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Am J Physiol Heart Circ Physiol 291: H216-H222, 2006. First published February 10, 2006; doi:10.1152/ajpheart.01383.2005
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Regional heterogeneity in acetylcholine-induced relaxation in rat vascular bed: role of calcium-activated K+ channels

Rob H. P. Hilgers, Joseph Todd, Jr., and R. Clinton Webb

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

Submitted 30 December 2005 ; accepted in final form 8 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ca+-activated K+-channels (KCa) regulate vasomotor tone via smooth muscle hyperpolarization and relaxation. The relative contribution of the endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation differs depending on vessel type and size. It is unknown whether these KCa channels are differentially distributed along the same vascular bed and hence have different roles in mediating the EDHF response. We therefore assessed the role of small- (SKCa), intermediate- (IKCa), and large-conductance (BKCa) channels in mediating acetylcholine-induced relaxations in both first- and fourth-order side branches of the rat superior mesenteric artery (MA1 and MA4, respectively). Two-millimeter segments of each MA were mounted in the wire myograph, incubated with N{omega}-nitro-L-arginine methyl ester (L-NAME, 100 µmol/l) and indomethacin (10 µmol/l), and precontracted with phenylephrine (10 µmol/l). Cumulative concentration-response curves to ACh (0.001–10 µmol/l) were performed in the absence or presence of selective KCa channel antagonists. Apamin almost completely abolished these relaxations in MA4 but only partially blocked relaxations in MA1. The selective IKCa channel blocker 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) caused a significantly greater inhibition of the ACh-induced relaxation in MA4 compared with MA1. Iberiotoxin had no inhibitory effect in MA4 but blunted relaxation in MA1. Relative mRNA expression levels of SKCa (rSK1, rSK3, and rSK4 = rIK1) were significantly higher in MA4 compared with MA1. BKCa (rBK{alpha}1 and rBKbeta1) genes were similar in both MA1 and MA4. Our data demonstrate regional heterogeneity in SKCa and IKCa function and gene expression and stress the importance of these channels in smaller resistance-sized arteries, where the role of EDHF is more pronounced.

THE ENDOTHELIUM plays a crucial role in the control of vascular tone via the release of nitric oxide (NO), prostacyclin, and the endothelium-derived hyperpolarizing factor (EDHF) (19). Ca2+-activated K+-channels (KCa) are key players in the control of this endothelium-mediated vasorelaxation through regulating endothelial membrane potential and Ca2+ homeostasis in response to hemodynamic stresses and vasoactive factors (1, 18). In large part, the small- and intermediate-conductance KCa channels (SKCa and IKCa, respectively) are generally believed to mediate endothelial hyperpolarization and smooth muscle hyperpolarization and relaxation in response to humoral stimulation (8, 10). Suppression of SKCa channel expression has been shown to result in elevated blood pressure (23). These observations indicate that endothelial KCa channels exert a profound effect on hyperpolarization in resistance arteries and suggest that the level of KCa channel expression is a fundamental determinant of vascular tone and blood pressure in both health and disease (11, 22).

Resistance-sized arteries, roughly having an internal diameter of <300 µm, are important regulators of vasomotor tone and hence blood pressure. Because the relative contribution of EDHF is dependent on the vessel type and size (21), being more important in smaller arteries and arterioles, we investigated whether there is a regional heterogeneity in the contribution of each KCa channel subtype in the mesenteric arterial bed by studying both small and large rat mesenteric arteries (fourth- and first-order branches of the superior mesenteric artery, respectively). To assess the functional importance of these KCa channels in EDHF-mediated vasorelaxations, we performed cumulative concentration-response curves for acetylcholine in phenylephrine-contracted mesenteric arteries by using the selective SKCa channel blocker apamin, the selective IKCa channel blocker 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34), and the selective BKCa channel blocker iberiotoxin. Expression levels of mRNA of the various KCa channels were measured via semiquantitative RT-PCR to study potential regional differences in mRNA expression of these KCa channels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Measurement of isometric tension. Male Sprague-Dawley rats (275–300 g) were anesthetized with pentobarbital sodium (50 mg/kg ip). The mesentery was rapidly excised and placed in cold physiological saline solution (PSS) of the following composition (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. First- and fourth-order segments (2 mm) of the superior mesenteric artery were isolated and mounted in the wire myograph (Danish MyoTech) filled with 5 ml PSS and continuously gassed with 95% O2-5% CO2 while temperature was maintained at 37°C. The arterial segments were stretched at a tension equivalent to that generated at 0.9 times the diameter of the vessel at 100 mmHg, this was 2.5 mN for fourth-order and 7.5 mN for first-order mesenteric arteries. The segments were allowed to incubate for 45 min. Arterial integrity was assessed by contracting arterial rings with phenylephrine (PE, 10 µmol/l) followed by relaxation with ACh (1 µmol/l). After washing was completed, the arterial rings were precontracted with PE (10 µmol/l). Endothelium-dependent relaxation to ACh under control conditions was examined by constructing cumulative dose-response curves by adding increasing concentrations of ACh (0.01–10 µmol/l) to the organ chamber. Cumulative concentration-response curves to PE (0.01–30 µmol/l) were constructed in the absence and presence of each KCa inhibitor. Contraction was expressed as percentage of the contraction induced by a depolarized high K+ (120 mmol/l) solution. To study the role of KCa channels in ACh-induced relaxations, we always determined EDHF-mediated relaxations in the combined presence of NO and prostacyclin inhibition to rule out any potential interferences of these two mediators with EDHF (3). Therefore, we incubated arterial segments with indomethacin (inhibitor of cyclooxygenase, 10 µmol/l) and N{omega}-nitro-L-arginine methyl ester (L-NAME, inhibitor of NO synthase, 100 µmol/l) for a period of 30 min. One single cumulative concentration-response curve for ACh was performed for each arterial segment incubated with L-NAME, indomethacin, and KCa channel inhibitor(s). Endothelium-independent relaxing responses to the NO donor sodium nitroprusside (0.001–10 µmol/l) were performed in PE (10 µmol/l)-contracted segments in the absence and presence of each KCa inhibitor.

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.

Semiquantitative RT-PCR amplification of KCa channel genes. First-order and fourth-order (and smaller branches along the gut wall) mesenteric arteries were carefully freed of adipose and connective tissue and quickly snap-frozen in liquid nitrogen. To generate sufficient amounts of total RNA, mesenteric artery segments (of either first- or fourth-order and higher) were pooled from 10 rats and subsequently homogenized. Total RNAs were isolated with TRIzol reagent (Invitrogen). The amount of RNA was determined spectrophotometrically at A260. 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). For analysis of expression of KCa channel subtypes in the rat mesenteric arterial bed, we designed specific primers for four rat SKCa subtypes [rSK1 to rSK4, the latter subtype encoded by the Kccn4 gene is actually the same as the rat IKCa subtype encoded by the rIK1 gene, see Genbank accession numbers AF190458 (SMIK) and NM023021 (rIK1)], two BKCa subtypes (BK{alpha}1 and BKbeta1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer sequences are shown in Table 1. The general protocol for PCR amplification was 35 cycles at 94°C for 1 min, at 56–65°C for 1 min, and 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 grey 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 gene of interest per GAPDH.


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  		Table 1. Specific primers used for semiquantitative RT-PCR

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

Drugs. The following drugs were used: L-phenylephrine hydrochloride, ACh, sodium nitroprusside, L-NAME, indomethacin, apamin, iberiotoxin, and TRAM-34 (all purchased from Sigma, St. Louis, MO). Indomethacin was dissolved in ethanol, and TRAM-34 was dissolved in DMSO, both in a stock solution of 10 mmol/l. All other stock solutions were prepared by using PSS.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Semiquantitative RT-PCR of SK, IK, and BK channels in the mesenteric arterial bed. Four members of the mammalian SKCa channel family have been identified, namely rSK1 to rSK4, the latter is also known as rIK1. To analyze which subtypes are expressed in the rat mesenteric arterial bed and whether these subtypes are differentially expressed along this vascular tree, total RNA isolated from first-order and fourth-order (and higher) branches (MA1 and MA4, respectively) from the superior mesenteric artery were pooled from 10 rats. Semiquantitative RT-PCR was performed using specific sets of primers (see Table 1). Only two SKCa channel subtypes, i.e., rSK1 and rSK3, seem to be expressed in the mesenteric arterial bed (Fig. 1A). KCa subtype rSK2 was not detected in rat mesenteric arteries. Two different sets of primers for the rSK2 gene were used, but none showed any detectable bands. The genes encoding for the SKCa subtypes (rSK1 and rSK3) are severalfold higher expressed in MA4 compared with MA1 (Fig. 1). The gene encoding for the IKCa subtype (rIK1) is 1.6-fold higher expressed in MA4 compared with MA1 (Fig. 1). BKCa channels consist of a core-forming {alpha}1-subunit (KCNMA1 gene) and an auxiliary beta-subunit (KCNMB1 gene). Both {alpha}- and beta-subunits are expressed in rat mesenteric arteries and are equally expressed in both MA1 and MA4 as depicted in Fig. 1.


Figure 1
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  		Fig. 1. Semiquantitative RT-PCR analysis of small- (SKCa), intermediate- (IKCa), and large-conductance (BKCa) channel subtypes in first-order and fourth-order rat mesenteric arteries. Total mRNAs were extracted and pooled from 10 rats. A: representative agarose gel image of amplicons derived from RT-PCR with first-order (MA1) and fourth-order (MA4) mesenteric artery cDNA amplified with primers specific for SK1, SK2, SK3, SK4 (=rIK1), BK{alpha}1, and BKbeta1 (see Table 1). Expected fragment sizes are 253 bp (rSK1), 391 bp (rSK2), 602 bp (rSK3), 198 bp (rSK4 = rIK1), 251 bp (BK{alpha}1), and 320 bp (BKbeta1). B: quantified densitometric analysis of fragments derived from MA1 (open bars) and MA4 (solid bars). Relative mRNA expression levels are normalized to GAPDH. Results are shown as means ± SE (n = 4–6). P values are shown compared with the normalized expression level of MA1.

 
ACh-induced relaxation in first- and fourth-order rat mesenteric arteries in control conditions. Basal tensions under control conditions averaged 7.2 ± 0.1 mN in MA1 and 2.5 ± 0.1 mN in MA4. PE (10 µmol/l) provoked a stable contraction in all arteries. In MA1 tension after contraction with PE (10 µM) averaged 31.3 ± 1.0 mN compared with 18.5 ± 0.6 mN in MA4. ACh (0.001–10 µmol/l) caused concentration-dependent relaxations and resulted in almost complete relaxations in MA1 and MA4 (93 ± 3% and 86 ± 3%, respectively, Fig. 2). MA1 were more sensitive to ACh compared with MA4 (pD2 value 7.08 ± 0.03 vs. 6.61 ± 0.05, respectively). The combined treatment of the cyclo-oxygenase inhibitor indomethacin (10 µmol/l) and NO synthase inhibitor L-NAME (100 µmol/l) did not result in any significant changes in tension during contraction with PE (10 µmol/l) MA4 and MA1. The concentration-response curves to ACh in the presence of L-NAME and indomethacin showed a similar rightward shift in MA1 as well as MA4 (Fig. 2). Maximal relaxation to 10 µmol/l ACh was comparable between MA4 and MA1 (Emax from 86 ± 3% to 76 ± 4% vs. 93 ± 3% to 75 ± 7%, respectively, Fig. 2). However, the difference in maximal relaxation during treatment with indomethacin and L-NAME tended to be higher in MA1 compared with MA4 (18% vs. 10%, respectively).


Figure 2
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  		Fig. 2. Concentration-responses curves (CRC) to ACh in phenylephrine-contracted (PE; 10 µmol/l) first-order rat mesenteric resistance arteries (A) and fourth-order mesenteric arteries (B). CRC under control situations are shown in open circles, whereas those constructed in the presence with N{omega}-nitro-L-arginine methyl ester (L-NAME) and indomethacin (INDO) are shown in closed circles. Values are shown as means ± SE; n = 10–12 arterial segments. *P < 0.05.

 
Involvement of SKCa channels in vascular relaxation. Apamin (0.5 µmol/l), the selective inhibitor of SKCa channels, did not result in any changes in the contractile response to PE and the vasorelaxing response to sodium nitroprusside (Table 2). However, tension values during contraction with PE (10 µmol/l) were lower in both MA1 (from 29.5 ± 1.7 to 25.1 ± 1.4 mN) and MA4 (from 16.1 ± 0.8 to 15.1 ± 1.0 mN). Maximal relaxation to ACh was significantly attenuated in the presence of apamin in MA1 (Emax 30 ± 7%; P < 0.001, Fig. 3A). In MA4, the relaxation was completely abolished in the presence of apamin (Emax 3 ± 3%; P < 0.001, Fig. 3B).


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  		Table 2. Contractile responses to PE and relaxing responses to SNP in MA4 and MA1

 

Figure 3
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  		Fig. 3. Effect of SKCa inhibition on endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations. A: CRC to ACh in first-order mesenteric arteries contracted with PE (10 µmol/l) in the combined presence of L-NAME (100 µmol/l) and INDO (10 µmol/l) and in the additional presence of apamin (0.5 µmol/l). B: CRC to ACh in fourth-order mesenteric arteries contracted with PE in the combined presence of L-NAME and INDO and in the additional presence of apamin. Values are shown as means ± SE; n = 11 arterial segments. #P < 0.001.

 
Involvement of IKCa channels in vascular relaxation. TRAM-34 (10 µmol/l), a potent and selective inhibitor of IKCa channels, lowered PE-induced (10 µmol/l) tensions in both arteries (from 29.5 ± 1.7 to 24.6 ± 1.1 mN in MA1 and from 16.1 ± 0.8 to 14.1 ± 0.8 mN in MA4), despite similar maximal contraction and sensitivity to PE compared with untreated segments (Table 2). Furthermore, TRAM-34 did not affect endothelium-independent vasorelaxations to sodium nitroprusside (Table 2). TRAM-34 did not result in any significant changes in ACh-induced relaxations of MA1 (Emax 47 ± 11, Fig. 4A). However, TRAM-34 significantly reduced the maximal relaxation to ACh in MA4 (Emax 29 ± 11%; P < 0.001, Fig. 4B).


Figure 4
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  		Fig. 4. Effect of IKCa inhibition on EDHF-mediated relaxations. A: CRC to ACh in first-order mesenteric arteries contracted with PE (10 µmol/l) in the combined presence of L-NAME (100 µmol/l) and INDO (10 µmol/l) and in the additional presence of 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34, 10 µmol/l). B: CRC to ACh in fourth-order mesenteric arteries contracted with PE in the combined presence of L-NAME and INDO and in the additional presence of TRAM-34. Values are shown as means ± SE; n = 14 arterial segments. #P < 0.001.

 
Involvement of BKCa channels in vascular relaxation. Iberiotoxin (0.1 µmol/l), the selective BKCa channel blocker, caused a greater sensitivity to PE in MA1, but not MA4, compared with untreated MA1 segments (Table 2). Plateau values of the tension induced by PE (10 µmol/l) in MA1 (31.5 ± 2.4 mN) and MA4 (17.7 ± 0.9 mN) were comparable. Endothelium-independent relaxation to sodium nitroprusside was unaffected at the concentration of iberiotoxin used (Table 2). In MA1, endothelium-dependent ACh-induced relaxation was significantly blunted in the presence of iberiotoxin (Emax 33 ± 16%; P < 0.001, Fig. 5A). However, iberiotoxin had no effect on ACh-induced relaxations in MA4 (Fig. 5B).


Figure 5
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  		Fig. 5. Effect of BKCa inhibition on EDHF-mediated relaxations. A: CRC to ACh in first-order mesenteric arteries contracted with PE (10 µmol/l) in combined presence of L-NAME (100 µmol/l) and INDO (10 µmol/l) and in the additional presence of iberiotoxin (IBTX, 0.1 µmol/l). B: CRC to ACh in fourth-order mesenteric arteries precontracted with PE in presence of L-NAME (100 µM) and INDO (10 µM) and in additional presence of IBTX (0.1 µM). Values are shown as means ± SE; n = 11 arterial segments. *P < 0.05.

 
The combined treatment of TRAM-34 and apamin completely abolished ACh-induced relaxations in MA1 and MA4 (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We investigated the relative contribution of KCa on ACh-induced vasorelaxation in regionally different arterial segments of the rat mesenteric arterial bed. The main findings of this study were the following: 1) pharmacological inhibition of SKCa and IKCa by apamin and TRAM-34, respectively, caused a greater reduction in ACh-induced relaxation in small fourth-order (ID 150–200 µm) compared with larger first-order (ID >400 µm) mesenteric arteries, 2) mRNA expression levels of SKCa channels (rSK1 and rSK3) and IKCa channels (rIK1) are higher in fourth-order compared with first-order mesenteric arteries, and 3) BKCa blockade by iberiotoxin only affected larger first-order mesenteric arteries, despite similar BKCa channel subtype expression compared with fourth-order mesenteric arteries.

Endothelial cells cause vasorelaxation in response to several agonists and hemodynamic stimuli through the release of NO, prostacyclin, and EDHF (20). EDHF causes endothelial hyperpolarization with subsequent vascular smooth muscle cell hyperpolarization, which is blocked by depolarization of smooth muscle cells by high extracellular K+ or inhibitors of endothelial KCa channels (15, 17). The relative contribution of EDHF varies among species, vascular bed, and vessel size (7, 12, 14, 21). Particularly in arterioles and small arteries, EDHF appears to be of major importance, whereas in larger arteries the role of NO is more pronounced. An inverse correlation exists between the degree of vasomotor tone and the release of EDHF (16). The experiments in the present study were therefore designed to investigate the role of KCa channels in both small and large mesenteric arteries (fourth- and first-order branches of the superior mesenteric artery, respectively) via both quantitative and qualitative measurements.

EDHF-mediated relaxation is particularly apparent when NO and prostaglandin production is blocked. Therefore, the effect of KCa channel blockade, and hence EDHF action, was always studied on arteries treated with both L-NAME and indomethacin. We found that both large and small mesenteric arteries had a comparable residual non-NO and non-PGI2-dependent, ACh-induced vasorelaxation. However, the difference in maximal vasorelaxation between arteries under control conditions and arteries treated with both L-NAME and indomethacin tended to be higher in larger mesenteric arteries, suggesting that the contribution of the vasoactive factors inhibited by L-NAME and indomethacin to vasorelaxation were higher in these larger mesenteric arteries. A hallmark of the EDHF-mediated response is its abolition by the combination of apamin and the nonselective IKCa, BKCa, and some voltage-dependent K+-channel blocker charybdotoxin (8, 15). In this study, the combined inhibition of apamin and the more selective inhibitor TRAM-34 completely abolished ACh-induced relaxation in all arterial segments, providing more convincing evidence that SKCa and IKCa channels are responsible for EDHF responses in the mesenteric vascular bed (8, 10).

We were interested in the contribution of each KCa channel subtype alone on ACh-induced and EDHF-mediated vasorelaxations in the rat mesenteric vascular bed, and whether there existed a regional heterogeneity in both KCa channel expression and function. We therefore used selective pharmacological inhibitors of each KCa channel subtype. To assess whether the concentrations of each KCa channel blocker used did not have any nonselective effects on relaxing responses that might explain any differences observed in ACh-induced relaxations, we performed concentration-response curves to the endothelium-independent vasodilator sodium nitroprusside. Each KCa channel inhibitor resulted in a similar maximal relaxation and sensitivity to the NO donor compared with relaxations in the absence of any inhibitor, indicating that the inhibitors did not change the relaxation response at the concentration used.

Apamin, the selective inhibitor of endothelial SKCa channels, almost completely blocked the ACh-induced relaxation in fourth-order mesenteric arteries but only partially in first-order mesenteric arteries. Murphy and Brayden (17) also found that apamin completely abolished ACh-mediated relaxations in rat mesenteric arteries, indicating that SKCa are involved in EDHF-mediated relaxations. The site of action of apamin has been demonstrated to be on the endothelium and not the smooth muscle (9), indicating that the blunted ACh-induced vasorelaxation in the presence of apamin is due to blockade of endothelial SKCa channels, hereby preventing the hyperpolarization. Semiquantitative reverse transcriptase PCR analysis showed only faint bands of rSK1 and rSK3 but not rSK2 in rat mesenteric arteries. Expression of rSK1 and rSK3 mRNA was severalfold higher in fourth-order compared with first-order mesenteric arteries, indicating that apamin-sensitive SKCa channels may be more expressed in smaller resistance-sized mesenteric arteries.

To study the effect of endothelial IKCa channels on EDHF-mediated vasorelaxation, we used the selective blocker TRAM-34. This compound has been shown to have no blocking effects on the cloned BKCa channel, several types of cloned voltage-gated K+- and inward-rectifying K+ channels (25). In the fourth-order mesenteric artery, EDHF-mediated relaxations were significantly reduced, in contrast, to first-order mesenteric arteries where TRAM-34 had no significant effect. This observation was supported by the fact that mRNA expression of the gene encoding for the IKCa channel (rIK1 or rSK4) was 1.6-fold higher in fourth-order compared with first-order mesenteric arteries. Although we did not perform any patch-clamp experiments to measure hyperpolarization more directly, we believe that both SKCa and IKCa channels are involved in EDHF-mediated vasorelaxations in response to ACh, and that their role is more pronounced in smaller resistance-sized mesenteric arteries. A differentially observed expression of endothelial SKCa and IKCa channel subtypes could be explained by a different intima-to-media ratio along the mesenteric vascular bed. We believe that this should not necessarily be the case because a greater lumen diameter, as is true for first-order mesenteric arteries compared with fourth-order vessels, would mean a higher surface area of endothelial cells. Therefore, the ratio of vascular smooth muscle to endothelial cells might be constant in both first- and fourth-order mesenteric arteries. To our knowledge this is the first study demonstrating differential expression of SKCa and IKCa channels in the rat mesenteric vascular bed.

Unlike apamin and TRAM-34, the selective BKCa channel blocker iberiotoxin caused no inhibition of ACh-mediated relaxation in fourth-order mesenteric arteries, suggesting that BKCa are not involved in the EDHF response in small mesenteric arteries. This is in agreement with previous studies showing that EDHF responses are resistant to blockade with iberiotoxin in small rat mesenteric arteries (8, 13). Because epoxyeicosatrienoic acids have been shown to mediate EDHF-like responses via BKCa channels in coronary arteries (5), it rules out any involvement of epoxyeicosatrienoic acids as a putative EDHF candidate in this vessel type (4, 6). However, we found that in first-order mesenteric arteries, ACh-induced relaxations could be blocked by iberiotoxin. Although both BKCa subunits are expressed in small and large mesenteric arteries with comparable mRNA expression levels, we currently have no explanation why iberiotoxin causes a greater inhibition in ACh-induced relaxation in larger mesenteric arteries.

Our results demonstrate regional heterogeneity in EDHF-mediated responses via KCa channel activation. Small resistance-sized mesenteric arteries show enhanced reactivity toward SKCa and IKCa channel antagonism compared with larger mesenteric arteries. Furthermore, we have demonstrated that small mesenteric arteries express more mRNA encoding for SKCa and IKCa gene subtypes, emphasizing the greater EDHF-mediated relaxation via these KCa channels in smaller arteries. They further stress the importance of SKCa and IKCa channels in regulating vascular tone and blood pressure, particularly in resistance-sized arteries.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. H. P. Hilgers, Dept. of Physiology, Medical College of Georgia, Augusta, GA 30912 (e-mail: rhilgers{at}mail.mcg.edu)

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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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R. H. P. Hilgers and R. C. Webb
Reduced expression of SKCa and IKCa channel proteins in rat small mesenteric arteries during angiotensin II-induced hypertension
Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2275 - H2284.
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