María Ruiz

Adenosine A1 receptor down-regulation in mothers and fetal brain after caffeine and theophylline treatments to pregnant rats

Pregnant rats were treated daily with 1 g/L of caffeine or theophylline in their drinking water during pregnancy and the effect of these methylxanthines on adenosine A1 receptor was assayed using binding and reverse transcription polymerase chain reaction (RT–PCR) assays in brains from both mothers and full‐term fetuses. In plasma membranes from pregnant rat brain, caffeine and theophylline caused a significant decrease in total receptor numbers, of the same order in both cases (30%), with no significant changes on receptor affinity. The effect of these adenosine receptor antagonists on plasma membranes from fetal brains was more marked, being detected at approximately 50% of the total receptors detected in control conditions. However, in this tissue, a significant increase in the receptor affinity, of the same order in both cases, was also detected after antagonist administration. No significant variation on the potency of caffeine and theophylline as antagonists was detected after treatments in mothers; however, higher affinities were detected in fetuses. A decrease in the total receptor numbers in fetal brain was associated with an increase in the mRNA coding A1 receptor, as determined by RT–PCR assays, not having detected any mRNA difference in maternal brain. No variation in the levels of mRNA coding A2A receptor was detected in any case. These results suggest that maternal caffeine or theophylline intake modulates adenosine A1 receptor, causing a down‐regulation of adenosine A1 receptor in brain in both mothers and fetuses.

Adenosine A1 receptor in cultured neurons from rat cerebral cortex : Colocalization with Adenosine deaminase

Adenosine A1 receptors (A1Rs) have been characterized in primary cultures of neurons from cerebral cortex. The specific adenosine A1 antagonist 8‐cyclopentyl‐1,3‐[3H]dipropylxanthine bound to both membranes and intact cells. When saturation experiments were performed in membranes, a KD value of 0.76 nM and a Bmax of 57 fmol/mg of protein were obtained. Competition assays revealed a pharmacological profile characteristic of A1Rs. The presence of this receptor was further confirmed by RT‐PCR analysis. The expression of the receptor showed no significant changes during the period of culture studied, up to 12 days in vitro. A1R agonist inhibited forskolin‐stimulated adenylyl cyclase, showing the functional coupling of these receptors with the effector. αGi1,2 protein level, detected by immunoblot, presented an increase during the period of culture. This increase correlated with an increase in the mRNA level of αGi1 but not αGi2. By immunochemical assays, it is shown that these receptors are expressed in both the neuronal cell body and the proximal dendrites. Colocalization of A1Rs with microtubule‐associated protein 2 and cell surface adenosine deaminase was shown by confocal microscopy. The high degree of colocalization observed between A1Rs and ectoadenosine deaminase in neurons could suggest an important role of the enzyme in adenosine‐mediated neuromodulation.

Diastereoselective synthesis of piperidine imino sugars using aldol additions of metalated bislactim ethers to threose and erythrose acetonides.

A general strategy for the synthesis of 1-deoxy-azasugars from a chiral glycine equivalent and 4-carbon building blocks is described. Diastereoselective aldol additions of metalated bislactim ethers to matched and mismatched erythrose or threose acetonides and intramolecular N-alkylation (by reductive amination or nucleophilic substitution) were used as key steps. The dependence of the yield and the asymmetric induction of the aldol addition with the nature of the metallic counterion of the azaenolate and the gamma-alkoxy protecting group for the erythrose or threose acetonides has been studied. The stereochemical outcome of the aldol additions with tin(II) azaenolates has been rationalized with the aid of density functional theory (DFT) calculations. In accordance with DFT calculations with model glyceraldehyde acetonides, high trans,syn,anti-selectivitity for the matched pairs and moderate to low trans,anti,anti-selectivity for the mismatched ones may originate from (1) the intervention of solvated aggregates of tin(II) azaenolate and lithium chloride as the reactive species and (2) favored chair-like transition structures with a Cornforth-like conformation for the aldehyde moiety. DFT calculations indicate that aldol additions to erythrose acetonides proceed by an initial deprotonation, followed by coordination of the alkoxy-derivative to the tin(II) azaenolate and final reorganization of the intermediate complex through pericyclic transition structures in which the erythrose moiety is involved in a seven-membered chelate ring. The preparative utility of the aldol-based approach was demonstrated by application in concise routes for the synthesis of the glycosidase inhibitors 1-deoxy-d-allonojirimycin, 1-deoxy-L-altronojirimycin, 1-deoxy-D-gulonojirimycin, 1-deoxy-D-galactonojirimycin, 1-deoxy-L-idonojirimycin and 1-deoxy-D-talonojirimycin.

Modulation of adenosine A1 and A2A receptors in C6 glioma cells during hypoxia: involvement of endogenous adenosine.

During hypoxia, extracellular adenosine levels are increased to prevent cell damage, playing a neuroprotective role mainly through adenosine A1 receptors. The aim of the present study was to analyze the effect of hypoxia in both adenosine A1 and A2A receptors endogenously expressed in C6 glioma cells. Two hours of hypoxia (5% O2) caused a significant decrease in adenosine A1 receptors. The same effect was observed at 6 h and 24 h of hypoxia. However, adenosine A2A receptors were significantly increased at the same times. These effects were not due to hypoxia‐induced alterations in cells number or viability. Changes in receptor density were not associated with variations in the rate of gene expression. Furthermore, hypoxia did not alter HIF‐1α expression in C6 cells. However, HIF‐3α, CREB and CREM were decreased. Adenosine A1 and A2A receptor density in normoxic C6 cells treated with adenosine for 2, 6 and 24 h was similar to that observed in cells after oxygen deprivation. When C6 cells were subjected to hypoxia in the presence of adenosine deaminase, the density of receptors was not significantly modulated. Moreover, DPCPX, an A1 receptor antagonist, blocked the effects of hypoxia on these receptors, while ZM241385, an A2A receptor antagonist, was unable to prevent these changes. These results suggest that moderate hypoxia modulates adenosine receptors and cAMP response elements in glial cells, through a mechanism in which endogenous adenosine and tonic A1 receptor activation is involved.

Access to iminosugars by aldol additions of metalated bis-lactim ethers to l-erythrose derivatives

Abstract Aldol additions of metalated bis-lactim ethers derived from cyclo -[Gly- d -Val] 3A – E* to mismatched l -erythrose-derivatives 4a and 4b have been studied. Reactions of titanium(IV) and tin(II) azaenolates with the lactol derivative 4b allow direct and moderate ( syn , syn )- or highly ( anti , anti )-selective access to polyhydroxy amino acids that have been efficiently transformed into 1-deoxy- d -gulonojirimycin or 1-deoxy- d -allonojirimycin.

Effect of chronic gestational treatment with caffeine or theophylline on Group I metabotropic glutamate receptors in maternal and fetal brain

Pregnant rats were treated throughout the gestational period with either caffeine or theophylline, and its effect on the metabotropic glutamate receptor (mGluRs) signal transduction pathway was studied in both maternal and fetal brain. In maternal brain, radioligand binding assays showed that chronic treatment with methylxanthines caused a significant decrease in the total number of mGluRs. This decrease was accompanied by an increase in receptor affinity. Immunodetection showed that mGluR1a and phospholipase C β1 (PLCβ1) were significantly decreased in response to chronic methylxanthine treatment, whereas αGq/11 was not affected. A loss was also detected of PLC stimulation mediated by (S)‐3,5‐dihydroxyphenylglycine (DHPG), a selective Group I mGluR agonist, suggesting desensitization of the mGluR/PLC pathway. In fetal brain, a loss in total mGluRs was observed in fetuses from mothers treated with caffeine or theophylline, without variation in receptor affinity. A decrease in mGluR1a, αGq/11 and PLCβ1 levels was also observed in response to treatment. However, changes detected in this immature tissue were not associated with variations in PLC activity. These results suggest that chronic caffeine or theophylline treatment down‐regulates several mGluR/PLC transduction pathway components in both maternal and fetal brain, causing a loss of receptor responsiveness only in maternal brain.

Metabotropic glutamate receptor/phospholipase C system in female rat heart

Glutamate is the main excitatory neurotransmitter in the central nervous system. This amino acid mediates learning and memory processes acting through ionotropic and metabotropic receptor binding. Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that stimulate phospholipase C (PLC) or inhibit adenylyl cyclase (AC). MGluRs have been widely described in CNS. However, little is known about these receptors in peripheral system. The present work describes the mGluR/PLC pathway in membranes from pregnant and non-pregnant rat heart by radioligand binding, Western-blot assays and PLC activity determination. Furthermore, mRNA coding mGluR1, mGluR5, alphaGq/11 and PLCbeta1 was identified by RT-PCR. Binding assays indicated total mGlu receptor numbers of 4.7+/-0.2 pmol/mg protein and 4.2+/-1.0 pmol/mg protein in non-pregnant and pregnant rats respectively, and their corresponding KD values were 545.3+/-85.6 nM and 1062.8+/-393.6 nM. Western blots revealed bands corresponding to mGluR1 and mGluR5 receptors, confirming that these receptors are expressed in heart. The beta1 isoform of PLC, which mediates group I mGluRs (mGluR I) response, was also expressed in rat heart. Moreover, PLC activity was modulated by calcium in a dose-dependent manner. Finally, specific agonists for mGluRs increased the PLC activity and the increase was prevented by specific mGluR antagonists. These results demonstrate the presence of group I mGlu receptors and their functional coupling to the PLC stimulation in female rat heart, suggesting a possible role of mGluR/PLC pathway in this tissue.

Down‐regulation of rat brain adenosine A1 receptors at the end of pregnancy

The status of the adenosine A1 receptor/adenylyl cyclase (A1R/AC) transduction pathway in rat brain was analysed at the end of pregnancy using different approaches. Pregnancy at term caused a significant decrease in the Bmax value obtained by saturation binding assays using [3H]DPCPX as radioligand, suggesting a down‐regulation of adenosine A1 receptor. Moreover, A1 receptor immunodetection in pregnant rat membranes and the level of mRNA coding A1 receptor were significantly decreased. This loss of A1 receptor was associated with a significant increase in receptor affinity, since the KD value from the [3H]DPCPX saturation curve and Ki for N6‐cyclohexyladenosine (CHA) were decreased in pregnant rats. Surprisingly, CHA‐mediated inhibition of adenylyl cyclase was increased, reflecting enhanced receptor responsiveness. On the other hand, immunoblotting of different αGi‐protein isoforms revealed a significant increase in αGi3 level in membranes from pregnant rats. Pre‐incubation of membranes with anti‐αGi3 antibody blocked the guanosine triphosphate (GTP) or CHA inhibitory effect on adenylyl cyclase in both pregnant and non‐pregnant rats, pointing to αGi3 as the main isoform involved in the A1 receptor response. These results suggest that, at the end of pregnancy, there is a down‐regulation of adenosine A1 receptors counterbalanced with a strengthened functionality, probably due to an increase in both αGi3 protein and receptor affinity.

Adenosine A1 receptor agonist treatment up-regulates rat brain metabotropic glutamate receptors.

Chronic R-N(6)-phenylisopropiladenosine (R-PIA) subcutaneous injection for 6 days significantly increased total glutamate receptor number (180% of control) in rat brain synaptic plasma membranes (SPM), without affecting receptor affinity. A higher increase in metabotropic glutamate (mGlu) receptor number (258% of control) was also detected, indicating that mGlu is the main type of glutamate receptor affected by this treatment. On the other hand, the observed increase in basal and calcium- and Gpp(NH)p-stimulated phospholipase C (PLC) activity after treatment was associated with a significant increase in PLC beta(1) isoform, detected in SPM by immunoblotting assays. Moreover, an increase in PLC activity stimulation with trans-ACPD, in the absence and in the presence of Gpp(NH)p, was detected after R-PIA treatment. These results show that mGlu receptors and its effector system, PLC activity, are up-regulated by chronic exposure to an adenosine A(1) receptor agonist and suggest the existence of a cross-talk mechanism between both signal transduction pathways in rat brain.

Chronic intake of caffeine during gestation down regulates metabotropic glutamate receptors in maternal and fetal rat heart

Summary.Caffeine is the most widely consumed substance in the world which antagonizes adenosine effects. Adenosine acting through A1 receptors inhibits glutamate release which binds to metabotropic glutamate receptors (mGluRs). Recently, we have shown that maternal caffeine intake during gestation causes down-regulation of A1 and metabotropic glutamate receptors in the brain of both rat mothers and fetuses. In the present work we provide evidence that caffeine also affects receptors in hearts, causing a decrease in mGluRs from both maternal and fetal hearts. A decrease in Gq/11 and PLC β1 proteins level was also observed in both tissues. However, phospholipase C activity was only affected in fetal heart, being significantly decreased. These results suggest an in vivo cross-talk mechanism between adenosine and glutamate receptors in peripheral tissues. Therefore, special attention should be paid to caffeine ingestion during gestation.