The role of the lower esophageal sphincter (LES) is characterized by the ability to maintain tone and to relax allowing the passage of a bolus. It is known that LES relaxation during swallowing may be induced by the cessation of the tonic neural excitation and the activation of non-adrenergic, non-cholinergic (NANC) inhibitory neurons. Furthermore, it is generally accepted that the relaxation of the smooth muscle is mediated primarily by the elaboration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and guanosine 3',5'-cyclic monophosphate (cyclic GMP) via activation of adenylate cyclase and guanylate cyclase, respectively. It is thus possible that cyclic nucleotides might be a second messenger involved in neural stimulation-induced relaxation of LES, although a relationship between relaxation and changes in cyclic nucleotides after neural stimulation has not been established. The present study was performed to define the participation of cyclic nucleotides in the relaxation of LES of dog in response to neural stimulation. Electrical field stimulation (EFS) caused relaxation of the canine isolated LES strips in a frequency-dependent manner, which was eliminated by pretreatment with tetrodotoxin (1 micrometer), but not by atropine (100 micrometer), guanethidine (100 micrometer) and indomethacin (10 micrometer). The nitric oxide synthase inhibitors, N-G-nitro-L-arginine, N-G-nitro-L-arginine methyl ester and N-G-monomethyl-L-arginine inhibited EFS-induced relaxation. Additions of sodium nitroprusside, a nitrovasodilator and forskolin, a direct adenylate cyclase stimulant, caused a dose-dependent relaxation of LES smooth muscle. Effects of sodium nitroprusside and forskolin were selectively blocked by the corresponding inhibitors, methylene blue for guanylate cyclase and N-ethylmaleimide (NEM) for adenylate cyclase, respectively. Dibutyryl cyclic AMP and dibutyryl cyclic GMP caused a concentration-dependent relaxation of the LES smooth muscle tone, which was not blocked by NEM or methylene blue, respectively. However, both NEM and methylene blue caused significant antagonism of the relaxation in LES tone in response to EFS. EFS increased the tissue cyclic GMP content by 124%, whereas it did not affect the tissue level of cyclic AMP. Based on these results, it is suggested that one of the components of canine LES smooth muscle relaxation in response to neural stimulation is mediated by an increase of cyclic GMP via the activation of guanylate cyclase. Additionally, an activation of cyclic AMP generation system was, in part, involved in the EFS-induced relaxation.
Adenosine ; Adenylyl Cyclases ; Animals ; Atropine ; Bucladesine ; Colforsin ; Cyclic AMP ; Cyclic GMP ; Deglutition ; Dibutyryl Cyclic GMP ; Dogs* ; Esophageal Sphincter, Lower* ; Ethylmaleimide ; Guanethidine ; Guanosine ; Guanylate Cyclase ; Indomethacin ; Methylene Blue ; Muscle, Smooth ; Neurons ; Nitric Oxide Synthase ; Nitroprusside ; Nucleotides, Cyclic* ; Relaxation* ; Second Messenger Systems ; Tetrodotoxin

No abstract available.
Calcium* ; Cyclic GMP* ; Muscle Cells*

Agents that elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD). We investigated whether PLD can be phosphorylated by cAMP-dependent protein kinase (PKA) and PKA-mediated phosphorylation affects the interaction between PLD and RhoA, a membrane regulator of PLD. PLD1, but not PLD2 was found to be phosphorylated in vivo by the treatment of dibutyryl cAMP (dbcAMP) and in vitro by PKA. PKA inhibitor (KT5720) abolished the dbcAMP-induced phosphorylation of PLD1, but dibutyryl cGMP (dbcGMP) failed to phosphorylate PLD1. The association between PLD1 and Val14RhoA in an immunoprecipitation assay was abolished by both dbcAMP and dbcGMP. Moreover, RhoA but not PLD1 was dissociated from the membrane to the cytosolic fraction in dbcAMP-treated cells. These results suggest that both PLD1 and RhoA are phosphorylated by PKA and the interaction between PLD1 and RhoA is inhibited by the phosphorylation of RhoA rather than by the phosphorylation of PLD1.
Bucladesine/pharmacology ; Carbazoles/pharmacology ; Cell Line, Tumor ; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors/*metabolism ; Dibutyryl Cyclic GMP/pharmacology ; Enzyme Inhibitors/pharmacology ; Humans ; Indoles/pharmacology ; Phospholipase D/*metabolism ; Phosphorylation/drug effects ; Pyrroles/pharmacology ; Research Support, Non-U.S. Gov't ; rhoA GTP-Binding Protein/*metabolism

No abstract available.
Cyclic GMP* ; Cytomegalovirus* ; Gene Expression* ; Humans*

Injury or inflammation affecting sensory neurons in the dorsal root ganglia (DRG) causes hyperexcitability of DRG neurons that can lead to spinal central sensitization and neuropathic pain. Recent studies have indicated that, following chronic compression of DRG (CCD) or acute dissociation of DRG (ADD) treatment, both hyperexcitability of neurons in intact DRG and behaviorally expressed hyperalgesia are maintained by activity in cGMP-PKG signaling pathway. Here, we provide evidence supporting the idea that CCD or ADD treatment activates cGMP-PKA signaling pathway in the DRG neurons. The results showed that CCD or ADD results in increase of levels of cGMP concentration and expression of PKG-I mRNA, as well as PKG-I protein in DRG. CCD or ADD treated-DRG neurons become hyperexcitable and exhibit increased responsiveness to the activators of cGMP-PKG pathway, 8-Br-cGMP and Sp-cGMP. Hyperexcitability of the injured neurons is inhibited by cGMP-PKG pathway inhibitors, ODQ and Rp-8-pCPT-cGMPS. In vivo delivery of Rp-8-pCPT-cGMPS into the compressed ganglion within the intervertebral foramen suppresses CCD-induced thermal hyperalgesia. These findings indicate that the in vivo CCD or in vitro ADD treatment can activate the cGMP-PKG signaling pathway, and that continuing activation of cGMP-PKG pathway is required to maintain DRG neuronal hyperexcitability and/or hyperalgesia after these two dissimilar forms of injury-related stress.
Animals ; Cyclic GMP ; analogs & derivatives ; metabolism ; Cyclic GMP-Dependent Protein Kinases ; metabolism ; Ganglia, Spinal ; physiopathology ; Hyperalgesia ; physiopathology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Thionucleotides ; metabolism

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 explore the relationship of aging with the changes of endogenous carbon monoxide (CO), cGMP and cAMP contents in the penile tissues of rats.

METHODSTwenty-four male rats were equally divided into an 8-month, a 16-month and a 24-month group, and their penile erection was detected by injecting apomorphine, their penile cavernous body harvested, and the contents of CO, cAPM and cGMP detected by improved dual wavelength spectrophotometry.

RESULTSThe contents of CO, cAPM and cGMP were reduced with the increase of age, with statistically significant differences between the three age groups (P < 0.01).

CONCLUSIONAging significantly decreased the contents of CO, cAMP and cGMP in the penile tissues of the rats, which suggests that aging might play an important role in erectile dysfunction.

Aging ; physiology ; Animals ; Carbon Monoxide ; metabolism ; Cyclic AMP ; metabolism ; Cyclic GMP ; metabolism ; Male ; Penis ; metabolism ; Rats ; Rats, Wistar

Animals ; Cyclic AMP ; metabolism ; Cyclic GMP ; metabolism ; Male ; Myocytes, Cardiac ; metabolism ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley ; Swimming

In order to investigate the correlation between traumatic servity and blood cAMP and cGMP levels in the patients with acute trauma and its clinical significance, 120 cases of trauma were randomly selected and divided into 4 groups (n = 30 in each group): mildly traumatic group (ISS < or = 9), moderately traumatic group (ISS = 10-16), severely traumatic group (ISS = 17-25) and dangerously traumatic group (> 25). The cAMP and cGMP levels were assayed in sera, leucocytes and platelets respectively in 6 h and 24 h after trauma. The results showed that cAMP and cGMP levels were elevated significantly in sera and platelets (P < 0.05 or P < 0.01), meanwhile cGMP levels in leucocytes (P < 0.05). It was concluded that cAMP and cGMP might play an important role in traumatic stress, participate in the cellular signal transducation and promote the immune function of leucocytes and the coagulation founction of platelets. Serum cAMP and cGMP levels were upregulated correspondingly as ISS increased, and positively correlated to the traumatic severity.
Blood Platelets/metabolism ; Cyclic AMP/*blood ; Cyclic GMP/*blood ; *Injury Severity Score ; Leukocytes/metabolism ; Wounds and Injuries/*blood