Published online before print January 11, 2007, doi: 10.1161/01.RES.0000257744.23795.93
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© 2007 American Heart Association, Inc.
Cellular Biology |
Ca2+ and Inositol 1,4,5-Trisphosphate–Mediated Signaling Across the Myoendothelial Junction
From the Department of Molecular Physiology and Biological Physics (B.E.I., B.R.D.), Robert M. Berne Cardiovascular Research Center (B.E.I., B.R.D.), and Cardiology Division of Internal Medicine (S.I.R.), University of Virginia School of Medicine, Charlottesville.
Correspondence to Brant E. Isakson, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, PO Box 801394, Charlottesville, VA 22908. E-mail bei6n{at}virginia.edu
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Abstract |
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Second messenger signaling between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) is poorly understood, but intracellular Ca2+ concentrations ([Ca2+]i) in the 2 cells are coordinated, possibly through gap junctions at the myoendothelial junction. To study heterocellular calcium signaling, we used a vascular cell coculture model composed of monolayers of ECs and VSMCs. Stimulation of either cell type leads to an increase in [Ca2+]i in the stimulated cell and a secondary increase in [Ca2+]i in the other cell type that was blocked by gap junction inhibitors. To determine which second messengers are involved, we initially depleted Ca2+ stores in the endoplasmic reticulum Ca2+ with thapsigargin in ECs or VSMCs, but this had no effect on heterocellular calcium signaling. Alternatively, we loaded ECs or VSMCs with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) to buffer changes in [Ca2+]i. BAPTA loading of ECs inhibited agonist-induced increases in intracellular calcium concentration ([Ca2+]i), in both ECs and VSMCs. In contrast, BAPTA loading of the VSMCs blunted the VSMC response but did not alter the secondary increase in EC [Ca2+]i. Xestospongin C (an inositol 1,4,5-trisphosphate receptor inhibitor) had no effect on the secondary Ca2+ response, but when xestospongin C or thapsigargin was loaded into ECs and BAPTA into VSMCs, intercellular Ca2+ signaling was completely blocked. We conclude that 1,4,5-trisphosphate and Ca2+ originating in the VSMCs induces the secondary increase in EC [Ca2+]i but stimulation of the ECs generates a Ca2+ dependent response in the VSMCs.
Key Words: calcium signaling • inositol-1,4,5-trisphosphate • connexins • gap junctions • myoendothelial junctions • heterocellular
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Introduction |
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Communication between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) is an essential component of vascular function.1 Much work has focused on the role of paracrine-mediated pathways in this communication (eg, NO),2 but it is now evident that gap junction–mediated pathways play a pivotal role as well.3 Gap junctions linking VSMCs and ECs are found in locations where dense cytoplasmic processes of the 2 cell types penetrate the internal elastic lamina (IEL),4 ie, the myoendothelial junction (MEJ).5,6 It is well established that gap junctions at the MEJ can provide pathways for coupling between the 2 cells,7 but little is known of the second messenger signaling processes. The discoveries that the gap junctional proteins can be selectively placed within the cell8–10 and that the different gap junctional proteins can show selective permeability to second messengers such as Ca2+ and/or inositol 1,4,5-trisphosphate (IP3)11 suggest that intercellular signaling across the MEJ might be more complicated than usually appreciated.
Diffusion of second messengers through gap junctions and subsequent activation of receptors (eg, ryanodine [RyR] or [IP3-R] IP3 receptors) on the endoplasmic reticulum (ER) of the recipient cell leads to a secondary rise in intracellular calcium concentration ([Ca2+]i) in the recipient cell12–15 and the rise in [Ca2+]i can induce a variety of physiological responses.13,16 For example, a phenylephrine (PE)-induced increase in VSMC [Ca2+]i causes an increase in EC [Ca2+]i, which, in turn, induces the production of NO by the ECs.17,18 The intercellular signaling leading to the secondary rise in EC [Ca2+]i has been assumed to be attributable to second messengers traversing gap junctions at the MEJ. In support of this idea is the fact that inhibition of phospholipase C in VSMCs caused a reduction in a PE-induced increase in EC [Ca2+]i,19 suggesting a possible role for IP3. However, movement of the second messenger from one cell type to another has not been demonstrated.
The interchange of second messengers between ECs and VSMCs has not been adequately studied, attributable, largely, to the inherent difficulty in selective manipulation of either ECs or VSMCs in vivo. We therefore used a vascular cell coculture (VCCC) system that (1) forms MEJs,9 (2) permits selective stimulation of either the VSMCs or ECs, and (3) allows the resulting Ca2+ responses of either cell type to be examined. We show that the responses of the Ca2+ pools of the 2 cells are linked, and we report which of 2 second messengers, Ca2+ or IP3, is critical in linking the response of ECs and VSMCs. We present evidence showing that an agonist-induced rise in EC Ca2+ is sufficient to explain the secondary rise in VSMC [Ca2+]i, whereas both IP3 and Ca2+ appear to be involved in coupling the agonist-induced increase in VSMC [Ca2+]i to the changes in EC [Ca2+]i. These data have important implications for understanding heterocellular signaling and associated physiological responses in the vasculature.
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Materials and Methods |
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An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Vascular Cell Coculture
VCCCs were prepared according to methods previously described.9 VSMCs were cultured on the bottom of a porous Transwell insert (0.4 µm; Corning), and ECs were cultured on the top of the insert allowing for selective manipulation of cell types. The cells form MEJs with a density comparable with that found in arterioles5 and manifest a comparable murine connexin 40 (Cx40)/Cx43 expression profile.9,20
Measurement of Cytoplasmic Intracellular Calcium
The ECs or the VSMCs could be selectively loaded with fluo-4. The acetomethoxyester form was dissolved in Hank’s buffered saline solution (2.5 µmol/L; with 0.0025% Pluronic F127 and 0.1% DMSO [Molecular Probes]; 25 mmol/L HEPES), incubated for 20 minutes at 37°C and allowed to deesterify in the Hank’s buffered saline solution for 20 minutes at 37°C. The VCCC was mounted on an Olympus FV200 confocal microscope for calcium imaging (Figure 1A). ECs were stimulated with 35 µmol/L ATP,21 and VSMCs were stimulated with 10 µmol/L PE22 applied from a micropipette (0.5-mm tip) positioned 1 mm over the cell surface (Figure 1A). Both stimuli have been shown to cause an IP3-mediated increase in [Ca2+]i in their respective cells.21,23 A background image of the Transwell devoid of cells was subtracted from the pixel intensity of each image (Fsub). Maximum fluorescence intensity (Fmax) of both cell types was determined at the termination of each experiment by application of 10 µmol/L ionomycin and stimulation with 100 µmol/L ATP.24 Relative [Ca2+]i values are plotted as the Fmax percentage.
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Inhibitors
Application of 18 
-glycyrrhetinic acid (50 µmol/L; Sigma) or gap2737,43 and gap2740 (190 µmol/L, respectively; Sigma-Genosys or ADI) were performed as previously described.9,25 The acetomethoxyester form of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (20 µmol/L; Molecular Probes18; dose response, Figure IA in the online data supplement) was loaded into cells following loading of fluo-4 for 20 minutes at room temperature, with 15 minutes allowed for deesterification. Xestospongin C (XPC) (20 µmol/L; Sigma26; dose response, supplemental Figure IB) was added to fluo-4–loaded cells for 10 minutes at room temperature before experiments. Thapsigargin (1.5 µmol/L; Sigma12) was added 30 minutes before experiments.
ER Visualization
On the VCCC, ER was visualized with calnexin (Chemicon). Mouse cremaster tissue was embedded in Embed 812 (EMS), and viewed on a Zeiss 900 electron microscope.
Statistics
Significance was at P<0.05 or P<0.01 and determined by one-way ANOVA (Tukey post-hoc test); error bars are ±SE.
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Results |
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Figure 1 shows a typical experiment demonstrating the method for studying MEJ-mediated Ca2+ signaling between the 2 cell types. ECs stimulated with 35 µmol/L ATP responded with an increase in [Ca2+]i, which was followed by a secondary increase in VSMC [Ca2+]i (Figures 1B and 2
A). Similarly, VSMC stimulation with 10 µmol/L PE showed the expected increase in VSMC [Ca2+]i, followed by a secondary increase in EC [Ca2+]i (Figures 1C and 2B). The secondary rise in intercellular Ca2+ in both cases was blocked by 18 -glycyrrhetinic acid or connexin-mimetic peptides (Figures 2C through 2F). Bath application of apyrase did not prevent the secondary increase in [Ca2+]i (data not shown).
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Role of the ER
The secondary Ca2+ rise and the delay between the responses in the 2 cell types represents the time required for intercellular diffusion (Ca2+, IP3, or both) and/or second messenger activation of receptors in the unstimulated cell, presumably attributable to release of Ca2+ from the ER. Using transmission electron microscopy, we examined the MEJ from mouse cremasteric arterioles in situ (Figure 3A). In these samples, an extensive ER network was found near the MEJ. In addition, at the light level, calnexin reflecting the presence of ER could be detected along the entire length of the cellular extensions of both cell types in the VCCC model (Figure 3B through 3D), suggesting that pools of calcium lies in proximity to the gap junctions at the MEJ.
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To test the role of the ER in mediating the increases in [Ca2+]i involved in heterocellular signaling, we selectively loaded either the EC or VSMC monolayer with the SERCA-pump inhibitor thapsigargin, thereby depleting the intracellular Ca2+ pools in the treated cell (supplemental Figure II). When ECs were loaded with thapsigargin, and VSMCs were stimulated, an increase in EC [Ca2+]i was still evident (Figure 4A); however, the response was less than control conditions (Figure 2B). An identical effect was observed when VSMCs were loaded with thapsigargin (Figure 4B). These findings indicate that heterocellular signaling between ECs and VSMCs was not completely dependant on IP3.
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Intercellular Ca2+ Signaling Is Dependent on the Stimulated Cell Type
Based on the preceding data, we hypothesized that heterocellular Ca2+ signaling might depend on diffusion of Ca2+ down its concentration gradient from the stimulated cell, through the MEJ and into the other cell leading to Ca2+-dependent Ca2+ release from the ER. This could account for the undiminished rise in [Ca2+]i in the unstimulated cell type when treated with thapsigargin. We attempted further exploration of this hypothesis by loading ECs or VSMCs with ryanodine but found that the high lipid solubility of ryanodine allowed the drug to block both cell types and rendered them unresponsive to agonist stimulation (data not shown).
We therefore took the alternate approach of buffering the agonist induced rise in [Ca2+]i with the Ca2+ buffer BAPTA (Figure 5). Selective loading of BAPTA was confirmed by demonstrating constancy of the agonist-induced responses in the cell type not loaded with the BAPTA (Figure 5B and 5C). BAPTA effectively eliminated the agonist induced Ca2+ responses when loaded into either cell type (Figure 5A and 5D), demonstrating the efficacy of [Ca2+]i buffering. Moreover, the secondary increase in VSMC [Ca2+]i was inhibited after stimulation of BAPTA-loaded ECs with ATP (Figure 5A), suggesting that intercellular calcium signaling from ECs to VSMCs could be explained by Ca2+ diffusion from ECs to VSMCs down a concentration gradient. In sharp contrast, although BAPTA loading of the VSMCs sharply reduced the direct agonist-induced VSMC Ca2+ response, it did not prevent the secondary increase in EC [Ca2+]i (Figure 5D). A slight delay in the EC response was observed (compare Figure 2B with 5D). These data suggest that second messengers other than Ca2+ are involved in the heterocellular signaling from VSMCs to ECs.
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Effects of IP3 on ECs and VSMCs
To test the hypothesis that a second messenger other than Ca2+ affects ECs after VSMC stimulation, we blocked IP3-R with XPC in either ECs (Figure 6A and 6B) or VSMCs (Figure 6C and 6D). XPC loaded into the stimulated cell type blocked the Ca2+ responses of both the stimulated and unstimulated cell types (Figure 6A and 6D), indicating that IP3 is a required initial component of some part of the signaling pathway and that for both of the agonists used did not produce different second messengers in VSMCs and ECs. Stimulation of the unblocked cell types resulted in the expected rise in [Ca2+]i, demonstrating selective loading of the cell types (Figure 6B and 6C). However, in these conditions, there was no inhibition of an increase in [Ca2+]i in either unstimulated cell types (Figure 6B and 6C). We interpret these data to indicate that IP3 alone does not mediate heterocellular signaling between ECs and VSMCs.
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Roles for Ca2+ and IP3 in ECs
We examined the possibility that a combination of Ca2+ and IP3 are involved in heterocellular signaling from VSMCs to ECs by loading the VSMCs with BAPTA and ECs with XPC. Under these circumstances, both the primary and secondary responses were blocked, regardless of the cell type stimulated (Figure 7A and 7B). The IP3-R inhibitors decavanadate and heparin showed similar effects (supplementary Figure III). Because IP3-induced Ca2+ release requires IP3 binding to IP3-R on the ER, we hypothesized that depleting the EC ER Ca2+ pool with thapsigargin would have the same effect as IP3-R inhibitors, and this was found to be the case (Figure 7C). In summary, the data indicate that there is directionality in the signaling between the 2 vascular cell types; heterocellular signaling from ECs to VSMCs is dependent on Ca2+ alone, whereas signaling from VSMCs to ECs appears to have both a Ca2+ and an IP3 component.
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Discussion |
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Heterocellular communication between the ECs and VSMCs modulates many aspects of vascular function, including control of vessel tone,7 regulation of blood pressure,6 VSMC proliferation,27 vascular development,28 response to shear stress,29 and normal and pathological angiogenesis.30 Both paracrine and gap junctional transmission are involved in the signaling but, to date, it is unknown whether electrical signals, Ca2+, IP3, or other second messengers traverse the gap junctions. Using the VCCC, we have been able to explore systematically which second messengers are involved in heterocellular signaling. The key observation is that stimulation of 1 of the cell types results in a calcium change in both cells. Here, we report data that indicate that the Ca2+ change of the secondary cell type is mediated by different second messengers.
Calcium Signaling From VSMCs to ECs
It has previously been demonstrated that PE stimulation of VSMCs causes an increase in EC [Ca2+]i.7,31 This may result from diffusion of Ca2+ from 1 cell to the other, or alternatively IP3 movement from VSMCs to ECs might trigger the secondary increase in EC [Ca2+]i after VSMC stimulation. This proposition is reasonable in view of the facts that IP3 can diffuse through gap junctions to elicit an increase in [Ca2+]i by binding to IP3-R on the ER12,13 and that a variety of IP3-R isoforms are found in ECs and VSMCs.32 Recent work from rat mesenteric arteries has demonstrated that inhibiting phospholipase C production in VSMCs and stimulating VSMCs with PE causes an inhibition of an increase in [Ca2+]i in ECs,19 indicating that it was possible for IP3 to diffuse through gap junctions linking VSMCs and ECs. In our system, we used XPC to inhibit IP3 only after it had crossed the MEJ from the stimulated to the unstimulated cell type. By stimulating BAPTA-loaded VSMCs with PE, we inhibited Ca2+, but not IP3 movement to the unstimulated cell (Figure 5D). We know that IP3 is produced after PE (or ATP) stimulation (Figure 6A and 6D), and, therefore, we assumed when BAPTA-loaded VSMCs were stimulated, only IP3 could traverse the MEJ. Therefore, we blocked IP3-R in the ECs with XPC (Figure 7B; as well as decavanadate and heparin, supplemental Figure III). In each case, the EC Ca2+ response originally seen in Figure 5D was inhibited. Using a different approach, we depleted the EC ER Ca2+ pools, thereby blocking any release of Ca2+ from IP3-R once IP3 was bound. These experiments had the same effect as the IP3-R inhibitors (Figure 7C). The most parsimonious interpretation of these findings is that after VSMC stimulation with PE, IP3 moves through gap junctions at the MEJ and binds to IP3-R in the ECs.
As noted above, stimulation of BAPTA-loaded VSMCs produced a delayed increase in EC [Ca2+]i (Figure 5D), suggesting the involvement of other downstream processes in the secondary response. Ca2+ ions have also been shown to traverse gap junctions in the vasculature,14 and RyRs located on ER are found throughout the ECs and VSMCs.14,33,34 We present evidence for Ca2+ acting as a second messenger in Figure 6B where XPC in ECs failed to inhibit an increase in [Ca2+]i after VSMC stimulation. When Figure 6B is compared with Figure 5D, the delayed increase in [Ca2+]i in ECs when BAPTA-loaded VSMCs were stimulated is not present. This may indicate that Ca2+ ions are responsible for the initial increase in [Ca2+]i in ECs, whereas IP3 is a secondary component that can sustain the increase in EC [Ca2+]i.
It should also be noted that IP3-Rs are also considered Ca2+-dependant Ca2+ channels,35 and thus BAPTA treatment may have altered the IP3-dependant Ca2+ release. This seems unlikely, however, because the BAPTA was not loaded into the cell type in which we observed changes in IP3-R effects and our data indicate that within our time frame of use, BAPTA was confined to specific cell types (Figure 5B and 5C). It is also possible that the buffering of Ca2+ with BAPTA had an effect on phospholipase C
that would limit IP3 production.13 In our system, the increase in EC [Ca2+]i after stimulation of BAPTA-loaded VSMCs has a time course similar to that seen in vivo,19 and the response could be eliminated with an IP3-R inhibitor in the ECs or depletion of ER calcium pools (Figure 7B and 7C). Our data thus strongly support the contention that IP3 and Ca2+ from VSMCs elicit an increase in EC [Ca2+]i.
In control conditions, we cannot exclude the possibility that IP3 and Ca2+ act in parallel. The mouse IP3-R1 has been shown to have Ca2+ binding sites, and IP3-R1 has been hypothesized to be close to the EC side of the MEJ.32 It is therefore plausible that cooperation of the two second messengers may be important for the EC response after PE stimulation of VSMCs.
We hypothesize that movement of IP3 and Ca2+ from VSMCs to ECs provides a means by which to ensure NO activation17,34,36 or modulate changes in EC permeability.37 Evidence for this concept is found in supplemental Figure IV, where the EC response to VSMC stimulation is significantly faster than the VSMC response following EC stimulation. In addition, as demonstrated in supplementary Figure V, the increase in EC [Ca2+]i after VSMC stimulation is longer than the increase in VSMC [Ca2+]i after EC stimulation. Therefore, the increase in EC [Ca2+]i is not only sustained but is more rapidly induced, possibly for activation of these physiological processes.
Calcium Signaling From ECs to VSMCs
There is strong evidence that EC–VSMC communication used gap junctions at the MEJ, although the nature of the signal remains to be defined.7,38,39 Our work demonstrates that Ca2+ ions can diffuse through the MEJ from ECs to the VSMCs following an increase in EC [Ca2+]i. This is shown by the fact that the secondary increase in [Ca2+]i in VSMCs is completely eliminated by BAPTA-loading of the ECs (Figure 5A). We hypothesize that Ca2+ ions entering the VSMCs activate RyR on the ER, which is found within the MEJ, thereby inducing an increase in [Ca2+]i. Activation of RyR on ER has been shown to induce Ca2+ sparks in VSMCs,40 and Ca2+ ions could potentially activate Ca2+-activated intermediate K+ channels found at the MEJ,41 in both cases inducing vasodilation. Taken together, it is clear that Ca2+ ions entering the VSMCs from ECs could have numerous physiological consequences.
Our experiments differ in the description of coordinated Ca2+ changes in VSMCs and ECs from reports in vivo, in which agonist stimulation of ECs is accompanied by a decrease in VSMC [Ca2+]i. A possible explanation for this difference is that expression of L-type and T-type calcium channels is reduced in cultured VSMCs,42–44 and thus active Ca2+ responses in the VCCC would be diminished, thereby unmasking the important means of communication that we report here. We have confirmed these observations by growing VSMCs in low serum and monitoring a response by the cells to K+, indicating the presence of voltage-gated Ca2+ channels (data not shown). Future experiments in vivo and in the VCCC in which the VSMCs are maintained in low serum will help to clarify this additional component to the interactions between the Ca2+ pools in the 2 cell types.
Different Second Messengers
Using murine vascular cells, we have previously demonstrated that the gap junctions at the MEJ in the VCCC are Cx40/Cx43 heterotypic.9 The gap junction composition at the MEJ from in vivo murine vessels is unknown. Using a rat model, other labs have come to a similar conclusion concerning a heterotypic gap junction, albeit a gap junction composed of Cx37 and Cx4041 at the MEJ, whereas others have suggested that only Cx40 may be present.45 It is also possible that because of rapid turnover rate of connexins, the gap junction composition at the MEJ is plastic and may be responsive to different physiological conditions or stimuli (e.g.,46). Clearly, the expression of connexins at the MEJ, as well as the vasculature as a whole, remains incomplete.6 This is important to understand as the connexin composition of the gap junctions confers unique permeability properties including differences in second messenger, dye, and electrical coupling.11,47 It is thus conceivable that differential second messenger signaling described herein is the result of the particular connexin composition of the gap junctions formed at the MEJ.
However, other possibilities exist, including qualitative or quantitative differences in the production of second messengers or signaling microdomains on either side of the MEJ. It is unlikely that there is a different second messenger involved following agonist stimulation of the 2 cell types, as both agonists are reported to act through similar G protein–coupled pathways.21,22 We cannot exclude the possibility that different concentrations of IP3 are generated by the 2 cell types; however, the Fmax percentage responses after direct stimulation appear to be similar in both cell types (Figure 2A and 2B). In terms of signaling microdomains, we have demonstrated the presence of ER at the MEJ in vivo and in the VCCC (Figure 3), which is a prerequisite for expression of IP3-R. Further examination of the receptor expression on the sides of the MEJ is an exciting prospect for future research.
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Conclusion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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Using the VCCC as a means to study heterocellular signaling, we have demonstrated that stimulation of either ECs with ATP or VSMCs with PE induces a secondary increase in [Ca2+]i in the unstimulated cell type and that this heterocellular Ca2+ signaling is mediated via gap junctions. Stimulation of VSMCs is followed by a Ca2+- and IP3-mediated response in the ECs, whereas the secondary response of the VSMCs following EC stimulation requires only Ca2+. These findings bear important implications for understanding how ECs and VSMCs coordinate increases in [Ca2+]i, across cell types, and demand future research into the organization of signaling proteins at the MEJ.
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Acknowledgments |
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We thank A. V. Somlyo, S. Boitano, A. S. Stevenson, and X. F. Figueroa for critical review of the manuscript; M. Koval, D. Locke, I. Rubio-Gayosso, G. J. Seedorf, K. H. Day, and S. N. Thornton for insightful comments; the University of Virginia Research Histology Core; the Robert M. Berne Cardiovascular Research Center for use of the confocal microscope; and the University of Virginia Department of Pathology for use of the ultramicrotome and electron microscope.
Sources of Funding
Supported by American Heart Association Beginning Grant-in-Aid 0565319U (to B.E.I.), a Robert M. Berne Cardiovascular Research Center Partners Fund grant (to B.E.I.), and Public Health Service grant HL53318 (to B.R.D.).
Disclosures
None.
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Footnotes |
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Original received August 20, 2006; resubmission received October 16, 2006; revised resubmission received December 19, 2006; accepted January 2, 2007.
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References |
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