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Am J Physiol Renal Physiol 293: F1248-F1255, 2007. First published August 1, 2007; doi:10.1152/ajprenal.00278.2007
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Descending vasa recta endothelia express inward rectifier potassium channels

Chunhua Cao,1 Whaseon Lee-Kwon,1 Kristie Payne,1 Aurélie Edwards,2 and Thomas L. Pallone1

1Division of Nephrology, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland; and 2Department of Chemical and Biological Engineering, Tufts University, Medford, Massachusetts

Submitted 18 June 2007 ; accepted in final form 25 July 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Descending vasa recta (DVR) are capillary-sized microvessels that supply blood flow to the renal medulla. They are composed of contractile pericytes and endothelial cells. In this study, we used the whole cell patch-clamp method to determine whether inward rectifier potassium channels (KIR) exist in the endothelia, affect membrane potential, and modulate intracellular Ca2+ concentration ([Ca2+]cyt). The endothelium was accessed for electrophysiology by removing abluminal pericytes from collagenase-digested vessels. KIR currents were recorded using symmetrical 140 mM K+ solutions that served to maximize currents and eliminate cell-to-cell coupling by closing gap junctions. Large, inwardly rectifying currents were observed at membrane potentials below the equilibrium potential for K+. Ba2+ potently inhibited those currents in a voltage-dependent manner, with affinity k = 0.18, 0.33, 0.60, and 1.20 µM at –160, –120, –80, and –40 mV, respectively. Cs+ also blocked those currents with k = 20, 48, 253, and 1,856 µM at –160, –120, –80, and –40 mV, respectively. In the presence of 1 mM ouabain, increasing extracellular K+ concentration from 5 to 10 mM hyperpolarized endothelial membrane potential by 15 mV and raised endothelial [Ca2+]cyt. Both the K+-induced membrane hyperpolarization and the [Ca2+]cyt elevation were reversed by Ba2+. Immunochemical staining verified that both pericytes and endothelial cells of DVR express KIR2.1, KIR2.2, and KIR2.3 subunits. We conclude that strong, inwardly rectifying KIR2.x isoforms are expressed in DVR and mediate K+-induced hyperpolarization of the endothelium.

kidney; medulla; microcirculation; electrophysiology; potassium channel; endothelium

INWARD RECTIFIER POTASSIUM channels (KIR) were first described in the cerebral arterioles by Edwards et al. (9) and subsequently identified in many other small generation resistance vessels (2, 5, 8, 10, 13, 14, 26). KIR are named for their characteristic ability to conduct K+ current more readily into than out of the cell when membrane potential is held below the equilibrium potential for K+ (Keq). In smooth muscle, KIR are generally characterized by strong inward rectification and voltage-dependent inhibition by low concentrations of either Ba2+ or Cs+ (11, 18, 24). Although many studies have demonstrated the expression and function of KIR channels in vascular smooth muscle cells, the functional role of KIR in endothelia has been less well characterized. It has been postulated that a primary function of vascular KIR channels is to mediate membrane hyperpolarization and vasodilatation in response to small elevations of extracellular K+ concentration (7, 9, 14, 24). For example, stimulation of endothelia by vasodilators like acetylcholine or bradykinin typically raises cytoplasmic Ca2+ concentration ([Ca2+]cyt) and hyperpolarizes membrane potential (1, 11, 19, 28). The rise in [Ca2+]cyt is thought to activate calcium-depended K+ channels (KCa), driving K+ efflux into the perivascular interstitium to create a localized "K+ cloud." The K+ cloud may activate smooth muscle KIR channels to favor membrane hyperpolarization and vasodilatation through inhibition of voltage-gated Ca2+ channels (VOCa). In addition, membrane hyperpolarization might spread between cells and along the vessel axis via gap junctions to cause remote vasodilatation (28, 29).

Descending vasa recta (DVR) are 12- to 15-µm-diameter vessels that arise from juxtamedullary efferent arterioles to supply blood flow to the renal medulla. DVR are composed of smooth muscle-like pericytes and an endothelial monolayer. Study of channel architecture in these vessels (3, 4, 17, 20, 21, 25, 29–31) has verified the presence of voltage-gated cation channels and led to recent identification of a strong, inwardly rectifying K+ conductance in the pericytes (3). To date, there has been no identification of KIR in the endothelium of DVR or any other renal microvessel. We hypothesized that expression of KIR in DVR endothelia might contribute to [Ca2+]cyt elevation by hyperpolarizing the cells to increase the electrochemical driving force favoring Ca2+ entry. To test this, we employed a method for removal of abluminal pericytes from DVR to access the endothelia for whole cell patch-clamp current recording (25). We readily identified inwardly rectifying K+ currents that are inhibited by external Ba2+ and Cs+ in a voltage-dependent manner. Elevation of external K+ from 5 to 10 mM hyperpolarized DVR endothelial membrane potential and increased [Ca2+]cyt. Immunohistochemical study verified expression of KIR2.1, KIR2.2, and KIR2.3 in both DVR pericytes and endothelial cells. Together, these data strongly support a role for KIR channels to regulate DVR endothelial cell membrane potential and Ca2+ signaling and thus affect renal medullary perfusion.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of endothelia by pericytes stripping. Investigations were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Maryland. Kidneys were harvested from Sprague-Dawley rats (120–200 g; Harlan) that had been anesthetized with an intraperitoneal injection of ketamine (80 mg/kg)-xylazine (10 mg/kg). Kidneys were sliced into sections along the corticomedullary axis and stored at 4°C in a physiological saline solution (PSS; in mM): 145 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, and 10 glucose, pH 7.4. As previously described, small wedges of renal medulla were separated from kidney slices by dissection and transferred to CaCl2-free PSS containing collagenase 1A (0.45 mg/ml), protease XIV (0.4 mg/ml), and albumin (1.0 mg/ml) (21, 25). These were incubated at 37°C for 22 min and then returned to 1 mM CaCl2 containing PSS and held at 4°C in a petri dish. DVR were isolated by microdissection as needed and transferred to an inverted microscope (Nikon TE300) for patch-clamp recording. Access to endothelial cells for patch clamp necessitated removal of pericytes from the abluminal surface. This was done, as previously described in detail (25), by drawing the vessel into the mouth of a micropipette that had been heat-polished to 5–10 µm. The aspirated, pericyte-denuded vessel was ejected from the pipette to yield a preparation of DVR endothelia.

Whole cell patch-clamp current recording. Whole cell currents were acquired by nystatin perforated patch recording. Patch pipettes were made from borosilicate glass capillaries (PG52151-4; external diameter 1.5 mm, internal diameter 1.0 mm; World Precision Instruments, Sarasota, FL) using a two-stage vertical pipette puller (Narishige PP-830) and heat polished. The pipette solution contained (in mM) 120 K-aspartate, 20 KCl, 10 NaCl, 10 HEPES, pH 7.2, and nystatin (100 µg/ml with 0.1% DMSO) in ultrapure water. Patch pipettes were backfilled from a light-protected syringe through a 0.2-µm filter. Whole cell currents were sampled at 10 kHz. Measurements were obtained using a CV201AU head stage and Axopatch 200B amplifier (Axon Instruments, Foster City, CA) and Clampex as previously described (21, 25). To maximize inward K+ currents, the extracellular KCl concentration was increased to 140 mM by isosmotic substitution of NaCl for KCl. To minimize other currents, the extracellular solution contained tetraethylammonium (TEA; 1 mM), glibenclamide (10 µM), and niflumic acid (100 µM) to inhibit KCa, ATP-sensitive, and Ca2+-dependent Cl– channels, respectively (3, 4, 20, 21).

Whole cell membrane potential recording. Membrane potential recordings were acquired by nystatin perforated patch in current-clamp mode (I = 0) at a sampling rate of 10 Hz using 8–10 M{Omega} pipettes. The extracellular solution was PSS without TEA, niflumic acid, or glibenclamide. Where needed, ouabain (1 mM) was included in the bath to inhibit Na+/K+-ATPase. The data have been corrected for junction potentials, as previously described (21).

Immunofluorescent labeling of isolated DVR. Using methods previously described (16, 17), we performed immunofluorescent labeling to detect expression of KIR2.1, KIR2.2, and KIR2.3 in the DVR wall. Monoclonal antibody directed against {alpha}-smooth muscle actin (SMA; 1:500 dilution to 9 µg/ml; A2547, Sigma, St. Louis, MO) was used to delineate the pericytes (7, 8). Rabbit polyclonal antibodies directed against KIR2.1, KIR2.2, and KIR2.3 (1:50 dilution; Sigma) were used to identify their respective distributions within pericytes and endothelia. Microdissected DVR were transferred onto slides and fixed with 3% paraformaldehyde in 100 mM cacodylate buffer, pH 7.4, and then rinsed three times. Vessels were then permeabilized with 0.1% Triton in PSS buffer for 2 min, blocked with 5% bovine serum albumin in PSS with 0.1% Triton (10 min, room temperature), and exposed to the primary antibody for 1 h at room temperature and then overnight at 4°C. After three washes with PBS containing 0.1% Triton X-100, the vessels were incubated with secondary antibodies conjugated to Alexa Fluor 488 goat anti-rabbit IgG (1:400 dilution to 5 µg/ml; Molecular Probes A-11034) and Alexa Fluor 568 goat anti-mouse IgG (1:400 dilution to 5 µg/ml; Molecular Probes A-11031) for 1 h at room temperature. After several additional washes, coverslips were mounted with Vectorshield (Vector Laboratories, Burlingame, CA). Immunofluorescent images were captured with a Zeiss LSM410 confocal fluorescence microscope. Images were captured at 512 x 512-pixel resolution with z-axis sectioning at 0.5-µm intervals. To verify specificity, negative controls were performed in which primary antibodies were omitted.

Measurement of endothelial [Ca2+]cyt. DVR were loaded with fura-2 AM (2 µM) at 37°C for 15 min. We previously showed that this protocol results in preferential fura-2 loading into endothelial cells and negligible fluorescent signal from pericytes (23). The vessels were visualized with a Nikon Fluor x40 (numerical aperture 1.3) oil-immersion objective. Fura-2 was alternately excited at 350 and 380 nm using a computer-controlled monochromator (PTI). Background-subtracted fluorescence emission ratios (F350/380) were isolated using a 510WB40 (Omega optical) filter and directed to a photon-counting photomultiplier assembly.

Reagents. Glibenclamide, niflumic acid, ouabain, nystatin, collagenase 1A, protease XIV, and other chemicals were obtained from Sigma. The enzyme digestion solution was prepared in 50-ml batches, frozen in 2-ml aliquots, and thawed daily as needed. Glibenclamide and niflumic acid were dissolved in DMSO. Reagents were thawed and diluted 1:1,000 on the day of the experiment. Fura-2 (Molecular Probes) was stored at 1 mM in anhydrous DMSO. Reagents were thawed once, and the excess was discarded at the end of the day.

Statistics. Data are means ± SE. The significance of differences was evaluated with SigmaStat 3.11 (Systat Software, Point Richmond, CA) using parametric or nonparametric tests as appropriate for the data. Comparisons between two groups were performed using Student's t-test (paired or unpaired, as appropriate) or the rank sum test (nonparametric). Comparisons between multiple groups employed repeated-measures ANOVA or repeated-measures ANOVA on ranks (nonparametric). Post hoc comparisons were performed using Tukey's or Holm-Sidak tests. P < 0.05 was used to reject the null hypothesis.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Concentration and voltage dependence of Ba2+ blockade. DVR endothelia are a highly coupled electrical syncytium so that inhibition of gap junctions is required to measure single-cell currents in intact vessels (29). We found that raising the bath K+ concentration from 5 to 140 mM always tended to close those junctions in a reversible manner, manifest as a sharp reduction of the time required for decay of capacitance transients (Fig. 1, A–C). We previously measured the effects of gap junction blockers to inhibit cell-to-cell coupling by quantifying the time required for the initial capacitance transient to decay by 85% toward baseline (29). When that approach was applied to these data, the decay time fell from 26 ± 4 to 0.9 ± 0.1 ms when the extracellular solution was switched from 5 to 140 mM KCl (Fig. 1D).


Figure 1
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  		Fig. 1. Capacitance transients in descending vasa recta (DVR) endothelium. A–C: examples of whole cell currents recorded from nystatin-patched DVR endothelial cells. Arrows point to the regions of the trace that represent the capacitance transient. The cell was held at –70 mV and pulsed to levels between –160 and +40 mV for 2 s. External [K+] was 5 (A) or 140 mM (B). Reduction of the time required for decay of the capacitance transient was reversible when external [K+] returned to 5 mM (compare B and C). To expand the region of the capacitance transients, only the first 250 ms of the 2,000-ms records are displayed. D: summary of the time required for the capacitance transient to change from its peak to 85% of final baseline in either 5 or 140 mM K+. The data are derived from analysis of the pulse to –80 mV from the holding level of –70 mV. High (140 mM) external [K+] dramatically reduced the decay time, indicating closure of gap junctions (P < 0.01, n = 12).

 
DVR endothelial cells were held at –70 mV and pulsed to levels between –160 and +40 mV in 20-mV increments for 2.0 s (Fig. 2A). Strong, inwardly rectifying currents were present when pulse potential was below Keq. Those currents were dramatically inhibited by Ba2+ (100 µM; Fig. 2, B and C). We examined the concentration dependence of the block by Ba2+. DVR endothelial cells were bathed in 140 mM KCl, and extracellular Ba2+ concentration was sequentially increased from 0 to 0.1, 1, 10, and 100 µM. End-pulse currents for Ba2+ concentrations of 0, 0.1, 1.0, 10, and 100 µM are summarized in Fig. 2D. These are also displayed as the Ba2+-sensitive currents (Fig. 2E) obtained by subtracting records of individual cells in Ba2+ from the currents that were present before Ba2+ inhibition. At negative membrane potentials, inward currents were very sensitive to Ba2+; concentrations at or above 1 µM produced near-complete inhibition. Little additional inhibition was observed when Ba2+ concentration was increased from 10 to 100 µM.


Figure 2
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  		Fig. 2. Concentration dependence of inhibition of endothelial inward rectifier K+ channel (KIR) current by Ba2+. A: protocol used to measure whole cell currents. Endothelial cells were changed from a holding level of –70 mV to pulse potentials between –160 and +40 mV for 2 s. B and C: examples of endothelial cell currents in 140 mM KCl at baseline (B) and after inhibition by 100 µM Ba2+ (C). Arrow indicates zero current. D: summary of means ± SE of end pulse currents (Im) measured in Ba2+ at 0, 0.1, 1, 10, and 100 µM. E: summary of means ± SE of Ba2+-sensitive end pulse currents calculated by subtracting currents in Ba2+ from those at baseline in individual cells. Complete inhibition of inward currents was achieved by [Ba2+] > 1 µM.

 
As summarized in Fig. 3, inhibition by Ba2+ was voltage dependent. The normalized Ba2+ currents were fit to the equation IBa/I0 = 1/(1 + [Ba2+]/k), where IBa and I0 are whole cell current in the presence and absence of Ba2+, respectively, and k is the binding constant (Fig. 3A). The binding constants for Ba2+ were k = 0.18 ± 0.04, 0.28 ± 0.07, 0.33 ± 0.08, 0.47 ± 0.09, 0.60 ± 0.14, and 0.95 ± 0.28 µM at –160, –140, –120, –100, –80, and –60 mV, respectively (Fig. 3B).


Figure 3
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  		Fig. 3. Voltage dependence of inhibition of KIR current by Ba2+. A: relationship between [Ba2+] and inward current (IBa) normalized to that in the absence of Ba2+ (I0) for pulse potentials between –40 and –160 mV. Lines are the least-squares fit of the data to the equation IBa/I0 = 1/(1 + [Ba2+]/k). B: apparent Ba2+ affinity (k) vs. pulse potentials. Vm, membrane potential. The values for k are strongly voltage dependent: 0.18, 0.28, 0.33, 0.47, 0.60, and 0.95 µM at –160, –140, –120, –100, –80, and –60 mV, respectively (n = 6).

 
Concentration and voltage dependence of Cs+ blockade. KIR currents are also characterized by sensitivity to Cs+ (18, 24). We tested the effect of Cs+ on DVR endothelial KIR current using the protocol shown in Fig. 4A. Examples of the currents elicited in the absence and presence of Cs+ are shown in Fig. 4, B and C, respectively. In these experiments, KIR current was recorded in 140 mM KCl with external Cs+ concentrations of 0, 1, 10, 100, and 1,000 µM. End-pulse currents are summarized in Fig. 4D. Cs+-sensitive currents were calculated by subtracting records in Cs+ from those elicited before the introduction of Cs+ into the extracellular buffer (Fig. 4E). Cs+ inhibited the inward current, but at higher concentrations than Ba2+.


Figure 4
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  		Fig. 4. Concentration dependence of inhibition of endothelial KIR current by Cs+. A: protocol used to examine whole cell currents. Endothelial cells were changed from a holding level of –70 mV to pulse potentials between –160 and +40 mV for 2 s. B and C: examples of endothelial cell currents in 140 mM KCl at baseline (B) and after inhibition by 1 mM Cs+ (C). D: summary of means ± SE of end pulse currents measured in Cs+ at 0, 1, 10, 100, and 1,000 µM. E: summary of means ± SE of Cs+-sensitive end pulse currents calculated by subtracting currents in Cs+ from those at baseline in individual cells.

 
Like Ba2+ (Fig. 3), inhibition by Cs+ was voltage dependent (Fig. 5). The ratio of the membrane current in Cs+ (ICs), normalized to that in its absence (I0), is shown in Fig. 5A. The binding constant for Cs+, fit to ICs/I0 = 1/(1 + [Cs+]/k), yielded k = 19.8 ± 4.5, 32.3 ± 12.9, 47.8 ± 15.3, 117.9 ± 28.2, 253.3 ± 29.6, and 732.5 ± 176.6 µM at –160, –140, –120, –100, –80, and –60 mV, respectively (Fig. 5B).


Figure 5
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  		Fig. 5. Voltage-dependent inhibition of inward rectifier current by Cs+. A: relationship between [Cs+] and inward current (ICs) between –40 and –160 mV. Lines are the least-squares fit of the data to the equation ICs/I0 = 1/(1 + [Cs+]/k). B: Cs+ binding constant k vs. pulse potentials. The values for k are strongly voltage dependent: 19.8, 32.3, 47.8, 117.9 2, 253.3, and 732.5 µM at –160, –140, –120, –100, –80, and –60 mV, respectively (n = 5).

 
Contributions of KIR to endothelial cell membrane potential and Ca2+ signaling. A postulated function of KIR is to induce hyperpolarization in response to small elevations of extracellular K+ concentration. In addition to K+-induced increases in KIR conductance, stimulation of the activity of electrogenic 3Na+:2K+ exchange by Na+/K+-ATPase can contribute to K+-induced hyperpolarization. Accordingly, to measure the effects of KIR activation, stimulated by K+ elevation, on membrane potential, we measured membrane potential in the presence of ouabain. As shown in Fig. 6A and summarized in Fig. 6B, ouabain alone depolarized DVR endothelial cells from –53 ± 3.0 to –29 ± 2.7 mV (P < 0.05), verifying a large role for Na+/K+-ATPase activity in the generation of DVR endothelial membrane potential. Subsequent elevation of extracellular K+ concentration from 5 to 10 mM in the presence of ouabain repolarized membrane potential to –45 ± 2.9 mV (P < 0.05), supporting the ability of KIR activation to reduce it. The repolarization by 10 mM KCl was reversed by Ba2+ (30 µM).


Figure 6
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  		Fig. 6. Effects of extracellular K+ and ouabain on DVR endothelial membrane potential. A: example of the effect of ouabain (1 mM) and extracellular K+ elevation (5 to 10 mM) on DVR endothelial membrane potential. Ouabain depolarized the cell, after which an increase in [K+] in the presence of ouabain repolarized the membrane potential. The repolarization by 10 mM K+ was reversed by Ba2+ (30 µM). The effects of ouabain, K+ elevation, and Ba2+ were reversible upon washout. B: summary of membrane potential measurements at baseline, in ouabain, in 10 mM K+ + ouabain, and in 10 mM K+ + ouabain + Ba2+. Open circles show results for individual cells, and the filled circle shows means ± SE (*P < 0.05 for comparison among the periods, n = 5).

 
It is generally accepted that endothelia lack VOCa isoforms so that Ca2+ entry occurs through nonselective cation channels (1, 11, 19). Thus hyperpolarization favors an increase in endothelial [Ca2+]cyt, because the electrochemical driving forces that favor Ca2+ entry rise and transmembrane Ca2+ conductance is not inhibited (as in VOCa-expressing cells). Since external K+ can hyperpolarize the DVR endothelia (Fig. 6), we tested whether it also elevates [Ca2+]cyt. An increase of external K+ from 5 to 10 mM elevated [Ca2+]cyt in a Ba2+-sensitive manner (P < 0.05) (Fig. 7). The blockade by Ba2+ was reversible (P = 0.06, n = 8).


Figure 7
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  		Fig. 7. Effect of elevation of external K+ on DVR endothelial cytoplasmic [Ca2+] ([Ca2+]cyt). Background subtracted fluorescence ratios (F350/380) from fura-2-loaded endothelia vs. extracellular [K+] ([K+]o). Elevation of extracellular [K+] from 5 to 10 mM increased DVR endothelial [Ca2+]cyt (*P < 0.05), an effect that was reversibly blocked by 100 µM extracellular Ba2+ ([Ba2+]o) (P = 0.06, n = 8).

 
Expression of KIR2.1, KIR 2.2, and KIR 2.3 in DVR. To further verify expression of KIR2.x strongly rectifying isoforms in DVR, we performed immunochemical staining with antibodies directed against KIR2.1, KIR2.2, and KIR2.3 subunits. Immunofluorescent images identified their expression in both pericytes and endothelia (Fig. 8). Staining was eliminated by omission of the primary antibody (not shown).


Figure 8
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  		Fig. 8. Immunochemical identification of KIR2.x in the isolated DVR. Hand-dissected rat DVR were fixed, permeabilized, and immunolabeled with anti {alpha}-smooth muscle actin ({alpha}-SMA; red) or antibodies directed against KIR2.x isoforms (green). White light images at left were obtained with differential interference contrast (DIC) optics. Arrowheads point to pericyte cell bodies protruding from the abluminal surface (red, compare with DIC images). Both the luminal endothelial cells and the abluminal pericytes show KIR2.1, KIR2.2, and KIR2.3 expression. Similar to results from n = 5 experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
DVR are 12- to 15-µm-diameter branches of juxtamedullary efferent arterioles that supply the renal medulla with the blood flow. DVR contract and dilate in response to a variety of hormonal and paracrine agents. Membrane potential modulates Ca2+ entry into DVR pericytes and depolarization can induce DVR contraction. Several ion channels classes are expressed in the pericytes and contribute to the regulation of membrane potential, [Ca2+]cyt, and vasoactivity (3, 4, 16, 17, 20, 21, 25, 30–32). Recently, our laboratory (3) reported expression of a strong KIR conductance in DVR pericytes and verified its contribution to membrane potential. In the present study, we expanded those observations to show that KIR also play a role to set membrane potential and modulate [Ca2+]cyt in the DVR endothelium.

DVR express connexins 37, 40, and 43. We have previously shown that there is variable myoendothelial coupling between pericytes and endothelia and that the endothelium is highly coupled as an electrical syncytium (29). Despite this, the currents measured in this study could not have been conducted from pericytes to endothelia because the pericytes were stripped away before patch-clamp recording (25, 29). An interesting observation that facilitated these studies concerns the effect of external K+ elevation on interendothelial gap junction coupling. Elevation of extracellular K+ from 5 to 140 mM always reduced the prolonged capacitance transients that result from cell-to-cell coupling such that their decay fell to the submillisecond time scale that is consistent with single-cell recording (Fig. 1). The KIR-, Ba2+-, and Cs+-sensitive currents illustrated in Figs. 2 and 4 are at least twice as large as those previously measured in DVR pericytes (3). A mean of ~800 pA of endothelial KIR current was elicited at a membrane potential of –160 mV, corresponding to a conductance of 5 nS. We recognize that these studies were not designed to thoroughly document the regulation of coupling by K+ and verify it by independent means. As such, we cannot rule out the possibility that some of that KIR currents resulted from transfer of charge to the patched cell from its neighbors. Given the submillisecond duration of capacitance transients, however, such a contribution seems unlikely to have been predominant. The regulation of gap junction coupling was not the topic under study, and these protocols were not properly designed to optimally define capacitance transients. Nonetheless, the present observations clearly show substantial K+-induced closure of cell-to-cell communication. Given that K+ concentrations in the renal medulla can be high (12, 22), we speculate that K+ effects on gap junctions might have a physiological niche in vivo.

DVR pericytes and endothelia have at least some coupling via myoendothelial gap junctions (29) and tend to have similar membrane potential responses to constrictors and dilators such as bradykinin and angiotensin II (25). Based on those observations, it seems possible that effects of external K+ to augment KIR conductance and induce hyperpolarization in endothelia might lead to transfer of charge via connexins to hyperpolarize and relax pericytes via inhibition of their voltage-gated Ca2+ entry pathways (30, 32). The large KIR conductance apparent from the data in Fig. 2 also point to that as a possible mechanism through which K+ might influence contraction and renal medullary blood flow.

In smooth muscle, VOCa, activated by depolarization, conduct Ca2+ into the cytoplasm to stimulate contraction. Their voltage sensitivity assigns a regulatory role to membrane potential so that hyperpolarization favors inhibition of contraction by lowering [Ca2+]cyt. We have previously shown that pericytes, the smooth muscle remnants of the DVR wall, express voltage-gated cation entry pathways (16, 30, 32) and are hyperpolarized by vasodilators and K+ (3, 25). In this study, we extended observations concerning the role of KIR to regulate DVR by showing that their endothelial expression is also robust. Since specific organic chemical inhibitors of KIR are not available, identification of KIR relies on observation of their characteristic voltage- and Keq-dependent current and inhibition by very low concentrations of Ba2+. With regard to the former, KIR conduct K+ from the extracellular space to the cytoplasm well, whereas movement of K+ in the reverse direction is more or less impeded to a degree that depends on the KIR isoform under study. Those characteristics in DVR endothelia, shown in Figs. 2 and 3, are clear. When membrane potential was held below the equilibrium potential for K+ in our buffers (about –2 mV), a large inward current was generated that reached about –850 pA at holding potentials of –160 mV. At negative potentials, that current showed exquisite sensitivity to inhibition by Ba2+ with k < 1 µM between membrane potentials of –160 and –40 mV. Albeit with less sensitivity than Ba2+, Cs+ ions also inhibit KIR conductance (24), a characteristic that was also confirmed (Figs. 4 and 5) with k for Cs+ of <1 mM over the same range of membrane potentials.

There are seven known subtypes of KIR channels (15, 24). Among those, KIR2.x and KIR4.x exhibit strong inward rectification; the former is the one generally expressed in the vasculature (24). The characteristics of the DVR endothelial KIR current are similar to those described by other investigators in other vascular beds (1, 7, 10, 19). Given the robust KIR currents we have identified in DVR pericytes (3) and endothelia, we sought to confirm their expression in both cell types by immunostaining hand-dissected vessels using commercial antibodies directed against KIR2.1, KIR2.2, and KIR2.3 (antibody against KIR2.4 is not available). To aid localization, we counterstained DVR pericytes with an antibody directed against SMA. Immunofluorescent images verified that DVR express KIR2.1, 2.2, and 2.3 subunits and that they are present in both pericytes and the endothelium (Fig. 8).

Since the original verification that K+ can function as an endothelium-dependent hyperpolarizing factor, it has been speculated that an important function of vascular KIR is to mediate K+-induced hyperpolarization and vasodilatation. Elevation of extracellular K+ within the range of 5–20 mM is sufficient for such activity (1, 3, 5, 19, 24). Several studies have demonstrated K+-induced vasodilatation and/or hyperpolarization of smooth muscle in cerebral (6, 8, 13), coronary (27), and renal afferent arterioles (5) and DVR pericytes (3). The presence of KIR expression in DVR endothelial cells raises the possibility that their hyperpolarization by K+ might influence DVR contraction in vivo. Several mechanisms might be involved. Spread of ions to equilibrate membrane potential between pericytes and endothelia cannot be excluded. Investigators in our laboratory (25) have observed that when stimulated by constrictors and dilators, endothelia and pericytes depolarize and hyperpolarize in parallel. Our group (29) recently showed that endothelial cells of the DVR wall are highly coupled by gap junctions and that some, but not all, pericytes exhibit myoendothelial junctions capable of conducting diffusion of the 454-Da fluorescent probe Lucifer yellow so that movement of monovalent ions or signaling molecules seems quite possible.

Although specifically unexplored in DVR, it is generally accepted that endothelia lack VOCa (1, 11, 19). Based on that assumption, endothelial hyperpolarization favors enhancement of Ca2+ entry by an increase in its electrochemical gradient. Hypothetically, an increase in endothelial [Ca2+]cyt might favor activation of pathways that generate diffusible vasodilators, such as prostaglandins and NO, via Ca2+-sensitive isoforms of NO synthase. We tested whether small elevations of extracellular K+ induce membrane hyperpolarization in DVR endothelial cells. In the presence of the Na+/K+-ATPase inhibitor ouabain, raising K+ concentration 5–10 mM caused endothelial membrane hyperpolarization that was reversed by the KIR channel blocker Ba2+ (Fig. 6). As expected, the small increase in extracellular K+ also increased DVR endothelial [Ca2+]cyt (Fig. 7).

The origin of the extracellular K+ ions available to activate KIR of DVR pericytes and endothelia is a question of interest. Theories generally point to the possibility that elevation of extracellular K+ results from extrusion of K+ through ubiquitous KCa in response to [Ca2+]cyt elevation. Notably, in endothelia that also express KIR, this could represent positive feedback, because the concomitant increase in KIR conductance due to the extracellular K+ concentration elevation would favor further hyperpolarization. The overall process must be self-limiting, because the equilibrium potential for K+ would rise as the extracellular K+ cloud forms, thus reducing driving force for further K+ extrusion. In addition to release of K+ ions from vascular cells via KCa, medullary K+ recycling might play a role in extracellular K+ concentration elevation. Plasma sampled from vasa recta at the papillary tip has been shown to have a K+ concentration >30 mM (22), possibly resulting from K+ recycling between collecting duct and pars recta (12). Thus changes in the rate of K+ recycling might regulate K+ concentration in plasma and interstitium surrounding the vasa recta to modulate DVR vasoactivity through KIR and Na+/K+-ATPase-mediated mechanisms.

In summary, DVR endothelial cells express a strong KIR current that is highly sensitive to inhibition by Ba2+ and Cs+. We identify a major role for Na+/K+-ATPase in the setting of DVR endothelial membrane potential and show a clear capacity for KIR activation to hyperpolarize the cells and raise [Ca2+]cyt. These results are consistent with a putative role for K+ to act as a renal medullary vasodilator.


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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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Studies in our laboratory were supported by National Institutes of Health Grants R37 DK42495, R01 DK67621, P01 HL78870 (T. L. Pallone), and R01 DK53775 (A. Edwards) and by American Heart Association Postdoctoral Fellowship 0625404U (C. Cao).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. L. Pallone, Division of Nephrology, N3W143, 22 S. Greene St, Univ. of Maryland Medical System, Baltimore, MD 21201 (e-mail: tpallone{at}medicine.umaryland.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.


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 DISCUSSION
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