RESUMEN
Ca(2+) channel blockers, such as amlodipine, inhibit vascular smooth muscle cell (VSMC) growth through interactions with targets other than L-type Ca(2+) channels. The effects of amlodipine on Ca(2+) movements in thrombin- and thapsigargin-stimulated VSMCs were therefore investigated by determining the variations of intracellular free Ca(2+) concentration in fura 2-loaded cultured VSMCs. Results indicated that 10-1,000 nM amlodipine inhibited 1) thrombin-induced Ca(2+) mobilization from a thapsigargin-sensitive pool and 2) thapsigargin-induced Ca(2+) responses, including Ca(2+) mobilization from internal stores and store-operated Ca(2+) entry. These effects of amlodipine do not involve L-type Ca(2+) channels and could not be reproduced with 100 nM isradipine, diltiazem, or verapamil. The inhibition by amlodipine of Ca(2+) mobilization appears therefore to be a specific property of the drug, in addition to its Ca(2+) channel-blocking property. It is suggested that amlodipine acts in this capacity by interacting with Ca(2+)-ATPases of the sarcoplasmic reticulum, thus modulating the enzyme activity. This mechanism might participate in the inhibitory effect of amlodipine on VSMC growth.
Asunto(s)
Amlodipino/farmacología , Calcio/metabolismo , Músculo Liso Vascular/metabolismo , Tapsigargina/farmacología , Trombina/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Células Cultivadas , Diltiazem/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Líquido Intracelular/metabolismo , Isradipino/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Cloruro de Potasio/farmacología , Ratas , Ratas Endogámicas WKY , Retículo Sarcoplasmático/metabolismo , Transducción de Señal/efectos de los fármacos , Trombina/farmacología , Verapamilo/farmacologíaRESUMEN
The replication and activation of both vascular smooth muscle cells and macrophages, which have previously entered the arterial wall, are key events in the atherosclerotic process. The importance of macrophage colony-stimulating factor (MCSF) in control of the growth/proliferation of both cell types confers to this compound a central role in the development of vascular lesions. In order to gain insight into the mechanisms of macrophage proliferation, we investigated the effect of MCSF upon the proliferation of DEL cells. DEL cells constitute a monocyte/histiocytic cell line that differentiates along a macrophage lineage following exposure to phorbol ester. DEL cells constitutively express MCSF, and its receptor MCSFR is encoded by c-fms. We examined whether MCSF might play a role in the proliferation of cultured DEL cells. [3H]Thymidine or 5-bromo-2-deoxyuridine (BrdU) incorporation was measured following the addition of recombinant MCSF or L929 cell supernatant (as a source of MCSF) to quiescent DEL cells. In DEL cells, serum-free L929 cell supernatant induced DNA synthesis in a dose-dependent manner, and such an effect could be blunted by pretreatment of L929 cell supernatant with anti-mouse MCSF antibody. In these cells, DNA synthesis could also be triggered in a dose-dependent manner by the addition of recombinant human MCSF (rh MCSF) or thrombin. These findings clearly show that MCSF influences DEL cell proliferation and suggest an autocrine loop activation. They indicate that MCSF plays an important role in the development of vascular lesions, which occur during atherosclerotic progression.
Asunto(s)
Histiocitos/citología , Factor Estimulante de Colonias de Macrófagos/fisiología , Monocitos/citología , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Histiocitos/metabolismo , Humanos , Factor Estimulante de Colonias de Macrófagos/farmacología , Monocitos/metabolismo , Proteínas Recombinantes/farmacología , Trombina/farmacologíaRESUMEN
The abnormal proliferation of vascular smooth muscle cells (VSMCs) is closely related to vascular diseases. There is growing evidence that calcium antagonists inhibit VSMC growth/proliferation, yet their molecular mechanisms remain to be determined. Recent reports suggest that p42/p44 mitogen-activated protein kinases (MAPKs) play an important role in cell growth and proliferation induced by growth factors. This study was designed to determine whether these MAPKs are involved in VSMC proliferation induced by basic fibroblast growth factor (bFGF) and to examine the inhibitory effect of amlodipine. Human VSMCs were obtained from inner mammary artery. p42/p44 MAPKs activity was measured by immunoblotting assay using anti-p42/p44 phospho-MAPK antibody. 1) bFGF (20 ng/ml) significantly activated p42/p44 MAPKs with a peak time of 5-15 min, which was maintained for 3 h. PD98059 (100 nM-10 microM), a specific inhibitor of MAPK kinase, inhibited bFGF-induced p42/p44 MAPKs activation in a dose-dependent manner. 2) Amlodipine (1-100 nM) dose-dependently inhibited p42/p44 MAPKs activation by bFGF. 3) Amlodipine (10 nM) could inhibit both short-term and long-term p42/p44 MAPKs activation by bFGF. Our results indicate that bFGF could activate p42/p44 MAPKs. Amlodipine, which could inhibit bFGF-induced human VSMC proliferation, inhibited both short-term and sustained p42/p44 MAPKs activation by bFGF, suggesting that bFGF-induced VSMC proliferation may be related to p42/p44 MAPKs activation, and that the antiproliferative effect of amlodipine may be related to its inhibition of p42/p44 MAPKs activation.
Asunto(s)
Amlodipino/farmacología , Inhibidores Enzimáticos/farmacología , Músculo Liso Vascular/citología , División Celular/efectos de los fármacos , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Flavonoides/farmacología , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/enzimología , Factores de TiempoRESUMEN
OBJECTIVES: To determine the mechanisms whereby calcium channel blockers (CCBs) control the reactivity of vascular smooth muscle cells (VSMCs). BACKGROUND: Although CCBs are known to play an important role in the calcium homeostasis of VSMCs, they are suspected to exert additional effects in this cell type. Thus, the possibility that CCBs could affect VSMC growth/proliferation through a mechanism distinct from the inhibition of calcium channels was investigated. METHODS: VSMCs were isolated from rat aortae and cultured. The influence of nifedipine and amlodipine on basic fibroblast growth factor (bFGF)-stimulated DNA synthesis and proliferation was studied by measuring bFGF-induced BrdU incorporation into VSMCs and cell counts, respectively. The influence of amlodipine (and of isradipine) on the mobilization of intracellular Ca2+ stores was determined by studying the fluorescence of thapsigargin-stimulated VSMCs pre-labeled with the fluoroprobe Fura-2. RESULTS: Both nifedipine and amlodipine inhibited bFGF-induced VSMC growth/proliferation. In the case of nifedipine but not in that of amlodipine, this inhibitory effect could be accounted for by the L-type Ca(2+)-channel antagonist property of the drug. On the other hand, amlodipine but not isradipine, diltiazem, and verapamil, did inhibit thapsigargin-induced Ca2+ mobilization. CONCLUSIONS: These findings suggest that in addition to its L-type Ca(2+)-channel antagonist property, amlodipine also exerts a "thapsigargin-like" activity which, together with its particular antioxidant property, might participate in its antiatherogenic potency.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Calcio/metabolismo , División Celular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Amlodipino/farmacología , Animales , Células Cultivadas , Nifedipino/farmacología , Ratas , Ratas Endogámicas WKY , Relación Estructura-ActividadRESUMEN
Release of superoxide anion by cultured vascular cells was investigated with the use of selective microelectrodes. Local concentration of superoxide anion (O2*-) was followed by differential pulse amperometry on a carbon microfiber at 0.1 V/SCE. The oxidation current allows O2*- detection in the 10(-8) M concentration range without interference of the other major oxygen species. Interleukin-1beta-stimulated O2*- release that progressively increased to reach local concentrations at the cell membrane level of 76 +/- 11 nm 40-60 min after stimulation in human cord vein endothelial cells, and 131 +/- 18 nm 1-2 h after stimulation in internal mammary artery smooth muscle cells. In the two types of cells, the O2*- oxidation signal was suppressed in the presence of superoxide dismutase. Spontaneous O2*-release from unstimulated cells was undetectable. These results demonstrate that selective microelectrodes allow direct and real-time monitoring of local O2*- released from vascular endothelial as well as from smooth muscle cells submitted to an inflammatory stimulus.
Asunto(s)
Interleucina-1/farmacología , Músculo Liso Vascular/efectos de los fármacos , Superóxidos/análisis , Células Cultivadas , Electroquímica/métodos , Endotelio Vascular/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Microelectrodos , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismoRESUMEN
Angiotensin II (AII), an important hypertrophic factor in the cardiovascular system, exerts most of its known effects in vivo through the AII receptor type 1 (AT1) subclass of AII receptors. These receptors are also responsible for the growth-related effects of AII in cultured vascular smooth muscle cells (VSMCs). We presently investigated the effects of BAY 10-6734 (Embusartan), a new orally active AT1 antagonist, on VSMC growth and proliferation of cultured VSMCs isolated from the aortae of Wistar Kyoto rats and spontaneously hypertensive rats. BAY 10-6734 and losartan (considered as AT1 receptor antagonist of reference), as well as their respective active metabolites, were studied for their inhibition of: 1) [125I]AII binding to its receptors, 2) AII-induced DNA and protein synthesis (by measuring the incorporation of 5-bromo-2'-deoxyuridine and [3H]L-leucine, respectively), and 3) AII-induced variations in intracellular Ca2+ concentration, using cells labeled with Fura-2. All of the tested compounds inhibited the aforementioned parameters in a concentration-dependent manner. Half-maximal inhibitory concentration values indicated that BAY 10-6734 was significantly more potent than losartan and that spontaneously hypertensive rat-derived VSMCs were more sensitive than Wistar Kyoto rat-derived ones. Neither BAY 10-6734 nor losartan affected the intracellular Ca2+ concentration of unstimulated VSMCs but both compounds inhibited both AII-induced Ca2+ mobilization from internal stores and Ca2+ influx. Neither compound affected arginine-vasopressin-, basic fibroblast growth factor-, or serum-induced DNA and protein synthesis. BAY 10-6734 appears therefore as a potent and specific new inhibitor of AII-induced growth-related events in VSMCs.
Asunto(s)
Angiotensina II/metabolismo , Antagonistas de Receptores de Angiotensina , Dihidropiridinas/farmacología , Músculo Liso Vascular/efectos de los fármacos , Tetrazoles/farmacología , Animales , Calcio/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Citosol/efectos de los fármacos , Citosol/metabolismo , ADN/biosíntesis , Imidazoles/farmacología , Losartán/farmacología , Masculino , Músculo Liso Vascular/citología , Biosíntesis de Proteínas , Ensayo de Unión Radioligante , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Especificidad de la EspecieRESUMEN
Atherosclerosis, like several other vascular diseases, exhibits structural and functional abnormalities resulting partially from an exaggerated proliferation of vascular smooth-muscle cells (VSMCs). Ca2+ channel blockers, such as amlodipine, have been suggested to retard or even prevent the progression of atherosclerosis. To determine the mechanisms involved in these effects, we investigated the influence of amlodipine on VSMC proliferation by using rat aortic VSMCs in culture. Amlodipine (0.1-10 microM) inhibited serum-, basic fibroblast growth factor (bFGF)-, and thrombin-induced VSMC proliferation and DNA synthesis in a concentration-dependent manner, as demonstrated by cell count and bromodeoxyuridine (BrdU)-incorporation measurements, respectively. Delayed addition of amlodipine after VSMC stimulation showed that the drug exerted its effect early in G1 phase of the cell cycle. This observation was confirmed by the finding that amlodipine did not influence DNA synthesis in VSMCs arrested to the G1/S boundary by hydroxyurea treatment. Consistent with its effects on VSMC growth/proliferation, amlodipine also decreased c-myc, c-fos, and c-jun protooncogene expression induced by serum, thrombin, or bFGF within 1 h after cell activation, as assessed by semiquantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis. The calcium channel agonist Bay K 8644, which counteracted the inhibition by nifedipine of bFGF-, thrombin- or serum-induced DNA synthesis, was ineffective to antagonize the inhibitory effect of amlodipine. The aforementioned effects of amlodipine were of similar amplitude, irrespective of the growth-enhancing agent used. This strongly indicates that amlodipine acts downstream of receptor activation to exert its antiproliferative action, probably early in the G1 phase of the cell cycle. Moreover, the lack of antagonistic effect between amlodipine and Bay K 8644 suggests that, in addition to its L-type Ca2+ channel inhibitory effect, amlodipine inhibits other intracellular signaling pathways. Such an interference of amlodipine with mitogenic signaling pathways might contribute to confer a blood vessel-protecting potential on amlodipine.
Asunto(s)
Amlodipino/farmacología , Antihipertensivos/farmacología , Factores de Crecimiento de Fibroblastos/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Trombina/efectos de los fármacos , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , División Celular/efectos de los fármacos , ADN/biosíntesis , ADN/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/fisiología , Masculino , Músculo Liso Vascular/citología , Oncogenes/efectos de los fármacos , Ratas , Ratas Endogámicas WKY , Trombina/fisiologíaRESUMEN
Cell growth and proliferation were evaluated in cultured vascular smooth muscle cells (VSMCs) isolated from spontaneously hypertensive rat (SHR) and normotensive Wistar-Kyoto (WKY) rat aortae by measuring [3H]-thymidine incorporation into newly synthesized DNA and by determining cell number, respectively. The results showed that in cultures from both rat strains (1) serum-, basic fibroblast growth factor (bFGF)- and thrombin-induced DNA synthesis were inhibited by L-phenylalanine dose-dependently; (2) L-phenylalanine inhibited cell proliferation in response to serum in a concentration-dependent manner; (3) L-phenylalanine inhibited serum-induced proto-oncogene c-fos and c-myc expression; (4) L-tyrosine, L-histidine and D-phenylalanine failed to mimic the inhibitory effect of L-phenylalanine. All these data demonstrate that L-phenylalanine could exert a direct and specific antiproliferative effect on VSMCs suggesting that such effect can account for the antihypertensive action of this amino acid observed in SHR.
Asunto(s)
ADN/biosíntesis , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Músculo Liso Vascular/citología , Fenilalanina/farmacología , Animales , Aorta Torácica/citología , División Celular/efectos de los fármacos , Células Cultivadas , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
In both atherosclerosis and arterial hypertension, structural and functional abnormalities result in vascular hypertrophy that is associated with an increased ratio of vascular media thickness to lumen diameter and hyperreactivity of vascular smooth muscle cells (VSMCs), resulting in uncontrolled cell migration and growth in vivo. In culture, VSMCs isolated from the spontaneously hypertensive rat (SHR) also display exaggerated growth and/or proliferation compared to VSMCs isolated from normotensive control Wistar Kyoto (WKY) rats. In vitro studies of cultured VSMCs can therefore be used as a model to investigate the mechanisms whereby a drug such as amlodipine can exert its antihypertensive and antiatherogenic effects. The present in vitro investigations examine the mechanisms whereby amlodipine reduces VSMC growth/proliferation promoted by basic fibroblast growth factor (bFGF), a peptide growth factor likely to participate in the vascular smooth muscle hypertrophy of the SHR. VSMCs from SHR and/or WKY rat aortae were isolated, passaged, and cultured. The influence of amlodipine on VSMC growth/proliferation was studied by measuring DNA synthesis and cell number under experimental conditions, which allowed us to determine the cell cycle phase in which amlodipine exerts its effects. Amlodipine was found to inhibit growth and bFGF-induced DNA synthesis in a concentration-dependent manner. Delayed addition of amlodipine showed that the drug exerts its effect early in the G1 phase, a result that was confirmed by the finding that amlodipine could not inhibit bFGF-induced DNA synthesis in VSMCs arrested at the G1/S boundary. In comparative experiments, the inhibitory effect of amlodipine on both cell growth and DNA synthesis was found to be of similar magnitude in SHR- and WKY-derived VSMCs. It is therefore likely that by modulating cell growth/proliferation induced by bFGF, amlodipine may reduce the vascular hypertrophy of the SHR. Since amlodipine also has been found to inhibit VSMC migration, one may reasonably envisage that these characteristics are important components of the antiatherogenic properties of the drug.