Chronic morphine exposure potentiates p-glycoprotein trafficking from nuclear reservoirs in cortical rat brain microvessels

<div><p>The rates of opioid prescription and use have continued to increase over the last few decades resulting in a greater number of opioid tolerant patients. Treatment of acute pain from surgery and injury is a clinical challenge for these patients. Several pain management strategies including prescribing increased opioids are used clinically with limited success; all currently available strategies have significant limitations. Many opioids are a substrate for p-glycoprotein (p-gp), an efflux transporter at the blood-brain barrier (BBB). Increased p-gp is associated with a decreased central nervous system uptake and analgesic efficacy of morphine. Our laboratory previously found that acute peripheral inflammatory pain (PIP) induces p-gp trafficking from the nucleus to the luminal surface of endothelial cells making up the BBB concomitant with increased p-gp activity and decreased morphine analgesic efficacy. In the current study, we tested whether PIP-induced p-gp trafficking could contribute to decreased opioid efficacy in morphine tolerant rats. A 6-day continuous dosing of morphine from osmotic minipumps was used to establish morphine tolerance in female rats. PIP induced p-gp trafficking away from nuclear stores showed a 2-fold increase in morphine tolerant rats. This observation suggests that p-gp trafficking contributes to the decreased morphine analgesic effects in morphine tolerant rats experiencing an acute pain stimulus. Attenuating p-gp trafficking during an acute pain stimulus could improve pain management by increasing the amount of opioid that could reach CNS analgesic targets and decrease the need for the dose escalation that is a serious challenge in pain management.</p></div

Chronic morphine exposure has no effect on total p-gp expression in the whole brain or microvessel isolates.

<p><b>(A)</b> Representative immunoblot indicating p-gp and tubulin as a loading control in whole brain cortex. P-gp expresion normalized to tubulin in whole brain cortex samples. Values are the mean + SEM (n = 3 pools of 3 rats each) <b>(B)</b> Representative immune blot indicating p-gp and tubulin in microvessel isolates. P-gp expresion normalized to tubulin in microvessel isolate samples. Values are the mean + SEM (n = 3 pools of 3 rats each).</p

Prolonged morphine exposure eliminates the antinociceptive effect of acute morphine administration on mechanical sensitivity.

<p><b>(A)</b> Timeline of treatments used in these experiments. <b>(B)</b> Mechanical ipsilateral paw withdrawal threshold determined by the von Frey mechanical sensitivity test in rats exposed to 5 mg/kg/day of morphine or saline for 6 days, then treated with an acute dose (2.5 mg/kg) of morphine or saline (time = 0) 3 hours after injection of λ-carrageenan into the left hind paw. The symbols mean: SCS: Saline osmotic minipump (24 μL/day)/ λ-carrageenan hind paw injection (0.1 mL)/ Saline intraperitoneal injection (1 mL/kg);SCM: Saline osmotic minipump (24 μL/day)/ λ-carrageenan hind paw injection (0.1 mL)/ Morphine intraperitoneal injection (1mL/kg) (2.5 mg/kg);MCS: Morphine osmotic minipump (24 μL/day) (5mg/kg/day)/ λ-carrageenan hind paw injection (0.1 mL)/ Saline intraperitoneal injection (1 mL/kg);MCM: Morphine osmotic minipump (24 μL/day) (5mg/kg/day)/ λ-carrageenan hind paw injection (0.1 mL)/ Morphine intraperitoneal injection (1 mL/kg) (2.5 mg/kg). Values are the mean +/- SEM (n = 8). <b>(C)</b> The area under the curve for the animals treated with SCM and MCM during the peak observed morphine effect (between 20 minutes and 60 minutes). Values are the mean + SEM (n = 8). *** denotes significantly different (p<0.0001).</p

Prolonged morphine exposure eliminates the antinociceptive effect of acute morphine administration on thermal sensitivity.

<p><b>(A)</b> Thermal paw withdrawal threshold was determined by the Hargraves thermal sensitivity test in the ipsilateral paw of rats exposed to 5 mg/kg/day of morphine or saline for 6 days, then treated with an acute dose (2.5 mg/kg) of morphine or saline (time = 0) 3 hours after injection of λ-carrageenan into the left hind paw. The symbols mean: SCS: Saline osmotic minipump (24 μL/day)/ λ-carrageenan hind paw injection (0.1 mL)/ Saline intraperitoneal injection (1 mL/kg);SCM: Saline osmotic minipump (24 μL/day)/ λ-carrageenan hind paw injection (0.1 mL)/ Morphine intraperitoneal injection (1 mL/kg) (2.5 mg/kg);MCS: Morphine osmotic minipump (24 μL/day) (5 mg/kg/day)/ λ-carrageenan hind paw injection (0.1 mL)/ Saline intraperitoneal injection (1 mL/kg);MCM: Morphine osmotic minipump (24 μL/day) (5 mg/kg/day)/ λ-carrageenan hind paw injection (0.1 mL)/ Morphine intraperitoneal injection (1 mL/kg) (2.5 mg/kg). Values are the mean +/- SEM (n = 9). <b>(B)</b> The area under the curve for the animals treated with SCM and MCM during the peak observed morphine effect (between 30 minutes and 60 minutes). Values are mean + SEM (n = 9). *** denotes significantly different (p = 0.007).</p

Suggested model demonstrating p-gp trafficking in the presence of peripheral inflammatory pain.

<p>In the presense of saline <b>(A)</b>, PIP induces trafficking of p-gp away from the nucleus <b>(B)</b> to the endothelial cell membrane where p-gp can efflux substrate through the membrane <b>(C)</b>. In the presense of morhine <b>(D)</b>, this trafficking is increased <b>(E)</b> resulting in a greater presense of p-gp at the cell membrane and an increase in substrate efflux <b>(F)</b>.</p

Chronic morphine exposure has no effect on hind paw edema following λ-carrageenan injection.

<p><b>(A)</b> Animals tested for mechanical sensitivity. <b>(B)</b> Animals tested for thermal sensitivity. The symbols mean: SS: Saline osmotic minipump/ Saline hind paw injection (100 μL); SC: Saline osmotic minipump/ λ-carrageenan hind paw injection (100 μL); MS: Morphine (5 mg/kg/day) osmotic minipump/ Saline hind paw injection (100 μL); MC: Morphine (5 mg/kg/day) osmotic minipump/ λ-carrageenan hind paw injection (100 μL). Values are the mean + SEM (A: n = 16, B: n = 18). *** denotes significantly different (p<0.0001).</p

Chronic morphine exposure increases trafficking of p-gp away from the nucleus after peripheral inflammatory pain.

<p><b>(A)</b> P-gp expresion normalized to nucleoporin in the nuclear fractions. Values are the mean + SEM (n = 3 pools of 3 rats each) <b>(B)</b> Ratio of nuclear p-gp normalized to nulcoporin in CAR/SAL injected animals as a measure of p-gp trafficking. The dashed line indicates the value if the nuclear p-gp in saline and ʎ-carrageenan injected animals was equal. Values are the mean + SEM (n = 3 pools of 3 rats each). *** denotes significantly different from control (saline) (p<0.05). The symbols mean: SAL/SAL represents animals with an osmotic minipump filled with 0.9% saline and a 0.9% saline hind paw injection. MOR/SAL represents animals with an osmotic minipump filled with morphine (5 mg/kg/day) in 0.9% saline and a 0.9% saline hind paw injection.</p