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

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.