| IntroductionHydrogen sulfide (H2S) is a kind of noxious gas for its smell of rotten eggs. Hydrogen sulfide is recognized as an important gaseous signaling molecule and it could modulate functions of different systems via targeting different ion channels and receptors in mammalian tissues. H2S is also a beneficial gas and has great potential for the therapy of human diseases in the cardiovascular, nervous, reproductive, digestive and immune systems. H2S can be synthesized from L-cysteine by cystathionine (3-synthetase (CBS) or cystathionine γ-lyase (CSE). It has been reported recently that there is CES expression in rat adrenal medulla chromaffin cells (AMCs), so we concluded that AMCs can synthesize H2S. AMCs play an important role in the protective response to stress. However, it is still not known whether H2S can directly stimulate CA release in mammalian AMCs. In the present study, we combined amperometry and whole-cell patch-clamp recording to explore the direct effect of exogenous H2S on C A release in AMCs and the underlying ionic mechanism.Materials and MethodsAfter the deep anaesthesia with urethane (1.15mg/kg, i.p.), a medial laparotomy is done on female Wistar rats weighing200-250g.Four adrenal glands were quickly removed and placed into ice-cold D-Hanks solution (without Ca2+and Mg2+). The adrenal medulla were carefully dissected from the cortex under a microscope and dissociated by incubation (at37℃) for70min in the0.5ml D-Hanks’ solution containing3mg/ml collagenase,3mg/ml bovine serum albumin,0.2mg/ml Deoxyribonuclease and2.4mg/ml hyaluronidase. After incubation, the tissue was triturated with a fire-polished, glass Pasteur pipette. Dissociated cells were resuspended in Dulbecco’s modified eagle’s medium supplemented with10%fetal bull serum,50U/ml penicillin G, and50μg/ml streptomycin. The suspension of isolated cells was then placed onto a poly-L-lysine-coated coverslip for culture. The cells were allowed to settle and adhere on the coverslip for1hr, then kept in the incubator at37℃for up to24hr before being used. In the experiments, a piece of coverslip containing the cultured cells was placed in the recording chamber and perfused at approximately1ml/min with standard external solution. Five-micrometer carbon fiber electrodes (CFE; Dagan, Minneapolis, MN) were used to measure CA release from the cultured AMCs. Whole-cell recordings were made using an Axon700B amplifier (Axon Instruments, CA, USA), using pipettes with a resistance of3-5MQ. All the data here shown correspond to means±SEM. Differences were subjected to statistical analysis by one-way ANOVA and Student’s t test. Differences were considered significant when.P<0.05.Results1. Amperometric recordings showed that local application of100μM NaHS significantly induced a burst of amperometric spikes. As a positive control, local application of high-K+solution resulted in reliable amperometric responses. As a negative control, standard external solution did not evoke amperometric spikes. We noticed that100μM NaHS induced no amperometric spikes when the CFE was held at0mV, whereas the same amount of NaHS treatment induced amperometric spikes at780mV. When we replaced the standard external solution with Ca2+-free solution containing1mM EGTA for at least5min, H2S-induced CA release was completely abolished. These results demonstrate that the H2S-induced oxidation current indeed reflects a CA release from chromaffin cells.2. H2S-induced membrane depolarization accompanied by an increase in both the frequency of action potentials and of the CA quantal secretion.3. H2S-induced depolarization is generated by inhibition of K+ca in AMCs. Furthermore, the inhibitory effect of H2S on K+current is reduced dramatically in the Ca2+-free condition.ConclusionWe conclude that H2S is capable of directly inducing CA release by inhibiting the K(ca) current. This conclusion indicates that H2S may involve in the response of adrenal medulla to stress by modulating K(ca) current and CA release in mammalian animals. |
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