| Objective:Oxytocin (OT) is a nonapeptide of the neurohypophyseal protein family. The well-defined effects of OT are to regulate uterine contraction during labor and milk ejection reflex. In recent years, more and more reports indicated that both OT and OT receptor (OTR) expressed throughout the gastrointestinal (GI) tract. OT inhibited the contraction of longitudinal muscle strips in rats in vitro mediated by myenteric neurons, but the specific kind of neurons responding to OT and the underlying mechanisms were not clearly demonstrated. Based on electrophysiological characteristics, myenteric neurons are basically divided into two major forms termed as S cells and AH cells. AH cells are intrinsic primary afferent neurons (IPANs), and S cells are inter-and motor neurons. In preliminary experiments, we found OT (10-6M) hyperpolarized25of25recorded AH cells and some S cells (10/30). The present study was designed to investigate the mechanism underlying the hyperpolarizing effect of OT on AH cells.Methods:Rats were killed by cervical dislocation and the duodenum was immediately placed into a Silgard lined dissecting dish containing carbogen gassed Krebs saline. A segment was opened by cutting along the lines of mesenteric attachment and pinned flat with mucosal layer uppermost. The mucosa, submucosa and circular muscle layers were removed by dissection, leaving the myenteric plexus exposed. This longitudinal muscle myenteric plexus (LMMP) preparation was then incubated in Krebs saline containing papain (10mg/ml) at37℃for50min. The LMMP was then cut into pieces which were placed in Dulbecco's modified eagle's medium (DMEM) containing collagenase Ⅱ (1mg/ml) at37℃for55min, triturated with a suction pipe, and centrifuged at1000r/min at room temperature for6min. The sediment was collected and suspended in1ml DMEM containing10%fetal bovine serum (FBS). Cells were then cultured at37℃in a5%CO2incubator for16-24h 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 IP3in the LMMP and the OT secreted from the LMMP were measured using ELISA. The OTR and large-conductance calcium-activated potassium (BKCa) channels, as well as the expression of OT and the IPAN marker calbindin28K, on the myenteric plexus neurons were localized using double-immunostaining techniques.Results:1. Effect of OT on AH cell resting membrane potential The resting membrane potential of AH cells was-54mV±1mV (n=30). After three doses of OT administration for15s, the membrane potential hyperpolarized by4.4mV±0.5mV (P<0.05, n=9; OT10-/M),12.7mV±0.7mV (P<0.05, n=8; OT10-6M) and8.9mV±0.8mV (P<0.05, n=8; OT10-5M) respectively. After1min pre-incubation with the OTR antagonist atosiban (10-6M), the OT-induced membrane hyperpolarization was significantly reduced to1.3mV±0.9mV; and these were significantly smaller hyperpolarizations than those recorded for the OT only group.2. Effect of OT on AH cell action potential parameters Sixty seconds following the onset of OT application, the action potential half width (AP1/2) decreased from3.2ms±0.4ms to2.9ms±0.3ms (P=0.04, n=12), and threshold (100-ms duration) was increased from69pA±10pA to130pA±21pA (P=0.02. n=12). The maximal depolarization rate (APdv/dt) and the action potential amplitude (APamp) did not change following OT administration. 3. Effects of OT on outward currents and involvement of BKCa channelsThe cell was initially held at a-80mV command potential to close voltage sensitive outwardly rectifying conductances. Outward current was then activated using a100ms-voltage step from-80mV to+50mV. One minute administration of OT (10-7M-10-5M) evoked an increase in outward currents of552pA±72pA ((P<0.05, n=8; OT10-7M),1035pA±122pA((P<0.05, n=8; OT10-6M) and1116pA±100pA (P<0.05, n=8;OT10-5M). A lower dose of OT (10-8M) did not alter the outward current magnitude (P=0.16, n=8). Then AH cells were pretreated with one of the blockers of the three kinds of KCa present in IPANs, namely IbTX (a BKCa channel blocker), clotrimazole (an IKCa channel blocker) or apamin (an SKCa channel blocker). IbTX (10-7M) but not clotrimazole (2×10-5M) nor apamin (10-7M) blocked the effect of applying10-6M OT.To establish if BKCa channels are functional expressed on the plasma membrane of AH cells, we investigated the effect of the BKCa channel opener NS1619on the whole cell outward current. NS1619(10-5M) evoked an increase of outward current of748pA±141pA (P=0.002, n=8) and IbTX (10-7M) completely blocked this change.4. Effect of OT on [Ca2+]i of myenteric neurons OT increased [Ca2+]i in12/30myenteric neurons. The [Ca2+]i reached the highest level50s after OT administration, and returned to basal levels after100s (10-7M),150s (10-6M) or250s (10-5M). Fifty seconds following3doses of OT administration (10-7,10-6, and10-5M), the F/F0increased from1(baseline) to1.3±0.2(P<0.05, n=8),1.3±0.2(P<0.05, n=11) and1.6±0.3(P<0.05, n=11) respectively.To help elucidate how [Ca2+]i was increased, we tested the effects of the cell permeant intracellular Ca2+releaser thapsigargin (10-6M) or CdCl2(5×10-5M) pretreatment on the OT (10-6M) evoked increase of AH cell outward currents. Pretreatment with thapsigargin (10-6M) reduced the increase from678pA±78pA to182pA±34pA (P<0.001, n=8). Pretreatment with CdCl2(5×10-5M) reduced the increase from1042pA±96pA to440pA±94pA (P<0.001, n=10).5. Role of the PLC-IP3pathway in OT-induced increase of AH cell outward current Pretreatment with2-APB (10-4M) or U73122(10-5M) for10min blocked the OT-induced increase in AH cell outward current. One min following OT (10-6M) administration, IP3concentration in cell lysate from the duodenal LMMP increased from16.0ng/ml±1.1ng/ml to22.0ng/ml±1.4ng/ml (P=0.001, n=16). However, after atosiban (10-6M) pretreatment, IP3concentration following OT administration decreased to16.8ng/ml±0.7ng/ml (P=0.003, atosiban+OT vs. OT group; P=0.7, atosiban+OT vs. vehicle, n=8).6. Effect of atosiban and KCl on the secretion of endogenous OT from LMMP OT concentration in the Krebs saline in which LMMP was cultured for3min was33.2ng/ml±2.6ng/ml (n=7). Adding atosiban (10-6M) or KCl (6×10-2M) to the Krebs saline increased OT concentration to47.1ng/ml±3.5ng/ml (P=0.01, n=7; atosiban treatment vs. control) and45.4ng/ml±2.5ng/ml (P=0.02, n=7; KCl treatment vs. control). When the LMMP was treated with both KCl and atosiban, the concentration of OT increased to62.6ng/ml±5.0ng/ml (P=0.002, KCl+atosiban vs. KCl; P=0.005, KCl+atosiban vs. atosiban; n=7).7. Location of OT receptors and BKCa channels in LMMP OTR was expressed on the soma of the myenteric neurons. The shape of most (63/71) OTR-immunoreactive cells are round and oval, similar to that of the Dogiel type Ⅱ/AH cells. BKCa alpha was also expressed on the soma of some myenteric neurons. All (71/71) the OTR-immunoreactive cells were also BKCa alpha-immunoreactive.8. Location of OT and calbindin28K in LMMP OT and calbindin28K antibody were used to locate OT immunoreactive neuron and AH cells in the LMMP preparation. Most73.1%(122/167) of the OT-immunoreactive cells are AH cells that were also calbindin28K-immunoreactive, and82.4%(122/148) of the AH cells are OT-immunoreactive.Conclusions: OT hyperpolarized myenteric IPANs by activating BKCa channels via the OTR-PLC-IP3-Ca2+signal pathway. OT might modulate IPANs mediated ENS reflex by an autocrine and negative feedback manner. |
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