Nociception and the differential expression of cyclooxygenase-1 (COX-1), the COX-1 variant retaining intron-1 (COX-1v), and COX-2 in mouse dorsal root ganglia (DRG)

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Abstract

Prostaglandins (PGs) formed via the cyclooxygenase (COX) pathway mediate hyperalgesia in sensory nerve endings. To investigate the role of the COX isoforms in pain transmission we recently studied nociception in COX-isozyme-deficient mice using models of “sharp” rapidly transmitted pain (hot-plate) and slowly developing, diffuse pain (writhing) [Ballou L, Botting RM, Goorha S, Zhang J, Vane JR. Nociception in cyclooxygenase isozyme-deficient mice. Proc Natl Acad Sci USA 2000;97:10272]. Our results demonstrated that COX-1 (and not COX-2) was the primary isoform involved in nociception in both model systems. Given the importance of dorsal root ganglia (DRG) in pain transmission we examined the expression patterns of COX-1, -2 and the recently described variant of COX-1 retaining intron-1, originally referred to as “COX-3” but hereafter referred to as COX-1 variant (COX-1v), in mouse L4 or L5 DRG taken from normal and COX-isozyme-deficient mice. Messenger RNA and protein for COX isoforms from DRG, spinal cord as well as, heart, brain, kidney, spleen and skin of adult mice were isolated and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Patterns of COX-isoform expression were determined using immunohistochemical techniques. We found that COX-1 and COX-1v were both expressed in neurons while COX-2 expression was completely undetectable in the DRG. Immunohistochemical analysis of COX expression in DRG of mice exhibiting the chronic pain and inflammation associated with collagen-induced arthritis (CIA) expressed COX-1 and COX-1v while no COX-2 could be detected. For purposes of comparison, COX-1v mRNA was also expressed in heart, brain, spinal cord, kidney, spleen and skin. Together, these data support a role for COX-1 and perhaps COX-1v, not COX-2, as the primary producers of PGs in mouse DRG in normal and in mice subject to chronic pain and inflammation. These data also suggest potential alternative analgesic mechanisms of action for the newly developed, COX-2 selective inhibitors and the nonsteroidal anti-inflammatory drugs (NSAIDs) in pain transmission in the peripheral nervous system.

Introduction

Prostaglandins (PGs), primarily PGE2 and prostacyclin, are important mediators of inflammation, pain, and fever. They are synthesized in tissues by the constitutive enzyme cyclooxygenase-1 (COX-1) and by its isoform, COX-2, which can be induced with cytokines, growth factors, or other inflammatory stimuli [1], [2], [3], [4]. There is also a newly described variant of COX-1 retaining intron-1 (COX-1v), “COX-3” about which little is currently known [5]. PGs are hyperalgesic and enhance nociception produced by other mediators such as bradykinin [6], [7], [8].

COX-1, is an ubiquitously expressed gene present in many tissues including the central nervous system, whereas COX-2, inducible isoform which is a dynamically regulated, responding to inflammatory stimuli, although constitutively expressed in some regions of the CNS and in the kidneys [9], [10], [11], [12]. While, relatively little is known about the physiologic roles of COX-1v, pharmacologic evidence does suggest that it is involved in pain signaling pathways and does exhibit different pharmacologic responses to NSAIDs than COX-1 [5]. Our findings that COX-1 is a major nociceptive mediator in acute and diffuse mouse pain models also suggests a potential role for COX-1v in pain transmission since it would not be expressed in COX-1−/− mice.

Pain (mechanical, chemical, and thermal) sensation originating in peripheral receptors is transmitted to spinal cord and brainstem neurons by afferent fibers of dorsal root ganglion (DRG). These signals are then forwarded to thalamic nuclei and from there to the trunk and extremity representation areas in the primary somatosensory cortex [13]. Tissue injury or inflammation involves the production and release of a number of chemical mediators including bradykinin, serotonin and PGs [14], [15], [16], [17]. Of these, PGs are critical for the processing of pain not only by sensitizing peripheral nociceptors but also augmenting processing of pain information at spinal level [4]. Primary afferent fibers have their cell bodies in the DRG. The different sensory modalities are clearly mediated by distinct classes of neurons in DRG according to different physiological criteria [18], [19], [20], [21].

In a previous study we found that COX-1 (not COX-2) was primarily involved in pain transmission by using COX-isozyme-deficient mice in models of “sharp” rapidly transmitted pain (hot-plate) and slowly developing, diffuse pain (writhing) [22]. In order to examine the potential contributions of the COX isozymes in nociception we examined the expression and localization patterns of each COX isoform in DRG, spinal cord and selected other tissues using immunohistochemistry, RT-PCR, and Western blot techniques in tissues from normal and COX-isozyme-deficient mice. Because COX-2 plays a key role in the pathogenesis of inflammatory pain in tissues, we also examined whether the expression of COX isoforms in DRG and inflamed skin were altered in mice with collagen-induced arthritis (CIA) [23], [24]. An understanding of the role(s) of COX in pain transmission in the peripheral nervous system is critical for discernment of the analgesic mechanisms of action for the newly developed, COX-2 selective inhibitors and the nonsteroidal anti-inflammatory drugs (NSAIDs).

Section snippets

Animals

Strain C57/DBA1 of COX-1−/−, COX-2−/− and wild-type (WT) adult mice (18–30 g) [22], [25] used in this study were housed in Plexiglas cages at 25 ± 1 °C and kept on a 12 h:12 h light/dark cycle. Food and water were available ad libitum. The genotype of COX-1 or COX-2 null mice was determined by PCR as previously described [22], [25].

Tissue isolation and preparation

Mouse tissues were prepared from anesthetized animals by perfusion via the left ventricle with 30 ml of 20 mM PBS pre-warmed to 37 °C. DRG from the lumbar 4 and 5 (L4 and L5)

COX-1, -2, and -3 mRNA expression in control and arthritic mouse tissues

In order to test the effects of chronic inflammation on COX expression in selected tissues we immunized WT mice with CII. As expected the WT mice developed arthritis about 21 days after immunization, with a final incidence of 80% (16 of 20 mice). All 12 of the control WT mice immunized with PBS only did not develop any signs of arthritis (one or more swollen paws). The arthritic mice exhibited profound swelling of the ankle and/or paw joints and a marked disturbance of gait. Of 16 arthritic

Discussion

It is generally accepted that COX-1 is typically constitutively expressed and performs homeostatic functions, while COX-2 expression is induced in response to physiological stresses. However, there are a few notable examples in which COX-2 is constitutively expressed, namely in stomach, kidney, some regions of the brain and in spinal cord [9], [10], [11], [12]. COX-1v is a newly discovered variant of COX-1 retaining intron-1 (“COX-3”) that is primarily expressed in brain and heart [5], but

Acknowledgements

Grant support: This material is based upon work supported by the Office of R&D, Medical research Service, Department of Veterans Affairs (VA) and the Arthritis Foundation. We also thank Tim Higgins for preparation of all figures and photographs.

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