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Stanley A Thayer

Publications and source records attributed to Stanley A Thayer.

7 recordsLinked to original sources
PubMed2003

Altered distribution of mitochondria impairs calcium homeostasis in rat hippocampal neurons in culture.

The specificity of Ca2+ signals is conferred in part by limiting changes in cytosolic Ca2+ to subcellular domains. Mitochondria play a major role in regulating Ca2+ in neurons and may participate in its spatial localization. We examined the effects of changes in the distribution of mitochondria on NMDA-induced Ca2+ increases. Hippocampal cultures were treated with the microtubule-destabilizing agent vinblastine, which caused the mitochondria to aggregate and migrate towards one side of the neuron. This treatment did not appear to decrease the energy status of mitochondria, as indicated by a normal membrane potential and pH gradient across the inner membrane. Moreover, electron microscopy showed that vinblastine treatment altered the distribution but not the ultrastructure of mitochondria. NMDA (200 micro m, 1 min) evoked a greater increase in cytosolic Ca2+ in vinblastine-treated cells than in untreated cells. This increase did not result from impaired Ca2+ efflux, enhanced Ca2+ influx, opening of the mitochondrial permeability transition pore or altered function of endoplasmic reticulum Ca2+ stores. Ca2+ uptake into mitochondria was reduced by 53% in vinblastine-treated cells, as reported by mitochondrially targeted aequorin. Thus, the distribution of mitochondria maintained by microtubules is critical for buffering Ca2+ influx. A subset of mitochondria close to a Ca2+ source may preferentially regulate Ca2+ microdomains, set the threshold for Ca2+-induced toxicity and participate in local ATP production.

Adenosine Triphosphate↗
PubMed2002

Modulating Ca2+ clearance from neurons.

Neurons are exquisitely sensitive to the duration, amplitude and localization of transient increases in intracellular Ca2+ concentration ([Ca2+]i). Modulation of Ca2+ uptake into the mitochondrion and endoplasmic reticulum, and efflux via the plasma membrane Ca2+ pump and Na+/Ca2+ exchange profoundly affect the shape of [Ca2+]i signals. Ca2+ clearance mechanisms are modulated by other signaling pathways, are sensitive to metabolic state and have a memory of the recent history of cell activation. We present here examples of pharmacologic and endogenous regulation of Ca2+ sequestration and efflux in neurons. Ca2+ clearance mechanisms differentially shape [Ca2+]i signals based on their affinity, capacity and location; their modulation alters specific neuronal functions. The increasingly apparent diversity of the molecular entities that make up the [Ca2+]i regulatory system reveals new sites for modulation. Specialized Ca2+ clearance mechanisms participate in unique cellular functions and thus, are important targets for pharmacological and physiological regulation of the neuron.

Animals↗
PubMed2002

Bradykinin and ATP accelerate Ca(2+) efflux from rat sensory neurons via protein kinase C and the plasma membrane Ca(2+) pump isoform 4.

Modulation of Ca(2+) channels by neurotransmitters provides critical control of neuronal excitability and synaptic strength. Little is known about regulation of the Ca(2+) efflux pathways that counterbalance Ca(2+) influx in neurons. We demonstrate that bradykinin and ATP significantly facilitate removal of action potential-induced Ca(2+) loads by stimulating plasma membrane Ca(2+)-ATPases (PMCAs) in rat sensory neurons. This effect was mimicked in the soma and axonal varicosities by phorbol esters and was blocked by antagonists of protein kinase C (PKC). Reduced expression of PMCA isoform 4 abolished, and overexpression of isoform 4b enhanced, PKC-dependent facilitation of Ca(2+) efflux. This acceleration of PMCA4 underlies the shortening of the action potential afterhyperpolarization produced by activation of bradykinin and purinergic receptors. Thus, isoform-specific modulation of PMCA-mediated Ca(2+) efflux represents a novel mechanism to control excitability in sensory neurons.

Action Potentials↗
PubMed2002

Transient rise in intracellular calcium produces a long-lasting increase in plasma membrane calcium pump activity in rat sensory neurons.

The plasma membrane Ca2+ ATPase (PMCA) plays a major role in clearing Ca2+ from the neuronal cytoplasm. Calmodulin stimulates PMCA activity and for some isoforms this activation persists following clearance of Ca2+ owing to the slow dissociation of calmodulin. We tested the hypothesis that PMCA-mediated Ca2+ efflux from rat dorsal root ganglion (DRG) neurons in culture would remain stimulated following increases in intracellular Ca2+ concentration ([Ca2+]i). PMCA-mediated Ca2+ extrusion was recorded following brief trains of action potentials using indo-1-based photometry in the presence of cyclopiazonic acid. A priming stimulus that increased [Ca2+]i to 506 +/- 28 nm (>15 min) increased the rate constant for [Ca2+]i recovery by 47 +/- 3%. Ca2+ clearance from subsequent test stimuli remained accelerated for up to an hour despite removal of the priming stimulus and a return to basal [Ca2+]i. The acceleration depended on the magnitude and duration of the priming [Ca2+]i increase, but was independent of the source of Ca2+. Increases in [Ca2+]i evoked by prolonged depolarization, sustained trains of action potentials or activation of vanilloid receptors all accelerated Ca2+ efflux. We conclude that PMCA-mediated Ca2+ efflux in DRG neurons is a dynamic process in which intense stimuli prime the pump for the next Ca2+ challenge.

Action Potentials↗
PubMed2002

Desensitization of cannabinoid-mediated presynaptic inhibition of neurotransmission between rat hippocampal neurons in culture.

Prolonged exposure to cannabinoids results in tolerance in vivo and desensitization of cannabinoid receptors in vitro. We show here that cannabinoid-induced presynaptic inhibition of glutamatergic neurotransmission desensitized after prolonged exposure to the cannabinoid receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-napthalenyl)methanone monomethanesulfonate (Win55,212-2). Synaptic activity between hippocampal neurons in culture was determined from network-driven increases in intracellular Ca(2+) concentration ([Ca(2+)](i) spikes) and excitatory postsynaptic currents. Win55,212-2-induced (100 nM) inhibition partially desensitized after 2 h and completely desensitized after 18- to 24-h exposure. The desensitization could be overcome by higher concentrations of agonist as indicated by a parallel rightward shift of the concentration response curve from an EC(50) of 2.7 +/- 0.3 nM to 320 +/- 147 nM for inhibition of [Ca(2+)](i) spiking and from 43 +/- 17 nM to 4505 +/- 403 nM for inhibition of synaptic currents, suggesting that this phenomenon may underlie tolerance. Presynaptic expression of dominant negative G-protein-coupled-receptor kinase (GRK2-Lys220Arg) or beta-arrestin (319-418) reduced the desensitization produced by 18- to 24-h pretreatment with 100 nM, Win55,212-2 suggesting that desensitization followed the prototypical pathway for G-protein-coupled receptors. Prolonged treatment with Win55,212-2 produced a modest increase in the EC(50) for adenosine inhibition of synaptic transmission and pretreatment with cyclopentyladenosine produced a slight increase in the EC(50) for Win55,212-2, suggesting a reciprocal ability to produce heterologous desensitization. The long-term changes in synaptic function that accompany chronic cannabinoid exposure will be an important factor in evaluating the therapeutic potential of these drugs and will provide insight into the role of the endocannabinoid system.

Animals↗
PubMed2002

NMDA-induced calcium loads recycle across the mitochondrial inner membrane of hippocampal neurons in culture.

Mitochondria sequester N-methyl-D-aspartate (NMDA)-induced Ca(2+) loads and regulate the shape of intracellular Ca(2+) concentration ([Ca(2+)](i)) responses in neurons. When isolated mitochondria are exposed to high [Ca(2+)](,) Ca(2+) enters the matrix via the uniporter and returns to the cytosol by Na(+)/Ca(2+) exchange. Released Ca(2+) may re-enter the mitochondrion recycling across the inner membrane dissipating respiratory energy. Ca(2+) recycling, the continuous uptake and release of Ca(2+) by mitochondria, has not been described in intact neurons. Here we used single-cell microfluorimetry to measure [Ca(2+)](i) and mitochondrially targeted aequorin to measure matrix Ca(2+) concentration ([Ca(2+)](mt)) to determine whether Ca(2+) recycles across the mitochondrial inner membrane in intact neurons following treatment with NMDA. We used ruthenium red and CGP 37157 to block uptake via the uniporter and release via Na(+)/Ca(2+) exchange, respectively. As predicted by the Ca(2+) recycling hypothesis, blocking the uniporter immediately following challenge with 200 microM NMDA produced a rapid and transient increase in cytosolic Ca(2+) without a corresponding increase in matrix Ca(2+). Blocking mitochondrial Ca(2+) release produced the opposite effect, depressing cytosolic Ca(2+) levels and prolonging the time for matrix Ca(2+) levels to recover. The Ca(2+) recycling hypothesis uniquely predicts these reciprocal changes in the Ca(2+) levels between the two compartments. Ca(2+) recycling was not detected following treatment with 20 microM NMDA. Thus Ca(2+) recycling across the inner membrane was more pronounced following treatment with a high relative to a low concentration of NMDA, consistent with a role in Ca(2+)-dependent neurotoxicity.

Animals↗