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

Muscle strips and muscle cells from cat stomach were used to investigate whether spontaneously formed cyclic nucleotides were involved in the inhibition of gastric smooth muscle contraction. A phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), increased the levels of both cyclic GMP (cGMP) and cyclic AMP (cAMP) in resting state cells, while decreasing acetylcholine-induced muscle contraction. Under the influence of IBMX, SQ22536, an adenylyl cyclase inhibitor and methylene blue, a guanylyl cyclase inhibitor completely blocked increases in cAMP and cGMP respectively, without any effect on contraction. However, the combination of SQ22536 and methylene blue completely blocked increases in both cAMP and cGMP levels and stimulated contractions markedly even in the presence of IBMX. Muscle contraction inhibitors such as isoprenaline, vasoactive intestinal polypeptide and sodium nitroprusside also appeared to increase cyclic nucleotide levels which decreased contraction. Which nucleotide increased the most was dependent on the agonist used. Therefore, irrespective of the cyclic nucleotide class, the spontaneous formation of cyclic nucleotides should be considered in evaluating the mechanism of gastric smooth muscle relaxation.
1-Methyl-3-isobutylxanthine ; Adenylyl Cyclases ; Animals ; Cats* ; Cyclic AMP ; Cyclic GMP ; Guanylate Cyclase ; Isoproterenol ; Methylene Blue ; Muscle Cells ; Muscle Contraction ; Muscle Relaxation* ; Muscle, Smooth ; Nitroprusside ; Nucleotides, Cyclic* ; Relaxation ; Stomach ; Vasoactive Intestinal Peptide

The purpose of present study is to investigate the influence of a spinal gamma-aminobutyric acid B(GABA|B) receptor on a central regulation of blood pressure (BP) and heart rate (HR), and to define its mechanism in the spinal cord. In urethane-anesthetized, d-tubocurarine-paralyzed and artificially ventilated male Sprague-Dawley rats, intrathecal administration of drugs were carried out using injection cannula (33-gauge stainless steel) through the guide cannula (PE 10) which was inserted intrathecally at lower thoracic level through the puncture of a atlantooccipital membrane. Intrathecal injection of an GABA|B receptor agonist, baclofen (30, 60, 100 nmol) decreased both BP and HR dose-dependently. Pretreatment with 8-bromo-cAMP (50 nmol), a cAMP analog, or glipizide (50 nmol), a ATP-sensitive K+ channel blocker, attenuated the depressor and bradycardic effects of baclofen (100 nmol), but not with 8-bromo-cGMP (50 nmol), a cGMP analog. These results suggest that the GABA|B receptor in the spinal cord plays an inhibitory role in central cardiovascular regulation and that this depressor and bradycardic actions are mediated by the decrease of cAMP via the inhibition of adenylate cyclase and the opening of K+ channel.
8-Bromo Cyclic Adenosine Monophosphate ; Adenylyl Cyclases ; Animals ; Baclofen* ; Blood Pressure ; Catheters ; gamma-Aminobutyric Acid ; Glipizide* ; Heart Rate ; Humans ; Injections, Spinal ; Male ; Membranes ; Nucleotides, Cyclic* ; Punctures ; Rats* ; Rats, Sprague-Dawley ; Spinal Cord

BACKGROUND: Extracellular nucleotides act as agonists to regulate a broad range of physiological processes by interacting with P2 receptors in various tissues including the kidney tubules. This study was undertaken to evaluate the effect of P2 receptor activation on PTH-dependent regulation of phosphate transport in the renal proximal tubular cells. METHODS: Proximal tubular cells were isolated from the rabbit kidney and grown as monolayers on 24 well culture plates. Phosphate uptake was determined by measuring the uptake of radiolabeled phosphate into cell monolayers. Cyclic AMP content was determined by radioimmunoassay using [3H]cAMP assay kit. RESULTS: Activation of P2 receptors with ATP exerted differential effects on phosphate uptake and cAMP generation. In the absence of PTH, it inhibited phosphate uptake and stimulated cAMP generation. In contrast, in the presence of PTH, it attenuated PTH-induced stimulation of cAMP generation and inhibition of phosphate uptake. The profile of the effects of different P2 agonists suggested that P2Y1- and P2Y2-like receptors are involved in the effects of ATP. The effect of ATP to interfere with the PTH-induced regulation was significantly blocked by calphostin C, pertussis toxin or PKC-depletion, whereas, the effects of ATP in the absence of PTH were abolished by indomethacin. CONCLUSION: Our results suggest that PKC-dependent modification of Gi proteins and, subsequently, reduced responsiveness of adenylate cyclases is responsible for the attenuating effect of ATP on the PTH-dependent regulation of phosphate transport in rabbit proximal tubule cells.
Adenosine Triphosphate ; Adenylyl Cyclases ; Cyclic AMP ; Indomethacin ; Kidney ; Kidney Tubules ; Nucleotides ; Parathyroid Hormone ; Pertussis Toxin ; Physiological Processes ; Radioimmunoassay

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

The regulatory role of cyclic nucleotides in the expression of neutrophil responses has been examined. fMLP-stimulated superoxide production in neutrophils was inhibited by dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), histamine, adenosine + theophylline, cAMP elevating agents, and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) and sodium nitroprusside, cGMP elevating agents. Staurosporine, a protein kinase C inhibitor, genistein, a protein tyrosine kinase inhibitor and chlorpromazine, a calmodulin inhibitor, inhibited superoxide production by fMLP, but they did not further affect the action of DBcAMP on the stimulatory action of fMLP. DBcAMP, histamine, adenosine + theophylline and genistein inhibited myeloperoxidease release evoked by fMLP, whereas BrcGMP, sodium nitroprusside and staurosporine did not affect it. The elevation of (Ca2+)-i evoked by fMLP was inhibited by genistein and chlorpromazine but was not affected by staurosporine. DBcAMP exerted little effect on the initial peak in (Ca2+)-i response to fMLP but effectively inhibited the sustained rise. On the other hand, BrcGMP significantly inhibited both phases. fMLP-induced Mn-2+ influx was inhibited by either DBcAMP or BrcGMP. These results suggest that fMLP-stimulated neutrophil responses may be regulated by cAMP more than cGMP. cAMP and cGMP appear not affect stimulated responses by direct protein kinase C activation. Their regulatory action on the stimulated neutrophil responses may be not influenced by other activation processes.
Adenosine ; Bucladesine ; Calmodulin ; Chlorpromazine ; Genistein ; Hand ; Histamine ; Neutrophils* ; Nitroprusside ; Nucleotides, Cyclic ; Protein Kinase C ; Protein-Tyrosine Kinases ; Staurosporine ; Superoxides ; Theophylline

We cultured canine kidney (MDCK) cells stably expressing aquaporin-2 (AQP2) on collagen-coated permeable membrane filters and examined the effect of extracellular ATP on arginine vasopressin (AVP)-stimulated fluid transport and cAMP production. Exposure of cell monolayers to basolateral AVP resulted in stimulation of apical to basolateral net fluid transport driven by osmotic gradient which was formed by addition of 500 mM mannitol to basolateral bathing solution. Pre-exposure of the basolateral surface of cell monolayers to ATP (100 ?M) for 30 min significantly inhibited the AVP-stimulated net fluid transport. In these cells, AVP-stimulated cAMP production was suppressed as well. Profile of the effects of different nucleotides suggested that the P2Y2 receptor is involved in the action of ATP. ATP inhibited the effect of isoproterenol as well, but not that of forskolin to stimulate cAMP production. The inhibitory effect of ATP on AVP-stimulated fluid movement was attenuated by a protein kinase C inhibitor, calphostin C or pertussis toxin. These results suggest that prolonged activation of the P2 receptors inhibits AVP-stimulated fluid transport and cAMP responses in AQP2 transfected MDCK cells. Depressed responsiveness of the adenylyl cyclase by PKC-mediated modification of the pertussis-toxin sensitive Gi protein seems to be the underlyihng mechanism.
Adenosine Triphosphate ; Adenylyl Cyclases ; Aquaporin 2 ; Arginine Vasopressin ; Baths ; Cyclic AMP ; Forskolin ; Isoproterenol ; Kidney ; Madin Darby Canine Kidney Cells ; Mannitol ; Membranes ; Naphthalenes ; Nucleotides ; Pertussis Toxin ; Protein Kinase C ; Vasopressins

There are evidences that exogenous electric currents are capable of enhancing bone formation and resorption, and that the conversion of the bioelectric response to biochemical activity provides the directional component of orthodontic tooth movement. In addition, evidence has implicated cyclic nucleotides in alveolar bone cellular activation mechanism during orthodontic tooth movement. In view of these evidences, this study was performed to investigate the effects of exogenous electric currents in cyclic nuclotide levels in feline alveolar bone and the possible clinical applicaiton of electric currents as an additional orthodontic tool. In the first study, three groups of three adult cats were subjected to application of a constant direct current of 10+/-2 microamperes to gingival tissue near maxillary canine noninvasively for 1,3, and 7 days respectively. In the second study, three groups of three adult cats each were treated by an electric-orthodontic procedure for 1, 3, and 7 days respectively. The left maxillary (control) canine received an orthodontic force of 80gm alone at time of initiation, while the right maxillary (experimental) canine received combined force-electric stimulation (80gm of force and 10+/-2 microamperes of a constant D.C currents). Alveolar bone samples were obtained from the mesial (tension and / or cathode) and the distal (compression and/ or anode) sites surriunding maxillary canines as well as from contralateral control sites. The samples were extracted, boiled, homogenized, and the supernatants were assayed for cyclic nucleotides (cAMP, cGMP) by a radiommunoassay method. And also the amount of tooth movement was measured in the second study. On the basis of this study, the following conclusions can be drawn: 1. The fluctuation pattern of cyclic nucleotide levels in alveolar bone treated by exogenous electric currents was similar to that treated by orthodontic force. 2. The cAMP levels in alveolar bone of electrically treated teeth significantly elevated above the control values. And of electrically treated teeth, the values of the anode sites were higher than those of the cathode sites. 3. The cGMP levels in alveolar bone of electrically teeth elevated above the control values at the initation phase of treatment, but dropped below the control values at time of termination. And of electrically treated teeth, the values of the cathode sites were higher than those of the anode sites. 4. The rate of tooth movement in teeth treated by force-electric combination increased with the length of treatment as compared to that treated by mechanical force alone.
Adult ; Animals ; Cats ; Electrodes ; Humans ; Nucleotides, Cyclic* ; Osteogenesis ; Tooth ; Tooth Movement

As a DNA receptor in the cytoplasm,cyclic GMP-AMP synthase (cGAS) can recognize abnormal DNA in the cytoplasm and activate stimulator of interferon genes (STING) to regulate the immune response. The recent studies have demonstrated that this pathway plays a role in non-infectious inflammatory diseases by promoting the expression of type Ⅰ interferon and interferon-stimulated gene.This article reviews the activation and regulation of cGAS-STING pathway in multiple systems and the effect of this pathway on the occurrence and progression of non-infectious inflammatory diseases,providing theoretical reference for future application of cGAS-STING pathway-related drugs in non-infectious inflammatory diseases.
Humans ; Interferons ; Membrane Proteins/metabolism* ; Noncommunicable Diseases ; Nucleotides, Cyclic ; Nucleotidyltransferases/metabolism* ; Signal Transduction

To elucidate the mechanism of cyclic nucleotides, such as adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5' -cyclic monophosphate (cGMP), in the regulation of human gastric motility, we examined the effects of forskolin (FSK), isoproterenol (ISO) and sodium nitroprusside (SNP) on the spontaneous, high K+ and acetylcholine (ACh)-induced contractions of corporal circular smooth muscle in human stomach. Gastric circular smooth muscle showed regular spontaneous contraction, and FSK, ISO and SNP inhibited its phasic contraction and basal tone in a concentration-dependent manner. High K+ (50 mM) produced sustained tonic contraction, and ACh (10 micrometer) produced initial transient contraction followed by later sustained tonic contraction with superimposed phasic contractions. FSK, ISO and SNP inhibited high K+-induced tonic contraction and also ACh-induced phasic and tonic contraction in a reversible manner. Nifedipine (1 micrometer), inhibitor of voltage-dependent L-type calcium current (VDCC(L)), almost abolished ACh-induced phasic contractions. These findings suggest that FSK, ISO and SNP, which are known cyclic nucleotide stimulators, inhibit smooth muscle contraction in human stomach partly via inhibition of VDCCL.
Acetylcholine ; Adenosine ; Calcium ; Contracts ; Forskolin ; Guanosine ; Humans ; Isoproterenol ; Muscle, Smooth ; Nifedipine ; Nitroprusside ; Nucleotides, Cyclic ; Relaxation ; Stomach