Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.065
Filtrar
1.
Cells ; 10(8)2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34440874

RESUMO

2-Ethyl-3-O-sulfamoyl-estra-1,3,5(10)16-tetraene (ESE-16) is an in silico-designed estradiol analogue which has improved the parent compound's efficacy in anti-cancer studies. In this proof-of-concept study, the potential radiosensitizing effects of ESE-16 were investigated in an in vitro deconstructed bone metastasis model. Prostate (DU 145) and breast (MDA-MB-231) tumor cells, osteoblastic (MC3T3-E1) and osteoclastic (RAW 264.7) bone cells and human umbilical vein endothelial cells (HUVECs) were representative components of such a lesion. Cells were exposed to a low-dose ESE-16 for 24 hours prior to radiation at non-lethal doses to determine early signaling and molecular responses of this combination treatment. Tartrate-resistant acid phosphatase activity and actin ring formation were investigated in osteoclasts, while cell cycle progression, reactive oxygen species generation and angiogenic protein expression were investigated in HUVECs. Increased cytotoxicity was evident in tumor and endothelial cells while bone cells appeared to be spared. Increased mitotic indices were calculated, and evidence of increased deoxyribonucleic acid damage with retarded repair, together with reduced metastatic signaling was observed in tumor cells. RAW 264.7 macrophages retained their ability to differentiate into osteoclasts. Anti-angiogenic effects were observed in HUVECs, and expression of hypoxia-inducible factor 1-α was decreased. Through preferentially inducing tumor cell death and potentially inhibiting neovascularization whilst preserving bone physiology, this low-dose combination regimen warrants further investigation for its promising therapeutic application in bone metastases management, with the additional potential of limited treatment side effects.


Assuntos
Neoplasias Ósseas/metabolismo , Estrenos/farmacologia , Radiossensibilizantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Sulfonamidas/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proteína Morfogenética Óssea 7/metabolismo , Neoplasias Ósseas/prevenção & controle , Neoplasias Ósseas/secundário , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dano ao DNA , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteoclastos/efeitos da radiação , Superóxidos/metabolismo , Raios Ultravioleta
2.
ACS Chem Biol ; 16(7): 1288-1297, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34232635

RESUMO

Inducing the formation of new oligodendrocytes from oligodendrocyte progenitor cells (OPCs) represents a potential approach to repairing the loss of myelin observed in multiple sclerosis and other diseases. Recently, we demonstrated that accumulation of specific cholesterol precursors, 8,9-unsaturated sterols, is a dominant mechanism by which dozens of small molecules enhance oligodendrocyte formation. Here, we evaluated a library of 56 sterols and steroids to evaluate whether other classes of bioactive sterol derivatives may also influence mouse oligodendrocyte precursor cell (OPC) differentiation or survival. From this library, we identified U-73343 as a potent enhancer of oligodendrocyte formation that induces 8,9-unsaturated sterol accumulation by inhibition of the cholesterol biosynthesis enzyme sterol 14-reductase. In contrast, we found that mouse OPCs are remarkably vulnerable to treatment with the glycosterol OSW-1, an oxysterol-binding protein (OSBP) modulator that induces Golgi stress and OPC death in the low picomolar range. A subsequent small-molecule suppressor screen identified mTOR signaling as a key effector pathway mediating OSW-1's cytotoxic effects in mouse OPCs. Finally, evaluation of a panel of ER and Golgi stress-inducing small molecules revealed that mouse OPCs are highly sensitive to these perturbations, more so than closely related neural progenitor cells. Together, these studies highlight the wide-ranging influence of sterols and steroids on OPC cell fate, with 8,9-unsaturated sterols positively enhancing differentiation to oligodendrocytes and OSW-1 able to induce lethal Golgi stress with remarkable potency.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Precursoras de Oligodendrócitos/efeitos dos fármacos , Esteróis/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Colestenonas/farmacologia , Colestenonas/toxicidade , Avaliação Pré-Clínica de Medicamentos , Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estrenos/farmacologia , Complexo de Golgi/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Células Precursoras de Oligodendrócitos/metabolismo , Oligodendroglia/metabolismo , Pirrolidinonas/farmacologia , Saponinas/farmacologia , Saponinas/toxicidade , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/toxicidade , Esteróis/toxicidade
3.
Reprod Domest Anim ; 56(7): 1015-1023, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33914997

RESUMO

Aglepristone, a competitive progesterone antagonist, is successfully used in various progesterone-dependent conditions. This study investigated uterine histomorphometric analysis, and expressions of the oestrogen α receptor (ERα) and progesterone receptor (PR) in uteri of bitches following the single dose of aglepristone treatment. Twelve client-owned healthy diestrous bitches were used in the study. The single dose of aglepristone (Alizine® , 10 mg/kg) was injected subcutaneously 5 days before ovariohysterectomy in the treatment group (n = 6); bitches without treatment served as a control group (n = 6). Uteri were collected for histomorphometric analysis, ERα and PR gene, and protein expressions studies. The mRNA expressions of ERα and PR were determined by RT-qPCR. Immunohistochemical analysis was used to evaluate the ERα and PR protein expressions using an H-score in five parts of the uterus. The results demonstrated glandular epithelium height significantly decreased (p < .05) and ERα mRNA increased (p < .01) in treated dogs. Of the treated bitches, lower expression levels of ERα were observed in the luminal epithelium, crypt and glandular epithelium, with higher expression in the endometrial stroma and myometrium (p < .05); however, PR expression decreased in the luminal epithelium, crypt and glandular epithelium (p < .01). In conclusion, reduction of the uterine glandular epithelium and ERα mRNA upregulation together with changes in ERα and PR expressions were observed in the treated bitches. However, changes in uterine ERα and PR expressions in the treated bitches depended on tissue layers. The treatment had no effect on serum oestradiol and progesterone levels.


Assuntos
Cães , Estrenos/farmacologia , Receptor alfa de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Útero/efeitos dos fármacos , Animais , Estradiol/sangue , Feminino , Histerectomia/veterinária , Ovariectomia/veterinária , Progesterona/sangue , RNA Mensageiro , Transcriptoma , Útero/anatomia & histologia , Útero/metabolismo
4.
Molecules ; 26(5)2021 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-33800024

RESUMO

The endocannabinoid system (ECS) is involved in the modulation of several basic biological processes, having widespread roles in neurodevelopment, neuromodulation, immune response, energy homeostasis and reproduction. In the adult central nervous system (CNS) the ECS mainly modulates neurotransmitter release, however, a substantial body of evidence has revealed a central role in regulating neurogenesis in developing and adult CNS, also under pathological conditions. Due to the complexity of investigating ECS functions in neural progenitors in vivo, we tested the suitability of the ST14A striatal neural progenitor cell line as a simplified in vitro model to dissect the role and the mechanisms of ECS-regulated neurogenesis, as well as to perform ECS-targeted pharmacological approaches. We report that ST14A cells express various ECS components, supporting the presence of an active ECS. While CB1 and CB2 receptor blockade did not affect ST14A cell number, exogenous administration of the endocannabinoid 2-AG and the synthetic CB2 agonist JWH133 increased ST14A cell proliferation. Phospholipase C (PLC), but not PI3K pharmacological blockade negatively modulated CB2-induced ST14A cell proliferation, suggesting that a PLC pathway is involved in the steps downstream to CB2 activation. On the basis of our results, we propose ST14A neural progenitor cells as a useful in vitro model for studying ECS modulation of neurogenesis, also in prospective in vivo pharmacological studies.


Assuntos
Moduladores de Receptores de Canabinoides/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/fisiologia , Receptores de Canabinoides/metabolismo , Animais , Canabinoides/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Corpo Estriado/citologia , Estrenos/farmacologia , Células-Tronco Neurais/fisiologia , Neurogênese/efeitos dos fármacos , Pirrolidinonas/farmacologia , Ratos , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/genética , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/antagonistas & inibidores , Receptor CB2 de Canabinoide/genética , Receptores de Canabinoides/genética , Fosfolipases Tipo C/antagonistas & inibidores
5.
J Physiol Biochem ; 77(2): 321-329, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33704695

RESUMO

Lysophosphatidic acid (LPA) acts through the activation of G protein-coupled receptors, in a Ca2+-dependent manner. We show the effects of LPA on the plasma membrane Ca2+-ATPase (PMCA) from kidney proximal tubule cells. The Ca2+-ATPase activity was inhibited by nanomolar concentrations of LPA, with maximal inhibition (~50%) obtained with 20 nM LPA. This inhibitory action on PMCA activity was blocked by Ki16425, an antagonist for LPA receptors, indicating that this lipid acts via LPA1 and/or LPA3 receptor. This effect is PKC-dependent, since it is abolished by calphostin C and U73122, PKC, and PLC inhibitors, respectively. Furthermore, the addition of 10-8 M PMA, a well-known PKC activator, mimicked PMCA modulation by LPA. We also demonstrated that the PKC activation leads to an increase in PMCA phosphorylation. These results indicate that LPA triggers LPA1 and/or LPA3 receptors at the BLM, inducing PKC-dependent phosphorylation with further inhibition of PMCA. Thus, LPA is part of the regulatory lipid network present at the BLM and plays an important role in the regulation of intracellular Ca2+ concentration that may result in significant physiological alterations in other Ca2+-dependent events ascribed to the renal tissue.


Assuntos
Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Receptores de Ácidos Lisofosfatídicos/genética , Animais , Fracionamento Celular , Membrana Celular/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Estrenos/farmacologia , Regulação da Expressão Gênica , Transporte de Íons/efeitos dos fármacos , Isoxazóis/farmacologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Naftalenos/farmacologia , Fosforilação/efeitos dos fármacos , ATPases Transportadoras de Cálcio da Membrana Plasmática/antagonistas & inibidores , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Cultura Primária de Células , Propionatos/farmacologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Pirrolidinonas/farmacologia , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais , Suínos , Acetato de Tetradecanoilforbol/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
6.
Biochim Biophys Acta Mol Cell Res ; 1868(6): 118988, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33581218

RESUMO

T cell activation starts with formation of second messengers that release Ca2+ from the endoplasmic reticulum (ER) and thereby activate store-operated Ca2+ entry (SOCE), one of the essential signals for T cell activation. Recently, the steroidal 2-methoxyestradiol was shown to inhibit nuclear translocation of the nuclear factor of activated T cells (NFAT). We therefore investigated 2-methoxyestradiol for inhibition of Ca2+ entry in T cells, screened a library of 2-methoxyestradiol analogues, and characterized the derivative 2-ethyl-3-sulfamoyloxy-17ß-cyanomethylestra-1,3,5(10)-triene (STX564) as a novel, potent and specific SOCE inhibitor. STX564 inhibits Ca2+ entry via SOCE without affecting other ion channels and pumps involved in Ca2+ signaling in T cells. Downstream effects such as cytokine expression and cell proliferation were also inhibited by both 2-methoxyestradiol and STX564, which has potential as a new chemical biology tool.


Assuntos
2-Metoxiestradiol/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Estrenos/farmacologia , Fatores de Transcrição NFATC/metabolismo , Linfócitos T/citologia , 2-Metoxiestradiol/análogos & derivados , Animais , Cálcio/metabolismo , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Estrenos/síntese química , Estrenos/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
7.
Biomolecules ; 11(2)2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33578683

RESUMO

A mimetic of the BDNF loop 4, bis (N-monosuccinyl-L-seryl-L-lysine) hexamethylenediamide, named GSB-106, was designed and synthesized in our scientific group. The compound activated TrkB, MAPK/ERK, PI3K/AKT, and PLCγ in in vitro experiments. In vivo experiments with rodents revealed its antidepressant-like activity in the forced swim and the tail suspension tests at the dose range of 0.1-5.0 mg/kg (i.p., p.o.). However, GSB-106 was not studied in depression models modulating major depression in humans. In the present study, the GSB-106 antidepressant-like activity was revealed in mice at the depression model induced by 28-day social defeat stress with 21-days oral administration (0.1 mg/kg) after stress. At the same time, GSB-106 restored reduced locomotor activity and completely eliminated the anhedonia manifestations. The compound also restored reduced levels of synaptophysin and CREB in the hippocampus. In addition, the Trk receptor antagonist K252A, and the PLC inhibitor U73122, were found to completely block the antidepressant-like activity of GSB-106 in the forced swimming test in mice. Thus, the present results demonstrate the dipeptide BDNF mimetic GSB-106 reversed depressive-like behavior and restored hippocampal neuroplasticity in a rodent depression model. These effects of GSB-106 are probably regulated by TrkB signaling.


Assuntos
Antidepressivos/uso terapêutico , Materiais Biomiméticos/uso terapêutico , Fator Neurotrófico Derivado do Encéfalo/química , Transtorno Depressivo/tratamento farmacológico , Dipeptídeos/uso terapêutico , Peptidomiméticos/uso terapêutico , Administração Oral , Animais , Antidepressivos/química , Antidepressivos/farmacologia , Comportamento Animal/efeitos dos fármacos , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Carbazóis/farmacologia , Dipeptídeos/química , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Estrenos/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Alcaloides Indólicos/farmacologia , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Proteínas Tirosina Quinases/metabolismo , Pirrolidinonas/farmacologia , Comportamento Social , Sinaptofisina/metabolismo
8.
Molecules ; 26(3)2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33572896

RESUMO

The search for novel anti-cancer compounds which can circumvent chemotherapeutic drug resistance and limit systemic toxicity remains a priority. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)15-tetraene-3-ol-17one (ESE-15-one) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) are sulphamoylated 2-methoxyestradiol (2-ME) analogues designed by our research team. Although their cytotoxicity has been demonstrated in vitro, the temporal and mechanistic responses of the initiated intracellular events are yet to be determined. In order to do so, assays investigating the compounds' effects on microtubules, cell cycle progression, signalling cascades, autophagy and apoptosis were conducted using HeLa cervical- and MDA-MB-231 metastatic breast cancer cells. Both compounds reversibly disrupted microtubule dynamics as an early event by binding to the microtubule colchicine site, which blocked progression through the cell cycle at the G1/S- and G2/M transitions. This was supported by increased pRB and p27Kip1 phosphorylation. Induction of apoptosis with time-dependent signalling involving the p-JNK, Erk1/2 and Akt/mTOR pathways and loss of mitochondrial membrane potential was demonstrated. Inhibition of autophagy attenuated the apoptotic response. In conclusion, the 2-ME analogues induced a time-dependent cross-talk between cell cycle checkpoints, apoptotic signalling and autophagic processes, with an increased reactive oxygen species formation and perturbated microtubule functioning appearing to connect the processes. Subtle differences in the responses were observed between the two compounds and the different cell lines.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Estrona/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Antineoplásicos/farmacologia , Apoptose/genética , Autofagia/genética , Neoplasias da Mama/patologia , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Estrenos/farmacologia , Estrona/análogos & derivados , Estrona/química , Feminino , Células HeLa , Humanos , Microtúbulos/química , Metástase Neoplásica , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Neoplasias do Colo do Útero/patologia
9.
Biomolecules ; 11(2)2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33494474

RESUMO

Recent evidence suggests that the reason Extra Virgin Olive Oil (EVOO) lowers blood pressure and reduces the risk of developing hypertension is partly due to minor components of EVOO, such as phenols. However, little is still known about the mechanism(s) through which EVOO phenols mediate anti-hypertensive effects. The aim of the present study was to investigate the mechanisms of action of EVOO phenols on mesenteric resistance arteries. A pressure myograph was used to test the effect of EVOO phenols on isolated mesenteric arteries in the presence of specific inhibitors of: 1) BKca channels (Paxillin, 10-5 M); 2) L-type calcium channels (Verapamil, 10-5 M); 3) Ryanodine receptor, RyR (Ryanodine, 10-5 M); 4) inositol 1,4,5-triphosphate receptor, IP3R, (2-Aminoethyl diphenylborinate, 2-APB, 3 × 10-3 M); 5) phospholipase C, PLC, (U73122, 10-5 M), and 6) GPCR-Gαi signaling, (Pertussis Toxin, 10-5 M). EVOO phenols induced vasodilation of mesenteric arteries in a dose-dependent manner, and this effect was reduced by pre-incubation with Paxillin, Verapamil, Ryanodine, 2-APB, U73122, and Pertussis Toxin. Our data suggest that EVOO phenol-mediated vasodilation requires activation of BKca channels potentially through a local increase of subcellular calcium microdomains, a pivotal mechanism on the base of artery vasodilation. These findings provide novel mechanistic insights for understanding the vasodilatory properties of EVOO phenols on resistance arteries.


Assuntos
Microdomínios da Membrana/química , Artérias Mesentéricas/efeitos dos fármacos , Azeite de Oliva/química , Canais de Potássio/química , Fosfolipases Tipo C/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Compostos de Boro/farmacologia , Canais de Cálcio/química , Estrenos/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/química , Masculino , Paxilina/farmacologia , Toxina Pertussis/farmacologia , Fenol/química , Fenóis/farmacologia , Pirrolidinonas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Vasodilatação/efeitos dos fármacos , Verapamil/farmacologia
10.
J Cell Mol Med ; 25(3): 1531-1545, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33372388

RESUMO

Previous studies identified the involvement of phosphoinositide-specific phospholipase C (PLC) γ1 in some events of chondrocytes. This study aims to investigate whether and how PLCγ1 modulates autophagy to execute its role in osteoarthritis (OA) progression. Rat normal or human OA chondrocytes were pretreated with IL-1ß for mimicking or sustaining OA pathological condition. Using Western blotting, immunoprecipitation, qPCR, immunofluorescence and Dimethylmethylene blue assays, and ELISA and transmission electron microscope techniques, we found that PLCγ1 inhibitor U73122 enhanced Collagen II, Aggrecan and GAG levels, accompanied with increased LC3B-II/I ratio and decreased P62 expression level, whereas autophagy inhibitor Chloroquine partially diminished its effect. Meanwhile, U73122 dissociated Beclin1 from Beclin1-IP3R-Bcl-2 complex and blocked mTOR/ULK1 axis, in which the crosstalk between PLCγ1, AMPK, Erk and Akt were involved. Additionally, by haematoxylin and eosin, Safranin O/Fast green, and immunohistochemistry staining, we observed that intra-articular injection of Ad-shPLCγ1-1/2 significantly enhanced Collagen and Aggrecan levels, accompanied with increased LC3B and decreased P62 levels in a rat OA model induced by anterior cruciate ligament transection and medial meniscus resection. Consequently, PLCγ1 inhibition-driven autophagy conferred cartilage protection against OA through promoting ECM synthesis in OA chondrocytes in vivo and in vitro, involving the crosstalk between PLCγ1, AMPK, Erk and Akt.


Assuntos
Autofagia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Interleucina-1beta/farmacologia , Osteoartrite/etiologia , Osteoartrite/metabolismo , Fosfolipase C gama/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Autofagia/efeitos dos fármacos , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Células Cultivadas , Suscetibilidade a Doenças , Estrenos/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirrolidinonas/farmacologia , Ratos , Serina-Treonina Quinases TOR/metabolismo
11.
Gen Comp Endocrinol ; 300: 113637, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33017583

RESUMO

Allatotropin is a pleiotropic peptide originally characterized in insects. The existence of AT neuropeptide signaling was proposed in other invertebrates. In fact, we previously proposed the presence of an AT-like system regulating feeding behavior in Hydra sp. Even in insects, the information about the AT signaling pathway is incomplete. The aim of this study is to analyze the signaling cascade activated by AT in Hydra plagiodesmica using a pharmacological approach. The results show the involvement of Ca2+ and IP3 signaling in the transduction pathway of the peptide. Furthermore, we confirm the existence of a GPCR system involved in this pathway, that would be coupled to a Gq subfamily of Gα protein, which activates a PLC, inducing an increase in IP3 and cytosolic Ca2+. To the best of our knowledge, this work represents the first in vivo approach to study the overall signaling pathway and intracellular events involved in the myoregulatory effect of AT in Hydra sp.


Assuntos
Sinalização do Cálcio , Hydra/metabolismo , Hormônios de Inseto/metabolismo , Neuropeptídeos/metabolismo , Orexinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Indóis/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Maleimidas/farmacologia , Meliteno/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Pirrolidinonas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo
12.
Cancer Res ; 80(24): 5491-5501, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33115803

RESUMO

Chromophobe renal cell carcinoma (chRCC) accounts for approximately 5% of all renal cancers and around 30% of chRCC cases have mutations in TP53. chRCC is poorly supported by microvessels and has markably lower glucose uptake than clear cell RCC and papillary RCC. Currently, the metabolic status and mechanisms by which this tumor adapts to nutrient-poor microenvironments remain to be investigated. In this study, we performed proteome and metabolome profiling of chRCC tumors and adjacent kidney tissues and identified major metabolic alterations in chRCC tumors, including the classical Warburg effect, the downregulation of gluconeogenesis and amino acid metabolism, and the upregulation of protein degradation and endocytosis. chRCC cells depended on extracellular macromolecules as an amino acid source by activating endocytosis to sustain cell proliferation and survival. Inhibition of the phospholipase C gamma 2 (PLCG2)/inositol 1,4,5-trisphosphate (IP3)/Ca2+/protein kinase C (PKC) pathway significantly impaired the activation of endocytosis for amino acid uptakes into chRCC cells. In chRCC, whole-exome sequencing revealed that TP53 mutations were not related to expression of PLCG2 and activation of endocytosis. Our study provides novel perspectives on metabolic rewiring in chRCC and identifies the PLCG2/IP3/Ca2+/PKC axis as a potential therapeutic target in patients with chRCC. SIGNIFICANCE: This study reveals macropinocytosis as an important process utilized by chRCC to gain extracellular nutrients in a p53-independent manner.


Assuntos
Aminoácidos/metabolismo , Carcinoma de Células Renais/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Neoplasias Renais/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Compostos de Boro/farmacologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Estrenos/farmacologia , Gluconeogênese , Humanos , Indóis/farmacologia , Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Inositol 1,4,5-Trifosfato/metabolismo , Neoplasias Renais/patologia , Maleimidas/farmacologia , Metaboloma , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteoma , Pirrolidinonas/farmacologia
13.
Int J Oncol ; 57(4): 989-1000, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32945365

RESUMO

The increased tyrosine kinase activity of non­small cell lung cancer (NSCLC)­associated epidermal growth factor receptor (EGFR) mutants results in deregulated pathways that contribute to malignant cell survival, tumor progression and metastasis. Previous studies investigating lung cancer­associated EGFR have focused on the prognostic implications of receptor kinase mutations in patients with NSCLC; however, the role of EGFR mutations in tumor cell invasion and migration remains undetermined. The present study was designed to investigate the role of NSCLC­associated mutant EGFR­driven signaling pathways in cell proliferation and invasion. Non­endogenous EGFR­expressing 293 cells stably expressing EGFR mutants that are sensitive or resistant to Food and Drug Administration (FDA)­approved EGFR­targeted tyrosine kinase inhibitors (TKIs) were used in the present study. The experiments demonstrated an increased phosphorylation of phospholipase (PLC)γ1, c­Cbl, signal transducer and activator of transcription (Stat), extracellular regulated kinase (Erk)1/2, Akt, Shc and Gab1 proteins in cells expressing a mutant form, rather than the wild­type receptor. As PLCγ1 is a known regulator of metastatic development, mutant receptor­mediated PLCγ1 activation was further evaluated. To examine the effects of EGFR and PLCγ1 phosphorylation, the metastatic potential of cells expressing mutants was investigated using wound healing, Transwell cell migration and invasion assays. The inhibition of receptor phosphorylation with the 1st, 2nd and 3rd generation TKIs, gefitinib, afatinib, osimertinib, respectively, reduced PLCγ1 phosphorylation, and reduced the invasive and migratory potential of 293 cells, confirming PLCγ1 as one of the probable downstream effectors of mutant EGFR signaling. However, the PLC inhibitor, U73122, inhibited cell migration and invasion without affecting EGFR signaling and PLCγ1 phosphorylation. Notably, U73122 reduced Akt and Erk1/2 phosphorylation within 25 min of its application; however, 100% cell viability was recorded even after 48 h. Upon further investigation, proliferative signaling pathways remained active at 48 h, in accordance with cell viability. Therefore, the present study concludes that mutant receptor­mediated PLCγ1 activation may play a significant role in the migration and invasion of NSCLC tumors; however, its regulatory role in tumor cell proliferation warrants further investigation and validation in lung tumor cell lines harboring EGFR mutations.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/metabolismo , Mutação , Fosfolipase C gama/metabolismo , Afatinib/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/biossíntese , Receptores ErbB/genética , Receptores ErbB/metabolismo , Estrenos/farmacologia , Gefitinibe/farmacologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Invasividade Neoplásica , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirrolidinonas/farmacologia , Proteínas Recombinantes/metabolismo , Transdução de Sinais
14.
Biochem Biophys Res Commun ; 532(2): 292-299, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-32868075

RESUMO

Phosphatidylcholine-specific phospholipase Cγ1 (PLCγ1) is involved in regulating cell metabolism. However, little is known how PLCγ1 directs BMSC differentiation. Here, we investigated the role of PLCγ1 in rat BMSC differentiation into osteoblasts and chondrocytes. The results of Alizarin red and Alcian blue staining showed that PLCγ1 inhibitor U73122 significantly enhanced the mineralization capacity and proteoglycan deposition of BMSCs. The results of qPCR technique and Western blot analysis showed that long-term treatment of U73122 enhanced COL1A1 and OPG mRNA levels and Collagen 1A1, BMP2, and p-Smad1/5/9 protein levels and that short-term treatment of U73122 enhanced COL2A1 and SOX9 mRNA levels and Collagen 2, SOX9, Aggrecan, TGF-ß3, and p-Smad2/3 protein levels. Decreased p-mTOR and p-P38 contributed to enhanced osteogenic potentials of BMSCs and increased p-P38 contributed to enhanced chondrogenic potentials of BMSCs. The scaffold transplantation with U73122+BMSC was more efficacious than BMSC alone for osteochondral defect repair in a rat model. Therefore, suppressing PLCγ1 could improve the capacity to effectively use BMSCs for cell therapy of osteochondral defect.


Assuntos
Condrogênese/fisiologia , Células-Tronco Mesenquimais/citologia , Osteogênese/fisiologia , Fosfolipase C gama/antagonistas & inibidores , Alginatos , Animais , Diferenciação Celular , Células Cultivadas , Condrogênese/efeitos dos fármacos , Estrenos/farmacologia , Hidrogéis/administração & dosagem , Hidrogéis/química , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteocondrose/fisiopatologia , Osteocondrose/terapia , Osteogênese/efeitos dos fármacos , Patela/diagnóstico por imagem , Patela/patologia , Patela/cirurgia , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Pirrolidinonas/farmacologia , Ratos Sprague-Dawley , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
Molecules ; 25(16)2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32781695

RESUMO

Previous studies have reported that estrogen hormone promotes melanogenesis while progesterone inhibits it. A selective estrogen receptor modulator (SERM), tamoxifen, has been shown to promote melanogenesis; however, to date, there have been no reports on the effects of a selective progesterone receptor modulator (SPRM) on melanogenesis. In the present study, we hypothesized that asoprisnil (AP), a SPRM, inhibits melanogenesis. AP was tested for cytotoxicity to B16F10 mouse melanoma cells for screening the nontoxic concentrations using MTS cytotoxicity assay. Extracellular and intracellular melanin levels were estimated at nontoxic concentrations of AP. To evaluate the direct effect of AP on tyrosinase enzyme, tyrosinase activity and copper chelating activities were measured. Next, the effects of AP on melanogenesis were tested in normal human melanocytes, neonatal, darkly pigmented (HEMn-DP). Our results demonstrate that AP was nontoxic at a concentration range of 10-50 µM in B16F10 cells; AP at 50 µM significantly suppressed extracellular melanin levels comparable to kojic acid at 500 µM, with no significant effect on intracellular melanin levels. The mechanism of melanogenesis inhibition was studied to assess if AP downregulated tyrosinase activity in cell lysates or in a cell-free system. However, AP was found to increase intracellular tyrosinase activity without any effect on tyrosinase enzyme activity or copper chelating activity in a cell-free system, indicating that AP inhibits melanogenesis by mechanisms other than direct effects on tyrosinase enzyme activity. The capacity of AP to inhibit melanosome export was further validated in HEMn-DP cells; AP significantly suppressed dendricity at concentrations of 20 and 30 µM in the absence of effects on melanin synthesis or intracellular tyrosinase activity. In addition, AP was nontoxic to human keratinocytes (HaCaT) at these concentrations, validating its safety for topical use. Taken together, our preliminary results demonstrate that AP might be repurposed as a candidate therapeutic for treatment of hyperpigmentation disorders via a unique mechanism, which encompasses a selective inhibition of melanosome export.


Assuntos
Estrenos/farmacologia , Melanossomas/efeitos dos fármacos , Melanossomas/metabolismo , Oximas/farmacologia , Receptores de Progesterona/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Melanoma Experimental/patologia , Camundongos
16.
Cells ; 9(8)2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32751549

RESUMO

In addition to antigen presentation to CD4+ T cells, aggregation of cell surface major histocompatibility complex class II (MHC-II) molecules induces signal transduction in antigen presenting cells that regulate cellular functions. We previously reported that crosslinking of MHC-II induced the endocytosis of MHC-II, which was associated with decreased surface expression levels in murine dendritic cells (DCs) and resulted in impaired activation of CD4+ T cells. However, the downstream signal that induces MHC-II endocytosis remains to be elucidated. In this study, we found that the crosslinking of MHC-II induced intracellular Ca2+ mobilization, which was necessary for crosslinking-induced MHC-II endocytosis. We also found that these events were suppressed by inhibitors of Syk and phospholipase C (PLC). Treatments with a phorbol ester promoted MHC-II endocytosis, whereas inhibitors of protein kinase C (PKC) suppressed crosslinking-induced endocytosis of MHC-II. These results suggest that PKC could be involved in this process. Furthermore, crosslinking-induced MHC-II endocytosis was suppressed by inhibitors of clathrin-dependent endocytosis. Our results indicate that the crosslinking of MHC-II could stimulate Ca2+ mobilization and induce the clathrin-dependent endocytosis of MHC-II in murine DCs.


Assuntos
Clatrina/metabolismo , Células Dendríticas/imunologia , Endocitose/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/metabolismo , Proteína Quinase C/metabolismo , Animais , Apresentação do Antígeno/imunologia , Células da Medula Óssea/citologia , Cálcio/metabolismo , Células Cultivadas , Clatrina/antagonistas & inibidores , Reagentes para Ligações Cruzadas/metabolismo , Endocitose/imunologia , Estrenos/farmacologia , Masculino , Camundongos , Ésteres de Forbol/farmacologia , Proteína Quinase C/antagonistas & inibidores , Pirrolidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Organismos Livres de Patógenos Específicos , Estaurosporina/farmacologia , Estilbenos/farmacologia , Quinase Syk/antagonistas & inibidores , Quinase Syk/metabolismo , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo
17.
PLoS One ; 15(8): e0238155, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841278

RESUMO

Non-small cell lung cancer (NSCLC), one of the leading causes of cancer-related death, has a low 5-year survival rate owing to the inevitable acquired resistance toward antitumor drugs, platinum-based chemotherapy, and targeted therapy. Epidermal growth factor (EGF)-EGF receptor (EGFR) signaling activates downstream events leading to phospholipase C/inositol trisphosphate (IP3)/Ca2+ release from IP3-sensitive Ca2+ stores to modulate cell proliferation, motility, and invasion. However, the role of EGFR-mediated Ca2+ signaling in acquired drug resistance is not fully understood. Here, we analyzed alterations of intracellular Ca2+ ([Ca2+]i) responses between gefitinib-sensitive NSCLC PC-9 cells and gefitinib-resistant NSCLC PC-9/GR cells, and we found that acute EGF treatment elicited intracellular Ca2+ ([Ca2+]i) oscillations in PC-9 cells but not in PC-9/GR cells. PC-9/GR cells presented a more sustained basal [Ca2+]i level, lower endoplasmic reticulum Ca2+ level, and higher spontaneous extracellular Ca2+ ([Ca2+]e) influx than PC-9 cells. Notably, restricting [Ca2+]e in both cell types induced identical [Ca2+]i oscillations, dependent on phospholipase C and EGFR activation. Consequently, restricting [Ca2+]e in PC-9/GR cells upregulated gefitinib-mediated poly (ADP-ribose) polymerase cleavage, an increase in Bax/Bcl-2 ratio, cytotoxicity, and apoptosis. In addition, nuclear factor of activated T cell (NFAT1) induction in response to EGF was inhibited by gefitinib in PC-9 cells, whereas EGF-mediated NFAT1 induction in PC-9/GR cells was sustained regardless of gefitinib treatment. Restricting [Ca2+]e in PC-9/GR cells significantly reduced EGF-mediated NFAT1 induction. These findings indicate that spontaneous [Ca2+]e influx in NSCLC cells plays a pivotal role in developing acquired drug resistance and suggest that restricting [Ca2+]e may be a potential strategy for modulating drug-sensitivity.


Assuntos
Antineoplásicos/farmacologia , Sinalização do Cálcio , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Gefitinibe/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Estrenos/farmacologia , Humanos , Fatores de Transcrição NFATC/biossíntese , Pirrolidinonas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores
18.
Am J Physiol Cell Physiol ; 318(5): C954-C968, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32186932

RESUMO

The increase in cytosolic Ca2+ concentration ([Ca2+]cyt) and upregulation of calcium-sensing receptor (CaSR) and stromal interaction molecule 2 (STIM2) along with inhibition of voltage-gated K+ (KV) channels in pulmonary arterial smooth muscle cells (PASMC) have been implicated in the development of pulmonary arterial hypertension; however, the precise upstream mechanisms remain elusive. Activation of CaSR, a G protein-coupled receptor (GPCR), results in Ca2+ release from the endoplasmic/sarcoplasmic reticulum (ER/SR) and Ca2+ influx through receptor-operated and store-operated Ca2+ channels (SOC). Upon Ca2+ depletion from the SR, STIM forms clusters to mediate store-operated Ca2+ entry. Activity of KV channels, like KCNA5/KV1.5 and KCNA2/KV1.2, contributes to regulating membrane potential, and inhibition of KV channels results in membrane depolarization that increases [Ca2+]cyt by opening voltage-dependent Ca2+ channels. In this study, we show that activation of Notch by its ligand Jag-1 promotes the clustering of STIM2, and clustered STIM2 subsequently enhances the CaSR-induced Ca2+ influx through SOC channels. Extracellular Ca2+-mediated activation of CaSR increases [Ca2+]cyt in CASR-transfected HEK293 cells. Treatment of CASR-transfected cells with Jag-1 further enhances CaSR-mediated increase in [Ca2+]cyt. Moreover, CaSR-mediated increase in [Ca2+]cyt was significantly augmented in cells co-transfected with CASR and STIM2. CaSR activation results in STIM2 clustering in CASR/STIM2-cotransfected cells. Notch activation also induces significant clustering of STIM2. Furthermore, activation of Notch attenuates whole cell K+ currents in KCNA5- and KCNA2-transfected cells. Together, these results suggest that Notch activation enhances CaSR-mediated increases in [Ca2+]cyt by enhancing store-operated Ca2+ entry and inhibits KCNA5/KV1.5 and KCNA2/KV1.2, ultimately leading to voltage-activated Ca2+ entry.


Assuntos
Canal de Potássio Kv1.2/genética , Canal de Potássio Kv1.5/genética , Hipertensão Arterial Pulmonar/genética , Receptores de Detecção de Cálcio/genética , Molécula 2 de Interação Estromal/genética , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/genética , Sinalização do Cálcio/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Estrenos/farmacologia , Células HEK293 , Humanos , Indóis/farmacologia , Proteína Jagged-1/genética , Potenciais da Membrana/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Pirrolidinonas/farmacologia , Receptores de Detecção de Cálcio/efeitos dos fármacos , Receptores Notch/genética , Análise de Célula Única
19.
Eur J Pharmacol ; 875: 173036, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32101765

RESUMO

Ellagic acid, a naturally occurring phenol found in a variety of fruits and nuts has been shown to possess anti-inflammatory properties. However, the mechanism of action behind its anti-inflammatory action is unclear. Using human Jurkat T cells, our study examined the effects of ellagic acid (EA) on Ca2+ handling, in particular, store-operated Ca2+ entry (SOCE), a process critical to proper T cell function. We observed that the acute addition of EA-induced Ca2+ release with an EC50 of 63 µM. The Ca2+ release was significantly attenuated by Xestospongin C, a known inhibitor of the Inositol 1,4,5-trisphosphate receptor (IP3R) channel and was unaffected by the phospholipase C (PLC) inhibitor, U73122. Furthermore, chronic incubation of Jurkat T cells with EA not only decreased the ATP-induced Ca2+ release but also diminished the SOCE-mediated Ca2+ influx in a dose-dependent manner. This inhibition was confirmed by reduced Mn2+ entry rates in the EA-treated cells. The ATP-induced Ca2+ entry was also attenuated in EA-treated HEK293 cells transiently transfected with SOCE channel Orai1-myc and ER-sensor stromal interaction molecule (STIM1) (HEKSTIM/Orai). Moreover, EA treatment interfered with the Orai1 and STIM1 coupling by disrupting STIM1 puncta formation in the HEKSTIM/Orai cells. We observed that EA treatment reduced cytokine secretion and nuclear factor of activated T-cell transcriptional activity in stimulated T cells. Hence, by inhibiting SOCE mediated Ca2+ influx, EA decreased downstream activation of pro-inflammatory mediators. These results suggest a novel target for EA-mediated effects and provide insight into the mechanisms underlying EA-mediated anti-inflammatory effects.


Assuntos
Anti-Inflamatórios/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Ácido Elágico/farmacologia , Sinalização do Cálcio/imunologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estrenos/farmacologia , Células HEK293 , Humanos , Mediadores da Inflamação/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Células Jurkat , Compostos Macrocíclicos/farmacologia , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Oxazóis/farmacologia , Pirrolidinonas/farmacologia , Molécula 1 de Interação Estromal/metabolismo
20.
Physiol Rep ; 8(1): e14337, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31960592

RESUMO

Although absorption of di- and tripeptides into intestinal epithelial cells occurs via the peptide transporter 1 (PEPT1, also called solute carrier family 15 member 1 (SLC15A1)), the detailed regulatory mechanisms are not fully understood. We examined: (a) whether dipeptide absorption in villous enterocytes is associated with a rise in cytosolic Ca2+ ([Ca2+ ]cyt ), (b) whether the calcium sensing receptor (CaSR) is involved in dipeptide-elicited [Ca2+ ]cyt signaling, and (c) what potential consequences of [Ca2+ ]cyt signaling may enhance enterocyte dipeptide absorption. Dipeptide Gly-Sar and CaSR agonist spermine markedly raised [Ca2+ ]cyt in villous enterocytes, which was abolished by NPS-2143, a selective CaSR antagonist and U73122, an phospholipase C (PLC) inhibitor. Apical application of Gly-Sar induced a jejunal short-circuit current (Isc), which was reduced by NPS-2143. CaSR expression was identified in the lamina propria and on the basal enterocyte membrane of mouse jejunal mucosa in both WT and Slc15a1-/- animals, but Gly-Sar-induced [Ca2+ ]cyt signaling was significantly decreased in Slc15a1-/- villi. Clotrimazole and TRM-34, two selective blockers of the intermediate conductance Ca2+ -activated K+ channel (IKCa ), but not iberiotoxin, a selective blocker of the large-conductance K+ channel (BKCa ) and apamin, a selective blocker of the small-conductance K+ channel (SKCa ), significantly inhibited Gly-Sar-induced Isc in native tissues. We reveal a novel CaSR-PLC-Ca2+ -IKCa pathway in the regulation of small intestinal dipeptide absorption, which may be exploited as a target for future drug development in human nutritional disorders.


Assuntos
Sinalização do Cálcio/fisiologia , Dipeptídeos/metabolismo , Enterócitos/metabolismo , Absorção Intestinal/fisiologia , Jejuno/metabolismo , Transportador 1 de Peptídeos/genética , Canais de Potássio Cálcio-Ativados/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Animais , Sinalização do Cálcio/genética , Clotrimazol/farmacologia , Dipeptídeos/farmacologia , Enterócitos/efeitos dos fármacos , Estrenos/farmacologia , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Jejuno/efeitos dos fármacos , Camundongos , Camundongos Knockout , Membrana Mucosa/metabolismo , Naftalenos/farmacologia , Transportador 1 de Peptídeos/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Pirrolidinonas/farmacologia , Receptores de Detecção de Cálcio/agonistas , Receptores de Detecção de Cálcio/antagonistas & inibidores , Espermina/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...