RESUMO
The anticancer antibiotic heptelidic acid is a sesquiterpene lactone produced by the beneficial plant fungus Trichoderma virens. This species has been separated into two strains, referred to as P and Q, based on its biosynthesis of secondary metabolites; notably, only P-strains were reported to produce heptelidic acid. While characterizing a Q-strain of T. virens containing a directed mutation in the non-ribosomal peptide synthetase encoding gene Tex7, the appearance of an unknown compound in anomalously large quantities was visualized by TLC. Using a combination of HPLC, LC-MS/MS, and NMR spectroscopy, this compound was identified as heptelidic acid. This discovery alters the strain classification structure of T. virens. Additionally, the Tex7 mutants inhibited growth of maize seedlings, while retaining the ability to induce systemic resistance against the foliar fungal pathogen, Cochliobolus heterostrophus.
Assuntos
Antibióticos Antineoplásicos/metabolismo , Proteínas Fúngicas/genética , Peptídeo Sintases/genética , Trichoderma/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Genes Fúngicos , Família Multigênica , Peptídeo Sintases/metabolismo , Sesquiterpenos/metabolismo , Trichoderma/metabolismo , Zea mays/crescimento & desenvolvimento , Zea mays/microbiologiaRESUMO
Fungal siderophores are known to be involved in iron acquisition and storage, as well as pathogenicity of mammals and plants. As avirulent plant symbionts, Trichoderma spp. colonize roots and induce resistance responses both locally and systemically. To study the role of intracellular siderophore(s) in Trichoderma-plant interactions, we have obtained mutants in a non-ribosomal peptide synthetase, TvTex10, that was predicted to be involved in intracellular siderophore(s) biosynthesis. This gene has a detectable basal level of expression and is also upregulated under iron-deplete conditions. This is unlike two other siderophore-encoding genes, which are tightly regulated by iron. Disruption of tex10 gene using homologous recombination resulted in mutants with enhanced growth rate, reduced conidiation and hyper-sensitivity to oxidative stress as compared to wildtype strain. The mutants also produced reduced levels of gliotoxin and dimethyl gliotoxin but have enhanced ability to colonize maize seedling roots. The mutants were also impaired in induction of induced systemic resistance (ISR) in maize against the foliar pathogen Cochliobolus heterostrophus.
Assuntos
Ferricromo/análogos & derivados , Sideróforos/fisiologia , Trichoderma/crescimento & desenvolvimento , Trichoderma/genética , Zea mays/microbiologia , Resistência à Doença , Ferricromo/metabolismo , Gliotoxina/biossíntese , Mutação , Sideróforos/biossíntese , Esporos Fúngicos/crescimento & desenvolvimento , Trichoderma/metabolismoRESUMO
Chemical warfare nerve agents (CWNA) inhibit acetylcholinesterase and are among the most lethal chemicals known to man. Children are predicted to be vulnerable to CWNA exposure because of their smaller body masses, higher ventilation rates and immature central nervous systems. While a handful of studies on the effects of CWNA in younger animals have been published, exposure routes relevant to battlefield or terrorist situations (i.e. inhalation for sarin) were not used. Thus, we estimated the 24 h LC50 for whole-body (10 and 60 min) exposure to sarin using a stagewise, adaptive dose design. Specifically, male and female Sprague-Dawley rats were exposed to a range of sarin concentrations (6.2-44.0 or 1.6-12.5 mg/m³) for either 10 or 60 min, respectively, at six different times during their development (postnatal day [PND] 7, 14, 21, 28, 42 and 70). For male and female rats, the lowest LC50 values were observed for PND 14 and the highest LC50 values for PND 28. Sex differences were observed only for PND 42 for the 10 min exposures and PND 21 and 70 for the 60 min exposures. Thus, younger rats (PND 14) were more susceptible than older rats (PND 70) to the lethal effects of whole-body exposure to sarin, while adolescent (PND 28) rats were the least susceptible and sex differences were minimal. These results underscore the importance of controlling for the age of the animal in research on the toxic effects associated with CWNA exposure.
Assuntos
Substâncias para a Guerra Química/toxicidade , Inibidores da Colinesterase/toxicidade , Sarina/toxicidade , Fatores Etários , Animais , Relação Dose-Resposta a Droga , Feminino , Exposição por Inalação , Dose Letal Mediana , Masculino , Ratos Sprague-Dawley , Fatores Sexuais , Fatores de TempoRESUMO
Fungal spores, germlings, and mycelia adhere to substrates, including host tissues. The adhesive forces depend on the substrate and on the adhesins, the fungal cell surface proteins. Attachment is often a prerequisite for the invasion of the host, hence its importance. Adhesion visibly precedes colonization of root surfaces and outer cortex layers, but little is known about the molecular details. We propose that by starting from what is already known from other fungi, including yeast and other filamentous pathogens and symbionts, the mechanism and function of Trichoderma adhesion will become accessible. There is a sequence, and perhaps functional, homology to other rhizosphere-competent Sordariomycetes. Specifically, Verticillium dahliae is a soil-borne pathogen that establishes itself in the xylem and causes destructive wilt disease. Metarhizium species are best-known as insect pathogens with biocontrol potential, but they also colonize roots. Verticillium orthologs of the yeast Flo8 transcription factor, Som1, and several other relevant genes are already under study for their roles in adhesion. Metarhizium encodes relevant adhesins. Trichoderma virens encodes homologs of Som1, as well as adhesin candidates. These genes should provide exciting leads toward the first step in the establishment of beneficial interactions with roots in the rhizosphere.
RESUMO
Trichoderma virens is a well-known mycoparasitic fungal symbiont that is valued for its biocontrol capabilities. T. virens initiates a symbiotic relationship with a plant host through the colonization of its roots. To achieve colonization, the fungus must communicate with the host and evade its innate defenses. In this study, we explored the genes involved with the host communication and colonization process through transcriptomic profiling of the wild-type fungus and selected deletion mutants as they colonized maize roots. Transcriptome profiles of the T. virens colonization of maize roots over time revealed that 24 h post inoculation appeared to be a key time for plant-microbe communication, with many key gene categories, including signal transduction mechanisms and carbohydrate transport and metabolism, peaking in expression at this early colonization time point. The transcriptomic profiles of Sm1 and Sir1 deletion mutants in the presence of plants demonstrated that Sir1, rather than Sm1, appears to be the key regulator of the fungal response to maize, with 64% more unique differentially expressed genes compared to Sm1. Additionally, we developed a novel algorithm utilizing gene clustering and coexpression network analyses to select potential colonization-related gene targets for characterization. About 40% of the genes identified by the algorithm would have been missed using previous methods for selecting gene targets.
RESUMO
We have measured the expression of T-type Ca2+ channel mRNA in breast cancer cell lines (MCF-7 (ERalpha+) using Western blot and quantitative real-time PCR (Q-RT-PCR). These results revealed that the MCF-7 cells express both alpha1G and alpha1H isoforms of T-type Ca2+ channels. In order to further clarify the role of T-type Ca2+ channels in proliferation, we tested the effects of a selective T-type Ca2+ channel inhibitor NNC-55-0396 on cellular proliferation. MCF-7 (ERalpha+) cellular proliferation was inhibited by the compound. In contrast, NNC-55-0396 at same concentration had no effect on the proliferation of MCF-10A cells, a non-cancer breast epithelial cell line. We also found that message expression of the T-type Ca2+ channels were only expressed in rapidly growing non-confluent cells but not in the cytostatic confluent cells. Knocking down the expression of T-type Ca2+ channels with siRNA targeting both alpha1G and alpha1H resulted in growth inhibition as much as 45%+/-5.0 in MCF-7 cells as compared to controls. In conclusion, our results suggest that T-type Ca2+ channel antagonism/silencing may reduce cellular proliferation in mitogenic breast cells.
Assuntos
Benzimidazóis/farmacologia , Neoplasias da Mama/metabolismo , Canais de Cálcio Tipo T/metabolismo , Proliferação de Células/efeitos dos fármacos , Mama/efeitos dos fármacos , Mama/metabolismo , Neoplasias da Mama/patologia , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Ciclopropanos , Humanos , Naftalenos , RNA Interferente Pequeno/farmacologiaRESUMO
Regulation of intracellular calcium is an important signaling mechanism for cell proliferation in both normal and cancerous cells. In normal epithelial cells, free calcium concentration is essential for cells to enter and accomplish the S phase and the M phase of the cell cycle. In contrast, cancerous cells can pass these phases of the cell cycle with much lower cytoplasmic free calcium concentrations, indicating an alternative mechanism has developed for fulfilling the intracellular calcium requirement for an increased rate of DNA synthesis and mitosis of fast replicating cancerous cells. The detailed mechanism underlying the altered calcium loading pathway remains unclear; however, there is a growing body of evidence that suggests the T-type Ca(2+) channel is abnormally expressed in cancerous cells and that blockade of these channels may reduce cell proliferation in addition to inducing apoptosis. Recent studies also show that the expression of T-type Ca(2+) channels in breast cancer cells is proliferation state dependent, i.e. the channels are expressed at higher levels during the fast-replication period, and once the cells are in a non-proliferation state, expression of this channel is minimal. Therefore, selectively blocking calcium entry into cancerous cells may be a valuable approach for preventing tumor growth. Since T-type Ca(2+) channels are not expressed in epithelial cells, selective T-type Ca(2+) channel blockers may be useful in the treatment of certain types of cancers.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Sinalização do Cálcio/fisiologia , Neoplasias/metabolismo , Neoplasias/patologia , Cálcio/metabolismo , Ciclo Celular/fisiologia , HumanosRESUMO
Using a modified noninvasive volume-displacement plethysmography system, we investigated the effects of inhaled dichlorvos (2,2-dimethyl-dichlorovinyl phosphate, or DDVP) vapor on the respiratory mechanics and blood cholinesterase activity of guinea pigs. Data revealed significant dose-dependent changes in several pulmonary parameters. Animals exposed to a DDVP concentration of 35 mg/m(3) did not show any significant changes in frequency, tidal volume, or minute ventilation. However, animals exposed to 55 mg/m(3) DDVP showed significantly decreased respiratory frequency and significantly increased tidal volume with no significant changes in minute ventilation. Similarly, animals exposed to 75 mg/m(3) DDVP showed significantly decreased respiratory frequency along with significantly increased tidal volume. The decreased respiratory frequency was large enough in the high exposure group to offset the increased tidal volume. This effect resulted in significantly decreased minute ventilation by the end of exposure, which remained attenuated 10 min after exposure. An analysis of whole-blood cholinesterase activity revealed significantly decreased activity for both acetylcholinesterase (AChE) and butyl-cholinesterase (BChE). Peak inhibition occurred for both enzymes at the end of exposure for all three concentrations and rapidly recovered within several minutes of exposure. Analysis of blood samples using gas chromatography-mass spectroscopy (GC-MS) revealed that minute ventilation may only play a minimal role in the dosimetry of inhaled DDVP vapor.
Assuntos
Acetilcolinesterase/sangue , Diclorvós/toxicidade , Exposição por Inalação/efeitos adversos , Mecânica Respiratória/efeitos dos fármacos , Doença Aguda , Animais , Câmaras de Exposição Atmosférica , Diclorvós/administração & dosagem , Diclorvós/farmacocinética , Relação Dose-Resposta a Droga , Cobaias , Masculino , Mecânica Respiratória/fisiologia , Fatores de Tempo , VolatilizaçãoRESUMO
A sensitive method for determining exposure to the chemical warfare agent VX is described in which the biomarker ethyl methylphosphonofluoridate (VX-G) is measured in red blood cells (RBCs) following treatment with fluoride ion using isotope-dilution gas chromatography-tandem mass spectrometry. The analyte was isolated via solid-phase extraction and detected using ammonia chemical ionization in the multiple reaction monitoring mode. A good linear relationship was obtained in the quantitative concentration range of 4 ng/mL to 1000 ng/mL with an absolute detection limit of < 1 pg on column. The method has been applied to the analysis of RBCs from a laboratory worker accidentally exposed to VX vapor. Detection and quantitation of VX-G were possible in samples taken as late as 27 days following exposure.
Assuntos
Monitoramento Ambiental/métodos , Eritrócitos/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Compostos Organotiofosforados/sangue , Espectrometria de Massas em Tandem/métodos , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Biomarcadores/análise , Biomarcadores/sangue , Substâncias para a Guerra Química/análise , Substâncias para a Guerra Química/metabolismo , Inibidores da Colinesterase/análise , Inibidores da Colinesterase/sangue , Inibidores da Colinesterase/metabolismo , Exposição Ambiental/análise , Eritrócitos/enzimologia , Fluoretos/química , Humanos , Compostos Organotiofosforados/análise , Compostos Organotiofosforados/metabolismo , Compostos de Potássio/química , Extração em Fase Sólida/métodosRESUMO
The lack of data in the open literature on human exposure to the nerve agent O-ethyl-S-(2-diisopropylaminoethyl) methylphosphonothioate (VX) gives a special relevance to the data presented in this study in which we report the quantification of VX-butyrylcholinesterase adduct from a relatively low-level accidental human exposure. The samples were analyzed by gas chromatography-high resolution mass spectrometry using the fluoride ion regeneration method for the quantification of multiple nerve agents including VX. Six human plasma samples from the same individual were collected after the patient had been treated once with oxime immediately after exhibiting signs of exposure. Detection limits of approximately 5.5 pg/mL plasma were achieved for the G-analogue of VX (G-VX). Levels of the G-VX ranged from 81.4 pg/mL on the first day after the exposure to 6.9 pg/mL in the sample taken 27 days after the exposure. Based on the reported concentration of human butyrylcholinesterase in plasma of approximately 80 nM, it can be calculated that inhibition levels of >or= 0.05% of BuChE can be accurately quantified. These data further indicate that the fluoride ion regeneration method is a potentially powerful tool that can be used to assess low-level exposure to VX.
Assuntos
Butirilcolinesterase/metabolismo , Monitoramento Ambiental/métodos , Compostos Organotiofosforados/sangue , Biomarcadores/análise , Biomarcadores/sangue , Butirilcolinesterase/química , Calibragem , Substâncias para a Guerra Química/análise , Substâncias para a Guerra Química/metabolismo , Inibidores da Colinesterase/sangue , Inibidores da Colinesterase/metabolismo , Exposição Ambiental/análise , Fluoretos/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Compostos Organotiofosforados/química , Compostos Organotiofosforados/metabolismo , Compostos de Potássio/químicaRESUMO
The present study was undertaken to investigate the miotic potency of soman vapor in the rat, as well as gender differences in the miotic response to soman vapor that have been reported previously for other nerve agents. The results of the present study demonstrate that the miotic potency of soman vapor is significantly less than that of other nerve agents, and that female rats are 2.5-3.0 times more sensitive to soman vapor than male rats. The results also demonstrate that ocular acetylcholinesterase and butyrylcholinesterase activities differ between males and females, although this difference is not likely large enough to account for the observed gender difference.
Assuntos
Inibidores da Colinesterase/farmacologia , Olho/efeitos dos fármacos , Miose/induzido quimicamente , Soman/farmacologia , Acetilcolinesterase/sangue , Análise de Variância , Animais , Butirilcolinesterase/sangue , Substâncias para a Guerra Química/farmacologia , Olho/anatomia & histologia , Olho/enzimologia , Feminino , Raios Infravermelhos , Masculino , Modelos Animais , Compostos Organofosforados/farmacologia , Compostos Organotiofosforados/farmacologia , Pupila/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Caracteres SexuaisRESUMO
In the present study, we demonstrate that lung microvascular endothelial cells express a Cav3.1 (alpha1G) T-type voltage-gated Ca2+ channel, whereas lung macrovascular endothelial cells do not express voltage-gated Ca2+ channels. Voltage-dependent activation indicates that the Cav3.1 T-type Ca2+ current is shifted to a positive potential, at which maximum current activation is -10 mV; voltage-dependent conductance and inactivation properties suggest a "window current" in the range of -60 to -30 mV. Thrombin-induced transitions in membrane potential activate the Cav3.1 channel, resulting in a physiologically relevant rise in cytosolic Ca2+. Furthermore, activation of the Cav3.1 channel induces a procoagulant endothelial phenotype; eg, channel inhibition attenuates increased retention of sickled erythrocytes in the inflamed pulmonary circulation. We conclude that activation of the Cav3.1 channels selectively induces phenotypic changes in microvascular endothelial cells that mediate vaso-occlusion by sickled erythrocytes in the inflamed lung microcirculation.
Assuntos
Anemia Falciforme/fisiopatologia , Canais de Cálcio Tipo T/fisiologia , Endotélio Vascular/fisiopatologia , Eritrócitos Anormais/patologia , Pulmão/irrigação sanguínea , Sequência de Aminoácidos , Anemia Falciforme/sangue , Animais , Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/genética , Adesão Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Estimulação Elétrica , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Flunarizina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Mibefradil/farmacologia , Microcirculação/fisiopatologia , Dados de Sequência Molecular , Neurotoxinas/farmacologia , Níquel/farmacologia , Pimozida/farmacologia , RNA/genética , RNA/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Venenos de Escorpião/farmacologia , Homologia de Sequência de AminoácidosRESUMO
Improving the quality of physiologic data collected from research animals is most easily accomplished by collecting as much information as possible from a single subject, thereby reducing animal use and error associated with satellite groups. We investigated the feasibility of using a large-animal implantable telemetry device in New Zealand white rabbits (n = 6). The first task was to develop an implantation technique that yielded calibrated tidal volume (Vt) measurements that were within 10% of those obtained simultaneously from a pneumotachograph, a low-noise electrocardiogram, and stable blood pressure. The second task was to challenge implanted rabbits with the respiratory stimulant doxapram to assess linearity of the calibration across a range of Vt. Of the 3 electrode placements attempted, only one resulted in calibrations consistently below 10% error. Optimal electrode placement resulted in calibrated Vt measurements within 1.7% ± 0.3% of those obtained from a pneumotachograph during normal tidal breathing, 7.3% ± 0.7% of those after saline injection, and 6.0% ± 0.5% of those after doxapram injection. The Vt range was 9 to 15 mL for normal tidal breathing and saline injection and 25 to 30 mL after doxapram injection. Increases in mean arterial pressure of 25.0 ± 6.82 mm Hg and decreases in heart rate of 56.3 ± 6.82 bpm were associated with doxapram injection only. Our findings represent the first time that multiple cardiopulmonary endpoints have been assessed by telemetry in conscious, restrained rabbits. Whether animal position affects calibration accuracy warrants investigation.
Assuntos
Coelhos/fisiologia , Testes de Função Respiratória/veterinária , Telemetria/veterinária , Animais , Doxapram/administração & dosagem , Impedância Elétrica , Eletrocardiografia , Frequência Cardíaca/efeitos dos fármacos , Masculino , Testes de Função Respiratória/métodos , Medicamentos para o Sistema Respiratório/administração & dosagem , Telemetria/métodos , Volume de Ventilação Pulmonar , Testes de ToxicidadeRESUMO
The present study investigated the effect of the organophosphate, dichlorvos (DDVP), on ocular function and cholinesterase activity in guinea pigs, using a single-animal-head-only vapor exposure system. All animals exhibited signs of mild organophosphate poisoning (e.g., salivation, chewing, lacrimation, urination, defecation, and rhinorrhea) after the 20-min exposure, regardless of the DDVP exposure concentration (e.g., 35 mg/m(3), 55 mg/m(3), and 75 mg/m(3)). Pupil constriction or miosis was the most pronounced effect seen after vapor exposure. The postexposure pupil size for the 35 mg/m(3) group was 45.8 +/- 3.68% of the preexposure baseline measurement. Postexposure pupil size in the 55- (38 +/- 1.36%) and 75 mg/m(3) (38.1 +/- 1.72%) groups was significantly less than both the preexposure baseline level and the 35 mg/m(3) group. All groups exhibited enhanced an pupillary response to light after DDVP exposure. The enhanced light response remained even after recovery from miosis (approximately 1 h after exposure). Measurement of cholinesterase activity revealed that even though pupil size had recovered, acetyl- and butyrylcholinesterase remained significantly inhibited in the blood.
Assuntos
Sistema Nervoso Autônomo/efeitos dos fármacos , Inibidores da Colinesterase/toxicidade , Diclorvós/toxicidade , Olho/efeitos dos fármacos , Acetilcolinesterase/sangue , Acetilcolinesterase/metabolismo , Animais , Butirilcolinesterase/sangue , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/administração & dosagem , Inibidores da Colinesterase/farmacocinética , Diclorvós/administração & dosagem , Diclorvós/farmacocinética , Relação Dose-Resposta a Droga , Olho/inervação , Cobaias , Exposição por Inalação , Masculino , Reflexo Pupilar/efeitos dos fármacos , VolatilizaçãoRESUMO
High-voltage-activated (HVA) calcium channels are known to be the primary source of calcium for glucose-stimulated insulin secretion. However, few studies have investigated how these channels can be regulated by chronically elevated levels of glucose. In the present study, we determined the level of expression of the four major HVA calcium channels (N-type, P/Q-type, L(C)-type, and L(D)-type) in rat pancreatic beta-cells. Using quantitative real-time PCR (QRT-PCR), we found the expression of all four HVA genes in rat insulinoma cells (INS-1) and in primary isolated rat islet cells. We then determined the role of each channel in insulin secretion by using channel-selective antagonists. Insulin secretion analysis revealed that N- and L-type channels are both involved in immediate glucose-induced insulin secretion. However, L-type was preferentially coupled to secretion at later time points. P/Q-type channels were not found to play a role in insulin secretion at any stage. It was also found that long-term exposure to elevated glucose increases basal calcium in these cells. Interestingly, chronically elevated glucose decreased the mRNA expression of the channels involved with insulin secretion and diminished the level of stimulated calcium influx in these cells. Using whole cell patch clamp, we found that N- and L-type channel currents increase gradually subsequent to lower intracellular calcium perfusion, suggesting that these channels may be regulated by glucose-induced changes in calcium.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Linhagem Celular Tumoral , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Mibefradil/farmacologia , Nifedipino/farmacologia , Técnicas de Patch-Clamp , RNA/química , RNA/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
NNC 55-0396 is a structural analog of mibefradil (Ro 40-5967) that inhibits both T-type and high-voltage-activated (HVA) Ca2+ channels with a higher selectivity for T-type Ca2+ channels. The inhibitory effect of mibefradil on HVA Ca2+ channels can be attributed to a hydrolyzed metabolite of the drug: the methoxy acetate side chain of mibefradil is removed by intracellular enzymes, thus it forms (1S,2S)-2-(2-(N-[(3-benzoimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl hydroxy dihydrochloride (dm-mibefradil), which causes potent inhibition of HVA Ca2+ currents. By replacing the methoxy acetate chain of mibefradil with cyclopropanecarboxylate, a more stable analog was developed (NNC 55-0396). The acute IC50 of NNC 55-0396 to block recombinant Cav3.1 T-type channels expressed in HEK293 cells is approximately 7 muM, whereas 100 microM NNC 55-0396 has no detectable effect on high voltage-activated currents in INS-1 cells. Block of T-type Ca2+ current was partially reduced by membrane hyperpolarization and was enhanced at high stimulus frequency. Washing NNC 55-0396 out of the recording chamber did not reverse the T-type Ca2+ current activity, suggesting that the compound dissolves in or passes through the plasma membrane to exert its effect; however, intracellular perfusion of the compound did not block T-type Ca2+ currents, arguing against a cytoplasmic route of action. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of L-type Ca2+ channel channels, thus rendering it more selective to T-type Ca2+ channels.
Assuntos
Benzimidazóis/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/metabolismo , Ciclopropanos/farmacologia , Naftalenos/farmacologia , Animais , Linhagem Celular , Desenho de Fármacos , Humanos , Mibefradil/farmacologiaRESUMO
Infants develop hypertrophic cardiomyopathy in approximately 30% of diabetic pregnancies. We have characterized the effects of glucose on voltage-gated T-type Ca2+ channels and intracellular free calcium concentration, [Ca2+]i in neonatal rat cardiomyocytes. We found that T-type Ca2+ channel current density increased significantly in primary culture neonatal cardiac myocytes that were treated with 25 mM glucose for 48 h when compared with those that were treated with 5 mM glucose. High-glucose treatment also caused a higher Ca2+ influx elicited by 50 mM KCl in the myocytes. KCl-induced Ca2+ influx was attenuated when nickel was present. Real-time PCR studies demonstrated that mRNA levels of both alpha1G (Ca(v)3.1) and alpha1H (Ca(v)3.2) T-type Ca2+ channels were elevated after high-glucose treatment. High-glucose also significantly increased ventricular cell proliferation as well as the proportion of cells in the S-phase of the cell cycle; both effects were reversed by nickel or mibefradil. These results indicate that high glucose causes a rise in [Ca2+]i in neonatal cardiac myocytes by a mechanism that is associated with the regulation of the T-type Ca2+ channel activity.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Proliferação de Células , Glucose/metabolismo , Miócitos Cardíacos/fisiologia , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Canais de Cálcio Tipo T/genética , Ciclo Celular , Células Cultivadas , Cloretos/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/citologia , Oligonucleotídeos Antissenso/metabolismo , Técnicas de Patch-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RatosRESUMO
Stimulation of vascular endothelial muscarinic receptors by acetylcholine (ACh) leads to the formation of an endothelium-derived relaxing factor (EDRF), which is generally accepted to be nitric oxide (NO). Recent evidence, however, suggests that NO may be only one of several EDRFs mediating the vasodilator response to ACh. Since this NO-independent vasodilator response to ACh has been hypothesized to be dependent upon K(+) channel activation, the current study was undertaken to investigate the role of K(+) channels in mediating the hindlimb vasodilator responses to ACh in vivo. Additionally, since variations in vascular tone can complicate the analysis of responses, the level of vascular tone was maintained at a similar level throughout the study so that responses could be compared directly. The results of the present study demonstrate that the vasodilator response to ACh possesses a significant component that is independent of NO production. The K(Ca) channel blockers charybdotoxin and apamin, but not K(+)-ATP channel blocker U37883A or the COX antagonist meclofenamate, attenuated the NO-independent component of the vasodilator response to ACh. This suggests that K(Ca) channels, but not K(+)-ATP channels or COX products, are involved in mediating the L-NAME resistant response to ACh. Further, the inhibition of the ACh vasodilator response by the K(+)-ATP opener BRL55834 suggests that the response is dependent upon membrane hyperpolarization. These data suggest that the mechanism mediating ACh responses in the hindlimb vascular bed of the rat are complex and may involve several signaling pathways.
Assuntos
Acetilcolina/farmacologia , Óxido Nítrico/fisiologia , Canais de Potássio/fisiologia , Vasodilatação/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Vasodilatação/fisiologiaRESUMO
Chronic exposure of pancreatic beta-cells to high concentrations of glucose impairs the insulin secretory response to further glucose stimulation. This phenomenon is referred to as glucose desensitization. It has been shown that glucose desensitization is associated with abnormal elevation of beta-cell basal intracellular free Ca2+ concentration ([Ca2+]i). We have investigated the relationship between the basal intracellular free Ca2+ and the L-type (Cav1.3) Ca2+ channel translocation in insulin-secreting cells. Glucose stimulation or membrane depolarization induced a nifedipine-sensitive Ca2+ influx, which was attenuated when the basal [Ca2+]i was elevated. Using voltage-clamp techniques, we found that changing [Ca2+]i could regulate the amplitude of the Ca2+ current. This effect was attenuated by drugs that interfere with the cytoskeleton. Immunofluorescent labeling of Cav1.3 showed an increase in the cytoplasmic distribution of the channels under high [Ca2+]i conditions by deconvolution microscopy. The [Ca2+]i-dependent translocation of Cav1.3 channel was also demonstrated by Western blot analysis of biotinylation/NeutrAvidin-bead-eluted surface proteins in cells preincubated at various [Ca2+]i. These results suggest that Cav1.3 channel trafficking is involved in glucose desensitization of pancreatic beta-cells.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Insulina/metabolismo , Membranas Intracelulares/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Transporte Biológico/fisiologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , Membrana Celular/fisiologia , Células Cultivadas , Citoplasma/metabolismo , Condutividade Elétrica , Eletrofisiologia , Glucose/farmacologia , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Nifedipino/farmacologia , Concentração Osmolar , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
Mibefradil is a Ca2+ channel antagonist that inhibits both T-type and high-voltage-activated Ca2+ channels. We previously showed that block of high-voltage-activated channels by mibefradil occurs through the production of an active metabolite by intracellular hydrolysis. In the present study, we modified the structure of mibefradil to develop a nonhydrolyzable analog, (1S, 2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride (NNC 55-0396), that exerts a selective inhibitory effect on T-type channels. The acute IC(50) of NNC 55-0396 to block recombinant alpha(1)G T-type channels in human embryonic kidney 293 cells was approximately 7 microM, whereas 100 microM NNC 55-0396 had no detectable effect on high-voltage-activated channels in INS-1 cells. NNC 55-0396 did not affect the voltage-dependent activation of T-type Ca2+ currents but changed the slope of the steady-state inactivation curve. Block of T-type Ca2+ current was partially relieved by membrane hyperpolarization and enhanced at a high-stimulus frequency. Washing NNC 55-0396 out of the recording chamber did not reverse the T-type Ca2+ current activity, suggesting that the compound dissolves in or passes through the plasma membrane to exert its effect; however, intracellular perfusion of the compound did not block T-type Ca2+ currents, arguing against a cytoplasmic route of action. After incubating cells from an insulin-secreting cell line (INS-1) with NNC 55-0396 for 20 min, mass spectrometry did not detect the mibefradil metabolite that causes L-type Ca2+ channel inhibition. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of L-type Ca2+ channels, thus rendering it more selective to T-type Ca2+ channels.