RESUMEN
BACKGROUND AND PURPOSE: Stem cell transplantation therapy is a promising option for treatment of severe ischaemic heart disease. Dimethyl sulphoxide (DMSO) differentiates P19CL6 embryonic carcinoma cells into cardiomyocyte-like cells, but with low differentiation capacity. To improve the degree of this differentiation, we have assessed several derivatives of the differentiation-inducing factor-1 (DIF-1), originally found in the cellular slime mould Dictyostelium discoideum, on P19CL6 cells. EXPERIMENTAL APPROACH: P19CL6 cells were cultured with each derivative and 1% DMSO for up to 16 days. Differentiation was assessed by measuring the number of beating and non-beating aggregates, and the expression of genes relevant to cardiac tissue. The mechanism of action was investigated using a T-type Ca(2+) channel blocker. KEY RESULTS: Of all the DIF-1 derivatives tested only Br-DIF-1 showed any effects on cardiomyocyte differentiation. In the presence of 1% DMSO, Br-DIF-1 (0.3-3 µM) significantly and dose-dependently increased the number of spontaneously beating aggregates compared with 1% DMSO alone, by day 16. Expression of mRNA for T-type calcium channels was significantly increased by Br-DIF-1 + 1% DMSO compared with 1% DMSO alone. Mibefradil (a T-type Ca(2+) channel blocker; 100 nM) and a small interfering RNA for the T-type Ca(2+) channel both significantly decreased the beating rate of aggregates induced by Br-DIF-1 + 1% DMSO. CONCLUSIONS AND IMPLICATIONS: Br-DIF-1 accelerated the differentiation, induced by 1% DMSO, of P19CL6 cells into spontaneously beating cardiomyocyte-like cells, partly by enhancing the expression of the T-type Ca(2+) channel gene.
Asunto(s)
Canales de Calcio Tipo T/fisiología , Diferenciación Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Hexanonas/farmacología , Miocitos Cardíacos/efectos de los fármacos , Animales , Bloqueadores de los Canales de Calcio/farmacología , Línea Celular Tumoral , Dimetilsulfóxido , Mibefradil/farmacología , Ratones , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiologíaRESUMEN
Ossification of the posterior longitudinal ligament (OPLL) of the spine is characterized by progressive ectopic bone formation in the spinal ligament. To identify the genes related to ossification affected by mechanical stress during OPLL, analyses using cDNA microarray were carried out using cultured human spinal ligament cells that had been subjected to uniaxial cyclic stretching. Samples were obtained from a total of 14 patients: seven cervical or thoracic OPLL patients and seven control patients. Spinal ligament cells derived from tissues of OPLL (OPLL cells) and control (non-OPLL cells) patients were subjected to uniaxial sinusoidal cyclic stretching (0.5 Hz, 20% stretch) for various time periods (0-9 hours). cDNA microarrays revealed that ranges of distribution of both up- and downregulated genes evoked by cyclic stretching were significantly wider in OPLL cells than in non-OPLL cells. Increases in the mRNA expression of endothelin-1 (ET-1) as well as various marker genes related to ossification were also observed. mRNA expression of ET-1 and alkaline phosphatase was increased by mechanical stress in a time-dependent manner, while addition of ET-1 to static cultures of OPLL cells increased mRNA expression of alkaline phosphatase in a dose-dependent manner. During 9 hours of cyclic stretching, ET-1 release increased to about sixfold the amount observed in nonstretched cells. In non-OPLL cells, neither cyclic stretching nor ET-1 induced any increase in alkaline phosphatase expression. These results suggest that mechanical stress promotes the progression of ossification in OPLL cells through autocrine and/or paracrine mechanisms of ET-1.
Asunto(s)
Endotelina-1/metabolismo , Ligamentos Longitudinales/metabolismo , Osificación del Ligamento Longitudinal Posterior/metabolismo , Anciano , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Calcinosis/tratamiento farmacológico , Calcinosis/metabolismo , Células Cultivadas , Vértebras Cervicales , Endotelina-1/antagonistas & inhibidores , Endotelina-1/genética , Endotelina-1/farmacología , Éteres/farmacología , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Hidrocarburos Fluorados/farmacología , Ligamentos Longitudinales/efectos de los fármacos , Ligamentos Longitudinales/fisiopatología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligopéptidos/farmacología , Osificación del Ligamento Longitudinal Posterior/fisiopatología , Piperidinas/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estrés Mecánico , Vértebras TorácicasRESUMEN
Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments. Mechanical stress, which acts on the posterior ligaments, is thought to be an important factor in the progression of OPLL. To clarify this mechanism, we investigated the effects of in vitro cyclic stretch (120% peak to peak, at 0.5 Hz) on cultured spinal ligament cells derived from OPLL (OPLL cells) and non-OPLL (non-OPLL cells) patients. The mRNA expressions of Cbfa1 (an osteoblast-specific transcription factor), type I collagen, alkaline phosphatase (ALP), osteocalcin and integrin beta1 (a mechanotransducer) were increased by cyclic stretch in OPLL cells, whereas no change was observed in non-OPLL cells. The effects of cyclic stretch on the spinal ligament tissues derived from OPLL and non-OPLL patients were also analyzed by immunohistochemistry using an antibody against Cbfa1. The expression of Cbfa1 was increased by cyclic stretch at the center of the spinal ligament tissues of OPLL patients, whereas no change was observed in the tissues of non-OPLL patients. Furthermore, U0126, a specific inhibitor of MAPK kinase (MEK), suppressed the stretch-induced mRNA expressions of Cbfa1, ALP and type I collagen in OPLL cells. These results suggest that in OPLL cells, mechanical stress is converted by integrin beta1 into intracellular signaling and that Cbfa1 is activated through the MAP kinase pathway. Therefore, we propose that mechanical stress plays a key role in the progression of OPLL through an increase in Cbfa1 expression.
Asunto(s)
Ligamentos Longitudinales/metabolismo , Proteínas de Neoplasias/biosíntesis , Osificación del Ligamento Longitudinal Posterior/metabolismo , Osificación Heterotópica/metabolismo , Estrés Mecánico , Factores de Transcripción/biosíntesis , Anciano , Fosfatasa Alcalina/biosíntesis , Fosfatasa Alcalina/efectos de los fármacos , Butadienos , Células Cultivadas , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Inmunohistoquímica , Cadenas beta de Integrinas/biosíntesis , Cadenas beta de Integrinas/efectos de los fármacos , Masculino , Persona de Mediana Edad , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Nitrilos , Osificación del Ligamento Longitudinal Posterior/fisiopatología , Osteocalcina/biosíntesis , Osteocalcina/efectos de los fármacos , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments. Mechanical stress, which acts on the posterior ligaments, is thought to be an important factor in the progression of OPLL. To elucidate this mechanism, we investigated the effects of in vitro sinusoidal cyclic stretch (120% peak to peak, at 1 Hz) on cultured spinal ligament cells derived from OPLL and non-OPLL patients. The mRNA expressions of alkaline phosphatase (ALP), osteopontin, bone morphogenetic protein (BMP)-2, BMP-4, and BMP receptors as well as ALP activity in cell layers and production of BMPs into the conditioned medium were significantly increased by cyclic stretch in OPLL cells, whereas no change was observed in non-OPLL cells. A stretch-activated Ca(2+) channel blocker, Gd(3+), the voltage-dependent L-type Ca(2+) channel blockers diltiazem and nifedipine, and Ca(2+)-free medium suppressed stretch-induced ALP activity, which suggests a role of Ca(2+) influx in the signal transduction of mechanical stress to the osteogenic response of OPLL cells. Our study provides first evidences that mechanical stress plays a key role in the progression of OPLL through the induction of osteogenic differentiation in spinal ligament cells and the promotion of the autocrine/paracrine mechanism of BMPs in this lesion.
Asunto(s)
Proteínas Morfogenéticas Óseas/biosíntesis , Ligamentos Longitudinales/metabolismo , Ligamentos Longitudinales/patología , Osificación del Ligamento Longitudinal Posterior/metabolismo , Osificación del Ligamento Longitudinal Posterior/patología , Osteogénesis/fisiología , Factor de Crecimiento Transformador beta , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Secuencia de Bases , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Receptores de Proteínas Morfogenéticas Óseas , Proteínas Morfogenéticas Óseas/genética , Canales de Calcio Tipo L/genética , Diferenciación Celular , Células Cultivadas , ADN/genética , Expresión Génica , Humanos , Osificación del Ligamento Longitudinal Posterior/etiología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteopontina , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento/genética , Sialoglicoproteínas/genética , Estrés MecánicoRESUMEN
Corticotropin-releasing factor (CRF) has a coronary vasodilator effect and a positive inotropic effect on the isolated rat heart. Recently, expression of CRF receptor type 2 (CRF-R2) has been demonstrated in the heart. In addition, urocortin (Ucn), a new member of the CRF family, has been reported to have much greater affinity for CRF-R2 than CRF. It is suggested that the cardiac effects of Ucn may be more potent than those of CRF. We compared the effect of Ucn with that of CRF on isolated rat heart. The effects of Ucn were then analyzed to determine whether these effects were mediated by CRF receptors and/or any other mediators under the following conditions: perfusion buffer containing (1) alpha-helical CRF 9-41, (2) indomethacin, (3) N(G)-nitro-l -arginine methylester and (4) propranolol. Ucn exhibited a greater effect with a longer duration of action than CRF. Indomethacin significantly attenuated the vasodilator effects of Ucn (P<0.05). CRF receptor antagonist diminished both coronary vasodilation and the positive inotropic effects of Ucn (P<0.05). These results suggest that the cardiac effects of Ucn may be mediated by a CRF receptor, and prostaglandins may be involved in the vasodilator effect.
Asunto(s)
Vasos Coronarios/efectos de los fármacos , Hormona Liberadora de Corticotropina/farmacología , Resistencia Vascular/efectos de los fármacos , Vasodilatadores/farmacología , Análisis de Varianza , Animales , Vasos Coronarios/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores Enzimáticos/farmacología , Indometacina/farmacología , Masculino , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Perfusión , Propranolol/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Hormona Liberadora de Corticotropina/metabolismo , UrocortinasRESUMEN
For the elucidation of the diversity of secondary metabolites of Dictyostelium cellular slime molds, we investigate the constituent of three species of slime molds. From the methanol extract of their fruit bodies, we obtained three novel compounds, dictyopyrone A (1) and B (2) from D. discoideum and D. rhizoposium and dictyopyrone C (3) from D. longosporum. They possess a unique alpha-pyrone moiety with a side chain at the C-3 position. Their relative structures were elucidated by spectral means, and the absolute configuration was confirmed by asymmetric synthesis of 1. Since these compounds were obtained from different species of Dictyostelium slime molds, they may be a type of compound common to this genus.
Asunto(s)
Dictyostelium/química , Pironas/química , Animales , Dicroismo Circular , Dictyostelium/clasificación , Contracción Isométrica/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Conformación Molecular , Músculo Liso/efectos de los fármacos , Pironas/síntesis química , Pironas/aislamiento & purificación , Pironas/farmacología , Ratas , Ratas WistarRESUMEN
Thiotetromycin isolated from the culture broth of Streptomyces sp. strain OM-674 slightly enhanced the superprecipitation and the ATPase activity of myosin B from skeletal muscle. The ATPase activity of troponin-tropomyosin-free myosin B was inhibited by thiotetromycin. The inhibitory effect of thiotetromycin was significantly attenuated by troponin-tropomyosin complex. The ATPase activity of actomyosin reconstituted from actin and myosin was inhibited by pretreatment of actin with thiotetromycin. Thiotetromycin induced a concentration-dependent decrease in the fluorescence intensity of actin and pyrenyl-F-actin. By using surface plasmon resonance (SPR), it was proved that thiotetromycin bound to actin. Thiotetromycin caused a concentration-dependent decrease in sedimentation of F-actin by hard centrifugation. This was a cross-correlation among the concentration-inhibition curves for thiotetromycin in the activity of actomyosin ATPase and the fluorescence intensity. These results suggest that thiotetromycin binds to actin to cause a conformational change, resulting in modulation of the interaction between actin and myosin, and in depolymerization of F-actin.
Asunto(s)
Actinas/efectos de los fármacos , Antibacterianos/farmacología , Miosinas/efectos de los fármacos , Actinas/química , Animales , Miosinas/metabolismo , Conformación Proteica/efectos de los fármacos , Conejos , Streptomyces , Tiofenos/farmacologíaRESUMEN
We developed a sensitive and nonradioactive fluorometric assay for cyclic guanosine 3',5'-monophosphate (cGMP). Guanine nucleotides except cGMP were enzymatically phosphorylated to GTP. cGMP, absorbed into a Sep-Pak amino propyl cartridge, was eluted separately from GTP. Purified cGMP was enzymatically converted to GTP, which was applied to the GTP-GDP cycle using succinic thiokinase and pyruvate kinase. When pyruvic acid produced by the GTP-GDP cycle was reduced by lactate dehydrogenase, a reduced form of nicotinamide adenine dinucleotide (NADH) was equivalently oxidized to NAD(+). NAD(+) was further converted into fluorescent compound, which was excited at 370 nm and emitted fluorescence at 460 nm, by a strong alkali. When 20 nmol NADH was used for this assay, the calibration curve over 50 to 500 fmol cGMP became sufficiently linear. The detection limit for cGMP was ca. 5 fmol (signal to noise ratio >3). Using this assay, we confirmed that the cGMP content in the left atrial strip of dog was changed from 11.4 +/- 3.8 to 19.3 +/- 2.6 fmol/mg wet wt of tissue (mean +/- SE, n = 6) by electrical driving at 1 Hz. Carbachol (1 microM) further increased the cGMP to 45.6 +/- 9.2 fmol/mg wet wt of tissue. From these results, it is suggested that this novel assay for cGMP is highly sensitive and can be applied to various biological samples.
Asunto(s)
GMP Cíclico/análisis , Corazón/fisiología , Miocardio/metabolismo , Animales , Función del Atrio Izquierdo , Calibración , Carbacol/farmacología , Coenzima A Ligasas , Perros , Femenino , Guanosina Trifosfato/análisis , Guanosina Trifosfato/aislamiento & purificación , Corazón/efectos de los fármacos , Indicadores y Reactivos , L-Lactato Deshidrogenasa , Masculino , NAD , Fosforilación , Piruvato Quinasa , Sensibilidad y Especificidad , Espectrometría de Fluorescencia/instrumentación , Espectrometría de Fluorescencia/métodosRESUMEN
Gamma-mangostin, purified from the fruit hull of the medicinal plant Garcinia mangostana caused a parallel rightwards shift of the concentration/response curve for the contraction elicited by 5-hydroxytryptamine (5-HT) in the rabbit aorta (pA2 = 8.2) without affecting the contractile responses to KCl, phenylephrine (alpha1) or histamine (H1). The perfusion pressure response of rat coronary artery to 5-HT (5-HT2A) was reduced concentration dependently by gamma-mangostin (IC50 = 0.32 microM). 5-HT amplified, ADP-induced aggregation of rabbit platelets (5-HT2A) was inhibited by gamma-mangostin (IC50 = 0.29 microM), whereas that induced by thrombin was not affected, nor did gamma-mangostin affect 5-HT-induced contraction of the guinea-pig ileum (5-HT3)in the presence of 5-HT1, 5-HT2 and 5-HT4 receptor antagonists. Furthermore, 5-HT-induced contraction of the rat fundus (5-HT2B) and 5-HT-induced relaxation of the rabbit aorta in the presence of ketanserin (5-HT1) and carbachol-induced contraction of the guinea-pig ileum (muscarinic M3) were not affected by gamma-mangostin (5 microM). Gamma-mangostin inhibited [3H]spiperone binding to cultured rat aortic myocytes (IC50 = 3.5 nM). The Kd for [3H]spiperone binding was increased by gamma-mangostin (3 nM) from 11.7 to 27.4 nM without affecting Bmax. These results suggest that gamma-mangostin is a novel competitive antagonist, free from a nitrogen atom, for the 5-HT2A receptors in vascular smooth muscles and platelets.
Asunto(s)
Inhibidores de la Proteasa del VIH/farmacología , Músculo Liso Vascular/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Receptores de Serotonina/efectos de los fármacos , Antagonistas de la Serotonina/farmacología , Xantenos/farmacología , Xantonas , Animales , Unión Competitiva , Relación Dosis-Respuesta a Droga , Cobayas , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Perfusión , Extractos Vegetales/farmacología , Conejos , Ratas , Ratas Wistar , Receptor de Serotonina 5-HT2A , Receptores de Serotonina/metabolismo , Espiperona/metabolismoRESUMEN
2,5-Di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), an inhibitor of endoplasmic reticulum Ca2+-ATPase, caused aggregation accompanied by a marked increase in intracellular Ca2+ concentrations ([Ca2+]i) in washed rabbit platelets. Treatment of platelets with SK&F96365 or removal of extracellular Ca2+ inhibited both Ca2+ influx and platelet aggregation induced by tBuBHQ. These responses of platelets to tBuBHQ were also inhibited by genistein. Western blots using antibody against phosphorylated amino acid residues revealed that tBuBHQ activated tyrosine kinase independently of extracellular Ca2+. These results suggest that tBuBHQ induces Ca2+ influx into platelets probably through activation of tyrosine kinase, resulting in platelet aggregation.
Asunto(s)
ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Retículo Endoplásmico/enzimología , Inhibidores Enzimáticos/farmacología , Hidroquinonas/farmacología , Agregación Plaquetaria/efectos de los fármacos , Animales , Plaquetas/efectos de los fármacos , Plaquetas/enzimología , Plaquetas/metabolismo , Western Blotting , Calcio/sangre , Retículo Endoplásmico/efectos de los fármacos , Imidazoles/farmacología , Técnicas In Vitro , Inhibidores de Agregación Plaquetaria/farmacología , Proteínas Tirosina Quinasas/metabolismo , ConejosRESUMEN
The importance of the Ca2+ -ATPases of the surface and endoplasmic reticulum membranes in regulating the intracellular Ca2+ concentrations in living cells has been recognized as a result of the development of biophysical techniques and finding of useful pharmacological tool drugs. The number of possible isoforms of these Ca2+ -ATPases are increased by a complex pattern of alternative splicing and the knowledge of the functional difference and tissue distribution of these isoforms has been accumulated. In this review, we discuss recent progress in our understanding of the roles of the Ca2+ -ATPases in physiological and pathophysiological conditions.
Asunto(s)
ATPasas Transportadoras de Calcio , Animales , Calcio/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , ATPasas Transportadoras de Calcio/fisiología , Humanos , Hipertensión/etiología , Hipertensión/metabolismoRESUMEN
By using a patch clamp technique, we examined the effect of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G kinase) on Ca(2+)-activated maxi K+ channels in canine coronary artery smooth muscle cells. Maxi K+ channels (274 +/- 4 pS in symmetrical 140 mM KCl at 24-26 degrees C) were activated by cytoplasmic Ca2+ and were completely blocked by 100 nM charybdotoxin (CTX). G kinase (300 U/ml) added to the cytoplasmic face of the membrane patch shifted the voltage dependence of these channels by about 25 mV in the negative direction in the presence of 1 microM Ca2+, 50 microM cGMP and 1 mM magnesium adenosine triphosphate. At -50 mV and 1 microM Ca2+, G kinase treatment increased the mean number of open channels 4.5-fold compared with the control. alpha-Human atrial natriuretic peptide (ANP, 100 nM) reduced the isometric tension of coronary arterial rings elicited by 14 or 24 mM KCl, but failed to relax the artery contracted by 34 mM KCl. Addition of 100 nM CTX augmented tension development elicited by 24 mM KCl and totally prevented ANP from relaxing the arterial rings. These results indicate that G kinase-dependent protein phosphorylation activates maxi K+ channels in canine coronary smooth muscle, and further suggest that the G kinase-induced activation of maxi K+ channels may cause hyperpolarization and relaxation of coronary artery.
Asunto(s)
Vasos Coronarios/enzimología , GMP Cíclico/farmacología , Músculo Liso Vascular/enzimología , Canales de Potasio/fisiología , Proteínas Quinasas/metabolismo , Adenosina Trifosfato/farmacología , Animales , Factor Natriurético Atrial/farmacología , Calcio/farmacología , Caribdotoxina , Perros , Femenino , Masculino , Relajación Muscular/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Cloruro de Potasio/farmacología , Venenos de Escorpión/farmacologíaRESUMEN
SH-1 thiol of S-1 was modified with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetoamide spin label (IASL). The extent of dissociation, alpha, of spin-labeled myosin subfragment-1 (IASL-S-1) from acto-IASL-S-1 by a nucleotide was measured by an ultracentrifugal separation method, a light-scattering method, and a saturation transfer EPR method. The alpha values obtained by these three methods were the same within the limits of the experimental errors. The dependence of alpha on the concentrations of AMPPNP, [S], and F-actin, [A], could be described by the equation: alpha-1 = 1 + (1 + Ks/[S])[A]/KA. The Ks and KA values were 0.65-1.2 mM and 1.7-2.7 mg/ml, respectively, in 0.5 M KCl and 4 mM MgCl2 at pH 7.0 and 20 degrees C. The height of the weakly immobilized peak of the conventional EPR spectrum of IASL-S-1, W, increased linearly with increase in the ATP or AMPPNP concentration, and became saturated at 1 mol nucleotide/mol IASL-S-1. No change in W was observed upon the binding of IASL-S-1 with F-actin. The dependence of the extent of change in W, delta W, on [A] and [S] was given by delta W-1 = 1 + Ks/[S], where Ks = Ks/(1 + KA/[A]). This finding indicates that the delta W value is proportional to the amount of a nucleotide bound to IASL-S-1 and independent of the binding of F-actin to IASL-S-1.