Cerebral and cerebellar pseudoatrophy associated with valproic acid. Report of three pediatric cases. / Pseudoatrofia cerebral y cerebelosa asociada a ácido valproico. Descripción de tres casos pediátricos.

INTRODUCTION: Cerebral and cerebellar pseudoatrophy is a rare adverse effect of valproic acid (VPA) that we need to be aware of, due to its diagnostic and therapeutic implications. CASE REPORT: We report three cases of children between 5 and 9 years old, with epilepsy and previous normal brain magnetic resonance imaging, who were taking the drug at correct doses. Pseudoatrophy manifests subacutely with symptoms and images of cerebral and/or cerebellar atrophy, reversible after drug withdrawal. DISCUSSION AND CONCLUSIONS: This is a type of VPA-related encephalopathy, different from dose-dependent toxic encephalopathy, hyperammonaemic encephalopathy or encephalopathy related to liver failure. In children, it causes cognitive, motor, mood and behavioral deterioration, and may be accompanied by epileptic decompensation. Withdrawing the drug leads to complete clinical-radiological recovery, and reducing the dose leads to improvement.

TITLE: Pseudoatrofia cerebral y cerebelosa asociada a ácido valproico. Descripción de tres casos pediátricos.Introducción. La pseudoatrofia cerebral y cerebelosa es un efecto adverso infrecuente del ácido valproico (VPA) que debemos conocer por sus implicaciones diagnósticas y terapéuticas. Caso clínico. Presentamos tres casos de niños de entre 5 y 9 años, con epilepsia y resonancia magnética craneal previa normal, que llevaban el fármaco con dosis correctas. La pseudoatrofia se manifiesta de forma subaguda con síntomas e imagen de atrofia cerebral y/o cerebelosa, reversible tras la retirada del fármaco. Discusión y conclusiones. Se trata de un tipo de encefalopatía relacionada con VPA diferente a la encefalopatía tóxica dependiente de la dosis, la encefalopatía hiperamoniémica o la relacionada con fallo hepático. En niños, cursa con deterioro cognitivo, motor, anímico y conductual, y puede acompañarse de descompensación epiléptica. La retirada del fármaco conlleva una recuperación completa clinicorradiológica, y la disminución de dosis, una mejoría.

Kinetic characterisation and thermal inactivation study of red alga (Mastocarpus stellatus) peroxidase.

Peroxidase (POD) was extracted from red alga (Mastocarpus stellatus) using Triton X-114 and characterised by UV-spectrophotometry. Optimum activity using 2,2´-azinobis(3-ethylbenzothiazolinesulphonic acid) (ABTS) as the H-donor was obtained at pH 5.0. In the presence of the anionic detergent, sodium dodecyl sulphate (SDS), however, POD was inactivated at all the pH values studied and totally inactivated at 1mM SDS. When the enzyme was kinetically characterised, the KM and Vm values for ABTS were found to be 13mM and 40µM/min, respectively. In addition, when the H2O2 concentration was increased, at a fixed concentration of ABTS, the activity was inhibited at the highest H2O2 concentrations. In a study of the effect of several reducing agents, l-cysteine was found to be the most active. A thermal inactivation study showed a first-order inactivation kinetic, and the Arrhenius plot yielded a straight line with a slope equivalent to an activation energy of 121.6kJ/mol. Significant inactivation occurred at temperatures of>35°C, with>90% of the relative activity being lost after only 5min of incubation at 48.4°C.

A sustainable approach by using microalgae to minimize the eutrophication process of Mar Menor lagoon.

The present study evaluates the removal capacity of microalgae photobioreactors of environmental pollutants present in wastewater from the dry riverbed El Albujón, as a way to minimize the eutrophication process of the Mar Menor. Particularly, the capacity of four autochthonous microalgae consortia collected from different locations of the salty lagoon to remove emerging contaminants (simazine, atrazine, terbuthylazine, adenosine and ibuprofen), nitrates, and phosphates, was evaluated. Among the four microalgae consortia, consortium 1 was the best in terms of biomass productivity (0.11 g L-1 d-1) and specific growth rate (0.14 d-1), providing 100% removal of emerging contaminants (simazine, atrazine, terbuthylazine, adenosine and ibuprofen), and a maximal reduction and consumption of macronutrients, especially nitrates and phosphates, reaching levels below 28 mg L-1, that is, a decrease of 89.90 and 99.70% of nitrates and phosphates, respectively. Therefore, this consortium (Monoraphidium sp., Desmodesmus subspicatus, Nannochloris sp.) could be selected as a green filter for successful large-scale applications. This study is the first one that combines the successful removal of herbicides, ibuprofen and adenosine as emerging contaminants, and nitrate removal.

Removal and toxicity evaluation of a diverse group of drugs from water by a cyclodextrin polymer/pulsed light system.

The presence of pharmaceutical compounds (PhCs) in the effluents of wastewater treatment plants (WWTPs) is an ecological concern. The issue could be alleviated by trapping those substances by cyclodextrin (CD) polymers or photolyzing them by pulsed light (PL). Consequently, a sequential CD polymer/PL system was tested for the removal of PhCs. Firstly, a survey detected the presence of recurrent PhCs in the effluents of local WWTPs. Then, pure water was spiked with 21 PhCs, 100 µg/L each one. The three-dimensional network provides amphiphilic features to the CD polymer that reduced the pollutant concentration by 77 %. Sorption involves a plead of physical and chemical mechanisms hindering the establishment of a general removal model for all compounds. The performed simulations hint that the retention capacity mainly correlates with the computed binding energies, so that theoretical models are revealed as valuable tools for further improvements. The complementary action of PL rose the elimination to 91 %. The polymer can be reused at least 10 times for ibuprofen (model compound) removal, and was able to eliminate the ecotoxicity of an ibuprofen solution. Therefore, this novel sequential CD polymer/PL process seems to be an efficient alternative to eliminate PhCs from wastewater.

ORAC-fluorescein assay to determine the oxygen radical absorbance capacity of resveratrol complexed in cyclodextrins.

The effect of the complexation of resveratrol with hydroxypropyl-beta-cyclodextrins (HP-beta-CDs) on the antioxidant capacity of the polyphenol is studied for the first time by means of the oxygen radical absorbance capacity (ORAC) method, using fluorescein (FL) as the fluorescent probe. The method is validated through its linearity, precision, and accuracy for measuring the ORAC of resveratrol in the absence or presence of cyclodextrins (CDs). The complexation of resveratrol in CDs increased the net area under the FL decay curve (net AUC) of resveratrol up to its saturation level, at which the polyphenol showed almost double the antioxidant activity it shows in the absence of CDs. The complexation constant ( K c) between resveratrol and HP-beta-CDs was calculated by linear regression of the phase solubility diagram ( K c = 18048 M (-1)). The antioxidant activity of resveratrol was dependent on the complexed resveratrol because CDs acts as a controlled dosage reservoir that protects resveratrol against rapid oxidation by free radicals. In this way, its antioxidant activity is prolonged and only reaches its maximum when all the resveratrol is complexed.

Ehlers-Danlos syndrome and periventricular nodular heterotopia in a Spanish family with a single FLNA mutation.

BACKGROUND: The Ehlers-Danlos syndrome (EDS) comprises a group of hereditary connective tissue disorders. Periventricular nodular heterotopia (PNH) is a human neuronal migration disorder characterised by seizures and conglomerates of neural cells around the lateral ventricles of the brain, caused by FLNA mutations. FLNA encodes filamin A, an actin binding protein involved in cytoskeletal organisation. The amino-terminal actin binding domain (ABD) of filamins contains two tandem calponin homology domains, CHD1 and CHD2. OBJECTIVE: To report clinical and genetic analyses in a Spanish family affected by a connective tissue disorder suggestive of EDS type III and PNH. METHODS: A clinical and molecular study was undertaken in the three affected women. Clinical histories, physical and neurological examinations, brain magnetic resonance imaging studies, and skin biopsies were done. Genetic analysis of the FLNA gene was undertaken by direct sequencing and restriction fragment length polymorphism analysis. RESULTS: Mutation analysis of the FLNA gene resulted in the identification of a novel mutation in exon 3 (c.383C-->T) segregating with the combination of both syndromes. This mutation results in a substitution of an alanine residue (A128V) in CHD1. CONCLUSIONS: The findings suggest that the Ala128Val mutation causes the dual EDS-PNH phenotype. This association constitutes a new variant within the EDS spectrum. This is the first description of a familial EDS-PNH association with a mutation in FLNA.

Effects of insulin on glucose transporters and metabolic patterns in Harding-Passey melanoma cells.

The effect of insulin on glucose metabolism through different pathways and the glucose transporters in Harding-Passey melanoma cells have been studied. Glucose was utilized at a rate of 6.9 +/- 2.3 (SD) mumol X g-1 X h-1 with 86% transformed into lactate and pyruvate and only 0.43 and 3% metabolized through the tricarboxylic acid cycle and the pentose phosphate pathway, respectively. Of the total glucose consumed 2% was used in protein synthesis and 2% was used for lipid synthesis. Hexokinase isoenzyme was type I and enolase was present mainly in the alpha gamma hybrid form. The glucose transporters were cytochalasin B sensitive. The number of high affinity cytochalasin B binding sites was 175,000 receptors/cell (about 0.6 pmol/mg protein) and Kd = 1 X 10(-7) M. Insulin increased glucose utilization and lactate production by about 70% and caused a 56% increase in transport without alterations in the Kd of the site. Insulin receptors were quantified by binding assay using 125I-insulin. Kd was 11 X 10(-9) M with the number of receptors calculated as 11,500/cell. Harding-Passey melanoma cells could thus be a useful model to study basic metabolic events and their modulation by hormones or other effectors.

Protective effect of cyclodextrins on the quality parameters of roast preserved pepper.

Total phenolics (TP), vitamin C, antioxidant activity and colour of preserved peppers were evaluated at 4, 25 and 50 ℃ storage during 30-day intervals. Except for 4 ℃, TP decreased during storage at 25 ℃ and 50 ℃, being softer for fortified samples with ß-CDs. The protective effect was evident, since 50 ℃ samples containing ß-CDs exhibited lower TP loss (19%) than control samples (38%) for 5 months storage. A decrease in the vitamin C content was observed for both samples as time and temperature progressed. In samples stored at 50 ℃ the protective effect of ß-CD only was evident at the first month, since fortified samples showed lower vitamin C loss (10%) than control samples. The fortified samples with ß-CDs exhibited lowest antioxidant activity loss (40%) during 90-day storage at 50 ℃, than control samples (64%). The colour changes were in line with those observed for total phenolics and at the end of study, the presence of 1% ß-CDs delayed the darkening of samples at both (25 and 50 ℃) storage conditions.

A kinetic study of the one-electron oxidation of Trolox C by the hydroperoxidase activity of lipoxygenase.

The oxidation of Trolox C (a vitamin E analog) by the hydroperoxidase activity of lipoxygenase was studied. Trolox C was oxidized to its corresponding phenoxyl radical in the presence of hydrogen peroxide, evolving through a ketodiene intermediate to the Trolox C quinone. The H2O2/Trolox C quinone molar ratio was 1.0. The overall reaction followed an enzymatic-chemical second-order system and involved a substrate regeneration mechanism. From the equations derived from this mechanism, the dismutation constant of the Trolox C radical was evaluated by non-linear regression as 4 x 10(5) M(-1) x s(-1). The accumulation curve of Trolox C quinone was found to be linear, with no lag period, and dependent on enzyme concentration. No phenoxyl radical was detected when the reaction was carried out in the presence of ascorbate. This synergistic reaction between the Trolox C radical and ascorbate was quantitative and depended on the respective concentrations of enzyme, Trolox C and hydrogen peroxide. The results presented in this paper suggest that the diferences observed in the kinetic behaviour of monophenols (one-electron donors) and diphenols (two-electron donors) stem from the fact that the latter evolve directly into ferric form without taking the slow pathway once the steady state is reached, whereas the monophenols are always forced take the slow way, even in the steady state. This peroxidative oxidation of a vitamin E analog by the hydroperoxidase activity of lipoxygenase together with the oxidation produced by dioxygenase activity suggests that lipoxygenase might be a key enzyme in destroying the lipophilic antioxidant barrier against the reactive oxygen species in membranes.

Adenosine transporters in chromaffin cells: subcellular distribution and characterization.

Adenosine transporters in freshly isolated and cultured chromaffin cells were quantified by the [3H]dipyridamole binding technique, showing a maximal bound capacity of 0.4 +/- 0.05 pmol/10(6) cells (240,000 +/- 20,000 transporters by cell). Scatchard analysis showed a similar affinity for [3H]dipyridamole in isolated cells and subcellular fractions (Kd = 5 +/- 0.6 nM). For enriched plasma membrane preparations and chromaffin granule membranes, the maximal binding capacities were also very similar, 2.3 +/- 0.3 and 1.8 +/- 0.4 pmol/mg protein, respectively. When [3H]nitrobenzylthioinosine was employed as a radioligand, the maximal bound capacity in cultured chromaffin cells was 0.053 +/- 0.004 pmol/10(6) cells (32,000 +/- 3000 transporters per cell) with a high affinity constant (Kd = 0.25 +/- 0.03 nM); similar values were obtained in all subcellular fractions (Kd = 0.1 +/- 0.01). Also, plasma and chromaffin granule membranes showed similar maximal binding values (0.4 +/- 0.06 pmol/mg protein). Photoincorporation studies with [3H]nitrobenzylthioinosine into plasma membrane polypeptides showed the presence of three molecular species of 115 +/- 10; 58 +/- 6 and 42 +/- 5 kDa. Chromaffin granule membranes showed only the 105 +/- 9 and 51 +/- 4 molecular species.

Down-regulation and recycling of the nitrobenzylthioinosine-sensitive nucleoside transporter in cultured chromaffin cells.

The dynamics of the nitrobenzylthioinosine (NBTI)-sensitive nucleoside transporter were studied in cultured chromaffin cells. Photolabelling of transporters with [3H]NBTI induced a down-regulation of this protein from the plasma membrane with a half-life value of 2.31 +/- 0.61 h, measured by specific isolation of plasma membrane on polycationic beads. In this internalization step 50-60% of transporters were destroyed. The remaining labelled protein reappeared in plasma membranes and underwent a new disappearance cycle with a longer half-life period (34.65 +/- 3.9 h). A similar pattern of internalization and reappearance of nucleoside transporters was observed in cells cross-linked with non-labelled NBTI, with a half value of reappearance of 33 h. Chromaffin cells cultured in the presence of the protein synthesis inhibitor, cycloheximide, had a component of disappearance for NBTI binding sites with a half-life value of 24.6 +/- 1.4 h.

Hydroperoxidase activity of lipoxygenase: a kinetic study of isoproterenol oxidation.

The hydroperoxidase activity of soybean lipoxygenase, a non-heme protein, oxidized isoproterenol using H2O2 at pH 6.0. This oxidation was enzymatic, since neither heat-denaturated enzyme or iron ions in the presence of H2O2 produced an increase in absorbance. The initial rate was not linear and showed a characteristic lag period whose length depended on the enzyme and substrate concentration. The lag was decreased if the enzyme and isoproterenol concentration were increased, whereas it increased if the H2O2 concentration was increased. Lipoxygenase showed the typical low specificity for electron donor characteristic of this hydroperoxidase activity (26 mM), but a high affinity for H2O2 (94 microM), although with substrate inhibition (ksi = 3.6 mM). The chemical intermediates produced during the oxidation of isoproterenol were characterized in order to determine the origin of the lag period. A plausible kinetic mechanism is proposed to explain the observed lag period and inhibition by high concentrations of H2O2.

Glucose transporters in chromaffin cells: subcellular distribution and characterization.

The glucose transporter was identified and characterized by cytochalasin B binding in subcellular membrane fractions of chromaffin tissue. The binding was saturable with Kd of about 0.3 microM for each subcellular fraction. The Bmax capacity was 12-16 pmol/mg protein for enriched plasma membrane fractions, 6.3 pmol/mg protein for microsomal membrane preparations and 5.4 pmol/mg protein for chromaffin granule membranes. Irreversible photoaffinity labelling of the glucose-protectable binding sites with [3H]cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis from enriched plasma membrane preparations demonstrated the presence of three molecular species: 97 +/- 10, 51.5 +/- 6 and 30 +/- 4 kDa. The chromaffin granule membranes showed only a molecular species of 80 +/- 10 kDa.

Flow cytometric studies of nucleoside transport regulation in single chromaffin cells.

The present paper reveals that a fluorescent derivative of nitrobenzylthioinosine, 5-(SAENTA-x8)-fluorescein, is a highly specific inhibitor of the neural NBTI-sensitive nucleoside transporter. 5-(SAENTA-x8)-fluorescein inhibited adenosine transport and [3H]NBTI binding with a Ki of 4 nM in cultured chromaffin cells. Flow cytometry demonstrated that 5-(SAENTA-x8)-fluorescein specifically interacted with the NBTI-sensitive nucleoside transporters with high affinity (K[D] = 6 nM). Activation of protein kinases A and C with forskolin or nicotinic receptor agonists, respectively, resulted in 50% inhibition of the fluorescence bound to the cells. Flow cytometry will allow studying nucleoside transport in single cells from heterogeneous neural cell populations.

Differential modulation of nucleoside transport types in neuroblastoma cells by protein kinase activation.

Nucleoside transport regulation in undifferentiated Neuro-2A cells has been studied and found to include Na+-dependent adenosine transport and facilitated diffusion adenosine transport. The latter corresponded to nitrobenzylthioinosine-sensitive nucleoside transport. Short-term treatment of Neuro-2A cells with physiologically relevant signals only modulated the facilitated diffusion component. The stimulation of undifferentiated cells with forskolin or other activators of the protein kinase A pathway, decreased NBTI-sensitive adenosine transport. Treatment of cells with an inactive analogue of forskolin, 1,9-dideoxi-forskolin, had no effect on NBTI-sensitive nucleoside transport. Therefore, the inhibition of protein kinase A activity by pre-incubation with H-89 or the cAMP antagonist, Rp-8-Br-cAMPS, completely prevented the inhibitory effect of forskolin. Similarly, the activation of protein kinase C with phorbol 12,13-dibutyrate (PDBu) and the calcium ionophore A-23187 decreased NBTI-sensitive adenosine transport. The effect of PDBu was reversed by pre-incubation of cells with staurosporine. Maximal transport inhibition was obtained by the simultaneous stimulation of cells with a phorbol ester and A-23187 or a phorbol ester and forskolin. The modulation of NBTI-sensitive nucleoside transport corresponded to changes in specific [3H]NBTI binding to Neuro-2A cells. Maximal inhibition correlated well with a maximal enhancement of cAMP production. However, the Na+-dependent adenosine transport in Neuro-2A cells was not modulated by any of these signals.

P2X2 characteristics of the ATP receptor coupled to [Ca2+]i increases in cultured Purkinje neurons from neonatal rat cerebellum.

P2X receptors present in cerebellar Purkinje cells have been studied by recording ATP-elicited [Ca2+]i signals from immuno-identified (calbindin+) cells in culture using fura-2 microfluorescence. The [Ca2+]i increases evoked by ATP were mimicked by 2MeSATP but not by alpha, beta-meATP and other purinoceptor agonists. The selective P2X1 antagonist diinosine pentaphosphate failed to inhibit ATP-elicited [Ca2+]i transients, but suramin and PPADS rapidly and reversibly blocked the [Ca2+]i responses to ATP and 2MeSATP. The IC50 values for suramin and PPADS inhibition were 48.7 +/- 4.4 and 5.9 +/- 0.3 microM, respectively. Both antagonists blocked completely the signal elicited by ATP, revealing that there was not a separate antagonist-insensitive P2X receptor population in Purkinje cells. The effect of ATP was potentiated by Zn2+ and H+ ions. A one unit acidification from pH 7.4 to 6.4 enhanced by 172% the [Ca2+]i transient elicited by an intermediate concentration of ATP. Conversely, alkalinization of the medium to pH 8.4 reduced the ATP response by 88%. This combination of pharmacological and modulatory properties indicates that endogenous P2X receptors present in Purkinje neurons are formed by P2X2 subunits, rather than the more abundantly expressed P2X4 purinoceptor subunits.

Effect of tuamine, heptaminol and two analogues on uptake and release of catecholamines in cultured chromaffin cells.

The effects of tuamine (1-methylhexylamine), a sympathomimetic compound with hypertensive properties, heptaminol (6-amino-2-methyl-2-heptanol), an aliphatic amine with pressor properties, and two structural analogues of tuamine on high-affinity Na(+)-dependent noradrenaline uptake and on nicotine-evoked release were examined in bovine chromaffin cells maintained in primary culture for 3 to 6 days. Tuamine was found to be a potent competitive inhibitor of noradrenaline uptake with an effect similar to that of cocaine. Its inhibition constant (Ki) was 1.1 +/- 0.1 microM while Ki values of heptaminol, of 1-methylamino-5-pentanol oxalate and of 5-amino-2-methylhexanol oxalate, which were also found to be competitive inhibitors of noradrenaline uptake, were 60 +/- 2 microM, 260 +/- 28 microM and 48 +/- 76 microM, respectively. Tuamine, hepataminol and 5-amino-2-methyl-2-hexanol were also shown to be inhibitors of nicotine-induced release of catecholamines, with IC50 values of 26 +/- 2 microM, 650 +/- 11 microM and 500 +/- 10 microM, respectively. Tuamine and hepataminol did not inhibit noradrenaline release evoked by 59 mM K+, suggesting that it acts at a step prior to calcium entry. The pharmacological properties of heptaminol as an anti-hypotension agent may partially account for its inhibitory effect on catecholamine uptake and release.

Effect of forskolin and cyclic AMP analog on adenosine transport in cultured chromaffin cells.

The adenosine transport in cultured chromaffin cells was inhibited by the presence of the adenylate cyclase activator, forskolin, and a cAMP analog. The V(max) values of this transport obtained for control and in the presence of 8-(-4-chlorophenylthio)adenosine-3?:5?-monophosphate cyclic (ClPhcAMP, 100 ?M) or forskolin (0.5 ?M) were 85 +/- 5; 45 +/- 1.5 and 38 +/- 3 pmol/10(6) cells/min, respectively. The K(m) values were not significantly modified. The number of adenosine transporters in cultured chromaffin cells, measured by nitrobenzylthioinosine (NBTI) binding, were decreased by the above mentioned effectors. The values of binding sites per cell were 30,000 +/- 3200; 12,000 +/- 1000 and 21,300 +/- 2000 for control, ClPhcAMP and forskolin, respectively; without changing the dissociation constant. When the binding studies were conducted with cellular homogenates, a significant decrease in the maximal binding capacity for nitrobenzylthioinosine was obtained. The values were as follows: 0.087 +/- 0.01 pmol/mg protein for control, 0.044 +/- 0.02 pmol/mg protein for ClPhcAMP; and 0.032 +/- 0.01 pmol/mg protein for forskolin. In this neural tissue, the adenosine transport system seems to be inhibited by stimulation of the adenylate cyclase or by the cyclic AMP analogue that enters the cells. These results suggest that this inhibition could be mediated by a molecular modification of adenosine transporters, the binding with NBTI is therefore a possible parameter of this modification.

Adenosine 5'-triphosphate modulation of nitrobenzylthioinosine binding sites in plasma membranes of bovine chromaffin cells.

Nitrobenzylthioinosine (NBTI) is a high affinity probe for facilitated diffusion nucleoside transporters. Kinetic analysis of the binding of [3H]NBTI to plasma membranes of chromaffin cells was conducted in the presence or absence of adenosine 5'-triphosphate (ATP). Similar curvilinear plots with a Hill number of 1.32 were obtained in both conditions. ATP significantly increased the number of NBTI binding sites in these preparations showing Bmax values of 1.62 +/- 0.20 pmol/mg protein for controls and 3.22 +/- 0.31 pmol/mg protein in the presence of ATP. However, the affinity constant (KD) was not significantly modified. The non-metabolizable ATP analogue, 5'-adenylyl imidodiphosphate (AMP-PNP) and diadenosine tetraphosphate (Ap4A) can mimic the stimulatory ATP effect, but adenosine monophosphate (AMP) has no effect on the NBTI binding to plasma membranes. These results indicate a modulatory role for ATP, non-hydrolysis dependent, on nucleoside transport in chromaffin cells. Therefore, a nucleotide binding site on the nucleoside transporter similar to that described for glucose transporter could be suggested.

Potentiation of adenosine 5'-triphosphate calcium responses by diadenosine pentaphosphate in individual rat cerebellar astrocytes.

The calcium responses induced by adenosine 5'-triphosphate (ATP) were examined in single cerebellar type 1 astrocytes by fura-2 microfluorometry. ATP elicited fast and transient increases of intracellular calcium concentration ([Ca2+]i) even in the absence of extracellular calcium, indicating the involvement of metabotropic purinoceptors. The co-stimulation with P1P5-di(adenosine-5')pentaphosphate (Ap5A; 0.1 microM) potentiated ATP-metabotropic calcium responses. After this preincubation ineffective concentrations of ATP triggered 40% of maximal response. Co-stimulation with Ap5A and ATP was mandatory. The potentiated response to ATP was also independent of extracellular Ca2+ and was maintained for long periods of time (h). These results show a relevant interaction of purinoceptors that may imply a novel mechanism of action for diadenosine polyphosphates.