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1.
Acta Virol ; 63(1): 117-120, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30879321

RESUMO

The phospholipase C (PLC) is a family of kinases that hydrolyze phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to generate two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which stimulate distinct downstream signaling. Recently, it has been reported that PLC signaling is activated by multiple viruses for efficient replication and the virus-induced inflammatory response. In this study, we demonstrated that PLC-specific inhibitor U73122 strongly suppressed porcine reproductive and respiratory syndrome virus (PRRSV) productive infection in cell cultures. The inhibitor affected both viral post-binding cell entry and post-entry processes. The virus infection led to an early transient activation of PLCγ-1 at 0.5 h post-infection (hpi), and sustained event at a stage from 4 to 16 hpi in MARC-145 cells. In addition, U73122 inhibited the activation of p38 MAPK signaling stimulated by PRRSV infection, suggesting that PLC signaling may be associated with the virus infection-induced inflammatory response. Taken together, these studies suggested that PLC signaling played an important role in PRRSV infection or pathogenesis. Keywords: PRRSV; U73122; phospholipase C; PLCγ-1.


Assuntos
Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Fosfolipases Tipo C , Animais , Linhagem Celular , Estrenos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Pirrolidinonas/farmacologia , Transdução de Sinais , Suínos , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/fisiologia , Internalização do Vírus/efeitos dos fármacos
2.
Artigo em Inglês | MEDLINE | ID: mdl-30373804

RESUMO

Stagnation in antimicrobial development has led to a serious threat to public health because some Acinetobacter baumannii infections have become untreatable. New therapeutics with alternative mechanisms of action to combat A. baumannii are therefore necessary to treat these infections. To this end, the virulence of A. baumannii isolates with various antimicrobial susceptibilities was assessed when the isolates were treated with miltefosine, a phospholipase C inhibitor. Phospholipase C activity is a contributor to A. baumannii virulence associated with hemolysis, cytolysis of A549 human alveolar epithelial cells, and increased mortality in the Galleria mellonella experimental infection model. While the effects on bacterial growth were variable among strains, miltefosine treatment significantly reduced both the hemolytic and cytolytic activity of all treated A. baumannii strains. Additionally, scanning electron microscopy of polarized A549 cells infected with bacteria of the A. baumannii ATCC 19606T strain or the AB5075 multidrug-resistant isolate showed a decrease in A549 cell damage with a concomitant increase in the presence of A549 surfactant upon administration of miltefosine. The therapeutic ability of miltefosine was further supported by the results of G. mellonella infections, wherein miltefosine treatment of animals infected with ATCC 19606T significantly decreased mortality. These data demonstrate that inhibition of phospholipase C activity results in the overall reduction of A. baumannii virulence in both in vitro and in vivo models, making miltefosine a viable option for the treatment of A. baumannii infections, particularly those caused by multidrug-resistant isolates.


Assuntos
Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/patogenicidade , Antibacterianos/uso terapêutico , Fosforilcolina/análogos & derivados , Células A549 , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Animais , Linhagem Celular , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Testes de Sensibilidade Microbiana , Mariposas/microbiologia , Fosforilcolina/uso terapêutico , Fosfolipases Tipo C/antagonistas & inibidores , Virulência/efeitos dos fármacos
3.
Environ Toxicol ; 34(2): 203-209, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30421542

RESUMO

Lung cancer is one of the most common cancer in cancer-related deaths worldwide, which is characterized by its strong metastatic potential. The melatonin hormone secreted by the pineal grand has an antioxidant effect and protects cells against carcinogenic substances. However, the effects of melatonin in lung cancer stemness are largely unknown. We found that melatonin reduces CD133 expression by ~50% in lung cancer cell lines, while results of a sphere formation assay showed that melatonin inhibits lung cancer stemness. These effects of melatonin were reversed when the cell lines were incubated with phospholipase C (PLC), ERK/p38, and a ß-catenin activator. Transfection with Twist siRNA augmented the inhibitory effects of melatonin, indicating that melatonin suppresses lung cancer stemness by inhibiting the PLC, ERK/p38, ß-catenin, and Twist signaling pathways. We also found CD133 expression is positively correlated with Twist expression in lung cancer specimens. Melatonin shows promise in the treatment of lung cancer stemness and deserves further study.


Assuntos
Neoplasias Pulmonares/patologia , Melatonina/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células A549 , Antígeno AC133/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição Twist/antagonistas & inibidores , Fosfolipases Tipo C/antagonistas & inibidores , beta Catenina/antagonistas & inibidores
4.
Neuroscience ; 396: 66-72, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30458219

RESUMO

Drosophila phototransduction occurs in light-sensitive microvilli arranged in a longitudinal structure of the photoreceptor, termed the rhabdomere. Rhodopsin (Rh), isomerized by light, couples to G-protein, which activates phospholipase C (PLC), which in turn cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG), inositol trisphosphate and H+. This pathway opens the light-dependent channels, transient receptor potential (TRP) and transient receptor potential like (TRPL). PLC and TRP are held together in a protein assembly by the scaffold protein INAD. We report that the channels can be photoactivated in on-cell rhabdomeric patches and in excised patches by DAG. In excised patches, addition of PLC-activator, m-3M3FBS, or G-protein-activator, GTP-γ-S, opened TRP. These reagents were ineffective in PLC-mutant norpA and in the presence of PLC inhibitor U17322. However, DAG activated TRP even when PLC was pharmacologically or mutationally suppressed. These observations indicate that PLC, G-protein, and TRP were retained functional in these patches. DAG also activated TRP in the protein kinase C (PKC) mutant, inaC, excluding the possibility that PKC could mediate DAG-dependent TRP activation. Labeling diacylglycerol kinase (DGK) by fusion of fluorescent mCherry (mCherry-DGK) indicates that DGK, which returns DAG to dark levels, is highly expressed in the microvilli. In excised patches, TRP channels could be light-activated in the presence of GTP, which is required for G-protein activation. The evidence indicates that the proteins necessary for phototransduction are retained functionally after excision and that DAG is necessary and sufficient for TRP opening. This work opens up unique possibilities for studying, in sub-microscopic native membrane patches, the ubiquitous phosphoinositide signaling pathway and its regulatory mechanisms in unprecedented detail.


Assuntos
Ativação do Canal Iônico/efeitos da radiação , Luz , Microvilosidades/metabolismo , Microvilosidades/efeitos da radiação , Células Fotorreceptoras de Invertebrados/citologia , Canais de Receptores Transientes de Potencial/metabolismo , Canais de Receptores Transientes de Potencial/efeitos da radiação , Animais , Diacilglicerol Quinase/biossíntese , Diglicerídeos/farmacologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/efeitos da radiação , Drosophila melanogaster , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Proteína Quinase C/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sulfonamidas/farmacologia , Canais de Receptores Transientes de Potencial/isolamento & purificação , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/genética
5.
Int J Mol Sci ; 20(1)2018 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-30587833

RESUMO

Epidermal growth factor (EGF) is a potent regulator of cell function in many cell types. In mammals, the EGF/EGFR system played an important role in both pituitary physiology and pathology. However, it is not clear about the pituitary action of EGF in lower vertebrates. In this study, using grass carp as a model, we found that EGF could stimulate NK3R mRNA and protein expression through pituitary ErbB1 and ErbB2 coupled to MEK/ERK and PI3K/Akt/mTOR pathways. In addition, EGF could also induce pituitary somatolactin α (SLα) secretion and mRNA expression in a dose- and time-dependent manner in vivo and in vitro. The stimulatory actions of EGF on SLα mRNA expression were also mediated by PI3K/Akt/mTOR and MEK/ERK pathways coupled to ErbB1 and ErbB2 activation. Our previous study has reported that neurokinin B (NKB) could also induce SLα secretion and mRNA expression in carp pituitary cells. In the present study, interestingly, we found that EGF could significantly enhance NKB-induced SLα mRNA expression. Further studies found that NK3R antagonist SB222200 could block EGF-induced SLα mRNA expression, indicating an NK3R requirement. Furthermore, cAMP/PKA inhibitors and PLC/PKC inhibitors could both abolish EGF- and EGF+NKB-induced SLα mRNA expression, which further supported that EGF-induced SLα mRNA expression is NK3R dependent.


Assuntos
Carpas/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Proteínas de Peixes/metabolismo , Hipófise/efeitos dos fármacos , Hormônios Hipofisários/metabolismo , Receptores da Neurocinina-3/metabolismo , Animais , AMP Cíclico/metabolismo , Sinergismo Farmacológico , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Peixes/genética , Fosfatidilinositol 3-Quinases/metabolismo , Hipófise/metabolismo , Hormônios Hipofisários/genética , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , RNA Mensageiro/metabolismo , Receptores da Neurocinina-3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo
6.
Eur J Pharmacol ; 838: 1-10, 2018 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-30171854

RESUMO

Trans-resveratrol was earlier shown to lower intraocular pressure (IOP) in rats; however, its mechanisms of action remain unclear. It has been shown to modulate adenosine receptor (AR) and TGF-ß2 signaling, both of which play a role in regulating IOP. Hence, we investigated effects of trans-resveratrol on AR and TGF-ß2 signaling. Steroid-induced ocular hypertensive (SIOH) rats were pretreated with A1AR, phospholipase C (PLC) and ERK1/2 inhibitors and were subsequently treated with single drop of trans-resveratrol. Metalloproteinases (MMP)-2 and -9 were measured in aqueous humor (AH). In another set of experiments, effect of trans-resveratrol on AH level of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) was determined after single and multiple drop administration in SIOH rats. Effect of trans-resveratrol on ARs expression, PLC and pERK1/2 activation and MMPs, tPA and uPA secretion was determined using human trabecular meshwork cells (HTMC). Further, effect of trans-resveratrol on TGF-ß2 receptors, SMAD signaling molecules and uPA and tPA expression by HTMC was determined in the presence and absence of TGF-ß2. Pretreatment with A1AR, PLC and ERK1/2 inhibitors antagonized the IOP lowering effect of trans-resveratrol and caused significant reduction in the AH level of MMP-2 in SIOH rats. Trans-resveratrol increased A1AR and A2AAR expression, cellular PLC, pERK1/2 levels and MMP-2, tPA and uPA secretion by HTMC. Additionally, it produced TGFßRI downregulation and SMAD 7 upregulation. In conclusion, IOP lowering effect of trans-resveratrol involves upregulation of A1AR expression, PLC and ERK1/2 activation and increased MMP-2 secretion. It downregulates TGFßRI and upregulates SMAD7 hence, inhibits TGF-ß2 signaling.


Assuntos
Pressão Intraocular/efeitos dos fármacos , Hipertensão Ocular/tratamento farmacológico , Resveratrol/farmacologia , Transdução de Sinais/efeitos dos fármacos , Antagonistas do Receptor A1 de Adenosina/farmacologia , Administração Oftálmica , Animais , Células Cultivadas , Dexametasona/farmacologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Masculino , Hipertensão Ocular/induzido quimicamente , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P1/metabolismo , Resveratrol/uso terapêutico , Malha Trabecular/citologia , Malha Trabecular/efeitos dos fármacos , Fator de Crescimento Transformador beta2/metabolismo , Resultado do Tratamento , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo , Regulação para Cima/efeitos dos fármacos
7.
J Food Sci ; 83(9): 2394-2401, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30088839

RESUMO

Nutrients regulate the secretion of gut satiety hormones, which is related to the modulation of food intake and blood glucose levels. Calcium-sensing receptor (CaSR) is involved in regulating gut hormone secretion in response to l-amino acids and multivalent cations. Rodents are often used to investigate the effect of nutrients on these hormonal release. However, results obtained using rodent models are difficult to be applied in humans, we used pigs as a model in this study because their physiology is similar to that of humans. In this study, we investigated whether l-Arginine (l-Arg) could induce gut hormones cholecystokinin (CCK) and glucose-dependent insulinotropic peptide (GIP) secretion in the porcine duodenum and if so, whether CaSR mediated l-Arg-regulated gut satiety hormone secretion. Our data showed that treatment with 20 and 50 mM l-Arg induced CCK and GIP secretion compared with 0 mM l-Arg. However, treatment with d-Arg (an inactive isomer) failed to elicit this response. The potency of l-Arg to induce CCK and GIP secretion was enhanced in the presence of extracellular Ca2+ and CaSR agonist cinacalcet. However, the effect of Arg on CCK and GIP secretion was attenuated by blocking CaSR and its downstream signaling molecules adenylate cyclase (AC) and phospholipase C (PLC). Taken all together, pig duodenum provides an appropriate model to explore the effects of l-Arg on the secretion of the satiety-related gut hormones CCK and GIP and the role of CaSR in this effect. Further investigations are needed to verify the effect of l-Arg on food intake and blood glucose in human study. PRACTICAL APPLICATION: l-Arginine is able to modulate cholecystokinin and glucose-dependent insulinotropic peptide secretion through the CaSR in pig model, which has a potential role in regulating food intake and blood glucose levels.


Assuntos
Regulação do Apetite/efeitos dos fármacos , Arginina/farmacologia , Colecistocinina/metabolismo , Duodeno/efeitos dos fármacos , Polipeptídeo Inibidor Gástrico/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Adenilil Ciclases/metabolismo , Animais , Glicemia/metabolismo , Cálcio/metabolismo , Cinacalcete/farmacologia , Dieta , Duodeno/metabolismo , Ingestão de Alimentos/fisiologia , Humanos , Isomerismo , Modelos Animais , Saciação/fisiologia , Suínos , Fosfolipases Tipo C/antagonistas & inibidores
8.
Am J Physiol Gastrointest Liver Physiol ; 315(4): G618-G630, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30001145

RESUMO

ATP-sensitive K+ (KATP) channels are expressed in gastrointestinal smooth muscles, and their activity is regulated by muscarinic receptor stimulation. However, the physiological significance and mechanisms of muscarinic regulation of KATP channels are not fully understood. We examined the effects of the KATP channel opener cromakalim and the KATP channel blocker glibenclamide on electrical activity of single mouse ileal myocytes and on mechanical activity in ileal segment preparations. To explore muscarinic regulation of KATP channel activity and its underlying mechanisms, the effect of carbachol (CCh) on cromakalim-induced KATP channel currents ( IKATP) was studied in myocytes of M2 or M3 muscarinic receptor-knockout (KO) and wild-type (WT) mice. Cromakalim (10 µM) induced membrane hyperpolarization in single myocytes and relaxation in segment preparations from WT mice, whereas glibenclamide (10 µM) caused membrane depolarization and contraction. CCh (100 µM) induced sustained suppression of IKATP in cells from both WT and M2KO mice. However, CCh had a minimal effect on IKATP in M3KO and M2/M3 double-KO cells. The Gq/11 inhibitor YM-254890 (10 µM) and PLC inhibitor U73122 (1 µM), but not the PKC inhibitor calphostin C (1 µM), markedly decreased CCh-induced suppression of IKATP in WT cells. These results indicated that KATP channels are constitutively active and contribute to the setting of resting membrane potential in mouse ileal smooth muscles. M3 receptors inhibit the activity of these channels via a Gq/11/PLC-dependent but PKC-independent pathways, thereby contributing to membrane depolarization and contraction of smooth muscles. NEW & NOTEWORTHY We systematically investigated the regulation of ATP-sensitive K+ channels by muscarinic receptors expressed on mouse ileal smooth muscles. We found that M3 receptors inhibit the activity of ATP-sensitive K+ channels via a Gq/11/PLC-dependent, but PKC-independent, pathway. This muscarinic suppression of ATP-sensitive K+ channels contributes to membrane depolarization and contraction of smooth muscles.


Assuntos
Íleo/fisiologia , Canais KATP/metabolismo , Contração Muscular , Miócitos de Músculo Liso/metabolismo , Receptores Muscarínicos/metabolismo , Transdução de Sinais , Potenciais de Ação , Animais , Carbacol/farmacologia , Cromakalim/farmacologia , Estrenos/farmacologia , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Íleo/metabolismo , Canais KATP/genética , Masculino , Camundongos , Agonistas Muscarínicos/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Peptídeos Cíclicos/farmacologia , Pirrolidinonas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo
9.
BMB Rep ; 51(8): 418-423, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30021674

RESUMO

Emergency granulopoiesis is a very important strategy to supply efficient neutrophil number in response to infection. However, molecular mechanism involved in this process remains unclear. Here, we found that administration of WKYMVm, an immune modulating peptide, to septic mice strongly increased neutrophil number through augmented emergency granulopoiesis. WKYMVm-induced emergency granulopoiesis was blocked not only by a formyl peptide receptor 2 (FPR2) antagonist (WRW4), but also by FPR2 deficiency. As progenitors of neutrophils, Lin-c-kit+Sca-1- cells expressed FPR2. WKYMVm-induced emergency granulopoiesis was also blocked by a phospholipase C inhibitor (U-73122). These results suggest that WKYMVm can stimulate emergency granulopoiesis via FPR2 and phospholipase C enzymatic activity. [BMB Reports 2018; 51(8): 418-423].


Assuntos
Hematopoese/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Receptores de Formil Peptídeo/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Interações Medicamentosas , Estrenos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Pirrolidinonas/farmacologia , Receptores de Formil Peptídeo/fisiologia , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/antagonistas & inibidores
10.
Cell Rep ; 23(3): 866-877, 2018 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-29669290

RESUMO

Chronic itch or pruritus is a debilitating disorder that is refractory to conventional anti-histamine treatment. Kappa opioid receptor (KOR) agonists have been used to treat chronic itch, but the underlying mechanism remains elusive. Here, we find that KOR and gastrin-releasing peptide receptor (GRPR) overlap in the spinal cord, and KOR activation attenuated GRPR-mediated histamine-independent acute and chronic itch in mice. Notably, canonical KOR-mediated Gαi signaling is not required for desensitizing GRPR function. In vivo and in vitro studies suggest that KOR activation results in the translocation of Ca2+-independent protein kinase C (PKC)δ from the cytosol to the plasma membrane, which in turn phosphorylates and inhibits GRPR activity. A blockade of phospholipase C (PLC) in HEK293 cells prevented KOR-agonist-induced PKCδ translocation and GRPR phosphorylation, suggesting a role of PLC signaling in KOR-mediated GRPR desensitization. These data suggest that a KOR-PLC-PKCδ-GRPR signaling pathway in the spinal cord may underlie KOR-agonists-induced anti-pruritus therapies.


Assuntos
Receptores Opioides kappa/genética , Transdução de Sinais , Medula Espinal/metabolismo , Animais , Membrana Celular/metabolismo , Cloroquina/toxicidade , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/metabolismo , Prurido/induzido quimicamente , Prurido/patologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores da Bombesina/metabolismo , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/deficiência , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo
11.
Mol Cell Biochem ; 447(1-2): 21-32, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29363059

RESUMO

Glucagon-like peptide-1 (GLP-1) is involved in the regulation of insulin secretion and glucose homeostasis. GLP-1 release is stimulated when berberine interacts with a novel G protein family (TAS2Rs) in enteroendocrine cells. In this study, we used STC-1 cells and examined a marked increase in Ca2+ in response to various bitter compounds. Ca2+ responses to traditional Chinese medicine extracts, including berberine, phellodendrine and coptisine, in STC-1 cells were suppressed by the phospholipase C (PLC) inhibitor U-73122, suggesting the involvement of bitter taste receptors in changing the physiological status of enteroendocrine cells in a PLC-dependent manner. STC-1 cells showed berberine-up-regulated preproglucagon (GLP-1 precursor) mRNA and GLP-1 secretion. A QPCR analysis demonstrated that TAS2R38, a subtype of the bitter taste receptor, was associated with GLP-1 secretion. Berberine-mediated GLP-1 secretion was attenuated in response to small interfering RNA silencing of TAS2R38. The current studies demonstrated that Gα-gustducin co-localized with GLP-1 and Tas2r106 in the STC-1 cells. We further utilized inhibitors of PLC and TRPM5, which are known to participate in taste signal transduction, to investigate the underlying pathways mediated in berberine-induced GLP-1 secretion. Berberine-induced GLP-1 release from enteroendocrine cells is modulated in a PLC-dependent manner through a process involving the activation of bitter taste receptors. Together, our data demonstrated a berberine-mediated GLP-1 secretion pathway in mouse enteroendocrine cells that could be of therapeutic relevance to hyperglycemia and the role of bitter taste receptors in the function of the small intestine.


Assuntos
Berberina/farmacologia , Células Enteroendócrinas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptores Acoplados a Proteínas-G/biossíntese , Regulação para Cima/efeitos dos fármacos , Linhagem Celular Tumoral , Células Enteroendócrinas/patologia , Estrenos/farmacologia , Humanos , Hiperglicemia/tratamento farmacológico , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Pirrolidinonas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo
12.
J Physiol ; 596(5): 921-940, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29280494

RESUMO

KEY POINTS: Neurotransmitter release is inhibited by metabotropic glutamate type 7 (mGlu7 ) receptors that reduce Ca2+ influx, yet synapses lacking this receptor also produce weaker release, suggesting that mGlu7 receptors may also prime synaptic vesicles for release. Prolonged activation of mGlu7 receptors with the agonist l-AP4 first reduces and then enhances the amplitude of EPSCs through a presynaptic effect. The inhibitory response is blocked by pertussis toxin, while the potentiating response is prevented by a phospholipase C inhibitor (U73122) and an inhibitor of diacylglycerol (DAG) binding (calphostin C), suggesting that this receptor also couples to pathways that generate DAG. Release potentiation is associated with an increase in the number of synaptic vesicles close to the plasma membrane, which was dependent on the Munc13-2 and RIM1α proteins. The Glu7 receptors activated by the glutamate released following high frequency stimulation provoke a bidirectional modulation of synaptic transmission. ABSTRACT: Neurotransmitter release is driven by Ca2+ influx at synaptic boutons that acts on synaptic vesicles ready to undergo exocytosis. Neurotransmitter release is inhibited when metabotropic glutamate type 7 (mGlu7 ) receptors provoke a reduction in Ca2+ influx, although the reduced release from synapses lacking this receptor suggests that they may also prime synaptic vesicles for release. These mGlu7 receptors activate phospholipase C (PLC) and generate inositol trisphosphate, which in turn releases Ca2+ from intracellular stores and produces diacylglycerol (DAG), an activator of proteins containing DAG-binding domains such as Munc13 and protein kinase C (PKC). However, the full effects of mGlu7 receptor signalling on synaptic transmission are unclear. We found that prolonged activation of mGlu7 receptors with the agonist l-AP4 first reduces and then enhances the amplitude of EPSCs, a presynaptic effect that changes the frequency but not the amplitude of the mEPSCs and the paired pulse ratio. Pertussis toxin blocks the inhibitory response, while the PLC inhibitor U73122, and the inhibitor of DAG binding calphostin C, prevent receptor mediated potentiation. Moreover, this DAG-dependent potentiation of the release machinery brings more synaptic vesicles closer to the active zone plasma membrane in a Munc13-2- and RIM1α-dependent manner. Electrically evoked release of glutamate that activates mGlu7 receptors also bidirectionally modulates synaptic transmission. In these conditions, potentiation now occurs rapidly and it overcomes any inhibition, such that potentiation prevails unless it is suppressed with the PLC inhibitor U73122.


Assuntos
Região CA1 Hipocampal/fisiologia , Diglicerídeos/metabolismo , Ácido Glutâmico/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sinapses/fisiologia , Transmissão Sináptica , Animais , Proteínas de Ligação ao GTP/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Naftalenos/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Toxina Pertussis/farmacologia , Transdução de Sinais , Membranas Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Fosfolipases Tipo C/antagonistas & inibidores
13.
Cell Biochem Funct ; 36(1): 13-17, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29277915

RESUMO

Recent evidence suggested a positive correlation between environmental estrogens (EEs) and high incidence of abnormalities in male urogenital system, but the mechanism remains unclear. Diethylstilbestrol (DES) is a nonsteroidal synthetic estrogen that disrupts the morphology and proliferation of gubernaculum testis cells, but the underlying mechanism is unclear. In this study, mouse gubernaculum testis cells were pretreated with phospholipase C (PLC) inhibitor U-73122 and then treated with DES. The results demonstrated that U-73122 impaired DES-evoked intracellular Ca2+ mobilization in gubernaculum testis cells and inhibited DES-induced proliferation of gubernaculum testis cells. Mechanistically, we found that U-73122 inhibited DES-induced activation of cAMP-response element binding protein (CREB) in gubernaculum testis cells. In conclusion, these data suggest that the effects of DES on mouse gubernaculum testis cells are mediated by PLC-Ca2+ -CREB pathway. SIGNIFICANCE OF THE STUDY: Environmental estrogens remain a serious threat to male reproductive health, and it is important to understand the mechanism by which EEs affect the male productive system. Here we explore potential mechanisms how the proliferation and contractility of gubernaculum testis cells are regulated by diethylstilbestrol. Our findings provide the first evidence that PLC-Ca2+ -CREB signalling pathway mediates the nongenomic effects of diethylstilbestrol on gubernaculum testis cells. These findings provide new insight into the role of diethylstilbestrol in the aetiology of male reproductive dysfunction and will help develop better approaches for the prevention and therapy of male reproductive malformation.


Assuntos
Cálcio/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dietilestilbestrol/farmacologia , Gubernáculo/efeitos dos fármacos , Testículo/efeitos dos fármacos , Fosfolipases Tipo C/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Estrenos/farmacologia , Gubernáculo/citologia , Gubernáculo/metabolismo , Masculino , Camundongos , Pirrolidinonas/farmacologia , Testículo/citologia , Testículo/metabolismo , Fosfolipases Tipo C/antagonistas & inibidores
14.
Med Sci Monit ; 23: 5951-5959, 2017 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-29247156

RESUMO

BACKGROUND Melatonin therapy shows positive effects on neuroprotective factor brain-derived neurotrophic factor (BDNF) expression and neuronal apoptosis in neonatal hemolytic hyperbilirubinemia. We hypothesized that melatonin promotes BDNF expression and anti-apoptotic effects in neonatal hemolytic hyperbilirubinemia through a phospholipase (PLC)-mediated mechanism. MATERIAL AND METHODS A phenylhydrazine hydrochloride (PHZ)-induced neonatal hemolytic hyperbilirubinemia model was constructed in neonatal rats. Four experimental groups - a control group (n=30), a PHZ group (n=30), a PHZ + melatonin group (n=30), and a PHZ + melatonin+U73122 (a PLC inhibitor) group (n=30) - were constructed. Trunk blood was assayed for serum hemoglobin, hematocrit, total and direct bilirubin, BDNF, S100B, and tau protein levels. Brain tissue levels of neuronal apoptosis, BDNF expression, PLC activity, IP3 content, phospho- and total Ca2+/calmodulin-dependent protein kinase type IV (CaMKIV) expression, and phospho- and total cAMP response element binding protein (CREB) expression were also assayed. RESULTS PHZ-induced hemolytic hyperbilirubinemia was validated by significantly decreased serum hemoglobin and hematocrit as well as significantly increased total and direct serum bilirubin (p<0.05). Neonatal bilirubin-induced neurotoxicity was validated by significantly decreased serum BDNF, brain BDNF, and serum S100B, along with significantly increased serum tau protein (p<0.05). PHZ-induced hemolytic hyperbilirubinemia significantly decreased serum BDNF, brain BDNF, and PLC/IP3/Ca2+ pathway activation while increasing neuronal apoptosis levels (p<0.05), all of which were partially rescued by melatonin therapy (p<0.05). Pre-treatment with the PLC inhibitor U73122 largely abolished the positive effects of melatonin on PLC/IP3/Ca2+ pathway activation, downstream BDNF levels, and neuronal apoptosis (p<0.05). CONCLUSIONS Promotion of BDNF expression and anti-apoptotic effects in neonatal hemolytic hyperbilirubinemia by melatonin largely operates via a PLC-mediated mechanism.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Hiperbilirrubinemia Neonatal/tratamento farmacológico , Hiperbilirrubinemia Neonatal/metabolismo , Melatonina/farmacologia , Fosfolipases Tipo C/metabolismo , Animais , Apoptose/efeitos dos fármacos , Bilirrubina/sangue , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Estrenos/farmacologia , Hemólise/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Pirrolidinonas/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Fosfolipases Tipo C/antagonistas & inibidores
15.
Mol Hum Reprod ; 23(8): 521-534, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28521061

RESUMO

STUDY QUESTION: Is extracellular cAMP involved in the regulation of signalling pathways in bovine sperm capacitation? SUMMARY ANSWER: Extracellular cAMP induces sperm capacitation through the activation of different signalling pathways that involve phospholipase C (PLC), PKC/ERK1-2 signalling and an increase in sperm Ca2+ levels, as well as soluble AC and cAMP/protein kinase A (PKA) signalling. WHAT IS KNOWN ALREADY: In order to fertilize the oocyte, ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, known as capacitation. This correlates with a number of membrane and metabolic modifications that include an increased influx of bicarbonate and Ca2+, activation of a soluble adenylyl cyclase (sAC) to produce cAMP, PKA activation, protein tyrosine phosphorylation and the development of hyperactivated motility. We previously reported that cAMP efflux by Multidrug Resistance Protein 4 (MRP4) occurs during sperm capacitation and the pharmacological blockade of this inhibits the process. Moreover, the supplementation of incubation media with cAMP abolishes the inhibition and leads to sperm capacitation, suggesting that extracellular cAMP regulates crucial signalling cascades involved in this process. STUDY DESIGN, SIZE, DURATION: Bovine sperm were selected by the wool glass column method, and washed by centrifugation in BSA-Free Tyrode's Albumin Lactate Pyruvate (sp-TALP). Pellets were resuspended then diluted for each treatment. For in vitro capacitation, 10 to 15 × 106 SPZ/ml were incubated in 0.3% BSA sp-TALP at 38.5°C for 45 min under different experimental conditions. To evaluate the role of extracellular cAMP on different events associated with sperm capacitation, 10 nM cAMP was added to the incubation medium as well as different inhibitors of enzymes associated with signalling transduction pathways: U73122 (PLC inhibitor, 10 µM), Gö6983 (PKC inhibitor, 10 µM), PD98059 (ERK-1/2 inhibitor, 30 µM), H89 and KT (PKA inhibitors, 50 µM and 100 nM, respectively), KH7 (sAC inhibitor, 10 µM), BAPTA-AM (intracellular Ca2+ chelator, 50 µM), EGTA (10 µM) and Probenecid (MRPs general inhibitor, 500 µM). In addition, assays for binding to oviductal epithelial cells and IVF were carried out to test the effect of cAMP compared with other known capacitant agents such as heparin (60 µg/ml) and bicarbonate (40 mM). PARTICIPANTS/MATERIALS, SETTING, METHODS: Straws of frozen bovine semen (20-25 × 106 spermatozoa/ml) were kindly provided by Las Lilas, CIALE and CIAVT Artificial Insemination Centers. The methods used in this work include western blot, immunohistochemistry, flow cytometry, computer-assisted semen analysis, live imaging of Ca2+ and fluorescence scanning. At least three independent assays with bull samples of proven fertility were carried. MAIN RESULTS AND THE ROLE OF CHANCE: In the present study, we elucidate the molecular events induced by extracellular cAMP. Our results showed that external cAMP induces sperm capacitation, depending upon the action of PLC. Downstream, this enzyme increased ERK1-2 activation through PKC and elicited a rise in sperm Ca2+ levels (P < 0.01). Moreover, extracellular cAMP-induced capacitation also depended on the activity of sAC and PKA, and increased tyrosine phosphorylation, indicating that the nucleotide exerts a broad range of responses. In addition, extracellular cAMP-induced sperm hyperactivation and concomitantly increased the proportion of spermatozoa with high mitochondrial activity (P < 0.01). Finally, cAMP increased the in vitro fertilization rate compared to control conditions (P < 0.001). LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study performed with bovine cryopreserved spermatozoa. Studies in other species and with fresh samples are needed to extrapolate these data. WIDER IMPLICATIONS OF THE FINDINGS: These findings strongly suggest an important role of extracellular cAMP in the regulation of the signalling pathways involved in the acquisition of bull sperm fertilizing capability. The data presented here indicate that not only a rise, but also a regulation of cAMP levels is necessary to ensure sperm fertilizing ability. Thus, exclusion of the nucleotide to the extracellular space might be essential to guarantee the achievement of a cAMP tone, needed for all capacitation-associated events to take place. Moreover, the ability of cAMP to trigger such broad and complex signalling events allows us to hypothesize that cAMP is a self-produced autocrine/paracrine factor, and supports the emerging paradigm that spermatozoa do not compete but, in fact, communicate with each other. A precise understanding of the functional competence of mammalian spermatozoa is essential to generate clinical advances in the treatment of infertility and the development of novel contraceptive strategies. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas [PIP0 496 to S.P.-M.], Agencia Nacional de Promoción Científica y Tecológica [PICT 2012-1195 and PICT2014-2325 to S.P.-M., and PICT 2013-2050 to C.D.], Boehringer Ingelheim Funds, and the Swedish Farmers Foundation [SLF-H13300339 to J.M.]. The authors declare there are no conflicts of interests.


Assuntos
AMP Cíclico/metabolismo , Transdução de Sinais , Capacitação Espermática , Animais , Cálcio/metabolismo , Bovinos , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Fertilidade , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Capacitação Espermática/efeitos dos fármacos , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo
16.
PLoS One ; 12(4): e0176108, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28423060

RESUMO

BACKGROUND: The chemokine receptor CXCR4 plays a crucial role in tumors, including glioblastoma multiforme (GBM), the most aggressive glioma. Phosphatidylcholine-specific phospholipase C (PC-PLC), a catabolic enzyme of PC metabolism, is involved in several aspects of cancer biology and its inhibition down-modulates the expression of growth factor membrane receptors interfering with their signaling pathways. In the present work we investigated the possible interplay between CXCR4 and PC-PLC in GBM cells. METHODS: Confocal microscopy, immunoprecipitation, western blot analyses, and the evaluation of migration and invasion potential were performed on U87MG cells after PC-PLC inhibition with the xanthate D609. The intracellular metabolome was investigated by magnetic resonance spectroscopy; lactate levels and lactate dehydrogenase (LDH) activity were analyzed by colorimetric assay. RESULTS: Our studies demonstrated that CXCR4 and PC-PLC co-localize and are associated on U87MG cell membrane. D609 reduced CXCR4 expression, cell proliferation and invasion, interfering with AKT and EGFR activation and expression. Metabolic analyses showed a decrease in intracellular lactate concentration together with a decrement in LDH activity. CONCLUSIONS: Our data suggest that inhibition of PC-PLC could represent a new molecular approach in glioma biology not only for its ability in modulating cell metabolism, glioma growth and motility, but also for its inhibitory effect on crucial molecules involved in cancer progression.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/farmacologia , Neuroglia/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Receptores CXCR4/genética , Tionas/farmacologia , Fosfolipases Tipo C/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação da Expressão Gênica , Glicólise/efeitos dos fármacos , Glicólise/genética , Humanos , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Ácido Láctico/metabolismo , Metaboloma/genética , Neuroglia/efeitos dos fármacos , Neuroglia/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo
17.
Cell Physiol Biochem ; 41(1): 399-412, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28214885

RESUMO

BACKGROUND/AIMS: Endothelin-1 (ET-1) and the α1-adrenoceptor agonist phenylephrine (PE) activate cAMP response element binding protein (CREB), a transcription factor implicated in cardiac hypertrophy. The signaling pathway involved in CREB activation by these hypertrophic stimuli is poorly understood. We examined signaling pathways for ET-1- or PE-induced cardiac CREB activation. METHODS: Western blotting was performed with pharmacological and genetic interventions in rat ventricular myocytes. RESULTS: ET-1 and PE increased CREB phosphorylation, which was inhibited by blockade of phospholipase C, the extracellular-signal-regulated kinase 1/2 (ERK1/2) pathway, protein kinase C (PKC) or Ca2+-calmodulin-dependent protein kinase II (CaMKII). Intracellular Ca2+ buffering decreased ET-1- and PE-induced CREB phosphorylation by ≥80%. Sarcoplasmic reticulum Ca2+ pump inhibitor, inositol 1,4,5-trisphosphate receptor (IP3R) blockers, or type 2 IP3R (IP3R2) knock-out abolished ET-1- or PE-induced CREB phosphorylation. ET-1 and PE increased phosphorylation of CaMKII and ERK1/2, which was eliminated by IP3R blockade/knock-out or PKC inhibition. Activation of CaMKII, but not ERK1/2, by these agonists was sensitive to Ca2+ buffering or to Gö6976, the inhibitor of Ca2+-dependent PKC and protein kinase D (PKD). CONCLUSION: CREB phosphorylation by ET-1 and PE may be mainly mediated by IP3R2/Ca2+-PKC-PKD-CaMKII signaling with a minor contribution by ERK1/2, linked to IP3R2 and Ca2+-independent PKC, in ventricular myocytes.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Endotelina-1/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Fenilefrina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Carbazóis/farmacologia , Células Cultivadas , Flavonoides/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/deficiência , Receptores de Inositol 1,4,5-Trifosfato/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo
18.
Am J Physiol Lung Cell Mol Physiol ; 312(3): L326-L333, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28062485

RESUMO

Bitter taste receptors (T2Rs), a G protein-coupled receptor family capable of detecting numerous bitter-tasting compounds, have recently been shown to be expressed and play diverse roles in many extraoral tissues. Here we report the functional expression of T2Rs in rat pulmonary sensory neurons. In anesthetized spontaneously breathing rats, intratracheal instillation of T2R agonist chloroquine (10 mM, 0.1 ml) significantly augmented chemoreflexes evoked by right-atrial injection of capsaicin, a specific activator for transient receptor potential vanilloid receptor 1 (TRPV1), whereas intravenous infusion of chloroquine failed to significantly affect capsaicin-evoked reflexes. In patch-clamp recordings with isolated rat vagal pulmonary sensory neurons, pretreatment with chloroquine (1-1,000 µM, 90 s) concentration dependently potentiated capsaicin-induced TRPV1-mediated inward currents. Preincubating with diphenitol and denatonium (1 mM, 90 s), two other T2R activators, also enhanced capsaicin currents in these neurons but to a lesser extent. The sensitizing effect of chloroquine was effectively prevented by the phospholipase C inhibitor U73122 (1 µM) or by the protein kinase C inhibitor chelerythrine (10 µM). In summary, our study showed that activation of T2Rs augments capsaicin-evoked TRPV1 responses in rat pulmonary nociceptors through the phospholipase C and protein kinase C signaling pathway.


Assuntos
Pulmão/metabolismo , Nociceptores/metabolismo , Proteína Quinase C/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Paladar , Fosfolipases Tipo C/metabolismo , Anestesia , Animais , Benzofenantridinas/farmacologia , Capsaicina/farmacologia , Cloroquina/administração & dosagem , Cloroquina/farmacologia , Estrenos/farmacologia , Infusões Intravenosas , Pirrolidinonas/farmacologia , Ratos Sprague-Dawley , Reflexo/efeitos dos fármacos , Respiração/efeitos dos fármacos , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Paladar/efeitos dos fármacos , Fosfolipases Tipo C/antagonistas & inibidores
19.
J Immunol ; 198(3): 1285-1296, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28039302

RESUMO

The adenylate cyclase toxin-hemolysin (CyaA) plays a key role in immune evasion and virulence of the whooping cough agent Bordetella pertussis. CyaA penetrates the complement receptor 3-expressing phagocytes and ablates their bactericidal capacities by catalyzing unregulated conversion of cytosolic ATP to the key second messenger molecule cAMP. We show that signaling of CyaA-generated cAMP blocks the oxidative burst capacity of neutrophils by two converging mechanisms. One involves cAMP/protein kinase A-mediated activation of the Src homology region 2 domain-containing phosphatase-1 (SHP-1) and limits the activation of MAPK ERK and p38 that are required for assembly of the NADPH oxidase complex. In parallel, activation of the exchange protein directly activated by cAMP (Epac) provokes inhibition of the phospholipase C by an as yet unknown mechanism. Indeed, selective activation of Epac by the cell-permeable analog 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate counteracted the direct activation of phospholipase C by 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide. Hence, by inhibiting production of the protein kinase C-activating lipid, diacylglycerol, cAMP/Epac signaling blocks the bottleneck step of the converging pathways of oxidative burst triggering. Manipulation of neutrophil membrane composition by CyaA-produced signaling of cAMP thus enables B. pertussis to evade the key innate host defense mechanism of reactive oxygen species-mediated killing of bacteria by neutrophils.


Assuntos
Toxina Adenilato Ciclase/fisiologia , AMP Cíclico/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Neutrófilos/fisiologia , Explosão Respiratória , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/antagonistas & inibidores , Bordetella pertussis/imunologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , Fosfatidilinositol 3-Quinases/fisiologia , Proteína Quinase C/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Fosfolipases Tipo C/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia
20.
Plant Cell Environ ; 40(4): 585-598, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27272019

RESUMO

Cryptogein is a 10 kDa protein secreted by the oomycete Phytophthora cryptogea that activates defence mechanisms in tobacco plants. Among early signalling events triggered by this microbial-associated molecular pattern is a transient apoplastic oxidative burst which is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity of the RESPIRATORY BURST OXIDASE HOMOLOG isoform D (RBOHD). Using radioactive [33 P]-orthophosphate labelling of tobacco Bright Yellow-2 suspension cells, we here provide in vivo evidence for a rapid accumulation of phosphatidic acid (PA) in response to cryptogein because of the coordinated onset of phosphoinositide-dependent phospholipase C and diacylglycerol kinase (DGK) activities. Both enzyme specific inhibitors and silencing of the phylogenetic cluster III of the tobacco DGK family were found to reduce PA production upon elicitation and to strongly decrease the RBOHD-mediated oxidative burst. Therefore, it appears that PA originating from DGK controls NADPH-oxidase activity. Amongst cluster III DGKs, the expression of DGK5-like was up-regulated in response to cryptogein. Besides DGK5-like is likely to be the main cluster III DGK isoform silenced in one of our mutant lines, making it a strong candidate for the observed response to cryptogein. The relevance of these results is discussed with regard to early signalling lipid-mediated events in plant immunity.


Assuntos
Diacilglicerol Quinase/metabolismo , Proteínas Fúngicas/farmacologia , NADPH Oxidases/metabolismo , Explosão Respiratória , Tabaco/enzimologia , Linhagem Celular , Análise por Conglomerados , Ativação Enzimática/efeitos dos fármacos , Mutação com Ganho de Função/genética , Inativação Gênica , MicroRNAs/metabolismo , Padrões Moleculares Associados a Patógenos/metabolismo , Ácidos Fosfatídicos/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Inibidores de Proteínas Quinases/farmacologia , Explosão Respiratória/efeitos dos fármacos , Tabaco/efeitos dos fármacos , Tabaco/genética , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo
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