8 M; Strøaek et al., 2004) were recorded. The same segments were then incubated for 0.5 h with 10 M indomethacin (INDO; inhibitor of cyclooxygenases), 100 M N -nitro-L-arginine methyl ester (L-NAME; inhibitor of NO synthases), and 10 M 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; inhibitor of the NO-sensitive soluble guanylate cyclase), and relaxing responses to ACh and NS309 were repeated. To study the contribution of each KCa channel subtype, EDHF-mediated relaxations were always studied in the combined presence of L-NAME, INDO, and ODQ to rule out any potential interference of NO and prostaglandins with KCa channels (Bolotina et al., 1994). UCL 1684 at 1 M (Romey et al., 1984) was used to block small-conductance KCa channels, and 10 M TRAM-34 (Wulff et al., 2000) was used to block intermediate-conductance KCa channels. This concentration for TRAM-34 was chosen in accordance with our recently published article (Hilgers and Webb, 2007). According to the Guide to Receptors and Channels (Alexander et al., 2008), the nomenclature for small-conductance KCa channels is KCa2.x (SKx) and, for intermediate-conductance KCa channels, it is KCa3.1 (SK4, IK1). The endothelial SK3 isoform is expressed in arteries. For simplicity, SK3 and IK1 are used in the remainder of the text. These antagonists were tested individually or in combination. Because NS309 can activate both human KCa subtypes, but with a slight (2to 4-fold) selectivity for IK1 over SK3 (Strøaek et al., 2004), the individual contribution of a KCa channel subtype upon NS309 activation was always analyzed in the combined presence of L-NAME, INDO, and ODQ and the opposing KCa channel blocker. For example, to address the IK1-mediated responses, segments were incubated with L-NAME, INDO, ODQ, and UCL 1684. In a subset of segments, the endothelium was mechanically removed by gently rubbing the lumen with an equine hair (Osol et al., 1989). Western Blotting. At 1 day postflow-modifying surgery, three 10-mm-long first-order MA that had been exposed to LF, HF, or NF were freed of adipose and connective tissue and quickly snap-frozen in liquid nitrogen and kept at 80°C until protein expression analysis. Segments were homogenized in cold (4°C) radioimmunoprecipitation assay buffer [50 mM Tris-HCl, pH 7.4, 0.15 mM NaCl, 0.25% Modulation of KCa Channels by Flow 211 at A PE T Jornals on A ril 9, 2017 jpet.asjournals.org D ow nladed from deoxycholic acid, 1% Nonidet P-40, and 1 mM EDTA] enriched with 1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 1 g/ml aprotinin, 1 g/ml pepstatin, and 1 mM Na3VO4. The homogenate was centrifuged at 15,000g for 30 min at 4°C. The supernatant was kept on ice. Protein concentration was determined with the bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL). Twenty micrograms of protein was loaded and separated by SDS-polyacrylamide gel electrophoresis (10%) and subsequently transferred to nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked by treatment with 5% nondry fat milk in Tris-buffered saline containing 0.05% Tween 20, anti-KCa2.3 (Nterm; Alomone Labs, Jerusalem, Israel), anti-KCa3.1 (Alomone Labs), endothelial nitric-oxide synthase (anti-eNOS, BD Biosciences Transduction Laboratories, Erembodegem-Aalst, Belgium), or glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; Millipore Bioscience Research Reagents, Temecula, CA) and kept overnight at 4°C. After incubation with secondary antibodies, signals were revealed with chemiluminescence autoradiography and quantified densitometrically by determining the ratio between protein of interest and GAPDH pixel density. Immunostaining. After the passive pressure-diameter curves, the segments were fixed at a pressure of 80 mm Hg in 4% phosphatebuffered formalin. Fixed vessels were embedded in paraffin, and cross-sections (4 m) were subjected to immunohistochemistry using a peroxidase second step approach (swine anti-rabbit horseradish peroxidase). Endothelial and smooth muscle cell nuclei were stained with hematoxylin. Primary antibodies were directed against IK1 (anti-KCa3.1, 1:1600; Alomone Labs) or SK3 (anti-KCa2.3 N-term, 1:3000; Alomone Labs). Negative control stainings were performed where the primary antibody was omitted to check whether the staining was specific. Video images were taken from cross-sections using an axioscope (Carl Zeiss, Jena, Germany) and a standard chargecoupled digital camera (model DFC 280; Leica, Wetzlar, Germany). Drugs. Acetylcholine, phenylephrine, norepinephrine, and L-NAME were purchased from Sigma (Zwijndrecht, The Netherlands) and dissolved in KRB. TRAM-34 and NS309 (Sigma) were dissolved in DMSO. Indomethacin (Sigma) was dissolved in ethanol. ODQ (Calbiochem, Darmstadt, Germany) was dissolved in DMSO. UCL 1684 (Tocris Bioscience, Bristol, UK) was dissolved in DMSO. The concentrations of DMSO used never exceeded 0.3% (v/v) in the organ bath and were observed previously not to significantly modify contractile responses (Hilgers and De Mey, 2009). Data and Statistical Analysis. Contractile responses were expressed as a percentage of the maximal contractile response to 10 M NE before the administration of any pharmacological inhibitor. Relaxing responses were expressed as a percentage of the maximal contractile response to 20 M PHE. Individual concentration-response curves were fitted to a nonlinear sigmoid regression curve (Prism 5.0; GraphPad Software Inc., San Diego, CA). Sensitivity (pEC50) and maximal effect (Emax) are shown as mean S.E.M. Statistical significance of effects and differences were analyzed using either one-way analysis of variance (comparison of pEC50 and Emax) or twoway analysis of variance (comparison of concentration-response curves). A Bonferroni post hoc test was used to compare multiple groups. A P value 0.05 was considered statistically significant.