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| 048P London, UK Pharmacology 2017 |
Role of Kir3 potassium channel in opioid receptor-mediated inhibition of electrical-field stimulation-induced contraction of the guinea pig myenteric plexus longitudinal muscle
Introduction: Opioid receptors signal via Gi/o heterotrimeric proteins where dissociation and translocation of Gai/o mediates inhibition of adenylyl cyclase, while Gbgi/o can activate the “G-protein-gated inwardly rectifying K+channel” (GIRK or Kir3) or inhibit CaV2.2 (N-type voltage-gated calcium channels (VGCC), all of which can inhibit neurotransmitter release. This study investigated Kir3 activity in μ- and κ-opioid receptor induced inhibition of electrical field stimulation (EFS)-mediated contraction of guinea pig myenteric plexus longitudinal muscle (GP-MPLM).
Methods: Male Dunkin-Hartley guinea pigs were killed by cervical dislocation MPLM isolated and mounted in Linton organ baths under resting tension of 1g (in Krebs solution, gassed with 95/5% O2/CO2 at 37°C; stimulated continuously by EFS (0.1 Hz, 0.5 msec pulse width, 60 - 100 V). Muscle contraction was monitored by UF1 force transducer (LCM Systems), Powerlab 4/25 interface (AD instruments) and displayed using Lab chart 7. Inhibition of contractions by DAMGO (100 nM) and U69593 (10 nM), selective μ- and κ-opioid receptor agonists, respectively, was examined in the absence and presence of the Kir3 blockers tertiapin Q (100 nM), SCH23390 (30 μM) or the non-selective GIRK inhibitor barium (BaCl, 30 mM, 3 mM). In addition, the effect of the N-type VGCC blocker PD173212 (1 μM) and the PKA inhibitor KT5720 (1 μM). Results are expressed as % of the maximum inhibition (mean ± s.e.m; analysed by Student\'s t-test, p < 0.05 considered significant). Expression of opioid receptor and Kir3 mRNA was characterised in MPLM by PCR.
Results: Moderate levels of μ-, κ- and δ-opioid receptor and Kir3 mRNA were detected in MPLM. DAMGO and U69593 inhibited EFS-mediated contraction by 60.9 ± 2.5% (n = 17) and 67.8 ± 2.7% (n = 13), respectively. DAMGO- and U69593-induced inhibition was unaffected by tertiapin Q but was reduced to 28 ± 8.6% (n = 4, p = 0.0002) and 16.6 ± 2.8% (n = 5, p = 0.0001), respectively by SCH23390. Neither PD173212 nor KT5720 affected the EFS-induced contractions. BaCl at 30mM did not affect either DAMGO- or U69593-induced inhibition while 3 mM induced a sustained contraction.
Conclusions: The functional role of Kir3 in mediating opioid receptor-mediated inhibition of EFS-induced contraction remains inconclusive since SCH23390 but not tertiapin Q blocked this effect. The lack of effect of PD173212 and KT5720 indicates that N-type VGCCs are not involved in transmitter release and that PKA is not involved in opioid receptor mediated inhibition of EFS-induced contraction.