We investigated the role of large-conductance Ca2+-activated K+ (BKCa) channels for the basal renal vascular tone in vivo. Furthermore, the possible buffering by BKCa of the vasoconstriction elicited by angiotensin II (ANG II) or norepinephrine (NE) was investigated. The possible activation of renal vascular BKCa channels by cAMP was investigated by infusing forskolin. Renal blood flow (RBF) was measured in vivo using electromagnetic flowmetry or ultrasonic Doppler. Renal preinfusion of tetraethylammonium (TEA; 3.0 μmol/min) caused a small reduction of baseline RBF, but iberiotoxin (IBT; 0.3 nmol/min) did not have any effect. Renal injection of ANG II (1–4 ng) or NE (10–40 ng) produced a transient decrease in RBF. These responses were not affected by preinfusion of TEA or IBT. Renal infusion of the BKCa opener NS-1619 (90.0 nmol/min) did not affect basal RBF or the response to NE, but it attenuated the response to ANG II. Coadministration of NS-1619 with TEA or IBT abolished this effect. Forskolin caused renal vasodilation that was not inhibited by IBT. The presence of BKCa channels in the preglomerular vessels was confirmed by immunohistochemistry. Despite their presence, there is no indication for a major role for BKCa channels in the control of basal renal tone in vivo. Furthermore, BKCa channels do not have a buffering effect on the rat renal vascular responses to ANG II and NE. The fact that NS-1619 attenuates the ANG II response indicates that the renal vascular BKCa channels can be activated under certain conditions.
- vascular smooth muscle
- potassium channels
- Sprague-Dawley rats
the cytosolic free Ca2+ concentration ([Ca2+]i) is an important second messenger in vascular smooth muscles with respect to the basal tone and the contraction as a response to activation of cell-surface receptors and other physiological stimuli (3). The increase in [Ca2+]i is mediated by entry from the extracellular space and/or by mobilization from intracellular stores. In several resistance vessel preparations, the Ca2+ entry occurs via voltage-operated Ca2+ channels mostly of the dihydropyridine-sensitive L type. Our laboratory and others have previously shown that voltage-sensitive Ca2+ channels play a role in the renal responses to vasoactive agents such as angiotensin II (ANG II) and norepinephrine (NE) (29, 30, 32). Smooth muscle cell membrane potential is thus of key importance in the regulation of vascular tone. The plasma membrane potential is controlled by the relative ion permeability of the plasma membrane and the chemical gradient for different ions. Cl− and K+ channels in the plasma membrane of vascular smooth muscle cells (VSMC) have been shown to play an important role in this regulation in resistance vessels of different origin (17). Opening of K+ channels causes hyperpolarization and closure of voltage-sensitive Ca2+ channels. Thus, by controlling the membrane potential, and thereby Ca2+ permeability, these channels have the potential to control vascular tone. K+ channels are the most numerous cation channels in the smooth muscle cell membrane.
Four main classes of K+ channels have been identified in the vascular smooth muscle cells, namely ATP-sensitive K+ channels, Ca2+-activated K+ channels (KCa), voltage-activated K+ channels, and inward rectifier K+ channels. They are all supposed to participate in the regulation of vascular tone (17). Among the KCa channels, several subtypes have been characterized. In VSMC, the dominant KCa subtype is the large-conductance KCa (BKCa). These channels consist of a pore-forming unit and a subunit modulating the Ca2+ sensitivity (18). BKCa channels have also been reported to be stimulated by cAMP in smooth muscle (40, 45). Some reports indicate that there is no major contribution of BKCa channels to the resting membrane potential in several microvascular beds (18). On the other hand, BKCa channels are supposed to participate in negative feedback during microvascular vasoconstriction (4). Also, these channels are reported to contribute to the resting membrane potential during hypertension (18).
A few studies have dealt with the role of BKCa channels in renal vascular beds (8, 15, 27, 42). For example, Prior et al. (27) found that iberiotoxin (IBT), which is considered to be a specific blocker for BKCa channels, constricted isolated rabbit renal arcuate artery. In isolated preglomerular microvessels, Gebremedhin et al. (12) found evidence for two different Ca2+-activated tetraethylammonium (TEA)-sensitive K+ channels using the patch-clamp technique. In another study utilizing the in vitro blood-perfused juxtamedullary nephron technique, it was found that 1 mM TEA constricted afferent arterioles and that the BKCa channel opener NS-1619 caused a dose-dependent dilation of the same vessel (8). In knockout mice lacking the regulatory β-subunit of the BKCa channel (BKCa-β−/−), it was found that the glomerular filtration rate (GFR) and Na+ and K+ excretion were not different from its wild-type counterpart (26). Under volume expansion, however, GFR was significantly lower in the BKCa-β−/− mice. A functional role for BKCa channels has also been suggested in rat juxtaglomerular cells (9).
To investigate the functional role of BKCa channels in renal basal tone and the integrated renal vascular response to vasoconstrictors in vivo, we measured renal blood flow (RBF) using electromagnetic and ultrasonic flowmetry. The BKCa channels were stimulated with NS-1619 and blocked with TEA or IBT. ANG II or NE was used as vasoconstrictive agonists for cell-surface receptors. To increase the cAMP of renal VSMC, we used forskolin, an activator of adenylate cyclase. All drugs were infused directly into the renal artery to minimize systemic effects.
Experiments were performed on male Sprague-Dawley (SD) rats; their body weight averaged 284 ± 6 g (n = 37). The rats were obtained from Taconic (Lille Skensved, Denmark) and were fed standard rat laboratory chow and tap water ad libitum. All experimental protocols were approved by the Danish National Animal Experiments Inspectorate.
Anesthesia was induced with 5% isoflurane delivered in 65% nitrogen and 35% oxygen. Mean arterial blood pressure (MAP) was measured by a Statham P23-dB pressure transducer (Gould, Oxnard, CA) via a polyethylene catheter (PE-50) placed in the left carotid artery. Continuous infusion of isotonic saline at a rate of 20 μl/min was performed via a polyethylene catheter (PE-10) in the right jugular vein together with Pavulon (Organon, Oss, Holland) (0.6 mg/ml). A tracheotomy was performed, and the rat was placed on a servo-controlled heating table to maintain a constant body temperature of 37°C. A small-animal ventilator ensured that the rat was ventilated by a tidal volume of 1.7–2.5 ml and a frequency of 60 breaths/min. The final isoflurane concentration needed to maintain sufficient anesthesia was ∼2%. The abdominal aorta and the left kidney were exposed by midline and subcostal incisions as previously described (36). Thereafter, a tapered and curved polyethylene catheter (PE-10) was introduced into the left iliac artery and advanced ∼1 mm into the left renal artery via the abdominal aorta. The catheter did not interfere with RBF. This catheter was used to administer test agents directly into the renal artery to minimize systemic effects. The left ureter was catheterized (PE-10 connected to PE-50) to ensure free urine flow. A noncannulating precalibrated electromagnetic flow probe (model 101, Scalar Medical, Delft, Holland) or an ultrasonic flow probe (model T 420, Transonic) was placed around the freed left renal artery to measure RBF. After the initial preparation was completed, the rat was allowed to recover for 30–60 min before the experimental protocol was initiated.
An Upchurch six-port injection valve was used to introduce a 10-μl bolus of NE or ANG II into the renal artery infusion line. Four to five minutes before administration of the agonist, the rate of renal artery infusion was increased from 10 to 144 or 288 μl/min (see below). The higher perfusion rate was necessary to infuse NS-1619 at a sufficient rate to establish a renal plasma concentration of ∼30 μM of NS-1619. These rates of infusion allowed administration of the bolus in <5 s. Using saline solution, we established that the infusion did not cause a volume expansion of such a degree that it altered the agonist-induced RBF response.
After recovery of RBF to baseline level, the infusion rate was returned to 10 μl/min to avoid clotting of the catheter. In each rat, doses between 10 and 40 ng of NE and between 1 and 4 ng of ANG II were injected into the renal artery. These injections normally caused excellent dose-dependent responses. The dose that caused a 30–50% reduction in RBF was then used throughout the experiment. After a recovery period of ∼10 min, a second agonist bolus was administered. After an additional recovery period, the pretreatment drug was infused, and an agonist bolus was injected in the presence of the pretreatment solution. IBT, TEA, and NS-1619 were infused at rates of 0.3 nmol/min, 3.0 μmol/min, and 90.0 nmol/min, respectively. These infusion rates were calculated to cause plasma concentrations of ∼100 nM, 1 mM, and 30 μM, respectively. These concentrations have previously been shown to elicit their respective effects in vivo and in vitro (see discussion). The pretreatment agent was infused for 4–5 min. Then the agonist was administrated.
Forskolin (1.6 nmol/min) was administered as continuous infusions over 3 min because this exposure time was required for full effect. IBT was administered for 4 min before and throughout the duration of vasodilator infusion. It has been reported that TEA has an initial effect on arteriolar diameter that disappears after a few minutes (19). We therefore decided to test the effects of 1-, 2-, 3-, and 4-min treatment of this compound on the RBF responses to ANG II and NE. Normally, a control agonist injection was performed after recovery from the pretreatment experiment. If the response to the control agonist injections varied substantially, the experiment was discarded. Also, if the NE or ANG II bolus produced a substantial rise in MAP, the rat was discarded. Occasionally, a minor (<5 mmHg) increase in MAP was observed ∼5 s after the bolus infusion. This might reflect effects of either elevated renal resistance on arterial pressure or minor shunting of the agonist into the systemic circulation. During agonist stimulation, the RBF values presented are an average of registrations obtained during 10 s when the blood flow response to the agonist was maximal. The RBF responses are expressed as absolute values or normalized as a percentage of baseline values. These were calculated separately for each injection using the mean value observed during the 15-s time interval between introduction of agonist (NE or ANG II) and onset of the renal vascular response. The heart rate is analyzed from the continuous blood pressure recordings.
Drugs and chemicals.
NE, ANG II, IBT, forskolin, and TEA (Sigma Aldrich) were dissolved in saline to their final concentrations (10–40 ng/10 μl, 1–4 ng/10 μl, 2.08 μM and 20.8 mM, respectively). NS-1619 (generous gift from Professor Søren-Peter Olesen), nifedipine, and forskolin were dissolved in DMSO and thereafter dissolved in saline to their final concentrations. Our laboratory performed experiments (n = 4) to ensure that the DMSO infused into the kidney did not affect the baseline RBF, MAP, or responses to the agonists (data not shown). Our laboratory has previously tested the validity of the experimental setup by the ability of preinfused prazosin to block the NE response (36).
Twelve-micrometer SD rat kidney sections were fixed in 2% paraformaldehyde for 15 min, permeabilized in 0.2% Triton X-100 in PBS with 4% BSA for 15 min, and blocked in PBS-BSA. Thereafter, the sections were incubated for 45 min. at room temperature with the primary BKCa antibody (rabbit anti-K+ channel Maxi K, AB5228, Chemicon International, Temecular, CA), washed and incubated with a secondary antibody, conjugated to Alexa-488 and rhodamine-phalloidin (Molecular Probes, Eugene, OR) at room temperature for 45 min. After washing, the sections were mounted in Prolong Gold (Molecular Probes) mounting medium and imaged in a Leica TCS SP2 confocal microscope. A staining, in which the primary antibody had been preblocked with its peptide antigen, was performed in parallel to verify the specificity of the primary antibody. We detected no staining when using the preblocked antibody (data not shown). The resulting images were deblurred to enhance fine details using AutoDeblur version X 1.3.3 (AutoQuant Imaging).
Data are presented as means ± SE; n equals number of rats. When a maneuver was repeated in the same rat, the average value of the individual registrations represents the rat. The SigmaStat (Jandel Scientific/SPSS) software was used for data analysis. Statistical significance was evaluated within groups by Student's paired t-test or one-way ANOVA for repeated measurements with the Student-Newman-Keuls post hoc test. Unpaired Student's t-test was used between groups. A P value < 0.05 was considered statistically significant.
The initial RBF and MAP (n = 37) values averaged 6.9 ± 0.3 ml/min and 104 ± 1 mmHg, respectively.
Effects of TEA on baseline RBF and agonist responses.
Preinfusion of TEA (3.0 μmol/min) reduced baseline RBF significantly from 4.6 ± 0.5 to 4.2 ± 0.5 ml/min at 4 min, which corresponds to decrease of 9.5% (n = 6) (Fig. 1). The infusion of TEA was calculated to obtain a renal plasma concentration of ∼1 mM. After 4 min of TEA infusion, there was a significant MAP decrease (Table 1). The heart rate was not affected by the intrarenal TEA infusion. Infusion of NaCl for 4 min caused a minor (∼1 mmHg), but significant, increase in MAP (n = 37) (Table 1). The RBF responses to ANG II (1–4 ng) (n = 5) and NE (10–40 ng) (n = 5) were not affected by 1, 2, 3, or 4 min of TEA infusion (Fig. 2, Table 2). Because the effect of TEA infusion at the rate of 3.0 μmol/min was limited, we performed preliminary experiments with a higher infusion rate (30.0 μmol/min). This rate of infusion caused, however, an immediate large drop of both MAP and RBF making injection of the agonists impossible.
Effects of IBT on baseline RBF and vasoconstrictor responses.
Renal artery infusion of the specific BKCa channel blocker IBT at a rate of 0.3 nmol/min, corresponding to an approximate renal plasma concentration of 100 nM, for 4 min did not affect baseline RBF (n = 14) (Fig. 1). As was the case with TEA, the heart rate was not affected by IBT infusion. In agreement with the observation in the TEA infusion series, the RBF responses to ANG II (1–4 ng) or NE (10–40 ng) were unaltered by pretreatment with IBT (n = 6) (Fig. 3, Table 2).
Effects of IBT on forskolin responses.
Forskolin, an activator of adenylate cyclase, was used to test the role of BKCa channels in the cAMP-mediated renal vasodilation because it has been reported that these channels are regulated by cAMP (40, 45). We found that intrarenal infusion of forskolin (1.6 nmol/min) for 3 min caused a 19% (from 9.3 ± 0.5 to 11.0 ± 0.5 ml/min) increase in RBF during control conditions (Fig. 4) (n = 5). Four-minute pretreatment with IBT (0.3 nmol/min) does not attenuate the response to forskolin. On the other hand, IBT augments the forskolin-induced response to 27% (from 8.4 ± 0.5 to 10.7 ± 0.5 ml/min) (Fig. 4) (n = 5). Interestingly, in the control experiments, forskolin decreased MAP significantly from 99 ± 2 to 95 ± 2 mmHg. After IBT treatment forskolin does not affect MAP (102 ± 3 and 103 ± 3 mmHg, baseline vs. forskolin, respectively).
Effects of NS-1619 on baseline RBF and agonist responses.
To stimulate the BKCa channels, we infused the channel opener NS-1619 at a rate of 90.0 nmol/min for 5 min. This rate of infusion is calculated to give a renal plasma concentration of ∼30 μM. Higher concentrations have been reported to inhibit Ca2+ channels (8). This infusion did not cause any changes in baseline RBF or MAP (n = 10) (Table 1, Fig. 5). Also, the response to NE (10–40 ng) was unaffected by NS-1619 treatment (n = 5) (Fig. 6, Table 2). On the other hand, the response to ANG II (1–4 ng) was attenuated by NS-1619 pretreatment (n = 14) (Fig. 7, Table 3). This attenuation was abolished when TEA (3.0 μmol/min) (n = 8) or IBT (0.3 nmol/min) (n = 6) was administrated together with NS-1619 (Fig. 7, Table 3). This finding indicates that the effect of NS-1619 was not exerted via inhibition of L-type Ca2+ channels. When NS-1619 was coadministered with TEA (3.0 μmol/min), there was a small decrease in baseline RBF and MAP (Table 1, Fig. 5). The lack of effect of NS-1619 on baseline RBF could be due to the fact that voltage-sensitive Ca2+ channels are not activated under these conditions. However, when we infused the L-type Ca2+ channel blocker nifedipine (0.3, 3 nmol/min, and 30 nmol/min), we found a dose-dependent renal vasodilation as summarized in Fig. 8 (n = 5). This finding indicates that there is a potential for hyperpolarization-induced vasodilation under basal conditions.
To verify the presence of BKCa channels in renal afferent arterioles, we analyzed a series of immunohistological stainings of kidney sections by confocal microscopy. We detected a high BKCa channel expression in the media of afferent arterioles and interlobular arteries of the kidney. We also noted a low expression of BKCa channels in the tubules (Fig. 9).
In this study, we tested the hypothesis that BKCa channels are involved in the regulation of renal hemodynamics and that the response to activation with ANG II or NE is buffered by activation of these channels. We quantified RBF in vivo using electromagnetic or ultrasonic flowmetry. We also verified the existence of the BKCa channels in the rat renal microvasculature by immunohistochemistry.
We found that 4-min intrarenal infusion of TEA at a rate giving a calculated plasma concentration of ∼1 mM caused a significant fall in the basal RBF by 9.5%. This finding is in accord with earlier findings where 1 mM TEA caused an afferent arteriolar constriction in the blood perfused juxtamedullary nephron (8, 15). On the other hand, when we repeated the same maneuver with a more specific BKCa channel blocker, IBT, there was no change in basal RBF. Thus the BKCa channels do not appear to be involved in the maintenance of the basal RBF, indicating that these channels do not contribute to the normal resting potential under control conditions. This is in accordance with findings from other vascular beds (18). TEA or IBT failed to constrict hamster cremaster arterioles in vivo (19). Similarly, it was found that the resting diameter of cerebral arterioles was unaffected by 1–3 mM TEA or 50 nM IBT (25). Furthermore, it is reported that superfusion of guinea pig submucosal arteriole with 10 mM TEA for 60 min or with 88 nM IBT for 5 min had no effect on the diameter of this vessel (13).
The discrepancy between the effects of TEA and IBT on the basal renal blood flow might be explained by the fact that TEA blocks other K+ channels such as voltage- and ATP-sensitive K+ channels (34, 39). In rat aortic rings, TEA increases the frequency and amplitude of the oscillatory waves elicited by phenylephrine, whereas there is no effect of IBT in this regard (39). Although TEA is considered relatively specific at the plasma concentrations employed in this study, it is likely that the effect of TEA is explained by an effect on other channel types than the BKCa channels.
It might be argued that the concentration of IBT was insufficient to cause a block of the BKCa channels. The concentration in the renal plasma (∼100 nM) in the present study is, however, substantially higher than the IC50 value of 0.25–5 nM reported for IBT inhibition of BKCa channels from vascular smooth muscle cells (10, 20). Also, in in vivo experiments using pigs, it was found that an even lower dose of IBT (1 μg/kg) caused a 30–60% increase in total peripheral resistance (44). The fact that IBT, in the present study, abolished the NS-1619 induced attenuation of the ANG II response is another indication that the intrarenal concentration of IBT is sufficient to block BKCa channels (see below). Also, IBT totally abolished the elevated MAP associated with forskolin infusion (see below). This indicates that IBT has a systemic effect, and because the infusion is intrarenal, any hypothetical intrarenal targets should thus have been reached.
It is also worth commenting on the possible role of the volatile anesthetics. It has been reported that isoflurane and desflurane can cause renal vasodilation in cats (38). Gas anesthetic-induced vasodilation has also been found in other tissues such as the mesenteric and coronary circulation (11, 21). It has been suggested that the vasodilation induced by the gas anesthetics might, at least in part, be attributed to opening of Ca2+-activated K+ channels (21). Such an effect of gas anesthetics on the renal BKCa channels is not likely in the present study because IBT was without effect both with respect to baseline RBF and the action of NE and ANG II.
The cell membrane in smooth muscle cells has a high electrical resistance (18). Therefore, small decreases in the activity of K+ channels would lead to depolarization of the smooth muscle cell membrane. It has been suggested that the myogenic response in rat small mesenteric arteries (outer diameter 267–474 μm) is mediated by closure of charybdotoxin-sensitive Ca2+-activated K+ channels (43). Thus it is possible that the vascular smooth muscle depolarization, which is a link in the chain leading to agonist-induced vasoconstriction, might be at least partially mediated via closure of BKCa channels. Experimental evidence for ANG II-induced closure of BKCa channels from coronary vessels has also been presented (41). This might be one of the mechanisms behind ANG II-induced vasoconstriction. With respect to renal hemodynamics, combining results in the literature provide support for such a mechanism. Carroll et al. (6) showed that infusion of ANG II to the isolated perfused rabbit kidney induced an approximately sevenfold increase in the release of the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) (6). This finding was confirmed by subsequent observation in rat isolated renal arterioles (7). 20-HETE in turn has been shown to cause constriction of afferent arterioles that is inhibited by blockade of BKCa channels (15). It is also reported that 17-octadecynoic acid, an inhibitor of the formation of 20-HETE, attenuates the vasoconstrictor response to ANG II in isolated rat renal interlobular arteries (1). However, the results of the present in vivo study do not support the notion that agonist-induced constriction of renal resistance vessels is mediated via 20-HETE-induced action on BKCa channels, because pharmacological inhibition of these channels did not affect the renal vasoconstriction elicited by ANG II or NE. Still, we cannot exclude the possibility that an 20-HETE induced effect on BKCa channels under certain conditions might modulate renal vascular responses.
It has also been suggested that activation of BKCa channels might function as a negative-feedback mechanism to buffer the extent of contraction during activation with vasoactive agonists (4). Jackson and Blair (19) found in hamster cremaster arteriolar cells that IBT significantly increased the number of cells that contracted in response to 1 μM NE. In this regard, Stockand and Sansom (37) also found that ANG II increased the open probability of BKCa channels in cultured human mesangial cells. Another pathway by which vasoconstrictors such as ANG II and NE might act on BKCa channels is via activation of protein kinase C (PKC) (22). A role for PKC has also been suggested in ANG II and NE-mediated renal vasoconstriction (31). Several studies indicate that PKC inhibits BKCa in smooth muscle, but an opposite action has also been reported [see review by Ledoux et al. (22)]. Thus blockade of BKCa channels has a theoretical potential via several pathways for both augmentation and attenuation of the agonist induced renal vascular responses.
Regarding renal effects, it has previously been shown by Fallet et al. (8), utilizing the juxtamedullary nephron technique, that 1 mM TEA had no effect on the ANG II and arginine vasopressin (AVP)-induced contractions of the juxtamedullary afferent and efferent arterioles. The lack of effect on the efferent arteriole could be expected because this vascular segment does not respond with depolarization to ANG II treatment (23). The afferent arteriolar response to ANG II, however, involves smooth muscle cell depolarization and could therefore potentially be affected by modulation of K+ channels (23). The afferent arteriolar responses to AVP are also blocked by nifedipine, an inhibitor of voltage-sensitive Ca2+ channels (16). In the study by Fallet et al., only the juxtamedullary glomerular arterioles were studied. The blood flow via the juxtamedullary circulation represents only ∼15% of the total RBF. In the present study, we wanted to investigate whether the findings by Fallet et al. could be generalized to the entire renal circulation. We also investigated the effect on another vasoconstrictor, NE, which exerts its renal vascular action via slightly different and less potential-dependent intracellular pathways (30, 32). However, we found, in agreement with Fallet et al., that blockade of BKCa channels with either IBT or TEA did not affect the RBF responses to ANG II or NE.
It is possible that vasodilators exert their effect via activation of BKCa channels (18). It has, for example, been reported that cAMP, the concentration of which is increased by several vasodilators, might regulate the activity of BKCa channels (40, 45). We found that forskolin, an activator of adenylate cyclase, caused a significant renal vasodilation. This vasodilation was not attenuated by prior and concomitant intrarenal infusion of IBT. On the contrary, there was a small but significantly increased effect of forskolin after IBT treatment. We do not have a complete explanation for this unexpected effect. It should, however, be noted that the forskolin infusion caused a decrease in MAP from 99 to 95 mmHg under control conditions and that IBT treatment abolished the MAP effect. Thus, we cannot exclude the possibility that the fall in MAP elicits a reflex renal vasoconstriction that counteracts the forskolin- induced renal vasodilation. This also indicates that BKCa channels play a role in the forskolin-induced systemic vasodilation.
To our knowledge, the effects of direct stimulation of the BKCa channels in the renal circulation has never been tested in vivo before. A 5-min intrarenal infusion of the BKCa channel activator NS-1619 at a rate giving a calculated plasma concentration of ∼30 μM did not affect the basal RBF in vivo. This finding is surprising because we could demonstrate the presence of these channels in the afferent arterioles with immunohistochemistry. It could be argued that the doses used are insufficient. However, concentrations of NS-1619 as low as 3 μM relaxed phenylephrine precontracted rat aortic rings by 75% (5). Furthermore, superfusion of 10 μM of NS-1619 caused an 11% dilatation of rat cerebral arteriolar diameter in vivo (28). In addition to its stimulatory effects on BKCa channels, NS-1619 has been shown to inhibit Ca2+ channels (14). In the study by Fallet et al. (8), NS-1619 concentrations of 100 μM and higher also exerted an unspecific vasodilation that was not counteracted by TEA treatment. As a result of these observations and methodological limitations in our experimental setting, we refrained from using higher concentrations (see methods). However, in contrast to our study, Fallet et al. report that 30 μM of NS-1619 significantly dilated juxtamedullary afferent arterioles (8). Currently, we are not able to completely explain this discrepancy between the two studies. Two possible explanations should, however, be considered. First, as mentioned above, the juxtamedullary afferent arterioles might represent a subpopulation of arterioles sensitive to stimulation of BKCa channels with NS-1619. As the integrated response of all arterioles is measured in the present study, the possible effect on the juxtamedullary afferent arterioles might be masked by the absent response of the majority of the arterioles. Subtypes of BKCa channels with different sensitivities to membrane potential and Ca2+, but with the same single-channel conductance, has been found in bovine mesenteric arteries (33). This difference might be due to variation in BKCa channel β-subunits. BKCa channels consists of α- and β-subunits. Several splice variants of the slo gene encoding for the α-subunit have been identified, and in addition, there are four genes encoding for the β-subunit (22). BKCa channels containing different β-subunits have been reported to have different activation patterns with respect to voltage and Ca2+ sensitivity (22). Thus juxtamedullary and cortical afferent arterioles might contain BKCa channels with different β-subunits. Second, the different experimental conditions can play a role in this context. It is reported that activation of BKCa by NS-1619 does not occur in the hyperpolarized (−80 mV) state or in the absence of Ca2+ (24). Thus, NS-1619 does not under all conditions activate BKCa channels; it merely causes a leftward shift of the dose-response curve of the Ca2+ activation. Stimulation with ANG II depolarizes the plasma membrane and causes an elevation of the [Ca2+]i. This increase in [Ca2+]i was not sufficient to activate the BKCa channels because the response to ANG II was unaffected by IBT and TEA. When, on the other hand, the BKCa channels are sensitized with NS-1619, we observed an attenuation of the ANG II response. The combined effect of the ANG II-induced increase in [Ca2+]i and the sensitization caused by NS-1619 might lead to an activation of the BKCa channels. Opening of these channels will counteract the depolarization that would lead to further increase in [Ca2+]i and thus attenuate the contractile response of ANG II. This suggests that the renal VSMC under our control conditions are too hyperpolarized or have too low a Ca2+ concentration to enable activation of the BKCa channels. This might, at least in part, explain the lack of effect of NS-1619 on baseline RBF in the present study. Furthermore, because it has been reported that Ca2+ antagonists have a limited effect on basal RBF in vivo, the possibility that voltage-sensitive Ca2+ channels are not activated under our basal experimental conditions has to be taken into consideration (35). If these channels are inactive, hyperpolarization induced by opening of BKCa channels would be without effect on renal hemodynamics. However, in accord with other investigators, we found that nifedipine caused a significant renal vasodilation, which indicates that there is a potential for stimulation of BKCa channels to increase basal RBF (2).
As mentioned above, it has been argued that NS-1619 might inhibit Ca2+ channels. This could potentially explain the attenuating effect on the ANG II-induced renal response as this has been shown to be dependent on Ca2+ channels (16, 32). However, simultaneous renal infusion of TEA abolished the attenuation caused by NS-1619, which indicates that the effect is not mediated via activation of Ca2+ channels. The fact that the specific BKCa blocker IBT also abolished the attenuating effect of NS-1619 on ANG II responses strongly indicates that the effect of NS-1619 is mediated via activation of BKCa channels. The fact that the response to NE is not attenuated by NS-1619 might be explained by observations indicating that the renal preglomerular response to this compound is less dependent on Ca2+ entry via voltage-sensitive channels than the response to ANG II (16, 30).
To summarize, we confirmed the existence of BKCa channels in the renal preglomerular vasculature. Blockade of these channels with TEA or IBT via intrarenal infusion does not affect the reduction in RBF induced by the vasoactive agents NE and ANG II in vivo in rats. IBT does not affect baseline RBF, whereas TEA caused a fall that might be explained by blockade of other K+ channels. Pharmacological activation of renal BKCa channels with NS-1619 did not affect baseline RBF or the responses to NE. There is, however, a small but significant attenuation of the response to ANG II, which was abolished when TEA or IBT was administered together with NS-1619. The forskolin-induced renal vasodilation was not attenuated by BKCa channel blockade. It is therefore concluded that BKCa channels do not play a major role in maintenance of baseline RBF or buffering of the rat renal vascular responses to NE and ANG II. The attenuating effect of NS-1619 on the ANG II responses indicates, however, that the renal vascular BKCa channels can be activated under certain conditions.
The present study was supported by grants from the Danish Medical Research Council, the Novo-Nordisk Foundation, and the König-Petersen Foundation.
The technical assistance of Anni Salomonsson, Trine Eidsvold, and Ian Godfrey is gratefully acknowledged. We also thank Prof. Søren-Peter Olesen for fruitful discussions regarding the use of NS-1619.
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.
- Copyright © 2007 the American Physiological Society