PURPOSE: The effects of amiloride on cellular toxicity caused by tissue plasminogen activator (tPA) in mouse primary retinal cells were investigated. METHODS: Primary retinal cell cultures were maintained using glial conditioned medium. Commercial tPA and L-arginine were added, and the level of cyclic guanosine monophosphate (cyclic-GMP) in the culture supernatant was assessed using an ELISA assay. We measured the cell viability of cultured retinal cells pretreated with three different concentrations of amiloride (1, 10, and 100 microm) in addition to commercial tPA or L-arginine treatment. RESULTS: After exposing the cultured mouse retinal cells to tPA plus L-arginine or L-arginine alone, cyclic-GMP concentrations were 61.9 +/- 5.1 pmole/mL and 63.1 +/- 6.1 pmole/mL, respectively. However, the control group had a significantly lower concentration of cyclic-GMP (37.2 +/- 3.4 pmole/mL, p < 0.01). The cyclic GMP-dissolved solution did not cause retinal cell death. In the control group and the group treated with 1 microm amiloride and tPA containing L-arginine, the cell viability was 43.7% and 44.5%, respectively. However, cell viability increased to 70.6% with 10 microm amiloride and 78.4% with 100 microm amiloride (p = 0.015). CONCLUSIONS: L-arginine increases intracellular cyclic-GMP and may give rise to retinal cells through this mechanism. In addition, amiloride in concentrations greater than 10 microm protects against L-arginine-induced retinal cell death.
Amiloride/*pharmacology ; Analysis of Variance ; Animals ; Arginine/toxicity ; Cell Death/drug effects ; Cells, Cultured ; Cyclic GMP/pharmacology ; Enzyme-Linked Immunosorbent Assay ; Mice ; Retina/cytology/*drug effects ; Tissue Plasminogen Activator/*toxicity

BACKGROUNDBradykinin (BK) acts mainly on two receptor subtypes: B(1) and B(2), and activation of B(2) receptor mediates the most well-known cardioprotective effects of angiotensin converting enzyme inhibitors (ACEi), however, the role that B(1) receptor plays in ACEi has not been fully defined. We examined the role of B(1) receptor in the inhibitory effect of ACE inhibitor captopril on rat cardiomyocyte hypertrophy and cardiac fibroblast proliferation induced by angiotensin II (Ang II) and explored its possible mechanism.

METHODSNeonatal cardiomyocytes and cardiac fibroblasts (CFs) were randomly treated with Ang II, captopril, B(2) receptor antagonist (HOE-140) and B(1) receptor antagonist (des-Arg(10), Leu(9)-kallidin) alone or in combination. Flow cytometry was used to evaluate cell cycle, size and protein content. Nitric oxide (NO) and intracellular cyclic guanosine monophosphate (cGMP) level were measured by colorimetry and radioimmunoassay.

RESULTSAfter the CFs and cardiomyocytes were incubated with 0.1 micromol/L Ang II for 48 hours, the percentage of CFs in the S stage, cardiomyocytes size and protein content significantly increased (both P < 0.01 vs control), and these increases were inhibited by 10 micromol/L captopril. However, NO and cGMP levels were significantly higher than that with Ang II alone (both P < 0.01). 1 micromol/L HOE-140 or 0.1 micromol/L des-Arg(10), Leu(9)-kallidin attenuated the effects of captopril, which was blunted further by blockade of both B(1) and B(2) receptors.

CONCLUSIONSActing via B(2) receptor, BK contributes to the antihypertrophic and antiproliferative effects of captopril on cardiomyocytes and CFs. In the absence of B(2) receptor, B(1) receptor may act a compensatory mechanism for the B(2) receptor and contribute to the inhibition of cardiomyocyte hypertrophy and CFs proliferation by captopril. NO and cGMP play an important role in the effect of B(1) receptor.

Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Animals ; Animals, Newborn ; Captopril ; pharmacology ; Cardiomegaly ; prevention & control ; Cell Proliferation ; drug effects ; Cell Size ; drug effects ; Cyclic GMP ; analysis ; DNA ; biosynthesis ; Fibroblasts ; drug effects ; physiology ; Myocytes, Cardiac ; drug effects ; pathology ; Nitric Oxide ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptor, Bradykinin B1 ; physiology

BACKGROUNDTo date, there have been no reports on altered nitric oxide (NO) content in ischemia/reperfusion with regard to in vivo preconditioning procedures. These studies are important for understanding the mechanisms of NO during early myocardial ischemic preconditioning. The aim of the present study was to investigate the mechanisms of NO during early myocardial ischemic preconditioning by measuring levels of NO and cyclic guanosine monophosphate (cGMP), as well as activity of nitric oxide synthase (NOS) in ischemia/reperfusion with respect to preconditioning in rats.

METHODSSixty-six female Sprague-Dawley rats were randomly divided into four groups: ischemic preconditioning group (IP), ischemia/reperfusion group (I/R), control group (CON), and preconditioning procedure group (PC). In the PC group, rats were further divided into PC1-, PC1 +, PC2-, PC2 +, PC3-, and PC3 + subgroups. Rats underwent left coronary artery occlusion and reperfusion, and subsequently, NOS activity and levels were assessed with spectrophotometric analysis. cGMP contents were measured with radioimmunoassay.

RESULTSThe level of NO and cGMP, as well as the activity of NOS, were significantly higher in the IP group compared to the I/R and CON groups (P < 0.05). During preconditioning prior to prolonged ischemia, NO and cGMP levels varied markedly with ischemia and reperfusion. The levels of NO repeatedly increased when the heart was exposed to three episodes of 5-minute ischemia, and were almost completely reversed during each reperfusion period. NO and cGMP levels were significantly different between the 5-minute period of ischemia and the same period of reperfusion during preconditioning.

CONCLUSIONSNO plays an important role during early phase myocardial ischemic preconditioning in rats. NO and cGMP could be triggers and mediators of early phase myocardial ischemic preconditioning. Altered NOS activity following ischemic stress could be the primary inducer of higher NO levels detected. NO and cGMP fluctuations might be the trigger for protection during early phase myocardial ischemic preconditioning.

Animals ; Cyclic GMP ; analysis ; Female ; Ischemic Preconditioning, Myocardial ; Nitric Oxide ; analysis ; physiology ; Nitric Oxide Synthase ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To observe the curative effect of Jieminqufeng decoction to the rats of allergic rhinitis and study the mechanism by which it treats allergic rhinitis. METHOD: Forty wistar rats were divided into 4 groups at random. There are Jieminqufeng decoction group, cetirizine group, model control group and normal control group. The rats of allergic rhinitis were established with ovalbumin. We surveyed the behavioral changes of rats, searched eosinophilic granulocytes in the nasal secretion, detected the contents of cAMP and cGMP in the blood plasma and nasal mucosa. RESULT: The model control group had typical symptoms of allergic rhinitis and the eosinophilic granulocytes could be found more frequently. The contents of cAMP and cAMP/cGMP rose in the blood plasma and nasal mucosa (P < 0.01). However, the changes of jieminqufeng decoction group were small. CONCLUSION The jieminqufeng decoction is an effective drug to allergic rhinitis. Its possible mechanism is that it changes the contents of cAMP and cGMP, lessens inflammatory reaction and blocks up the allergy.
Animals ; Anti-Inflammatory Agents ; therapeutic use ; Cyclic AMP ; analysis ; Cyclic GMP ; analysis ; Drugs, Chinese Herbal ; therapeutic use ; Male ; Phytotherapy ; Rats ; Rats, Wistar ; Rhinitis, Allergic, Perennial ; drug therapy

OBJECTIVETo explore the effect and the mechanism of moxibustion for treatment of diabetic erectile dysfunction (ED).

METHODSDiabetes mellitus (DM) rat model was induced by streptozotozin (STZ) and then penis erectile experiment of apomorphine (APO) was used to select diabetic ED rats model, which were divided into 2 groups: a model group and a moxibustion group, with another normal control group set up. The moxibustion group were treated with moxibustion at "Shenshu" (BL 23) and "Sanyinjiao" (SP 6) with small moxa cone about the size of a wheat grain. The effects on penis erectile, blood sugar and total NOS, cNOS, iNOS, and cGMP were investigated.

RESULTSMoxibustion had a certain improving action on blood sugar, improved significantly erectile function, and more significantly increased NOS, cNOS, iNOS activities and cGMP contents in the penis.

CONCLUSIONMoxibustion has a certain action on the erectile function in the diabetic ED rats, which is related with improvement of blood sugar and promoting NO-cGMP pathway.

Animals ; Blood Glucose ; analysis ; Cyclic GMP ; physiology ; Diabetes Complications ; therapy ; Diabetes Mellitus, Experimental ; complications ; Erectile Dysfunction ; therapy ; Male ; Moxibustion ; methods ; Nitric Oxide ; physiology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

BACKGROUNDAngiotensin II (Ang II) acting at angiotensin AT(1) receptor (AT(1)R) has well documented effects on cardiovascular structure such as the promotion of cardiovascular hypertrophy and fibrosis, which are believed to be opposed by angiotensin AT(2) receptor (AT(2)R) stimulation. The expressions of AT(1)R and AT(2)R are up-regulated in senescent hearts. The purpose of this study was to investigate the interaction of signal transduction between AT(1)R and AT(2)R, and to detect whether there is any difference in the interaction in rat hearts of different age.

METHODSIn 3.5-, 12-, 18- and 24-month-old rats, the heart cell membrane activities of protein kinase C (PKC) and tyrosine kinase were measured when AT(1)R and AT(2)R were both activated by Ang II or just the AT(1)R was activated by Ang II and PD123319. The activities of cytosolic phospholipase A(2) (cPLA(2)) and the levels of cGMP were investigated when AT(1)R and AT(2)R were both activated by Ang II or just the AT(2)R was activated by Ang II and losartan.

RESULTSWhen AT(1)R and AT(2)R were both activated compared to when the AT(1)R was activated, the activities of PKC were not different in hearts from 3.5- and 12-month-old rats, but decreased significantly in 18- and 24-month-old rats; the activities of tyrosine kinase were not different in 3.5-month-old rats but decreased significantly in 12-, 18- and 24-month-old rats. The activities of cPLA(2) were all decreased significantly in rats of different age when AT(1)R and AT(2)R were both activated compared to when the AT(2)R was activated. Treatment with Ang II alone compared to Ang II and losartan decreased the levels of cGMP (fmol/mg) in rats of different age (102.7 +/- 12.7 versus 86.0 +/- 8.0 in 3.5-month-old rats, P < 0.05; 81.0 +/- 9.4 versus 70.0 +/- 6.3 in 12-month-old rats, P < 0.05; 69.8 +/- 5.6 versus 54.2 +/- 5.3 in 18-month-old rats, P < 0.01; 57.7 +/- 8.0 versus 39.0 +/- 3.0 in 24-month-old rats, P < 0.01).

CONCLUSIONSThe activation of AT(1)R inhibited the signal transduction of AT(2)R during the aging variation, and the activation of AT(2)R inhibited the signal transduction of AT(1)R in rat heart of different age.

Aging ; metabolism ; Animals ; Cyclic GMP ; analysis ; Male ; Myocardium ; metabolism ; Phospholipases A2 ; metabolism ; Protein Kinase C ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Angiotensin, Type 1 ; physiology ; Receptor, Angiotensin, Type 2 ; physiology ; Signal Transduction ; physiology

BACKGROUNDThe form deprivation (FD) reduces spatial contrasts and induces myopia. Nitric oxide and cyclic guanosine monophosphate (cGMP) are involved in visual signal transmission. This study investigated changes in nitric oxide synthase (NOS) activity and cGMP concentration in ocular tissues in acute and chronic form deprivation myopia.

METHODSGuinea pigs had one eye covered by translucent glass for 7, 14 or 21 days. Untreated litter mates were used as controls. NOS activity and cGMP concentrations in the retinal, choroidal and scleral tissues of FD eyes and control eyes were analyzed by radioimmunoassay after various durations of FD. The expression of NOS subtypes was identified by immunohistochemistry.

RESULTSMyopia was successfully induced in FD eyes after 14 days. Compared with control groups, the retinal NOS activity and cGMP concentrations in the FD eyes significantly increased after 14 and 21 days while the retinal NOS activity in the FD eyes was transiently suppressed by 7 days of FD. The NOS activity and cGMP concentrations of choroid and sclera in the FD eyes were higher than in the control groups at 21 days. The three isoenzymes of nitric oxide synthase were detected in the ocular tissues of guinea pigs.

CONCLUSIONSThe NOS activity and cGMP concentrations were upregulated after chronic FD and the retinal NOS activity was transiently suppressed at acute FD. The function of elevated NOS activity may be mediated by cGMP.

Animals ; Cyclic GMP ; analysis ; Guinea Pigs ; Immunohistochemistry ; Myopia ; metabolism ; Nitric Oxide ; physiology ; Nitric Oxide Synthase ; metabolism ; Refractive Errors ; Retina ; metabolism

To investigate the effect of S-nitrosoglutathione (GSNO), a nitric oxide donor, on platelet production from megakaryocytes differentiated from cord blood CD34(+) cells in vitro, the CD34 (+) cells from eight fresh umbilical cord blood samples by a high-gradient magnetic cell sorting (MACS) system were cultured in serum-free medium for 14 d with thrombopoietin (TPO) 50 ng/ml, IL-3 10 ng/ml, stem cell factor (SCF) 50 ng/ml and rHuGM-CSF 20 ng/ml. Then, CD61 (+) cells were purified by MACS system from these CD34 (+) cells, and were cultured in serum-free medium supplemented with TPO 50 ng/ml, IL-3 10 ng/ml and SCF 50 ng/ml in the presence (treatment group) and absence (control group) of GSNO for 30 min or 2 h. Platelet-sized particles were counted by flow cytometry; megakaryocyte structure was detected by scanning electron microscope. Aggregation of the thrombin-induced platelet particle was observed under inversion microscope. cGMP was assessed by commercial ELISA kit. The results showed that, compared with the control group, the number of platelet-sized particles significantly increased (P<0.05) in the treatment group, in which megakaryocytes presented significant pseudopod formation and extensive membrane blebbing. The platelet particle aggregation could be observed under microscope after thrombin induction. cGMP activity was significantly increased after treatment with GSNO (P<0.05). These results propose that GSNO can facilitate platelet production from megakaryocyte, and it may be partly through cGMP pathway.
Antigens, CD34 ; analysis ; Blood Platelets ; cytology ; Cell Differentiation ; drug effects ; Cyclic GMP ; blood ; Female ; Fetal Blood ; cytology ; Hematopoiesis ; drug effects ; Hematopoietic Stem Cells ; cytology ; Humans ; Megakaryocytes ; cytology ; Nitric Oxide ; physiology ; Platelet Aggregation ; drug effects ; Pregnancy ; S-Nitrosoglutathione ; pharmacology

AIMTo investigate the effect of icariin on erectile function and the expression of nitric oxide synthase (NOS) isoforms in castrated rats.

METHODSThirty-two adult male Wistar rats were randomly divided into one sham-operated group (A) and three castrated groups (B, C and D). One week after surgery, rats were treated with normal saline (groups A and B) or oral icariin (1 mg/[kg.day] for group C and 5 mg/[kg.day] for group D) for 4 weeks. One week after treatment, the erectile function of the rats was assessed by measuring intracavernosal pressure (ICP) during electrostimulation of the cavernosal nerve. The serum testosterone (ST) levels, the percent of smooth muscle (PSM) in trabecular tissue, and the expression of mRNA and proteins of neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and phosphodiesterase V (PDE5) in corpus cavernosum (CC) were also evaluated.

RESULTSICP, PSM, ST and the expression of nNOS, iNOS, eNOS and PDE5 were significantly decreased in group B compared with those in group A (P 0.01). However, ICP, PSM and the expression of nNOS and iNOS were increased in groups C and D compared with those in group B (P 0.05). Changes in ST and the expression of eNOS and PDE5 were not significant (P 0.05) in groups C and D compared with those in group B.

CONCLUSIONOral treatment with icariin ( 98.6 % purity) for 4 weeks potentially improves erectile function. This effect is correlated with an increase in PSM and the expression of certain NOS in the CC of castrated rats. These results suggest that icariin may have a therapeutic effect on erectile dysfunction.

3',5'-Cyclic-GMP Phosphodiesterases ; genetics ; metabolism ; Animals ; Blood Pressure ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Drugs, Chinese Herbal ; pharmacology ; Erectile Dysfunction ; drug therapy ; metabolism ; Flavonoids ; pharmacology ; Gene Expression Regulation, Enzymologic ; drug effects ; Male ; Muscle, Smooth ; drug effects ; physiology ; Nitric Oxide Synthase ; genetics ; metabolism ; Nitric Oxide Synthase Type I ; genetics ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Orchiectomy ; Penile Erection ; drug effects ; Penis ; drug effects ; enzymology ; Pressure ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Testosterone ; blood

OBJECTIVETo investigate the role of endogenous carbon monoxide (CO) in hypoxia.

METHODSAfter rats were inhaled with hypoxic gases and the heme oxygenase inhibitor ZnPPIX was administered, we measured the CO levels in plasma, liver, lung and kidney. Meanwhile plasma cGMP levels were observed. Furthermore, we recorded the change of hemodynamic and blood gases.

RESULTSAcute mild hypoxia (10% O2) significantly increaed CO levels in plasma as well as liver, kidney and lung, while acute severe hypoxia (5% O2) significantly decreased CO levels in plasma as well as liver, kidney and lung. In addition, the former significantly elevated cGMP levels in plasma while the latter markedly reduced cGMP levels in plasma. The hemodynamic change occurred in accordance with the changes carbon monoxide.

CONCLUSIONOur results indicate, for the first time, that the endogenous carbon monoxide plays an important role in regulating the vessel tone during hypoxia.

Acute Disease ; Animals ; Blood Gas Analysis ; Carbon Monoxide ; blood ; metabolism ; Cyclic GMP ; blood ; Heme Oxygenase (Decyclizing) ; antagonists & inhibitors ; Hemodynamics ; Hypoxia ; metabolism ; physiopathology ; Kidney ; metabolism ; Liver ; metabolism ; Lung ; metabolism ; Rats ; Rats, Wistar