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1 From the Departments of Ophthalmology and Visual Sciences and 2 Physiology, University of Michigan, Ann Arbor, Michigan.
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Abstract |
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METHODS. The perforated-patch configuration of the patch-clamp technique was used to monitor whole-cell currents of pericytes located on microvessels freshly isolated from the rat retina. To assay cell-to-cell coupling within retinal microvessels, a gap junction–permeant tracer was loaded through patch pipettes into pericytes and the spreading of the tracer detected by immunohistochemistry.
RESULTS. ET-1 acting through ETA receptors altered pericyte currents and caused depolarization of the membrane potential. The effects on pericyte currents were dynamic over time. Initially, the nonspecific cation (NSC) and calcium-activated chloride (ClCa) currents were activated and the adenosine triphosphate (ATP)–sensitive potassium (KATP) current inhibited. Subsequently, by a mechanism sensitive to a protein kinase C (PKC) inhibitor, the NSC, ClCa, and voltage-dependent potassium currents diminished as gap junction pathways closed within the microvessels.
CONCLUSIONS. ET-1 regulates pericyte conductances by multiple mechanisms. One process involves a PKC-dependent closure of gap junction pathways resulting in loss of electrotonic input from neighboring cells. Thus, ET-1 not only affects individual microvascular cells, but also regulates the effective size of the multicellular functional units that may serve to control capillary blood flow. This regulation of intercellular communication within pericyte-containing microvessels may be an important, previously unrecognized, action of ET-1.
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Introduction |
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A specialized feature of the retinal microvasculature is an absence of the smooth muscle sphincters3 that control capillary perfusion in other tissues. The responsiveness of retinal capillaries to vasoactive signals,4 5 despite the absence of precapillary myocytes, suggests that the control of retinal blood flow occurs, in part, at the capillary level. Regulation of microperfusion at distal locations in the vasculature may be an effective mechanism to efficiently distribute oxygen and nutrients to metabolically active neurons within the inner retina.
Candidates for playing a role in adjusting capillary blood flow are the pericytes, which envelope small blood vessels and change their contractile tone, at least in culture, during exposure to vasoactive molecules.6 7 8 Contraction and relaxation of these cells may adjust lumen size.4 9 Although pericytes are present in almost all vascular beds, the ratio of pericytes to endothelial cells is greatest in the retina,10 suggesting the particular importance of these cells in the retinal microcirculation. However, the mechanisms by which vasoactive molecules influence the function of these cells remain uncertain.
In this study, we focused on the effects of endothelin (ET)-1, which is a putative endothelial cell-to-pericyte signal. This molecule is expressed by retinal vascular endothelial cells,11 and pericytes have ET receptors.11 12 Consistent with its role as a vasoconstrictor, ET-1 increases calcium levels in pericytes,13 14 15 causes these cells to contract,13 14 and evokes a constriction of pericyte-containing microvessels of the retina.4
Because ion channels are likely to be important in mediating the functional responses of pericytes to vasoactive signals, we examined the effects of ET-1 on the ionic currents of these cells. Using the perforated-patch technique, we monitored currents in pericytes located on microvessels freshly isolated from the adult rat retina. We now report that exposure of retinal microvessels to ET-1 alters four types of ionic currents in the pericytes. Multiple mechanisms appeared to mediate the ET-induced changes in pericyte physiology. One process involved the regulation of electronic input from neighboring cells through gap junction pathways within the retinal microvasculature.
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Methods |
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Electrophysiology
A coverslip containing microvessels was placed in a recording
chamber that was perfused at 2 mL/min with solution A plus additives,
as noted. During the recording session, vessels were examined at x400
magnification with an inverted microscope equipped with phase-contrast
optics. As detailed previously,5
16
18
the
perforated-patch configuration of the patch-clamp technique was used to
monitor the ionic currents of pericytes located on microvessels that
had been isolated from a retina within 2 hours. The pipette solution
for these experiments contained 50 mM KCl, 65 mM
K2SO4, 6 mM
MgCl2, 10 mM K-HEPES, 240 µg/mL amphotericin B,
and 240 µg/mL nystatin at pH 7.4, with the osmolarity adjusted to 280
mOsm. Pipettes, which had resistances of approximately 5 M
, were
mounted in the holder of a patch-clamp amplifier (Dagan Corp.,
Minneapolis, MN). After the tip of a pipette was positioned with the
aid of a piezoelectric micromanipulator (Burleigh, Victor, NY) onto the
soma of a pericyte, application of gentle suction to the back end of
the pipette created a more than 10-G seal. As the
amphotericin-nystatin perforated the patch, the access resistance to
the pericytes studied decreased to less than 20 M. For the
generation of current–voltage (I-V) plots, currents were evoked by
voltage step protocols controlled by computer (pClamp 8 software; Axon
Instruments, Inc., Foster, CA) and were filtered at 1 kHz with a
four-pole Bessel filter and digitally sampled at 400-µs intervals,
using a data acquisition system (Digidata 1200B; Axon Instruments).
Digitized data were stored in a computer for data analysis and graphics
display (pClamp 8; Axon, and Origin 6.1; OriginLab, Northampton, MA).
Adjustment for the calculated19 liquid junction potential was made after data collection. The current during the last 200 ms of each voltage step was used to generate I-V plots, as described previously.16 Although cell coupling within a retinal microvessel20 may limit the ability of a micropipette sealed to a pericyte soma to fully clamp the membrane potential of all the cells that generate the currents detected in a sampled pericyte, our previous finding5 18 that the observed reversal potential for potassium currents is very close to the calculated potassium equilibrium potential suggests that voltage can be clamped reasonably well at the sites contributing the bulk of the detected current.21 As detailed elsewhere,16 the nonspecific cation (NSC) current was measured at -103 mV, which is near the equilibrium potential for potassium (EK). Potassium current was measured at 0 mV, which is close to ENSC. Chord conductances were calculated from these values. Similar to our previous description,5 the chord conductance of the transient chloride currents recorded during a 20- to 50-second sampling period was quantified at a holding potential of -58 mV by subtracting the amplitude of the current in the absence of any transient events from the average current amplitude, which was calculated by computer (pClamp 8; Axon, and Origin 6.1; OriginLab). Dividing this difference by 34 mV, which is the difference between the equilibrium potential for chloride (-24 mV) and the holding potential, yielded the chord conductance for the transient chloride currents. For analysis of the decay of membrane capacitive currents, a 10-mV step from -58 to -48 mV evoked currents that were filtered at 10 kHz and digitally sampled at 50-µs intervals; curve-fitting software (Origin 6.1; OriginLab) calculated exponential functions that described the decay of the membrane capacitive currents.
Tracer Labeling
As detailed previously,20
a patch pipette
containing 0.5% of the gap junction–permeant tracer
N-(2-aminoethyl) biotinamide chloride (Neurobiotin; Vector
Laboratories, Burlingame, CA) plus (in millimolar) 25 KCl, 105
potassium aspartate, 1.5 CaCl2, 2
MgCl2, 3 K-EGTA, and 10 K-HEPES at pH 7.4 and
osmolarity of approximately 280 mOsm was mounted in a holder. The tip
was positioned onto the soma of a retinal pericyte located on a freshly
isolated retinal microvessel, and an approximately 10-G seal was
created. After the patch of cell membrane at the tip of the pipette was
broken by applying progressively increasing amounts of suction through
a pneumatic transducer (Bio-Tek, Winooski, VT), a voltage of +50 mV was
applied to the pipette for 5 minutes to enhance movement of the tracer
from the pipette into the sampled pericyte. The tracer-containing
pipette was then removed, and the microvessel was left undisturbed for
40 minutes. The location of the sampled pericyte was documented in a
sketch. After approximately 18 hours of fixation at 4°C in
phosphate-buffered saline (PBS) containing 4% paraformaldehyde, the
microvessel-containing coverslip was washed in PBS, and the endogenous
peroxidase activity of the vessels was blocked by exposure to 0.3%
hydrogen peroxide in PBS for 30 minutes. After a 60-minute incubation
in 0.5% Triton X-100, the coverslip was exposed for 40 to 65 hours at
4°C to a horseradish peroxidase streptavidin solution (1 µg/mL,
RTU; Vector Laboratories) supplemented with 0.5% Triton X-100.
Microvessels were then developed for 5 minutes in diaminobenzidine plus
nickel (DAB kit; Vector Laboratories). After the vessels were
counterstained with methyl green, they were viewed at x100
magnification with a microscope equipped with bright-field optics, and
the extent of staining by the tracer was measured.
Chemicals
Chemicals were from Sigma/RBI (St. Louis, MO) unless noted
otherwise.
Statistics
Data are expressed as the mean ± SEM. Probability was
evaluated by Student’s t-test.
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Results |
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In the present study, we found that exposure of isolated microvessels to ET-1 altered both the sustained and transient currents of retinal pericytes. This is illustrated in Figure 1 and quantified in Figure 2 . Initially, ET-1 induced an increase in the amplitudes of the NSC and ClCa currents, but did not significantly (P = 0.4) affect the KV conductance. Associated with the early changes in the ionic conductances, the membrane potential of the pericytes decreased from -46 ± 1 to -31 ± 2 mV (n = 13). After approximately 5 minutes of exposure to ET-1, the amplitudes of the NSC, KV, and ClCa currents began to decrease. New steady state levels were reached within approximately the next 10 minutes (Figs. 1 2) . Despite these conductance changes, the mean membrane potential of the sampled pericytes remained at -31 ± 1 mV. The ET-induced changes in the NSC, KV, and ClCa currents were similar (P > 0.3) at ET-1 concentrations of 0.25 (n = 8), 1 (n = 13), and 100 nM (n = 8). The effects of ET-1 on the pericyte currents and membrane potential were reversible (Fig. 1) , although up to 1 hour or more was required to return to control values.
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0.25, n = 4) alter the NSC, KV,
ClCa, or KATP currents in
pericytes. Further support for an ETA, rather
than an ETB mechanism, was our observation that
IRL-1620 (100 nM), which is a specific ETB
receptor agonist,23
had no significant (P > 0.4, n = 3) effect on the pericyte currents. Taken
together, these findings indicate that ET-1 acts through
ETA receptors to regulate the conductances of
retinal pericytes.
Effect of a PKC Inhibitor
In a number of cell types,24
ET-1 activates protein
kinase C (PKC). To assess whether this enzyme is involved in mediating
the effects of ET-1 on the ionic conductances of pericytes, we tested
the effect of chelerythrine, which is a PKC inhibitor.25
In a series of experiments (Fig. 4)
, the early effects of ET-1 on pericyte conductances were not
significantly (P > 0.3, n = 5) altered when
isolated microvessels were exposed to 1 µM chelerythrine for 45 to 90
minutes before supplementing the bathing solution with 1 nM ET-1. This
finding suggests that during ET exposure, PKC does not have a critical
role in either the initial activation of the NSC and
ClCa channels or the early inhibition of
KATP channels.
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ET-Induced Inhibition of Cell Coupling
The question arose as to the mechanism by which PKC mediates the
late ET-induced decrease in NSC, KV, and
ClCa conductances. We considered the hypothesis
that ET-1 reduces electrotonic coupling between pericytes and
neighboring microvascular cells. This seemed to be a reasonable
possibility, because we have demonstrated that there is an extensive
network of cell-to-cell coupling within pericyte-containing
microvessels of the retina and that this intercellular communication is
markedly reduced in the presence of an activator PKC.20
The uncoupling of the pericyte-containing microvasculature may involve
a phosphorylation of connexin 43, which is expressed by retinal
pericytes26
and is regulated by PKC.27
Closure of gap junction pathways would prevent the electrotonic spread
to sampled pericytes of ionic currents that are generated in
neighboring cells.
To help assess the possibility that ET-1 reduces cell coupling, we analyzed the rate of decay of the transient membrane capacitive current evoked by a voltage step (Fig. 5) . A complex rate of decay is predicted for cells that are interconnected in series.28 29 30 In contrast, the decay of this capacitive current should be fit by a single exponential function for a single cell.31 Figure 5A shows an example of a recording under control conditions in which the decay of the capacitive current (solid line) in a pericyte was poorly fit by a single exponential function (dotted line). However, after 16 minutes of exposure to ET-1, a single exponential function (dotted line) described the pericyte’s capacitive current (Fig. 5B) . Similar results were observed in five other recordings from pericytes located on isolated microvessels. In this series of recordings, the decay of the capacitive current was fit by a single exponential function at 13 ± 3 minutes after the onset of ET exposure. However, when the perfusate also contained 500 nM BQ123, the ETA receptor antagonist, we did not detect a change in the capacitive current during an exposure of 12 minutes or more (n = 5) to ET-1 (1 nM). Consistent with a role for PKC in closing gap junction pathways during exposure to 1 nM ET-1, chelerythrine (1 µM) prevented the ET-induced transition to an exponentially decaying membrane capacitive current in each of five sampled pericytes. Thus, it appears likely that the ET-induced uncoupling of pericytes from neighboring vascular cells is mediated by a mechanism involving ETA receptors and PKC activation.
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Discussion |
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The initial inhibition of KATP currents and activation of NSC and ClCa currents in retinal pericytes is similar to the effects of ET-1 on these types of currents in vascular smooth muscle cells. For example, ET-1 inhibits KATP channels in cerebral pial arteries,32 as well as in other vascular myocytes.33 This vasoactive molecule is also known to activate NSC channels of cerebral arterioles34 and of other larger vessels.35 In addition, ET-1 increases ClCa currents in smooth muscle cells,36 including those of the choroidal vasculature in the eye.37 Thus, it appears that the initial response of retinal pericytes to this vasoconstrictor is similar to that of other contractile cells within the vascular system.
However, despite the initial similarities of pericyte and vascular smooth muscle responses, there appeared to be differences during prolonged exposure to ET-1. Specifically, our electrophysiological and tracer assays of intercellular communication revealed that sustained exposure to ET-1 was associated with a loss of cell-to-cell coupling within the pericyte-containing microvasculature. With the uncoupling of pericytes from neighboring cells, the currents recorded from the sampled pericytes decreased. This is the first report of an ET-mediated regulation of gap junction pathways in the vascular system.
Control of gap junction pathways within the retinal microvasculature may be an important mechanism by which ET-1 influences pericyte physiology. There is likely to be a causal link between the ET-induced loss of cell coupling and the decrease in the NSC, ClCa, and KV currents recorded in pericytes during exposure to this vasoconstrictor. Consistent with this possibility, we found that pericyte uncoupling, which occurred between approximately 5 and 15 minutes after the onset of exposure to ET-1, was temporally associated with a reduction in the pericyte conductances. Also supporting the hypothesis that closure of gap junction pathways causes a reduction in pericyte currents, exposure to the PKC inhibitor chelerythrine prevented both the uncoupling of pericytes and the decrease in the NSC, ClCa, and KV currents. A parsimonious explanation is that a PKC-dependent closure of gap junction pathways prevents currents generated in neighboring vascular cells from spreading electrotonically to the sampled pericytes.
Although it appears that control of cell-to-cell coupling plays an important role in the late decrease in pericyte NSC, ClCa, and KV currents, the rapid and sustained inhibition by ET-1 of the KATP current is not regulated by this mechanism. In agreement with this, the decrease in the KATP current was maximal before the loss of cell-to-cell coupling. Also, PKC activation appeared not to be essential for the ET-induced inhibition of KATP channels, because chelerythrine did not prevent the ET-induced decrease in KATP current. Thus, it seems clear that ET-1 regulates pericyte physiology by multiple mechanisms.
The significance of the ET-induced decrease in cell-to-cell coupling within the pericyte-containing microvasculature is uncertain. One likely consequence is to limit the number of vascular cells that are depolarized when ET-1 is released from a localized area of the vascular endothelium. Because of the extensive gap junction pathways interconnecting cells of the retinal capillaries, the ET-induced depolarization of a pericyte could initially spread electrotonically through widespread portions of the microvascular tree. This may cause a rather generalized vasoconstriction, which, we predict, would become significantly more restricted as gap junction pathways are closed during a sustained exposure to ET-1. Thus, ET-1 not only affects individual cells of the retinal microvasculature, but also regulates the effective size of the multicellular functional units that may serve to control capillary blood flow.20 However, more studies are needed to establish that the regulation of intercellular communication is an important mechanism for the autoregulation of retinal blood flow.
The ET-induced decrease in the membrane potential of pericytes is likely to have functional consequences. With depolarization, the membrane potential enters the "window of current" for the voltage-gated calcium channels (VGGCs) that are expressed by these cells.15 16 38 At voltages within this window (i.e., approximately -35 ± 15 mV16 ) there is sufficient depolarization to activate these calcium channels, but not enough to cause their complete inactivation. Consistent with ET-1 activating VGCCs, a substantial portion of the ET-induced increase in intracellular calcium levels in cultured retinal pericytes is blocked by an inhibitor of these ion channels.15
A likely response of pericytes to an ET-activated calcium influx is cellular contraction. The expression of calcium-sensitive contractile protein by pericytes39 40 and the demonstration of pericyte contraction, at least in culture,6 7 8 13 supports the idea that intracellular calcium levels regulate the contractile tone of these cells. A contraction of pericytes may constrict the microvascular lumen and thereby reduce capillary blood flow.
In addition to regulating pericyte function under physiological conditions, ET-1 may have pathophysiological effects. For example, elevated levels of ET-1 are detected in the retinas of diabetic animals.41 42 An excess of this vasoactive molecule may contribute to the closure of gap junctions20 and to the disruption of blood flow regulation observed early in the course of diabetes.43 Also, the ability of microvessels in the diabetic retina to respond to metabolic compromise may be limited if an overexpression of ET-1 prevents the activation of KATP channels by vasodilators, such as adenosine.18
Our use of isolated microvessels permitted electrophysiological studies of fresh, rather than cultured, pericytes. This may be particularly important in elucidating the mechanisms by which ET-1 regulates the function of the retinal microvasculature. For example, although this vasoactive molecule is reported to have no major effects on the membrane potential of cultured pericytes,44 we found that ET-1 reversibly induced a significant depolarization of these cells when they were located on freshly isolated microvessels. Of course, because an in vivo application of the electrophysiological techniques used in this study seems impractical at present, it remains to be demonstrated that the effects of ET-1 observed in isolated microvessels also occur in the retina in vivo. However, despite some limitations, experiments using freshly isolated retinal microvessels should help to clarify how vasoactive molecules regulate the function of the pericyte-containing microvasculature under physiological and pathophysiological conditions.
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Acknowledgements |
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Footnotes |
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Submitted for publication September 19, 2001; accepted November 27, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: Donald G. Puro, Department of Ophthalmology and Visual Sciences, University of Michigan, 1000 Wall Street, Ann Arbor, MI 48105; dgpuro{at}umich.edu
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 12 minutes
after the onset of exposure to 1 nM ET-1 (
), and 36 minutes after
ET-washout (
). (B, inset) I-V plots under
control conditions (
0.04, **P 
) and 6
minutes after the onset of exposure to 1 nM ET-1 (
