Subcellular mechanisms of angiotensin II and arginine vasopressin activation of area postrema neurons.

Angiotensin II (ANG II) and arginine vasopressin (AVP) act on area postrema (AP) neurons to modulate the baroreflex. Because activation of AP neurons by either ANG II or AVP increases intracellular free Ca2+ concentrations ([Ca2+]i), the goal of this study was to analyze the factors affecting the [Ca2+]i responses to ANG II and AVP. Neurons were recovered from 14- to 16-day old rats and studied after 8-14 days in culture by use of the microscopic digital image analysis for fura 2-loaded cells. The effects of ANG II (100 nM) and AVP (100 nM) on [Ca2+]i were determined in normal (2 mM) and low (< 10 nM) extracellular Ca2+ concentrations. In 143 of 240 neurons, ANG II increased [Ca2+]i 4.65-fold after 20 s, and a similar response was observed in the absence of extracellular Ca2+ (3.65-fold after 20 s). After 60 s of observation, steady-state levels of increased [Ca2+]i were still present under both conditions. Pretreatment with AT1 antagonist or pertussis toxin abolished the response to ANG II. AVP also increased [Ca2+]i (3.6-fold at peak, 20 s) in normal and low extracellular Ca2+. Pretreatment with AVP V1 antagonist or pertussis toxin abolished the response to AVP. This study indicates that ANG II-induced increases in [Ca2+]i are independent of extracellular Ca2+ concentrations and involve the activation of AT1 receptors and a pertussis toxin-sensitive G protein. Although AVP affects a fewer number of AP neurons, the mechanisms of activation are also independent of extracellular Ca2+ concentration and are mediated by a pertussis toxin-sensitive G protein.

An obligate role for oxygen in the early stages of glutamate-induced, delayed neuronal death.

In vitro models of hypoxic/hypoglycemic injury reveal common mechanisms with glutamate excitotoxicity, but glutamate-induced toxicity in the absence of oxygen has never been directly addressed. Therefore, we assessed neuronal survival and intracellular calcium concentrations ([Ca2+]i) in neonatal hippocampal cultures in response to glutamate in the presence and absence of oxygen. Twenty-four hours of hypoxia alone killed 40% of the initial population, attributable to glutamate receptor-stimulated osmotic lysis. A 5 min glutamate exposure in ambient air killed 80% of the initial population by 24 hr later. When cultures were deprived of oxygen during and for 2-24 hr after excitotoxin exposure, glutamate did not cause additional neuronal death beyond that induced by oxygen depletion alone. Toxicities caused by activation of NMDA, AMPA, or kainate receptors were each ameliorated by oxygen depletion. In the absence of oxygen, glutamate evoked normal increases in [Ca2+]i, indicating that glutamate receptors functioned normally. The glutamate-induced increases in [Ca2+]i were not toxic in the absence of oxygen. In a similar manner, oxygen-depletion prevented neuronal killing by the calcium ionophore, ionomycin. Neuronal death produced by hydrogen peroxide or iron sulfate was not ameliorated by oxygen removal. These oxidants maximally produced only a slow increase in [Ca2+]i as the plasma membrane permeability increased nonspecifically. Therefore, oxygen-based reactions were an essential component of calcium-mediated, delayed neuronal death.

On the probabilistic nature of excitotoxic neuronal death in hippocampal neurons.

In an attempt to distinguish hypothesized rapid and slow components, we have systematically studied the time course of hippocampal neuronal death in an in vitro model of excitotoxicity. In all paradigms involving glutamate, NMDA or AMPA as toxins, the population of trypan-blue excluding (live) neurons progressively declined over 48 hr. The percent survival over time could be fit mathematically using single exponential decay curves, implying that the death of any individual neuron was a stochastic event. One or two hours after glutamate exposure, prevention of further glutamate-receptor interactions by addition of MK-801 or MK-801 plus CNQX resulted in the survival of 60-80% of the original population at 24 hr. Thus delayed, continuous blockade of secondary glutamate receptor stimulation was protective, apparently interrupting the cyclic nature of the toxicity cascade. Twelve hours of MK-801 immediately following glutamate removal protected the majority of cells during the period of active receptor blockade. As soon as MK-801 was removed, the progressive decay in population size resumed, indicating that short term receptor blockade was insufficient to prevent expression of the initial injury. A kinetic model is proposed to place these experimental results into a framework for discussion and formulation of future experimentation.