Division of Nephrology, University of Maryland School of Medicine, Baltimore, Maryland 21201-1595
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ABSTRACT |
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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
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INTRODUCTION |
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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 (m) are depolarized by ANG II, and both are
hyperpolarized by the vasodilator BK.
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METHODS |
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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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Whole cell patch-clamp recording.
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.
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.
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RESULTS |
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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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Electrophysiological measurements of m in DVR
endothelia and pericytes.
We obtained recordings of
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
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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DISCUSSION |
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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
m response to those hormones.
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
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
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 m
changes and [Ca2+]i responses is hypothesized
on the basis of Ca2+ entry pathways in the two cell types.
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ACKNOWLEDGEMENTS |
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Studies in our laboratory were supported by National Institutes of Health Grants DK-42495, HL-62220, and HL-68686.
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FOOTNOTES |
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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.
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