Correlation of the time course of development and decay of tolerance to morphine with alterations in sodium pump protein isoform abundance

Abstract

Since the heterologous tolerance that develops after chronic morphine administration has been proposed to be an adaptive process, it follows that the time course of the change in the cellular components should coincide with the time course of the altered responsiveness. This study correlated the time course over which heterologous tolerance develops with changes in the abundance of selected proteins in the guinea-pig longitudinal muscle/myenteric plexus (LM/MP) preparation. Tissues were obtained at various times following a single surgical implantation procedure and heterologous tolerance confirmed by a significant reduction in the sensitivity of the LM/MP to inhibition of neurogenic twitches by morphine, DAMGO, and 2-CADO. Tolerance developed with a delayed onset (significant 2–5-fold reduction in sensitivity by day 4 after pellet implantation) that reached a maximum by 7 days (4–8-fold reduction in responsiveness) that was maintained through 14 days with normal sensitivity spontaneously returning by 21 days post-implantation. Dot blot analysis was used to examine the abundance of the alpha₁ and alpha₃ subunit isoforms of the Na⁺/K⁺ ATPase and beta-actin over the same time course. The results showed significant decreases in abundance of the alpha₃ subunit at 4, 7, and 10 days following pellet implantation but no change in beta-actin or the alpha₁ subunit at any time period. These data support the idea that heterologous tolerance following chronic morphine exposure results from a cellular adaptive change that may involve a change in the abundance of the alpha₃ subunit isoform of the Na⁺/K⁺ ATPase.

Introduction

Tolerance and dependence are phenomena associated with long-term opioid use. While a number of cellular mechanism(s) and/or proteins have been suggested to be fundamentally involved in the phenomena, no clear singular entity has been identified as being responsible and it is generally agreed that both result from some form of cellular adaptation to acute chronic mu-opioid receptor activation [1], [2], [3], [4], [5]. Tolerance is a complicated phenomenon often studied at the cellular and molecular level using in vitro tissues from chronically treated animals. Chronic treatment with morphine has been accomplished using two primary procedures: pellet implantation and subcutaneous (s.c.) injection [6], [7], [8]. However, no studies have evaluated whether tolerance produced by pellet implantation differs from that produced by other methods of administration in the same species. Thus, one goal was to determine if the characteristics of tolerance were impacted by the method of chronic drug exposure.

The foundation of cellular adaptation suggests that neurons have the ability to undergo long-term compensatory changes that develop over time in response to the acute actions of an agonist. In order to fully define a cellular entity as part of an adaptive change responsible for the development of tolerance, certain criteria should be met [1], [9]. These criteria were originally suggested to be as follows: “The proposed cellular change must: 1) be induced by experimental procedures identical to those that induce tolerance and/or dependence; 2) follow a similar time course as the tolerance and/or dependence in that tissue; 3) quantitatively account for the tolerance and/or dependence; 4) account for the qualitative characteristics of tolerance and/or dependence; and 5) occur in the very cells upon which the opioid is acting”.

Opioid tolerance in the guinea-pig ileum (LM/MP) is accompanied by the development of non-specific supersensitivity to excitatory agonists [7], [10] and non-specific sub-sensitivity to inhibitory agonists [11], [12] utilizing different receptor and signaling pathways and has been termed “heterologous”. The non-specific super- and sub-sensitivity correlate with the observation that chronic opioid exposure leads to membrane depolarization in myenteric ‘S’ neurons without changes to either the threshold for action potential generation or the magnitude of the hyperpolarizing effect of morphine and 2-CADO [13], [14]. These electrophysiological changes coincide with the decrease in the electrogenic contribution of the Na⁺, K⁺ pump to cellular membrane potential (15) and lead to an increased cellular excitability thus requiring lower concentrations of excitatory agonists to generate action potentials and higher concentrations of inhibitory agonists to reach the same level of inhibition. This membrane depolarization and decrease in Na⁺/K⁺ ATPase activity [15] is associated with a reduction in the abundance of the alpha₃ subunit isoform [16]. The alpha₃ subunit is the isoform that is specifically associated with neurons in the myenteric plexus [16]. Thus, the evidence suggests that in this model the decrease in pump activity, membrane depolarization, and heterologous tolerance may represent the same phenomenon [11], [13], [14], [15]. Therefore, the only criterion not yet tested is the time course over which these changes occur.

The hypotheses of the present experiment were that (1) single pellet implantation procedure produces tolerance that is qualitatively and quantitatively similar to other methods of chronic morphine administration and, therefore, can be employed to define the time course over which tolerance develops and decays; (2) that heterologous tolerance that develops following chronic morphine exposure exhibits a delay in onset and a spontaneous, slow return to normal responsiveness; and (3) that down-regulation of the alpha₃ subunit isoform but not other proteins follows a time course of decay and return similar to that of the decrease in responsiveness. Since the reduced abundance of the alpha₃ subunit isoform of the protein appeared to coincide with a decrease in sodium pump activity that produces the membrane depolarization observed after 7 days of chronic exposure, this change in cell function may represent a compensatory alteration that accounts for characteristics of tolerance observed in the guinea-pig ileum.