The Effect Of Hydrogen Sulfide On Catecholamine Secretion In Neonatal Rat Adrenal Chromaffin Cells And Its Underlying Mechanism

IntroductionHydrogen sulfide (H2S) is an important gaseous signaling molecule and act on many types of ionic channels in the cell membrane. Catecholamine (CA) secreted from chromaffin cells of the adrenal medulla plays an important role in the regulation of stress response. CA is involved in the initiation of breathing and cardiovascular functions, and helps the newborn to adjust to the environment outside the uterus. So Both CA release from adrenal chromaffin cells and H2S have been shown to play critical roles in the regulation of hypoxic stress response. Our previous study has demonstrated that exogenous H2S directly induced quantal CA released from adult rat adrenal chromaffin cells(ARACCs) by inhibiting Ca2+-activated K+current [IK(Ca)current]. However, it is not clear now whether H2S can also directly induce quantal CA release from NRACCs. In the present study, Using carbon-fiber amperometry and whole-cell patch clamping techniques, we investigated whether exogenous H2S can stimulate quantal CA release from NRACCs and whether there were differences in the kinetics of H2S-induced quantal CA release between ARACCs and NRACCs.Materials and MethodsSix adrenal glands were removed from3neonatal Wistar rats (P0-P7) and immediately immersed in ice-cold, Ca2+-and Mg2+-free D-Hanks solution. The cortex of the adrenal gland was removed and the medulla was isolated in D-Hanks solution. The isolated medullas were digested for20min at37℃in enzyme solution containing (in mg/ml):3collagenase type Ⅰ,3BSA,0.2DNase I and2.4hyaluronidase I-S. After digestion, the medullas were trituratedby200μl pipette tip. Finally, the dispersed NRACCs were placed on glass coverslipsin a CO2incubator at37℃with high-glucose DMEM containing250μg/ml streptomycin SO4,250μg/ml penicillin G and10%heat-inactivated FBS. The NRACCs were used within12h.In the experiments, apieces of coverslip containing the cultured cells was placed in the recording chamber and perfused at approximated1ml/min with standard external solution. Five-micrometercarbon fiber electrodes(CEF;Dagan,Minneapolis,MN) were used to measure CA release from the cultured NRACCs.Whole-cell recordings were made using an Axon700B amplifier(Axon Instruments,CA,USA), using pipettes with a resistance of6-10MΩ.All the data are shown as mean±SEM. Differences were analyzed by one-way ANOVA and Student’s t test. Differences were considered significant when P <0.05.Results1. H2S induced quantal CA release from NRACCs.2. H2S induced depolarization of membrane potential andinhibited IK+Ca in NRACCs.3. Kinetic differences of the H2S-induced quantal CArelease of NRACCs and ARACCs. compared with ARACCs, much smaller quantal size and faster quantal release were showed in NRACCs through the kinetic analysis of the single-vesicle secretion induced by Hydrogen sulfide.ConclusionWe conclude that exogenous H2S can directly induce quantal CA released from NRACCs. Compared with ARACCs, kinetic analysis shows much faster quantal CA release and smaller quantal size in NRACCs. Our research may indicate that the H2S-induced CA release possibly helps the newborn to avoid the hypoxic stress-induced injury.