Lisa Chilton and Rodger LoutzenhiserArterioles

Functional Evidence for an Inward Rectifier Potassium Current in Rat Renal Afferent

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Functional Evidence for an Inward Rectifier PotassiumCurrent in Rat Renal Afferent Arterioles

Lisa Chilton, Rodger Loutzenhiser

Abstract—An inward rectifier potassium current, Kir, has been identified in cerebral and coronary resistance vessels, whereit is considered to be an important determinant of resting membrane potential (RMP) and to play a role in blood flowregulation. We investigated the functional role of Kir in the renal afferent arteriole using the in vitro–perfusedhydronephrotic rat kidney. Increasing external KCl from 5 to 15 mmol/L induced afferent arteriolar vasodilation. Thisresponse was inhibited by 10 to 100mmol/L Ba21, concentrations selective for blockade of Kir, and by chloroethyl-clonidine (100mmol/L) but was not blocked by glibenclamide (10mmol/L) or ouabain (3 mmol/L). Reducing externalKCl from 5 to 1.5 mmol/L to enhance rectification of Kir caused vasoconstriction at low renal arterial pressure (40mm Hg) and vasodilation during myogenic vasoconstriction (120 mm Hg), suggesting that this current dominates RMPat low perfusion pressures. When administered to kidneys perfused at 40 mm Hg renal arterial pressure, 30mmol/L Ba21

elicited afferent arteriolar depolarization, reducing RMP from24762 to 23462 mV (n510, P,0.0001), andvasoconstriction, reducing diameters from 14.561 to 10.960.8mm (n510,P50.0016). Although Ba21 reduced restingdiameter, blockade of Kir did not prevent myogenic signaling in this vessel. Our findings thus demonstrate the presenceof Kir in rat renal afferent arterioles and suggest that this current is an important determinant of RMP in situ.(Circ Res.2001;88:152-158.)

Key Words: renal hemodynamicsn renal microcirculationn membrane potentialn barium n ouabain

Of the 4 classes of K1 channels known to regulatevascular smooth muscle tone, the inward rectifier, Kir,

is the least characterized. This is attributable in part to itsrestricted expression in resistance vessels. Kir is absent inmost large conduit vessels. However, the density of Kir andits physiological importance may increase with decreasingvessel caliber.1–3 Although Kir has been identified in submu-cosal,4 carotid,5 cerebral,2,6 and coronary arteries,6 the role ofKir in the renal microcirculation has not been studied. Prior etal7 concluded that Kir does not play a functional role inregulation of tone in rabbit renal arcuate arteries. However,the arcuate is not a resistance vessel. Renal vascular resis-tance is determined predominantly by the afferent and effer-ent arterioles.8 The afferent arteriole is the terminal resistancevessel upstream of the glomerulus and, by regulating preglo-merular resistance, plays the major role in renal autoregula-tion and the control of glomerular capillary pressure.

K1-induced vasodilation has long been recognized as amechanism for regulating local blood supply. In general,modest increases in local K1 concentration result in vasodi-lation and increased blood flow.9 This vasodilation is medi-ated, in part, by the outward current carried by Kir. Patch-clamp studies using smooth-muscle myocytes from vesselsexhibiting K1-induced dilation demonstrate that elevations inexternal K1 increase outward current through Kir.10–12 This

effect is attributable to a displacement by K1 of polyamine orMg21 binding within the pore, which reduces rectification andincreases outward current.13 An alternate mechanismwhereby external K1 can induce vasodilation is by stimulat-ing the ouabain-sensitive, electrogenic Na1-K1 ATPase.6,7,14

Ba21 blocks Kir at micromolar concentrations and can be usedto distinguish Kir-induced vasodilation from effects mediatedby other K1 channels or from ouabain-sensitive vasodila-tion.10–12,14In addition, thea-adrenoceptor agonist/antagonistchloroethylclonidine (CEC) has recently been shown to blockKir.15 Hence, the combination of K1-induced vasodilationtogether with micromolar sensitivity to Ba21 and lack ofeffect of ouabain provides reasonable evidence for a func-tional role of Kir in intact vessels.

In the present study, the role of Kir in the renal afferentarteriole was investigated using the in vitro–perfused hydro-nephrotic rat kidney preparation.16 We determined the abilityof 15 mmol/L KCl to dilate the afferent arteriole and theeffects of Ba21, CEC, and ouabain on this response. Thecontribution of Kir to resting membrane potential (RMP) wasassessed by simultaneously measuring afferent arteriolarRMP and diameter during blockade of Kir. Finally, weexamined the impact of blocking Kir on myogenic reactivity.Our findings provide the first evidence of a functional role forKir in the renal microcirculation.

Original received November 2, 2000; revision received November 28, 2000; accepted November 29, 2000.From the Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta, Canada.Correspondence to Rodger D. Loutzenhiser, PhD, Department of Pharmacology and Therapeutics, University of Calgary, Health Sciences Centre, 3330

Hospital Dr NW, Calgary, Alberta T2N 4N1, Canada. E-mail rloutzen@ucalgary.ca© 2001 American Heart Association, Inc.

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152

Integrative Physiology

Materials and MethodsThe in vitro hydronephrotic rat kidney16 was used to examine therole of Kir in the afferent arteriole. Use of animals complied withCanadian Council on Animal Care regulations. Unilateral hydrone-phrosis was induced in male Sprague-Dawley rats by ligating the leftureter under halothane anesthesia. Rats were obtained from theAnimal Resource Center breeding facility at the University ofCalgary. Kidneys were harvested after 6 weeks, when tubularatrophy had advanced to a stage allowing direct visualization of therenal microvasculature. The renal artery was cannulated, and thekidney was excised with continuous perfusion. Kidneys were per-fused with modified DMEM (GIBCO) at 37°C, equilibrated with 5%CO2, containing (in mmol/L) Ca21 1.6, bicarbonate 30, glucose 5,pyruvate 1, and HEPES 5. Endogenous prostanoids modulate vas-cular reactivity in both normal kidneys and in our model.16 There-fore, 10mmol/L ibuprofen was included in the perfusate. Perfusionpressure was monitored at the level of the renal artery and wascontrolled by adjusting pressure within the perfusion reservoir.Kidneys were allowed 1 hour to recover before initiation of exper-imental protocols. Diameters were measured by online imageprocessing.

Afferent arteriolar RMPs were measured as described previous-ly.17 Hydronephrotic kidneys were excised as described above. Thecapsule was removed, and the perfused kidney was mounted on aPlexiglas post, allowing access to the surface. A custom microscopewith a water-immersion ceramic objective, coupled to a projectionlens and video system, provided high magnification for impalementsand diameter measurements. A microelectrode (2 mmol/L KCl andbeveled at 30° to 80 to 100 MV) was placed within a patch-typepipette (tip diameter 10 to 20mm) using a custom holder withmotorized positioner. The patch pipette was then positioned againstthe vessel wall and held with suction to stabilize the impalement site.A piezo device was used to impale the vessel. We have shown thatRMP measurements obtained using this approach are similar to thoseobtained using conventional microelectrodes.17 An impalement wasconsidered successful if each of the following criteria were met:sudden negative deflection in membrane potential, stable recordingof membrane potential for at least 1 minute before treatment withBa21, and no change in microelectrode tip resistance on withdrawal.The effects of Ba21 on RMP and diameter were recorded simulta-neously using this approach.

Glibenclamide and ibuprofen were obtained from Research Bio-chemicals International. All other chemicals were obtained fromSigma. KCl solutions 10 and 15 mmol/L were prepared by isotonicsubstitution for NaCl. Ouabain was made fresh for each experiment(3 mmol/L in DMEM). Phentolamine and propranolol were addedduring the ouabain experiment to avoid effects mediated by neuro-transmitter release and to the CEC experiments to avoid effects ofadrenoceptor modulation.

Data are expressed as mean6SEM. The number of replicates (n)refers to the number of vessels. Generally, no more than 2 vesselswere studied per kidney (where appropriate, N refers to number ofkidneys). Differences between means were evaluated by Student’spaired t test unless otherwise noted.P,0.05 was consideredsignificant.

ResultsFigure 1 illustrates the vasodilator response of an afferentarteriole to 15 mmol/L KCl. Vasoconstriction was induced byelevating renal arterial pressure (RAP) from 80 to 160mm Hg, reducing diameters from 16.360.4 to 8.160.5 mm(n525, P,0.0001). In this setting, 15 mmol/L KCl causedafferent arteriolar dilation to 13.660.5 mm (P,0.0001).Returning to 5 mmol/L KCl restored myogenic vasoconstric-tion to previous levels (8.560.5 mm, P50.26).

We next assessed the effects of Ba21 and CEC on K1-induced vasodilation. The control studies presented in Figure2A illustrate that repetitive K1-induced responses were stable

over time. As shown in Figure 2B, Ba21 attenuated thisresponse in a concentration-dependent manner. Ba21 alsoreduced basal diameters (considered in detail below), pre-cluding a calculation of the ED50. Nevertheless, Ba21 reducedthe K1-induced dilation over concentrations that selectivelyblock Kir10–12 (10 to 100mmol/L). Complete blockade wasobserved at 100mmol/L (P50.54, n56). At 100 mmol/L,Ba21 may block ATP-sensitive K1 channels (KATP).14 How-ever, glibenclamide had no effect. Thus, 15 mmol/L KClincreased diameters from 861.4 to 15.162.1 mm in thepresence of vehicle (ethanol) and from 8.362.6 to 15.162.5mm in the presence of 10mmol/L glibenclamide (n53,P50.9). The K1-induced dilation was also blocked by 100mmol/L CEC, an agent recently shown to block Kir15 (Figure2C). Before application of CEC, 15 mmol/L K1 induceddilation in preconstricted arterioles (9.561.6 versus 15.560.9mm with 15 mmol/L KCl, RAP5140 mm Hg, n55). CECcaused additional constriction, reducing diameters to 6.461.1mm, and completely blocked the K1-induced dilation. Nota-bly, application of 15 mmol/L KCl during CEC treatmentcaused additional constriction (to 4.961.1 mm, n55), con-sistent with the change in the K1 equilibrium potential.

The above findings suggest that the K1-induced dilation ismediated by Kir on the basis of the effects of Ba21 and CEC.We next examined the effects of blockade of the Na1-K1

ATPase. Vessels were preconstricted by administering 3mmol/L ouabain (RAP 80 mm Hg), a concentration sufficientto block the rat isoform.14 Kidneys were pretreated withphentolamine and propranolol (10mmol/L) to prevent effectsattributable to the release of neurotransmitters. As shown inFigure 3, ouabain reduced diameters from 16.361.5 to4.261.5 mm (n54, P50.022), but K1-induced vasodilationwas preserved (increasing diameters from 4.261.5 to13.760.9 mm, P50.0246). In these studies, 10 mmol/L KClwas used to allow for the decrease in internal [K1] induced by

Figure 1. K1-induced dilation of afferent arterioles. A, Originaltracing illustrating RAP-induced vasoconstriction andK1-induced dilation in the afferent arteriole. B, Summary of dataobtained in 25 arterioles subjected to the protocol illustrated inpanel A. *P,0.0001 vs elevated RAP alone.

Chilton and Loutzenhiser Evidence for Kir in Renal Arterioles 153

ouabain. When kidneys were treated with both 100mmol/LBa21 and ouabain, diameters decreased from 1860.9 to3.861 mm (n54, P50.0033), and KCl-induced dilation wasblocked (3.961.2 mm, P50.56). The ability of Ba21 to blockKCl-induced dilation was readily reversed when Ba21 wasremoved from the perfusate (Figure 4A).

Kir is thought to contribute to RMP and resting tone inother vessel types.10–12 We thus determined the effects ofBa21 blockade of Kir on resting diameter and RMP in theafferent arteriole. As shown in Figure 4B, Ba21 elicited avasoconstrictor response of the afferent arteriole that wasreversed on Ba21 removal. Ba21 blockade of Kir eliciteddose-dependent vasoconstriction over concentrations of 1 to100mmol/L. At 80 mm Hg, 30mmol/L Ba21 reduced afferentarteriolar diameter from 15.660.5 to 7.661.6 mm (5169%decrease, n56, P50.0041). Half-maximal constriction (EC50)

was seen at 1963 mmol/L Ba21 (Figure 5). As describedbelow, Ba21 altered the threshold for myogenic vasoconstric-tion, shifting the response to lower pressures. We thereforecharacterized the effects of Ba21 at low perfusion pressure (40mm Hg). Under these conditions, 30mmol/L Ba21 reducedRMP from 24762 to 23462 mV (P,0.0001) and reduceddiameter from 14.561 to 10.960.8 mm (n510, N58,P50.002). These data are summarized in Figure 6.

Figure 2. Barium and CEC blockade of K1-induced vasodilation.A, Time controls illustrating reproducible vasodilatory responseof 4 vessels to 6 consecutive challenges with 15 mmol/L KCl. B,Effects of Ba21 on K1-induced vasodilation. Ba21 decreasedbasal diameter and blocked K1-induced vasodilation in aconcentration-dependent manner (1, 3, 10, 30, and 100 mmol/L;‚, ƒ, e, M, and V, respectively; n56). C, Blockade ofK1-induced dilation by 100 mmol/L CEC. Propranolol and phen-tolamine (10 mmol/L) were present to prevent adrenoceptor-mediated effects.

Figure 3. Lack of effect of ouabain on K1-induced vasodilation.Tracings illustrate K1-induced vasodilation in afferent arteriolepreconstricted with 3 mmol/L ouabain (A) and blockade of thisresponse by 100 mmol/L Ba21 (B). Mean data are depicted inpanel C (n54 each). All studies carried out in the presence of 10mmol/L phentolamine and propranolol.

Figure 4. Tracings illustrating reversibility of Ba21-induced inhi-bition of KCl-induced vasodilation (A) and Ba21-induced vaso-constriction (B). A, ▫ indicates exposure to 15 mmol/L KCl. Datarepresentative of 3 replicates each.

154 Circulation Research February 2, 2001

These findings suggest an important role of Kir in thesetting of RMP and basal tone in the afferent arteriole.However, this interpretation is based exclusively on thepremise that 30mmol/L Ba21 selectively blocks Kir. Toindependently test this postulate, we examined the effects ofreducing external K1 ([K 1]o). The inward rectification that ischaracteristic of Kir is attributed to polyamine or Mg21 block.

Elevated [K1]o reduces rectification by displacing theseblocking agents and enhancing outward current, therebycausing vasodilation.10–13 Conversely, reducing [K1]o willenhance the effects of the blocking agents and reduceoutward current carried by Kir. Thus, if Kir is the predomi-nant K1 channel influencing RMP, then reducing [K1]o

should cause vasoconstriction. On the other hand, reducing[K 1]o shifts the K1 equilibrium potential to more negativevalues and will enhance the hyperpolarizing current carriedby other K1 channel types. Kir is the only K1 channel invascular smooth muscle in which reduced [K1]o wouldenhance intrinsic block. Hence, if K1 channels other than Kirare dominant, reducing [K1]o would cause vasodilation, notvasoconstriction. As shown in Figure 7, reducing [K1]o from5 to 1.5 mmol/L induced a constriction at 40 mm Hg,reducing diameters from 1560.6 to 12.860.9 mm (n56,N55, P50.0088). To rule out the possibility that lowering[K1]o induced vasoconstriction by reducing activity of theelectrogenic Na1-K1 ATPase, we conducted additional ex-periments in the presence of 3 mmol/L ouabain. The ouabain-induced vasoconstriction was minimized by reducing RAP to30 mm Hg. In the presence of ouabain, lowering [K1]o from5 to 1.5 mmol/L caused additional constriction, reducingdiameters from 1468.2 to 8.261.2 mm (P50.0014, n55).We next examined the effects of lowering [K1]o at elevatedRAP, conditions in which channels other than Kir, such asvoltage- or Ca21-activated K1 channels (Kv and KCa), mightbe active. We have shown that KCa is activated at elevatedpressure, modulating myogenic vasoconstriction.18 ElevatingRAP from 80 to 120 mm Hg reduced afferent diameters from15.860.8 to 9.960.8 mm (n56, P50.0008). In this setting,reducing [K1]o to 1.5 mmol/L increased diameters from9.960.8 to 14.160.5 mm (n56, P50.0006). In concert withthe Ba21 data, these observations provide compelling evi-dence that Kir is the predominant K1 channel responsible forsetting resting afferent arteriolar tone at low levels of perfu-sion pressure.

Accordingly, alterations in Kir might contribute to myo-genic activation. Therefore, we examined the effects of Ba21

on the myogenic response. As shown in Figure 8A, gradedafferent vasoconstriction is elicited when RAP is increased

Figure 5. Ba21-induced constriction in afferent arterioles(mean6SEM, n56). *P,0.05 vs control. EC5051963 mmol/L;RAP580 mm Hg.

Figure 6. A, Original tracings of membrane potential and diame-ter of an afferent arteriole during the administration of 30 mmol/LBa21. B, Ba21 (30 mmol/L)-induced depolarization (left) andvasoconstriction (right) in afferent arterioles perfused at a renalarterial pressure of 40 mm Hg. V, individual measurements; F,mean6SEM (n510). C, Summary of individual measurements.

Figure 7. Effects of reducing [K1]o on afferent arteriolar diame-ter. Left panel depicts original tracings illustrating the vasocon-striction evoked by 1.5 mmol/L KCl at 40 mm Hg (top) and thevasodilation evoked in the same vessel at 120 mm Hg (bottom).Mean data (6SEM) depicted on right.

Chilton and Loutzenhiser Evidence for Kir in Renal Arterioles 155

from 20 to 180 mm Hg. Figure 8A additionally illustrates thatin a series of time controls, 6 repeated challenges to increasedrenal arterial pressure (administered over a 7-hour period)elicited identical myogenic responses (n54). As shown inFigure 8B, 1 to 100mmol/L Ba21 elicited vasoconstriction atlow RAP but did not prevent additional constriction whenRAP was increased. This is additionally illustrated in Figure8C, which depicts the response as the percent change indiameter. Ba21 caused a leftward shift in the activation curve,perhaps reflecting the impact of blockade of Kir on basal toneand RMP. However, despite the Ba21-induced preconstric-tion, the arterioles responded in an incremental manner toelevations in RAP, suggesting that Kir modulation is not arequisite component of myogenic signaling in this vessel.

DiscussionThe present study provides the first evidence for a functionalrole of Kir in the renal afferent arteriole. We demonstratedthat 15 mmol/L KCl elicits a vasodilation in this vessel andthat this response was not prevented by ouabain but wassensitive to CEC and Ba21 at concentrations reported toselectively block Kir. Moreover, manipulations that wouldreduce Kir currents under basal conditions (30mmol/L Ba21,low [K1]o) elicit both depolarization and vasoconstriction,suggesting that Kir is a significant determinant of RMP andresting tone in the afferent arteriole.

The properties of the K1-induced dilation in the afferentarteriole are similar to those attributed to Kir in other vesseltypes. Application of 6 to 16 mmol/L KCl to preconstrictedisolated posterior cerebral arteries,6,14 middle cerebral arter-ies,19 and coronary arteries6 results in dilation, and thisresponse is blocked by micromolar levels of Ba21. In thesepreparations, as in the present study, neither ouabain norglibenclamide prevented this response. Electrophysiologicalcharacterization of Kir in vascular smooth muscle was firstreported by Hirst and Neild,4 who described currents exhib-iting inward rectification in submucosal arterioles of guineapig ileum. Similar inwardly rectifying currents have beendescribed in carotid artery5 and middle cerebral arteriole.20,21

Studies using patch-clamp techniques have identified K1-induced increases in outward current and inhibition of thiscurrent by micromolar Ba21 in myocytes isolated fromposterior cerebral artery2,6 and coronary artery.3,6,22Recently,the targeted disruption of the Kir2.1 gene in mice was shownto be associated with a loss of both the inwardly rectifying K1

current and K1-induced dilation in cerebral arteries, suggest-ing its essential role in the response.23 Our finding that CECinhibited K1-induced vasodilation additionally supports a rolefor Kir. CEC is an irreversible agonist ata2 adrenoceptors andan antagonist ata1 adrenoceptors24 but was recently reportedto selectively inhibit inward rectifier K1 currents in skeletalmuscle fibers and whole-cell K1 currents in cultured cell linestransfected with Kir2.1.15 These effects did not involveadrenoceptors, because they were not mimicked or altered byadrenoceptor ligands and could be demonstrated in cells thatdo not express adrenoceptors.15 Similarly, the inhibition ofK1-induced dilation by CEC in the afferent arteriole that weobserved occurred in the presence of phentolamine andpropranolol, indicating that adrenoceptors are not involved.

Two previous studies have addressed the possible role ofKir in the renal circulation. Kurtz and Penner25 describedinward rectifying K1 currents in mouse juxtaglomerular cells,renin-secreting cells in the terminal segment of the afferentarteriole. They recorded whole-cell currents that were sensi-tive to changes in external K1 but did not assess Ba21

sensitivity. More recently, Prior et al7 evaluated the Ba21 andouabain sensitivity of K1-induced dilation of rabbit renalarcuate arteries. In contrast to our findings with the afferentarteriole, these authors found that K1-induced dilation of thearcuate artery was attenuated by ouabain but not blocked byBa21, suggesting that Kir is not involved. As mentionedpreviously, Kir is thought to be more prevalent in resistancevessels than in conduit arteries, such as the arcuate artery. Ourfindings, together with those of Prior et al,7 indicate that thispattern holds in the kidney.

In our studies, 30mmol/L Ba21 produced marked constric-tion of the afferent arteriole and membrane depolarization (13mV) at low RAP, suggesting a role for Kir in maintainingRMP and resting tone. A more modest Ba21-induced constric-tion (8%, from 20867 to 191619mm with 120mmol/L Ba21)was described in isolated perfused rat middle cerebral arteriesby Johnson et al.19 In our studies, Ba21 inhibited K1-inducedvasodilation at 10 to 100mmol/L and elicited vasoconstric-tion in the same concentration range (ED5051963 mmol/L).The ED50 for Ba21-induced vasoconstriction in the middle

Figure 8. Effect of Ba21 on the myogenic response. A, Timecontrols illustrating reproducible responses of 4 arterioles to 6consecutive challenges with stepped elevation in RAP. B,Increasing concentrations of Ba21 (1, 3, 10, 30, and 100 mmol/L;M, ‚, ƒ, e, and V, respectively) elicited vasoconstriction at lowRAP and augmented the maximal response to RAP (controls, F;n56). C, Data from panel B expressed as percent change rela-tive to the diameter at 40 mm Hg. Increasing Ba21 concentra-tions elicited a leftward shift in the pressure-activation curve.

156 Circulation Research February 2, 2001

cerebral artery is'60mmol/L (see Figure 6 in Johnson et al).Similarly, the IC50 for Ba21 block of K1-induced dilation inmiddle cerebral arteries was 20 to 40mmol/L (see Figure 3 inJohnson et al19). These values are close to the range for Ba21

blockade of Kir channels. At245 mV, the Kd for Ba21-induced inhibition of Kir in posterior cerebral artery myo-cytes was estimated at 8mmol/L.2,6 Similar Kd values, 3mmol/L6 Ba21 and 4.7 mmol/L22 Ba21 at 240 mV, werereported for coronary artery myocytes.

K1-induced vasodilation is an important physiologicalmechanism in the cerebral and coronary circulations but hasnot been studied in the renal circulation. Periods of highneural activity, such as seizure, and ischemic, hypoxic, orhypoglycemic conditions increase K1 levels in cerebral spinalfluid to .10 mmol/L.26 The resultant K1-induced dilationincreases blood supply to areas of high metabolic demand.14

Similarly, in the coronary circulation, interstitial K1 levelsmay rise during periods of ischemia to levels sufficient toactivate K1-induced dilation.27 In the canine kidney, eleva-tion of plasma [K1] to '10 mmol/L has been shown toincrease renal blood flow (RBF) and glomerular filtration rate(GFR).28 Similar effects of acute hyperkalemia on RBF andGFR have been reported in conscious sheep29 and in rats30

and rabbits.31 Although these investigations did not considerthe possible role of Kir, our finding that elevated K1

promotes afferent arteriolar vasodilation through Kir is con-sistent with the observed increase in RBF and GFR.

By modulating glomerular inflow resistance, the afferentarteriole regulates glomerular capillary pressure and GFRwhen renal perfusion pressure is altered. Pressure-inducedafferent vasoconstriction mediates this effect. Our observa-tion that Kir is a major determinant of RMP in this vesselprompted an investigation into the possible involvement ofKir in myogenic signaling. Our findings do not support thispostulate. Thus, although Ba21 elicited membrane depolariza-tion and vasoconstriction at low pressure, it did not preventelevations in RAP from causing additional vasoconstriction.These data agree with findings from Kir2.1 knockout mice.23

In this model, cerebral arteries showed normal myogenicreactivity but no K1-induced dilation. The observed changesin myogenic responses, including a leftward shift in themyogenic threshold, likely reflect the increase in basal toneand more positive RMP in the presence of Ba21.

Defining the determinants of RMP under physiologicalconditions is of critical importance in regard to potentialactivation mechanisms. Agonists have been shown to inhibitspecific K1 channels in isolated myocytes.10–12 However,such mechanisms could contribute to physiological responsesonly if the affected K1 channel contributes to RMP in theintact setting. The model we used allowed direct assessmentof RMP and contractions in the intact afferent arteriole in situunder physiological conditions in regard to perfusion andtransmural pressure. Our findings clearly indicate that Kir isa primary determinant of RMP under these conditions. Ourmodel requires the induction of hydronephrosis to visualizethe microvasculature. This preparation is well characterizedand exhibits responses that are not only comparable to thoseobserved using other renal microvascular models8,32 but alsosimilar to those of the normal, intact kidney.16,33 Thus, there

is no indication that hydronephrosis alters vascular K1

channel expression. Nevertheless, this possibility cannot beexcluded. As described above, K1-induced vasodilation isobserved in the normal kidney,28–31but the role of Kir in thisresponse has not been investigated. Moreover, barium-sensitive K1 currents have not been studied in myocytes orendothelial cells of the afferent arteriole. Information on theexpression and regulation of Kir in this vessel would be ofmajor interest.

In conclusion, this study provides the first functionalevidence for Kir in renal afferent arterioles. Our findingssuggest that Kir is a major determinant of in situ RMP andresting tone in this vessel. Accordingly, alterations in Kiractivity or expression would likely impact on renal microvas-cular reactivity and could contribute to physiological orpathophysiological alterations in renal hemodynamics.

AcknowledgmentsThis study supported by grants from the Canadian Institutes forHealth Research and the Alberta Heritage Foundation for MedicalResearch (AHFMR). R.L. is an AHFMR Senior Medical Scholar,and L.C. is an Honorary Killam Scholar. The authors wish to thankDr Wayne Giles and Dr William Cole for their comments andsuggestions.

References1. Hirst GDS, Edwards FR. Sympathetic neuroeffector transmission in

arteries and arterioles.Physiol Rev. 1989;69:546–604.2. Quayle JM, McCarron JG, Brayden JE, Nelson MT. Inward rectifier K1

currents in smooth muscle cells from rat resistance-sized cerebral arteries.Am J Physiol. 1993;265:C1363–C1370.

3. Quayle JM, Dart C, Standen NB. The properties and distribution ofinward rectifier potassium currents in pig coronary arterial smoothmuscle.J Physiol. 1996;494:715–726.

4. Hirst GDS, Neild TO. An analysis of excitatory junctional potentialsrecorded from arterioles.J Physiol. 1978;280:87–104.

5. Keatinge WR. Mechanism of slow discharges of sheep carotid artery.J Physiol. 1978;279:275–289.

6. Knot HJ, Zimmermann PA, Nelson MT. Extracellular K1-induced hyper-polarizations and dilatations of rat coronary and cerebral arteries involveinward rectifier K1 channels.J Physiol. 1996;492:419–430.

7. Prior HM, Webster N, Quinn K, Beech DJ, Yates MS. K1-induceddilation of a small renal artery: no role for inward rectifier K1 channels.Cardiovasc Res. 1998;37:780–790.

8. Navar LG, Inscho EW, Majid DSA, Imig JD, Harrison-Benard LM,Mitchell KD. Paracrine regulation of the renal microcirculation.PhysiolRev. 1996;76:425–536.

9. Haddy FJ, Scott JB. Metabolically linked vasoactive chemicals in localregulation of blood flow.Physiol Rev. 1968;48:688–707.

10. Nelson MT, Quayle JM. Physiological roles and properties of potassiumchannels in arterial smooth muscle.Am J Physiol. 1995;268:C799–C822.

11. Cole WC, Clément-Chomienne O. Properties, regulation, and role ofpotassium channels of smooth muscle. In: Barr L, Christ GJ, eds.Advances in Organ Biology. Stamford, Conn: JAI Press Inc; 2000:247–317.

12. Quayle JM, Nelson MT, Standen NB. ATP-sensitive, and inwardly rec-tifying potassium channels in smooth muscle.Physiol Rev. 1997;77:1165–1232.

13. Lopatin AN, CG Nichols. [K1] dependence of polyamine-induced recti-fication in inward rectifier potassium channels (IRK1, Kir2.1).J GenPhysiol. 1996;108:105–113.

14. McCarron JG, Halpern W. Potassium dilates rat cerebral arteries by twoindependent mechanisms.Am J Physiol. 1990;259:H902–H908.

15. Barrett-Jolley R, Dart C, Standen NB. Direct block of native and cloned(Kir2.1) inward rectifier K1 channels by chloroethylclonidine.Br JPharmacol. 1999;128:760–766.

16. Tang L, Loutzenhiser R. Biphasic actions of PGE2 on the renal afferentarteriole.Circ Res. 2000;86:663–670.

Chilton and Loutzenhiser Evidence for Kir in Renal Arterioles 157

17. Loutzenhiser R, Chilton L, Trottier G. Membrane potential measurementsin renal afferent and efferent arterioles: actions of angiotensin II.Am JPhysiol. 1997;273:F307–F314.

18. Loutzenhiser R, Parker M. Hypoxia inhibits myogenic reactivity of renalafferent arterioles by activating ATP-sensitive potassium channels.CircRes. 1994;74:861–869.

19. Johnson TD, Marrelli SP, Steenberg ML, Childres WF, Bryan RM Jr.Inward rectifier potassium channels in the rat middle cerebral artery.Am JPhysiol. 1998;274:R541–R547.

20. Hirst GDS, Silverberg GD, van Helden DF. The action potential andunderlying ionic currents in proximal rat middle cerebral arterioles.J Physiol. 1986;371:289–304.

21. Edwards FR, Hirst GDS, Silverberg GD. Inward rectification in ratcerebral arterioles; involvement of potassium ions in autoregulation.J Physiol. 1988;404:455–466.

22. Robertson BE, Bonev AD, Nelson MT. Inward rectifier K1 currents insmooth muscle cells from rat coronary arteries: block by Mg21, Ca21, andBa21. Am J Physiol. 1996;271:H696–H705.

23. Zaritsky JJ, Eckman DM, Wellman GC, Nelson MT, Schwarz TL.Targeted disruption of Kir2.1 and Kir2.2 genes reveals the essential roleof the inwardly rectifying K1 current in K1-mediated vasodilation.CircRes. 2000;87:160–166.

24. Docherty JR, O’Rourke M. Thea-adrenoceptor-mediated actions of chlo-roethylclonidine.Gen Pharmacol. 1997;28:197–201.

25. Kurtz A, Penner R. Angiotensin II induces oscillations of intracellularcalcium and blocks anomalous inward rectifying potassium current inmouse renal juxtaglomerular cells.Proc Natl Acad Sci U S A. 1989;86:3423–3427.

26. Syková E. Extracellular K1 accumulation in the central nervous system.Prog Biophys Molec Biol. 1983;42:135–189.

27. Weiss JN, Lamp ST, Shine KI. Cellular K1 loss and anion efflux duringmyocardial ischemia and metabolic inhibition.Am J Physiol. 1989;256:H1165–H1175.

28. Scott J, Emanuel D, Haddy F. Effect of potassium on renal vascularresistance and urine flow rate.Am J Physiol. 1959;197:305–308.

29. Budtz-Olsen OE, Clark RC, Cross RB, French TJ. Changes in renalhaemodynamics and electrolyte excretion during acute hyperkalemia inconscious adrenectomized sheep.Quart J Exp Physiol Cog Med Sci.1975;60:207–221.

30. Paller MS, Linas SL. Hemodynamic effects of alterations in potassium.Hypertension. 1982;4:III-20–III-26.

31. Lin H, Young DB. Impaired control of renal hemodynamics and reninrelease during hyperkalemia in rabbits.Am J Physiol. 1988;254:F704–F710.

32. Loutzenhiser R. In situ studies of renal arteriolar function using the invitro perfused hydronephrotic rat kidney.Int Rev Exp Pathol. 1996;36:145–160.

33. Cupples WA, Loutzenhiser R. Dynamic autoregulation in the in vitroperfused hydronephrotic rat kidney.Am J Physiol. 1998;275:F126–F130.

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