Department of Physiology, University of Arizona, Tucson, Arizona 85724
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ABSTRACT |
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In atherosclerosis and hypertension, vascular smooth muscle cells (SMCs) are stimulated to proliferate and exhibit enhanced gap junction protein expression. Our goal was to determine whether gap junction function differs in proliferating vs. growth-arrested SMCs. A7r5 cells (embryonic rat aortic SMCs) did not proliferate in media with reduced serum (~90% of cells in G0/G1 phase after 48-96 h in 1% fetal bovine serum). Dye coupling was less but electrical coupling was comparable in proliferating vs. growth-arrested A7r5 cells, suggesting differences in junctional permselectivity. In growth-arrested cells, junctional conductances measured with potassium glutamate, tetraethylammonium chloride, and KCl were well predicted by the conductivities of these solutions. In contrast, junctional conductances measured with potassium glutamate and tetraethylammonium chloride in proliferating cells were significantly greater than predicted by the conductivities of these solutions. These results suggest that junctions between growth-arrested cells are permeated equally well and simultaneously by anions and cations, whereas junctions between proliferating cells are poorly permeated by large molecules of either charge and equally well but not simultaneously by small anions and cations. The data indicate that A7r5 cells regulate chemical coupling independent of electrical coupling, a capacity that could facilitate growth control while protecting vasomotor responsiveness of vessels.
connexon; intercellular communication; permselectivity; cell cycle; growth control
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INTRODUCTION |
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GAP JUNCTION PROTEINS (connexins, Cx) are present in the cell membrane as hexameric hemichannels. Hemichannels between neighboring cells dock to form the functional channel, which provides a pathway for the diffusive movement between cells of ions, metabolites, and second-messenger molecules (10, 33). In the vasculature, intercellular communication likely functions in a dual role, coordinating vasomotor responses (11, 31, 32) and regulating cell growth (23, 42). Two scenarios relating cell growth and gap junction function have been advanced: one links enhanced growth with reduced or absent communication (23); the other links enhanced growth with elevated expression of connexin proteins and presumably increased coupling (5, 6, 16, 20).
Vascular wall cells appear to fit the second scenario, wherein increased cell growth and proliferation are associated with increased expression of connexin proteins. Proliferation of vascular cells in the adult animal occurs in response to physiological stimuli, leading to angiogenesis, and in response to pathophysiological stimuli, leading to wall thickening or plaque formation, as in hypertension and atherosclerosis, respectively. Comparatively little is known about gap junction protein expression or channel function in the angiogenic setting, but it is apparent in the pathophysiological setting that changes in gap junction expression occur. Smooth muscle cells (SMCs) from hypertensive animals placed in cell culture exhibit a significantly greater gap junctional area per cell than their nonhypertensive counterparts (15). Recent evidence supports and extends this observation by demonstrating increased expression of Cx43 in vivo in aortic smooth muscle of hypertensive animals (16, 35). Similarly, in the early stages of atherogenesis, Cx43 expression in intimal smooth muscle is enhanced (6). These studies did not examine the functional properties of gap junctions between SMCs, nor did they look for expression of Cx40, a gap junction protein coexpressed with Cx43 in SMCs of resistance vessels (22), large arteries (4), and possibly diseased vessels (25) or vessels undergoing vasculogenesis (24). Consequently, the functional ramifications of these changes in connexin expression are unknown.
A number of investigators have reported altered gap junction function in cells stimulated to proliferate with growth factors. Whereas several groups have reported decreased Lucifer yellow dye coupling in cells stimulated to proliferate (21, 29), other groups have reported increased dye coupling and connexin expression (12, 28, 30). The opposite character of the results in these reports could reflect temporal differences, or they could be indicative of cell-specific differences in coupling. Regardless, the dye-coupling technique provides only limited information about gap junction function. For example, a decrease in Lucifer yellow dye coupling could be indicative of 1) a decreased number of functional channels with no change in their overall permselectivity, 2) a selective decrease in anion permeability, 3) a selective decrease in large molecule permeability, or even 4) a selective decrease in cation permeability that also reduces, to a more limited extent, anion permeability. The implications of these scenarios for the types of molecules that are shared between cells and their importance in the regulation of proliferation and maintenance of normal vasomotor responsiveness of vessels could be quite different.
The purpose of the present study was to determine whether gap junction function differed in proliferating vs. growth-arrested SMCs. A7r5 cells, an embryonic rat aortic SMC line, were selected for these studies, because they express the connexins found in SMCs of resistance vessels, Cx40 and Cx43 (22). Proliferating A7r5 cells were found to be less well dye coupled than their growth-arrested counterparts. Despite reduced dye coupling, electrical coupling (measured with KCl patch solution) between proliferating and growth-arrested cells was comparable. Permselectivity studies indicate that gap junctions in proliferating cells are poorly permeated by large molecules (anions or cations), whereas in growth-arrested cells such molecules permeated the junction freely and in accord with their mobilities in bulk solution. The data indicate that gap junctions in SMCs undergo changes in permselectivity as a function of growth status, i.e., changes that would not be expected to compromise their capacity to share electrical signals but would significantly reduce their ability to mediate exchange of larger molecules with growth-regulatory potential.
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MATERIALS AND METHODS |
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Cell culture. A7r5 cells were obtained from American Type Culture Collection at passage 11 and maintained in DMEM (Sigma Chemical) supplemented with 10% fetal bovine serum (FBS) and antibiotics (5% streptomycin and 3% penicillin) in a humidified, 10% CO2 incubator at 37°C. Cells were passaged weekly with 0.25% trypsin in Ca2+-Mg2+-free PBS. In several experiments, lipoprotein-free serum (LPD-FBS) was used, thereby eliminating potential mitogenic effects of low-density lipoproteins. Lipoproteins were removed from the serum by gradient centrifugation (17, 18).
Growth curves. Cells were plated at a density of 0.5 × 104 cells/cm2 in 24-well plates and maintained for 24 h in DMEM containing 10% FBS to allow cells to adhere to the plate and recover from trypsinization. Growth curves were initiated at the end of this recovery period as follows. After cells were washed gently with PBS, cells were maintained for the duration of the growth curve experiment in DMEM containing 10% FBS, 10, 5, 1, or 0.1% LPD-FBS, or 0.1% BSA. Wells were washed daily with PBS, and fresh medium was restored. Every 24 or 48 h, three wells of cells in each treatment group were washed twice with PBS and fixed with 1% glutaraldehyde. Fixed cells were then washed with and stored in PBS until all wells in the experiment had been fixed. On the final day of the growth curve experiment, PBS was removed from all wells, and the fixed cells were incubated in 0.1% crystal violet for 40 min. Cells were then washed with water, dried, and dissolved in 2 ml of 10% acetic acid. The absorbance of the resultant solution, which is linearly related to cell number (data not shown), was determined spectrophotometrically at 590 nm with 10% acetic acid as a blank. Despite identical procedures, data from different experiments are not directly comparable because of variability in final plating density (density 24 h after plating) and staining efficiency, both of which can alter the position of the growth curves relative to the axes, despite identical growth rates. Thus the technique is ideally suited to identifying growth-inhibitory conditions but not to assessing relative rates of growth.
Cell cycle analysis. Fluorescence-activated cell sorting (FACS) was used to quantify the proportion of cells in G0/G1, S, and G2/M phases. A7r5 cells (passages 18-20) were plated at low or high density (~0.5 × 104 or ~1.5 × 104 cells/cm2) on gelatin-coated or uncoated six-well plates and maintained in 10% FBS-DMEM for 24 h. The cells were then washed three times with PBS and subsequently incubated in 0.1% BSA-DMEM, 1% FBS-DMEM, or 10% FBS-DMEM (each treatment at each time point was performed in triplicate). As in the growth curve experiments, to minimize effects of any autocrine growth factors, the medium was changed daily after three washes in PBS (14, 27, 41).
Cells were harvested for cell cycle analysis by using a modification of the procedure from Vindelov and Christensen (38). Briefly, cells were washed three times with PBS and incubated with gentle rocking for 15 min at room temperature in a solution containing trypsin (30 µg/ml), Nonidet P-40 (0.1% vol/vol), and spermine tetrahydrochloride (1.5 mM). After addition of trypsin inhibitor (50 µg/ml) and RNase A (100 µg/ml) to each well, cells were incubated for an additional 15 min at room temperature with gentle rocking. Cells were stained with propidium iodide (400 µg/ml) and filtered through a 50-µm nylon mesh, and their DNA content was quantified on a FACStar flow cytometer within 3 h of harvesting. Cell cycle data were analyzed for the proportion of cells in each stage of the cell cycle by using the WinMDI program.Dye coupling.
A7r5 cells were plated on glass coverslips and grown to confluence.
They were then treated with 10 or 1% FBS for 48 h (with PBS washes at
24 h). Coverslips were mounted in a chamber and bathed in Hanks'
buffered saline solution. Electrodes with resistances of ~15 M
were fabricated (Sutter Instruments) from thin-walled glass (1.0 mm;
Corning 6150, AM Systems). The tips of these electrodes were filled
with Lucifer yellow (5% in water, ~150 mM), and the remainder of the
electrode was backfilled with 150 mM LiCl. An electrode was positioned
on the surface of the desired cell, such that it "dented" the
membrane. The capacitance compensation feature of the amplifier (model
KS-700, WPI) was then overcompensated, which resulted in cell
impalement and simultaneous efflux of dye from the electrode into the
cell. The electrode was removed from the cell immediately after
impalement, and 5 min later the number of neighboring cells to which
the dye had diffused was visually determined.
Junctional conductance and permeability. For dual whole cell voltage-clamp experiments, A7r5 cells were plated at low density (0.4-0.5 × 104 cells/cm2) onto glass coverslips coated with 1% gelatin in PBS and maintained in 10% FBS-DMEM for 24 h. On the following day, some of the cells were used for determination of junctional conductance (gj); these cells composed the proliferating cell group. The remaining cells were washed three times with PBS and subsequently incubated in growth-arrest medium (1% FBS-DMEM) for 48 h (with PBS washes at 24 h). After 48 h in growth-arrest medium, gj of the cells was determined; these cells composed the growth-arrested cell group.
The dual whole cell voltage-clamp method was used to determine gj in proliferating and growth-arrested A7r5 cells. Coverslips with proliferating or growth-arrested cells were placed in an experimental chamber and bathed in extracellular solution containing (in mM) 142.5 NaCl, 4 KCl, 1 MgCl2, 5 glucose, 2 sodium pyruvate, 10 HEPES, 15 CsCl, 10 tetraethylammonium chloride (TEACl), 1 BaCl2, and 1 CaCl2; pH and osmolarity were adjusted to 7.2 and 322 mosM, respectively. Patch pipettes were filled with KCl, potassium glutamate (KGlut), or TEACl (PS-KCl, PS-KGlut, or PS-TEACl; Table 1) and fabricated (1.2-mm glass; Corning 6020, AM Systems) such that, irrespective of patch solution, they had resistances of ~8 M
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Statistical analysis.
Values are means ± SE. Student's
t-test was employed to compare
proliferating with growth-arrested cells tested via dye coupling. Significance was set at P 0.05. For
the permselectivity data derived with the three patch solutions, data
were also compared with t-test.
However, the Bonferroni adjustment was used to account for multiple
comparisons on the same data. This adjustment involves creating a
conservative family-wide error rate of
P
0.05 by dividing 0.05 by the
number of comparisons and using that value to determine significance
for each comparison.
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RESULTS |
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Growth arrest. To establish conditions that reproducibly arrested A7r5 cell proliferation, the dependence of proliferation on serum content of the growth medium was evaluated. Cell number as a function of time was determined for cells maintained in media containing 0.1, 1, 5, and 10% LPD-FBS, 0.1% BSA, or 10% FBS. Despite reports to the contrary (2, 3), the results (Fig. 1) suggested that 1% FBS was sufficient to produce growth arrest. To further verify this finding, we tested the effects of 1% LPD-FBS on proliferation over a period of 15 days. Figure 2 shows that A7r5 cells do not proliferate in 1% LPD-FBS medium.
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Cell cycle analysis. FACS was used to assess where in the cell cycle A7r5 cells arrested and to verify that 1% FBS (vs. the 1% LPD-FBS used in the experiments described above) also produced growth arrest (experimental paradigms are summarized in Table 2). Figure 3 shows the results of an FACS experiment in which the cells were plated at high density and maintained in 10% FBS for 96 h (A) or at low density and maintained in 1% FBS for 48 h (B). In 10% FBS, 79% of cells were in the G0/G1 phase, 6% in the S phase, and 15% in the G2/M phase. In contrast, 91% of cells cultured for 48 h in 1% FBS were in the G0/G1 phase, 5% in the S phase, and 4% in the G2/M phase. Data from multiple experiments in which the effects of 1% FBS and 0.1% BSA on cell cycle position were assessed on cells plated at low or high density for 48 or 96 h, respectively, are summarized in Fig. 4. The data indicate that cells plated at low or high density and arrested in 1% FBS vs. 0.1% BSA for 48 or 96 h were not different. In addition, no effect of gelatin coating could be detected (data not shown).
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Dye coupling. To determine whether gap junction function differed in proliferating vs. growth-arrested A7r5 cells, the number of neighboring cells to which intracellularly injected Lucifer yellow diffused was measured. After A7r5 cells achieved confluence, the medium bathing the cells was changed to 1% FBS to induce growth arrest or 10% FBS to maintain the proliferative state. After 48 h, Lucifer yellow was injected into individual cells and the extent of dye coupling was assessed. As illustrated in Fig. 5, growth-arrested A7r5 cells (in 1% FBS) were fivefold better coupled than proliferating cells (in 10% FBS).
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Junctional conductance. The dual whole cell voltage-clamp technique offers a more quantitative measure of cell-cell coupling. Consequently, this technique was used to assess gj in proliferating and growth-arrested A7r5 cells. Contrary to expectations arising from the dye-coupling experiments, gj (measured with PS-KCl) between proliferating cells (10% FBS) was not different (Fig. 5) from gj between arrested cells (1% FBS). Reconciliation of these electrical-coupling results with the dye-coupling results prompted us to investigate possible differences in permselectivity of the junctions in proliferating vs. growth-arrested cells.
Macroscopic junctional selectivity.
To determine whether junctional permselectivity differed in
growth-arrested vs. proliferating cells, we measured
gj with patch solutions that differed in the composition of primary current-carrying ions (Table 1). Substitution of
Glut for
Cl
distinguishes the
PS-KGlut solution from PS-KCl; comparison of gj values
measured with these solutions allows determination of anion
permeability. Substitution of TEA+
for K+ distinguishes the PS-TEACl
solution from PS-KCl; comparison of gj values
measured with these solutions allows determination of cation
permeability. Because of differences in the mobilities of the
constituent ions, the conductivities of these solutions differed as
summarized in Table 1.
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DISCUSSION |
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To better appreciate the potential role of gap junctions in growth properties of cells, the permeability of the junctions to molecules with growth-regulatory potential must be elucidated. The purpose of the present study was to characterize functional differences in macroscopic permeability of gap junctions between growth-arrested and proliferating A7r5 cells. We demonstrate that these cells do not proliferate and reside largely in the G0/G1 phase of the cell cycle when maintained in media with low serum content. The gj values, when measured with a KCl pipette solution, were equal in growth-arrested and proliferating cells, suggesting that electrical signals are shared equally well in both growth conditions. However, permeation of the junctions by large ions of either charge was restricted in proliferating cells, suggesting that metabolic and chemical signaling may be compromised in proliferating cells. These observations are discussed with respect to their possible mechanistic basis and their potential implications for growth control.
The dye-coupling studies presented here suggested that gap junctions in proliferating A7r5 cells were less well permeated by Lucifer yellow than their counterparts in growth-arrested cells. The decreased dye coupling in proliferating cells could occur as a result of 1) decreased activity or numbers of channels with selectivity comparable to those in growth-arrested cells, 2) decreased anion permeability with or without changes in cation permeability, 3) decreased large molecule permeability, irrespective of molecular charge, 4) decreased numbers of contacting cells, or 5) increased cytosolic expression of dye-binding proteins (8, 34). The pipette solutions used in our studies allowed us to distinguish between these possibilities as follows. If permeation of gap junction channels is dictated largely by the mobility of the current-carrying ions in bulk solution, then differences in gj measured with the three solutions should be predicted by their conductivities (Fig. 6). If a junction exhibits cation selectivity, then gj measured with PS-KGlut vs. PS-KCl should differ by less than predicted by their solution conductivities (above solid line in Fig. 6), whereas gj measured with PS-TEACl vs. PS-KCl should differ by more than predicted by their solution conductivities (below the solid line in Fig. 6). The opposite would be expected for a junction exhibiting anion selectivity. If junctions restrict the movement of large ions, irrespective of their charge, then gj measured with PS-KGlut and PS-TEACl should differ from that measured with PS-KCl by less than predicted by the respective solution conductivities (above the solid line in Fig. 6). Finally, electrical coupling between growth-arrested and proliferating cells should not differ if dye-coupling data are explained by increased expression of cytosolic dye-binding proteins or decreased numbers of contacting cells in the proliferating cell group. As discussed below and shown in Fig. 6, our data suggest that the junctions in proliferating cells restrict the permeation of large ions, irrespective of their charge.
For growth-arrested cells, gj values measured with PS-KCl, PS-KGlut, and PS-TEACl were well predicted by solution conductivities, consistent with the conclusion that permeation of gap junction channels in growth-arrested cells is dictated in large part by the mobilities of the ions in bulk solution. The data further suggest that anions and cations permeate the junction equally well and simultaneously (Fig. 7A). Thus these junctions exhibit the nonselective behavior stereotypically described for gap junctions in most textbooks. Very different conclusions are required to explain results obtained with proliferating cells.
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In proliferating cells, gj measured with PS-KGlut vs. PS-KCl differed by only 8% rather than the predicted 26%. In the absence of additional information, these data would suggest that gap junctions in proliferating cells exhibit significant cation selectivity. However, gj measurements with PS-TEACl vs. PS-KCl differed by only 15% rather than the predicted 32%, which would seem to suggest that gap junctions exhibit significant anion selectivity. Together, these data indicate that gap junction channels in proliferating cells restrict the movement of larger molecules irrespective of their charge but are permeated equally well (slight preference for cations) but not simultaneously by small anions and cations (Fig. 7B). Thus the data indicate that decreased dye coupling in proliferating vs. growth-arrested cells results from diminished permeation by large molecules irrespective of their charge.
Macroscopic junctional selectivity represents the summation of the
selectivities of all the individual channels in the junction. The
permselectivity characteristics for Cx40 and Cx43 channels have been
published (1, 36, 39), although a clear picture has yet to emerge. On
the basis of reversal potential measurements with asymmetric solutions,
Wang and Veenstra (39) concluded that
Cl/K+
selectivity of homomeric/homotypic Cx43 channels was 0.13. However, using symmetric solutions, Wang and Veenstra and Valiunas et al. (36)
found that the unitary conductance
(
j) of Cx43 channels in their
main state was dictated in large part by the mobility of the
current-carrying ions in bulk solution. Valiunas et al. found that
j measured with cesium
aspartate vs. KCl differed by 64% vs. a solution conductivity
difference of 67%. Similarly, Wang and Veenstra found that
j measured with KGlut and TEACl were 63 and 55% of the conductance measured with KCl, values
reasonably predicted by the mobility of ions in bulk solution. These
measurements suggest that the main state of Cx43 channels exhibits
nearly equal anion and cation permeability. In contrast, the residual
state of the Cx43 channel appears to exhibit significant cation
selectivity (36). Cx40 channels exhibit a more restricted permeation
pattern. Beblo and Veenstra (1) found that
j values measured with KGlut and TEACl were 113 and 44%, respectively, of the conductance measured with KCl. From these and other measurements, they conclude that Cx40
channels exhibit cation selectivity with poor permeation of the channel
by large anions. Again, cation permeation was approximated by ion
mobility in bulk solution with minor restriction by the channel.
Despite the poor permeation of Cx40 channels by anions suggested by
these studies, Elfgang and colleagues (13) demonstrated quite
convincingly that Lucifer yellow permeates Cx40 channels just as it
permeates Cx43 channels. The measurements of Valiunas et al. and
Veenstra and colleagues (1, 39) were made on cells exposed to growth
factors. No data are available as to how or even whether these
permeability properties might be altered by growth status. Nor
are there data regarding open probability as a function of growth status.
Are the junctional permeability characteristics described here for the growth-arrested or proliferating cells consistent with what would be expected for cells expressing Cx40 and Cx43? On the basis of the selectivities of homomeric/homotypic Cx40 vs. Cx43 channels reviewed above, at least four scenarios could be consistent with the data. 1) In proliferating cells, Cx43 might be downregulated and Cx40 upregulated, such that junctional permeability is reduced but electrical coupling maintained in proliferating vs. growth-arrested cells. 2) Cx43 channels in proliferating cells might be phosphorylated (26), such that their permeation by large molecules is restricted while permeation by small molecules and consequently electrical coupling is maintained. 3) In proliferating cells the open probability of Cx43 channels might be reduced [practically speaking to zero (9)] with a compensatory increase in open probability of Cx40 channels. 4) Cx40 and Cx43 could form heteromeric channels (7, 19), the permeation of which by large molecules decreases as the contribution of Cx40 to the channel increases. In this case, any change in connexin expression that resulted in an increased ratio of Cx40 to Cx43 in the proliferating cells would result in restricted permeation, possibly without major changes in electrical coupling. Further studies are necessary to resolve these possibilities.
The dye-coupling and permselectivity data presented here indicate that growth-arrested cells share large molecules much more readily than their proliferating counterparts. Large molecules of potential interest to cells include metabolites and second messengers (e.g., inositol trisphosphate, cAMP, cGMP). Focusing on the latter, our data suggest that these molecules would diffuse readily between growth-arrested cells but poorly between proliferating cells. Given the short half-life of second-messenger molecules, the limited diffusion of large molecules observed in the proliferating setting may functionally restrict second-messenger molecules to the cells in which they are being actively generated. The significance of such a restriction depends on whether the gap junction serves a growth-suppressing or -promoting role in the cell type being studied.
In a growth-promoting role, growth factors are envisioned to induce a rise in second-messenger concentration that is sufficient to trigger a growth response in the stimulated cell as well as in neighboring cells to which the stimulated cell is coupled. In a growth-suppressing role (23), a growth factor-induced rise in second-messenger levels fails to reach a critical growth-inducing concentration in the stimulated cell, because the molecule rapidly diffuses to neighboring cells (thereby diluting the signal). Our data suggest that in growth-arrested vascular SMCs, at least those expressing Cx40 and Cx43, the gap junction plays a suppressive role, wherein a rise in concentration of a potential growth-regulatory molecule is diluted out because of significant intercellular coupling, effectively shifting the dose-response curve for the growth factor to the right. At sufficiently high growth factor concentration, proliferation is triggered, despite high levels of coupling, perhaps as a consequence of transient uncoupling (21, 29). Maintenance of the proliferating state would then be facilitated by gap junctions that restrict diffusion of growth-regulatory molecules away from the stimulated cell, thereby ensuring sufficient concentration of those molecules for proliferation to continue.
In summary, whereas electrical signaling is preserved, chemical and metabolic signaling is compromised in proliferating A7r5 cells. These results suggest that vasomotor control should be maintained in vascular smooth muscle stimulated to proliferate, as in vasculogenesis, angiogenesis, injury, and disease. It will be interesting to determine whether the proliferation-induced changes in communication described here are unique to A7r5 cells or smooth muscle cells, common to cells coexpressing Cx40 and Cx43, or generic to proliferating vs. growth-arrested cells irrespective of connexin expression.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Institutional Training Grant 2T32 HL-07249 and Grants HL-31008 and HL-58732 and by Arizona Disease Control Research Commission Grant 5-090.
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FOOTNOTES |
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Present address of D. T. Kurjiaka: Dept. of Biological Sciences, Ohio University, Athens, OH 45701.
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. §1734 solely to indicate this fact.
Address for reprint requests: J. M. Burt, Dept. of Physiology, University of Arizona, PO Box 245051, Tucson, AZ 85724-5051.
Received 5 March 1998; accepted in final form 27 August 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Beblo, D. A.,
and
R. D. Veenstra.
Monovalent cation permeation through the connexin40 gap junction channel Cs, Rb, K, Na, Li, TEA, TMA, TBA, and effects of anions Br, Cl, F, acetate, aspartate, glutamate, and NO3.
J. Gen. Physiol.
109:
509-522,
1997
2.
Bennett, M. R.,
G. I. Evan,
and
A. C. Newby.
Deregulated expression of the c-myc oncogene abolishes inhibition of proliferation of rat vascular smooth cells by serum reduction, interferon-, heparin, and cyclic nucleotide analogues and induces apoptosis.
Circ. Res.
74:
525-536,
1994[Abstract].
3.
Bennett, M. R.,
T. D. Littlewood,
D. C. Hancock,
G. I. Evan,
and
A. C. Newby.
Down-regulation of the c-myc proto-oncogene in inhibition of vascular smooth-muscle cell proliferation: a signal for growth arrest?
Biochem. J.
301:
702-708,
1994.
4.
Beyer, E. C.,
K. E. Reed,
E. M. Westphale,
H. L. Kanter,
and
D. M. Larson.
Molecular cloning and expression of rat connexin40, a gap junction protein expressed in vascular smooth muscle.
J. Membr. Biol.
127:
69-76,
1992[Medline].
5.
Blackburn, J. P.,
J. L. Connat,
N. J. Severs,
and
C. R. Green.
Connexin43 gap junction levels during development of the thoracic aorta are temporally correlated with elastic laminae deposition and increased blood pressure.
Cell Biol. Int.
21:
87-97,
1997[Medline].
6.
Blackburn, J. P.,
N. S. Peters,
H.-I. Yeh,
S. Rothery,
C. R. Green,
and
N. J. Severs.
Upregulation of connexin43 gap junctions during early stages of human coronary atherosclerosis.
Arterioscler. Thromb. Vasc. Biol.
15:
1219-1228,
1995
7.
Brink, P. R.,
K. Cronin,
K. Banach,
E. Peterson,
E. M. Westphale,
K. H. Seul,
S. V. Ramanan,
and
E. C. Beyer.
Evidence for heteromeric gap junction channels formed from rat connexin43 and human connexin37.
Am. J. Physiol.
273 (Cell Physiol. 42):
C1386-C1396,
1997[Medline].
8.
Brink, P. R.,
and
S. V. Ramanan.
A model for the diffusion of fluorescent probes in the septate giant axon of earthworm. Axoplasmic diffusion and junctional membrane permeability.
Biophys. J.
48:
299-309,
1985[Abstract].
9.
Brink, P. R.,
S. V. Ramanan,
and
G. J. Christ.
Human connexin 43 gap junction channel gating: evidence for mode shifts and/or heterogeneity.
Am. J. Physiol.
271 (Cell Physiol. 40):
C321-C331,
1996
10.
Bruzzone, R.,
T. W. White,
and
D. L. Paul.
Connections with connexins: the molecular basis of direct intercellular signaling.
Eur. J. Biochem.
238:
1-27,
1996[Abstract].
11.
Christ, G. J.,
D. C. Spray,
M. E. El-Sabban,
L. K. Moore,
and
P. R. Brink.
Gap junctions in vascular tissues: role of heterotypic and homotypic intercellular communication in the modulation of vasomotor tone.
Circ. Res.
79:
631-646,
1996
12.
Doble, B. W.,
Y. Chen,
D. G. Bosc,
D. W. Litchfield,
and
E. Kardami.
Fibroblast growth factor-2 decreases metabolic coupling and stimulates phosphorylation as well as masking of connexin43 epitopes in cardiac myocytes.
Circ. Res.
79:
647-658,
1996
13.
Elfgang, C.,
R. Eckert,
H. Lichtenberg-Frate,
A. Butterweck,
O. Traub,
R. A. Klein,
D. Hulser,
and
K. Willecke.
Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells.
J. Cell Biol.
129:
805-817,
1995[Abstract].
14.
Gibbons, G. H.,
R. E. Pratt,
and
V. J. Dzau.
Vascular smooth muscle cell hypertrophy vs. hyperplasia. Autocrine transforming growth factor-1 expression determines growth response to angiotensin II.
J. Clin. Invest.
90:
456-461,
1992[Medline].
15.
Grunwald, J.,
H. Robenek,
J. Mey,
and
W. H. Hauss.
In vivo and in vitro cellular changes in experimental hypertension: electronmicroscopic and morphometric studies of aortic smooth muscle cells.
Exp. Mol. Pathol.
36:
164-176,
1982[Medline].
16.
Haefliger, J. A.,
E. Castillo,
G. Waeber,
G. E. Bergonzelli,
J. F. Aubert,
E. Sutter,
P. Nicod,
B. Waeber,
and
P. Meda.
Hypertension increases connexin43 in a tissue-specific manner.
Circulation
95:
1007-1014,
1997
17.
Hatch, F. T.,
and
R. S. Lees.
Practical methods for plasma lipoprotein analysis.
Adv. Lipid Res.
6:
1-68,
1968[Medline].
18.
Havel, R. J.,
H. A. Eder,
and
J. H. Bragdon.
The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.
J. Clin. Invest.
34:
1345-1353,
1955[Medline].
19.
He, D. S.,
and
J. M. Burt.
Function of gap junction channels formed in cells co-expressing connexins 40 and 43.
In: Gap Junctions, edited by R. Werner. Amsterdam: IOS, 1998, p. 40-44.
20.
Larson, D. M.,
M. J. Wrobleski,
G. D. V. Sagar,
E. M. Westphale,
and
E. C. Beyer.
Differential regulation of connexin43 and connexin37 in endothelial cells by cell density, growth, and TGF-1.
Am. J. Physiol.
272 (Cell Physiol. 41):
C405-C415,
1997
21.
Lau, A. F.,
M. Y. Kanemitsu,
W. E. Kurata,
S. Danesh,
and
A. L. Boynton.
Epidermal growth factor disrupts gap junctional communication and induces phosphorylation of connexin43 on serine.
Mol. Biol. Cell
3:
865-874,
1992[Abstract].
22.
Little, T. L.,
E. C. Beyer,
and
B. R. Duling.
Connexin43 and connexin40 gap junctional proteins are present in both arteriolar smooth muscle and endothelium in vivo.
Am. J. Physiol.
268 (Heart Circ. Physiol. 37):
H729-H739,
1995
23.
Loewenstein, W. R.
Junctional intercellular communication and the control of growth.
Biochim. Biophys. Acta
560:
1-65,
1979[Medline].
24.
Moore, L. K.,
E. C. Beyer,
and
J. M. Burt.
Characterization of gap junction channels in A7r5 vascular smooth muscle cells.
Am. J. Physiol.
260 (Cell Physiol. 29):
C975-C981,
1991
25.
Moore, L. K.,
and
J. M. Burt.
Gap junction function in vascular smooth muscle: influence of serotonin.
Am. J. Physiol.
269 (Heart Circ. Physiol. 38):
H1481-H1489,
1995
26.
Moreno, A. P.,
J. C. Saez,
G. I. Fishman,
and
D. C. Spray.
Human connexin43 gap junction channels: regulation of unitary conductances by phosphorylation.
Circ. Res.
74:
1050-1057,
1994[Abstract].
27.
Nilsson, J.,
M. Sjolund,
L. Palmberg,
J. Thyberg,
and
C. H. Heldin.
Arterial smooth muscle cells in primary culture produce a platelet-derived growth factor-like protein.
Proc. Natl. Acad. Sci. USA
82:
4418-4422,
1985[Abstract].
28.
Ozer, N. K.,
P. Palozza,
D. Boscoboinik,
and
A. Azzi.
d--Tocopherol inhibits low density lipoprotein induced proliferation and protein kinase C activity in vascular smooth muscle cells.
FEBS Lett.
322:
307-310,
1993[Medline].
29.
Pelletier, D. B.,
and
A. L. Boynton.
Dissociation of PDGF receptor tyrosine kinase activity from PDGF-mediated inhibition of gap junctional communication.
J. Cell. Physiol.
158:
427-434,
1994[Medline].
30.
Pepper, M. S.,
R. Montesano,
A. El Aoumari,
D. Gros,
L. Orci,
and
P. Meda.
Coupling and connexin 43 expression in microvascular and large vessel endothelial cells.
Am. J. Physiol.
262 (Cell Physiol. 31):
C1246-C1257,
1992
31.
Segal, S. S.,
and
B. R. Duling.
Conduction of vasomotor responses in arterioles: a role for cell-to-cell coupling?
Am. J. Physiol.
256 (Heart Circ. Physiol. 25):
H838-H845,
1989
32.
Segal, S. S.,
and
D. T. Kurjiaka.
Coordination of blood flow control in the resistance vasculature of skeletal muscle.
Med. Sci. Sports Exerc.
27:
1158-1164,
1997.
33.
Sneyd, J.,
B. T. R. Wetton,
A. C. Charles,
and
M. J. Sanderson.
Intercellular calcium waves mediated by diffusion of inositol trisphosphate: a two-dimensional model.
Am. J. Physiol.
268 (Cell Physiol. 37):
C1537-C1545,
1995
34.
Stewart, W. W.
Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer.
Cell
14:
741-759,
1978[Medline].
35.
Tsai, M. L.,
S. W. Watts,
R. Loch-Caruso,
and
R. C. Webb.
The role of gap junctional communication in contractile oscillations in arteries from normotensive and hypertensive rats.
J. Hypertens.
13:
1123-1133,
1995[Medline].
36.
Valiunas, V.,
F. Bukauskas,
and
R. Weingart.
Conductances and selective permeability of connexin43 gap junction channels examined in neonatal rat heart cells.
Circ. Res.
80:
708-719,
1997
37.
Van Rijen, H. V. M.,
R. Wilders,
A. C. G. Van Ginneken,
and
H. J. Jongsma.
Quantitative analysis of dual whole-cell voltage-clamp determination of gap junctional conductance.
Pflügers Arch.
436:
141-151,
1998[Medline].
38.
Vindelov, L. L.,
and
I. J. Christensen.
Detergent and proteolytic enzyme-based techniques for nuclear isolation and DNA content analysis.
In: Flow Cytometry, edited by Z. Darzynkiewicz,
J. P. Robinson,
and H. A. Crissman. San Diego, CA: Academic, 1994, p. 219-229.
39.
Wang, H.-Z.,
and
R. D. Veenstra.
Monovalent ion selectivity sequences of the rat connexin43 gap junction channel.
J. Gen. Physiol.
109:
491-507,
1997
40.
Wilders, R.,
and
H. J. Jongsma.
Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels.
Biophys. J.
63:
942-953,
1992[Abstract].
41.
Winkles, J. A.,
R. Friesel,
W. H. Burgess,
R. Howk,
T. Mehlman,
R. Weinstein,
and
T. Maciag.
Human vascular smooth muscle cells both express and respond to heparin-binding growth factor I (endothelial cell growth factor).
Proc. Natl. Acad. Sci. USA
84:
7124-7128,
1987[Abstract].
42.
Yamasaki, H.,
and
C. C. G. Naus.
Role of connexin genes in growth control.
Carcinogenesis
17:
1199-1213,
1996[Medline].