Neuronal nitric oxide plays an important role in nociception and opioid action in the central and peripheral nervous systems and the importance of the NMDA/nitric oxide cascade in opioid tolerance has been extensively studied. Early studies quickly established the ability of NOS inhibitors to block morphine tolerance (Kolesnikov, Y. A. et al. 1992
; Kolesnikov, Y. A. et al. 1993
). They also influence morphine analgesia directly. However, these actions are complex. Supraspinal NOArg enhances morphine analgesia whereas spinal NOArg decreases it (Kolesnikov, Y. A. et al. 1997
). Although useful, enzymatic inhibitors have limitations, particularly when examining isoforms of a protein. Antisense mapping provides a valuable approach that can downregulate individual isoforms by targeting specific exons (Standifer, K. M. et al. 1994
). Splice variants containing the targeted exon may be downregulated while those without the exon are unaffected. However, the approach does have some limitations, including limited downregulation of the mRNA being targeted. While activity of an antisense can be helpful, the absence of an effect might reflect the lack of involvement of the isoforms containing the targeted exon or it may simply be due to inactivity of the probe in the assay. Thus, these studies must be interpreted cautiously. In our earlier study, we observed opposing activities of nNOS-1 and nNOS-2 in morphine actions. Downregulation of nNOS-1 prevented the appearance of morphine tolerance. In contrast, selectively downregulating nNOS-2 with an antisense probe to a unique splice junction impaired morphine’s analgesic activity. Our current studies have extended this approach to the formalin test.
The current studies illustrate the complexity of the role of NO in the mediation of formalin–induced pain. Local application of NOArg did not affect either phase of the formalin assay. Yet, increasing NO production with the administration of the precursor L-arginine yielded a biphasic response, with low doses pro-nociceptive and higher doses anti-nociceptive. Thus, the lack of an effect with the inhibitor NOArg might reflect the simultaneous blockade of the opposing systems. Antisense studies illustrated the ability of antisense probes targeting exons 2 and 9 applied locally to lower the second phase of the response. There was a slight elevation in the phase I response that was significant with an antisense selective for nNOS-2, but this effect was modest. Together, these antisense results lend support to the possibility of opposing NOS systems in the periphery.
Supraspinal blockade of nNOS with NOArg lowered both phases in the formalin assay, implying a causal role for supraspinal NO in the production of the formalin response. Supraspinal antisense treatment also revealed a reduction in the pain response. The exon 18 antisense probe lowered both phases while the exon 2 and 9 antisense probes lowered only the second phase. The pro-nociceptive role of supraspinal NO is similar to the ability of supraspinal NOArg to enhance morphine analgesia (Kolesnikov, Y. A. et al. 1997
A number of spinal antisense oligodeoxynucleotide probes significantly lowered the second phase of the formalin response without affecting the first phase, including the antisense unique to nNOS-β. Thus, nNOS at the spinal level has a pro-nociceptive activity. However, the antisense probe targeting the exon 8–11 splice site that is unique to nNOS-2 showed a different response. This antisense probe increased both the first and second phase of the formalin response, implying that the endogenous activity of this isoform diminishes the intensity of formalin nociception, an anti-nociceptive action, and provides further evidence for opposing nNOS systems in pain modulation. Thus, at the spinal level, nNOS-2 and nNOS-β have opposing actions. The role of nNOS-1 in these actions remains a bit unclear since the other antisense probes are not selective for a single isoform.
These observations emphasize the importance of peripheral, spinal and supraspinal nNOS in mediating formalin-induced hyperalgesia. However, these enzymes also are involved with morphine tolerance. Earlier studies had implicated nNOS-1 in morphine tolerance with the tailflick assay, a thermal measure of nociception (Babey, A. M. et al. 1994
; Kolesnikov, Y. A. et al. 1992
; Kolesnikov, Y. A. et al. 1993
; Kolesnikov, Y. A. et al. 1997
). The current studies reveal similar effects on tolerance with the formalin assay. Chronic morphine dosing produced tolerance in both the thermal and formalin assays. Spinal antisense treatment blocked the development of tolerance in both tests. Thus, the NMDA/nitric oxide cascade is important in the development of tolerance in both pain models.
The current study separates the functional roles of alternatively spliced isoforms of nNOS in formalin–induced hyperalgesia. nNOS-1 appears to play a major role in the second phase of formalin pain supraspinally, spinally and peripherally while nNOS-β does the same spinally. In contrast, the nNOS-2 isoform has an opposite effect, as shown by the enhanced formalin response with the isoform was downregulated. These results correlate with the morphine study, in which we have shown that nNOS-1 diminishes the analgesic actions of morphine, while nNOS-2 enhances them (Kolesnikov 1997
). These studies demonstrate complex effects of NO in pain modulation and the utility of antisense mapping in exploring functional roles of NOS isoforms in the central nervous system.