RESUMEN
OBJECTIVE: The aim was to determine if endothelial VCAM-1 (eVCAM-1) expression in the common carotid artery (CCA) would correlate with predictive markers of atherosclerotic disease, would precede reduction of markers of endothelial cell function and would predict coronary artery disease (CAD). METHODS AND RESULTS: Carotid arterial segments (bifurcation, proximal and distal CCA) were harvested from 14 and 24 month-old male castrated familial hypercholesterolemic (FH) swine, a model of spontaneous atherosclerosis. Quantification of local expression of eVCAM-1, intimal macrophage accumulation, oxidative stress, intima-media (I/M) ratio, intima-media thickness (IMT), endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (p-eNOS) in selected regions of the carotids revealed a relationship between local inflammation and atheroscle-rotic plaque progression. Importantly, inflammation was not uniform throughout the CCA. Endo-thelial VCAM-1 expression was the greatest at the bifurcation and increased with age. Finally, eV-CAM-1 best estimated the severity of CAD compared to blood levels of glucose, hypercholestero-lemia, carotid IMT, and p-eNOS. CONCLUSION: Our data suggested that eVCAM-1 was closely associated with atherosclerotic plaque progression and preceded impairment of EDD. Thus, this study supported the use of carotid VCAM-1 targeting agents to estimate the severity of CAD.
Asunto(s)
Aterosclerosis/prevención & control , Enfermedad Coronaria/prevención & control , Ejercicio Físico , Actividad Motora , Conducta de Reducción del Riesgo , Aterosclerosis/etiología , Aterosclerosis/fisiopatología , Enfermedad Coronaria/etiología , Enfermedad Coronaria/fisiopatología , Humanos , Servicios Preventivos de Salud , Medición de Riesgo , Factores de RiesgoRESUMEN
The intermediate-conductance Ca(2+)-activated K(+) channel (K(Ca)3.1) was first described by Gardos in erythrocytes and later confirmed to play a significant role in T-cell activation and the immune response. More recently, K(Ca)3.1 has been characterized in numerous cell types which contribute to the development of vascular disease, such as T-cells, B-cells, endothelial cells, fibroblasts, macrophages, and dedifferentiated smooth muscle cells (SMCs). Physiologically, K(Ca)3.1 has been demonstrated to play a role in acetylcholine and endothelium-derived hyperpolarizing factor (EDHF) induced hyperpolarization, and thus control of blood pressure. Pathophysiologically, K(Ca)3.1 contributes to proliferation of T-cells, B-cells, fibroblasts, and vascular SMCs, as well as the migration of SMCs and macrophages and platelet coagulation. Recent studies have indicated that blockade of K(Ca)3.1, by specific blockers such as TRAM-34, could prove to be an effective treatment for vascular disease by inhibiting T-cell activation as well as preventing proliferation and migration of macrophages, endothelial cells, and SMCs. This vasculoprotective potential of K(Ca)3.1 inhibition has been confirmed in both rodent and swine models of restenosis. In this review, we will discuss the physiological and pathophysiological role of K(Ca)3.1 in cells closely associated with vascular biology, and the effect of K(Ca)3.1 blockers on the initiation and progression of vascular disease.
Asunto(s)
Canales de Potasio Calcio-Activados/antagonistas & inhibidores , Enfermedades Vasculares/tratamiento farmacológico , Animales , Endotelio Vascular , Humanos , Canales de Potasio Calcio-Activados/fisiología , Sustancias Protectoras/uso terapéutico , Enfermedades Vasculares/patología , Enfermedades Vasculares/fisiopatologíaRESUMEN
OBJECTIVE: We previously demonstrated that upregulation of intermediate-conductance Ca(2+)-activated K(+) channels (K(Ca)3.1) is necessary for mitogen-induced phenotypic modulation in isolated porcine coronary smooth muscle cells (SMCs). The objective of the present study was to determine the role of K(Ca)3.1 in the regulation of coronary SMC phenotypic modulation in vivo using a swine model of postangioplasty restenosis. METHODS AND RESULTS: Balloon angioplasty was performed on coronary arteries of swine using either noncoated or balloons coated with the specific K(Ca)3.1 blocker TRAM-34. Expression of K(Ca)3.1, c-jun, c-fos, repressor element-1 silencing transcription factor (REST), smooth muscle myosin heavy chain (SMMHC), and myocardin was measured using qRT-PCR in isolated medial cells 2 hours and 2 days postangioplasty. K(Ca)3.1, c-jun, and c-fos mRNA levels were increased 2 hours postangioplasty, whereas REST expression decreased. SMMHC expression was unchanged at 2 hours, but decreased 2 days postangioplasty. Use of TRAM-34 coated balloons prevented K(Ca)3.1 upregulation and REST downregulation at 2 hours, SMMHC and myocardin downregulation at 2 days, and attenuated subsequent restenosis 14 and 28 days postangioplasty. Immunohistochemical analysis demonstrated corresponding changes at the protein level. CONCLUSIONS: Blockade of K(Ca)3.1 by delivery of TRAM-34 via balloon catheter prevented smooth muscle phenotypic modulation and limited subsequent restenosis.
Asunto(s)
Estenosis Coronaria/prevención & control , Vasos Coronarios/efectos de los fármacos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Músculo Liso Vascular/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Pirazoles/farmacología , Angioplastia Coronaria con Balón/efectos adversos , Animales , Diferenciación Celular/efectos de los fármacos , Estenosis Coronaria/patología , Vasos Coronarios/metabolismo , Vasos Coronarios/patología , Modelos Animales de Enfermedad , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Masculino , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Fenotipo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Porcinos , Factores de Transcripción/metabolismoRESUMEN
Sex hormone status has emerged as an important modulator of coronary physiology and cardiovascular disease risk in both males and females. Our previous studies have demonstrated that testosterone increases protein kinase C (PKC) delta expression and activity in coronary smooth muscle (CSMC). Because PKCdelta has been implicated in regulation of proliferation and apoptosis in other cell types, we sought to determine if testosterone modulates CSMC proliferation and/or apoptosis through PKCdelta. Porcine CSMC cultures (passages 2-6) from castrated males were treated with testosterone for 24 h. Testosterone (20 and 100 nM) decreased [(3)H]thymidine incorporation in proliferating CSMC to 59 +/- 5.3 and 33.1 +/- 4.5% of control. Flow cytometric analysis demonstrated that testosterone induced G(1) arrest in CSMC with a concomitant reduction in the S phase cells. Testosterone reduced protein levels of cyclins D(1) and E and phosphorylation of retinoblastoma protein while elevating levels of p21(cip1) and p27(kip1). There were no significant differences in the levels of cyclins D(3), CDK2, CDK4, or CDK6. Testosterone significantly reduced kinase activity of CDK2 and -6, but not CDK4, -7, or -1. PKCdelta small interfering RNA (siRNA) prevented testosterone-mediated G(1) arrest, p21(cip1) upregulation, and cyclin D(1) and E downregulation. Furthermore, testosterone increased CSMC apoptosis in a dose-dependent manner, which was blocked by either PKCdelta siRNA or caspase 3 inhibition. These findings demonstrate that the anti-proliferative, pro-apoptotic effects of testosterone on CSMCs are substantially mediated by PKCdelta.
Asunto(s)
Apoptosis , Proliferación Celular , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteína Quinasa C-delta/metabolismo , Testosterona/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Inhibidores de Caspasas , Castración , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Vasos Coronarios/citología , Vasos Coronarios/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Fase G1/efectos de los fármacos , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/patología , Proteína Quinasa C-delta/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Fase S/efectos de los fármacos , Porcinos , Testosterona/farmacologíaRESUMEN
A hallmark of smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and restenosis is suppression of SMC differentiation marker genes, proliferation, and migration. Blockade of intermediate-conductance Ca(2+)-activated K(+) channels (IKCa1) has been shown to inhibit restenosis after carotid balloon injury in the rat; however, whether IKCa1 plays a role in SMC phenotypic modulation is unknown. Our objective was to determine the role of IKCa1 channels in regulating coronary SMC phenotypic modulation and migration. In cultured porcine coronary SMCs, platelet-derived growth factor-BB (PDGF-BB) increased TRAM-34 (a specific IKCa1 inhibitor)-sensitive K(+) current 20-fold; increased IKCa1 promoter histone acetylation and c-jun binding; increased IKCa1 mRNA approximately 4-fold; and potently decreased expression of the smooth muscle differentiation marker genes smooth muscle myosin heavy chain (SMMHC), smooth muscle alpha-actin (SMalphaA), and smoothelin-B, as well as myocardin. Importantly, TRAM-34 completely blocked PDGF-BB-induced suppression of SMMHC, SMalphaA, smoothelin-B, and myocardin and inhibited PDGF-BB-stimulated migration by approximately 50%. Similar to TRAM-34, knockdown of endogenous IKCa1 with siRNA also prevented the PDGF-BB-induced increase in IKCa1 and decrease in SMMHC mRNA. In coronary arteries from high fat/high cholesterol-fed swine demonstrating signs of early atherosclerosis, IKCa1 expression was 22-fold higher and SMMHC, smoothelin-B, and myocardin expression significantly reduced in proliferating vs. nonproliferating medial cells. Our findings demonstrate that functional upregulation of IKCa1 is required for PDGF-BB-induced coronary SMC phenotypic modulation and migration and support a similar role for IKCa1 in coronary SMC during early coronary atherosclerosis.
Asunto(s)
Vasos Coronarios/citología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/fisiología , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/fisiología , Regulación hacia Arriba , Actinas/genética , Animales , Becaplermina , Biomarcadores , Técnicas de Cultivo de Célula , Diferenciación Celular , División Celular , Movimiento Celular , Células Cultivadas , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Modelos Biológicos , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/citología , Cadenas Pesadas de Miosina/genética , Fenotipo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-sis , Pirazoles/farmacología , ARN Mensajero/metabolismo , Porcinos , Porcinos Enanos , Túnica Media/citologíaRESUMEN
Glucocorticoids (GC) exert diverse cellular effects in response to both acute and chronic stress, the functional consequences of which have been implicated in the development of cardiovascular pathology such as hypertension and atherosclerosis. However, the mechanisms by which GCs activate divergent signaling pathways are poorly understood. The present study examined the direct effects of natural (cortisol) and synthetic (dexamethasone) GCs on protein kinase C (PKC) isoform expression in coronary arteries. Porcine right coronary arteries were treated in vitro for 18 h in the presence and absence of either dexamethasone (10, 100, or 500 nM) or cortisol (50, 125, 250, or 500 nM). PKC isoform levels and subcellular distribution were determined by immmunoblotting of whole cell homogenates and immunocytofluorescence using PKC-alpha, -betaII, -epsilon, -delta, and -zeta specific antibodies. Dexamethasone caused a approximately 4-fold increase in PKC-alpha, a approximately 2.5-fold increase in PKC-betaII, and a 2-fold increase in PKC-epsilon (p<0.05). In contrast, dexamethasone had no effect on PKC-delta or PKC- zeta levels. Dexamethasone also caused an increase in the activity of PKC-alpha (285%), -betaII (170%), and -epsilon (210%). Cortisol produced similar effects on PKC isoform expression. Confocal microscopy revealed that while dexamethasone altered localization patterns for PKC-alpha, -betaII and -epsilon, no such effect was observed for PKC-delta or PKC-zeta. The stimulatory effects of dexamethasone and cortisol on coronary PKC levels and translocation were prevented by the GC receptor (GR) blocker, RU486. These results demonstrate, for the first time, that GCs modulate coronary PKC expression and subcellular distribution in an isoform-specific manner through a GR-dependent mechanism.
Asunto(s)
Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/enzimología , Glucocorticoides/farmacología , Proteína Quinasa C/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Isoenzimas/biosíntesis , Isoenzimas/metabolismo , Masculino , Técnicas de Cultivo de Órganos , Proteína Quinasa C/biosíntesis , Fracciones Subcelulares/enzimología , Fracciones Subcelulares/metabolismo , PorcinosRESUMEN
Hypercholesterolemic patients display reduced coronary flow reserve in response to adenosine infusion. We previously reported that voltage-dependent K+ (Kv) channels contribute to adenosine-mediated relaxation of coronary arterioles isolated from male miniature swine. For this study, we hypothesized that hypercholesterolemia attenuates Kv channel contribution to adenosine-induced vasodilatation. Pigs were randomly assigned to a control or high fat/high cholesterol diet for 20-24 wk, and then killed. After completion of the experimental treatment, arterioles (approximately 150 microm luminal diameter) were isolated from the left-ventricular free wall near the apical region of the heart, cannulated, and pressurized at 40 mmHg. Adenosine-mediated relaxation was significantly attenuated in both endothelium-intact and -denuded arterioles from hypercholesterolemic compared with control animals. The classic Kv channel blocker, 4-aminopyridine (1 mM), significantly attenuated adenosine-mediated relaxation in arterioles isolated from control but not hypercholesterolemic animals. Furthermore, the nonselective K+ channel blocker, tetraethylammonium (TEA; 1 mM) significantly attenuated adenosine-mediated relaxation in arterioles from control but not hypercholesterolemic animals. In additional experiments, coronary arteriolar smooth muscle cells were isolated, and whole cell Kv currents were measured. Kv currents were significantly reduced (approximately 15%) in smooth muscle cells from hypercholesterolemic compared with control animals. Furthermore, Kv current sensitive to low concentrations of TEA was reduced (approximately 45%) in smooth muscle cells from hypercholesterolemic compared with control animals. Our data indicate that hypercholesterolemia abolishes Kv channel contribution to adenosine-mediated relaxation in coronary arterioles, which may be attributable to a reduced contribution of TEA-sensitive Kv channels in smooth muscle of hypercholesterolemic animals.
Asunto(s)
Vasos Coronarios/fisiología , Hipercolesterolemia/fisiopatología , Canales de Potasio con Entrada de Voltaje/fisiología , Vasodilatación/fisiología , Adenosina/farmacología , Animales , Arteriolas/efectos de los fármacos , Arteriolas/fisiología , Colesterol en la Dieta/farmacología , Vasos Coronarios/efectos de los fármacos , Grasas de la Dieta/farmacología , Masculino , Potasio/metabolismo , Porcinos , Porcinos Enanos , Vasodilatación/efectos de los fármacosRESUMEN
Vascular smooth muscle cell (SMC) contraction is mediated in part by calcium influx through L-type voltage-gated Ca2+ channels (VGCC) and activation of the RhoA/Rho kinase (ROK) signaling cascade. We tested the hypothesis that Ca2+ influx through VGCCs regulates SMC differentiation marker expression and that these effects are dependent on RhoA/ROK signaling. Depolarization-induced activation of VGCCs resulted in a nifedipine-sensitive increase in endogenous smooth muscle myosin heavy chain (SMMHC) and SM alpha-actin expression and CArG-dependent promoter activity, as well as c-fos promoter activity. The ROK inhibitor, Y-27632, prevented depolarization-induced increase in SMMHC/SM alpha-actin but had no effect on c-fos expression. Conversely, the Ca2+/calmodulin-dependent kinase inhibitor, KN93, prevented depolarization-induced increases in c-fos expression with no effect on SMMHC/SM alpha-actin. Depolarization increased expression of myocardin, a coactivator of SRF that mediates CArG-dependent transcription of SMC marker gene promoters containing paired CArG cis regulatory elements (SMMHC/SM alpha-actin). Both nifedipine and Y-27632 prevented the depolarization-induced increase in myocardin expression. Moreover, short interfering RNA (siRNA) specific for myocardin attenuated depolarization-induced SMMHC/SM alpha-actin transcription. Chromatin immunoprecipitation (ChIP) assays revealed that depolarization increased SRF enrichment of the CArG regions in the SMMHC, SM alpha-actin, and c-fos promoters in intact chromatin. Whereas Y-27632 decreased basal and depolarization-induced SRF enrichment in the SMMHC/SM alpha-actin promoter regions, it had no effect of SRF enrichment of c-fos. Taken together, these results provide evidence for a novel mechanism whereby Ca2+ influx via VGCCs stimulates expression of SMC differentiation marker genes through mechanisms that are dependent on ROK, myocardin, and increased binding of SRF to CArG cis regulatory elements.
Asunto(s)
Canales de Calcio Tipo L/fisiología , Regulación del Desarrollo de la Expresión Génica , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Proteínas Serina-Treonina Quinasas/fisiología , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Actinas/fisiología , Animales , Aorta , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas/citología , Células Cultivadas/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes fos , Péptidos y Proteínas de Señalización Intracelular , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Cadenas Pesadas de Miosina/fisiología , Nifedipino/farmacología , Proteínas Nucleares/fisiología , Organoides/citología , Técnicas de Placa-Clamp , Cloruro de Potasio/farmacología , Transporte de Proteínas , ARN Interferente Pequeño/farmacología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Elemento de Respuesta al Suero/genética , Factor de Respuesta Sérica/fisiología , Transactivadores/fisiología , Transfección , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/fisiologíaRESUMEN
Evidence indicates that gender and sex hormonal status influence cardiovascular physiology and pathophysiology. We recently demonstrated increased L-type voltage-gated Ca2+ current (ICa,L) in coronary arterial smooth muscle (CASM) of male compared with female swine. The promoter region of the L-type voltage-gated Ca2+ channel (VGCC) (Cav1.2) gene contains a hormone response element that is activated by testosterone. Thus the purpose of the present study was to determine whether endogenous testosterone regulates CASM ICa,L through regulation of VGCC expression and activity. Sexually mature male and female Yucatan swine (7-8 mo; 35-45 kg) were obtained from the breeder. Males were left intact (IM, n=8), castrated (CM, n=8), or castrated with testosterone replacement (CMT, n=8; 10 mg/day Androgel). Females remained gonad intact (n=8). In right coronary arteries, both Cav1.2 mRNA and protein were greater in IM compared with intact females. Cav1.2 mRNA and protein were reduced in CM compared with IM and restored in CMT. In isolated CASM, both peak and steady-state ICa were reduced in CM compared with IM and restored in CMT. In males, a linear relationship was found between serum testosterone levels and ICa. In vitro, both testosterone and the nonaromatizable androgen, dihydrotestosterone, increased Cav1.2 expression. Furthermore, this effect was blocked by the androgen receptor antagonist cyproterone. We conclude that endogenous testosterone is a primary regulator of Cav1.2 expression and activity in coronary arteries of males.
Asunto(s)
Vasos Coronarios/metabolismo , Músculo Liso Vascular/metabolismo , Testosterona/metabolismo , Animales , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Femenino , Técnicas In Vitro , Masculino , Orquiectomía , ARN Mensajero/metabolismo , Caracteres Sexuales , Porcinos , Testosterona/sangreRESUMEN
Hypercholesterolemia (HC) is a mary risk factor for the development of coronary heart disease. Coronary ion regulation, especially calcium, is thought to be important in coronary heart disease development; however, the influence of high dietary fat and cholesterol on coronary arterial smooth muscle (CASM) ion channels is unknown. The purpose of this study was to determine the effect of diet-induced HC on CASM voltage-gated calcium current (I(Ca)). Male miniature swine were fed a high-fat, high-cholesterol diet (40% kcal fat, 2% wt cholesterol) for 20-24 wk, resulting in elevated serum total and low-density lipoprotein cholesterol. Histochemistry indicated early atherosclerosis in large coronary arteries. CASM were isolated from the right coronary artery (>1.0 mm ID), small arteries ( approximately 200 microm), and large arterioles ( approximately 100 microm). I(Ca) was determined by whole cell voltage clamp. L-type I(Ca) was reduced approximately 30% by HC compared with controls in the right coronary artery (-5.29 +/- 0.42 vs. -7.59 +/- 0.41 pA/pF) but not the microcirculation (small artery, -8.39 +/- 0.80 vs. -10.13 +/- 0.60; arterioles, -10.78 +/- 0.93 vs. -11.31 +/- 0.95 pA/pF). Voltage-dependent activation was unaffected by HC in both the macro- and microcirculation. L-type voltage-gated calcium channel (Ca(v)1.2) mRNA and membrane protein levels were unaffected by HC. Inhibition of I(Ca) by HC was reversed in vitro by the cholesterol scavenger methyl-beta-cyclodextrin and mimicked in control CASM by incubation with the cholesterol donor cholesterol:methyl-beta-cyclodextrin. These data indicate that CASM L-type I(Ca) is decreased in large coronary arteries in early stages of atherosclerosis, whereas I(Ca) in the microcirculation is unaffected. The inhibition of calcium channel activity in CASM of large coronary arteries is likely due to increases in membrane free cholesterol.
Asunto(s)
Canales de Calcio Tipo L/fisiología , Colesterol en la Dieta , Circulación Coronaria/fisiología , Regulación de la Expresión Génica/fisiología , Hipercolesterolemia/fisiopatología , Microcirculación/fisiopatología , Animales , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/genética , Masculino , Técnicas de Placa-Clamp , ARN Mensajero/genética , Porcinos , Porcinos EnanosRESUMEN
The intracellular mechanisms underlying enhanced myogenic contraction (MC) in coronary resistance arteries (CRAs) from exercise-trained (EX) pigs have not been established. The purpose of this study was to test the hypothesis that exercise-induced alterations in protein kinase C (PKC) signaling underlie enhanced MC. Furthermore, we sought to determine whether modulation of intracellular Ca(2+) signaling by PKC underlies enhanced MC in EX animals. Male Yucatan miniature swine were treadmill trained (n = 7) at approximately 75% of maximal O(2) uptake for 16 wk (6 miles/h, 60 min) or remained sedentary (SED, n = 6). Diameter measurements in response to intraluminal pressure (60, 75, and 90 cmH(2)O) or 60 mM KCl were determined in single, cannulated CRAs ( approximately 100 microm ID) with and without the PKC inhibitor chelerythrine (CE, 1 microM). Confocal imaging of Ca(2+) signaling [myogenic Ca(2+) (Ca(m))] was also performed in CRAs of similar internal diameter after abluminal loading of the Ca(2+) indicator dye fluo 4 (1 microM, 37 degrees C, 30 min). We observed significantly greater MC in CRAs isolated from EX than from SED animals at 90 cmH(2)O, as well as greater reductions in MC after CE at all pressures studied. At intraluminal pressures of 75 and 90 cmH(2)O, CE produced greater decreases in Ca(m) in CRAs from EX than from SED animals (64% vs. 25%, P < 0.05). Inhibition of KCl constriction and Ca(m) by CE was also greater in EX animals (P < 0.05). Western blotting revealed significant increases in Ca(2+)-dependent PKC-alpha ( approximately 50%) but not Ca(2+)-independent PKC-epsilon levels in CRAs isolated from EX animals (P < 0.05). We also observed significant group differences in phosphorylated PKC-alpha levels. Finally, voltage-gated Ca(2+) current (VGCC) was effectively blocked by CE, bisindolylmaleimide, and staurosporine in isolated smooth muscle cells from CRAs, providing evidence for a mechanistic link between VGCCs and PKC in our experimental paradigm. These results suggest that enhanced MC in CRAs from EX animals involves PKC-dependent modulation of intracellular Ca(2+), including regulation of VGCCs.
Asunto(s)
Vasos Coronarios/fisiología , Músculo Liso Vascular/fisiología , Proteína Quinasa C/metabolismo , Transducción de Señal/fisiología , Resistencia Vascular , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Electrofisiología , Membranas Intracelulares/metabolismo , Masculino , Condicionamiento Físico Animal , Proteína Quinasa C-alfa , Proteína Quinasa C-epsilon , Porcinos , Porcinos Enanos , Vasoconstricción/fisiologíaRESUMEN
Substantial evidence exists supporting the role of chronic exercise in reducing the incidence and severity of coronary vascular disease. Physical inactivity is an independent risk factor for coronary heart disease suggesting that the cardioprotective effect of exercise is due, in part, to an intrinsic adaptation within the coronary vasculature. Surprisingly, a paucity of information exists regarding the intrinsic cellular changes within the coronary vasculature associated with exercise training and even less is known regarding the effect of physical activity on long-term phenotypic modulation of coronary smooth muscle (CSM). The purpose of this symposium is to provide a concise update on the current knowledge regarding CSM adaptation to exercise training and the potential for these adaptations to contribute to exercise-induced cardioprotection. The potential role of CSM in exercise-induced cardioprotection will be approached from two perspectives. First, endurance exercise training effects on the regulation of coronary vasomotor tone via changes in CSM calcium regulation will be reviewed, i.e. short-term functional adaptation. Secondly, we will discuss potential long-term consequences of this altered calcium regulation, i.e. exercise-induced phenotypic modulation of CSM. We propose that exercise training alters CSM intracellular calcium regulation to reduce Ca2+-dependent activation of the contractile apparatus and Ca2+-dependent gene transcription and increase activation of sarcolemmal potassium channels. The overall effect is to increase the gain of the vasomotor system and maintain a stable, contractile CSM phenotype.
Asunto(s)
Enfermedad Coronaria/prevención & control , Ejercicio Físico/fisiología , Músculo Liso Vascular/fisiopatología , Adaptación Fisiológica , Calcio/metabolismo , Canales de Calcio/metabolismo , Circulación Coronaria/fisiología , Enfermedad Coronaria/fisiopatología , Vasos Coronarios/fisiopatología , Endotelinas/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Microcirculación/fisiopatología , Mitógenos/fisiología , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Fenotipo , Canales de Potasio/metabolismo , Vasoconstrictores/metabolismo , Sistema Vasomotor/fisiopatologíaRESUMEN
Physical inactivity is an independent risk factor for coronary heart disease, yet the mechanism(s) of exercise-related cardioprotection remains unknown. We tested the hypothesis that coronary smooth muscle after exercise training would have decreased mitogen-induced phenotypic modulation and enhanced regulation of nuclear Ca(2+). Yucatan swine were endurance exercise trained (EX) on a treadmill for 16-20 wk. EX reduced endothelin-1-induced DNA content by 40% compared with sedentary (SED) swine (P < 0.01). EX decreased single cell peak endothelin-1-induced cytosolic Ca(2+) responses compared with SED by 16% and peak nuclear Ca(2+) responses by 33% (P < 0.05), as determined by confocal microscopy. On the basis of these results, we hypothesized that sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and intracellular Ca(2+) stores in native smooth muscle are spatially localized to dissociate cytosolic Ca(2+) and nuclear Ca(2+). Subcellular localization of SERCA in living and fixed cells revealed a distribution of SERCA near the sarcolemma and on the nuclear envelope. These results show that EX enhances nuclear Ca(2+) regulation, possibly via SERCA, which may be one mechanism by which coronary smooth muscle cells from EX are less responsive to mitogen-induced phenotypic modulation.
Asunto(s)
Señalización del Calcio/fisiología , Núcleo Celular/metabolismo , Vasos Coronarios/metabolismo , Músculo Liso Vascular/metabolismo , Condicionamiento Físico Animal , Animales , Bromodesoxiuridina , Cafeína/farmacología , Calcio/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Núcleo Celular/ultraestructura , Separación Celular , Vasos Coronarios/citología , Vasos Coronarios/efectos de los fármacos , ADN/metabolismo , Endotelina-1/farmacología , Femenino , Colorantes Fluorescentes , Técnicas In Vitro , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/ultraestructura , Fenotipo , Esfuerzo Físico/fisiología , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Porcinos EnanosRESUMEN
Endurance exercise training increases smooth muscle L-type Ca(2+) current density in both resistance and proximal coronary arteries of female miniature swine. The purpose of the present study was to determine 1) whether gender differences exist in coronary smooth muscle (CSM) L-type Ca(2+) current density and 2) whether endurance training in males would demonstrate a similar adaptive response as females. Proximal, conduit (approximately 1.0 mm), and resistance [~200 microm (internal diameter)] coronary arteries were obtained from sedentary and treadmill-trained swine of both sexes. CSM were isolated by enzymatic digestion (collagenase plus elastase), and voltage-gated Ca(2+)-channel current (I(Ca)) was determined by using whole cell voltage clamp during superfusion with 75 mM tetraethylammonium chloride and 10 mM BaCl(2). Current-voltage relationships were obtained at test potentials from -60 to 70 mV from a holding potential of -80 mV, and I(Ca) was normalized to cell capacitance (pA/pF). Endurance treadmill training resulted in similar increases in heart weight-to-body weight ratio, endurance time, and skeletal muscle citrate synthase activity in male and female swine. I(Ca) density was significantly greater in males compared with females in both conduit (-7.57 +/- 0.58 vs. -4.14 +/- 0.47 pA/pF) and resistance arteries (-11.25 +/- 0.74 vs. -6.49 +/- 0.87 pA/pF, respectively). In addition, voltage-dependent activation of I(Ca) in resistance arteries was shifted to more negative membrane potentials in males. Exercise training significantly increased I(Ca) density in both conduit and resistance arteries in females (-7.01 +/- 0.47 and -9.73 +/- 1.13 pA/pF, respectively) but had no effect in males (-8.61 +/- 0.50 and -12.04 +/- 1.07 pA/pF, respectively). Thus gender plays a significant role in determining both the magnitude and voltage dependence of I(Ca) in CSM and the adaptive response of I(Ca) to endurance training.
Asunto(s)
Adaptación Fisiológica , Canales de Calcio Tipo L/fisiología , Vasos Coronarios/fisiología , Condicionamiento Físico Animal , Caracteres Sexuales , Animales , Arterias/fisiología , Conductividad Eléctrica , Femenino , Masculino , Músculo Liso Vascular/fisiología , Resistencia Física/fisiología , Porcinos , Porcinos Enanos , Resistencia VascularRESUMEN
The purpose of the present study was to examine the effect of short duration H(2)O(2) exposure on coronary artery endothelial cell [Ca(2+)](i) regulation. Freshly dispersed cells from porcine coronary artery were exposed to H(2)O(2) (300 micromol/L) for 3 min while monitoring [Ca(2+)](i) using fura-2 microfluorometry. H(2)O(2) increased [Ca(2+)](i) from 0.86 +/- 0.03 to 2.19 +/- 0.41 ratio units at 3 min of H(2)O(2) (P < 0.05). Intracellular Ca(2+) remained elevated 3 min following removal of H(2)O(2), yet H(2)O(2) had no effect on the subsequent [Ca(2+)](i) response to bradykinin (0.1 micromol/L). The H(2)O(2)-induced [Ca(2+)](i) increase was completely abolished either by removal of extracellular Ca(2+) or lowering extracellular Na(+). Cells exposed to the Na(+) ionophore, monensin, showed an increase in [Ca(2+)](i) with a time course similar to that seen with H(2)O(2). Furthermore, H(2)O(2)-induced Ca(2+) influx was not attenuated by either Ni(2+) (300 micromol/L) or econazole (10 micromol/L), excluding Ca(2+) influx via the agonist-sensitive pathway. Thus, in coronary arterial endothelial cells, H(2)O(2) increases Ca(2+) influx in an extracellular Na(+)-dependent manner via an agonist-insensitive pathway.
Asunto(s)
Arterias/metabolismo , Calcio/metabolismo , Circulación Coronaria/fisiología , Endotelio Vascular/metabolismo , Estrés Oxidativo/fisiología , Sodio/metabolismo , Animales , Transporte Biológico , Bradiquinina/farmacología , Cationes , Bovinos , Peróxido de Hidrógeno/farmacología , Ionóforos/farmacología , Monensina/farmacologíaRESUMEN
We hypothesized that enhanced voltage-gated Ca2+ channel current (VGCC) density in coronary smooth muscle cells of exercise-trained miniature Yucatan pigs is compensated by other cellular Ca2+ regulatory mechanisms to limit net myoplasmic free Ca2+ accumulation. Whole-cell voltage clamp experiments demonstrated enhanced VGCC density in smooth muscle cells freshly dispersed from coronary arteries of exercise-trained vs. sedentary animals. In separate experiments using fura-2 microfluorometry, we measured depolarization-induced (80 mM KCl) accumulation of myoplasmic free Ba2+ and free Ca2+. Both maximal rate and net accumulation of free Ba2+ in response to membrane depolarization were increased in smooth muscle cells isolated from exercise-trained pigs, consistent with an increased VGCC density. Depolarization also produced an enhanced maximal rate of free Ca2+ accumulation in cells of exercise-trained pigs; however, net accumulation of free Ca2+ was not significantly increased suggesting enhanced Ca2+ influx was compensated to limit net free Ca2+ accumulation. Inhibition of sarco-endoplasmic reticulum Ca2+-transporting ATPase (SERCA; 10 microM cyclopiazonic acid) and/or sarcolemmal Na+-Ca2+ exchange (low extracellular Na+) suggested neither mechanism compensated the enhanced VGCC in cells of exercise-trained animals. Local Ca2+-dependent inactivation of VGCC, assessed by buffering myoplasmic Ca2+ with EGTA in the pipette and using Ca2+ and Ba2+ as charge carriers, was not different between cells of sedentary and exercise-trained animals. Our findings indicate that increased VGCC density is compensated by other cellular Ca2+ regulatory mechanisms to limit net myoplasmic free Ca2+ accumulation in smooth muscle cells of exercise-trained animals. Further, SERCA, Na+-Ca2+ exchange and local Ca2+-dependent inactivation of VGCC do not appear to function as compensatory mechanisms. Additional potential compensatory mechanisms include Ca2+ extrusion via plasma membrane Ca2+-ATPase, mitochondrial uptake, myoplasmic Ca2+-binding proteins and other sources of VGCC inactivation.
Asunto(s)
Canales de Calcio Tipo L/fisiología , Calcio/metabolismo , Vasos Coronarios/fisiología , Músculo Liso Vascular/fisiología , Condicionamiento Físico Animal , Animales , Bario/farmacocinética , Calcio/fisiología , Conductividad Eléctrica , Femenino , Colorantes Fluorescentes , Fura-2 , Miofibrillas/metabolismo , Retículo Sarcoplasmático/metabolismo , Intercambiador de Sodio-Calcio/fisiología , Porcinos , Porcinos EnanosRESUMEN
Exercise training produces complex changes in intrinsic control of coronary vascular resistance. In smooth muscle, adaptations that alter Ca2+ regulation seem central, including changes in the function of sarcolemmal K+ and L-type Ca2+ channels and the sarcoplasmic reticulum. Exercise training also increases the ability of the endothelium to release vasoactive factors, with increased expression and activity of endothelial cell nitric oxide synthase playing a key role.
Asunto(s)
Adaptación Fisiológica/fisiología , Vasos Coronarios/fisiología , Endotelio Vascular/fisiología , Ejercicio Físico , Músculo Liso Vascular/fisiología , Femenino , Humanos , Masculino , Sensibilidad y Especificidad , Estrés MecánicoAsunto(s)
Adaptación Fisiológica , Ejercicio Físico/fisiología , Canales Iónicos/fisiología , Microcirculación , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Circulación Coronaria , Perros , Hemodinámica , Humanos , Transporte Iónico , Modelos Biológicos , Proteínas Musculares/efectos de los fármacos , Proteínas Musculares/fisiología , Músculo Esquelético/irrigación sanguínea , Fosforilación , Condicionamiento Físico Animal , Bloqueadores de los Canales de Potasio , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Procesamiento Proteico-Postraduccional , Ratas , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Porcinos , Resistencia VascularRESUMEN
Exercise training produces numerous adaptations in the coronary circulation, including an increase in coronary tone, both in conduit and resistance arteries. On the basis of the importance of voltage-gated Ca2+ channels (VGCC) in regulation of vascular tone, we hypothesized that exercise training would increase VGCC current density in coronary smooth muscle. To test this hypothesis, VGCC current was compared in smooth muscle from conduit arteries (>1.0 mm), small arteries (200-250 micrometer), and large arterioles (75-150 micrometer) from endurance-trained (Ex) or sedentary miniature swine (Sed). After 16-20 wk of treadmill training, VGCC current was determined using whole cell voltage-clamp techniques. In both Ex and Sed, VGCC current density was inversely related to arterial diameter, i.e., large arterioles > small arteries > conduit arteries. Exercise training increased peak inward currents approximately twofold in smooth muscle from all arterial sizes compared with those from Sed (large arteriole, -12.52 +/- 2.05 vs. -5.74 +/- 0.99 pA/pF; small artery, -6.20 +/- 0.97 vs. -3.18 +/- 0.44 pA/pF; and conduit arteries, -4.22 +/- 0.30 vs. -2.41 +/- 0.55 pA/pF; 10 mM Ba2+ external). Dihydropyridine sensitivity, voltage dependence, and inactivation kinetics identified this Ca2+ current to be L-type current in all arterial sizes from both Sed and Ex. Furthermore, peak VGCC current density was correlated with treadmill endurance in all arterial sizes. We conclude that smooth muscle L-type Ca2+ current density is increased within the coronary arterial bed by endurance exercise training. This increased VGCC density may provide an important mechanistic link between functional and cellular adaptations in the coronary circulation to exercise training.