Heteromultimeric Kv1 Channels Contribute to Myogenic Control of Arterial Diameter
http://www.100md.com
循环研究杂志 2005年第2期
the Smooth Muscle Research Group and CIHR Group in Regulation of Vascular Contractility (F.P., R.J., P.K., W.W., K.T., T.C., W.C.), Faculty of Medicine, University of Calgary, Canada
Division of Cardiovascular Pharmacology (K.I.), Yamagata University School of Medicine, Japan.
Abstract
Inhibition of vascular smooth muscle (VSM) delayed rectifier K+ channels (KDR) by 4-aminopyridine (4-AP; 200 eol/L) or correolide (1 eol/L), a selective inhibitor of Kv1 channels, enhanced myogenic contraction of rat mesenteric arteries (RMAs) in response to increases in intraluminal pressure. The molecular identity of KDR of RMA myocytes was characterized using RT-PCR, real-time PCR, and immunocytochemistry. Transcripts encoding the pore-forming Kv subunits, Kv1.2, Kv1.4, Kv1.5, and Kv1.6, were identified and confirmed at the protein level with subunit-specific antibodies. Kv transcript (1.1, 1.2, 1.3, and 2.1) expression was also identified. Kv1.5 message was 2-fold more abundant than that for Kv1.2 and Kv1.6. Transcripts encoding these three Kv1 subunits were 2-fold more abundant in 1st/2nd order conduit compared with 4th order resistance RMAs, and Kv1 was 8-fold higher than Kv2 message. RMA KDR activated positive to eC50 mV, exhibited incomplete inactivation, and were inhibited by 4-AP and correolide. However, neither -dendrotoxin or -dendrotoxin affected RMA KDR, implicating the presence of Kv1.5 in all channels and the absence of Kv1.1, respectively. Currents mediated by channels because of coexpression of Kv1.2, Kv1.5, Kv1.6, and Kv1.2 in human embryonic kidney 293 cells had biophysical and pharmacological properties similar to those of RMA KDR. It is concluded that KDR channels composed of heteromultimers of Kv1 subunits play a critical role in myogenic control of arterial diameter.
Key Words: delayed rectifier potassium channel KCNA vascular smooth muscle myogenic contraction arterial diameter
Introduction
The ability of small resistance arteries to develop myogenic tone in response to elevations in intraluminal (or transmural) pressure is an essential autoregulatory mechanism and an important determinant of peripheral vascular resistance, regional blood flow, and blood pressure. Pressure-induced depolarization of vascular smooth muscle (VSM) leading to increased intracellular Ca2+ ([Ca2+]i) via voltage-dependent activation of Ca2+ channels is required for myogenic tone development.1,2 However, the depolarization does not evoke regenerative action potentials, rather incremental changes in diameter are achieved by graded, steady-state depolarizations.3 Our understanding of the ionic basis of this precise control of myogenic depolarization is incomplete.
Increased intraluminal pressure is thought to activate nonselective cation or CleC channels of VSM cells,2 with the level of depolarization attributable to these channels precisely controlled by an activation of VSM K+ channels.3 Compelling evidence for a contribution of large Ca2+-activated K+ channels (BKCa) to this feedback control comes from studies using specific inhibitors (eg, iberiotoxin) and transgenic mice lacking the BKCa modulatory 1 subunit.4 However, whether other channels are also involved in controlling myogenic depolarization in VSM is not clear.
Voltage-gated delayed rectifier channels (KDR) are expressed by VSM cells of several vascular beds3 and could also play a role in control of myogenic contraction. This view is supported by studies of cerebral arteries; myogenic reactivity was enhanced by 4-aminopyridine (4-AP)eCinduced inhibition of KDR.5 Moreover, changes in the activity and/or expression of KDR channel subunits are reported to occur in diseases (eg, hypertension,6,7 pulmonary hypertension,8eC10 and diabetes11) characterized by increased myogenic reactivity.12eC14
Our understanding of arterial VSM KDR is incomplete and complicated by variations in the reported properties of the channels, as well as the channel subunits expressed.6eC10,15eC21 Whether these differences are attributable to regional variations in channel type and/or level of expression, or alternatively, to varied recording conditions, cell quality and/or selectivity of tools used is unknown. KDR are composed of pore-forming Kv and modulatory Kv subunits arranged in an octameric complex. We provided the first evidence that Kv1.2, Kv1.5, and Kv1.2 coassemble to form heteromultimeric KDR in rabbit portal vein (RPV) VSM.16,17 Expression of transcripts encoding these subunits was reported for resistance arteries, as well as additional Kv1 subunits, including Kv1.1 and Kv1.6.6,7,15,18eC23 However, parallel evidence of subunit protein expression and comparison of the properties of native and recombinant channels was not always performed to justify the conclusion that each subunit contributed to the functional channels and/or control of arterial function.6,7,15,18eC22
The aims of this study were 3-fold: (1) assess the role of Kv1-containing KDR in the myogenic response of rat mesenteric arteries (RMAs) using 4-AP (200 eol/L) and correolide23eC25 (1 eol/L) to inhibit the channels, (2) identify the expression profile of Kv1 and Kv subunits in RMAs, and (3) compare the properties of RMA KDR and channels resulting from heterologous coexpression of the subunits present in RMA myocytes.
Materials and Methods
Male Sprague-Dawley rats (Charles River, Montreal, Canada) were housed and killed according to the standards of the Canadian Council on Animal care and a protocol reviewed by the Animal Care Committee of the Faculty of Medicine, University of Calgary. Details of the methods and materials used in this study are similar to previous publications16,17,26 and/or are in the online data supplement available at http://circres.ahajournals.org.
Results
Control of Myogenic Response by Kv1-Containing KDR
Second order conduit RMAs dilated passively in response to increased transmural pressure (20 to 120 mm Hg) and did not develop active myogenic tone; ie, the pressure-response curves for control and nifedipine-treated (10 eol/L) tissues were identical (Figure 1A). KDR inhibition with 4-AP (200 eol/L) led to active myogenic tone development and arterial diameter was unchanged at pressures >40 mm Hg (no significant difference in the values >40 mm Hg). In contrast, 4th order resistance RMAs developed active tone at pressures >60 mm Hg, and the control pressure-response curve was different from the passive dilation in nifedipine (Figure 1B). 4-AP enhanced the magnitude of change in diameter with increasing pressure in these resistance RMAs, and in 6 of 7 vessels, spontaneous oscillations in diameter were observed (Figure 1B).
Similar results were obtained for RMAs in the presence of correolide (1 eol/L; gift of Merck & Co. Inc, Rahway, NJ) (Figure 2). Enhanced responses to increased intraluminal pressure were observed between 60 to 120 mm Hg in 2nd and 4th order RMAs after correolide treatment compared with control conditions. Oscillations in the diameter of 4th order vessels were observed in correolide, but less often than with 4-AP (ie, 2 of 6 tissues).
Expression of Kv1 and Kv Subunits by RMA VSM
Kv1 subunit expression by intact 4th order RMAs was examined by RT-PCR using subunit-specific primers. Products of appropriate size were obtained for Kv1.1, Kv1.2, Kv1.4, Kv1.5, and Kv1.6 after 35 cycles (Figure 3A). Consistent results were obtained except for Kv1.1, which was detected in only 3 of 6 arteries, but was always apparent in brain RNA. Kv1.1 and/or Kv1.6 expression was reported for cerebral and mesenteric arteries,6,7,15,19,24,25 but not for RPV.17 RNA samples were obtained from 200 to 300 individually selected RMA myocytes to confirm VSM cell-specific expression and control for contamination by message of non-VSM origin (eg, endothelium and neurons). Kv1.6 mRNA was consistently detected, but Kv1.1 expression was not evident (Figure 3B). Primers for the endothelial cell and neuronal markers, endothelin-1 and Erg3, respectively, confirmed the absence of contaminating message (Figure 3B). The expression of Kv1.1, 1.2, 1.3, and Kv2.1 in intact RMAs was also identified by RT-PCR (Figure 3C).
The abundance of Kv1 message normalized to -actin mRNA in 1st/2nd (combined) and 4th order RMAs was determined by real-time PCR (see online data supplement for method and primer verification). The relative abundance of Kv1.5 message was 2-fold greater than that for Kv1.2 and Kv1.6 in 1st/2nd and 4th order RMAs, and the abundance of all three transcripts was significantly higher in 1st/2nd order RMAs (Figure 3D). The similarity in nucleotide sequence of Kv1.1-1.3 and of Kv2.1-2.2 precluded generation of subunit-specific primers, so pan-Kv1 and -Kv2 primers were used. Kv1 message abundance was higher than that of Kv2 in 1st/2nd and 4th order RMAs (Figure 3D).
Kv1 protein expression in 4th order RMA myocytes was identified using subunit-specific antibodies. Anti-Kv1.1 and anti-Kv1.6 were first checked for specificity using human embryonic kidney (HEK293) cells cotransfected with cDNAs encoding the subunits (Kv1.1 and Kv1.6 cDNAs; gifts of Drs H. Duff [University of Calgary, Canada] and O. Pongs [Universitt Hamburg, Germany], respectively) and green fluorescent protein (GFP) (Figure 3E; the specificity of the anti-Kv1.2, -Kv1.4, and -Kv1.5 was previously confirmed).17,26 Polyclonal anti-Kv1.1 and anti-Kv1.6 from Calbiochem and Alomone, respectively, appropriately recognized Kv1.1 and Kv1.6 subunit expression, and the immunofluorescence was blocked by preabsorption with antigenic peptide. Two additional anti-Kv1.1s from Upstate Biotechnology (monoclonal) and Chemicon (polyclonal; Figure 3E) were also tested, but these only demonstrated nonspecific reactivity.
Kv1.2, Kv1.4, Kv1.5, and Kv1.6 immunofluorescence was detected in RMA myocytes and blocked by antigenic peptide or absent in 2° antibody alone (Figure 3F). However, no immunoreactivity was apparent for RMAs when Calbiochem anti-Kv1.1 was used (Figure 3F). Similarly, a lack of Kv1.1 protein expression by rat cerebral arterial myocytes was also evident using this antibody (Figure 3F), contrary to previous reports.15,22
Comparison of RMA KDR and Kv1 Currents
RMA myocytes (4th order) were isolated using methods (see online data supplement) that yielded healthy, relaxed cells or overdigested, contracted cells (Figure 4A and 4B). Mean cell capacitance of relaxed myocytes was 27.5±2.0 pF (n=31) and significantly higher than the 18.8±1.3 pF (n=35; P<0.01) identified for overdigested, contracted cells. Sustained KDR currents, similar to those of other VSM cells16,29 were apparent in both cell types, and a small transient outward current component (inactivation in less than 50 ms) was occasionally apparent (see inset in Figure 7). However, the density of KDR current (at +30 mV) was significantly higher at 27.4±1.5 (n=19) pA/pF for relaxed cells and it activated at a more negative voltage of eC40 mV compared with overdigested myocytes at 4.7±0.6 pA/pF (n=13; P<0.01) (Figure 4C) and eC30 mV (Figure 4C and 4E). This indicates that cell isolation can influence VSM KDR current, and for this reason, only relaxed myocytes with >20 pA/pF KDR current were used.
RMA KDR currents showed voltage-dependent activation and inactivation (Figure 4A and 4D). A single exponential Boltzmann function was used to fit normalized tail current amplitude and the voltage required for half-maximal (V0.5) activation was eC10 mV (Figures 4D and 5A and Table). Steady-state availability was assessed using a standard double-pulse protocol and inactivation was found to be incomplete, with a residual component of noninactivating net KDR remaining after 10 seconds at >+10 mV (Figure 4D and 4E). The V0.5 for inactivation determined by fits of normalized end-pulse amplitude with a Boltzmann function was eC39.4±1.8 mV (k=11.1; n=7). The time constants of activation, inactivation, and deactivation were determined by curve fitting with single or double exponential functions. Values for activation and inactivation are given in the Table.
The properties of currents attributable to cotransfection of combinations of cDNAs encoding Kv1.2, Kv1.5, Kv1.6, and Kv1.2 were determined (Table). Figure 5 shows data for coexpression of all four subunits; the V0.5 for activation was more negative, but RMA KDR and Kv1.2-Kv1.5-Kv1.6-Kv1.2 currents activated, inactivated, and deactivated with similar time constants (Table), and the tail currents were overlapping when scaled to an identical amplitude (Figure 5). In the absence of Kv1.2, the V0.5 and deactivation time constant values were both different from RMA KDR (Table 1). Currents attributable to coexpression of Kv1.2 and Kv1.5 or Kv1.5 and Kv1.6, expression of these pairs of subunits as tandem constructs (joined by a polyglycine linker of 7 or 5 residues, respectively),16 or coexpression of Kv1.2 and Kv1.6 did not deactivate at a rate consistent with RMA KDR, and the Kv1.5-Kv1.6 channels exhibited slower activation (Table).
Inhibition of VSM KDR by 4-AP is associated with an apparent positive shift in the voltage of activation (ie, the V0.5 shifts by +10 mV).16 4-AP (1 mmol/L) reduced RMA KDR current by 34% (Table) and caused a similar shift in V0.5 of +10.7±1.8 mV (n=7) (Figure 5). Kv1.2-Kv1.5-Kv1.6-Kv1.2 currents were also inhibited by 34% (Table) and showed a similar shift in activation of +12.1±0.7 mV (n=3) in the presence of 4-AP (Figure 5D). Paired combinations of coexpressed subunits exhibited a shift in V0.5, but the extent of current suppression by 4-AP was >50% (Table).
Figure 6 compares the inhibition by correolide (1 eol/L) of KDR currents of 2nd and 4th order RMA myocytes. The inhibition occurred at a voltage consistent with the level of membrane potential reported for the vessels, ie, eC30 to eC50 mV (Figure 6A and 6B). Although KDR current density (at +30 mV) for myocytes of 2nd (28.5±1.5 pA/pF, n=11) and 4th order arteries (32.9±2.5 pA/pF, n=10) was identical (P>0.05), the level of current suppression by correolide was significantly greater at 57.3±2.8% (n=3) compared with 37.4±3.6% (n=3; P<0.01), respectively (Figure 6C).
-Dendrotoxin (-DTX; 50 nmol/L) was used as it inhibits homo- and heteromultimeric channels composed of Kv1.2 and Kv1.6 (as well as Kv1.1), but not channels containing Kv1.5.30 -DTX suppressed Kv1.2-Kv1.6 current at 50 nmol/L (Figure 7A), but it had no effect on RMA KDR or currents attributable to coexpression of Kv1.2, Kv1.5, Kv1.6, and Kv1.2 (Figure 7B and 7C). The contribution of Kv1.1 to RMA KDR was assessed using -dendrotoxin (-DTX), a specific inhibitor of homo- and heteromultimeric Kv channels containing Kv1.1.27,28 -DTX (50 nmol/L) suppressed currents attributable to Kv1.1 expression in HEK293 cells, but it did not affect RMA KDR (Figure 7D and 7E).
Discussion
This study provides novel evidence for the contribution of heteromultimeric KDR composed of Kv1.2, Kv1.5, Kv1.6, and Kv subunits to the control of myogenic contraction of arterial resistance vessels. This view is based on an analysis of arterial myogenic responses in the presence of Kv1 channel inhibition, identification of Kv1 subunit message and protein expression, quantification of transcript abundance, and a comparative assessment of biophysical and pharmacological properties of RMA KDR and recombinant channels composed of the subunits expressed by RMA myocytes.
KDR currents of RMA myocytes are due, at least in part, to heteromultimeric channel complexes composed of Kv1.2, Kv1.5, Kv1.6, and Kv1 and/or Kv2.1, but not Kv1.1. Expression of Kv1.1 to 1.6 in arteries was previously shown by RT-PCR using RNA obtained from intact segments in several studies,6,7,15,18,20,22,23,31 but whether all of the identified subunits contributed to functional KDR was not determined. Our analysis of intact RMAs showed expression of Kv1.2, Kv1.4, Kv1.5, and Kv1.6 message and protein, with Kv1.5 message being 2-fold more abundant than that encoding Kv1.2 and Kv1.6. Expression of Kv1 (1.1, 1.2, and 1.3) and Kv2 (2.1) was also identified. Evidence for a contribution of Kv1.1 to RMA KDR was not obtained, however, contrary to studies of small arteries and arterioles.6,7,15,22,23,31 Message encoding Kv1.1 was inconsistently identified in RMAs and could not be identified in RNA of isolated RMA myocytes. Additionally, no evidence of Kv1.1 protein or functional contribution to RMA KDR was obtained with anti-Kv1.1 or -DTX, a toxin that inhibits Kv1.1-containing channels regardless of subunit composition or stoichiometry.27,28 Kv1.1 mRNA (unpublished observation, 2004) and/or protein expression as well could not be identified in rat cerebral arteries and isolated myocytes.
The properties of RMA KDR were characterized for comparison with currents attributable to coexpression of Kv1 and Kv subunits. The voltage-dependence and kinetics of RMA KDR were similar to those previously identified for arterial KDR,32,33 but the reported values exhibit considerable variability, eg, V0.5 values for activation range from eC33.7 mV23 to +3 mV.33 In this study, the amplitude of KDR current at +30 mV was significantly higher at >500 pA compared with previous studies at <500 pA,7,31,32,34,35 activation threshold was more negative at eC45 mV, and the kinetics of deactivation were slower than previously reported.7,31,32,34,35 We also did not observe the complete inactivation of KDR current described in some studies,31,32,35 rather 20% of the current persisted during prolonged steps to positive voltages. These differences could reflect a species- and/or vessel-dependent expression of KDR with varied subunit composition and properties. Alternatively, the variability may derive from differences in the quality of the isolated myocytes used. The importance of cell quality is indicated by our data showing that cell capacitance and KDR current density were lower and the voltage threshold for KDR activation more positive for contracted, overdigested cells compared with relaxed, healthy RMA myocytes. This suggests that careful attention must be paid to cell quality to achieve an accurate analysis of the properties of arterial KDR. In the absence of appropriate care, erroneous conclusions may be made regarding the operating voltage range over which the channels are active and contribute to control of membrane potential. Also, meaningful comparisons of the properties of native and recombinant channels may be precluded if the former are affected by the cell isolation procedure.
Detailed comparisons of currents attributable to native and recombinant channels can be complicated by variations in the biophysical properties of the recombinant channels in different heterologous expression systems, especially voltage-dependence of activation and inactivation.36 With the exception of the V0.5 of activation, the properties of channels attributable to coexpression of Kv1.2, Kv1.5, and Kv1.6 with Kv1.2 in HEK293 cells mimicked those of RMA KDR and provide evidence of the subunit composition of the native channels. (1) The kinetics of activation, deactivation, and inactivation of RMA KDR were similar to currents resulting from coexpression of Kv1.2, Kv1.5, Kv1.6, and Kv1.2. On the other hand, paired heteromultimeric combinations of these Kv subunits, or all three subunits in the absence of Kv1.2, deactivate more quickly and/or activate more slowly than RMA KDR implying that the native channels may contain all four subunits. (2) RMA KDR and channels attributable to coexpression of Kv1.2, Kv1.5, Kv1.6, and Kv1.2 displayed a lack of sensitivity to -DTX, a toxin that inhibits homo- and heteromultimeric Kv1.1, Kv1.2, and Kv1.6 channels, but not Kv1.5-containing channels.30 This indicates that homo- and heteromultimeric Kv1.2 and Kv1.6 channels are likely not present; ie, all RMA KDR and Kv1.2-Kv1.5-Kv1.6-Kv2.1 channels contained Kv1.5 and were insensitive to -DTX. That Kv1.5 contributes to RMA KDR is consistent with the demonstrated presence of this subunit in KDR of other vessels.15,16 (3) The extent of inhibition by 4-AP of RMA KDR and Kv1.2-Kv1.5-Kv1.6-Kv1.2 currents was identical at 34%, but 4-AP had a greater effect on channels attributable to other heteromultimeric combinations of these subunits at >50%. Significantly, RMA KDR were less sensitive to 4-AP compared with RPV KDR current (> 50% block with 1 mmol/L),16 but consistent with the findings of other studies of RMAs (5 to 10 mmol/L)31,34,35 and cerebral (5 mmol/L)37 arterial KDR. (4-AP almost completely inhibited human mesenteric KDR current, but the control current amplitude was only 100 pA).25 The differences in 4-AP sensitivity of VSM KDR currents may be attributable to varied levels of expression of 4-APeCsensitive Kv1-containing and 4-APeCresistant non-Kv1 channels, eg, Kv2 channels.33 Alternatively, differences in Kv1 subunit composition could be involved, eg, the presence of Kv1.6 which has a higher IC50 for 4-AP block.38 In agreement with this view, Kv1.2-Kv1.5-Kv1.2 channels expressed by RPV exhibit >50% block by 1 mmol/L 4-AP16,17 compared with the 34% inhibition shown here for Kv1.2-Kv1.5-Kv1.6-Kv1.2 channels. Interestingly, the sensitivity of Kv1.5-Kv1.6 and Kv1.2-Kv1.5 channels was identical and greater than RMA KDR and Kv1.2-Kv1.5-Kv1.6-Kv2.1 channels, suggesting the combination of Kv1.6 with Kv1.2 and Kv1.5 may affect 4-AP sensitivity rather than the presence of Kv1.6 alone. Further experiments are required to identify the mechanism involved. (4) 4-AP inhibition of RPV KDR was associated with a positive shift in the voltage-dependence of activation,16 and the same was shown here for RMA KDR. Homomultimeric Kv1.5 channels do not exhibit a shift in activation, but homomultimeric Kv1.216 and Kv1.6 (unpublished observation, 2004), as well as Kv1.2-Kv1.5,16 Kv1.5-Kv1.6 (unpublished observation, 2004), and Kv1.2-Kv1.5-Kv1.6-Kv1 (present study) channels all mimic VSM KDR. This indicates that RMA KDR must contain Kv1.5 in association with Kv1.2 and/or Kv1.6. (5) RMA KDR do not display fast inactivation as observed for Kv1 subunits when expressed with Kv1.4 or Kv1 subunits.38,39,40 This lack of fast inactivation is consistent with the presence of Kv1.6 in RMA KDR; the N-terminus of Kv1.6 has a motif, the NIP domain, that inhibits pore block by binding the positively charged inactivation ball of Kv1.4 and Kv1 subunits.39 Although Kv1.4 is expressed in RMA myocytes, we do not believe that this subunit contributes to RMA KDR as Kv1.4-containing channels display significantly faster activation.39,40
This study indicates for the first time the importance of KDR channels composed of Kv1 subunits to the normal function of arterial resistance vessels via their role in controlling myogenic reactivity. Previous studies indicating a role for KDR channels in control of myogenic contraction used 4-AP at 0.3 to 1 mmol/L.5 Kv1 channels are sensitive to this concentration of 4-AP, but so too are Kv channels of other subfamilies with overlapping and, in some cases, coincident sensitivities to 4-AP; eg, Kv3.38 For this reason, we also used the putative Kv1 channel blocker, correolide, to assess the role of Kv1-containing KDR in control of the myogenic response. Inhibition of KDR by correolide was previously shown to elicit depolarization and constriction of cerebral arteries and/or arterioles,5,15,24 but its effect on myogenic reactivity was not assessed. We found that correolide mimicked the ability of 4-AP to enhance myogenic reactivity of RMAs and to inhibit RMA KDR over a voltage range consistent with that associated with myogenic contraction of RMAs.1,5 Moreover, RMA KDR currents activated positive to eC50 mV and showed incomplete inactivation; this is consistent with the view that the channels are capable of steady-state activity and contribute to control of membrane potential over the range reported for myogenic depolarization.5 Taken together, these data provide evidence for the participation of Kv1 subuniteCcontaining KDR in controlling the response of resistance arterial VSM to changes in intraluminal pressure. Whether additional subunits from other Kv subfamilies (eg, Kv2, which is expressed in RMAs; unpublished observation, 2004) also contribute to control of myogenic reactivity must be addressed in the future.
The ability of resistance arteries to develop myogenic tone varies between vascular beds, the response being most prominent in areas with the greatest degree of autoregulation. For example, cerebral and skeletal muscle arteries show a pronounced decrease in diameter with increasing pressure,5,41 whereas RMAs of similar diameter do not.41,42 However, smaller resistance RMAs do show a pressure-induced increase in [Ca2+]i similar to that of cerebral vessels41 and their pressure-diameter curve is flattened at higher pressures (ie, 60 mm Hg). This indicates that although they do not display a decrease in diameter, they do show active tone development and maintenance of diameter rather than passive dilation.41,42 Consistent with previous reports,43 2nd order conduit RMAs did not exhibit active constriction and only dilated passively in response to elevated pressure. However, in the presence of 4-AP or correolide, these vessels maintained a stable diameter with increasing transmural pressure. This suggests that KDR activation inhibits pressure-induced alterations in diameter in conduit arteries; ie, the presence of KDR composed of Kv1 subunits is sufficient to prevent myogenic tone development. In contrast, 4th order resistance RMAs maintained a stable diameter when subjected to increased transmural pressure, and in the presence of KDR inhibition, myogenic tone development was enhanced and accompanied by rhythmic oscillations in diameter. This indicates that a negative feedback regulation of VSM depolarization via voltage-dependent activation of Kv1-containing KDR contributes to the control of myogenic contraction in resistance RMAs and may be necessary for stable, graded changes in arterial diameter. Interestingly, correolide produced a greater inhibition of KDR current of 2nd versus 4th order RMA myocytes. This implies that the component of KDR current attributable to Kv1-containing channels may be smaller in resistance compared with conduit RMAs, a view that is consistent with the lower abundance of Kv1 message in 4th compared with 1st/2nd order vessels as detected by real time PCR. It is possible that the amplitude of the negative feedback regulation provided by Kv1 subunit-containing KDR may be an important determinant of the level of myogenic reactivity exhibited by arteries of different size and/or vascular origin. A reduced contribution of Kv1-containing KDR would permit greater depolarization by inward current activated in response to increased transmural pressure, and thereby, greater active tone development. Whether differences in other ionic currents are also present and contribute to variability in myogenic reactivity requires further study.
In contrast to the specialized nature of the cerebral vasculature, the mesenteric arterial bed is relatively nonspecialized and representative of the peripheral vasculature in general. Our demonstration of a role for KDR in permitting stable myogenic alterations in the diameter of resistance RMAs suggests that this may be a mechanism of generalized importance for appropriate function of the peripheral arterial vasculature and, therefore, for normal blood pressure regulation.
Acknowledgments
This work was supported by the Canadian Institutes of Health Research (MT-13505), The Wellcome Trust (UK), a Natural Sciences and Engineering Research Council PSG A award to R.J. and AHFMR and HSFC studentship awards to T.C. We thank Claude Viellette and Susan Li for technical assistance. This work is dedicated to the memory of Prof Burton Horowitz, University of Nevada (Reno).
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Ohya S, Tanaka M, Watanabe M, Imaizumi Y. Diverse expression of delayed rectifier K+ channel subtype transcripts in several types of smooth muscle of the rat. J Smooth Muscle Res. 2000; 36: 101eC115.
Cheong A, Dedman AM, Xu SZ, Beech DJ. Kv1 channels in murine arterioles: differential expression and regulation of diameter. Am J Physiol Heart Circ Physiol. 2001; 281: H1087eCH1065.
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Fountain SJ, Cheong A, Flemming R, Mair L, Sivaprasadarao A, Beech DJ. Functional up-regulation of KCNA gene family expression in murine mesenteric resistance artery smooth muscle. J Physiol (Lond). 2004; 556: 29eC42.
Felix JP, Bugianesi RM, Schmalhofer WA, et al. Identification and biochemical characterization of a novel nortriterpene inhibitor of the human lymphocyte voltage-gated potassium channel, Kv1.3. Biochemistry. 1999; 38: 4922eC4930.
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Hayabuchi Y, Standen NB, Davies NW. Angiotensin II inhibits and alters kinetics of voltage-gated K+ channels of rat arterial smooth muscle. Am J Physiol Heart Circ Physiol. 2001; 281: H2480eCH2489.
Smirnov SV, Beck R, Tammaro P, Ishii T, Aaronson PI. Electrophysiologically distinct smooth muscle cell subtypes in rat conduit and resistance pulmonary arteries. J Physiol (Lond). 2002; 538: 867eC878.
Xu C, Lu Y, Tang G, Wang R. Expression of voltage-dependent K+ channel genes in mesenteric artery smooth muscle cells. Am J Physiol Gastrointest Liver Physiol. 1999; 277: G1055eCG1063.
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Luykenaar KD, Brett SE, Wu BN, Wiehler WB, Welsh DG. Pyrimidine nucleotides suppress KDR currents and depolarize rat cerebral arteries by activating Rho kinase. Am J Physiol Heart Circ Physiol. 2004; 286: H1088eCH1100.
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Chlopicki S, Nilsson H, Mulvany MJ. Initial and sustained phases of myogenic response of rat small mesenteric arteries. Am J Physiol Heart Circ Physiol. 2001; 281: H2176eCH2183.(Frances Plane, Rosalyn Jo)
Division of Cardiovascular Pharmacology (K.I.), Yamagata University School of Medicine, Japan.
Abstract
Inhibition of vascular smooth muscle (VSM) delayed rectifier K+ channels (KDR) by 4-aminopyridine (4-AP; 200 eol/L) or correolide (1 eol/L), a selective inhibitor of Kv1 channels, enhanced myogenic contraction of rat mesenteric arteries (RMAs) in response to increases in intraluminal pressure. The molecular identity of KDR of RMA myocytes was characterized using RT-PCR, real-time PCR, and immunocytochemistry. Transcripts encoding the pore-forming Kv subunits, Kv1.2, Kv1.4, Kv1.5, and Kv1.6, were identified and confirmed at the protein level with subunit-specific antibodies. Kv transcript (1.1, 1.2, 1.3, and 2.1) expression was also identified. Kv1.5 message was 2-fold more abundant than that for Kv1.2 and Kv1.6. Transcripts encoding these three Kv1 subunits were 2-fold more abundant in 1st/2nd order conduit compared with 4th order resistance RMAs, and Kv1 was 8-fold higher than Kv2 message. RMA KDR activated positive to eC50 mV, exhibited incomplete inactivation, and were inhibited by 4-AP and correolide. However, neither -dendrotoxin or -dendrotoxin affected RMA KDR, implicating the presence of Kv1.5 in all channels and the absence of Kv1.1, respectively. Currents mediated by channels because of coexpression of Kv1.2, Kv1.5, Kv1.6, and Kv1.2 in human embryonic kidney 293 cells had biophysical and pharmacological properties similar to those of RMA KDR. It is concluded that KDR channels composed of heteromultimers of Kv1 subunits play a critical role in myogenic control of arterial diameter.
Key Words: delayed rectifier potassium channel KCNA vascular smooth muscle myogenic contraction arterial diameter
Introduction
The ability of small resistance arteries to develop myogenic tone in response to elevations in intraluminal (or transmural) pressure is an essential autoregulatory mechanism and an important determinant of peripheral vascular resistance, regional blood flow, and blood pressure. Pressure-induced depolarization of vascular smooth muscle (VSM) leading to increased intracellular Ca2+ ([Ca2+]i) via voltage-dependent activation of Ca2+ channels is required for myogenic tone development.1,2 However, the depolarization does not evoke regenerative action potentials, rather incremental changes in diameter are achieved by graded, steady-state depolarizations.3 Our understanding of the ionic basis of this precise control of myogenic depolarization is incomplete.
Increased intraluminal pressure is thought to activate nonselective cation or CleC channels of VSM cells,2 with the level of depolarization attributable to these channels precisely controlled by an activation of VSM K+ channels.3 Compelling evidence for a contribution of large Ca2+-activated K+ channels (BKCa) to this feedback control comes from studies using specific inhibitors (eg, iberiotoxin) and transgenic mice lacking the BKCa modulatory 1 subunit.4 However, whether other channels are also involved in controlling myogenic depolarization in VSM is not clear.
Voltage-gated delayed rectifier channels (KDR) are expressed by VSM cells of several vascular beds3 and could also play a role in control of myogenic contraction. This view is supported by studies of cerebral arteries; myogenic reactivity was enhanced by 4-aminopyridine (4-AP)eCinduced inhibition of KDR.5 Moreover, changes in the activity and/or expression of KDR channel subunits are reported to occur in diseases (eg, hypertension,6,7 pulmonary hypertension,8eC10 and diabetes11) characterized by increased myogenic reactivity.12eC14
Our understanding of arterial VSM KDR is incomplete and complicated by variations in the reported properties of the channels, as well as the channel subunits expressed.6eC10,15eC21 Whether these differences are attributable to regional variations in channel type and/or level of expression, or alternatively, to varied recording conditions, cell quality and/or selectivity of tools used is unknown. KDR are composed of pore-forming Kv and modulatory Kv subunits arranged in an octameric complex. We provided the first evidence that Kv1.2, Kv1.5, and Kv1.2 coassemble to form heteromultimeric KDR in rabbit portal vein (RPV) VSM.16,17 Expression of transcripts encoding these subunits was reported for resistance arteries, as well as additional Kv1 subunits, including Kv1.1 and Kv1.6.6,7,15,18eC23 However, parallel evidence of subunit protein expression and comparison of the properties of native and recombinant channels was not always performed to justify the conclusion that each subunit contributed to the functional channels and/or control of arterial function.6,7,15,18eC22
The aims of this study were 3-fold: (1) assess the role of Kv1-containing KDR in the myogenic response of rat mesenteric arteries (RMAs) using 4-AP (200 eol/L) and correolide23eC25 (1 eol/L) to inhibit the channels, (2) identify the expression profile of Kv1 and Kv subunits in RMAs, and (3) compare the properties of RMA KDR and channels resulting from heterologous coexpression of the subunits present in RMA myocytes.
Materials and Methods
Male Sprague-Dawley rats (Charles River, Montreal, Canada) were housed and killed according to the standards of the Canadian Council on Animal care and a protocol reviewed by the Animal Care Committee of the Faculty of Medicine, University of Calgary. Details of the methods and materials used in this study are similar to previous publications16,17,26 and/or are in the online data supplement available at http://circres.ahajournals.org.
Results
Control of Myogenic Response by Kv1-Containing KDR
Second order conduit RMAs dilated passively in response to increased transmural pressure (20 to 120 mm Hg) and did not develop active myogenic tone; ie, the pressure-response curves for control and nifedipine-treated (10 eol/L) tissues were identical (Figure 1A). KDR inhibition with 4-AP (200 eol/L) led to active myogenic tone development and arterial diameter was unchanged at pressures >40 mm Hg (no significant difference in the values >40 mm Hg). In contrast, 4th order resistance RMAs developed active tone at pressures >60 mm Hg, and the control pressure-response curve was different from the passive dilation in nifedipine (Figure 1B). 4-AP enhanced the magnitude of change in diameter with increasing pressure in these resistance RMAs, and in 6 of 7 vessels, spontaneous oscillations in diameter were observed (Figure 1B).
Similar results were obtained for RMAs in the presence of correolide (1 eol/L; gift of Merck & Co. Inc, Rahway, NJ) (Figure 2). Enhanced responses to increased intraluminal pressure were observed between 60 to 120 mm Hg in 2nd and 4th order RMAs after correolide treatment compared with control conditions. Oscillations in the diameter of 4th order vessels were observed in correolide, but less often than with 4-AP (ie, 2 of 6 tissues).
Expression of Kv1 and Kv Subunits by RMA VSM
Kv1 subunit expression by intact 4th order RMAs was examined by RT-PCR using subunit-specific primers. Products of appropriate size were obtained for Kv1.1, Kv1.2, Kv1.4, Kv1.5, and Kv1.6 after 35 cycles (Figure 3A). Consistent results were obtained except for Kv1.1, which was detected in only 3 of 6 arteries, but was always apparent in brain RNA. Kv1.1 and/or Kv1.6 expression was reported for cerebral and mesenteric arteries,6,7,15,19,24,25 but not for RPV.17 RNA samples were obtained from 200 to 300 individually selected RMA myocytes to confirm VSM cell-specific expression and control for contamination by message of non-VSM origin (eg, endothelium and neurons). Kv1.6 mRNA was consistently detected, but Kv1.1 expression was not evident (Figure 3B). Primers for the endothelial cell and neuronal markers, endothelin-1 and Erg3, respectively, confirmed the absence of contaminating message (Figure 3B). The expression of Kv1.1, 1.2, 1.3, and Kv2.1 in intact RMAs was also identified by RT-PCR (Figure 3C).
The abundance of Kv1 message normalized to -actin mRNA in 1st/2nd (combined) and 4th order RMAs was determined by real-time PCR (see online data supplement for method and primer verification). The relative abundance of Kv1.5 message was 2-fold greater than that for Kv1.2 and Kv1.6 in 1st/2nd and 4th order RMAs, and the abundance of all three transcripts was significantly higher in 1st/2nd order RMAs (Figure 3D). The similarity in nucleotide sequence of Kv1.1-1.3 and of Kv2.1-2.2 precluded generation of subunit-specific primers, so pan-Kv1 and -Kv2 primers were used. Kv1 message abundance was higher than that of Kv2 in 1st/2nd and 4th order RMAs (Figure 3D).
Kv1 protein expression in 4th order RMA myocytes was identified using subunit-specific antibodies. Anti-Kv1.1 and anti-Kv1.6 were first checked for specificity using human embryonic kidney (HEK293) cells cotransfected with cDNAs encoding the subunits (Kv1.1 and Kv1.6 cDNAs; gifts of Drs H. Duff [University of Calgary, Canada] and O. Pongs [Universitt Hamburg, Germany], respectively) and green fluorescent protein (GFP) (Figure 3E; the specificity of the anti-Kv1.2, -Kv1.4, and -Kv1.5 was previously confirmed).17,26 Polyclonal anti-Kv1.1 and anti-Kv1.6 from Calbiochem and Alomone, respectively, appropriately recognized Kv1.1 and Kv1.6 subunit expression, and the immunofluorescence was blocked by preabsorption with antigenic peptide. Two additional anti-Kv1.1s from Upstate Biotechnology (monoclonal) and Chemicon (polyclonal; Figure 3E) were also tested, but these only demonstrated nonspecific reactivity.
Kv1.2, Kv1.4, Kv1.5, and Kv1.6 immunofluorescence was detected in RMA myocytes and blocked by antigenic peptide or absent in 2° antibody alone (Figure 3F). However, no immunoreactivity was apparent for RMAs when Calbiochem anti-Kv1.1 was used (Figure 3F). Similarly, a lack of Kv1.1 protein expression by rat cerebral arterial myocytes was also evident using this antibody (Figure 3F), contrary to previous reports.15,22
Comparison of RMA KDR and Kv1 Currents
RMA myocytes (4th order) were isolated using methods (see online data supplement) that yielded healthy, relaxed cells or overdigested, contracted cells (Figure 4A and 4B). Mean cell capacitance of relaxed myocytes was 27.5±2.0 pF (n=31) and significantly higher than the 18.8±1.3 pF (n=35; P<0.01) identified for overdigested, contracted cells. Sustained KDR currents, similar to those of other VSM cells16,29 were apparent in both cell types, and a small transient outward current component (inactivation in less than 50 ms) was occasionally apparent (see inset in Figure 7). However, the density of KDR current (at +30 mV) was significantly higher at 27.4±1.5 (n=19) pA/pF for relaxed cells and it activated at a more negative voltage of eC40 mV compared with overdigested myocytes at 4.7±0.6 pA/pF (n=13; P<0.01) (Figure 4C) and eC30 mV (Figure 4C and 4E). This indicates that cell isolation can influence VSM KDR current, and for this reason, only relaxed myocytes with >20 pA/pF KDR current were used.
RMA KDR currents showed voltage-dependent activation and inactivation (Figure 4A and 4D). A single exponential Boltzmann function was used to fit normalized tail current amplitude and the voltage required for half-maximal (V0.5) activation was eC10 mV (Figures 4D and 5A and Table). Steady-state availability was assessed using a standard double-pulse protocol and inactivation was found to be incomplete, with a residual component of noninactivating net KDR remaining after 10 seconds at >+10 mV (Figure 4D and 4E). The V0.5 for inactivation determined by fits of normalized end-pulse amplitude with a Boltzmann function was eC39.4±1.8 mV (k=11.1; n=7). The time constants of activation, inactivation, and deactivation were determined by curve fitting with single or double exponential functions. Values for activation and inactivation are given in the Table.
The properties of currents attributable to cotransfection of combinations of cDNAs encoding Kv1.2, Kv1.5, Kv1.6, and Kv1.2 were determined (Table). Figure 5 shows data for coexpression of all four subunits; the V0.5 for activation was more negative, but RMA KDR and Kv1.2-Kv1.5-Kv1.6-Kv1.2 currents activated, inactivated, and deactivated with similar time constants (Table), and the tail currents were overlapping when scaled to an identical amplitude (Figure 5). In the absence of Kv1.2, the V0.5 and deactivation time constant values were both different from RMA KDR (Table 1). Currents attributable to coexpression of Kv1.2 and Kv1.5 or Kv1.5 and Kv1.6, expression of these pairs of subunits as tandem constructs (joined by a polyglycine linker of 7 or 5 residues, respectively),16 or coexpression of Kv1.2 and Kv1.6 did not deactivate at a rate consistent with RMA KDR, and the Kv1.5-Kv1.6 channels exhibited slower activation (Table).
Inhibition of VSM KDR by 4-AP is associated with an apparent positive shift in the voltage of activation (ie, the V0.5 shifts by +10 mV).16 4-AP (1 mmol/L) reduced RMA KDR current by 34% (Table) and caused a similar shift in V0.5 of +10.7±1.8 mV (n=7) (Figure 5). Kv1.2-Kv1.5-Kv1.6-Kv1.2 currents were also inhibited by 34% (Table) and showed a similar shift in activation of +12.1±0.7 mV (n=3) in the presence of 4-AP (Figure 5D). Paired combinations of coexpressed subunits exhibited a shift in V0.5, but the extent of current suppression by 4-AP was >50% (Table).
Figure 6 compares the inhibition by correolide (1 eol/L) of KDR currents of 2nd and 4th order RMA myocytes. The inhibition occurred at a voltage consistent with the level of membrane potential reported for the vessels, ie, eC30 to eC50 mV (Figure 6A and 6B). Although KDR current density (at +30 mV) for myocytes of 2nd (28.5±1.5 pA/pF, n=11) and 4th order arteries (32.9±2.5 pA/pF, n=10) was identical (P>0.05), the level of current suppression by correolide was significantly greater at 57.3±2.8% (n=3) compared with 37.4±3.6% (n=3; P<0.01), respectively (Figure 6C).
-Dendrotoxin (-DTX; 50 nmol/L) was used as it inhibits homo- and heteromultimeric channels composed of Kv1.2 and Kv1.6 (as well as Kv1.1), but not channels containing Kv1.5.30 -DTX suppressed Kv1.2-Kv1.6 current at 50 nmol/L (Figure 7A), but it had no effect on RMA KDR or currents attributable to coexpression of Kv1.2, Kv1.5, Kv1.6, and Kv1.2 (Figure 7B and 7C). The contribution of Kv1.1 to RMA KDR was assessed using -dendrotoxin (-DTX), a specific inhibitor of homo- and heteromultimeric Kv channels containing Kv1.1.27,28 -DTX (50 nmol/L) suppressed currents attributable to Kv1.1 expression in HEK293 cells, but it did not affect RMA KDR (Figure 7D and 7E).
Discussion
This study provides novel evidence for the contribution of heteromultimeric KDR composed of Kv1.2, Kv1.5, Kv1.6, and Kv subunits to the control of myogenic contraction of arterial resistance vessels. This view is based on an analysis of arterial myogenic responses in the presence of Kv1 channel inhibition, identification of Kv1 subunit message and protein expression, quantification of transcript abundance, and a comparative assessment of biophysical and pharmacological properties of RMA KDR and recombinant channels composed of the subunits expressed by RMA myocytes.
KDR currents of RMA myocytes are due, at least in part, to heteromultimeric channel complexes composed of Kv1.2, Kv1.5, Kv1.6, and Kv1 and/or Kv2.1, but not Kv1.1. Expression of Kv1.1 to 1.6 in arteries was previously shown by RT-PCR using RNA obtained from intact segments in several studies,6,7,15,18,20,22,23,31 but whether all of the identified subunits contributed to functional KDR was not determined. Our analysis of intact RMAs showed expression of Kv1.2, Kv1.4, Kv1.5, and Kv1.6 message and protein, with Kv1.5 message being 2-fold more abundant than that encoding Kv1.2 and Kv1.6. Expression of Kv1 (1.1, 1.2, and 1.3) and Kv2 (2.1) was also identified. Evidence for a contribution of Kv1.1 to RMA KDR was not obtained, however, contrary to studies of small arteries and arterioles.6,7,15,22,23,31 Message encoding Kv1.1 was inconsistently identified in RMAs and could not be identified in RNA of isolated RMA myocytes. Additionally, no evidence of Kv1.1 protein or functional contribution to RMA KDR was obtained with anti-Kv1.1 or -DTX, a toxin that inhibits Kv1.1-containing channels regardless of subunit composition or stoichiometry.27,28 Kv1.1 mRNA (unpublished observation, 2004) and/or protein expression as well could not be identified in rat cerebral arteries and isolated myocytes.
The properties of RMA KDR were characterized for comparison with currents attributable to coexpression of Kv1 and Kv subunits. The voltage-dependence and kinetics of RMA KDR were similar to those previously identified for arterial KDR,32,33 but the reported values exhibit considerable variability, eg, V0.5 values for activation range from eC33.7 mV23 to +3 mV.33 In this study, the amplitude of KDR current at +30 mV was significantly higher at >500 pA compared with previous studies at <500 pA,7,31,32,34,35 activation threshold was more negative at eC45 mV, and the kinetics of deactivation were slower than previously reported.7,31,32,34,35 We also did not observe the complete inactivation of KDR current described in some studies,31,32,35 rather 20% of the current persisted during prolonged steps to positive voltages. These differences could reflect a species- and/or vessel-dependent expression of KDR with varied subunit composition and properties. Alternatively, the variability may derive from differences in the quality of the isolated myocytes used. The importance of cell quality is indicated by our data showing that cell capacitance and KDR current density were lower and the voltage threshold for KDR activation more positive for contracted, overdigested cells compared with relaxed, healthy RMA myocytes. This suggests that careful attention must be paid to cell quality to achieve an accurate analysis of the properties of arterial KDR. In the absence of appropriate care, erroneous conclusions may be made regarding the operating voltage range over which the channels are active and contribute to control of membrane potential. Also, meaningful comparisons of the properties of native and recombinant channels may be precluded if the former are affected by the cell isolation procedure.
Detailed comparisons of currents attributable to native and recombinant channels can be complicated by variations in the biophysical properties of the recombinant channels in different heterologous expression systems, especially voltage-dependence of activation and inactivation.36 With the exception of the V0.5 of activation, the properties of channels attributable to coexpression of Kv1.2, Kv1.5, and Kv1.6 with Kv1.2 in HEK293 cells mimicked those of RMA KDR and provide evidence of the subunit composition of the native channels. (1) The kinetics of activation, deactivation, and inactivation of RMA KDR were similar to currents resulting from coexpression of Kv1.2, Kv1.5, Kv1.6, and Kv1.2. On the other hand, paired heteromultimeric combinations of these Kv subunits, or all three subunits in the absence of Kv1.2, deactivate more quickly and/or activate more slowly than RMA KDR implying that the native channels may contain all four subunits. (2) RMA KDR and channels attributable to coexpression of Kv1.2, Kv1.5, Kv1.6, and Kv1.2 displayed a lack of sensitivity to -DTX, a toxin that inhibits homo- and heteromultimeric Kv1.1, Kv1.2, and Kv1.6 channels, but not Kv1.5-containing channels.30 This indicates that homo- and heteromultimeric Kv1.2 and Kv1.6 channels are likely not present; ie, all RMA KDR and Kv1.2-Kv1.5-Kv1.6-Kv2.1 channels contained Kv1.5 and were insensitive to -DTX. That Kv1.5 contributes to RMA KDR is consistent with the demonstrated presence of this subunit in KDR of other vessels.15,16 (3) The extent of inhibition by 4-AP of RMA KDR and Kv1.2-Kv1.5-Kv1.6-Kv1.2 currents was identical at 34%, but 4-AP had a greater effect on channels attributable to other heteromultimeric combinations of these subunits at >50%. Significantly, RMA KDR were less sensitive to 4-AP compared with RPV KDR current (> 50% block with 1 mmol/L),16 but consistent with the findings of other studies of RMAs (5 to 10 mmol/L)31,34,35 and cerebral (5 mmol/L)37 arterial KDR. (4-AP almost completely inhibited human mesenteric KDR current, but the control current amplitude was only 100 pA).25 The differences in 4-AP sensitivity of VSM KDR currents may be attributable to varied levels of expression of 4-APeCsensitive Kv1-containing and 4-APeCresistant non-Kv1 channels, eg, Kv2 channels.33 Alternatively, differences in Kv1 subunit composition could be involved, eg, the presence of Kv1.6 which has a higher IC50 for 4-AP block.38 In agreement with this view, Kv1.2-Kv1.5-Kv1.2 channels expressed by RPV exhibit >50% block by 1 mmol/L 4-AP16,17 compared with the 34% inhibition shown here for Kv1.2-Kv1.5-Kv1.6-Kv1.2 channels. Interestingly, the sensitivity of Kv1.5-Kv1.6 and Kv1.2-Kv1.5 channels was identical and greater than RMA KDR and Kv1.2-Kv1.5-Kv1.6-Kv2.1 channels, suggesting the combination of Kv1.6 with Kv1.2 and Kv1.5 may affect 4-AP sensitivity rather than the presence of Kv1.6 alone. Further experiments are required to identify the mechanism involved. (4) 4-AP inhibition of RPV KDR was associated with a positive shift in the voltage-dependence of activation,16 and the same was shown here for RMA KDR. Homomultimeric Kv1.5 channels do not exhibit a shift in activation, but homomultimeric Kv1.216 and Kv1.6 (unpublished observation, 2004), as well as Kv1.2-Kv1.5,16 Kv1.5-Kv1.6 (unpublished observation, 2004), and Kv1.2-Kv1.5-Kv1.6-Kv1 (present study) channels all mimic VSM KDR. This indicates that RMA KDR must contain Kv1.5 in association with Kv1.2 and/or Kv1.6. (5) RMA KDR do not display fast inactivation as observed for Kv1 subunits when expressed with Kv1.4 or Kv1 subunits.38,39,40 This lack of fast inactivation is consistent with the presence of Kv1.6 in RMA KDR; the N-terminus of Kv1.6 has a motif, the NIP domain, that inhibits pore block by binding the positively charged inactivation ball of Kv1.4 and Kv1 subunits.39 Although Kv1.4 is expressed in RMA myocytes, we do not believe that this subunit contributes to RMA KDR as Kv1.4-containing channels display significantly faster activation.39,40
This study indicates for the first time the importance of KDR channels composed of Kv1 subunits to the normal function of arterial resistance vessels via their role in controlling myogenic reactivity. Previous studies indicating a role for KDR channels in control of myogenic contraction used 4-AP at 0.3 to 1 mmol/L.5 Kv1 channels are sensitive to this concentration of 4-AP, but so too are Kv channels of other subfamilies with overlapping and, in some cases, coincident sensitivities to 4-AP; eg, Kv3.38 For this reason, we also used the putative Kv1 channel blocker, correolide, to assess the role of Kv1-containing KDR in control of the myogenic response. Inhibition of KDR by correolide was previously shown to elicit depolarization and constriction of cerebral arteries and/or arterioles,5,15,24 but its effect on myogenic reactivity was not assessed. We found that correolide mimicked the ability of 4-AP to enhance myogenic reactivity of RMAs and to inhibit RMA KDR over a voltage range consistent with that associated with myogenic contraction of RMAs.1,5 Moreover, RMA KDR currents activated positive to eC50 mV and showed incomplete inactivation; this is consistent with the view that the channels are capable of steady-state activity and contribute to control of membrane potential over the range reported for myogenic depolarization.5 Taken together, these data provide evidence for the participation of Kv1 subuniteCcontaining KDR in controlling the response of resistance arterial VSM to changes in intraluminal pressure. Whether additional subunits from other Kv subfamilies (eg, Kv2, which is expressed in RMAs; unpublished observation, 2004) also contribute to control of myogenic reactivity must be addressed in the future.
The ability of resistance arteries to develop myogenic tone varies between vascular beds, the response being most prominent in areas with the greatest degree of autoregulation. For example, cerebral and skeletal muscle arteries show a pronounced decrease in diameter with increasing pressure,5,41 whereas RMAs of similar diameter do not.41,42 However, smaller resistance RMAs do show a pressure-induced increase in [Ca2+]i similar to that of cerebral vessels41 and their pressure-diameter curve is flattened at higher pressures (ie, 60 mm Hg). This indicates that although they do not display a decrease in diameter, they do show active tone development and maintenance of diameter rather than passive dilation.41,42 Consistent with previous reports,43 2nd order conduit RMAs did not exhibit active constriction and only dilated passively in response to elevated pressure. However, in the presence of 4-AP or correolide, these vessels maintained a stable diameter with increasing transmural pressure. This suggests that KDR activation inhibits pressure-induced alterations in diameter in conduit arteries; ie, the presence of KDR composed of Kv1 subunits is sufficient to prevent myogenic tone development. In contrast, 4th order resistance RMAs maintained a stable diameter when subjected to increased transmural pressure, and in the presence of KDR inhibition, myogenic tone development was enhanced and accompanied by rhythmic oscillations in diameter. This indicates that a negative feedback regulation of VSM depolarization via voltage-dependent activation of Kv1-containing KDR contributes to the control of myogenic contraction in resistance RMAs and may be necessary for stable, graded changes in arterial diameter. Interestingly, correolide produced a greater inhibition of KDR current of 2nd versus 4th order RMA myocytes. This implies that the component of KDR current attributable to Kv1-containing channels may be smaller in resistance compared with conduit RMAs, a view that is consistent with the lower abundance of Kv1 message in 4th compared with 1st/2nd order vessels as detected by real time PCR. It is possible that the amplitude of the negative feedback regulation provided by Kv1 subunit-containing KDR may be an important determinant of the level of myogenic reactivity exhibited by arteries of different size and/or vascular origin. A reduced contribution of Kv1-containing KDR would permit greater depolarization by inward current activated in response to increased transmural pressure, and thereby, greater active tone development. Whether differences in other ionic currents are also present and contribute to variability in myogenic reactivity requires further study.
In contrast to the specialized nature of the cerebral vasculature, the mesenteric arterial bed is relatively nonspecialized and representative of the peripheral vasculature in general. Our demonstration of a role for KDR in permitting stable myogenic alterations in the diameter of resistance RMAs suggests that this may be a mechanism of generalized importance for appropriate function of the peripheral arterial vasculature and, therefore, for normal blood pressure regulation.
Acknowledgments
This work was supported by the Canadian Institutes of Health Research (MT-13505), The Wellcome Trust (UK), a Natural Sciences and Engineering Research Council PSG A award to R.J. and AHFMR and HSFC studentship awards to T.C. We thank Claude Viellette and Susan Li for technical assistance. This work is dedicated to the memory of Prof Burton Horowitz, University of Nevada (Reno).
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