ABSTRACT
AIM: To investigate the primary culture method for coronary artery smooth muscle cells (CASMCs), and to establish the endoplasmic reticulum stress ( ERS) model in CASMCs of SD rats.METHODS:CASMCs were cultured by tissue explant method .The morphological characteristics were observed under optical micro-scope.The marker proteins of CASMCs , including α-SMA and SM-MHC, were identified by immunofluorescence tech-nique.The protein expression levels of BiP and CHOP , the marker molecules of ERS, were determined by Western blot . RESULTS:The spindle-shaped CASMCs climbed out from the edge of coronary artery tissues after 6 d, and formed the typical hill and valleygrowth pattern of CASMCs at 9~10 d.The result of immunofluorescence technique showed that α-SMA and SM-MHC were positively expressed .The results of Western blot showed that the protein expression of BiP and CHOP in TG ( 1 and 2 μmol/L ) treatment groups was increased compared with control group .Compared with control group, the protein expression of BiP and CHOP was significantly increased after 1 μmol/L TG treatment for 24 and 48 h. CONCLUSION:CASMCs can be successfully cultured by tissue explant method .ERS model of CASMCs was established by 1 μmol/L TG treatment for 24 h.
ABSTRACT
AIM: To study the effect of caffeine on the large conductance calcium activated potassium (K_(Ca)) channels by patch-clamp technique on smooth muscle cells enzymatically isolated from the porcine coronary artery (PCASMC),and to investigate the effect of ryanodine on K_(Ca) being activated by caffeine.METHODS: Using the single channel patch-clamp technique,single PCASMC was isolated by collagenase,the activity of single K_(Ca) channel was recorded in porcine coronary artery smooth muscle cells.RESULTS: Caffeine (0.1-10 mmol/L) enhanced the open probability (Po) of K_(Ca) channels in a dose-dependent manner in the intracellular side of inside-out patches and its effect was almost completely abolished by washout. Caffeine decreased the mean close time markedly,but had no effect on the amplitude of K_(Ca) channels. However,ryanodine (10-40 μmol/L) decreased Po of K_(Ca) channels activated by caffeine in a dose-dependent manner in cell-attached patches. The mean open time also decreased.CONCLUSION: Caffeine directly activates K_(Ca) channels of porcine coronary artery smooth muscle cells in inside-out patches,the activity of single K_(Ca) channel is inhibited by ryanodine indirectly in cell-attached patches.
ABSTRACT
AIM:To study the effect of caffeine on the large conductance calcium activated potassium (KCa) channels by patch-clamp technique on smooth muscle cells enzymatically isolated from the porcine coronary artery (PCASMC),and to investigate the effect of ryanodine on KCa being activated by caffeine.METHODS:Using the single channel patch-clamp technique,single PCASMC was isolated by collagenase,the activity of single KCa channel was recorded in porcine coronary artery smooth muscle cells.RESULTS:Caffeine (0.1-10 mmol/L) enhanced the open probability (Po) of KCa channels in a dose-dependent manner in the intracellular side of inside-out patches and its effect was almost completely abolished by washout. Caffeine decreased the mean close time markedly,but had no effect on the amplitude of KCa channels. However,ryanodine (10-40 ?mol/L) decreased Po of KCa channels activated by caffeine in a dose-dependent manner in cell-attached patches. The mean open time also decreased.CONCLUSION:Caffeine directly activates KCa channels of porcine coronary artery smooth muscle cells in inside-out patches,the activity of single KCa channel is inhibited by ryanodine indirectly in cell-attached patches.