cyclic-gmp and 8-chloro-cyclic-adenosine-monophosphate

The ubiquitous bacterial second messenger c-di-GMP is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). Pseudomonas putida has dozens of DGC/PDE-encoding genes in its genome, but the phenotypical-genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterize function and transcriptional regulation of a P. putida c-di-GMP-metabolizing enzyme, GcsA. GcsA consists of two per-ARNT-sim (PAS) domains, followed by a canonical conserved central sequence pattern (GGDEF) domain and a truncated EAL domain. In vitro analysis confirmed the DGC activity of GcsA. The phenotypic observation revealed that GcsA inhibited swimming motility in an FlgZ-dependent manner. In terms of transcriptional regulation, gcsA was found to be cooperatively regulated by c-di-GMP and cAMP via their effectors, FleQ and Crp respectively. The transcription of gcsA was promoted by c-di-GMP and inhibited by cAMP. In vitro binding analysis revealed that FleQ indirectly regulated the transcription of gcsA, while Crp directly regulated the transcription of gcsA by binding to its promoter. Besides, an inverse relationship between the cellular c-di-GMP and cAMP levels in P. putida was confirmed. These findings provide basic knowledge regarding the function and transcriptional regulation of GcsA and demonstrate a crosstalk between c-di-GMP and cAMP in the regulation of the expression of GcsA in P. putida.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Bacterial Proteins; Conserved Sequence; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Promoter Regions, Genetic; Pseudomonas putida; Second Messenger Systems

The effects of the Pseudomonas aeruginosa virulence factor pyocyanin (PCN) on the contractile function of porcine coronary arteries was investigated in vitro. Artery rings (5 mm) were suspended in organ baths containing Krebs' solution for the measurement of isometric tension. The effect of PCN on resting and precontracted coronary arteries was initially investigated with various agents. Arteries were precontracted with prostaglandin (PG) F2α or potassium chloride and endothelium-dependent relaxations were induced by various agents in the presence of PCN. Pyocyanin (0.1-10 μmol/L) evoked small-amplitude, dose-dependent contractions in resting porcine coronary arteries. In addition, PCN amplified the contractile response to PGF2α , but did not alter responses to carbachol. Pyocyanin (0.1-10 μmol/L) significantly inhibited endothelium-dependent relaxations evoked by neurokinin A. Pyocyanin also inhibited relaxations evoked by diethylamine nitric oxide (a nitric oxide donor), forskolin (an adenylate cyclase activator), dibuytyryl-cAMP (a cAMP analogue), 8-bromo-cGMP (a cGMP analogue) and P1075 (a KATP channel activator), but not isoprenaline (β-adrenoceceptor agonist). These results indicate that physiological concentrations of PCN interfere with multiple intracellular processes involved in vascular smooth muscle relaxation, in particular pathways downstream of nitric oxide release. Thus, PCN may alter normal vascular function in patients infected with P. aeruginosa.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Colforsin; Coronary Vessels; Cyclic AMP; Cyclic GMP; Diethylamines; Dinoprost; Female; Isoproterenol; Male; Muscle Contraction; Muscle Relaxation; Nitric Oxide; Pseudomonas aeruginosa; Pyocyanine; Swine; Vasodilation

During capacitation of mammalian sperm intracellular [Ca(2+)] and cyclic nucleotides increase, suggesting that CNG channels play a role in the physiology of sperm. Here we study the effect of capacitation, 8Br-cAMP (8-bromoadenosine 3',5'-cyclic monophosphate) and 8Br-cGMP (8-bromoguanosine 3',5'-cyclic monophosphate) on the macroscopic ionic currents of mouse sperm, finding the existence of different populations of sperm, in terms of the recorded current and its response to cyclic nucleotides. Our results show that capacitation and cyclic nucleotides increase the ionic current, having a differential sensitivity to cGMP (cyclic guanosine monophosphate) and cAMP (cyclic adenosine monophosphate). Using a specific inhibitor we determine the contribution of CNG channels to macroscopic current and capacitation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Dose-Response Relationship, Drug; Drug Resistance; Male; Membrane Potentials; Mice; Sperm Capacitation; Spermatozoa

Effects of cyclic nucleotides 8-Bromo-cAMP and 8-Bromo-cGMP (membrane permeable analogs of cAMP and cGMP) were examined on action potential (AP) configuration and rate of spontaneous firing of the freshly isolated 3-day embryonic chick ventricle (ECV) to assess the role of L-type slow Ca2+ channels in upstroke of AP and spontaneous electrical activity (pacemaker potential). The 3-day ECV exhibited prominent automaticity and spontaneous APs characterized by maximum upstroke velocity (+V(max)), maximum diastolic potential (MDP), overshoot (E(ov)), AP duration at -20 mV (APD20) and cycle length (CL) of 33.09 +/- 3.18 V/sec, -63.77 +/- 1.17 mV, 17.40 +/- 0.91 mV, 51.20 +/- 3.05 m sec and 795 +/- 150 m sec, respectively (n = 10 preparations). 8-Br-cAMP (1 mM) caused significant increase in E(ov) and APD20 (37% and 56%, respectively, p < 0.01), but failed to produce any stimulatory effect on +V(max) and MDP. Surprisingly, 8-Br-cAMP produced negative chronotropic effect on spontaneous firing (automaticity) and enhanced the CL significantly by 43% (p < 0.05). 8-Br-cGMP, however, had no effect on AP configuration and the rate of spontaneous firing. The present findings with 8-Br-cAMP suggest that L-type slow Ca2+ channels do not contribute to upstroke of AP and pacemaker potential of spontaneously firing freshly isolated 3-day ECV. The negative chronotropic effect of 8-Br-cAMP suggests that the ionic mechanism underlying pacemaker potential is [Ca]i-dependent. However, the lack of any effect of 8-Br-cGMP on spontaneous electrical activity of freshly isolated 3-day ECV indicates that cGMP does not modulate the basal Ca2+ channel activity in young embryonic myocardium.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Action Potentials; Animals; Calcium; Calcium Channels, L-Type; Chick Embryo; Cyclic GMP; Heart Ventricles; Ventricular Function