The role of phosphodiesterase isoforms 2, 5, and 9 in the regulation of NO-dependent and NO-independent cGMP production in the rat cervical spinal cord

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Abstract

NO-responsive, cGMP-producing structures are abundantly present in the cervical spinal cord. NO-mediated cGMP synthesis has been implicated in nociceptive signaling and it has been demonstrated that cGMP has a role establishing synaptic connections in the spinal cord during development. As cGMP levels are controlled by the activity of soluble guanylyl cyclase (synthesis) and the phosphodiesterase (PDE) activity (breakdown), we studied the influence of PDE activity on NO-stimulated cGMP levels in the rat cervical spinal cord.

cGMP-immunoreactivity (cGMP-IR) was localized in sections prepared from slices incubated in vitro. A number of reported PDE isoform-selective PDE inhibitors was studied in combination with diethylamineNONOate (DEANO) as a NO-donor including isobutyl-methylxanthine (IBMX) as a non-selective PDE inhibitor. We studied 8-methoxy-IBMX as a selective PDE1 inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and BAY 60-7550 as selective PDE2 inhibitors, sildenafil as a selective PDE5 inhibitor, dipyridamole as a mixed type PDE5 and PDE10 inhibitor, rolipram as a PDE4 inhibitor, and SCH 81566 as a selective PDE9 inhibitor. cGMP-IR structures (nerve fibers, axons, and terminals) were characterized using the following neurochemical markers: vesicular transporter molecules for acetylcholine, GABA, and glutamate (type 1 and type 2), parvalbumin, glutamate transporter molecule EAAT3, synaptophysin, substance P, calcitonin gene-related peptide, and isolectin B4. Most intense cGMP-IR was observed in the dorsal lamina. Ventral motor neurons were devoid of cGMP-IR. cGMP-IR was observed in GABAergic, and glutamatergic terminals in all gray matter laminae. cGMP-IR was abundantly colocalized with anti-vesicular glutamate transporter 2 (vGLUT2), however not with the anti-vesicular glutamate transporter 1 (vGLUT1), suggesting a functional difference between structures expressing vGLUT1 or vGLUT2. cGMP-IR did not colocalize with substance P- or calcitonin-gene related peptide-IR structures, however did partially colocalize with isolectin B4 in the dorsal horn. cGMP-IR in cholinergic structures was observed in dorsal root fibers entering the spinal cord, occasionally in laminae 1–3, in laminae 8 and 9 in isolated boutons and in the C-type terminals, and in small cells and varicosities in lamina 10. This latter observation suggests that the proprioceptive interneurons arising in lamina 10 are also NO-responsive.

No region-specific nor a constant co-expression of cGMP-IR with various neuronal markers was observed after incubation of the slices with one of the selected PDE inhibitors. Expression of the mRNA of PDE2, 5, and 9 was observed in all lamina. The ventral motor neurons and the ependymal cells lining the central canal expressed all three PDE isoforms.

Incubation of the slices in the presence of IBMX, DEANO in combination with BAY 41-2272, a NO-independent activator of soluble guanylyl cyclase, provided evidence for endogenous NO synthesis in the slice preparations and enhanced cGMP-IR in all lamina. Under these conditions cGMP-IR colocalized with substance P in a subpopulation of substance P-IR fibers.

It is concluded that NO functions as a retrograde neurotransmitter in the spinal cord but that also postsynaptic structures are NO-responsive by producing cGMP. cGMP-IR in a subpopulation of isolectin B4 positive fibers and boutons is indicative for a role of NO-cGMP signaling in nociceptive processing. cGMP levels in the spinal cord are controlled by the concerted action of a number of PDE isoforms, which can be present in the same cell.

Introduction

NO has an important messenger function in the nervous system of probably all animals species (Alonso et al., 2000, Scholz and Truman, 2000). NO synthesis is catalyzed by the enzyme nitric oxide synthase (NOS). In the mammalian central nervous system, the constitutively expressed neuronal isoform of the enzyme (nNOS) has a wide-spread localization and can be observed in virtually every (sub)region. Nevertheless, there are large differences in the amount of NOS expressed in the various regions of the CNS (Bredt et al., 1990, Vincent and Kimura, 1992, Southam and Garthwaite, 1993, De Vente et al., 1998). The target molecule for NO is the so-called soluble isoform of guanylyl cyclase sGC (Murad, 1994), also designated NOGCR (Denninger and Marletta, 1999, Friebe and Koesling, 2003, Koesling et al., 2004).

NO-independent cGMP signaling can occur through activation of the receptor-operated, membrane-bound particulate GC (pGC) by natriuretic peptides (Tamura et al., 2001) (see the accompanying paper by De Vente et al.). In addition, compounds like YC-1 (Wu et al., 1995a, Wu et al., 1995b) and BAY 41-2272 and related compounds (Becker et al., 2001, Stasch et al., 1991) are NO-independent activators of sGC and potentiate the action of NO on the enzyme. It has been proposed that these compounds can be used to sense endogenous NO production (Ott et al., 2004, Van Staveren et al., 2005).

NO-cGMP signaling in the brain has been observed in virtually every region of the brain (e.g. Southam and Garthwaite, 1993, De Vente et al., 1998). Less data are available on NO-cGMP signaling in the spinal cord. Using in vitro experiments with spinal cord slices NO-mediated cGMP production was demonstrated throughout the gray matter of the spinal cord (Vles et al., 2000). These experiments were all performed in the presence of isobutyl-methylxanthine (IBMX) as a non-specific phosphodiesterase (PDE) inhibitor in order to inhibit cGMP hydrolysis, as PDE activity in neural tissue is high, and it is almost impossible to localize the sites of synthesis of cGMP by means of cGMP-immunocytochemistry (cGMP-ICC) without inhibiting PDE activity. Presently, 11 subfamilies of PDE's are known. Subfamilies have been classified on the basis of structural data, and on the basis of substrate and inhibitor profiles (Beavo, 1995, Conti and Jin, 1999, Francis et al., 2001). PDE's which are important in controlling cGMP metabolism in the CNS are PDE1, PDE2, PDE5, PDE6, PDE9, and PDE10. Of these, PDE6 is only present in the retina (Beavo, 1995). Specific isoforms of the PDE's show a selective regional localization in the brain and it has become evident that several PDE isoforms can be expressed in the same cell (Juilfs et al., 1997, Andreeva et al., 2001, Bellamy and Garthwaite, 2001, Van Staveren et al., 2003).

There is evidence that cGMP is involved in (anti)nociceptive processing in the spinal cord (Kitto et al., 1992, Siegan et al., 1996, Inoue et al., 1998, Ferreira et al., 1999, Tegeder et al., 2004). It has been demonstrated that during development cGMP is involved in patterning of the sensory input into the spinal cord and also in synaptic arrangements in the gray matter (Inglis et al., 1998, Schmidt et al., 2002). As cGMP levels are controlled by PDE activity, and there is no information on the cellular localization of PDE's in the spinal cord, we investigated the localization of the mRNA of the cGMP-hydrolyzing PDE's2, 5, and 9. In addition, in an attempt to study the contribution of these three PDE's to NO-cGMP signaling in the spinal cord, we investigated the effects of a number of PDE inhibitors of different selectivity on the localization of NO-mediated cGMP synthesis in the slices of the rat cervical spinal cord and attempted to characterize the cGMP-immunoreactive (cGMP-IR) structures in terms of known neurotransmitters in the spinal cord.

Section snippets

Materials

DiethylamineNONOate (DEANO), dipyridamole, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), Nω-nitro-l-arginine (l-NAME), isobutyl-methylxanthine, 8-methoxy-IBMX, and TRITC-conjugated isolectin B4 were from Sigma–Aldrich. Rolipram was from RBI. 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ) was from Tocris Cookson Ltd. Sildenafil was a gift of Pfizer, Sandwich, UK. SCH 51866 was a gift of Schering-Plough, Kenilworth, NJ, USA. 5-Cyclopropyl-2-[1-(2-fluoro-benzyl0-1H

Non-specific PDE inhibition using IBMX

cGMP-IR in slices of the cervical spinal cord incubated in vitro was virtually absent if no PDE inhibitor was present (Fig. 2(a)). Addition of 1 mM IBMX resulted in the appearance of cGMP-IR in varicosities and fibers in the gray matter of the slices, especially in lamina 1–3 (Fig. 2(b)). The effect of IBMX could be inhibited by 10 μM ODQ or 0.1 mM l-NAME. These results suggest ongoing NO synthesis in the spinal cord slices (see also the section on the effects of BAY 41-2272). Addition of 100 μM

Discussion

In the spinal cord, cGMP has been implicated in the development and maintenance of hyperalgesia (Kawabata et al., 1993, Meller et al., 1994, Salter et al., 1996, Siegan et al., 1996, Ferreira et al., 1999, Sousa and Prado, 2001, Tao et al., 2000, Tao and Johns, 2002, Schmidtko et al., 2003, Tegeder et al., 2004). NO-cGMP signaling in the spinal cord is wide-spread and has been observed in all laminae of the spinal cord (Vles et al., 2000). Nevertheless, the role of cGMP in spinal

Acknowledgements

This study was financially supported by the Phelps Stichting, Profileringsfonds azM, and Ipsen BV.

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