KATP channel conductance of descending vasa recta pericytes

【关键词】  recta

    1Division of Nephrology, Department of Medicine
    2Department of Physiology, University of Maryland School of Medicine, Baltimore
    3Department of Biology, Towson University, Towson, Maryland

    ABSTRACT

    Using nystatin-perforated patch-clamp and whole cell recording, we tested the hypothesis that KATP channels contribute to resting conductance of rat descending vasa recta (DVR) pericytes and are modulated by vasoconstrictors. The KATP blocker glybenclamide (Glb; 10 μM) depolarized pericytes and inhibited outward currents of cells held at 40 mV. KATP openers pinacidil (Pnc; 10 μM) and P-1075 (1 μM) hyperpolarized pericytes and transiently augmented outward currents. All effects of Pnc and P-1075 were fully reversed by Glb. Inward currents of pericytes held at 60 mV in symmetrical 140 mM K+ were markedly augmented by Pnc and fully reversed by Glb. Ramp depolarizations in symmetrical K+, performed in Pnc and Pnc + Glb, yielded a Pnc-induced, Glb-sensitive KATP difference current that lacked rectification and reversed at 0 mV. Immunostaining identified both KIR6.1, KIR6.2 inward rectifier subunits and sulfonurea receptor subtype 2B. ANG II (1 and 10 nM) and endothelin-1 (10 nM) but not vasopressin (100 nM) significantly lowered holding current at 40 mV and abolished Pnc-stimulated outward currents. We conclude that DVR pericytes express KATP channels that make a significant contribution to basal K+ conductance and are inhibited by ANG II and endothelin-1.

    kidney; medulla; microcirculation; electrophysiology; vasoconstriction

    MEMBRANE POTENTIAL plays a pivotal role in the regulation of voltage-gated Ca2+ influx into smooth muscle cells. Depolarization leads to elevation of cytoplasmic Ca2+ that activates myosin light chain kinase to augment cross-bridge formation and induce contraction. ATP-sensitive K+ channels (KATP), originally discovered in cardiac myocytes (42), are widely distributed in many cell types, including vascular smooth muscle (41, 51). KATP channels are a tetrameric association of inward rectifier (KIR) K+ channel subtypes KIR6.1 or KIR6.2 and the sulfonylurea receptor (SUR) (1, 7). Studies have shown that KATP channels participate in regulation of resting potential and K+ conductance of smooth muscle and the contractile state of various vascular beds (2, 3, 9, 12, 14, 18, 19, 24, 29, 30, 35, 41, 51).

    Blood flow to the renal medulla traverses afferent and efferent arterioles of juxtamedullary glomeruli. Descending vasa recta (DVR) are 12- to 15-μm-diameter branched continuations of juxtamedullary efferent arterioles and are the sole source of blood flow to the renal medulla (47). Infusions of KATP channel inhibitors have been shown to reduce renal medullary blood flow (MBF) (43, 55), implying probable induction of vasoconstriction through KATP channel inhibition at some site along the medullary microvascular circuit. Contractile smooth muscle/pericytes surround DVR and could be a site for KATP channel-mediated effects.

    It is generally accepted that shunting of oxygen by countercurrent exchange leads to regional medullary hypoxia. The renal medulla has been shown to be vulnerable to hypoxic insult, so that regulation of its blood supply has been the subject of much investigation (4, 5, 10, 47, 48). In this study, we tested the general hypothesis that KATP channels contribute to overall K+ conductance of the pericyte plasma membrane and that vasoconstrictors regulate KATP channel activity. We found that KATP channel inhibition or activation depolarizes and hyperpolarizes pericytes, respectively, implying tonic contribution to K+ conductance. Immunochemical staining verified expression of KIR6.1 and KIR6.2 pore-forming and SUR2B-regulatory KATP subunits in DVR. Finally, ANG II and endothelin (ET)-1 potently reduced resting pericyte membrane conductance to K+ and inhibited the ability of the KATP opener, pinacidil (Pnc), to activate K+ currents. KATP inhibition is specific to vasoconstrictors because vasopressin was less effective than ET-1 and ANG II to inhibit DVR pericyte KATP channels.

    METHODS

    Isolation of DVR. 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 harvested from Sprague-Dawley rats (100150 g; Harlan) that had been anesthetized by an intraperitoneal injection of ketamine/xylazine (80 mg/kg; 10 mg/kg). Tissue slices were stored at 4°C in a physiological saline solution (PSS; in mM: 155 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, and 10 glucose, pH 7.4). Small wedges of renal medulla were dissected 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 and transferred to a perfusion chamber for patch-clamp recording.

    Whole cell patch-clamp recording. Patch pipettes were made from borosilicate glass capillaries (PG521514, 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 kaspartate, 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. Measurements were obtained as previously described (45, 46). 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 8- to 10-M pipettes. Whole cell current recording was performed with 6- to 8-M pipettes. Whole cell currents were sampled at 10 kHz during 100-ms ramp depolarizations and at 10 Hz during continuous recordings. To calculate cell capacitance (Cm), 10-mV pulses from a holding potential of 70 mV were applied using the "membrane test" feature of Clampex (Axon Instruments). Some pericytes showed prolonged capacitance transients indicative of cell-to-cell coupling via gap junctions. Such cells were abandoned, and recordings were not obtained from them. To ensure that selected pericytes were uncoupled, we compared their capacitances to those of pericytes preexposed for at least 5 min to gap junction blockers (100 μM carbenoxolone, 1 mM heptanol). All data have been corrected for junction potentials (39, 45, 46).

    These studies focused on the putative expression of KATP channels in DVR pericytes. To generate electrochemical forces favoring a K+ outward current, the holding potential of the cell (Vh) was increased above the K+ equilibrium potential (Keq) of our bath and electrode buffers (89 mV, PSS and electrode solutions, see above) by shifting Vh from 80 to 40 mV. To generate conditions favoring a K+ inward current, Vh was held at 60 mV, whereas Keq was shifted from 89 to 0 mV by raising bath KCl concentration from 5 to 140 mM by isosmotic substitution of KCl for NaCl. In the former case, a holding potential of 40 mV is close to the Cl equilibrium potential (Cleq) of our buffers, minimizing contamination of recordings with Cl currents. In the latter case, Vh = 60 mV is below the threshold for global activation of the Ca2+-dependent Cl current of DVR pericytes (46). Chord conductance (GK) was calculated from the expression IK = GK x (V Keq) where IK is current carried by K+ ion, V is membrane potential, and Keq is the K+ equilibrium potential for the electrode and extracellular buffers.

    Immunochemical staining of renal medulla and isolated DVR. Immunochemical staining of isolated DVR was performed as previously described (28). Hand-dissected DVR were placed on glass coverslips, fixed with 3% paraformaldehyde in 0.1 M cacocodylate for 5 min at 25°C, and washed with PSS solution. The fixed DVR were washed with PSS buffer, permeabilized for 30 min with 0.1% Triton X-100 in PSS, and placed in 5% BSA blocking solution for 30 min at room temperature. Primary antibodies were incubated overnight at 4°C in blocking solution. The primary antibodies directed against inward rectifier K+ channels, KIR6.1, KIR6.2, and sulfonurea receptor subunits SUR1, SUR2B were obtained from Santa Cruz Biotechnology (sc11225, sc11228, sc5789, sc5793, respectively, 1:30 dilution) (36). Pericytes were identified with primary monoclonal antibody against -smooth muscle actin (Sigma, diluted 1:500). DVR were subsequently washed three times with PSS and incubated with Alexa-conjugated donkey anti-goat or anti-mouse secondary antibodies (Molecular Probes, diluted 1:200). The vessels were again washed with PSS and then mounted in Vectashield (Vector Laboratories). Negative controls were performed by omitting the incubation with primary antibody. 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.

    Reagents. ANG II, ET-1, vasopressin, Pnc, glybenclamide (Glb), heptanol, carbenoxolone, nystatin, and other chemicals were from Sigma (St. Louis, MO). The KATP channel activator, N-cyano-N'-(1,1-dimethylpropyl)-N''-3-pyridylguanidine (P-1075), was from Tocris Cookson (Ellisville, MO). Liberase Blenzyme 1 was from Roche Applied Science. Pnc and P-1075 were dissolved in ethanol and Glb in DMSO. ANG II, ET-1, and vasopressin were stored as 10-μm aliquots in water. Reagents were thawed and diluted 1:1,000 on the day of the experiment. The excess was discarded daily. 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.

    Statistics. Data in the text and figures are reported as 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 with Student’s t-test (paired or unpaired, as appropriate) or the Rank Sum Test (nonparametric). Comparisons between multiple groups employed one-way ANOVA, 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

    Cell capacitance measurements in DVR pericytes. As previously described, we studied pericytes on the abluminal surface of isolated intact DVR (46). Table 1 provides measurements of Cm from the pericytes we studied. We did not measure currents of cells that showed prolonged capacitance transients indicative of gap junction coupling. Peptide constrictors such as ANG II and ET-1 have been shown to close gap junctions in other preparations (22). Cm exposed to ANG II (10 nM) or ET-1 (10 nM) as part of these protocols was not significantly different from baseline (Table 1). Also, for comparison, the capacitance of pericytes exposed to high concentrations of gap junction blockers, carbenoxolone (100 mM) or heptanol (2 mM), is provided. Table 1 shows that pericytes selected for study were electrically isolated from the adjacent cells of this intact microvessel preparation.

    Role of KATP channels in the determination of pericyte membrane potential. DVR pericyte membrane potential, under conditions of nystatin-perforated patch recording, typically lies between 50 and 65 mV (45, 46, 53), implying contribution of K+ conductance to resting membrane potential. We first tested whether KATP channels contribute to that K+ conductance by examining the ability of the KATP channel blocker Glb (10 μM) to depolarize pericytes (Fig. 1). Glb depolarized most cells (for example, Fig. 1, A and B) and, on average, raised membrane potential from 60.4 ± 2.0 to 56.4 ± 2.8 mV (Fig. 1C, n = 10). Thus KATP channels contribute to resting K+ conductance.

    In separate series, we tested the ability of the KATP channel openers, Pnc (10 μM) and P-1075 (1 μM), to hyperpolarize DVR perictyes. The selectivity of those reagents for KATP channels was also verified by testing their sensitivity to Glb. Pnc reliably hyperpolarized DVR pericytes and the effect was completely reversed by Glb (Fig. 2, A and B). On average, Pnc lowered membrane potential from 59.8 ± 5.0 to 76.6 ± 3.0 mV (Fig. 2C, n = 6). The effect of Pnc was maximal at a concentration of 10 μM (Fig. 2D, n = 5) (69). The KATP channel opener P-1075 also hyperpolarized DVR pericytes in a concentration-dependent manner and was reversed by Glb (Fig. 2E, n = 5). Taken together, the findings verify that KATP channels are available to be activated and increase pericyte membrane conductance to K+.

    Participation of KATP channels in K+ outward currents of DVR pericytes. We next tested whether outward currents, induced by clamping DVR pericytes to Cleq (Vh = 40 mV), are carried by K+ efflux through KATP channels. When holding potential was shifted from 80 to 40 mV (Fig. 3, A and B), whole cell current reversed sign from net inward to outward. The mean currents at 80 and 40 mV were 54 ± 9.8 and 115 ± 18 pA, respectively (Fig. 3C). Application of Glb (10 μM) reversibly lowered the mean outward current from 115 ± 18 to 65 ± 18 pA (n = 15). Because 40 mV is near Cleq, those currents are largely carried by K+ ion. Glb reduced the chord conductance for K+ from 2.37 ± 0.39 to 1.34 ± 0.39 nS. Thus KATP channels contribute 43% of K+ conductance at 40 mV. In separate series, we measured the ability of the KATP channel openers, Pnc and P-1075, to augment outward currents of pericytes held at Vh = 40 mV. As illustrated in Fig. 4A, Pnc (10 μM) markedly enhanced outward current but its effect spontaneously reversed. Pnc increased the mean outward current from 102 ± 13 to a peak of 324 ± 37 pA (Fig. 4B, n = 24). To test specificity, we verified that preapplication of Glb prevented Pnc augmentation of outward current (Fig. 4, C and D). P-1075 (1 μM) had a similar effect, transiently increasing outward current from 142 ± 21 to 295 ± 33 pA (Fig. 4, E and F, n = 9). P-1075-induced currents were also completely blocked by Glb.

    Inward currents conducted by KATP channels in DVR pericytes. The protocols of the experiments reported in Figs. 3 and 4 examined Pnc, P-1075, and Glb effects on outward current. KATP channels typically exhibit linear "ohmic" current-voltage (I-V) relationships with little rectification, i.e., they carry K+ outward or inward current with similar conductance (41, 51). To test for that behavior, DVR pericytes were held at 60 mV as external KCl was increased from 5 to 140 mM to create an electrochemical gradient favoring a K+ inward current. The ability of Pnc (10 μM) to augment the inward current and the ability of Glb (10 μM) to block the Pnc-augmented current were examined by sequentially introducing Pnc and then Pnc + Glb into the bath (Fig. 5, A-C). During the recording, at 5-s intervals, pericytes were subjected to ramp depolarizations from 60 to +40 mV over 100 ms while sampling at 10 kHz so that I-V characteristics could be repeatedly examined in Pnc and Pnc + Glb.

    An example is shown in Fig. 5, A and B. The I-V traces labeled as "1" and "2" in Fig. 5B correspond to ramp depolarizations in Pnc and Pnc + Glb, respectively (times 1 and 2, Fig. 5A). Their difference (1 2) provides the I-V characteristic of Glb-sensitive KATP current in symmetrical 140 mM KCl. At the holding potential (60 mV), Pnc increased chord conductance from a baseline of 2.25 ± 0.32 to 6.71 ± 0.66 nS, i.e., overall conductance can be increased 198% by stimulating Pnc-sensitive KATP channels. The Glb-sensitive current calculated from ramp depolarizations had a mean conductance of 4.2 ± 0.74 nS for n = 10 cells (Fig. 5D). The current did not rectify and it reversed near 0 mV as expected for conductance of KATP channels in symmetrical K+. Application of Glb with Pnc reduced conductance from 6.71 ± 0.66 to 1.47 ± 0.27 nS, a value that is less than the pre-Pnc baseline. The latter implies that 35% of the resting conductance in symmetrical KCl at 60 mV is accounted for by Glb-sensitive KATP channels. The concentration dependence of Pnc stimulation of inward current is shown in Fig. 5E (n = 4 to 10 cells at each Pnc concentration) and is similar to effects on membrane potential Fig. 2D.

    Several features warrant attention. Raising external KCl from 5 to 140 mM induced a large inward current that rapidly waned. After completion of the waning phase, the inward current that persisted was markedly augmented by Pnc and fully reversed by Glb. The inhibition was reversible on Glb washout, showing that Pnc augmentation of the inward current does not wane on the time scale of these experiments (Fig. 5, A and C). The latter contrasts with the transient nature of Pnc-stimulated outward currents (Fig. 4A).

    Expression of Kir6.1, Kir6.2, SUR1, and SUR2B in DVR. The sensitivity of pericyte membrane current to KATP channel openers (Pnc, P-1075) and the blocker, Glb (Figs. 14), coupled with linear I-V characteristics, and reversal potential consistent with K+ ion as the charge carrier (Fig. 5) provide strong evidence for robust KATP expression in DVR pericytes. For further verification, we performed immunochemical staining of isolated DVR to identify KIR6.x and SUR regulatory KATP channel subunits. Microfluorescent images verified that DVR express both KIR6.1 and KIR6.2 subtypes (n = 4) and that they are present in both pericytes and endothelium (Fig. 6). Fluorescence was not observed when the primary antibody was omitted (not shown). Immunochemical staining for SUR subunits (n = 3) revealed predominant expression of SUR2B (Fig. 7).

    Inhibition of KATP channels by vasoconstrictors. In a recent communication, we showed that TEA and Ba2+-sensitive K+ currents are blocked by ANG II (45). We extended those observations by determining whether KATP channels partially carry the ANG II-sensitive K+ current and by measuring the concentration dependence of the ANG II effect. In addition, we tested whether two other DVR vasoconstrictors, ET-1 and AVP, can inhibit KATP currents. To perform those experiments, we used the conditions illustrated in Figs. 3 and 4. The effects of the constrictors on baseline and Pnc (10 μM)-induced outward currents of pericytes held at Vh = 40 mV were measured.

    Examples of records obtained after 10-min pretreatment with ANG II are provided in Fig. 8, A-C. ANG II, at 10 pM (Fig. 8A) and 100 pM (Fig. 8B), did not block Pnc stimulation of outward current. Pnc tended to induce oscillations in cells pretreated with 100 pM ANG II (Fig. 8B). At 1 nM (Fig. 8C) and 10 nM, ANG II completely prevented Pnc stimulation of outward current. The summary of the effects of ANG II pretreatment on holding current and Pnc-induced outward currents is provided in Fig. 8, D and E, respectively.

    In same context, AVP, at the vasoconstrictor concentration of 100 nM (63), was less effective than ANG II to block K+ current. Ten-minute pretreatment with AVP failed to lower holding current or reduce Pnc-stimulated outward current in some cells (Fig. 9A) but seemed to markedly affect it in others (Fig. 9B). On average, significant Pnc stimulation of outward current persisted after vasopressin pretreatment (Fig. 9C).

    ET-1, at the maximal vasoconstrictor concentration of 10 nM (58), had potent effects to inhibit outward current. As illustrated in Fig. 10A, ET-1 promptly lowered holding current and rendered pericytes insensitive to subsequent application of Pnc (10 μM). Figure 10B shows the effect of pretreatment of a pericyte, held at 80 mV, with ET-1 (10 nM). ET-1 application was followed by repetitive, spiking, spontaneous, transient inward currents (STICs; Fig. 10Ba) similar to those previously reported after ANG II stimulation (46). Shifting Vh to 40 mV (near Cleq) reduced their amplitude, implying Cl efflux as the likely charge carrier of the STICs. At Vh = 40 mV, Pnc failed to elicit outward currents (Fig. 10Bb). The average holding and Pnc-augmented currents observed after ET-1 exposure are summarized in Fig. 10, C and D, along with the means ± SE of those measured in ANG II (10 nM) and vasopressin (100 nM). High concentrations of ANG II and ET-1, but not vasopressin, lowered pericyte holding current and eliminated Pnc-inducible outward current. Similarly, after ANG II (10 nM) or ET-1 (10 nM) pretreatment, the reduction in holding current at Vh = 40 mV was accompanied by a loss of sensitivity to Glb (10 μM) implying inhibition of basal activity of KATP channels (Fig. 10E).

    DISCUSSION

    DVR pericytes, like smooth muscle cells from many vascular beds, increase intracellular cytoplasmic Ca2+ through activation of voltage-gated Ca2+ channels. We previously showed that depolarization, whether mediated by contractile agonists or extracellular K+, raises pericyte cytoplasmic Ca2+ and induces contraction (48, 53, 68, 69). KATP channels are the tetrameric association of SUR-regulatory subunits and KIR6.1 or KIR6.2 pore-forming subunits. Despite their importance in the control of DVR vasoactivity and MBF, the classes of K+ channels responsible for control of membrane potential of DVR pericytes have not been explored. In this study, we verified KATP channel subunit expression and quantified the role of KATP channels in the maintenance of membrane potential and K+ conductance. Apart from effects on membrane potential (Figs. 1 and 2), evidence that KATP channels contribute to the conductance of DVR pericytes to K+ is provided by the observation that K+ outward currents are inhibited by Glb (Fig. 3) and transiently enhanced by K+ channel openers, Pnc and P-1075 (Fig. 4). On exposure to Glb, the resting conductance of pericytes held at 40 mV in 5 mM KCl, fell from 2.37 ± 0.39 to 1.34 ± 0.39 nS (Fig. 3). In symmetrical K+, at 60 mV, Glb induced a similar reduction of conductance from 2.25 ± 0.32 to 1.47 ± 0.27 nS (Fig. 5). Those values correspond to a 43 and 35% contribution of KATP channels to conductance under the respective experimental conditions. In contrast, stimulation with high concentration of the channel opener Pnc in symmetrical KCl increased K+ conductance by 4.46 to 6.71 ± 0.66 nS (Fig. 5). The Pnc-induced increase can be used to bracket the number of channels expressed on the surface of DVR pericytes. Assuming that Pnc stimulates open probability to near unity, and taking a high estimate for the average single channel conductance(s) of 100 pS, channel number could be as low as 44 per cell. Alternately, if channel conductance is only 10 pS, 444 channels per cell would be present. Those channel numbers are similar to estimates summarized by Quayle et al. (51) for smooth muscle of other origin.

    In this study, we quantified KATP currents by pharmacological maneuvers. Specificity of the agonists, Pnc and P-1075, for KATP channel activation is supported by their complete reversal by Glb, a reagent that is highly specific for KATP channels (41). Waning of currents after stimulation with KATP openers, similar to that observed in Fig. 4, has been observed in renin-secreting cells (54) and smooth muscle of the pig urethra (61). The mechanisms responsible for the spontaneous reduction of KATP current stimulated by openers are uncertain, but they are unlikely to be explained by run-down attributable to loss of intracellular signaling molecules. The latter problem is largely prevented during nystatin-perforated patch recording. Furthermore, Pnc activated durable inward currents in symmetrical K+ (Fig. 5).

    Unlike currents mediated by KIR2.x and KIR4.x subclasses, KATP (KIR6.x) channel currents do not rectify (51). Using ramp depolarizations, we confirmed that Pnc-activated, Glb-sensitive currents in DVR pericytes reverse at Keq = 0 mV, as expected for selective transport of K+ ion in symmetrical KCl. We also confirmed that the Glb-sensitive currents do not inwardly rectify (Fig. 5, B and D). Finally, KATP channel expression was verified with immunochemical staining. Commercial antibodies against KIR6.1 and KIR6.2 identified targets in DVR pericytes and endothelia (Fig. 6). Antibody directed at SUR2B identified it as a predominant KATP regulatory subunit in DVR (Fig. 7). Taken together, the prior and current evidence point to a role for KATP channels to modulate DVR pericyte membrane potential, K+ conductance, and contractility.

    The hypothesis that metabolically induced reduction of ATP stimulates KATP channels to favor vasodilation has received much support, including demonstration of vulnerability of murine knockout models to ischemic insult (57). In the renal outer medulla, oxygen is consumed to transport NaCl by the thick ascending limb of Henle, a nephron segment that is vulnerable to ischemic insult (4, 5). Given that oxygen tensions in the renal medulla may be as low as 1025 mmHg (5) and that DVR are its sole blood supply, it is inviting to speculate that KATP channel activation in response to metabolic demand is an important regulatory mechanism in DVR pericytes. Also, in view of the ability of Glb to depolarize DVR pericytes (Fig. 1) and reduce MBF (49, 55), the popularity of Glb and similar hypoglycemic agents in the treatment of diabetes raises concerns about their use in diabetic patients that undergo procedures, such as surgery and radiocontrast dye administration, which can precipitate acute renal failure.

    KATP channels are present in smooth muscle cells of various vascular beds. Along with voltage-dependent (Kv), Ca2+-activated (KCa), and other classes of inward rectifier (KIR) channels, they regulate membrane potential and vascular tone (3, 6, 18, 23, 25, 38). Evidence favors basal KATP channel activity in mesenteric, coronary, and carotid arteries (12, 14, 24, 38). Other investigations in cerebral and pulmonary arteries have favored the interpretation that KATP channels are largely dormant in the resting state but are available to be activated when vasodilation is required (31). In this study, we showed that the KATP channel blocker Glb, and the activators, Pnc or P-1075, depolarize and hyperpolarize DVR pericytes, respectively (Figs. 1 and 2). Glb has also been described to depolarize smooth muscle cells from isolated mesenteric (31), coronary (12, 24), and carotid arteries (8). The conditions to which DVR pericytes are exposed are unique because osmolality and extracellular K+ concentration in the renal medullary interstitium are higher than that of systemic plasma (20). Thus the intracellular and extracellular K+ concentrations to which pericyte KATP channels are exposed in vivo are uncertain and probably vary with the location of the parent vessel along the corticomedullary axis. Nonetheless, the current data make it clear that KATP channels are expressed in DVR and contribute substantially to K+ conductance. Their activation and inhibition are expected to favor hyperpolarization and depolarization, respectively, as a target for hormones and paracrine agents to mediate vasodilation and constriction.

    KATP channel regulation of vasoactivity in vascular beds, including the renal medulla, has been examined by testing the ability of Glb to lower blood flow. Gardiner and colleagues (14, 24) found that Glb infusion into rats induced mesenteric, skeletal muscle, and renal vasoconstriction. A similar effect was observed in splanchnic and portal circulations (24, 35) and the canine diaphragm (64). In the kidney, a role for modulation of arteriolar tone by KATP channels has been repeatedly observed (30, 49, 55, 60). DVR, the subject of the current study, supply the renal medulla with blood flow. Tonic effects of KATP channel activity to regulate MBF have been reported. Using laser-Doppler flowmetry, Parekh and Zou (49) reported that Glb reduced MBF by 19% while the KATP opener lemakalim increased MBF by 27%. With different methods, Sadowski et al. (55) reported a 15% reduction of MBF by Glb.

    KATP channels are named for their characteristic inhibition by intracellular ATP (42) and activation by nucleotide diphosphates (NDPs). Reduction of intracellular ATP concentration increases KATP open probability, an action that probably serves to hyperpolarize smooth muscle, reduce Ca2+ entry via voltage-gated channels, dilate blood vessels, and increase the supply of oxygen to meet metabolic demands (3, 41, 51). In vivo, KATP channel inhibition also increases resistance to blood flow in mesentery (56), renal cortex and medulla (32, 43, 55), and skeletal muscle (6). Conversely, endogenously released vasodilators inhibit vascular smooth contraction by activating KATP channels (23, 40, 41, 59). Variation of intracellular ATP may not be the sole or primary modulator of KATP channel activity. KATP channels are the targets of a variety of signaling cascades. Endogenous vasodilators such as adenosine, -adrenergic agonists, vasoactive intestinal peptide, adrenomedullin, and calcitonin gene-related peptide activate them, generally via protein kinase A-mediated phosphorylation (15, 23, 40, 51, 52, 59, 60). KATP channels are also the target of vasoconstrictors that activate protein kinase C (PKC). Although reports of PKC studies in the vasculature have largely identified inhibition of channel activity by vasoconstrictors and PKC (17, 21, 26, 33, 34, 50, 62, 6567), reports of PKC-mediated KATP activation in ventricular myocytes and insulin-secreting islet cells also exist (51).

    Within smooth muscle cells, constrictors can either inhibit K+ channels or activate them as a braking mechanism that limits vasoconstriction (13, 41). In a prior study, we found that K+ currents sensitive to the nonselective K+ channel blockers, Ba2+ and tetraethylammonium, were inhibited by 10 nM ANG II (45). In this study, we verified that KATP channels are at least one of the subtypes so affected (Fig. 8). We extended our observations by determining the concentration range over which ANG II mediates KATP inhibition and by examining parallel effects of AVP and ET-1. With regard to concentration dependence of ANG II, at 100 pM, oscillations were induced and there was partial inhibition of Pnc-stimulated currents. At 1 and 10 nM, ANG II fully blocked Pnc-induced KATP currents. Thus the ANG II concentrations that block KATP currents coincide well with those that induce DVR vasoconstriction, the EC50 of which is 500 pM (44). ANG II inhibition of KATP currents has been reported in smooth muscle of coronary and mesenteric arteries (16, 17, 26, 33).

    The most potent constrictor of DVR studied to date is ET-1 (48, 58). Like ANG II, ET-1 promptly lowers whole cell K+ currents and eliminates the ability of Pnc to activate KATP currents (Fig. 10). ET-1 has also been described to inhibit KATP channels in porcine pial arteries and arterioles (21), rabbit coronary and pulmonary arterioles (50), and cardiac myocytes (67), whereas in the pulmonary bed, endothelin type B receptor stimulation can be associated with KATP channel activation and vasodilation (37). It is interesting to note that ANG II (10 nM) and ET-1 (10 nM) virtually abolish the outward current of pericytes held at 40 mV (Figs. 8 and 10). Because Glb did not have as potent an effect to lower outward currents (Fig. 3), we conclude that other classes of K+ channels probably also blocked ANG II and ET-1. Efflux of Cl is unlikely to be involved because the holding potential for those experiments (Figs. 8 and 10) was the equilibrium potential for Cl ion. The complete elimination of Pnc-inducible currents by ANG II and ET-1 also strongly favors the interpretation that KATP-mediated K+ current is potently blocked by those constrictors. The elimination of the ability of Glb to inhibit outward K+ current (Fig. 10E) also indicates suppression of basal KATP channel activity in the cells. A similar effect has been reported by Lahaye et al. (27). The potency of ANG II and ET-1 to depolarize pericytes, inhibit K+ channels, activate Ca2+-dependent Cl channels, and constrict DVR probably does not fully reflect the influences to which DVR are exposed. Dickhout and colleagues (10) showed that renal medullary vasoconstrictors such as ANG II and ET-1 can activate a variety of compensatory pathways including those on endothelia and nearby nephrons to release paracrine vasodilators and influence vasoactivity.

    Compared with ET-1 and ANG II, AVP is a weak vasoconstrictor of isolated DVR (48, 63). Based on the current results, it seems possible that the lack of potency of arginine vasopressin to induce vasoconstriction is related to its inability to inhibit K+ channel activity in pericytes (Fig. 9). At picomolar concentrations, vasopressin exerts hydro-osmotic effects by activating collecting duct V2 receptors. At nanomolar concentrations, it stimulates V1 receptors to induce vasoconstriction. We previously showed that vasopressin-mediated constriction of isolated DVR maximizes at 10 nM (63). In this study, we used 100 nM vasopressin to test whether it induces KATP channel inhibition as part of that action. The data showed a tendency toward partial inhibition of basal and Pnc-stimulated outward currents but neither achieved statistical significance (Fig. 10). Examination of vasopressin-mediated inhibition of KATP channels in other preparations has also produced variable results (11, 65, 66).

    In summary, we showed that KATP channels are present in rat DVR pericytes and contribute substantially to resting K+ conductance. They mediate a substantial portion of the outward current observed in pericytes held at 40 mV. Both outward currents at 40 mV and inward currents in 140 mM external KCl are markedly augmented by KATP channel agonists, Pnc and P-1075. Glb-sensitive currents in DVR pericytes do not rectify, a characteristic expected for KATP channels. By immunochemistry, KIR6.1 and KIR6.2 pore-forming subunits are expressed along with the SUR2B. High concentrations of ANG II and ET-1 inhibit KATP channels as part of their action. The identification of robust KATP expression in DVR pericytes is consistent with prior reports of Glb-mediated reduction of renal MBF.

    GRANTS

    These studies have been supported by National Institutes of Health Grants R37-DK-42495, R01-DK-68492, and P01-HL-78870.

    FOOTNOTES

    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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