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CONTEXT: ECa 233 is the standardized extract of Centella asiatica (L.) Urban. (Apiaceae). It contains at least 85% of triterpenoid glycosides and yields neuroprotective and memory-enhancing effects. However, the exact molecules exerting the effects might be triterpenic acid metabolites reproduced through gut metabolism after orally ingesting C. asiatica, not triterpenoid glycosides. OBJECTIVE: This study demonstrates the effect of unmetabolized ECa 233 on hippocampal synaptic plasticity after directly perfusing ECa 233 over acute brain slices. MATERIALS AND METHODS: The brain slices obtained from 7-week-old male Wistar rats were randomly divided into 4 groups. We perfused either artificial cerebrospinal fluid (ACSF), 0.01% DMSO, 10 µg/mL ECa 233, or 100 µg/mL on brain slices, and measured the long-term potentiation (LTP) magnitude to determine the synaptic plasticity of hippocampal circuits in each group. RESULTS: The LTP magnitude of ACSF, DMSO, 10 ug/mL ECa 233, and 100 ug/mL ECa 233 groups increased from 100% to 181.26 ± 38.19%, 148.74 ± 5.40%, 273.71 ± 56.66%, 182.17 ± 18.61%, respectively. ECa 233 at the concentration of 10 µg/mL robustly and significantly enhanced hippocampal LTP magnitude. The data indicates an improvement of the hippocampal synaptic plasticity. DISCUSSION AND CONCLUSIONS: This study emphasizes the effectiveness of triterpenoid glycosides in ECa 233 on synaptic plasticity enhancement. Therefore, this study supported and complimented the known effects of C. asiatica extract on the enhancement of synaptic plasticity, and subsequently, learning and memory, suggesting that ECa 233 could be a promising memory enhancing agent.
Assuntos
Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Relação Dose-Resposta a Droga , Glicosídeos/isolamento & purificação , Glicosídeos/farmacologia , Hipocampo/metabolismo , Masculino , Memória/efeitos dos fármacos , Extratos Vegetais/administração & dosagem , Ratos , Ratos Wistar , Triterpenos/isolamento & purificação , Triterpenos/farmacologiaRESUMO
Gastric motility disturbance is commonly found in long-standing hyperglycemia. Both delayed and rapid gastric emptying has been reported in diabetes. However, very few studies have followed the changes in gastric emptying during disease progression in diabetes because of technical limitations. 13C-Acetic acid breath test is a validated method which is non-invasive and can be used repeatedly or serially to evaluate gastric emptying changes in animal. We investigated the gastric emptying changes in different stages of diabetes using 13C-acetic acid breath test, as well as its related mechanisms involving interstitial cells of Cajal (ICCs), and stem cell factor (SCF) in streptozotocin-induced diabetic rats. The results showed that gastric emptying was accelerated at the early stage (12 weeks of diabetes) whereas intramuscular ICCs (ICC-IM) networks were not different from normal group. At long-term stage (28 weeks of diabetes), gastric emptying had returned to normal pattern with no delayed. ICC-IM networks were decreased in the diabetic group compared to 12th weeks, and were lower than in the normal group at the same time point. SCF levels were constantly high in the diabetic group than in the normal group. This result indicated that 13C-acetic acid breath test is useful to track the alteration in gastric emptying during disease progression. The change of gastric emptying was not found to be significantly associated with ICC-IM. Elevated SCF may help to preserve ICC-IM, especially in the early phase of diabetes.
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Ácido Acético/análise , Testes Respiratórios/métodos , Diabetes Mellitus Experimental/fisiopatologia , Esvaziamento Gástrico , Animais , Glicemia/análise , Peso Corporal , Contagem de Células , Diabetes Mellitus Experimental/patologia , Progressão da Doença , Células Intersticiais de Cajal/patologia , Masculino , Monitorização Fisiológica , Proteínas Proto-Oncogênicas c-kit/biossíntese , Ratos , Ratos Sprague-Dawley , Fator de Células-Tronco/biossínteseRESUMO
BACKGROUND: This study explored Bacopa monnieri, a medicinal Ayurvedic herb, as a cardioprotectant against ischemia/reperfusion injury using cardiac function and coronary flow as end-points. METHODS: In normal isolated rat hearts, coronary flow, left ventricular developed pressure, heart rate, and functional recovery were measured using the Langendorff preparation. Hearts were perfused with either (i) Krebs-Henseleit (normal) solution, (control), or with 30, 100 µg/ml B. monnieri ethanolic extract (30 min), or (ii) with normal solution or extract for 10 min preceding no-perfusion ischemia (30 min) followed by reperfusion (30 min) with normal solution. Infarct volumes were measured by triphenyltetrazolium staining. L-type Ca2+-currents (ICa, L) were measured by whole-cell patching in HL-1 cells, a mouse atrial cardiomyocyte cell line. Cytotoxicity of B. monnieri was assessed in rat isolated ventricular myocytes by trypan blue exclusion. RESULTS: In normally perfused hearts, B. monnieri increased coronary flow by 63 ± 13% (30 µg/ml) and 216 ± 21% (100 µg/ml), compared to control (5 ± 3%) (n = 8-10, p < 0.001). B. monnieri treatment preceding ischemia/reperfusion improved left ventricular developed pressure by 84 ± 10% (30 µg/ml), 82 ± 10% (100 µg/ml) and 52 ± 6% (control) compared to pre- ischemia/reperfusion. Similarly, functional recovery showed a sustained increase. Moreover, B. monnieri (100 µg/ml) reduced the percentage of infarct size from 51 ± 2% (control) to 25 ± 2% (n = 6-8, p < 0.0001). B. monnieri (100 µg/ml) reduced ICa, L by 63 ± 4% in HL-1 cells. Ventricular myocyte survival decreased at higher concentrations (50-1000 µg/ml) B. monnieri. CONCLUSIONS: B. monnieri improves myocardial function following ischemia/reperfusion injury through recovery of coronary blood flow, contractile force and decrease in infarct size. Thus this may lead to a novel cardioprotectant strategy.
Assuntos
Bacopa , Coração/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Fitoterapia , Extratos Vegetais/uso terapêutico , Fluxo Sanguíneo Regional/efeitos dos fármacos , Animais , Fármacos Cardiovasculares/farmacologia , Fármacos Cardiovasculares/uso terapêutico , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/fisiopatologia , Coração/fisiopatologia , Frequência Cardíaca , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/fisiopatologia , Técnicas In Vitro , Masculino , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/patologia , Extratos Vegetais/farmacologia , Substâncias Protetoras/farmacologia , Substâncias Protetoras/uso terapêutico , Ratos Wistar , Pressão VentricularRESUMO
[This corrects the article DOI: 10.1177/1535370220962708.].
RESUMO
Chronic inflammatory temporomandibular disorder (TMD) pain has a high prevalence, and available nonspecific treatments have adverse side effects. ECa 233, a standardized Centella asiatica extract, is highly anti-inflammatory and safe. We investigated its therapeutic effects by injecting complete Freund's adjuvant (CFA) into right temporomandibular joint of mice and administering either ibuprofen or ECa 233 (30, 100, and 300 mg/kg) for 28 days. Inflammatory and nociceptive markers, bone density, and pain hypersensitivity were examined. CFA decreased ipsilateral bone density, suggesting inflammation localization, which ipsilaterally caused immediate calcitonin gene-related peptide elevation in the trigeminal ganglia (TG) and trigeminal subnucleus caudalis (TNC), followed by late increase of NaV1.7 in TG and of p-CREB and activation of microglia in TNC. Contralaterally, only p-CREB and activated microglia in TNC showed delayed increase. Pain hypersensitivity, which developed early ipsilaterally, but late contralaterally, was reduced by ibuprofen and ECa 233 (30 or 100 mg/kg). However, ibuprofen and only 100-mg/kg ECa 233 effectively mitigated marker elevation. This suggests 30-mg/kg ECa 233 was antinociceptive, whereas 100-mg/kg ECa 233 was both anti-inflammatory and antinociceptive. ECa 233 may be alternatively and safely used for treating chronic inflammatory TMD pain, showing an inverted U-shaped dose-response relationship with maximal effect at 100 mg/kg.
Assuntos
Centella , Hipersensibilidade , Transtornos da Articulação Temporomandibular , Animais , Masculino , Ibuprofeno , Dor , Adjuvante de Freund , Modelos Animais de Doenças , AnalgésicosRESUMO
Ceftriaxone (CTX) exerts a neuroprotective effect by decreasing glutamate excitotoxicity. We further studied the underlying mechanisms and effects of CTX early post-treatment on behavior in a cerebral hypoperfusion rats. The rats' common carotid arteries (2VO) were permanently ligated. CTX was treated after ischemia. Biochemical studies were performed to assess antioxidative stress and inflammation. Behavioral and histological studies were then tested on the ninth week after vessel ligation. The 2VO rats showed learning and memory deficits as well as working memory impairments without any motor weakness. The treatment with CTX was found to attenuate white matter damage, MDA production, and interleukin 1 beta and tumor necrosis factor alpha production, mainly in the hippocampal area. Moreover, CTX treatment could increase the expression of glia and the glial glutamate transporters, and the neuronal glutamate transporter. Taken together, our data indicate the neuroprotective mechanisms of CTX involving the upregulation of glutamate transporters' expression. This increased expression contributes to a reduction in glutamate excitotoxicity and oxidative stress as well as pro-inflammatory cytokine production, thus resulting in the protection of neurons and tissue from further damage. The present study highlights the mechanism of the effect of CTX treatment and of the underlying ischemia-induced neuronal damage.
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SNARE-mediated exocytosis is a multistage process central to synaptic transmission and hormone release. Complexins (CPXs) are small proteins that bind very rapidly and with a high affinity to the SNARE core complex, where they have been proposed recently to inhibit exocytosis by clamping the complex and inhibiting membrane fusion. However, several other studies also suggest that CPXs are positive regulators of neurotransmitter release. Thus, whether CPXs are positive or negative regulators of exocytosis is not known, much less the stage in the vesicle life cycle at which they function. Here, we systematically dissect the vesicle stages leading up to exocytosis using a knockout-rescue strategy in a mammalian model system. We show that adrenal chromaffin cells from CPX II knockout mice exhibit markedly diminished releasable vesicle pools (comprising the readily and slowly releasable pools), while showing no change in the kinetics of fusion pore dilation or morphological vesicle docking. Overexpression of WT CPX II-but not of SNARE-binding-deficient mutants-restores the size of the the releasable pools in knockout cells, and in WT cells it markedly enlarges them. Our results show that CPXs regulate the size of the primed vesicle pools and have a positive role in Ca(2+)-triggered exocytosis.
Assuntos
Cálcio/metabolismo , Células Cromafins/fisiologia , Exocitose/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Vesículas Secretórias/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Animais , Catecolaminas/metabolismo , Células Cromafins/metabolismo , Células Cromafins/ultraestrutura , Feminino , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Mutantes , Microscopia Eletrônica , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Proteínas SNARE/metabolismo , Vesículas Secretórias/ultraestruturaRESUMO
OBJECTIVE: Mirror-image pain is a kind of pain that occurs on the contralateral side, but its pathogenesis remains unclear. To develop an osteoarthritis mouse model for investigating mirror-image pain through observing nocifensive behaviors, histological changes, and nociceptive activity at days 3, 7, 14, 21, and 28 after the chemical induction of unilateral temporomandibular joint (TMJ) osteoarthritis. METHODOLOGY: We randomly divided 6-week-old mice into sham and complete Freund adjuvant groups. To induce nocifensive behaviors, we applied 0.04 g of von Frey filament, 10 psi of air puff, and cold acetone on both sides of whisker pads at different days. The histology of TMJ on both sides was observed by hematoxylin/eosin staining and microcomputed tomography scanning. Furthermore, the nociceptive activity was evaluated using the phosphorylated cyclic AMP response element binding protein (pCREB) and a microglia marker at different days in the trigeminal subnucleus caudalis. RESULTS: Nocifensive behaviors against mechanical and temperature stimuli on the contralateral side became stronger than the baseline on day 28, in agreement with the elevation of the pCREB and the microglia marker in the trigeminal subnucleus caudalis. Thus, hypernociception on the contralateral side occurred at day 28. CONCLUSIONS: Clearly, the TMJ model with unilateral osteoarthritis exhibited mirror-image pain. Therefore, this model is useful in investigating the pathogenesis of pain and in developing treatments.
Assuntos
Osteoartrite , Articulação Temporomandibular , Animais , Adjuvante de Freund , Camundongos , Osteoartrite/diagnóstico por imagem , Dor , Microtomografia por Raio-XRESUMO
Obesity is one of major risk factors increasing chronic diseases including type II diabetes, cardiovascular diseases, and hypertension. The effects of epigallocatechin gallate (EGCG), the major active compound in green tea, on reduced obesity and improved metabolic profiles are still controversial. Furthermore, the effects of EGCG on human adipocyte lipolysis and browning of white adipocytes have not been elucidated. This study aimed to investigate the effects of EGCG on obesity, lipolysis, and browning of human white adipocytes. The results showed that, when compared to the baseline values, EGCG significantly decreased fasting plasma triglyceride levels (P < 0.05), systolic blood pressure (P < 0.05), diastolic blood pressure (P < 0.05), and serum kisspeptin levels (P < 0.05) after 8 weeks of supplement. On the other hand, supplement of EGCG in obese human subjects for 4 or 8 weeks did not decrease body weight, body mass index, waist and hip circumferences, nor total body fat mass or percentage when compared to their baseline values. The study in human adipocytes showed that EGCG did not increase the glycerol release when compared to vehicle, suggesting that it had no lipolytic effect. Furthermore, treatment of EGCG did not enhance uncoupling protein 1 (UCP1) mRNA expression in human white adipocytes when compared with treatment of pioglitazone, the peroxisome proliferator-activated receptor γ (PPAR-γ) agonist, suggesting that EGCG did not augment the browning effect of PPAR-γ on white adipocytes. This study revealed that EGCG reduced 2 metabolic risk factors which are triglyceride and blood pressure in the human experiment. We also showed a novel evidence that EGCG decreased kisspeptin levels. However, EGCG had no effects on obesity reduction in humans, lipolysis, nor browning of human white adipocytes.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Catequina/análogos & derivados , Kisspeptinas/sangue , Obesidade/sangue , Obesidade/fisiopatologia , Triglicerídeos/sangue , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Marrons/metabolismo , Adipócitos Brancos/efeitos dos fármacos , Adipócitos Brancos/metabolismo , Adiponectina/sangue , Adulto , Glicemia/metabolismo , Catequina/farmacologia , Humanos , Rim/efeitos dos fármacos , Rim/fisiopatologia , Leptina/sangue , Lipólise , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Pessoa de Meia-Idade , Obesidade/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMO
Obesity is associated with the growth and expansion of adipocytes which could be decreased via several mechanisms. Cissus Quadrangularis (CQ) extract has been shown to reduce obesity in humans; however, its effect on human white adipocytes (hWA) has not been elucidated. This study aimed to investigate the effects of CQ on obesity, lipolysis, and browning of hWA. CQ treatment in obese humans significantly decreased waist circumference at week 4 and week 8 when compared with the baseline values (p < 0.05 all) and significantly decreased hip circumference at week 8 when compared with the baseline and week 4 values (p < 0.05 all). Serum leptin levels of the CQ-treated group were significantly higher at week 8 compared to baseline levels (p < 0.05). In hWA, glycerol release was reduced in the CQ-treated group when compared with the vehicle-treated group. In the browning experiment, pioglitazone, the PPAR-γ agonist, increased UCP1 mRNA when compared to vehicle (p < 0.01). Interestingly, 10, 100, and 1000 ng/ml CQ extract treatment on hWA significantly enhanced UCP1 expression in a dose-dependent manner when compared to pioglitazone treatment (p < 0.001 all). In conclusion, CQ decreased waist and hip circumferences in obese humans and enhanced UCP1 mRNA in hWA suggestive of its action via browning of hWA.
Assuntos
Cissus/química , Obesidade Abdominal/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Proteína Desacopladora 1/genética , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Brancos/efeitos dos fármacos , Adulto , Feminino , Humanos , Leptina/genética , Lipólise/efeitos dos fármacos , Masculino , Obesidade Abdominal/patologia , Extratos Vegetais/química , RNA Mensageiro/genéticaRESUMO
BACKGROUND: Vibratory angioedema (VA) is a subtype of chronic inducible urticaria that manifests with erythematous wheals or angioedema after skin exposure to vibration. Because the condition is rare, the available information is limited. OBJECTIVE: To systematically review the clinical manifestations and treatment options of VA. METHODS: Relevant literature published until August 2020 was searched using the terms "vibratory urticaria," "vibratory angioedema," "vibratory-induced angioedema," and "vibratory-induced urticaria." Preferred Reporting Items for Systematic Reviews and Meta-analysis recommendations were applied to this systematic review. RESULTS: On the basis of review of 22 studies (16 case reports, 4 case series, and 2 cohort studies) that had a combined total of 83 patients, we propose that VA be classified as hereditary VA (33.7%) and acquired VA (66.3%). Vibration-induced itching was frequent in both subgroups. Patients with hereditary VA more commonly had wheals and systemic symptoms, whereas patients with acquired VA more frequently had angioedema, burning, pain, or tingling. Although many VA treatments are used, there is little information on their efficacy. Most patients do not achieve complete control. CONCLUSIONS: The novel VA classification proposed could help clinicians with the diagnostic workup of patients with VA. Because of the paucity of reported cases, firm recommendations for the treatment of VA are currently not possible. For patients with acquired VA, we suggest second-generation H1-antihistamines as the first-line treatment. Controlled therapeutic trials are needed and should be performed.
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Angioedema , Urticária Crônica , Hipersensibilidade Imediata , Urticária , Angioedema/diagnóstico , Angioedema/terapia , Humanos , Hipersensibilidade Imediata/congênito , Urticária/diagnóstico , Urticária/terapiaRESUMO
The herb Centella asiatica has long been considered a memory tonic. A recent review found no strong evidence for improvement of cognitive function, suggesting negative results were due to limitations in dose, standardization and product variation. We used a standardized extract of C. asiatica (ECa 233) to study behavioral, cellular and molecular effects on learning and memory enhancement. ECa 233 (10, 30, and 100 mg/kg) was given orally to normal rats twice a day for 30 days. We used the Morris water maze to test spatial learning and performed acute brain slice recording to measure changes of synaptic plasticity in the hippocampus, a core brain region for memory formation. Plasticity-related protein expressions (NR2A, NR2B, PSD-95, BDNF and TrkB) in hippocampus was also measured. Rats receiving 10 and 30 mg/kg doses showed significantly enhanced memory retention, and hippocampal long-term potentiation; however, only the 30 mg/kg dose showed increased plasticity-related proteins. There was an inverted U-shaped response of ECa 233 on memory enhancement; 30 mg/kg maximally enhanced memory retention with an increase of synaptic plasticity and plasticity-related proteins in hippocampus. Our data clearly support the beneficial effect on memory retention of a standardized extract of Centella asiatica within a specific therapeutic range.
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Centella/química , Memória/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Proteína 4 Homóloga a Disks-Large , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Plasticidade Neuronal/efeitos dos fármacos , Ratos Wistar , Receptor trkB/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Aprendizagem Espacial/efeitos dos fármacos , Memória Espacial/efeitos dos fármacos , Triterpenos/sangueRESUMO
Bacopa monnieri has been used in Ayurvedic medicine as a memory enhancer for a long time; however, its direct effect on synaptic plasticity has not been investigated. To the best of our knowledge, this study is the first to report the effect of B. monnieri on long-term synaptic potentiation in acute hippocampal slices. Adult male Wistar rats were orally administered either sterile water or the ethanolic extract of B. monnieri for 60 days. The extracellular recording was performed to measure the field excitatory postsynaptic potential in the acute hippocampal slices of these rats. Our results showed that B. monnieri extract significantly increased long-term potentiation magnitude compared with the control group, whereas there was no change in basal synaptic transmission. The data support the beneficial mnemonic effect of B. monnieri, and suggest that this effect might be because of the increase of learning-associated synaptic machinery, resulting in the long-term potentiation enhancement and strengthening of hippocampal synapses, which plays a critical role in learning and memory formation.
Assuntos
Bacopa , Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Nootrópicos/farmacologia , Extratos Vegetais/farmacologia , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Masculino , Nootrópicos/administração & dosagem , Extratos Vegetais/administração & dosagem , Ratos , Ratos Wistar , Transmissão Sináptica/efeitos dos fármacosRESUMO
Abstract Mirror-image pain is a kind of pain that occurs on the contralateral side, but its pathogenesis remains unclear. Objective To develop an osteoarthritis mouse model for investigating mirror-image pain through observing nocifensive behaviors, histological changes, and nociceptive activity at days 3, 7, 14, 21, and 28 after the chemical induction of unilateral temporomandibular joint (TMJ) osteoarthritis. Methodology We randomly divided 6-week-old mice into sham and complete Freund adjuvant groups. To induce nocifensive behaviors, we applied 0.04 g of von Frey filament, 10 psi of air puff, and cold acetone on both sides of whisker pads at different days. The histology of TMJ on both sides was observed by hematoxylin/eosin staining and microcomputed tomography scanning. Furthermore, the nociceptive activity was evaluated using the phosphorylated cyclic AMP response element binding protein (pCREB) and a microglia marker at different days in the trigeminal subnucleus caudalis. Results Nocifensive behaviors against mechanical and temperature stimuli on the contralateral side became stronger than the baseline on day 28, in agreement with the elevation of the pCREB and the microglia marker in the trigeminal subnucleus caudalis. Thus, hypernociception on the contralateral side occurred at day 28. Conclusions Clearly, the TMJ model with unilateral osteoarthritis exhibited mirror-image pain. Therefore, this model is useful in investigating the pathogenesis of pain and in developing treatments.
Assuntos
Animais , Camundongos , Osteoartrite/diagnóstico por imagem , Articulação Temporomandibular , Dor , Adjuvante de Freund , Microtomografia por Raio-XRESUMO
In using chromaffin cells as a model for studying the mechanism of regulated exocytosis, there is a requirement for an efficient, safe, and robust system for the transduction and expression of heterologous cDNA in these cells. We have used Semliki Forest virus to transduce cDNAs encoding various proteins fused to enhanced green fluorescent protein (EGFP) into cultured bovine adrenal cells. Transduction is highly efficient but has no significant effect on the steady state levels of several endogenous proteins or of catecholamines in the transfected cells. Furthermore, the transfected cells show depolarization-induced calcium currents and nicotine-induced catecholamine release. We present data to show that virally transduced proteins are targeted to their intracellular locations correctly in chromaffin cells. The fusion protein pro-ANF-EGFP is specifically targeted to large dense-core vesicles as shown by its colocalization with acidophilic dyes and chromogranin A, making this a useful system for the study of secretory vesicle dynamics.
Assuntos
Células Cromafins/metabolismo , Técnicas de Transferência de Genes , Vírus da Floresta de Semliki/genética , Transdução Genética , Animais , Bovinos , Células Cultivadas , DNA Complementar/metabolismo , Exocitose , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Proteínas Recombinantes de Fusão/metabolismo , Fatores de TempoRESUMO
Fluorescent fusion proteins are an important tool for the study of vesicle trafficking and exocytosis, especially when combined with newer types of microscopy. We previously reported that the design of a vesicle-targeted fluorescent fusion construct strongly influences the kinetics of fluorescence change at exocytosis. In the present study we demonstrate that the cell in which a construct is expressed also affects the kinetics of fluorescence change at exocytosis. We fused enhanced green fluorescent protein to the carboxy terminus of the vesicular cargo protein rodent islet amyloid polypeptide. The two proteins were separated by a "linker" sequence of 18 amino acids. We then compared kinetics of fluorescence change at exocytosis for this fluorescent cargo protein expressed in three different types of peptidergic endocrine cell: pancreatic alpha cell, pancreatic beta cell, and adrenal chromaffin cell. In resting cells of all three types, fluorescent spots of similar size and membrane-proximal density appeared near the plasma membrane as expected if the probe is stored in large dense-core secretory vesicles. Upon stimulation, the fluorescent spots displayed sudden changes in fluorescence intensity that were consistent with exocytosis. In beta and alpha cells the fluorescent spots consistently brightened and persisted, whereas in chromaffin cells the fluorescent spots always dispersed rapidly. Thus, for fluorescent cargo proteins in peptidergic endocrine cells, cell type influences the kinetics of fluorescence change at exocytosis. Together with our previous findings, this observation strongly highlights the fact that the behavior of vesicle-targeted fluorescent cargo may be unrelated to that of native cargo, and it emphasizes the need for caution in interpreting fluorescence kinetics in terms of an exocytosis mechanism.