Identification of Factors Associated with the Development and Decay of Heterologous Tolerance to Morphine in the Guinea Pig

Abstract

It is believed that the heterologous tolerance that develops upon chronic exposure to morphine is the result of adaptive changes in the cellular signaling pathways associated with the [mu] opioid receptor. Proposed adaptive changes that occur in these signaling elements should occur as the changes in function appear in the whole animal and should spontaneously reverse as function returns to normal. The length of time it takes for these adaptations to reverse should be proportional to the magnitude of tolerance that has developed. To test these hypotheses, it is necessary to establish a complete time course for the development and decay of heterologous tolerance. Such a time course has been documented in the LM/MP of the guinea pig following pellet implantation. However, that method of administration only allows for a qualitative assessment of the nature of the decay of tolerance because the time at which morphine exposure ends is unknown. We therefore, assessed the capacity of two different twice daily injection administrations (subcutaneous and intraperitoneal) to generate a time course for the development and decay of heterologous tolerance that was reproducible. Animals were treated for 1, 2, 4, 7, or 10 days and then assessed for the appearance of tolerance via paw pressure testing in the whole animal and by measuring the response of the LM/MP to DAMGO and 2-CADO either 0, 1, 2, or 4 days after the last treatment day. Administration of morphine via intraperitoneal injection was found to produce tolerance that was highly variable, presumably due to variable absorption from the gut, making it unsuitable for the time course study. Subcutaneous injection was shown to reproducibly induce tolerance in both the whole animal via paw pressure analgesic testing and in the response of the guinea pig LM/MP to DAMGO and 2-CADO. The results of this study indicated that a significant level of tolerance begins to develop by 2 days of treatment which appears to become maximal and plateau by 4 days of treatment. In addition, the tolerance observed after 2 days is not heterologous as evidenced by the lack of a significant change in the IC50 value for 2-CADO in the LM/MP. By 4 days of treatment tolerance had become heterologous. Despite the plateau after 4 days of treatment, LM/MP responses from animals treated for 7 or 10 days of treatment took longer (4 days) to return to baseline than animals treated for 4 days (2 days). These data suggest that more extensive adaptations occur as treatment length increases despite the fact that the magnitude of change in the responses from the LM/MP and whole animal were not significantly different from each other after 4, 7, or 10 days of treatment. Western blot analysis of LM/MP and brainstem homogenates from these same animals revealed that heterologous tolerance in these tissues is not the result of a decrease in the receptor number of either the [mu], A1, A2a, or [alpha]2b receptors. A trend was observed for the [alpha]3 subunit isoform of the Na+/K+ ATPase in the LM/MP to decrease in abundance as tolerance develops and return to baseline as tolerance decays. Although it was not statistically significant, the change in abundance is of a similar magnitude to significantly changed values previously reported in the literature. A trend for an increase in the abundance of PKC[epsilon] was also observed in the LM/MP. This trend, though not significant, is in agreement with reported literature values in other tissues. Contrary to the LM/MP results, PKC[gamma] and PKC[epsilon] showed a trend for a decrease in the brainstem while there was no change in the abundance of the [alpha]3 subunit of the Na+/K+ ATPase there. No change was observed in the brainstem for the [alpha]1 and [beta]1 subunits of the Na+/K+ ATPase as well. The trends observed may indicate that the brainstem and the LM/MP develop tolerance by distinct mechanisms but more research will be necessary elucidate the role of the [alpha]3 subunit of the Na+/K+ ATPase and PKC in the development of tolerance to morphine.