Localization of cyclooxygenase-2 and prostaglandin E2 receptor EP3 in the rat lumbar spinal cord
Introduction
Prostaglandins (PGs), peripheral mediators of inflammatory stimuli, are also known to be involved in regulatory processes in the central nervous system like the sleep–wake regulation (Hayashi, 1991), febrile response (Wolfe and Coceani, 1979), pain control (Poddubiuk, 1976) and nociceptive processing (Ferreira et al., 1978). Of the two known isoforms of cyclooxygenases (COX), enzymes essential for prostanoid synthesis, the otherwise mostly induced COX-2 isozyme seems to be predominantly constitutively expressed in the central nervous system: in the normal rat brain, COX-2 was primarily observed in neurons involved in processing and integration of nociceptive, visceral and special sensory input and in the integration of autonomic, endocrine and behavioral responses (Breder et al., 1995). By immunocytochemistry, COX-2 was detected in dendrites and neuronal cell bodies localized chiefly in the cerebral cortex and allocortical structures such as hippocampus and amygdala (Yamagata et al., 1993; Breder et al., 1995; Kaufmann et al., 1996). Colocalization with glutamate and increased expression after seizures suggest a role in excitatory synaptic activity (Yamagata et al., 1993; Kaufmann et al., 1996).
In the spinal cord, neuronal pain transmission at some stage involves activation of PG biosynthesis, as various studies could demonstrate in recent years: hyperalgesic effects were observed after intrathecal administration of PGs (Malmberg and Yaksh, 1992a; Meller et al., 1994) and a spinal site of action is proposed for the analgesic actions of non-steroidal anti-inflammatory drugs (Geisslinger et al., 1994; Neugebauer et al., 1995). Furthermore, release of PGs in the spinal cord was observed after stimulation of afferent nerves (Ramwell et al., 1966), noxious thermal stimulation (Coderre et al., 1990) and by increased potassium levels (Yaksh, 1982). Indirect evidence for the presence of cyclooxygenases in the spinal cord has thus existed for some time. Recently, we could demonstrate mRNA expression of both COX isoforms in the rat spinal cord with COX-2 presumably as the predominant isoform (Beiche et al., 1996).
Prostanoids exert their manifold effects via a variety of seven transmembrane domain receptors classified as DP, EP, FP, IP and TP receptors, according to their respective prostanoid ligand (Negishi et al., 1995). In the central nervous system, the prostaglandin D2 receptor DP (Morii and Watanabe, 1993) and prostaglandin F receptor FP (Li et al., 1995) as well as different subtypes of the prostaglandin E2 receptor EP have been described (Minami et al., 1994). The EP3 receptor exists in several carboxyterminal splice variants (Takeuchi et al., 1994) and is expressed in neurons and sensory ganglia in large areas of the brain (Sugimoto et al., 1994). However, localization of the prostanoid receptor subtypes with specific antibodies has not been demonstrated to this date. As PGE2 clearly is one of the major spinally produced prostanoids (Hay et al., 1996; Goppelt-Struebe and Beiche, 1998), we tried to correlate spinal COX-2 expression with the localization of PGE2 receptor EP3 subtype against which a polyclonal antibody was raised.
In the present study, we document the widespread presence and localization of COX-2 immunoreactivity (IR) in different areas of the rat lumbar spinal cord, along with a spatially confined expression of PGE2 receptor EP3 in the superficial laminae of the dorsal horns.
Section snippets
Tissue preparation
Spinal cord tissue samples were obtained from male Sprague–Dawley rats weighing 200–400 g (Charles River, Sulzfeld, Germany). Animals were deeply anesthesized with pentobarbital and perfused transcardially with a 2% paraformaldehyde/phosphate buffered saline (PBS) solution. Segments L4–L5 of the lumbar enlargement were removed separately, postfixed in the same fixative for 60 min, cryoprotected in 20% sucrose/PBS at 4°C overnight and frozen at −70°C. 10 μm sections were cut on a cryostat,
Specificity of the antibodies
The specificity of the anti-COX-2 antibody in neuronal tissue was verified by Western blot analysis. Fig. 1 shows a COX-2 stained immunoblot with purified COX-1 and COX-2 enzymes (Cayman) and different microsomal preparations. Distinct COX-2 immunoreactive bands were detected at approximately 72 kDa in the lanes representing purified COX-2 isozyme and microsomal preparations of 4-h fetal calf serum (5%)-stimulated rat renal mesangial cells (Stroebel and Goppelt-Struebe, 1994) and the spinal
Discussion
In the present study, we could demonstrate for the first time the presence of COX-2-immunoreactivity (IR) in neurons and astrocytes of the rat lumbar spinal cord. We determined the exact location of COX-2 IR which was prominent in neurons of the superficial dorsal horn and motoneurons of lamina IX. Furthermore, COX-2 IR was matched to a highly selective expression of PGE2 receptor EP3 in the afferent nerve terminating area of the superficial dorsal horn laminae I and II.
In cells of peripheral
Acknowledgements
This work was supported by Deutsche Forschungsgemeinschaft (SFB 353/A1). Special thanks to C. Reichel, C. Labahn and G. Geisslinger for their support.
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