Inhibition of K + conductance in descending vasa recta pericytes by ANG II

【摘要】  We tested whether K + channel inhibition accompanies ANG II-induced depolarization of descending vasa recta (DVR) pericytes. An increase in extracellular K + concentration ([K + ] o ) from 5 to 100 mM depolarized resting pericytes but had no effect after prolonged (10 nM, 20 min) ANG II exposure. In contrast, reduction of extracellular Cl - concentration ([Cl - ] o ) from 154 to 34 mM had a minor effect on resting membrane potential but strongly depolarized pericytes treated with ANG II. The K + channel blockers BaCl 2 (0.1, 1 mM) and tetraethylammonium (TEA; 30 mM) depolarized resting pericytes but did not affect membrane potential of ANG II-treated pericytes. Pericyte whole cell currents were reduced by ANG II and nearly eliminated by combined ANG II exposure and the Cl - channel blocker niflumic acid (100 µM). Augmentation of inward current induced by raising [K + ] O from 5 to 50 mM was eliminated by preexposure to ANG II. TEA- and BaCl 2 -sensitive outward currents, generated by depolarizing pericytes from -80 to -40 mV, were eliminated by ANG II. We conclude that ANG II depolarizes DVR pericytes by a combination of Cl - channel activation and K + channel inhibition.

【关键词】  patch clamp microcirculation kidney medulla

BLOOD FLOW TO THE MEDULLA of the kidney is supplied by descending vasa recta (DVR). DVR are 15-µm-diameter arteriolar microvessels that arise predominantly from juxtamedullary efferent arterioles to form outer medullary vascular bundles. The radial arrangement of DVR within the bundles leads to the conclusion that DVR participate in the transport processes essential to countercurrent exchange and to the control of blood flow distribution within the medulla ( 9, 24, 27 ). Pericytes are smooth muscle-like cells that surround DVR and impart contractile function. As such, the DVR pericyte is a key regulator of medullary perfusion. DVR are vasoactive and respond to a wide variety of systemic and paracrine vasoconstrictors and dilators ( 27 ).

A growing body of literature points to regulation of perfusion of the renal medulla in determination of diuretic state, blood pressure, sodium balance, and extracellular fluid volume ( 9, 24 ). Despite the importance of the DVR pericyte in this scheme, fundamental information concerning the channel architecture and signaling processes that govern its behavior has been lacking. To bridge that gap, we adapted electrophysiological methods to permit investigations of the control of membrane potential in pericytes of DVR explanted by microdissection. We found that ANG II constriction of DVR is presaged by activation of a 16.8-pS Ca 2+ -dependent Cl - channel and an increase in Ca 2+ -activated, niflumic acid-sensitive Cl - conductance. Using patch-clamp techniques with artificial buffers, we observed Cl - channel activation that depolarizes membrane potential from resting values (-50 to -70 mV) toward the equilibrium potential of the Cl - ion ( 26, 30, 38 ), an action that favors voltage-gated Ca 2+ entry into the pericyte cytoplasm ( 39 ). In other smooth muscle cells, ANG II has also been shown to modulate the activity of K + channels. ANG II activates K + conductance in mesangial cells and inhibits K + conductance in smooth muscle of the renal artery ( 6, 11, 32 ). ANG II activation of K + channels could serve as a braking mechanism that serves to limit depolarization, cytoplasmic Ca 2+ entry, and vasoconstriction ( 10, 18, 25 ). In contrast, K + channel deactivation has been also been demonstrated in small arterioles of the renal and cerebral circulation, mediated by signaling through 20-hydroxyeicosatrienoic acid ( 2, 23, 33 ). K + channel inhibition would be predicted to favor smooth muscle depolarization and intensification of vasoconstriction.

In this study, we tested whether ANG II-induced depolarization of DVR pericytes is accompanied by modulation of pericyte K + conductance. The results show that prolonged ANG II exposure renders pericyte membrane potential insensitive to extracellular K + concentration ([K + ] o ) changes or K + channel blockers. Similarly, whole cell K + currents in DVR pericytes are reduced by prolonged ANG II stimulation. The data support the hypothesis that ANG II-induced depolarization of DVR pericytes occurs through combined stimulation of a Ca 2+ -activated Cl - conductance and inhibition of K + channels.

METHODS

Whole cell patch-clamp recording. All investigations involving animal use were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Maryland. Kidneys were removed from Sprague-Dawley rats (70-150 g; Harlan), sliced, and stored at 4°C in a physiological saline solution (PSS; in mM: 145 NaCl, 5 KCl, 1 MgCl 2, 1 CaCl 2, 10 HEPES, and 10 glucose, pH 7.4) at room temperature. As previously described, membrane potential and whole cell currents were monitored by patch-clamp recording from pericyte cell bodies on isolated vessels at room temperature ( 26, 30, 38, 39 ). Small wedges of renal medulla were separated from kidney slices by dissection and transferred to Blendzyme 1 (Roche) at 0.27 mg/ml in high-glucose DMEM media (Invitrogen), incubated at 37°C for 45 min, transferred to PSS, and stored at 4°C. At intervals, DVR were isolated from the enzyme-digested renal tissue by microdissection and transferred to a perfusion chamber on the stage of an inverted microscope (Nikon Diaphot).

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 (Narshige PP-830), and heat polished. For whole cell perforated patch-clamp recording, the pipette solution contained (in mM) 120 K aspartate, 20 KCl, 10 NaCl, and 10 HEPES, pH 7.2, as well as nystatin (100 µg/ml with 0.1% DMSO) in ultrapure water. Nystatin was dissolved in DMSO, and the excess was discarded daily. Nystatin stock was dispersed into the K aspartate pipette solution at 37°C by vigorous vortexing for 1 min and protected from light. To clear any slight remaining nystatin precipitate, patch pipettes were backfilled from a syringe via a 0.2-µm filter.

Membrane potential was measured using a CV201AU headstage and Axopatch 200 amplifier (Axon Instruments, Foster City, CA) in current-clamp mode ( I = 0) at a sampling rate of 10 Hz using pipettes of 8- to 10-M resistance. Whole cell current recording in voltage-clamp mode was accomplished with 4- to 6-M pipettes. Due to the small size of the pericytes, lower resistance pipettes proved difficult to use. Pipettes with nystatin-containing electrode solution were inserted into the bath under positive pressure, positioned near the cell with a piezoelectric manipulator (Burleigh PCS-5000), and the offset of the amplifier was adjusted to null the junction and electrode potentials. After gigaseal and nystatin pore formation, final access resistance was between 15 and 40 M. Cell capacitance and access resistance compensations were applied as appropriate. Whole cell currents were sampled at 2 kHz during pulse protocols and at 10 Hz during continuous recording.

To examine the effects of [K + ] o or Cl - ion concentration ([Cl - ] o ) changes, either [Cl - ] o was lowered by substituting NaCl with Na gluconate or [K + ] o was raised by isosmotic substitution of NaCl with KCl. In those experiments, to avoid errors resulting from a change in the reference electrode-bath interface, a 3 M KCl-3% agar bridge was substituted for a Ag/AgCl wire as the reference electrode. The low-Cl - buffer contained (in mM) 130 Na gluconate, 25 NaCl, 5 KCl, 1 CaCl 2, 1 MgCl 2, 10 HEPES, and 10 glucose, pH 7.4, at room temperature. This buffer has a final Cl - concentration of 34 mM. As previously described, after accounting for Donnan effects, this yields a Cl - concentration that is approximately symmetrical across the cell membrane ( 17, 26 ). The high-K + buffer contained (in mM) 50 NaCl, 100 KCl, 1 CaCl 2, 1 MgCl 2, 10 HEPES, and 10 glucose, pH 7.4, at room temperature. Junction potential and Donnan potential effects were corrected as previously described ( 26 ). The predicted offset adjustment resulting from the change in diffusion potential between the 3 M KCl reference electrode and PSS vs. the low-Cl - or KCl buffers is <2 mV and was neglected.

Reagents. ANG II, nystatin, niflumic acid, and other chemicals were from Sigma (St. Louis, MO). Liberase Blendzyme 1 was from Roche Applied Science. Blendzyme was stored in 40-µl aliquots of 4.5 mg/ml in water and diluted into high-glucose DMEM on the day of the experiment. ANG II was stored as 10 µM aliquots in water, niflumic acid was stored in 100 µM aliquots, and both were frozen at -20°C and diluted into PSS on the day of the experiment. Stock reagents were thawed once, and the excess was discarded each day.

Statistics. Data are given as means ± SE. The significance of differences between means was calculated using Student's t -test (paired or unpaired, as appropriate) and analysis of variance. Some data sampled at 10 Hz were averaged 10 values at a time for display at 1 Hz. In figures that show averaged current or membrane potential records, the majority of error bars are suppressed to optimize graphic display of the data.

RESULTS

Effect of alterations in K + and Cl - equilibrium potentials on pericyte membrane potential. The central hypothesis tested by this study is that, in addition to activation of pericyte Cl - conductance ( 26, 39 ), ANG II depolarizes pericytes by inhibiting conductance to K +. To examine the relationship between the membrane potential and conductance to K + and Cl -, we compared the effects of altering the equilibrium potentials (K eq, Cl eq ) before and after ANG II treatment. [K + ] o was raised from 5 to 100 mM (electrode K +, intracellular K + concentration = 140 mM) by isosmotic substitution for Na +, a maneuver that changed K eq from -89 to -9 mV. Similarly, [Cl - ] o was lowered from 154 to 34 mM by isosmotic substitution for Na gluconate (electrode Cl -, intracellular Cl - concentation = -34 mM), changing the Cl eq from -43 to -3 mV. Bath K + and Cl - concentration changes were for 2 and 3 min, respectively. The effects of [K + ] o and [Cl - ] o changes are shown in Fig. 1. Raising K eq from -89 to -9 mV before ANG II depolarized pericytes from resting level to about -10 mV. Raising Cl eq to -3 mV was substantially less effective; pericyte membrane potential remained well below Cl eq = -3 mV, increasing to only -42 mV. Those results are largely as expected if resting membrane potential is predominantly determined by conductance to the K + ion. Before ANG II exposure, reduction of [Cl - ] o to 34 mM led to rapid depolarization, followed by some tendency toward repolarization. The duration of exposure to low [Cl - ] o (3 min) was too short to fully define the response, but an effect of changes in [Cl - ] o on Cl - channel activity might exist.

Fig. 1. Effect of K + and Cl - ion substitution on descending vasa recta (DVR) pericyte membrane potential. A : membrane potential, measured as extracellular K + concentration ([K + ] o ), was isosmotically increased from 5 to 100 mM before and then after addition of ANG II (10 nM) to the extracellular buffer. Values are means ± SE; n = 8, 7, 6, and 5 observations, respectively, during successive periods (0-5, 10-15, and 20-25 min) after addition of ANG II to the bath. The initial period from 0-5 min includes ANG II "wash-in." The number of observations decreased with time due to loss of some seals. K eq is the theoretical Nernst equilibrium potential for the K + ion (-9 mV, [K + ] o = 100 mM, intracellular K + concentration = 140 mM). B : membrane potential was recorded as extracellular Cl - was isosmotically reduced from 154 to 34 mM at intervals before and after ANG II as in A. Values are means ± SE; n = 8, 7, 5, and 5 observations, respectively. Cl eq is the theoretical Nernst Cl - equilibrium potential for the Cl - ion (-3 mV, extracellular Cl - concentration = 34 mM, intracellular Cl - concentration = 30 mM). Data were sampled at 10 Hz and averaged to 1 Hz for display. Solid horizontal bars represent 2 min of 100 mM K + exposure in A and 3 min of 34 mM Cl - exposure in B. Most error bars have been suppressed for clarity.

After the effects of [K + ] o and [Cl - ] o changes on resting potential were measured, pericytes were exposed to ANG II (10 nM). As previously reported ( 26 ), the membrane depolarized to about -30 mV. At intervals after exposure to ANG II, [K + ] o and [Cl - ] o were again changed to 100 and 34 mM, respectively. The magnitude of responses to the associated change in K eq and Cl eq changed with the time period that elapsed since ANG II application. At 5, 15, and 25 min, raising [K + ] o to 100 mM became progressively less effective to induce further depolarization toward K eq ( Fig. 1 A ). In contrast, low [Cl - ] o became progressively more effective to depolarize membrane potential toward Cl eq ( Fig. 1 B ). Time controls in which the effects of altering [K + ] o and [Cl - ] o were tested before and after 25 min of perforated patch formation revealed stable responses (data not shown), ruling out K + channel rundown as an explanation for the decline in response to raising [K + ] o in Fig. 1 A.

The experiments in Fig. 1 have two potential interpretations. First, after ANG II exposure, pericyte Cl - conductance might have progressively increased to become overwhelmingly large, making K + conductance insignificant by comparison. A second explanation is that pericyte K + conductance was inhibited so that pericyte membrane potential was determined by conductance to Cl - and other ions. Further experiments were performed to distinguish those possibilities.

Effect of K + channel blockers on pericyte membrane potential. We examined the effects of the nonspecific K + channel blockers Ba 2+ and tetraethylammonium (TEA) on membrane potential before and after ANG II exposure. Figure 2, A and B, shows the respective effects of 0.1 and 1.0 mM Ba 2+ on resting pericyte membrane potential. At both concentrations, Ba 2+ induced substantial depolarization. In contrast, after ANG II exposure (10 nM, 20 min), 1.0 mM Ba 2+ had no effect ( Fig. 2 C ). Experiments with Ba 2+ are summarized in Fig. 2, D - F. We also tested the effect of 30 mM TEA on membrane potential ( Fig. 3 ). TEA depolarized pericytes before ( Fig. 3 A ) but not after 20 min of ANG II ( Fig. 3 B ). Results with TEA are summarized in Fig. 3, C and D.

Fig. 2. Effect of Ba 2+ on DVR pericyte membrane potential. A and B : sample tracings showing the effect of extracellular Ba 2+ (0.1, 1.0 mM) on resting membrane potential of DVR pericytes. The membrane potential (in mV) at the beginning of the record is noted under each trace. C : example of a record demonstrating the lack of effect of Ba 2+ (1.0 mM) on pericyte membrane potential after prolonged exposure to ANG II (10 nM, 20 min). D and E : summaries showing membrane potential measurements from different cells before, during, and after exposure to Ba 2+ at either 0.1 or 1.0 mM. Values shown above the graph are means ± SE of measurements before, during, and after Ba 2+ application. * P < 0.05, Ba 2+ vs. baseline. F : summary of membrane potential measurements from cells previously exposed to ANG II (10 nM, 20 min). Measurements were taken before, during, and after exposure to Ba 2+ (1.0 mM).

Fig. 3. Effect of tetraethylammonium (TEA + ) on DVR pericyte membrane potential. A : sample tracing showing the effect of extracellular TEA + (30 mM) on resting potential of a DVR pericyte. The membrane potential at the beginning of the record (in mV) is shown under each trace. B : example of a record demonstrating the lack of effect of TEA + (30 mM) on pericyte membrane potential after prolonged exposure to ANG II (10 nM, 20 min). C : summary of membrane potential measurements from different cells before, during, and after exposure to TEA + (30 mM). * P < 0.05, TEA + vs. baseline. D : summary of membrane potential measurements from cells previously exposed to ANG II (10 nM, 20 min). Measurements were taken before, during, and after exposure to TEA +.

In two cells, ANG II induced membrane potential oscillations. Those results were excluded from the summaries shown in Figs. 2 F and 3 D. During the oscillations, the effects of Ba 2+ and TEA to augment ANG II-induced depolarization remained unaltered over time ( Fig. 4 ), suggesting that ANG II might be less effective in inhibiting K + conductance when pericyte membrane potential is oscillating. Serendipitous occurrences of oscillations were too few to permit further exploration of that issue.

Fig. 4. Effect of TEA + and Ba 2+ during ANG II-induced membrane potential oscillations. Tracings show 2 records in which pericyte membrane potential undergoes sustained oscillations after ANG II exposure. In each case, exposure to either TEA (30 mM) or Ba 2+ (1.0 mM) augments depolarization. These K + channel blockers do not appear to lose effectiveness even after a prolonged exposure to ANG II (contrast with Figs. 2 and 3 ). The resting membrane potential (in mV) before ANG II exposure is shown at the beginning of each trace.

Effect of ANG II on whole cell currents. In a series of pericytes, whole cell current was measured during voltage clamp before and after 20-min exposure to ANG II (10 nM). The protocol employed is illustrated in Fig. 5 A. Cells were held at -80 mV and pulsed from -150 to 40 mV (corrected for junction potential) in 20-mV increments for 500 ms (10 s between pulses). Figure 5 B shows the currents ( I m ) elicited by that protocol. For all V m -90 mV, where electrochemical forces favor K + efflux from the cell, ANG II caused a reduction of I m. Note that in the range of V m that spans physiological membrane potential, the reduction of I m after ANG II was a large fraction of the total current that was present before ANG II. That implies that ANG II has an important effect on ensemble ion conductance in the pericytes. If ANG II inhibits K + channels, then residual currents after should largely be due to transport of Cl - ion. To test this, we measured the effect of the Cl - channel blocker niflumic acid (100 µM) on post-ANG II currents. Niflumic acid nearly eliminated post-ANG II currents and shifted the reversal potential from -48 to -5 mV ( Fig. 5, C and D ).

Fig. 5. Effect of ANG II on whole cell current. A : protocol used for depolarizations. Pericytes were depolarized from a holding potential of -80 mV to pulse potentials ranging from -150 to +40 mV (1,000 ms, 20-mV increments). B : ordinate shows means ± SE of cell current ( I m; n = 6) as a function of pulse potential ( V m ) at baseline and after exposure to ANG II (10 nM, 20 min). The abscissa is corrected for junction potential. The arrow indicates the theoretical equilibrium potential for the Cl - ion in our buffers (-43 mV). * P < 0.05 vs. baseline. C : example of cell current traces in ANG II (10 nM, 20 min) before and after inhibition by niflumic acid (100 µM). D : ordinate shows means ± SE of cell current ( n = 7) vs. pulse potential in cells that have been exposed to ANG II (10 nM, 20 min). Data show end pulse current before and after addition of niflumic acid (Nifl A; 100 µM) to the bath. The abscissa is corrected for junction potential. * P < 0.05 vs. ANG II.

Effect of increasing [K + ] o on whole cell currents. To test whether ANG II inhibition of K + conductance is responsible for the reduction of whole cell current in Fig. 5 B, we augmented the electrochemical driving force favoring K + entry by increasing [K + ] o from 5 to 50 mM. Cells were held at -80 mV and pulsed to a range of -150 to +40 mV (10-mV increments). As shown in Fig. 6 C, 50 mM K + increased the inward current at negative pulse potentials. ANG II exposure (10 nM, 20 min) eliminated the augmentation of inward currents by 50 mM KCl ( Fig. 6 D ), favoring the interpretation that conductance(s) specific to the K + ion are suppressed by ANG II.

Fig. 6. Effect of ANG II on K + inward currents. A and B : tracings showing whole cell current in 5 ( A ) and 50 mM KCl ( B ). Pericytes were depolarized from a holding potential of -80 mV to pulse potentials ranging from -150 to +40 mV (500 ms, 10-mV increments). C : ordinate shows means ± SE of cell current ( n = 6) as a function of pulse potential in PSS (KCl, 5 mM) and after extracellular KCl concentration was increased to 50 mM by isosmotic substitution for NaCl. The abscissa is corrected for junction potential. At negative pulse potentials, inward current increased when [K + ] o was raised to 50 mM. * P < 0.05 vs. 5 mM KCl. D : ordinate shows means ± SE of cell current ( n = 6) as a function of pusle potential in PSS (KCl, 5 mM) and after extracellular KCl concentration was increased to 50 mM by isosmotic substitution for NaCl. Cells had been preexposed to ANG II (10 nM, 20 min). The abscissa is corrected for junction potential. After ANG II treatment, inward currents at negative pulse potentials are not enhanced by increasing [K + ] o from 5 to 50 mM (compare with C ).

To further confirm that ANG II inhibits K + conductance, parallel protocols were performed in which inward current was continuously monitored while pericytes were held at -80 mV and [K + ] o was raised from 5 to 50 mM by isosmotic substitution for extracellular Na + concentration. In one group, the [K + ] o change was performed 20 min after patch formation. As expected, raising [K + ] o to 50 mM induced a reversible inward current ( Fig. 7 A ). In another group, pericytes were treated with ANG II for 20 min before patch formation and then exposed to 50 mM [K + ] o. The inward currents were abolished whether cells were exposed to ANG II before or after establishment of electrical access to the cytoplasm via nystatin ( Fig. 7, B and C; summarized in Fig. 7 D ). The results in Fig. 7 confirm inhibition of pericyte K + conductance by ANG II and show that the duration of nystatin pore formation does not influence the results; i.e., K + channel rundown cannot explain the insensitivity of inward currents to [K + ] o in the experiments.

Fig. 7. Effect of ANG II on K + inward currents. A : pericytes were held at -80 mV (after junction potential correction) for 20 min while cell current was measured continuously. After 20 min, [K + ] o was transiently increased from 5 to 50 mM by isosmotic substitution for NaCl. The example shows that increasing [K + ] o induced an inward current, reinforcing the findings obtained with pulse protocols in Fig. 6. B : pericytes were held at -80 mV for 20 min in the presence of ANG II (10 nM). After exposure to ANG II, increasing [K + ] o failed to induce an inward current. C : pericytes were exposed to ANG II (10 nM) for 20 min before electrical access for patch-clamp recording was obtained. Subsequently, current was monitored in cells held at -80 mV as [K + ] o was transiently increased from 5 to 50 mM. After exposure to ANG II, increasing [K + ] o failed to induce an inward current. D : summary of experiments performed using protocols illustrated in A - C. The ordinate shows the change in current induced by increasing [K + ] o from 5 to 50 mM. * P < 0.01 vs. controls.

Inhibition of outward currents by K + channel blockers. Pericytes held at -80 mV have membrane potential clamped near the predicted K eq (-89 mV) for our buffers. Depolarizing the pericytes by voltage clamp from a holding potential of -80 to -40 mV generated an outward current ( Fig. 5 ). It is likely that a portion of that outward current is carried by the K + ion because -40 mV is near Cl eq. We tested whether ANG II could reduce the outward current of pericytes held at -40 mV and whether any residual post-ANG II currents remain sensitive to K + channel blockade. The means ± SE of holding currents at -80 and -40 mV are summarized for control pericytes ( n = 34) and pericytes treated with ANG II (10 nM, 20 min, n = 21) in Fig. 8. Net currents at -40 mV were much lower in ANG II-treated pericytes, a finding that is consistent with inhibition of an outward current carried by K +. The effects of K + channel blockade in those cells are provided in Figs. 9 and 10. The examples in Fig. 9, A - C, show continuous measurement of whole cell current in pericytes shifted from a holding potential of -80 to -40 mV. Inward currents at -80 mV were -62 or -216 pA ( Fig. 9 A ) and -58 or -62 pA ( Fig. 9 B ). Outward currents generated when the holding potential of the cells was shifted to -40 mV (arrows) were inhibited by 0.1 ( Fig. 9 A ) or 1.0 mM ( Fig. 9 B ) Ba 2+. When similar experiments were performed after ANG II treatment (10 nM, 20 min, Fig. 9 C ), currents generated by the shift to -40 mV remained negative (summarized in Fig. 8 ) and were insensitive to Ba 2+. Summaries of the results of all such experiments are provided in Fig. 9, D - F. Analogous experiments using TEA (30 mM) as the K + channel blocker produced similar results ( Fig. 10 ), but TEA was less effective than Ba 2+ to inhibit the outward currents in control cells.

Fig. 8. ANG II reduces whole cell currents in pericytes held ( V hold ) at -40 mV. Whole cell current measurements are shown from DVR pericytes held at -80 and -40 mV in the control state ( n = 34) and after prolonged ANG II exposure ( n = 21). Currents at -40 mV were much lower in ANG II-treated cells (* P < 0.01 vs. controls at -40 mV).

Fig. 9. Effect of ANG II on outward current inhibition by Ba 2+. A and B : pericytes were held at -80 mV (near K eq = -89 mV) while cell current was continuously measured. Holding potential was abruptly depolarized to -40 mV (arrows) to approximate Cl eq (-43 mV), inducing an outward current. Subsequently, the K + channel blocker Ba 2+ was transiently added to the bath at 0.1 ( A ) or 1.0 mM ( B ). Ba 2+ effectively reduced the outward current. Current values at the start of the traces are initial measurements at the holding potentials of -40 and -80 mV. C : after exposure to ANG II (10 nM, 20 min), increasing the holding potential from -80 to -40 mV generated a smaller current (compare with A and B ) that was insensitive to 1.0 mM Ba 2+. D and E : graphs showing summaries of pericyte cell current at a holding potential ( V h ) of -40 mV before, during addition of Ba 2+ to the bath, and after washout. F : graph showing a summary of cell current measured in ANG II-exposed pericytes at a holding potential of -40 mV. Values are before, during, and after washout of Ba 2+ (1 mM).

Fig. 10. Effect of ANG II on outward current inhibition by TEA +. A : pericytes were held at -80 mV (near K eq = -89 mV) while cell current was continuously measured. Holding potential was abruptly depolarized to -40 mV (arrows) to approximate Cl eq (-43 mV), inducing an outward current. Subsequently, the K + channel blocker TEA + (30 mM) was transiently added to the bath. TEA + reduced the outward current. B : after exposure to ANG II (10 nM, 20 min), the outward current was insensitive to TEA +. C : graph showing a summary of pericyte cell currents at a holding potential of -40 mV before, during, and after addition of TEA + (30 mM) to the bath. TEA + inhibited the outward current induced by depolarization ( P < 0.05). D : graph showing a summary of currents measured in ANG II-exposed pericytes at a holding potential of -40 mV. Values are before, during, and after washout of TEA + (30 mM).

DISCUSSION

Membrane potential of smooth muscle controls Ca 2+ entry into the cytoplasm through voltage-gated channels and is an important determinant of contractility ( 18, 25 ). Depolarization, coupled with intracellular events leading to Ca 2+ sensitization, is often the means by which vasoconstrictors such as ANG II activate myosin light chain kinase to favor myosin cross-bridge formation and contraction ( 29, 31 ). Depolarization of smooth muscle generally occurs by increasing Cl - conductance. At membrane potentials below the Cl - equilibrium potential, activation of Cl - channels favors Cl - efflux from the cell, shifting membrane potential away from K eq (about -90 mV) toward Cl eq (-20 to -40 mV). It has been frequently shown that smooth muscle of renal cortical afferent arterioles behaves in that way ( 4, 5, 12, 14, 21, 22, 36 ); however, studies of the contraction of the efferent arteriole have produced varied results. Several investigations found that efferent smooth muscle cells do not always depolarize and that depolarization induced by increasing [K + ] o does not consistently raise cytoplasmic Ca 2+ concentration ([Ca 2+ ] CYT ) or induce vasoconstriction ( 5, 7, 8 ). Strong evidence has recently been obtained that the channel architecture of efferent smooth muscle varies with the depth of the parent glomerulus in the renal cortex. Voltage-gated Ca 2+ channels are important to control [Ca 2+ ] CYT in juxtamedullary but not superficial efferent arteriolar smooth muscle ( 13 ). DVR are branches of juxtamedullary efferent arterioles, and we have repeatedly observed that DVR pericytes are, in fact, depolarized by ANG II and that Ca 2+ entry is modulated by membrane potential change ( 26, 38, 39 ).

Resting membrane potential of the DVR pericyte, measured with artificial buffers and nystatin-perforated patches, is in the range of -50 to -70 mV, similar to that of other smooth muscle ( 25, 26, 39 ). Measurement of membrane potential with patch-clamp techniques necessitates dialysis of the cell interior with the buffer in the electrode. As such, we cannot ensure that current or prior membrane potential measurements replicate exact levels that exist in vivo. Despite this, we are confident that ANG II can depolarize pericytes by activating a Ca 2+ -sensitive, niflumic acid-inhibitable Cl - conductance ( 26 ). The goal of this study was to determine whether inhibition of pericyte K + conductance is also a mechanism by which ANG II acts to induce depolarization. Our first approach to that question was to study the influence of changing K eq and Cl eq on membrane potential. When [K + ] o was raised to increase K eq, the membrane potential of pericytes shifted from its preexisting baseline to a close approximation of K eq ( Fig. 1 A ). That finding simply verifies that K + conductance dominates over other ions in the determination of resting membrane potential. After treatment with ANG II, membrane potential depolarized, as previously described ( 26, 38, 39 ), but became increasingly insensitive to raising K eq ( Fig. 1 A ). As responsiveness to raising K eq was lost, responses to raising Cl eq from -43 to -3 mV increased ( Fig. 1 B ). That finding implies that, after ANG II, Cl - conductance rather than K + conductance is dominant in the determination of membrane potential. We also tested whether K + channel blockers (Ba 2+ and TEA + ) affect membrane potential before and after ANG II exposure. Those agents depolarized resting pericytes but had no effect after prolonged ANG II treatment ( Figs. 2 and 3 ). Taken together, the data in Figs. 1 - 3 fully support concomitant activation of Cl - channels and inhibition of K + channels by ANG II, but they do not rigorously prove the latter because an overwhelming increase in Cl - conductance relative to a stable K + conductance could hypothetically yield similar results.

The relative magnitude of K + vs. Cl - conductance after ANG II treatment was investigated by measuring whole cell current in pericytes subjected to depolarizing pulses ( Fig. 5 ). That approach proved to be technically challenging, because it necessitated measurement of the relatively small currents that occur within the range of physiological membrane potential while maintaining stability of the gigaseals and preparation for the 20 min needed to eliminate sensitivity to K eq, as established in Fig. 1. At pulse potentials that flank the Cl eq of our buffers (-50 to -10 mV, Fig. 5 B ), ANG II significantly reduced outward currents, a finding that further supports inhibition of K + conductance. Stated another way, because Cl - current is eliminated by voltage clamp near Cl eq, alterations of Cl - conductance cannot explain the reduction of whole cell current by ANG II in Fig. 5 B; transport of K + or other ions must be affected. In support of the overall dominance of Cl - conductance in ANG II-treated cells, blockade with with niflumic acid reduced residual currents in the vicinity of Cl eq to nearly zero and shifted reversal of the current toward 0 mV ( Fig. 5 D ).

To specifically establish that the above effects are attributable to changes in conductance to the K + ion, additional protocols were employed. We increased [K + ] o from 5 to 50 mM to raise K eq from -89 to -30 mV, a maneuver that favors a K + inward current at pulse potentials less than -30 mV ( Fig. 6 C ). The inward currents so generated were eliminated after prolonged ANG II treatment ( Fig. 6 F ). To ensure that nonspecific effects of nystatin-perforated patches do not account for K + channel inhibition, the seals in Fig. 6 C were held for 20 min before KCl was raised from 5 to 50 mM so that the results in Fig. 6 C provide a proper time control for those in Fig. 6 F. In addition, we examined the effect of short- and long-term maintenance of perforated patches on the inhibition of K + inward current. Whether ANG II was applied 20 min before or 20 min after establishment of electrical access to the cytoplasm via nystatin patches, the inward current that resulted from raising [K + ] o from 5 to 50 mM was abrogated ( Fig. 7 ).

In a final series, we examined the ability of ANG II to reduce outward current in pericytes voltage clamped near Cl eq at -40 mV ( Figs. 8 - 10 ). In pericytes that had not been exposed to ANG II, depolarization from -80 to -40 mV generated a net outward current, raising I m from a mean of -103 to +102 pA ( Fig. 8 ). In voltage-clamped cells exposed to ANG II for 20 min, the same depolarization only increased current from -117 to -40 pA, a finding that is consistent with elimination of K + efflux by ANG II. To verify that the outward current generated by depolarization to -40 mV is at least partially carried by K +, we tested whether the classic, nonselective K + channel blockers Ba 2+ and TEA inhibit it. Both agents were effective in doing so in resting cells ( Figs. 9 E and 10 C ) but not in cells preexposed to ANG II ( Figs. 9 F and 10 D ).

The results of this study do not establish which classes of K + channels control resting potential or are modulated by vasoconstrictors such as ANG II. K + channel architecture of smooth muscle is generally complex; multiple classes of K + channels contribute to the ensemble of currents that affect cell function ( 18, 25 ). Ca 2+ -activated K + channels (K Ca ) and voltage-operated K + channels (Kv) activate with depolarization, probably serving to limit microvessel contraction by holding the membrane potential low enough to reduce voltage-gated Ca 2+ entry. Large- and small-conductance K Ca channels are widely expressed in smooth muscle ( 25 ). If they are present in DVR pericytes, it is likely that they are inactivated by ANG II because ANG II elevates pericyte [Ca 2+ ] CYT ( 30, 39 ) to activate K Ca channels if present. ANG II-mediated inhibition of K Ca channels in other renal microvessels does occur. Experiments by Roman and colleagues ( 2, 23, 33 ) established the existence of K Ca inhibition through the cytochrome P -450A -hydroxylase product 20-HETE. ANG II inhibition of K Ca channels mediated by protein kinase C and c-SRC has also been verified ( 1, 3 ). Kv channels are common in smooth muscle, and inhibition of their activity by ANG II occurs in various cell types, including arterial smooth muscle ( 15, 28, 35 ). K ATP channels formed by association of inward rectifier (K IR ) 6.x with sulfonurea receptors are widely expressed in smooth muscle ( 18, 25 ). We have shown that the K ATP channel activator pinacidil hyperpolarizes DVR pericytes and dilates preconstricted vessels ( 39 ). Precedent for ANG II inhibition of K ATP channels is well established ( 16, 20, 34, 37 ). It seems probable that ANG II inhibits K ATP channels in DVR pericytes, but extensive investigations will be needed to support that hypothesis. Inward rectifier K + channels (K IR ) are expressed in smooth muscle. Strongly rectifying classes of K IR would be expected to conduct an inward K + current below K eq. The experiments in Figs. 5 - 7 failed to identify a strong pattern of inward rectification below K eq = -89 mV. The ability of Ba 2+, at a concentration of 0.1 mM, to depolarize the cells ( Fig. 2 ) and block currents ( Fig. 9 ) favors their existence ( 25 ). The extent to which K IR channels exhibit inward rectification varies with subclass, and we cannot dismiss the possibility that K IR subtypes or splice variants, present in DVR pericytes, are blocked by ANG II. ANG II inhibition of inward rectifier currents has been reported in juxtaglomerular cells of the afferent arteriole ( 19 ).

In summary, it is likely that multiple classes of K + channels are expressed in DVR pericytes and that prolonged exposure to ANG II leads to inhibition of one or more of them. The strong inhibition of inward and outward currents ( Figs. 5 - 10 ) and complete elimination of the effectiveness of high concentrations of the broad K + channel blockers Ba 2+ and TEA ( Figs. 9 and 10 ) favor that interpretation. These studies have been performed in DVR explanted from vascular bundles of collagenase-digested rat kidney. The preparation offers the advantage of access to native cells. It also has the broad disadvantage that insufficient tissue is harvested to enable the use of the biochemical approaches needed to define protein expression and phosphorylation events responsible for modulation of channel activity. The need to harvest vessels throughout the day makes assignment of measured currents to a specific ion channel difficult to investigate through antisense or RNA silencing techniques that require days to have effects. As such, in the absence of a well-characterized cell culture model, future efforts to study Cl - channel and K + channel modulation in DVR pericytes by vasoactive agents will rely on technically demanding experiments involving ion substitutions and pharmacological inhibitors.

GRANTS

Studies in our laboratory were supported by National Institutes of Health Grants DK-42495, DK-68492, DK-67621, and HL-68686.

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作者单位：Division of Nephrology, Department of Medicine, and Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201-1595