AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 292: H2275-H2284, 2007. First published January 5, 2007; doi:10.1152/ajpheart.00949.2006
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/5/H2275    most recent
00949.2006v2
00949.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hilgers, R. H. P.
Right arrow Articles by Webb, R. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hilgers, R. H. P.
Right arrow Articles by Webb, R. C.

Reduced expression of SKCa and IKCa channel proteins in rat small mesenteric arteries during angiotensin II-induced hypertension

Rob H. P. Hilgers and R. Clinton Webb

Department of Physiology, Medical College of Georgia, Augusta, Georgia

Submitted 31 August 2006 ; accepted in final form 28 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ca2+-activated K+ channels (KCa), in particular, the small and intermediate KCa (SKCa and IKCa, respectively) channels, are key players in endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation in small arteries. Hypertension is characterized by an endothelial dysfunction, possibly via reduced EDHF release and/or function. We hypothesize that during angiotensin II (14 days)-induced hypertension (ANG II-14d), the contribution of SKCa and IKCa channels in ACh-induced relaxations is reduced due to decreased expression of SKCa and IKCa channel proteins in rat small mesenteric arteries (MAs). Nitric oxide- and prostacyclin-independent vasorelaxation to ACh was similar in small MAs of sham-operated and ANG II-14d rats. Catalase had no inhibitory effects on these relaxations. The highly selective SKCa channel blocker UCL-1684 almost completely blocked these responses in MAs of sham-operated rats but partially in MAs of ANG II-14d rats. These changes were pressure dependent since UCL-1684 caused a greater inhibition in MAs of 1-day ANG II-treated normotensive rats compared with ANG II-14d rats. Expression levels of both mRNA and protein SK3 were significantly reduced in MAs of ANG II-14d rats. The IKCa channel blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) resulted in comparable reductions in the relaxation responses to ACh in MAs of sham-operated and ANG II-14d rats. Relative mRNA expression levels of IK1 were significantly reduced in MAs of ANG II-14d rats, whereas protein levels of IK1 were not but tended to be lower in MAs of ANG II-14d rats. The findings demonstrate that EDHF-like responses are not compromised in a situation of reduced functional activity and expression of SK3 channels in small MAs of ANG II-induced hypertensive rats. The role of IK1 channels is less clear but might compensate for reduced SK3 activity.

calcium-activated potassium channels; endothelium

CALCIUM-ACTIVATED K+ CHANNELS (KCa) are key players in the control of endothelium-dependent vasorelaxation. The endothelial cell intermediate- and small-conductance KCa channels (IKCa and SKCa, respectively) are especially important in endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation and hyperpolarization in resistance-sized arteries (1, 7, 13, 18, 28, 36, 37). This hyperpolarization is communicated to adjacent smooth muscle cells likely through myoendothelial gap junctions (30, 37) or via diffusible factor(s) (11, 17, 34, 41). These KCa channels regulate membrane potential and Ca2+ homeostasis in response to hemodynamic stresses and vasoactive factors. For instance, an increase in intracellular Ca2+ leads to an increase in the activity of KCa channels, causing smooth muscle cell hyperpolarization and prevention of further influx of Ca2+ (2, 4, 29).

The relative contribution of endothelium-derived relaxing factors regulating arterial tone depends largely on the vessel type and the (patho)physiological state. An inverse correlation between the arterial diameter and the relative contribution of EDHF has been proposed (42). We recently observed a regional heterogeneity in the expression level of SKCa and IKCa channels and their contribution in ACh-induced relaxations in rat mesenteric arteries, with higher expression levels in fourth-order mesenteric arteries (MAs) compared with first-order vessels, stressing the importance of these channels in smaller vessels where the role of EDHF is more pronounced (27). A number of cardiovascular diseases, including diabetes, atherosclerosis, and hypertension, are characterized by a reduced EDHF response (20, 21). Recently, a reduced expression of SKCa channel mRNA, but not IKCa, was observed in MAs of streptozotocin-induced diabetic apolipoprotein E-deficient mice (16). The role of SKCa and IKCa channels during hypertension is far from clear. A study using transgenic SK3T/T mice, in which the SK3 expression levels can be manipulated with dietary doxycycline, showed that SK3 channel suppression caused a pronounced hypertension, indicating the significance of this SK3 channel in regulating vasomotor tone and blood pressure (45). Knowledge of the mechanisms underlying EDHF and KCa channel function would improve our understanding of the regulation of arterial tone in health and disease. Changes in molecular composition of KCa channels may therefore be a fundamental event contributing to the progression of arterial dysfunction during hypertension. Whether these changes in KCa channel function and expression during hypertension occur at the level of resistance-sized arteries, where they are needed in regulating vascular tone and hence local blood flow, is not completely understood and requires further studies.

We measured EDHF-mediated vasorelaxing responses in the absence or presence of specific antagonists of IKCa and SKCa channels in small (fourth order) MAs of sham-operated normotensive and angiotensin II (ANG II)-induced (14 days, chronic infusion; 60 ng·kg–1·min–1) hypertensive rats. To distinguish between rapid (hours–days) ANG II-induced and slow (days–weeks) hypertension-related changes in KCa channel function, we included a third group of rats treated with ANG II (60 ng·kg–1·min–1) via osmotic minipumps for 24 h. Since these rats still remained normotensive (22), the effects of ANG II, independently of pressure on SKCa and IKCa channel function, could be studied. Furthermore, RT-PCR and immunoprecipitation followed by Western blot analysis were performed to analyze the expression of both IKCa and SKCa channel mRNA and protein levels in isolated small MAs of sham-operated and 14-day ANG II-treated rats. We hypothesized that after 14 days of ANG II treatment, but not after 1 day, the contribution of SKCa and IKCa channels in ACh-induced relaxations is reduced, due to decreased expression of SKCa and IKCa channel proteins in small rat MAs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal preparation and blood pressure measurement. Sprague-Dawley rats (225–250 g, Harlan, Indianapolis, IN) were implanted subcutaneously with osmotic minipumps (Alzet, Alza) after they were anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg). The rats were divided into three groups: a control group infused with saline only, and two groups of rats infused with ANG II (60 ng·kg–1·min–1) for a period of 1 and 14 days, respectively. Systolic blood pressure was measured by tail-cuff plethysmography in conscious rats to monitor the progression of hypertension. The basal level of systolic blood pressure was measured on two consecutive days before the implantation of minipumps. All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were reviewed and approved by the Institutional Animal Care and Use Committee of the Medical College of Georgia.

Measurement of isometric tension. Rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and the MA was rapidly excised and placed in cold physiological saline solution (PSS) of the following composition (in mmol/l) 118 NaCl, 4.7 KCl, 1.18 KH2PO4, 1.6 CaCl2·2H2O, 1.6 MgSO4·7H2O, 25 NaHCO3, 5.5 dextrose, and 0.03 EDTA. Fourth-order segments (2 mm) of the superior MA were isolated and mounted in the wire myograph (Danish MyoTech) filled with 5 ml PSS and continuously gassed with 95% O2-5% CO2 while maintaining temperature at 37°C. The segments were stretched to a diameter resulting in a resting wall tension of 0.63 N/m. The segments were allowed to incubate for 45 min. Arterial integrity was assessed by contracting the segments with phenylephrine (10 µmol/l) followed by relaxation with ACh (1 µmol/l). After being washed, the arterial rings were contracted with the thromboxane analog 9,11-dideoxy-9{alpha},11{alpha}-methanoepoxyprostaglandin F2{alpha} (U-46619, 0.1 µmol/l). U-46619 was observed to produce stable contractions for a long period in MAs of both normo- and hypertensive rats, unlike phenylephrine, which showed unstable contractions, in particular, in MAs of hypertensive rats (personal observations). Cumulative concentration-response curves (CRCs) to ACh were performed under control conditions in the combined presence of indomethacin (inhibitor of cyclooxygenase, 10 µmol/l) and N{omega}-nitro-L-arginine methyl ester [L-NAME, inhibitor of nitric oxide (NO) synthase, 100 µmol/l]. To study the contribution of H2O2 derived from superoxide anions resulting in vasorelaxation, CRCs to ACh were performed in the combined presence of L-NAME and indomethacin and catalase (100 U/ml). To study the role of KCa channels in ACh-induced relaxations, we always determined EDHF-mediated relaxations in the combined presence of L-NAME and indomethacin to rule out any potential interference of NO and prostaglandins with KCa channels (3). Since U-46619 has been shown to cause an inactivation of SKCa channels in small rat MAs (14), one single cumulative CRC for ACh was performed for each arterial segment incubated with L-NAME, indomethacin, and KCa channel inhibitor(s).

Semiquantitative RT-PCR amplification of KCa channel genes. Fourth-order (and smaller branches along the gut wall) MAs were carefully freed of adipose and connective tissue and quickly snap frozen in liquid nitrogen. To generate sufficient amounts of total RNA, isolated MA segments were pooled from 10 rats and subsequently homogenized. This pool was considered as n = 1, three individual pools of a total of 30 different rats were used to isolate total RNA, which yielded a total of n = 3. Total RNAs were isolated with TRIzol reagent (Invitrogen). The amount of RNA was determined spectrophotometrically at an absorbance of 260 nm. An amount of 0.5 µg of total RNAs was reverse transcribed [oligo(dT)12–18 primer using Moloney murine leukemia virus reverse transcriptase, Amersham Biosciences], and cDNAs were subsequently amplified in Ready-to-Go RT-PCR beads in a two-step procedure (Amersham Biosciences). Primer sequences for the SK3 gene (Genbank accession number AF292389) were as follows: forward, nt: 1389–1406; 5'-CCTCTACATCAGCCTGGA-3'; reverse, nt: 2090–2073: 5'-ACTTTGGCGTGGTCAATC-3'. The PCR product generated a fragment of 702 bp. Primer sequences for the SK4 gene, which is encoded by the Kccn4 gene, are actually the same as the rat IKCa subtype encoded by the rat (r)IK1 gene [see Genbank accession numbers AF190458 (SMIK) and NM023021 (rIK1)], were as follows: forward, nt: 1193–1212; 5'-GAACAAGTGAATTCCATGGT-3'; reverse, nt: 1390–1372: 5'-CTATGTGGCCTCCTGGATG-3'. The PCR product generated a fragment of 198 bp. The primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Genbank accession number AF106860) were as follows: forward, nt: 84–104; 5'-GGCTGCCTTCTCTTGTGACAA-3'; reverse, nt: 282–263; 5'-CGCTCCTGGAGGATGGTGAT-3'. The PCR product generated a fragment of 199 bp. The general protocol for PCR amplification was 35 cycles at 94°C for 1 min, at 60°C for 1 min, and at 72°C for 1 min, followed by a final extension at 72°C for 10 min. Amplified DNA fragments were separated in a 2% agarose gel. The gel images were recorded by video camera (Sony Video Camera Module CCD, Tokyo, Japan) connected to an IBM AT computer (New York, NY) with a 512 x 512-pixel array imaging board with 256 gray levels. The PCR products were quantified by densitometric scanning of gel images using UN-SCAN-IT software (Silk Scientific). Results were then expressed as the densitometric ratio of the gene of interest to GAPDH.

Immunoprecipitation of SK3 and IK1 proteins. Fourth-order and higher-order MAs running along the gut were carefully freed of adipose and connective tissue and quickly snap frozen in liquid nitrogen and kept at –80°C until protein expression analysis. Proteins were isolated from arteries pooled from 10 rats to ensure a sufficient amount of proteins. This pool was considered as n = 1, three individual pools of a total of 30 different rats were used to isolate total protein extract, which yielded a total of n = 3. Arteries were homogenized in cold RIPA buffer [50 mmol/l Tris·HCl (pH 7.4), 0.15 mmol/l NaCl, 0.25% deoxycholic acid, 1% Nonidet P-40, and 1 mmol/l EDTA] enriched with 1 mmol/l PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin, and 1 mmol/l Na3VO4. The homogenate was separated by centrifugation at 15,000 g for 30 min at 4°C. The supernatant was kept on ice. Protein concentration was determined via the BSA protein assay kit (Pierce Chemical, Rockford, IL). Aliquot samples of 100 µg of total protein extract were then incubated with 1 µg of antibody (anti-SK3, Sigma-Aldrich, St. Louis, MI, or anti-IK1, Santa Cruz Biotechnology) for 6 h at 4°C under constant rotation, followed by the addition of Sepharose A and B beads (1:1) for 6 h at 4°C (Upstate Biotechnology, Lake Placid, NY). Immunoprecipitates were collected by centrifugation (12,000 rpm; 5 min), washed three times with phosphate-buffered saline solution, and resuspended in Laemmli's sample buffer with beta-mercaptoethanol (Bio-Rad, Hercules, CA). Samples were loaded and separated by SDS-PAGE (10%) and subsequently transferred to nitrocellulose membrane (Bio-Rad). Membranes were blocked by treatment with 5% nondry-fat milk in Tris-buffered saline containing 0.05% Tween 20, probed with anti-SK3 or anti-IK1 (1:1,000), and kept overnight at 4°C. After incubation with secondary antibody, signals were revealed with chemiluminescence autoradiography and quantified densitometrically.

Data analyses and statistics. Experimental values were calculated relative to the maximal changes from the contraction produced by U-46619 in each segment, which was taken as 100%. The phospho-EC50 (pEC50) values for ACh were expressed as –log of the molar concentration to produce 50% of the maximal response. Data are expressed as means ± SE of n arterial segments. Statistical analysis was performed by using a two-way analysis of ANOVA to compare the CRCs between the groups. The analyses were performed by using GraphPad Prism software. Values of P < 0.05 were considered a statistically significant difference.

Drugs. L-Phenylephrine hydrochloride, ACh, sodium nitroprusside, L-NAME, indomethacin, sodium nitroprusside, catalase, and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) were all purchased from Sigma-Aldrich. 9,11-Dideoxy-9{alpha},11{alpha}-methanoepoxyprostaglandin F2{alpha} (U-46619) was purchased from Calbiochem (San Diego, CA). 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 ditrifluoroacetate (UCL-1684) was purchased from Tocris (Ellisville, MI). Indomethacin was dissolved in ethanol, and TRAM-34 and UCL-1684 were dissolved in DMSO. All other stock solutions were prepared by using PSS.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Increased blood pressure in ANG II-treated rats. Systolic blood pressure was elevated in ANG II (14 days)-treated rats compared with sham-operated rats. The systolic blood pressure after 14 days of treatment was 191 ± 6 mmHg in hypertensive rats compared with 120 ± 2 mmHg in normotensive rats (n = 8, P < 0.01). There were no significant changes of the systolic blood pressure in sham-operated rats during the 14-day treatment and in rats after 1-day infusion with ANG II (data not shown).

ACh-induced vasorelaxations. U-46619 (0.1 µmol/l) resulted in stable contractions in fourth-order MAs. The active force generated with this concentration of U-46619 was similar in isolated MAs of sham-operated and ANG II (14 days)-treated rats (15.6 ± 0.7 vs. 14.6 ± 1.3 mN, respectively). Under control conditions, ACh (0.001–10 µmol/l) caused concentration-dependent relaxations and resulted in almost complete relaxations in U-46619 (0.1 µmol/l)-contracted MAs of both groups of rats (Fig. 1). Sensitivity for ACh was comparable for MAs of sham-operated and ANG II (14 days)-treated rats (pEC50 value 7.02 ± 0.04 vs. 7.21 ± 0.06, respectively). The CRCs to ACh in the presence of L-NAME and indomethacin showed a similar rightward shift in MAs of both groups of rats (Fig. 1). Maximal relaxation to ACh (10 µmol/l) was comparable between MAs of both groups of rats (Fig. 1).


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 1. Concentration-response curves to ACh in U-46619-contracted (0.1 µmol/l) 4th-order mesenteric arteries of sham-operated normotensive (A) and ANG II 14-day (14-d)-treated rats (B) under control conditions (vehicle) and in the presence of N{omega}-nitro-L-arginine methyl ester (L-NAME) and indomethacin (L-NAME + Indo).

 
Involvement of H2O2 in non-NO, non-PGI2-mediated ACh-induced relaxations. The involvement of H2O2 in non-NO, non-PGI2-mediated ACh-induced relaxations was assessed by incubating MAs of sham-operated and ANG II (14 days)-treated rats with catalase (100 U/ml) in the combined presence of L-NAME and indomethacin. In both groups of rats, catalase did not result in any significant changes in the sensitivity and maximal relaxation to ACh, but they tended to be reduced in the MAs of ANG II (14 days)-treated rats compared with sham-operated rats [maximum effect (Emax), 79 ± 5% vs. 89 ± 2%, respectively; P = 0.08; Fig. 2].


Figure 2
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 2. Concentration-response curves to ACh in U-46619-contracted (0.1 µmol/l) 4th-order mesenteric arteries of sham-operated normotensive (A) and ANG II (14 day)-treated (B) rats in the presence of L-NAME and indomethacin (L-NAME + Indo) and in the presence of catalase (100 U/ml) and L-NAME + Indo.

 
Involvement of SKCa channels in vascular relaxation. UCL-1684 (0.1 µmol/l), the selective inhibitor of SKCa channels, did not alter the vasorelaxing response to the NO donor sodium nitroprusside (data not shown). Maximal tension to U-46619 (0.1 µmol/l) in the presence of UCL-1684 (0.1 µmol/l) did not differ between values from L-NAME and indomethacin-incubated MAs of both groups of rats (data not shown). CRCs to ACh in the presence of L-NAME, indomethacin, and UCL-1684 (0.1 µmol/l) were virtually abolished in MAs of sham-operated rats (Fig. 3A). However, a residual vasorelaxation was evident in MAs of ANG II (14 days)-treated rats (Fig. 3B). Maximal vasorelaxation to ACh (10 µmol/l) was significantly higher in MAs of ANG II (14 days)-treated rats compared with sham-operated rats (53 ± 11% vs. 0 ± 2%, respectively). CRCs to ACh were also assessed in MAs of rats that were treated with ANG II for 1 day to dissociate between ANG II-induced and hypertension-related effects on SKCa channel function. UCL-1684 (0.1 µmol/l) resulted in a greater blunted ACh-induced relaxation in ANG II (1 day)-treated rats compared with ANG II (14 days)-treated rats (Emax, 18 ± 16% vs. 53 ± 11%, respectively; Fig. 3, B and C).


Figure 3
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 3. Effect of small-conductance Ca2+-activated K+ channel (SKCa) inhibition on endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations. Concentration-response curves to ACh in 4th-order mesenteric arteries from sham-operated normotensive (A), ANG II (14 day)-treated (B), and ANG II (1 day)-treated rats incubated with L-NAME (100 µmol/l) and Indo (10 µmol/l) and contracted with U-46619 (0.1 µmol/l) in the absence (vehicle) and in the presence of UCL-1684 (n = 7 artery segments). Values are means ± SE. #P < 0.001.

 
Involvement of IKCa channels in vascular relaxation. TRAM-34 (10 µmol/l), the selective inhibitor of IKCa channels, did not result in any changes in the vasorelaxing response to sodium nitroprusside (data not shown). Maximal tension to U-46619 (0.1 µmol/l) in the combined presence of L-NAME, indomethacin, and TRAM-34 (10 µmol/l) were lower but did not differ significantly from values in L-NAME and indomethacin-treated MAs of sham-operated and ANG II (14 days)-treated rats (data not shown). CRCs to ACh were partially inhibited in the presence of TRAM-34 (10 µmol/l) compared with vehicle-treated segments (Fig. 4). Maximal responses to ACh were significantly blunted in L-NAME, indomethacin, and TRAM-34-incubated MAs of sham-operated and ANG II (14-days)-treated rats (Emax, 51 ± 16% vs. 44 ± 11%, respectively; Fig. 4, A and B) compared with L-NAME and indomethacin-treated vessels (Emax, 79 ± 6% vs. 87 ± 4%, respectively; Fig. 4, A and B). This blunted relaxation tended to be greater in MAs of ANG II (14 days)-treated rats ({Delta}43% vs. {Delta}28%). In 1-day ANG II-infused rats, maximal responses to ACh were similar for L-NAME, indomethacin, and TRAM-34-incubated (Emax, 72 ± 10%, Fig. 4C) and L-NAME and indomethacin-treated MAs (Emax, 82 ± 5%, Fig. 4C).


Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 4. Effect of intermediate-conductance KCa (IKCa) inhibition on EDHF-mediated relaxations. Concentration-response curves to ACh in 4th-order mesenteric arteries from sham-operated normotensive (A), ANG II (14 day)-treated (B), and ANG II (1 day)-treated rats incubated with L-NAME (100 µmol/l) and Indo (10 µmol/l) and contracted with U-46619 (0.1 µmol/l) in the absence (vehicle) and in the presence of 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; n = 8 artery segments). Values are means ± SE. #P < 0.001; **P < 0.01.

 
ACh-induced relaxations were completely abolished in the combined presence of L-NAME, indomethacin, UCL-1684, and TRAM-34 in MAs of sham-operated and ANG II (14 days)-treated rats (Fig. 5).


Figure 5
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 5. Effect of combined SKCa and IKCa inhibition on EDHF-mediated relaxations. Concentration-response curves to ACh in 4th-order mesenteric arteries from sham-operated normotensive (A) and ANG II (14 day)-treated (B) rats incubated with L-NAME (100 µmol/l) and Indo (10 µmol/l) and contracted with U-46619 (0.1 µmol/l) in the absence (vehicle) and in the presence of UCL-1684 and TRAM-34 (n = 7 artery segments).

 
Semiquantitative RT-PCR of SKCa and IKCa mRNA expression. Expression of SK3 and IK1, the pore-forming subunits of SKCa and IKCa channels, respectively, was quantified in total mRNA obtained from sham-operated and ANG II (14 days)-treated rats. Relative expression of mRNA levels of rat SK3 was significantly reduced in isolated small MAs of ANG II (14 days)-treated rats compared with sham-operated rats (Fig. 6A). As seen in Fig. 6B, relative mRNA expression levels of the rat SK4 or IK1 subunit were also significantly reduced in isolated small MAs of ANG II (14 days)-treated rats.


Figure 6
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 6. Semiquantitative RT-PCR analysis of SK3 (A) and SK4 (IK1; B) KCa channel subtypes from 4th-order mesenteric arteries from sham-operated (sham) and ANG II (14 days)-treated rats. Total mRNAs were extracted and pooled from 10 rats. Representative agarose gel image of amplicons derived from RT-PCR reactions with cDNA amplified with primers specific for SK3, SK4 (IK1), and GAPDH (see MATERIALS AND METHODS) is shown. Expected fragment sizes are 701 bp (SK3), 195 bp (IK1), and 198 bp (GAPDH). Quantified densitometric analysis of SK3 (A) and SK4 (IK1; B) fragments derived from sham-operated and ANG II (14 days)-treated rats are shown. Relative mRNA expression levels are normalized to GAPDH. rSK3, rat SK3; rIK1, rat IK1. Results are expressed as means ± SE from 3 separate experiments. *P < 0.05 vs. sham.

 
Immunoprecipitation of SKCa and IKCa proteins. Analysis of protein expression levels of endothelial SK3 and IK1 subunits was performed via immunoprecipitation. Protein expression levels of SK3 were drastically reduced (>10-fold) in MA cell lysates of ANG II (14 days)-treated rats compared with sham-operated rats (Fig. 7A). Protein expression levels of IK1 were lower in MAs of ANG II (14 days)-treated rats compared with sham-operated rats but did not reach a significant difference (Fig. 7B).


Figure 7
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 7. Protein expression levels of SK3 (A) and IK1 (B) subunits from 4th-order mesenteric arteries from sham-operated and ANG II (14 days)-treated rats. Total protein was extracted and pooled from 10 rats, and this pool was considered n = 1. Quantified densitometric analysis of SK3 (A) and IK1 (B) protein expression derived from sham-operated and ANG II (14 days)-treated rats. Relative protein expression levels are normalized to beta-actin. Results are expressed as means ± SE from 3 separate experiments. #P < 0.001 vs. sham.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We demonstrate reduced expression of SKCa and IKCa channel proteins, SK3 and IK1, respectively, despite normal ACh-induced endothelium-dependent vasorelaxations in small MAs of ANG II-induced hypertensive rats compared with normotensive rats.

The resting membrane potential of vascular smooth muscle cells are more depolarized, associated with enhanced contractile sensitivity, in arteries from hypertensive animals (6, 26). KCa channel blockade has been shown to prevent sustained pressure-induced depolarization in cannulated rat small MAs (44). The effect of high blood pressure on membrane potential may be more pronounced in smaller resistance-sized arteries that play a significant role in the regulation of blood pressure. We previously observed a greater mRNA expression of genes encoding for SKCa and IKCa channel subunits in smaller MAs compared with larger MAs in normotensive rats (27), stressing the importance of these KCa channels in small resistance-sized arteries. In small MAs, the EDHF response, classically regarded as the non-NO and non-prostacyclin-dependent relaxation, can be completely blocked by a combination of specific SKCa and IKCa channel blockers (13, 18, 28), which is in line with the earlier conclusion that large-conductance KCa (BKCa) channels do not contribute to the EDHF response in rat MAs. A study using transgenic mice (SK3T/T), in which the SK3 expression levels can be manipulated with dietary doxycycline, showed that endothelial SK3 channels exert a profound hyperpolarizing influence in resistance arteries and that suppression of SK3 channel expression caused a pronounced and reversible hypertension (45). Given the importance of SKCa and IKCa channels in EDHF-mediated responses and hence blood pressure regulation, we hypothesized that the functional and molecular expression of these KCa channels would be reduced in small MAs of ANG II-induced hypertensive rats compared with normotensive rats. We have chosen the experimental model of chronic ANG II infusion using an osmotic minipump implanted in the neck of rats, which leads to a considerable rise in systolic blood pressure measured after 14 days compared with sham-operated rats. This model is an experimental model of acquired hypertension and differs from the genetic models of hypertension, such as the spontaneously hypertensive rat (SHR). This model of hypertension enables us to study the effect of a sudden drastic rise in blood pressure at adult age on changes in the expression of SKCa and IKCa channels in small resistance-sized arteries. Because of the limited amount of small MA tissue, we pooled isolated small MA segments collected from 10 rats. With this method, we were able to generate sufficient amounts of total RNA and protein extract to perform RT-PCR and Western blot analysis.

We observed no apparent impairment in ACh-induced relaxations in small MAs of ANG II-induced hypertensive rats. Furthermore, the non-NO, nonprostacyclin (PGI2)-mediated endothelium-dependent relaxation induced by ACh, which can be attributed to the EDHF pathway, was similar in MAs of both normotensive and hypertensive rats. ANG II is a potent activator of NADPH oxidase in vascular smooth muscle cells. The formation of reactive oxygen species (ROS) generated by NADPH oxidase plays an essential role in endothelial dysfunction (23, 24). Superoxide anions can be reduced to the uncharged H2O2, where it can act as an EDHF depending on the species and artery (34, 41). To assess whether small MAs from ANG II (14 days)-treated rats had a greater contribution of non-NO, non-PGI2-mediated ACh-induced relaxation caused by H2O2, we incubated small MAs of sham-operated and ANG II-treated rats with catalase to dismutate H2O2. We did not observe any significant changes in the CRCs to ACh in the presence of catalase compared with L-NAME and indomethacin-incubated segments, suggesting no significant contribution of H2O2 and/or other ROS in the ACh-induced relaxation of resistance-sized arteries of ANG II-treated hypertensive rats.

Studies using the same vessels obtained from SHRs and stroke-prone SHRs have observed a reduced EDHF component (31, 44). The decrease in the EDHF-mediated response has been associated to a change in the expression of gap junctional proteins, such as connexins 37 and 40 (19, 38). In SHRs, the release of vasoconstrictor prostanoids at higher doses of ACh, and which seemed to progress with advancing age, resulted in endothelial dysfunction (46). The difference in results between our observations and results obtained in SHRs may be due to differences in the duration of the hypertension and/or compensatory mechanisms.

We next addressed the role of SKCa and IKCa in ACh-induced EDHF responses in these artery segments. UCL-1684 is one of the most potent nonpeptidic blockers of SKCa channels, with an IC50 value of 3 nmol/l (12), with little inhibitory action on IKCa channels (32). In small MAs of sham-operated rats, EDHF-mediated responses were completely blocked by UCL-1684, whereas a residual relaxation was evident in similar vessels of ANG II-induced hypertensive rats. The concentration of UCL-1684 (100 nmol/l) was well above the IC50 value of 3 nmol/l, suggesting a full blockade of SKCa channels. The smaller inhibition by UCL-1684 in MAs of ANG II-induced rats could not be attributed to differences in tension levels generated by U-46619. We and others have also observed a complete abolition of ACh-induced vasorelaxation in MAs incubated with the peptidic SKCa blocker apamin (27, 36). However, this complete blockade of the EDHF-mediated response during SKCa inhibition is in contrast with other studies using small arteries, where only the combined blockade of SKCa and IKCa channels abolished ACh-induced EDHF-mediated responses (7, 8, 13, 17, 18, 28, 35). However, in these studies, MAs of third order and lower were used. We recently observed regional heterogeneity in the contribution of SKCa channel function between large and small rat MAs (27). In the large (first-order branches of the superior MA) arteries, we observed that apamin did not abolish ACh-induced EDHF-mediated responses but almost completely blocked these responses in the smaller (fourth order) segments. The lower inhibition by UCL-1684 in SKCa-mediated vasorelaxation in small MAs of ANG II (14 days)-treated rats coincided with a downregulation of the SK3 subunit at the level of mRNA and protein.

Blockade of IKCa channels by TRAM-34 resulted in a lower reduction of the ACh-induced relaxation in MAs of ANG II (14 days)-induced hypertensive rats compared with sham-operated rats. Expression of mRNA encoding for the IK1 subunit was significantly reduced in small MAs of ANG II (14 days)-treated rats, whereas protein levels of IK1 tended to be lower in these segments compared with sham-operated rats.

ANG II has numerous actions on the vascular wall, including DNA and protein synthesis in vascular smooth muscle cells independently from pressure changes in both large and small arteries (25, 43), as well as modulation of vasomotor tone via increased ROS by activating NADPH oxidase (22). The latter has been associated with the pathology of hypertension. ROS have been proposed to be regulators of KCa channel function in various tissues (5, 9, 10). NO directly stimulated endothelial BKCa in renal arteries, whereas ROS, especially H2O2, inhibited these KCa channels (5). This ROS-induced inhibition of BKCa channels is unlikely in rat small MAs since EDHF responses are resistant to blockade with the BKCa channel antagonist iberiotoxin in the MA (27, 49). However, the inhibitory effect of ROS on SKCa and IKCa channels is not well known. To distinguish between ANG II-induced and pressure-related changes in KCa channel function, we included a third group of rats, which were treated with ANG II (60 ng·kg–1·min–1) via osmotic minipumps and were euthanized 24 h later. Since these ANG II-infused rats still remained normotensive (22) and since nonpressor doses of ANG II have been shown to increase protein synthesis after 24 h (15), we could examine the pressure-independent effects of ANG II on KCa channel function. We observed a greater inhibitory effect of SKCa channel inhibition on ACh-induced EDHF-mediated responses in MAs of 1-day ANG II-treated rats compared with 14-day ANG II-treated rats. This blunted relaxation was almost comparable with the sham-operated rats, suggesting that it is unlikely that elevated circulating ANG II concentrations cause inactivation of SKCa channels and/or downregulation of SK3 channel expression. Inhibition of IKCa channels by TRAM-34 resulted in comparable ACh-induced and EDHF-mediated responses in sham-operated and 1-day ANG II-treated rats. Further studies are needed to clarify whether elevated and prolonged exposures to ROS inactivate SKCa and IKCa channels and whether the downregulation of SK3 and IK1 channels is caused by a pressure-dependent mechanism.

The observations presented in this study in depolarized MAs suggest that each KCa type may have separate physiological roles, which become particularly clear when they are activated individually. Indeed, Crane and colleagues (13) suggested that true endothelium-dependent hyperpolarization of smooth muscle cells in response to ACh is attributable to SKCa channels, whereas IKCa channels play an important role during ACh-induced repolarization following depolarization. Furthermore, they suggested that this differential activation likely reflects in distinct subcellular localization within endothelial cells (13). Recently, high-resolution immunohistochemistry demonstrated colocalization of SKCa channels and connexins 37, 40, and 43 at adjacent endothelial cell gap junctions, whereas IKCa and connexin 37 were found at myoendothelial gap junctions (39). This spatial separation of KCa channels suggests a relationship between connexin function and the differential functional activity of SKCa and IKCa. The IKCa channel may allow for additional fine-tuning of the endothelial hyperpolarizing response in situations of reduced SK3 expression.

It is of interest to study the consequences of reduced expression of SK3 and IK1 in more physiological situations such as shear stress-induced vasodilatation. Hemodynamic stresses, such as circumferential wall stress and shear stress, which are altered during hypertension, may be a mechanism to alter KCa channel expression. Indeed, flow-induced arterial remodeling has been shown to activate signaling events leading to smooth muscle cell dedifferentiation in rat small MAs (47).

In summary, we have demonstrated maintained EDHF-like responses despite reduced expression of both SK3 and IK1 mRNA and protein in small MAs of ANG II-induced hypertensive rats. Endothelial KCa channels, in particular the SK3 channel expression, may be a novel target for therapeutic treatment or molecular manipulation for the treatment of many cardiovascular diseases such as diabetes, preeclampsia, and hypertension.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. H. P. Hilgers, Dept. of Pharmacology and Toxicology, Cardiovascular Research Institute Maastricht, Univ. of Maastricht, Universiteitssingel 50, 6200 MD Maastrict, The Netherlands (e-mail: rhilgers{at}farmaco.unimaas.nl)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Adams DJ, Barakeh J, Laskey R, Van Breemen C. Ion channels and regulation of intracellular calcium in vascular endothelial cells. FASEB J 3: 2389–2400, 1989.[Abstract]
  2. Blatz AL, Magleby KL. Single apamin-blocked Ca-activated K+ channels of small conductance in cultured rat skeletal muscle. Nature 323: 718–720, 1986.[CrossRef][Medline]
  3. Bolotina VM, Najibi S, Palacino JJ, Pagano PJ, Cohen RA. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 368: 850–853, 1994.[CrossRef][Medline]
  4. Bond CT, Maylie J, Adelman JP. Small-conductance calcium-activated potassium channels. Ann NY Acad Sci 868: 370–378, 1999.[Abstract/Free Full Text]
  5. Brakemeier S, Eichler I, Knorr A, Fassheber T, Köhler R, Hoyer J. Modulation of Ca2+-activated K+ channel in renal artery endothelium in situ by nitric oxide and reactive oxygen species. Kidney Int 64: 199–207, 2003.[CrossRef][ISI][Medline]
  6. Bratz IN, Dick GM, Partridge D, Kanagy NL. Reduced molecular expression of K+ channel proteins in vascular smooth muscle from rats made hypertensive with N{omega}-nitro-L-arginine. Am J Physiol Heart Circ Physiol 289: H1277–H1283, 2005.[Abstract/Free Full Text]
  7. Burnham MP, Bychkov R, Feletou M, Richards GR, Vanhoutte PM, Weston AH, Edwards G. Characterization of an apamin-sensitive small-conductance Ca2+-activated K+ channel in porcine coronary artery endothelium: relevance to EDHF. Br J Pharmacol 135: 1133–1143, 2002.[CrossRef][ISI][Medline]
  8. Bychkov R, Burnham MP, Richards GR, Edwards G, Weston AH, Feletou M, Vanhoutte PM. Characterization of a charybdotoxin-sensitive intermediate conductance Ca2+-activated K+ channel in porcine coronary endothelium: relevance to EDHF. Br J Pharmacol 138: 1346–1354, 2002.[CrossRef]
  9. Bychkov R, Pieper K, Ried C, Milosheva M, Bychkov E, Luft FC, Haller H. Hydrogen peroxide, potassium currents, and membrane potential in human endothelial cells. Circulation 99: 1719–1725, 1999.[Abstract/Free Full Text]
  10. Cai S, Sauve R. Effects of thiol-modifying agents on a KFormula channel of intermediate conductance in bovine aortic endothelial cells. J Membr Biol 158: 147–158, 1997.[CrossRef][ISI][Medline]
  11. Campbell WB, Gebremedhin D, Pratt PF, Harder DR. Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ Res 78: 415–423, 1996.[Abstract/Free Full Text]
  12. Campos Rosa J, Galanakis D, Piergentili A, Bhandari K, Ganellin CR, Dunn PM, Jenkinson DH. Synthesis, molecular modeling, and pharmacological testing of bis-quinolinium cyclophanes: potent, non-peptidic blockers of the apamin-sensitive Ca2+-activated K+ channel. J Med Chem 43: 420–431, 2000.[CrossRef][ISI][Medline]
  13. Crane GJ, Gallagher N, Dora KA, Garland CJ. Small- and intermediate-conductance calcium-activated K+ channels provide different facets of endothelium-dependent hyperpolarization in rat mesenteric artery. J Physiol 553: 183–189, 2003.[Abstract/Free Full Text]
  14. Crane GJ, Garland CJ. Thromboxane receptor stimulation associated with loss of SKCa activity and reduced EDHF responses in the rat isolated mesenteric artery. Br J Pharmacol 142: 43–50, 2004.[CrossRef][ISI][Medline]
  15. Daigle C, Martens FM, Girardot D, Dao HH, Touyz RM, Moreau P. Signaling of angiotensin II-induced vascular protein synthesis in conduit and resistance arteries in vivo. BMC Cardiovasc Disord 10: 4–6, 2004.[Medline]
  16. Ding H, Hashem M, Wiehler WB, Lau W, Martin J, Reid J, Triggle C. Endothelial dysfunction in the streptozotocin-induced diabetic apoE-deficient mouse. Br J Pharmacol 146: 1110–1118, 2005.[CrossRef][ISI][Medline]
  17. 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.[CrossRef][Medline]
  18. Eichler I, Wibawa J, Grgic I, Knorr A, Brakemeier S, Pries A, Hoyer J, Kohler R. Selective blockade of endothelial Ca2+-activated small- and intermediate-conductance K+-channels suppresses EDHF-mediated vasodilation. Br J Pharmacol 138: 594–601, 2003.[CrossRef][ISI][Medline]
  19. Figueroa XF, Isakson BE, Duling BR. Vascular gap junctions in hypertension. Hypertension 48: 804–811, 2006.[Free Full Text]
  20. Fujii K, Tominaga M, Ohmori S, Kobayashi K, Koga T, Takata Y, Fujishima M. Decreased endothelium-dependent hyperpolarization to acetylcholine in smooth muscle of the mesenteric artery of spontaneously hypertensive rats. Circ Res 70: 660–669, 1992.[Abstract/Free Full Text]
  21. Fukao M, Hattori Y, Kanno M, Sakuma I, Kitabatake A. Alterations in endothelium-dependent hyperpolarization and relaxation in mesenteric arteries from streptozotocin-induced diabetic rats. Br J Pharmacol 121: 1383–1391, 1997.[CrossRef][ISI][Medline]
  22. Fukui T, Ishizaka N, Rajagopalan S, Laursen JB, Capers Q 4th, Taylor WR, Harrison DG, de Leon H, Wilcox JN, Griendling KK. P22phox mRNA expression and NADPH oxidase activity are increased in aortas from hypertensive rats. Circ Res 80: 45–51, 1997.[Abstract/Free Full Text]
  23. Griendling KK, FitzGerald GA. Oxidative stress and cardiovascular injury. Part I. Basic mechanisms and in vivo monitoring of ROS. Circulation 108: 1912–1916, 2003.[Free Full Text]
  24. Griendling KK, FitzGerald GA. Oxidative stress and cardiovascular injury. Part II. Animal and human studies. Circulation 108: 2034–2040, 2003.[Free Full Text]
  25. Griffin SA, Brown WCB, Macpherson F, McGrawth JC, Wilson VG, Korsgaard N, Mulvany MJ, Lever AF. Angiotensin II causes vascular hypertrophy in part by a non-pressor mechanism. Hypertension 17: 626–635, 1991.[Abstract/Free Full Text]
  26. Harder DR, Smeda J, Lombard J. Enhanced myogenic depolarization in hypertensive cerebral arterial muscle. Circ Res 57: 319–322, 1985.[Abstract/Free Full Text]
  27. Hilgers RH, Todd J Jr, Webb RC. Regional heterogeneity in acetylcholine-induced relaxation in rat vascular bed: role of calcium-activated K+ channels. Am J Physiol Heart Circ Physiol 291: H216–H222, 2006.[Abstract/Free Full Text]
  28. Hinton JM, Langton PD. Inhibition of EDHF by two new combinations of K+-channel inhibitors in rat isolated mesenteric arteries. Br J Pharmacol 138: 1031–1035, 2003.[CrossRef][ISI][Medline]
  29. Jaggar JH, Porter VA, Lederer WJ, Nelson MT. Calcium sparks in smooth muscle. Am J Physiol Cell Physiol 278: C235–C256, 2000.[Abstract/Free Full Text]
  30. Laursen JB, Rajagopalan S, Galis T, Tarpey M, Freeman BA, Harrison DG. Role of superoxide in angiotensin II-induced but not catecholamine-induced hypertension. Circulation 95: 588–595, 1997.[Abstract/Free Full Text]
  31. Lüscher TF, Vanhoutte PM. Endothelium-dependent contractions to acetylcholine in the aorta of the spontaneously hypertensive rat. Hypertension 8: 344–348, 1986.[Abstract/Free Full Text]
  32. Malik-Hall M, Ganellin CR, Galanakis D, Jenkinson DH. Compounds that block both intermediate-conductance (IKCa) and small-conductance (SKCa) calcium-activated potassium channels. Br J Pharmacol 129: 1431–1438, 2000.[CrossRef][ISI][Medline]
  33. Mather S, Dora KA, Sandow SL, Winter P, Garland CJ. Rapid endothelial cell-selective loading of connexin 40 antibody blocks endothelium-derived hyperpolarizing factor dilation in rat small mesenteric arteries. Circ Res 97: 399–407, 2005.[Abstract/Free Full Text]
  34. Matoba T, Shimokawa H, Nakashima M, Hirakawa Y, Mukai Y, Hirano K, Kanaida H, Takeshita A. Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice. J Clin Invest 106: 1521–1530, 2000.[ISI][Medline]
  35. McNeish AJ,Sandow SL, Neylon CB, Chen MX, Dora KA, Garland CJ. Evidence for involvement of both IKCa and SKCa channels in hyperpolarizing responses of the rat middle cerebral artery. Stroke 37: 1277–1282, 2006.[Abstract/Free Full Text]
  36. Murphy ME, Brayden JE. Apamin-sensitive K+ channels mediate an endothelium-dependent hyperpolarization in rabbit mesenteric arteries. J Physiol 489: 723–734, 1995.[Abstract/Free Full Text]
  37. Nilius B, Droogmans G. Ion channels and their functional role in vascular endothelium. Physiol Rev 81: 1415–1459, 2001.[Abstract/Free Full Text]
  38. Rummery NM, Hill CE. Vascular gap junctions and implications for hypertension. Clin Exp Pharmacol Physiol 31: 659–667, 2004.[CrossRef][ISI][Medline]
  39. Sandow SL, Neylon CB, Chen MX, Garland CJ. Spatial separation of endothelial small- and intermediate-conductance calcium-activated potassium channels (KCa) and connexins: possible relationship to vasodilator function? J Anat 209: 689–698, 2006.[CrossRef][ISI][Medline]
  40. Sandow SL, Tare M, Coleman HA, Hill CE, Parkington HC. Involvement of myoendothelial gap junctions in the actions of endothelium-derived hyperpolarizing factor. Circ Res 90: 1108–1113, 2002.[Abstract/Free Full Text]
  41. Shimokawa H, Matoba T. Hydrogen peroxide as an endothelium-derived hyperpolarizing factor. Pharmacol Res 49: 543–549, 2004.[CrossRef][ISI][Medline]
  42. Shimokawa H, Yasutake H, Fujii K, Owada M, Nakaike R, Fukumoto Y, Takayanagi T, Nagao T, Egashira K, Fujishima M, Takeshita A. The importance of the hyperpolarizing mechanism increases as the vessel size decreases in endothelium-dependent relaxations in rat mesenteric circulation. J Cardiovasc Pharmacol 28: 703–711, 1996.[CrossRef][ISI][Medline]
  43. Su EJ, Lombardi DM, Siegal J, Schwartz SM. Angiotensin II induces vascular smooth muscle cell replication independent of blood pressure. Hypertension 31: 1331–1337, 1998.[Abstract/Free Full Text]
  44. Sunano S, Watanabe H, Tanaka S, Sekiguchi F, Shimamura K. Endothelium-derived relaxing, contracting and hyperpolarizing factors of mesenteric arteries of hypertensive and normotensive rats. Br J Pharmacol 126: 709–716, 1996.[CrossRef]
  45. Taylor MS, Bonev AD, Gross TP, Eckman DM, Brayden JE, Bond CT, Adelman JP, Nelson MT. Altered expression of small-conductance Ca2+ activated K+ (SK3) channels modulates arterial tone and blood pressure. Circ Res 93: 124–131, 2003.[Abstract/Free Full Text]
  46. Watt PAC, Thurston H. Endothelium-dependent relaxation in resistance vessels from the spontaneously hypertensive rats. J Hypertens 7: 661–666, 1989.[CrossRef][ISI][Medline]
  47. Wesselman JP, Kuijs R, Hermans JJ, Janssen GM, Fazzi GE, van Essen H, Evelo CT, Struijker-Boudier HA, De Mey JG. Role of the Rhoa/Rho kinase system in flow-related remodeling of rat mesenteric small arteries in vivo. J Vasc Res 41: 277–290, 2004.[CrossRef][ISI][Medline]
  48. Wesselman JPM, Schubert R, Vanbavel E, Nilsson H, Mulvany MJ. KCa-channel blockade prevents sustained pressure-induced depolarization in rat mesenteric small arteries. Am J Physiol Heart Circ Physiol 272: H2241–H2249, 1997.[Abstract/Free Full Text]
  49. Zygmunt PM, Högestatt ED. Role of potassium channels in endothelium-dependent relaxation resistant to nitroarginine in the rat hepatic artery. Br J Pharmacol 117: 1600–1606, 1996.[ISI][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
A. Makino, O. Platoshyn, J. Suarez, J. X.-J. Yuan, and W. H. Dillmann
Downregulation of connexin40 is associated with coronary endothelial cell dysfunction in streptozotocin-induced diabetic mice
Am J Physiol Cell Physiol, July 1, 2008; 295(1): C221 - C230.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/5/H2275    most recent
00949.2006v2
00949.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hilgers, R. H. P.
Right arrow Articles by Webb, R. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hilgers, R. H. P.
Right arrow Articles by Webb, R. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.