Inhibition of N-, P/Q- and other types of Ca channels in rat hippocampal nerve terminals by the adenosine A1 receptor

The effects of the adenosine A₁ receptor agonist, N⁶-cyclopentyladenosine (CPA), on both the increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and on the release of endogenous glutamate in rat hippocampal synaptosomes were studied. The inhibitory effect of CPA on the increase in [Ca²⁺]_i stimulated with 4-aminopyridine was neutralized by the adenosine A₁ receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The inhibitory effect of CPA was greater in synaptosomes from the CA1 subregion than in whole hippocampal synaptosomes. The inhibitory effects of both CPA and of the Ca²⁺ channel blockers, ω-conotoxin GVIA, ω-conotoxin MVIIC or ω-conotoxin GVIA plus ω-conotoxin MVIIC, were greater than those caused by the Ca²⁺ channel blockers. The release of endogenous glutamate was inhibited by 41% by CPA. The inhibition observed when CPA and ω-conotoxin GVIA or CPA and ω-conotoxin MVIIC were present was also greater than the inhibition by the Ca²⁺ channel blockers alone. The presence of both ω-conotoxin GVIA and ω-conotoxin MVIIC did not completely inhibit the release of glutamate, and CPA significantly enhanced this inhibition. The membrane potential and the accumulation of [

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]tetraphenylphosphonium of polarized or depolarized synaptosomes was not affected by CPA, suggesting that adenosine did not increase potassium conductances. The present results suggest that, in hippocampal glutamatergic nerve terminals, adenosine A₁ receptor activation partly inhibits P/Q- and other non-identified types of Ca²⁺ channels.     

Uptake inhibitors do not change the effect of imidazoline α2-adrenoceptor agonists on transmitter release evoked by single pulse stimulation in mouse vas deferens

Summary The present study was undertaken to compare the presynaptic interaction of neuronal noradrenaline uptake inhibitors with imidazoline and phenylethylamine ₂adrenoceptor agonists under two different conditions: at low and high noradrenaline concentrations in the biophase.Isolated mouse vasa deferentia were stimulated with trains of 7 pulses given at 0.2 Hz and the inhibition by the ₂-adrenoceptor agonists clonidine, -methylnoradrenaline, and UK-14,304 of twitch responses was measured in the absence and in the presence of either cocaine (12 mol/l) or desipramine (40 nmol/l). The effects were determined for the first (equivalent to single pulse stimulation) and the last stimulus of each train. Both uptake inhibitors antagonized the presynaptic inhibitory effects of imidazolines (clonidine and UK-14,304) on the last twitch; the effects on the first twitch remained unchanged. In contrast, the uptake inhibitors potentiated the inhibitory effect of the phenylethylamine (-methylnoradrenaline) on both the first and the last twitches.These results support the view that the concentration of noradrenaline in the biophase plays a decisive role in the inhibition by a₂-adrenoceptor agonists of the electrically evoked release of noradrenaline. Agonists that are not substrates of neuronal uptake (i.e., clonidine, UK-14,304) become less effective when noradrenaline is present in the biophase while substrates of neuronal uptake (i. e., -methylnoradrenaline) do not. The results argue against the hypothesis that uptake inhibitors interact directly with presynaptic ₂-adrenoceptors or act at some link between uptake and receptor sites. Send offprint requests to S. Guimarães at the above address     

Reflex increases in heart-rate induced by perfusing the hind leg of the rat with solutions containing lactic acid

The hypothesis that metabolic receptors in skeletal muscle influence heart-rate during exercise was tested by means of a perfused preparation of the rat's hind legs. The isolated leg was connected to the body only by nerve and bone and was perfused with tyrode solution. The humoral changes of exercise were simulated by perfusing with modified tyrode solutions in which concentration of K⁺, osmolality, concentrations of lactic acid, and inorganic phosphate were changed to reflect to those occurring during heavy exercise. Only perfusion with a solution enriched with lactic acid elicited a significant increase in heart-rate. The response disappeared when the nerve supply to the leg was cooled or sectioned. 20–60 s after the start of perfusion with solution of high [lactic acid] heart-rate began to increase reaching a maximum ( ± SE = 20.2 ± 8.2,n = 7) after about 2 min. The effect on heart-rate increased when the venous concentration of lactic acid was increased the range from 3 to 10 mmol/l. In further experiments, we tried to separate the effects of pH and lactate. Heart-rate responses were induced only at low pH and at low pH the extent to which heart-rate changed increased with increases in lactate concentration.     

Early-onset gastric carcinomas display molecular characteristics distinct from gastric carcinomas occurring at a later age

Gastric cancer is thought to result from a combination of environmental factors and accumulation of specific genetic alterations, and consequently mainly affects older patients (>50 years of age). Fewer than 10% of patients present with the disease before 45 years of age and these young patients are thought to develop carcinomas with a different molecular genetic profile from that of sporadic carcinomas occurring at a later age. Forty early-onset gastric carcinoma resection specimens were characterized for microsatellite instability (MSI) and loss of heterozygosity status using 22 polymorphic microsatellite markers. Twenty-four biopsies were additionally evaluated for the presence of MSI. No MSI was observed in any of the cases analysed. Losses were infrequent, but were most common for the D1S234 (26.1%) and D1S1676 (17.4%) markers, flanking the RUNX3 gene; for the p53ALU (23.1%) and TP53 (15.4%) markers, near the TP53 gene; and for the D16S2624 (17.2%) marker, near the E-cadherin (CDH1) gene. All cases with loss of CDH1, as well as 6/7 cases with loss of TP53, displayed aberrant staining of the corresponding proteins, pointing to a functional role for these proteins in early-onset gastric carcinogenesis. No germline CDH1, TP53 or RUNX3 mutations were detected in any of the cases analysed. No correlation was observed between non-functional E-cadherin and the histological type of the tumours analysed. Finally, Epstein-Barr virus was not detected in any of the cases analysed. On the basis of these results, early-onset gastric carcinomas appear to have characteristics distinct from gastric carcinomas occurring at a later age.     

Effect of resin hydrophilicity and water storage on resin strength

This study evaluated the change in the ultimate tensile strength (UTS) of five polymerised resin blends of increasing hydrophilicity, after ageing in distilled water or silicon oil. Resin blocks were prepared from each resin blend by dispensing the uncured resin into a flexible, embedding mould, containing multiple cavities. The resins were polymerised in the moulds under nitrogen at 551.6 kPa and light-activated at 125 degrees C for 10 min. After dry ageing for 24 h at 37 degrees C, the middle third of each resin specimen was trimmed into an 'I' shape. Fifteen control specimens were randomly selected from each resin blend for baseline UTS evaluation. The UTS of the experimental specimens were determined after 1, 3, 6 and 12 months of ageing in water or oil. The UTS of each group of resins at different storage periods in water or oil were analysed using the Friedman multiple ANOVA on ranks and Dunn's multiple comparison tests at 95% confidence level. Significant reduction (p < 0.01) in UTS was observed in Groups II-V resins after 12-month storage in water, while the most hydrophobic Group I resin showed no significant change (p > 0.05) in the same period. The percentage reduction in UTS increased with the hydrophilicity of the resin blends. Long-term water storage of hydrophilic resin blends such as those employed in dentine adhesives, resulted in a marked reduction in their mechanical strength that may compromise the durability of resin-dentine bonds.     

Association between atopy and recurrent vaginal candidiasis

To determine whether there is an association between atopy and recurrent vaginal candidiasis (RVC) and to evaluate the type-2 immune response in patients with RVC. Evaluation of immediate hypersensitivity skin tests to aeroallergens, measurement of total IgE and Candida albicans specific IgE and levels of IL-5 in 44 women with RVC and 26 with sporadic vaginal candidiasis (SVC). Statistical analyses were performed by Mann-Whitney test and chi(2) test with Yates correction. History of atopy (68%) and positive skin test (42%) were higher (P < 0.05) in RVC than in patients with SVC. No significant difference was found in total IgE, C. albicans specific IgE and IL-5 levels. There was a strong association between atopy and RVC, but type-2 immune response to C. albicans antigen was absent or similar in the two groups of patients.     

Intracellular calcium mobilization by muscarinic receptors is regulated by micromolar concentrations of external Ca2+

Carbachol-induced contractions of rat stomach fundus strips, obtained in a nutrient solution containing 1.8 mM Ca2+, were resistant to Ca2+ withdrawal, even after 1 h of bathing the tissues in a nominal 0 Ca2+ solution. This was not observed when K+ was used to evoke contractions, which were rapidly inhibited after Ca2+ removal (t1/2=2 min). The effect of carbachol in 0 Ca2+ solution was reduced by using drugs that reduce intracellular pools of Ca2+, such as caffeine (1-3 mM), ryanodine (30 microM) or thapsigargin (1 microM), corroborating the involvement of intracellular Ca2+ stores. On the other hand, when the 0 Ca2+ solution contained EGTA, a complete decline of carbachol effects was observed within about 8 min, indicating the involvement of extracellular Ca2+. Atomic absorption spectrometry showed that our 0 Ca2+ solution still contained 45 microM Ca2+, which was drastically reduced to 5.9 nM in the presence of EGTA. Taken together, our results indicate that the effects of carbachol are due to the mobilization of caffeine-, ryanodine- and thapsigargin-sensitive intracellular Ca2+ stores, and that these stores are not inactivated or depleted if micromolar concentrations (45 microM), but not nanomolar concentrations (5.9 nM) of Ca2+ are maintained in the extracellular milieu.     

Comparison of C(18)-carboxypropylbetaine and standard N-acetyl-L-cysteine-NaOH processing of respiratory specimens for increasing tuberculosis smear sensitivity in Brazil

Techniques to improve the sensitivity of smear microscopy would facilitate early tuberculosis (TB) diagnosis and disease control, especially in low-income countries where the positive predictive value is high. C(18)-carboxypropylbetaine (CB-18) is a zwitterionic detergent that helps to compensate for the innate buoyancy of mycobacteria, potentially enhancing recovery by centrifugation. Previous data suggest that CB-18 may increase the sensitivity of smear, culture, and molecular amplification diagnostic testing. The goal of the present study was to evaluate if the sensitivity of the smear technique using light microscopy could be improved by treating respiratory samples with CB-18. In the first phase, respiratory specimens were collected consecutively from patients with suspected pulmonary tuberculosis in a tertiary-care hospital in Rio de Janeiro, Brazil (236 specimens were analyzed). After protocol modifications, another 120 respiratory specimens were evaluated. The standard technique was N-acetyl-L-cysteine with sodium hydroxide (NALC-NaOH) treatment, smear concentration with centrifugation, and Ziehl-Neelsen staining. Culture on L?wenstein-Jensen slants was performed on all specimens for use as the "gold standard." No specimens from patients undergoing active TB treatment were included. The initial protocol for CB-18 processing resulted in a sensitivity of 59.6% and specificity of 96.8% compared to standard processing with a sensitivity of 66.0% and specificity of 96.8%. Using the modified protocol, the sensitivity of CB-18 increased to 71.4% with a specificity of 97.0% versus standard processing with a sensitivity of 61.9% and a specificity of 99.0%. The diagnostic yield of acid-fast bacillus smear with CB-18 in the absence of fluorescence microscopy and PCR compared to standard processing with NALC-NaOH was not significantly different, although the power to detect a difference by the modified assay was low.