Intracellular Concentration Of Cyclic Nucleotides Research Articles

CUMP hydrolysis by PDE3A.

As previously reported, the cardiac phosphodiesterase PDE3A hydrolyzes cUMP. Moreover, cUMP-degrading activity was detected in cow and dog hearts several decades ago. Our aim was to characterize the enzyme kinetic parameters of PDE3A-mediated cUMP hydrolysis and to investigate whether cUMP and cUMP-hydrolyzing PDEs are present in cardiomyocytes. PDE3A-mediated cUMP hydrolysis was characterized in time course, inhibitor, and Michaelis-Menten kinetics experiments. Intracellular cyclic nucleotide (cNMP) concentrations and the mRNAs of cUMP-degrading PDEs were quantitated in neonatal rat cardiomyocytes (NRCMs) and murine HL-1 cardiomyogenic cells. Moreover, we investigated cUMP degradation in HL-1 cell homogenates and intact cells. Educts (cNMPs) and products (NMPs) of the PDE reactions were detected by HPLC-coupled tandem mass spectrometry. PDE3A degraded cUMP (measurement of UMP formation) with a K M value of ~143μM and a V max value of ~42μmol/min/mg. PDE3A hydrolyzed cAMP with a K M value of ~0.7μM and a V max of ~1.2μmol/min/mg (determination of AMP formation). The PDE3 inhibitor milrinone inhibited cUMP hydrolysis (determination of UMP formation) by PDE3A (K i=57nM). Significant amounts of cUMP as well as of PDE3A mRNA (in addition to PDE3B and PDE9A transcripts) were detected in HL-1 cells and NRCMs. Although HL-1 cell homogenates contain a milrinone-sensitive cUMP-hydrolyzing activity, intact HL-1 cells may use additional PDE3-independent mechanisms for cUMP disposal. PDE3A is a low-affinity and high-velocity PDE for cUMP. Future studies should investigate biological effects of cUMP in cardiomyocytes and the role of PDE3A in detoxifying high intracellular cUMP concentrations under pathophysiological conditions.

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4-/- fibroblast, compared to Mrp4+/+fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration.

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4(-/-) fibroblast, compared to Mrp4(+/+)fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

Cyclic Nucleotide Phosphodiesterase 3 Signaling Complexes

The superfamily of cyclic nucleotide phosphodiesterases is comprised of 11 gene families. By hydrolyzing cAMP and cGMP, PDEs are major determinants in the regulation of intracellular concentrations of cyclic nucleotides and cyclic nucleotide-dependent signaling pathways. Two PDE3 subfamilies, PDE3A and PDE3B, have been described. PDE3A and PDE3B hydrolyze cAMP and cGMP with high affinity in a mutually competitive manner and are regulators of a number of important cAMP- and cGMP-mediated processes. PDE3B is relatively more highly expressed in cells of importance for the regulation of energy homeostasis, including adipocytes, hepatocytes, and pancreatic β-cells, whereas PDE3A is more highly expressed in heart, platelets, vascular smooth muscle cells, and oocytes. Major advances have been made in understanding the different physiological impacts and biochemical basis for recruitment and subcellular localizations of different PDEs and PDE-containing macromolecular signaling complexes or signalosomes. In these discrete compartments, PDEs control cyclic nucleotide levels and regulate specific physiological processes as components of individual signalosomes which are tethered at specific locations and which contain PDEs together with cyclic nucleotide-dependent protein kinases (PKA and PKG), adenylyl cyclases, Epacs (guanine nucleotide exchange proteins activated by cAMP), phosphoprotein phosphatases, A-Kinase anchoring proteins (AKAPs), and pathway-specific regulators and effectors. This article highlights the identification of different PDE3A- and PDE3B-containing signalosomes in specialized subcellular compartments, which can increase the specificity and efficiency of intracellular signaling and be involved in the regulation of different cAMP-mediated metabolic processes.

Quantification of cAMP and cGMP analogs in intact cells: pitfalls in enzyme immunoassays for cyclic nucleotides

Immunoassays are routinely used as research tools to measure intracellular cAMP and cGMP concentrations. Ideally, this application requires antibodies with high sensitivity and specificity. The present work evaluates the cross-reactivity of commercially available cyclic nucleotide analogs with two non-radioactive and one radioactive cAMP and cGMP immunoassay. Most of the tested cyclic nucleotide analogs showed low degree competition with the antibodies; however, with Rp-cAMPS, 8-Br-cGMP and 8-pCPT-cGMP, a strong cross-reactivity with the corresponding cAMP and cGMP, respectively, immunoassays was observed. The determined EIA-binding constants enabled the measurement of the intracellular cyclic nucleotide concentrations and revealed a time- and lipophilicity-dependent cell membrane permeability of the compounds in the range of 10–30% of the extracellular applied concentration, thus allowing a more accurate prediction of the intracellular analog levels in a given experiment.Electronic supplementary materialThe online version of this article (doi:10.1007/s00210-011-0662-6) contains supplementary material, which is available to authorized users.

A protein phosphorylation-based assay for screening and monitoring of drugs modulating cyclic nucleotide pathways

Cyclic nucleotide regulation is an important target for drug development, particularly for treatment and prophylaxis of cardiovascular diseases. Determination of cyclic nucleotide levels for screening and monitoring of cyclic nucleotide modulating drug action is necessary, yet the techniques available are cumbersome and not sufficiently accurate. Here we present an approach based on the detection of cyclic nucleotide-dependent protein phosphorylation. By use of a common substrate of cyclic nucleotide-dependent protein kinases, the protein vasodilator-stimulated phosphoprotein (VASP) featuring two phosphorylation sites specifically phosphorylated by these kinases, an assay was developed for the monitoring of intracellular cyclic nucleotide levels. The assay was tested with human platelets ex vivo treated with stimulants of nucleotide cyclases, kinases, and phosphodiesterase inhibitors. Phosphorylation of the protein VASP correlates with intracellular cyclic nucleotide concentration ( R 2 > 0.90 for cGMP and cAMP); however, VASP phosphorylation is more sensitive to elevated cyclic nucleotide levels and significantly more stable over time. Quantification of VASP phosphorylation offers a reliable and robust tool for fast and easy monitoring of cyclic nucleotide levels and is also applicable to unprocessed biological matrices. Owing to these properties, VASP is a promising biomarker for screening and monitoring of cyclic nucleotide modulating drugs.

Cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase is functional in bovine mammary gland

Previous studies have shown that using nonspecific phosphodiesterase (PDE) inhibitors such as caffeine improved milk production, supporting the premise that modulation of intracellular concentration of cyclic nucleotides (cyclic AMP, cyclic guanosine 3′-5′-monophosphate) is involved. Intracellular cyclic nucleotides are degraded by the PDE enzyme family. The contribution of type IV PDE (PDE4) in the secretion of casein has been reported in rat mammary gland. The objective of this study was to demonstrate the functional presence of the PDE4 family in the bovine mammary gland. To understand the enzymatic expression pattern in the mammary gland, tissue samples were taken randomly from udders obtained from a local slaughterhouse. Reverse transcription PCR revealed that the PDE4D transcript was amplified, and the expected size fragment was obtained in a 1% agarose gel. Sequence analysis of the amplicon resulted in 99% homology to PDE4D. Moreover, Western blotting using a specific PDE4D antibody has confirmed that the protein of the isoenzyme PDE4D1 is present. A clear immunoreactive signal was also observed within the acini where epithelial cells are located. Assaying cyclic AMP PDE activity reported a total activity of 38.71±3.22 fmol/min per μg of total protein. Rolipram, a specific PDE4 inhibitor, showed a sensitive activity of 8.48±1.28 fmol/min per μg of total protein, indicating that PDE4 is responsible for one-fifth of the total enzymatic activity of PDE in the mammary gland. To further validate the presence of PDE4D in the bovine mammary epithelial cells, protein extracts from bovine mammary epithelial cells were separated on SDS-PAGE gels, and PDE4D protein was detected. The PDE assays reported a total activity of 30.16±4.82 fmol/min per μg of total protein. Rolipram showed a sensible activity of 11.91±5.93 fmol/min per μg of total protein. In conclusion, these results not only demonstrate the presence of PDE4D transcript and protein, but also show an active enzyme, suggesting a functional role of PDE4D in bovine mammary gland.

S2063 Calcium Sensing Receptor (CaSR) Function in Intestine Epithelial Cells of Rodents and Humans

Membranous calcium-sensing receptors (CaSR) on intestinal epithelial cells are difficult to study, due to fast loss of function following isolation of the cells. We established a chemical isolation process to maintain receptor function, being able to measure receptor-activity in a fluorescence spectrometry setup. In diarrhea, high intracellular concentrations of cyclic nucleotides in intestine epithelial cells lead to net fluid excretion due to enhanced serosal to mucosal fluid-transport. We showed earlier the reversibility of this effect via activation of CaSR with calcimimetic agents including natural and synthetic small molecules such as the compound R-568. Exposure to these allosteric modifiers in the presence of calcium resulted in a cessation of fluid excretion, and enhanced absorption. In the present study we develop a new isolation method for single cells of all intestinal segments and demonstrate that CaSR remains viable and active upon stimulationwith allostericmodifiers or by increasing the extracellular calcium concentration. These results give an important new tool for intestinal transport screening. METHODS We generated functional individual intestinal epithelial cells from the duodenum, jejunum, ileum and colon of female rats and human colon, using EDTA digestion solution and light mechanical force at 37°C for 20 minutes. Cells were loaded with 2nM Fluo-4 and Fluo-8, calcium-sensing fluorescent dyes. After multiple washout-steps and sedimentation for two hours, the activity of the CaSRwas determined, using a fluorescence spectrometer measuring the fluorescent excitation at 516nm. RESULTS We were able to show the functionality of epithelial CaSR on epithelial cells from four sections of the intestine of the rat: duodenum, jejunum, ileum and colon, and also human colon. The CaSR shows a dose dependent activation over a concentration range of 0.125mM to 2.5mM. The EC50 for calcium for the freshly isolated cells was comparable to that previously obtained for intact native tissue. In a separate study isolated cells were exposed to R-568 prior to the calcium dose curve, resulting in a left phase shift of the activation curve indicative of enhanced calcium binding to the receptor. CONCLUSION Our studies demonstrate that it is now possible to develop viable isolated cells from the entire digestive tract that maintain CaSR density. This technique provides an important new screening tool for use in testing pharmacological agents on intestinal epithelia.

S2064 Intestinal Alkaline Phosphatase Maintains the Normal Homeostasis of Gut Microbiota

Membranous calcium-sensing receptors (CaSR) on intestinal epithelial cells are difficult to study, due to fast loss of function following isolation of the cells. We established a chemical isolation process to maintain receptor function, being able to measure receptor-activity in a fluorescence spectrometry setup. In diarrhea, high intracellular concentrations of cyclic nucleotides in intestine epithelial cells lead to net fluid excretion due to enhanced serosal to mucosal fluid-transport. We showed earlier the reversibility of this effect via activation of CaSR with calcimimetic agents including natural and synthetic small molecules such as the compound R-568. Exposure to these allosteric modifiers in the presence of calcium resulted in a cessation of fluid excretion, and enhanced absorption. In the present study we develop a new isolation method for single cells of all intestinal segments and demonstrate that CaSR remains viable and active upon stimulationwith allostericmodifiers or by increasing the extracellular calcium concentration. These results give an important new tool for intestinal transport screening. METHODS We generated functional individual intestinal epithelial cells from the duodenum, jejunum, ileum and colon of female rats and human colon, using EDTA digestion solution and light mechanical force at 37°C for 20 minutes. Cells were loaded with 2nM Fluo-4 and Fluo-8, calcium-sensing fluorescent dyes. After multiple washout-steps and sedimentation for two hours, the activity of the CaSRwas determined, using a fluorescence spectrometer measuring the fluorescent excitation at 516nm. RESULTS We were able to show the functionality of epithelial CaSR on epithelial cells from four sections of the intestine of the rat: duodenum, jejunum, ileum and colon, and also human colon. The CaSR shows a dose dependent activation over a concentration range of 0.125mM to 2.5mM. The EC50 for calcium for the freshly isolated cells was comparable to that previously obtained for intact native tissue. In a separate study isolated cells were exposed to R-568 prior to the calcium dose curve, resulting in a left phase shift of the activation curve indicative of enhanced calcium binding to the receptor. CONCLUSION Our studies demonstrate that it is now possible to develop viable isolated cells from the entire digestive tract that maintain CaSR density. This technique provides an important new screening tool for use in testing pharmacological agents on intestinal epithelia.

Association of PDE11A global haplotype with major depression and antidepressant drug response

Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze the intracellular second messengers cAMP and cGMP to their corresponding monophosphates. PDEs play an important role in signal transduction by regulating the intracellular concentration of cyclic nucleotides. We have previously shown that the individual haplotype GAACC in the PDE11A gene was associated with major depressive disorder (MDD) based on block-by-block analysis. There are two PDE genes, PDE11A and PDE1A, located in chromosome 2q31-q32. In this study, we have further explored whether the whole region 2q31-q32 contribute to MDD or antidepressant response 278 depressed Mexican-American participants and 321 matched healthy controls. Although there is no significant interaction between the two genes, the remission rate of individual carrying the combination genotype at rs1880916 (AG/AA) and rs1549870 (GG) is significantly increased. We analyzed the global haplotype by examining 16 single-nucleotide polymorphisms (SNPs) in PDE11A and six SNPs in PDE1A. None of the haplotypes consisting of six SNPs in the PDE1A have a significant difference between depressed and control groups. Among haplotypes consisting of 16 SNPs across 440 kb in the PDE11A gene, 18 common haplotypes (with frequency higher than 0.8%) have been found in the studied population. Six haplotypes showed significantly different frequencies between the MDD group and the control group. The phylogenetic network result for the 16 SNPs showed that several historic recombination events have happened in the PDE11A gene. The frequency of one haplotype is significantly lower in the remitter group than in the nonremitter group for the depressed participants treated with either desipramine or fluoxetine. Thus, our data suggest that the PDE11A global haplotype is associated with both MDD and antidepressant drug response.

Overview of PDEs and Their Regulation

Contraction and relaxation of vascular smooth muscle and cardiac myocytes are key physiological events in the cardiovascular system. These events are regulated by second messengers, cAMP and cGMP, in response to extracellular stimulants. The strength of signal transduction is controlled by intracellular cyclic nucleotide concentrations, which are determined by a balance in production and degradation of cAMP and cGMP. Degradation of cyclic nucleotides is catalyzed by 3',5'-cyclic nucleotide phosphodiesterases (PDEs), and therefore regulation of PDEs hydrolytic activity is important for modulation of cellular functions. Mammalian PDEs are composed of 21 genes and are categorized into 11 families based on sequence homology, enzymatic properties, and sensitivity to inhibitors. PDE families contain many splice variants that mostly are unique in tissue-expression patterns, gene regulation, enzymatic regulation by phosphorylation and regulatory proteins, subcellular localization, and interaction with association proteins. Each unique variant is closely related to the regulation of a specific cellular signaling. Thus, multiple PDEs function as a particular modulator of each cardiovascular function and regulate physiological homeostasis.

Inhibition of cGMP-Dependent Protein Kinase Reduces the Response to Sonic Hedgehog in Neuralized Embryoid Bodies

Previously, we have shown that increasing the intracellular cGMP concentration enhances the sonic hedgehog (Shh) response in neural plate cells. The use of two mouse embryonic stem (ES) cell lines allowed a highly sensitive and reproducible quantification of the Shh response in neuralized embryoid bodies. Here we demonstrate that the specific, membrane-permeable cGMP-dependent protein kinase G-Ialpha (PKG-Ialpha) inhibitor DT-2 prevents an efficient Shh response, indicating that the effects of cGMP on the Shh response are mediated via PKG. We also demonstrate that the PKG acts upon the Shh response upstream of the Ptc1 promoter, which is up-regulated invariably and early in response to Shh, significantly limiting the targets for PKG phosphorylation to molecules involved in the early steps of the Shh response. These effects of cGMP and PKG are antagonistic to those of cAMP and PKA, and thus provide a mechanism by which the sensitivity of cells to the effects of Shh can be regulated, by modulating the intracellular cyclic nucleotide concentration.

Access of Quaternary Ammonium Blockers to the Internal Pore of Cyclic Nucleotide-gated Channels: Implications for the Location of the Gate

Cyclic nucleotide-gated (CNG) channels play important roles in the transduction of visual and olfactory information by sensing changes in the intracellular concentration of cyclic nucleotides. We have investigated the interactions between intracellularly applied quaternary ammonium (QA) ions and the α subunit of rod cyclic nucleotide-gated channels. We have used a family of alkyl-triethylammonium derivatives in which the length of one chain is altered. These QA derivatives blocked the permeation pathway of CNG channels in a concentration- and voltage-dependent manner. For QA compounds with tails longer than six methylene groups, increasing the length of the chain resulted in higher apparent affinities of ∼1.2 RT per methylene group added, which is consistent with the presence of a hydrophobic pocket within the intracellular mouth of the channel that serves as part of the receptor binding site. At the single channel level, decyltriethyl ammonium (C10-TEA) ions did not change the unitary conductance but they did reduce the apparent mean open time, suggesting that the blocker binds to open channels. We provide four lines of evidence suggesting that QA ions can also bind to closed channels: (1) the extent of C10-TEA blockade at subsaturating [cGMP] was larger than at saturating agonist concentration, (2) under saturating concentrations of cGMP, cIMP, or cAMP, blockade levels were inversely correlated with the maximal probability of opening achieved by each agonist, (3) in the closed state, MTS reagents of comparable sizes to QA ions were able to modify V391C in the inner vestibule of the channel, and (4) in the closed state, C10-TEA was able to slow the Cd2+ inhibition observed in V391C channels. These results are in stark contrast to the well-established QA blockade mechanism in Kv channels, where these compounds can only access the inner vestibule in the open state because the gate that opens and closes the channel is located cytoplasmically with respect to the binding site of QA ions. Therefore, in the context of Kv channels, our observations suggest that the regions involved in opening and closing the permeation pathways in these two types of channels are different.

Influence of pentoxifylline on natural cytotoxicity and expression of granzymes and PI-9, a specific granzyme B inhibitor

Pentoxifylline (PTX) is an unspecific inhibitor of phosphodiesterase activity that increases intracellular concentration of cyclic nucleotides, mainly cAMP. Since PTX improves microcirculatory blood flow, it is commonly and often chronically used in peripheral vascular diseases. On the other hand PTX also displays a variety of immunomodulatory activities. PTX inhibits natural cytotoxicity and it has previously been suggested that it could partially act also through its influence on perforin/granzyme-dependent pathways. However, the underlying mechanisms are obscure and it remains unknown whether PTX inhibits natural cytotoxicity influencing only leukocytes or also acting on target cells. In this study, we show that PTX inhibits expression of granzyme A in human leukocytes probably due to suppression of phosphodiesterase activity. Contrary, PTX does not affect expression of granzyme B and H. On the other hand we hypothesized that PTX could inhibit natural cytotoxicity not only affecting leukocytes but also due to generation of resistance to leukocyte-mediated cytotoxicity in target cells e.g. through overexpression of PI-9, a specific granzyme B inhibitor. We found that at the mRNA level, PTX stimulates expression of PI-9 in K562 cells. However, we did not observe such an influence at the protein level, in either K562 cells or in human leukocytes. It may suggest that other PTX-triggered molecular events may interfere with PI-9 overexpression in these cells at the further, post-transcriptional levels. According to these results, PTX did not affect resistance of target cells to natural cytotoxicity. Altogether, PTX inhibits natural cytotoxicity affecting mainly effector but not target cells and in case of the effector cells, besides previously reported mechanisms, it can also inhibit granzyme A expression.

Activation of olfactory cyclic‐nucleotide gated channels revisited

Ion channels that are gated by cyclic nucleotides (CNG channels) have been best studied for their roles in visual and olfactory sensory transduction. Sensory stimulation of vertebrate photoreceptors by light and olfactory receptor neurones (ORNs) by odorants activates 7-transmembrane receptors, and triggers, via a G protein, changes in the intracellular concentration of second messenger cyclic nucleotides. In the case of photoreceptors, the G protein Gt stimulates phosphodiesterase leading to the hydrolysis of cGMP, which in turn promotes closure of cGMP-gated channels and hyperpolarization. In the case of ORNs, the G protein Golf stimulates adenylyl cyclase to produce cAMP, which promotes opening of cAMP-gated channels and depolarization. The ability of CNG channels to be gated by cyclic nucleotides is therefore crucial for organisms to monitor their visual and odorant environments.

Influence of pentoxifylline on perforin expression in human PBMC.

Pentoxifylline (PTX) is a methylxanthine derivative that unspecifically inhibits phosphodiesterase activity and thus, it increases intracellular concentration of cyclic nucleotides. Currently, PTX is commonly and chronically used in peripheral blood vessel diseases. Besides its well-known influence on rheologic properties of blood, PTX has also been found to decrease secretion of some cytokines such as IL-12, TNF and IFN-gamma and thus it could exert immunomodulatory activity. Furthermore, PTX inhibits lymphocyte cytotoxicity affecting the perforin-dependent pathway, both in humans and animals. It has also been shown recently that in some murine models, PTX promotes tumor growth. Such a phenomenon, at least partially, could result from PTX-dependent inhibition of natural cytotoxicity. However, the detailed mechanism of PTX influence on cytotoxic activity in humans has not been established. We hypothesized that PTX-dependent inhibition of natural cytotoxicity could result from decrease in perforin expression. In this study, it was shown that pentoxifylline only moderately inhibits perforin expression at the mRNA level in human peripheral blood mononuclear cells (PBMC), and this effect seems to be independent of intracellular cAMP concentration. On the other hand, PTX did not significantly influence perforin expression at the protein level. These results suggest that in humans, contrary to murine models, inhibition of perforin-dependent natural cytotoxicity through pentoxifylline does not result from changes in perforin production. Presumably, influence of PTX on natural cytotoxicity could be caused by e.g. interference with lymphocyte degranulation. Moreover, also other possibilities, such as PTX influence on conformational changes of perforin molecules and/or its influence on susceptibility of target cells to pore-forming of polyperforins cannot be excluded.

Characterization of the MRP4- and MRP5-mediated Transport of Cyclic Nucleotides from Intact Cells

Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3',5'-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4- and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20-600 microm, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A(1) and E(1), the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterase-mediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.

Influence of permeant ions on gating in cyclic nucleotide-gated channels.

Cyclic nucleotide–gated channels are key components in the transduction of visual and olfactory signals where their role is to respond to changes in the intracellular concentration of cyclic nucleotides. Although these channels poorly select between physiologically relevant monovalent cations, the gating by cyclic nucleotide is different in the presence of Na+ or K+ ions. This property was investigated using rod cyclic nucleotide–gated channels formed by expressing the subunit 1 (or α) in HEK293 cells. In the presence of K+ as the permeant ion, the affinity for cGMP is higher than the affinity measured in the presence of Na+. At the single channel level, subsaturating concentrations of cGMP show that the main effect of the permeant K+ ions is to prolong the time channels remain open without major changes in the shut time distribution. In addition, the maximal open probability was higher when K+ was the permeant ion (0.99 for K+ vs. 0.95 for Na+) due to an increase in the apparent mean open time. Similarly, in the presence of saturating concentrations of cAMP, known to bind but unable to efficiently open the channel, permeant K+ ions also prolong the time channels visit the open state. Together, these results suggest that permeant ions alter the stability of the open conformation by influencing of the O→C transition.

First Computational Evidence for a Catalytic Bridging Hydroxide Ion in a Phosphodiesterase Active Site

Phosphodiesterases are clinical targets for a variety of biological disorders, because this superfamily of enzymes regulates the intracellular concentration of cyclic nucleotides that serve as the second messengers playing a critical role in a variety of physiological processes. Understanding the structure and mechanism of a phosphodiesterase will provide a solid basis for rational design of the more efficient therapeutics. Although a three-dimensional X-ray crystal structure of the catalytic domain of human phosphodiesterase 4B2B was recently reported, it is uncertain whether a critical bridging ligand in the active site is a water molecule or a hydroxide ion. The identity of this bridging ligand is theoretically determined by performing first-principles quantum chemical calculations on models of the active site. All the results obtained indicate that this critical bridging ligand in the active site of the reported X-ray crystal structure is a hydroxide ion, rather than a water molecule, expected to serve as the nucleophile to initialize the catalytic degradation of the intracellular second messengers.

Serotonin-induced protein kinase C activation in cultured rat heart endothelial cells

This study examined whether serotonin can activate protein kinase C in rat heart endothelial cells. Protein kinase C isozyme translocation was examined by Western blot analysis with isozyme-specific anti-protein kinase C antibody. In this study, only α protein kinase C isozyme was found to be translocated from the cytosolic to the particulate fractions after serotonin stimulation. The effect of serotonin on the incorporation of 32P from [γ- 32P]ATP into peptide substrate was studied as another indicator of protein kinase C activation. The experiments in this study demonstrated that the Ca 2+-phospholipid-dependent protein kinase, protein kinase C, was activated by serotonin. By investigating [ 3H]phorbol 12,13-dibutyrate binding to protein kinase C and trypsin-treated protein kinase C activity, we demonstrated that the site of action of serotonin is probably the regulatory domain of protein kinase C. Finally, we also demonstrated that serotonin had no effect on the intracellular concentration of cyclic nucleotides (cAMP, cGMP). These findings support the hypothesis that protein kinase C may be an important participant in serotonin-induced endothelial cell contraction and barrier dysfunction.