Department of Physiology and Biophysics, University of Nebraska College of Medicine, Omaha, Nebraska 68198-4575
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ABSTRACT |
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Experiments were performed to evaluate the hypothesis that opening of Ca2+-activated K+ channels (BKCa channels) promotes juxtamedullary arteriolar dilation and curtails constrictor responses to depolarizing agonists. Under baseline conditions, afferent and efferent arteriolar lumen diameters averaged 23.4 ± 0.9 (n = 36) and 22.8 ± 1.1 (n = 13) µm, respectively. The synthetic BKCa channel opener NS-1619 evoked concentration-dependent afferent arteriolar dilation. BKCa channel blockade (1 mM tetraethylammonium; TEA) decreased afferent diameter by 15 ± 3% and prevented the dilator response to 30 µM NS-1619. ANG II (10 nM) decreased afferent arteriolar diameter by 44 ± 4%, a response that was reduced by 30% during NS-1619 treatment; however, TEA failed to alter afferent constrictor responses to either ANG II or arginine vasopressin. Neither NS-1619 nor TEA altered agonist-induced constriction of the efferent arteriole. Thus, although the BKCa channel agonist was able to curtail afferent (but not efferent) arteriolar constrictor responses to ANG II, BKCa channel blockade did not allow exaggerated agonist-induced arteriolar constriction. These observations suggest that the BKCa channels evident in afferent arteriolar smooth muscle do not provide a prominent physiological brake on agonist-induced constriction under our experimental conditions.
afferent arteriole; angiotensin II; arginine vasopressin; efferent arteriole; NS-1619; tetraethylammonium
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INTRODUCTION |
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CALCIUM INFLUX INTO arteriolar myocytes through dihydropyridine-sensitive voltage-gated channels and the resulting increase in intracellular Ca2+ concentration ([Ca2+]i) are important processes for maintaining renal vascular tone and evoking agonist-induced vasoconstriction. These events are especially prominent in the preglomerular afferent arteriole, exerting little impact on efferent arteriolar function (32). Because the open probability of voltage-gated Ca2+ channels is governed primarily by membrane potential, regulation of this parameter is crucial for appropriate control of preglomerular microvascular tone. The membrane potential of vascular smooth muscle is determined largely by sarcolemmal K+ conductance, with opening of K+ channels promoting hyperpolarization and closing of K+ channels favoring depolarization. Patch-clamp studies have revealed several types of K+ channel in renal preglomerular vascular smooth muscle cells (15, 16, 18, 37, 44), including the large-conductance Ca2+-activated K+ channel (BKCa channel). Expressed in smooth muscle cells from a variety of sources, the open probability of the BKCa channel is elevated exponentially by increases in [Ca2+]i and by membrane depolarization (5).
Although involvement of BKCa channels in vascular control can be envisioned in several scenarios, their functional role is most widely viewed as providing negative-feedback regulation of intrinsic vascular tone (3). This phenomenon arises by virtue of the effect of vasoconstrictor stimuli to induce membrane depolarization and increase [Ca2+]i, events that promote opening of BKCa channels, thereby preventing excessive depolarization and limiting Ca2+ influx through voltage-gated channels. Patch-clamp studies have demonstrated that ANG II provokes negative-feedback activation of BKCa channels in human mesangial cells (41) and vascular smooth muscle from canine renal artery (16). Conversely, a reduction in BKCa channel activity may contribute to agonist-induced membrane depolarization under some conditions. Such a process is indicated by the ability of ANG II to inhibit BKCa channels from coronary artery smooth muscle (43), a phenomenon proposed to involve channel phosphorylation (5). The ability of depolarizing and Ca2+ mobilizing agonists such as ANG II to variably increase or decrease BKCa channel activity could reflect functional differences between vascular beds (12, 26) or between macrovessels and microvessels (22).
The role of BKCa channels in regulating renal microvascular function has been scrutinized primarily with regard to the ability of P-450 metabolites of arachidonic acid to alter channel activity (44, 45). However, little information is available regarding the involvement of BKCa channels in promoting or limiting agonist-induced renal vasoconstriction. Pressurized intrarenal arteries (150-250 µm diameter) isolated from rat kidney and studied under conditions of phenylephrine-induced tone have been shown to constrict in response BKCa channel blockade (37, 44), indicating that negative-feedback opening of BKCa channels tempers the constrictor impact of phenylephrine on the renal vasculature. In addition, electrophysiological studies have shown that endothelin-1 provokes negative-feedback activation of BKCa channels in smooth muscle cells isolated from interlobar and arcuate arteries (18). The impact of BKCa channel activity on afferent and efferent arteriolar responsiveness to peptide vasoconstrictor agonists remains unexplored. Accordingly, experiments were performed to test the hypothesis that opening of BKCa channels limits the afferent arteriolar contractile response to peptide agonists known to provoke depolarization and Ca2+ mobilization. Because efferent arteriolar function is relatively independent of membrane potential, we further postulated that BKCa channel activity would have little impact on contractile responses in this vascular segment.
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METHODS |
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Animals
The procedures used in this study were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee and were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats (SAS:VAF strain) were purchased from Charles River Laboratories (Wilmington, MA) and were provided ad libitum access to food and water before study.In Vitro Blood-Perfused Juxtamedullary Nephron Technique
Arteriolar contractile function was assessed in experiments performed using the in vitro blood-perfused juxtamedullary nephron technique (10). Each rat was anesthetized with pentobarbital sodium (50 mg/kg ip). For some experiments, enalaprilat (2 mg ia) was administered to suppress endogenous ANG II formation and its impact on vascular tone. Thirty minutes later, the right renal artery was cannulated via the superior mesenteric artery. This procedure initiated in situ perfusion of the kidney with Tyrode solution containing 52 g/l dialyzed BSA. The rat was then exsanguinated via a carotid arterial cannula into a heparinized syringe, and the kidney was harvested for in vitro study. Renal perfusion was maintained throughout the dissection procedure needed to reveal the tubules, glomeruli, and vasculature of juxtamedullary nephrons. Ligatures were placed around the distal segments of the large arterial branches that supplied the exposed microvasculature.The collected blood was processed to remove leukocytes and platelets, as detailed previously (20). The resulting perfusate was stirred continuously in a closed reservoir that was pressurized under 95% O2-5% CO2, thus providing both oxygenation and the driving force for perfusion of the dissected kidney at a constant renal arterial pressure of 110 mmHg. The renal perfusion chamber was warmed, and the tissue surface was superfused with Tyrode solution containing 10 g/l BSA at 37°C. The renal microvasculature was transilluminated on the stage of a compound microscope, and a single afferent or efferent arteriole was selected for study based on visibility and blood flow. Video images of each microvessel were generated continuously and stored on videotape for later analysis. In three experiments, two vessels could be visualized clearly within the same field of view, a situation that allowed responses of both vessels to be recorded simultaneously and analyzed separately during videotape playback.
Experimental Protocols
Effects of BKCa channel manipulation on afferent arteriolar diameter. The ability of BKCa channels to elicit afferent arteriolar dilation was assessed in experiments employing NS-1619, a synthetic BKCa channel agonist (36). NS-1619 was dissolved in ethanol to a concentration of 69 mM and diluted in Tyrode bath on the day of the experiment. Afferent arteriolar lumen diameter was monitored under baseline conditions and during sequential exposure to increasing concentrations of NS-1619 via the bathing solution (10-300 µM; 5-7 min at each concentration), followed by a recovery period. The extent to which the dilator response to NS-1619 could be attributed to opening of BKCa channels was examined based on responses to NS-1619 during exposure to the BKCa channel blocker tetraethylammonium chloride (TEA, 1 mM; included in both the perfusate blood and the superfusate bath). Recovery of TEA-treated arterioles from NS-1619 was followed by exposure to the organic Ca2+ channel blocker diltiazem (10 µM) during the continued presence of TEA. Other studies assessed the ability of charybdotoxin (CbTX; 50 and 100 nM) to reverse the afferent arteriolar response to 30 µM NS-1619.
Effect of BKCa channel activation on ANG II-induced arteriolar vasoconstriction. These experiments evaluated the ability of pharmacological BKCa channel activation to suppress agonist-induced arteriolar constriction. With the use of tissue harvested from enalaprilat-treated rats, afferent and efferent arteriolar diameter responses to increasing concentrations of ANG II were evaluated by exposing the juxtamedullary microvasculature to the following superfusate bathing solutions: 1) Tyrode solution alone (5-10 min); 2) Tyrode solution containing 1 and 10 nM ANG II (3 min at each concentration); and 3) Tyrode solution alone (10 min). After this recovery period, NS-1619 was added to the Tyrode bathing solution to achieve a final concentration of 30 µM. A 5-min NS-1619 treatment period preceded initiation of the second ANG II exposure sequence (1 and 10 nM ANG II; 3 min each) in the continued presence of NS-1619. This was followed by a recovery period during which the tissue was exposed to Tyrode solution containing NS-1619 alone. Imposition of two consecutive ANG II exposure sequences according to this protocol evokes indistinguishable juxtamedullary arteriolar diameter responses, even at peptide concentrations 10-fold higher than those employed in the present study (6). The NS-1619-containing bathing solutions employed in this protocol contained 0.04% ethanol. We previously found no effect of 0.1% ethanol on afferent arteriolar basal diameter or responsiveness to changes in perfusion pressure (42).
Effect of BKCa channel blockade on agonist-induced contractile responses. To determine the extent to which agonist-induced constriction is blunted by physiological (negative-feedback) activation of BKCa channels, TEA treatment was employed to block BKCa channels during exposure to vasoconstrictor agonists. With the use of tissue harvested from enalaprilat-treated rats, afferent or efferent arteriolar diameter responses to ANG II (1 and 10 nM) were documented before and during TEA exposure (1 mM in both perfusate blood and superfusate bath) according to the protocol described above. To determine the agonist specificity of this phenomenon, additional experiments determined the effect of TEA on afferent and efferent arteriolar constrictor responses to 0.1 and 1 nM arginine vasopressin (AVP). AVP responsiveness studies employed tissue harvested from rats not subjected to enalaprilat pretreatment.
Chemicals and Reagents
Enalaprilat was a gift from Merck Research Laboratories (Rahway, NJ). NS-1619 was purchased from RBI (Natick, MA), and diltiazem hydrochloride was from Marion Merrell Dow (Kansas City, MO). All other reagents were purchased from Sigma Chemical (St. Louis, MO).Data Analysis
Arteriolar lumen diameter was measured from videotaped images at 12-s intervals from a single point along the length of the vessel. The average diameter (in µm) during the final minute of each treatment period was used for statistical analysis by repeated-measures ANOVA or Friedman's repeated-measures ANOVA on ranks, as appropriate, followed by the Newman-Keuls test. Between-group comparisons employed the unpaired t-test. Statistical computations were performed using the SigmaStat 2.03 software package (SPSS, Chicago, IL), with statistical significance defined as P < 0.05. All data are reported as means ± SE (n = number of arterioles). ![]() |
RESULTS |
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Acute enalaprilat treatment was employed to suppress endogenous
ANG II formation for experiments examining arteriolar responsiveness to
ANG II, whereas other experiments used tissue from rats not receiving
enalaprilat. As detailed in Table 1,
neither afferent nor efferent arteriolar baseline diameter differed
significantly between untreated and enalaprilat-treated groups.
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Effects of BKCa Channel Manipulation on Afferent Arteriolar Diameter
Figure 1 shows the effect of the BKCa agonist NS-1619 on afferent arteriolar lumen diameter. Baseline afferent diameter averaged 24.3 ± 1.9 µm (n = 5). Addition of NS-1619 to the bathing solution produced a concentration-dependent increase in diameter, with 30 µM NS-1619 increasing diameter by 2.8 ± 0.8 µm (P < 0.05 vs. baseline) and 300 µM NS-1619 further increasing diameter to a value averaging 6.5 ± 0.8 µm greater than baseline (P < 0.05 vs. baseline; P < 0.05 vs. 30 µM NS-1619). Removal of NS-1619 from the bathing solution restored afferent arteriolar lumen diameter to 25.5 ± 1.9 µm (P > 0.05 vs. baseline). The reversible, concentration-dependent afferent arteriolar dilation evoked by NS-1619 is consistent with the previously described actions of this agent to increase the open probability of BKCa channels in vascular smooth muscle (36).
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Figure 2 summarizes data regarding the
extent to which the afferent arteriolar diameter response to NS-1619
can be attributed to opening of BKCa channels in our
experimental setting. For comparison purposes, the dilator response to
30 µM NS-1619 in the absence of any BKCa blocker (Fig. 1)
is also included in Fig. 2. In experiments performed in their entirety
with 1 mM TEA present in both the perfusate blood and the superfusate
bath, 30 µM NS-1619 failed to provoke afferent arteriolar dilation
(0.5 ± 1.1 µm, n = 4; Fig. 2). Subsequent
exposure to 100 and 300 µM NS-1619 increased afferent diameter by
3.0 ± 1.1 and 5.4 ± 1.1 µm, respectively, responses that
did not differ significantly from those observed in experiments
performed in the absence of TEA (Fig. 1). After recovery from NS-1619,
diltiazem (10 µM) evoked a 6.1 ± 2.5 µm increase in afferent
diameter during TEA treatment (n = 4), confirming that this
BKCa channel blocker does not provoke a nonspecific abolition of vasodilator responsiveness. In other experiments, the
afferent arteriolar dilation elicited by 30 µM NS-1619 [change (
) = 1.3 ± 0.4 µm; n = 4] was reversed
upon addition of 50 nM CbTX to the NS-1619-containing bath (
0.2 ± 0.6 µm vs. baseline; Fig. 2), and 100 nM CbTX further reduced
arteriolar diameter to a value averaging 1.8 ± 0.7 µm below
baseline. Because the CbTX- and TEA-sensitive nature of the afferent
dilation provoked by 30 µM NS-1619 indicates the involvement of
BKCa channels in this response, this concentration was
employed in all further experiments involving pharmacological
BKCa channel activation.
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Effect of BKCa Channel Activation on ANG II-Induced Arteriolar Vasoconstriction
Figure 3 summarizes the effects of NS-1619 on afferent and efferent arteriolar diameter responses to exogenous ANG II in kidneys harvested from enalaprilat-treated rats. Afferent arteriolar lumen diameter averaged 23.0 ± 1.4 µm (n = 8) under untreated baseline conditions and declined by 3.9 ± 1.2 and 10.5 ± 1.3 µm during exposure to 1 and 10 nM ANG II, respectively (Fig. 3A). Upon removal of ANG II from the bathing solution, arteriolar diameter was restored to 102 ± 4% of baseline. Subsequent exposure to 30 µM NS-1619 increased afferent diameter to 25.2 ± 1.7 µm (P < 0.05 vs. untreated baseline). During continued NS-1619 exposure, ANG II reduced afferent diameter to values averaging 2.6 ± 1.8 µm (1 nM ANG II) and 7.3 ± 1.1 µm (10 nM ANG II) below the NS-1619 baseline. The response to 10 nM ANG II during NS-1619 treatment was diminished significantly compared with the response observed in the same arterioles before NS-1619 treatment. On average, pharmacological opening of BKCa channels by NS-1619 exposure suppressed afferent arteriolar responsiveness to 10 nM ANG II by 29 ± 7%.
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Figure 3B summarizes the efferent arteriolar vasoconstrictor
responses to ANG II in the absence and presence of NS-1619. Efferent arteriolar baseline diameter averaged 24.8 ± 2.2 µm
(n = 5) under untreated baseline conditions. ANG II (1 nM) tended to decrease efferent diameter ( =
1.2 ± 0.6 µm), and 10 nM ANG II decreased lumen diameter by 5.7 ± 0.7 µm
(P < 0.05 vs. baseline). Efferent diameter was
restored to 24.1 ± 2.5 µm during recovery from ANG II
exposure. Subsequent exposure to 30 µM NS-1619 did not significantly alter efferent diameter (23.7 ± 2.7 µm) or constrictor responses to
ANG II (1 nM,
=
0.7 ± 0.3 µm; 10 nM,
=
4.5 ± 0.6 µm; both P > 0.05 vs. untreated). Thus,
in contrast to responses observed in afferent arterioles, treatment
with the BKCa channel opener failed to alter efferent
arteriolar constrictor responses to exogenous ANG II.
Effect of BKCa Channel Blockade on Agonist-Induced Contractile Responses
To determine the impact of physiological opening of BKCa channels on agonist-induced arteriolar constriction, ANG II responses were assessed before and during BKCa channel blockade. Afferent arteriolar diameter averaged 25.5 ± 3.3 µm (n = 6) under baseline conditions. As shown in Fig. 4A, afferent diameter decreased by 4.3 ± 1.6 µm upon exposure to 1 nM ANG II (P < 0.05) and further declined to a value averaging 10.6 ± 2.0 µm below baseline when exposed to 10 nM ANG II (P < 0.05 vs. baseline). Removal of ANG II from the bath restored diameter to 25.4 ± 3.1 µm. Addition of 1 mM TEA to both the perfusate blood and the superfusate bath reduced afferent arteriolar diameter by 3.4 ± 1.0 µm (P < 0.05 vs. untreated baseline). During TEA treatment, ANG II reduced afferent arteriolar lumen diameter by 2.4 ± 0.7 µm (1 nM) and 9.4 ± 1.7 µm (10 nM). These responses to ANG II in the presence of TEA did not differ from those observed in the same arterioles before TEA treatment. In additional experiments, responses to 1 and 10 nM ANG II were assessed before and during exposure to either 100 nM iberiotoxin (IbTX; n = 2) or 50 nM CbTX (n = 1). ANG II (10 nM) decreased afferent diameter by 11.6 ± 1.5 µm before and 11.4 ± 0.4 µm during exposure to these highly specific BKCa antagonists. Because these studies failed to reveal any tendency for BKCa blockade to alter ANG II-induced afferent arteriolar constriction, they were not pursued further because of the virtually prohibitive expense of using the scorpion toxins in our experimental setting.
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The effect of TEA treatment on efferent arteriolar ANG II responsiveness is shown in Fig. 4B. Baseline efferent arteriolar diameter averaged 21.9 ± 1.1 µm (n = 3), and 1 and 10 nM ANG II reduced lumen diameter by 6.0 ± 3.6 and 13.8 ± 3.5 µm (P < 0.05 vs. baseline). During subsequent TEA treatment, baseline diameter averaged 20.5 ± 2.1 µm, and ANG II reduced efferent diameter by 4.6 ± 2.9 and 13.7 ± 3.1 µm, respectively (both P > 0.05 vs. responses before TEA). Thus, although the BKCa channel agonist (NS-1619) suppressed afferent arteriolar diameter responses to ANG II, neither the afferent nor the efferent arteriolar ANG II response was exaggerated after BKCa channel blockade.
The impact of BKCa channel blockade (TEA) on AVP-induced
renal arteriolar constriction is shown in Fig.
5. These experiments were performed using
tissue harvested from rats not receiving enalaprilat treatment.
Afferent arteriolar diameter averaged 21.6 ± 2.3 µm
(n = 6) under untreated baseline conditions and was
diminished by 4.6 ± 1.8 and 15.3 ± 2.6 µm, respectively,
during exposure to 0.1 and 1.0 nM AVP (Fig. 5A). Upon
removal of AVP from the bathing solution, afferent diameter was
restored to 20.4 ± 2.0 µm (P > 0.05 vs.
baseline). Subsequent addition of TEA to both the perfusate blood and
the superfusate bath reduced afferent diameter to 18.2 ± 1.8 µm. In the continued presence of TEA, 0.1 and 1 nM AVP reduced
afferent diameter by 3.9 ± 1.0 and 13.2 ± 1.2 µm,
respectively. These responses did not differ significantly from those
exhibited by the same arterioles before TEA treatment. Hence, as was
the case for ANG II, AVP-induced afferent arteriolar constriction was
not exaggerated by TEA treatment.
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Efferent arteriolar AVP responsiveness in the absence and presence of 1 mM TEA is summarized in Fig. 5B. Efferent diameter averaged
21.5 ± 1.6 µm under untreated baseline conditions
(n = 5), tended to decline upon exposure to 0.1 nM AVP
( =
0.8 ± 0.2 µm), and significantly decreased in
response to 1 nM AVP (
=
10.9 ± 2.1 µm). TEA
treatment failed to alter efferent diameter (20.2 ± 2.1 µm) or
the change in diameter elicited by AVP (0.1 nM,
=
0.5 ± 0.2 µm; 1 nM,
=
10.9 ± 1.4 µm). Thus the
efferent arteriolar constrictor response to AVP was unaffected by TEA treatment.
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DISCUSSION |
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The results of the present study confirm and extend previous reports concerning the role of BKCa channels in regulating renal microvascular function. Afferent arteriolar diameter was reduced by BKCa channel blockade (TEA) and increased by a synthetic BKCa channel agonist (NS-1619), indicating the ability of resident BKCa channels to influence preglomerular resistance. Pharmacological opening of BKCa channels attenuated the afferent arteriolar contractile response to ANG II; however, BKCa channel blockade did not alter the constrictor response to either ANG II or AVP. Efferent arteriolar function was unaffected by pharmacological manipulation of BKCa channels. These observations suggest that renal arteriolar responses to these peptide vasoconstrictor agonists do not normally involve changes in BKCa channel activity. The validity of this reasoning relies on the efficacy and specificity of the pharmacological agents employed to manipulate BKCa channel activity in our experimental setting.
Low-millimolar extracellular concentrations of TEA are widely employed to achieve BKCa channel blockade. In rat mesenteric arterial myocytes, TEA inhibits mean unitary BKCa current with a dissociation constant (Kd) of 0.2 mM (27). The effect of 1 mM TEA is relatively specific, inhibiting KCa channels by ~90% but exerting little or no impact on other K+ channels (Kd > 7 mM for ATP-sensitive, voltage-dependent, and inward rectifier K+ channels; Ref. 34). Although the present studies relied heavily on the BKCa blocking effect of 1 mM TEA, similar effects of CbTX and IbTX were confirmed in a few experiments. Nanomolar concentrations of these peptidyl scorpion toxins provide potent and specific blockade of BKCa channels in a variety of experimental settings (25).
In recent years, several types of small-molecule compounds have been shown to possess BKCa channel agonist activity. Although some of these compounds act at intracellular sites and exhibit poor membrane permeability, benzimidazolone analogs (e.g., NS-1619) have proven useful in physiological studies because of their ability to reversibly stimulate BKCa channel activity in intact cells (36). NS-1619-induced hyperpolarization of basilar artery smooth muscle is rapidly reversed by IbTX, indicating the critical role of BKCa channels in this response, whereas NS-1619-induced relaxation of the basilar artery is only partially blocked by IbTX (19). This phenomenon reflects the ability of NS-1619 to inhibit L-type Ca2+ channels under some conditions (14, 19) and introduces the possibility that the NS-1619-induced afferent arteriolar dilation observed in the present study might arise not only by direct activation of the BKCa channel (which subsequently decreases voltage-gated Ca2+ influx) but also via direct inhibition of the Ca2+ channel. L-type Ca2+ channels contribute significantly to basal afferent arteriolar tone in our experimental setting (8). Hence, if NS-1619 influences the afferent arteriole via direct inhibition of the L-type Ca2+ channel, the vasodilator response to this agent would remain evident in the presence of TEA (which does not block Ca2+ channels). Indeed, the TEA-insensitive nature of the afferent arteriolar dilator response to high concentrations (100 and 300 µM) of NS-1619 is consistent with the possibility that this dilation arises via inhibition of L-type Ca2+ channels. However, it is critical to note that the dilator response to 30 µM NS-1619 was prevented by 1 mM TEA and completely reversed by 50 µM CbTX. These observations provide compelling support for the contention that the afferent vasodilator response to 30 µM NS-1619 arises predominantly via opening of BKCa channels in our experimental setting. Accordingly, this concentration of NS-1619 was employed in all further experiments involving pharmacological BKCa channel activation.
Imig et al. (21) were the first to report that 1 mM TEA
produces afferent arteriolar constriction (~10% decline in diameter) in the juxtamedullary microvasculature studied during perfusion with
physiological salt solution. Quantitatively similar responses (15 ± 3% decrease in afferent diameter; n = 12 arterioles
pooled from the ANG II and AVP responsiveness experiments) were evident in the present study using the blood-perfused juxtamedullary nephron technique. These observations confirm a contribution of
BKCa channels to basal tone of afferent arterioles in this
experimental setting that preserves myogenic, tubuloglomerular feedback
and paracrine influences on the microvasculature. The stimulus
underlying significant tonic BKCa channel activity under
these conditions remains uncertain. In the isolated perfused
hydronephrotic rat kidney, afferent arteriolar smooth muscle membrane
potential is 40 mV (28). Previous reports indicate that
BKCa channel blockers have no effect on membrane potential
in the absence of induced hyperpolarization (4, 17) and
that a background (cGMP-dependent) dilator influence is necessary to
allow an effect of BKCa channel inhibition on mesangial
cell function (40). Our experimental setting exhibits a
significant nitric oxide (NO)-dependent tonic dilator influence on both
afferent and efferent arterioles, as evidenced by constrictor responses to NO synthase inhibition (20, 35). Assuming that the
juxtamedullary arteriolar dilator impact of NO arises, at least in
part, via a cGMP-dependent mechanism (1), activation of
BKCa channels by cGMP-dependent protein kinase
(38) may contribute to the tonic impact of
BKCa channels on afferent arteriolar resistance. BKCa channel activation counteracting myogenic tone may
also occur under our experimental conditions, in accord with the
ability of 1 mM TEA to potentiate the afferent arteriolar myogenic
response in the isolated perfused hydronephrotic kidney
(30). Regardless of the mechanism underlying tonic
BKCa channel activation in the afferent arteriole, the
capacity for further augmentation of the BKCa channel
influence on afferent arteriolar tone is revealed by the CbTX- and
TEA-sensitive dilator response to 30 µM NS-1619. Thus the results of
the present study support the contention that BKCa channels
exert a tonic dilator influence on the juxtamedullary afferent
arteriole and that pharmacological opening of BKCa channels can further dilate this vascular segment. Efferent arteriolar diameter
was unaffected by either TEA or NS-1619, in accord with the relative
insensitivity of this vascular segment to changes in membrane potential
(7, 29).
Further evidence that BKCa channels can influence afferent arteriolar function is provided by our observations concerning the effect of NS-1619 on ANG II-induced vasoconstrictor responsiveness. The afferent arteriolar smooth muscle response to ANG II involves membrane depolarization (28) and Ca2+ influx through voltage-gated channels (8); hence, pharmacological opening of BKCa channels to promote membrane hyperpolarization can be expected to temper the constrictor response to ANG II. Indeed, to the extent that the effects of 30 µM NS-1619 can be attributed to opening of BKCa channels, the ability of this agent to significantly attenuate afferent arteriolar ANG II responsiveness suggests that BKCa channels are capable of modulating agonist-induced vasoconstriction of this vascular segment. Efferent arteriolar responses to ANG II were unaffected by NS-1619, in accord with the absence of ANG II-induced membrane depolarization (28) and minimal involvement of voltage-gated Ca2+ influx in the response of this vascular segment to ANG II (8).
Although the effect of NS-1619 on afferent arteriolar ANG II
responsiveness suggests that resident BKCa channels are
capable of influencing vasoconstrictor responsiveness, it does not
indicate the extent to which this phenomenon occurs physiologically.
Indeed, ANG II and other peptide agonists that provoke membrane
depolarization and increases in [Ca2+]i may
either inhibit BKCa channel activity to promote
constriction or activate BKCa channels to temper
constriction. We reasoned that pharmacological BKCa channel
blockade should inhibit ANG II-induced constriction if BKCa
channel closure normally contributes to ANG II-induced membrane
depolarization. Conversely, if BKCa channels activate in a
negative-feedback fashion to temper vasoconstriction, pharmacological
BKCa channel blockade should allow an exaggerated vasoconstrictor response to ANG II. Because pharmacological
BKCa channel blockade did not curtail ANG II-induced
vasoconstriction, inhibition of BKCa channels does not
appear to be a prominent mechanism for eliciting ANG II-induced
depolarization of afferent arteriolar smooth muscle. This conclusion is
in accord with evidence that ANG II-induced depolarization of
preglomerular microvascular smooth muscle arises primarily via opening
of Ca2+-activated Cl channels (6, 23,
24). To our surprise, pharmacological blockade of
BKCa channels (TEA, IbTX, or CbTX) did not allow
exaggerated afferent arteriolar constrictor responses to either ANG II
or AVP. These observations argue against a significant
negative-feedback role of BKCa channels in tempering
afferent arteriolar constrictor responses to depolarizing and
Ca2+-mobilizing peptide agonists in our experimental setting.
Given the known effects of membrane potential and [Ca2+]i on the BKCa channel, and the effects of ANG II and AVP to provoke depolarization and increase [Ca2+]i in preglomerular microvascular smooth muscle, our observations might be explained if these peptides provoke additional signaling events that act to prevent or counteract negative-feedback activation of the BKCa channel. 20-HETE is released from isolated perfused kidney exposed to ANG II (9) and is known to inhibit BKCa channels in renal microvascular smooth muscle (44); hence, this P-450 arachidonic acid metabolite may act to prevent negative-feedback BKCa channel activation in response to ANG II. However, this mechanism is not likely involved in the response to AVP, which does not stimulate renal release of 20-HETE (9). Recently, protein kinase C (PKC) has been demonstrated to inhibit BKCa channels (2). Both ANG II and AVP stimulate PKC activity in rat vascular smooth muscle (11), and PKC blockade attenuates afferent arteriolar contractile and [Ca2+]i responses to ANG II (31, 39). The ability of ANG II and AVP to provoke afferent arteriolar vasoconstriction may critically rely on the receptor-dependent PKC activation and subsequent phosphorylation events that prevent negative-feedback activation of BKCa channels. Nelson et al. (33) have suggested that agonist-induced sparks of Ca2+ release from intracellular stores cause spatially localized transient increases in [Ca2+]i that, while not influencing the contractile apparatus or global [Ca2+]i, provoke opening of nearby BKCa channels to temper membrane depolarization. If this represents the primary mechanism responsible for negative-feedback activation of BKCa channels, the strong dependence of afferent arteriolar ANG II and AVP responsiveness on membrane depolarization and Ca2+ influx, with lesser involvement of Ca2+ mobilization from intracellular stores, may generate insufficient Ca2+ spark activity to activate negative-feedback BKCa channel activation. These postulates require further investigation at the single cell level.
Because endothelial cells possess BKCa channels, the possibility exists that TEA acts on the endothelium in a manner that masks the impact of BKCa channel blockade in afferent arteriolar smooth muscle. Blockade of endothelial BKCa channels should depolarize the membrane, decrease Ca2+ influx through effects on the electrochemical driving force, decrease NO release, and provoke vasoconstriction (13). If TEA influences the juxtamedullary microvasculature by decreasing NO release, it should mimic the effect of NO synthase inhibition. In our experimental setting, NO synthase inhibition provokes afferent and efferent arteriolar constriction and allows exaggerated vasoconstrictor responses to ANG II and AVP in both vascular segments (20). In contrast, TEA constricts only afferent arterioles and does not modify vasoconstrictor responses to ANG II or AVP in either vascular segment. Thus it is unlikely that the effects of TEA on arteriolar diameter in our experimental setting result from a diminution of NO release, although we cannot rule out the possibility that endothelial BKCa blockade has other consequences that might mask direct effects of TEA on arteriolar smooth muscle function.
In summary, the in vitro blood-perfused juxtamedullary nephron technique was employed to explore the impact of BKCa channels on renal arteriolar function. Pharmacological opening of BKCa channels provoked afferent arteriolar dilation and reduced constrictor responsiveness to ANG II. Blockade of BKCa channels caused afferent arteriolar constriction but did not alter vasoconstrictor responsiveness to either ANG II or AVP. Efferent arteriolar diameter and vasoconstrictor responsiveness was unaltered by pharmacological manipulation of BKCa channel activity. These observations indicate that the BKCa channels influence afferent arteriolar tone, presumably by virtue of their effect on smooth muscle membrane potential. However, changes in BKCa channel activity do not appear to be prominently involved in engendering or modulating afferent arteriolar constrictor responses to ANG II and AVP under our experimental conditions.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-39202. The Nebraska Affiliate of the American Heart Association provided fellowship support for J. P. Bast (predoctoral).
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. K. Carmines, Dept. of Physiology & Biophysics, Univ. of Nebraska College of Medicine, 984575 Nebraska Medical Center, Omaha, NE 68198-4575 (E-mail: pkcarmin{at}unmc.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.
Received 11 September 2000; accepted in final form 21 November 2000.
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