Profiling Endogenous Opioid Peptide Release from Adrenal Chromaffin Cells

Prolonged inflammatory pain modifies corticotropin-releasing factor-induced opioid peptide release in the hypothalamus

In inflammation, pain is regulated by a balance of pro- and analgesic mediators. Analgesic mediators include opioid peptides which are secreted by neutrophils at the site of inflammation, leading to activation of opioid receptors on peripheral sensory neurons. In humans, local opioids and opioid peptides significantly downregulate postoperative as well as arthritic pain. In rats, inflammatory pain is induced by intraplantar injection of heat inactivated Mycobacterium butyricum, a component of complete Freund's adjuvant. We hypothesized that mycobacterially derived formyl peptide receptor (FPR) and/or toll like receptor (TLR) agonists could activate neutrophils, leading to opioid peptide release and inhibition of inflammatory pain. In complete Freund's adjuvant-induced inflammation, thermal and mechanical nociceptive thresholds of the paw were quantified (Hargreaves and Randall-Selitto methods, respectively). Withdrawal time to heat was decreased following systemic neutrophil depletion as well as local injection of opioid receptor antagonists or anti-opioid peptide (i.e. Met-enkephalin, β-endorphin) antibodies indicating an increase in pain. In vitro, opioid peptide release from human and rat neutrophils was measured by radioimmunoassay. Met-enkephalin release was triggered by Mycobacterium butyricum and formyl peptides but not by TLR-2 or TLR-4 agonists. Mycobacterium butyricum induced a rise in intracellular calcium as determined by FURA loading and calcium imaging. Opioid peptide release was blocked by intracellular calcium chelation as well as phosphoinositol-3-kinase inhibition. The FPR antagonists Boc-FLFLF and cyclosporine H reduced opioid peptide release in vitro and increased inflammatory pain in vivo while TLR 2/4 did not appear to be involved. In summary, mycobacteria activate FPR on neutrophils, resulting in tonic secretion of opioid peptides from neutrophils and in a decrease in inflammatory pain. Future therapeutic strategies may aim at selective FPR agonists to boost endogenous analgesia.

Immune Mechanisms in Pain Control

Antinociception by neutrophil-derived opioid peptides in noninflamed tissue—Role of hypertonicity and the perineurium

Opioid peptide release in the hippocampus was shown to be increased immediately following amygdala kindling stimulation in freely moving rats using microdialysis combined with a universal opioid peptide radioimmunoassay (RIA). Extracellular opioid peptide levels were elevated (55% above basal levels) within the first 10 min after electrical stimulation-induced partial seizures in previously nonkindled animals. Fully kindled rats showed lower extracellular opioid peptide levels (40% reduction) during the interictal period [16 +/- 2.1 days (mean +/- SEM) after the last stage V seizure], in comparison with values obtained from the sham-kindled group under basal conditions. However, opioid peptide release in fully kindled rats increased above 152% of interictal levels within the first 20 min after onset of fully kindled seizures, attaining peak levels equal to that of the partial kindled group and returning to prestimulation conditions 40-60 min following the ictal events. The majority of the immunoreactive material recovered from the hippocampus within the first 20 min following partial and generalized kindled seizures coeluted with dynorphin-A (1-6), dynorphin-A (1-8), and Leu-enkephalin by HPLC/RIA analysis. It is proposed that the enhanced opioid peptide release in hippocampus induced by amygdala kindling stimulation might be associated with either enhanced excitability or seizure suppression as seizure susceptibility fluctuates. The reduced interictal opioid peptide levels may also underlie some interictal behavioral disturbances.

Morphine treatment during juvenile isolation increases social activity and opioid peptides release in the adult rat

Opioid peptides and noradrenaline co-exist in large dense-cored vesicles from sympathetic nerve

<b>Abstract ID 28409</b> <b>Poster Board 144</b> Opioid peptides are key modulators of natural reward and threat processing. It has been shown in the rodent models that enkephalinergic neurons in the nucleus accumbens (NAc) shell may play a modulatory role in stressful situations specifically as anti-stress agents, for example after exposure to predator odor and in the vulnerability to social defeat stress. However, it has been challenging to identify the specific roles of Met- and Leu-enkephalins due to the difficulty in their detection. Here, we show that enkephalinergic neurons in the ventral NAc shell are activated following exposure to predator odor and experimenter handling using fiber photometry. To get a deeper insight into enkephalin release dynamics we couple microdialysis and nano-liquid chromatography/mass spectrometry (nLC-MS) to allow the detection of endogenous Leu- and Met-enkephalin <i>in&nbsp;vivo</i>. With this approach, we can detect and distinguish between Met- and Leu- Enkephalin release in awake behaving mice. We show that enkephalins are released following exposure to predator or handling stress. We also demonstrate the dynamics of Met- and Leu-Enkephalin release as well as how they correlate to one another in the ventral NAc shell. Due to the success of this method in measuring peptide dynamics following stress, we wanted to expand our investigation into how exogenous opioids alter the release of enkephalins. Since enkephalins activate the mu and delta opioid receptors, we chose to focus on fentanyl, a mu-opioid receptor agonist that has high abuse liability. Using fiber photometry, we show that fentanyl silences enkephalinergic neurons. We also show that fentanyl attenuates the release of enkephalins using nLC-MS, and that this effect is reversible by artificially evoking neuronal activity in the ventral NAc shell, thereby driving peptide release. Our findings suggest that exogenous opioids alter endogenous opioid peptide release dynamics which may alter reward circuitry. Overall, we show distinct roles for Met- and Leu-enkephalins following acute stressors and exogenous opioid administration, allowing us for the first time to correlate real-time behavioral manipulations with opioid peptide release. Support/Funding Information: •Cognitive, Computational, and Systems Neuroscience Fellowship (WUSTL Med) •Al-Hasani R21DA048650 •Al-Hasani R00DA038725

It has been suggested that kainic acid enhances opioid peptide release. However, no direct evidence exists to support this hypothesis. The main aim of the present study was to determine whether such release occurs in the hippocampus of the rat after status epilepticus induced by kainic acid. Microdialysis experiments revealed significant opioid peptide release in the hippocampus 90-150 min (100%) and 270-300 min (50%) after kainic acid-induced status epilepticus. The peptides released were identified by high-performance liquid chromatography linked to radioimmunoassay as Met-enkephalin, Leu-enkephalin, Dynorphin-A (1-6), and Dynorphin-A (1-8). Reduced extracellular opioid peptide immunoreactivity was detected 28 days after status epilepticus (38% compared with control situation). The present results indicate an important activation of opioid peptide systems by kainic acid-induced status epilepticus. In addition, the reduced hippocampal extracellular opioid peptide levels long-term after kainic acid administration could have important implications for the progressive nature of epileptogenesis.

Effects of chronic treatment with atypical neuroleptics on the biosynthesis and release of opioid peptides in guinea-pig ileum.

Munc13-1 is required for the sustained release of insulin from pancreatic β cells

Neuroleptic drugs increase the biosynthesis and release of opioid peptides from the myenteric plexus of guinea-pig ileum. In the present work, the involvement of dopamine receptors or alpha-adrenoceptors in the release of opioids from the myenteric plexus of guinea-pig was investigated. Acute or chronic treatment with prazosin, an alpha 1-blocking drug, produced no changes in the release of these peptides. Release was also unchanged after acute or chronic treatment with the alpha 2-blocking drug yohimbine. However, treatment with domperidone, a selective dopamine receptor antagonist, resulted in an increase in the release of opioids, as did treatment with (-)-3-(3-hydroxyphenyl)-N-n-propylpiperidine ((-)-3-PPP), a dopamine autoreceptor stimulant. It is concluded that the effect of neuroleptics on the release of opioids from myenteric plexus is due to the blockade of dopamine receptors, and that interruption of dopaminergic transmission produces an increase in opioid release at this level.

Histamine activates proenkephalin A mRNA but not phenylethanolamine N-methyltransferase mRNA expression in cultured bovine adrenal chromaffin cells

Investigation of Nociceptive Endogenous Opioid Dynamics in the Periaqueductal Gray

Sixteen anesthetized foxhounds were instrumented for hemodynamic measurements. The adrenolumbar vein was cannulated, and hemorrhagic hypotension (MAP = 40 mmHg for 3h) was induced by bleeding. The plasma levels of beta-endorphin (beta-END), methionine-enkephalin (M-ENK), and leucine-enkephalin (L-ENK) were determined in systemic and adrenal venous blood by specific RIA. Five dogs received an i.v. bolus of naloxone (2 mg/kg) and a subsequent naloxone infusion of 2 mg/kg per hour 1 h after onset of hypovolemia. Eleven dogs served as controls and received equivalent volumes (1 ml/kg per hour) of saline. Hemorrhage resulted in a sharp increase in plasma concentrations of all measured opioid peptides, particularly of M-ENK (26-fold) and L-ENK (24-fold) in the adrenal effluent. Systemic beta-END levels remained 3-fold increased, whereas the ENK release decreased spontaneously. Naloxone treatment inhibited the spontaneous fall of adrenal ENK release during the hypotensive phase; the ENK values remained elevated 20- to 35-fold. Reinfusion of the autologous blood resulted in a normalization of the concentrations of all peptides in both groups. These data demonstrate that hemorrhagic hypotension will cause stimulation of release of endogenous opioid peptides. The high levels of ENK in the adrenal effluent indicate that the adrenal gland is the main source of these peptides in the circulation. In addition to beta-END, the ENK have therefore to be considered as possible factors perpetuating circulatory shock.