RESUMEN
The flavonoid myricetin is found in several sedative herbs, for example, the St. John's Wort, but its influence on sedation and its possible mechanism of action are unknown. Using patch-clamp technique on a brain slice preparation, the present study found that myricetin promoted GABAergic activity in the neurons of hypothalamic paraventricular nucleus (PVN) by increasing the decay time and frequency of the inhibitory currents mediated by GABA(A) receptor. This effect of myricetin was not blocked by the GABA(A) receptor benzodiazepine- (BZ-) binding site antagonist flumazenil, but by KN-62, a specific inhibitor of the Ca(2+)/calmodulin-stimulated protein kinase II (CaMK-II). Patch clamp and live Ca(2+) imaging studies found that myricetin could increase Ca(2+) current and intracellular Ca(2+) concentration, respectively, via T- and L-type Ca(2+) channels in rat PVN neurons and hypothalamic primary culture neurons. Immunofluorescence staining showed increased phosphorylation of CaMK-II after myricetin incubation in primary culture of rat hypothalamic neurons, and the myricetin-induced CaMK-II phosphorylation was further confirmed by Western blotting in PC-12 cells. The present results suggest that myricetin enhances GABA(A) receptor activity via calcium channel/CaMK-II dependent mechanism, which is distinctively different from that of most existing BZ-binding site agonists of GABA(A) receptor.
RESUMEN
BACKGROUND: The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in electrolyte and fluid transport in epithelial cells, and women with cystic fibrosis (CF), caused by CFTR gene mutations, have a higher incidence of infertility. METHODS: In the present study, we investigated the expression of CFTR in porcine oviduct and its functional role in oviductal HCO(3)(-) secretion and embryo development with RT-PCR, western blot, patch-clamp, short-circuit current (I(sc)), pH measurement and embryo culture. RESULTS: RT-PCR and western blot analysis showed the expression of CFTR mRNA and protein in the oviduct with its localization demonstrated by immunohistochemistry. The whole-cell patch-clamp recording revealed a forskolin (FSK)-activated current with electrophysiological and pharmacological characteristics of CFTR. The I(sc) measurement showed that FSK-stimulated an increase in the I(sc), which could be significantly reduced by CFTR inhibitor or removal of both CO(2) and HCO(3)(-). pH measurement showed a FSK stimulated alkalization at the apical surface, which could be inhibited by CFTR inhibitor, indicating CFTR-mediated HCO(3)(-) secretion. Mouse embryo development from 2-cell to morula or blastocyst stage was significantly inhibited in the absence of HCO(3)(-) or when co-cultured with HCO(3)(-) secretion-deficient CFTR mutant cells as compared with the wild-type. RT-PCR, western blot and immunostaining showed the expression of soluble adenylate cyclase (sAC), the known HCO(3)(-) sensor, in embryos. Treatment with its inhibitors, 2-hydroxyestradiol and KH7, prevented the HCO(3)(-) dependent embryo development. CONCLUSION: The present results suggest that CFTR-mediated oviductal HCO(3)(-) secretion may be vital for sAC-dependent early embryo development, a defect of which may contribute to the reduced fertility seen in women with CF.
Asunto(s)
Adenilil Ciclasas/fisiología , Bicarbonatos/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Trompas Uterinas/metabolismo , Animales , Western Blotting , Colforsina/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Conductividad Eléctrica , Técnicas de Cultivo de Embriones , Femenino , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Ratones , Técnicas de Placa-Clamp , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Since contractility of the uterus appears to be the major source of pain during dysmenorrhoea, alleviation of the contractions is believed to be a possible treatment strategy. Bak Foong Pills, a traditional Chinese formulation for use in gynaecological disorders, has long been thought as effective in the treatment of dysmenorrhoeal symptoms. The present study thus aims to investigate whether ethanol extract of Bak Foong Pills (BFP-Ex) or its constituent herbs may have direct effects on alleviating dysmenorrhoeal symptoms by altering uterine tone. This was investigated using isolated uterine preparations and intracellular messenger analysis of adenylate cyclase, via [(3)H]-adenine assay, and calcium, with fluorometry imaging, in myometrial cultures. BFP-Ex can stimulate uterine relaxation following oxytocin-induced contractions ex-vivo. Attempted inhibition of BFP-Ex's relaxatory response with a nitric oxide inhibitor and adenylate cyclase inhibitor, however, had no significant effect, suggesting that most of BFP-Ex's relaxatory response was not due to increases in NO or cAMP. Further studies on tetramethylpyrazine (TMP), a major active ingredient of BFP-Ex, indicated that TMP could modulate intracellular calcium levels in favour of uteri relaxation. The ability of Bak Foong Pills to alleviate menstrual pain may be due to direct regulation of uterine tone.
Asunto(s)
Medicamentos Herbarios Chinos/farmacología , Medicina Tradicional China , Contracción Uterina/efectos de los fármacos , Útero/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Medicamentos Herbarios Chinos/uso terapéutico , Dismenorrea/tratamiento farmacológico , Femenino , Fluorometría , Ratones , Ratones Endogámicos ICR , Relajación Muscular/efectos de los fármacos , Miometrio/enzimología , Miometrio/patología , Pirazinas/farmacología , Ratas , Ratas Sprague-Dawley , Útero/enzimologíaRESUMEN
Prior to fertilization sperm has to undergo an activation process known as capaciation, leading to the acrosome reaction. Till now, little is known about the mechanism for preventing premature capacitation in sperm although decapacitation factors from various sources have been thought to be involved. In this study, we report that NYD-SP27, an isoform of phospholipase C Zeta 1 (PLCZ1), is localized to the sperm acrosome in mouse and human spermatozoa by immunofluorescence using a specific antibody. Western blot and double staining analyses show NYD-SP27 becomes detached from sperm, as they undergo capacitation and acrosome reaction. The absence of HCO3-, a key factor in activating capacitation, from the capacitation-inducing medium prevents the loss of NYD-SP27 from sperm. The anti-NYD-SP27 antibody also prevents the loss of NYD-SP27 from sperm, reduced the number of capacitated sperm, inhibited the acrosome reaction induced by ATP and progesterone, and inhibited agonist-induced PLC-coupled Ca2+ mobilization in sperm, which can be mimicked by the PLC inhibitor, U73122. These data strongly suggest that NYD-SP27 is a physiological inhibitor of PLC that acts as an intrinsic decapacitation factor in sperm to prevent premature capacitation and acrosome reaction.
Asunto(s)
Reacción Acrosómica/fisiología , Acrosoma/metabolismo , Fosfoinositido Fosfolipasa C/metabolismo , Capacitación Espermática/fisiología , Acrosoma/efectos de los fármacos , Adulto , Animales , Calcio/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Sueros Inmunes/farmacología , Masculino , Ratones , Persona de Mediana Edad , Fosfoinositido Fosfolipasa C/inmunología , Capacitación Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismoRESUMEN
Müllerian inhibiting substance (MIS) has recently been implicated in multiple cellular functions including promotion of cell survival, but the receptor(s) and signaling pathways involved remain elusive. We have investigated the possibility of YWK-II protein, previously shown to interact physically with MIS and G(o) protein, being a receptor mediating the cell survival effect of MIS. In YWK-II-overexpressing CHO cells, MIS activates the G(o)-coupled ERK1/2 signaling pathway and promotes cell survival with altered levels of p53 and caspase-3. YWK-II antibody is found to interfere with the ability of MIS to promote viability of mouse sperm and affect MIS-activated ERK1/2 phosphorylation. In vivo studies involving injection of YWK-II antibody into the seminiferous tubule of the mouse testis, where MIS is known to be produced, show significant reduction in the sperm count with accumulation of p53 and cleaved caspase-3 in testicular nuclei. Taken together, the present study has demonstrated a new G(o)-coupled receptor for MIS in mediating ERK1/2 activation leading to anti-apoptotic activity or cell survival.
Asunto(s)
Precursor de Proteína beta-Amiloide/fisiología , Apoptosis/fisiología , Glicoproteínas/farmacología , Proteínas del Tejido Nervioso/fisiología , Receptores Acoplados a Proteínas G/fisiología , Hormonas Testiculares/farmacología , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Hormona Antimülleriana , Apoptosis/efectos de los fármacos , Células CHO , Células COS , Caspasa 3/metabolismo , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Cricetinae , Cricetulus , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosforilación/efectos de los fármacos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Receptores de Péptidos/fisiología , Receptores de Factores de Crecimiento Transformadores beta , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Testículo/citología , Testículo/efectos de los fármacos , Testículo/metabolismo , TransfecciónRESUMEN
In the present study we investigated the role of Ca(2+) in tetramethylpyrazine (TMP)-induced anion secretion in the human colonic epithelial cell line, Caco-2, using the short-circuit current (I(SC)) technique in conjunction with intracellular Ca(2+) measurements. The results showed that TMP-induced I(SC) response was significantly reduced by 58.8% and 38.3% after inhibiting Ca(2+) ATPase of endoplasmic reticulum (ER) with thapsigargin and mobilizing ER stored Ca(2+) release with ATP, respectively. Conversely, thapsigargin- and ATP-evoked I(SC) responses were also significantly reduced by pretreatment with TMP by 43.2% and 38.5%, respectively. Conversely, removal of extracellular Ca(2+), apical but not basolateral, or the presence of the Ca(2+) chelator (EGTA) significantly increased TMP-induced I(SC) by 47.1% and 37.8%, respectively. Similar to TMP, thapsigargin-induced current increase was also enhanced by chelating extracellular Ca(2+) or in Ca(2+) free solution; however, removal of extracellular Ca(2+) did not significantly affect 3-isobutyl-1-methylxanthine (IBMX)- and forskolin-induced transepithelial current. Measurement of the intracellular concentration of free Ca(2+) ([Ca(2+)](i)) with fura-2/AM showed that TMP could induce an increase in [Ca(2+)](i) but pretreatment with TMP significantly reduced thapsigargin-evoked, but not ATP-induced, [Ca(2+)](i) increase. These results suggest that the effect of TMP on colonic anion secretion is partly mediated by TMP-increased [Ca(2+)](i) by acting on a target similar to thapsigargin. The observed inhibitory effect of extracellular Ca(2+) on Ca(2+)-dependent anion secretion represents a novel mechanism by which Ca(2+)-dependent regulation of epithelial electrolyte transport may be fine-tuned by extracellular Ca(2+) in the apical domain.
Asunto(s)
Aniones/metabolismo , Calcio/metabolismo , Colon/metabolismo , Mucosa Intestinal/metabolismo , Pirazinas/farmacología , Adenosina Trifosfato/farmacología , Células CACO-2 , Señalización del Calcio , Colon/efectos de los fármacos , Humanos , Mucosa Intestinal/efectos de los fármacos , Tapsigargina/farmacologíaRESUMEN
We have previously demonstrated that tetramethylpyrazine (TMP) could stimulate colonic and pancreatic anion secretion. The present study investigated the signaling pathways and cellular mechanisms underlying the effect of TMP using human colonic Caco-2 cells, with permeabilized apical or basolateral membranes, in conjunction with Ussing chamber technique, intracellular cAMP and Ca2+ measurements as well as competitive RT-PCR for mRNA expression of cystic fibrosis transmembrane conductance regulator (CFTR) and Ca(2+)-dependent Cl- channels (CACC). Basolateral addition of TMP induced a short circuit current (I(SC)) response, which could be mimicked by forskolin and 3-isobutyl-1-methylxanthine (IBMX). Adenylate cyclase inhibitor, MDL12330A, and intracellular Ca2+ chelator, BAPTA-AM, significantly inhibited the TMP-induced I(SC). In basolateral membrane-permeabilized cells, TMP, as well as forskolin and IBMX, induced an I(SC) response, which was sensitive to MDL-12330A, H89, and specific channel blocker CFTR(inh-172), but insensitive to apical application of 4-4'-didsothiocyanostilbene-2, 2'-disulfonic acid (DIDS) and basolateral pretreatment with BAPTA-AM. In apical membrane-permeabilized cells, TMP, similar to forskolin and IBMX, produced a very small current increase, which was sensitive to K+ channel blockers, BaCl2 and tetraethylammonium (TEA), but not Chromanol 293B and charybdotoxin (ChTX), alone or combined. However, in intact Caco-2 monolayers, the TMP-induced I(SC) could be partially inhibited by ChTX. TMP (5 mM) could stimulate intracellular cAMP production. Intracellular Ca2+ was also increased by TMP (5 mM) in both Ca(2+)-containing and Ca(2+)-free bathing solutions. RT-PCR showed that the expression of CFTR in Caco-2 cells was 5.2 fold higher than that of Ca(2+)-activated Cl- channel (CACC). In conclusion, TMP stimulates Cl- secretion by activating cAMP and [Ca2+]i signaling pathways leading to subsequent activation of apical CFTR and basolateral K+ channels.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Canales de Potasio Calcio-Activados/metabolismo , Pirazinas/farmacología , Secuencia de Bases , Células CACO-2 , Calcio/metabolismo , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Colon/efectos de los fármacos , Colon/metabolismo , AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , ADN/genética , Humanos , Transporte Iónico/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
AIM: Menoease Pills (MP), a Chinese medicine-based new formula for postmenopausal women, has been shown to modulate the endocrine and immune systems. The present study investigated the effects of MP and one of its active ingredients, ligustrazine, on epithelial barrier and ion transport function in a human colonic cell line, T84. METHODS: Colonic transepithelial electrophysiological characteristics and colonic anion secretion were studied using the short circuit current (ISC) technique. RT-PCR was used to examine the expression of cytoplasmic proteins associated with the tight junctions, ZO-1 (zonula occludens-1) and ZO-2 (zonula occludens-2). RESULTS: Pretreatment of T84 cells with MP (15 microg/mL) for 72 h significantly increased basal potential difference, transepithelial resistance and basal ISC. RT-PCR results showed that the expressions of ZO-1 and ZO-2 were significantly increased after MP treatment, consistent with improved epithelial barrier function. Results of acute stimulation showed that apical addition of MP produced a concentration-dependent (10-5,000 microg/mL, EC50 = 293.9 microg/mL) increase in ISC. MP-induced ISC was inhibited by basolateral treatment with bumetanide (100 micromol/L), an inhibitor of the Na+-K+-2Cl- cotransporter, apical addition of Cl- channel blockers, diphenylamine-2, 2'-dicarboxylic acid (1 mmol/L) or glibenclamide (1 mmol/L), but not 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid or epithelial Na+ channel blocker, amiloride. The effect of MP on ZO-1 and ZO-2 was mimicked by Ligustrazine and the ligustrazine-induced ISC was also blocked by basolateral application of bumetanide and apical addition of diphenylamine-2, 2'-dicarboxylic acid or glibenclamide, and reduced by a removal of extracellular Cl-. CONCLUSION: The results of the present study suggest that MP and lligustrazine may improve epithelial barrier function and exert a stimulatory effect on colonic anion secretion, indicating the potential use of MP and its active ingredients for improvement of GI tract host defense and alleviation of constipation often seen in the elderly.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/fisiología , Medicina Tradicional China , Pirazinas/farmacología , Aniones/metabolismo , Transporte Biológico/efectos de los fármacos , Línea Celular , Cloruros/metabolismo , Colon/citología , Expresión Génica , Humanos , Mucosa Intestinal/citología , Potenciales de la Membrana/efectos de los fármacos , Proteínas de la Membrana/genética , Fosfoproteínas/genética , Posmenopausia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Uniones Estrechas/fisiología , Proteína de la Zonula Occludens-1 , Proteína de la Zonula Occludens-2RESUMEN
Although the role of the epididymis, a male accessory sex organ, in sperm maturation has been established for nearly four decades, the maturation process itself has not been linked to a specific molecule of epididymal origin. Here we show that Bin1b, a rat epididymis-specific beta-defensin with antimicrobial activity, can bind to the sperm head in different regions of the epididymis with varied binding patterns. In addition, Bin1b-expressing cells, either of epididymal origin or from a Bin1b-transfected cell line, can induce progressive sperm motility in immotile immature sperm. This induction of motility is mediated by the Bin1b-induced uptake of Ca(2+), a mechanism that has a less prominent role in maintaining motility in mature sperm. In vivo antisense experiments show that suppressed expression of Bin1b results in reduced binding of Bin1b to caput sperm and in considerable attenuation of sperm motility and progressive movement. Thus, beta-defensin is important for the acquisition of sperm motility and the initiation of sperm maturation.