Measurement Of Cyclic Nucleotides Research Articles

Identification of bacterial guanylate cyclases.

The ability of bacteria to use cGMP as a second messenger has been controversial for decades. Recently, nucleotide cyclases from Rhodospirillum centenum, GcyA, and Xanthomonas campestris, GuaX, have been shown to possess guanylate cyclase activities. Enzymatic activities of these guanylate cyclases measured in vitro were low, which makes interpretation of the assays ambiguous. Protein sequence analysis at present is insufficient to distinguish between bacterial adenylate and guanylate cyclases, both of which belong to nucleotide cyclases of type III. We developed a simple method for discriminating between guanylate and adenylate cyclase activities in a physiologically relevant bacterial system. The method relies on the use of a mutant cAMP receptor protein, CRPG, constructed here. While wild-type CRP is activated exclusively by cAMP,_CRPG can be activated by either cAMP or cGMP. Using CRP- and CRPG-dependent lacZ expression in two E. coli strains, we verified that R. centenum GcyA and X. campestris GuaX have primarily guanylate cyclase activities. Among two other bacterial nucleotide cyclases tested, one, GuaA from Azospillrillum sp. B510, proved to have guanylate cyclase activity, while the other one, Bradyrhizobium japonicum CyaA, turned out to function as an adenylate cyclase. The results obtained with this reporter system were in excellent agreement with direct measurements of cyclic nucleotides secreted by E. coli expressing nucleotide cyclase genes. The simple genetic screen developed here is expected to facilitate identification of bacterial guanylate cyclases and engineering of guanylate cyclases with desired properties.

Quantitation of Cyclic Nucleotides in Biological Samples by Negative Electrospray Tandem Mass Spectrometry Coupled to Ion Suppression Liquid Chromatography

Measurement of cyclic nucleotides by electrospray ionization mass spectrometry constitutes an unequivocal quantitative and qualitative technique superior to the established estimation methods of UV spectroscopy, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and other mass spectrometric ionization techniques such as electron impact, thermospray and fast-atom bombardment. Optimal conditions for negative-ion electrospray tandem mass spectrometry showed a linear detection range using solutions with a concentration ranging from 10−6 and 5×10−9 M with an absolute detection limit of 100 fmol injected. Measurement of diagnostic product ions derived from low-energy collision-activated dissociation of the [M−H]− ions provided unambiguous identification. Quantitation of cyclic nucleotides present in biological samples in the femtomole region, needed further purification using on-line ion suppression high-performance liquid chromatography in order to avoid reduced sensitivity caused by matrix quenching.

Factors involved in the relaxation of female pig urethra evoked by electrical field stimulation.

1. Non-adrenergic, non-cholinergic (NANC) relaxations induced by electrical field stimulation (EFS) were studied in pig isolated urethra. The mechanism for relaxation was characterized by measurement of cyclic nucleotides and by study of involvement of different subsets of voltage-operated calcium channels (VOCCs). 2. EFS evoked frequency-dependent and tetrodotoxin-sensitive relaxations in the presence of propranolol (1 microM), phentolamine (1 microM) and scopolamine (1 microM). At low frequencies (< 12 Hz), relaxations were rapid, whereas at high (> 12 Hz) frequencies distinct biphasic relaxations were evoked. The latter consisted of a rapidly developing first phase followed by a more long-lasting second phase. 3. Treatment with the NO-synthesis inhibitor NG-nitro-L-arginine (L-NOARG; 0.3 mM) inhibited relaxations at low frequencies of stimulation. At high frequencies (> 12 Hz) only the first relaxation phase was affected. 4. Measurement of cyclic nucleotides in preparations subjected to continuous nerve-stimulation, revealed an increase in guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels from 1.3 +/- 0.3 to 3.0 +/- 0.4 pmol mg-1 protein (P < 0.01). In the presence of L-NOARG, there was a significant decrease in cyclic GMP content to control. However, there was no increase in cyclic GMP content in response to EFS. Levels of cyclic AMP remained unchanged following EFS. 5. Treatment with the N-type VOCC-inhibitor, omega-conotoxin GVIA (0.1 microM) reduced NO-dependent relaxations, the effect being most pronounced at low frequencies (1-4 Hz) of stimulation. The NO independent second phase of the relaxation, studied in the presence of L-NOARG (0.3 mM) at 16-30 Hz, was however markedly reduced or abolished by omega-conotoxin GVIA. omega-Conotoxin MVIIC (1 microM)or omega-agatoxin IVA (30 nM) had no effect on electrically evoked relaxations.6. These results suggest that NANC-nerve derived urethral relaxation in the pig consists of two apparently independent components. One is mediated by NO and associated with an increase in cyclic GMP content. The other mediator is unknown and produces relaxations not associated with changes in levels of cyclic nucleotides. The release of this mediator seems to involve the N-type VOCC, since the relaxation was markedly reduced or abolished by omega-conotoxin GVIA.

Cyclic AMP-dependent up-regulation of the taurine transporter in a human retinal pigment epithelial cell line.

This investigation was undertaken to study the role of cAMP in the regulation of the taurine transporter expressed in a human retinal pigment epithelial (HRPE) cell line. Treatment of the HRPE cells with cholera toxin for 24 h was found to stimulate the taurine transporter activity, as measured by taurine transport into the cells in the presence of NaCl, to a significant extent. The stimulation was 50-60% at 100 ng/ml cholera toxin. This stimulation was specific to the taurine transporter since the transport of two other amino acids (leucine and alanine), which are not substrates for the taurine transporter, was not affected by cholera toxin under similar conditions. Exposure of the cells to cholera toxin for a time period > 4 h was needed to elicit the stimulatory effect. The cholera toxin-induced stimulation of the taurine transporter activity was associated with an increase in the maximal velocity of the transport system. The affinity of the transporter for taurine was not altered by the treatment. The stimulatory effect was markedly blunted when the treatment of the cells with cholera toxin was done in the presence of actinomycin D, an inhibitor of transcription, or cycloheximide, an inhibitor of translation. The increase in the taurine transporter activity induced by cholera toxin was associated with a 2.6-fold increase in the steady state levels of the transporter mRNA. Measurement of cyclic nucleotides in control and cholera toxin-treated cells revealed that the toxin caused a 20-fold increase in the cellular levels of cAMP, the levels of cGMP remaining unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of cyclic nucleotides in amniotic fluid

Measurement of cyclic nucleotides in amniotic fluid

14] Microdissection of retina and measurement of cyclic nucleotides

This chapter discusses the microdissection of retina and measurement of cyclic nucleotides. It reveals that guanosine monophosphate (cyclic GMP) and adenosine monophosphate (cyclic AMP) have been shown to be present in all vertebrate retinas studied. In those animals examined, cyclic AMP is fairly uniform in distribution throughout the retina, whereas cyclic GMP is concentrated in the photoreceptor cells, with the highest levels observed in the outer segments. Tissue samples are obtained by removing eyes immediately after decapitation and freezing them in liquid N 2 , which has been chilled to its freezing point by rapid evaporation with a vacuum pump. Keeping N 2 below its boiling point prevents violent bubbling when the tissue is immersed, which would delay freezing. Dissection and weighing of the tissue samples are done in a temperature and humidity-controlled room. Depending on the concentration of cyclic nucleotide, one of two methods of sample preparation can be used. Cyclic AMP in all retinal layers and cyclic GMP in layers containing photoreceptor cells are sufficiently concentrated so that samples in a range of 50–500 ng dry weight may be used.

Cyclic nucleotides in the cone-dominant ground squirrel retina

Dark-adapted cone-dominant ground squirrel retina has an average concentration of guanosine 3′,5′-monophosphate (cyclic GMP) of 9·0 μmol/kg dry wt. The average concentration of adenosine 3′,5′-monophosphate (cyclic AMP) is 6·6 μmol/kg dry wt. Measurement of cyclic nucleotides in individual layers of the ground squirrel retina demonstrates that the highest concentrations of cyclic GMP are found in layers containing photoreceptor cells. These levels are 10–40 times those found in the inner portions of the retina. Cyclic AMP, on the other hand, is more evenly distributed and similar to that found in other regions of the CNS. The levels of cyclic GMP in the outer segment layer of the ground squirrel are 10 times that of cyclic AMP. In contrast, in the inner retinal layers cyclic AMP concentrations are 7–8 times greater than those of cyclic GMP. Light-adaptation does not influence the average levels of either cyclic GMP or cyclic AMP or their concentrations in any layer of the ground squirrel retina. These data indicate that cyclic GMP is the predominant cyclic nucleotide in the cone photoreceptor and that its distribution is similar to the reported previously for roddominant retinas. The failure of long periods of light-adaptation to modify the levels of either cyclic GMP or cyclic AMP in the ground squirrel retina indicates either a fundamental difference in the way these cyclic nucleotides are metabolized in cone-dominant retinas, or simply a quantitative difference in the rate at which their concentrations are altered.

Experimental canine anaphylaxis: cyclic nucleotides, histamine, and lung function

We studied the effects of Ascaris suum antigen (iv) in 32 natively allergic, anesthetized dogs. After thoracotomy, bilateral cervical vagotomy, and propranolol, samples of peripheral lung frozen in situ were obtained for measurement of cyclic nucleotides and histamine. Following Ascaris, lung histamine decreased 20.4 +/- 3.7% (mean +/- SE), cAMP increased 391 +/- 122%, and cGMP increased 110 +/- 20% with increased plasma histamine and physiological changes of anaphylaxis. No significant changes occurred in 10 control dogs. Release of histamine, reflecting immunological degranulation of mast cells, correlated closely with the physiological effects of anaphylaxis. beta-Adrenergic stimulation with isoproterenol prevented these physiological effects of anaphylaxis in three dogs studied. Furthermore, the level of cAMP induced in lung tissue by beta-adrenergic stimulation in these dogs correlated with the degree of inhibition of immunologically induced histamine release. These results illustrate the suitability of this experimental preparation to study the biochemical and physiological mechanisms of anaphylaxis in vivo.

Distribution of Cyclic Nucleotides in the Organ of Corti

Recent developments in radioimmunoassay technology have made possible measurements of cyclic nucleotides in individual specimens of the organ of Corti and its subdivisions. Steep longitudinal and transverse gradients of glycogen are known to exist in the organ of Corti of the guinea pig, with preferential accumulation in the outer hair cells of the apical turns. However, no significant longitudinal gradient of cyclic AMP was detectable in the organ of Corti, and the concentration of the compound was found to be nearly equal in the inner and outer hair cell layers. This is indirect evidence against the concept that cyclic AMP plays a role as "second messenger" in the control of glycogen metabolism of the organ of Corti. By contrast, the concentration of cyclic GMP was found to be consistently higher in the inner layer than in the outer layer of the organ of Corti, and to increase significantly in basal direction. This trend is remarkably similar to the distribution patterns of acetylcholinesterase, which may be considered as indirect evidence in favor of a possible role of cyclic GMP in the mediation of cholinergic effects.

Measurement of cyclic nucleotides in histologically defined samples of brain and retina

Abstract A method is described which allows measurement of cyclic AMP and cyclic GMP in histologically defined tissue samples containing as little as 0.6 fmol of either compound. This permits, for example, assays of freeze-dried samples as small as 0.03 μg dry weight from individual retina layers or 4-μg samples from individual layers of cerebellum. The procedure involves a modified radioimmunoassay at reduced volume for acetylated cyclic nucleotides in combination with some of the techniques previously developed for quantitative histochemical assays.

An examination of baseline and drug-induced levels of cyclic nucleotides in the cerebrospinal fluid of control and psychiatric patients

Baseline c-AMP levels, and probenecid-induced accumulations of c-AMP and c-GMP in the lumbar CSF of depressed, manic, and schizophrenic patients failed to show differences when compared to controls screened to exclude affective, schizophrenic, and CNS neurological disorders. Cyclic-GMP baseline levels in all three psychiatric groups did approach a level of significance when compared to controls. The administration of psychotropic drugs to these patients failed to show significant differences in paired comparisons. Levels of c-GMP from lumbar CSF were found to be positively correlated to concentrations of c-AMP. Categorization of data according to sex, and then age did not result in a discernible trend. It seems unlikely on the basis of this investigation that measurement of cyclic nucleotides in lumbar CSF will be of significance in differentiating psychiatric disorders.