OBJECTIVETo study the effect of cGMP-dependent protein kinase (PKG) on the pathogenesis of septic shock.

METHODSConfluent endothelial cells were disintegrated and centrifugated to obtain cell lysates after being treated with LPS or PKG activator 8-Br-cGMP. PKG activity of lysates was measured with radioactive isotope label method in a reaction system of phosphorylation of specific substrate H2B by PKG, and the shape and the distribution of intracellular filamentous actin were detected by specific fluorescence staining. For the control study, the PKG specific inhibitor KT5823 was used to pretreat the endothelial cells before the administration of LPS or PKG activator 8-Br-cGMP.

RESULTSExposure to LPS for 5, 10, 30 and 60 minutes led to a rapid time-dependent increase in endothelial PKG activity (P < 0.01 compared to the blank) and the polar distribution of intracellular filamentous actin and preincubation with KT5823 abolished these effects. 8-Br-cGMP was similar to LPS.

CONCLUSIONSThe results suggested that LPS can mediate PKG activation and the stress variety of filamentous actin in the vascular endothelial cells, which probably induce the endothelial hyperpermeability after septic shock.

Capillary Permeability ; Cyclic GMP ; analogs & derivatives ; pharmacology ; Cyclic GMP-Dependent Protein Kinases ; physiology ; Cytoskeleton ; metabolism ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Lipopolysaccharides ; pharmacology ; Shock, Septic ; metabolism ; Signal Transduction

OBJECTIVESTo investigate the effects of antisense oligodeoxynucleotide(ASON) on the cyclic nucleotide monophosphates (cNMP) in smooth muscle cells of human corpus cavernosum, and provide experimental groundwork for the gene therapy of erectile dysfunction.

METHODSPDE5 gene ASON(containing exon 1) was transfected into the corpus cavernosum smooth muscle cells with the presence of liposome DOTAP. Another sense oligodeoxynucleotide(SON) and 1% of bovine serum were also transducted into the cells as controls. Two of cNMP, cAMP and cGMP, were probed and measured by ELISA at 1, 2, 4, 6, 10, 24 and 48 h after transfection.

RESULTSAfter transfection, the level of cGMP(1-6 h) in human corpus cavernosum smooth muscle cells was significantly higher than that in controls(P < 0.01).

CONCLUSIONSThe PDE5 gene ASON had been showed to manifest stimulative effect on the cGMP in smooth muscle cells of human corpus cavernosum in vitro, and it provides experimental groundwork for the gene therapy of erectile dysfunction.

3',5'-Cyclic-GMP Phosphodiesterases ; antagonists & inhibitors ; genetics ; Cyclic AMP ; metabolism ; Cyclic GMP ; metabolism ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Humans ; Male ; Muscle, Smooth ; drug effects ; metabolism ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Penis ; cytology

OBJECTIVETo investigate the effect of phosphodiesterase type 5 (PDE5) small interfering RNA (siRNA) on the cGMP in the smooth muscle cells of human corpus cavernosum, and to provide an experimental groundwork for the gene therapy of erectile dysfunction (ED).

METHODSSmall interfering RNAs targeting PDE5 gene were synthesized by using web design software provided by Ambion, three siRNAs and a control siRNA were synthesized by Ambion. siRNAs were transfected into the smooth muscle cells of human corpus cavernosum by using siPORT Lipid reagent. cGMP was detected by ELISA at different times (24, 48, 72 and 96 h) after transfection.

RESULTSThe cGMP levels of the siRNA1, siRNA2 and siRNA3 groups were significantly higher than those of the siRNA control and blank control groups (P < 0.05), and so was it in the siRNA1 group than the siRNA2 and siRNA3 groups (P < 0.05), with significant difference between the siRNA control and the blank control group (P > 0.05).

CONCLUSIONThe synthesized siRNAs in vitro are capable of increasing the level of cGMP in the smooth muscle cells of human corpus cavernosum, different siRNAs with different capabilities. The siRNA technique could provide not only an extremely powerful tool for the functional analysis of genome but also a new approach to ED gene therapy.

3',5'-Cyclic-GMP Phosphodiesterases ; genetics ; Cells, Cultured ; Cyclic GMP ; metabolism ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Gene Silencing ; Humans ; Male ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Penis ; metabolism ; RNA, Small Interfering ; pharmacology ; Transfection

No abstract available.
Animal ; Cyclic AMP/metabolism ; Cyclic GMP/metabolism ; Neurotransmitters/physiology ; Pharmacology, Clinical/trends* ; Receptors, Adrenergic/physiology ; Receptors, Drug/physiology* ; Receptors, Opioid/physiology

OBJECTIVETo further investigate the action mechanisms of berberine (Ber), and assess the effects of Ber on the in vitro formation of cGMP and cAMP in the isolated rabbit corpus cavernosum.

METHODSIsolated segments of the rabbit corpus cavernosum were exposed to different concentrations of Ber, and, the dosage-dependent accumulations of cGMP and cAMP were determined in the tissue samples by means of 125I radioimmunoassay. Responses of the isolated tissue preparations to Ber were compared with those obtained with the reference compound sildenafil (Sil).

RESULTSBer increased cGMP concentrations directly (P < 0.05). In the presence of sodium nitroprusside (SNP), a stimulatory agent of cGMP, both Ber and Sil increased cGMP with increasing dosage (P < 0.01), the EC, values being 1.32 and 0.67 micromol/L respectively. With the same concentration, neither Ber nor Sil influenced the cAMP level significantly (P > 0.05). In the presence of PGE1, a stimulator of cAMP, Ber and Sil also raised the cAMP level concentration (P < 0.01 ), the EC, values being 4.90 (Ber) and 6.53 (Sil) micromol/L respectively.

CONCLUSIONBer can increase cGMP and cAMP concentrations in the corpus cavernosum smooth muscles, which may contribute to its action of relaxing corpus cavernosum smooth muscles.

Animals ; Berberine ; pharmacology ; Cyclic AMP ; metabolism ; Cyclic GMP ; metabolism ; Dose-Response Relationship, Drug ; In Vitro Techniques ; Male ; Muscle, Smooth ; drug effects ; metabolism ; Penis ; drug effects ; metabolism ; Rabbits ; Radioimmunoassay

OBJECTIVETo observe the effects of signal factors of corticosterone (CS), cAMP, cGMP, Ca2+ andprotein kinase C (PKC) on lymphocyte apoptosis in mouse thymus induced by X-rays of 4 Gy in vitro.

METHODSThe DNA lytic rate for thymocytes was measured by fluorospectrophotometry.

RESULTSThe DNA lyric rate for thymocytes 4-8 hours after irradiation with 2-8 Gy was significantly higher than that in the control (P<0.01). As compared with the control, the DNA lytic rate for thymocytes treated with 0.01 micromol/L CS (P<0.01), 50 ng/mL cAMP (P<0.01), 0.05-0.4 microg/mL ionomycin (Iono, P<0.05 or P<0.01) or 0.05-0.4 ng/mL phorbol myristate acetate (PMA, P<0.05 or P<0.01), respectively, was significantly increased, while the rate for thymocytes treated with 50 ng/mL cGMP was not significantly increased. The DNA lytic rate for thymocytes treated with 0.01 micromol/L CS (P<0.01), 50 ng/mL cAMP (P<0.01), 0.2 and 0.4 microg/mL Iono (P<0.05), and 0.2 and 0.4 ng/mL PMA (P<0.05) plus 4-Gy irradiation, respectively, was significantly higher than that treated with single 4-Gy irradiation, while the rate for thymocytes treated with 50 ng/mL cGMP plus 4-Gy irradiation was not increased. When both 0.4 microg/mL Iono and 0.4 ng/mL PMA acted on the thymocytes, the DNA lytic rate for thymocytes was significantly higher than that in the control (P<0.01), the DNA lytic rate for thymocytes treated with both 0.4 microg/mL Iono and 0.4 ng/mL PMA plus 4-Gy irradiation was significantly higher than that treated with single 4-Gy irradiation (P<0.05), but was not significantly higher than that treated with 0.4 microg/mL Iono plus 4-Gy irradiation or 0.4 ng/mL PMA plus 4-Gy irradiation.

CONCLUSIONCS, cAMP, Ca2+, and PKC signal factors can promote thymocyte apoptosis induced by larger dose X-rays.

Animals ; Apoptosis ; drug effects ; radiation effects ; Calcium ; pharmacology ; Corticosterone ; pharmacology ; Cyclic AMP ; pharmacology ; Cyclic GMP ; pharmacology ; Ionomycin ; pharmacology ; Male ; Mice ; Protein Kinase C ; metabolism ; Spectrometry, Fluorescence ; Tetradecanoylphorbol Acetate ; pharmacology ; Thymus Gland ; cytology ; drug effects ; X-Rays

We have previously shown that the vasodilator effect of protopine (Pro) on rabbit aorta is related to the elevations of cAMP and cGMP. In the present study, the vasodilator mechanisms of Pro were further explored by recording the isotonic contraction of the rat aortic strips, detecting directly the intracellular free Ca(2+) concentration ([Ca(2+)](i)) with Fura-2/AM loaded vascular smooth muscle cells (VSMCs) of rat aorta, and determining the activity of protein kinase C (PKC) in rat aortic tissue with radioactive isotope gamma-32P -ATP-catalyzing assay. By recording the aortic strips contraction induced by noradrenaline (NA) and high potassium (K(+)), Pro shifted nonparallelly the concentration-response curves of NA and high K(+) to right, in which the maximal response was depressed in the presence of Pro (30 and 100 micromol/L), and the values of pD'(2) were 3.70-/+0.25 and 3.97-/+0.15 for NA and high K(+), respectively. In the Fura-2/AM loaded VSMCs, Pro (50 and 100 micromol/L) could not produce any significant change on the resting [Ca(2+)](i), but significantly decreased the [Ca(2+)](i) elevated by NA and high K(+). Pro (30 and 100 micromol/L) had no significant effect on the activity of the cytosolic and membrane PKC in the aortic strips inpretreated by NA. However, in the aortic strips pretreated by NA, the activity of membrane PKC was significantly increased and the activity of cytosolic PKC tended to be decreased by Pro, while the activity of total PKC did not change. These results suggest that Pro seems to promote the translocation of PKC from the cytosol to the membrane in the presence of NA, its vasodilator effect may be the comprehensive result of its decreasing effect on the [Ca(2+)](i) and the increasing effect on cAMP and cGMP, as well as its influence on the PKC.
Animals ; Aorta, Thoracic ; cytology ; Benzophenanthridines ; pharmacology ; Berberine Alkaloids ; pharmacology ; Calcium ; metabolism ; Cells, Cultured ; Cyclic AMP ; metabolism ; Cyclic GMP ; metabolism ; In Vitro Techniques ; Male ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Norepinephrine ; pharmacology ; Protein Kinase C ; metabolism ; Rats ; Rats, Wistar ; Vasodilator Agents ; pharmacology

OBJECTIVETo investigate the vasorelaxation effect of emodin and its relationship with NO-cGMP signal pathway.

METHODSChanges of tension of rat thoracic aortic rings were measured by MedLab biologic signal collection system, and the activity of total nitric oxide synthase (tNOS), constitutive NOS (cNOS) and inducible NOS (iNOS) in endothelium after being treated with emodin was determined with nitric acid reductase method.

RESULTSEmodin relaxed the phenylephrine and potasium chlorate induced contraction of aortic rings, either with or without intact endothelium, in a concentration-dependent manner. Pretreatment of no-specific potassium channel blocker strontium chloride (CsCL) could attenuate the vasorelaxation effect of emodin on aortic rings without intact endothelium, but it could not inhibit vasorelaxation of emodin on aortic rings with intact endothelium. This vasorelaxation action of emodin (40 micromol/L) could be partial blocked by NOS inhibitor L-NAME and guanylate cyclase inhibitor ODQ, with the vasorelaxation range dropped to 64.76 +/- 13.73% and 6.28 +/- 4.79% respectively. Moreover, emodin (40 micromol/L) increased iNOS activity significantly.

CONCLUSIONThe concentration-dependent vasorelaxation effect of emodin might act by activating the NO-cGMP pathway in vascular endothelium.

Animals ; Aorta, Thoracic ; cytology ; Cyclic GMP ; metabolism ; Emodin ; pharmacology ; Endothelium, Vascular ; metabolism ; Male ; Nitric Oxide ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Vasodilator Agents ; pharmacology

This study was undertaken to observe the effects of organic or inorganic calcium antagonists and to investigate the involvement of cyclic nucleotides in regulating the vascular tone in the chorionic artery from normal or preeclamptic placenta. KCI and prostaglandin (PG) F2 alpha produced marked and constant contractions in chorionic arterial preparations of both normal and preeclamptic placentas. Nifedipine (NIF), verapamil (VER) and diltiazem (DIL) reduced the tension that had been produced by KCI and PGF2 alpha in a concentration-dependent fashion in both preparations, and the potency order of the three agents was NIF> VER > DIL. In preeclamptic arteries, however, the magnitudes of vasodilatation induced by NIF and DIL were much smaller than those in normal chorionic arteries. Mg2+ and Cd2+ also relaxed the tension induced by KCI and PGF2 alpha. In preeclamptic chorionic artery, the vasodilatation induced by Mg2+ was significantly potentiated, while that by Cd2+ was not. Removing endothelium did not alter cyclic GMP content in both preparations. In both preparations contracted by PGF2 alpha, nitroprusside markedly increased cyclic GMP content, but neither cyclic GMP nor cyclic AMP content was affected by acetylcholine, NIF, isopro-terenol, or Mg2+. The above results suggest that neither cyclic AMP nor cyclic GMP is involved in regulating the vascular tone of chorionic artery and that sensitivity of the artery in preeclampsia to the inhibitory action of calcium antagonist might be different from that in normal placenta.
Arteries/physiopathology ; Calcium Channel Blockers/*pharmacology ; Cyclic AMP/analysis ; Cyclic GMP/analysis ; Dinoprost/pharmacology ; Female ; Human ; Placenta/*blood supply ; Potassium Chloride/pharmacology ; Pre-Eclampsia/*physiopathology ; Pregnancy ; Vasoconstriction/*drug effects

Scutellarin (SCU), a flavonoid from a traditional Chinese medicinal plant. Our previous study has demonstrated that SCU relaxes mouse aortic arteries mainly in an endothelium-depend-ent manner. In the present study, we investigated the vasoprotective effects of SCU against HR-induced endothelial dysfunction (ED) in isolated rat CA and the possible mechanisms involving cyclic guanosine monophosphate (cGMP) dependent protein kinase (PKG). The isolated endothelium-intact and endothelium-denuded rat CA rings were treated with HR injury. Evaluation of endothelium-dependent and -independent vasodilation relaxation of the CA rings were performed using wire myography and the protein expressions were assayed by Western blotting. SCU (10-1 000 μmol·L(-1)) could relax the endothelium-intact CA rings but not endothelium-denuded ones. In the intact CA rings, the PKG inhibitor, Rp-8-Br-cGMPS (PKGI-rp, 4 μmol·L(-1)), significantly blocked SCU (10-1 000 μmol·L(-1))-induced relaxation. The NO synthase (NOS) inhibitor, NO-nitro-L-arginine methylester (L-NAME, 100 μmol·L(-1)), did not significantly change the effects of SCU (10-1 000 μmol·L(-1)). HR treatment significantly impaired ACh-induced relaxation, which was reversed by pre-incubation with SCU (500 μmol·L(-1)), while HR treatment did not altered NTG-induced vasodilation. PKGI-rp (4 μmol·L(-1)) blocked the protective effects of SCU in HR-treated CA rings. Additionally, HR treatment reduced phosphorylated vasodilator-stimulated phosphoprotein (p-VASP, phosphorylated product of PKG), which was reversed by SCU pre-incubation, suggesting that SCU activated PKG phosphorylation against HR injury. SCU induces CA vasodilation in an endothelium-dependent manner to and repairs HR-induced impairment via activation of PKG signaling pathway.
Animals ; Apigenin ; pharmacology ; Cell Adhesion Molecules ; drug effects ; Cell Hypoxia ; Coronary Vessels ; drug effects ; Cyclic GMP ; analogs & derivatives ; metabolism ; pharmacology ; Cyclic GMP-Dependent Protein Kinases ; Glucuronates ; pharmacology ; Microfilament Proteins ; drug effects ; NG-Nitroarginine Methyl Ester ; metabolism ; pharmacology ; Phosphoproteins ; drug effects ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; complications ; physiopathology ; Signal Transduction ; drug effects ; Thionucleotides ; metabolism ; pharmacology ; Vasodilation ; drug effects ; physiology