Subunit Of cAMP-dependent Protein Kinase Research Articles

Evolution of PKA Signaling: Structure of Yeast Regulatory Subunit

cAMP is known to mediate, in both prokaryotes and eukaryotes, a wide variety of cellular responses to external stimuli. In eukaryotes, the major receptor for cAMP signaling is the regulatory subunit (R) of cAMP‐dependent protein kinase (PKA). In mammals there are two major classes of R subunits (I and II), however, Saccharomyces cerevisiae has only one R gene (Bcy1). As the yeast is far from mammals in the phylogenetic tree the study of this protein is important from the point of view of molecular evolution. Sequence analysis of the cyclic‐AMP binding domain (CNB) has shown that the fungal R subunits belong to a distinct group, yet exhibits some similarity to both mammalian RI and RII. In order to a molecular understanding of the mechanism of activation of PKA by cAMP in yeast and to provide insight into the evolution of cAMP receptor, we purified and crystalized a deletion mutant of Bcy1, Bcy1(168‐416) bound to cAMP and then compared it with the previous structures of mammalian RIα and RII ? cAMP‐bound isoforms. Surprisingly the relative orientation of the two CNB domains in Bcy1 is very different from RIα and RIIβ, although each domain superimposes very well. This structure serves as a hallmark for the yeast PKA. Closer examination of the structural differences between mammalian R subunits and Bcy1 identifies a hinge point in the kink between the B and C helices of the A domain that accounts for the major deviation in tertiary structure of the different R subunits. The similarity and differences of the interface between the two CNB domains in Bcy1 was also described and compared with the mammalian isoforms. (Supported in part by NIH GM34921 to SST)

Enzymatic characteristics of a Ser/Thr protein kinase, SpkA, from Myxococcus xanthus

Two Ser/Thr protein kinases, SpkA and SpkB, selected from Myxococcus xanthus based on amino acid sequence similarities with the catalytic subunits of cAMP-dependent protein kinases (PKA) were synthesized using a cell-free protein synthesis system. In various protein kinase assays, purified StkA and StkB showed their highest protein kinase activities in a PKA assay using the selective PKA substrate Kemptide and in a protein kinase C (PKC) assay using the selective PKC substrate neurogranin((28-43)), respectively. SpkA had apparent K(m) values of 45 microM and 37 microM for Kemptide and ATP, respectively. Phosphorylation of Kemptide was inhibited by a specific PKA inhibitor peptide, PKI(5-24), and the IC(50) and K(i) values for inhibition of the SpkA activity were 117 nM and 36 nM, respectively.

Structural Analysis of ARC-Type Inhibitor (ARC-1034) Binding to Protein Kinase A Catalytic Subunit and Rational Design of Bisubstrate Analogue Inhibitors of Basophilic Protein Kinases

The crystal structure of a complex of the catalytic subunit (type alpha) of cAMP-dependent protein kinase (PKA C alpha) with ARC-type inhibitor (ARC-1034), the presumed lead scaffold of previously reported adenosine-oligo-arginine conjugate-based (ARC-type) inhibitors, was solved. Structural elements important for interaction with the kinase were established with specifically modified derivatives of the lead compound. On the basis of this knowledge, a new generation of inhibitors, conjugates of adenosine-4'-dehydroxymethyl-4'-carboxylic acid moiety and oligo(D-arginine), was developed with inhibitory constants well into the subnanomolar range. The structural determinants of selectivity of the new compounds were established in assays with ROCK-II and PKBgamma.

Regulatory subunits of PKA define an axis of cellular proliferation/differentiation in ovarian cancer cells

BackgroundThe regulatory subunit of cAMP-dependent protein kinase (PKA) exists in two isoforms, RI and RII, which distinguish the PKA isozymes, type I (PKA-I) and type II (PKA-II). Evidence obtained from a variety of different experimental approaches has shown that the relative levels of type I and type II PKA in cells can play a major role in determining the balance between cell growth and differentiation. In order to characterize the effect of PKA type I and type II regulatory subunits on gene transcription at a global level, the PKA regulatory subunit genes for RIα and RIIβ were stably transfected into cells of the ovarian cancer cell line (OVCAR8).ResultsRIα transfected cells exhibit hyper-proliferative growth and RIIβ transfected cells revert to a relatively quiescent state. Profiling by microarray revealed equally profound changes in gene expression between RIα, RIIβ, and parental OVCAR cells. Genes specifically up-regulated in RIα cells were highly enriched for pathways involved in cell growth while genes up-regulated in RIIβ cells were enriched for pathways involved in differentiation. A large group of genes (~3600) was regulated along an axis of proliferation/differentiation between RIα, parental, and RIIβ cells. RIα/wt and RIIβ/wt gene regulation was shown by two separate and distinct gene set analytical methods to be strongly cross-correlated with a generic model of cellular differentiation.ConclusionOverexpression of PKA regulatory subunits in an ovarian cancer cell line dramatically influences the cell phenotype. The proliferation phenotype is strongly correlated with recently identified clinical biomarkers predictive of poor prognosis in ovarian cancer suggesting a possible pivotal role for PKA regulation in disease progression.

Case report of familial Carney complex due to novel frameshift mutation c.597del C (p.Phe200LeufsX6) in PRKAR1A

Carney complex is an autosomal dominantly inherited disease characterized by skin pigmentation, myxoma, primary pigmented nodular adrenocortical disease (PPNAD), and acromegaly. However, only a few incidences of PPNAD combined with acromegaly are observed in patients. The type 1α regulatory subunit of cAMP-dependent protein kinase ( PRKAR1A) has been identified in patients as a causative gene for Carney complex by a positional cloning approach. Here, we report a female patient diagnosed with Cushing’s syndrome and a GH-producing pituitary adenoma without otherwise evident acromegaly that could be diagnosed only by specialized endocrinological tests. Based on family history of acromegaly (mother and sister) and the fact that the combination of both diseases is very rare, genetic diagnosis involving Carney complex was considered to be appropriate. The 10 exons and flanking regions of PRKAR1A were screened for mutations by direct DNA sequencing. The patient and her mother and sister were found to have the same, novel frameshift mutation resulting from a single base deletion in exon 6 coding cAMP-binding domain A, denoted c.597delC in PRKAR1A. This single base deletion generated an immature stop codon at the sixth codon (p.Phe200LeufsX6). Even family members with the same mutation can show distinct phenotypes, suggesting that Carney complex is a multifactorial disorder comprising various genetic and environmental factors. Genetic diagnosis makes it possible to prepare more effective therapeutic strategies for patients and gene carriers and to avoid unnecessary tests for non-carriers in the family of the patient.

Detection of somatic β‐catenin mutations in primary pigmented nodular adrenocortical disease (PPNAD)

Primary pigmented nodular adrenocortical disease (PPNAD) leads to Cushing syndrome (CS) and is often associated with Carney complex (CNC). Genetic alterations of the type 1-alpha regulatory subunit of cAMP-dependent protein kinase A (PRKAR1A) and phosphodiesterase 11A4 (PDE11A) genes have been found in PPNAD. Recent studies have demonstrated that beta-catenin mutations are frequent in adrenocortical adenomas and carcinomas and that the Wnt-signalling pathway is involved in PPNAD tumorigenesis. We hypothesized that adrenocortical adenomas that form in the context of PPNAD may harbour beta-catenin mutations. We studied 18 patients with CS secondary to PPNAD who were screened for germline PRKAR1A and PDE11A mutations. Tumor DNA was extracted from pigmented adrenocortical adenoma and nodular adrenal hyperplasia. Mutation analysis of exons 3 and 5 of beta-catenin was performed using polymerase chain reaction and direct sequencing. Sections from formalin-fixed, paraffin-embedded tumour samples were studied by immunohistochemistry with an antibody against beta-catenin. Nine patients were carrying germline PRKAR1A mutations and one patient had a PDE11A mutation. We found somatic beta-catenin mutations in 2 of 18 patients (11%). In both cases, the mutations occurred in relatively large adenomas that had formed in the background of PPNAD. Tumor DNA analysis revealed a heterozygous ACC-to-GCC missense mutation in codon 41 (T41A) and a TCT-to-CCT missense mutation in codon 45 (S45P) of exon 3 of the beta-catenin gene that was confirmed at the cDNA level. There were no alterations in the DNA of PPNAD-adjacent tissues and lymphocytes from the patients, indicating somatic events. Immunohistochemistry showed nuclear accumulation of beta-catenin in more than 90% of cells in adenomatous tissue whereas no nuclear immunoreactivity was detected in adjacent PPNAD nodular cells. Nuclear translocation of beta-catenin protein in the PPNAD adenoma suggests activation of the Wnt-beta-catenin pathway in PPNAD. We report, for the first time, beta-catenin mutations in adenomas associated with PPNAD, further implicating Wnt-beta-catenin signalling in tumorigenesis linked to bilateral adrenal hyperplasias.

Deciphering the Role of Disulfide Bonds in RIalpha

The dimeric regulatory subunits (R) of cAMP‐dependent protein kinase (PKA) are multidomain proteins with diverse functions. Other than inhibiting the catalytic subunit of PKA, one isoform (RIalpha) has been implicated in diseases. Though much is known about RIalpha, the role of the disulfide bond at the N‐terminus is poorly understood. Studies have shown that the cysteines involved in the disulfide bond contribute to its binding to A Kinase Anchoring Proteins (AKAPs) and may influence its localization to discrete sub‐cellular locations. One mutant of RIalpha, R74C, has been found in Carney complex patients. To date, the mechanism of action of the R74C mutation remains unknown. We determined if the disulfide bonding is altered in the R74C mutant and found a novel dimer. We are using mass spectrometry to analyze the disulfide bonding profile of this dimer. Additionally, studies are currently underway to see if there are changes in its binding to proteins, such as AKAPs, and whether there are changes in the sub‐cellular localization of this mutant.

A Deletion in the PRKARIA Gene is Associated with Carney Complex

Mutations of the PRKAR1A gene are an important cause of Carney complex (CC). The PRKAR1A gene encodes the type 1A regulatory subunit of cAMP-dependent protein kinase A. We have identified one mutation of PRKAR1A (553delG) in three members of the same family affected by CC. This mutation was not identified in six unaffected family members, 12 patients with sporadic cardiac myxoma and 100 non-related healthy individuals. The novel mutation (553delG) is predicted to produce a frameshift leading to a premature stop codon. RNA analysis in the index patient showed normal size transcripts in RT-PCR amplicons of several exons, but an overall tendency to lower amounts of transcripts in relation to GAPDH controls. In Western blot analyses only full-length protein was present without any evidence of truncated product. These data suggest that the mutant allele might be a null allele due to degradation of the mutant mRNA via nonsense-mediated decay.

Signaling through cAMP and cAMP-dependent protein kinase: Diverse strategies for drug design

The catalytic subunit of cAMP-dependent protein kinase has served as a prototype for the protein kinase superfamily for many years while structures of the cAMP-bound regulatory subunits have defined the conserved cyclic nucleotide binding (CNB) motif. It is only structures of the holoenzymes, however, that enable us to appreciate the molecular features of inhibition by the regulatory subunits as well as activation by cAMP. These structures reveal for the first time the remarkable malleability of the regulatory subunits and the CNB domains. At the same time, they allow us to appreciate that the catalytic subunit is not only a catalyst but also a scaffold that mediates a wide variety of protein:protein interactions. The holoenzyme structures also provide a new paradigm for designing isoform-specific activators and inhibitors of PKA. In addition to binding to the catalytic subunits, the regulatory subunits also use their N-terminal dimerization/docking domain to bind with high affinity to A Kinase Anchoring Proteins using an amphipathic helical motif. This targeting mechanism, which localizes PKA near to its protein substrates, is also a target for therapeutic intervention of PKA signaling.

R-subunit Isoform Specificity in Protein Kinase A: Distinct Features of Protein Interfaces in PKA Types I and II by Amide H/ 2H Exchange Mass Spectrometry

The two isoforms (RI and RII) of the regulatory (R) subunit of cAMP-dependent protein kinase or protein kinase A (PKA) are similar in sequence yet have different biochemical properties and physiological functions. To further understand the molecular basis for R-isoform-specificity, the interactions of the RIIβ isoform with the PKA catalytic (C) subunit were analyzed by amide H/ 2H exchange mass spectrometry to compare solvent accessibility of RIIβ and the C subunit in their free and complexed states. Direct mapping of the RIIβ-C interface revealed important differences between the intersubunit interfaces in the type I and type II holoenzyme complexes. These differences are seen in both the R-subunits as well as the C-subunit. Unlike the type I isoform, the type II isoform complexes require both cAMP-binding domains, and ATP is not obligatory for high affinity interactions with the C-subunit. Surprisingly, the C-subunit mediates distinct, overlapping surfaces of interaction with the two R-isoforms despite a strong homology in sequence and similarity in domain organization. Identification of a remote allosteric site on the C-subunit that is essential for interactions with RII, but not RI subunits, further highlights the considerable diversity in interfaces found in higher order protein complexes mediated by the C-subunit of PKA.

Regulation of the Na+/Ca2+ exchanger (NCX) in the murine embryonic heart

The Na+/Ca2+ exchanger (NCX) is involved in embryonic heart development and function demonstrated by the abnormal myofibrillar organization, defects in heartbeat, and early embryonic death of NCX-null embryos. It was therefore the aim of our study to identify key functional regulators of the embryonic NCX. NCX current (I(NCX)) density was measured as the Ni2+ (5 mM)-sensitive current applying the whole-cell patch-clamp technique in early (EDS, E10.5V) and late developmental stage (LDS, E16.5V) mouse ventricular cardiomyocytes. Compared to LDS, cardiomyocytes derived from EDS showed a significantly higher basal I(NCX) density for the I(NCX) forward (-120 mV: -4.1+/-1 pA/pF, n=15 versus -1.7+/-0.4, n=11, p<0.05) and reverse modes (+60 mV: 4.0+/-0.9 pA/pF, n=15 versus 1.8+/-0.4, n=11, p<0.05). There was 2-3-fold elevation of forward and reverse current in LDS on application of ATP-gamma-S (2 mM) together with forskolin (1 microM) as well as intracellular application of the catalytic subunit of cAMP-dependent protein kinase (cPKA, 200 U/mL), cAMP (200 microM), phorbol 12-myristate-13-acetate (PMA), a direct activator of protein kinase C (PKC), and 8-Br-cGMP, a membrane permeable analog of cGMP. The specific PKC inhibitor Ro 31-8220 significantly reduced I(NCX) by 70%. Co-application of 20 microM PKA inhibitor Fragment 14-22 (PKI), a specific inhibitor of PKA, and cAMP significantly reduced the exchanger activity by approx 60%. Despite these obvious effects in LDS we could not detect a significant impact of these compounds on I(NCX) in EDS-derived cardiomyocytes. Application of the alkaline phosphatase to test for constitutive phosphorylation of NCX did not affect I(NCX) density in LDS but led to an approx 80% reduction of I(NCX) in EDS. In EDS cardiomyocytes I(NCX) density is upregulated, at least in part by the high phosphorylation of the exchanger protein. At LDS, embryonic cardiomyocytes showed a strong increase of I(NCX) density upon stimulation by PKC- and PKA-dependent signalling pathways.

CK2-mediated phosphorylation of a type II regulatory subunit of cAMP-dependent protein kinase from the mollusk Mytilus galloprovincialis

Two isoforms of regulatory (R) subunit of cAMP-dependent protein kinase (PKA), named R myt1 and R myt2, were identified so far in the sea mussel Mytilus galloprovincialis. Out of them, only R myt2 was phosphorylated in vitro by casein kinase 2 (CK2) using GTP as phosphate donor. CK2 catalytic subunit (CK2α) itself was sufficient to phosphorylate R myt2, but phosphorylation was enhanced by the presence of the regulatory subunit CK2β. Even in the absence of CK2, R myt2 was phosphorylated to a certain extent when it was incubated with GTP. This basal phosphorylation was partially abolished by the known inhibitors apigenin and emodin, which suggests the presence of a residual amount of endogenous CK2 in the preparation of purified R subunit. CK2-mediated phosphorylation significantly decreases the ability of R myt2 to inhibit PKA catalytic (C) subunit activity in the absence of cAMP. On the other hand, the sequence of several peptides obtained from the tryptic digestion of R myt2 showed that mussel protein contains the signature sequence common to all PKA family members, within the “phosphate binding cassette” (PBC) A and B. Moreover, the degree of identity between the sequences of peptides from R myt2, as a whole, and those from type II R subunits was 68–75%, but the global identity percentage with type I R subunits was only about 30%, so that R myt2 can be classified as a type II R subunit.

The Drosophila DCO mutation suppresses age-related memory impairment without affecting lifespan

The study of age-related memory impairment (AMI) has been hindered by a lack of AMI-specific mutants. In a screen for such mutants in Drosophila melanogaster, we found that heterozygous mutations of DCO (DCO/+), which encodes the major catalytic subunit of cAMP-dependent protein kinase (PKA), delay AMI more than twofold without affecting lifespan or memory at early ages. AMI is restored when a DCO transgene is expressed in mushroom bodies, structures important for olfactory memory formation. Furthermore, increasing cAMP and PKA activity in mushroom bodies causes premature AMI, whereas reducing activity suppresses AMI. In Drosophila AMI consists of a specific reduction in memory dependent on the amnesiac (amn) gene. amn encodes putative neuropeptides that have been proposed to regulate cAMP levels in mushroom bodies. Notably, both the memory and AMI defects of amn mutants are restored in amn;DCO/+ double mutants, suggesting that AMI is caused by an age-related disruption of amn-dependent memory via PKA activity in mushroom bodies.

Comparative thermodynamic analysis of cyclic nucleotide binding to protein kinase A

We have investigated the thermodynamic parameters and binding of a regulatory subunit of cAMP-dependent protein kinase (PKA) to its natural low-molecular-weight ligand, cAMP, and analogues thereof. For analysis of this model system, we compared side-by-side isothermal titration calorimetry (ITC) with surface plasmon resonance (SPR). Both ITC and SPR analyses revealed that binding of the protein to cAMP or its analogues was enthalpically driven and characterised by similar free energy values (DeltaG=-9.4 to -10.7 kcal mol-1) for all interactions. Despite the similar affinities, binding of the cyclic nucleotides used here was characterised by significant differences in the contribution of entropy (-TDeltaS) and enthalpy (DeltaH) to DeltaG. The comparison of ITC and SPR data for one cAMP analogue further revealed deviations caused by the method. These equilibrium parameters could be complemented by thermodynamic data of the transition state (DeltaHnot equal, DeltaGnot equal, DeltaSnot equal) for both association and dissociation measured by SPR. This direct comparison of ITC and SPR highlights method-specific advantages and drawbacks for thermodynamic analyses of protein/ligand interactions.

Prostanoid receptors regulate the volume‐sensitive efflux of osmolytes from murine fibroblasts via a cyclic AMP‐dependent mechanism

The ability of prostanoid receptors to regulate the volume-dependent efflux of taurine from murine fibroblasts (L cells) via a cAMP-dependent mechanism has been examined. Incubation of L cells under hypoosmotic conditions resulted in a time-dependent efflux of taurine, the threshold of release occurring at 250 mOsM. Addition of prostaglandin E1 (PGE1) potently (EC50 = 2.5 nM) enhanced the efflux of taurine and increased the threshold for osmolyte release to 290 mOsM. Inclusion of agents known to inhibit volume-sensitive organic osmolyte and anion channels blocked the ability of PGE1 to enhance taurine release. The ability of PGE1 to increase osmolyte release from L cells was mimicked by the addition of agents that inhibit cAMP breakdown, directly activate adenylyl cyclase, or are cell-permeant analogs of cAMP. Taurine release elicited by either PGE1 or 8-(4-chlorophenylthio)-cAMP was attenuated by >70% in L cells that had been stably transfected with a mutant regulatory subunit of cAMP-dependent protein kinase (PKA). PGE1 stimulation of taurine efflux was not attenuated by either depletion of intracellular calcium or inhibition of protein kinase C. The results indicate that activation of prostanoid receptors on murine fibroblasts enhances osmolyte release via a cAMP and PKA-dependent mechanism. Supported by NIH NS23831(SKF) and NIH Training Grant GM 07767(DJF)

Surface-plasmon-resonance-based biosensor with immobilized bisubstrate analog inhibitor for the determination of affinities of ATP- and protein-competitive ligands of cAMP-dependent protein kinase

Interactions between adenosine–oligoarginine conjugates (ARC), bisubstrate analog inhibitors of protein kinases, and catalytic subunits of cAMP-dependent protein kinase (cAPK Cα) were characterized with surface-plasmon-resonance-based biosensors. ARC-704 bound to the immobilized kinase with subnanomolar affinity. The immobilization of ARC-704 to the chip surface via streptavidin–biotin complex yielded a high-affinity surface ( K D = 16 nM). The bisubstrate character of ARC-704 was demonstrated with various ligands targeted to ATP-binding pocket (ATP and inhibitors H89 and H1152P) and protein-substrate-binding domain of Cα (RIIα and GST–PKIα) in competition assays. The experiments performed on surfaces with different immobilization levels of ARC-704 produced similar results. The closeness of the obtained affinities of the tested compounds to the inhibitory potencies and affinities of the compounds measured with other methods demonstrates the applicability of the chip with the immobilized biligand inhibitor for the characterization of both ATP- and substrate protein-competitive ligands of basophilic protein kinases.

Role of phosphorylated Thr-197 in the catalytic subunit of cAMP-dependent protein kinase

Protein phosphorylation of Thr-197 in the activation loop of the catalytic subunit (C-subunit) of cAMP-dependent protein kinase (PKA) is an essential step for its proper biological function. In order to explore the influences triggered by phosphorylation of Thr-197, comparative molecular dynamics (MD) simulation studies with or without phosphate group on Thr-197 and Ser-338 were performed on the complex of C-subunit bound to ATP and two Mg 2+ ions (C/Mg 2ATP complex) and on the complex of C-subunit bound to substrate peptide (C/substrate complex). The results present a decreased flexibility of the activation loop in the phosphorylated state, because of several critical interactions formed by the phosphorylated Thr-197 with His-87, Arg-165, Lys-189 and Thr-195. The unphosphorylated activation loop does not block the substrate-binding site, which is in good agreement with experimental result. In unphosphorylated state, the crucial interaction between Thr-197 and His-87 of C-helix does not exist, which eventually makes glycine-rich loop run away from its original position and displaces the phosphates group of ATP. We suggest that the reduction in rate of phosphoryl transfer may be caused by the displacement of γ-phosphate group that demolish phosphoryl transfer in-line mechanism.

A purine at +2 rather than +1 adjacent to the human U6 promoter is required to prepare effective short hairpin RNAs

The human U6 (hU6) promoter is widely used to express short hairpin RNAs (shRNAs) in mammalian cells. To verify the validity of the generalized concept—the hU6 promoter essentially requires a purine (usually guanine) at +1 for transcription, we enzymatically constructed an arbitrary shRNA library with the following features: (1) to have any one of adenine, cytosine, guanine, and thymine at the site; (2) to comprise shRNAs of 25–30 nucleotides in stem length which are transcribed through the promoter. cDNA of the catalytic subunit of cAMP-dependent protein kinase (PKACα) was used as material for library construction. We then used luciferase reporter cell lines to screen shRNAs which effectively reduced PKACα activity. Consequently, a purine was mostly present at +2, not at +1, of the clones isolated, suggesting that a purine at +2 rather than +1 adjacent to the hU6 promoter provides effective shRNAs for target gene silencing.

Phosphorylation of SPICK2, an AKT2 channel homologue from Samanea motor cells

SPICK2, a homologue of the weakly-inward-rectifying Shaker-like Arabidopsis K channel, AKT2, is a candidate K+-influx channel participating in light- and clock-regulated leaf movements of the legume, Samanea saman. Light and the biological clock regulate the in situ K+-influx channel activity differentially in extensor and flexor halves of the pulvinus (the S. saman leaf motor organ), and also-though differently-the transcript level of SPICK2 in the pulvinus. This disparity between the in situ channel activity versus its candidate transcript, along with the sequence analysis of SPICK2, suggest an in situ regulation of the activity of SPICK2, possibly by phosphorylation and/or by interaction with cAMP. Consistent with this (i) the activity of the voltage-dependent K+-selective fraction of the inward current in extensor and flexor cells was affected differentially in whole-cell patch-clamp assays promoting phosphorylation (using the protein phosphatase inhibitor okadaic acid); (ii) several proteins in isolated plasma membrane-enriched vesicles of the motor cells underwent phosphorylation without an added kinase in conditions similar to patch-clamp; and (iii) the SPICK2 protein was phosphorylated in vitro by the catalytic subunit of the broad-range cAMP-dependent protein kinase. All of these results are consistent with the notion that SPICK2 is the K+-influx channel, and is regulated in vivo directly by phosphorylation.

Prostanoid Receptors Regulate the Volume-Sensitive Efflux of Osmolytes from Murine Fibroblasts via a Cyclic AMP-Dependent Mechanism

The ability of prostanoid receptors to regulate the volume-dependent efflux of the organic osmolyte taurine from murine fibroblasts (L cells) via a cAMP-dependent mechanism has been examined. Incubation of L cells under hypoosmotic conditions resulted in a time-dependent efflux of taurine, the threshold of release occurring at 250 mOsM. Addition of prostaglandin E(1) (PGE(1)) potently (EC(50) = 2.5 nM) enhanced the volume-dependent efflux of taurine at all time points examined and increased the threshold for osmolyte release to 290 mOsM. Maximal PGE(1) stimulation (250-300% of basal) of taurine release was observed at 250 mOsM. Of the PGE analogs tested, only the EP(2)-selective agonist butaprost (9-oxo-11alpha,16S-dihydroxy-17-cyclobutyl-prost-13E-en-1-oic acid) was able to enhance taurine efflux. Inclusion of 1,9-dideoxyfoskolin, 5-nitro-2-(3-phenylpropylamino) benzoic acid, or 4-[(2-butyl-6,7-dicloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]-butanoic acid blocked the ability of PGE(1) to enhance taurine release, indicating the mediation of a volume-sensitive organic osmolyte and anion channel. The ability of PGE(1) to increase osmolyte release from L cells was mimicked by the addition of agents that inhibit cAMP breakdown, directly activate adenylyl cyclase, or are cell-permeant analogs of cAMP. Taurine release elicited by either PGE(1) or 8-(4-chlorophenylthio)-cAMP was attenuated by >70% in L cells that had been stably transfected with a mutant regulatory subunit of cAMP-dependent protein kinase (PKA). PGE(1) stimulation of taurine efflux was not attenuated by either depletion of intracellular calcium or inhibition of protein kinase C. The results indicate that activation of prostanoid receptors on murine fibroblasts enhances osmolyte release via a cAMP and PKA-dependent mechanism.