RESUMEN
1. Oxytocin and vasopressin secretion from the neurohypophysis (NHP) is evoked by strongly patterned bursts of action potentials. We studied excitation-secretion coupling in single isolated terminals of rat NHP using patch clamp and capacitance detection techniques. 2. The secretory response evoked by trains of depolarizing pulses consisted of two discrete phases. Ca2+ entry during pulses early in the train did not elicit secretion. Exocytotic responses began only after a characteristic amount of total Ca2+ entry called "threshold". 3. In the postthreshold secretory phase, exocytotic events occurred during or immediately after depolarizing pulses, indicating that the final Ca(2+)-dependent step is triggered by high Ca2+ concentrations near the plasma membrane that dissipate rapidly after channel closure. Secretion was sensitive to both the concentration and species of Ca2+ chelator. BAPTA, a Ca2+ chelator with rapid Ca2+ binding kinetics, was more effective than EGTA in diminishing secretion. 4. The "threshold" amount of Ca2+ was determined by the concentration, but not species, of Ca2+ chelator. The threshold value was constant even when Ca2+ entry parameters were varied over a broad range of current amplitudes, pulse durations, and number of pulses, indicating that it did not require high Ca2+ concentrations near the plasma membrane. 5. These results suggest that the secretory response to a train of pulses consists of a Ca(2+)-dependent preparatory step that must be completed before subsequent Ca2+ entry can elicit exocytosis. 6. Exocytotic responses during single trains showed strong depression at a step subsequent to Ca2+ entry. Recovery from depression required 30-60 sec. 7. The properties of threshold secretion observed in NHP terminals are discussed in terms of current models of secretion.
Asunto(s)
Terminaciones Nerviosas/metabolismo , Terminaciones Nerviosas/fisiología , Neurohipófisis/metabolismo , Animales , Bario/farmacología , Canales de Calcio/metabolismo , Células Cultivadas , Quelantes/farmacología , Células Cromafines/efectos de los fármacos , Células Cromafines/metabolismo , Conductividad Eléctrica , Potenciales Evocados/efectos de los fármacos , Masculino , Terminaciones Nerviosas/efectos de los fármacos , Sistemas Neurosecretores/fisiología , Técnicas de Placa-Clamp , Neurohipófisis/citología , Neurohipófisis/efectos de los fármacos , RatasRESUMEN
Stimulus-secretion coupling was monitored with capacitance detection in bovine chromaffin cells recorded in perforated patch mode and stimulated with trains of depolarizing pulses. A subset of stimulus trains evoked a response with a Ca2+-exocytosis relationship identical to that obtained for single depolarizing pulses (Engisch and Nowycky, 1996). Other trains evoked responses with enhanced or diminished Ca2+ efficacy relative to this input-output function. The probability of obtaining a particular Ca2+-exocytosis relationship was correlated with the amount of Ca2+ entry per pulse, such that shorter pulses or smaller currents were associated with the greatest efficacy, and longer pulses and larger currents with the lowest efficacy. Apparent enhancements in Ca2+ efficacy were not caused by residual Ca2+ summing between pulses, because decreasing the interval between pulses usually reduced efficacy in the same cell; conversely, increasing the interval between pulses did not prevent an enhanced Ca2+-exocytosis relationship. Apparent decreases in Ca2+ efficacy were not caused by depletion of an available pool of release-ready vesicles, because an equivalent amount of total Ca2+ entry during a single long depolarizing pulse usually evoked a much larger secretory response in the same cell. Finally, there were no striking differences in global Ca2+ levels monitored with the fluorescent indicator Fura Red that could account for apparent changes in Ca2+ efficacy during repetitive stimulus protocols. It appears that in chromaffin cells, the Ca2+-exocytosis relationship is subject to activity-dependent changes during a stimulus train and can be modulated up or down from a basal state accessed by single pulse stimulations.
Asunto(s)
Médula Suprarrenal/citología , Canales de Calcio/fisiología , Calcio/fisiología , Catecolaminas/metabolismo , Células Cromafines/metabolismo , Exocitosis/fisiología , Potenciales de Acción , Animales , Bovinos , Células Cultivadas , Conductividad Eléctrica , Estimulación Eléctrica , Activación del Canal Iónico , Transporte Iónico , Técnicas de Placa-ClampRESUMEN
The ability of cell adhesion molecules (CAMs) to transduce cell surface signals into intracellular responses is critical for developing neurons, particularly during axonal pathfinding and targeting. It has been suggested that different CAMs can promote neuronal outgrowth via activation of common neuronal CAM-specific second-messenger pathways, although the elements involved in this cascade could differ. Limbic system-associated membrane protein (LAMP), a member of the Ig superfamily, is a molecule that promotes cell adhesion and neurite outgrowth from specific populations of fetal neurons. In the present study, we show that LAMP can induce several types of calcium (Ca2+) signals. Neurite outgrowth is promoted if fetal hippocampal neurons are grown on lamp-transfected CHO cells. This LAMP-induced outgrowth of neurons is mediated in part through activation of L-type Ca channels. Application of soluble LAMP to cultures of fetal hippocampal neurons caused a sustained (up to 60 min) elevation of intracellular Ca2+ as measured by fluo-3 fluorescence on a confocal microscope. The number of responding hippocampal neurons was initially low, but increased with age in culture and the [Ca2+]i elevation was only partially decreased by an L-type Ca(2+)-channel blocker. In contrast, at all times in culture, only a small fraction of neurons from visual cortex responded to LAMP application and only with transient elevation of cytosolic Ca2+ (< 15 min). Based on these observations, LAMP appears to function primarily through homophilic interactions and acts in part by modulating intracellular Ca2+ levels during neurite outgrowth by increasing the Ca2+ influx through L-type calcium channels, but has additional effects on intracellular Ca2+ signaling at later developmental stages.
Asunto(s)
Calcio/metabolismo , Moléculas de Adhesión Celular Neuronal/farmacología , Membranas Intracelulares/metabolismo , Neuritas/efectos de los fármacos , Neuritas/fisiología , Neuronas/metabolismo , Animales , Células CHO , Bloqueadores de los Canales de Calcio/farmacología , Cricetinae , Electrofisiología , Feto/citología , Proteínas Ligadas a GPI , Concentración Osmolar , Cloruro de Potasio/farmacología , Ratas/embriología , Proteínas Recombinantes/farmacologíaRESUMEN
The coupling between divalent cations and exocytosis of large dense-cored vesicles (LDCV) was studied with capacitance-detection techniques in nerve terminals of the rat neurohypophysis (NHP) and bovine chromaffin cells. Ba2+ substitution for Ca2+ produced kinetically distinct responses in the two preparations. In NHP terminals, Ba2+ ions behave as weak substitutes for Ca2+. Exocytotic events occur principally during depolarizing pulses, i.e., events are "stimulus-coupled" to Ba2+ entry through voltage-gated Ca2+ channels. Stimulus-coupled exocytosis apparently requires elevated submembrane cation concentrations that dissipate rapidly on hyperpolarization-induced Ca(2+)-channel closure. Intracellular dialysis of NHP terminals with Ba2+ does not evoke exocytosis, nor does it interfere with depolarization-evoked Ca2+ influx and exocytosis. In chromaffin cells, Ba2+ ions evoke a small quantity of stimulus-coupled secretion, but the dominant response is an additional pronounced poststimulus capacitance increase that outlasts channel closures by 20-50 sec. "Stimulus-decoupled" exocytosis is slow (approximately 25-40 fF/sec) compared with Ca(2+)-evoked stimulus-coupled exocytosis (approximately 1000 fF/sec). Decoupled secretion is not attributable to Ba2+ displacement of intracellular Ca2+ ions, because it is insensitive to 10 mM EGTA or thapsigargin. Slow exocytosis is initiated by inclusion of Ba2+ ions in the recording pipette and continues steadily for 5-12 min, producing a total increase of several thousand fF, which ultimately doubles or triples the original cell-surface area. We propose that two pathways of regulated exocytosis with distinct kinetics and divalent cation sensitivity exist in chromaffin cells. Only a single kinetic pattern is detected in NHP terminals, suggesting that mechanisms for secretion are not universally distributed in excitable cells.
Asunto(s)
Bario/farmacología , Sistema Cromafín/efectos de los fármacos , Exocitosis/fisiología , Neurohipófisis/efectos de los fármacos , Animales , Calcio/farmacología , Cationes/farmacología , Bovinos , Cinética , Factores de TiempoRESUMEN
The link between electrical activity, Ca2+ entry through voltage-gated channels, and transmitter or hormone secretion is a central issue in neurobiology. In peptidergic nerve terminals of the mammalian neurohypophysis (NHP), secretion is elicited by patterned bursts of action potentials (APs). All parameters of the bursts are important to elicit efficient secretion, including AP frequency, AP broadening, burst duration, and interburst interval (Leng, 1988). We have studied Ca(2+)-secretion coupling of peptide-containing large dense-core vesicles (LDCV) in isolated rat NHP terminals. Ca2+ influx through voltage-gated Ca2+ channels was elicited and recorded by the whole-cell patch-clamp technique. Exocytosis was monitored on line with high temporal resolution by the capacitance detection technique (Neher and Marty, 1982). AP bursts were simulated by depolarizing pulse trains that mimic pulsatile submembrane Ca2+ elevations predicted for physiological stimuli. The characteristic capacitance response (delta Cm) to a train of depolarizing pulses was triphasic. It consisted of a threshold phase during which early pulses did not elicit secretion, a subsequent secretory phase during which Cm increases were coupled to depolarizing pulses, and a fatigued or inactivated state during which additional Ca2+ entry was ineffective. Both the threshold phase and secretory phase were correlated with the integrals of Ca2+ current. Ca2+ chelators affect both the threshold and secretory phase at submillimolar concentrations. Thus, a "shell" rather than "microdomain" model of Ca2+ elevation is appropriate for analyzing Ca(2+)-secretion coupling in NHP terminals (Nowycky and Pinter, 1993). We propose a two-step model, with a ca(2+)-dependent preparatory step followed by a final exocytotic step that is coupled to active Ca2+ influx. The results suggest that under physiological conditions, APs early in a burst prepare an NHP terminal for secretion, but later APs actually trigger exocytosis. Since NHP terminals do not possess a readily releasable pool of vesicles that require only a single Ca2+ step for exocytosis as seen in chromaffin cells (Neher and Zucker, 1993) and melanotrophs (Thomas et al, 1993a), Ca(2+)-secretion coupling mechanisms may be heterologous even within a single class of vesicles.
Asunto(s)
Potenciales de Acción/fisiología , Canales de Calcio/fisiología , Calcio/metabolismo , Exocitosis , Terminaciones Nerviosas/fisiología , Neurohipófisis/fisiología , Animales , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Estimulación Eléctrica , Activación del Canal Iónico , Cinética , Terminaciones Nerviosas/efectos de los fármacos , Ratas , Factores de TiempoRESUMEN
The modulation of voltage-dependent calcium channels by various neurotransmitters has been demonstrated in many neurons. Because of the critical role of Ca2+ in transmitter release and, more generally, in transmembrane signalling, this modulation has important functional implications. Hippocampal neurons possess low-threshold (T-type) Ca2+ channels and both L- and N-type high voltage-activated Ca2+ channels. N-type Ca2+ channels are blocked selectively by omega-conotoxin and adenosine. These substances both block excitatory synaptic transmission in the hippocampus, whereas dihydropyridines, which selectively block L-type channels, are ineffective. Excitatory synaptic transmission in the hippocampus displays a number of plasticity phenomena that are initiated by Ca2+ entry through ionic channels operated by N-methyl-D-aspartate (NMDA) receptors. Here we report that NMDA receptor agonists selectively and effectively depress N-type Ca2+ channels which are involved in neurotransmitter release from presynaptic sites. The inhibitory effect is eliminated by the competitive NMDA antagonist D-2-amino-5-phosphonovalerate, does not require Ca2+ entry into the cell, and is probably receptor-mediated. This phenomenon may provide a negative feedback between the liberation of excitatory transmitter and entry of Ca2+ into the cell, and could be important in presynaptic inhibition and in the regulation of synaptic plasticity.