Megalencephalic leukoencephalopathy with subcortical cysts protein-1: A new calcium-sensitive protein functionally activated by endoplasmic reticulum calcium release and calmodulin binding in astrocytes.

BACKGROUND: MLC1 is a membrane protein highly expressed in brain perivascular astrocytes and whose mutations account for the rare leukodystrophy (LD) megalencephalic leukoencephalopathy with subcortical cysts disease (MLC). MLC is characterized by macrocephaly, brain edema and cysts, myelin vacuolation and astrocyte swelling which cause cognitive and motor dysfunctions and epilepsy. In cultured astrocytes, lack of functional MLC1 disturbs cell volume regulation by affecting anion channel (VRAC) currents and the consequent regulatory volume decrease (RVD) occurring in response to osmotic changes. Moreover, MLC1 represses intracellular signaling molecules (EGFR, ERK1/2, NF-kB) inducing astrocyte activation and swelling following brain insults. Nevertheless, to date, MLC1 proper function and MLC molecular pathogenesis are still elusive. We recently reported that in astrocytes MLC1 phosphorylation by the Ca2+/Calmodulin-dependent kinase II (CaMKII) in response to intracellular Ca2+ release potentiates MLC1 activation of VRAC. These results highlighted the importance of Ca2+ signaling in the regulation of MLC1 functions, prompting us to further investigate the relationships between intracellular Ca2+ and MLC1 properties. METHODS: We used U251 astrocytoma cells stably expressing wild-type (WT) or mutated MLC1, primary mouse astrocytes and mouse brain tissue, and applied biochemistry, molecular biology, video imaging and electrophysiology techniques. RESULTS: We revealed that WT but not mutant MLC1 oligomerization and trafficking to the astrocyte plasma membrane is favored by Ca2+ release from endoplasmic reticulum (ER) but not by capacitive Ca2+ entry in response to ER depletion. We also clarified the molecular events underlining MLC1 response to cytoplasmic Ca2+ increase, demonstrating that, following Ca2+ release, MLC1 binds the Ca2+ effector protein calmodulin (CaM) at the carboxyl terminal where a CaM binding sequence was identified. Using a CaM inhibitor and generating U251 cells expressing MLC1 with CaM binding site mutations, we found that CaM regulates MLC1 assembly, trafficking and function, being RVD and MLC-linked signaling molecules abnormally regulated in these latter cells. CONCLUSION: Overall, we qualified MLC1 as a Ca2+ sensitive protein involved in the control of volume changes in response to ER Ca2+ release and astrocyte activation. These findings provide new insights for the comprehension of the molecular mechanisms responsible for the myelin degeneration occurring in MLC and other LD where astrocytes have a primary role in the pathological process.

Rapid maxillary expansion for interceptive orthodontic treatment of palatally displaced canine: A systematic review.

AIM: The present systematic review aims to summarise the relevant randomised clinical trials and estimate the efficacy of interceptive orthodontic intervention, in particular if an interceptive treatment with rapid maxillary expansion could be successful in managing of palatally displaced canines (PDCs). MATERIALS AND METHODS: A search strategy was developed on electronic databases including Medline, Web of Science, Scopus and Cochrane Collaboration Trial from 1925 to 2019. Two reviewers independently reviewed the sources deciding for a full reading according to the inclusion and exclusion criteria. Methodological quality criteria were applied to the selected articles. RESULTS: Three randomised clinical trials (RCTs) and one prospective longitudinal study were included in the systematic review. Generally, the intervention groups showed a higher incidence of successful eruption of PDCs (45.1% - 65.7%) compared with the control groups (13.1% - 13.6%). CONCLUSIONS: Based on the literature published, authors reached a reasonable conclusion that rapid maxillary expansion can facilitate the eruption of PDCs. Authors suggest to associate maxillary expansion with deciduous canine extraction or prevention of mesial movement of the upper first molars.

Driving under the influence of drugs: Prevalence in road traffic accidents in Italy and considerations on per se limits legislation.

OBJECTIVE: The objective of this study was to present the prevalence and concentrations of drugs in blood samples of drivers involved in road traffic accidents (RTAs) and to discuss the effects of adopting different concentration cutoff values proposed or applied in other European countries on the number of driving under the influence of drugs (DUID) offenses. METHODS: Blood samples from drivers involved in RTAs in Padova province from 2014 to 2017 were analyzed for the presence of alcohol and drugs. The prevalence of positive subjects was reported for each substance adopting the limits of quantification (LOQs) of our laboratory and the concentration cutoff values proposed and/or used in other European countries. The reduction of cases of driving under the influence of illicit drugs in applying different cutoffs was calculated. RESULTS: Four thousand four hundred forty-three blood samples were analyzed: 23.7% were positive for alcohol and 19.9% for psychoactive drugs, with prevalences of polydrug and alcohol-drug abuse of 4.5 and 6%, respectively. The most frequently detected drugs were cannabinoids (9.7%) and cocaine (7.2%), followed by benzodiazepines (4.1%), opiates (1.9%), and other opioids (1.7%). Barbiturates, amphetamines, and ketamine were identified in a much smaller number of cases. The overall decrease in DUID cases when adopting different cutoffs with respect to cases above the LOQs was between 8 and 84%. The adoption of high LOQs such as those used in the European Union's research project on Driving Under the Influence of Drugs, Alcohol and Medicines (DRUID) decreases the hypothetical number of DUID offenses by a quarter, and per se limits proposed as broadly equivalent to a blood alcohol concentration (BAC) between 0.2 and 0.8 g/L dramatically reduce the cases of DUID (cocaine -81%, cannabis -79%, opioids -97%, opiates -96%, and amphetamines -77%); no ketamine-positive samples were above the cutoff. CONCLUSIONS: The implementation of high analytical limits or per se limits based on impairing concentrations in the Italian legislation could result in the prosecution of a much lower number of drugged drivers involved in RTAs, with a decrease from 25% to more than 80% depending on the limits.

Charged cyclic hexapeptides: Updating molecular descriptors for permeability purposes.

Three polar cyclic hexapeptides differently charged at physiological pH (1 = neutral, 2 = anionic, 3 = cationic) were synthesized and their cell permeability measured. Lipophilicity in octanol/water didn't account for the higher permeability of the cationic derivative but three chromatographic indexes (log KwIAM, log k' HILIC and log k' c-HILIC) were more efficient to this respect. NMR amide chemical shift temperature coefficients (ΔδNH/ΔT) were used to explore the IMHB network of the backbone. MD simulations in different environments (water, chloroform and DMPC lipid bilayer) highlighted that the charged amino group of the lysine moiety of 3 is not involved in the formation of any IMHB in water whereas a different behavior is registered in chloroform and DMPC lipid bilayer. Overall this paper highlights how a combination of experimental and computational approaches could help in comparing permeability and physicochemical properties of neutral and charged cyclic peptides.

Innovative clinical pathways for obese pregnant women: design and feasibility of the Padua project (North-Eastern Italy).

BACKGROUND: Obesity during pregnancy can adversely affect the wellbeing of the mother and the newborn, as well as the latter's long-term health. Preconception counseling, careful prenatal management, and strict follow-up during pregnancy are, therefore, essential for obese fertile women in order to prevent the negative effects of obesity. METHODS: In this setting, we developed a project that consisted in creating an integrated network of primary, secondary, and tertiary care providers and designing new clinical pathways for managing pregnancy in obese women. RESULTS: Two distinct pathways were devised: a Pre-Gestational Pathway for programming a pregnancy in obese women; and a Gestational Pathway for the clinical management of their pregnancy. DISCUSSION: Judging from the preliminary results of our study, the latter (Gestational) pathway seems to be successful, since there has been a gradual increase in the number of women using it, and these women have reported having no difficulty in accessing the services involved. It is noteworthy that immigrant women (who accounted for 60% of the women using the pathway) also reported no access issues. The pre-gestational pathway was very little used, however, accounting for only 2% of the appointments made with the services involved. In conclusion, the key to success in managing pregnancy in obese women lies in sharing the various different health care competences required and taking the local resources into account. The prevention of obesity in women of fertile age remains the main problem, however, and further efforts are needed in this setting.

Docosahexaenoic acid promotes oligodendrocyte differentiation via PPAR-γ signalling and prevents tumor necrosis factor-α-dependent maturational arrest.

Docosahexaenoic acid (DHA) is an essential omega-3 fatty acid known to be neuroprotective in several models of human diseases, including multiple sclerosis. The protective effects of DHA are largely attributed to its ability to interfere with the activity of transcription factors controlling immune and inflammatory responses, including the agonist-dependent transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ). In this study, we used primary oligodendrocyte progenitor (OP) cultures from neonatal rat brain to investigate whether DHA could influence OP maturation and directly promote myelination, as previously reported for selective PPAR-γ agonists. We show that, similarly to the selective PPAR-γ agonist pioglitazone (PGZ), DHA promotes OP maturation and counteracts the maturational arrest induced by TNF-α, used to mimic inflammatory conditions. The PPAR-γ antagonist GW9662 prevented both DHA-induced OP maturation and PPAR-γ nuclear translocation, supporting the hypothesis that DHA acts through the activation of PPAR-γ. In addition, both PGZ and DHA induced the phosphorylation of extracellular signal-regulated-kinase 1-2 (ERK1/2), in a PPAR-γ-dependent manner. ERK1/2 activity is known to regulate the transition from OPs to immature oligodendrocytes and the presence of specific inhibitors of ERK1/2 phosphorylation (U0126 or PD98059) prevented the differentiating effects of both DHA and PGZ. These results indicate that DHA might influence the process of OP maturation through its PPAR-γ agonistic activity and provide novel molecular mechanisms for the action of this dietary fatty acid, further supporting the nutritional intervention in demyelinating diseases such as multiple sclerosis.

3D Mass Spectrometry Imaging Reveals a Very Heterogeneous Drug Distribution in Tumors.

Mass Spectrometry Imaging (MSI) is a widespread technique used to qualitatively describe in two dimensions the distribution of endogenous or exogenous compounds within tissue sections. Absolute quantification of drugs using MSI is a recent challenge that just in the last years has started to be addressed. Starting from a two dimensional MSI protocol, we developed a three-dimensional pipeline to study drug penetration in tumors and to develop a new drug quantification method by MALDI MSI. Paclitaxel distribution and concentration in different tumors were measured in a 3D model of Malignant Pleural Mesothelioma (MPM), which is known to be a very heterogeneous neoplasm, highly resistant to different drugs. The 3D computational reconstruction allows an accurate description of tumor PTX penetration, adding information about the heterogeneity of tumor drug distribution due to the complex microenvironment. The use of an internal standard, homogenously sprayed on tissue slices, ensures quantitative results that are similar to those obtained using HPLC. The 3D model gives important information about the drug concentration in different tumor sub-volumes and shows that the great part of each tumor is not reached by the drug, suggesting the concept of pseudo-resistance as a further explanation for ineffective therapies and tumors relapse.

Is it possible to study the kinetic parameters of interaction between PNA and parallel and antiparallel DNA by stopped-flow fluorescence?

Peptide nucleic acids (PNAs) are among the most interesting and versatile artificial structural mimics of nucleic acids and exhibit peculiar and important properties (i.e. high chemical stability, and a high resistance to cellular enzymes and nucleases). Despite their unnatural structure, they are able to recognize and bind DNA and RNA in a very high, specific and selective manner. One of the most popular, easy and reliable method to measure the stability of PNA-DNA hybrid systems is the melting temperature but the thermodynamic data are obtained using a big quantity of materials failing to provide information on the kinetics of the interaction. In the present work, the PNA decamer 6, with the TCACTAGATG sequence of nucleobases, and the corresponding fluorescent PNA-FITU (fluorescein isothiourea) decamer 8 were synthesized with standard manual Boc-based chemistry. The interaction of the PNA-FITU with parallel and antiparallel DNA has been studied by stopped-flow fluorescence, which is proposed as an alternative technique to obtain the kinetic parameters of the binding. The great advantage of using the stopped-flow technique is the possibility of studying the kinetics of the PNA-DNA duplex formation in a physiological environment. In particular, fluorescence stopped-flow technique has been exploited to compare the affinity of two PNA-DNA duplexes since it can discriminate between parallel and antiparallel DNA binding.

Stimulation of adenosine A2A receptors reduces intracellular cholesterol accumulation and rescues mitochondrial abnormalities in human neural cell models of Niemann-Pick C1.

Niemann Pick C 1 (NPC1) disease is an incurable, devastating lysosomal-lipid storage disorder characterized by hepatosplenomegaly, progressive neurological impairment and early death. Current treatments are very limited and the research of new therapeutic targets is thus mandatory. We recently showed that the stimulation of adenosine A2A receptors (A2ARs) rescues the abnormal phenotype of fibroblasts from NPC1 patients suggesting that A2AR agonists could represent a therapeutic option for this disease. However, since all NPC1 patients develop severe neurological symptoms which can be ascribed to the complex pathology occurring in both neurons and oligodendrocytes, in the present paper we tested the effects of the A2AR agonist CGS21680 in human neuronal and oligodendroglial NPC1 cell lines (i.e. neuroblastoma SH-SY5Y and oligodendroglial MO3.13 transiently transfected with NPC1 small interfering RNA). The down-regulation of the NPC1 protein effectively resulted in intracellular cholesterol accumulation and altered mitochondrial membrane potential. Both effects were significantly attenuated by CGS21680 (500 nM). The protective effects of CGS were prevented by the selective A2AR antagonist ZM241385 (500 nM). The involvement of calcium modulation was demonstrated by the ability of Bapta-AM (5-7 µM) in reverting the effect of CGS. The A2A-dependent activity was prevented by the PKA-inhibitor KT5720, thus showing the involvement of the cAMP/PKA signaling. These findings provide a clear in vitro proof of concept that A2AR agonists are promising potential drugs for NPC disease.

Lipophilicity of amyloid ß-peptide 12-28 and 25-35 to unravel their ability to promote hydrophobic and electrostatic interactions.

The growing interest for peptide therapeutics calls for new strategies to determine the physico-chemical properties responsible for the interactions of peptides with the environment. This study reports about the lipophilicity of two fragments of the amyloid ß-peptide, Aß 25-35 and Aß 12-28. Firstly, computational studies showed the limits of log D(7.4)oct in describing the lipophilicity of medium-sized peptides. Chromatographic lipophilicity indexes (expressed as log k', the logarithm of the retention factor) were then measured in three different systems to highlight the different skills of Aß 25-35 and Aß 12-28 in giving interactions with polar and apolar environments. CD studies were also performed to validate chromatographic experimental conditions. Results show that Aß 12-28 has a larger skill in promoting hydrophobic and electrostatic interactions than Aß 25-35. This finding proposes a strategy to determine the lipophilicity of peptides for drug discovery purposes but also gives insights in unraveling the debate about the aminoacidic region of Aß responsible for its neurotoxicity.

Peroxisome proliferator activated receptor-γ agonists protect oligodendrocyte progenitors against tumor necrosis factor-alpha-induced damage: Effects on mitochondrial functions and differentiation.

The activation of the nuclear receptor peroxisome proliferator-activated receptor-γ (PPAR-γ) is known to exert anti-inflammatory and neuroprotective effects and PPAR-γ agonists are considered potential therapeutic agents in brain diseases including those affecting myelin. In demyelinating diseases such as multiple sclerosis (MS), inflammation is one of the causes of myelin and axonal damage. Oligodendrocyte (OL) differentiation is highly dependent on mitochondria, which are major targets of inflammatory insult. Here we show that PPAR-γ agonists protect OL progenitors against the maturational arrest induced by the inflammatory cytokine TNF-α by affecting mitochondrial functions. We demonstrate that the inhibition of OL differentiation by TNF-α is associated with i) increased mitochondrial superoxide production; ii) decreased mitochondrial membrane potential (mMP); and iii) decreased ADP-induced Ca(2+) oscillations, which we previously showed to be dependent on efficient mitochondria. The TNF-α effects were comparable to those of the mitochondrial toxin rotenone, further suggesting that TNF-α damage is mediated by mitochondrial function impairment. PPAR-γ agonists protected OL progenitors against the inhibitory activities of both TNF-α and rotenone on mMP, mitochondrial ROS production, Ca(2+) oscillations and OL differentiation. Finally, the PPAR-γ agonist pioglitazone increased the expression of PGC-1α (a mitochondrial biogenesis master regulator), UCP2 (a mitochondrial protein known to reduce ROS production), and cytochrome oxidase subunit COX1. These findings confirm the central role of mitochondria in OL differentiation and point to mitochondria as major targets of PPAR-γ agonist protection against TNF-α damage.

Estimation of prenatal aorta intima-media thickness from ultrasound examination.

Prenatal events such as intrauterine growth restriction and increased cardiovascular risk in later life have been shown to be associated with an increased intima-media thickness (aIMT) of the abdominal aorta in the fetus. In order to assess and manage atherosclerosis and cardiovascular disease risk in adults and children, in recent years the measurement of abdominal and carotid artery thickness has gained a growing appeal. Nevertheless, no computer aided method has been proposed for the analysis of prenatal vessels from ultrasound data, yet. To date, these measurements are being performed manually on ultrasound fetal images by skilled practitioners. The aim of the presented study is to introduce an automatic algorithm that identifies abdominal aorta and estimates its diameter and aIMT from routine third trimester ultrasonographic fetal data.The algorithm locates the aorta, then segments it and, by modeling the arterial wall longitudinal sections by means of a gaussian mixture, derives a set of measures of the aorta diameter (aDiam) and of the intima-media thickness (aIMT). After estimating the cardiac cycle, the mean diameter and the aIMT at the end-diastole phase are computed.Considering the aIMT value for each subject, the correlation between automatic and manual end-diastolic aIMT measurements is 0.91 in a range of values 0.44-1.10 mm, corresponding to both normal and pathological conditions. The automatic system yields a mean relative error of 19%, that is similar to the intra-observer variability (14%) and much lower that the inter-observer variability (42%).The correlation between manual and automatic measurements and the small error confirm the ability of the proposed system to reliably estimate aIMT values in prenatal ultrasound sequences, reducing measurement variability and suggesting that it can be used for an automatic assessment of aIMT.

[Infantile visceral leishmaniasis, an etiology of easily curable hemophagocytic lymphohistiocytosis syndrome]. / La leishmaniose viscérale infantile, une cause de syndrome d'hémophagocytose lympho-histiocytaire facilement curable.

Infantile visceral leishmaniasis associated hemophagocytic lymphohistiocytosis (HLH) is a rare clinicopathological entity, difficult to diagnose and fatal if untreated. The diagnosis should be considered in young infants with fever and splenomegaly. We report two cases of HLH caused by visceral leishmaniasis. In the first case, a 3-month-old boy was admitted with fever and pancytopenia, leading to the diagnosis of HLH based on complete clinical and biological features including hemophagocytosis on bone marrow smears. Investigations for an underlying genetic, metabolic disease and an infectious trigger were negative. Primary or genetic hemophagocytic syndrome was suspected and immunosuppressive treatment (steroids and cyclosporin) was instituted. A second bone marrow examination performed 1 month later revealed leishmania. The boy was treated with liposomal amphotericin and recovered rapidly. In the second case, a 10-year-old child was hospitalized with fever, pancytopenia, and a tumoral syndrome. He had a history of recurrent infections. The bone marrow biopsy showed leishmania and treatment with liposomal amphotericin was delivered. After 3 days of treatment, the improvement was judged inadequate and the boy presented biological signs of HLH. He was treated with steroids. An underlying primary immunodeficiency (interleukin-12/interferon-γ axis disorder) was secondarily diagnosed.

Pharmacological activity of a Bv8 analogue modified in position 24.

BACKGROUND AND PURPOSE: The amphibian peptide Bv8 induces potent nociceptive sensitization in rodents. Its mammalian homologue, prokineticin 2 (PROK2), is strongly up-regulated in inflamed tissues and is a major determinant in triggering inflammatory pain. Bv8 and PROK2 activate two closely related GPCRs, PK(1) and PK(2) , in a relatively non-selective fashion. To characterize better the roles of the two receptors in hyperalgesia and to obtain ligands whose binding affinity and efficacy differed for the two receptors, we modified the Bv8 molecule in regions essential for receptor recognition and activation. EXPERIMENTAL APPROACH: We modified the Bv8 molecule by substituting Trp in position 24 with Ala (A-24) and compared it with Bv8 for binding and activating PK(1) and PK(2) receptors in cell preparations and in affecting nociceptive thresholds in rodents. KEY RESULTS: A-24 preferentially bound to PK(2) receptors and activated them with a lower potency (5-fold) than Bv8. When systemically injected, A-24 induced Bv8-like hyperalgesia in rats and in mice, at doses 100 times higher than Bv8. Locally and systemically injected at inactive doses, A-24 antagonized Bv8-induced hyperalgesia. In rat and mouse models of inflammatory and post-surgical pain, A-24 showed potent and long-lasting anti-hyperalgesic activity. Unlike Bv8, A-24 increased ß-endorphin levels in mouse brain. CONCLUSIONS AND IMPLICATIONS: A-24 induced its anti-hyperalgesic effect in rodents by directly blocking nociceptor PK(1) receptors and by activating the central opioid system and the descending pain control pathway through brain PK(2) receptors.

[Treatment of gastrointestinal bleeding in neonates: a case report]. / Scelta terapeutica nelle emorragie intestinali pediatriche: un controverso caso neonatale.

Gastrointestinal bleeding in infants and children is an uncommon and potentially serious problem, but fortunately is usually limited and most cases resolve with close medical attention. The therapeutic criteria is often difficult particularly in neonates. In this work we examine the case of a neonate with serious gastrointestinal bleeding and the delayed treatment for diagnostic difficulties.

Hypertrophic obstructive cardiomyopathy and pregnancy: anesthesiological observations and clinical series.

Hypertrophic obstructive cardiomyopathy represents a genetic disorder characterized by hypertrophy, usually asymmetrical, of the ventricular musculature at the base of the septum in the left ventricular efflux tract. Patients suffering from this disorder can be extremely sensitive to small alterations in ventricular volumes, arterial pressure, cardiac frequency and rhythm. This disorder is found in pregnancy with an incidence of 0.1-0.5% and, because of its gravity, represents a contraindication which is often absolute to pregnancy. Hemodynamic variations such as those found in pregnancy, labor and delivery have complex influences on hypertrophic cardiomyopathy. Our clinical series includes 2 pregnant patients suffering from hypertrophic obstructive cardiomyopathy who both underwent caesarian section in general anesthesia, the first due to the gravity of cardiac obstruction and the second due to the emergent need to proceed after the beginning of labor. The small number of clinical cases in the literature, especially in the last few years, clearly underlines the difficulty of defining both the most correct method for delivery and the most appropriate anesthesiological techniques. In accordance with the literature and our clinical experience, we can conclude that a carefully managed pregnancy can proceed without complications in patients with moderate obstruction and that a regional anesthesiological approach is also possible with careful hemodynamic monitoring. General anesthesia, however, remains the safest method and has fewer risks for patients with serious obstruction or with worsening of their clinical condition during pregnancy.

Rat Schwann cells express M1-M4 muscarinic receptor subtypes.

The expression of different muscarinic receptor subtypes was analyzed in immature Schwann cells obtained from sciatic nerve of 2-day neonatal rats. By using RT-PCR analysis, we demonstrated the presence of M1, M2, M3, and M4 receptor subtypes in cultured Schwann cells, with M2 displaying the highest expression levels. Muscarinic subtypes were also quantified by immunoprecipitation and [3H]QNB binding. With this approach, we found the levels of receptor expression to be M2 > M3 > M1. M4 is expressed at very low levels, and M5 receptor was not detectable. Moreover, we also demonstrated that stimulation of the receptors by muscarinic agonists activates previously described signal transduction pathways, leading to a decrease of cAMP and an increase of IP3 levels not associated with an efficient intracellular Ca2+ release. The presence and activity of particular muscarinic receptors in immature Schwann cells suggest that ACh may play an important role in Schwann cell development.

Different patterns of Ca²âº signals are induced by low compared to high concentrations of P2Y agonists in microglia.

Brain-resident macrophages (microglia) are key cellular elements in the preservation of tissue integrity. On the other hand, they can also contribute to the development of pathological events by causing an extensive and inappropriate inflammatory response. A growing number of reports indicate the involvement of nucleotides in the control of microglial functions. With this study on P2Y receptors in rat microglia, we want to contribute to the definition of their expression profile and to the characterisation of their signalling mechanisms leading to Ca²âº movements. Endogenous nucleotides, when applied at a concentration of 100 muM, elicited robust Ca²âº transients, thanks to a panel of metabotropic receptors comprising mainly P2Y2, P2Y6 and P2Y12 subtypes. The involvement of P2Y12 receptors in Ca²âº responses induced by adenine nucleotides was confirmed by the pharmacological and pertussis toxin sensitivity of the response induced by adenosine diphosphate (ADP). Beside the G protein involved, Gi and Gq respectively, adenine and uracil nucleotides differed also for induction by the latter of a capacitative Ca²âº plateau. Moreover, when applied at low (sub-micromolar) concentrations with a long-lasting challenge, uracil nucleotides elicited oscillatory Ca²âº changes with low frequency of occurrence (