To confirm the existence of heme oxygenase (HO)-carbon monoxide (CO)- cyclic guanosine monophosphate (cGMP) pathway in the cultured human trabecular meshwork cells (HTMCs) in vitro, and to evaluate the inductive role...To confirm the existence of heme oxygenase (HO)-carbon monoxide (CO)- cyclic guanosine monophosphate (cGMP) pathway in the cultured human trabecular meshwork cells (HTMCs) in vitro, and to evaluate the inductive role of hemin on this pathway, HTMCs of the third to fourth generation were cultured in vitro. Reverse transcripase-polymerase chain reaction (RT-PCR) was employed for detection of HO-1 and HO-2 mRNA. Immunohistochemical staining was used to detect HO-1 and HO-2 proteins. Hemin was added into the culture solution. The HO-1 mRNA levels were quantified by RT-PCR. The relative amount of carbon monoxide released into the media was measured with the quantifying carbon monoxide hemoglobin (HbCO) by spectrophotometry. Radioimmunoassay was used to determine changes of cGMP in HTMCs. The results showed that cultured cells had the specific characteristics of HTMCs. Both HO-1 and HO-2 genes were expressed in HTMCs, as well as HO-1 and HO-2 proteins in HTMCs. Hemin induced HO-1 mRNA, HbCO and cGMP in a dose-dependent manner. In conclusion, HO-CO-cGMP pathway exists in the cultured HTMCs and can be induced by hemin. Pharmacological stimulation of HO-CO-cGMP pathway may constitute a novel therapeutic approach to rescuing glaucoma.展开更多
Carbon monoxide (CO) has recently proven to be an important bioactive or signaling molecule in mammalian cells. Its effects are mainly mediated by nitric oxide (NO) and cyclic GMP (cGMP). In Vicia faba leaves, CO prod...Carbon monoxide (CO) has recently proven to be an important bioactive or signaling molecule in mammalian cells. Its effects are mainly mediated by nitric oxide (NO) and cyclic GMP (cGMP). In Vicia faba leaves, CO production and heme oxygenase (HO) activity, an important CO synthetic enzyme, are first reported to increase in response to ABA treatment, which could result in stomatal closure. Inter- estingly, ABA-induced stomatal closure in V. faba guard cells is partially blocked when the synthetic CO inhibitor ZnPP, or the CO/NO scavenger Hb is added. Furthermore, we show that, exogenously applied CO donor, hematin, and CO aqueous solution not only result in the enhancement of CO release, but also time-dependently induce stomatal closure, and the latter is mimicked by the application of an NO donor SNP. The above-mentioned stomatal closure effects are differentially reversed by the addition of tungstate, a potent inhibitor of NO synthetic enzyme nitrate reductase (NR), the specific NO scavenger cPTIO, ZnPP, or Hb. During treatment for 4 h, SNP, 0.01% CO aqueous solution or hematin significantly triggers NO synthesis, whereas cPTIO, or tungstate approximately fully inhibits NO fluorescence. Ad- ditionally, application of the GC inhibitor ODQ blocks CO-induced stomatal closure. This inhibition could be reversed when 8-Br-cGMP is added. Thus, the above results suggest that CO produced by HO is involved in ABA-induced stomatal closure, and NO and cGMP may function as downstream interme- diates in the CO signaling responsible for stomatal closure.展开更多
文摘To confirm the existence of heme oxygenase (HO)-carbon monoxide (CO)- cyclic guanosine monophosphate (cGMP) pathway in the cultured human trabecular meshwork cells (HTMCs) in vitro, and to evaluate the inductive role of hemin on this pathway, HTMCs of the third to fourth generation were cultured in vitro. Reverse transcripase-polymerase chain reaction (RT-PCR) was employed for detection of HO-1 and HO-2 mRNA. Immunohistochemical staining was used to detect HO-1 and HO-2 proteins. Hemin was added into the culture solution. The HO-1 mRNA levels were quantified by RT-PCR. The relative amount of carbon monoxide released into the media was measured with the quantifying carbon monoxide hemoglobin (HbCO) by spectrophotometry. Radioimmunoassay was used to determine changes of cGMP in HTMCs. The results showed that cultured cells had the specific characteristics of HTMCs. Both HO-1 and HO-2 genes were expressed in HTMCs, as well as HO-1 and HO-2 proteins in HTMCs. Hemin induced HO-1 mRNA, HbCO and cGMP in a dose-dependent manner. In conclusion, HO-CO-cGMP pathway exists in the cultured HTMCs and can be induced by hemin. Pharmacological stimulation of HO-CO-cGMP pathway may constitute a novel therapeutic approach to rescuing glaucoma.
基金Supported by the National Natural Science Foundation of China (Grant No. 30671248)"Blue Project" of Jiangsu Province and the Student Research Train-ing (SRT) Project of Nanjing Agricultural University (Grant No. 0506A11)
文摘Carbon monoxide (CO) has recently proven to be an important bioactive or signaling molecule in mammalian cells. Its effects are mainly mediated by nitric oxide (NO) and cyclic GMP (cGMP). In Vicia faba leaves, CO production and heme oxygenase (HO) activity, an important CO synthetic enzyme, are first reported to increase in response to ABA treatment, which could result in stomatal closure. Inter- estingly, ABA-induced stomatal closure in V. faba guard cells is partially blocked when the synthetic CO inhibitor ZnPP, or the CO/NO scavenger Hb is added. Furthermore, we show that, exogenously applied CO donor, hematin, and CO aqueous solution not only result in the enhancement of CO release, but also time-dependently induce stomatal closure, and the latter is mimicked by the application of an NO donor SNP. The above-mentioned stomatal closure effects are differentially reversed by the addition of tungstate, a potent inhibitor of NO synthetic enzyme nitrate reductase (NR), the specific NO scavenger cPTIO, ZnPP, or Hb. During treatment for 4 h, SNP, 0.01% CO aqueous solution or hematin significantly triggers NO synthesis, whereas cPTIO, or tungstate approximately fully inhibits NO fluorescence. Ad- ditionally, application of the GC inhibitor ODQ blocks CO-induced stomatal closure. This inhibition could be reversed when 8-Br-cGMP is added. Thus, the above results suggest that CO produced by HO is involved in ABA-induced stomatal closure, and NO and cGMP may function as downstream interme- diates in the CO signaling responsible for stomatal closure.
