[7] Purification and assay of cAMP, cGMP, and cyclic nucleotide analogs in cells treated with cyclic nucleotide analogs

doi:10.1016/0076-6879(88)59009-2

Methods in Enzymology

Volume 159, 1988, Pages 74-82

https://doi.org/10.1016/0076-6879(88)59009-2 Get rights and content

Publisher Summary

To determine cAMP, cGMP, or cyclic nucleotide analog levels, this chapter develops a method to first separate the analog(s) from cAMP or cGMP by a simple chromatographic procedure followed by functionally directed assay of the respective cyclic nucleotide by either cAMP- or cGMP-dependent protein kinase activation. The sensitivity is much better than that of the cyclic nucleotide binding assays, and approaches that of the radioimmunoassays. Because high concentrations of cyclic nucleotide analogs are usually used for intact tissue incubations, trace cyclic nucleotide contaminants in commercial cyclic nucleotides may not be separated during purification and would thus interfere with the assay for the nucleotide of interest. It is therefore necessary to purify the analogs before use. Alternatively, sensitive and specific cAMP- and cGMP-dependent protein kinase activation assays are described in the chapter.

References (16)

J.P. Miller et al.
Biochem. Pharmacol.
(1981)
B.E. Kemp et al.
J. Biol. Chem.
(1977)
D.B. Glass et al.
J. Biol. Chem.
(1982)
T.F. Walseth et al.
Biochim. Biophys. Acta
(1979)
M. Bradford
Anal. Biochem.
(1976)
S.J. Beebe et al.
J. Biol. Chem.
(1984)
T.M. Lincoln et al.
J. Biol. Chem.
(1978)
J.D. Corbin et al.
J. Biol. Chem.
(1986)

There are more references available in the full text version of this article.

Cited by (29)

Expression and distribution of cyclic GMP-dependent protein kinase-1 isoforms in human penile erectile tissue
2008, Journal of Sexual Medicine
Citation Excerpt :
To verify the localization of cGKI within the smooth musculature, double stainings using the monoclonal smooth muscle α-actin antibody (A5228, Sigma Chemical Co., St. Louis, MO, USA; 1:1000) [25] were carried out. Moreover, an anti-cGMP rabbit polyclonal antibody (Calbiochem, 1:500) [26] and the nitric oxide synthase III (eNOS) polyclonal antibody (Chemicon International, Temecula, CA, USA; 1:500) were used in order to show the colocalization of cGKI and its effector cGMP or cGKI and eNOS, respectively. The following Alexa Fluor (Molecular Probes Europe BV) secondary antibodies were used: #A11001, #A11008, #A11011, #A11055, and #A11057.
Besides the bioavailability of nitric oxide (NO), downstream guanine monophosphate (cGMP) effector proteins are also considered to play a significant role in penile vascular disease. In animal studies, a downregulation of the cGMP-dependent protein kinase-1 (cGKI) α isoform has been linked to erectile dysfunction and diabetes mellitus. So far, the expression of cGKI α and β isoforms has not been evaluated in human penile erectile tissue.
To evaluate the expression of cGKI α and β isoforms in relation to smooth muscle α-actin, cGMP, and endothelial NO synthase (eNOS) in human cavernous arteries (HCAs) and human corpus cavernosum (HCC).
Cryostat sections of HCA and HCC were incubated with primary antibodies directed against α-actin, cGMP, eNOS, cGKI, cGKI α, and cGKI β. Visualization of double-labeled immunofluorescent stainings was achieved by laser microscopy. Western blot analysis was performed in order to confirm the expression of cGKI isoforms.
Expression of cGKI α and β isoforms in relation to smooth muscle α-actin, cGMP, and eNOS in human penile erectile tissue.
Immunoreactivities specific for cGKI, cGKI α, and cGKI β were observed within the smooth musculature and the endothelium of cavernous arteries and sinusoids. Double stainings revealed the colocalization of alpha-actin, cGMP, eNOS, and cGKI isoforms. The expression of cGKI isoforms was confirmed by Western blot analysis.
Our results demonstrate, for the first time, the expression of both cGKI α and β isoforms in the smooth musculature of HCA and HCC. Corresponding to recent findings from animal studies, the presence of cGKI α and β provides further evidence for a significant role of these enzymes in the control of smooth muscle function in human penile erectile tissue. Waldkirch E, Uckert S, Sigl K, Imkamp F, Langnaese K, Richter K, Jonas U, Sohn M, Stief C, Wolf G, and Hedlund P. Expression and distribution of cyclic GMP-dependent protein kinase-1 isoforms in human penile erectile tissue
Immunohistochemical Distribution of Cyclic GMP-Dependent Protein Kinase-1 in Human Prostate Tissue
2007, European Urology
Citation Excerpt :
To verify the localization of cGKI within the smooth musculature, double-staining using the monoclonal smooth muscle α-actin antibody (A5228, Sigma Chemical Co., St. Louis, MO, USA; 1:1000) [16] was carried out. In addition, an anti-cGMP rabbit polyclonal antibody (Calbiochem; 1:500) [17] was used to show the colocalization of cGKI and its effector protein cGMP. Fresh frozen tissue samples of the transition zone of human prostate tissue were sectioned into 20-μm slices and collected in Eppendorf tubes.
Phosphodiesterase 5 (PDE5) inhibitors improve smooth muscle relaxation and therefore are considered for pharmacotherapy of benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS). Cyclic guanosine monophosphate (cGMP)-dependent protein kinase-1 (cGKI) has been identified as one of the downstream targets for cGMP. The aim of the present study was to evaluate, by means of immunohistochemistry and Western blot analysis, the expression and localization of cGKI isoforms in relation to smooth muscle α-actin and cGMP in the human prostate.
Cryostat sections of tissue segments excised from the transition zone of human prostates from 11 patients (aged 54–68 yr) were incubated with primary antibodies directed against smooth muscle α-actin, cGMP, cGKI, cGKIα, and cGKIβ. Visualization of double-labelled immunofluorescent staining was achieved by laser microscopy. Western blot analysis was performed to confirm the expression of cGKI isoforms.
Immunoreactivities specific for cGKI, cGKIα, and cGKIβ were observed in the smooth musculature of the transition zone. Double-staining revealed the colocalization of smooth muscle α-actin, cGMP, and cGKI isoforms in smooth muscle cells of the fibromuscular stroma. The expression of cGKI isoforms was confirmed by Western blot analysis.
Our results confirm the presence of cGKI isoforms α and β in the transition zone of human prostate tissue. In addition, the colocalization of α-actin, cGMP, and cGKI isoforms provides further evidence for a significant role of the nitric oxide/cGMP pathway in the regulation of smooth muscle contractility in human prostate tissue and therefore could provide additional targets for pharmacotherapy of BPH and LUTS.
Ethanol stimulates ciliary beating by dual cyclic nucleotide kinase activation in bovine bronchial epithelial cells
2003, American Journal of Pathology
Citation Excerpt :
The papers were then washed five times manually for 1 minute each, rinsed 1 minute in EtOH, dried, and counted in nonaqueous scintillant.38 The assay for cGMP levels was performed similarly to that for cAMP.36 The PKG used was partially purified from bovine lung as previously described.39
Previously, we have shown that ethanol (EtOH) stimulates a rapid increase in the ciliary beat frequency (CBF) of bovine bronchial epithelial cells (BBECs) via the activation of PKA. We have also shown that inhibitors of nitric oxide synthase block EtOH-stimulated increases in CBF. We hypothesize that EtOH acutely stimulates CBF via the activation of both PKA and PKG pathways. Using chemiluminescence detection of nitric oxide (NO), we directly measured increases in NO production in BBECs treated with 100 mmol/L of EtOH beginning at 25 minutes. Pretreatment of BBECs with guanylyl cyclase inhibitors, ODQ or LY83583, resulted in the inhibition of EtOH-stimulated CBF. Low concentrations (1 nmol/L) of cyclic nucleotide analogues do not stimulate CBF increases. However, a combination of both 1 nmol/L of 8Br-cAMP and 8Br-cGMP stimulates a significant increase over baseline CBF. This effect could be blocked by pretreating BBECs with inhibitors of either PKA or PKG. Very high concentrations of either 8Br-cAMP or 8Br-cGMP (≥100 μmol/L) were required to cross-activate both PKA and PKG. This suggests that cross-activation of PKA by cGMP is not occurring at the concentrations (1 nmol/L) capable of stimulating CBF. 8-pCPT-cGMPS, an antagonist analogue to cGMP, blocked EtOH-stimulated PKA activity increases. These data support that EtOH-stimulated increases in CBF require the dual activation of both PKA (via cAMP) and PKG (via NO).
A conserved serine juxtaposed to the pseudosubstrate site of type I cGMP-dependent protein kinase contributes strongly to autoinhibition and lower cGMP affinity
2002, Journal of Biological Chemistry
Citation Excerpt :
This value was within 1.5–2-fold of the value obtained from the cGMP kinase activation studies (Table I). As expected, the WT and S64A cGMP binding affinities were greater at 4 °C (34). Interestingly, the KD of either S64D or S64N was similar at 30 and 4 °C (2.0 ± 0.3 and 2.1 ± 0.4 nm; 1.7 ± 0.5 and 2.6 ± 1.1 nm, respectively).
Serines 64 and 79 are homologous residues that are juxtaposed to the autoinhibitory pseudosubstrate site in cGMP-dependent protein kinase type Iα and type Iβ (PKG-Iα and PKG-Iβ), respectively. Autophosphorylation of this residue is associated with activation of type I PKGs. To determine the role of this conserved serine, point mutations have been made in PKG-Iα (S64A, S64T, S64D, and S64N) and PKG-Iβ (S79A). In wild-type PKG-Iα, basal kinase activity ratio (−cGMP/+cGMP) is 0.11, autophosphorylation increases this ratio 3-fold, and theK_a and K_D values for cGMP are 127 and 36 nm, respectively. S64A PKG-Iα basal kinase activity ratio increases 2-fold, cGMP binding affinity increases ∼10-fold in both K_a and K_D, and activation by autophosphorylation is slight. S64D and S64N mutants are nearly constitutively active in the absence of cGMP, cGMP binding affinity in each increases 18-fold, and autophosphorylation does not affect the kinase activity of these mutants. Mutation of the homologous site in PKG-Iβ (S79A) increases the basal kinase activity ratio 2-fold and cGMP binding affinity 5-fold over that of wild-type PKG-Iβ. The combined results demonstrate that a conserved serine juxtaposed to the pseudosubstrate site in type I PKGs contributes importantly to enzyme function by increasing autoinhibition and decreasing cGMP binding affinity.
In vitro PKA phosphorylation-mediated human PDE4A4 activation
2002, FEBS Letters
The PDE4 catalytic machinery comprises, in part, two divalent cations in a binuclear motif. Here we report that PDE4A4 expressed in Sf9 cells exhibits a biphasic Mg²⁺ dose–response (EC₅₀ of ∼0.15 and >10 mM) in catalyzing cAMP hydrolysis. In vitro phosphorylation of PDE4A4 by the PKA-catalytic subunit increases the enzyme’s sensitivity to Mg²⁺, leading to 4-fold increased cAMP hydrolysis without affecting its K_m. The phosphorylation also increases the potencies of (R)- and (S)-rolipram without affecting CDP-840 and SB-207499. The results support that modulating the cofactor binding affinity of PDE4 represents a mechanism for regulating its activity.
Cyclic nucleotide phosphodiesterases in rabbit detrusor smooth muscle
2002, Urology
Objectives. To identify the phosphodiesterase (PDE) isoenzymes in the rabbit detrusor and to evaluate their roles in regulating detrusor muscular tone. Cyclic nucleotides are important secondary messengers involved in modulating the contractility of various smooth muscles. Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are synthesized by their respective cyclases and degraded by various PDEs.
Methods. PDE isoenzymes from male and female rabbit detrusor were isolated by the Mono-Q anion exchange column and identified with various inhibitors. Detrusor strips from both sexes were precontracted with carbachol and relaxed with PDE inhibitors and adenylate and guanylyl cyclase activators in a tissue bath. Cyclic nucleotide concentrations in strips from male rabbits were determined after the compound treatment.
Results. Similar results were obtained from both sexes in the experiments in which both sexes were used. The activities of PDE1, 2, 3, 4, and 5 were identified. Forskolin induced a dramatic rise in the cAMP levels and was the most effective relaxant. Papaverine generated moderate increases in the cAMP and cGMP levels and induced very good relaxation. Vinpocetine produced no detectable changes in the cyclic nucleotide levels but elicited good relaxation. Sildenafil caused an increase in the cGMP levels and had a similar relaxation effect as vinpocetine. Sodium nitroprusside induced some increase in cGMP and had no relaxation effect. Rolipram raised the cAMP levels significantly, yet had a moderate effect on relaxation.
Conclusions. Our results demonstrated the presence of PDE1 through 5 in rabbit detrusor muscle and supported their involvement in regulating detrusor muscle tone. The relaxation of rabbit detrusor was mainly mediated by the cAMP pathway.

View all citing articles on Scopus

View full text

[7] Purification and assay of cAMP, cGMP, and cyclic nucleotide analogs in cells treated with cyclic nucleotide analogs

Publisher Summary

Biochem. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.