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.

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.

Objective:To reduce the immunogenicity of vaccinia virus vector by replacing the D8L region, which is a neutralizing antibody epitope in vaccinia virus, with an exogenous gene.Methods:A gene fragment encoding influenza virus hemagglutinin (HA) was inserted into the D8L region to replace it using homologous recombination technique. Then, a recombinant vaccinia virus influenza vaccine was constricted. A recombinant vaccinia virus vaccine with the TK region expressing HA was used as a control. The expression of HA was validated by Western blot. BALB/c mice were immunized with the vaccines and the serum antibody titers two weeks after each immunization were evaluated by ELISA and hemagglutination inhibition assay. The protective efficacy of the recombinant vaccinia virus was assessed through a challenge experiment.Results:Western blot confirmed the successful expression of HAD8L protein in the constructed recombinant vaccines. ELISA and hemagglutination inhibition assay showed that after the primary immunization, the anti-HA antibody titer induced by the recombinant vaccinia virus with D8L region mutation was slightly higher than that induced by the vaccine with TK region mutation, and the difference was statistically significant with the increase of immunization times ( P<0.05). The recombinant vaccinia virus with D8L region mutation showed significantly lower immunogenicity than the recombinant virus with TK region mutation after the primary immunization, but there was no significant difference between them with the increase of immunization times ( P>0.05). After H1N1pdm challenge, no virus was detected in the mice immunized with the recombinant vaccinia virus with D8L region mutation and the mice showed mild lung inflammation and less tissue damage. Conclusions:This study indicated that inserting exogenous genes into the D8L region of the neutralizing antibody epitope in the vaccinia virus vector could help to reduce the immunogenicity of the vector itself and enhance the immunogenicity of the exogenous genes. This provided a reference for the use of the vaccinia virus vector as a delivery tool in the field of vaccines or gene therapy.