RESUMO
Recently, it has been suggested that progesterone affects the contractile activity of pregnant myometrium via nongenomic pathways; therefore, we aimed to clarify whether progesterone causes and/or inhibits pregnant myometrial contractions via nongenomic pathways. Our in vitro experiments using myometrial strips obtained from rats at 20 days of gestation revealed that progesterone caused myometrial contractions in a concentration- and time-dependent manner at concentrations up to 5 × 10-7 M; however, this effect decreased at concentrations higher than 5 × 10-5 M. Similarly, progesterone enhanced oxytocin-induced contractions up to 5 × 10-7 M and inhibited contractions at concentrations higher than 5 × 10-5 M. Conversely, progesterone did not enhance high-KCl-induced contractions but inhibited contractions in a concentration- and time-dependent manner at concentrations higher than 5 × 10-7 M. We also found that RU486 did not affect progesterone-induced contractions or the progesterone-induced inhibition of high-KCl-induced contractions; however, progesterone-induced contractions were blocked by calcium-free phosphate saline solution, verapamil, and nifedipine. In addition, FPL64176, an activator of L-type voltage-dependent calcium channels, enhanced high-KCl-induced contractions and rescued the decrease in high-KCl-induced contractions caused by progesterone. Together, these results suggest that progesterone exerts conflicting nongenomic effects on the contractions of pregnant myometrium via putative L-type voltage-dependent calcium channels.
Assuntos
Miométrio/fisiologia , Progesterona/fisiologia , Contração Uterina/fisiologia , Animais , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Feminino , Antagonistas de Hormônios/farmacologia , Mifepristona/farmacologia , Miométrio/efeitos dos fármacos , Nifedipino/farmacologia , Técnicas de Cultura de Órgãos , Ocitocina/farmacologia , Cloreto de Potássio/farmacologia , Gravidez , Progesterona/farmacologia , Pirróis/farmacologia , Ratos Wistar , Contração Uterina/efeitos dos fármacos , Verapamil/farmacologiaRESUMO
The role of calcium, but not of other intracellular signaling molecules, in the release of pituitary hormones by exocytosis is well established. Here, we analyzed the contribution of phosphatidylinositol kinases (PIKs) to calcium-driven prolactin (PRL) release in pituitary lactotrophs: PI4Ks - which control PI4P production, PIP5Ks - which synthesize PI(4, 5)P2 by phosphorylating the D-5 position of the inositol ring of PI4P, and PI3KCs - which phosphorylate PI(4, 5)P2 to generate PI(3, 4, 5)P3. We used common and PIK-specific inhibitors to evaluate the strength of calcium-secretion coupling in rat lactotrophs. Gene expression was analyzed by single-cell RNA sequencing and qRT-PCR analysis; intracellular and released hormones were assessed by radioimmunoassay and ELISA; and single-cell calcium signaling was recorded by Fura 2 imaging. Single-cell RNA sequencing revealed the expression of Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c, in lactotrophs. Wortmannin, a PI3K and PI4K inhibitor, but not LY294002, a PI3K inhibitor, blocked spontaneous action potential driven PRL release with a half-time of ~20 min when applied in 10 µM concentration, leading to accumulation of intracellular PRL content. Wortmannin also inhibited increase in PRL release by high potassium, the calcium channel agonist Bay K8644, and calcium mobilizing thyrotropin-releasing hormone without affecting accompanying calcium signaling. GSK-A1, a specific inhibitor of PI4KA, also inhibited calcium-driven PRL secretion without affecting calcium signaling and Prl expression. In contrast, PIK93, a specific inhibitor of PI4KB, and ISA2011B and UNC3230, specific inhibitors of PIP5K1A and PIP5K1C, respectively, did not affect PRL release. These experiments revealed a key role of PI4KA in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4, 5)P2-dependent calcium signaling.
Assuntos
Cálcio/metabolismo , Lactotrofos/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Prolactina/metabolismo , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Agonistas dos Canais de Cálcio/farmacologia , Sinalização do Cálcio , Exocitose , Lactotrofos/efeitos dos fármacos , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Prolactina/biossíntese , Prolactina/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ratos , Análise de Sequência de RNA , Análise de Célula Única , Wortmanina/farmacologiaRESUMO
The present study explored the modulating apoptosis effect of hydrogen sulfide (H2S) in subarachnoid hemorrhage (SAH) rats and its exact mechanism. A rat SAH model established by intravascular puncturing was used for the present study. After giving NaHS (donor of H2S), an L-type calcium channel opener (Bay K8644), or a calcium channel agonist (nifedipine), the neurological function of the rats, associated pathological changes, and expression of apoptosis-related proteins (Bcl-2, Bax, and caspase-3) and microtubule-associated protein (MAP-2) were examined. The concentration of H2S and expression of cystathionine beta synthase in the hippocampus changed upon early brain injury (EBI) after SAH. Compared with the SAH group, the neurological function of the rats and microstructure observed by electron microscopy were better in the SAH + NaHS group and SAH + Bay K8644 group. It was observed that apoptosis was more obvious in the SAH group than in the control group and was alleviated in the SAH + NaHS group. Furthermore, the alleviating effect of NaHS was partially weakened by nifedipine, indicating that the effect of anti-apoptosis in H2S might be correlated with the calcium channel. The expression of Bax and caspase-3 was elevated, while the expression of Bcl-2 decreased in the SAH group but improved in the SAH + NaHS and SAH + Bay K8644 group. Compared with the SAH + NaHS group, the expression of pro-apoptotic proteins was higher in the SAH + NaHS + nifedipine group. Therefore, upon EBI following SAH, the H2S system plays an important neurological protective effect by modulating the function of the L-type calcium channel and inhibiting apoptosis.
Assuntos
Lesões Encefálicas/metabolismo , Encéfalo/metabolismo , Canais de Cálcio Tipo L/metabolismo , Sulfeto de Hidrogênio/metabolismo , Neuroproteção/fisiologia , Hemorragia Subaracnóidea/metabolismo , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Lesões Encefálicas/prevenção & controle , Agonistas dos Canais de Cálcio/farmacologia , Masculino , Neuroproteção/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Hemorragia Subaracnóidea/prevenção & controle , Sulfitos/farmacologiaRESUMO
Neuronal control of the energy homeostasis requires the arcuate nucleus of the hypothalamus. This structure integrates peripheral and central signals concerning the energy state of the body. It comprises two populations of neurons releasing anorexigenic and orexigenic peptides, among others. Both populations are regulated by leptin, an anorexigenic hormone, released by white adipose tissue. Voltage-gated calcium entry is critical to promote neurotransmitter and hormone release. It is already known that calcium channel current is inhibited by leptin in orexigenic neurons. However, fine-tuning details of calcium channel regulation in arcuate nucleus by leptin remain to be elucidated. This work aimed to investigate whether 5' adenosine monophosphate-activated protein kinase (AMPK) underlies the leptin-induced inhibition of calcium channels. By using patch-clamping methods, immunocytochemical, and biochemical reagents, we recorded calcium channel currents in orexigenic neuropeptide Y neurons of the arcuate nucleus of rats. Consistently, leptin inhibition of the calcium channel current was not only prevented by AMPK inhibition with Compound C but also hampered with 5-aminoimidazole-4-carboxamide ribonucleoside. Furthermore, leptin selectively inhibited L-type calcium channel current amplitude without major changes in voltage dependence or current kinetics. These results support for the first time the key role of AMPK in the maintenance and regulation of voltage-gated calcium channels. Together, they advance our understanding of the regulation of calcium channels in the central nervous system and emerging questions concerning food intake and energy balance.NEW & NOTEWORTHY Our results readily support the hypothesis that AMPK is responsible for the maintenance of the calcium current and mediates the fine-tuning modulation of the leptin response. The novelty of these results strengthens the critical role of AMPK in the general energy balance and homeostasis.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Núcleo Arqueado do Hipotálamo/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Leptina/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Aminoimidazol Carboxamida/farmacologia , Animais , Núcleo Arqueado do Hipotálamo/citologia , Células Cultivadas , Metabolismo Energético/efeitos dos fármacos , Cinética , Masculino , Neuropeptídeo Y/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos WistarRESUMO
(1) Background: Vitamin D compounds (VDC) are extensively studied in the field of anticancer properties, including breast cancer. Previously, we showed that calcitriol and its analogs (PRI-2191 and PRI-2205) stimulate metastasis in 4T1 murine mammary gland cancer models in young mice, whereas the reverse effect was observed in aged ovariectomized (OVX) mice; (2) Methods: We determined the phenotype of monocytes/macrophages using FACS and examined the expression of selected genes and proteins by Real-Time PCR and ELISA; (3) Results: Activities of VDC are accompanied by an increase in the percentage of Ly6Clow anti-inflammatory monocytes in the spleen of young and a decrease in aged OVX mice. Treatment of young mice with VDC resulted in an increase of CCL2 plasma and tumor concentration and Arg1 in tumor. In later stage of tumor progression the expression of genes related to metastasis in lung tissue was decreased or increased, in old OVX or young mice, respectively; (4) Conclusions: Pro- or anti-metastatic effects of calcitriol and its analogs in young or aged OVX mice, respectively, can be attributed to the differences in the effects of VDC on the tumor microenvironment, as a consequence of differences in the immunity status of young and aged mice.
Assuntos
Calcitriol/química , Calcitriol/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacos , Animais , Apoptose , Agonistas dos Canais de Cálcio/química , Agonistas dos Canais de Cálcio/farmacologia , Movimento Celular , Proliferação de Células , Feminino , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Ovariectomia , Células Tumorais CultivadasRESUMO
Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand.
Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Macrófagos/metabolismo , Cloridrato de Raloxifeno/farmacologia , Animais , Benzilisoquinolinas/farmacologia , Cálcio/metabolismo , Agonistas dos Canais de Cálcio/química , Canais de Cálcio/genética , Flufenazina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Ionomicina/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , NADP/análogos & derivados , NADP/metabolismo , Fosfatos de Fosfatidilinositol/farmacologia , Imagem Individual de Molécula , Sódio/metabolismoRESUMO
BACKGROUND: Dystrophin deficiency sensitizes skeletal muscle of mice to eccentric contraction (ECC)-induced strength loss. ECC protocols distinguish dystrophin-deficient from healthy, wild type muscle, and test the efficacy of therapeutics for Duchenne muscular dystrophy (DMD). However, given the large lab-to-lab variability in ECC-induced strength loss of dystrophin-deficient mouse skeletal muscle (10-95%), mechanical factors of the contraction likely impact the degree of loss. Therefore, the purpose of this study was to evaluate the extent to which mechanical variables impact sensitivity of dystrophin-deficient mouse skeletal muscle to ECC. METHODS: We completed ex vivo and in vivo muscle preparations of the dystrophin-deficient mdx mouse and designed ECC protocols within physiological ranges of contractile parameters (length change, velocity, contraction duration, and stimulation frequencies). To determine whether these contractile parameters affected known factors associated with ECC-induced strength loss, we measured sarcolemmal damage after ECC as well as strength loss in the presence of the antioxidant N-acetylcysteine (NAC) and small molecule calcium modulators that increase SERCA activity (DS-11966966 and CDN1163) or lower calcium leak from the ryanodine receptor (Chloroxine and Myricetin). RESULTS: The magnitude of length change, work, and stimulation duration ex vivo and in vivo of an ECC were the most important determinants of strength loss in mdx muscle. Passive lengthening and submaximal stimulations did not induce strength loss. We further showed that sarcolemmal permeability was associated with muscle length change, but it only accounted for a minimal fraction (21%) of the total strength loss (70%). The magnitude of length change also significantly influenced the degree to which NAC and small molecule calcium modulators protected against ECC-induced strength loss. CONCLUSIONS: These results indicate that ECC-induced strength loss of mdx skeletal muscle is dependent on the mechanical properties of the contraction and that mdx muscle is insensitive to ECC at submaximal stimulation frequencies. Rigorous design of ECC protocols is critical for effective use of strength loss as a readout in evaluating potential therapeutics for muscular dystrophy.
Assuntos
Contração Muscular , Força Muscular , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Acetilcisteína/farmacologia , Aminoquinolinas/farmacologia , Animais , Antioxidantes/farmacologia , Benzamidas/farmacologia , Cálcio/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Cloroquinolinóis/farmacologia , Flavonoides/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Estresse MecânicoRESUMO
Remifentanil is commonly used clinically for perioperative pain relief, but it may induce postoperative hyperalgesia. Low doses of ketamine have remained a common choice in clinical practice, but the mechanisms of ketamine have not yet been fully elucidated. In this study, we examined the possible effects of ketamine on calcium/calmodulin-dependent protein kinase II α (CaMKIIα) and N-methyl-d-aspartate receptor (NMDAR) subunit NR2B in a mouse model of remifentanil-induced postoperative hyperalgesia (RIPH) in the primary somatosensory cerebral cortex (SI) region. The paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were used to assess mechanical allodynia and thermal hyperalgesia, respectively, before and after intraoperative remifentanil administration. Before surgery, mice received intrathecal injections of the following drugs: ketamine, NMDA, BayK8644 (CaMKII activator), and KN93 (CaMKII inhibitor). Immunofluorescence was performed to determine the anatomical location and expression of activated CaMKIIα, phosphorylated CaMKIIα (p-CaMKIIα). Additionally, western blotting was performed to assess p-CaMKIIα and NMDAR expression levels in the SI region. Remifentanil decreased the PWMT and PWTL at 0.5â¯h, 2â¯h, and 5â¯h and increased p-CaMKIIα expression in the SI region. Ketamine increased the PWMT and PWTL and reversed the p-CaMKIIα upregulation. Both BayK8644 and NMDA reversed the effect of ketamine, decreased the PWMT and PWTL, and upregulated p-CaMKIIα expression. In contrast, KN93 enhanced the effect of ketamine by reducing hyperalgesia and downregulating p-CaMKIIα expression. These results suggested that ketamine reversed RIPH by inhibiting the phosphorylation of CaMKIIα and the NMDA receptor in the SI region in mice.
Assuntos
Analgésicos/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/efeitos dos fármacos , Hiperalgesia/induzido quimicamente , Ketamina/farmacologia , Complicações Pós-Operatórias/induzido quimicamente , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Remifentanil/efeitos adversos , Córtex Somatossensorial/efeitos dos fármacos , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Analgésicos Opioides/efeitos adversos , Animais , Benzilaminas/farmacologia , Western Blotting , Agonistas dos Canais de Cálcio/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Imunofluorescência , Hiperalgesia/fisiopatologia , Camundongos , Limiar da Dor/efeitos dos fármacos , Dor Pós-Operatória/tratamento farmacológico , Complicações Pós-Operatórias/fisiopatologia , Inibidores de Proteínas Quinases/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Córtex Somatossensorial/metabolismo , Sulfonamidas/farmacologiaRESUMO
Intestine is a major target of vitamin D and several studies indicate an association between vitamin D deficiency and inflammatory bowel diseases (IBD), but also increased colorectal cancer (CRC) risk and mortality. However, the putative effects of 1α,25-dihydroxyvitamin D3 (calcitriol), the active vitamin D metabolite, on human colonic stem cells are unknown. Here we show by immunohistochemistry and RNAscope in situ hybridization that vitamin D receptor (VDR) is unexpectedly expressed in LGR5+ colon stem cells in human tissue and in normal and tumor organoid cultures generated from patient biopsies. Interestingly, normal and tumor organoids respond differentially to calcitriol with profound and contrasting changes in their transcriptomic profiles. In normal organoids, calcitriol upregulates stemness-related genes, such as LGR5, SMOC2, LRIG1, MSI1, PTK7, and MEX3A, and inhibits cell proliferation. In contrast, in tumor organoids calcitriol has little effect on stemness-related genes while it induces a differentiated phenotype, and variably reduces cell proliferation. Concordantly, electron microscopy showed that calcitriol does not affect the blastic undifferentiated cell phenotype in normal organoids but it induces a series of differentiated features in tumor organoids. Our results constitute the first demonstration of a regulatory role of vitamin D on human colon stem cells, indicating a homeostatic effect on colon epithelium with relevant implications in IBD and CRC.
Assuntos
Calcitriol/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Colo/citologia , Neoplasias do Colo/patologia , Organoides/citologia , Receptores de Calcitriol/metabolismo , Células-Tronco/citologia , Apoptose , Proliferação de Células , Células Cultivadas , Colo/efeitos dos fármacos , Colo/metabolismo , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Humanos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Receptores de Calcitriol/deficiência , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
Mammalian two-pore-channels (TPC1, 2; TPCN1, TPCN2) are ubiquitously- expressed, PI(3,5)P2-activated, Na+-selective channels in the endosomes and lysosomes that regulate luminal pH homeostasis, membrane trafficking, and Ebola viral infection. Whereas the channel activity of TPC1 is strongly dependent on membrane voltage, TPC2 lacks such voltage dependence despite the presence of the presumed 'S4 voltage-sensing' domains. By performing high-throughput screening followed by lysosomal electrophysiology, here we identified a class of tricyclic anti-depressants (TCAs) as small-molecule agonists of TPC channels. TCAs activate both TPC1 and TPC2 in a voltage-dependent manner, referred to as Lysosomal Na+ channel Voltage-dependent Activators (LyNa-VAs). We also identified another compound which, like PI(3,5)P2, activates TPC2 independent of voltage, suggesting the existence of agonist-specific gating mechanisms. Our identification of small-molecule TPC agonists should facilitate the studies of the cell biological roles of TPCs and can also readily explain the reported effects of TCAs in the modulation of autophagy and lysosomal functions.
Assuntos
Antidepressivos Tricíclicos/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Lisossomos/enzimologia , Animais , Canais de Cálcio/genética , Linhagem Celular , Análise Mutacional de DNA , Humanos , Mutagênese Sítio-Dirigida , Fosfatos de Fosfatidilinositol/farmacologiaRESUMO
The type 2 ryanodine receptor (RyR2) is responsible for releasing Ca2+ from the sarcoplasmic reticulum of cardiomyocytes, subsequently leading to muscle contraction. Here, we report 4 cryo-electron microscopy (cryo-EM) structures of porcine RyR2 bound to distinct modulators that, together with our published structures, provide mechanistic insight into RyR2 regulation. Ca2+ alone induces a contraction of the central domain that facilitates the dilation of the S6 bundle but is insufficient to open the pore. The small-molecule agonist PCB95 helps Ca2+ to overcome the barrier for opening. FKBP12.6 induces a relaxation of the central domain that decouples it from the S6 bundle, stabilizing RyR2 in a closed state even in the presence of Ca2+ and PCB95. Although the channel is open when PCB95 is replaced by caffeine and adenosine 5'-triphosphate (ATP), neither of the modulators alone can sufficiently counter the antagonistic effect to open the channel. Our study marks an important step toward mechanistic understanding of the sophisticated regulation of this key channel whose aberrant activity engenders life-threatening cardiac disorders.
Assuntos
Regulação Alostérica/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Cálcio/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Microscopia Crioeletrônica , Modelos Moleculares , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , SuínosRESUMO
Cryoelectron microscopy and mutational analyses have shown that type 1 ryanodine receptor (RyR1) amino acid residues RyR1-E3893, -E3967, and -T5001 are critical for Ca2+-mediated activation of skeletal muscle Ca2+ release channel. De novo missense mutation RyR1-Q3970K in the secondary binding sphere of Ca2+ was reported in association with central core disease (CCD) in a 2-yr-old boy. Here, we characterized recombinant RyR1-Q3970K mutant by cellular Ca2+ release measurements, single-channel recordings, and computational methods. Caffeine-induced Ca2+ release studies indicated that RyR1-Q3970K formed caffeine-sensitive, Ca2+-conducting channel in HEK293 cells. However, in single-channel recordings, RyR1-Q3970K displayed low Ca2+-dependent channel activity and greatly reduced activation by caffeine or ATP. A RyR1-Q3970E mutant corresponds to missense mutation RyR2-Q3925E associated with arrhythmogenic syndrome in cardiac muscle. RyR1-Q3970E also formed caffeine-induced Ca2+ release in HEK293 cells and exhibited low activity in the presence of the activating ligand Ca2+ but, in contrast to RyR1-Q3970K, was activated by ATP and caffeine in single-channel recordings. Computational analyses suggested distinct structural rearrangements in the secondary binding sphere of Ca2+ of the two mutants, whereas the interaction of Ca2+ with directly interacting RyR1 amino acid residues Glu3893, Glu3967, and Thr5001 was only minimally affected. We conclude that RyR1-Q3970 has a critical role in Ca2+-dependent activation of RyR1 and that a missense RyR1-Q3970K mutant may give rise to myopathy in skeletal muscle.
Assuntos
Cálcio/metabolismo , Ativação do Canal Iônico , Músculo Esquelético/metabolismo , Mutação de Sentido Incorreto , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Sítios de Ligação , Cafeína/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Células HEK293 , Humanos , Potenciais da Membrana , Músculo Esquelético/efeitos dos fármacos , Ligação Proteica , Conformação Proteica , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
The accumulation of misfolded proteins is a common pathological feature of many neurodegenerative disorders, including synucleinopathies such as Parkinson's disease (PD), which is characterized by the presence of α-synuclein (α-syn)-containing Lewy bodies. However, although recent studies have investigated α-syn accumulation and propagation in neurons, the molecular mechanisms underlying α-syn transmission have been largely unexplored. Here, we examined a monogenic form of synucleinopathy caused by loss-of-function mutations in lysosomal ATP13A2/PARK9. These studies revealed that lysosomal exocytosis regulates intracellular levels of α-syn in human neurons. Loss of PARK9 function in patient-derived dopaminergic neurons disrupted lysosomal Ca2+ homeostasis, reduced lysosomal Ca2+ storage, increased cytosolic Ca2+, and impaired lysosomal exocytosis. Importantly, this dysfunction in lysosomal exocytosis impaired α-syn secretion from both axons and soma, promoting α-syn accumulation. However, activation of the lysosomal Ca2+ channel transient receptor potential mucolipin 1 (TRPML1) was sufficient to upregulate lysosomal exocytosis, rescue defective α-syn secretion, and prevent α-syn accumulation. Together, these results suggest that intracellular α-syn levels are regulated by lysosomal exocytosis in human dopaminergic neurons and may represent a potential therapeutic target for PD and other synucleinopathies.SIGNIFICANCE STATEMENT Parkinson's disease (PD) is the second most common neurodegenerative disease linked to the accumulation of α-synuclein (α-syn) in patient neurons. However, it is unclear what the mechanism might be. Here, we demonstrate a novel role for lysosomal exocytosis in clearing intracellular α-syn and show that impairment of this pathway by mutations in the PD-linked gene ATP13A2/PARK9 contributes to α-syn accumulation in human dopaminergic neurons. Importantly, upregulating lysosomal exocytosis by increasing lysosomal Ca2+ levels was sufficient to rescue defective α-syn secretion and accumulation in patient neurons. These studies identify lysosomal exocytosis as a potential therapeutic target in diseases characterized by the accumulation of α-syn, including PD.
Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Neurônios Dopaminérgicos/metabolismo , Exocitose/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Lisossomos/metabolismo , alfa-Sinucleína/toxicidade , Linhagem Celular Tumoral , Células Cultivadas , Neurônios Dopaminérgicos/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/genética , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismoRESUMO
Store-operated channels activated in response to intracellular calcium store depletion represent the main pathway of calcium entry from the extracellular space in nonelectroexcitable cells. Adapter proteins organize the components of this system into integral complex. We studied the influence of adapter proteins of the Homer family on endogenous store-operated calcium Imin channels in A431 cells. Monomeric Homer 1a proteins increase activity of Imin channels, but did not modulate their electrophysiological properties. Recombinant Homer 1c protein did not block the induced calcium currents.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Arcabouço Homer/fisiologia , Potenciais de Ação/efeitos dos fármacos , Agonistas dos Canais de Cálcio/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Citoplasma/metabolismo , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Proteínas de Arcabouço Homer/farmacologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Técnicas de Patch-Clamp , Multimerização Proteica/fisiologia , Proteínas Recombinantes/farmacologia , Células Tumorais CultivadasRESUMO
BACKGROUND AND AIMS: Vitamin D signaling is involved in infectious and non-infectious liver diseases, yet the natural vitamin D metabolites are suboptimal therapeutic agents. In the present study, we therefore aimed to explore the potential and mechanism of selected calcitriol analogs to regulate the hepatocellular transcriptome and to inhibit hepatitis C virus (HCV) in comparison with calcitriol. METHODS: Human hepatoma cell lines and primary human macrophages were stimulated with calcitriol and selected calcitriol analogs. The effect of calcitriol and its derivatives on hepatocellular gene expression and vitamin D receptor (VDR) signaling as well as on replication of HCV were assessed by quantitative PCR, microarray analyses and in silico analyses of ligand-VDR complexes. RESULTS: The structurally related vitamin D analogs calcipotriol and tacalcitiol, but not calcitriol itself, suppressed HCV replication in a VDR-dependent manner. Using a residue-interaction network approach we outline structural and functional differences between VDR-ligand complexes. In particular we find characteristics in the VDR structure bound to calcipotriol with distinct local residue interaction patterns that affect key functional residues that pertain to the VDR charge clamp, H397 and F422, a VDR regulatory element for interaction with co-activators and -repressors. As a consequence, we show calcipotriol in comparison to calcitriol to induce stronger regulatory actions on the transcriptome of hepatocytes and macrophages including key antimicrobial peptides. CONCLUSION: Calcipotriol induces local structure rearrangements in VDR that could possibly translate into a superior clinical potential to execute important non-classical vitamin D effects such as inhibition of HCV replication.
Assuntos
Calcitriol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Imunidade Inata/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Replicação Viral/efeitos dos fármacos , Calcitriol/análogos & derivados , Agonistas dos Canais de Cálcio/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/virologia , Fármacos Dermatológicos/farmacologia , Hepacivirus/imunologia , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/virologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/virologia , Receptores de Calcitriol/metabolismo , Transdução de Sinais , TranscriptomaRESUMO
Cutaneous melanoma is the most serious type of skin cancer, so new cytotoxic weapons against novel targets in melanoma are of great interest. Euplotin C (EC), a cytotoxic secondary metabolite of the marine ciliate Euplotes crassus, was evaluated in the present study on human cutaneous melanoma cells to explore its anti-melanoma activity and to gain more insight into its mechanism of action. EC exerted a marked cytotoxic effect against three different human melanoma cell lines (A375, 501Mel and MeWo) with a potency about 30-fold higher than that observed in non-cancer cells (HDFa cells). A pro-apoptotic activity and a decrease in melanoma cell migration by EC were also observed. At the molecular level, the inhibition of the Erk and Akt pathways, which control many aspects of melanoma aggressiveness, was shown. EC cytotoxicity was antagonized by dantrolene, a ryanodine receptor (RyR) antagonist, in a concentration-dependent manner. A role of RyR as a direct target of EC was also suggested by molecular modelling studies. In conclusion, our data provide the first evidence of the anti-melanoma activity of EC, suggesting it may be a promising new scaffold for the development of selective activators of RyR to be used for the treatment of melanoma and other cancer types.
Assuntos
Organismos Aquáticos/metabolismo , Euplotes/metabolismo , Melanoma/tratamento farmacológico , Sesquiterpenos/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Agonistas dos Canais de Cálcio/isolamento & purificação , Agonistas dos Canais de Cálcio/farmacologia , Agonistas dos Canais de Cálcio/uso terapêutico , Linhagem Celular Tumoral , Dantroleno/farmacologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Oncogênica v-akt/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/uso terapêuticoRESUMO
Methylglyoxal, a highly reactive dicarbonyl compound, is formed as a by-product of glycolysis and plays an important role in the pathogenesis of diabetic complications, including diabetic retinopathy. However, it remains to be determined how methylglyoxal affects the regulatory mechanisms of retinal blood flow. In this study, we examined the effects of methylglyoxal on ß2-adrenoceptor-mediated vasodilatory mechanisms in rat retinal arterioles. The retinal vasodilator responses were assessed by measuring the diameter of retinal arterioles in the fundus images. Intravitreal injection of methylglyoxal significantly diminished the vasodilation of retinal arterioles induced by the ß2-adrenoceptor agonist salbutamol. The vasodilator effect of BMS-191011, a large-conductance Ca2+-activated K+ (BKCa) channel opener, on retinal arterioles was also attenuated by methylglyoxal. In contrast, methylglyoxal had no significant effect on retinal vasodilator response to forskolin. Methylglyoxal attenuated retinal vasodilator response to salbutamol under blockade of BKCa channels with iberiotoxin, an inhibitor of the channels. These results suggest that methylglyoxal attenuates ß2-adrenoceptor-mediated retinal vasodilation by impairing the coupling of the ß2-adrenoceptor to the guanine nucleotide-binding protein (Gs protein) and the function of the BKCa channel. Increased methylglyoxal in the eyes may contribute to the impairment of regulatory mechanisms of retinal blood flow in patients with diabetic retinopathy.
Assuntos
Antagonistas de Receptores Adrenérgicos beta 2/metabolismo , Arteríolas/metabolismo , Aldeído Pirúvico/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Retina/metabolismo , Vasos Retinianos/metabolismo , Vasodilatação , Agonistas de Receptores Adrenérgicos beta 2/administração & dosagem , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Antagonistas de Receptores Adrenérgicos beta 2/administração & dosagem , Albuterol/administração & dosagem , Albuterol/farmacologia , Animais , Arteríolas/efeitos dos fármacos , Agonistas dos Canais de Cálcio/administração & dosagem , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Colforsina/administração & dosagem , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Subunidades alfa Gs de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Injeções Intravenosas , Injeções Intravítreas , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/agonistas , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Oxidiazóis/administração & dosagem , Oxidiazóis/farmacologia , Peptídeos/farmacologia , Aldeído Pirúvico/administração & dosagem , Ratos Sprague-Dawley , Receptores Adrenérgicos beta 2/química , Retina/efeitos dos fármacos , Vasos Retinianos/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/administração & dosagem , Vasodilatadores/farmacologiaRESUMO
BACKGROUND: Circulating fibrocytes (CFs) have been shown to participate in subepithelial fibrosis of asthma with chronic airflow limitation by acting as an important source of fibroblasts deposited beneath airway epithelia. Serum amyloid P (SAP) is an innate inhibitor of fibrocytes differentiation. Store-operated Ca2+ entry (SOCE) is the major Ca2+ influx of non-excitable cells. In this study, the role of SOCE in the regulation of fibrocytes differentiation and the effects of Th2 cytokine IL-4 and SAP on SOCE of fibrocytes were investigated. METHODS: Peripheral blood mononuclear cells or monocytes were cultured in serum-free medium for 7days to differentiate into fibrocytes; the expression of SOC channels was determined with PCR, SOCE was measured with Ca2+ fluorescence imaging. RESULTS: IL-4 significantly promoted monocyte derived fibrocytes differentiation in vitro. It also increased both SOCE which was induced by thapsigargin or UTP and molecules STIM1 and Orai1 which were related to expression of SOC channels in fibrocytes. Fibrocytes differentiation induced by IL-4 and SOC channels activity could be inhibited by SOC channel blocker SKF-96365. As expected, SAP significantly inhibited IL-4-induced differentiation of fibrocytes, the activity of SOCE and the expression of STIM1 and Orai1 in IL-4-treated fibrocytes. CONCLUSION: IL-4 and SAP reversely regulates cultured fibrocytes differentiation in vitro by respectively promoting or inhibiting the expression and activity of SOC channels in fibrocytes.
Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Interleucina-4/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteínas de Neoplasias/efeitos dos fármacos , Proteína ORAI1/efeitos dos fármacos , Componente Amiloide P Sérico/farmacologia , Molécula 1 de Interação Estromal/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Imidazóis/farmacologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Fatores de TempoRESUMO
Voltage-gated calcium channels (VGCCs) are critical regulators of many cellular functions, including the activity-dependent release of chemical neurotransmitter from nerve terminals. At nerve terminals, the Cav2 family of VGCCs are closely positioned with neurotransmitter-containing synaptic vesicles. The relationship between calcium ions and transmitter release is such that even subtle changes in calcium flux through VGCCs have a strong influence on the magnitude of transmitter released. Therefore, modulators of the calcium influx at nerve terminals have the potential to strongly affect transmitter release at synapses. We have previously developed novel Cav2-selective VGCC gating modifiers (notably GV-58) that slow the deactivation of VGCC current, increasing total calcium ion flux. Here, we describe ten new gating modifiers based on the GV-58 structure that extend our understanding of the structure-activity relationship for this class of molecules and extend the range of modulation of channel activities. In particular, we show that one of these new compounds (MF-06) was more efficacious than GV-58, another (KK-75) acts more quickly on VGCCs than GV-58, and a third (KK-20) has a mix of increased speed and efficacy. A subset of these new VGCC agonist gating modifiers can increase transmitter release during action potentials at neuromuscular synapses, and as such, show potential as therapeutics for diseases with a presynaptic deficit that results in neuromuscular weakness. Further, several of these new compounds can be useful tool compounds for the study of VGCC gating and function.
Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/metabolismo , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Biofísica , Agonistas dos Canais de Cálcio/química , Canais de Cálcio Tipo N/genética , Linhagem Celular , Relação Dose-Resposta a Droga , Estimulação Elétrica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/genética , Neuroblastoma/patologia , Neurotransmissores/metabolismo , Técnicas de Patch-Clamp , Purinas/química , Purinas/farmacologia , Transmissão Sináptica/genética , Tiofenos/química , Tiofenos/farmacologia , Fatores de Tempo , TransfecçãoRESUMO
The axon initial segment (AIS), the domain responsible for action potential initiation and maintenance of neuronal polarity, is targeted for disruption in a variety of central nervous system pathological insults. Previous work in our laboratory implicates oxidative stress as a potential mediator of structural AIS alterations in two separate mouse models of central nervous system inflammation, as these effects were attenuated following reactive oxygen species scavenging and NADPH oxidase-2 ablation. While these studies suggest a role for oxidative stress in modulation of the AIS, the direct effects of reactive oxygen and nitrogen species (ROS/RNS) on the stability of this domain remain unclear. Here, we demonstrate that oxidative stress, as induced through treatment with 3-morpholinosydnonimine (SIN-1), a spontaneous ROS/RNS generator, drives a reversible loss of AIS protein clustering in primary cortical neurons in vitro. Pharmacological inhibition of both voltage-dependent and intracellular calcium (Ca2+) channels suggests that this mechanism of AIS disruption involves Ca2+ entry specifically through L-type voltage-dependent Ca2+ channels and its release from IP3-gated intracellular stores. Furthermore, ROS/RNS-induced AIS disruption is dependent upon activation of calpain, a Ca2+-activated protease previously shown to drive AIS modulation. Overall, we demonstrate for the first time that oxidative stress, as induced through exogenously applied ROS/RNS, is capable of driving structural alterations in the AIS complex.