Role Of Arachidonic Acid In Hyposmotic Membrane Stretch-induced Increase In I_K(Ca) In Gastric Myocytes

With the development of patch-clamp techniques, many studies have found that hyposmotic stretch regulates activities of ionic channels which exist widely in the membrane of various cells and activates many signal transduction pathways. In our previous study, it was reported that voltage-operated calcium current (ICa) , the volume-sensitive chloride current (ICl), I_K(Ca), muscarinic currents(ICCh) were all activated by hyposmotic stretch in gastric antral circular myocytes of the guinea-pig .In the process of the activation of I_K(Ca), Ca²⁺ release is not caused by IP3 .We, therefore, had to take other second messengers into consideration. Recent reports indicate that hyposmotic stretch activates membrane bound phospholipases and generates lipid soluble endogenous fatty acids such as AA, which can regulates ionic channels and mediates signal transduction. The present study is to investigate if arachidonic acid mediate the increase in I_K(Ca) during hyposmotic stretch in gastric antral circular myocytes of the guinea-pig. The myocytes were isolated by collagenase â…¡, the majority of which possessed an intact structure . The membrane current was recorded with the conventional whole cell patch-clamp technique.Results are as follow:I : Role of Arachidonic acid on hyposmotic membrane stretch -increased I_K(Ca) in guinea pig gastric myocytes1. Effects of hyposmotic stretch and AA on I_K(Ca) and STOCs.Under whole-cell configuration, membrane potential was clamped at -60mV, and/K(Ca) was elicited by a single depolarizing step pulse (depolarized from -60 to +60mV, 15s intervals, 400s ).we have observed the duration of hyposmotic stretch and AA increased-/ic(ca) , founding that /|<(Ca) began to increase 139.28571 ±11.33893s after cells were exposed to hyposmotic solution, and 165 ±25.0998s after administration of lOuM AA. Hyposmotic solution (200 mOsm) and 10(iM AA markedly increased /K(Ca) to 168.246 ± 16.106%, 158.529 + 20.477% at +60 mV, respectively.In whole cell configuration, the holding potential was clamped at -20mV; STOCs were recorded and found to be enhanced by hyposmotic solution (200 mOsm) and lOuM AA.2. Effects of exogenous arachidonic acid on /K(Ca) and STOCs increased by hyposmotic stretch.Under whole-cell configuration, the membrane potential was clamped at -60mV, and /K(Ca) was elicited by step voltage command pulse from -40 to lOOmV for 400ms with a 20mV increment at 10s intervals. When cells were superfused in turn with isosmotic solution (290 mOsm), isosmotic solution containing 10(aM AA, hyposmotic solution containing lO^MAA (200mOsm) , we observed that /K(Ca) was increased tol71.78±20.30 % by 10|aMAA , then to 311.54 + 44.36 % by hyposmotic stretch at +60 mV. Cells were superfused in turn with isosmotic solution (290 mOsm), hyposmotic solution (200 mOsm), hyposmotic solution containing lOuMAA. It was found that /K(Ca) was first increased to 184.20+17.66 % % by hyposmotic stretch, then to 281.28 ±28.27 % by AA at +60 mV. There is no significant difference between the increased effect of AA on hyposmotic stretch induced-activation of /K(Ca) in the two different conditions.Under the same configuration, the holding potential was clamped at -20mV; STOCs were recorded. 1 OuM AA could potentiate the incrasing effect of hyposmotic stretch(200 mOsm) on STOCs3. Effects of endogenous arachidonic acid and its metabolites on /i?ca) increased by hyposmotic stretch.When cells have pretreated for 15min with 10\iM NDGA, a lipoxygenase inhibitor of AA, AA and hyposmotic stretch-induced increase in It^ca) could besignificantly inhibited. The increased percentage decreased from 145 + 9% to 110 + 4%, from 170 ± 10% to 142 + 3% , respectively.When pipette solution containing lOOuMDEDA, a nonselective inhibitor of phospholipase A2, we recorded currents lOmin after breakthrough into whole-cell configuration, finding that DEDA (lOOuM in pipette) significantly suppressed hyposmotic membrane stretch-induced increase in /K(Ca), and the increased percentage decreased from 164.3 + 9.8 % of control to 113.4 + 3.6%.II: Mechanism of Arachidonic acid in hyposmotic stretch increased /k(Ca) in guinea pig gastric myocytes.l.Effects of extracellular Ca + on AA-induced increase in /R(Ca)We removed extracellular calcium and added lOuMEGTA in bath solution to observe the effect of AA on /K(Ca), and found the increase in /K(ca) was completely blocked.Nicardipine (5uM) , an L-type calcium channel blocker, did not block AA induced increase in /n(Ca)Gadolilium(20uM), a blocker of SA channel, completely suppressed AA induced increase in /iqca).2 . Effects of intracellular Ca2+ on AA-induced increase in /i<(Ca) Heparin, a potent inhibitor of IP3 induced calcium release (IICR), which could inhibited STOCs significantly, did not suppress AA-induced increase in STOCs.Ryanodine (lOuM), a specific inhibitor of calcium induced calcium release(CICR), firstly increased STOCs and subsequently blocked them completely , not increased by caffeine lmg/ml, an incitant of CTCR . In the present of ryanodine, lOuMAA did not increase STOCs again.Above results suggest that hyposmotic membrane stretch maybe activate phospholipase A2 (PLA2) which hydrolyzes membrane phospholipids to ultimately produce AA; AA as a second messenger mediates Ca2+ influx that triggers Ca2+-induced Ca2+ release, thereby increasing /R(ca) in gastric antral circular myocytes of guinea-pig.