Caffeine has a dual influence on NMDA receptor–mediated glutamatergic transmission at the hippocampus

Caffeine, a stimulant largely consumed around the world, is a non-selective adenosine receptor antagonist, and therefore caffeine actions at synapses usually, but not always, mirror those of adenosine. Importantly, different adenosine receptors with opposing regulatory actions co-exist at synapses. Through both inhibitory and excitatory high-affinity receptors (A₁R and A₂R, respectively), adenosine affects NMDA receptor (NMDAR) function at the hippocampus, but surprisingly, there is a lack of knowledge on the effects of caffeine upon this ionotropic glutamatergic receptor deeply involved in both positive (plasticity) and negative (excitotoxicity) synaptic actions. We thus aimed to elucidate the effects of caffeine upon NMDAR-mediated excitatory post-synaptic currents (NMDAR-EPSCs), and its implications upon neuronal Ca²⁺ homeostasis. We found that caffeine (30–200 μM) facilitates NMDAR-EPSCs on pyramidal CA1 neurons from Balbc/ByJ male mice, an action mimicked, as well as occluded, by 1,3-dipropyl-cyclopentylxantine (DPCPX, 50 nM), thus likely mediated by blockade of inhibitory A₁Rs. This action of caffeine cannot be attributed to a pre-synaptic facilitation of transmission because caffeine even increased paired-pulse facilitation of NMDA-EPSCs, indicative of an inhibition of neurotransmitter release. Adenosine A_2ARs are involved in this likely pre-synaptic action since the effect of caffeine was mimicked by the A_2AR antagonist, SCH58261 (50 nM). Furthermore, caffeine increased the frequency of Ca²⁺ transients in neuronal cell culture, an action mimicked by the A₁R antagonist, DPCPX, and prevented by NMDAR blockade with AP5 (50 μM). Altogether, these results show for the first time an influence of caffeine on NMDA receptor activity at the hippocampus, with impact in neuronal Ca²⁺ homeostasis.

     

Long Withdrawal of Methylphenidate Induces a Differential Response of the Dopaminergic System and Increases Sensitivity to Cocaine in the Prefrontal Cortex of Spontaneously Hypertensive Rats

Methylphenidate (MPD) is one of the most prescribed drugs for alleviating the symptoms of Attention Deficit/Hyperactivity Disorder (ADHD). However, changes in the molecular mechanisms related to MPD withdrawal and susceptibility to consumption of other psychostimulants in normal individuals or individuals with ADHD phenotype are not completely understood. The aims of the present study were: (i) to characterize the molecular differences in the prefrontal dopaminergic system of SHR and Wistar strains, (ii) to establish the neurochemical consequences of short- (24 hours) and long-term (10 days) MPD withdrawal after a subchronic treatment (30 days) with Ritalin® (Methylphenidate Hydrochloride; 2.5 mg/kg orally), (iii) to investigate the dopaminergic synaptic functionality after a cocaine challenge in adult MPD-withdrawn SHR and Wistar rats. Our results indicate that SHR rats present reduced [³H]-Dopamine uptake and cAMP accumulation in the prefrontal cortex (PFC) and are not responsive to dopaminergic stimuli in when compared to Wistar rats. After a 24-hour withdrawal of MPD, SHR did not present any alterations in [³H]-Dopamine Uptake, [³H]-SCH 23390 binding and cAMP production; nonetheless, after a 10-day MPD withdrawal, the results showed a significant increase of [³H]-Dopamine uptake, of the quantity of [³H]-SCH 23390 binding sites and of cAMP levels in these animals. Finally, SHR that underwent a 10-day MPD withdrawal and were challenged with cocaine (10 mg/kg i.p.) presented reduced [³H]-Dopamine uptake and increased cAMP production. Wistar rats were affected by the 10-day withdrawal of MPD in [³H]-dopamine uptake but not in cAMP accumulation; in addition, cocaine was unable to induce significant modifications in [³H]-dopamine uptake and in cAMP levels after the 10-day withdrawal of MPD. These results indicate a mechanism that could explain the high comorbidity between ADHD adolescent patients under methylphenidate treatment and substance abuse in adult life.     

Norepinephrine acts as D1-dopaminergic agonist in the embryonic avian retina: late expression of beta1-adrenergic receptor shifts norepinephrine specificity in the adult tissue

Dopamine is the main catecholamine found in the chick retina whereas norepinephrine is only found in trace amounts. We compared the effectiveness of dopamine and norepinephrine in promoting cyclic AMP accumulation in retinas at embryonic day 13 (E13) and from post-hatched chicken (P15). Dopamine (EC(50)=10microM) and norepinephrine (EC(50)=30microM), but not the beta(1)-adrenergic agonist isoproterenol, stimulated over seven-fold the production of cyclic AMP in E13 retina. The cyclic AMP accumulation induced by both catecholamines in embryonic tissue was entirely blocked by 2microM SCH23390, a D(1) receptor antagonist, but not by alprenolol (beta-adrenoceptor antagonist). In P15 retinas, 100microM isoproterenol stimulated five-fold the accumulation of cAMP. This effect was blocked by propanolol (10microM), but not by 2microM SCH23390. Embryonic and adult retina display beta(1) adrenergic receptor mRNA as detected by RT-PCR, but the beta(1) adrenergic receptor protein was detected only in post-hatched tissue. We conclude that norepinephrine cross-reacts with D(1) dopaminergic receptor with affinity similar to that of dopamine in the embryonic retina. In the mature retina, however, D(1) receptors become restricted to activation by dopamine. Moreover, as opposed to the embryonic tissue, norepinephrine seems to stimulate cAMP accumulation via beta(1)-like adrenergic receptors in the mature tissue.     

Purification and characterization of aprotinin from porcine lungs

Aprotinin, the most studied serine proteinase inhibitor, was isolated from porcine lung for the first time. The purified porcine aprotinin had an Mr value of ∼7 kDa. It cross-reacted with polyclonal serum anti-commercial aprotinin. About 1 μg porcine aprotinin inhibited 6 μg trypsin whereas 1 μg commercial soybean inhibitor inhibited only 1 μg trypsin. The aprotinin gene was also isolated from porcine lung: the deduced amino acid sequence showed 74% identity to bovine aprotinin.     

Effect of heavy metals on chromatin supraorganization, nuclear phenotypes, and survival ofTriatoma infestans

The nuclear phenotypes and survival of the hemipteran,Triatoma infestans (Hemiptera, Reduviidae), were studied in specimens treated with copper sulfate and methylmercury. The objective was to determine whether changes in chromatin supraorganization and insect survival similar to those promoted by other stressing agents could also be induced by heavy metals. At the concentrations used, copper sulfate and methylmercury were toxic to the cells, mainly inducing nuclear degenrration in the Malpighian tubules and being lethal to a large part of the insect population. Although some individual resistance was found, especially in fasted specimens, heavy metals were found to be much more lethal than was, for instance, a thermal shock at 0°C for 12 h. The nuclear phenotypes detected after heavy metal treatment were similar to those reported under other stressing conditions. However, the frequency at which nuclei exhibited aspects of heterochromatin unraveling was much higher than that found in fasted and thermal-shocked specimens, and was independent of the heavy metal type used. If this phenotype represents an attempt to improve opportunities for extensive cell and insect survival, it was not sufficiently effective. In 5th instar nymphs, the effect of CuSO₄ on chromatin supraorganization was detected at early steps of spermatogenesis but not in the cells which were at late spermiogenesis when the metal was administered. This is probably due to changes in nuclear protein composition and to the tightly packed state of the DNA-protein complexes occurring at spermiogenesis, which may protect chromatin from damages. However, when CuSO₄ was supplied to 4th instar nymphs, it slowed down the spermiogenesis process, possibly due to several factors including Cu²⁺ binding to DNA phosphates.     

Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells

Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1–10mM) showed that 5–10mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50mM KCl (labeled as β_III tubulin positive cells). BBG 100nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70μM and MK-801 20μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H₂O₂. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit.     

Ultrastructural chemical reaction to detect saturated phospholipids of a natural surfactant from pig lungs

The ultrastructure of the natural surfactant from pig lungs fixed by the tricomplex method with Pb(NO₃)₂ and K₃Fe(CN)₆ reveals its lamellar phase (ordered configuration). The lamellar forms are usually associated with being surface active.     

L-DOPA supply to the neuro retina activates dopaminergic communication at the early stages of embryonic development

DOPA decarboxylase (DDC; aromatic-l-amino acid decarboxylase; EC 4.1.1.28) is absent in retinas from 6-day-old chicken embryos (E6) but is expressed in retina of E8 embryos, in the presumptive outer plexiform layer. Thereafter, DDC appears in cell bodies of presumptive amacrine cells. The dopamine (DA) content of E9/10 and E15/16 retinas, pre-incubated with l-DOPA for 1 h, increased 250- and 600-fold, respectively, showing that DDC is active since early in development. Intercellular communication, measured by endogenous cyclic AMP accumulation, was observed when retinas from E9/10 to E15/16 were pre-incubated for 1 h with 1 mm l-DOPA, washed and followed by incubation in the presence of 0.5 mm 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. Cyclic AMP accumulation was prevented when pre-incubation with l-DOPA was carried out in the presence of carbidopa. Moreover, the accumulation of cyclic AMP was inhibited by SCH 23390 (2 micro m). The incubation of retinas in medium previously conditioned by retina-pigmented epithelium (RPE) also increased its cyclic AMP content with the characteristics described for l-DOPA. Our results show that dopaminergic communication takes place in the embryonic retina, before tyrosine hydroxylase expression, provided l-DOPA is supplied to the tissue. It also shows that RPE is a potential source of l-DOPA early in development.