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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 12, 2002 as doi:10.1096/fj.01-0971fje. |
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Neuroscience Research Centre, Guy’s, King’s and St Thomas’ School of Biomedical Sciences, King’s College London, London SE1 1UL, UK;
* Vision and Ophthalmology Research Group, Rayne Institute, St Thomas’ Hospital, London SE1 7EH, UK; and
The William Harvey Research Institute, Charterhouse Square, London EC1M 6BQ, UK
2Correspondence: Sensory Function, Centre for Neuroscience, Hodgkin Building, KCL, Guy’s Campus, London Bridge, London SE1 1UL. E-mail: Marzia.Malcangio{at}kcl.ac.uk
SPECIFIC AIMS
Small-diameter sensory neurones found in the rat dorsal root ganglion (DRG) include cells sensitive to glial cell line-derived neurotrophic factor (GDNF) that express the inhibitory non-opioid peptide somatostatin (SOM). We wanted to assess whether GDNF would acutely influence activity-induced release of SOM from sensory neurones in the rat dorsal horn isolated in vitro and to test the effect of single GDNF treatment in animal models of inflammation and thermal hypersensitivity in which sensory neurone-derived SOM has been shown to display inhibitory properties.
PRINCIPAL FINDINGS
1. GDNF modulates sensory neurone synaptic activity in dorsal horn
We used adult rat isolated spinal cord with dorsal roots attached. High-intensity electrical stimulation was applied to the dorsal roots and the release of neuropeptides substance P (SP) and SOM, as well as the amino acid glutamate (Glu), in spinal cord superfusates was quantified as a measure of sensory neurone activation. SOM basal outflow values were not altered after electrical stimulation of the dorsal roots in control preparations (Fig. 1
A). However, the presence of GDNF in the superfusing medium, though not changing SOM basal outflow, promoted SOM release during electrical stimulation (Fig. 1A
). The effect of GDNF was concentration dependent with a calculated EC50 of 15.8 ng/ml (500 pM; Fig. 1B
). The excitatory neuropeptide SP was released significantly over basal outflow from spinal cord preparations after electrical stimulation of the attached dorsal roots (Fig. 1C
); the presence of GDNF did not alter SP release pattern (Fig. 1C
). When Glu was quantified in the superfusates, neither electrical stimulation nor GDNF superfusion modified amino acid content (Fig. 1D
). GDNF-induced release of SOM was not observed when calcium ions (plus EDTA) were omitted from the superfusing medium and was blocked by tyrosine kinase inhibitor K-252a at 100 nM. Data with isolated dorsal horn preparations show that topical application of GDNF selectively promoted activity-induced release of SOM from sensory neurone central terminals. Data in the literature suggest that SOM released from sensory neurones acts as a functional antagonist of the proinflammatory and pronociceptive peptide SP in models of neurogenic inflammation. Here, we evaluated GDNF’s ability to modulate SP-induced neutrophil migration and hyperalgesia and whether endogenous SOM was involved. The selective agonist of neurokinin type 1 (NK1) receptors Sar9-SP was used.
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2. GDNF inhibits SP-induced neutrophil migration
Direct application of the SOM analog Octreotide (25 µg/mouse) in the air pouch reduced neutrophil recruitment promoted by zymosan or Sar9-SP but not carrageenin (Fig. 2
A). Local application of GDNF (3 µg/mouse) reduced neutrophil migration induced by Sar9-SP (Fig. 2C
). Again, GDNF’s effect was selective since it did not affect cell influx induced by nonspecific inflammogens such as carrageenin or zymosan (Fig. 2B
). Neuronal SOM involvement in GDNF antimigratory activity against Sar9-SP was substantiated by the observation that GDNF’s effect was absent when the endogenous SOM neuronal pool had been depleted by 
90% with systemic administration of cysteamine (100 mg/kg) (Fig. 2C
). Finally, a single injection of GDNF into the air pouch of naive mice at a dose (3 µg) that inhibited neutrophil migration significantly reduced SOM content in spinal cords measured 4 h later. SOM-LI content was 35.8 ± 1.5 pg/mg wet tissue in control mice treated with vehicle (n=4) and was reduced to 15.8 ± 5.0 pg/mg wet tissue in mice receiving GDNF (n=4, P<0.05 Student’s t test).
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3. Intrathecal GDNF reaches the dorsal horn and inhibits intrathecal SP-induced thermal hypersensitivity
Using intrathecal Sar9-SP (10 µg) -induced thermal hypersensitivity as a behavioral model, we found that a single intrathecal injection of GDNF (12 µg/rat) in the rat lumbar spinal cord opposed Sar9-SP-induced hyperalgesia. This effect was reversed by the SOM antagonist c-SOM (0.3 µg/rat) and mimicked by the SOM analog Octreotide (3.2 µg/rat). To prove intrathecal GDNF injection had reached its proposed site of action in the rat dorsal horn, we demonstrated activation of the extracellular signal-regulated kinase (ERK). ERK phosphorylation is a downstream event that occurs after GDNF interaction with its receptor RET and subsequent activation of mitogen-activated protein kinase.
CONCLUSION AND SIGNIFICANCE
It has become clear that neurotrophic factors are strong regulators of synaptic efficacy of adult primary sensory neurones. The specialized primary sensory neurones that respond to tissue injury (nociceptors) express receptors for two distinct neurotrophic factors: nerve growth factor (NGF) or GDNF. The NGF-sensitive group of nociceptors expresses SP; indeed, SP levels are regulated by NGF availability. The GDNF-sensitive group of nociceptors expresses SOM. We show that topical application of GDNF promoted SOM release from the central terminals of activated sensory neurones in the dorsal horn of the spinal cord. GDNF differs from NGF, which does not act locally to influence SP release in the dorsal horn. This is the first report with evidence for short-term GDNF modulation of synaptic transmission. The effect of GDNF on sensory neurones was marked, rapid, and dependent on the continued presence of neurotrophin. In the absence of GDNF, release of SOM from activated sensory neurones was undetectable and could not be attributed to a failure of electrical stimulation to recruit high threshold fibers that contain the peptide: under the same conditions, a significant amount of SP (also contained in high threshold fibers) was released.
We have begun to determine the intracellular mechanism by which GDNF promoted the release of SOM. A potential pathway could involve activation of GDNF receptor-coupled tyrosine kinase activity that can lead to MAP-kinase activation, based on two observations. 1) The effect of GDNF on SOM release in the dorsal horn was prevented by K-252a, a tyrosine kinase inhibitor. 2) Intrathecal injection of GDNF to anesthetized rats induced ERK phosphorylation in the superficial laminae where primary afferent fibers containing SOM terminate. The calcium dependency of GDNF’s effect on SOM release suggests that GDNF promoted a substantial buildup in cytosolic calcium ion concentration that is required for release of peptides from large-dense core vesicles. It remains to be established which SOM neuronal pool in the dorsal horn is targeted by GDNF treatment; it is likely, however, that GDNF acted on primary afferents because GFR
-1 (GDNF family of receptors) immunoreactivity is found on axon terminals of sensory neurones in dorsal horn lamina II, some of which contain SOM and express RET. It is unlikely that GDNF promoted release of SOM from spinal cord interneurones, as there is a lack of evidence for RET mRNA expression in these neurones.
Because of its ability to modulate SOM release, it is conceivable that GDNF could influence phenomena in which sensory neurone-derived peptides play an important physiopathological role; an example is neurogenic inflammation. After tissue injury, the SOM released by peripheral terminals of sensory neurones acts as an anti-inflammatory peptide that directly opposes the actions of proinflammatory peptides such as SP. SP activates NK1 receptors on the venular endothelium to increase microvascular permeability and promote plasma extravasation. SP also stimulates leukocyte adhesion to the vessel wall and their emigration into the inflamed tissue, again brought about by NK1 receptors. Neutrophils and human mononuclear cells express receptors for SP and SOM. We show that the SOM stable analog OCT locally applied to the mouse air pouch inhibited Sar9-SP- and zymosan-induced neutrophil migration. Similar to OCT, GDNF injected locally into the mouse air pouch inhibited Sar9-SP-induced PMN migration. That depletion of neuronal SOM pools by cysteamine treatment prevented the effect of GDNF strongly suggests that GDNF was counteracting the effect of Sar9-SP by activating the endogenous SOM system. However, sensory neurones are bipolar cells that, once activated, can release the content of their central terminals (including SOM) in the dorsal horn of the spinal cord. Activation of SOM receptors would depress nociceptive neurones, antagonizing the action of SP that, after activation of NK1 receptors, induces a response by neurones otherwise unresponsive to noxious stimuli. Accordingly, intrathecal injection of Sar9-SP reduced rat hind paw threshold to thermal stimulation and intrathecal OCT inhibited Sar9-SP-induced thermal sensitivity. GDNF counteracted Sar9SP-induced thermal hypersensitivity. These data show that GDNF opposed the pronociceptive effect of SP. In all models used, GDNF appeared to modulate SOM release exclusively when sensory neurones were activated. This experimental evidence supports a modulatory role for GDNF in the complex scenario characteristic of neurogenic inflammation. We propose that GDNF modulation of activity-induced release of SOM is a novel mechanism (Fig. 3
) that deserves exploration as a potential therapeutic strategy based on local release of SOM to control two major features of inflammation: pain and leukocyte recruitment.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0971fje; to cite this article, use FASEB J. (March 12, 2002) 10.1096/fj.01-0971fje 
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