Dye Tracers Define Differential Endothelial and Smooth Muscle Coupling Patterns Within the Arteriolar Wall

Materials and Methods

All procedures and protocols in this study were approved by the University of Virginia Animal Care and Use Committee. Male Golden hamsters were anesthetized with pentobarbital sodium (50 mg/kg IP), and the cheek pouches were excised. Individual arterioles, together with a thin sheet of surrounding connective tissue, were dissected and pinned flat. The preparation was superfused with bicarbonate buffer (pH 7.4, 37°C). Probenecid (2 mmol/L; Sigma) was added to the superfusate during injections of negatively charged dyes to inhibit the nonspecific leak of the dyes via anion exchange. Heptanol, when used to uncouple intercellular communication, was added to the superfusate at a final concentration of 1 or 3 mmol/L. We found that 1 mmol/L heptanol blocked dye transfer, and this concentration was used subsequently.

Dyes (Molecular Probes), aniline-2-sulfonic acid (Aldrich Co), and potassium sulfate (Sigma) were stored as frozen stock solutions, which were diluted each day (see Table 1⇑) in 0.1 mol/L KCl (0.1 mol/L LiCl for Lucifer yellow), 0.05 mol/L Tris, pH 7.8. Injection of biocytin by negative current was facilitated by the high pH of the electrode solution, which served to increase the percent of negatively charged molecules. High-molecular-weight anionic FITC- or TRITC-dextran (3000 Da) was coinjected with the low-molecular-weight dyes, providing a marker of the injected cell.

Individual smooth muscle or endothelial cells were loaded with dye using microiontophoresis. Electrode resistance ranged from 300 to 350 MΩ for dye electrodes (80 to 100 MΩ when filled with 3 mol/L KCl). Square wave current pulses (1 Hz, 500 milliseconds) were used (−5 nA for negatively charged dyes, +0.3 nA for positively charged dyes) to inject the dyes. Injection times ranged from 5 to 20 minutes. Times from injection to observation ranged from 5 to 90 minutes. Multiple injections were performed in each arteriole, with each dye tested in a minimum of 3 individual arterioles. Following injections, most arterioles were fixed with 4% paraformaldehyde. Biocytin-labeled arterioles were permeabilized with 0.02% Triton-X-100 and stained overnight with a 1:100 dilution of Neutralite avidin–Texas red (Molecular Probes).

Arterioles were visualized using confocal fluorescence microscopy. The MRC 600 laser scanning confocal microscope (Biorad, Inc) was used with filter sets T1 and T2, allowing simultaneous measurement of fluorescein/Lucifer yellow–wavelength and rhodamine/Texas red–wavelength dyes. Arterioles were oriented horizontally on the microscope slide and optically sectioned. Nikon Fluor ×40 (NA 1.3) and Nikon ×100 (NA 1.3) oil immersion objectives were used, providing optical section thicknesses of 0.70 μm and 0.35 μm, respectively. Previous studies have shown that these conditions allow separate imaging of smooth muscle and endothelial cell layers in whole-mount arterioles.¹¹ Silver nitrate staining of arterioles has shown that endothelial cells, which are oriented parallel with the long axis of the arteriole, can be readily distinguished from smooth muscle cells, which encircle the arteriole, perpendicular to the long vessel axis.²⁰ Images of stained arterioles were processed using Image 1 (Universal Imaging), Picture Publisher, and Designer (Micrografx). Standard epifluorescence video microscopy was used to record diffusion of nonfixable dyes (carboxyfluorescein). A Nikon ×40 (NA 0.55) water immersion objective and fluorescein- and rhodamine-wavelength filter packages were used. A DAGE-MTI SIT 66 intensified camera was used to obtain video images, which were recorded on VHS cassette and analyzed using Image 1.

Results

Charge Selectivity and Junctional Coupling

Adjacent cells in the endothelial cell layer appeared well coupled in each case (100%, n=7 injections) following intracellular injection of biocytin, a zwitterionic molecule that is 98% neutral at pH 7.4 (Fig 1A⇓). The dye diffused extensively both transversely and longitudinally, distributing homogeneously within the cytoplasm of each endothelial cell. Furthermore, in all endothelial injections of biocytin, dye diffused homogeneously into numerous overlying smooth muscle cells (Fig 1B⇓). Smooth muscle cell staining was seen both immediately above and also several hundred micrometers distant from the injected endothelial cell. This movement of dye is in agreement with the hypothesized presence of a direct intercellular pathway linking endothelial cells and smooth muscle cells through myoendothelial junctions and represents the first such observation in an intact arteriole.

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Figure 1.

Zwitterionic biocytin shows the existence of three coupling pathways and polarity in the myoendothelial junctions. A, In an endothelial cell injection, dye spreads diffusely within the endothelial layer, such that individual cell boundaries are not readily apparent (solid arrow). Biocytin staining is shown in red pseudocoloring; dextran-injected cells are green and marked with an asterisk. Two endothelial cells (approximately 140 μm in length) have been injected, as evidenced by the forked appearance of the dextran stain, which also suggests that the two cells lie on opposite sides of the flattened arteriole. B, At the same injection site, dye movement from the endothelial cells into overlying smooth muscle cells (dotted arrow) is clearly seen. C, Smooth muscle cell injection results in heterogeneous spread of biocytin to various adjacent smooth muscle cells (solid arrow), but no dye is visible in underlying endothelial cells. Avidin (used to detect biocytin) binds with high affinity to components of the mast cells lining arterioles, as can be seen in several of the images. Panels A and B are confocal composites of 2 confocal slices (0.35 μm thickness); panel C is a confocal composite of 20 slices (0.79 μm thickness). Scale bars represent 10 μm.

Biocytin injected into a single smooth muscle cell diffused from the injected cell into multiple adjacent smooth muscle cells (Fig 1C⇑) in 95% of injections (20 of 21 cells). Dye coupling between smooth muscle cells appeared more heterogeneous than in the endothelium, implying a lower overall level of coupling and suggesting that not all smooth muscle cells are equally well coupled. In contrast to the observations of endothelial cells, biocytin was confined to the smooth muscle layer, with no detectable dye movement into underlying endothelial cells.

Ethidium bromide was used to test for charge selectivity of the junctions. This cationic dye binds with high affinity to DNA, and its fluorescence emission intensity increases greatly upon binding. We reasoned that the high level of localized nuclear fluorescence emitted by ethidium bromide would greatly increase our ability to detect low levels of dye diffusion from smooth muscle into the underlying endothelial cells. Ethidium bromide injected into endothelial cells brightly stained neighboring endothelial cell nuclei, as well as multiple overlying smooth muscle cell nuclei (100%, n=5) (Fig 2A⇓). Ethidium bromide disclosed coupling between smooth muscle cells as effectively as biocytin (91%, 10 of 11 cells) (Fig 2B⇓). Of the 11 smooth muscle cell injections, only 2 showed very weak smooth muscle–endothelial cell coupling. Thus, although ethidium bromide allowed visualization of low levels of coupling from smooth muscle to endothelium, dye movement from endothelium to smooth muscle was much more prominent.

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Figure 2.

Cationic ethidium bromide also demonstrates polarity in myoendothelial junctions. A, Endothelial cell injection of ethidium bromide (red) and dextran (green) results in the staining of multiple endothelial cells (solid arrow) and smooth muscle cells (dotted arrow). B, Smooth muscle cell injection stains only adjacent smooth muscle cells (solid arrow) and not the underlying endothelial cells. Injected cells are marked with an asterisk. Images are confocal composites of optical slices (0.79 μm thickness) through the entire arteriolar wall. Scale bars represent 10 μm.

We confirmed previous reports that anionic Lucifer yellow is capable of tracing coupling only between endothelial cells. Following most endothelial cell injections of Lucifer yellow (Fig 3A⇓), dye spread to multiple adjacent cells (75%, 3 of 4 cells) but never diffused into smooth muscle cells. Furthermore, Lucifer yellow injected into smooth muscle cells remained confined to the cell of injection (n=9 cells) regardless of the time provided for injection or diffusion (Fig 3B⇓).

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Figure 3.

Lucifer yellow block of dye movement in smooth muscle cells can be attributed to an action of sulfated compounds. A, Lucifer yellow (yellow) injected into a single endothelial cell diffused within the endothelial cell layer (solid arrow) but never into overlying smooth muscle cells. B, Lucifer yellow (yellow) injected into smooth muscle cells did not diffuse from the injected cell. C, Coinjection of Lucifer yellow (yellow) with biocytin (red) resulted in blockade of biocytin diffusion from the injected cell. D, Carboxyfluorescein (green) and dextran-TRITC (red) coinjected into a smooth muscle cell resulted in carboxyfluorescein diffusion into multiple adjacent smooth muscle cells (solid arrow) but not into underlying endothelial cells. Coinjection of sulfate (nonfluorescent) with carboxyfluorescein (green) and dextran-TRITC (red) into an endothelial cell (panel E) did not block the diffusion of carboxyfluorescein, whereas in smooth muscle cell injections (panel F), carboxyfluorescein movement was completely blocked. All injected cells are marked with an asterisk; examples of cells into which dye has diffused are marked with a solid arrow. In panel E, endothelial cell nuclei are clearly visible, whereas cytoplasmic staining is dim, because of limits in endothelial cell signal detectability when using video microscopy. In this and several other endothelial cell injections, longitudinal coupling of cells appears much greater than transverse coupling. Panels A through C are confocal composites of optical slices (0.79 μm thickness) through the entire arteriolar wall. Panels D through F are video microscopy images. Scale bars represent 10 μm.

We also injected cells with anionic carboxyfluorescein, which has the same net charge as Lucifer yellow. Endothelial cell injections (100%, n=5) showed coupling between endothelial cells and also from endothelium to smooth muscle (60%, 3 of 5 cells), as seen with biocytin and ethidium bromide. In contrast to results seen with Lucifer yellow, smooth muscle–smooth muscle cell coupling was evident with carboxyfluorescein (94%, 16 of 17 cells) (Fig 3D⇑). Coupling patterns seen with carboxyfluorescein closely resembled those seen with injected biocytin or ethidium bromide. Again, dye was not visible within the underlying endothelium following smooth muscle injection.

Because all dyes except Lucifer yellow demonstrated similar coupling patterns, it appeared likely that in each case cell-cell movement occurred by the same mechanism. Heptanol, a well-established uncoupler of gap junctional communication,^{21 22} was used to test for involvement of gap junctions in dye spread. Heptanol treatment (1 mmol/L) completely blocked carboxyfluorescein coupling following smooth muscle (n=3) or endothelial (n=3) cell injections (not shown), thus providing strong evidence for involvement of gap junctions in the movement of dye through each of the detected pathways.

Discussion

Our studies provide strong evidence for each of the three hypothesized cell-cell communication pathways. Homocellular coupling exists within the endothelial cell layer, and contrary to previous results reported with Lucifer yellow and in affirmation of electrical recordings, homocellular coupling exists within the smooth muscle cell layer. We have also shown evidence for a novel heterocellular dye coupling between endothelial and smooth muscle cells. This pathway is unique in the sense that it is strongly biased in the direction of dye movement from endothelium to smooth muscle. Finally, we have shown that Lucifer yellow can produce a selective blockade of dye movement either through homocellular or heterocellular smooth muscle junctions.

Coinjections of high-molecular-weight dextran with each tracer dye allowed us to dismiss the possibility that dye transfer occurred as a result of cytoplasmic bridging between cells, because at no time did we see the high-molecular-weight dextran fill cells adjacent to the injected cell. Furthermore, dye transfer was reversibly blocked by heptanol, a putative uncoupler of junctional communication, providing strong evidence for involvement of gap junctions in the movement of dye from cell to cell.

The tracers were selected to reveal any charge selectivities of the detected pathways, since certain gap junctional channels (Cx40, Cx37, Cx45) are known to be cation selective.^{17 18 19} However, neutral (biocytin), anionic (carboxyfluorescein), and cationic (ethidium bromide) dyes all showed similar extents of coupling in both smooth muscle and endothelial cell layers (see Table 2⇑), arguing against prominent charge selectivities of the channels involved.

Movement of molecules through gap junctions is thought to be directionally symmetrical. Although this was the case within smooth muscle and endothelial cell layers, “polar” coupling from endothelium to smooth muscle was demonstrated by each of the above tracers. These observations raise the provocative idea that small molecules can move from endothelium to smooth muscle, but that diffusion in the opposite direction is limited. A similar degree of polarity existing across gap junctions of two different cell types has been previously reported in a neuronal cell population.¹⁵ There are two possible explanations for this phenomenon. The first is that the polarity results from heterotypic gap junction formation within the myoendothelial junctional complex. Cx43 and Cx40 are present in both smooth muscle and endothelium of hamster cheek pouch arterioles,¹¹ and Cx37 has been reported within some vascular endothelial preparations.^{23 24 25} Localization of particular connexin proteins to smooth muscle versus endothelial sides of the junction could result in the formation of multi-connexin myoendothelial junctions, which results in the nontypical rectifying properties seen, as has been modeled by Loewenstein.²⁶ This model is dependent on the occurrence of a unique site on the “restrictive” (smooth muscle) side of the junction, which interacts with diffusing molecules and creates an asymmetrical free-energy barrier to diffusion of molecules, thus favoring movement from the opposite (endothelial) direction through the channel. The smooth muscle–specific site could be either a component of the junctional pore (a function of the specific connexin amino acid sequence) or a modification of the channel (occurring only within smooth muscle cells).

Second, apparent polarity in dye movement might be explained by differential patterns of dye dilution in endothelium compared with smooth muscle. Although endothelial cells have average lengths of 140 μm, they are very thin and the calculated cell volume is much smaller than that of a smooth muscle cell (length 65 μm).²⁰ The comparatively small volume of endothelial cells may allow dye or second-messenger molecules within the endothelium to diffuse to large numbers of adjacent endothelial cells before the molecules are greatly diluted. Therefore, many endothelial cells could provide a source for dye movement into overlying smooth muscle cells. On the other hand, the larger cell volumes of smooth muscle cells would result in rapid dilution of molecules into only several adjacent smooth muscle cells. The diffusion of molecules from smooth muscle to endothelium would then be highly dependent on the frequency of myoendothelial connections and thus might result in an apparent polarity in the movement of molecules between the two cell layers.

A volume differential between the two cell types might contribute to the observed polarity, but it does not account for the behavior of Lucifer yellow in smooth muscle as compared with endothelium, strongly suggesting that a difference in the junctions comprising each cell type is the cause of the directional coupling. Although the failure of Lucifer yellow to pass through certain gap junctions has been reported previously in several different cell systems,^{14 15 16} a mechanistic explanation for the poor dye passage is lacking. Through use of structurally similar dyes, we have shown that a specific structural component of Lucifer yellow interferes with the movement of dye through smooth muscle gap junctions but not through endothelial junctions. The blocking characteristic of Lucifer yellow could be mimicked by the structurally similar fluorescent dye brilliant sulfoflavin, by nonfluorescent aniline-5-sulfonic acid, and by sulfate itself. Because of the apparently specific action of sulfate on smooth muscle–endothelial and smooth muscle–smooth muscle junctions, with no detectable effect on membrane potential, sulfate may present a powerful tool for manipulating the blockade of smooth muscle junctional pathways while leaving the endothelial pathway intact.

The patterns of movement of Lucifer yellow and other sulfate-containing molecules are consistent with the behavior of a molecule that either interacts directly with junctional proteins or causes junctional uncoupling. Although gap junctional channels have been extensively modeled as large nonspecific aqueous pores, recent data from electrophysiological studies suggest that fixed charges within the pore may strongly influence the movement of charged molecules through the channel.^{18 19} Binding of Lucifer yellow and other sulfated compounds to sites in the channel would explain the low incidence of Lucifer yellow passage through the junctions, as well as the blockade of transfer of coinjected dyes. It is also conceivable that these molecules interact with a cytoplasmic site on the connexin protein, resulting in channel gating to a lower conductance state or perhaps complete channel closure. A third explanation would be a sulfate-specific activation of a regulatory second-messenger cascade within the smooth muscle cells, resulting in channel gating or closure. Clearly, it must also be proposed that endothelial cells lack the same mechanisms. At this time, the available molecular data are insufficient to either refute or support any one model.

The direct applicability of dye movement to that of ion movement remains to be shown, as limits in dye movement may not necessarily represent limits in current flow. However, the dye tracers do mimic, in size and charge characteristics, common second-messenger molecules and thus allow us to make inferences about potential signaling pathways within the arteriole. Many cytoplasmic molecules that have been implicated in signaling pathways (Ca²⁺, IP₃, cAMP) are of a size compatible with gap junctional transfer. Ca²⁺ (40 Da) and/or IP₃ (420 Da) diffusion between cells has been reported in a number of cultured cell systems,^{9 10 27} potentially mediating and synchronizing a host of vasoactive and growth-related responses involving large numbers of cells.

Lucifer yellow and similar molecules define a clear distinction in the junctions found between smooth muscle cells compared with those found between endothelial cells. Our data provide novel evidence for heterocellular dye coupling between endothelial and smooth muscle cells. The high level of coupling within the endothelial layer, as well as the demonstrated existence of direct intercellular communication from endothelium to smooth muscle, suggests a dominant role for the endothelium in integrating and coordinating vascular, blood-borne signals, which can then be passed to large numbers of overlying smooth muscle cells. The limited capacity for hydrophilic molecules to diffuse from smooth muscle to endothelial cells may imply a hierarchy of signal transduction within the cells of the arteriolar wall.