On cholecystokinin-opioid interaction in the spinal dorsal horn following peripheral nerve injury and inflammation

Sammanfattning: The peptide cholecystokinin (CCK) antagonizes opioid-induced analgesia. Conversely, antagonists of CCK receptors enhance the analgesic effect of opioids. Together, these results support a role for CCK as an anti-opioid peptide in pain mechanisms. In pathological pain states the CCK-opioid interaction may be altered resulting in changes in the effectiveness of opioids. Thus, the reduced antinociceptive potency of morphine after nerve injury cae be reversed by CCK antagonists. After inflammation however, the potency of opioids is enhanced. The present thesis, using histological, behavioural, electrophysiological and biochemical techniques, addressed the mechanisms of CCK-opioid interaction under normal and pathological conditions. With double-coloured immunofluorescence method partial co-localization between CCK-like immunoreactivity (LI) and [my]-opioid receptor (MOR)-LI in neurons in superfical dorsal horn was observed. The presence of MOR in CCK containing neurons suggests a possible direct influence of opioids on CCK release in the spinal cord. In addition, we showed that axotomy, but not inflammation, induced a moderate decrease in CCK- and MOR-LI in the dorsal horn. Intravenous (i.v.) morphine further reduced CCK- and MOR-Lls in axotomized, but not in normal or inflamed, rats. While the effect of morphine on CCK-LI can be interpreted as the result of increased CCK release, the effect on MOR-LI is more difficult to explain and may be related to changes in the microenvironment of the dorsal horn induced by nerve injury. Peripheral nerve injury induces an upregulation of mRNA for CCK and CCK-B receptors in sensory neurons. We have studied the involvement of CCK in spinal nociception in normal and axotomized rats. Intrathecal (i.t.) CCK facilitated the flexor reflex in rats with intact or sectioned nerves. However, the facilitation induced by CCK in axotomized rats was significantly reduced compared to rats with intacted nerves. The CCK-B receptor antagonist CI-988 did not depress the baseline flexor reflex or facilitation of the reflex induced by repetitive C-fiber stimulation in normal or axotomized rats. These results suggest that despite a excitatory effect of exogenous CCK, this peptide is unlikely to be directly involved in mediating nociceptive transmission at spinal level. By using in vivo microdialysis K+-stimulation induced an increase in the extracellular level of CCK in the dorsal horn. CCK was also released by i.v. or i.t. morphine in a naloxone reversible manner. No detectable change on the basal levels of CCK after nerve injury or inflammation was observed. However while morphine was still able to release CCK after axotomy, moprhine produced no effect in inflamed rats. In contrast, K+-stimulation failed to increase extracellular levels of CCK in axtomized rats, an effect that was reversed by CI-988. K+-stimulation, however, released CCK in inflamed rats. In order to study whether CCK is involved in the development and maintenance of morphine tolerance, we measured spontaneous and K+-evoked release of CCK in the spinal dorsal horn in morphine tolerant rats. Basal CCK levels were below the detection limit of the assay in the majority of tolerant and normal rats, with no difference between the two groups. Similarly, K+-stimulation induced comparable release of CCK in normal or tolerant rats. The ability of morphine to release CCK under normal conditions provides further evidence for a physiological role for CCK as an opioid modulating peptide and for the concept that CCK-opioid interaction represents a limiting factor for opioid analgesia. The variable effects of axotomy and inflammation on morphine-induced CCK release support the idea that changes in opioid sensitivity may involved activity of the CCK system. Moreover, these data suggest that the mechanisms of CCK release by K+ and by morphine are different and modulated in different ways after nerve injury or inflammation. It is possible that axotomy may induce transsynaptic changes in the spinal cord leading to (1) effects by morphine on MOR-LI, (2) spinal hyposensitivity to CCK, and (3) changes in the mechanism of K+-evoked CCK release. Finally, the extracellular level of CCK may not play a major role in the development of morphine tolerance.