In Vitro Cell Sensitivity to Palytoxin Correlates with High Gene Expression of the Na+/K+-ATPase ß2 Subunit Isoform.

The marine polyether palytoxin (PLTX) is one of the most toxic natural compounds, and is involved in human poisonings after oral, inhalation, skin and/or ocular exposure. Epidemiological and molecular evidence suggest different inter-individual sensitivities to its toxic effects, possibly related to genetic-dependent differences in the expression of Na+/K+-ATPase, its molecular target. To identify Na+/K+-ATPase subunits, isoforms correlated with in vitro PLTX cytotoxic potency, sensitivity parameters (EC50: PLTX concentration reducing cell viability by 50%; Emax: maximum effect induced by the highest toxin concentration; 10-7 M) were assessed in 60 healthy donors' monocytes by the MTT (methylthiazolyl tetrazolium) assay. Sensitivity parameters, not correlated with donors' demographic variables (gender, age and blood group), demonstrated a high inter-individual variability (median EC50 = 2.7 × 10-10 M, interquartile range: 0.4-13.2 × 10-10 M; median Emax = 92.0%, interquartile range: 87.5-94.4%). Spearman's analysis showed significant positive correlations between the ß2-encoding ATP1B2 gene expression and Emax values (rho = 0.30; p = 0.025) and between Emax and the ATP1B2/ATP1B3 expression ratio (rho = 0.38; p = 0.004), as well as a significant negative correlation between Emax and the ATP1B1/ATP1B2 expression ratio (rho = -0.30; p = 0.026). This toxicogenetic study represents the first approach to define genetic risk factors that may influence the onset of adverse effects in human PLTX poisonings, suggesting that individuals with high gene expression pattern of the Na+/K+-ATPase ß2 subunit (alone or as ß2/ß1 and/or ß2/ß3 ratio) could be highly sensitive to PLTX toxic effects.

Azaspiracids Increase Mitochondrial Dehydrogenases Activity in Hepatocytes: Involvement of Potassium and Chloride Ions.

BACKGROUND: Azaspiracids (AZAs) are marine toxins that are produced by Azadinium and Amphidoma dinoflagellates that can contaminate edible shellfish inducing a foodborne poisoning in humans, which is characterized by gastrointestinal symptoms. Among these, AZA1, -2, and -3 are regulated in the European Union, being the most important in terms of occurrence and toxicity. In vivo studies in mice showed that, in addition to gastrointestinal effects, AZA1 induces liver alterations that are visible as a swollen organ, with the presence of hepatocellular fat droplets and vacuoles. Hence, an in vitro study was carried out to investigate the effects of AZA1, -2, and -3 on liver cells, using human non-tumor IHH hepatocytes. RESULTS: The exposure of IHH cells to AZA1, -2, or -3 (5 × 10-12-1 × 10-7 M) for 24 h did not affect the cell viability and proliferation (Sulforhodamine B assay and 3H-Thymidine incorporation assay), but they induced a significant concentration-dependent increase of mitochondrial dehydrogenases activity (MTT reduction assay). This effect depends on the activity of mitochondrial electron transport chain complex I and II, being counteracted by rotenone and tenoyl trifluoroacetone, respectively. Furthermore, AZAs-increased mitochondrial dehydrogenase activity was almost totally suppressed in the K+-, Cl--, and Na+-free media and sensitive to the specific inhibitors of KATP and hERG potassium channels, Na+/K+, ATPase, and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. CONCLUSIONS: These results suggest that AZA mitochondrial effects in hepatocytes derive from an imbalance of intracellular levels of K+ and, in particular, Cl- ions, as demonstrated by the selective reduction of toxin effects by CFTR chloride channel inhibition.

Ovatoxin-a, A Palytoxin Analogue Isolated from Ostreopsis cf. ovata Fukuyo: Cytotoxic Activity and ELISA Detection.

This study provides the first evaluation of the cytotoxic effects of the recently identified palytoxin (PLTX) analog, ovatoxin-a (OVTX-a), the major toxin produced by Ostreopsis cf. ovata in the Mediterranean Sea. Its increasing detection during Ostreopsis blooms and in seafood highlights the need to characterize its toxic effects and to set up appropriate detection methods. OVTX-a is about 100 fold less potent than PLTX in reducing HaCaT cells viability (EC50 = 1.1 × 10(-9) M vs 1.8 × 10(-11) M, MTT test) in agreement with a reduced binding affinity (Kd = 1.2 × 10(-9) vs 2.7 × 10(-11) M, saturation experiments on intact cells). Similarly, OVTX-a hemolytic effect is lower than that of the reference PLTX compound. Ost-D shows the lowest cytotoxicity toward HaCaT keratinocytes, suggesting the lack of a hydroxyl group at C44 as a critical feature for PLTXs cytotoxic effects. A sandwich ELISA developed for PLTX detects also OVTX-a in a sensitive (LOD = 4.2 and LOQ = 5.6 ng/mL) and accurate manner (Bias = 0.3%), also in O. cf. ovata extracts and contaminated mussels. Although in vitro OVTX-a appears less toxic than PLTX, its cytotoxicity at nanomolar concentrations after short exposure time rises some concern for human health. The sandwich ELISA can be a viable screening method for OVTXs detection in monitoring program.

Pro-inflammatory effects of palytoxin: an in vitro study on human keratinocytes and inflammatory cells.

Palytoxin (PLTX) is one of the most harmful marine toxins known so far. Although the ingestion of contaminated seafood is the most dangerous exposure route for humans, cutaneous and inhalational exposures are far more frequent, and can cause strong inflammatory reactions. However, little is known about the inflammatory events that follow the cutaneous exposure to the toxin. In this study, we investigated (1) the effects of both short (2 h) and long (24 h) term exposures of HaCaT keratinocytes to a sub-cytotoxic PLTX concentration on pro-inflammatory mediator gene expression and release and (2) the effect of PLTX-conditioned HaCaT cell media on undifferentiated (monocytes) and differentiated (macrophages; immature dendritic cells, iDCs; mature dendritic cells, mDCs) THP-1 cells. At 10-11 M, PLTX induced interleukin (IL)-6 and IL-8 release from HaCaT keratinocytes after 24 h of continuous exposure to the toxin, as well as after 23 h in toxin-free medium preceded by 1 h exposure to PLTX. Under the same experimental conditions, release of the inflammatory mediators prostaglandin-E2 and histamine was also found after both short and long exposures to the toxin. The conditioned media collected from HaCaT cells treated with PLTX increased the migration of the differentiated and undifferentiated THP-1 cells (potency rank order: monocytes ≥ iDCs > mDCs > macrophages) but did not induce cell differentiation. These results indicate that keratinocytes can be actively involved in the recruitment of inflammatory cells in response to cutaneous contact with PLTX. The lack of a significant effect on monocyte differentiation towards mature immune cells suggests that PLTX is endowed with irritant rather than sensitizing properties.

Titanium Dioxide Nanoparticle Penetration into the Skin and Effects on HaCaT Cells.

Titanium dioxide nanoparticles (TiO2NPs) suspensions (concentration 1.0 g/L) in synthetic sweat solution were applied on Franz cells for 24 h using intact and needle-abraded human skin. Titanium content into skin and receiving phases was determined. Cytotoxicity (MTT, AlamarBlue(®) and propidium iodide, PI, uptake assays) was evaluated on HaCat keratinocytes after 24 h, 48 h, and seven days of exposure. After 24 h of exposure, no titanium was detectable in receiving solutions for both intact and damaged skin. Titanium was found in the epidermal layer after 24 h of exposure (0.47 ± 0.33 µg/cm(2)) while in the dermal layer, the concentration was below the limit of detection. Damaged skin, in its whole, has shown a similar concentration (0.53 ± 0.26 µg/cm(2)). Cytotoxicity studies on HaCaT cells demonstrated that TiO2NPs induced cytotoxic effects only at very high concentrations, reducing cell viability after seven days of exposure with EC50s of 8.8 × 10(-4) M (MTT assay), 3.8 × 10(-5) M (AlamarBlue(®) assay), and 7.6 × 10(-4) M (PI uptake, index of a necrotic cell death). Our study demonstrated that TiO2NPs cannot permeate intact and damaged skin and can be found only in the stratum corneum and epidermis. Moreover, the low cytotoxic effect observed on human HaCaT keratinocytes suggests that these nano-compounds have a potential toxic effect at the skin level only after long-term exposure.

Cobalt Oxide Nanoparticles: Behavior towards Intact and Impaired Human Skin and Keratinocytes Toxicity.

Skin absorption and toxicity on keratinocytes of cobalt oxide nanoparticles (Co3O4NPs) have been investigated. Co3O4NPs are commonly used in industrial products and biomedicine. There is evidence that these nanoparticles can cause membrane damage and genotoxicity in vitro, but no data are available on their skin absorption and cytotoxicity on keratinocytes. Two independent 24 h in vitro experiments were performed using Franz diffusion cells, using intact (experiment 1) and needle-abraded human skin (experiment 2). Co3O4NPs at a concentration of 1000 mg/L in physiological solution were used as donor phase. Cobalt content was evaluated by Inductively Coupled-Mass Spectroscopy. Co permeation through the skin was demonstrated after 24 h only when damaged skin protocol was used (57 ± 38 ng·cm⁻²), while no significant differences were shown between blank cells (0.92 ± 0.03 ng cm⁻²) and those with intact skin (1.08 ± 0.20 ng·cm⁻²). To further investigate Co3O4NPs toxicity, human-derived HaCaT keratinocytes were exposed to Co3O4NPs and cytotoxicity evaluated by MTT, Alamarblue and propidium iodide (PI) uptake assays. The results indicate that a long exposure time (i.e., seven days) was necessary to induce a concentration-dependent cell viability reduction (EC50 values: 1.3 × 10-4 M, 95% CL = 0.8-1.9 × 10⁻4 M, MTT essay; 3.7 × 10⁻5 M, 95% CI = 2.2-6.1 × 10⁻5 M, AlamarBlue assay) that seems to be associated to necrotic events (EC50 value: 1.3 × 10⁻4 M, 95% CL = 0.9-1.9 × 10⁻4 M, PI assay). This study demonstrated that Co3O4NPs can penetrate only damaged skin and is cytotoxic for HaCat cells after long term exposure.

The marine toxin palytoxin induces necrotic death in HaCaT cells through a rapid mitochondrial damage.

Palytoxin (PLTX) is one of the most toxic algal biotoxin known so far. It transforms the Na(+)/K(+)-ATPase into a cationic channel inducing a massive intracellular Na(+) influx. However, from a mechanistic point of view, the features and the intracellular pathways leading to PLTX-induced cell death are still not completely characterized. This study on skin HaCaT keratinocytes demonstrates that PLTX induces necrosis since propidium iodide uptake was observed already after 1 h toxin exposure, an effect that was not lowered by toxin removal. Furthermore, necrotic-like morphological alterations were evidenced by confocal microscopy. Apoptosis occurrence was excluded since no caspases 3/7, caspase 8, and caspase 9 activation as well as no apoptotic bodies formation were recorded. Necrosis was preceded by a very early mitochondrial damage as indicated by JC-1 fluorescence shift, recorded already after 5 min toxin exposure. This shift was totally abolished when Na(+) and Ca(2+) ions were withdrawn from culture medium, whereas cyclosporine-A was ineffective, excluding the occurrence of a controlled biochemical response. These results clearly establish necrosis as the primary mechanism for PLTX-induced cell death in HaCaT cells. The rapidity of mitochondrial damage and the consequent irreversible necrosis rise serious concerns about the very fast onset of PLTX toxic effects.

Characterization of palytoxin binding to HaCaT cells using a monoclonal anti-palytoxin antibody.

Palytoxin (PLTX) is the reference compound for a group of potent marine biotoxins, for which the molecular target is Na+/K+-ATPase. Indeed, ouabain (OUA), a potent blocker of the pump, is used to inhibit some PLTX effects in vitro. However, in an effort to explain incomplete inhibition of PLTX cytotoxicity, some studies suggest the possibility of two different binding sites on Na+/K+-ATPase. Hence, this study was performed to characterize PLTX binding to intact HaCaT keratinocytes and to investigate the ability of OUA to compete for this binding. PLTX binding to HaCaT cells was demonstrated by immunocytochemical analysis after 10 min exposure. An anti-PLTX monoclonal antibody-based ELISA showed that the binding was saturable and reversible, with a K(d) of 3 × 10-10 M. However, kinetic experiments revealed that PLTX binding dissociation was incomplete, suggesting an additional, OUA-insensitive, PLTX binding site. Competitive experiments suggested that OUA acts as a negative allosteric modulator against high PLTX concentrations (0.3-1.0 × 10-7 M) and possibly as a non-competitive antagonist against low PLTX concentrations (0.1-3.0 × 10-9 M). Antagonism was supported by PLTX cytotoxicity inhibition at OUA concentrations that displaced PLTX binding (1 × 10-5 M). However, this inhibition was incomplete, supporting the existence of both OUA-sensitive and -insensitive PLTX binding sites.

Oxidative stress induced by palytoxin in human keratinocytes is mediated by a H+-dependent mitochondrial pathway.

In the last decades, massive blooms of palytoxin (PLTX)-producing Ostreopsis cf. ovata have been observed along Mediterranean coasts, usually associated to human respiratory and cutaneous problems. At the molecular level, PLTX induces a massive intracellular Na(+) influx due to the transformation of Na(+)/K(+) ATPase in a cationic channel. Recently, we have demonstrated that Na(+) overload is the crucial step in mediating overproduction of reactive oxygen species (ROS) and cell death in human HaCaT keratinocytes, tentatively explaining PLTX-induced skin irritant effects. In the present study the molecular mechanisms of ROS production induced by PLTX-mediated Na(+) intracellular overload have been investigated. In HaCaT cells, PLTX exposure caused accumulation of superoxide anion, but not of nitric oxide or peroxynitrite/hydroxyl radicals. Even if RT-PCR and western blot analysis revealed an early NOX-2 and iNOS gene and protein over-expressions, their active involvement seemed to be only partial since selective inhibitors did not completely reduce O(2)(-) production. A significant role of other enzymes (COX-1, COX-2, XO) was not evidenced. Nigericin, that counteracts Na(+)-mediated H(+)-imbalance, dissipating ΔpH across mitochondrial inner membrane, and the uncouplers DNP significantly reduced O(2)(-) production. These inhibitions were synergistic when co-exposed with complex-I inhibitor rotenone. These results suggest a novel mechanism of O(2)(-) production induced by PLTX-mediated ionic imbalance. Indeed, the H(+) intracellular overload that follows PLTX-induced intracellular Na(+) accumulation, could enhance ΔpH across mitochondrial inner membrane, that seems to be the driving force for O(2)(-) production by reversing mitochondrial electron transport.

Sanitary problems related to the presence of Ostreopsis spp. in the Mediterranean Sea: a multidisciplinary scientific approach.

The increased presence of potentially toxic microalgae in the Mediterranean area is a matter of great concern. Since the end of the last century, microalgae of the genus Ostreopsis have been detected more and more frequently in the Italian coastal waters. The presence of Ostreopsis spp. has been accompanied by the presence of previously undetected marine biotoxins (palytoxins) into the ecosystem with the increased possibility of human exposure. In response to the urgent need for toxicity characterization of palytoxin and its congeners, an integrated study encompassing both in vitro and in vivo methods was performed.

The stretch-activated channel blocker Gd(3+) reduces palytoxin toxicity in primary cultures of skeletal muscle cells.

Palytoxin (PLTX) is one of the most toxic seafood contaminants ever isolated. Reports of human food-borne poisoning ascribed to PLTX suggest skeletal muscle as a primary target site. Primary cultures of mouse skeletal muscle cells were used to study the relationship between Ca(2+) response triggered by PLTX and the development of myotoxic insult. Ca(2+) imaging experiments revealed that PLTX causes a transitory intracellular Ca(2+) response (transient phase) followed by a slower and more sustained Ca(2+) increase (long-lasting phase). The transient phase is due to Ca(2+) release from intracellular stores and entry through voltage-dependent channels and the Na(+)/Ca(2+) exchanger (reverse mode). The long-lasting phase is due to a massive and prolonged Ca(2+) influx from the extracellular compartment. Sulforhodamine B assay revealed that the long-lasting phase is the one responsible for the toxicity in skeletal muscle cells. Our data analyzed, for the first time, pathways of PLTX-induced Ca(2+) entry and their correlation with PLTX-induced toxicity in skeletal muscle cells. The cellular morphology changes induced by PLTX and the sensitivity to gadolinium suggest a role for stretch-activated channels.

Silver nanoparticles exert a long-lasting antiproliferative effect on human keratinocyte HaCaT cell line.

For their antibacterial activity, silver nanoparticles (Ag NPs) are largely used in various commercially available products designed to come in direct contact with the skin. In this study we investigated the effects of Ag NPs on skin using the human-derived keratinocyte HaCaT cell line model. Ag NPs caused a concentration- and time-dependent decrease of cell viability, with IC(50) values of 6.8 ± 1.3 µM (MTT assay) and 12 ± 1.2 µM (SRB assay) after 7 days of contact. A 24h treatment, followed by a 6 day recovery period in Ag NPs-free medium, reduced cell viability with almost the same potency (IC(50)s of 15.3 ± 4.6 and 35 ± 20 µM, MTT and SRB assays, respectively). Under these conditions, no evidence of induction of necrotic events (propidium iodide assay) was found. Apocynin, NADPH-oxidase inhibitor, or N(G)-monomethyl-L-argynine, nitric oxide synthase inhibitor, did not prevent NPs-induced reduction of cell viability. TEM analysis of cells exposed to NPs for 24h revealed alteration of nuclear morphology but only a marginal presence of individual NPs inside the cells. These results demonstrate that on HaCaT keratinocytes a relatively short time of contact with Ag NPs causes a long-lasting inhibition of cell growth, not associated with consistent Ag NPs internalization.

A 3D-pharmacophore model for sigma2 receptors based on a series of substituted benzo[d]oxazol-2(3H)-one derivatives.

In this work we developed a 3D-pharmacophore model for sigma(2) receptor based on 19 benzooxazolone derivatives. The best 3D-pharmacophore hypothesis, consisting of five features: a positive ionizable, a hydrogen bond acceptor, a hydrophobic aromatic, a hydrophobic aliphatic, and a generic hydrophobic provided a 3D-QSAR model with a correlation coefficient of 0.97 and a RMSD of 0.48.

Design, synthesis and SAR analysis of novel selective sigma1 ligands (Part 2).

In order to investigate the molecular features involved in sigma receptors (sigma-Rs) binding, new compounds based on arylalkylaminoalcoholic, arylalkenyl- and arylalkylaminic scaffolds were synthesized and their affinity towards sigma(1)- and sigma(2)-Rs subtypes was evaluated. The most promising compounds were also screened for their affinity at micro-opioid, delta-opioid and kappa-opioid receptors. Biological results are herein presented and discussed.

Stereostructure and biological activity of 42-hydroxy-palytoxin: a new palytoxin analogue from Hawaiian Palythoa subspecies.

This paper reports on the analysis of the toxin content from Palythoa tuberculosa and Palythoa toxica samples collected off of the Hawaiian coast. Our work, based on in-depth high-resolution liquid chromatography-mass spectrometry analysis along with extensive NMR study, led us to structurally characterize 42-hydroxy-palytoxin, a new palytoxin congener. This toxin and palytoxin itself appeared to be the major components of toxic extract from a P. tuberculosa sample, while 42-hydroxy-palytoxin was proven by far to be the main palytoxin derivative in P. toxica. Functional studies on this new palytoxin-like compound suggest that the new palytoxin analogue and palytoxin itself present similar biological activities.

Synthesis, biological evaluation, and three-dimensional in silico pharmacophore model for sigma(1) receptor ligands based on a series of substituted benzo[d]oxazol-2(3H)-one derivatives.

Novel benzo[d]oxazol-2(3H)-one derivatives were designed and synthesized, and their affinities against sigma receptors were evaluated. On the basis of 31 compounds, a three-dimensional pharmacophore model for the sigma(1) receptor binding site was developed using the Catalyst 4.9 software package. The best 3D pharmacophore hypothesis, consisting of one positive ionizable, one hydrogen bond acceptor, two hydrophobic aromatic, and one hydrophobic features provided a 3D-QSAR model with a correlation coefficient of 0.89. The best hypothesis was also validated by three independent methods, i.e., the Fisher randomization test included in the CatScramble functionality of Catalyst, the leave-one-out test, and activity prediction of an additional test set. The achieved results will allow researchers to use this 3D pharmacophore model for the design and synthesis of a second generation of high affinity sigma(1) ligands, as well as to discover other lead compounds for this class of receptors.

Nanoparticle dermal absorption and toxicity: a review of the literature.

INTRODUCTION: Nanotechnologies are among the fastest growing areas of scientific research and have important applications in a wide variety of fields. The data suggest that in the future workers and consumers exposed to nanoparticles will significantly increase. DERMAL ABSORPTION AND TOXICITY OF NANOPARTICLES: At now there are gaps in understanding about the human and environmental risk that manufactured nanoparticles pose for occupational exposed people and for consumers. There is a need for assessing the health and environmental impacts, the nanoparticles life cycle, the human exposure routes, the behavior of nanoparticles in the body, and the risk for workers. Possible routes of entry into the body include inhalation, absorption through the skin or digestive tract, injection, and absorption or implantation for drugs delivery systems. In particular, dermal absorption and skin penetration of nanoparticles needs a better evaluation because few and contradictory data are present in the literature, mainly on titanium dioxide. CONCLUSIONS: There are limited data on carbon-based nanoparticles and very few data on other metal nanoparticles increasingly used in industry. The article reviews the literature on the percutaneous absorption of nanoparticles and their effect on skin.

Substituted benzo[d]oxazol-2(3H)-one derivatives with preference for the sigma1 binding site.

We describe here the synthesis and the binding interaction with sigma(1) and sigma(2) receptors of a series of new benzo[d]oxazol-2(3H)-one derivatives variously substituted on the N-benzyl moiety. The results of binding studies confirm the notion that the benzoxazolone moiety confers preference towards sigma(1) sites and establish that the ability to bind to sigma(1), but not to sigma(2) receptors, is strongly affected by the kind and the position of the substituents introduced in the N-benzyl ring. In fact, compounds with substitutions in para-position with atoms of Cl, H or F or with a CH(3) group exhibit a higher affinity for sigma(1) receptors than the corresponding ortho-substituted compounds. The highest affinity and selectivity, with K(i) values of 0.1 and 427 nM for sigma(1) and sigma(2) receptors, respectively, and a corresponding K(i)sigma(2)/K(i)sigma(1) selectivity ratio of 4270 were found for the Cl-substituted compound. These results indicate that benzo[d]oxazol-2(3H)-one derivatives are among the most selective and sigma(1) receptor-preferring ligands currently available.

In vitro effects of yessotoxin on a primary culture of rat cardiomyocytes.

Oral administration of yessotoxin (YTX) has been reported to induce ultrastructural alterations in rodent cardiac muscle. To study its effects on various fundamental aspects of cardiac muscle cells activity, that is, cell beating, Ca(2+) and cyclic adenosine 3',5'-monophosphate (cAMP) levels, as well as cell vitality, a primary culture of rat cardiomyocytes was used. Patch-clamp recordings, Ca(2+) imaging, and cAMP assays were performed on cultured cardiomyocytes to characterize YTX effects on the cell beating frequency. 3-(4,5-Dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) and sulforhodamine B (SRB) tests were carried out to determine its effect on cardiomyocytes viability. Videoimaging techniques showed a time- and concentration-dependent reduction in the beating frequency after 1, 5, and 24 h incubation with YTX (0.1-1 microM). This effect was neither associated to the uncoupling between the membrane electrical activity and Ca(2+) release from intracellular stores nor to the impairment of the mechanisms controlling the Ca(2+) homeostasis. In addition, 1 microM YTX did not modify basal cAMP levels in cardiomyocytes. MTT and SRB assays revealed that incubation of cardiomyocytes with YTX (0.01-1 microM; 24, 48, and 72 h) caused a decrease in cell viability in a concentration- and time-dependent way. This effect was still evident in cardiomyocytes exposed to YTX for 1, 5, and 24 h and cultured up to 72 h in YTX-free medium. Our results demonstrate that, at nanomolar concentrations, a short incubation with YTX causes an inhibition of the beating activity and an irreversible reduction of viability of cardiac cells in vitro.

Cyclic AMP in rat ileum: evidence for the presence of an extracellular cyclic AMP-adenosine pathway.

BACKGROUND & AIMS: Extracellular adenosine plays a relevant role in regulating intestinal motility and preventing inflammatory processes. Adenosine 3',5'-cyclic monophosphate (cAMP) extruded from cells may be converted to adenosine monophosphate and then to adenosine by ecto-phosphodiesterase and CD73/ecto-5'nucleotidase, respectively, thus representing a source of adenosine. Our purpose was to assess the existence of a functional extracellular cAMP-adenosine pathway in intestinal tissue, obtaining evidence for CD73 expression and evaluating the effect of cAMP on ileum motility. METHODS: The formation of cAMP metabolites in rat ileum strips incubated with exogenous cAMP or [(3)H]cAMP was monitored by high-performance liquid chromatography. CD73 was detected by immunoprecipitation and immunofluorescence. The functional activity of exogenous cAMP on ileum strips was recorded by measuring tension changes. RESULTS: In ileum strips, the generation of cAMP-derived adenosine monophosphate, adenosine, and inosine was time and concentration dependent and was blocked by phosphodiesterase or CD73 inhibitors in a manner consistent with exogenous cAMP being processed through the extracellular cAMP-adenosine pathway. Accordingly, [(3)H]cAMP uptake in ileum strips was negligible. Immunofluorescence revealed CD73 surface expression on intestinal smooth muscle cells and intact smooth muscle. Exogenous cAMP concentration-dependently increased ileum muscle tension partially inhibited by adenosine inactivation or receptor blockade. Forskolin-stimulated endogenous cAMP induced concentration-dependent ileum relaxations. CONCLUSIONS: A functioning extracellular cAMP-adenosine pathway featuring CD73 expression is present in rat ileum and affects intestinal motility. Extracellular cAMP may therefore act on intestinal muscle both directly by binding to specific smooth muscle cell membrane sites and indirectly through its degradation products.