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Ca2+ signaling and membrane potential in descending vasa recta pericytes and endothelia

Kristie Rhinehart, Zhong Zhang, and Thomas L. Pallone

Division of Nephrology, University of Maryland School of Medicine, Baltimore, Maryland 21201-1595


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We devised a method for removal of pericytes from isolated descending vasa recta (DVR). After enzymatic digestion, aspiration of a descending vas rectum into a micropipette strips the pericytes from the abluminal surface. Pericytes and denuded endothelia can be recovered for separate study. Using fura 2-loaded preparations, we demonstrated that 10 nM angiotensin II (ANG II) elevates pericyte intracellular Ca2+ concentration ([Ca2+]i) and suppresses endothelial [Ca2+]i. The anion transport blocker probenecid helps retain fura 2 in the pericyte cytoplasm. DVR endothelia were accessed for membrane potential measurement by perforated-patch whole cell recording by using the pericyte-stripping technique and by turning nondigested vessels inside out with concentric micropipettes. By either method of access, 10 nM ANG II depolarized (n = 20) and 100 nM bradykinin hyperpolarized (n = 25) the endothelia. We conclude that isolated endothelia and pericytes remain functional for study of [Ca2+]i responses and that ANG II and bradykinin receptors exist separately on each cell type.

medulla; kidney; microcirculation; patch clamp; fura 2


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

RENAL MEDULLARY BLOOD FLOW is supplied by branches of juxtamedullary glomerular efferent arterioles called descending vasa recta (DVR). DVR traverse the renal outer medulla sequestered with ascending vasa recta into vascular bundles. The radial arrangement of DVR within the bundles suggests that DVR function to regionally distribute blood flow within the outer and inner medulla of the kidney (10, 15, 17). Pericytes surround DVR and impart contractile function and are therefore an important regulatory element in determination of medullary perfusion. Vasomotor tone of isolated DVR responds to a large number of agents (15).

In addition to the key role of pericytes, DVR endothelia are important in modulation of transport functions and vasomotor tone. In vitro studies have shown that they generate nitric oxide at a rate influenced by oxygen free radical formation (21). In addition, DVR endothelia exhibit unusual Ca2+-signaling events (16, 18). Angiotensin II (ANG II), a hormone that is expected to elevate intracellular Ca2+ concentration ([Ca2+]i) by activating the G protein-coupled angiotensin type 1 (AT1) receptor, has been shown to suppress basal [Ca2+]i and [Ca2+]i responses to bradykinin (BK) or the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) blocker thapsigargin (18).

Renal medullary perfusion is a determinant of diuretic state and appears to influence blood pressure, sodium balance, and extracellular fluid volume (3, 12). Despite the role that DVR pericytes and endothelia play in this scheme, information concerning the ion channel activity and signaling processes that govern their function has been lacking. For this purpose, we adapted electrophysiological and microfluorescent methods to investigate that regulation (14, 16, 18, 24). Those efforts have been hampered by two technical barriers. First, when the Ca2+-sensitive fluorescent probe fura 2 is loaded into isolated vessels, an exclusively endothelial signal is obtained, so that pericyte [Ca2+]i responses have yet to be reported (16). Second, patch clamp of abluminal pericytes on isolated vessels is possible, but the luminal endothelial cells are inaccessible to study. Thus fura 2-based [Ca2+]i studies and electrophysiological measurements have been restricted to endothelia and pericytes, respectively. We describe two methods that overcome those technical barriers. After collagenase digestion, a micropipette can be used to strip pericytes from isolated vessels to isolate either cell population. Patch clamp can be performed on DVR endothelia isolated after pericyte stripping or on DVR "everted" by turning the vessels inside out with a micropipette (5, 23). In the presence of the anion transport blocker probenecid, pericytes load well with fura 2, so that their [Ca2+]i responses can be examined (4). By using these methods, we have shown that ANG II suppresses endothelial [Ca2+]i and increases pericyte [Ca2+]i. Pericyte and endothelial membrane potentials (Psi m) are depolarized by ANG II, and both are hyperpolarized by the vasodilator BK.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of endothelia by pericyte stripping. Kidneys were removed from Sprague-Dawley rats (70-150 g; Harlan), sliced into sections along the corticomedullary axis, and stored at 4°C in a physiological saline solution (PSS) composed of (in mM) 145 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, and 10 glucose, pH 7.4, at room temperature. 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; Sigma), protease XIV (0.4 mg/ml; Sigma), and albumin (1.0 mg/ml) (14). 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. At intervals, vessels were isolated from the digested renal tissue by microdissection and transferred to a perfusion chamber on an inverted microscope (Nikon Diaphot). In the chamber, the vessels were captured by a micropipette and positioned onto the coverslip, and after 15-30 min were allowed for cell attachment and stabilization, bath flow was initiated. We previously showed that this approach to enzymatic digestion allows gigaohm seals to be obtained on pericytes for electrophysiological recording (14, 24).

To separate the pericytes from the enzymatically digested vessels, the maneuver illustrated in Fig. 1 was performed. Isolated DVR were aspirated into a microperfusion-style holding pipette (19) with an opening of 5-10 µm. During the aspiration, pericytes strip from the abluminal surface of the vessel and are retained at the pipette tip (Fig. 1, A and B). Once the vessel has been completely drawn into the pipette, a preparation of pericytes remains as a group of cells suspended in the bath, adherent to the pipette. Finally, the aspirated, pericyte-denuded vessel can be ejected from the pipette to yield a preparation of DVR endothelia that is free of pericytes (Fig. 1C). The ejected endothelia were positioned on the chamber coverslip and given 15-30 min to adhere for stabilization before bath flow was initiated.


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Fig. 1.   Separation of descending vasa recta (DVR) endothelia and pericytes. A: a single descending vas rectum microdissected from enzymatically digested renal outer medulla. Pericytes are visible on abluminal surface. B: vessel is aspirated into a microperfusion-style collection pipette. During the process, pericytes are stripped from abluminal surface and collect into a mass of cells near pipette tip. Horizontal bar, ~10 µm. C: after the process described in B is completed, pericyte-stripped vessel can be ejected to yield a monolayer of DVR endothelial cells. D: fluorescent emission of fura 2 loaded into pericytes isolated by the method described in B. Values on ordinate have been normalized to time 0. Fura 2 was excited at its Ca2+-insensitive isosbestic point (360 nm), while emissions were measured by photon-counting photomultiplier tube at 510 nm. Fura 2 emission falls more rapidly in the absence (n = 7) than in the presence of the anion transport inhibitor probenecid (1 mM, n = 6). Difference between groups was continuously significant after 2 min (P < 0.05). F360 and F0, fluorescence at 360 nm and 0 nm, respectively.

Whole cell patch-clamp recording. Psi m was monitored by patch-clamp recording from endothelial cells or pericytes at room temperature. To accomplish this, outer medullary DVR were digested to remove basement membrane as described above and to enable establishment of gigaohm seals with patch pipettes (14, 24). Patch-clamp studies of pericytes were always done on intact vessels. Stripped pericytes were used for Ca2+ measurements but not for electrophysiological studies. Patch pipettes were made from borosilicate glass capillaries (model PG52151-4, World Precision Instruments, Sarasota, FL; 1.5 mm OD, 1.0 mm ID) using a two-stage vertical pipette puller (model PP-830, Narshige) and heat polished. To obtain electrical access for whole cell perforated patch-clamp recording, nystatin was used as the pore-forming agent (6, 8). The pipette solution contained 120 mM potassium aspartate, 20 mM KCl, 10 mM NaCl, 10 mM HEPES, pH 7.2, and nystatin (100 µg/ml with 0.1% DMSO) in ultrapure water. Nystatin in DMSO was kept frozen at -20°C and renewed weekly. Each day, the nystatin stock was thawed, dispensed into the potassium aspartate pipette solution at 37°C by vigorous vortexing for 1 min, and subsequently protected from light. To clear slight remaining nystatin precipitate from the saturated electrode solution, pipettes were backfilled from a syringe via a 0.2-µm filter.

Psi m was measured using a CV201AU head stage and Axopatch 200A amplifier (Axon Instruments, Foster City, CA) in current-clamp mode (current = 0) at a sampling rate of 10 Hz. Psi m was recorded with pipettes of 8- to 15-MOmega resistance. Lower resistance pipettes proved technically difficult to use and led to premature loss of seals. Pipettes with nystatin-containing electrode solution were inserted into the bath under positive pressure and positioned near the cell, and the offset of the amplifier was adjusted to null the junction and electrode potentials. The final approach to the cell was controlled with a piezoelectric drive (model PCS-5000, Burleigh). Gigaseals were established by pressing the pipette tip against the cell and applying light suction. The progress of seal formation was followed on a digital oscilloscope (model M305, Hameg) by observing the current elicited by test pulses of 5-mV amplitude. Seal formation was facilitated by gradually reducing the holding potential from 0 to -70 mV. After seal formation, the appearance of the cell capacitance transient and the access resistance were monitored using a Digidata analog-to-digital converter and Clampex 7.0 (Axon Instruments, Union City, CA) with 10-mV pulses at a holding potential of -70 mV. Final access resistance was generally 15-40 MOmega . Junction and Donnan potential corrections were applied as previously described (8, 14).

Measurement of [Ca2+]i. Incubation in 10 µM fura 2-AM (Molecular Probes, Eugene, OR) for 20 min at 37°C yields a strong signal when loaded into a collection of pericytes isolated as shown in Fig. 1B from a single vessel. Figure 1D shows the fluorescent emission of fura 2-loaded pericytes (510 nm) during excitation of fura 2 at the isosbestic point (360 nm). Results are shown for vessels incubated and then maintained in the presence or absence of the anion transport inhibitor probenecid (1 mM). Probenecid has a marked effect of preventing the leak of fura 2 from the pericyte cytoplasm. Endothelial cells were loaded with fura 2 as previously described (16) by exposure to bath containing 2 µM fura 2-AM. In this study, the fura 2 incubation buffer and all solutions involved in [Ca2+]i signaling studies of both cell types contained 1 mM probenecid.

A photon-counting photomultiplier assembly was employed to measure fluorescent emission at 510 nm. Light for excitation of fura 2 was provided from a 75-W xenon arc lamp. Fura 2-loaded cells were excited using 350/380-nm wavelength combinations. The excitation wavelengths were isolated with a computer-controlled monochrometer (PTI, Lawrenceville, NJ). A Nikon CF fluor ×40 oil-immersion objective with numerical aperture of 1.3 was used for fura 2/[Ca2+]i measurement. Fluorescent emission was isolated with a 510WB40 filter (Omega Optical, Brattleboro, VT). The background-subtracted ratio of fluorescent emission (R350/380) was converted to the equivalent [Ca2+]i with the assumption of a dissociation constant of 224 nM for fura 2 at 37°C (10). Maximum and minimum fluorescence ratios were measured as previously described by exposing vessels to buffer containing 5 mM CaCl2 or 0 CaCl2-0.5 mM EGTA, respectively, along with 10 µM ionomycin (16).

Reagents. ANG II, BK, probenecid, ionomycin, bovine serum albumin (A2153, Cohn fraction V), nystatin, collagenase 1A, and protease XIV were obtained from Sigma (St. Louis, MO). ANG II or BK in water was stored in 200-µl aliquots at -20°C and diluted on the day of an experiment. The enzyme digestion solution was prepared in 50-ml batches, frozen in 2-ml aliquots, and thawed daily as needed. Cyclopiazonic acid (CPA; Calbiochem, San Diego, CA) was stored in DMSO at 10 mM. 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. Values are means ± SE. The significance of differences between means was calculated using Student's t-test (paired or unpaired, as appropriate) and analysis of variance. Where sampling rates were high, the majority of error bars were suppressed to clarify display of data.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ca2+ signaling in DVR pericytes and endothelia. We previously showed that ANG II reduces basal [Ca2+]i and suppresses BK- or thapsigargin-induced [Ca2+]i elevations in DVR endothelia. We had been unable to examine ANG II-induced [Ca2+]i changes in pericytes because of poor loading of fura 2 (16). Experiments verifying the endothelial [Ca2+]i responses and showing previously undocumented pericyte responses are shown in Figs. 2 and 3. These data were obtained in enzymatically isolated pericytes and endothelia in the presence of probenecid. Figure 2A shows the effect of abluminal application of 10 nM ANG II on [Ca2+]i in isolated pericytes (mean ± SE, n = 10). A classic peak-and-plateau response was observed. The contrasting effect of 10 nM ANG II on endothelial [Ca2+]i is shown in Fig. 2B. In pericyte-stripped vessels, basal endothelial [Ca2+]i, which is between 50 and 100 nM, fell to a mean of ~20 nM after exposure to ANG II. This effect of ANG II reproduces prior results obtained in isolated DVR that had not been subjected to enzymatic digestion or pericyte stripping (18).


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Fig. 2.   [Ca2+]i responses of DVR pericytes and endothelia. A: intracellular Ca2+ concentration ([Ca2+]i) response of fura 2-loaded pericytes isolated by the process described in Fig. 1B. After baseline recording for 1 min, 10 nM ANG II was introduced into the bath for 10 min. [Ca2+]i increased from a baseline of ~65 nM to a peak of 555 ± 100 nM and then fell to a plateau near 200 nM (n = 10). B: [Ca2+]i response of fura 2-loaded endothelia isolated as described in Fig. 1C. After 2 min, 10 nM ANG II or vehicle (n = 7, each group) was introduced into the bath. [Ca2+]i fell from resting value to ~20 nM in the ANG II-treated group (P < 0.05, ANG II vs. vehicle, continuously after 5 min). C: [Ca2+]i response of fura 2-loaded endothelia to 100 nM bradykinin (BK). Endothelia were studied by stripping pericytes and then loading fura 2 (n = 3) or by loading fura 2, stripping, and immediately proceeding with [Ca2+]i measurements without equilibration (n = 5). BK induced a response in both cases, but the peak was blunted in the latter group.



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Fig. 3.   [Ca2+]i inhibition in DVR endothelia treated with cyclopiazonic acid (CPA). A: DVR pretreated with vehicle or 10 nM ANG II for 5 min were exposed to 10 µM CPA. Inhibition of internal store sarcoplasmic/endoplasmic reticulum Ca2+-ATPase by this agent resulted in elevation of [Ca2+]i. Final [Ca2+]i was much lower in ANG II-pretreated group. These experiments are a continuation of those in Fig. 2B, with abscissa relabeled to time 0. P < 0.05, ANG II vs. vehicle, continuously after 4 min. B: endothelial [Ca2+]i was measured for 1 min, and then 10 µM CPA was introduced into the bath. After 5 min of CPA exposure, the bath was exchanged to also introduce 10 nM ANG II. DVR endothelial [Ca2+]i was markedly suppressed after ANG II exposure.

Endothelial [Ca2+]i responses to BK were also reexamined in isolated endothelia (Fig. 2C). BK stimulated the expected peak-and-plateau [Ca2+]i elevation. In vessels that had been stripped and then equilibrated at 37°C during fura 2 loading, the response was similar to that previously observed (16). When vessels were loaded with fura 2 and then stripped of pericytes so that [Ca2+]i was immediately examined after the stripping process, the peak response to BK was blunted (Fig. 2C). This suggests that the stripping process might cause intracellular Ca2+ store depletion and points to the need for an equilibration period after the mechanical trauma of stripping.

The ability of ANG II to inhibit endothelial [Ca2+]i responses is difficult to demonstrate in resting vessels, because basal [Ca2+]i is low and ANG II-induced changes are small (Fig. 2B). We also reexamined the ability of ANG II to block [Ca2+]i elevations in isolated endothelia treated with the SERCA inhibitor CPA (10 µM; Fig. 3). CPA markedly increased endothelial [Ca2+]i. Compared with vehicle-treated controls, ANG II reduced the endothelial [Ca2+]i elevation achieved by CPA (Fig. 3A). Similarly, addition of 10 nM ANG II to the bath after treatment with 10 µM CPA markedly reduced endothelial [Ca2+]i (Fig. 3B). These results reproduce our prior observations that were obtained in intact vessels when thapsigargin was used as the SERCA inhibitor (18).

Electrophysiological measurements of Psi m in DVR endothelia and pericytes. We obtained recordings of Psi m from DVR endothelia by two methods. First, pericyte-denuded vessels (Fig. 1C) were ejected onto a coverslip and accessed for whole cell recording by patches formed on their abluminal surface. Second, intact, nondigested microdissected DVR were everted by turning them inside out (Fig. 4A). This was done by deliberately crushing one end during microdissection. The crushed end was then cannulated, as typically done for microperfusions (5, 23). By continuously advancing an elongated "perfusion pipette," portions of the endothelial surface were exposed and made accessible for patch-clamp recording on their luminal surface. For better visualization of the process, most patch pipettes were bent ~90° by heating on a microforge. This permitted orientation of the tip so that the approach of the patch pipette tip to the endothelial cell could be visualized. Alternately, some gigaseals were formed with unaltered patch pipettes by lowering the tip onto the preparation until variations in the current visualized as pulses on the oscilloscope showed that the cell membrane had been touched. With the latter method, the perfusion pipette obscures visualization on an inverted microscope, making choice of the cell impossible. During recording, incremental variations in Psi m were sometimes observed that indicated single-channel or groups of channel openings. A particularly dramatic example is shown in Fig. 4B.


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Fig. 4.   Eversion of DVR for endothelial patch-clamp recording. A: a single descending vas rectum in which an elongated perfusion-style pipette (eversion pipette) has been advanced through a holding pipette to turn the vessel inside out and expose endothelial cells at its distal end. A patch-clamp pipette deformed under heat to bend its tip ~90° has been advanced to form a seal with luminal endothelial surface. B: endothelial membrane potential (Psi m) recordings often showed incremental variations indicative of channel openings.

The effect of 10 nM ANG II on endothelial Psi m is illustrated in Fig. 5. Most often, spiking, rapid oscillations of Psi m occurred that were often superimposed on some elevation of the baseline (Fig. 5, A-D). Reversibility after ANG II washout was variable but often present. Summaries of the effects of ANG II on Psi m are provided in Fig. 5, E and F, for stripped and everted vessels, respectively. The baseline, ANG II, and recovery values are shown as 10-s averages at the end of each period. In pericyte-stripped vessels, Psi m depolarized from -53.1 ± 3.2 to -43.4 ± 4.4 mV (P < 0.05) and recovered to -48.3 ± 3.7 mV (P = 0.07, ANG II vs. recovery). ANG II tended to depolarize the endothelia more in everted than in stripped vessels [-56.3 ± 4.2 (baseline) to -35.8 ± 3.8 mV (ANG II)], but the difference in the ANG II-depolarized values (-43.4 ± 4.4 vs. -35.8 ± 3.8 mV) did not achieve significance (P = 0.24) for the two configurations.


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Fig. 5.   Effect of ANG II on Psi m in DVR endothelia. A-D: individual recordings of Psi m from endothelial cells as 10 nM ANG II was introduced into and then removed from the bath (10-min exposure). A and B are from everted DVR; C and D are from pericyte-stripped DVR. Depolarization was observed. Voltage shown at the beginning of each trace is resting potential at time 0. Trace in C shows evidence of incremental changes due to channel activity. E and F: summary of endothelial Psi m changes induced by ANG II in pericyte-stripped (n = 12) and everted (n = 8) vessels. NS, not significant.

The effect of 100 nM BK on endothelial Psi m is illustrated in Fig. 6. The response was characterized by transient hyperpolarization, which tended to return to baseline before BK washout (Fig. 6, A-D). Summaries of the effects of BK on Psi m are provided in Fig. 6, E and F, for stripped (n = 16) and everted (n = 9) vessels, respectively. Values show 10-s averages for the baseline and recovery periods. Because the BK effect was transient, the minimum Psi m achieved during the hyperpolarization, rather than a time average, is displayed. In stripped vessels, Psi m changed from -39.9 ± 3.4 mV to a minimum of -62.7 ± 2.8 mV (P < 0.01) and recovered to -45.2 ± 4.5 mV (Fig. 6E; P < 0.01, BK vs. recovery). Everted vessels showed similar hyperpolarization (Fig. 6F). The baseline of the summaries in Figs. 5 and 6 differs, because vessels with higher resting Psi m were consciously selected for exposure to BK. The mean resting Psi m of all endothelia was -45.5 ± 2.2 mV (n = 45). Resting endothelial Psi m in pericyte-stripped and everted vessels was similar: -45.6 ± 2.7 (n = 28) and -45.3 ± 4.0 mV (n = 17), respectively.


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Fig. 6.   Effect of BK on Psi m in DVR endothelia. A-D: individual recordings of Psi m from endothelial cells as 100 nM BK was exchanged into and then removed from the bath (5- or 10-min exposure). A and B are from everted DVR; C and D are from pericyte-stripped DVR. Hyperpolarization occurred and was most often transient. Voltage shown at the beginning of each trace is resting potential at time 0. E and F: summary of endothelial Psi m changes induced by BK in pericyte-stripped (n = 16) and everted (n = 9) vessels.

To test whether variation of Psi m in DVR pericytes might be part of the mechanism by which BK dilates these vessels, we obtained eight records from pericytes. As shown in Fig. 7, baseline Psi m was low: -65.0 ± 1.8 mV. Despite the low resting potential of this group of pericytes, 100 nM BK hyperpolarized them to -72.0 ± 1.0 mV (P < 0.05), an effect that reversed to -62.7 ± 3.6 mV after washout (P = 0.05, BK vs. recovery).


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Fig. 7.   Effect of BK on Psi m in DVR pericytes. A and B: individual recordings of Psi m from pericytes are shown as 100 nM BK was introduced into and then removed from the bath (10-min exposure). Voltage shown at the beginning of each trace is resting potential at time 0. C: summary of pericyte Psi m changes induced by BK (n = 8). Pericytes on abluminal surface of digested vessels (Fig. 1A) were patched for recording as previously described (14).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We previously demonstrated that complex Ca2+-signaling events occur in DVR endothelia. The endothelium-dependent vasodilator BK produces classic peak-and-plateau elevations of [Ca2+]i, as expected for stimulation of a G protein-coupled receptor that signals through inositol trisphosphate-mediated Ca2+ release-and-entry mechanisms (2). Also as expected, BK enhances production of nitric oxide (NO), probably through stimulation of NO synthase type III (21). In most cell types, ANG II acts through the AT1 receptor to increase [Ca2+]i (22). Furthermore, infusion of ANG II leads to NO production within the kidney, particularly in the renal medulla (25). On the basis of those expectations, our prior finding, reproduced in this study (Figs. 2 and 3), that ANG II suppresses [Ca2+]i signaling in DVR endothelia is particularly surprising. On the basis of this finding, we have speculated that the source of ANG II-stimulated NO production in the renal medulla is epithelial, rather than endothelial, in origin and that this enables NO to provide a feedback mechanism through which oxygen-consuming outer medullary interbundle nephrons can modulate their own perfusion (18). The receptor subtype and signaling mechanisms that mediate this process are unknown.

Given the importance of Ca2+ signaling and NO production in the preservation of medullary blood flow, we were motivated to develop methods that would enable determination of the mechanisms by which DVR cellular processes are regulated. We were previously unable to examine pericyte [Ca2+]i signaling, because fura 2 failed to load into pericytes on isolated DVR (16). In contrast, electrophysiological examination of pericytes has been possible because of their abluminal location (14, 24). Conversely, because fura 2 loads avidly into DVR endothelia, we have been readily able to measure endothelial [Ca2+]i changes but unable to access those cells for electrophysiological recording. In this effort, we developed two methods for bridging those gaps. First, enzymatic digestion of the basement membrane makes it possible to form gigaseals on the pericytes for patch clamp (14) and to strip the pericytes from the vessels simply by aspirating a vas rectum into a holding pipette (Fig. 1). The result is that either cell type can be isolated for Ca2+ studies or electrophysiological recording. As a second, somewhat arduous, approach to endothelial electrophysiology, isolated DVR that have not been enzymatically digested can be everted to expose the luminal endothelial surface (5, 23) (Fig. 4). Gigaseals form on the luminal endothelial surface without prior enzymatic treatment.

Studies of endothelial [Ca2+]i signaling in pericyte-denuded vessels differ from our prior examinations in isolated vessels in important ways. First, the endothelial cells have been subjected to enzymatic digestion and the trauma required to denude the abluminal surface of pericytes. Second, endothelial cell responses are studied in the absence of pericytes, so that pericyte-endothelial communications via diffusible factors or myoendothelial junctions would have been disrupted. Motivated by this, we first determined whether we could reproduce our prior finding that BK elevates DVR endothelial [Ca2+]i and that ANG II suppresses it (16, 18). Despite the trauma of isolation, enzymatic digestion, and the absence of pericytes, both responses were intact (Figs. 2 and 3), but an equilibration period after stripping is probably needed to achieve full responsiveness (Fig. 2C). This supports the notion that cellular signaling and receptors for those ligands are preserved. We previously showed that perfusion of ANG II into the lumen of isolated DVR fails to constrict them but still modulates [Ca2+]i responses to BK (18). That finding, coupled with the strong vasoconstriction elicited by abluminal ANG II, was interpreted to show that separate ANG II receptors probably exist on DVR endothelia and pericytes. The present observation that ANG II suppresses [Ca2+]i in pericyte-denuded DVR (Figs. 2B and 3) strongly supports the presence of an endothelial ANG II receptor.

The removal of pericytes from the abluminal surface of the DVR makes the underlying endothelia accessible for whole cell patch-clamp recording (Fig. 1C). By this method and by eversion to expose the luminal surface (Fig. 4), ANG II and BK were found to have opposite effects to depolarize and hyperpolarize DVR endothelia, respectively (Figs. 5 and 6). Thus, as with [Ca2+]i responses (Figs. 2 and 3), neither the enzymatic digestion nor the removal of pericytes eliminated the Psi m response to those hormones.

Psi m is an important determinant of smooth muscle contractility, because it regulates transport of Ca2+ into the cytoplasm via voltage-gated channels. Depolarization has been found to be an important component of ANG II constriction of the renal afferent arteriole as well as DVR (11, 24). The generally accepted paradigm is that constrictors depolarize the cells, enhance Ca2+ entry, increase [Ca2+]i, activate myosin light chain kinase, and increase myosin cross-bridge formation, resulting in cellular contraction (9, 20). Variation in Psi m is also responsible for modulating contraction through its effects in endothelial cells (1, 9, 13). Endothelium-dependent vasodilators such as BK and acetylcholine generally hyperpolarize endothelial cells. Unlike smooth muscle, where hyperpolarization is expected to decrease Ca2+ entry by inhibiting voltage-gated channels, hyperpolarization of endothelia is thought to enhance Ca2+ entry by increasing the electrochemical driving force for influx via nonselective cation channels (1, 13). On the basis of that hypothesis, our finding that ANG II depolarizes DVR pericytes and endothelia might partially explain the opposite [Ca2+]i responses of those cells to ANG II. Perhaps ANG II-induced depolarization enhances pericyte plasmalemmal Ca2+ entry via voltage-gated channels while reducing endothelial Ca2+ entry via nonselective cation channels. The recent demonstration of expression of L- and T-type voltage-operated Ca2+ channels in the juxtamedullary efferent arteriole and DVR is consistent with that possibility (7). We demonstrated that ANG II decreases the rate of refilling of Ca2+ into store-depleted DVR endothelia and decreases the rate of Mn2+ entry into Ca2+-replete cells. Those findings do not differentiate between ANG II-induced blockade of the Ca2+ entry pathway and Psi m change, because either could inhibit divalent cation influx (16). Further studies are required to study the effects of ANG II on endothelial currents in voltage-clamp experiments.

In summary, we devised a relatively simple method for separating and isolating DVR pericytes and endothelia for examination by electrophysiological and microfluorescent methods. We have, for the first time, verified that ANG II increases [Ca2+]i in the DVR pericyte cytoplasm and reduces [Ca2+]i in DVR endothelia. When pericytes are removed from DVR, the endothelial response to ANG II is preserved, supporting the existence of an endothelial ANG II receptor. ANG II and BK have opposite effects to depolarize and hyperpolarize pericytes and endothelia. A relationship between Psi m changes and [Ca2+]i responses is hypothesized on the basis of Ca2+ entry pathways in the two cell types.


    ACKNOWLEDGEMENTS

Studies in our laboratory were supported by National Institutes of Health Grants DK-42495, HL-62220, and HL-68686.


    FOOTNOTES

Address for reprint requests and other correspondence: T. L. Pallone, Div. of Nephrology, N3W143, University of Maryland at Baltimore, Baltimore, MD 21201-1595 (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.

April 23, 2002;10.1152/ajprenal.00065.2002

Received 14 February 2002; accepted in final form 16 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adams, DJ, Barakeh J, Laskey R, and van Breemen C. Ion channels and regulation of intracellular calcium in vascular endothelial cells. FASEB J 3: 2389-2400, 1989[Abstract/Free Full Text].

2.   Berridge, MJ. Inositol trisphosphate and calcium signalling. Nature 361: 315-325, 1993[ISI][Medline].

3.   Cowley, AW, Jr. Role of the renal medulla in volume and arterial pressure regulation. Am J Physiol Regul Integr Comp Physiol 273: R1-R15, 1997[Abstract/Free Full Text].

4.   Di Virgilio, F, Steinberg TH, and Silverstein SC. Inhibition of fura-2 sequestration and secretion with organic anion transport blockers. Cell Calcium 11: 57-62, 1990[ISI][Medline].

5.   Engbretson, BG, Beyenbach KW, and Stoner LC. The everted renal tubule: a methodology for direct assessment of apical membrane function. Am J Physiol Renal Fluid Electrolyte Physiol 255: F1276-F1280, 1988[Abstract/Free Full Text].

6.   Hamill, OP, Marty A, Neher E, Sakmann B, and Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85-100, 1981[ISI][Medline].

7.   Hansen, PB, Jensen BL, Andreasen D, and Skott O. Differential expression of T- and L-type voltage-dependent calcium channels in renal resistance vessels. Circ Res 89: 630-638, 2001[Abstract/Free Full Text].

8.   Horn, R, and Marty A. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol 92: 145-159, 1988[Abstract].

9.   Jackson, WF. Ion channels and vascular tone. Hypertension 35: 173-178, 2000[Abstract/Free Full Text].

10.   Lemley, KV, and Kriz W. Cycles and separations: the histotopography of the urinary concentrating process. Kidney Int 31: 538-548, 1987[ISI][Medline].

11.   Loutzenhiser, R, Chilton L, and Trottier G. Membrane potential measurements in renal afferent and efferent arterioles: actions of angiotensin II. Am J Physiol Renal Physiol 273: F307-F314, 1997[Abstract/Free Full Text].

12.   Mattson, DL, and Wu F. Control of arterial blood pressure and renal sodium excretion by nitric oxide synthase in the renal medulla. Acta Physiol Scand 168: 149-154, 2000[ISI][Medline].

13.   Nilius, B, Viana F, and Droogmans G. Ion channels in vascular endothelium. Annu Rev Physiol 59: 145-170, 1997[ISI][Medline].

14.   Pallone, TL, and Huang JMC Control of descending vasa recta pericyte membrane potential by angiotensin II. Am J Physiol Renal Physiol. 282: F1064-F1074, 2002[Abstract/Free Full Text].

15.   Pallone, TL, and Silldorff EP. Pericyte regulation of renal medullary blood flow. Exp Nephrol 9: 165-170, 2001[ISI][Medline].

16.   Pallone, TL, Silldorff EP, and Cheung JY. Response of isolated rat descending vasa recta to bradykinin. Am J Physiol Heart Circ Physiol 274: H752-H759, 1998[Abstract/Free Full Text].

17.   Pallone, TL, Silldorff EP, and Turner MR. Intrarenal blood flow: microvascular anatomy and the regulation of medullary perfusion. Clin Exp Pharmacol Physiol 25: 383-392, 1998[ISI][Medline].

18.   Pallone, TL, Silldorff EP, and Zhang Z. Inhibition of calcium signaling in outer medullary descending vasa recta by angiotensin II. Am J Physiol Heart Circ Physiol 278: H1248-H1255, 2000[Abstract/Free Full Text].

19.   Pallone, TL, Work J, Myers R, and Jamison RL. Transport of NaCl and urea in outer medullary vascular bundles. J Clin Invest 93: 212-222, 1994[ISI][Medline].

20.   Pfitzer, G. Regulation of myosin phosphorylation in smooth muscle. J Appl Physiol 91: 497-503, 2001[Abstract/Free Full Text].

21.   Rhinehart, K, and Pallone TL. Modulation of nitric oxide synthesis in isolated descending vasa recta. Am J Physiol Heart Circ Physiol 281: H316-H324, 2001[Abstract/Free Full Text].

22.   Timmermans, PB, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JA, and Smith RD. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev 45: 205-251, 1993[ISI][Medline].

23.   Turner, MR, Silldorff EP, and Pallone TL. Effects of angiotensin II on descending vasa recta isolated from rats (Abstract). FASEB J 12: A388, 1998.

24.   Zhang, Z, Huang JM, Turner MR, Rhinehart KL, and Pallone TL. Role of chloride in constriction of descending vasa recta by angiotensin II. Am J Physiol Regul Integr Comp Physiol 280: R1878-R1886, 2001[Abstract/Free Full Text].

25.   Zou, AP, Wu F, and Cowley AW, Jr. Protective effect of angiotensin II-induced increase in nitric oxide in the renal medullary circulation. Hypertension 31: 271-276, 1998[Abstract/Free Full Text].


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