3664
Research Article
Localized expression of an Ins(1,4,5)P3 receptor at the
myoendothelial junction selectively regulates
heterocellular Ca2+ communication
Brant E. Isakson
Robert M. Berne Cardiovascular Research Center, and Department of Molecular Physiology and Biological Physics, University of Virginia School
of Medicine, P.O. Box 801394, Charlottesville, VA 22908, USA
e-mail: [email protected]
Journal of Cell Science
Accepted 6 August 2008
Journal of Cell Science 121, 3664-3673 Published by The Company of Biologists 2008
doi:10.1242/jcs.037481
Summary
Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] originating in the
vascular smooth-muscle cells (VSMCs) has been shown to
modulate the Ca2+ stores in endothelial cells (ECs). However,
the reverse is not found, suggesting that Ins(1,4,5)P3 movement
might be unidirectional across gap junctions at the
myoendothelial junction (MEJ), or that distribution of the
Ins(1,4,5)P3 receptor [Ins(1,4,5)P3-R] is different between
the two cell types. To study trans-junctional communication at
the MEJ, we used a vascular-cell co-culture model system and
selectively modified the connexin composition in gap junctions
in the two cell types. We found no correlation between
modification of connexin expression and Ins(1,4,5)P3 signaling
between ECs and VSMCs. We next explored the distribution
of Ins(1,4,5)P3-R isoforms in the two cell types and found that
Ins(1,4,5)P3-R1 was selectively localized to the EC side of the
MEJ. Using siRNA, selective knockdown of Ins(1,4,5)P3-R1 in
ECs eliminated the secondary Ins(1,4,5)P3-induced response in
these cells. By contrast, siRNA knockdown of Ins(1,4,5)P3-R2
or Ins(1,4,5)P3-R3 in ECs did not alter the EC response to
VSMC stimulation. The addition of 5-phosphatase inhibitor
Introduction
The functional integration of endothelial cells (ECs) and vascular
smooth-muscle cells (VSMCs) within arterioles is important for
several physiological processes, including the control of blood flow
and response to vascular wounding (Figueroa et al., 2004; Figueroa
et al., 2006; Michel et al., 1995). The integration of ECs and VSMCs
can occur via the release of paracrine factors [e.g. nitric oxide (NO)]
or through direct cell-cell contact via gap junctions at sites termed
myoendothelial junctions (MEJs) (e.g. Sandow and Hill, 2000).
Gap junctions are dodecameric channels composed of two
hexameric hemichannels that allow movement of current and
solutes (<1000 Da) directly from the cytoplasm of one cell to the
cytoplasm of an adjacent cell (Figueroa et al., 2004). Connexin (Cx)
proteins compose the hemichannels and are found in over 20
different isoforms, each conferring electrical or solute selectivity
on the hemichannel or gap junction (Saez et al., 2003). Although
hemichannels composed of a single connexin isoform (homomeric)
have been studied extensively, the mixing of connexin isoforms into
hemichannels (heteromeric) is probably a common occurrence
because cells express multiple connexin isoforms (Koval, 2006).
The mixing of connexin isoforms in hemichannels and in gap
(5-PI) to ECs that were transfected with Ins(1,4,5)P3-R1 siRNA
rescued the Ins(1,4,5)P3 response, indicating that metabolic
degradation of Ins(1,4,5)P3 is an important part of EC-VSMC
coupling. To test this concept, VSMCs were loaded with 5-PI
and BAPTA-loaded ECs were stimulated, inducing an
Ins(1,4,5)P3-mediated response in VSMCs; this indicated that
Ins(1,4,5)P3 is bidirectional across the gap junction at the MEJ.
Therefore, localization of Ins(1,4,5)P3-R1 on the EC side of the
MEJ allows the ECs to respond to Ins(1,4,5)P3 from VSMCs,
whereas Ins(1,4,5)P3 moving from ECs to VSMCs is probably
metabolized before binding to a receptor. This data implicates
the MEJ as being a unique cell-signaling domain in the
vasculature.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/121/21/3664/DC1
Key words: Inositol (1,4,5)-trisphosphate, Calcium, Connexin,
5-phosphatase, Endothelium, Smooth muscle
junctions probably has important implications regarding the
regulation of solute movement (Locke et al., 2004), which might
be especially important in MEJs, where the presence of multiple
connexin isoforms might lead to the formation of heterotypic gap
junctions. For example, in rat mesenteric arteries, both Cx37 (also
known as Cxa4 and Gja4) and Cx40 (also known as Cxa5 and Gja5)
have been demonstrated to be present between ECs and VSMCs,
and, in mouse cremaster arterioles, Cx40 and Cx43 (also known as
Cxa1 and Gja1) predominate (Isakson et al., 2008). Because certain
heterotypic gap junctions have been shown to be electrically
rectifiable (Kreuzberg et al., 2005) and some heteromeric
hemichannels have been shown to be less permeable than
homomeric channels to the second messenger inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] (Ayad et al., 2006; Locke et al., 2004),
it is possible that heterotypic gap junctions at the MEJ are capable
of selectively regulating the movement of solutes.
Several laboratories have now demonstrated that stimulation of
VSMCs with phenylephrine (PE) via α1D-adrenoceptors causes an
increase in VSMC intracellular Ca2+ concentration ([Ca2+]i), with
a subsequent increase in EC [Ca2+]i (Dora et al., 1997; Isakson et
al., 2007; Jackson et al., 2008; Kansui et al., 2008; Lamboley et
Journal of Cell Science
Expression and function of Ins(1,4,5)P3-R at the MEJ
al., 2005). This intercellular Ca2+ communication is probably due
to the movement of Ins(1,4,5)P3 from VSMCs, through gap
junctions at the MEJ, to ECs (Isakson et al., 2007; Kansui et al.,
2008; Lamboley et al., 2005). However, stimulation of ECs with
agonists that are known to generate Ins(1,4,5)P3 produces no
observable increase in Ins(1,4,5)P3 within VSMCs (e.g. de Wit et
al., 2006), suggesting unidirectional movement of Ins(1,4,5)P3
through the gap junctions at the MEJ. An attractive hypothesis is
that gap junctions at the MEJ provide directionality for the
movement of Ins(1,4,5)P3.
The half-life of Ins(1,4,5)P3 is approximately 60 seconds (Sims
and Allbritton, 1998) before being metabolized [e.g. by type-I 5phosphatases (Gaspers and Thomas, 2005; Rottingen and Iversen,
2000; Safrany et al., 1994; Speed et al., 1999)]. However, numerous
reports have demonstrated that Ins(1,4,5)P3 is sufficiently stable to
survive movement through gap junctions and thus increase [Ca2+]i
in neighboring cells (e.g. Boitano et al., 1992; Carter et al., 1996).
The secondary response to Ins(1,4,5)P3 in neighboring cells is
dependant on Ins(1,4,5)P3 binding to Ins(1,4,5)P3-receptors
[Ins(1,4,5)P3-Rs] on the endoplasmic reticulum (ER) of the
neighboring cell and a subsequent release of Ca2+. Three isoforms
of Ins(1,4,5)P3-R exist [Ins(1,4,5)P3-R1 (also known as ITPR1),
Ins(1,4,5)P3-R2 (also known as ITPR2) and Ins(1,4,5)P3-R3 (also
known as ITPR3)], each with distinct Ca2+-release dynamics (Patel
et al., 1999) and isoform localization within cellular domains
(Colosetti et al., 2003). We previously demonstrated that ER
extends into the MEJ both in vivo and in vitro (Isakson et al., 2007),
so it is possible that Ins(1,4,5)P3-Rs reside close to the gap
junctions at the EC-VSMC interface, providing a pool of releasable
Ca2+ for Ins(1,4,5)P3 moving from VSMCs to ECs through gap
junctions at the MEJ.
In this paper, we test two hypotheses: (1) that gap junctions
impose directionality on Ins(1,4,5)P3 and (2) localization of
Ins(1,4,5)P3-Rs at the MEJ is responsible for the observed
unidirectional Ins(1,4,5)P3 response. We test these hypotheses
using a vascular-cell co-culture (VCCC) for Ca2+ measurements in
conjunction with selective application of short interference
(si)RNAs. Our results demonstrate a novel heterocellular signaling
mechanism implicating the MEJ as a unique signaling domain in
the vasculature.
Results
Cremasteric vascular-cell co-culture
Phenotypically distinct cremasteric EC and VSMC (supplementary
material Figs S1 and S2) were assembled in a VCCC (Isakson and
Duling, 2005) with ECs retaining a microvasculature bio-marker
(supplementary material Fig. S3) (King et al., 2004). Phenotypic
confirmation demonstrated an EC monolayer on the top of the
Transwell (Fig. 1A) and a VSMC monolayer on the bottom of the
Transwell (Fig. 1B), with each cell type growing F-actin cellular
extensions to create in vitro MEJs. This therefore represents a similar
cell-type configuration to an arteriole, in which MEJs are numerous.
Altering connexins at the MEJ does not alter intercellular Ca2+
communication
Using the cremasteric VCCC, we examined connexin expression.
Neither Cx37 or Cx45 were detected at the in vitro MEJ, whereas
Cx40 and Cx43 were found in the cellular extensions on the EC
side of the MEJ and Cx43 was found in the cellular extensions on
the VSMC side of the MEJ (Fig. 1C-F); this arrangement
corresponded to mouse cremasteric arterioles in vivo (Isakson et
3665
al., 2008). We next examined Cx40 and Cx43 in the cremasteric
VCCC in control conditions, in conditions in which ECs had Cx43
siRNA applied and in conditions in which ECs lacked Cx40 and
could not demonstrate any re-arrangement of the connexins (Fig.
1G). To determine whether these different connexin organizations
resulted in selective gap junctions at the MEJ, we examined Cy3
coupling from ECs to VSMCs and found that, in controls or
conditions in which Cx43 siRNA was applied to ECs, heterotypic
gap junctions organized at the MEJ. This was indicated by the
presence of biocytin, but not Cy3 dye transfer. By contrast, when
Cx40 was deleted from the ECs, Cx43 homotypic gap junctions
organized at the MEJ and allowed Cy3 movement to the VSMCs
(supplementary material Fig. S4) (e.g. Cottrell et al., 2002; Isakson
and Duling, 2005).
On the basis of the selectivity of the gap junctions, we examined
whether this could affect Ca2+ signaling. In control conditions,
cremasteric ECs and VSMCs were able to communicate an increase
in [Ca2+]i to unstimulated cells (Fig. 2A,B). When the VCCCs were
plated with Cx40–/– ECs, stimulation of ECs or VSMCs did not
result in any changes in intercellular Ca2+ communication when
compared with controls (Fig. 2C,D). Furthermore, there were no
changes in EC Cx43 knockdown when compared with controls (Fig.
2E,F). The only time that intercellular Ca2+ communication was
altered was when connexins composing the gap junctions at the
MEJ were eliminated: Cx40 and Cx43 in ECs (Fig. 2G,H) or Cx43
in VSMCs (Fig. 2I,J). These results are consistent with the inhibition
of intercellular Ca2+ communication when 18 α-glycyrrhetinic acid
(18 α-GA) was added to the VCCC (Fig. 2K,L). On the basis of
these observations, altering connexin composition of the gap
junctions at the MEJ does not alter heterocellular Ca2+
communication.
Because Ins(1,4,5)P3 has been shown to act unidirectionally from
VSMCs to ECs in vivo and in our culture model (Isakson et al.,
2007; Kansui et al., 2008; Lamboley et al., 2005), we surmised that
it was possible that gross changes in intercellular Ca2+
communication mask differences in second-messenger movement
when the connexin composition at the MEJ is altered. We therefore
loaded ECs with BAPTA and stimulated them with ATP so that
only the Ins(1,4,5)P3 response in VSMCs would be visible if it was
present (Isakson et al., 2007). Regardless of the connexin
composition at the MEJ, there were no conditions that could
demonstrate Ins(1,4,5)P3 movement from ECs to VSMCs (Fig. 3AC). Next, we loaded VSMCs with BAPTA, stimulated them with
PE and found that no changes in the connexin composition at the
MEJ could alter the Ins(1,4,5)P3-induced increase in [Ca2+]i in ECs
(Fig. 3D-F), except when the Ins(1,4,5)P3-Rs in the ECs were
blocked with Xestospongin C (XPC) (Fig. 3G-I). Thus, altering
connexin composition of the gap junctions at the MEJ does not
alter the apparent unidirectional movement of Ins(1,4,5)P3 from
VSMCs to ECs.
Ins(1,4,5)P3-R localization at the MEJ
An alternative hypothesis to gap junctions imposing directionality
on Ins(1,4,5)P3 signaling is selective localization of Ins(1,4,5)P3Rs on the MEJ. Using immunocytochemistry and immunoblots, we
probed for each Ins(1,4,5)P3-R isoform on transverse sections of
mouse cremaster arterioles and from freshly isolated cremasteric
ECs and VSMCs, and found expression of each isoform within both
ECs and VSMCs (Fig. 4A-F). However, only Ins(1,4,5)P3-R1 was
apparent on actin bridges between ECs and VSMCs (Fig. 4G). This
was confirmed in transmission electron microscopy (TEM) sections
Journal of Cell Science
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Journal of Cell Science 121 (21)
Fig. 1. Phenotypic verification and connexin expression in cremasteric vascular-cell co-culture. En face staining for acetylated low-density lipoprotein (LDL)
demonstrated ECs on the top of the Transwell insert, with no observed staining for α-actin (A). (A) Transverse sections of the VCCC (top is ECs, bottom is
VSMCs) double stained with phalloidin (F-actin, green) and VE-cadherin (red) demonstrated distinct EC localization in the pores of the Transwell. In the adjacent
immunoblot, CD31 stained only the EC isolates. (B) When examining VSMCs en face, acetylated LDL was not detected; however, α-actin was found throughout
the monolayers. Transverse sections of VCCC revealed VSMC extensions, which stained with desmin (red), following the F-actin (green) within the pores of the
Transwell. The immunoblot, which was probed for desmin, demonstrates that only VSMC isolates express this phenotypic marker. (C-F) In transverse sections of
the cremasteric VCCCs, Cx37 (C), Cx40 (D), Cx43 (E) and Cx45 (F) were observed. Arrows demonstrate connexin expression within the pores of the Transwell.
(G) Single pores of the Transwell were double stained for both Cx40 (red) and Cx43 (green) to demonstrate a lack of re-arrangement of connexins at the in vitro
MEJ when Cx43 or Cx40 was eliminated. Scale bars: 75 μm (en face view in A,B); 10 μm (transverse view from A,B; C-F); 2.5 μm (G).
probed for Ins(1,4,5)P3-R1 (Fig. 4H). Interestingly, Ins(1,4,5)P3R1 appeared to be selectively localized to the EC side of the MEJ
(Fig. 4H).
Because of the localization of Ins(1,4,5)P3-R1 at the MEJ in vivo,
we attempted to remove the receptor from the ECs in the VCCC
by applying Ins(1,4,5)P3-R1 siRNA to the EC monolayer. The
siRNA did not alter Ins(1,4,5)P3-R1 expression in VSMCs, nor
expression of the other Ins(1,4,5)P3-R isoforms in either cell type
(Fig. 5A,B). Using immunocytochemistry, we visualized
Ins(1,4,5)P3-R1 in control conditions or in conditions in which the
Ins(1,4,5)P3-R1 siRNA was applied to the ECs, and demonstrated
selective knockdown of the receptor within the EC (Fig. 5C,D).
Although both Ins(1,4,5)P3-R2 and Ins(1,4,5)P3-R3 were in EC and
VSMC monolayers, neither isoform was detected within the EC or
VSMC cellular extensions at the MEJ (Fig. 5E,F). Similar to that
found in vivo, using the VCCC we localized Ins(1,4,5)P3-R1 to the
EC side of the MEJ; we were capable of selectively downregulating
Ins(1,4,5)P3-R1 expression.
Effect of Ins(1,4,5)P3-R localization on intercellular Ca2+
communication
To test whether Ins(1,4,5)P3-R1 localization influences the
directionality of Ins(1,4,5)P3 signaling from VSMCs to ECs, a
VCCC was prepared with BAPTA in VSMCs and Ins(1,4,5)P3-R1
siRNA in ECs. Under these conditions, stimulation of VSMCs was
unable to induce an increase in EC [Ca2+]i, demonstrating that
directionality of Ins(1,4,5)P3 had been lost (Fig. 6A). Knockdown
of Ins(1,4,5)P3-R2 or Ins(1,4,5)P3-R3 within ECs (Fig. 6B) did not
inhibit the Ins(1,4,5)P3-mediated signal (Fig. 6C,D). Therefore,
localization of Ins(1,4,5)P3-R1 at the MEJ might play a major role
in mediating the EC [Ca2+]i response after VSMC stimulation.
Based on the above observations, it was hypothesized that,
without Ins(1,4,5)P3-R1 on the EC side of the MEJ, the Ins(1,4,5)P3
must be metabolized, or it would be capable of activating
Ins(1,4,5)P3-R2 and/or Ins(1,4,5)P3-R3 in the EC monolayer. To
determine whether Ins(1,4,5)P3 is metabolized by 5-phosphatase
within the MEJ before it reaches the monolayer, ECs were preloaded with Ins(1,4,5)P3-R1 siRNA and 5-phosphatase inhibitor (5PI). When the BAPTA-loaded VSMCs were stimulated, an increase
in EC [Ca2+]i was evident (Fig. 7A), which was inhibited with XPC
(Fig. 7B). This demonstrated that degradation of Ins(1,4,5)P3 might
be an important part of the heterocellular signaling process.
If this is the case, we hypothesize that the lack of an identifiable
Ins(1,4,5)P3-R isoform on the VSMC side of the MEJ might be
preventing Ins(1,4,5)P3 from activating Ins(1,4,5)P3-R isoforms in
the VSMC. To test this idea, VSMCs were loaded with 5-PI, and
BAPTA-loaded EC were stimulated. In this instance, an increase
in VSMC [Ca2+]i was evident (Fig. 7C), which was inhibited by
XPC (Fig. 7D). Therefore, it appears that degradation of Ins(1,4,5)P3
in the VSMCs, after it moved through gap junctions at the MEJ
Expression and function of Ins(1,4,5)P3-R at the MEJ
3667
Journal of Cell Science
Fig. 2. Effects of different connexins on Ca2+ communication between ECs
and VSMCs. In the cartoon at the top of the figure, the pipette indicates the
stimulated cell type (ECs top, VSMCs bottom). In control conditions (A,B),
stimulation of ECs (A) resulted in an increase in EC [Ca2+]i and, after a short
delay, an increase in VSMC [Ca2+]i. (B) Stimulation of VSMCs resulted in an
increase in VSMC [Ca2+]i and, after a short delay, an increase in EC [Ca2+]i.
(C,D) When ECs from Cx40–/– animals were used, stimulation of ECs (C)
resulted in an increase in VSMC [Ca2+]i and stimulation of VSMCs (D) caused
an increase in EC [Ca2+]i. (E,F) ECs with Cx43 siRNA were also tested and
EC stimulation of these cells resulted in an increase in VSMC [Ca2+]i (E).
Stimulation of VSMCs increased EC [Ca2+]i (F). (G,H) When both Cx40 and
Cx43 were deleted from the ECs, stimulation of ECs resulted in an increase in
EC [Ca2+]i, but not VSMC [Ca2+]i (G), and stimulation of VSMCs caused only
an increase in VSMC [Ca2+]i, and not EC [Ca2+]i (H). (I,J) This observation
was also evident when Cx43 was deleted from VSMCs: after stimulation of
ECs, there was no increase in [Ca2+]i in VSMCs (I) and, after stimulation of
VSMCs, no increase in EC [Ca2+]i was observed (J). (K,L) Lastly, when the
gap-junction inhibitor 18 α-GA was used, stimulation of ECs was unable to
produce an increase in VSMC [Ca2+]i (K) and stimulation of VSMCs was
unable to produce an increase in EC [Ca2+]i (L). *P<0.05.
from ECs, might be the reason that no apparent Ins(1,4,5)P3-induced
increase in VSMC [Ca2+]i is observed after EC stimulation.
So as to better understand the localization of Ins(1,4,5)P3
degradation, we stained transverse sections of the VCCC for
Ins(1,4,5)P3-R1 and 5-phosphatase (Fig. 7E). Line scans through
the pores of the Transwell demonstrate 5-phosphatase found
through the pore, but to a lesser degree where the Ins(1,4,5)P3-R1
was localized on the EC side of the MEJ (Fig. 7F). When taken
together, these data indicate that Ins(1,4,5)P3 is bidirectional across
gap junctions at the MEJ, but cellular localization of the
Ins(1,4,5)P3-R on the EC side of the MEJ, and possibly increased
expression of 5-phosphatase on the VSMC side of the MEJ,
determines the heterocellular [Ca2+]i response.
Discussion
In this study, we put forth two hypotheses to explain the observation
that Ins(1,4,5)P3 can move across gap junctions at the MEJ from
VSMCs to ECs, but not from ECs to VSMCs: (1) that gap junctions
themselves impose directionality on Ins(1,4,5)P3 (e.g. the gap
junctions are rectifiable) and (2) that localization of an Ins(1,4,5)P3R at the MEJ is responsible for the bidirectional Ins(1,4,5)P3
response. Our data support the second hypothesis, that Ins(1,4,5)P3
is bidirectional through gap junctions at the MEJ and that the
directionality is probably due to the proximity of the Ins(1,4,5)P3R on the EC side of the MEJ. We came to this determination by
examining the localization of Ins(1,4,5)P3-R and the functional
effect of Ins(1,4,5)P3-R deletion on Ca2+ communication, as
compared with the functional effect of specific connexin-isoform
deletion or knockdown. Our experiments in relation to each
hypothesis are discussed below.
The role of connexins in facilitating Ins(1,4,5)P3 movement
across the MEJ
It is generally assumed that coordination of chemical signals
between ECs and VSMCs regulates arteriolar reactivity. Although
multiple paracrine factors (e.g. NO) certainly play a major part
in controlling arteriolar reactivity, solid evidence has emerged that
gap junctions linking the two cell types at the MEJ might also
have a role in the coordination of arteriolar activity [e.g. VSMCto-EC communication and EDHF (Haddock et al., 2006; Kansui
et al., 2008)]. Both Cx37 and Cx40 have been reported to be the
connexins composing the gap junctions at the MEJ in some
vascular beds (Sandow et al., 2006), and Cx40 and Cx43 in others
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Fig. 3. Directional Ins(1,4,5)P3 persists regardless of connexin deletion. In the cartoons on the left, the pipette indicates the stimulated cell type (EC top, VSMC
bottom), blue represents BAPTA and green represents XPC. In control conditions (A), stimulation of BAPTA-loaded ECs resulted in no increase in [Ca2+]i in either
cell type. There was also no increase in [Ca2+]i in either cell type when ECs with Cx43 siRNA that were loaded with BAPTA were stimulated (B), or when Cx40–/–
ECs were loaded with BAPTA and stimulated (C). However, after stimulation of VSMCs loaded with BAPTA in control conditions (D), ECs responded with an
increase in [Ca2+]i. When Cx43 siRNA was added to the ECs (E), an increase in EC [Ca2+]i was still observed after VSMC stimulation. When ECs from Cx40–/–
mice were used (F), an increase in EC [Ca2+]i was observed after VSMC stimulation. The addition of XPC to the ECs (G-I) eliminated the increase in EC [Ca2+]i
after VSMC stimulation in control conditions (G), when Cx43 siRNA was applied to ECs (H) or when Cx40–/– ECs were used (I). *P<0.05.
(Isakson et al., 2008), with the common link being Cx40 that is
almost exclusively derived from the ECs. Therefore, when Cx40–/–
mice exhibited hypertension (de Wit et al., 2003), and EC-specific
Tie-2/Cre Cx43–/– mice exhibited hypotension (Liao and Duling,
2000), an attractive hypothesis was that the observed alterations
in blood pressure were due to alterations in solute movement
across the gap junctions at the MEJ, causing impaired
communication between ECs and VSMCs. Because Ins(1,4,5)P3
has been demonstrated to move through gap junctions (Boitano
et al., 1992) and to move between VSMCs and ECs (Isakson et
al., 2007; Lamboley et al., 2005), we focused on this solute. Our
data demonstrate that the deletion of Cx40 from the ECs had no
effect on gross changes in intercellular Ca2+ communication or
on the movement of Ins(1,4,5)P3 from VSMCs to ECs (Figs 2
and 3). There was also no change when Cx43 was knocked down
from the ECs (Figs 2 and 3). We interpret this data to mean that
heterocellular communication between ECs and VSMCs is not
due to a rectifiable gap junction. Recent evidence now indicates
that Cx40 is also found between renin-secreting cells in the kidneys
and the Cx40 genetic deletion results in increased renin secretion
(Wagner et al., 2007). It is possible that this is an explanation for
the hypertension observed in Cx40–/– mice. Based on our evidence,
impaired EC and VSMC communication due to Cx40 or Cx43
deletion at the MEJ is not the cause for the observed changes in
blood pressure; however, impairments in homocellular EC or
VSMC communication after connexin deletion cannot be
discounted (e.g. Figueroa et al., 2003).
Although the data presented herein indicates that connexin
composition of the gap junction at the MEJ does not influence
second-messenger directionality, there are additional ways in which
connexins may regulate second-messenger movement. For example,
we cannot discount alterations in the rate of Ins(1,4,5)P3 or Ca2+
flux across the gap junctions that are outside of our temporal
detection limit, or other untested solutes that might be more heavily
Expression and function of Ins(1,4,5)P3-R at the MEJ
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Fig. 4. Ins(1,4,5)P3-R1 is selectively localized to the EC side of the myoendothelial junction in vivo. Immunocytochemistry on cremasteric muscles (A,C,E) and
immunoblots of freshly isolated cremasteric ECs and VSMCs (cre-EC, cre-VSMC; B,D,F) were stained with Ins(1,4,5)P3-R1 (A,B), Ins(1,4,5)P3-R2 (C,D) or
Ins(1,4,5)P3-R3 (E,F). (A,C,E) Red is the Ins(1,4,5)P3-R isoform and green is the autofluorescence of the internal elastic lamina. (G) Using protein quantification
of antibody detection on actin bridges from in situ cremasteric arterioles (e.g. Isakson et al., 2008), only the Ins(1,4,5)P3-R1 isoform was present in significant
quantities. *P<0.05. (H) At the TEM level, Ins(1,4,5)P3-R1 (10-nm gold particles) was localized to the EC side of the MEJ, but not the VSMC (insert of enlarged
MEJ; H). Scale bars: 20 μm (A,C,E); 2 μm (H), 1 μm (insert in H).
influenced by connexin composition (e.g. K+ or cAMP). In addition,
we have previously demonstrated in mouse cremaster arterioles that
Cx43 appears capable of being phosphorylated at the serine-368
residue (Cx43-S368) on actin bridges between ECs and VSMCs
[presumably the MEJ (Isakson et al., 2008)]. In the cremasteric
VCCC, there is no detectable Cx43-S368 phosphorylation (data not
shown), which is probably due to cell-culture conditions and so this
variable was not tested in the present study. Cx43-S368
phosphorylation appears to be capable of altering gap-junction
permeability, without closing the channel completely (Lampe et al.,
2000). It is therefore possible that, if connexin phosphorylation
Fig. 5. Selective knockdown of the Ins(1,4,5)P3-R1
isoform from the ECs by using the cremasteric VCCC.
(A) Immunoblots of ECs or VSMCs from the VCCC that
were stained for Ins(1,4,5)P3-R1, Ins(1,4,5)P3-R2 or
Ins(1,4,5)P3-R3. Representative β-actin staining from
stripped immunoblots is also shown. Experiments
included lanes loaded with samples from control
conditions, the addition of Ins(1,4,5)P3-R1 siRNA to ECs
only, or the addition of control siRNA (c-siRNA) to ECs
only. (B) Quantification of ratioed immunoblots. *P<0.05.
(C,D) Immunostain for Ins(1,4,5)P3-R1 on transverse
sections of cremasteric VCCC before (C) and after (D)
application of Ins(1,4,5)P3-R1 siRNA to the ECs; note the
sparse staining for Ins(1,4,5)P3-R1 in the ECs, but the
isoform is still observed in the VSMCs. Arrows
demonstrate Ins(1,4,5)P3-R1 expression within the pores
of the Transwell. (E,F) On the cremasteric VCCC,
Ins(1,4,5)P3-R2 and Ins(1,4,5)P3-R3 show expression
within the monolayers of the cells only. Scale bar: 10 μm.
occurs at the MEJ, another layer of control over second-messenger
movement could be present.
The role of Ins(1,4,5)P3-R in facilitating Ins(1,4,5)P3 movement
across the MEJ
A pool of releasable Ca2+ in the form of ER extends down into the
MEJ from the ECs (Isakson et al., 2007). In Fig. 4, we present
evidence that Ins(1,4,5)P3-R1 is on the EC side of the MEJ, both
by identification on actin bridges and by immunohistochemistry on
TEM sections. The distribution pattern of Ins(1,4,5)P3-Rs was
replicated in the VCCC. The polarized localization of Ins(1,4,5)P3-
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Journal of Cell Science 121 (21)
Journal of Cell Science
Fig. 6. Selective knockdown of the Ins(1,4,5)P3-R1
isoform eliminates the Ins(1,4,5)P3 response in ECs. In
the cartoons on the left, the pipette indicates the
stimulated cell type (EC top, VSMC bottom), blue is
BAPTA and ‘+’ indicates the cell type that is loaded with
siRNA. (A) When Ins(1,4,5)P3-R1 was selectively deleted
from the ECs, stimulation of BAPTA-loaded VSMCs no
longer elicited an increase in EC [Ca2+]i. (B) Knockdown
of Ins(1,4,5)P3-R2 and Ins(1,4,5)P3-R3 using siRNA is
demonstrated by the immunoblot, with β-actin loading
controls shown directly underneath. (C,D) When
Ins(1,4,5)P3-R2 was knocked down, a polarized increase
in EC [Ca2+]i was still present (C), and the deletion of
Ins(1,4,5)P3-R3 also failed to inhibit increases in EC
[Ca2+]i (D). *P<0.05.
Rs has also been demonstrated by Colosetti et al., who showed that
Ins(1,4,5)P3-R1–EGFP [and Ins(1,4,5)P3-R3–EGFP], but not
Ins(1,4,5)P3-R2–EGFP, localized to the tight-junction region in
MCDK cells when cellular polarity was achieved (Colosetti et al.,
2003). It is not clear exactly why only Ins(1,4,5)P3-R1 was found
at the MEJ, although it might be due to the differential properties
of each Ins(1,4,5)P3-R. For example, mice with Ins(1,4,5)P3-R1
deletion die of ataxia (Matsumoto and Nagata, 1999), whereas
Ins(1,4,5)P3-R2- and Ins(1,4,5)P3-R3-deleted mice do not exhibit
a vascular phenotype (Futatsugi et al., 2005). In addition, Ca2+
binding to Ins(1,4,5)P3-R1 has been shown to have a high affinity
and serves to control both the opening and closing of the channel
(Bosanac et al., 2004). It is possible then that the Ca2+ we previously
demonstrated to move from VSMCs to ECs serves to modulate the
Ins(1,4,5)P3-R (Isakson et al., 2007). It is clear that more research
into the biophysics of the Ins(1,4,5)P3-R at the MEJ is required.
Ins(1,4,5)P3 is known to be discreetly activated in signaling
domains within the cytoplasm and near the plasma membrane of
cells to control Ca2+ signaling events (Delmas et al., 2002; Delmas
and Brown, 2002). We expand on this by demonstrating functional
relevance of the polarized placement of Ins(1,4,5)P3-R1 at the MEJ.
Using Ins(1,4,5)P3-R1 siRNA, we knocked down the protein from
the MEJs of the ECs, which eliminated the VSMC-induced
Ins(1,4,5)P3 response in the ECs (Figs 5 and 6). However,
knockdown of Ins(1,4,5)P3-R2 or -R3 had no effect on Ca2+
communication from VSMCs to ECs (Fig. 6). In mesenteric
arteries, recent work has shown that an Ins(1,4,5)P3-mediated Ca2+
event originates from holes in the internal elastic lamina [presumably
MEJs (Kansui et al., 2008)], lending strong correlative evidence to
our hypothesis that selective localization of Ins(1,4,5)P3-R on the
EC side of the MEJ is responsible for initiating increases in [Ca2+]i
in ECs after VSMC stimulation.
The enzyme 5-phosphatase causes degradation of Ins(1,4,5)P3
to Ins(1,4,5)P2, significantly reducing its ability to bind to and
activate Ins(1,4,5)P3-Rs (Sims and Allbritton, 1998). The addition
of 5-PI to ECs with Ins(1,4,5)P3-R1 siRNA ‘rescued’ the Ca2+
response in ECs after VSMC stimulation (Fig. 7). Presumably, this
was accomplished by preventing Ins(1,4,5)P3 degradation in ECs
after crossing the gap junctions at the MEJ from VSMCs. The
Ins(1,4,5)P3 could then reach the EC monolayer and activate either
Ins(1,4,5)P3-R2 or Ins(1,4,5)P3-R3 (Fig. 7). This ‘rescue’ was
inhibited by XPC in ECs, indicating that it was due to activation
of the EC Ins(1,4,5)P3-Rs after VSMC stimulation. In control
conditions, VSMCs did not express Ins(1,4,5)P3-Rs at the MEJ
(Fig. 4). For this reason, we tested whether 5-PI could ‘rescue’ a
VSMC response after EC stimulation. As demonstrated in Fig. 7,
the addition of 5-PI enabled the VSMCs to respond to ECs with
an Ins(1,4,5)P3-mediated response (as noted by the inhibition of
the Ca2+ response with XPC). Based on these experiments, we
hypothesize that Ins(1,4,5)P3 is bidirectional across gap junctions
at the MEJ. This concept was strengthened by the apparent
overexpression of 5-phosphatase in the VSMC extensions as
compared with the EC extensions of the VCCC (Fig. 7), which
we interpreted as a method by which VSMCs ensure that
Ins(1,4,5)P3 is metabolized. Therefore, the location of the
Ins(1,4,5)P3-R on the EC side of the MEJ enables the EC to respond
to Ins(1,4,5)P3, whereas the VSMC, devoid of Ins(1,4,5)P3-Rs at
the MEJ, cannot.
Our results are not without concerns. For example, the length of
the cellular extensions composing the in vitro MEJ are greater than
that observed at the MEJ in vivo, which might have had a role in
exaggerating the responses that we observed. In addition, the
temporal limits by our microscope might have not been fast enough
to detect rapid Ca2+ signaling events in Figs 2 and 3 relating to the
alterations in connexin composition. Lastly, it is unclear why 5phosphatase appears to be slightly more expressed on the VSMC
side of the MEJ, which we predict would actually hinder
Ins(1,4,5)P3 moving from the VSMCs to ECs. This implies that
Ins(1,4,5)P3 in VSMCs should be activated near the MEJ to avoid
degradation. However, with the localization of the Ins(1,4,5)P3-R
and reduced 5-phosphatase on the EC side of the MEJ, this would
ensure that any Ins(1,4,5)P3 arising from the VSMCs through the
Expression and function of Ins(1,4,5)P3-R at the MEJ
3671
Journal of Cell Science
Fig. 7. Metabolic degradation of Ins(1,4,5)P3 determines
directional responses. In the cartoons, the pipette indicates the
stimulated cell type (EC top, VSMC bottom), blue is BAPTA,
yellow is 5-PI, orange is XPC with 5-PI and ‘+’ indicates the cell
type that is loaded with siRNA. (A,B) BAPTA-loaded VSMCs
were stimulated. When 5-PI was loaded into ECs treated with
Ins(1,4,5)P3-R1 siRNA, an increase in EC [Ca2+]i was observed
(A) and, when XPC was added to these cells, an increase in EC
[Ca2+]i was no longer present (B). (C,D) BAPTA-loaded ECs were
stimulated. With the addition of 5-PI to VSMCs, an increase in
VSMC [Ca2+]i was observed after EC stimulation (C) and, when
XPC was added to the VSMCs with 5-PI, the VSMC Ca2+
response to EC stimulation was abolished (D). *P<0.05. (E) In
transverse sections of the cremasteric VCCC, pores of the
Transwell demonstrate the presence of Ins(1,4,5)P3-R1 (green) and
5-phosphatase (red). The vertical white line indicates the location
of the line scan. (F) The line scans correspond to the fluorescent
intensity from each of the proteins tested in E. Scale bar: 10 μm.
gap junction at the MEJ would have the capability to
rapidly induce Ca2+ release, and so large amounts of
Ins(1,4,5)P3 are not necessarily required. Indeed, it is
possible that signaling from VSMCs to ECs is influenced
by the amount of Ins(1,4,5)P3 produced with different
concentrations of PE (e.g. Dora et al., 2008). The
increased 5-phosphatase levels and lack of Ins(1,4,5)P3R on the VSMC side of the MEJ would ensure that
Ins(1,4,5)P3 from ECs does not affect VSMCs. It is clear
that this metabolic aspect of the spatial heterocellular Ca2+
signaling will require more investigation.
Conclusion
Protein placement at the MEJ is not a random occurrence
(e.g. Dora et al., 2008; Isakson et al., 2007; Isakson and
Duling, 2005; Sandow et al., 2006). The data presented
herein demonstrate selective localization of and
functionally relevant Ins(1,4,5)P3-R1 on the EC side of
the MEJ, which might provide an explanation for the
unidirectional response seen with Ins(1,4,5)P3 between
ECs and VSMCs and provide a mechanism by which
heterocellular Ca2+ communication between the two cell
types occurs. Taken together, the functional data imply
that the MEJ is capable of being a unique cellular
signaling domain in the vasculature.
Materials and Methods
Mice
All C57/Bl6 mice (Taconic) or Cx40–/– mice were males between 6 to 10 weeks of
age and used according to the University of Virginia Animal Care and Use Committee
guidelines. Mice were euthanized with an intraperitoneal injection of 60-90 mg/kg
pentobarbital.
Cremasteric microvascular EC and VSMC isolation
For each isolation (at least three), ten cremaster muscles from five male mice were
rapidly removed and pooled in ice-cold recovery buffer (HBSS, 5% BSA, 1% sodium
pyruvate, 25 mM HEPES, 1% glutamine, 0.5 mM L-ascorbic acid and 0.1%
fungizone). The cremasters were moved to isolation buffer [85% Ca2+, magnesiumfree-HBSS, 15% DMEM, 15 μg/ml elastase (Sigma), 1.25 mg/ml collagenase VII
(Sigma), 5% BSA, 1% sodium pyruvate, 25 mM HEPES, 1% glutamine, 0.5 mM
L-ascorbic acid and 0.1% fungizone at 37°C] and vortexed before being put on a
rotator in a 37°C oven for 8 minutes. After 8 minutes, the cremaster samples were
removed and vortexed again for 30 seconds, then placed back in the oven. This
process was repeated a total of four times. After the last vortex, the samples were
rapidly strained over an 80-mesh sieve into an autoclaved beaker, rinsed with FBS
and centrifuged at 25 g for 5 minutes. Upon supernatant removal, recovery buffer
at 37°C was added back with 8⫻107 CD31-coated magnetic beads (Dynal,
Invitrogen) or NG2 (Murfee et al., 2005)-coated magnetic beads [made by incubating
1.34⫻108 anti-rabbit magnetic beads (Dynal) with 100 μl rabbit anti-NG2 (1 μg/μl;
Chemicon)]. The cells and the magnetic beads were rotated inside a 37°C incubator
for 30 minutes and separated by a magnet. The supernatant was removed and culture
medium (see below) was applied. This process yields approximately 1.1⫻106 cells
with the CD31-coated beads and 1.4⫻106 cells with the NG2-coated beads. Trypanblue exclusion assays were always greater than 90% or the isolated cells were
discarded. These results demonstrated a highly enriched population of rapidly isolated
cremasteric microvascular ECs and VSMCs (supplementary material Figs S1-S3;
Fig. 1).
Cremasteric microvascular EC and VSMC culture
ECs were grown in MCDB-131 (Invitrogen) supplemented with 20% FBS, 1%
glutamine (Invitrogen), 1% penicillin/streptomycin (Invitrogen), 1% sodium pyruvate
(Invitrogen), 40 μg/ml heparin and 20 μg/ml endothelial cell growth supplement;
VSMCs were grown in MEM supplemented with 10% FBS, 1% penicillin/
streptomycin, 1% glutamine and 1% non-essential amino acids.
Vascular-cell co-culture
The cremasteric ECs and VSMCs were assembled into a VCCC as originally described
(Isakson and Duling, 2005). VSMCs were plated at 1⫻105 on the bottom of each
3672
Journal of Cell Science 121 (21)
Table 1. siRNA of Ins(1,4,5)P3-R isoforms
Name
IP3-R1A
IP3-R1B
IP3-R1C
IP3-R2A
IP3-R2B
IP3-R2C
IP3-R3A
IP3-R3B
IP3-R3C
Sense (5⬘-3⬘)
Anti-sense (5⬘-3⬘)
GGGUCCUGGUUUUACAUUC
CCAUUCAAACAGCAUUCAU
GCUGGAAGAUAGUGCUUUU
GAAUCCUCCUGAAUCGGUA
GCUUAAUCCUGACUAUCGA
CAUCCGAACUUGUCAUCGA
CGUGAAUCACUGCUACGUA
GCAUGGAGCAGAUCGUGUU
GCAUAUGAAGAGCAACAAA
GAAUGUAAAACCAGGACCC
AUGAAUGCUGUUUGAAUGG
AAAAGCACUAUCUUCCAGC
UACCGAUUCAGGAGGAUUC
UCGAUAGUCAGGAUUAAGC
UCGAUGACAAGUUCGGAUG
UACGUAGCAGUGAUUCACG
AACACGAUCUGCUCCAUG
UUUGUUGCUCUUCAUAUGC
Transwell insert (polyester, 0.4-μm pore diameter; Corning) before ECs were plated
on the top of insert (1.5⫻105).
Journal of Cell Science
Calcium imaging
Either the ECs or the VSMCs were loaded with the acetomethoxyester form of Fluo4 (2.5 mM with 0.0025% pluronic F127 and 0.1% DMSO; all supplied by Invitrogen)
and the VCCC was mounted on an Olympus FV200 confocal microscope (for details,
see Isakson et al., 2007). The pixel intensity from each image was subtracted from
a background image of the Transwell insert devoid of cells after experiments were
finished (Fsub). Maximum fluorescence intensity (Fmax) of both cell types was
determined at the termination of each experiment by application of 10 mM ionomycin
and stimulation with 100 mM ATP (Dora and Duling, 1998). Relative [Ca2+]i values
are plotted as the % Fmax (Isakson et al., 2007). In all experiments, when stimulated
ECs were used, 35 mM ATP was applied (Beny, 2004; Isakson et al., 2007) and,
when stimulated VSMCs were used, 10 mM PE was applied (Isakson et al., 2007;
Langlands and Diamond, 1990). Statistics for determining significance between EC
and VSMC Ca2+ responses was at P<0.05 and determined by one-way ANOVA (Tukey
post-hoc test); error bars are ± s.e.
Application of 18 α-GA (35 mM; Sigma) was performed as previously described
(Isakson et al., 2007). The acetomethoxyester form of BAPTA [20 mM; Invitrogen
(Yashiro and Duling, 2000)] was loaded into cells following loading of Fluo-4.
Controls for BAPTA loading of a specific cell type were obtained by stimulating the
unloaded cell type (e.g. Isakson et al., 2007) (also data not shown). The cell-permeate
selective Ins(1,4,5)P3-R blocker XPC [20 μM; Sigma (Oka et al., 2002)] was added
to Fluo-4-loaded cells. Cells were loaded with 5-PI (Ki=4 μM; 500 μM dissolved in
0.5% DMSO; NMR indicated purity >95%, catalog number 524620, lot number
746693, Calbiochem) using a pinocytotic kit (Invitrogen) (e.g. Isakson and Duling,
2005) after loading with Fluo-4.
Short interference RNA
All siRNA was applied directly to either the ECs or VSMCs on the Transwell insert.
Negative control siRNA (25 nM) and Cx43 siRNA duplexes (15 nM for both duplex
sequences) have previously been verified (Isakson and Duling, 2005) and
demonstrated to have no effect on other connexins. We used three different
Ins(1,4,5)P3-R duplex sequences per isoform (Table 1), transfected simultaneously
(siLentFect, Bio-Rad) at 10 nM each. In each case, cells were used 48 hours after
transfection.
Antibodies
Griffonia simplicifolia conjugated to Alexa Fluor 594, Helix pomatia conjugated
to Alexa Fluor 594, acetylated low-density lipoprotein (LDL) conjugated to Alexa
Fluor 594, phalloidin conjugated to Alexa Fluor 488 and all secondary antibodies
were Alexa fluorphores obtained from Invitrogen. Desmin, Cx43, α-actin and βactin (all monoclonal) were from Sigma; VE-cadherin and type-I 5-phosphatase
were from Santa Cruz Biotechnologies; Cx40 and Cx37 were from ADI; Cx45 was
a kind gift from Thomas H. Steinberg (Lecanda et al., 1998). Two anti-Ins(1,4,5)P3R1 antibodies were used [NeuroMab facility (UC DavisNINDS/NIMH), and
Alomone] and anti-Ins(1,4,5)P3-R2 and anti-Ins(1,4,5)P3-R3 were obtained from
Millipore. Owing to the reported cross-reactivity of anti-Ins(1,4,5)P3-R antibodies,
the specificity of these antibodies was tested by siRNA knock down (Figs 5
and 6). Anti-rabbit and anti-mouse 12-nm gold beads were from Jackson
Laboratories.
Immunostaining
Immunohistochemistry was performed as previously described on the VCCC (Isakson
and Duling, 2005) and cremasteric arterioles (Isakson et al., 2008). Quantification
of protein on actin bridges in mouse cremaster arterioles was performed as previously
described (Isakson et al., 2008).
Electron microscopy
Mouse cremaster tissue was fixed with 4% paraformaldehyde and embedded in LR
White. Ultra-thin sections were washed with Tris-buffer, blocked with 1% ovalbumin
and viewed on a Zeiss 900 electron microscope.
Immunoblots
Immunoblots [lysate buffer: PBS with 1% SDS, 1% protease inhibitor cocktail
(Sigma), 50 mM NaF and 2 mM PMSF] were performed as previously described
(Isakson and Duling, 2005) and quantified as described (Olsen et al., 2005) using
ImageJ software (NIH) using at least three different preparations.
We thank Brian Duling for critical feedback; Scott R. Johnstone,
Angela Best, Katherine Heberlein and Michael Rizzo for evaluations
of the manuscript; and Susan Ramos for electron microscopy images.
This work is supported by NIH ROI HL088554 (B.E.I.) and an
American Heart Association Scientist Development Grant (B.E.I.).
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