Comparison of tolerance characteristics in the guinea pig following chronic in-vivo exposure to opioid versus cannabinoid receptor agonists

Few studies have compared the nature of tolerance that develops following chronic in vivo opioid treatment with that which develops after chronic cannabinoid exposure in the same tissue and species. Based on similarities in signaling and overlapping neuroanatomical receptor localization the candidate determined whether the tolerance that develops to hypothermic, analgesic and inhibitory action on neurogenic contractions of the longitudinal muscle-myenteric plexus (LM/MP) in the guinea pig is qualitatively similar regardless of the agonist employed. Since previous in vitro drug exposure studies using the LM/MP model have reported bidirectional heterologous tolerance, it was hypothesized: 1) that in vivo exposure to either agonist would result in heterologous tolerance 2) that the type of tolerance could be used to define the underlying cellular mechanisms; 3) that homologous tolerance would employ a mechanism that involved receptor regulation; 4) that co-localization of opioid and cannabinoid receptors may provide a basis for some cross-tolerance between agonists; and 5) that the mechanisms that underlie the development of tolerance can vary among different tissues or models. Specific aim 1 assessed the effect of chronic cannabinoid or opioid exposure on the sensitivity of the LM/MP to inhibitory agonists (WIN-55,212-2-2, CADO or morphine) or an excitatory agent (nicotine). Animals pretreated with morphine in vivo developed an increased responsiveness to nicotine and tolerance to all inhibitory agonists tested: the magnitude of rightward shift (i.e. ratio of mean IC50 values) or loss of sensitivity of the treated compared to the control group was 4.8-fold for DAMGO, 3.5-fold for CADO, and 5.2-fold for WIN-55,212-2. In contrast, in vivo WIN-55,212-2 pretreatment resulted in subsensitivity to WIN-55,212-2 only (factor of rightward shift of the IC50 values was 9.8) and reduced maximum responses to WIN-55,212-2 and DAMGO; no shift was observed in the dose response curves for DAMGO, CADO and nicotine. Specific aim 2 sought to determine the effect of chronic WIN-55,212-2 or morphine exposure on the levels of both mu-opioid receptor (MOR) and cannabinoid receptor 1 (CB1) protein in homogenates of the LM/MP. WIN-55,212-2 treatment resulted in a selective reduction in CB1 receptor protein levels by 35% while MOR levels remained unchanged whereas morphine exposure altered neither MOR nor CB1 receptor protein levels. Specific aim 3 sought to determine the qualitative nature of tolerance that develops in analgesic (thermal and mechanical) and hypothermic models. Chronic morphine treatment resulted in heterologous tolerance to the thermal analgesic effect of morphine and WIN-55,212-2 but did not alter the sensitivity to the hypothermic effect of WIN-55,212-2. The nature of tolerance observed in the hot plate test corresponds closely to that observed in the LM/MP studies where chronic morphine treatment produced heterologous tolerance and WIN-55,212-2 pretreatment resulted in homologous tolerance. In contrast to the results in the LM/MP studies, WIN-55,212-2 pretreatment resulted in tolerance to the analgesic effect of morphine in the paw pressure model despite the fact that WIN-55,212-2 did not produce analgesia in this model. Unlike chronic treatment with WIN-55,212-2, chronic morphine treatment did not induce tolerance to the hypothermic effect of WIN-55,212-2. However, since only a very modest hypothermia was observed in response to a morphine challenge, tolerance to this effect was difficult to assess and may not be pharmacologically relevant. For specific aim 4 the candidate explored the distribution of MOR and CB1 receptor expressing neurons in the LM/MP and hypothalamus. Immunofluorescence assessment of the distribution of neurons expressing MOR and CB1 receptors in the LM/MP revealed significant co-localization of receptors on myenteric plexus neurons thus raises the possibility of intracellular crosstalk between the two receptor systems. Furthermore, neither opioid nor cannabinoid treatment altered the density or distribution pattern of neurons expressing MOR or CB1 receptors. Assessment of neurons expressing MOR and CB1 receptors in the preoptic anterior hypothalamus revealed extensive co-localization suggesting possible interaction of the two receptor systems in the regulation of body temperature. In conclusion, the variable tolerance expression observed in different models affirms the notion that nature and potential cellular mechanisms of tolerance can vary depending on the model system, the drug, the species, and regimen used to establish the phenomenon. The data also suggest that multiple cellular effects may play a role in the induction of functional tolerance in different model systems.  Ph.D

Time Course of Altered Sensitivity to Inhibitory and Excitatory Agonist Responses in the Longitudinal Muscle–Myenteric Plexus and Analgesia in the Guinea Pig after Chronic Morphine Treatment

Tolerance that develops after chronic morphine exposure has been proposed to be an adaptive response that develops and decays over a defined time course. The present study examined the development of tolerance to the acute hypothermic and analgesic effects of morphine and correlated the time course for the desensitization in vivo with the reduced responsiveness to DAMGO and 2-CADO and increased responsiveness to nicotine of the longitudinal muscle/myenteric plexus (LM/MP) preparation in vitro. Assessment was performed at various times after morphine or placebo pellet implantation. Morphine produced a modest hypothermic response to which no tolerance developed. However, the development of tolerance to the analgesic effect of morphine, the inhibitory effect of DAMGO and CADO on neurogenic twitches of the LM/MP and hypersensitivity to the contractile response to nicotine was observed to occur in a time-dependent manner. The alterations in sensitivity to DAMGO, nicotine, and responsiveness to morphine analgesia occurred between days 4 and 10 and returned to normal by day 14 post-implantation. In contrast, sensitivity of LM/MP preparations to 2-CADO displayed a similar time-dependent onset but the tolerance persisted beyond 14 days after implantation. These data suggest that the heterologous tolerance that develops after chronic morphine treatment is time-dependent and persistent but, ultimately returns to normal in the absence of any intervention. Furthermore, the data suggest that the basis of the adaptive phenomenon may involve multiple cellular mechanisms including the modulation of cell excitability and normal physiology but the consequences of the adaptation extend to all effects of the agonist

Specific Localization of β-Arrestin2 in Myenteric Plexus of Mouse Gastrointestinal Tract

Abstract β-arrestin2 is a key molecule involved in signaling and internalization of activated G protein-coupled receptors including µ-opioid receptors (MOR). Previously we have shown that decreased expression of β-arrestin2 upon chronic morphine is associated with the development of opioid tolerance in the gastrointestinal tract. However, the localization of β-arrestin2 within the gastrointestinal wall is not known. In this study we found that β-arrestin2 is localized in the soma of a select group of neurons in the myenteric ganglia but not in smooth muscle. The density of β-arestin2 was significantly higher in the ileum than the colon. We identified four variants of β-arrestin2 in the ileum, with ARRB-005 and ARRB-013 being the most abundant. Further, the current study utilized multiple-labeling immunofluorescence to characterize the chemical coding of neurons expressing β-arrestin2 in the murine myenteric plexus and the co-localization of MOR1 and β-arrestin2. β-arrestin2 co-localized with choline acetyltransferase and calretinin. In contrast, β-arrestin2 neither co-localized with substance P, nitric oxide synthase nor calbindin. Genetic deletion of β-arrestin2 did not affect cholinergic neuron activation by nicotine in the isolated ileum (-log M EC50: wild type = 5.8 vs. β-arrestin2 knockout = 5.9). Our findings suggest specificity in the localization of β-arrestin2 in the myenteric plexus within MOR1-expressing neurons and provide a relation for direct intracellular crosstalk between MOR1 receptor activation and β-arrestin2 signaling in the myenteric neurons. β-arrestin2 deletion does not directly alter basal enteric cholinergic neuronal function

Time Course of Altered Sensitivity to Inhibitory and Excitatory Agonist Responses in the Longitudinal Muscle–Myenteric Plexus and Analgesia in the Guinea Pig after Chronic Morphine Treatment

Tolerance that develops after chronic morphine exposure has been proposed to be an adaptive response that develops and decays over a defined time course. The present study examined the development of tolerance to the acute hypothermic and analgesic effects of morphine and correlated the time course for the desensitization in vivo with the reduced responsiveness to DAMGO and 2-CADO and increased responsiveness to nicotine of the longitudinal muscle/myenteric plexus (LM/MP) preparation in vitro. Assessment was performed at various times after morphine or placebo pellet implantation. Morphine produced a modest hypothermic response to which no tolerance developed. However, the development of tolerance to the analgesic effect of morphine, the inhibitory effect of DAMGO and CADO on neurogenic twitches of the LM/MP and hypersensitivity to the contractile response to nicotine was observed to occur in a time-dependent manner. The alterations in sensitivity to DAMGO, nicotine, and responsiveness to morphine analgesia occurred between days 4 and 10 and returned to normal by day 14 post-implantation. In contrast, sensitivity of LM/MP preparations to 2-CADO displayed a similar time-dependent onset but the tolerance persisted beyond 14 days after implantation. These data suggest that the heterologous tolerance that develops after chronic morphine treatment is time-dependent and persistent but, ultimately returns to normal in the absence of any intervention. Furthermore, the data suggest that the basis of the adaptive phenomenon may involve multiple cellular mechanisms including the modulation of cell excitability and normal physiology but the consequences of the adaptation extend to all effects of the agonist

Time Course of Altered Sensitivity to Inhibitory and Excitatory Agonist Responses in the Longitudinal Muscle - Myenteric Plexus and Analgesia in the Guinea Pig after Chronic Morphine Treatment

Tolerance that develops after chronic morphine exposure has been proposed to be an adaptive response that develops and decays over a defined time course. The present study examined the development of tolerance to the acute hypothermic and analgesic effects of morphine and correlated the time course for the desensitization in vivo with the reduced responsiveness to DAMGO and 2-CADO and increased responsiveness to nicotine of the longitudinal muscle/myenteric plexus (LM/MP) preparation in vitro. Assessment was performed at various times after morphine or placebo pellet implantation. Morphine produced a modest hypothermic response to which no tolerance developed. However, the development of tolerance to the analgesic effect of morphine, the inhibitory effect of DAMGO and CADO on neurogenic twitches of the LM/MP and hypersensitivity to the contractile response to nicotine was observed to occur in a time-dependent manner. The alterations in sensitivity to DAMGO, nicotine, and responsiveness to morphine analgesia occurred between days 4 and 10 and returned to normal by day 14 post-implantation. In contrast, sensitivity of LM/MP preparations to 2-CADO displayed a similar time-dependent onset but the tolerance persisted beyond 14 days after implantation. These data suggest that the heterologous tolerance that develops after chronic morphine treatment is time-dependent and persistent but, ultimately returns to normal in the absence of any intervention. Furthermore, the data suggest that the basis of the adaptive phenomenon may involve multiple cellular mechanisms including the modulation of cell excitability and normal physiology but the consequences of the adaptation extend to all effects of the agonist

Representative images of LMMP whole mounts comparing expression of calbindin and calretinin in β-arrestin2 immunopositive neurons.

<p><b>A</b>: β-arrestin2-immunopositive neurons (green). <b>B</b>: calbindin-immunopositive neurons (red). <b>C</b>: merged image depicting expression of β-arrestin2 and calbindin. <b>D</b>: β-arrestin2-immunopositive neurons (green). <b>E</b>: calretinin -immunopositive neurons (red). <b>F</b>: merged image depicting colocalization of β-arrestin2 and calretinin (yellow). Scale bars: Upper panel: 50 µm, bottom panel: 20 µm.</p

Image of myenteric ganglia expression of MOR₁ mRNA in β-arrestin2 immunopositive neurons.

<p>β-arrestin2-immunopositive neurons (grey) with punctate mRNA for MOR₁ (red) determined my mRNA-FISH. A) High magnification of merged image showing expression of MOR1 (red) within cell bodies labeled with β-arrestin2 (grey). B) grey scale image of β-arrestin2, C) fluorescent Cy5 labeled MOR1 – mRNA. Arrows are non-specific staining of cells outside the ganglia. D) Cy5 labelling of MOR1 mRNA in isolated ileum smooth muscle cell. Nuclear stain (blue). Middle panel indicates the absence of Cy5 fluorescent in smooth muscle. Bright field image of smooth muscle cell on the right panel. Scale bar: 20 µm.</p

Mean concentration-response curves for nicotine in longitudinal ileum preparation obtained from WT or β-arrestin2 KO mice.

<p>A slight but insignificant change to the sensitivity to nicotine was observed in the β-arrestin2 knockout mice (A). B) representative isometric tension recording tracing of the longitudinal ileum preparation from C57BL/6 WT mice showing the non-cumulative dose response effect of repeated exposure with nicotine. Statistically significant differences (p≤0.05) are identified by *.</p

Shows a bar graph comparing the maximal tension values attained by nicotine (A) or acetylcholine (B) stimulation in the longitudinal ileum preparation from WT or β-arrestin2 KO mice.

<p>No significant increase in maximal efficacy to nicotine or acetylcholine. Statistically significant differences (p≤0.05) are identified by *.</p

β-arrestin2-immunopositive neurons (green) and ChAT -immunopositive neurons (red), merged image depicting co-localization of β-arrestin2 and ChAT (yellow).

<p>Bottom bar graph are total β-arrestin2 positive and ChAT positive neurons from 6 ganglia. Scale bars: 20 µm.</p