Increase in Cx45 Gap Junction Channels in Cerebral
Smooth Muscle Cells from SHR
Xing Li, J. Marc Simard
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Abstract—We recently reported the novel finding of expression and function of connexin45 (Cx45) in cerebrovascular
smooth muscle cells. We examined the hypothesis that Cx45 is altered in hypertension. Immunoblots for Cx45 showed
a significant increase in Cx45 in cerebral arteries from adult spontaneously hypertensive rats (SHR) compared with adult
Wistar-Kyoto (WKY) rats, with no difference in aorta or femoral artery. Patch-clamp of cerebral smooth muscle cells
pairs from SHR versus WKY showed a significantly steeper voltage dependence of deactivation and partial block of
junctional currents by quinine and by a peptide that interferes with docking of Cx45, consistent with dominance of
functional Cx45 channels in SHR. We examined potential roles of blood pressure versus angiotensin in elevated Cx45
in SHR by measuring Cx45 protein in 4 groups: (1) long-term administration in Wistar rats of the nitric oxide synthase
inhibitor L-NAME; (2) long-term administration in SHR of the ACE inhibitor captopril; (3) long-term administration
in Wistar rats of angiotensin; and (4) exposure of basilar artery segments in organ culture to angiotensin. Blood pressure
was significantly elevated in groups 1 and 3 and was normal in group 2. In groups 1, 2, and 4, there was no significant
change in Cx45 protein. In group 3, there was a modest but insignificant increase in Cx45 protein but no change in
voltage dependence of deactivation of junctional currents. Overall, our data show increased Cx45 in SHR that is unlikely
to be due to either elevated blood pressure or to angiotensin. Relative dominance of Cx45 over Cx43 in cerebral vessels
may predispose SHR to ischemic stroke. (Hypertension. 2002;40:●●●-●●●.)
Key Words: hypertension, experimental 䡲 angiotensin II 䡲 ion channels 䡲 muscle, smooth vascular 䡲 stroke
G
amount of Cx43 mRNA is similar to that in control WistarKyoto (WKY) rats,11 whereas endothelium from tail arteries
of spontaneously hypertensive rats (SHR) shows a significant
decrease in Cx40, Cx43, and Cx37.12 Thus, to date, reports of
alterations of connexins in hypertension have been limited,
and specific findings have depended on the particular model
under study.
All of the work on connexins in hypertension has focused
on Cx43, Cx40, and Cx37, the three most widely recognized
connexins in blood vessels. Recently, we3 and others2,13
discovered that Cx45 is also a prominent member of the
connexin family in blood vessels. In the current study, we
sought to determine whether Cx45 might be altered in
hypertension. We studied vessels from normotensive rats and
from three models of hypertension, SHR, hypertension induced by long-term administration of NG-nitro-L-arginine
methyl ester (L-NAME), and hypertension induced by longterm administration of angiotensin-II (Ang), ie, Ang hypertensive rat (AHR). We compared expression of Cx45 and
Cx43 protein, and we measured electrophysiological properties in SMC pairs. Our data reveal that Cx45 expression was
significantly increased in cerebral but not in noncerebral
vessels from SHR and that this led to demonstrable alter-
ap junctions are formed from an assembly of transmembrane channels that are dodecamers of proteins of the
connexin (Cx) family. Depending on which specific connexins are involved, these channels may allow direct exchange of
ions or small signaling molecules between adjacent cells. For
the endothelial and smooth muscle cells (SMC) of the vessel
wall, gap junctions are formed from Cx37, Cx40, Cx43, and
Cx45, each of which have electrical and conductive properties that are different from one another.1–3 When compared
with Cx43, homotypic Cx45 channels exhibit a small conductance, low permeability to Ca2⫹, anions, and Lucifer
yellow and are likely to contribute much less to metabolic
coupling.4 – 6 Thus, the relative proportion of each connexinforming gap junction is important for determining functional
connectivity.
Surprisingly, little has been found of changes in connexins
associated with hypertension. In two models of acquired
hypertension, the DOCA-salt model and the 2-kidney, 1-clip
Goldblatt model, Cx43 protein and mRNA are increased in
aorta, whereas in hypertension induced by inhibition of nitric
oxide synthase, Cx43 is decreased in aorta.7–10 Findings in
genetic hypertension are more limited. In mesenteric arteries
from stroke-prone spontaneously hypertensive rats, the
Received June 7, 2002; first decision July 9, 2002; revision accepted October 4, 2002.
From the Departments of Neurosurgery (X.L., J.M.S.), Pathology (X.L., J.M.S.), and Physiology (J.M.S.), University of Maryland School of Medicine,
Baltimore, Md.
Correspondence to Dr J. Marc Simard, Department of Neurosurgery, University of Maryland School of Medicine, 22 South Greene St, Baltimore, MD
21201–1595. E-mail [email protected]
© 2002 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/01.HYP.0000041882.39865.A8
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Hypertension
December 2002
ations in properties of electrical coupling between SMC. Our
data also indicate that acquired hypertension, induced by
either L-NAME or Ang, did not lead to similar changes,
suggesting that neither elevated blood pressure nor Ang can
be directly implicated in the increase in Cx45 in SHR.
Methods
Hypertension Models
All studies were conducted with female rats. SHR and their genetic
controls, WKY rats, were obtained from Charles River Laboratory.
For L-NAME–induced hypertension and for AHR, Wistar rats (250
g) were implanted subcutaneously with an ALZET osmotic pump
(Durect Corporation) filled either with L-NAME (Sigma), delivered
at 200 mg/kg per day for 4 weeks,14 or with Ang (Sigma), delivered
at 265 ␮g/kg per day for 4 weeks.15 Anesthesia for survival surgery
was obtained by intraperitoneal administration of ketamine (60
mg/kg) plus zylazine (8 mg/kg). Blood pressure was monitored by
means of tail-cuff plethysmography.
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Smooth Muscle Cells
Basilar and posterior cerebral arteries were dissected from WKY rats
(16 to 20 weeks), SHR (16 to 20 weeks), Wistar rats (250 to 300 g),
and AHR (12 to 14 weeks). SMC pairs were isolated by enzymatic
digestion16 and used for electrophysiological experiments within 24
hours. In some preparations of SHR SMC, cerebral arteries were
isolated in the presence of anti-Cx45 peptide.3
Western Blots
Cerebral arteries were pooled from 3 to 6 age-matched rats in each
of the following 9 groups: young WKY rats (5 weeks); adult WKY
rats (18 weeks); young SHR (5 weeks); adult SHR (18 weeks); adult
SHR treated with captopril (10 mg/kg per day for 4 weeks, through
osmotic pump), which were normotensive; adult Wistar rats (12
weeks); adult AHR; adult Wistar rats with L-NAME–induced
hypertension; adult Wistar rats treated with captopril (10 mg/kg per
day for 4 weeks, through osmotic pump). For some experiments,
cerebral vessels were harvested from adult Wistar rats and segments
were incubated with Ang (0, 30 nmol/L, 300 nmol/L) at 37°C for 6
hours before further processing. Aortas and femoral arteries were
pooled from 4 adult WKY rats and SHR.
Tissues were homogenized in lysate buffer containing 10 mmol/L
Tris-HCl, 1 mmol/L EDTA, 1% Nonidet-P40, 250 mmol/L sucrose,
1% protease inhibitor cocktail (Sigma), 1% phosphatase inhibitor
cocktail I and II (Sigma), and 1% 2-mercaptoethanol, pH 6.8.
Subconfluent HeLa and F9 cells were lysed in RIPA buffer. Aliquots
(40 ␮g total protein) were separated on SDS-PAGE and transferred
to PVDF membranes. Cx43 was probed with mouse anti-Cx43
monoclonal antibody (1:1000, Chemicon); Cx45 was probed with
rabbit anti-Cx45 polyclonal antibody (1:5000, generously provided
by Dr T.H. Steinberg, Washington University, St Louis); ␤-actin was
probed with mouse anti–␤-actin monoclonal antibody (1:5000,
Sigma). Probes were labeled with horseradish peroxidase–labeled
species-appropriate IgG (1:1000 or 1:5000, Amersham) and were
detected by chemiluminescence (ECL, Amersham). Autoradiographs
were scanned and quantified by densitometry (Scion Image).
Immunofluorescence Labeling
and Electrophysiology
Immunolabeling of vessel segments and isolated cells as well as dual
perforated patch-clamp experiments were carried out as previously
described.3
Data Analysis
Data were fit to Boltzmann3 or gaussian functions by using Origin
7.0 (Microcal Software, Inc). Data are given as mean⫾SEM except
where otherwise indicated. Statistical significance was assessed by
using ANOVA, t test, or ␹2 analysis.
Figure 1. Western blots for Cx45 in cerebral and noncerebral
arteries from WKY rats and SHR. A, Western blots for Cx45 on
lysates from HeLa cells (negative control for Cx45), F9 cells
(positive control for Cx45), cerebral arteries (CA), femoral arteries (FA), and aortas of adult WKY and SHR. B, Densitometric
analyses giving the relative abundance of Cx45 protein, with
relative abundance being determined after normalization to
␤-actin; each bar represents mean⫾SEM of 2 to 3 experiments.
*P⬍0.05.
Results
Spontaneously Hypertensive Rats
Initial experiments were aimed at determining expression of
Cx45 protein in blood vessels from adult WKY and SHR. We
studied cerebral (basilar and posterior cerebral) arteries and
compared these to similarly sized femoral arteries as well as
aorta, with HeLa and F9 cells serving as negative and positive
controls,3 respectively. Cx45 was visibly more prominent in
cerebral vessels from SHR compared with WKY (Figure 1A).
After quantification by densitometry and normalization to
levels of ␤-actin, Cx45 protein was found to be significantly
elevated (by ANOVA, P⬍0.05) in cerebral vessels from SHR
compared with WKY rats (Figure 1B). By comparison, levels
of Cx45 were similar in aorta and femoral artery of SHR
compared with WKY (Figures 1A and 1B).
Immunofluorescence study of cerebral vessels indicated
abundant labeling for Cx45 in basilar and posterior cerebral
arteries from SHR (Figure 2A). Cx45 labeling was confined
to smooth muscle layers, with no labeling of endothelial or
adventitial layers. By comparison, Cx43 was present in
medial layers of SHR but was less prominent than Cx45, and
Cx43 was apparent in both endothelial and adventitial layers
(Figure 2B). Nuclear labeling with DAPI was used to confirm
the identity of cell layers (Figure 2C).
Double labeling of freshly dissociated pairs of SMC from
SHR revealed that Cx45 and Cx43 could be coexpressed in
the same cells, although in different patterns. Cx45 labeling
was arranged linearly along SMC membranes and was often
particularly prominent at cell-to-cell interfaces (Figure 2D).
Cx43 tended to be more punctate in appearance and was more
Li and Simard
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Figure 2. Immunofluorescence imaging for Cx45 and Cx43 in
intact vessels and isolated SMC from cerebral arteries of SHR.
A through C, Labeling for Cx45 (A), Cx43 (B), and DAPI (4⬘,6diamidino-2-phenylindole) (C) in a longitudinal section of posterior cerebral artery from SHR; lumen is arranged vertically near
middle. D through F, Labeling for Cx45 (D), Cx43 (E), and DAPI
(F) in a cell pair isolated from the basilar artery of SHR. Scale
bar, 10 ␮m.
diffusely distributed (Figure 2E). DAPI labeling of nuclei
confirmed the presence of two cells (Figure 2F).
In additional separate experiments, we confirmed increased
expression of Cx45 protein in cerebral vessels from SHR
compared with WKY (Figures 3A and 3B). Cx43 was also
present in tissue from cerebral vessels (Figure 3A), but no
significant difference in level of expression was found in
WKY versus SHR (Figure 3C).
We also examined cerebral vessels from 5-week-old WKY
versus SHR, with the latter not yet hypertensive. In both
groups, levels of Cx45 were comparable to adult SHR (Figure
3B). Similarly, in both groups, levels of Cx43 were similar to
those in adults (Figure 3C).
We next determined whether the increase in Cx45 protein
found in cerebral vessels from adult SHR would be reflected
in altered electrophysiological properties of gap junctions in
isolated SMC pairs. In Wistar rats, we previously found that
one third (10 of 26) of cell pairs exhibited junctional
conductances with rapid deactivation and steep voltagedependence consistent with dominance of Cx45 channels,
with the remaining pairs showing slower deactivation and
weaker voltage dependence consistent with dominance of
Cx43 or Cx40 channels.3 Here, we carried out a similar
analysis of junctional currents in WKY versus SHR. Original
records of junctional current obtained in a cell pair from SHR
Cx45 in Cerebral SMC
3
Figure 3. Western blots for Cx45 and Cx43 in cerebral arteries
from WKY rats and SHR. A, Western blots for Cx45, Cx43, and
␤-actin on lysates from HeLa cells (negative control for Cx45),
F9 cells (positive control for Cx45), heart (positive control for
Cx43), adult and 5-week-old WKY and SHR, and adult SHR
treated with captopril (CAPT), as indicated. B, Densitometric
analyses giving the relative abundance of Cx45. C, Cx43 protein
in cerebral arteries from the same groups, with relative abundance being determined after normalization to ␤-actin. Each bar
represents mean⫾SEM of 2 to 5 experiments. *P⬍0.05.
exhibiting rapid deactivation are shown in Figure 4A. At low
transjunctional potentials, little deactivation was apparent,
but as transjunctional potential increased beyond 10 mV,
strong, rapid deactivation was evident. A plot of the normalized steady-state (6-second) conductance, G⬘j-ss as a function
of junctional potential, Vj, is shown for this cell pair (Figure
4B). Similar data obtained from 31 and 46 cell pairs from
Figure 4. Gap junction currents in SMC pairs from cerebral vessels from SHR. A, Recording of typical gap junction current,
showing fast deactivation at ⫺10 mV⬎Vj⬎10 mV. B, Normalized
G⬘j-ss/Vj relation and Boltzmann fit of the gap junction current
shown in A, with Gmin⫽0.15, V1/2⫽⫺15.9 mV, and k⫽5.1 mV. C
and D, Normalized G⬘j-ss/Vj relation in 28 of 31 SMC pairs (data
from 3 pairs not shown) from 9 WKY rats (C) and 33 of 46 SMC
pairs (data from 13 pairs not shown) from 15 SHR (D).
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Figure 5. Effect of quinine and anti-Cx45 peptide on gap junctional conductance in SHR SMC. A, Reversible block of gap
junction current by quinine. B, Concentration-response relation
for block of gap junctional conductance by quinine at the concentrations indicated. C, Values of junctional conductance at
Vj⫽10 mV in 46 untreated SHR SMC pairs and 15 pairs in which
cerebral arteries were isolated in the presence of 500 ␮mol/L
anti-Cx45 peptide for 3 hours. *P⬍0.05. D, Normalized G⬘j-ss/Vj
relation in 12 of 15 SHR SMC pairs (data from 3 pairs not
shown) isolated with anti-Cx45 peptide.
WKY rats and from SHR, respectively, are plotted in Figures
4C and 4D, respectively. Although both groups contained
pairs with strong and weak voltage dependence of deactivation, pairs with strong voltage dependence of deactivation
dominated the SHR group. Overall, a significantly different
proportion (by ␹2, P⬍0.01) of pairs, 13 of 31 versus 33 of 46
from WKY versus SHR rats, respectively, showed sufficiently strong voltage dependence that 55% or less of the
steady-state current remained at ⫹30 mV.
Based on our previous work,3 we considered that the
strongly voltage-dependent deactivation evident in many cell
pairs from SHR was likely to be due to Cx45. To evaluate
this, we tested the effect of quinine, which has been shown to
partially but selectively block junctional currents caused by
Cx45.17 At concentrations of 0.3 mmol/L and 1 mmol/L,
quinine reversibly blocked up to half of the junctional
conductance in pairs from SHR exhibiting strong voltagedependent deactivation (Figures 5A and 5B). We also tested
the effect of an oligopeptide that selectively interferes with
docking of Cx45 hemichannels.3 Treatment with anti-Cx45
peptide significantly decreased (by t test, P⬍0.05) the macroscopic junctional conductance (Figure 5C) and led to
weaker voltage dependence of deactivation (Figure 5D). In
SHR pairs treated with anti-Cx45 peptide, only 5 of 15 pairs
showed sufficiently strong voltage dependence that 55% or
less of the steady-state current remained at ⫹30 mV, a
proportion that was significantly different (by ␹2, P⬍0.02)
from 33 of 46 in untreated pairs. Together, these data
showing strong voltage dependence of deactivation, combined with sensitivity to quinine and to anti-Cx45 peptide,
provided evidence that the Cx45 protein identified in Western
blots was forming functional gap junction channels in SMC
pairs from SHR.
L-NAME
The significant augmentation of Cx45 protein and of functional channels in cerebral SMC pairs from SHR raised the
possibility that elevated blood pressure could be responsible
for increasing Cx45. To assess this, we measured Cx45 after
long-term administration of the nitric oxide synthase inhibitor
L-NAME in Wistar rats.14 These animals responded to
long-term L-NAME administration by elevation of systolic
blood pressure (BP sys ) to values (mean⫾SD) of
237⫾6 mm Hg, compared with values of 141⫾12 mm Hg in
untreated animals. However, levels of Cx45 protein, as well
as those of Cx43 protein, were not appreciably changed in
cerebral vessels (Figures 6A through 6C), suggesting that
high blood pressure per se was not the cause of augmented
Cx45 in SHR.
Angiotensin
Hypertension in SHR may be treated successfully with the
use of Ang ACE inhibitors such as captopril,18 suggesting
Figure 6. Western blots for Cx45 and Cx43 in
cerebral arteries and gap junction currents in
SMC pairs from Wistar rats and AHR. A,
Western blots for Cx45, Cx43, and ␤-actin on
lysates from controls, Wistar rats, and AHR
and Wistar rats treated with L-NAME or captopril (CAPT), as indicated. B and C, Densitometric analyses giving the relative abundance
of Cx45 (B) and Cx43 (C) protein in cerebral
arteries from the same groups, with relative
abundance being determined after normalization to ␤-actin. Each bar represents
mean⫾SEM of 2 to 5 experiments. *P⫽0.05.
D and E, Normalized G⬘j-ss/Vj relation in 33 of
45 SMC pairs (data from 12 pairs not shown)
from 20 Wistar rats (D) and 25 of 47 SMC
pairs (data from 22 pairs not shown) from 21
AHR (E).
Li and Simard
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involvement of Ang in the pathophysiology of hypertension
in SHR. We thus sought to determine whether Ang could be
implicated in augmented Cx45 in cerebral vessels of SHR.
We examined Cx45 protein in two in vivo models relevant to
Ang: (1) in SHR after long-term administration of captopril;
and (2) in Wistar rats after long-term administration of
subpressor Ang (AHR).
In SHR treated with captopril, values (mean⫾SD) of BPsys
were reduced to 134⫾8 mm Hg, compared with values of
191⫾14 mm Hg in untreated SHR. However, levels of Cx45
protein, as well as those of Cx43 protein, were not significantly changed in cerebral vessels (Figures 3A through 3C).
Captopril significantly reduced (by ANOVA, P⫽0.05) Cx43
in cerebral vessels from Wistar rats (Figures 6A and 6C), but
this was not investigated further.
In AHR, values (mean⫾SD) of BPsys were elevated to
225⫾32 mm Hg, compared with values of 141⫾12 mm Hg in
untreated Wistar rats. In these animals, a modest increase in
both Cx45 and Cx43 was found in cerebral vessels, but these
increases were not statistically significant (by ANOVA,
P⫽0.09; Figures 6A through 6C).
To further examine the possible relation between Ang and
Cx45, we measured the electrophysiological properties of gap
junction channels in isolated SMC pairs. Data on the G⬘j-ss-Vj,
relation obtained from 45 and 47 cell pairs from Wistar rats
and from AHR, respectively, are plotted in Figures 6D and
6E, respectively. Both groups contained pairs with strong as
well as weak voltage dependence of deactivation. Overall, 15
of 45 versus 18 of 47 pairs from Wistar rats versus AHR,
respectively, showed sufficiently strong voltage dependence
that 55% or less of the steady-state current remained at ⫹30
mV. These proportions (15 of 45 and 18 of 47) were not
significantly different (by ␹2, P⬎0.05) and were similar to
our previously published observations in Wistar rats (10 of
26).3 Thus, despite the tendency to exhibit more Cx45 as well
as Cx43 protein in AHR, these changes were not reflected in
any apparent change in electrical connectivity between isolated cell pairs.
Given the suggestion above of an elevation in Cx45 in
AHR, we also measured Cx45 expression in isolated basilar
arteries exposed to Ang for 6 hours in vitro. In these
experiments, Ang did not appreciably alter Cx45 at concentrations up to 300 mmol/L (by ANOVA, P⬎0.05; not shown).
Strong Versus Weak Voltage Dependence
of Deactivation
The electrophysiological data presented above were obtained
from a total of 169 cell pairs. This large number of observations allowed us to assess the validity of a criterion we
previously established for assessing gap junctional connectivity in SMC pairs. In our previous report,3 we cited the
proportion of cell pairs “showing sufficiently strong voltage
dependence that half or more of the current is deactivated at
⫹30 mV.” This simple measure appeared to correlate well
with junctions dominated by Cx45 versus those dominated by
Cx43 and Cx40. Here, we compiled a frequency histogram of
the normalized steady-state junctional conductance at ⫹30
mV (G⬘j-30 mV) in the 169 pairs pooled from WKY, SHR,
Wistar rats, and AHR (Figure 7A). The data were distributed
Cx45 in Cerebral SMC
5
Figure 7. Distribution of normalized steady-state gap junctional
conductance at ⫹30 mV in 169 individual SMC pairs pooled
from WKY rats, SHR, Wistar rats, and AHR. A, Double-peak
gaussian fit of histogram (bin width, 0.05) gave values
(mean⫾SD) of 0.29⫾0.13 and 0.96⫾0.05. B, Distribution
according to strong (G⬘j-30 mV⬍0.55) and weak (G⬘j-30 mV⬎0.86)
voltage dependence of deactivation in the 4 groups indicated,
with ␹2 analysis indicating significantly different proportions
(P⬍0.01) for SHR only.
into two groups, and the histogram was well fit with the sum
of two gaussian functions, with values (mean⫾SD) of
0.29⫾0.13 and 0.96⫾0.05. Thus, a discriminating cutoff, the
mean plus 2 SD, would be 0.86 for the group with weak
voltage dependence and 0.55 for the group with strong
voltage dependence, with the latter agreeing with the value of
0.5 that we previously used. With the use of these criteria to
segregate observations into two groups, those with strong or
weak voltage dependence of deactivation, SHR was found to
have a significantly higher (by ␹2, P⬍0.01) proportion of
pairs exhibiting strong voltage dependence, compared with
WKY, Wistar, and AHR (Figure 7B).
Discussion
The principal finding of this study was that Cx45 was
markedly increased in cerebral but not noncerebral vessels
from SHR compared with WKY, resulting in a significant
alteration in electrical connectivity between cerebral SMC. In
cerebral vascular SMC from Wistar rats, we previously
showed coexistence of multiple functional connexin channels, including homotypic Cx43, Cx45, and Cx40 channels,
as well as nonhomotypic channels, which together lead to
complex connectivity between cells.3,16 Similarly, complex
connectivity caused by expression of the same connexins may
exist in vessels from SHR, but the relative dominance of
Cx45 in SHR resulted in a different electrophysiological
picture.
The signature finding of gap junctions dominated by Cx45
was strong voltage dependence of deactivation. We used the
electrophysiological criterion of 55% or less of the steadystate current remaining at ⫹30 mV to discriminate between
junctions dominated by Cx45 and those dominated by Cx43
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or Cx40. This approach was justified by our previous3 as well
as present experiments using anti-Cx45 peptide and quinine17
and was further substantiated by our observation of two
distinct populations of SMC pairs, based on an analysis of
recordings from 169 pairs. This electrophysiological characteristic appears to be a robust correlate of Cx45 dominance.
No alteration of any specific connexin has ever been linked
with hypertension. Expression of Cx43 may be either increased or decreased, depending on the specific model,7–10
suggesting that blood pressure per se is not a direct regulatory
factor for Cx43. Based on our findings in this study, the same
appears to be true for Cx45. For Cx43, expression is linked to
the phenotypic or proliferative status of SMC,19,20 but
whether this is true for Cx45 remains to be determined.
Our data indicating that Ang was not a regulator of Cx43
or Cx45 in SMC were surprising, given that Ang is known to
upregulate Cx43 synthesis in rat ventricular myocytes.21,22
Although mechanisms regulating Cx45 expression are not
well understood, this connexin is known to be commonly
downregulated after early development. In cardiac and motor
neurons, maturation is associated with reduced expression of
Cx45.23,24 In the vascular system, Cx45 expression is critical
during early development,13 but any decline in expression
after development had not been examined. Our data indicated
that Cx45 levels were elevated in cerebral vessels from both
WKY rats and SHR at 5 weeks and that levels diminished
with maturation in WKY rats but not SHR, suggesting that
elevated levels of Cx45 in adult SHR may represent failure of
normal developmental downregulation.
The relative dominance of Cx45 in SHR is expected to
influence the nature of the signals transmitted between cells.
Cx43 and Cx45 form junctional channels with different
molecular permeabilities, and thus the consequence of a shift
from Cx43 dominance to Cx45-dominance is likely to be
significant. Homotypic Cx43 channels are highly permeable
to cations and anions, including Ca2⫹ and the divalent anion
Lucifer yellow, consistent with an ability to mediate intercellular exchange of anionic molecules such as cAMP, cGMP,
and IP3.4,6 Conversely, homotypic Cx45 channels exhibit a
small conductance, low permeability to Ca2⫹, anions, and dye
molecules, and thus probably contribute much less to metabolic coupling.4 – 6
Whether the difference that we observed in Cx45 in SHR
versus WKY can account for any specific aspect of altered
function in cerebral vessels remains to be determined, but a
potentially relevant functional corollary may be the susceptibility of SHR to ischemic stroke.25,26 Patency and opening
of collateral vessels are crucial determinants of infarct
size.27,28 In SHR, the increase in susceptibility to infarction is
attributed to a lack of collateral flow and narrower anastomoses.29,30 Cx43 gap junctions, with their weak voltage
dependence and nondiscriminating permeability, are critical
for enhancing opening of collaterals, as shown by the fact that
Cx43-null mice exhibit an exaggerated susceptibility to focal
cerebral ischemia.31 Thus, Cx45 dominance, as in SHR, may
result in the functional equivalence of a deficiency in Cx43,
impairing the opening of collateral channels and thereby
worsening cerebral ischemia.
Perspective
In this study, we showed that cerebral vessels from SHR
express greater levels of functional Cx45 compared with
noncerebral vessels and compared with cerebral vessels from
their genetic controls, WKY rats. Moreover, our data indicate
that this is likely to be due to a genetic defect rather than to
hypertension per se. This is the first demonstration of a
difference in gap junction channel expression and function in
cerebral vessels from hypertensive animals. Given the known
differences in permeability between Cx45 and Cx43, we
speculate that this finding may be an important correlate of
the tendency of these animals to have greater damage from
cerebral ischemia.
Acknowledgments
This work was supported by grants (to J.M.S.) from the National
Heart, Lung, and Blood Institute (HL51932), the National Institute
for Neurological Diseases and Stroke (NS39956), and a Bugher
award from the American Heart Association.
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Xing Li and J. Marc Simard
Hypertension. published online November 4, 2002;
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