| Background:Hydrogen sulfide(H2S),a gasotransmitter,can be endogenously produced from rat colonic tissues and by bacteria in the gastrointestinal(GI)lumen.Both exogenous and endogenous H2S play critical roles in the GI tract,including exerting anti-nociceptive effects,regulating the pacemaker activity of interstitial cells of Cajal(ICC),as well as regulating GI motility.L-type calcium channels are expressed in colonic smooth muscle and submucosal layers.The channels are involved in regulation of smooth muscle contraction and cell excitability.Some GI motility disorders are related to the abnormal expression of L-type calcium channels.Thus,L-type calcium channels may be potential therapeutic targets for the treatment of GI motility disorders.L-type calcium channels are modulated by H2S,but the effects of H2S differs depending on tissues or species.Previously,we have found that sodium hydrosulfide(NaHS)inhibited steady-state activation of L-type calcium channels,but the molecular mechanism by which H2S modulates L-type calcium channels remains unclear.NaHS and diallyl trisulfide(DATS)are regarded as exogenous donors of H2S,which are used in studies.This study is designed to investigate the effects of NaHS and DATS on modulating L-type calcium channels in colonic smooth muscle cells and colonic motility.Part I Mechanism of sodium hydrosulfide modulation of L-type calcium channels in rat colonic smooth musclesObjective:To investigate the possible mechanisms underlying the modulation of L-type calcium channels by sodium hydrosulfide(NaHS)in rat colonic smooth muscle cells.Methods:The midcolic tissue of the healthy male Wistar rats was selected and the smooth muscle cells were isolated by collagenase digestion.Using the whole-cell patch clamp technique,the effects of NaHS on the channel current of L-type calcium channels and on the steady-state activation of the channels were observed under the voltage-clamp mode.Then cells were pre-incubated with the sulfhydryl modifiers diamide(DM),DL-dithiothreitol(DTT)and reduced L-glutathione(GSH)before adding NaHS.Changes are recorded.Results:The current of L-type calcium channel of colonic smooth muscle cells reached the peak value at 0 mV,the average peak current density was-2.54 ± 0.41 pA/pF.300 ?m NaHS decreases the current density at 0 mV to-1.31 ± 0.34 pA/pF(P<0.05,n=6).The membrane potential at which the L-type calcium current reached the peak value changed to +20 mV,the peak value at +20 mV was-4.16 ± 0.62 pA/pF(P<0.05,n=6).Thus,the shape of current-voltage(I-V)curve changed obviously and shifted rightward after incubation with NaHS,meanwhile,the steady-state activation curve of L-type calcium channels was shifted to the right.V1/2 changed to 4.94 ± 1.89 mV from-22.13 ± 3.41 mV,while ? value changed to 6.57 ± 1.19 from 4.02 ± 2.25(P<0.05,n=6).DTT significantly enhanced the effects of NaHS,and DM and GSH weakened its effects.The membrane potential at which the L-type calcium current reached the peak changed to +30 mV after incubated with 200 ?M DTT + 300?M NaHS,while changed to +10 mV after incubated withl00?M DM + 300?M NaHS or 200?M GSH + 300?M NaHS.Conclusion:NaHS inhibits the steady-state activation of L-calcium channel in colonic smooth muscle cells and changes the I-V curve.The possible mechanism of NaHS regulation of calcium channel is potentially by modifying the free sulfhydryl groups of L-type calcium channels,which changes the structure and function of the channel protein.Part ? Effects of sodium hydrosulfide and sulfhydryl modifiers on rat colonic muscle stripsObjective:To study the effects of NaHS on spontaneous contraction of colonic smooth muscle strips and its possible mechanism.Methods:The midcolic tissue of healthy male Wistar rats were obtained to prepare the longitudinal and circular muscle strips.The effects of NaHS on spontaneous contraction of muscle strips of colonic smooth muscle were recorded by multichannel physiological signal acquisition system(RM6240B)and a four-chamber bath experimental system.The muscle strips were incubated with DM,DTT and GSH,and the alteration on the effects of NaHS was recorded.Results:Both longitudinal and circular muscle strips experienced a series of regular high-amplitude contractile waves with low frequencies.In addition,circular muscle strips experienced low-amplitude waves with high frequencies.NaHS inhibited the spontaneous high-amplitude contractions of both longitudinal and circular smooth muscle strips in a dose-dependent manner.The inhibitory effects were reversible.After adding 500 ?M,1.0 mM and 1.5 mM NaHS,the average amplitudes of longitudinal muscle strips were 0.99 ± 0.16 g,0.79 ± 0.20 g and 0.29 ± 0.11 g,compared with the basal amplitude 1.31 ± 0.23 g;the average amplitudes of circular muscle strips were 1.23 ± 0.17 g,0.93 ± 0.11 g and 0.66 ± 0.12 g,compared with the basal amplitude 1.32 ± 0.13 g(P<0.05,n=6).There was no obvious change after 100?M DM,200 ?M DTT or 200 ?M GSH incubation in both the longitudinal and circular muscle strips.But pretreatment with DM attenuated the effects of NaHS,while DTT and GSH enhanced the effects of NaHS.Conclusion:NaHS significantly inhibits the contraction of colonic smooth muscle by a dose-dependent manner,which is related to the sulfhydryl modification.Part ? Modulation of diallyl trisulfide in L-type calcium channels of rat colonic smooth muscle cells and colonic motilityObjective:To investigate the modulatory effects of diallyl trisulfide(DATS)on L-type calcium channels in rat colonic smooth muscle and colonic contraction.Methods:The midcolic tissue of the healthy male Wistar rats was selected and the smooth muscle cells were isolated by collagenase digestion.the whole-cell patch clamp technique was used to examine the effects of DATS on L-type calcium channels in colonic smooth muscle cells.Then smooth muscle tissue was cut into 4 mm×10 mm strips along or vertically to the mesenteric border to obtain longitudinal and circular muscle strips.Using a biological signal acquisition system RM6240B and rat colonic smooth muscle contraction and a four-chamber bath experimental system to recode the effects of DATS on spontaneous contractile activity,and the effects of glutathione(GSH)and indoacetamide(IAM)on the action of DATS.Results:DATS dose-dependently attenuated the currents of L-type calcium channels without changing steady-state activation or inactivation kinetics.The peak current density at 0 mV decreased to 93.59%± 9.49%,85.66%± 7.26%and 79.56%± 9.43%,respectively(P<0.05,n = 7).Moreover,DATS inhibited the spontaneous contractions of both longitudinal and circular smooth muscle strips,this inhibitory effect was dose-and time-dependent.The inhibition induced by DATS was much slower than NaHS.The amplitude of longitudinal muscle strips changed to 1.45 ±0.07 g,1.23 ± 0.09 g,1.08 ± 0.07 g and 0.61 ± 0.08 g while incubated with10,100,200 and 400?M DATS form basal amplitude 1.61 ± 0.14 g;similarly,the amplitude of circular muscle strips changed to 1.38 ± 0.13 g,1.02 ± 0.08 g,0.77 ±0.29 g and 0.57± 0.06 g while incubated with 10,100,200 and 400?M DATS form basal amplitude 1.41 ± 0.12 g(P<0.05,n = 5).GSH enhanced the effect of DATS,while IAM blocked the effect of DATS.Conclusion:DATS inhibited the current of L-type calcium channel,without shifting the steady-state activation and inactivation curves.DATS has an inhibitory effect on the contractions of colonic muscle strips that is related to its regulation of L-type calcium channels as well as the release of H2S. |
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