RESUMEN
BACKGROUND: FMSP is a synthesized ferrocene derivative with anti-cancer characteristics on tumor cells. Naringenin is a polyphenolic flavonoid with anti-tumor ability. METHODS: Cell viability and proliferation of two cancer cells and a normal cell line after treatment with these agents were determined with MTT assay. To predict the possible interaction between calmodulin (CaM) and FMSP and naringenin, docking studies were performed. By using fluorescence emission spectra, the effects of FMSP and naringenin on CaM structure and activity were studied. CaM-dependent activation of phosphodiesterase 1 (PDE1) by FMSP in relation to naringenin and their combination were compared. Effects of these compounds on PDE1 inhibition, cAMP accumulation, and cAMP-dependent protein kinase A (PKA) activation were assayed. RESULTS: The combination of FMSP and naringenin had more inhibitory effects on CaM structure than FMSP and naringenin alone. Results of docking analyses also confirmed efficient interaction of the two compounds with a hydrophobic pocket of calmodulin active site. Kinetic analyses of these agents' interaction with CaM showed FMSP and naringenin both competitively inhibited PDE1 activation without changing the Vmax parameter. FMSP and naringenin synergistically increased Km values at a higher level compared to FMSP or naringenin alone. The combination of these two agents also had more cytotoxic effects on cancer cells than FMSP alone. CONCLUSIONS: It was shown that mechanism of proliferation inhibition in both cancer cells by these compounds is based on CaM and consequent PDE inhibition followed by intracellular cAMP level elevation and increased PKA activity in a dose-dependent manner.
Asunto(s)
Calmodulina/metabolismo , Flavanonas/farmacología , Animales , Antineoplásicos/farmacología , Calmodulina/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Compuestos Ferrosos/farmacología , Flavonoides/farmacología , Humanos , Metalocenos/farmacología , Hidrolasas Diéster Fosfóricas/metabolismoRESUMEN
AIMS: The Ca2+-binding protein calmodulin (CaM) modulates numerous target proteins but is produced insufficiently to bind all of them, generating a limiting CaM equilibrium. Menopause increases cardiac morbidity; however, it is unknown if the cardiac CaM equilibrium is affected by estrogen. We devised an assay to assess the effects of ovariectomy and estrogen treatment on the cardiac CaM equilibrium. MATERIALS AND METHODS: Sprague-Dawley rats received sham surgery or ovariectomy, followed by 2-week treatment with vehicle or 17ß-estradiol. Ca2+-saturated left ventricular (LV) lysates were processed through CaM sepharose columns, which retained CaM-binding proteins unoccupied by endogenous CaM. Eluants therefrom were subjected to a competitive binding assay against purified CaM and a CaM biosensor to assess the amounts of unoccupied CaM-binding sites. LV cellular composition was assessed by immunohistochemistry. KEY FINDINGS: LV eluants processed from sham animals reduce biosensor response by ~32%, indicating baseline presence of unoccupied CaM-binding sites and a limiting CaM equilibrium. Ovariectomy exacerbates the limiting CaM equilibrium, reducing biosensor response by ~65%. 17ß-estradiol treatment equalizes the difference between sham and ovariectomized animals. These changes reflect whole tissue responses and are not mirrored by changes in total surface areas of cardiomyocytes and fibroblasts. Consistently, Ca2+-dependent, but not Ca2+-independent, interaction between CaM and the cardiac inositol trisphosphate receptor (IP3R) is reduced following ovariectomy and is restored by subsequent 17ß-estradiol treatment. SIGNIFICANCE: Our assay provides a new parameter to assess tissue CaM equilibrium. The exacerbated limiting CaM equilibrium following estrogen loss may contribute to cardiac morbidity and is prevented by estrogen treatment.
Asunto(s)
Calmodulina/metabolismo , Estradiol/farmacología , Miocitos Cardíacos/metabolismo , Animales , Sitios de Unión , Señalización del Calcio/efectos de los fármacos , Calmodulina/efectos de los fármacos , Estradiol/metabolismo , Estrógenos/metabolismo , Estrógenos/farmacología , Femenino , Ventrículos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Ovariectomía , Posmenopausia/fisiología , Unión Proteica , Ratas , Ratas Sprague-DawleyRESUMEN
AIM: We previously revealed that Caï¼ï¼-activated calmodulin binds to ABCA1 by the region near the PEST sequence and retards its calpain-mediated degradation to increase HDL biogenesis. Calmodulin activity is reportedly modulated also by other nutritional divalent cations; thus, we attempted to determine whether Znï¼ï¼ is involved in the regulation of ABCA1 stability through the modulation of calmodulin activity. METHODS: The effects of Znï¼ï¼ on ABCA1 expression was investigated in J774 mouse macrophage cell-line cells and HepG2 human hepatoma cell-line cells. RESULTS: Znï¼ï¼ increased ABCA1 expression, not by increasing the mRNA but by attenuating its decay rate, more prominently in the presence of cAMP. Accordingly, it enhanced cell cholesterol release with extracellular apolipoprotein A-I. Calmodulin binding to ABCA1 was increased by Znï¼ï¼ and Caï¼ï¼. Znï¼ï¼ suppressed calpain-mediated hydrolysis of the peptide of ABCA1 cytosolic loop, including the PEST sequence and the calmodulin-binding site, in a calmodulin- dependent fashion, in the presence of the minimum amount of Caï¼ï¼ to activate calpain, but not calmodulin. Calpain activity was not directly inhibited by Znï¼ï¼ at the concentration for enhancing calmodulin binding to ABCA1. CONCLUSION: Nutritional divalent cation Znï¼ï¼ is involved in the regulation of ABCA1 activity and biogenesis of HDL through the modulation of calmodulin activity. The results were consistent with previous clinical findings that Znï¼ï¼ increased plasma HDL in the conditions of sympathetic activation, such as type 2 diabetes and chronic hemodialysis.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Calmodulina/efectos de los fármacos , Macrófagos/efectos de los fármacos , Proteolisis/efectos de los fármacos , Zinc/farmacología , Animales , Apolipoproteína A-I/metabolismo , Calmodulina/metabolismo , Calpaína/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Colesterol/metabolismo , AMP Cíclico , Humanos , Hidrólisis , Macrófagos/metabolismo , Ratones , ARN MensajeroRESUMEN
Ischemic brain damage is triggered by glutamate excitotoxicity resulting in neuronal cell death. Previous research has demonstrated that N-methly-D-aspartate (NMDA) receptor activation triggers downstream calcium-dependent signaling pathways, specifically Ca2+/calmodulin-dependent protein kinase II (CaMKII). Inhibiting CaMKII is protective against hippocampal ischemic injury, but there is little known about its role in the cerebellum. To examine the neuroprotective potential of CaMKII inhibition in Purkinje cells, we subjected C57BL/6 or CaMKIIα KO male mice (8-12 weeks old) to cardiac arrest followed by cardiopulmonary resuscitation (CA/CPR). We performed a dose-response study for tat-CN19o and cerebellar injury was analyzed at 7 days after CA/CPR. Acute signaling was assessed at 6 h after CA/CPR using Western blot analysis. We observed increased phosphorylation of the T286 residue of CaMKII, suggesting increased autonomous activation. Analysis of Purkinje cell density revealed a decrease in cell density at 7 days after CA/CPR that was prevented with tat-CN19o at doses of 0.1 and 1 mg/kg. However, neuroprotection in the cerebellum required doses that were 10-fold higher than what was needed in the hippocampus. CaMKIIα KO mice subjected to sham surgery or CA/CPR had similar Purkinje cell densities, suggesting CaMKIIα is required for CA/CPR-induced injury in the cerebellum. We also observed a CA/CPR-induced activation of death-associated protein kinase (DAPK1) that tat-CN19o did not block. In summary, our findings indicate that inhibition of autonomous CaMKII activity is a promising therapeutic approach that is effective across multiple brain regions.
Asunto(s)
Calcio/metabolismo , Calmodulina/efectos de los fármacos , Sustancias Protectoras/farmacología , Células de Purkinje/efectos de los fármacos , Animales , Señalización del Calcio/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Masculino , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Células de Purkinje/metabolismoRESUMEN
OBJECTIVE: We recently reported that the heat shock response played a major role in the protection of hair cells against stress. Oral administration of the heat shock inducer, geranylgeranylacetone (GGA) protected hair cells against intense noise. In our present study, we investigated the effect of GGA on vestibular hair cell death induced by an aminoglycoside. METHODS: We used CBA/N mice aged 4-6 weeks. The mice were divided into two groups, GGA and control. Mice in the GGA group were fed a diet containing GGA (0.5%) for 4 weeks, and those in the control group were fed a standard diet. Immunohistochemical analyses for Hsp70 were performed in four animals. The utricles of the remaining animals were cultured in medium for 24h with neomycin to induce hair cell death. After fixation, the vestibular hair cells were immunohistochemically stained against calmodulin, and hair cell survival was evaluated. RESULTS: The vestibular hair cells of mice in the GGA group expressed Hsp70. In addition, after exposure to neomycin, vestibular hair cell survival was higher in the GGA group than in the control group. CONCLUSION: Our results demonstrated the oral administration of GGA induced the heat shock response in the vestibule and could protect sensory cells.
Asunto(s)
Antibacterianos/toxicidad , Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Diterpenos/farmacología , Células Ciliadas Vestibulares/efectos de los fármacos , Neomicina/toxicidad , Administración Oral , Animales , Calmodulina/efectos de los fármacos , Calmodulina/metabolismo , Supervivencia Celular/efectos de los fármacos , Proteínas del Choque Térmico HSP72/efectos de los fármacos , Proteínas del Choque Térmico HSP72/metabolismo , Células Ciliadas Vestibulares/metabolismo , Respuesta al Choque Térmico/efectos de los fármacos , Ratones , Ratones Endogámicos CBA , Sáculo y Utrículo/citología , Sáculo y Utrículo/metabolismoRESUMEN
Taurochenodeoxycholic acid (TCDCA), a natural bioactive substance in animal bile, has anti-inflammatory and immunoregulatory effects. This study evaluates the effects of TCDCA on calcium/calmodulin (Ca2+/CaM) signalling mediated by G Protein Coupled Bile Acid Receptor 1 (TGR5) to provide preliminary information on the mechanism of TCDCA in immune regulation and also to benefit future research. After treatment of NR8383 and high TGR5 expression cell (TGR5-NR8383) with TCDCA (10-6 mol/L, 10-5 mol/L, and 10-4 mol/L) for 1 h, we measured TGR5 and CaM gene and protein levels by quantitative reverse transcription-polymerase chain reaction (qPCR) and western blotting, respectively. The inositol triphosphate (IP3) concentration was measured by Enzyme-linked Immunosorbent Assay (ELISA), and the Ca2+ concentration was measured by calcium fluorescent probe (Fluo-3 AM). The present study showed that the expression levels of IP3, Ca2+, and CaM in NR8383 cells were increased by TCDCA at concentrations ranging from 10-6 mol/L to 10-4 mol/L. TCDCA (10-4 mol/L) increased both the gene and protein expression of IP3and CaM through TGR5. TCDCA (10-4 mol/L or 10-5 mol/L) also increased the Ca2+ concentration via the TGR5 receptor. Our data suggest TCDCA activates Ca2+/CaM signalling via the TGR5 signalling pathway.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Calmodulina/efectos de los fármacos , Receptores Acoplados a Proteínas G/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Ácido Tauroquenodesoxicólico/farmacología , Animales , Señalización del Calcio/genética , Calmodulina/biosíntesis , Calmodulina/genética , Línea Celular , Expresión Génica/efectos de los fármacos , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Calmodulin (CaM) is a calcium-sensing protein that can bind to and activate various target enzymes. Here, electrospray ionization mass spectrometry (ESI-MS) was used to investigate calcium-induced structural changes of CaM, as well as binding to the model target melittin (Mel). Nonspecific metalation artifacts were eliminated by conducting the experiments in negative ion mode and with calcium tartrate as metal source [Pan et al. (2009) Anal. Chem. 81, 5008]. Two coexisting CaM subpopulations can be distinguished on the basis of their ESI charge state distributions, namely, relatively disordered conformers (CaM(D), high charge states) and more tightly folded proteins (CaM(F), low charge states). Calcium titration experiments on isolated CaM reveal that the transition from apo-CaM(D) to Ca(4).CaM(F) proceeds with apparent K(d) values of 10, 14, 30, and 12 microM. In the presence of Mel, a gradual [Ca(2+)] increase results in an overall population shift from apo-CaM(D) to Ca(4).CaM(F).Mel. This transition involves various intermediates, Ca(n).CaM(F).Mel with n = 0, ..., 3, as well as apo-CaM(D).Mel. Thus, neither the binding of four Ca(2+) nor the existence of a tightly folded CaM conformation is a prerequisite for target binding. Millisecond time-resolved ESI-MS experiments were conducted to monitor the response of a premixed CaM-Mel solution to a calcium concentration jump, thereby mimicking the conditions encountered in a cellular signaling context. The resulting data suggest that the formation of Ca(4).CaM(F).Mel proceeds along three parallel kinetic pathways: (1) metal binding to CaM(D) followed by formation of a compact protein-target complex, (2) folding of the apoprotein, then target binding, followed by metal complexation, (3) target binding to apo-CaM(D) followed by sequential metal binding. The exact structural properties of the various metal-unsaturated CaM species, as well as their physiological roles, remain to be elucidated.
Asunto(s)
Calcio/farmacología , Calmodulina/química , Meliteno/química , Sitios de Unión , Calcio/química , Calmodulina/efectos de los fármacos , Calmodulina/metabolismo , Cinética , Meliteno/metabolismo , Metales/química , Metales/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-G(q/11)alpha-binding, catalytically-inactive (D110A,K220R)G protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCepsilon, but not Ca2+/diacylglycerol-sensitive eGFP-PKCbetaII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.
Asunto(s)
Hipocampo/metabolismo , Neuronas/metabolismo , Proteínas Quinasas/metabolismo , Receptor Muscarínico M1/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Animales , Animales Recién Nacidos , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Calmodulina/efectos de los fármacos , Calmodulina/metabolismo , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Antagonistas del GABA/farmacología , Hipocampo/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Neuronas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/metabolismo , Proteínas Quinasas/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Ratas , Receptor Muscarínico M1/efectos de los fármacos , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacosRESUMEN
Electrophysiological recording reveals that chronic nicotine treatment prevents stress-induced impairment of long-term potentiation (LTP) in the CA1 region of the hippocampus of anesthetized rats. We investigated the molecular mechanism of this action of nicotine in the CA1 region. Immunoblot analysis showed that chronic nicotine treatment (1 mg/kg, 2 times/day) normalized the stress-induced decrease in the basal levels of BDNF, CaMKII (total and phosphorylated; P-CaMKII), and calmodulin. Additionally, nicotine reversed the stress-induced increase in calcineurin basal levels. Chronic nicotine treatment also markedly increased the basal levels of BDNF in naïve rats. Furthermore, high-frequency stimulation (HFS), which increased the levels of P-CaMKII in control as well as nicotine-treated stressed rats, failed to increase P-CaMKII levels in untreated stressed rats. Compared to unstimulated control, the levels of both total CaMKII and calcineurin were increased after HFS in all groups including the stressed, but no changes were detected after HFS in the levels of BDNF and calmodulin. These results indicate that normalization by nicotine of the stress-induced changes in the levels of signaling molecules including BDNF may contribute to the recovery of LTP.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/efectos de los fármacos , Estimulantes Ganglionares/farmacología , Hipocampo/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Nicotina/farmacología , Estrés Psicológico/fisiopatología , Animales , Western Blotting , Calcineurina/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/efectos de los fármacos , Calmodulina/efectos de los fármacos , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Masculino , Psicología , Ratas , Ratas Wistar , Estrés Psicológico/prevención & controlRESUMEN
Lanthanum ion (La3+) was generally regarded as calcium antagonist and was used as calcium channel blocker. However, its potential biological effects on cells were poorly understood. In the present work, it was found that La3+ could induce rapid extracellular signal-regulated kinase (ERK) phosphorylation in both HeLa cells and NIH 3T3 cells, but different mechanisms were involved. At a concentration of 30 microM or higher, La3+ enters the cells and activates ERK through a mechanism involving calmodulin activation inside the cells, which is similar to the action of intracellular Ca2+. However, at lower concentration, free La3+ promoted ERK phosphorylation in NIH 3T3 cells outside the cells through an unknown La3+ sensing mechanism, while Ca2+ exerted much weaker effect. The present results suggested that the biological effects of La3+ on cells maybe involve mechanisms beyond calcium antagonist.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Lantano/farmacología , Animales , Calmodulina/efectos de los fármacos , Calmodulina/metabolismo , Membrana Celular/efectos de los fármacos , Células Cultivadas , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Fosforilación/efectos de los fármacosAsunto(s)
Antineoplásicos Fitogénicos/farmacología , Medicamentos Herbarios Chinos/farmacología , Neoplasias/patología , Transducción de Señal/efectos de los fármacos , Animales , Calmodulina/efectos de los fármacos , Calmodulina/fisiología , Comunicación Celular/efectos de los fármacos , AMP Cíclico/fisiología , Humanos , Neoplasias/fisiopatología , Células Tumorales CultivadasRESUMEN
In this study we aimed to (1). screen phenothiazines for cytotoxic activity in glioma, neuroblastoma, and primary mouse brain tissue; and (2). determine the mechanism of the cytotoxic effect (apoptosis, necrosis) and the roles of calmodulin inhibition and sigma receptor modulation. Rat glioma (C6) and human neuroblastoma (SHSY-5Y) cell lines were treated with different phenothiazines. All agents induced a dose-dependent decrease in viability and proliferation, with the highest activity elicited by thioridazine. Sensitivity to thioridazine of glioma and neuroblastoma cells was significantly higher (p < 0.05) than that of primary mouse brain culture (IC50 11.2 and 15.1 microM vs 41.3 microM, respectively). The N-mustard fluphenazine induced significantly lower cytotoxicity in glioma cells, compared to fluphenazine. The sigma receptor selective ligand (+)-SK&F10047 increased viability slightly while combined with fluphenazine; SK&F10047 did not alter fluphenazine activity. Flow cytometry of propidium iodide (PI)-stained glioma cells treated with thioridazine, fluphenazine, or perphenazine (6-50 microM) resulted in a concentration-dependent increase of fragmented DNA up to 94% vs 3% in controls by all agents. Thioridazine (12.5 microM)-treated glioma cells costained with PI and Hoechst 33342 revealed a red fluorescence of fragmented nuclei in treated cells and a blue fluorescence of intact control nuclei. After 4-h exposure to thioridazine (25 and 50 microM), a 25- to 30-fold increase in caspase-3 activity in neuroblastoma cells was noted. Overall, the marked apoptotic effect of phenothiazines in brain-derived cancer cells, and the low sensitivity of primary brain tissue suggest the potential use of selected agents as therapeutic modalities in brain cancer.
Asunto(s)
Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Glioma/tratamiento farmacológico , Neuroblastoma/tratamiento farmacológico , Fenazocina/análogos & derivados , Fenotiazinas/toxicidad , Animales , Antineoplásicos/uso terapéutico , Apoptosis/fisiología , Calmodulina/efectos de los fármacos , Calmodulina/metabolismo , Caspasa 3 , Caspasas/efectos de los fármacos , Caspasas/metabolismo , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/fisiología , Relación Dosis-Respuesta a Droga , Flufenazina/toxicidad , Humanos , Ratones , Ratones Endogámicos ICR , Fenazocina/farmacología , Fenotiazinas/uso terapéutico , Ratas , Receptores sigma/efectos de los fármacos , Receptores sigma/metabolismo , Tioridazina/toxicidad , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
The denaturation of calmodulin (CaM) induced by urea has been studied by small-angle X-ray scattering, which is a direct way to evaluate the shape changes in a protein molecule. In the absence of Ca(2+), the radii of gyration (R(g)) of CaM are 20.8+/-0.3 A in the native state and about 34+/-1.0 A in the unfolded state. The transition curve derived from Kratky plots indicates a bimodal transition via a stable unfolding intermediate around 2.5 M urea. In the presence of Ca(2+) and in the presence of both Ca(2+) and a target peptide, the R(g) values are 21.5+/-0.3 and 18.1+/-0.3 A in the native state and 26.7+/-0.4 and 24.9+/-0.4 A at 9 M urea, respectively. The results indicate that a stable unfolding intermediate still persists in 9 M urea. The present results suggest that the shape of unfolding intermediates is an asymmetric dumbbell-like structure, one in the folded and one in the unfolded state.
Asunto(s)
Calmodulina/química , Urea/farmacología , Calmodulina/efectos de los fármacos , Desnaturalización Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Difracción de Rayos XRESUMEN
We examined the influence of 3,5-diisopropylsalicylic acid (3,5-DIPS) and calcium(II)3 (3,5-diisopropylsalicylate)6 (H2 O)6 [Ca(II)3 (3,5-DIPS)6 ], a new activator of calcium-dependent calmodulin-triggered nitric oxide synthase, on thrombin-induced platelet P-selectin expression. Citrated whole blood samples were incubated with either ethanol vehicle, 3,5-DIPS, or Ca(II)3 (3,5-DIPS)6. These whole blood samples were also co-incubated with thrombin receptor activating peptide (TRAP) or adenosine diphosphate (ADP), to up-regulate P-selectin (CD62P) on platelets. Both TRAP and ADP up-regulated P-selectin on platelets compared with platelets in whole blood samples that were not incubated with either platelet activator. Co-incubation of whole blood samples with TRAP, ADP together with 3,5-DIPS, or Ca(II)3 (3,5-DIPS)6 revealed that Ca(II)3 (3,5-DIPS)6 caused a decrease in platelet P-selectin expression for TRAP, ADP, and no-activator co-incubated samples of whole blood. Incubation of platelets with 3,5-DIPS also caused a decrease in ADP-induced up-regulation of P-selectin but failed to affect TRAP or no-activator-treated platelets. Incubation of whole blood with Ca(II)3 (3,5-DIPS)6 induced some hemolysis. We found that hemolysis increases basal P-selectin expression on platelets. We therefore conclude that Ca(II)3 (3,5-DIPS)6 decreased not only basal, but also hemolysis-induced P-selectin expression on platelets. In contrast, incubation of haemolysed whole blood with SIN-1 (standard nitric oxide-releasing drug) had no effect on P-selectin expression. In summary, Ca(II)3 (3,5-DIPS)6, a new calmodulin-dependent nitric oxide synthase activator, decreases P-selectin expression of human platelets in response to thrombin receptor activation. Improved calcium-dependent calmodulin activators may become useful drugs for the treatment of disorders associated with platelet activation, and P-selectin may decrease expression due to hemolysis.
Asunto(s)
Calcio/farmacología , Calmodulina/metabolismo , Molsidomina/análogos & derivados , Selectina-P/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Salicilatos/farmacología , Adenosina Difosfato/farmacología , Adulto , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Calcio/química , Calmodulina/efectos de los fármacos , Estudios Transversales , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Femenino , Citometría de Flujo , Humanos , Técnicas In Vitro , Masculino , Molsidomina/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Selectina-P/sangre , Selectina-P/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Salicilatos/química , omega-N-Metilarginina/farmacologíaRESUMEN
The aim of the presented study was to compare the effect of 17-beta-estradiol on the hydrolytic activity of plasma membrane calcium pump (PMCA) purified from excitable (rat cortical synaptosomes) and non-excitable (human erythrocytes) cells. Both types of cell membranes contained different composition of the PMCA isoforms. To elucidate if the hormone action could depend on structure of PMCA protein, we assayed the hormone effect on Ca(2+)-ATP-ases pretreated for 15 and 40 min with trypsin. The full length and trypsin-treated Ca(2+)-ATP-ases were next incubated with 17-beta-estradiol at a concentration of 10(-9) and 10(-7) M. In addition, stimulation of calcium pumps by naturally existing activator, calmodulin, was tested. The activity of synaptosomal and erythrocyte Ca(2+)-ATP-ases was differently altered in the trypsin-treated samples. At physiologically relevant concentration of estradiol (10(-9) M), a significant enhancement of the activity was observed for synaptosomal Ca(2+)-ATP-ase, and a further increase occurred in the enzyme treated with trypsin for 15 min. The highest activity of erythrocyte calcium pump was induced after 40 min of incubation with protease. Moreover, the potency of the truncated calcium pump to promote ATP hydrolysis was approximately 2-fold elevated in the presence of 17-beta-estradiol. Calmodulin significantly stimulated the Ca(2+)-ATP-ase, but only the erythrocyte enzyme digested with trypsin for 15 min. It may be suggested that PMCA is a target for estradiol, that shows different mechanisms of action depending on isoform compositions and structural features of the enzyme.
Asunto(s)
ATPasas Transportadoras de Calcio/efectos de los fármacos , ATPasas Transportadoras de Calcio/fisiología , Membrana Celular/efectos de los fármacos , Estradiol/farmacología , Animales , Calcio/metabolismo , ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/metabolismo , Calmodulina/efectos de los fármacos , Calmodulina/farmacología , Membrana Celular/química , Membrana Celular/enzimología , Fenómenos Fisiológicos Celulares , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/enzimología , Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/enzimología , Humanos , Isoenzimas/química , Isoenzimas/efectos de los fármacos , Isoenzimas/metabolismo , Ratas , Sinaptosomas/efectos de los fármacos , Sinaptosomas/enzimología , Factores de Tiempo , Tripsina/farmacologíaRESUMEN
Bioassay-guided fractionation of a phytotoxic extract of Prionosciadium watsoni led to the isolation of three new pyranocoumarins and two pyranochromones. The new compounds were characterized as propionic acid (9R,10R)-9-acetoxy-8,8-dimethyl-9,10-dihydro-2H,8H-benzo[1,2-b:3,4-b']dipyran-2-one-10-yl ester (1), isobutyric acid (9R,10R)-9-hydroxy-8,8-dimethyl-9,10-dihydro-2H,8H-benzo[1,2-b:3,4-b']dipyran-2-one-10-yl ester (2), isobutyric acid (9R)-8,8-dimethyl-9,10-dihydro-2H,8H-benzo[1,2-b:3,4-b']dipyran-2-one-9-yl ester (10), 2-methylbut-(2Z)-enoic acid (3R)-5-methoxy-3,4-dihydro-2,2,8-trimethyl-6-oxo-2H,6H-benzo[1,2-b:5,4-b']dipyran-3-yl ester (11), and isobutyric acid (3R)- 5-methoxy-3,4-dihydro-2,2,8-trimethyl-6-oxo-2H,6H-benzo[1,2-b:5,4-b']dipyran-3-yl ester (12) by spectroscopic and chemical methods. The stereochemistry at the stereogenic centers was established by applying the Mosher ester methodology. The structures of 1 and 2 were corroborated by single-crystal X-ray diffraction studies. The phytotoxic activity of the isolated compounds was assessed on Amaranthus hypochondriacus, Echinochloa crus-galli, and Lemna pausicostata. The phytotoxins also modified the electrophoretic mobility of calmodulin from both bovine-brain and spinach.
Asunto(s)
Apiaceae/química , Compuestos Heterocíclicos con 3 Anillos/aislamiento & purificación , Plantas Medicinales/química , Plantas Tóxicas/química , Amaranthus/efectos de los fármacos , Animales , Encéfalo , Calmodulina/efectos de los fármacos , Bovinos , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Compuestos Heterocíclicos con 3 Anillos/química , Compuestos Heterocíclicos con 3 Anillos/farmacología , Espectroscopía de Resonancia Magnética , México , Conformación Molecular , Estructura Molecular , Spinacia oleracea/química , EstereoisomerismoRESUMEN
The pineal hormone, melatonin, has been shown to inhibit the proliferation of the estrogen receptor alpha (ERalpha)-positive macrophage chemotactic factor (MCF)-7 human breast cancer cells. Previous studies from other systems indicate that melatonin modulates the calcium (Ca2+)/calmodulin (CaM) signaling pathway either by changing intracellular calcium concentration ([Ca2+]i) via activation of its G-protein coupled membrane receptors, or through a direct interaction with CaM. In this study, although melatonin alone had no effect on basal [Ca2+]i in MCF-7 cells, it significantly enhanced the elevation of [Ca2+]i induction by extracellular adenosine triphosphate (ATP), which increases [Ca2+]i via the G protein-coupled P2y-purinoceptor and the phospholipase C (PLC) pathway. Pretreatment of MCF-7 cells with 10(-7) M melatonin increased the 10(-5) M ATP-induced [Ca2+]i peak change from 79.4 +/- 11.6 nM to 146.2 +/- 22.3 nM. Furthermore, without changing total cellular CaM levels, melatonin markedly increased the amount of membrane-bound CaM to 237 and 162% of control levels after I and 6 hr of treatment, respectively. Cytosolic CaM levels were also elevated to 172% of control after 6 hr of melatonin treatment. Correlative growth studies demonstrated that ATP (10(-5) M) can stimulate MCF-7 cell growth, that melatonin can suppress MCF-7 cell proliferation, but that pretreatment of MCF-7 cells with melatonin followed by ATP(10(-5) M), like 10(-4) M ATP can further suppress MCF-7 cell proliferation; this indicates that melatonin's potentiation of ATP induced [Ca2+]i may be above the threshold for cell growth. Given the important role of [Ca2+]i and CaM in tumor cell homeostasis and proliferation and melatonin's modulation of [Ca2+]i, melatonin's effects on the Ca2+/CaM signaling pathway may play an important role in mediating the growth-inhibitory effect of melatonin on MCF-7 human breast cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Melatonina/farmacología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Calmodulina/efectos de los fármacos , División Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Humanos , Transducción de Señal , Fracciones Subcelulares , Células Tumorales CultivadasRESUMEN
Calmodulin redistribution in MDCK and HeLa cells subjected to microtubule perturbations by antimitotic drugs was followed using a calmodulin-EGFP fusion protein that preserves the Ca(2+) affinity, target binding and activation properties of native calmodulin. CaM-EGFP targeting to spindle structures in normal cell division and upon spindle microtubule disruption allows evaluation of the dynamic redistribution of calmodulin in cell division. Under progressive treatment of stably transfected mammalian cells with nocodazole or vinblastine, the centrosomal matrix at the mitotic poles subdivides into numerous small 'star-like' structures, with the calmodulin concentrated centrally, and partially distinct from the reduced microtubule mass to which kinetochores and chromosomes are attached. Prolonged vinblastine treatment causes the release of localised calmodulin into a uniform cytoplasmic distribution, and tubulin paracrystal formation. By contrast, paclitaxel treatment of metaphase cells apparently causes limited disassembly of the pericentriolar material into a number of multipolar 'ring-like' structures containing calmodulin, each one having multiple attached microtubules terminating in the partially disordered kinetochore/chromosome complex. Thus drugs with opposite effects in either destabilising or stabilising mitotic microtubules cause subdivision of the centrosomal matrix into two distinctive calmodulin-containing structures, namely small punctate 'stars' or larger polar 'rings' respectively. The 'star-like' structures may represent an integral subcomponent for the attachment of kinetochore microtubules to the metaphase centrosome complex. The results imply that microtubules have a role in stabilising the structure of the pericentriolar matrix, involving interaction, either direct or indirect, with one or more proteins that are targets for binding of calmodulin. Possible candidates include the pericentriolar matrix-associated coiled-coil proteins containing calmodulin-binding motifs, such as myosin V, kendrin (PCNT2) and AKAP450.
Asunto(s)
Calmodulina/metabolismo , División Celular/fisiología , Centrosoma/metabolismo , Células Eucariotas/metabolismo , Microtúbulos/metabolismo , Animales , Antineoplásicos/farmacología , Calmodulina/efectos de los fármacos , División Celular/efectos de los fármacos , Centrosoma/efectos de los fármacos , Células Eucariotas/citología , Células Eucariotas/efectos de los fármacos , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes , Microtúbulos/efectos de los fármacos , Nocodazol/farmacología , Paclitaxel/farmacología , Proteínas Recombinantes de Fusión , Vinblastina/farmacologíaRESUMEN
Spin-label electron paramagnetic resonance (EPR) provides optimal resolution of dynamic and conformational heterogeneity on the nanosecond time-scale and was used to assess the structure of the sequence between Met(76) and Ser(81) in vertebrate calmodulin (CaM). Previous fluorescence resonance energy transfer and anisotropy measurements indicate that the opposing domains of CaM are structurally coupled and the interconnecting central sequence adopts conformationally distinct structures in the apo-form and following calcium activation. In contrast, NMR data suggest that the opposing domains of CaM undergo independent rotational dynamics and that the sequence between Met(76) and Ser(81) in the central sequence functions as a flexible linker that connects two structurally independent domains. However, these latter measurements also resolve weak internuclear interactions that suggest the formation of transient helical structures that are stable on the nanosecond time-scale within the sequence between Met(76) and Asp(80) in apo-CaM (H. Kuboniwa, N. Tjandra, S. Grzekiek, H. Ren, C. B. Klee, and A. Bax, 1995, Nat. Struct. Biol. 2:768-776). This reported conformational heterogeneity was resolved using site-directed mutagenesis and spin-label EPR, which detects two component spectra for 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate spin labels (MTSSL) bound to CaM mutants T79C and S81C that include a motionally restricted component. In comparison to MTSSL bound within stable helical regions, the fractional contribution of the immobilized component at these positions is enhanced upon the addition of small amounts of the helicogenic solvent trifluoroethanol (TFE). These results suggest that the immobilized component reflects the formation of stable secondary structures. Similar spectral changes are observed upon calcium activation, suggesting a calcium-dependent stabilization of the secondary structure. No corresponding changes are observed in either the solvent accessibility to molecular oxygen or the maximal hyperfine splitting. In contrast, more complex spectral changes in the line-shape and maximal hyperfine splitting are observed for spin labels bound to sites that undergo tertiary contact interactions. These results suggest that spin labels at solvent-exposed positions within the central sequence are primarily sensitive to backbone fluctuations and that either TFE or calcium binding stabilizes the secondary structure of the sequence between Met(76) and Ser(81) and modulates the structural coupling between the opposing domains of CaM.
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Calcio/metabolismo , Calmodulina/genética , Metionina/metabolismo , Serina/metabolismo , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , Animales , Calcio/farmacología , Calmodulina/química , Calmodulina/efectos de los fármacos , Pollos , Espectroscopía de Resonancia por Spin del Electrón/métodos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/fisiología , Conformación Proteica/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína/fisiología , Marcadores de Spin , Trifluoroetanol/farmacología , VertebradosRESUMEN
Calmodulin-binding sites on target proteins show considerable variation in primary sequence; hence compounds that block the access of calmodulin to these binding sites may be more selective than compounds that inactivate calmodulin. Suramin and its analogue NF307 inhibit the interaction of calmodulin with the ryanodine receptor. We have investigated whether inhibition of calmodulin binding to target proteins is a general property of these compounds. Suramin inhibited binding of [(125)I]calmodulin to porcine brain membranes and to sarcoplasmic reticulum from skeletal muscle (IC(50)=4.9+/-1.2 microM and 19.9+/-1.8 microM, respectively) and blocked the cross-linking of [(125)I]calmodulin to some, but not all, target proteins in brain membranes by [(125)I]calmodulin. Four calmodulin-binding proteins were purified [ryanodine receptor-1 (RyR1) from rabbit skeletal muscle, neuronal NO synthase (nNOS) from Sf9 cells, G-protein betagamma dimers (Gbetagamma) from porcine brain and a glutathione S-transferase-fusion protein comprising the C-terminal calmodulin-binding domain of the metabotropic glutamate receptor 7A (GST-CmGluR7A) from bacterial lysates]. Three of the proteins employed (Gbetagamma, GST-CmGluR7A and RyR1) display a comparable affinity for calmodulin (in the range of 50-70 nM). Nevertheless, suramin and NF307 only blocked the binding of Gbetagamma and RyR1 to calmodulin-Sepharose. In contrast, the association of GST-CmGluR7A and nNOS was not impaired, whereas excess calmodulin uniformly displaced all proteins from the matrix. Thus suramin and NF307 are prototypes of a new class of calmodulin antagonists that do not interact directly with calmodulin but with calmodulin-recognition sites. In addition, these compounds discriminate among calmodulin-binding motifs.