Nitric Oxide Synthesis and cGMP Production Is Important for Neurite Growth and Synapse Remodeling after Axotomy

Nitric oxide (NO) is an important signaling molecule with a variety of functions in the CNS, including a potential role in modulating neuronal growth and synapse formation. In the present study, we used tractable, identified neurons in the CNS of the pond snail Lymnaea stagnalis to study the role of endogenous NO signaling in neuronal growth and synaptic remodeling after nerve injury. Axonal damage of L. stagnalis neurons B1 and B2 induces extensive central growth of neurites that is accompanied by changes in existing electrical connections, the transient formation of novel electrical connections, and the formation of a novel excitatory chemical synapse from B2 to B1 neurons. Partial chronic inhibition of endogenous NO synthesis reduces neurite growth in NO-synthase-expressing B2, but has only minor effects on NOS-negative B1 neurons. Chronic application of an NO donor while inhibiting endogenous NO synthesis rescues neurite extension in B2 neurons and boosts growth of B1 neurons. Blocking soluble guanylate cyclase activity completely suppresses neurite extension and synaptic remodeling after nerve crush, demonstrating the importance of cGMP in these processes. Interestingly, inhibition of cGMP-dependent protein kinase only suppresses chemical synapse formation without effects on neuronal growth and electrical synapse remodeling. We conclude that NO signaling via cGMP is an important modulator of both neurite growth and synaptic remodeling after nerve crush. However, differential effects of cGMP-dependent protein kinase inhibition on neurite growth and synaptic remodeling suggest that these effects are mediated by separate signaling pathways

Nitric Oxide Synthesis and cGMP Production Is Important for Neurite Growth and Synapse Remodeling after Axotomy

Nitric oxide (NO) is an important signaling molecule with a variety of functions in the CNS, including a potential role in modulating neuronal growth and synapse formation. In the present study, we used tractable, identified neurons in the CNS of the pond snail Lymnaea stagnalis to study the role of endogenous NO signaling in neuronal growth and synaptic remodeling after nerve injury. Axonal damage of L. stagnalis neurons B1 and B2 induces extensive central growth of neurites that is accompanied by changes in existing electrical connections, the transient formation of novel electrical connections, and the formation of a novel excitatory chemical synapse from B2 to B1 neurons. Partial chronic inhibition of endogenous NO synthesis reduces neurite growth in NO-synthase-expressing B2, but has only minor effects on NOS-negative B1 neurons. Chronic application of an NO donor while inhibiting endogenous NO synthesis rescues neurite extension in B2 neurons and boosts growth of B1 neurons. Blocking soluble guanylate cyclase activity completely suppresses neurite extension and synaptic remodeling after nerve crush, demonstrating the importance of cGMP in these processes. Interestingly, inhibition of cGMP-dependent protein kinase only suppresses chemical synapse formation without effects on neuronal growth and electrical synapse remodeling. We conclude that NO signaling via cGMP is an important modulator of both neurite growth and synaptic remodeling after nerve crush. However, differential effects of cGMP-dependent protein kinase inhibition on neurite growth and synaptic remodeling suggest that these effects are mediated by separate signaling pathways

No evidence for altered intracellular calcium-handling in airway smooth muscle cells from human subjects with asthma

Background: Asthma is characterized by airway hyper-responsiveness and variable airflow obstruction, in part as a consequence of hyper-contractile airway smooth muscle, which persists in primary cell culture. One potential mechanism for this hyper-contractility is abnormal intracellular Ca2+ handling. Methods: We sought to compare intracellular Ca2+ handling in airway smooth muscle cells from subjects with asthma compared to non-asthmatic controls by measuring: i) bradykinin-stimulated changes in inositol 1,4,5-trisphosphate (IP3) accumulation and intracellular Ca2+ concentration, ii) sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) expression, iii) mechanisms of cytoplasmic Ca2+ clearance assessed following instantaneous flash photolytic release of Ca2+ into the cytoplasm. Results: We found no differences in airway smooth muscle cell basal intracellular Ca2+ concentrations, bradykinin-stimulated IP3 accumulation or intracellular Ca2+ responses. Quantification of SERCA2 mRNA or protein expression levels revealed no differences in ASM cells obtained from subjects with asthma compared to non-asthmatic controls. We did not identify differences in intracellular calcium kinetics assessed by flash photolysis and calcium uncaging independent of agonist-activation with or without SERCA inhibition. However, we did observe some correlations in subjects with asthma between lung function and the different cellular measurements of intracellular Ca2+ handling, with poorer lung function related to increased rate of recovery following flash photolytic elevation of cytoplasmic Ca2+ concentration. Conclusions: Taken together, the experimental results reported in this study do not demonstrate major fundamental differences in Ca2+ handling between airway smooth muscle cells from non-asthmatic and asthmatic subjects. Therefore, increased contraction of airway smooth muscle cells derived from asthmatic subjects cannot be fully explained by altered Ca2+ homeostasis

Apparent binding affinities (expressed as −log <i>K</i>_B values) for various mAChR agonists and antagonists at the M₁-cam5 AChR, determined by [³H]NMS competition binding.

<p>Data are shown as means ± s.e.m. for duplicate determinations in the number of separate experiments (n) indicated.</p

Cellular localization of M₁-cameleons transiently expressed in HEK293 cells.

<p>HEK293 cells were transiently transfected with M₁-cam1 (<b>A</b>), M₁-cam2 (<b>B</b>), M₁-cam3 (<b>C</b>), M₁-cam4 (<b>D</b>) or M₁-cam5 (<b>E</b>). Images were acquired by confocal microscopy and show fluorescence emission at >530 nm following excitation at 514 nm. Scale bar, 15 µm.</p

Comparisons of maximal FRET changes and rate constants for a variety of orthosteric and allosteric ligands in HEK293-M₁-cam5 cells.

<p>Cells were stimulated with a maximally effective concentration of each agonist and FRET changes (<b>A</b>) and K<i>_obs</i> values (<b>B</b>) were determined as described above. Data are presented as means ± s.e.m. from at least three independent experiments. One-way AVOVA (*<i>p</i><0.05; **<i>p</i><0.005; ***<i>p</i><0.0001).</p

Internalization characteristics of the M₁-cam5 mAChR stably expressed in HEK293 cells.

<p>HEK293-M₁-cam5 cells were treated with various concentration of MCh for 45 min (to assess the concentration-dependency of receptor internalization), or with MCh (300 µM) for 0–60 min (to assess the time-dependency of receptor internalization). Cellular distributions of M₁-cam5 mAChR were monitored by confocal microscopy. For quantification of intracellular fluorescence at least 10 individual cells in five random fields of view were examined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029946#s2" target="_blank">Methods</a> section. Data represent means ± s.e.m. from three independent experiments.</p

Acceptor photobleaching of the three M₁ mAChR cameleons showing primarily plasma membrane localization.

<p>HEK293 cells were transiently transfected with M₁-cam1, M₁-cam2 or M₁-cam5 and imaged by confocal microscopy (458 nm excitation, 470–500 nm emission). Areas of the plasma membrane (delineated by red lines in each image) were bleached using repeated brief exposures to high intensity 514 nm illumination. The graphs show the signal at 458 nm excitation for 470–500 nm emission (ECFPc, cyan line) and >530 nm (EYFP^F46L emission, yellow line), with acceptor photobleach initiated at the arrow. The fluorescence signals from a non-photobleached region were also assessed as a control, which was comparable for all constructs, but only shown for M₁-cam1 (area outlined in cyan within the image and fluorescence within the graph for ECFPc (dark blue) and EYFP^46L (red)). These findings are representative of photobleaching experiments from at least 3 separate transfections for each construct.</p

Overview of M₁ AChR chimeric constructs.

<p>Primers used for introducing <i>AgeI</i> site into the i3 loop are shown where applicable.</p

MCh-induced changes in FRET in HEK293 M₁-cam5 cells.

<p>HEK293 cells stably expressing M₁-cam5 were observed using fluorescence imaging with single wavelength excitation (436 nm) and dual wavelength emission (436 nm to detect ECFPc and 535 nm to detect EYFP^46L. <b>A</b>. Representative images showing plasma membrane distribution of ECFPc (480 nm, left panel) and EYFP^46L (535 nm, middle panel), which overlap (right panel) as expected for signals from the same population of receptors. <b>B–D</b>, right panels: blue and yellow traces represent signals from ECFP and EYFP, respectively; left panels: red traces represent the FRET signal (ratio of F_EYFP/F_ECFP). Addition of MCh (100 µM) induced decreases in FRET, which remained constant throughout the application period (30–40 s; <b>B</b>); this effect was reversed on addition of atropine (1 µM; <b>C</b>); and the MCh-induced change in FRET ratio could be completely prevented by pre-addition of atropine (<b>D</b>). FRET data have been normalized so that the initial FRET signal is 100%. Emission traces are expressed as the change in fluorescence intensity from the basal fluorescence level (<i>F</i>/<i>F</i>₀). Representative traces of at least three independent experiments are shown.</p