RESUMO
The lamina cribrosa (LC) in glaucoma is with augmented production of extracellular matrix proteins (ECM) and connective tissue fibrosis. Fundamental pathological mechanisms for this fibrosis comprise fibrotic growth factors and oxidative stress. Transient receptor potential canonical channels (TRPC) channels play a key role in ECM fibrosis. Here, we study TRPC expression in glaucomatous LC cells, and investigate the role of TRPC in oxidative stress induced-profibrotic ECM gene transcription and cell proliferation in normal LC cells. Age-matched human LC cells (normal, n = 3 donors; glaucoma, n = 3 donors) were used. Hydrogen peroxide (H2O2, 100 µM), was used to induce oxidative stress in LC cells in the presence or absence of the pan TRPC inhibitor SKF96365 (10 µM) or knockdown of TRPC1/6 with siRNA. After treatments, ECM gene transcription, LC cell viability and proliferation and the phosphorylation of the transcription factor NFATc3, were measured using real time RT-PCR, colorimetric cell counting with the methyl-thiazolyl tetrazolium salt (MTS) assay, and Western immunoblotting, respectively. Results showed that TRPC1/C6 transcript and protein expression levels were significantly (p < 0.05) enhanced in glaucoma LC cells. Both SKF96365 and siRNA-TRPC1/C6 treatments significantly reduced the oxidative stress induced-ECM gene expression (transforming growth factor-ß1 (TGFß1), alpha smooth muscle actin (α-SMA), and collagen type 1A1 (Col1A1)), and cell proliferation in normal and glaucoma LC cells. Also, SKF96365 treatment inhibited the H2O2-induced NFATc3 protein dephosphorylation in LC cells. In conclusion, TRPC1/C6 expression is enhanced in glaucoma LC cells. These channels may contribute to oxidative stress-induced ECM gene transcription and cell proliferation in normal and glaucoma LC cells through Ca2+-NFATc3 signaling pathway mechanism. TRPC1 and TRPC6 channels could be important therapeutic targets to prevent ECM remodeling and fibrosis development in glaucoma optic neuropathy.
Assuntos
Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica , Glaucoma/genética , Disco Óptico/patologia , RNA/genética , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6/genética , Western Blotting , Proliferação de Células , Células Cultivadas , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Glaucoma/metabolismo , Glaucoma/patologia , Humanos , Disco Óptico/metabolismo , Canais de Cátion TRPC/biossíntese , Canal de Cátion TRPC6/biossíntese , Transcrição GênicaRESUMO
PURPOSE: This study examines the effect of the L-type calcium channel blocker verapamil on mechanical strain-induced extracellular matrix genes in optic nerve head lamina cribrosa (LC) cells. METHODS: Changes in LC cell intracellular calcium [Ca(2+)]i following hypotonic cell membrane stretch were measured with the fluorescent probe fura-2/AM. Fluorescence intensity was measured, after labelling, by calcium (Ca2+) imaging confocal microscopy. Confluent human LC cell cultures were serum starved for 24â h prior to exposure to cyclical mechanical strain (1â Hz, 15%) for 24â h in the presence or absence of verapamil (10â mm). Transforming growth factor-ß 1 (TGF-ß1), collagen 6A3 (COL6A3) and chondroitin sulfate proteoglycan 2 (CSPG2) mRNA expression levels were assessed by quantitative RT-PCR. RESULTS: Hypotonic cell membrane stretch of LC cells from normal donors significantly increased [Ca2+]i (p<0.05). Exposure to cyclical mechanical strain (15% strain) produced a statistically significant increase in the three matrix genes that were examined (TGF-ß1, COL6A3 and CSPG2). This response in both cyclical and mechanical stretch was significantly reduced by pretreating LC cells with the L-type calcium channel blocker verapamil (p<0.05). CONCLUSIONS: This study provides evidence of a novel mechanotransduction pathway linking mechanical strain, cation channel function and the induction of LC cell matrix gene transcription. This highlights the potential involvement of calcium influx in the activation of matrix remodelling responses in the optic nerve head and supports the rationale that calcium channel blockers may attenuate disease progression in glaucoma.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/fisiologia , Matriz Extracelular/genética , Regulação da Expressão Gênica/fisiologia , Mecanotransdução Celular/efeitos dos fármacos , Verapamil/farmacologia , Idoso , Idoso de 80 Anos ou mais , Cálcio/metabolismo , Células Cultivadas , Colágeno Tipo VI/genética , Fura-2/análogos & derivados , Fura-2/metabolismo , Humanos , Mecanotransdução Celular/fisiologia , Microscopia Confocal , Disco Óptico/citologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Estresse Mecânico , Fator de Crescimento Transformador beta1/genética , Versicanas/genéticaRESUMO
PURPOSE: Oxidative stress is implicit in the pathological changes associated with glaucoma. The purpose of this study was to compare levels of oxidative stress in glial fibrillary acid-negative protein (GFAP) lamina cribrosa (LC) cells obtained from the optic nerve head (ONH) region of 5 normal (NLC) and 4 glaucomatous (GLC) human donor eyes and to also examine mitochondrial function and calcium homeostasis in this region of the ONH. METHODS: Intracellular reactive oxygen species (ROS) production was examined by a thiobarbituric acid reactive substances (TBARS) assay which measures malondialdehyde (MDA), a naturally occurring product of lipid peroxidation and is used as an indicator of oxidative stress. Mitochondrial membrane potential (MMP) and intracellular calcium ([Ca(2+)](i)) levels were evaluated by flow cytometry using the JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetrabenzimidazolecarbocyanine iodide) and fluo-4/AM probes respectively. Anti-oxidant and Ca(2+) transport system gene and protein expression were determined by real time polymerase chain reaction (RT-PCR) using gene-specific primer/probe sets and western immunoblotting, respectively. RESULTS: Intracellular ROS production was increased in GLC compared to NLC (27.19 ± 7.05 µM MDA versus 14.59 ± 0.82 µM MDA, p < 0.05). Expression of the anti-oxidants Aldo-keto reductase family 1 member C1 (AKR1C1) and Glutamate cysteine ligase catalytic subunit (GCLC) were significantly lower in GLC (p = 0.02) compared to NLC control. MMP was lower in GLC (57.5 ± 6.8%) compared to NLC (41.8 ± 5.3%). [Ca(2+)](i) levels were found to be higher (p < 0.001) in GLC cells compared to NLC. Expression of the plasma membrane Ca(2+)/ATPase (PMCA) and the sodium-calcium (NCX) exchangers were lower, while intracellular sarco-endoplasmic reticulum Ca(2+)/ATPase 3 (SERCA) expression was significantly higher in GLC compared to NLC. Subjection of NLC cells to oxidative stress (200 µM H(2)0(2)) reduced expression of Na(+)/Ca2(+) exchanger 1 (NCX 1), plasma membrane Ca2+ ATPase 1 (PMCA 1), and PMCA 4 as determined by RT-PCR. CONCLUSIONS: Our data finds evidence of oxidative stress, mitochondrial dysfunction and impaired calcium extrusion in GLC cells compared to NLC cells and suggests their importance in the pathological changes occurring at the ONH in glaucoma. Future therapies may target reducing oxidative stress and / or [Ca(2+)](i).
Assuntos
Astrócitos/metabolismo , Cálcio/metabolismo , Lâmina Limitante Posterior/metabolismo , Glaucoma/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Mitocôndrias/metabolismo , Disco Óptico/metabolismo , 20-Hidroxiesteroide Desidrogenases/genética , 20-Hidroxiesteroide Desidrogenases/metabolismo , Idoso , Idoso de 80 Anos ou mais , Astrócitos/citologia , Western Blotting , Estudos de Casos e Controles , Técnicas de Cultura de Células , Lâmina Limitante Posterior/citologia , Lâmina Limitante Posterior/patologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Glaucoma/patologia , Proteína Glial Fibrilar Ácida/genética , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Potencial da Membrana Mitocondrial , Disco Óptico/patologia , Estresse Oxidativo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Activation of opioid receptors in the CNS evokes a dramatic decrease in heart rate which is mediated by increases in inhibitory parasympathetic activity to the heart. Injection of opiates into the nucleus ambiguus, where premotor cardiac parasympathetic nucleus ambiguus neurons are located elicits an increase in parasympathetic cardiac activity and bradycardia. However, the mechanisms responsible for altering the activity of premotor cardiac parasympathetic nucleus ambiguus neurons is unknown. This study examined at the electron microscopic level whether premotor cardiac parasympathetic nucleus ambiguus neurons possess postsynaptic opioid receptors and whether mu-opioid receptor agonists alter voltage-gated calcium currents in these neurons. Premotor cardiac parasympathetic nucleus ambiguus neurons were identified in the rat using retrograde fluorescent tracers. One series of experiments utilized dual-labeling immunocytochemical methods combined with electron microscopic analysis to determine if premotor cardiac parasympathetic nucleus ambiguus neurons contain mu-opioid receptors. In a second series of experiments whole cell patch clamp methodologies were used to determine whether activation of postsynaptic opioid receptors altered voltage-gated calcium currents in premotor cardiac parasympathetic nucleus ambiguus neurons in brainstem slices. The perikarya and 78% of the dendrites of premotor cardiac parasympathetic nucleus ambiguus neurons contain mu-opioid receptors. Voltage-gated calcium currents in premotor cardiac parasympathetic nucleus ambiguus neurons were comprised nearly entirely of omega-agatoxin-sensitive P/Q-type voltage-gated calcium currents. Activation of mu-opioid receptors inhibited these voltage-gated calcium currents and this inhibition was blocked by pretreatment with pertusis toxin. The mu-opioid receptor agonist endomorphin-1, but not the mu-opioid receptor agonist endomorphin-2, inhibited the calcium currents. In summary, mu-opioid receptors are located postsynaptically on premotor cardiac parasympathetic nucleus ambiguus neurons. The mu-opioid receptor agonist endomorphin1 inhibited the omega-agatoxin-sensitive P/Q-type voltage-gated calcium currents in premotor cardiac vagal nucleus ambiguus neurons. This inhibition is mediated via a G-protein mediated pathway which was blocked by pretreatment with pertusis toxin. It is possible that the inhibition of calcium currents may act to indirectly facilitate the activity of premotor cardiac parasympathetic nucleus ambiguus neurons by disinhibition, such as by a reduction in inhibitory calcium activated potassium currents.
Assuntos
Analgésicos Opioides/farmacologia , Canais de Cálcio/fisiologia , Bulbo/fisiologia , Oligopeptídeos/farmacologia , Sistema Nervoso Parassimpático/fisiologia , Receptores Opioides mu/fisiologia , Animais , Barorreflexo/fisiologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Coração/inervação , Frequência Cardíaca/fisiologia , Bulbo/citologia , Bulbo/efeitos dos fármacos , Microscopia Eletrônica , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Sistema Nervoso Parassimpático/citologia , Sistema Nervoso Parassimpático/efeitos dos fármacos , Toxina Pertussis/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Opioides mu/análise , Sinapses/química , Sinapses/fisiologia , Sinapses/ultraestrutura , ômega-Agatoxina IVA/farmacologiaRESUMO
The nucleus ambiguus is an area containing cardiac vagal neurons, from which originates most of the parasympathetic control regulating heart rate and cardiac function. GABAergic pathways to these neurons have recently been described, yet modulation of this GABAergic input and its impact upon cardiac vagal neurons is unknown. The nucleus ambiguus has been shown to contain mu-opioid receptors and endomorphin-1 and endomorphin-2, the endogenous peptide ligands for the mu-receptor, whilst microinjections of opioids in the ambiguus area evoke bradycardia. The present study therefore examined the effects of endomorphin-1, endomorphin-2 and DAMGO (a synthetic, mu-selective agonist) on spontaneous GABAergic IPSCs in cardiac parasympathetic neurons. Only endomorphin-2 (100 microM) produced a significant inhibition, of both the frequency (-22.8%) and the amplitude (-30.5%) of the spontaneous IPSCs in cardiac vagal neurons. The inhibitory effects of endomorphin-2 were blocked by naloxonazine (10 microM), a selective mu(1) receptor antagonist. Naloxonazine alone (10 microM) had a potentiating effect on the frequency of the GABAergic IPSCs (+161.43%) but not on the amplitude, indicating that GABA release to cardiac vagal neurons may be under tonic control of opioids acting at the mu(1) receptor. Endomorphin-2 did not reduce the responses evoked by exogenous application of GABA. These results indicate that endomorphin-2 acts on mu(1) receptors located on precedent neurons to decrease GABAergic input to cardiac vagal neurons located in the nucleus ambiguus. The subsequent increase in parasympathetic outflow to the heart may be one mechanism by which mu-selective opioids act to induce bradycardia.
Assuntos
Neurônios/efeitos dos fármacos , Oligopeptídeos/farmacologia , Fibras Parassimpáticas Pós-Ganglionares/efeitos dos fármacos , Rombencéfalo/efeitos dos fármacos , Ácido gama-Aminobutírico/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Animais Recém-Nascidos , Relação Dose-Resposta a Droga , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Coração/efeitos dos fármacos , Coração/fisiologia , Técnicas In Vitro , Neurônios/fisiologia , Oligopeptídeos/fisiologia , Fibras Parassimpáticas Pós-Ganglionares/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores Opioides mu/fisiologia , Rombencéfalo/fisiologia , Nervo Vago/efeitos dos fármacos , Nervo Vago/fisiologiaRESUMO
Previous studies have shown that arginine vasopressin is an important neuropeptide that can modulate the reflex control of blood pressure and heart rate. The nucleus ambiguus, where cardiac parasympathetic neurons are located, receives dense arginine vasopressin projections. However the mechanisms by which arginine vasopressin alters cardiac parasympathetic activity are unknown. We tested the hypothesis that arginine vasopressin can alter the activity of cardiac parasympathetic neurons by altering the spontaneous GABAergic input to these neurons. Experiments were conducted using whole cell patch clamp recordings of cardiac parasympathetic neurons in an in vitro slice preparation in rats. The results of this study demonstrate that arginine vasopressin increases the frequency and amplitude of GABAergic inhibitory post-synaptic currents in cardiac parasympathetic neurons. Arginine vasopressin did not alter the GABAergic currents evoked by exogenous application of GABA. Similarly, in the presence of tetrodotoxin, arginine vasopressin did not alter the frequency, amplitude or decay time of GABAergic miniature synaptic events evoked by high osmolarity. These results indicate that arginine vasopressin likely acts on neurons precedent to cardiac parasympathetic neurons and that arginine vasopressin likely acts not at the synaptic terminal but at the soma or dendrites of the precedent neuron. Oxytocin and agonists for the V(2)-arginine vasopressin and V(1b)-arginine vasopressin receptors had no effect. By contrast, the arginine vasopressin-evoked responses were completely abolished by a selective V(1a)-arginine vasopressin receptor antagonist indicating arginine vasopressin responses are mediated by V(1a)-arginine vasopressin receptors. We conclude that the V(1a)-arginine vasopressin receptor-mediated increase in frequency and amplitude of inhibitory GABAergic activity to cardiac parasympathetic neurons may be at least one mechanism by which central arginine vasopressin may increase heart rate and inhibit reflex bradycardia.
Assuntos
Arginina Vasopressina/farmacologia , Coração/inervação , Inibição Neural/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Sistema Nervoso Parassimpático/efeitos dos fármacos , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/farmacologia , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Inibição Neural/fisiologia , Neurônios/metabolismo , Concentração Osmolar , Ocitocina/farmacologia , Sistema Nervoso Parassimpático/citologia , Sistema Nervoso Parassimpático/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Receptores de Vasopressinas/metabolismo , Tetrodotoxina/farmacologia , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
Cardiac vagal neurons play a critical role in the control of heart rate and cardiac function. These neurons, which are primarily located in the nucleus ambiguus (NA) and the dorsal motor nucleus of the vagus (DMNX), dominate the neural control of heart rate under normal conditions. Cardiac vagal activity is diminished and unresponsive in many disease states, while restoration of parasympathetic activity to the heart lessens ischemia and arrhythmias and decreases the risk of sudden death. Recent work has demonstrated that cardiac vagal neurons are intrinsically silent and therefore rely on synaptic input to control their firing. To date, three major synaptic inputs to cardiac vagal neurons have been identified. Stimulation of the nucleus tractus solitarius evokes a glutamatergic pathway that activates both NMDA and non-NMDA glutamatergic postsynaptic currents in cardiac vagal neurons. Acetylcholine excites cardiac vagal neurons via three mechanisms, activating a direct ligand-gated postsynaptic nicotinic receptor, enhancing postsynaptic non-NMDA currents, and presynaptically by facilitating transmitter release. This enhancement by nicotine is dependent upon activation of pre- and postsynaptic P-type voltage-gated calcium channels. Additionally, there is a GABAergic innervation of cardiac vagal neurons. The transsynaptic pseudorabies virus that expresses GFP (PRV-GFP) has been used to identify, for subsequent electrophysiologic study, neurons that project to cardiac vagal neurons. Bartha PRV-GFP-labeled neurons retain their normal electrophysiological properties, and the labeled baroreflex pathways that control heart rate are unaltered by the virus.
Assuntos
Tronco Encefálico/fisiologia , Coração/inervação , Neurônios/fisiologia , Neurotransmissores/fisiologia , Sinapses/fisiologia , Nervo Vago/fisiologia , Animais , Ácido Glutâmico/fisiologia , Humanos , Receptores Nicotínicos/fisiologia , Nervo Vago/citologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
Superior laryngeal motoneurons control muscles in the larynx and recent work has shown they also have axon collaterals that project to cardiac vagal neurons in the nucleus ambiguus. The present study was undertaken to identify and examine the firing properties of superior laryngeal neurons (SLNs) in the rat. SLNs typically fired spontaneously and repetitively at a rate of 4-7 Hz. The firing was continuous and showed little bursting activity. Firing evoked afterhyperpolarizations were insensitive to apamin but blocked by charybdotoxin. The voltage-gated currents in SLNs consist of a TTX-sensitive Na current and a 4-aminopyridine sensitive K current. It is likely that the activity of these neurons not only control respiratory laryngeal muscles, but may also provide an interaction between the respiratory system and the control of the heart rate.
Assuntos
Potenciais de Ação/fisiologia , Nervos Laríngeos/fisiologia , 2-Amino-5-fosfonovalerato/farmacologia , 4-Aminopiridina/farmacologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Antagonistas de Aminoácidos Excitatórios/farmacologia , Nervos Laríngeos/efeitos dos fármacos , Ratos , Transmissão Sináptica/fisiologia , Tetrodotoxina/farmacologiaRESUMO
Despite the importance of GABAergic input to cardiac vagal neurons the electrophysiological properties and possible origins of this innervation have not yet been studied. Individual cardiac vagal neurons were identified by a retrograde fluorescent tracer and were studied in an in vitro slice preparation using patch-clamp electrophysiology. Cardiac vagal neurons received spontaneous GABAergic inhibitory post-synaptic currents (IPSCs) that were blocked by the GABA(A) receptor antagonist bicuculline. The spontaneous presynaptic GABAergic input to cardiac vagal neurons in the nucleus ambiguus occurred at a significantly lower frequency than that recorded in cardiac vagal neurons in the dorsal motor nucleus of the vagus. To identify a possible source of the GABAergic innervation to cardiac vagal neurons the nucleus tractus solitarius was electrically stimulated. GABAergic synaptic currents in cardiac vagal neurons, in both the dorsal motor nucleus of the vagus (DMNX) and the nucleus ambiguus (NA), were consistently evoked upon stimulation of the nucleus tractus solitarius and these responses were also blocked by bicuculline.
Assuntos
Coração/inervação , Neurônios/fisiologia , Sinapses/fisiologia , Nervo Vago/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Bicuculina/farmacologia , Eletrofisiologia , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Antagonistas GABAérgicos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/efeitos dos fármacos , Ratos , Reflexo Monosináptico/efeitos dos fármacos , Reflexo Monosináptico/fisiologia , Sinapses/efeitos dos fármacos , Tetrodotoxina/farmacologia , Nervo Vago/efeitos dos fármacosRESUMO
Whole cell currents and miniature glutamatergic synaptic events (minis) were recorded in vitro from cardiac vagal neurons in the nucleus ambiguus using the patch-clamp technique. We examined whether voltage-dependent calcium channels were involved in the nicotinic excitation of cardiac vagal neurons. Nicotine evoked an inward current, increase in mini amplitude, and increase in mini frequency in cardiac vagal neurons. These responses were inhibited by the nonselective voltage-dependent calcium channel blocker Cd (100 microM). The P-type voltage-dependent calcium channel blocker agatoxin IVA (100 nM) abolished the nicotine-evoked responses. Nimodipine (2 microM), an antagonist of L-type calcium channels, inhibited the increase in mini amplitude and frequency but did not block the ligand gated inward current. The N- and Q-type voltage-dependent calcium channel antagonists conotoxin GVIA (1 microM) and conotoxin MVIIC (5 microM) had no effect. We conclude that the presynaptic and postsynaptic facilitation of glutamatergic neurotransmission to cardiac vagal neurons by nicotine involves activation of agatoxin-IVA-sensitive and possibly L-type voltage-dependent calcium channels. The postsynaptic inward current elicited by nicotine is dependent on activation of agatoxin-IVA-sensitive voltage-dependent calcium channels.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , Neurônios/metabolismo , Agonistas Nicotínicos/farmacologia , Nervo Vago/fisiologia , Animais , Cloreto de Cádmio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Conotoxinas/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Técnicas In Vitro , Bulbo/citologia , Bulbo/efeitos dos fármacos , Bulbo/fisiologia , Neurônios/efeitos dos fármacos , Nicotina/farmacologia , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/fisiologia , Ratos , Nervo Vago/citologia , ômega-Agatoxina IVA/farmacologiaRESUMO
A fluorescent transneuronal marker capable of labeling individual neurons in a central network while maintaining their normal physiology would permit functional studies of neurons within entire networks responsible for complex behaviors such as cardiorespiratory reflexes. The Bartha strain of pseudorabies virus (PRV), an attenuated swine alpha herpesvirus, can be used as a transsynaptic marker of neural circuits. Bartha PRV invades neuronal networks in the CNS through peripherally projecting axons, replicates in these parent neurons, and then travels transsynaptically to continue labeling the second- and higher-order neurons in a time-dependent manner. A Bartha PRV mutant that expresses green fluorescent protein (GFP) was used to visualize and record from neurons that determine the vagal motor outflow to the heart. Here we show that Bartha PRV-GFP-labeled neurons retain their normal electrophysiological properties and that the labeled baroreflex pathways that control heart rate are unaltered by the virus. This novel transynaptic virus permits in vitro studies of identified neurons within functionally defined neuronal systems including networks that mediate cardiovascular and respiratory function and interactions. We also demonstrate superior laryngeal motorneurons fire spontaneously and synapse on cardiac vagal neurons in the nucleus ambiguus. This cardiorespiratory pathway provides a neural basis of respiratory sinus arrhythmias.
Assuntos
Herpesvirus Suídeo 1/metabolismo , Proteínas Luminescentes/biossíntese , Rede Nervosa/anatomia & histologia , Respiração , Sinapses/metabolismo , Animais , Barorreflexo/efeitos dos fármacos , Barorreflexo/fisiologia , Vias Eferentes/anatomia & histologia , Vias Eferentes/metabolismo , Vias Eferentes/virologia , Feminino , Corantes Fluorescentes , Proteínas de Fluorescência Verde , Herpesvirus Suídeo 1/genética , Imuno-Histoquímica , Técnicas In Vitro , Nervos Laríngeos/citologia , Nervos Laríngeos/metabolismo , Nervos Laríngeos/virologia , Proteínas Luminescentes/genética , Masculino , Potenciais da Membrana/fisiologia , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Neurônios Motores/virologia , Rede Nervosa/metabolismo , Rede Nervosa/virologia , Técnicas de Patch-Clamp , Pericárdio/inervação , Fenilefrina/farmacologia , Ratos , Ratos Sprague-Dawley , Sinapses/virologia , Nervo Vago/citologia , Nervo Vago/metabolismo , Nervo Vago/virologia , Replicação ViralRESUMO
Monoclonal antibodies (mAbs) were produced against gastric H,K-ATPase using a theoretical and experimental strategy based on prediction of linear epitopes by molecular modelling followed by production of anti-peptide antibodies. By analysing the alpha subunit sequence, we predicted several epitopes corresponding to amino acids K519-L533, E543-Y553 and S786-L798 and produced monoclonal antibodies HK519, HK543 and HK786. All three react against gastric H,K-ATPase in RaLISA, immunohistochemistry and Western blots demonstrating that they recognize the native and the SDS-denatured ionic pump and that the epitopes are located at the surface of the native ATPase. Antibody Kd are in the range 6-10x10(-8) M. Monoclonal antibody HK519 is a competitive inhibitor of ATP, in agreement with ATP binding to K519. Neither mAb 543, nor mAb 786 inhibit the ATPase activity. Monoclonal antibody 95111, whose epitope is mapped between residues C529 and E561, competes with mAb HK543 but not with the other two. We suggest that the 95111 epitope is overlapping or very close to the HK543-553 sequence. Induction of E1 conformer by binding FITC to K519 increases the number of mAb 95111 and mAb HK543 epitopes but not that of mAb 786, supporting the fact that the fragment E543-Y553 changes accessibility, maybe during the E1-E2 transconformation.