RESUMEN
AIMS/HYPOTHESIS: We set out to test the hypothesis that insulin secretion from beta cells is targeted towards the vasculature. METHODS: The spatial location of granule fusion was identified by live-cell two-photon imaging of mouse pancreatic beta cells within intact islets, using sulforhodamine B labelling. Three-dimensional (3D) immunofluorescence of pancreatic slices was used to identify the location of proteins associated with neuronal synapses. RESULTS: We demonstrated an asymmetric, non-random, distribution of sites of insulin granule fusion in response to glucose and focal targeting of insulin granule secretion to the beta cell membrane facing the vasculature. 3D immunofluorescence of islets showed that structural proteins, such as liprin, piccolo and Rab2-interacting molecule, normally associated with neuronal presynaptic targeting, were present in beta cells and enriched at the vascular face. In contrast, we found that syntaxin 1A and synaptosomal-associated protein 25 kDa (SNAP25) were relatively evenly distributed across the beta cells. CONCLUSIONS/INTERPRETATION: Our results show that beta cells in situ, within intact islets, are polarised and target insulin secretion. This evidence for an 'endocrine synapse' has wide implications for our understanding of stimulus-secretion coupling in healthy islets and in disease.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Sinapsis/metabolismo , Animales , Exocitosis/efectos de los fármacos , Exocitosis/fisiología , Glucosa/farmacología , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Ratones , Sinapsis/efectos de los fármacos , Proteína 25 Asociada a Sinaptosomas/metabolismoRESUMEN
Both intracellular pH (pHi) and synaptic cleft pH change during neuronal activity yet little is known about how these pH shifts might affect synaptic transmission by influencing vesicle fusion. To address this we imaged pH- and Ca(2+) -sensitive fluorescent indicators (HPTS, Oregon green) in boutons at neuromuscular junctions. Electrical stimulation of motor nerves evoked presynaptic Ca(2+) i rises and pHi falls (â¼0.1 pH units) followed by recovery of both Ca(2+) i and pHi. The plasma-membrane calcium ATPase (PMCA) inhibitor, 5(6)-carboxyeosin diacetate, slowed both the calcium recovery and the acidification. To investigate a possible calcium-independent role for the pHi shifts in modulating vesicle fusion we recorded post-synaptic miniature end-plate potential (mEPP) and current (mEPC) frequency in Ca(2+) -free solution. Acidification by propionate superfusion, NH(4)(+) withdrawal, or the inhibition of acid extrusion on the Na(+)/H(+) exchanger (NHE) induced a rise in miniature frequency. Furthermore, the inhibition of acid extrusion enhanced the rise induced by propionate addition and NH(4)(+) removal. In the presence of NH(4)(+), 10 out of 23 cells showed, after a delay, one or more rises in miniature frequency. These findings suggest that Ca(2+) -dependent pHi shifts, caused by the PMCA and regulated by NHE, may stimulate vesicle release. Furthermore, in the presence of membrane permeant buffers, exocytosed acid or its equivalents may enhance release through positive feedback. This hitherto neglected pH signalling, and the potential feedback role of vesicular acid, could explain some important neuronal excitability changes associated with altered pH and its buffering.
Asunto(s)
Drosophila/metabolismo , Fusión de Membrana , Unión Neuromuscular/metabolismo , Neuronas/metabolismo , Vesículas Sinápticas/metabolismo , Compuestos de Amonio/farmacología , Animales , Calcio/metabolismo , Drosophila/crecimiento & desarrollo , Drosophila/fisiología , Concentración de Iones de Hidrógeno , Membranas Intracelulares/metabolismo , Larva/metabolismo , Larva/fisiología , Potenciales Postsinápticos Miniatura , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/antagonistas & inhibidores , Propionatos/farmacología , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidoresRESUMEN
Tools that allow acute and selective silencing of synaptic transmission in vivo would be invaluable for understanding the synaptic basis of specific behaviors. Here, we show that presynaptic expression of the proton pump archaerhodopsin enables robust, selective, and reversible optogenetic synaptic silencing with rapid onset and offset. Two-photon fluorescence imaging revealed that this effect is accompanied by a transient increase in pH restricted to archaerhodopsin-expressing boutons. Crucially, clamping intracellular pH abolished synaptic silencing without affecting the archaerhodopsin-mediated hyperpolarizing current, indicating that changes in pH mediate the synaptic silencing effect. To verify the utility of this technique, we used trial-limited, archaerhodopsin-mediated silencing to uncover a requirement for CA3-CA1 synapses whose afferents originate from the left CA3, but not those from the right CA3, for performance on a long-term memory task. These results highlight optogenetic, pH-mediated silencing of synaptic transmission as a spatiotemporally selective approach to dissecting synaptic function in behaving animals.
Asunto(s)
Proteínas Arqueales/genética , Región CA1 Hipocampal/fisiología , Región CA3 Hipocampal/fisiología , Memoria a Largo Plazo/fisiología , Terminales Presinápticos/fisiología , Transmisión Sináptica/fisiología , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Concentración de Iones de Hidrógeno , Inyecciones Intraventriculares , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Plasticidad Neuronal/fisiología , Imagen Óptica , Optogenética/métodos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Técnicas EstereotáxicasRESUMEN
A major limitation to the effectiveness of grafts of fetal ventral mesencephalic tissue for parkinsonism is that about 90-95% of grafted dopaminergic neurones die. In rats, many of the cells are dead within 1 day and most cell death is complete within 1 week. Our previous results suggest that a major cause of this cell death is the release of toxins from the injured CNS tissue surrounding the graft, and that many of these toxins have dissipated within 1 h of inserting the grafting cannula. In the present experiments we measured the change over time in the concentration of several potential toxins around an acutely implanted grafting cannula. We also measured the additional effect of injecting suspensions of embryonic mesencephalon, latex microspheres, or vehicle on these concentrations. Measurements of glutamate, aspartate, and dopamine by microdialysis showed elevated levels during the first 20-60 min, which then declined to baseline. In the first 20 min glutamate levels were 10.7 times, aspartate levels 5 times, and dopamine levels 24.3 times baseline. Potassium levels increased to a peak of 33 +/- 10.6 mM 4-5 min after cannula insertion, returning to baseline of <5 mM by 30 min. Injection of cell suspension, latex microspheres, or vehicle had no significant effect on these levels. We then assayed the effect of high concentrations of glutamate, aspartate, dopamine, and potassium on dopaminergic neuronal survival in E14 ventral mesencephalic cultures. In monolayer cultures only dopamine at 200 microM showed toxicity. In three-dimensional cultures only the combination of raised potassium, dopamine, glutamate, and aspartate together decreased dopaminergic neuronal survival. We conclude that toxins other than the ones measured are the main cause of dopaminergic cell death after transplantation, or the effects of the toxins measured are enhanced by anoxia and metabolic challenges affecting newly inserted grafts.
Asunto(s)
Cuerpo Estriado/cirugía , Supervivencia de Injerto/fisiología , Neuronas/metabolismo , Trastornos Parkinsonianos/terapia , Trasplante de Células Madre/métodos , Células Madre/metabolismo , Sustancia Negra/trasplante , Animales , Ácido Aspártico/metabolismo , Ácido Aspártico/toxicidad , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Cuerpo Estriado/inmunología , Cuerpo Estriado/fisiopatología , Dopamina/metabolismo , Dopamina/toxicidad , Aminoácidos Excitadores/metabolismo , Aminoácidos Excitadores/toxicidad , Espacio Extracelular/metabolismo , Femenino , Ácido Glutámico/metabolismo , Ácido Glutámico/toxicidad , Supervivencia de Injerto/efectos de los fármacos , Neuronas/efectos de los fármacos , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/fisiopatología , Potasio/metabolismo , Potasio/toxicidad , Ratas , Ratas Endogámicas , Células Madre/efectos de los fármacos , Sustancia Negra/citología , Sustancia Negra/embriologíaRESUMEN
BACKGROUND: Phototransduction in microvillar photoreceptors is mediated via G protein-coupled phospholipase C (PLC), but how PLC activation leads to the opening of the light-sensitive TRPC channels (TRP and TRPL) remains unresolved. In Drosophila, InsP(3) appears not to be involved, and recent studies have implicated lipid products of PLC activity, e.g., diacylglycerol, its metabolites, or the reduction in PIP(2). The fact that hydrolysis of the phosphodiester bond in PIP(2) by PLC also releases a proton is seldom recognized and has neither been measured in vivo nor implicated previously in a signaling context. RESULTS: Following depletion of PIP(2) and other phosphoinositides by a variety of experimental manipulations, the light-sensitive channels in Drosophila photoreceptors become remarkably sensitive to rapid and reversible activation by the lipophilic protonophore 2-4 dinitrophenol in a pH-dependent manner. We further show that light induces a rapid (<10 ms) acidification originating in the microvilli, which is eliminated in mutants of PLC, and that heterologously expressed TRPL channels are activated by acidification of the cytosolic surface of inside-out patches. CONCLUSIONS: Our results indicate that a combination of phosphoinositide depletion and acidification of the membrane/boundary layer is sufficient to activate the light-sensitive channels. Together with the demonstration of light-induced, PLC-dependent acidification, this suggests that excitation in Drosophila photoreceptors may be mediated by PLC's dual action of phosphoinositide depletion and proton release.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , 2,4-Dinitrofenol/farmacología , Animales , Línea Celular , Drosophila/efectos de los fármacos , Drosophila/efectos de la radiación , Proteínas de Drosophila/efectos de los fármacos , Proteínas de Drosophila/efectos de la radiación , Concentración de Iones de Hidrógeno , Fosfatidilinositoles/metabolismo , Fotones , Células Fotorreceptoras de Invertebrados/efectos de los fármacos , Células Fotorreceptoras de Invertebrados/efectos de la radiación , Canales de Potencial de Receptor Transitorio/efectos de los fármacos , Canales de Potencial de Receptor Transitorio/efectos de la radiación , Visión Ocular/fisiologíaRESUMEN
The relationship between cell volume (V(c)) and membrane potential (E(m)) in Rana temporaria striated muscle fibres was investigated under different conditions of intracellular acidification. Confocal microscope xz-scanning monitored the changes in V(c), whilst conventional KCl and pH-sensitive microelectrodes measured E(m) and intracellular pH (pH(i)), respectively. Applications of Ringer solutions with added NH(4)Cl induced rapid reductions in V(c) that rapidly reversed upon their withdrawal. These could be directly attributed to the related alterations in extracellular tonicity. However: (1) a slower and persistent decrease in V(c) followed the NH(4)Cl withdrawal, leaving V(c) up to 10% below its resting value; (2) similar sustained decreases in resting V(c) were produced by the addition and subsequent withdrawal of extracellular solutions in which NaCl was isosmotically replaced with NH(4)Cl; (3) the same manoeuvres also produced a marked intracellular acidification, that depended upon the duration of the preceding exposure to NH(4)Cl, of up to 0.53 +/- 0.10 pH units; and (4) the corresponding reductions in V(c) similarly increased with this exposure time. These reductions in V(c) persisted and became more rapid with Cl(-) deprivation, thus excluding mechanisms involving either direct or indirect actions of pH(i) upon Cl(-)-dependent membrane transport. However they were abolished by the Na(+),K(+)-ATPase inhibitor ouabain. The E(m) changes that accompanied the addition and withdrawal of NH(4)(+) conformed to a Nernst equation modified to include realistic NH(4)(+) permeability terms, and thus the withdrawal of NH(4)(+) restored E(m) to close to control values despite a persistent change in V(c). Finally these E(m) changes persisted and assumed faster kinetics with Cl(-) deprivation. The relative changes in V(c), E(m) and pH(i) were compared to predictions from the recent model of Fraser and Huang published in 2004 that related steady-state values of V(c) and E(m) to the mean charge valency (z(x)) of intracellular membrane-impermeant anions, X(-)(i). By assuming accepted values of intracellular buffering capacity (beta(i)), intracellular acidification was shown to produce quantitatively predictable decreases in V(c). These findings thus provide experimental evidence that titration of the anionic z(x) by increased intracellular [H(+)] causes cellular volume decrease in the presence of normal Na(+),K(+)- ATPase activity, with Cl(-)-dependent membrane fluxes only influencing the kinetics of such changes.
Asunto(s)
Líquido Intracelular/química , Potenciales de la Membrana/fisiología , Células Musculares/química , Células Musculares/citología , Músculo Esquelético/química , Músculo Esquelético/citología , Cloruro de Amonio/farmacología , Animales , Tamaño de la Célula , Células Cultivadas , Simulación por Computador , Concentración de Iones de Hidrógeno , Líquido Intracelular/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Modelos Biológicos , Células Musculares/efectos de los fármacos , Células Musculares/fisiología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , Rana temporaria , Estadística como Asunto , Equilibrio Hidroelectrolítico/efectos de los fármacos , Equilibrio Hidroelectrolítico/fisiologíaRESUMEN
Neuronal electrical activity causes only modest changes in global intracellular pH (pH(i)). We have measured regional pH(i) differences in isolated patch-clamped neurones during depolarization, using confocal imaging of 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) fluorescence. The pH(i) shifts in the soma were as expected; however, substantially larger shifts occurred in other regions. These regional differences were still observed in the presence of CO(2)-HCO(3)(-), they decayed over many seconds and were associated with changes in calcium concentration. Lamellipodial HPTS fluorescence fell by 8.7 +/- 1.3 % (n = 9; approximately 0.1 pH unit acidification) following a 1 s depolarization to 0 mV; this was more than 4-fold greater than the relative shift seen in the soma. Depolarization to +40 mV for 1 s caused a 46.7 +/- 7.0 % increase (n = 11; approximately 0.4 pH unit alkalinization) in HPTS fluorescence in the lamellipodia, more than 6-fold that seen in the soma. Application of 5 % CO(2)-20 mM HCO(3)(-) did not significantly reduce the size of the +40 mV-evoked local pH shifts despite carbonic anhydrase activity. The pH(i) gradient between regions approximately 50 microm apart, resulting from acid shifts, took 10.3 +/- 3.1 s (n = 6) to decay by 50 %, whereas the pH(i) gradient resulting from alkaline shifts took only 3.7 +/- 1.4 s (n = 12) to decay by 50 %. The regional rates of acidification and calcium recovery were closely related, suggesting that the acidic pH microdomains resulted from Ca(2+)-H(+) pump activity. The alkaline pH microdomains were blocked by zinc and resulted from proton channel opening. It is likely that the microdomains result from transmembrane acid fluxes in areas with different surface area to volume ratios. Such neuronal pH microdomains are likely to have consequences for local receptor, channel and enzyme function in restricted regions.
Asunto(s)
Calcio/metabolismo , Hidrógeno/metabolismo , Neuronas/fisiología , Animales , Arilsulfonatos , Bicarbonatos/farmacología , Calibración , Dióxido de Carbono/farmacología , Anhidrasas Carbónicas/metabolismo , Separación Celular , Electrofisiología , Colorantes Fluorescentes , Caracoles Helix , Concentración de Iones de Hidrógeno/efectos de los fármacosRESUMEN
Our aim was to test the hypothesis that depolarization-induced intracellular pH (pH(i)) shifts in restricted regions (dendrites) of mammalian neurones might be larger and faster than those previously reported from the cell soma. We used confocal imaging of the pH-sensitive dye, HPTS, to measure pH changes in both the soma and dendrites of whole-cell patch-clamped rat cerebellar Purkinje cells. In the absence of added CO(2)-HCO(3)(-), depolarization to +20 mV for 1 s caused large (approximately 0.14 pH units) and fast dendritic acid shifts, whilst the somatic acidifications were significantly smaller (approximately 0.06 pH units) and slower. The pH(i) shifts were smaller in the presence of 5 % CO(2)-25 mM HCO(3)(-)-buffered saline (approximately 0.08 pH units in the dendrites and approximately 0.03 pH units in the soma), although a clear spatiotemporal heterogeneity remained. Acetazolamide (50 microM) doubled the size of the dendritic acid shifts in the presence of CO(2)-HCO(3)(-), indicating carbonic anhydrase activity. Removal of extracellular calcium or addition of the calcium channel blocker lanthanum (0.5 mM) inhibited the depolarization-evoked acid shifts. We investigated more physiological pH(i) changes by evoking modest bursts of action potentials (approximately 10 s duration) in CO(2)-HCO(3)(-)-buffered saline. Such neuronal firing induced an acidification of approximately 0.11 pH units in the fine dendritic regions, but only approximately 0.03 pH units in the soma. There was considerable variation in the size of the pH(i) shifts between cells, with dendritic acid shifts as large as 0.2-0.3 pH units following a 10 s burst of action potentials in some Purkinje cells. We postulate that these large dendritic pH(i) changes (pH microdomains) might act as important signals in synaptic function.
Asunto(s)
Dendritas/metabolismo , Hidrógeno/metabolismo , Células de Purkinje/fisiología , Ácidos/metabolismo , Potenciales de Acción , Animales , Calcio/metabolismo , Dióxido de Carbono/farmacología , Carbonatos/farmacología , Anhidrasas Carbónicas/metabolismo , Estimulación Eléctrica , Electrofisiología , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Células de Purkinje/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
This study investigated membrane transport mechanisms influencing relative changes in cell volume (V) and resting membrane potential (E(m)) following osmotic challenge in amphibian skeletal muscle fibres. It demonstrated a stabilization of E(m) despite cell shrinkage, which was attributable to elevation of intracellular [Cl(-)] above electrochemical equilibrium through Na(+)-Cl(-) and Na(+)-K(+)-2Cl(-) cotransporter action following exposures to extracellular hypertonicity. Fibre volumes (V) determined by confocal microscope x z - scanning of cutaneous pectoris muscle fibres varied linearly with [1/extracellular osmolarity], showing insignificant volume corrections, in fibres studied in Cl(-)-free, normal and Na(+)-free Ringer solutions and in the presence of bumetanide, chlorothiazide and ouabain. The observed volume changes following increases in extracellular tonicity were compared with microelectrode measurements of steady-state resting potentials (E(m)). Fibres in isotonic Cl(-)-free, normal and Na(+)-free Ringer solutions showed similar E(m) values consistent with previously reported permeability ratios P(Na)/P(K)(0.03-0.05) and P(Cl)/P(K) ( approximately 2.0) and intracellular [Na(+)], [K(+)] and [Cl(-)]. Increased extracellular osmolarities produced hyperpolarizing shifts in E(m) in fibres studied in Cl(-)-free Ringer solution consistent with the Goldman-Hodgkin-Katz (GHK) equation. In contrast, fibres exposed to hypertonic Ringer solutions of normal ionic composition showed no such E(m) shifts, suggesting a Cl(-)-dependent stabilization of membrane potential. This stabilization of E(m) was abolished by withdrawing extracellular Na(+) or by the combined presence of the Na(+)-Cl(-) cotransporter (NCC) inhibitor chlorothiazide (10 microM) and the Na(+)-K(+)-2Cl(-) cotransporter (NKCC) inhibitor bumetanide (10 microM), or the Na(+)-K(+)-ATPase inhibitor ouabain (1 or 10 microM) during alterations in extracellular osmolarity. Application of such agents after such increases in tonicity only produced a hyperpolarization after a time delay, as expected for passive Cl(-) equilibration. These findings suggest a model that implicates the NCC and/or NKCC in fluxes that maintain [Cl(-)](i) above its electrochemical equilibrium. Such splinting of [Cl(-)](i) in combination with the high P(Cl)/P(K) of skeletal muscle stabilizes E(m) despite volume changes produced by extracellular hypertonicity, but at the expense of a cellular capacity for regulatory volume increases (RVIs). In situations where P(Cl)/P(K) is low, the same co-transporters would instead permit RVIs but at the expense of a capacity to stabilize E(m).
Asunto(s)
Fibras Musculares Esqueléticas/fisiología , Animales , Calibración , Tamaño de la Célula/fisiología , Electrofisiología , Inhibidores Enzimáticos/farmacología , Soluciones Hipertónicas/farmacología , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Microscopía Confocal , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/ultraestructura , Concentración Osmolar , Técnicas de Placa-Clamp , Rana temporaria , Sodio/fisiología , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico , Simportadores de Cloruro de Sodio-Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidoresRESUMEN
Ventricular arrhythmogenesis leading to sudden cardiac death remains responsible for significant mortality in conditions such as cardiac failure and the long-QT syndrome (LQTS). Arrhythmias may be accentuated by beta-adrenergic stimulation and, accordingly, the present study explored the possible effects of beta-adrenergic stimulation and L-type Ca(2+) channel blockade on ventricular arrhythmogenesis and Ca(2+) handling using the mouse heart as an experimental system. Studies in whole, Langendorff-perfused hearts using programmed electrical stimulation protocols adapted from clinical practice demonstrated sustained ventricular tachycardia following addition of 0.1 microM isoprenaline (n=15), whilst no arrhythmias were observed in the absence of the drug (n=15). Arrhythmias were suppressed by nifedipine or diltiazem pre-treatment (both 1 microM) (n=8 and 4 respectively) and were also induced by elevating external [Ca(2+)] (n=3). At the cellular level, 0.1 microM isoprenaline significantly increased normalized fluorescence (F/F(0)) in field-stimulated fluo-3-loaded mouse ventricular myocytes imaged using confocal microscopy, reflecting increases in sarcoplasmic reticulum Ca(2+) release (n=8). Elevated external [Ca(2+)] also increased F/F(0) (n=4) whilst 0.1 microM nifedipine or 0.1 microM diltiazem significantly decreased F/F(0) (n=13 and 6 respectively). Pre-treatment with 0.1 microM nifedipine or 0.1 microM diltiazem suppressed the increases in F/F(0) induced by 0.1 microM isoprenaline alone (n=14 and 6 respectively). The findings thus paralleled suppression of isoprenaline-induced arrhythmias seen with nifedipine or diltiazem at the whole-heart level. Taken together, the findings may have implications for the use of L-type Ca(2+) channel blockade in conditions associated with beta-adrenergically driven ventricular arrhythmias such as cardiac failure and LQTS.