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Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906-0011
Submitted 24 July 2002 ; accepted in final form 12 March 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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2-mm length).
Change in membrane potential always preceded dilation, both at the site of
stimulation and at distant sites along the arteriole. In contrast, an increase
in endothelial cell [Ca2+]i was observed only at the
application site. Micropipette application of KCl, which can depolarize both
smooth muscle and endothelial cells (250 mM, 2.5-s pulse), also caused a
rapid, spreading response consisting of depolarization followed by
vasoconstriction. With KCl stimulation, in addition to changes in membrane
potential, increases in endothelial cell [Ca2+]i were
observed at distant sites not directly exposed to KCl. The rapid longitudinal
spread of both hyperpolarizing and depolarizing responses support electrical
coupling as the mode of signal transmission along the arteriolar length. In
addition, the relatively short distance between heterologous cell types
enables the superimposed radial Ca2+ signaling between smooth
muscle and endothelial cells to modulate vasomotor responses.
gap junctions; acetylcholine; hyperpolarization; depolarization; intracellular calcium ion concentration
The complex relationship between membrane potential and Ca2+ in the two cell types of the arteriolar wall led us to investigate the relative contributions to arteriolar responses made by either 1) membrane potential change or 2) Ca2+ flux. Changes in endothelial cell [Ca2+]i and membrane potential were measured in isolated, perfused arterioles with photometric and electrophysiological techniques. We made measurements at a site of stimulation and at distant sites along the arteriolar length. We examined the temporal patterns of changes in [Ca2+]i, membrane potential, and diameter in response to brief pulsatile stimulation with either a receptor-mediated agonist (ACh) or a depolarizing agent (KCl).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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3 mm in length without visible branches was
dissected and transferred to a temperature-controlled experimental chamber
mounted on an inverted microscope (Olympus IMT-2). Arterioles were cannulated
at both ends with glass pipettes and attached to MOPS-BSA-filled reservoirs
(13). The heights of the
upstream and downstream reservoirs were initially set 40 mmHg above the vessel
with a pressure gradient of 2.6 mmHg between the two ends, thereby
establishing flow within the range found in vivo
(11). A 30- to 60-min
equilibration period followed, during which temperature in the tissue chamber
was gradually raised to 37°C. All vessels developed spontaneous tone by
constricting to 60–80% of maximum diameter. Throughout the
experiment, the tissue bath was continuously flushed with MOPS-buffered saline
solution at a rate of 2 ml/min, equivalent to a bath turnover rate of 1.5
times/min. At the end of each experiment, maximal diameter was determined by
exposure of the vessel to nominally Ca2+-free MOPS-buffered saline
solution with 0.1 mM adenosine at room temperature.
Stimulating pipettes (tip diameter 5 µm) were positioned near the vessel
(20 µm distant) with the aid of a motor-driven micromanipulator
(STM/III), and agonists were pressure ejected onto the vessel with a brief
pressure pulse by using a Picospritzer (General Valve), thereby allowing rapid
and precise control of delivery. Vascular responses were observed at the site
of the stimulating pipette (designated the local site) or at sites 500 and
1,000 µm upstream from the stimulating pipette. Both the superfusion and
perfusion flows were directed along the length of the vessel toward the
stimulating pipette (Fig.
1).
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Arterioles were visualized with either bright-field (Olympus halogen illuminator) or fluorescence (LEP Hg lamp with 1.75% neutral density filter in light path) microscopy at a magnification of x900 (Olympus x40, 0.7 NA objective). In fluorescence studies, a slit placed at an object plane limited the field of illumination to a 100-µm vessel segment, thereby reducing photo damage, a critical issue in studies of this type. The image of an arteriolar segment produced by an intensified charge-coupled device camera (Dage-MTI, Gen II or Gen III with extended resolution and blue response, Stanford Photonics) was displayed on a video monitor (Dage-MTI) and recorded (model BV-1000, Mitsubishi) for subsequent image analysis (Image 1, Universal Imaging). The video signal was also used for diameter measurement (video caliper, Microcirculation Research Institute). An image splitter (Nikon, with appropriate filters) was mounted on the microscope side port to direct fluorescent light to a pair of photomultiplier tubes (PMTs; Hamamatsu) for measurement of fluorescence from the potential-sensitive or Ca2+-sensitive dyes. Videocaliper and PMT voltages were sampled by an analog-to-digital board, and the data were processed and stored with data acquisition software (Workbench PC, Strawberry Tree). This system allowed simultaneous measurement of both vessel diameter and changes in fluorescence intensity. The video and PMT measurements were synchronized by a signal from the pipette pressure-pulse trigger, allowing the establishment of precise temporal correlation between changes in fluorescence intensity and diameter.
Measurement of endothelial cell [Ca2+]i. Fluo 3 was selectively loaded into endothelial cells by perfusing arterioles with MOPS-BSA containing 2.2 µM fluo 3-AM (in 0.1% DMSO; Molecular Probes) for 40–60 min, followed by a 20-min wash. The fluo 3 was excited at 488 nm, and the resultant emissions sampled at 530 nm were five- to sixfold above the vessel autofluorescence observed before dye loading. Changes in endothelial cell fluorescence intensity in response to stimuli were assessed with software allowing high dynamic range (Workbench PC; measured at 5 Hz). Ca2+ calibrations were performed on completion of experiments. Agents were added either to the superfusion solution or to the luminal perfusion solutions. Minimum and maximum fluorescence (Fmin and Fmax) were determined with concentrations of 10 µM ionomycin and solutions containing either 0 mM Ca2+ and 5 mM EGTA or 2 mM Ca2+. Adenosine (0.1 mM) was used to maximally dilate the vessels to eliminate motion artifact. After Fmax was obtained, 0 Ca2+ and 0.05% saponin were added to permeabilize the membranes to release free cytosolic Ca2+ and determine the extent of membrane-bound dye. This treatment was found to consistently return the fluorescence intensity to Fmin.
Measurement of endothelial cell membrane potential. The emission spectrum of the voltage-sensitive dye di-8-ANEPPS has a broad peak centered in the range of 590 nm and shifts toward longer wavelengths in response to increases in membrane potential. Comparison of the ratio of the emission intensities at 560 and 620 nm (F620/F560) with simultaneous microelectrode recordings has shown that the ratio changes linearly with potential over the range of -56 to -6 mV and that a 1% decrease in the ratio corresponds roughly to a 10-mV depolarization (2).
Di-8-ANEPPS was selectively loaded into endothelial cell membranes by perfusing arterioles with MOPS-BSA containing 6 µM Pluronic (Molecular Probes) and 1 µM di-8-ANEPPS-AM (in 0.02% DMSO; Molecular Probes) for 20 min, followed by a 20-min wash. Resultant levels of fluorescence (excitation 488 nm, emission 560 and 620 nm) were at least 10-fold above the vessel autofluorescence observed before dye loading. Fluorescence ratios were calculated on-line (Workbench PC software) at a rate of 10 Hz. In these experiments it was not possible to obtain absolute value calibrations of the membrane potentials measured with di-8-ANEPPS. To ensure that the changes reflected changes in membrane potential as accurately as possible, we performed numerous controls during each experiment. The excitation light intensity was changed by placement of neutral-density filters in the light path, and it was established that the absolute light level had no effect on F620/F560.
In a few experiments, intracellular microelectrodes were used to confirm the findings obtained with di-8-ANEPPS. Arterioles were isolated as described above and were transferred to a dual-vessel chamber (model CH/2/A; Living System Instrumentation) for cannulation. One end of the arteriole was cannulated and tied with a thread onto the cannulating pipette mounted on one side of the chamber under a Zeiss dissection microscope. The chamber was then transferred to the stage of an Olympus BH2-RFCA microscope for cannulation of the free end of the arterioles with a modified V-track, double-pipette cannulating system (as above). The microelectrode was passed through the cannulating pipette to impale cells from the luminal surface of the vessel (Fig. 1).
Membrane potential was recorded with glass microelectrodes pulled from
borosilicate glass tubes with an inner filament (1.5-mm OD, 0.86-mm ID;
GC150F-15, Clark Electromechanical) on a Brown-Flaming micropipette puller
(model P-77, Sutter). Microelectrodes filled with 2 M KCl had a tip resistance
of 80–120 M
and were connected to an Axoclamp-2 amplifier. A
reference electrode (a Ag/AgCl pellet) was connected to the bath via a
flexible salt bridge tube (WPI) filled with 3 M KCl. The output of the
Axoclamp-2 was displayed on an oscilloscope (V-650F, Hitachi) and stored to
disk. To minimize the effects of capacitance in the recording system, a
minimal fluid level superfusion solution in the tissue bath was maintained.
Cells in the arteriolar wall were impaled by lowering the micropipette with a
Leitz manipulator at 70–80° to horizontal. Successful penetration of
the cell was marked by a sharp deflection of the potential to a stable level
and a depolarization response to pulse application of KCl.
This approach allowed successful cell penetration, but the capacitance between perfusion solution and recording electrode was very high because of the immersion of the outer surface of the recording pipette in MOPS-buffered saline solution. Capacitance could not be fully compensated for by using the capacitance neutralization function of the AxoClamp 2B amplifier, and as a result, the electrical recordings were somewhat damped in both time and amplitude.
The arteriole was viewed with a x10 eyepiece and a Leitz x32 UM objective. The image was projected to a television camera (model SIT 66, Dage-MTI) and displayed on a video monitor (model PMM-935, Lenco). The diameter of the arteriole was continuously measured with Diamtrak software (Monteck).
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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8 mV and at 1.2 Hz.
The resting membrane potential was taken at the most hyperpolarized level and
had a mean value of -37.6 ± 1.1 mV (n = 14). As previously shown (9, 10, 29), luminal perfusion of the fluorescent dyes confined loading to the endothelial cells and changes in arteriolar diameter did not affect the fluorescence intensity except as a function of a change in [Ca2+]i or membrane potential. Cell selectivity of dye loading was assessed by examining the orientation of stained cells (10). Di-8-ANEPPS appeared to localize to the cell membranes, whereas fluo 3 was largely intracellular as indicated by the strong fluorescence signal from the nuclear region. Under no circumstances were experiments performed when circumferential outlines (smooth muscle cells) were visible.
Local responses. Application of ACh caused an increase in endothelial cell Ca2+ and an increase in the fluorescence ratio (F620/F560, i.e., hyperpolarization) followed by vasodilation (Fig. 2). Changes in endothelial cell [Ca2+]i occurred well in advance of changes in arteriolar diameter (Figs. 3 and 4), supporting the conclusion that the fluorescence change originated from the endothelium and that the change in endothelial cell Ca2+ initiated the vasomotor response. Hyperpolarization followed the rise in [Ca2+]i and clearly preceded changes in diameter (Figs. 2 and 3). In contrast, the endothelium-independent vasodilator sodium nitroprusside (SNP) caused only a slight decrease in endothelial cell [Ca2+]i and a small hyperpolarization despite stimulating vasodilation of the same magnitude as observed with ACh (Figs. 2 and 4). This was confirmed with microelectrode experiments, in which SNP (0.5-s pulse) also stimulated an increase in diameter (8.2 ± 0.6 µm) and only a negligible change in membrane potential (0.3 ± 0.4 mV; n = 3).
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To establish a link between these results and those published previously (2, 9), KCl was also used to alter membrane potential. As shown in Fig. 5, pipette application of KCl caused vasoconstriction, a decrease in the fluorescence ratio (i.e., a depolarization), and a sharp transient increase in endothelial cell [Ca2+]i. Because KCl would likely alter the membrane potential in both smooth muscle and endothelium, we cannot be certain which cell type was depolarized during KCl application. In any case, blockade of L-type, voltage-gated Ca2+ channels with nifedipine sharply reduced the magnitude of the KCl-induced rises in [Ca2+]i and the associated contractile responses. The membrane potential changes were unaltered (Fig. 6; Ref. 27).
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The sensitivity of these vessels to K+ was assessed in experiments in which the concentration of KCl in the bath was modified. Elevations >30 mM evoked depolarization and vasoconstriction, whereas small rises in extracellular [KCl] (to 10 or 15 mM) caused hyperpolarization and relaxation of arterioles (Fig. 7).
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To determine whether a change in diameter per se influenced fluorescence intensity, indolactam, which acts by sensitizing the contractile proteins to Ca2+ (17), was used to cause vasoconstriction without a change in Ca2+ or membrane potential (Fig. 6). The observations indicate that the two fluorescent dyes were responding to changes in Ca2+ and membrane potential directly rather than to movement of the arteriole.
Conducted responses. The vasodilation induced by ACh spread along the length of the arteriole and was always preceded by hyperpolarization (Figs. 2 and 3). Endothelial cell [Ca2+]i, in contrast, increased at the local site but not at sites 500 and 1,000 µm upstream, clearly dissociating changes in endothelial cell [Ca2+]i from the vasodilation (Fig. 2). The sensitivity of the endothelial cells to muscarinic agonists at these upstream sites was tested by applying ACh directly. In all cases, application of ACh to a vessel segment induced an increase in endothelial cell [Ca2+]i at that site (data not shown), the expected outcome if endothelial cell [Ca2+]i rather than smooth muscle cell [Ca2+]i was being measured (9).
The pattern seen with KCl was quite different, with the changes in Ca2+ and membrane potential in response to KCl being qualitatively similar along the arteriolar length. KCl caused a depolarization and a constriction that roughly mirrored the change in membrane potential. Both membrane potential and diameter change spread rapidly along the vessel length. Shortly after the onset of the depolarization, there was a transient elevation in endothelial cell [Ca2+]i (Fig. 5).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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The use of a potential-sensitive dye instead of microelectrodes has experimental advantages and disadvantages. An advantage is that the fluorescence measurement is relatively easy, whereas it is technically very difficult to penetrate (and hold for long periods of time) either smooth muscle or endothelial cells with microelectrodes. A major disadvantage of the fluorescence ratio technique is that absolute calibration is difficult or impossible, although our data do seem quite reasonable, showing a 2–3% increase in the fluorescence ratio in response to ACh that would be roughly equivalent to a hyperpolarization of 20–30 mV (2). This is quite consistent with the membrane potential changes observed in the cannulated arteriole (Fig. 3).
The finding that ACh elevates endothelial cell [Ca2+]i at the site of stimulation but not at distant sites (Fig. 2), despite the rapid spread of hyperpolarization and vasodilation along the arteriolar length, has two important implications. First, it supports the suggestion that the vasodilation at distant sites is due to a conducted response intrinsic to the arteriolar wall and not to diffusion of the agonist. We previously demonstrated (18) the presence of gap junctions between the longitudinally orientated endothelial cells, which could serve as a path for cell-cell communication of these signals. Second, the absence of a change in Ca2+ at the distant sites suggests that neither Ca2+ nor inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is the signal responsible for the cell-cell communication that leads to relaxation of the smooth muscle. Indeed, the rapid spread of conduction of the mechanical responses along the vessel axis makes the diffusional transfer of a signaling molecule such as Ca2+ or Ins(1,4,5)P3 unlikely as the underlying mode of transmission. Root mean squared diffusion times for Ca2+ and/or Ins(1,4,5)P3 over a 1-mm distance would likely be on the order of minutes, not the fractions of a second observed here (3, 8). In this study, we were unable to determine the distance of interendothelial cell Ca2+ diffusion. However, if we assume that in a 500-µm segment of vessel there could be as few as 3 endothelial cell lengths (16), the lack of endothelial cell response at this distance from the site of application indicates that the short-pulse stimulation with ACh does not stimulate rapid intercellular Ca2+ spread. Overall, the data presented here strongly support the proposal that electrical current spread was the longitudinally communicated signal.
The presence of myoendothelial gap junctions in these arterioles (12, 18) may provide the pathway responsible for the movement of current from the endothelial cells to the smooth muscle cells at the distant sites. The receptor-independent hyperpolarization of the endothelium at the distant sites would be expected to cause a small rise in endothelial cell [Ca2+]i (4, 19, 21, 23). However, the fall in smooth muscle Ca2+ associated with dilation at the distant site would contribute to a lowering of the endothelial cell Ca2+. The fact that SNP-induced dilation was associated with a small decrease in endothelial cell [Ca2+]i (Fig. 4; Ref. 9) is consistent with the idea that a decrease in smooth muscle cell [Ca2+]i is readily transferred to the endothelium. A decrease in smooth muscle cell [Ca2+]i is also associated with the hyperpolarization evoked by ACh (9). Thus, at the upstream sites in the absence of endothelial cell muscarinic receptor stimulation, this decrease in smooth muscle [Ca2+]i may transfer to the underlying endothelial cells and negate any drive from membrane potential to increase intracellular Ca2+ levels.
An endothelium-derived hyperpolarizing factor (EDHF) might be involved in ACh-induced hyperpolarization of the smooth muscle at both the local and distant sites, but because the release of EDHF is thought to be dependent on an increase in endothelial cell [Ca2+]i (5), and endothelial cell [Ca2+]i decreased at the distant sites (Fig. 4), this seems unlikely. Further evidence to support our data that endothelial cell [Ca2+]i did not rise at the distant sites is the observation that L-arginine analogs do not block the conducted vasodilation mediated by short pulses of ACh (11). A rise in endothelial cell [Ca2+]i at the distant sites would be expected to evoke a release of nitric oxide and thus dilation. A similar observation was made in hamster cheek pouch arterioles in situ (26), a preparation in which it was proposed that the release of an EDHF at the upstream sites contributes to a conducted dilation (1), as it appeared myoendothelial junctions were not open (1, 25, 26). If the myoendothelial junctions are not open at the distant sites, it is possible that there is a small rise in endothelial cell [Ca2+]i associated with hyperpolarization. Whether this occurs and whether it is sufficient to stimulate the release of an EDHF remain to be established.
An additional possibility to explain the distant dilation in response to ACh is the release of K+ from endothelial cells during ACh-induced hyperpolarization. When released into the restricted space between the smooth muscle and endothelial cells, the K+ might cause smooth muscle hyperpolarization and thus relaxation (14). Only small rises in extracellular [KCl] are necessary to evoke both hyperpolarization and relaxation (Fig. 7). The mechanism responsible for the release of K+ at the distant sites has not been defined, but opening of inwardly rectifying K channels may contribute (20).
The time course of the changes in endothelial cell [Ca2+]i in response to depolarizing concentrations of KCl (Fig. 5) is an important observation and is difficult to explain. As mentioned above, the rapidity of the rise in endothelial cell [Ca2+]i along the length of the arterioles argues against longitudinal intercellular spread of Ca2+ and/or Ins(1,4,5)P3 as the mechanism. Furthermore, the onset of the spreading contraction to KCl is extremely rapid, ruling out a major contribution by smooth muscle-smooth muscle Ca2+ signaling, as again this would be too slow (7). An alternative explanation is that the K+ induces a rapid spread of depolarizing current through gap junctions to the neighboring smooth muscle cells and activates voltage-gated Ca2+ channels, causing a rise in smooth muscle [Ca2+]i, and that this drives radial Ca2+ movement from smooth muscle cells to endothelial cells through the myoendothelial cell junctions. The radial Ca2+ signaling might involve simple diffusion of Ca2+, be secondary to elevation of Ins(1,4,5)P3 (9, 22, 29), or be the result of the extracellular diffusion of an as yet unidentified factor (9).
In view of the fact that the recovery of endothelial cell calcium is so rapid compared with the arteriolar contraction and depolarization (Fig. 5), additional factors must be involved in determining the time course of the endothelial cell Ca2+ transient. Application of nifedipine produced almost complete blockade of the contractile response (and presumably the changes in smooth muscle Ca2+) but left a small portion of the endothelial cell Ca2+ response intact (Fig. 6). Thus the endothelial cell Ca2+ may not be simply tracking the pattern set by the rise in smooth muscle cell calcium after activation of L-type Ca2+ channels. One possible explanation for the abrupt termination of the Ca2+ response to KCl is that the endothelial cell membrane Ca2+ pumps, working with the endoplasmic reticulum, reduce the cytoplasmic Ca2+ very rapidly after the initial Ca2+ transient in the smooth muscle (30). This could result in substantial dissociation between the time course of recovery of the smooth muscle cytoplasmic Ca2+ and that of the endothelium.
A second possible contributor to the time course of the KCl-induced Ca2+ transient shown in Fig. 5 is movement of a conducted depolarization along the arteriolar length through endothelial cells, with associated activation of voltage-sensitive Ca2+ channels in either or both cells. Although it is often assumed that voltage-sensitive Ca2+ channels are not present in the endothelium (6), T-type Ca2+ channels have now been reported (see, e.g., Ref. 24) and these could explain the brief calcium transient shown in Fig. 6. This is certainly an important avenue for future investigation. Regardless of the mechanism, as current flows longitudinally down the arteriole through smooth muscle and/or endothelium, there is an elevation in endothelial cell [Ca2+]i that can in turn, modulate arteriolar contraction (9, 29).
An effect of a change in endothelial cell [Ca2+]i is often masked by the Ca2+ signaling from activated smooth muscle (9, 29). Indeed, in the case of depolarization mediated by phenylephrine, the local rise in endothelial cell [Ca2+]i attenuates the local contraction and manifests itself as conducted vasodilation (29). Simply by either blocking the rise in endothelial cell [Ca2+]i or blocking the endothelial cell Ca2+-activated K+ channels, which are crucially important for endothelial cell hyperpolarization, the response to phenylephrine reverts to one of conducted constriction (29). Together, these findings underline the importance of the endothelium in modulating vasoconstriction, even under circumstances that are not usually thought to be "endothelial cell-dependent mechanisms."
In summary, these results reveal the complex interactions that occur between cells in the arteriole wall before the onset of vasomotor responses. The current coupling and Ca2+ signaling between smooth muscle cells and endothelial cells, when considered both separately and in unison, afford signaling processes that can augment or attenuate the magnitude of responses, depending on the signaling processes activated. The observation that changes in membrane potential precede Ca2+ responses at the distant sites supports the view that the rapid spread of current signals a change in smooth muscle Ca2+ and hence the onset of vasomotor responses. Secondary to this activation, smooth muscle cells can influence the endothelium to modulate vasomotor responses.
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ACKNOWLEDGMENTS |
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The work reported here was supported by National Heart, Lung, and Blood Institute Grant HL-53318; American Heart Association, Virginia Affiliate, Grant 96-F-25; and a CJ Martin Fellowship from the National Health and Medical Research Council (Australia).
Portions of this work were presented in preliminary form at the 1997 Microcirculatory Society meeting.
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FOOTNOTES |
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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.
Present address of K. A. Dora: Dept. of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK.
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) and membrane potential (
) in
response to cumulative concentrations of KCl. Extracellular [KCl] was raised
from 4.7 mM (resting membrane potential -38.0 ± 7.1 mV; resting
diameter 74.3 ± 2.5 µm) by addition of KCl to the bath. Values are
means ± SD of steady-state responses from 2 arterioles.