RESUMEN
Steatosis is the most important prognostic histologic feature in the setting of liver procurement. The currently utilized diagnostic methods, including gross evaluation and frozen section examination, have important shortcomings. Novel techniques that offer advantages over the current tools could be of significant practical utility. The aim of this study is to evaluate the accuracy of surface color spectrophotometry in the quantitative assessment of steatosis in a murine model of fatty liver. C57BL/6 mice were divided into a control group receiving normal chow (n = 19), and two steatosis groups receiving high-fat diets for up to 20 weeks-mild steatosis (n = 10) and moderate-to-severe steatosis (n = 19). Mouse liver surfaces were scanned with a hand-held spectrophotometer (CM-600D; Konica-Minolta, Osaka, Japan). Spectral reflectance data and color space values (L*a*b*, XYZ, L*c*h*, RBG, and CMYK) were correlated with histopathologic steatosis evaluation by visual estimate, digital image analysis (DIA), as well as biochemical tissue triglyceride measurement. Spectral reflectance and most color space values were very strongly correlated with histologic assessment of total steatosis, with the best predictor being % reflectance at 700 nm (r = 0.91 [0.88-0.94] for visual assessment, r = 0.92 [0.88-0.95] for DIA of H&E slides, r = 0.92 [0.87-0.95] for DIA of oil-red-O stains, and r = 0.78 [0.63-0.87] for biochemical tissue triglyceride measurement, p < 0.0001 for all). Several spectrophotometric parameters were also independently predictive of large droplet steatosis. In conclusion, hepatic steatosis can accurately be assessed using a portable, commercially available hand-held spectrophotometer device. If similarly accurate in human livers, this technique could be utilized as a point-of-care tool for the quantitation of steatosis, which may be especially valuable in assessing livers during deceased donor organ procurement.
Asunto(s)
Hígado Graso , Hígado , Espectrofotometría/métodos , Animales , Modelos Animales de Enfermedad , Hígado Graso/diagnóstico por imagen , Hígado Graso/patología , Técnicas Histológicas , Hígado/diagnóstico por imagen , Hígado/patología , Trasplante de Hígado , Masculino , Ratones , Ratones Endogámicos C57BL , Espectrofotometría/instrumentaciónRESUMEN
Cyclic adenosine diphospho-ribose (cADPR) triggers Ca2+ release from intracellular stores and is therefore proposed to function as a second messenger in cellular signaling; however, an extracellular stimulus, i.e., first messenger (hormone or autacoid) that modulates cADPR metabolism has not been identified. We discovered that all-trans-retinoic acid (atRA) is a potent stimulus to increase cADPR synthesis by cultured LLC-PK1 cells. The stimulation of cADPR synthesis by atRA is dose dependent between 0.1 nM and 1 microM (maximum increase approximately delta + 600%), while atRA does not alter the rate of cADPR hydrolysis by LLC-PK1 cells. The activity of other intrinsic apical membrane enzymes was not significantly altered. The stimulation of cADPR synthesis by atRA occurs after a lag period of 6-8 h, and the stimulation is inhibited by actinomycin D and by cycloheximide. Our results therefore demonstrate that atRA in physiological concentrations is a potent extracellular stimulus, first messenger, that enhances cADPR synthesis, and the effect of atRA requires de novo protein synthesis. We suggest that some of the diverse biologic actions of atRA such as morphogenetic and cell differentiation may be mediated via cADPR.
Asunto(s)
Adenosina Difosfato Ribosa/análogos & derivados , Antígenos CD , Tretinoina/farmacología , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Adenosina Difosfato Ribosa/biosíntesis , Compuestos de Anilina , Animales , Antígenos de Diferenciación/metabolismo , Calcio/metabolismo , División Celular , Membrana Celular/enzimología , ADP-Ribosa Cíclica , Cicloheximida/farmacología , ADN/biosíntesis , Dactinomicina/farmacología , Femenino , Cinética , Células LLC-PK1 , N-Glicosil Hidrolasas/metabolismo , Oocitos/fisiología , Erizos de Mar , Porcinos , Factores de Tiempo , XantenosRESUMEN
We studied interactions between the mitogen-activated protein kinase (MAPK) signalling pathway and cAMP-protein kinase (PKA) signaling pathway in regulation of mitogenesis of mesangial cells (MC) determined by [3H]thymidine incorporation, with or without added EGF. Forskolin or dibutyryl cAMP strongly (by 60-70%) inhibited [3H]thymidine incorporation into MC. Cilostamide, lixazinone or cilostazol selective inhibitors of cAMP-phosphodiesterase (PDE) isozyme PDE-III, inhibited mitogenesis to similar extent as forskolin and DBcAMP and activated in situ PKA, but without detectable increase in cAMP levels. Cilostamide and cilostazol were more than three times more effective at inhibiting mesangial mitogenesis than rolipram and denbufylline, inhibitors of isozyme PDE-IV, even though PDE-IV was two times more abundant in MC than was PDE-III. On the other hand, when incubated with forskolin, rolipram-enhanced cAMP accumulation was far greater (10-100x) than with cilostamide. EGF increased MAPK activity (+300%); PDE isozyme inhibitors which suppressed mitogenesis also inhibited MAPK. PDE isozyme inhibitors also suppressed PDGF-stimulated MC proliferation. We conclude that cAMP inhibits the mitogen-dependent MAPK-signaling pathway probably by decreasing the activity of Raf-1 due to PKA-catalyzed phosphorylation. Further, we surmise that minor increase in the cAMP pool metabolized by PDE-III is intimately related to regulation of mesangial proliferation. Thus, PDE isozyme inhibitors have the potential to suppress MC proliferation by a focused effect upon signaling pathways.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Mesangio Glomerular/efectos de los fármacos , Isoenzimas/antagonistas & inhibidores , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas , Factor de Crecimiento Epidérmico/farmacología , Mesangio Glomerular/citología , Mesangio Glomerular/enzimología , Masculino , Factor de Crecimiento Derivado de Plaquetas/farmacología , Pirrolidinonas/farmacología , Quinolonas/farmacología , Ratas , Ratas Sprague-Dawley , Rolipram , Timidina/metabolismoRESUMEN
We investigated whether ADP-ribosyl cyclase (ADPR-cyclase) in rat vascular smooth muscle cells (VSMCs) has enzymatic properties that differ from the well-characterized CD38-antigen ADPR-cyclase, expressed in HL-60 cells. ADPR-cyclase from VSMCs, but not CD38 ADPR-cyclase from HL-60 cells, was inhibited by gangliosides (10 micromol/L) GT(1B), GD(1), and GM(3). Preincubation of membranes from CD38 HL-60 cells, but not from VSMCs, with anti-CD38 antibodies increased ADPR-cyclase activity; CD38 antigen was detected both in VSMCs and in HL-60 cells. ADPR-cyclase in VSMC membranes was more sensitive than CD38 HL-60 ADPR-cyclase to inactivation by N-endoglycosidase F and to thermal inactivation at 45 degrees C. The specific activity of ADPR-cyclase in membranes from VSMCs was >20-fold higher than in membranes from CD38 HL-60 cells. Most importantly, VSMC ADPR-cyclase was inhibited by Zn(2+) and Cu(2+) ions; the inhibition by Zn(2+) was dose dependent, noncompetitive, and reversible by EDTA. In contrast, Zn(2+) stimulated the activity of CD38 HL-60 ADPR-cyclase and other known types of ADPR-cyclases. Retinoids act either via the nuclear receptor retinoic acid receptor or retinoid X receptor, including all-trans retinoic acid (atRA), and panagonist 9-cis-retinoic acid-upregulated VSMC ADPR-cyclase; the stimulatory effect of atRA was blocked by actinomycin D and cycloheximide. 1,25(OH)(2)-Vitamin D(3) (calciferol) stimulated VSMC ADPR-cyclase dose dependently at subnanomolar concentrations (ED(50) congruent with 56 pmol/L). Oral administration of atRA to rats resulted in an increase of ADPR-cyclase activity in aorta ( congruent with+60%) and, to a lesser degree, in myocardium of left ventricle (+18%), but atRA had no effect on ADPR-cyclases in lungs, spleen, intestinal smooth muscle, skeletal muscle, liver, or testis. Administration of 3,5,3'-triiodothyronine (T(3)) to rats resulted in an increase of ADPR-cyclase activity in aorta ( congruent with+89%), but not in liver or brain. We conclude the following: (1) ADPR-cyclase in VSMCs has enzymatic properties distinct from "classic" CD38 ADPR-cyclase, especially sensitivity to inhibition by Zn(2+) and Cu(2+); (2) ADPR-cyclase in VSMCs is upregulated by various retinoids, calcitriol, and T(3) in vitro; and (3) administration of atRA and T(3) increases ADPR-cyclase in aorta in vivo. We suggest that the cADPR signaling system plays an important role in the regulation of VSMC functions in response to steroid superfamily hormones.
Asunto(s)
Antígenos CD , Antígenos de Diferenciación/metabolismo , Músculo Liso Vascular/enzimología , NAD+ Nucleosidasa/metabolismo , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Animales , Calcitriol/farmacología , Células Cultivadas , Cobre/farmacología , Células HL-60/enzimología , Humanos , Masculino , Glicoproteínas de Membrana , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Retinoides/farmacología , Distribución Tisular , Tretinoina/farmacología , Triyodotironina/farmacología , Regulación hacia Arriba , Zinc/farmacologíaRESUMEN
Store-operated Ca2+ entry plays an important role in Ca2+ homeostasis in cells but the mechanisms of control of these channels are not completely understood. We describe an investigation of the role of the CD38-cyclic-ADP-ribose (cADPR)-ryanodine-channel (RyR) signaling pathway in store-operated Ca2+ entry in human smooth muscle. We observed that human myometrial cells have a functional store-operated Ca2+ entry mechanism. Furthermore, we observed the presence of transient receptor potential 1, 3, 4, 5, and 6 ion channels in human myometrial cells. Store-operated Ca2+ transient was inhibited by at least 50-70% by several inhibitors of the RyR, including ryanodine (10 microM), dantrolene (10 microM), and ruthenium red (10 microM). Furthermore, the cell permeable inhibitor of the cADPR-system, 8-Br-cADPR (100 microM), is a potent inhibitor of the store-operated entry, decreasing the store operated entry by 80%. Pre-incubation of cells with 100 microM cADPR and the hydrolysis-resistant cADPR analog 3-deaza-cADPR (50 microM), but not with ADP-ribose (ADPR) leads to a 1.6-fold increase in the store-operated Ca2+ transient. In addition, we observed that nicotinamide (1-10 mM), an inhibitor of cADPR synthesis, also leads to inhibition of the store-operated Ca2+ transient by 50-80%. Finally, we observed that the transient receptor potential channels, RyR, and CD38 can be co-immunoprecipitated, indicating that they interact in vivo. Our observations clearly implicate the CD38-cADPR-ryanodine signaling pathway in the regulation of store-operated Ca2+ entry in human smooth muscle cells.
Asunto(s)
Calcio/metabolismo , ADP-Ribosa Cíclica/metabolismo , Miocitos del Músculo Liso/metabolismo , Miometrio/citología , ADP-Ribosil Ciclasa 1 , Western Blotting , Calcio/farmacología , ADP-Ribosa Cíclica/antagonistas & inhibidores , ADP-Ribosa Cíclica/farmacología , Femenino , Humanos , Inmunohistoquímica , Inmunoprecipitación , Miocitos del Músculo Liso/efectos de los fármacos , Miometrio/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Transducción de Señal , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
The (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum catalyzes the hydrolysis of acetyl phosphate in the presence of Mg2+ and EGTA and is stimulated by Ca2+. The Mg2(+)-dependent hydrolysis of acetyl phosphate measured in the presence of 6 mM acetyl phosphate, 5 mM MgCl2, and 2 mM EGTA is increased 2-fold by 20% dimethyl sulfoxide. This activity is further stimulated 1.6-fold by the addition of 30 mM KCl. In this condition addition of Ca2+ causes no further increase in the rate of hydrolysis and Ca2+ uptake is reduced to a low level. In leaky vesicles, hydrolysis continues to be back-inhibited by Ca2+ in the millimolar range. Unlike ATP, acetyl phosphate does not inhibit phosphorylation by Pi unless dimethyl sulfoxide is present. The presence of dimethyl sulfoxide also makes it possible to detect Pi inhibition of the Mg2(+)-dependent acetyl phosphate hydrolysis. These results suggest that dimethyl sulfoxide stabilizes a Pi-reactive form of the enzyme in a conformation that exhibits comparable affinities for acetyl phosphate and Pi. In this conformation the enzyme is transformed from a Ca2(+)- and Mg2(+)-dependent ATPase into a (K+ + Mg2+)-ATPase.
Asunto(s)
ATPasa de Ca(2+) y Mg(2+)/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Magnesio/farmacología , Músculos/enzimología , Organofosfatos/metabolismo , Potasio/farmacología , Retículo Sarcoplasmático/enzimología , Animales , ATPasa de Ca(2+) y Mg(2+)/antagonistas & inhibidores , Calcio/metabolismo , Calcio/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Dimetilsulfóxido/farmacología , Ácido Egtácico/farmacología , Hidrólisis , Organofosfatos/farmacología , Fosfatos/farmacología , Fosfoproteínas/metabolismo , Conformación Proteica , ConejosRESUMEN
Volatile anesthetics have multiple actions on intracellular Ca(2+) homeostasis, including activation of the ryanodine channel (RyR) and sensitization of this channel to agonists such as caffeine and ryanodine. Recently it has been described that the nucleotide cADP-ribose (cADPR) is the endogenous regulator of the RyR in many mammalian cells, and cADPR has been proposed to be a second messenger in many signaling pathways. I investigated the effect of volatile anesthetics on the cADPR signaling system, using sea urchin egg homogenates as a model of intracellular Ca(2+) stores. Ca(2+) uptake and release were monitored in sea urchin egg homogenates by using the fluo-3 fluorescence technique. Activity of the ADP-ribosyl cyclase was monitored by using a fluorometric method using nicotinamide guanine dinucleotide as a substrate. Halothane in concentrations up to 800 microM did not induce Ca(2+) release by itself in sea urchin egg homogenates. However, halothane potentiates the Ca(2+) release mediated by agonists of the ryanodine channel, such as ryanodine. Furthermore, other volatile anesthetics such as isoflurane and sevoflurane had no effect. Halothane also potentiated the activation of the ryanodine channel mediated by the endogenous nucleotide cADPR. The half-maximal concentration for cADPR-induced Ca(2+) release was decreased about three times by addition of 800 microM halothane. The reverse was also true: addition of subthreshold concentrations of cADPR sensitized the homogenates to halothane. In contrast, all the volatile anesthetics used had no effect on the activity of the enzyme that synthesizes cADPR. I propose that the complex effect of volatile anesthetics on intracellular Ca(2+) homeostasis may involve modulation of the cADPR signaling system.
Asunto(s)
Adenosina Difosfato Ribosa/farmacología , Anestésicos por Inhalación/farmacología , Calcio/metabolismo , Adenosina Difosfato Ribosa/análogos & derivados , Animales , ADP-Ribosa Cíclica , Halotano/farmacología , Masculino , Ratas , Ratas Sprague-Dawley , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/fisiologíaRESUMEN
Trout and rabbit (Ca2+ + Mg2+)-ATPases from sarcoplasmic reticulum were compared for differences in thermal inactivation and susceptibility to trypsin digestion. The trout ATPase is more heat-sensitive than the rabbit ATPase and is stabilized by Ca2+, Na+, K+ and nucleotides. Solubilization of both ATPases shows that the two ATPases have different protein-intrinsic inactivation kinetics. When digested by trypsin, the two ATPases display different cleavage patterns. The present results indicate that the trout and rabbit ATPases have dissimilarities in protein structure that may explain the differences in thermal inactivation kinetics.
Asunto(s)
ATPasa de Ca(2+) y Mg(2+)/metabolismo , Retículo Sarcoplasmático/enzimología , Animales , ATPasa de Ca(2+) y Mg(2+)/fisiología , Relación Dosis-Respuesta a Droga , Activación Enzimática/fisiología , Estabilidad de Enzimas/efectos de los fármacos , Estabilidad de Enzimas/fisiología , Calor , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Litio/farmacología , Peso Molecular , Músculo Esquelético/enzimología , Músculo Esquelético/metabolismo , Octoxinol/farmacología , Potasio/farmacología , Conformación Proteica/efectos de los fármacos , Conejos , Retículo Sarcoplasmático/metabolismo , Sodio/farmacología , Solubilidad , Espectrometría de Fluorescencia , Trucha , Tripsina/metabolismoRESUMEN
The disaccharides trehalose and sucrose, and to a lesser extent the monosaccharides glucose and fructose, decrease the apparent Km of the Ca2+, Mg(2+)-ATPase of sarcoplasmic reticulum for Pi. This effect is more pronounced at pH 7.4 than at pH 6.2. The enzyme is not phosphorylated by Pi when the temperature of the medium is decreased to 0 degree C, but when 1.5 M trehalose or sucrose is present phosphoenzyme formation increases to 0.5 mumol E-P/g protein.
Asunto(s)
ATPasa de Ca(2+) y Mg(2+)/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Fructosa/farmacología , Glucosa/farmacología , Fosfatos/metabolismo , Retículo Sarcoplasmático/enzimología , Sacarosa/farmacología , Trehalosa/farmacología , Animales , ATPasa de Ca(2+) y Mg(2+)/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Cinética , FosforilaciónRESUMEN
We have recently found that nicotinate adenine dinucleotide phosphate (NAADP) is a potent agonist that triggers Ca2+ release from intracellular stores of sea urchin eggs, and that its action is distinct from effects of IP3 and cyclic ADP-ribose (J. Biol. Chem. 270:3216, 1995). Now we report that extracts from rat brain, heart, liver, and spleen but not kidney cortex contain enzymatic activity which catalyzes NAADP synthesis by exchange of nicotinamide for nicotinic acid and which is probably catalyzed by NAD(P)-glycohydrolase. Extracts from these tissues also inactivate NAADP in the rank inverse to their ability for NAADP synthesis. These results suggest that NAADP, a Ca(2+)-releasing agent, can be generated in mammalian tissues, namely in brain.
Asunto(s)
Calcio/metabolismo , NADP/análogos & derivados , Animales , Transporte Biológico , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Encéfalo/metabolismo , Calcio/agonistas , Corazón/efectos de los fármacos , Corteza Renal/efectos de los fármacos , Corteza Renal/enzimología , Corteza Renal/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/metabolismo , Masculino , Miocardio/enzimología , Miocardio/metabolismo , N-Glicosil Hidrolasas/metabolismo , NAD+ Nucleosidasa , NADP/biosíntesis , NADP/metabolismo , NADP/farmacología , Niacina/farmacología , Ratas , Ratas Sprague-Dawley , Erizos de Mar , Bazo/efectos de los fármacos , Bazo/enzimología , Bazo/metabolismoRESUMEN
We investigated the effect of glycolytic pathway intermediaries upon Ca(2+) release induced by cyclic ADP-ribose (cADPR), inositol 1',4', 5-trisphosphate (IP(3)), and nicotinate adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenate. Fructose 1,6, -diphosphate (FDP), at concentrations up to 8 mM, did not induce Ca(2+) release by itself in sea urchin egg homogenate. However, FDP potentiates Ca(2+) release mediated by agonists of the ryanodine channel, such as ryanodine, caffeine, and palmitoyl-CoA. Furthermore, glucose 6-phosphate had similar effects. FDP also potentiates activation of the ryanodine channel mediated by the endogenous nucleotide cADPR. The half-maximal concentration for cADPR-induced Ca(2+) release was decreased approximately 3.5 times by addition of 4 mM FDP. The reverse was also true: addition of subthreshold concentrations of cADPR sensitized the homogenates to FDP. The Ca(2+) release mediated by FDP in the presence of subthreshold concentrations of cADPR was inhibited by antagonists of the ryanodine channel, such as ruthenium red, and by the cADPR inhibitor 8-Br-cADPR. However, inhibition of Ca(2+) release induced by IP(3) or NAADP had no effect upon Ca(2+) release induced by FDP in the presence of low concentrations of cADPR. Furthermore, FDP had inhibitory effects upon Ca(2+) release induced by both IP(3) and NAADP. We propose that the state of cellular intermediary metabolism may regulate cellular Ca(2+) homeostases by switching preferential effects from one intracellular Ca(2+) release channel to another.
Asunto(s)
Señalización del Calcio/fisiología , Glucólisis/fisiología , Adenosina Difosfato Ribosa/análogos & derivados , Adenosina Difosfato Ribosa/farmacología , Animales , Señalización del Calcio/efectos de los fármacos , ADP-Ribosa Cíclica , Femenino , Fructosadifosfatos/farmacología , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/farmacología , Cinética , NADP/análogos & derivados , NADP/farmacología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Erizos de MarRESUMEN
BACKGROUND: FK506 binding protein is closely associated with the sarcoplasmic reticulum ryanodine receptor-channel and can modulate its function. The ryanodine receptor is stabilized by its association with FK506 binding protein. The immunosuppressant drugs FK506 (tacrolimus) and rapamycin can promote dissociation of FK506 binding protein from the ryanodine receptor 1 and by this mechanism increase sensitivity of ryanodine receptor 1 to agonists such as caffeine. Furthermore, it was shown recently that treatment of normal human skeletal muscle with FK506 and rapamycin increased halothane-induced contracture. The authors investigated the effect of the immunosuppressants FK506 and rapamycin on halothane-induced Ca2+ release in skeletal muscle sarcoplasmic reticulum vesicles. METHODS: Skeletal muscle terminal cisterns were isolated from New Zealand White rabbits. Ca2+ uptake and release was monitored in skeletal muscle sarcoplasmic reticulum vesicles using the fluo-3 fluorescent technique. Western Blot analysis of FK506 binding protein was performed using standard protocol. RESULTS: The authors observed that treatment of skeletal muscle sarcoplasmic reticulum vesicles with FK506 and rapamycin enhances halothane-induced Ca2+ release by about five times. Furthermore, the Ca2+ release induced by halothane in the presence of FK506 was inhibited by several antagonists of the ryanodine receptor, such as ruthenium red, spermine, and Mg2+. CONCLUSION: Dissociation of FK506 binding protein from its binding site in skeletal muscle sarcoplasmic reticulum vesicles can modulate halothane-induced Ca2+ release through the ryanodine receptor. Data are discussed in relation to the role of the FK506 binding protein in modulating the effect of halothane on the ryanodine receptor and the development of malignant hyperthermia phenotype.
Asunto(s)
Anestésicos por Inhalación/farmacología , Calcio/metabolismo , Halotano/farmacología , Inmunofilinas/metabolismo , Inmunosupresores/farmacología , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Tacrolimus/farmacología , Animales , Sinergismo Farmacológico , Contracción Muscular/efectos de los fármacos , Conejos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Sirolimus/farmacología , Proteínas de Unión a TacrolimusRESUMEN
Cyclic ADP-ribose (cADPR) is a potent mediator of Ca2+ mobilization from intracellular stores in sea urchin eggs that ultimately activates the ryanodine channel. We now report that certain long-chain acyl-CoA derivative metabolites (14-18 carbons in length), such as palmitoyl-CoA, greatly potentiate the effect of cADPR on Ca2+ release. Furthermore, in higher concentrations, palmitoyl-CoA and other closely related long-chain acyl-CoA derivatives trigger Ca2+ release apparently through the ryanodine channel in sea urchin egg homogenates. Palmitoyl-CoA-induced Ca2+ release was suppressed by ruthenium red, spermine, and the calmodulin antagonist N-(6-aminohexyl)-1-naphthalenesulfonamide, which all prevent activation of the ryanodine channel, but not by heparin or thionicotinamide-NADP. In addition, cADPR was able to desensitize the sea urchin egg homogenates to the subsequent Ca2+ release induced by palmitoyl-CoA and vice versa. In contrast, neither inositol 1,4,5-trisphosphate (IP3) nor the newly identified Ca2+ release agonist nicotinate adenine dinucleotide phosphate was able to desensitize the homogenate to palmitoyl-CoA, indicating that palmitoyl-CoA probably acts selectively by activating the ryanodine channel, but, unlike cADPR, palmitoyl-CoA might act directly on this channel. Finally, we found that palmitoyl-CoA was able to counteract the inhibitory effect of Mg2+ and spermine, which, in physiological concentrations, suppress specifically the cADPR-induced Ca2+ release. We propose that palmitoyl-CoA, present in micromolar concentrations, may trigger Ca2+ release through the ryanodine channel and, in lower concentrations, may increase the sensitivity of the Ca2+ release system to cADPR. Thus palmitoyl-CoA may serve as a regulatory link between the intermediary metabolism and the cADPR-induced Ca2+ release signaling pathway.
Asunto(s)
Adenosina Difosfato Ribosa/análogos & derivados , Calcio/metabolismo , Palmitoil Coenzima A/farmacología , Adenosina Difosfato Ribosa/farmacología , Animales , Agonistas de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , ADP-Ribosa Cíclica , Sinergismo Farmacológico , Magnesio/farmacología , Proteínas Musculares/metabolismo , Canal Liberador de Calcio Receptor de Rianodina , Erizos de Mar/metabolismo , Espermina/farmacologíaRESUMEN
We investigated the dependence of nicotinate-adenine dinucleotide phosphate (NAADP)-induced Ca2+ release from intracellular stores of sea urchin egg homogenates, upon extravesicular Ca2+. In contrast to the Ca2+ release induced inositol 1',4',5'-triphosphate (IP3) or cyclic ADP-ribose (cADPR), the Ca2+ release induced by NAADP was completely independent of the free extravesicular Ca2+ over a wide range of concentrations (0-0.1 mM). The Ca2+ release triggered by either cADPR or IP3 was biphasically modulated by extravesicular Ca2+, and the Ca2+ release by these agents was abolished when the extravesicular Ca2+ was removed by chelation with 2 mM EGTA. On the other hand, NAADP-triggered Ca2+ release was not influenced by EGTA. These data indicate that while both cADPR and IP3 systems behave as functional Ca(2+)-induced Ca2+ release mechanisms, NAADP activates a Ca2+ release mechanism which is independent of the presence of extravesicular Ca2+. Therefore, the NAADP-sensitive Ca2+ release mechanisms may have a unique regulatory impact upon intracellular Ca2+ homoeostasis.
Asunto(s)
Calcio/metabolismo , Calcio/farmacología , NADP/análogos & derivados , Óvulo/metabolismo , Adenosina Difosfato Ribosa/análogos & derivados , Adenosina Difosfato Ribosa/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ADP-Ribosa Cíclica , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacología , Cinética , NADP/farmacología , Óvulo/efectos de los fármacos , Erizos de Mar , Terpenos/farmacología , TapsigarginaRESUMEN
Transient fluxes of intracellular ionized calcium (Ca2+) from intracellular stores are integral components of regulatory signaling pathways operating in numerous biological regulations, including in early stages of egg fertilization. Therefore, we explored whether NADP, which is rapidly generated by phosphorylation of NAD upon fertilization may, directly or indirectly, exert a regulatory role as a trigger of Ca2+ release from intracellular stores in sea urchin eggs. NADP had no effect, but we found that the deamidated derivative of NADP, nicotinate adenine dinucleotide phosphate (beta-NAADP), is a potent and specific stimulus (ED50 16 nM) for Ca2+ release in sea urchin egg homogenates. NAADP triggers the Ca2+ release via a mechanism which is distinct from the well-known Ca2+ release systems triggered either by inositol-1,4,5-triphosphate (IP3) or by cyclic adenosine diphospho-ribose (cADPR). The NAADP-induced release of Ca2+ is not blocked by heparin, an antagonist of IP3, or by procaine or ruthenium red, antagonists of cADPR. However, it is selectively blocked by thionicotinamide-NADP which does not inhibit the actions of IP3 or cADPR. NAADP produced by heating of NADP in alkaline (pH = 12) medium or synthetized enzymatically by nicotinic acid-NADP reaction catalyzed by NAD glycohydrolase have identical properties. The results presented herein thus describe a novel endocellular Ca(2+)-releasing system controlled by NAADP as a specific stimulus. The NAADP-controlled Ca2+ release system may be an integral component of multiple intracellular regulations occurring in fertilized sea urchin eggs, which are mediated by intracellular Ca2+ release, and may also have similar role(s) in other tissues.
Asunto(s)
Calcio/metabolismo , NADP/análogos & derivados , NADP/farmacología , Óvulo/metabolismo , Animales , Femenino , Fertilización , Cinética , Espectrometría de Masas , NADP/química , Óvulo/efectos de los fármacos , Erizos de Mar , Relación Estructura-ActividadRESUMEN
Release of Ca2+ from intracellular stores is a widespread mechanism in regulation of cell function. Two hitherto unknown adenine diphosphonucleotides were recently identified, which trigger Ca2+ release from intracellular stores via channels that are distinct from the well-known receptor/channel controlled by inositol 1,4,5,-trisphosphate (IP3): cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Here we review synthesis of cADPR from beta-NAD, its hydrolysis to adenosine diphosphoribose (noncyclic) by cADPR glycohydrolase, as well as our knowledge about the metabolism of NAADP. The Ca2+ release triggered by cADPR, NAADP, or IP3 can be distinguished by the action of inhibitors and by desensitization studies. Evidence now emerges that cADPR synthesis from beta-NAD can be stimulated, at least in some cell types by all-trans-retinoic acid as a first messenger. We then review the properties of cADPR and NAADP as potential second messengers in the intracrine regulation of cell functions. Although their exact role in signaling sequences is not yet known, cADPR and NAADP are likely to play important intracellular regulatory functions, as extensively documented for the process of egg fertilization.
Asunto(s)
Adenosina Difosfato Ribosa/análogos & derivados , Calcio/fisiología , NADP/análogos & derivados , Sistemas de Mensajero Secundario , Adenosina Difosfato Ribosa/fisiología , Animales , Canales de Calcio/fisiología , Compartimento Celular , ADP-Ribosa Cíclica , Homeostasis , Proteínas Musculares/fisiología , NADP/fisiología , Canal Liberador de Calcio Receptor de Rianodina , Erizos de Mar/fisiología , Transducción de Señal , Tretinoina/farmacologíaRESUMEN
We investigated the pH dependence and the effects of thimerosal and dithiothreitol (DTT) upon the Ca2+ release induced by cADP-ribose (cADPR) and nicotinate-adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenates. Both Ca2+ release triggered by cADPR and the binding of [3H]cADPR to sea urchin egg homogenates were decreased by alkalization of the assay media from pH 7.2 to 8.9. In contrast, NAADP-triggered Ca2+ release was not influenced by changes in pH. The Ca2+ release induced by cADPR was potentiated by thimerosal and inhibited by DTT, but neither thimerosal nor DTT had any effect upon the Ca2+ release induced by NAADP. We conclude that cADPR-sensitive Ca2+-release mechanisms are dependent on pH of the assay media and are sensitive to thiol group modification. On the other hand, these functional properties are not shared by NAADP-regulated Ca2+ channels.
Asunto(s)
Adenosina Difosfato Ribosa/análogos & derivados , Calcio/metabolismo , NADP/análogos & derivados , Oocitos/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Adenosina Difosfato Ribosa/farmacología , Animales , Cafeína/farmacología , ADP-Ribosa Cíclica , Femenino , Concentración de Iones de Hidrógeno , Cinética , NADP/farmacología , Erizos de Mar , Timerosal/farmacología , TritioRESUMEN
Urea, in nondenaturing concentrations, inhibited Ca2+ uptake by sarcoplasmic reticulum vesicles with no concomitant effect on ATP hydrolysis. This inhibition was antagonized by 5 mM oxalate and 20 mM orthophosphate. At concentrations of 0.2 to 1.0 M, urea induced an increase in the Ca2+ efflux from preloaded vesicles diluted in a medium at pH 7.0 containing 2 mM ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid, 0.1 mM orthophosphate, and 0.1 mM MgCl2. The urea-induced efflux was arrested by ligands of the (Ca(2+)-Mg2+) ATPase, namely, K+, Mg2+, Ca2+, and ADP, and by ruthenium red and the polyamines spermine, spermidine, and putrescine. In the case of polyamines a dissociation between the effect on the efflux and the net Ca2+ uptake was observed, as only the efflux could be blocked by the drugs. Glycine betaine, trimethylamine-N-oxide, and sucrose antagonized the effects of urea on both the net Ca2+ uptake and the rate of Ca2+ efflux.
Asunto(s)
Calcio/metabolismo , Retículo Sarcoplasmático/metabolismo , Urea/farmacología , Adenosina Difosfato/farmacología , Animales , Betaína/farmacología , Cinética , Magnesio/farmacología , Músculos/metabolismo , Oxalatos/farmacología , Ácido Oxálico , Fosfatos/farmacología , Poliaminas/farmacología , Potasio/farmacología , Conejos , Rojo de Rutenio/farmacología , Retículo Sarcoplasmático/efectos de los fármacos , Urea/antagonistas & inhibidoresRESUMEN
Cyclic ADP-ribose (cADPR) is a potent mediator of Ca2+ mobilization from intracellular stores in sea urchin eggs. However, the regulation of the cADPR-induced Ca2+ release system is not yet fully elucidated. We now report that spermine and related polyamines, in physiological concentrations, were able to inhibit the Ca2+ release induced by cADPR in sea urchin egg homogenate bioassays, as measured using the Ca2+ indicator fluo 3, but had no effect on the Ca2+ release induced by D-myo-inositol 1,4,5-trisphosphate (IP3) or by nicotinate adenine dinucleotide phosphate (NAADP). Spermine was a more potent inhibitor of the cADPR-induced Ca2+ release than spermidine and putrescine. Spermine inhibited not only the release induced by cADPR but also the Ca2+ release induced by caffeine and ryanodine. Finally, pretreatment of the sea urchin egg homogenates with caffeine or Sr2+ and Ca2+ prevented the inhibitory effect of spermine on cADPR-induced Ca2+ release. We propose that polyamines, which are present in millimolar concentrations in fertilized eggs, are specific inhibitors of the ryanodine channel and perhaps may serve as endogenous regulators of the cADPR-induced Ca2+ release system.
Asunto(s)
Adenosina Difosfato Ribosa/análogos & derivados , Calcio/metabolismo , Poliaminas/farmacología , Adenosina Difosfato Ribosa/farmacología , Animales , Cafeína/farmacología , ADP-Ribosa Cíclica , Inositol 1,4,5-Trifosfato/farmacología , NADP/análogos & derivados , NADP/farmacología , Óvulo/metabolismo , Rianodina/farmacología , Erizos de Mar/metabolismo , Espermina/farmacologíaRESUMEN
The molecular mechanism that initiates Ca2+ signaling in sea urchin egg fertilization has not yet been clarified. To determine whether sea urchin sperm may generate and possibly supply cyclic ADP-ribose (cADPR) as a Ca2+-releasing factor in the course of sea urchin egg fertilization, we determined cADPR content and the capacity for cADPR synthesis in sea urchin sperm. cADPR content was determined using the sea urchin egg homogenate Ca2+-release bioassay combined with high-performance liquid chromatography (HPLC). We found that sperm homogenates synthesized cADPR from beta-NAD but did not synthesize cADPR when alpha-NAD was the substrate. The identity of cADPR generated by sperm homogenates was verified by HPLC analysis, use of specific Ca2+-release antagonists, and homologous desensitization of the sea urchin egg homogenate Ca2+-release bioassay. The ambient content of cADPR was approximately 0.3 nmol cADPR/g wet wt sea urchin sperm. Our results show that sperm can synthesize cADPR and that they contain cADPR levels comparable to other tissues.