Intercellular communication in cultured human vascular smooth
muscle cells
Hong-Zhan
Wang1,
Nancy
Day1,
Mira
Valcic1,
Ken
Hsieh1,
Scott
Serels1,
Peter R.
Brink2, and
George J.
Christ1,3
Departments of 1 Urology and 3 Physiology and
Biophysics, Institute for Smooth Muscle Biology, Albert Einstein
College of Medicine, Bronx 10461; and 2 Department of Physiology
and Biophysics, State University of New York at Stony Brook,
Stony Brook, New York 11794
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ABSTRACT |
Intercellular communication through gap junction channels
plays a fundamental role in regulating vascular myocyte tone. We investigated gap junction channel expression and activity in myocytes from the physiologically distinct vasculature of the human internal mammary artery (IMA, conduit vessel) and saphenous vein (SV,
capacitance vessel). Northern and Western blots documented the presence
of connexin43 (Cx43) in frozen tissues and cultured cells from both vessels. Northern blots also confirmed the presence of Cx40 mRNA in
cultured IMA and SV myocytes. Dual whole cell patch-clamp experiments revealed that macroscopic junctional conductance was voltage dependent and characteristic of that observed for Cx43. In the majority of
records, in both vessels, single-channel activity was dominated by a
main-state conductance of 120 pS, with subconducting events comprising
less than 10% of the amplitude histograms. However, some records
showed "atypical" unitary events that had a conductance similar to
Cx40 (~140-160 pS), but gating behavior like that of Cx43. As
such, it is conceivable that the presence and coexpression of Cx40 and
Cx43 in IMA and SV myocytes may result in heteromeric channel
formation. Nonetheless, in terms of gating, Cx43-like behavior clearly dominates.
connexins; internal mammary artery; saphenous vein; connexin40; connexin43
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INTRODUCTION |
INTERCELLULAR
COMMUNICATION through gap junction channels plays an important
role in modulating vascular smooth muscle tone in blood vessels
throughout the vascular tree. Evidence for this supposition has been
catalogued, mostly in laboratory animals, by numerous investigators
using a variety of in vitro as well in vivo techniques (14,
21). In the smaller vessels, such as skeletal muscle resistance
arterioles, gap junctions are important to coordination of vasodilation
and constriction and thus to regional alterations (increases or
decreases, respectively) in blood flow (38). In irideal
arterioles, intercellular communication through gap junctions seems
essential to coordination of spontaneous rhythmic contractions of
myogenic origin (22). In medium-sized vessels such as the
coronary artery, an important role for gap junctions in the spread of
hyperpolarization among smooth muscle cells appears to be involved in
vascular relaxation responses (2). In still larger, more
muscular conduit arteries, such as the isolated superior mesenteric
artery, intercellular communication between vascular smooth muscle
cells via gap junctions is thought to be essential for synchronized
rhythmic activity (8). In addition, gap junctions may also
be important to tonic contractile responses in rabbit mesenteric artery
(9). In even larger elastic and muscular arteries such as
the rat aorta, an important role for gap junctions in modulating tonic
agonist-induced contractile responses is well documented (10, 11,
13).
Despite their widespread cardiovascular distribution, a role for gap
junction-mediated intercellular communication in modulating vascular
contractility seems most obvious in small- to medium-sized vessels with
rhythmic or dynamically modulated alterations in tone. However, the
relevance of intercellular communication to modulating vascular tone in
the larger elastic and muscular arteries, which are more tonically
contracted, should seem equally obvious. In this regard, it is quite
clear that diffusion distances, neurotransmitter volatility, tissue
tortuosity factors, and enzymatic degradation and tissue uptake
processes all seemingly converge to limit the effective diffusion
radius of neuronally and endothelially derived substances in the
vascular wall (13). That is, presumably there are local
restrictions to the direct actions on vascular myocytes of both
neurotransmitters released from varicosities at the adventitial-medial smooth muscle border, as well as endothelially derived relaxing and
contracting factors released from the luminal surface. As such, the
extant experimental evidence in these larger vessels also points toward
the importance of intercellular communication in coordinating vascular
smooth muscle tone across the vessel wall (14, 34). Not
surprisingly then, an important role for gap junctions in modulating
endothelium-dependent vasorelaxation appears to span the spectrum of
vessels from resistance arterioles (18, 35, 47) to
muscular arteries (17, 20, 23) to large elastic arteries
(24).
In light of the fact that cardiovascular disease is still a leading
cause of human morbidity and/or mortality, rigorous evaluation of
vascular regulatory mechanisms, such as intercellular communication, would seem critical to the improved understanding of human vascular physiology and/or disease. In this regard, with the possible exception of patch-clamp studies in cultured vascular myocytes of the human corpus cavernosum (14, 15, 41), there is still a relative dearth of information concerning the biophysical characteristics of
intercellular communication when the connexin channels are expressed in
their native human vascular tissue/cell type. Therefore, as a first
step toward an improved understanding of the role of intercellular
communication to human vascular physiology and function, we have begun
a detailed biophysical investigation of cultured myocytes derived from
surgical specimens (excess tissue from coronary artery bypass grafts)
of the internal mammary artery (IMA) and saphenous vein (SV).
Because the IMA is nominally a conduit/muscular artery, whereas the SV
is a capacitance vein, the selected vessels also provide an appropriate
physiological contrast for initial investigations into the potential
role of differential connexin expression, regulation or physiology, to
differential human vascular function. To this end, dual whole cell
patch-clamp (DWCP) studies were conducted on homogeneous explant
cultures of myocytes obtained from patients undergoing coronary artery
bypass surgery. Many similarities were observed between IMA and SV
myocytes in the voltage-dependent behavior of the recorded macroscopic
whole cell currents for Cx43, as well as in the distribution and
single-channel conductance of the majority of unitary events. However,
more careful inspection of unitary recordings in cell pairs from both
vessels also revealed evidence for the presence of unique or
"atypical" single-channel events with a conductance similar to Cx40
(i.e., ~140-160 pS) (1, 21) but gating behavior
more typical of Cx43 (5, 10, 30). Because coexpression of
Cx40 and Cx43 mRNA was verified in vitro (i.e., Northern blot analysis
in cell cultures) from both vessels, this observation raised the
possibility of heteromeric channel formation (i.e., more than one
connexin type present in a connexon or hemichannel) in these vessels
(4, 6, 25, 40). The apparent low frequency of occurrence
of these "hybrid/heteromeric" channels might be considered vestigial.
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MATERIALS AND METHODS |
Obtainment of surgical specimens.
Excised pieces of human IMA (5 patients: 3 male, 2 female) and SV (6 patients, all male) were obtained from a total of 11 patients,
according to an institutional review board protocol approved by the
Committee on Clinical Investigation of the Albert Einstein College of
Medicine/Montefiore Medical Center. All vascular tissue was
obtained from surplus vessels available after coronary artery bypass
surgery. The mean patient age was 67 ± 3 yr (range 52-79).
Cell preparation.
Homogeneous explant smooth muscle cell cultures were developed from
rings of IMA and SV by a procedure identical to that described previously for the preparation of human corporal vascular smooth muscle
cells (5, 10, 12, 31, 48). Briefly, sections of human IMA
and SV were placed in Dulbecco's medium (DME, GIBCO, Grand Island, NY)
containing antibiotics (100 U/ml penicillin and 100 µg/ml
streptomycin). Tissue was washed and cut into 1-2-mm pieces and
placed in tissue culture dishes with sufficient nutrient medium to
prevent drying. After the explants had attached to the substrate,
usually within 1-2 days, more culture medium was added. When the
cells had migrated from the explant and undergone division, they were
detached with 0.05% trypsin and 0.02% EDTA at 37°C for 5 min. Cells
were subsequently grown in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum, 2 mM glutamine, and antibiotics. Cellular homogeneity was verified by immunofluorescent staining with
monoclonal antibodies to human smooth muscle myosin. Only passages 1-5 were used for this study. Cultures were
prepared from a total of 11 patients, 9 male and 2 female, as described in Obtainment of surgical specimens.
Immunostaining.
Smooth muscle cells were grown on 10-mm coverslips in 100-mm petri
dishes for 1-2 days (i.e., until ~60-80% confluent). At this time the media was removed, and cells were washed three times with
PBS. Cells were permeabilized with 70% ethanol at
20°C for 10 min,
thoroughly washed with PBS, and then incubated for 10 min in PBS
containing 0.25% BSA before addition of the primary antibody. Cells
were then incubated at room temperature for 2 h in the dark. The
cells were subsequently washed in 1× PBS for 5-min intervals (a total
of 4 times). The coverslips were then placed face down on slides with
p-phenylenediamine-glycerol solution. Immunoreactivity was
examined on a fluorescence microscope. Note that prior control
experiments revealed no immunoreactivity in the absence of the primary
myosin antibody or in the presence of blocking peptide (data not shown).
Electrophysiological recording mode and solutions.
The standard DWCP technique was used. For all DWCP experiments, the
bathing solution was a cesium saline containing (in mM): 165 CsCl, 30 tetraethylammonium-Cl, 1 CoCl2, 1 NiCl2, 1 MgCl2, 2 CaCl2, 1 aminopyridine, 10 HEPES, and
0.5 ZnCl2 at a pH of 7.0. The pipette solution was
a cesium saline solution identical to the bathing solution, except that
the CaCl2 was reduced to 0.1 mM, 0.6 mM EGTA was added, and
ZnCl2 was removed (pH 7.0). The reason for using the cesium
solution for both the bath and pipette is to achieve maximum inhibition
of nonjunctional channel activity in the absence of lipophilic
uncoupling agents (5, 10). The voltage protocol was
generated by pCLAMP6 (Axon Instruments), and unless otherwise stated
the experimental protocol was as follows. Initially, both cells of the
pair were clamped at 0 mV, and then one cell remained clamped at 0 mV,
while the other cell was stepped to ± 100 mV in 10-mV increments.
The pulse duration was 2.5 s with 5-s intervals. For
single-channel events, the transjunctional voltage
(Vj) ranged from 20 to 60 mV with a step
duration of 30 s to 5 min. All current and voltage recordings were
stored as pCLAMP6 files and simultaneously stored on videotape with a
four-channel digitizing unit and videocassette recorder for off-line
analysis. Note that cell pairs with a conductance >20 nS or cell pairs
that lost their voltage dependence were excluded from this study.
Data analysis.
All analog signals were low-pass filtered (8-pole Bessel, LPF-30; WPI,
Sarasota, FL) at 100 Hz and digitized at 2 kHz using a DT2801A A/D
board (Data Translation, Marlboro, MA) installed in an IBM
personal computer/ AT clone. The dead time of the recording instrumentation was 1.8 ms. Off-line analysis of all digitized junctional current traces was performed with the DOSTAT and PATCH programs developed by Dr. S. V. Ramanan in the laboratory of Dr. P. R. Brink. The gaussian distributions present in the all-points current amplitude histogram were fitted with a probability density function (pdf) that assumes that multichannel records reflect the
activity of independent and identical channel types (32, 33, 44,
45). The solid line represents the best fit of the data,
assuming a two-state Boltzmann distribution of the form
where Gmax is the normalized maximum
conductance (=1), Gmin is the normalized minimum
conductance measured from Vj ± 100 mV,
V0 is the voltage where
Gss, steady-state junctional
conductance, lies halfway between Gmax
and Gmin, and A is a parameter
expressing the slope of the curve (32). The constant
A can be defined as zq/kT, where z is
the valence of charge q that acts as the voltage sensor in
the membrane to effect the transition from the open to closed
conductance states, and k and T represent
Boltzmann's constant and absolute temperature, respectively.
RNA preparation.
Total cellular RNA was isolated from tissues and cultured (3 × 107 cells) cells by using TRIzol total RNA isolation
reagent (Life Technologies). Briefly, tissues were homogenized in
TRIzol reagent, whereas cultured cells were directly lysed in culture
dishes by adding TRIzol reagent. The lysates from tissues or cultures
were transferred to a polypropylene round-bottom tube (Falcon, Becton Dickinson) and then incubated for 5 min at room temperature. RNA was
recovered in the aqueous phase from the TRIzol and chloroform mixture.
RNA was precipitated in 0.1 volume of sodium acetate and 2.5 volumes of ethanol.
Northern blot analysis for Cx43 mRNA levels.
Twenty micrograms of total RNA from each sample was size fractionated
on a denaturing 1% agarose-formaldehyde gel and then was immobilized
on GeneScreen filters (New England Nuclear). The baked
filters (2 h, 80°C) were prehybridized in 5× SSC, 7% SDS, 10×
Denhardt's solution, 20 mM sodium phosphate, pH 7.4, and 10% dextran
sulfate with 100 µg/ml denatured salmon sperm DNA and then hybridized
in the same solution with the addition of a radiolabeled oligonucleotide probe of human Cx43 and human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The blots were washed
three times in 2× SSC and 0.5% SDS and subjected to autoradiography
at
70°C. The sequence of the Cx43-specific probe utilized was
5'-GCAGGGCTCAGCGCACCACTG-3', corresponding to nucleotides 952-972
of human Cx43 cDNA (19). The hybridization and washing
temperature was 55°C. The sequence of the GAPDH probe used was
5'-AGGACGTGGTGGTTGACGAAT, corresponding to nucleotides
445-465 of the human GAPDH, and was used at 57°C.
Northern blot analysis for Cx40 mRNA levels.
The cDNA probe specific to human Cx40 (GenBank accession no. L34954;
base 412-953) was cloned from RT-PCR. The oligonucleotide primer
set used for PCR was 5'-gaagggaatggaaggattgc-3' and
5'-ccataacgaacctggatgaaac-3'. The human Cx40-specific cDNA fragment was
then subcloned into pCRII (Invitrogen) by TA cloning. RNA samples (20 µg) were denatured and electrophoresed through 1% agarose gels
containing 2.2 M formaldehyde and were transferred onto
nylon membranes by capillary blotting. The positions of the 28S
and 18S ribosomal RNA bands on the ethidium-stained gels were observed
under ultraviolet illumination before transblotting. RNA was fixed to
the filter by ultraviolet irradiation at 254 nm. Hybridization was then
carried out in Rapid-hyb buffer (Amersham, Arlington Heights, IL) at
50°C for 2 h. Filters were washed two times in 1× SSC and 0.1%
SDS at room temperature, followed by one wash in 1× SSC and 0.1% SDS
at 50°C. After being washed, the membranes underwent detection steps
using streptavidin and biotin alkaline phosphatase with CDP-Star
substrate according to the manufacturer's instructions. After
incubation with CDP-Star substrate, the membranes were removed and
exposed to the Hyperfilm (Amersham) in an intensifying screen. The film
was developed with time adjustments, and the bands were analyzed.
Western blot analysis for Cx43 protein levels.
Western blots were performed as described elsewhere (15).
Frozen human SV and IMA smooth muscle tissues were homogenized in 25 mM
Tris · HCl buffer, pH 7.4, containing 1 mM EDTA, 2 mM dithiothreitol, and 10 µg/ml each of leupeptin, aprotinin,
phenylmethylsulfonyl fluoride, and centrifuged at 800 g
for 10 min. The supernatant was concentrated to a final protein
concentration of ~5 µg/µl. Protein samples (30 µg each lane)
were dissolved in SDS-PAGE sample buffer and loaded into 10%
polyacrylamide gels. After electrophoresis, the proteins were
transferred to a Zeta-Probe membrane (Bio-Rad). The membranes were then
blocked overnight with 5% (wt/vol) nonfat milk in 1× PBS and probed
with an anti-human Cx43 antibody (Chemicon) at a 1:1,000 dilution for
2 h. After incubation, the membranes were washed three times for
10 min each with 1× PBS and then incubated with anti-mouse secondary
antibody for 1 h. After 3 more washes with 1× PBS (again, 10 min
each), the Cx43 bands were detected with enhanced chemiluminescence
reagents (Amersham).
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RESULTS |
Molecular identification of Cx43 mRNA and protein in homogeneous
short-term cultures as well as frozen tissue from human IMA and SV.
As shown in Fig. 1, immunostaining with a
human smooth muscle-specific myosin antibody revealed homogeneous
populations of smooth muscle cells after establishment of short-term
(i.e., passages 1-5) explant cultures of both IMA and
SV. Consistent with previous reports for human corpus cavernosum, Cx43
mRNA is found in frozen tissues and cultured myocytes from both vessels
(Fig. 2). The Western blots displayed in
Fig. 3 reveal two protein bands (45 and
41 kDa) in frozen tissue from both IMA and SV smooth muscle tissue. The
45-kDa bands were significantly stronger than the 41-kDa bands in both
tissue preparations, suggesting that the phosphorylated Cx43 isoform
was predominant in these tissues.

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Fig. 1.
Immunocytochemical staining of cultured smooth muscle cells from
human internal mammary artery (IMA) and saphenous vein (SV).
A: phase contrast image of IMA myocytes. B:
fluorescent image of myosin immunostaining in IMA myocytes.
C: phase-contrast image of SV myocytes. D:
fluorescent image of myosin-immunostaining SV myocytes. All cells were
passage 2 and were photographed at ×400 magnification.
Cells were stained for the presence of myosin with a primary myosin
anti-human antibody (see MATERIALS AND METHODS). Note the
homogeneous staining and distribution of myosin filaments in cells from
both vessels.
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Fig. 2.
Northern blot analysis for identification of connexin43
(Cx43) mRNA in vascular smooth muscle cells and the vascular tissues
from human SV or IMA. Total RNA was extracted from frozen tissues and
cultured cells, and 20 µg RNA from each sample was electrophoresed in
1% agarose-formaldehyde gels that were capillary blotted onto
GeneScreen and probed with oligonucleotides complementary to human Cx43
cDNA or to human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA
(see MATERIALS AND METHODS). In this blot, as illustrated,
3.1-kb mRNA bands correspond to Cx43 mRNA. The bands below Cx43 are
GAPDH. Cells, cultured IMA and SV smooth muscle cells of the second
passage; tissue, frozen IMA and SV tissue.
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Fig. 3.
Western blot analysis for identification of Cx43 protein
in frozen vascular tissue from human SV and IMA. Thirty micrograms of
protein was loaded per lane in a 10% SDS-PAGE gel. The 41-kDa (that
is, largely unphosphorylated) and 45-kDa (that is, largely
phosphorylated) Cx43 protein species are denoted. The results indicate
that the Cx43 gap junction channels are present in both SV and IMA
smooth muscle and that the 45-kDa doublet is predominant in these
tissues.
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Distribution of macroscopic junctional currents in IMA and SV.
Macroscopic junctional conductance was examined in IMA and SV cell
pairs, respectively, immediately after establishment of the double
whole cell configuration. Macroscopic activity was monitored anywhere
from 10 to 40 min. As shown in Fig. 4,
macroscopic junctional conductance of IMA (Fig. 4A) and SV
(Fig. 4B) cell pairs ranged from 1 to 14 nS with a mean ± SE of 4.7 ± 3.1 nS (n = 52) for IMA and
3.1 ± 2.5 nS (n = 43) for SV.

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Fig. 4.
Frequency distribution of macroscopic junctional
conductances (Gj) in IMA and SV cell pairs. The
instantaneous Gj was measured from 52 IMA and 43 SV cell pairs, respectively, and was grouped in 1-nS bins. The average
Gj was 4.7 ± 3.1 nS for IMA cell pairs and
3.1 ± 2.5 nS for SV cell pairs, respectively.
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Characteristics of macroscopic currents in IMA and SV.
To evaluate the voltage (Vj) dependence of the
observed junctional currents, an identical voltage protocol was applied
to all cell pairs measured (see MATERIALS AND METHODS).
Representative examples of macroscopic junctional currents
(Ij) obtained from an IMA and SV cell pair,
respectively, are shown in Fig. 5,
A and B. In both experiments, cell 1 was stepped from a holding potential of 0 mV in 10-mV increments to
produce Vj values ranging from
100 to +100 mV.
Each Vj pulse was 2.5 s with a 5-s recovery interval. As illustrated, typical for Cx43 macroscopic recordings, instantaneous Ij increases linearly with
Vj, however, the steady-state Ij clearly undergoes a time-dependent decay when
Vj exceeds ± 50 mV.

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Fig. 5.
Voltage dependence of macroscopic junctional currents
(Ij) in IMA (A) and SV (B)
cell pairs. Ij for IMA and SV were recorded
during 2.5-s pulse duration at 10-mV increments ranging from 100 to
+100 mV, with each pulse followed by a 5-s recovery period. In
C (IMA) and D (SV), the instantaneous (inst.) and
steady-state (SS) Ij shown in A and
B, respectively, were replotted as a function of
transjunctional voltage (Vj). As shown, the
instantaneous Ij-Vj
relationships were linear in the ± 100 mV ranges with
corresponding slope conductance values of 4.8 and 3.7 nS, respectively
(C and D). However, the SS
Ij-Vj relationships did
deviate from linearity above ± 50 mV. All currents were low-pass
filtered at a frequency of 1,000 Hz and digitized at 4 kHz.
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Plotting the instantaneous and steady-state Ij
values for both cell types as a function of Vj
(see Fig. 5, C and D) better illustrates the
Vj dependence of Ij. As
shown in Fig. 5C, the instantaneous
Ij-Vj relationship in IMA
approximates a straight line at all recorded voltages with a slope of
4.8 nS. In contrast, the steady-state
Ij-Vj relationship is
linear within the Vj range of ± 50 mV but
decreases above that voltage range. A similar relationship exists in
SV, as evidenced by the
Ij-Vj curve shown in Fig.
5D that was characterized by a linear instantaneous slope
conductance of 3.7 nS. However, as observed for the IMA cell pairs,
once again, the steady-state
Ij-Vj relationship in SV
cell pairs is linear within the Vj range of ± 50 mV but decreases above that voltage range. The
Vj-dependent relaxation appears to be
symmetrical about the origin, which indicates a bilateral voltage-gated mechanism.
Characteristics of macroscopic junctional conductance.
To further characterize the relationship between steady-state
junctional conductance (Gss) and
Vj, Gss was normalized to
the instantaneous junctional conductance (Ginst)
of each pulse (Fig. 6). That the
instantaneous Ij-Vj
curves were always linear provided a convenient method to normalize
data from cell pairs with different junctional conductance values. The
results of 24 IMA cell pairs are summarized in Fig. 6A. As
illustrated, the Gss decreased between Vj ± 40 and ± 90 mV, leveling off
near 0.35 at Vj ± 100 mV. This value is similar to one previously reported for Cx43 in human corporal
vascular smooth muscle (3, 5). Analysis of the data shown
in Fig. 6A revealed the following Boltzmann parameter values: maximal junctional conductance
(Gmax) = 1; minimal junctional conductance
(Gmin)
± 0.35; A = 0.121 (which corresponds to an equivalent gating charge of 3); and
V0 = ± 67 mV (see MATERIALS AND
METHODS). Similar results were obtained from 21 SV cell pairs; the data are plotted in Fig. 6B. Analysis of this data set
revealed the following Boltzmann parameter estimates:
Gmax = 1; Gmin
is
± 0.32 for negative and positive
Vj; V0 = ± 61 mV,
and A = 0.107 (z = 2.7). These data are
summarized in Table 1.

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Fig. 6.
Conductance-voltage relationships for SS
Ij in IMA (A) and SV (B).
Shown are the ratios of the SS to inst. Gj taken
from 24 IMA and 21 SV cell pairs, respectively. The voltage protocols
used were identical to those described in Fig. 5 legend. Each point
represents the normalized Gj at the
corresponding Vj. Note that
Gj declines symmetrically in both ± Vj directions, with the greatest decrease in
Gj occurring when Vj
exceeds ± 50 mV. The solid lines represent the theoretical fit of
the data to a two-state Boltzmann distribution (see Data
analysis). The Boltzmann parameters derived from such fits are
listed in Table 1.
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Single-channel conductance.
Unitary channel activity was also recorded in cultured cell pairs from
both IMA and SV in a symmetrical 165-mM CsCl solution (5,
10). The high cesium concentration in the presence of nonjunctional ion channel blockers minimizes contamination of the
observed Ij, thus improving the signal-to-noise
ratio of the recordings. A representative example of single-channel
currents in IMA under these recording conditions is illustrated in Fig. 7A, which shows a
representative ~20-s record. Figure 7B shows the
corresponding all-points histogram compiled from the same record, where
the dashed line represents the pdf fit to the digitized data assuming
two main states (open current amplitudes of 7.4 and 6.6 pA,
respectively) and one substate (current amplitude of 1.74 pA). The open
probabilities of the two main-state conductances were 0.45 and 0.13, respectively, whereas the open probability of the substate was 0.007. Figure 7C shows the unitary I-V curve for the
same cell pair with a calculated slope conductance of 117 pS for the
fully open channel and 31 pS for the substate. Similar results were
obtained from 18 other cell pairs, yielding a mean population slope
conductance value of 119 ± 4.3 pS.

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Fig. 7.
Representative recording of unitary junctional activity in an IMA
cell pair. Shown in A is a 20-s record of unitary activity
observed during a 60-mV voltage step applied to cell 1 from a holding potential of 0 mV (A, bottom
trace). Junctional currents (A, top trace)
appear as equal amplitude and opposite polarity signals. The zero
current level (labeled by C) and the 4 open-channel current levels are
depicted by the dashed lines. Both current traces were low-pass
filtered at 100 Hz and digitized at 1 kHz. B shows the
corresponding all-points histogram compiled from the same 20-s record.
The solid line indicates actual digitized data point count, and the
dashed line represents the probability density function (pdf) fit
assuming two independent channels and two substates with open currents,
7.4 and 6.6 pA, and open probability of 0.45 and 0.13 for the fully
open channels. For the channel substates, the open-channel currents are
1.74 pA and channel open probability are 0.007. C
illustrates construction of the single-channel current-voltage
relationship from the same cell pair revealed a slope conductance of
117 pS for the fully open channel and 31 pS for the substate.
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A representative example of unitary channel activity in an SV cell pair
is shown in Fig. 8, which displays
currents recorded during a
40 mV, 60-s pulse applied to cell
1 (Fig. 8A, bottom). Each channel current
level on the Ij trace (Fig. 8A,
top) is denoted by the dashed line. Figure 8B
shows the corresponding amplitude histogram for the same record, where
the dashed line represents the pdf fit to the digitized data, assuming
two independent channels and two substates. The main-state and substate
current amplitudes were 5.7 and 1.2 pA, respectively. The open
probabilities for the two independent main-state channels were 0.65 and
0.35, and 0.02 and 0.04 for the substates. The single-channel
I-V relationship with a linear regression fit from the same
cell pair with different voltages yields a unitary slope conductance
value of ~123 pS for the fully open channel and 31 pS for the
substates (Fig. 8C). Similar results were observed in other
16-cell pairs, with a mean population slope conductance value of
123 ± 5.2 pS.

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Fig. 8.
Representative record of unitary activity in an SV smooth muscle
cell pair. A shows a 60-s record at 40 mV applied to
cell 1 (trace at bottom, A). The
Ij (trace at top, A) show
two main-state channels, two substates, and one closed state. Current
amplitude levels are depicted by the dashed lines. Both current traces
were low-pass filtered at 100 Hz and digitized at 1 kHz. B
shows the all-points histogram compiled from the entire current trace,
where once again the solid line indicates the actual digitized data
point count, and the dashed line represents the pdf fit (see
Single-channel conductance). C shows the
single-channel current-voltage relationship with a linear regression
fit from the same cell pair with corresponding slope conductance values
of ~123 and ~31 pS for the main-state and substate conductance
values, respectively.
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The presence of "atypical" high-conductance unitary events in
both IMA and SV cell pairs.
As described above, the macroscopic whole cell current recordings, as
well as a majority of the observed unitary events, were consistent with
the presence of homotypic Cx43-derived intercellular communication.
However, closer inspection of the database clearly reveals, albeit more
infrequently, the presence of atypical high-conductance unitary events,
that is, unitary conductances significantly greater than expected for
the Cx43 main state (in all cases, the atypical high-conductance events
were
3 SE more than
120 pS Cx43 main state; see above).
Specifically, in myocytes from both IMA and SV, unitary events were
observed that had a conductance value similar to that of Cx40
(~140-160 pS) but exhibited a voltage-dependent gating behavior
that was uncharacteristic of Cx40 and, furthermore, more typical of
that expected for Cx43.
Figure 9 shows some representative
examples of such high-conductance events in cell pairs from IMA. For
example, the equal-amplitude, but opposite-polarity, unitary events
displayed in the dual whole cell recording at +40 mV (Fig.
9A) clearly illustrates a single large-conductance channel
with a single substate. The corresponding all-points histogram for this
data set, as well the pdf, fit to the digitized data (depicted by the
dashed lines), is shown in Fig. 9B. As illustrated, three
distinct peaks were detected, corresponding to one closed- and two
open-channel current peaks. The open-channel current amplitudes were
6.0 (~150 pS) and 1.3 pA (~33 pS) for the main state and substate,
respectively. The corresponding channel open probability calculated by
computer fitting the histogramic data was 0.91 for the main state and
0.06 for the substate (6).

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Fig. 9.
Large-conductance unitary activity in IMA cell pairs. A
shows ~10-s record of a large-conductance channel not characteristic
of the Cx43 unitary activity typically observed during a +40-mV step.
B shows the all-points histogram and pdf fit composed from a
longer record of 55 s at the same voltage and in the same cell
pair. Three distinct current peaks, corresponding to closed- and two
open-channel current peaks, were observed. The open-channel current
amplitudes were 6.0 and 1.3 pA. The channel open probability calculated
by fitting the histogram was 0.91 for the main state and 0.06 for the
substate (5). C shows two other records on
distinct cell pairs at a 40-mV step. Distinct main-state current
levels of 5.7 and 4.6 pA are indicated by the dashed lines, along with
the closed state, and graphically represented on the side bar.
D shows an all-points histogram composed from another 60-s
segment at a 40-mV step.
|
|
Figure 9C (left and right) shows two
different segments of the same record during a
40-mV step but on a
distinct cell pair from that shown in Fig. 9A. In Fig.
9C, distinct main-state current levels of 5.7 pA (~143 pS)
and 4.6 pA (~115 pS) are indicated by the dashed lines, along with
the closed state, and graphically represented on the side bar in both
panels. Figure 9D shows the all-points histogram composed
from a 60-s segment on the same cell pair shown in Fig. 9A,
also at a
40-mV step. In this record, three distinct current
peaks with corresponding open-current levels of 5.7, 4.6, and 1.4 pA
(~35 pS) were observed, in addition to the peak for the closed state.
The channel open probabilities calculated by computer fitting the
histogram were 0.4, 0.3, and 0.02 for the two fully open channels and
the substate, respectively.
Figure 10 shows a similar
electrophysiological scenario for an SV cell pair. As illustrated at
center, unique high-conductance unitary events
(~140-160 pS) that are uncharacteristic of Cx43 channels were
also observed in SV myocytes at both ± 40 mV. The inset
clearly illustrates that these events were also of equal amplitude and
opposite polarity. As illustrated in Fig.
11, the histogramic distribution of
thousands of unitary events recorded on 18 IMA and 16 SV cells pairs is
consistent with the presence of a smaller fraction of these
high-conductance unitary events, relative to the number of Cx43-like
conductance states (i.e., events of 30, 60, 90, or 120 pS) typically
observed for homotypic Cx43 recordings. Note that the unitary
conductance of the events recorded in both IMA and SV is reminiscent of
that reported for Cx40; however, the gating behavior is not that
expected of Cx40 channels at these voltages (1).

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Fig. 10.
Large-conductance unitary activity in SV cell pairs. Whole cell
current from an SV cell pair during a 67-s voltage step at ± 40 mV (center). The distinct current levels are indicated by
the dashed lines, with the closed current level labeled C. To better
illustrate individual channel transitions, the record has been expanded
at both ± 40-mV directions as indicated by arrows. Inset
(top right): Ij tracings from both cells of
the same cell pair.
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Fig. 11.
Histogramic distribution of the unitary events observed
in cell pairs from IMA (A, n = 18 cell
pairs) and SV (B, n = 16 cell pairs).
|
|
Molecular identification of Cx40 in IMA and SV cell pairs.
To explore the possibility that these unique high-conductance events
might be related to the coexpression of a distinct myocyte connexin, we
performed Northern blots on short-term cell cultures of both IMA and SV
myocytes. As shown in Fig. 12, Northern
blot analysis clearly revealed a detectable Cx40 mRNA signal with an apparent size of 3.3 kb in cultured cells from both vessels.

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Fig. 12.
Northern blot for Cx40 in cultured IMA and SV cells.
Each lane contains 20 µg of total RNA. The blot was hybridized with a
human Cx40-specific 542-bp cDNA probe (GenBank accession no. L34954).
Consistent loading of total RNA was checked by hybridization to a
GAPDH-specific probe. Migration of ribosomal RNA (28S and 18S) is
indicated by adjacent lines.
|
|
Mean macroscopic whole cell currents largely reflect expectations
for Cx43 only.
Figure 13 shows a comparison of the
plots of the mean macroscopic current data from IMA and SV (derived
from the data displayed in Figs. 5 and 6, respectively) vs. the mean
macroscopic current data derived from Cx43-transfected N2A cells. As
illustrated, it is clear that despite the presence of Cx40 mRNA and the
detection of Cx40-like unitary conductances, the macroscopic data
clearly reflect the contributions of primarily Cx43.

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Fig. 13.
Graphical depiction of the mean macroscopic
Ij observed in IMA, SV, and neuroblastoma
(Neuro-2A, N2A) cells expressing rat Cx43. Note the overt similarity in
the shape of the curves.
|
|
 |
DISCUSSION |
Intercellular communication through gap junctions plays a major
role in the coordination of myocyte responses in vascular tissues
throughout the vascular tree. Despite the identification of more than a
dozen mammalian connexins, only three are generally reported to be
present in vasculature, and they are Cx37, Cx40, and Cx43 (14,
21, 27, 28, 37). Recent reports have also documented the
presence of a fourth myocyte connexin, that is, Cx45 (16,
26). When one considers connexin expression in most vascular
myocytes, this number is further reduced to primarily two connexins,
Cx40 and Cx43. Moreover, Cx43 expression and function seem dominant in
many vascular myocytes (5, 10, 14, 21, 27, 30, 37). A
major goal of this investigation, therefore, was to evaluate and
compare the presence and potential functional relevance of Cx43-derived
gap junction channels in physiologically distinct human vascular myocytes.
To this end, we developed homogeneous short-term cell cultures of human
IMA and SV myocytes (Fig. 1). Northern blots revealed the presence of
Cx43 transcripts in cultured myocytes and frozen tissues from both
human blood vessels (Fig. 2). Western blotting revealed the presence of
Cx43 protein in frozen IMA and SV tissue (Fig. 3). In addition, this
report provides the first electrophysiological (Figs. 9-11) and
molecular (Fig. 12) data documenting that Cx43- and Cx40-derived gap
junction channels can coexist in vitro in both human IMA and SV myocytes.
On the virtually indistinguishable nature of macroscopic
junctional currents in physiologically diverse human vascular tissues.
A main conclusion of these studies is that nearly all aspects of
the recorded macroscopic junctional activity in cell pairs from both
vessels (Figs. 4-6) are very characteristic of those previously reported for Cx43-dominated junctional currents in many other cell
types in both endogenous and exogenous expression systems (3, 5,
7, 10, 21, 30, 36, 39, 41, 43, 46). Moreover, the macroscopic
currents reported here for both IMA and SV are also virtually
indistinguishable from those previously observed for the specialized
vascular myocytes of the human corpus cavernosum (nominally a
resistance vascular myocyte) (5, 10, 30). They are also
indistinguishable from the macroscopic records of Cx43-transfected cell
lines (6). Figure 13 illustrates the steady-state
G-V relationship for IMA and SV compared with data obtained
in transfected neuroblastoma cells (4). The main
implication of this important observation is that, at least at the
macroscopic level, the biophysical characteristics of Cx43-mediated
intercellular communication seem remarkably similar in vessels with
vastly different physiological functions.
Cx43-mediated unitary conductance and activity in IMA and SV are
also comparable and similar to previous reports.
Figures 7 and 8 clearly illustrate the presence of Cx43 main-state and
substate channel conductances that are very reminiscent of previous
reports in human corpus cavernosum myocytes (5, 10, 30).
Once again, despite the notable frequency of substate conductance
events, their contribution to the weighted amplitude histograms is
generally so minor that it calls into question their potential
physiological relevance (10). As such, the data shown in
Figs. 7 and 8 provide compelling evidence that the Cx43 main-state conductance is the major determinant of intercellular coupling in
cultured IMA and SV myocytes.
The presence of atypical high-conductance single-channel events.
Despite all of the aforementioned biophysical similarities to previous
reports on Cx43, some very important differences were also noted in the
present records. The most significant was the appearance, albeit
infrequently, of high conductance unitary events that were clearly
quite atypical for Cx43-mediated Ij. More
specifically, a relatively small but consistent fraction of main-state
events observed in both IMA and SV had conductance values that were
well outside the range of values typically reported for the Cx43 main state. To further illustrate the point, let us consider the fact that
the upper portion of the 95% confidence interval for Cx43 main-state
conductance values, in both IMA and SV, are well below the
140-160-pS conductance values for the atypical unitary events reported herein. That is, the Cx43 main-state slope conductance value
is ~120 ± 5 pS in myocytes from both vessels. Therefore, the
upper 95% confidence interval for the Cx43 main-state conductance value would be ~130 pS, with an upper 99.9% confidence limit of ~135 pS. It follows logically from such considerations that the 140-160-pS events displayed in Figs. 9-11 clearly reflect the
contribution of a separate and unique population of high-conductance
main-state events in both IMA and SV myocytes.
Interestingly, although the unitary conductance of these channels is
consistent with that previously reported for Cx40 (1, 14,
29), the gating characteristics were very atypical. In fact,
previous investigations indicate that the vast majority of homomeric
Cx40 channels should be closed at the voltages at which we consistently
observed them in these studies (i.e., ± 40 mV; see Figs. 9 and 10).
Therefore, these atypical channels are easily distinguished from Cx43
based on their main-state conductance, but moreover, they can also be
readily distinguished from Cx40 based on their gating characteristics.
As shown in Fig. 12, there is compelling molecular evidence for the
coexpression of Cx40 in both IMA and SV myocytes.
Other investigators using in situ hybridization techniques also
reported the presence of Cx43, but not Cx40 mRNA, in human IMA
(27). The reason for the apparent discrepancy between
these prior observations in situ and our current studies in vitro is not certain but may be related to the relatively low Cx40 mRNA signal
present in the IMA, as indicated by our in vitro observations (Figs. 9
and 11). In addition, we cannot at present exclude the possibility that
Cx40 expression is associated with our cell culture conditions.
Moreover, although other connexins have been recently identified in
myocytes (i.e., Cx45; see Refs. 16 and 26), we saw no
evidence for the existence of additional connexins at the
single-channel level, and thus the possibility of the expression of
additional myocyte connexins in IMA and SV will be explored in future experiments.
What might Cx40/43 coexpression imply about the observed atypical
channel behaviors?
Evidence that coexpression of connexins can result in mixed hemichannel
channels, referred to as heteromeric channel forms, has been previously
reported (4, 6, 25, 40). Such observations have indicated
that at least some of the biophysical properties of mixed gap junction
channels are different from those characteristic of their homotypic
counterparts. For example, electrophysiological studies clearly showed
that for cells coexpressing Cx37 and Cx43 there was an apparently
weaker voltage dependence of the macroscopic currents typically
observed for Cx37, as well as a subpopulation of single-channel
conductances that could not be explained as arising from homotypic or
heterotypic gap junction channels formed of either Cx37 or Cx43. This
possibility has also been validated in an endogenous expression system
(21). Specifically, the cultured rat aortic vascular
smooth muscle cell line, A7r5, normally coexpresses Cx40 and Cx43, and,
as might be expected of heteromeric channel forms, the observed
macroscopic voltage dependence was weaker than either of the homotypic
forms; moreover, some of the single-channel conductances were not
easily explained as either homotypic or heterotypic forms
(21).
Pertinent to the current report, the symmetrical voltage dependence of
the observed macroscopic and unitary recordings would seem to rule out
the possibility of a large population of heterotypic Cx40/43 channels
(i.e., a homomeric Cx40 hemichannel in one cell and a homomeric Cx43
hemichannel in the other cell) (42). Therefore, a cogent
interpretation of our present observations is that the expression level
of Cx40 mRNA results in the formation of a small number of Cx43/Cx40
heteromeric channels. Furthermore, the recordings illustrated in Figs.
9 and 10 are consistent with previous reports in which heteromeric
connexins display properties that are not truly characteristic of
homotypic channels of either of the connexins of interest (4, 6,
21, 42). As such, the Cx40-like conductance and Cx43-like gating
behavior might reflect a "hybrid" biophysical manifestation of
heteromeric connexins in an endogenous human vascular smooth muscle
cell line. Although previous reports have documented the presence of
Cx43 in human internal mammary artery (27), this is the
first report we are aware of that provides evidence for the
coexpression of Cx40 and 43 in human vascular smooth muscle from IMA
and SV and, moreover, for the putative presence of heteromeric gap
junction channels composed of such.
What might be the role of heteromeric channels in vivo?
Finally, to put things in proper perspective, it is clear that the
frequency and dwell time of the observed high-conductance events
reported here are such that they would not be expected to make any
significant contributions to the extent of intercellular communication
between myocytes from these two vessels under the conditions utilized
for these studies. However, we cannot rule out the possibility that
agonist-mediated alterations in cellular activation or the presence of
disease states could create a physiological circumstance, in vitro or
in vivo, in which the frequency and distribution of these
high-conductance events became of functional importance. Moreover,
another interesting issue, which this study cannot yet resolve with
regard to permselectivity, is whether or not heteromeric biophysical
properties (if present at higher levels in vivo, for
example) are sufficiently different from their homotypic counterparts
to affect the cell-to-cell diffusion of critical substances that are
able to alter and/or trigger cellular responses (e.g., inositol
1,4,5-trisphosphate, cAMP, cGMP, Ca2+, etc.)
(6).
Therefore, although the precise physiological significance in vivo of
these observations in vitro is uncertain, these cultured human myocytes
appear to represent an excellent model system for exploring the
physiological boundary conditions of endogenous connexin coexpression
in human vascular myocytes. Determination of the true functional
relevance of these purported "heteromeric" channel forms to the
control of human vasomotor tone in vivo will necessarily remain the
province of future investigations.
 |
ACKNOWLEDGEMENTS |
We are extremely grateful to the surgeons, nurses, and other
operating room staff who were responsible for supplying and/or collecting and storing the excess human blood vessels after an otherwise long day of work. We also acknowledge the excellent secretarial assistance of Diane Ditrapani. The authors gratefully acknowledge the technical assistance of Dr. Yan Ping Wen with the
Northern blot analysis illustrated in Fig. 2.
 |
FOOTNOTES |
This work was supported in part by National Institutes of Health United
States Public Health Service Grants DK-42027, DK-46379, and GM-55263
(to P. R. Brink).
Address for reprint requests and other correspondence: G. J. Christ, Depts. of Urology and Physiology & Biophysics, Rm. 716S, Forchheimer Bldg., Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: christ{at}aecom.yu.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 31 March 2000; accepted in final form 15 February 2001.
 |
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