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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
G<SUB>ss</SUB><IT>=</IT>{(<IT>G</IT><SUB>max</SUB><IT>−G</IT><SUB>min</SUB>)<IT>/</IT>[<IT>1+</IT>exp(<IT>A</IT>[<IT>V</IT><SUB>j</SUB><IT>−V<SUB>0</SUB></IT>])]}<IT>+G</IT><SUB>min</SUB>
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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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.

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.

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 (Gminapprox  ± 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 approx  ± 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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Table 1.   SV and IMA

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.

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.

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 approx 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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