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Cgmp-Dependent Protein Kinase in Dorsal Root Ganglion: Relationship with Nitric Oxide Synthase and Nociceptive Neurons

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Cgmp-Dependent Protein Kinase in Dorsal Root Ganglion: Relationship with Nitric Oxide Synthase and Nociceptive Neurons The Journal of Neuroscience, May 15, 1996, W(10):3130-3138 cGMP-Dependent Protein Kinase in Dorsal Root Ganglion: Relationship with Nitric Oxide Synthase and Nociceptive Neurons Yifang Qian,l Daniel S. Chao,’ Daniel R. Santillano,l Trudy L. Cornwell, Angus C. Nairn,4 Paul Greengard,d Thomas M. Lincoln,3 and David S. BredV* 1Deparfment of Physiology and *Program in Biomedical Sciences, University of California San Francisco, San Francisco, California 94 143, 3Deparlmen t of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294, and 4DepaHment of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York IO02 1 Nitric oxide and cGMP influence plasticity of nociceptive pro- and roof plates. Neuronat nitric oxide synthase (nNOS) is co- cessing in spinal cord. However, effecters for cGMP have not expressed with cGKI in sensory neurons during embryonic been identified in sensory pathways. We now demonstrate that development and after peripheral nerve axotomy. The primary cGMP-dependent protein kinase I (cGKI) occurs in the DRGs at target for cGKl in cerebellum, G-substrate, is not present in levels comparable to that in cerebellum, the richest source of developing, mature, or regenerating sensory neurons, indicat- cGKI in the body. Immunohistochemical studies reveal that ing that other proteins serve as effecters for cGKI in sensory cGKI is concentrated in a subpopulation of small- and medium- processing. These data establish sensory neurons as a primary diameter DRG neurons that partially overlap with substance P locus for cGMP actions during development and suggest a role and calcitonin gene-related polypeptide containing cells. Dur- for cGKI in plasticity of nociception. ing development, cGKl expression throughout the embryo is Key words: nociceptive; dorsal root ganglion; nitric oxide; essentially restricted to sensory neurons and to the spinal floor development; substance P; cGMP Nitric oxide (NO) serves as a cellular mediator for diverse devel- sensory processing derives from pharmacological studies. NOS opmental and physiological processes (Marletta, 1993; Moncada antagonists do not alter baseline responses indicating that NO and Higgs, 1993; Bredt and Snyder, 1994a; Nathan and Xie, 1994; does not function as a fast transmitter at primary afferent syn- Garthwaite and Boulton, 1995). Neuronal type NO synthase apses. NOS antagonists, however, do prevent spinal sensitization (nNOS, or type I) is localized to a discrete population of neurons of nociceptive reflexes (Moore et al., 1991; Kitto et al., 1992; in the embryonic and mature nervous system (Bredt et al., 1991; Meller et al., 1992; Meller and Gebhart, 1993). This NO- Bredt and Snyder, 1994b). nNOS is a calcium/calmodulin- dependent sensitization contributes to hyperalgesia after periph- dependent enzyme that is stimulated by activation of NMDA type eral injury and is also blocked by NMDA receptor antagonists glutamate receptors (Garthwaite et al., 1988; Knowles et al., 1989; (Dickenson, 1990). Bredt and Snyder, 1990). NO formed during excitatory transmis- NO signaling occurs primarily through activation of soluble sion participates in synaptic plasticity mediated by NMDA recep- isoforms of guanylyl cyclase. In the vascular and nervous systems, tor activity (Schuman and Madison, 1994). cGMP-dependent protein kinases (cGKs) serve as major effecters A role for NO in sensory signaling was initially suggested based for NO and cGMP (Lincoln et al., 1994). Endothelial-derived NO on the specific localization of nNOS in sensory pathways (Bredt et stimulates guanylyl cyclase and subsequently cGK in vascular al., 1990; Aimi et al., 1991). In normal adult mammals, nNOS is smooth muscle leading to blood vessel relaxation (Ignarro, 1993; expressed in a few small- and medium-diameter DRG neurons, Moncada and Higgs, 1993; Warner et al., 1994). Similarly, phys- and in most of these cells, nNOS coexists with calcitonin gene- iological actions of NO in sensory pathways appear to involve related peptide (CGRP) and rarely with substance P @hang et al., cGMP (Meller and Gebhart, 1993). NO-mediated hyperalgesia is 1993). By contrast a large percentage of DRG neurons contain blocked by extracellular hemoglobin (Kitto et al., 1992), indicating nNOS transiently during embryonic development (Bredt and Sny- a role for intercellular NO signaling. Furthermore, membrane der, 1994b) and after peripheral nerve damage @hang et al., permeable cGMP analogs mimic the effects of NO on sensory 1993). Direct evidence for involvement of NO in plasticity of neuron responsiveness (McGehee et al., 1992; Niedbala et al., -_--l--“-.--.------ - --~.- . .“_-_~-.-...- 1995), Downstream effecters for cGMP, however, have not pre- Received Oct. 5, 1995; revised Feb. 12, 1996; accepted Feb. 22, 1996. viously been characterized in sensory neurons. This work was supported by grants (to D.S.B.) from the NationaI Science Foun- Molecular studies have identified two genetic loci encoding dation, the Lucille P. Markey Charitable Trust, and the National Association for Research on Schizophrenia and Depression, the McKnight Endowment Fund for mammalian cGKs. The first gene product characterized, cGKI, Neuroscience, the Esther A. and Joseph Klingenstein Fund, and National Institutes was purified and sequenced from soluble extracts of vascular of Health Grants HL 34646 (T.M.L.) and MH-40899 (P.G.). We thank Allan smooth muscle from bovine lung (Lincoln et al., 1977; Flockerzi et Basbaum for helpful discussions and for review of this manuscript. Correspondence should be addressed to David S. Bredt, Department of Physiol- al., 1978; Takio et al., 1984). Molecular cloning of the cDNA ogy, School of Medicine, University of California at San Francisco, 513 Parnassus demonstrated N-terminal alternative splicing yielding cGKIa Avenue, San Francisco, CA 94143-0444. Dr. Qian’s present address: Department of Psychiatry, San Mateo County General and cGKI/3 isoforms (Wernet et al., 1989). More recently, a Hospital, San Mateo, CA 94113. membrane-associated cGKI1 has been cloned from brain (Uhler, Copyright 0 19% Society for Neuroscience 0270-6474/96/i 63 130-09 $0500/O 1993) and from the epithelium of intestinal mucosa (Jarchau et Qian et al. l cGMP-Dependent Protein Kinase in Dorsal Root Ganglion J. Neurosci., May 15, 1996, 76(10):3130-3138 3331 al., 1994). In the CNS cGK enzyme activity is selectively enriched Cb Cx DRG in the cerebellum where high levels of cGKI protein are histo- A chemically apparent in Purkinje neurons (Lohmann et al., 1981). 200- Low levels of cGK activity found in forebrain tissues appear to primarily represent cGKI from vascular smooth muscle (Loh- 97- mann et al., 1981). A prominent target for cGK, G-substrate, is - - exclusively found in cerebellum (Detre et al., 1984). The prominent functions for NO and cGMP in sensory plastic- 68- ity led us to investigate the possible expression of cGKs in sensory neurons. Strikingly, we demonstrate that cGKI occurs in DRG at levels equivalent to that in cerebellum. cGKI is found in small- 43- and medium-diameter neurons of DRG and is concentrated in axonal processes in laminae I and II of spinal cord, consistent with a-cGK1 known roles for NO and cGMP in nociceptive processing. During B embryogenesis, cGKI is found only in DRG neurons and cells of 200- the spinal floor and roof plates. Whereas cerebellar Purkinje cell -- soma contain both cGKI and G-substrate, sensory neurons lack 97- G-substrate, suggesting that novel targets mediate actions of cGKI in sensory neuron development and plasticity. 68- MATERIALS AND METHODS Antisera. The following primary polyclonal rabbit antibodies were used: cGKI (Lohmann et al., 1981; Wyatt et al., 1991), nNOS (Bredt et al., 1990), substance P (Peninsula Laboratories), CGRP (Peninsula Labora- 43- tories). For double-immunofluorescence labeling, a polyclonal guinea pig antiserum to substance P was used (Too and Maggio, 1991). a-nNOS Ajinity purification of G-substrate antibody. Polyclonal antibody to G-substrate (Dolphin et al., 1983) was further affinity purified by presorb- ing nonspecific antibodies with a crude cerebral cortical extract. Rat cerebral cortex was homogenized in 10 volumes of 25 mM Tris-HCl buffer, pH 7.4, 1 mM EDTA, 100 PM PMSF and diluted to 5 mg protein/ml. Ten micrograms of antibody to G-substrate (Dolphin et al., 1983) were incubated overnight with 5 mg of cortical extract. Soluble antibodies were recovered by filtration through a 0.44 pm mesh. Western blotting. Tissues were dissected and homogenized in 10 vol- umes of Tris-HCl buffer, pH 7.4, containing 1 mM EDTA and 100 PM PMSF. Acid soluble extracts were prepared using the method of Aswad and Greengard (1981). Proteins were separated by SDS-PAGE and transferred to PVDF membranes, and blots were incubated in blocking solution containing 3% bovine serum albumin in PBS and 0.1% Tween 20. Antibodies were diluted in this same buffer and hybridized to blots overnight. Immunoreactive bands were visualized by ECL according to the manufacturer’s specifications (Arnersham, Arlington Heights, IL). Immunohistochemistry. Adult and embryonic rats were anesthetized a-G sub with pentobarbital and perfused with 4% freshly depolymerized parafor- maldehyde in 0.1 M phosphate buffer. Tissues were removed and postfixed Figure 1. cGMP-dependent protein kinase but not nNOS or G-substrate in paraformaldehyde for 1 hr at 4°C. Tissues were cryoprotected over- is enriched in dorsal root ganglion extracts. A, Western blotting shows that night in 20% sucrose. Thin sections were cut on a cryostat (6 pm), and cGKI occurs at similar levels in crude extracts of DRGs as in cerebellum free-floating sections (40 pm) were cut on a sliding microtome. Endog- (Cb) but is only weakly detectable in cerebracortical (Cx) tissue. B, nNOS enous peroxidase activity was inactivated by incubating sections in 0.5% is most enriched in cerebellum, is moderately expressed in cerebral cortex, H,O, for 15 min. Sections were blocked for 1 hr in PBS containing 1.5% and is only faintly detectable in DRG. C, G-substrate is only detectable in NGS and then incubated overnight in the same buffer containing diluted cerebellar extracts and does not occur in cortex or DRG.
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