Stimulation of Imidazoline Receptors Inhibits Proliferation of Human Coronary Artery Vascular Smooth Muscle Cells
Abstract Vascular smooth muscle cells of rat aorta express imidazoline receptors whose stimulation, by drugs or an endogenous ligand, agmatine, inhibits serum-stimulated proliferation. We investigated whether imidazoline receptors are expressed in human vascular smooth muscle cells if their stimulation is antiproliferative. Cultured human coronary artery vascular smooth muscle cells express a nonadrenergic binding site for 3H-idazoxan and an imidazoline receptor–binding protein as revealed by immunocytochemical and immunoblot analyses with a specific antibody. Idazoxan and agmatine dose-dependently inhibited serum-stimulated proliferation of these cells as measured by the incorporation of 3H-thymidine (IC50: 5 and 70 μmol/L, respectively) and serum-stimulated expression of proliferating cell nuclear antigen and cell numbers. The agents inhibited proliferation of human and rat (aorta) smooth muscle cells stimulated by either norepinephrine (6560±440 disintegrations per minute norepinephrine versus 3345±220 norepinephrine and idazoxan), angiotensin II (7680±335 disintegrations per minute angiotensin II versus 3769±450 angiotensin II and idazoxan), or platelet-derived growth factor (IC50: 3 μmol/L for idazoxan and 40 μmol/L for agmatine), indicating inhibition of mitosis mediated by G-protein or receptor tyrosine kinase pathways. We conclude that human vascular smooth muscle cells express imidazoline-receptors whose activation inhibits proliferation by interacting at a distal step in an intracellular signal cascade common to G-protein and receptor tyrosine kinase mitogenic pathways. Agmatine, synthesized in endothelium, may act as a paracrine regulator of vascular smooth muscle cell proliferation through imidazoline receptors, and agents acting at this site may be useful in treating vascular hyperplasia.
Imidazoline receptors (I-receptors) are nonadrenergic sites that bind clonidine, idazoxan, and related (“imidazoline”) drugs and also the endogenous ligand agmatine (decarboxylated arginine).1 2 We have recently reported that VSMC of rat aorta express I-receptors of the I2-subclass in vitro and ex vitro.3 The vascular I-receptors are 10 times more prevalent than α2-adrenergic receptors to which all I-receptor ligands also bind.4
Although stimulation of I-receptors has modest, if any, direct action on vascular contraction,5 6 we have discovered that agmatine and some imidazoline drugs can markedly inhibit the growth and proliferation of VSMC of rat aorta, which is stimulated by FCS.3 The antimitotic effects of the agents directly correlate with their affinities at I2-subtype of I-receptors but not I1-subtype or α2-adrenergic receptors. Moreover, the fact that agmatine is synthesized and stored in endothelium7 8 and is present in normal serum9 10 has led us to propose that agmatine and I-receptors may form a natural endothelial/vascular smooth muscle system controlling vascular growth.
In the present study, we sought to determine whether I-receptors are expressed in human VSMC and whether their activation also inhibits stimulated proliferation. Since mitogenic responses can be initiated at the cell surface by receptors coupled to G-protein (eg, angiotensin II, norepinephrine) pathways or by those using receptor tyrosine kinase–linked intracellular pathways (eg, PDGF),11 12 13 we also asked whether the antiproliferative actions are specific to one or both pathways. We report that cultured human coronary artery VSMC express I-receptors and that their activation by idazoxan or agmatine inhibits proliferation whether stimulated by a G-protein or receptor tyrosine kinase pathway.
Isolation and Culturing of VSMC
Primary cultures of rat VSMC were prepared from rat aorta by our modification7 8 of the method of Meyer-Lehnert and Schrier,14 and the viability of the cells was established by exclusion of trypan blue. Cultured VSMC were only used after 5 to 6 passages and then were replaced by freshly prepared cells.
Primary normal VSMC of human coronary artery were obtained from Clonetics Corp and cultured in medium provided by the supplier. These cells are well characterized and were monitored for phenotypic changes by immunostaining with antibodies to α-smooth muscle actin. These cells generally maintain their smooth muscle phenotype for 3 to 4 passages. To obtain sufficient cell membranes, confluent VSMC were used for receptor binding, immunocytochemistry, and immunoblot analysis. Because receptor expression does not change with stages of confluency,3 semiconfluent cultures were used for proliferation studies.
Ligand Binding Assay
Membranes from cultured cells were prepared, and ligand binding assays were performed as described previously.3 Briefly, cells were homogenized in PBS to disrupt the plasma membrane and centrifuged at 600g to remove nucleus and cell debris. The supernatant was centrifuged at 30 000g to obtain the plasma membrane pellet. The membrane fraction was then homogenized in Tris-HCl buffer (pH 7.7) using a Polytron and centrifuged at 30 000g. The membrane pellet was washed three times with Tris-HCl buffer, and the final washed pellet was used for binding assays. Binding assays were performed using 3H-idazoxan (5 nmol/L) as ligand, and nonspecific binding was defined with 10 μmol/L cold idazoxan. Agents that selectively bind to adrenergic receptors such as epinephrine and rauwolscine were used to classify the binding site as adrenergic or nonadrenergic. All binding data were analyzed using the nonlinear curve fitting program ligand.
Western blot analysis of the solubilized membranes was carried out as described previously3 15 with the use of antiserum to I-receptor protein.3 This antiserum was produced against a partially-purified I-receptor protein that has been extensively characterized in previous reports.15 Membranes were prepared from cultured VSMC and solubilized in 50 mmol/L Tris buffer (pH 7.4) that contained 5% sodium dodecyl sulfate and proteins separated on a 10% sodium dodecyl sulfate–polyacrylamide gel. After electrophoresis had been performed, the proteins were electrotransferred onto Immobilon-P membranes. The transferred membranes were washed with PBS/0.05% Tween-20 and incubated with 5% milk at 37°C for 30 minutes to block the remaining protein binding sites. After blocking, membranes were washed twice with PBS and incubated overnight at 4°C with antiserum to I-receptor protein (1/5000 dilution) or preimmune serum.
After exposure to primary antiserum, blots were extensively washed and incubated for 1 hour with horseradish peroxidase–conjugated goat anti-rabbit IgG (1/10 000 dilution). Blots were then extensively washed and incubated for 1 minute in a 1:1 mixture of Amersham Enhanced Chemiluminescence detection solution. Blots were exposed to X-Omat film (Kodak) to visualize the immunoreactive bands.
Cultured VSMC were immunostained with specific antibodies to I-receptor protein,15 PCNA (Sigma), or α-smooth muscle actin (Sigma) by the immunoperoxidase method. Briefly, cultures were fixed with 4% paraformaldehyde and blocks prepared with 0.5% BSA in 0.1 mol/L Tris-saline. Cultures were incubated overnight with antiserum to I-receptor protein, PCNA, or α-smooth muscle actin in 0.1% BSA containing Tris-saline at 4°C. Immunoreactivity was visualized by the sequential addition of (1) biotinylated anti-rabbit or anti-mouse IgG, 1:50 for 30 minutes; (2) avidin-biotin complex, 1:100 for 30 minutes in 0.1% BSA containing Tris-saline, pH 7.4; and (3) 3,3′-diaminobenzidine, 0.44 mg/100 mL Tris-saline with 10 μL of 30% hydrogen peroxide. Triton X-100 (0.1% to 0.3%) was included in all steps to permeabilize the tissue and improve antibody penetration. Cultures were rinsed in Tris-saline between each incubation, dehydrated, and mounted.
Measurement of Proliferation
Proliferation of VSMC was assessed by measuring the replication of DNA by 3H-thymidine incorporation, the expression of proliferating cell nuclear antigen (PCNA), and the assay of cell number by MTT.
Methods for measurement of the incorporation of 3H-thymidine (DNA synthesis) and cell proliferation assays are described elsewhere.3 Briefly, cells were plated onto 24-well plates at a density of approximately 30 000 cells/well in Dulbecco’s modified Eagle’s medium in 10% FCS. After 48 hours, the cells were starved in 0.5% FCS for another 48 hours to decrease proliferation and induce quiescence. Drugs were added for a total period of 72 hours and 3H-thymidine (2 μCi/mL) added during the last 4 hours of incubation. The medium was aspirated and the cells washed three times with PBS and twice with ice-cold 10% trichloroacetic acid. Fixed cells were then solubilized in 0.2 mol/L NaOH and sonicated. An aliquot was used for scintillation counting.
Expression of PCNA, a prerequisite for DNA replication, was assessed by immunocytochemistry and immunoblot analysis using antibodies to PCNA (Sigma). In some experiments, proliferation of cells was determined by measuring the viable cell numbers by cell proliferation assays (Promega) based on the colorimetric measurement of formazon dye formed from tetrazolium salt (MTT) by mitochondrial dehydrogenases. All statistical analyses were performed by Student’s t test.
Binding of 3H-Idazoxan to Membranes of Human Coronary Artery VSMC
In initial studies, binding of 3H-idazoxan to membranes of human coronary artery VSMC was measured to establish the presence of nonadrenergic binding sites. When 5 nmol/L 3H-idazoxan and 200 μg of membrane protein were used, the specific binding was >60% (2200±150 dpm total versus 900±110 dpm nonspecific), indicating significant expression of the binding site. To determine the extent of nonadrenergic binding, the ability of rauwolscine and epinephrine, selective adrenergic agents, to inhibit the binding of 3H-idazoxan was assessed. As shown in Fig 1⇓, whereas idazoxan potently and completely inhibited the binding, rauwolscine and epinephrine failed to inhibit the binding even at very high concentrations. Thus, the binding of 3H-idazoxan was exclusively to nonadrenergic sites that are probably I-receptors of the I2-subtype as observed in rat VSMC.3
Expression of I-Receptor Binding Protein in Human Coronary Artery VSMC
To establish whether human coronary artery VSMC, like rat aorta,3 express I-receptor protein, cultured cells were immunostained or immunoblotted with a polyclonal antiserum to an I-receptor binding protein isolated from bovine adrenal chromaffin cells.16 This antiserum specifically labels I-receptor protein in rat brain and cultured astrocytes, rat aortic VSMC, bovine pulmonary artery endothelial cells, and human brain cells.3 17 18 19 In immunocytochemical analysis, most human coronary artery VSMC were positively stained when exposed to specific antiserum (Fig 2A⇓) but not to preimmune serum (Fig 2C⇓).The staining was cytoplasmic and closely associated with the nucleus (Fig 2B⇓) as in rat.3 The labeled cells were VSMC since all cells in sister cultures were positively stained with antibodies to α-smooth muscle actin (Fig 2D⇓).
For immunoblot (Western) analysis, solubilized membranes of human coronary artery VSMC were exposed to I-receptor antibody or preimmune serum. Antiserum, but not preimmune serum, stained a prominent band of approximately 60 kD (Fig 3⇓). The size of the immunoreactive protein is comparable to that obtained in membranes of rat aortic VSMC, bovine adrenal chromaffin cells,15 and human brain19 indicating that receptors in these species share common structural elements.
Inhibition of Proliferation in Human Coronary Artery VSMC
We investigated whether agmatine or idazoxan would inhibit the stimulated proliferation of human coronary artery VSMC as they do in rat VSMC. Two indices of proliferation were used: incorporation of 3H-thymidine and expression of PCNA, a protein whose expression is a prerequisite for DNA replication.
Idazoxan and agmatine each dose-dependently inhibited the incorporation of 3H-thymidine into human coronary artery VSMC when cells were exposed to 5% FCS (Fig 4⇓). Idazoxan was the more potent and partially, yet significantly, inhibited replication at 1 μmol/L and completely inhibited it at 100 μmol/L. In contrast, agmatine reached significance at 10 μmol/L but even at 1 mmol/L did not totally block the response.
Expression of PCNA was measured by immunocytochemistry using a monoclonal antibody to the protein. As expected, serum-starved human VSMC sparsely express PCNA (Fig 5A⇓). After 24 hours’ exposure to FCS (5%), most of the cells were immunostained with PCNA antiserum (Fig 5B⇓). However, after exposure of serum-starved cells to FCS (5%) in the presence of idazoxan (10 μmol/L), PCNA immunostaining was restricted to only a few cells (Fig 5C⇓). The effect of agmatine was relatively less marked than idazoxan, although smaller numbers of cells express PCNA (Fig 5D⇓) compared with FCS-treated controls.
To determine whether the inhibition of DNA replication results in a decreased number of cells, the viable cell number was measured by the MTT proliferation assay. Stimulation of proliferation by FCS (5%) for 24 hours resulted in approximately a twofold increase in the number of cells compared with control (serum-free) cultures (Fig 6⇓). Exposure of cells to FCS (5%), idazoxan (10 μmol/L), or agmatine (100 μmol/L) significantly reduced the number of cells compared with FCS-treated cultures (Fig 6⇓). Thus, the inhibition of DNA replication by idazoxan or agmatine actually results in a decreased number of viable cells.
To assess whether the reduction of cell numbers was attributable to toxicity of idazoxan or agmatine, we measured the release of lactate dehydrogenase and changes of cellular morphology in cells immunostained with α-smooth muscle actin after drug treatment. Exposure of human coronary artery VSMC to idazoxan (100 μmol/L) or agmatine (1 mmol/L) for 24 hours did not increase the concentration of lactate dehydrogenase in the medium (data not shown) or the morphology of cells stained with α-smooth muscle actin (Fig 7⇓). These results confirm our observations in rat VSMC, in which trypan blue exclusion was used as an index, that neither drug is cytotoxic.
Effects of Idazoxan and Agmatine on Proliferation of VSMC Stimulated by G-Protein–Coupled Receptors or PDGF
VSMC proliferation can be stimulated by agents that act through one of the two principal classes of intracellular signal transduction pathways: those coupled to G-proteins or to receptor tyrosine kinases.20 21 We investigated whether the antimitogenic actions of idazoxan and/or agmatine are specific for one or the other of these pathways.
To differentiate between the two signaling pathways, we investigated the effects of idazoxan or agmatine on the proliferation of rat aortic and human coronary artery VSMC stimulated by (1) norepinephrine or angiotensin II, which act via G-protein coupling, or (2) PDGF, which activates receptor tyrosine kinase.
As expected, norepinephrine (10 μmol/L) and angiotensin II (1 μmol/L) increased the incorporation of 3H-thymidine into quiescent VSMC of rat aorta and human coronary artery by about twofold (Table⇓). The responses were significantly inhibited by idazoxan (10 μmol/L) or agmatine (100 μmol/L) in both species (Table⇓).
Stimulation of quiescent VSMC by PDGF (10 ng/mL) increased 3H-thymidine incorporation by more than eightfold (550±140 dpm control versus 4500±850 dpm PDGF-treated). Idazoxan and agmatine dose-dependently inhibited PDGF-stimulated incorporation in human VSMC (Fig 8⇓). Similar results were obtained using rat aortic VSMC (data not shown). Thus, the antiproliferative actions of idazoxan and agmatine appear to be mediated at a downstream step in the signal transduction cascade from cell surface receptors.
In the present study we sought to determine whether the nonadrenergic binding sites for imidazoline/guanidinium agents, the I-receptor,1 are expressed in VSMC of human arteries and whether exposure of these cells to idazoxan and agmatine, I-receptor ligands, will inhibit VSMC proliferation in vitro. We have discovered that human coronary artery VSMC express I-receptors and that both ligands inhibit proliferation initiated by FCS or by mitogenic agents stimulating proliferation by either G-protein–coupled receptors or PDGF receptor tyrosine kinase–coupled intracellular pathways.
That cultured human coronary artery VSMC express I-receptors has been demonstrated by the facts that cells express a nonadrenergic binding site for 3H-idazoxan, a high-affinity ligand for I-receptors, and a protein cross-reacting with an antibody raised against an I-receptor binding protein, isolated from bovine adrenal medulla.15 The protein appears to be a constituent of I-receptor complex by virtue of its distinct localization and the fact that it shares comparable binding kinetics and drug selectivity with native receptors.16 Moreover, this antiserum recognizes I-receptor protein of various species such as rodent, bovine, and human, suggesting common epitope. While ligand binding studies revealed that I-receptors of rat aortic VSMC are of the I2-subclass, the paucity of human material has not permitted us to fully define the receptor subclass. However, initial ligand binding studies in which 3H-idazoxan was used suggest the presence of a nonadrenergic site in human VSMC that are probably of the I2-subclass.
As in rat aortic VSMC, idazoxan and agmatine inhibit proliferation of human VSMC in vitro. This was demonstrated by a reduction in the incorporation of 3H-thymidine into DNA, by expression of PCNA, and by a reduction in total cell number unattributable to any toxic action of these agents. Idazoxan, which has a higher affinity than agmatine for the I2 receptor, was also a more potent mitogenic inhibitor. Although agmatine, an endogenous polyamine, dose-dependently inhibited proliferation, only partial inhibition was achieved even at 1 mmol/L agmatine. Conceivably, the reason for the partial block could be that agmatine may be metabolized to putrescine22 and to higher polyamines and that these metabolites may stimulate cellular proliferation and thus counteract the antimitotic actions. Moreover, such metabolic turnover of agmatine may also explain why relatively higher concentrations of agmatine are required to observe significant antiproliferative response. Although idazoxan and agmatine both bind to α2-adrenergic receptors, the antiproliferative responses cannot be attributed to that interaction since α2-adrenergic agonists (eg, clonidine) andantagonists (eg, rauwolscine) have no antiproliferative actions.3
The site of action at which I-receptor agents impair stimulated mitogenesis is unknown. Mitogenic stimulation of VSMC can be initiated by two broad classes of cell membrane receptors that couple to two distinct intracellular signal transduction pathways.20 21 Growth factors, particularly PDGF, primarily activate a membrane receptor tyrosine kinase to trigger a series of intracellular events, including activation of PKC and several MAPK. The final enzyme in the cascade is MAPK (ERK 1 and ERK 2), which activates nuclear cyclin kinases (eg, p34 cdc2kinase) to generate expression of Fos and Myc proteins and, hence, promote DNA synthesis and cell division.12 23 24 25 26 27 In contrast, neurotransmitters and hormones,20 including ATP,28 activate G-protein–coupled receptors to generate intracellular second messengers including Ca2+ and diacylglycerol. Increases in diacylglycerol and intercellular Ca2+ activate PKC to promote proliferation either directly by an action at the nucleus or by activating the MAPK signaling cascade.26 The fact that idazoxan and agmatine inhibited proliferation stimulated by norepinephrine or angiotensin II indicates that the antiproliferative response is probably not at the receptor level. Moreover, since these agents also inhibited PDGF-stimulated proliferation, the site of action is not at the cell membrane but rather lies downstream in the signal transduction cascade, probably at a common intracellular site. Whether this site is within the cytosolic signaling pathways (eg, Ca2+ and PKC or MAPK cascade) or at the nuclear transcriptional level (eg, Fos or Myc) is not known.
In conclusion, we have now provided further evidence that human VSMC express I-receptors and that agonists at this site, including endogenous agmatine, inhibit proliferation of human and rat VSMC. These findings support the hypothesis that I-receptor/agmatine may be a novel system regulating growth and proliferation of VSMC. Thus, selective activation of I2 imidazoline sites by idazoxan and related compounds may be a novel strategy for the treatment of disorders resulting from increased proliferation of vascular VSMC.
Selected Abbreviations and Acronyms
|BSA||=||bovine serum albumin|
|dpm||=||disintegrations per minute|
|FCS||=||fetal calf serum|
|MAPK||=||mitogen-activated protein kinases|
|MTT||=||3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide|
|PCNA||=||proliferating cell nuclear antigen|
|PDGF||=||platelet-derived growth factor|
|PKC||=||protein kinase C|
|VSMC||=||vascular smooth muscle cells|
Reprint requests to S. Regunathan, PhD, Division of Neurobiology, Cornell University Medical College, 411 E 69th St, Room KB410, New York, NY 10021.
- Received August 18, 1996.
- Revision received October 12, 1996.
- Accepted January 16, 1997.
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