OBJECTIVETo prepare rabbit monoclonal antibody (RabMab) against guanosine 3', 5'-cyclic monophosphate (cGMP) and to develop a competitive ELISA for the detection of cGMP.

METHODSNew Zealand white rabbits were immunized with synthesized cGMP-keyhole limpet hemoeyanin (cGMP-KLH) to prepared a RabMAb with monoclonal antibody technique of Epitomics. A competitive ELISA kit was produced with cGMP RabMAb. The specificity, the precision and the recoveries of the method were determined.

RESULTSThe RabMAb with high sensitivity towards cGMP were prepared with an antibody timer of 3.1 ng/mL and 50% inhibitive concentration (IC50) of 12.57 ng/mL. The cGMP RabMAb had 33% cross-reactivity to inosine 3', 5'-cyclic monophosphate (cIMP) and little or no cross-reactivity to other compounds. A competitive ELISA was developed for detection of cGMP. The range of detection was 0~120 ng/mL with a minimal limit of 1.95 ng/mL. The recovery of assay was 89%~103%. The inter-assay and intra-assay coefficient variations were below 11.68% and 13.85%, respectively.

CONCLUSIONThe RabMab against cGMP with high affinity and high specificity has been generated successfully, and a competitive ELISA for detection of cGMP has been developed with the prepared cGMP RabMAb.

Animals ; Antibodies, Monoclonal ; biosynthesis ; Antibody Specificity ; Cross Reactions ; Cyclic GMP ; immunology ; Enzyme-Linked Immunosorbent Assay ; Rabbits

To confirm the existence of heme oxygenase (HO)- carbon monoxide (CO)- cyclic guanosine monophosphate (cGMP) pathway in the cultured human trabecular meshwork cells (HT-MCs) 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.
Carbon Monoxide/*metabolism ; Cells, Cultured ; Cyclic GMP/*biosynthesis ; Cyclic GMP/genetics ; Heme Oxygenase (Decyclizing)/*biosynthesis ; Heme Oxygenase (Decyclizing)/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Signal Transduction ; Trabecular Meshwork/cytology ; Trabecular Meshwork/*metabolism

Animals ; Cyclic GMP ; metabolism ; Ischemic Preconditioning, Myocardial ; Myocytes, Cardiac ; metabolism ; Nitric Oxide ; biosynthesis ; Rats ; Rats, Sprague-Dawley

To confirm the existence of heme oxygenase (HO)- carbon monoxide (CO)- cyclic guanosine monophosphate (cGMP) pathway in the cultured human trabecular meshwork cells (HT-MCs) 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.
Carbon Monoxide ; metabolism ; Cells, Cultured ; Cyclic GMP ; biosynthesis ; genetics ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Humans ; RNA, Messenger ; biosynthesis ; genetics ; Signal Transduction ; Trabecular Meshwork ; cytology ; metabolism

The incidence of erectile dysfunction (ED) significantly increases in aging men, which may be associated with the decrease of the serum growth hormone (GH) level. GH may play an important role in the maintenance of penile erectile function, perhaps by promotion of the NO-cGMP pathway, stimulation of the regeneration of NOS-containing nerve fibers and the augmentation of androgenic action.
Adult ; Age Factors ; Aged ; Animals ; Cyclic GMP ; biosynthesis ; Erectile Dysfunction ; physiopathology ; Growth Hormone ; metabolism ; Humans ; Male ; Middle Aged ; Nitric Oxide Synthase ; biosynthesis ; Rats ; Signal Transduction ; physiology

To investigate the expression of the HO-1 gene in PC12 cells in hypoxic environment and gain further insight to the role of HO-1 in cerebral ischemia, PC12 cells were exposed to hypoxia environment (95% N2, 5% CO2) for 0.5 h, 1 h, 4 h, 8 h, 12 h, 24 h respectively. The level of HO-1 mRNA was examined by reverse transcriptase polymerase chain reaction (RT-PCR); the volume of COHb in the media were measured spectrophotometrically and the cGMP concentration of PC12 cell extracts was determined by radioimmunoassay. We found that after exposure to hypoxia for 1 h, 4 h, 8 h, 12 h, 24 h, HO-1 mRNA increased by 3%, 4%, 17%, 31% 36% as compared with that in control group respectively (P < 0.01 or P < 0.05); the COHb increased by 12%, 29%, 59%, 88%, 94% as compared with that in control group respectively (P < 0.01 or P < 0.05), and the cGMP concentration were 2.2, 3.4, 5.2, 8.1, 10.9-fold as that of the control group (P < 0.01). We are led to conclude that hypoxia induced the expression of HO-1 gene, the production of endogenous CO, and the concentration of cGMP was elevated as well.
Animals ; Carbon Monoxide ; metabolism ; Cell Hypoxia ; Cyclic GMP ; metabolism ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Heme Oxygenase-1 ; PC12 Cells ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Up-Regulation

OBJECTIVETo further investigate the action mechanisms of berberine (Ber) and to assess the effects of Ber on the mRNA expression of phosphodiesterase type 5 (PDE5) in rat corpus cavernosum.

METHODSAfter incubating with Ber for 1 or 3 h respectively, we examined the levels of PDE5 mRNA by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSThere were PDE5A1 and PDE5A2 mRNA expressions in the rat corpus cavernosum with PDE5A2 as the dominant isoform. Ber could obviously inhibit the mRNA expression of the two isoforms in the rat penis and bring on a pronounced decrease in PDE5A2 (P < 0.01).

CONCLUSIONThe present study indicates that the inhibitory effect of Ber on PDE5 mRNA expression, especially on PDE5A2, might account for its molecular mechanism for treating ED.

3',5'-Cyclic-GMP Phosphodiesterases ; biosynthesis ; genetics ; Animals ; Berberine ; pharmacology ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Male ; Penis ; drug effects ; metabolism ; RNA, Messenger ; biosynthesis ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction

The present work was to investigate the effects of vasonatrin peptide (VNP) on cardiomyocyte protein synthesis induced by moderate hypoxia. In cultured neonatal rat cardiomyocytes, MTT methods, total protein measurement and (3)H-leucine incorporation were used to calculate the cell number and measure the protein synthesis of cardiomyocytes. Furthermore, radioimmunoassay was undertaken to observe the effects of VNP on the intracellular levels of cAMP, cGMP and the concentration of endothelin (ET) in the culture medium. The results showed that both the cell number and protein synthesis decreased with severe hypoxia for 24 h. In contrast, under moderate hypoxia, cardiomyocyte hypertrophy developed; the protein synthesis as evidenced by total protein content and 3H-eucine incorporation increased significantly. VNP reduced cardiomyocyte protein synthesis induced by moderate hypoxia in a dose-dependent manner. Furthermore, VNP increased the intracellular level of cGMP and decreased the concentration of ET in the culture medium under moderate hypoxia, but had no effect on the level of cAMP. These results suggest that VNP inhibits moderate hypoxia-induced protein synthesis in cultured neonatal rat cardiac myocytes. This effect is mediated, at least in part, by an increase in intracellular cGMP, a reduction in synthesis, and/or a release in ET of cardiomyocytes.
Animals ; Animals, Newborn ; Atrial Natriuretic Factor ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Cyclic AMP ; metabolism ; Cyclic GMP ; metabolism ; Dose-Response Relationship, Drug ; Endothelins ; biosynthesis ; Myocytes, Cardiac ; metabolism ; Protein Biosynthesis ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo observe the expression and effect of human calcitonin gene-related peptide (hCGRP) gene mediated by recombinant adeno-associated virus (rAAV) in primary cultured corporal cavernosum smooth muscle cells of the rat and explore the possibility of using CGRP gene for gene therapy in erectile dysfunction.

METHODSThe primary cultured corporal cavernosum smooth muscle cells of the rat were randomly divided into 4 groups and infected with recombinant virus VssHGCMV-hCGRP, VssHGCMV, VssC-MV-GFP and the untreated, respectively. CGRP-like immunoreactivity was measured by protein dot blot assay in the 24 h-culture medium, and intracellular cAMP and cGMP levels in the cultured cells were also determined using radioimmunoassay to ascertain bioactivity of transduced CGRP.

RESULTSThe exogenous gene was transferred into primary corporal cavernosum smooth muscle cells by VssHGCMV-hCGRP infection and efficiently expressed. Compared with the control group, intracellular cAMP level in the cell infected by VssHGCMV-hCGRP was significantly increased (48.7 +/- 1.1 nmol/L vs 7.8 +/- 1.4 nmol/L, P < 0.01), whereas cGMP level remained unchanged in two groups, and CGRP-like immunoreactivity was also detected in the culture medium infected by VssHGCMV- hCGRP.

CONCLUSIONThe system of secretory expressing bioactive peptide rAAV mediated gene transfer may be used to express efficiently exogenous gene in corporal cavernosum smooth muscle cells and affect cAMP level in the corporal cavernosum smooth muscle cells of the rat.

Animals ; Calcitonin Gene-Related Peptide ; biosynthesis ; genetics ; Cells, Cultured ; Cyclic AMP ; metabolism ; Cyclic GMP ; metabolism ; Dependovirus ; genetics ; Male ; Muscle, Smooth ; cytology ; metabolism ; Penis ; metabolism ; Rats ; Rats, Sprague-Dawley ; Recombination, Genetic ; Transfection

To investigate the expression of the HO-1 gene in PC12 cells in hypoxic environment and gain further insight to the role of HO-1 in cerebral ischemia, PC12 cells were exposed to hypoxia environment (95% N2, 5% CO2) for 0.5 h, 1 h, 4 h, 8 h, 12 h, 24 h respectively. The level of HO-1 mRNA was examined by reverse transcriptase polymerase chain reaction (RT-PCR); the volume of COHb in the media were measured spectrophotometrically and the cGMP concentration of PC12 cell extracts was determined by radioimmunoassay. We found that after exposure to hypoxia for 1 h, 4 h, 8 h, 12 h, 24 h, HO-1 mRNA increased by 3%, 4%, 17%, 31% 36% as compared with that in control group respectively (P < 0.01 or P < 0.05); the COHb increased by 12%, 29%, 59%, 88%, 94% as compared with that in control group respectively (P < 0.01 or P < 0.05), and the cGMP concentration were 2.2, 3.4, 5.2, 8.1, 10.9-fold as that of the control group (P < 0.01). We are led to conclude that hypoxia induced the expression of HO-1 gene, the production of endogenous CO, and the concentration of cGMP was elevated as well.
Carbon Monoxide/metabolism ; Cell Hypoxia ; Cyclic GMP/metabolism ; Heme Oxygenase (Decyclizing)/biosynthesis ; Heme Oxygenase (Decyclizing)/*genetics ; Heme Oxygenase-1 ; PC12 Cells ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Up-Regulation