RESUMO
BACKGROUND: Medicine is a high-status, high-skill occupation which has traditionally provided access to good quality jobs and relatively high salaries. In Ireland, historic underfunding combined with austerity-related cutbacks has negatively impacted job quality to the extent that hospital medical jobs have begun to resemble extreme jobs. Extreme jobs combine components of a good quality job-high pay, high job control, challenging demands, with those of a low-quality job-long working hours, heavy workloads. Deteriorating job quality and the normalisation of extreme working is driving doctor emigration from Ireland and deterring return. METHODS: Semi-structured qualitative interviews were conducted with 40 Irish emigrant doctors in Australia who had emigrated from Ireland since 2008. Interviews were held in July-August 2018. RESULTS: Respondents reflected on their experiences of working in the Irish health system, describing hospital workplaces that were understaffed, overstretched and within which extreme working had become normalised, particularly in relation to long working hours, fast working pace, doing more with less and fighting a climate of negativity. Drawing on Hirschman's work on exit, voice and loyalty (1970), the authors consider doctor emigration as exit and present respondent experiences of voice prior to emigration. Only 14/40 respondent emigrant doctors intend to return to work in Ireland. DISCUSSION: The deterioration in medical job quality and the normalisation of extreme working is a key driver of doctor emigration from Ireland, and deterring return. Irish trained hospital doctors emigrate to access good quality jobs in Australia and are increasingly likely to remain abroad once they have secured them. To improve doctor retention, health systems and employers must mitigate a gainst the emergence of extreme work in healthcare. Employee voice (about working conditions, about patient safety, etc.) should be encouraged and used to inform health system improvement and to mitigate exit.
Assuntos
Atitude do Pessoal de Saúde , Emigrantes e Imigrantes/psicologia , Médicos Graduados Estrangeiros/psicologia , Médicos Graduados Estrangeiros/estatística & dados numéricos , Satisfação no Emprego , Área de Atuação Profissional/estatística & dados numéricos , Adulto , Austrália , Emigrantes e Imigrantes/estatística & dados numéricos , Emigração e Imigração/estatística & dados numéricos , Feminino , Humanos , Entrevistas como Assunto , Irlanda/etnologia , Masculino , Médicos/psicologia , Médicos/estatística & dados numéricos , Carga de Trabalho/psicologia , Carga de Trabalho/estatística & dados numéricos , Adulto JovemRESUMO
Endochondral ossification during long bone development and natural fracture healing initiates by mesenchymal cell condensation, directed by local morphogen signals and mechanical cues. Here, we aimed to mimic development for regeneration of large bone defects. We hypothesized that engineered human mesenchymal condensations presenting transforming growth factor-ß1 (TGF-ß1) and/or bone morphogenetic protein-2 (BMP-2) from encapsulated microparticles promotes endochondral defect regeneration contingent on in vivo mechanical cues. Mesenchymal condensations induced bone formation dependent on morphogen presentation, with BMP-2 + TGF-ß1 fully restoring mechanical function. Delayed in vivo ambulatory loading significantly enhanced the bone formation rate in the dual morphogen group. In vitro, BMP-2 or BMP-2 + TGF-ß1 initiated robust endochondral lineage commitment. In vivo, however, extensive cartilage formation was evident predominantly in the BMP-2 + TGF-ß1 group, enhanced by mechanical loading. Together, this study demonstrates a biomimetic template for recapitulating developmental morphogenic and mechanical cues in vivo for tissue engineering.
Assuntos
Desenvolvimento Ósseo/fisiologia , Osso e Ossos/fisiologia , Morfogênese/fisiologia , Osteogênese/fisiologia , Animais , Biomimética/métodos , Osso e Ossos/metabolismo , Células Cultivadas , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Ratos , Engenharia Tecidual , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
International advocacy of patient-centred healthcare delivery has led to emphasis on the (re)design and evaluation of healthcare processes and outcomes from a patient perspective. Patient-reported outcome measures (PROMs) have significant potential to inform such attempts. However there is limited understanding of the processes by which this can be achieved. This exploratory study followed attempts to utilise two different PROMs measures to support service quality improvement in clinical genetics. PROMs used were the Genetic Counseling Outcome Scale (GCOS-24), a well-validated clinical genetics-specific PROM and Euroqol (EQ-5D), a generic PROM favoured by the UK National Institute for Health and Excellence (NICE). Both of these PROMs enable pre/post intervention comparison. A service audit tool was also used, premised on a patient-reported experience measure. In addition, the study draws on interviews with clinical staff to identify challenges associated with the use of PROMs (response rate, data collection, analysis). Benefits are also explored and include the provision of insight into patients' needs; complementing clinical judgement; identification of needs being met, evidencing the benefit of services provided; prompting consideration of areas requiring attention; and encouraging professional development.
Assuntos
Aconselhamento Genético/organização & administração , Medidas de Resultados Relatados pelo Paciente , Melhoria de Qualidade/organização & administração , Testes Genéticos , Humanos , Assistência Centrada no Paciente/organização & administração , Qualidade de VidaRESUMO
We describe the case of a 37-year-old man with a slowly enlarging neck lump and compressive symptoms. He presented to a separate institution 10 years prior where an observational approach was advocated. Following preoperative investigations and embolization, an 11cm vagal schwannoma was excised and vagus nerve was sacrificed. Although conservative management is appropriate for a select patient population, surgical excision is treatment of choice for cervical neurogenic tumours and paraganglionomas and must be considered in young patients or rapidly expanding tumours to avoid compressive symptoms, as in this case.
RESUMO
Antimicrobial peptides (AMPs) are part of the immune system in a wide range of organisms. They generally carry positive charges under physiological conditions, allowing them to accumulate on the negatively charged bacterial membrane as the first step of bactericidal action. The concentration range of AMPs necessary for rapid killing of bacteria tested in vitro is much higher than levels found at epithelial surfaces and body fluids in vivo, and close to the a level that is toxic to the host cells. It is likely that AMPs in vivo are localized and act cooperatively to enhance antimicrobial activity, while the global concentration is low thus demonstrating low toxicity to host cells. Herein we employed well-defined mixed self-assembled monolayers (SAMs) to localize LL-37, one of the most studied AMPs, via electrostatic interactions. We systematically varied the surface density of LL-37, and found that the immobilized AMPs not only attracted bacteria Pseudomonas aeruginosa to the surface, but also killed nearly all bacteria when above a threshold density. More significantly, the AMPs displayed low toxicity to human corneal epithelial cells. The results indicated that localization of AMPs on suitable polyanion substrates facilitated the bactericidal activity while minimizing the cytotoxicity of AMPs.
Assuntos
Ânions/química , Peptídeos Catiônicos Antimicrobianos/química , Ácidos Carboxílicos/química , Catelicidinas/química , Células Epiteliais/química , Pseudomonas aeruginosa/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Catelicidinas/metabolismo , HumanosRESUMO
INTRODUCTION: Diabetes is a chronic disease amenable to management in the community and outpatient setting. The increasing incidence of diabetes places outpatient endocrinology services under pressure to provide a quality service in a timely manner. Our aim was to apply lean thinking to the diabetes clinic in a tertiary referral centre in the West of Ireland to improve flow, as reflected in reduced patient journey times. METHODS: The project lasted 6 months, from January to June 2011. An introductory seminar on lean thinking was arranged to inform and motivate the Diabetes Day Centre staff. Two 'rapid improvement events' took place. Value stream mapping (VSM) was the predominant lean tool employed. Patient journeys were mapped and quantified (minutes) using timesheets allocated to each step in the process at baseline, and following intervention. Data were analysed using Minitab V.16.0. RESULTS: VSM allowed the value-adding and problem-causing steps in the patient journey through the diabetes clinic process to be identified and addressed. Total patient journey time through the clinic was significantly reduced from 118 (± 38.02) min to 58 (± 18.30) min (p<0.001). CONCLUSIONS: This project reflects the successful application of VSM as a lean tool in a pilot study at our institution as evidenced by improved patient flow and a significant reduction in patient journey time through the clinic. Through the incorporation of Lean into the ethos of the hospital, we have the potential to deliver excellent care in a safe environment and in an efficient manner, while benefiting the patient, employees and tax-payer.
Assuntos
Diabetes Mellitus/terapia , Centros de Atenção Terciária/organização & administração , Gestão da Qualidade Total/métodos , Fluxo de Trabalho , Assistência Ambulatorial , Humanos , Irlanda , Projetos Piloto , Melhoria de QualidadeAssuntos
Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Infecções Oculares/tratamento farmacológico , Olho/microbiologia , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Modelos Animais de Doenças , Olho/imunologia , Previsões , Humanos , Camundongos , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacosRESUMO
BACKGROUND/AIMS: Pseudomonas aeruginosa is a major cause of severe bacterial keratitis and remains a difficult clinical entity to treat successfully with the current arsenal of antimicrobial agents. Defensins are small cationic peptides with broad in vitro antimicrobial activity and are potential ocular therapeutic agents. The authors characterised the in vitro activity of defensins NP-1 and NP-3a against P aeruginosa in the presence of human tears. METHODS: A clinical Pseudomonas isolate was grown to mid-log phase, and 1 x 10(6 )colony forming units were exposed to the peptides (200 microg/ml) for up to 2 hours in the presence of varying concentrations (10-70%) of human tears. RESULTS: For both peptides in the presence of 10% tears, >3 log units of killing was achieved within 30 minutes. In 70% tears, NP-1 produced >1 log unit of killing at 2 hours, indicating that, although reduced, its activity remained significant. In 20% tears, NP-3a demonstrated 2 log units of killing at 2 hours; however, the antimicrobial activity of this defensin was completely inhibited in the presence of 70% tears. CONCLUSION: These in vitro data suggest that while the microbicidal activity of some defensins may be diminished at the ocular surface in vivo, significant activity is still possible with certain peptides.
Assuntos
Antibacterianos/farmacologia , Defensinas/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Lágrimas , Animais , Humanos , Testes de Sensibilidade Microbiana , Coelhos , alfa-Defensinas/farmacologiaRESUMO
PURPOSE: Human beta-defensins 1 and 2 (hBD-1, -2) are antimicrobial peptides found in several epithelia including corneal epithelium. Breach of the epithelium leaves the cornea vulnerable to infection so we sought to determine if there is an increase in defensin expression after injury. METHODS: The epithelium from human cadaver corneas was collected by scraping (original samples). The corneas were then placed into organ culture to permit regeneration of the epithelium. Samples of re-grown epithelium were collected when healing was partially and 100% complete as determined by fluorescein staining. Total RNA was extracted from original and re-grown samples and used in RT-PCR reactions using primers specific for hBD-1 and hBD-2 and the constitutively expressed gene glyceraldehyde-3-phosphate dehydrogenase. Immunoblotting was performed to detect defensin peptide in original and re-grown samples. RESULTS: hBD-1 mRNA was detected in all original epithelial tissue samples (n = 10) examined suggesting that it is constitutively expressed. hBD-2 mRNA was detectable in only two of the ten samples. Of six corneas placed in to organ culture, hBD-1 mRNA expression was unchanged in the re-grown epithelial samples compared to the original epithelium samples, however the expression of hBD-2 mRNA was markedly increased. hBD-1 and hBD-2 peptides showed the same pattern of expression as their respective transcripts. CONCLUSIONS: These data show that hBD-2 mRNA and peptide is up-regulated in the corneal epithelium during re-epithelialization which is in keeping with the role of this defensin as an antimicrobial peptide.
Assuntos
Anti-Infecciosos/metabolismo , Epitélio Corneano/fisiologia , Proteínas do Olho/metabolismo , beta-Defensinas/metabolismo , Idoso , Primers do DNA/química , Epitélio Corneano/citologia , Proteínas do Olho/genética , Humanos , Técnicas de Cultura de Órgãos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima , beta-Defensinas/genéticaRESUMO
Organosilicone molecules are important surfactant ingredients used in formulating pesticides. These methylated silicones are considered inert ingredients, but their superior surfactant properties allow them to wet, and either suffocate or disrupt important physiological processes in mites and insects. Aqueous solutions of the tri-siloxane surfactants Silwet L-77, Silwet 408, and Silwet 806 were bioassayed against adult female two-spotted spider mites, Tetranychus urticae Koch, with leaf dip methods to compare their toxicity with organosilicone molecules containing bulkier hydrophobic components. All three tri-siloxanes in aqueous solutions were equivalently toxic (LC50 = 5.5-8.9 ppm), whereas Silwet L-7607 solutions were less toxic (LC50 = 4,800 ppm) and Silwet L-7200 was nontoxic to mites. In another experiment, the toxicity of Silwet L-77 was affected by the wettability of leaf surfaces. The LC50 shifted from 22 to 84 ppm when mites were tested on bean and strawberry leaf disks, respectively. Droplet spreading on paraffin and surface tension were both related to the toxicity of surfactant solutions. Surface tensions of solutions below 23 mN/m caused > 90% mite mortality in leaf dip bioassays. A field test of Conserve SC and its formulation blank, with and without Dyne-Amic adjuvant (a vegetable oil-organosilicone surfactant mixture) revealed that Dyne-Amic had the greatest miticidal contribution, reducing mite populations by 70%, followed by formulation inactive ingredients. Spinosad, the listed active ingredient in Conserve, only contributed miticidal activity when synergized by Dyne-Amic. Researchers should include appropriate surfactant or formulation blank controls when testing insecticides or miticides, especially when using high spray volumes.
Assuntos
Ácaros , Compostos de Organossilício , Controle de Pragas , Tensoativos , Animais , Feminino , Controle de Pragas/métodos , Folhas de Planta , SoluçõesRESUMO
PURPOSE: To examine the effect of elevated extracellular glucose, thus simulating diabetes, on migration, adhesion and proliferation of SV40 transformed human corneal epithelial (HCE) cells. METHODS: HCE cells were maintained in serum supplemented media containing 5 mM, 17.5 mM or 38 mM D-glucose. Cell migration was determined using Blind well chambers fitted with fibronectin/collagen I coated filters. In adhesion experiments, cells were allowed to adhere to extracellular matrix protein-coated wells for 90 min at 37 degrees C. Non-adherent cells were removed by washing, then the fluorochrome calcein-AM was added to quantitate the number of attached cells. Proliferation was determined by plating the cells at low density, then quantitating viable cells with calcein-AM 5 to 7 days later. RESULTS: Raising extracellular glucose from 5 mM to 17.5 mM significantly increased cell migration by 42%. When glucose was further raised to 38 mM, migration was not significantly different from that in 5 mM glucose. Adhesion to fibronectin and collagen I (but not IV) was significantly increased (62% and 32% respectively) when cells were cultured in 17.5 mM glucose. Similarly, proliferation was increased by 44%. Adhesion and proliferation tended to be decreased at 38 mM compared to 17.5 mM glucose, but not significantly so. In the presence of 5 mM glucose and mannitol (12.5 mM or 33 mM), neither migration, adhesion nor proliferation were significantly different from that in 5 mM glucose alone. CONCLUSION: Elevated extracellular glucose modulates migration, adhesion and proliferation of HCE cells. The effects are dependent on the concentration of glucose and are not due to changes in osmolality since mannitol failed to produce similar results. Our in vitro findings suggest that high-glucose effects may directly contribute to the etiology of impaired corneal wound healing in diabetes.
Assuntos
Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Córnea/citologia , Glucose/farmacologia , Linhagem Celular Transformada , Células Cultivadas , Colágeno/metabolismo , Córnea/fisiologia , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Fibronectinas/metabolismo , Fluoresceínas , Corantes Fluorescentes , Humanos , Vírus 40 dos SímiosRESUMO
PURPOSE: The purpose of the study was to examine the effect of topical application of substance P (SP), insulin-like growth factor-1 (IGF-1) and vasoactive intestinal polypeptide (VIP) on corneal re-epithelialization in galactosemic rats. METHODS: Experimental galactosemia was induced by feeding a diet containing 30% galactose for 4-6 months. The corneal epithelium was debrided bi-laterally by scraping with a blunted scalpel blade. One eye (control) received only a saline solution whilst the other eye received a solution of SP and/or IGF-1 or VIP. A single drop of control or test solution was administered 4 times daily until wound closure. Corneas were stained with fluorescein and videotaped under ultraviolet illumination at regular time intervals after debridement. After digitizing the video image, the wound area was calculated using an image analysis program (NIH Image). RESULTS: Corneal re-epithelialization was significantly delayed in galactosemic compared to normal animals. Rates of healing were 1.3 +/- 0.07 mm2/hour and 1.02 +/- 0.02 mm2/hour for normal and galactosemic animals, respectively. Topical application of SP in concentrations ranging from 25 pg/ml up to 250 microg/ml had no significant effect on the rate of corneal re-epithelialization. Similarly, IGF-1 (1 microg/ml) or VIP (1 microg/ml) when applied alone did not affect re-epithelialization. Furthermore, resurfacing of the debrided area was not affected by co-application of SP (250 microg/ml) and IGF-1 or VIP. CONCLUSIONS: Independent or combined topical application of SP, VIP or IGF-1 at the concentrations tested, does not modulate corneal epithelial wound healing in rats with galactosemia induced by 30% galactose.
Assuntos
Córnea/fisiologia , Galactosemias/fisiopatologia , Fator de Crescimento Insulin-Like I/farmacologia , Regeneração/fisiologia , Substância P/farmacologia , Peptídeo Intestinal Vasoativo/farmacologia , Cicatrização/fisiologia , Animais , Córnea/citologia , Córnea/efeitos dos fármacos , Desbridamento , Quimioterapia Combinada , Epitélio/lesões , Epitélio/fisiologia , Galactose , Galactosemias/induzido quimicamente , Processamento de Imagem Assistida por Computador , Fator de Crescimento Insulin-Like I/administração & dosagem , Masculino , Soluções Oftálmicas , Ratos , Ratos Sprague-Dawley , Regeneração/efeitos dos fármacos , Substância P/administração & dosagem , Peptídeo Intestinal Vasoativo/administração & dosagem , Cicatrização/efeitos dos fármacosRESUMO
The major substrate of protein kinase C(PKC) in platelets is the 40 kDa protein, pleckstrin. Addition of the homobifunctional reagent, bis(sulphosuccinimidyl)suberate (BS3), to platelet lysate, cytosol fraction or to electropermeabilized platelets resulted in cross-linking of pleckstrin to give higher-molecular-mass complexes of 68 kDa, 90 kDa and 100-120 kDa respectively, which were visualized by immunoblotting with an anti-pleckstrin antibody. Higher levels of cross-linking were observed in permeabilized platelets than in platelet lysates. The yields of the cross-linked complexes were much reduced after dilution of platelet lysate or lysis of electropermeabilized platelets and, in the case of the 90 kDa and 100-120 kDa species, after activation of PKC by phorbol 12-myristate 13-acetate. Similar experiments with purified pleckstrin indicated that the 90 kDa and 100-120 kDa species consist, at least in part, of pleckstrin dimers and higher oligomers. After incubation of purified pleckstrin (0.45 mg/ml) for 1 h with 2 mM BS3, about 25% of the protein was present in cross-linked species. The results indicate that pleckstrin undergoes a reversible self-association that can be prevented by phosphorylation of the protein, and also interacts with an unidentified platelet protein of about 28 kDa.
Assuntos
Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Fosfoproteínas , Proteína Quinase C/metabolismo , Plaquetas/química , Proteínas Sanguíneas/química , Permeabilidade da Membrana Celular , Reagentes de Ligações Cruzadas , Ativação Enzimática , Humanos , Conformação Proteica , Proteína Quinase C/efeitos dos fármacos , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Succinimidas , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Male Sprague-Dawley rats were injected ip with 1 microgram pertussis toxin (PTX)/100 g BW. The rats were killed 24, 48, and 72 h after injection, and their pancreases were removed. At each time point, insulin secretion by isolated islets was measured under basal and glucose-stimulated conditions and in the absence and presence of norepinephrine. cAMP levels were measured under basal and forskolin-stimulated conditions in the absence and presence of norepinephrine. PTX-induced ADP ribosylation of Gi/Go proteins in vivo was monitored by ADP ribosylation in vitro using PTX and 32P-labeled NAD and also by Western blotting. At 24 h, 1 microM norepinephrine inhibited glucose-stimulated insulin secretion by 92% in the control islets, but by only 53% in the PTX-treated islets; at 48 h, norepinephrine still inhibited secretion (by 40%) in the PTX-treated islets; at 72 h, the inhibitory effect of norepinephrine was abolished. Therefore, contrary to recent suggestions, all of the effect of norepinephrine to inhibit insulin release is PTX sensitive. The effects of PTX on the ability of norepinephrine to lower cAMP levels were similar to those observed for the inhibition of insulin release. PTX partially blocked the effect of norepinephrine to lower cAMP levels at 24 and 48 h, and the block was complete after 72 h. The extent of the in vivo ADP ribosylation of the Gi/Go proteins, monitored at each time point by in vitro [32P]ADP-ribosylation and Western blotting, demonstrated a profound ADP ribosylation at 48 and 72 h. As detected by Western blotting, the rates of ADP ribosylation by PTX and the onset of decreased expression varied among the different G-proteins. G alpha o was virtually eliminated after only 24 h of PTX treatment. G alpha i2 was markedly affected by 48 h; G alpha i3 was little affected until 72 h.
Assuntos
Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Norepinefrina/farmacologia , Toxina Pertussis , Fatores de Virulência de Bordetella/farmacologia , Adenosina Difosfato Ribose/metabolismo , Animais , Western Blotting , Colforsina/farmacologia , AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/isolamento & purificação , Proteínas de Ligação ao GTP/metabolismo , Glucose/farmacologia , Técnicas In Vitro , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Masculino , NAD/metabolismo , Norepinefrina/antagonistas & inibidores , Propranolol/farmacologia , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Inhibition of adenylyl cyclase activity is one of at least four mechanisms by which the neuropeptide galanin inhibits insulin secretion from pancreatic beta-cells. In a membrane preparation of the insulin-secreting cell line RINm5F, a maximally effective concentration of galanin inhibited forskolin-stimulated adenylyl cyclase activity by 30%. Pretreatment of the cells with pertussis toxin abolished the inhibitory effect of galanin, indicating the involvement of Gi or Go guanine nucleotide binding proteins (G-proteins). Because galanin receptors interact with four G-proteins (Gi1, Gi2, Gi3, and Go1), any or all of these may inhibit adenylyl cyclase. Therefore, to identify the G-protein(s) involved, antibodies raised against various G-protein alpha-subunits were used to block the inhibition of forskolin-stimulated adenylyl cyclase activity by galanin in RINm5F membrane preparations. Antisera AS/7 and EC/2, specific for G alpha i1/alpha i2 and G alpha i3, respectively, were able to significantly attenuate the inhibitory effect of galanin, whereas antisera specific for Go proteins were not. The use of additional antisera specific for the various subtypes of Gi proteins indicated that Gi2 and Gi3, but not Gi1, are involved. Simultaneous application of antisera AS/7 and EC/2 resulted in a greater attenuation of the effect of galanin than application of either antiserum alone. Thus, galanin inhibition of adenylyl cyclase activity in these cells is selectively mediated by two inhibitory G-proteins, Gi2 and Gi3.
Assuntos
Inibidores de Adenilil Ciclases , Proteínas de Ligação ao GTP/fisiologia , Peptídeos/farmacologia , Toxina Adenilato Ciclase , Animais , Western Blotting , Linhagem Celular , Membrana Celular/enzimologia , Colforsina/farmacologia , Galanina , Técnicas In Vitro , Neuropeptídeos/farmacologia , Toxina Pertussis , Ratos , Transdução de Sinais , Fatores de Virulência de Bordetella/farmacologiaRESUMO
In the insulin-secreting beta cell line RINm5F, sodium fluoride stimulated exocytosis in a concentration (5-15 mM)- and temperature-dependent manner. Depletion of aluminum with the chelator deferoxamine or addition of aluminum to the buffer failed to affect the NaF-stimulated insulin release. This suggests that stimulation of heterotrimeric G proteins or inhibition of phosphatases or other enzymes by fluoroaluminate, an analog of the phosphate moiety, is not involved in the insulinotropic action of NaF. Removal of extracellular Ca2+ suppressed the NaF-stimulated insulin release. However, nitrendipine, a blocker of L-type voltage-dependent Ca2+ channels, did not inhibit the NaF-stimulated insulin release and NaF did not cause any changes in the cytosolic free calcium concentration ([Ca2+]i). Decreasing [Ca2+]i with thapsigargin or increasing [Ca2+]i with ionomycin or a depolarizing concentration of KCl resulted in suppression or enhancement of NaF-stimulated insulin release, respectively. Furthermore, NaF enhanced Ca(2+)-induced insulin release in electrically permeabilized RINm5F cells. These findings indicate that the effect of NaF on exocytosis is dependent on [Ca2+]i, although NaF itself does not change [Ca2+]i. Inhibitors of protein kinase C, such as staurosporine and bisindolylmaleimide, in concentrations sufficient to block the effects of phorbol esters, did not attenuate the NaF-stimulated insulin release. Neither cellular cAMP content nor [3H]arachidonic acid release was increased by NaF. NaF-stimulated insulin release was synergistically enhanced by the activation of protein kinases A and C. Finally, trifluoperazine, an inhibitor of calmodulin and other Ca(2+)-binding proteins, inhibited the insulinotropic action of NaF in a concentration-dependent manner. Trifluoperazine (50 microM) and W-7 (100 microM) nullified the 10 mM NaF-stimulated insulin release. It is concluded that NaF evokes exocytosis by a novel mechanism of sensitization to Ca2+, possibly on a Ca(2+)-responsive protein that is sensitive to trifluoperazine and W-7, leading to exocytosis. Protein kinases A and C also act at this site or at a more distal point.
Assuntos
Proteínas de Ligação ao Cálcio/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Exocitose/efeitos dos fármacos , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Fluoreto de Sódio/farmacologia , Alumínio/fisiologia , Animais , Ácido Araquidônico/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Linhagem Celular , Colforsina/farmacologia , AMP Cíclico/metabolismo , Desferroxamina/farmacologia , Exocitose/fisiologia , Espaço Extracelular/metabolismo , Secreção de Insulina , Ionomicina/farmacologia , Cinética , Meliteno/farmacologia , Nitrendipino/farmacologia , Cloreto de Potássio/farmacologia , Proteína Quinase C/antagonistas & inibidores , Taxa Secretória/efeitos dos fármacos , Estimulação Química , Sulfonamidas/farmacologia , Temperatura , Terpenos/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Tapsigargina , Trifluoperazina/farmacologiaRESUMO
In some cells a supersensitive adenylyl cyclase response follows hormonal inhibition of the enzyme. We have compared the effect of pre-treatment with noradrenaline in RINm5F cells to that in cells known to demonstrate supersensitivity. In NG108-15 cells, 12 or 24 hours pre-treatment with 1-10 microM noradrenaline markedly enhanced forskolin (1 microM) stimulated cAMP accumulation compared to that in untreated cells (12 h, 429 +/- 123 vs 72 +/- 8 pmol/mg protein; 24 h 190 +/- 32 vs 82 +/- 10 for treated and untreated cells, respectively). In a second cell, HT29-18, 30 min pre-treatment with 10 microM noradrenaline enhanced forskolin (10 microM) stimulated cAMP accumulation from 47 +/- 13 pmol/mg protein to 405 +/- 109 pmol/mg protein. In contrast, pre-treatment of RINm5F cells with noradrenaline under the same conditions did not enhance the forskolin response. These data indicate that noradrenaline which induces a supersensitive adenylyl cyclase response in NG108-15 and HT29-18 cells does not induce the response in the insulin secreting cell RINm5F.
Assuntos
Adenilil Ciclases/metabolismo , Norepinefrina/farmacologia , Inibidores de Adenilil Ciclases , Animais , Linhagem Celular , Colforsina/farmacologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Polipeptídeo Inibidor Gástrico/farmacologia , Glioma , Humanos , Células Híbridas , Insulinoma , Cinética , Neuroblastoma , Neoplasias Pancreáticas , Células Tumorais CultivadasRESUMO
Mastoparan, a tetradecapeptide from wasp venom, stimulated exocytosis in a concentration-dependent manner, which was enhanced by pertussis toxin pre-treatment, in the insulin secreting beta-cell line RINm5F. Mastoparan (3-20 microM) also elevated cytosolic free calcium concentration ([Ca2+]i), a rise that was not attenuated by nitrendipine. Divalent cation-free Krebs-Ringer bicarbonate (KRB) medium with 0.1 mM EGTA nullified the mastoparan-induced increase in [Ca2+]i, suggesting that the peptide increased Ca2+ influx but not through the L-type voltage-dependent Ca2+ channel. Depletion of the intracellular Ca2+ pool did not affect the mastoparan-induced elevation of [Ca2+]i. Remarkably, in divalent cation-free KRB medium with 0.1 mM EGTA and 2 microM thapsigargin in which mastoparan reduced [Ca2+]i, the mastoparan-stimulated insulin release was similar to that in normal Ca(2+)-containing KRB medium. Inhibitors of protein kinase C, such as bisindolylmaleimide, staurosporine, and 1-O-hexadecyl-2-O-methyl-rac-glycerol did not suppress the mastoparan-stimulated insulin release. Mastoparan at 10-20 microM did not increase cellular cAMP levels, nor did mastoparan at 5-10 microM affect [3H]arachidonic acid release. In conclusion, although mastoparan increased [Ca2+]i, this increase was not involved in the stimulation of insulin release. Rather, the data suggest that mastoparan directly stimulates exocytosis in a Ca(2+)-independent manner. As GTP-binding proteins (G proteins) are thought to be involved in the process of exocytosis and as mastoparan is known to exert at least some of its effects by activation of G proteins, an action of mastoparan to activate the putative stimulatory Ge (exocytosis) protein is likely.
Assuntos
Cálcio/metabolismo , Exocitose/efeitos dos fármacos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Venenos de Vespas/farmacologia , Animais , Linhagem Celular , Colforsina/farmacologia , Hidroquinonas/farmacologia , Técnicas In Vitro , Secreção de Insulina , Peptídeos e Proteínas de Sinalização Intercelular , Nitrendipino/farmacologia , Peptídeos , Toxina Pertussis , Ratos , Taxa Secretória/efeitos dos fármacos , Terpenos/farmacologia , Tapsigargina , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Insulin secretion from the beta-cells of the endocrine pancreas is subject to a pot-pourri of stimulatory, modulatory and inhibitory influences. beta-Cell secretion is reduced or blocked by a variety of inhibitors (including galanin, somatostatin and noradrenaline) which reach the cells either via the islet vascular system or are released locally from sympathetic and peptidergic nerves terminating in the pancreas. It is now becoming clear that among these many inhibitors, several have multiple mechanisms by which they inhibit release at the cellular level. Indeed, with multiple inhibitors (some of which are co-secreted) and multiple mechanisms of inhibition, the latter including a late effect in stimulus secretion coupling (perhaps on the exocytotic step per se), inhibition of insulin secretion has the characteristics of a fail-safe system.
Assuntos
Exocitose , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/fisiologia , Adenilil Ciclases/fisiologia , Animais , Glicemia/fisiologia , Cátions/metabolismo , Exocitose/efeitos dos fármacos , Proteínas de Ligação ao GTP/fisiologia , Galanina , Secreção de Insulina , Canais Iônicos/fisiologia , Ilhotas Pancreáticas/efeitos dos fármacos , Neuropeptídeos/fisiologia , Peptídeos/fisiologia , Receptores de Galanina , Receptores dos Hormônios Gastrointestinais/fisiologia , Taxa Secretória/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Whilst the CNS effects of thyrotropin releasing hormone (TRH) may result from a direct action on neurones it is also a possibility that another cell type may mediate an indirect action. A potential candidate for such a role is the astrocyte. The ability of TRH and a stable analogue RX77368 (di-methyl proline TRH) to stimulate phosphoinositide turnover has been investigated in cultured astrocytes from a number of CNS regions. Significant increases in turnover were noted in three of the four regions studied. Percentage values were: in spinal cord, 33% TRH, 31% RX77368 (n = 15); in brain stem, 33% TRH, 37% RX77368 (n = 6); in cerebellum, 72% TRH, 73% RX77368 (n = 6); in cortex, 4% TRH, 13% RX77368 (n = 6). EC50 values for both peptides were in the picomolar range. These results indicate that some astrocytes in vitro possess functional TRH receptors linked to the phosphoinositide second messenger system and hence this cell type would potentially be capable of mediating an indirect action of the peptide. Also, because the response was limited to certain regions, heterogeneity in receptor expression is implied. Furthermore, in the light of other evidence to support astrocyte heterogeneity within a region, we suggest that certain characteristics of the response seen in our experiments may be the result of TRH receptors being restricted to a subpopulation of astrocytes within a given culture. Thus, our data show that astrocytes prepared from some CNS regions possess functionally coupled TRH receptors.