title: Oxytocin hyperpolarizes cultured duodenum myenteric intrinsic primary afferent neurons by opening BK(Ca) channels through IP₃ pathway.
abstract: Oxytocin (OT) is clinically important in gut motility and constitutively reduces duodenum contractility. Intrinsic primary afferent neurons (IPANs), whose physiological classification is as AH cells, are the 1st neurons of the peristaltic reflex pathway. We set out to investigate if this inhibitory effect is mediated by IPANs and to identify the ion channel(s) and intracellular signal transduction pathway that are involved in this effect. Myenteric neurons were isolated from the longitudinal muscle myenteric plexus (LMMP) preparation of rat duodenum and cultured for 16-24 h before electrophysiological recording in whole cell mode and AH cells identified by their electrophysiological characteristics. The cytoplasmic Ca²⁺ concentration ([Ca²⁺](i) ) of isolated neurons was measured using calcium imaging. The concentration of IP(3) in the LMMP and the OT secreted from the LMMP were measured using ELISA. The oxytocin receptor (OTR) and large-conductance calcium-activated potassium (BK(Ca)) channels, as well as the expression of OT and the IPAN marker calbindin 28 K, on the myenteric plexus neurons were localized using double-immunostaining techniques. We found that administration of OT (10⁻⁷ to 10⁻⁵ M) dose dependently hyperpolarized the resting membrane potential and increased the total outward current. The OTR antagonist atosiban or the BK(Ca) channel blocker iberiotoxin (IbTX) blocked the effects of OT suggesting that the increased outward current resulted from BK(Ca) channel opening. OTR and the BK(Ca) α subunit were co-expressed on a subset of myenteric neurons at the LMMP. NS1619 (10⁻⁵ M, a BK(Ca) channel activator) increased the outward current similar to the effect of OT. OT administration also increased [Ca²⁺](i) and the OT-evoked outward current was significantly attenuated by thapsigargin (10⁻⁶ M) or CdCl₂. The effect of OT on the BK(Ca) current was also blocked by pre-treatment with the IP₃ receptor antagonist 2-APB (10⁻⁴ M) or the PLC inhibitor U73122 (10⁻⁵ M). OT (10⁻⁶ M) also increased the IP₃ concentration within the LMMP. Both of the spontaneous and KCl-induced secretion of OT was enhanced by atosiban. Most of OT-immunoreactive cells are also immunoreactive for calbindin 28 K. In summary, we concluded that OT hyperpolarized myenteric IPANs by activating BK(Ca) channels via the OTR-PLC-IP₃-Ca²⁺ signal pathway. OT might modulate IPANs mediated ENS reflex by an autocrine and negative feedback manner.
authors.affiliation: Department of Physiology, Shandong Provincial Key Laboratory of Mental Disorders, Shandong University School of Medicine, Jinan, China.
authors.forename: Tongtong
authors.initials: T
authors.lastname: Che
authors.forename: Hui
authors.initials: H
authors.lastname: Sun
authors.forename: Jingxin
authors.initials: J
authors.lastname: Li
authors.forename: Xiao
authors.initials: X
authors.lastname: Yu
authors.forename: Dexiao
authors.initials: D
authors.lastname: Zhu
authors.forename: Bing
authors.initials: B
authors.lastname: Xue
authors.forename: Kejing
authors.initials: K
authors.lastname: Liu
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Kunze
authors.forename: Chuanyong
authors.initials: C
authors.lastname: Liu
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D019898:Autocrine Communication / Q000187:drug effects; D002469:Cell Separation; D002478:Cells, Cultured; D004386:Duodenum / Q000187:drug effects / Q000294:innervation*; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology*; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009197:Myenteric Plexus / Q000187:drug effects*; D009475:Neurons, Afferent / Q000187:drug effects* / Q000648:ultrastructure; D010121:Oxytocin / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D018045:Receptors, Oxytocin / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects*; D010738:Type C Phospholipases / Q000502:physiology; D014668:Vasotocin / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D051036:Large-Conductance Calcium-Activated Potassium Channels; D018045:Receptors, Oxytocin; C047046:atosiban; D010121:Oxytocin; D011189:Potassium Chloride; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D014668:Vasotocin