Magdalena Torres

Presence of diadenosine polyphosphates— Ap4A and Ap5A— In rat brain synaptic terminals. Ca2+ dependent release evoked by 4-aminopyridine and veratridine

The study of the adenine nucleotides in middle brain synaptosomes from rat showed the presence of two diadenosine polyphosphates, Ap4A and Ap5A. HPLC techniques and phosphodiesterase digestion were employed in order to characterize and quantify the dinucleotides. The Ap4A content per mg of protein was 169 +/- 25 pmol and 159 +/- 22 pmol for Ap5A. The study of the exocytotic release of these compounds was carried out with 100 microM 4-aminopyridine or 10 microM veratridine in the presence and in the absence of calcium. 4-Aminopyridine released 14.5 +/- 3.0 pmol/mg protein of Ap4A and 11.6 +/- 2.4 pmol/mg protein of Ap5A in a calcium dependent process. Veratridine in the presence of calcium released 19.9 +/- 3.0 and 16.6 +/- 2.8 pmol/mg of protein of Ap4A and Ap5A respectively. The ratios of exocytosis were close to 7-9% and 10-12% of the total synaptosomal content in the presence of 4-aminopyridine and veratridine, respectively.

Subcellular Distribution Studies of Diadenosine Polyphosphates—Ap4A and Ap5A—in Bovine Adrenal Medulla: Presence in Chromaffin Granules

Abstract: Diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) have been identified in bovine adrenal medullary tissue using an HPLC method. The values obtained were 0.1 ± 0.05 μmol/g of tissue for both compounds. The subcellular fraction where Ap4A and Ap5A were present in the highest concentration was chromaffin granules: 32 nmol/mg of protein for both compounds (∼6 mM intragranularly). This value was 30 times higher than in the cytosolic fraction. Enzymatic degradation of Ap4A and Ap5A, isolated from chromaffin granules, with phosphodiesterase produces AMP as the final product. The Ap4A and Ap5A obtained from this tissue were potent inhibitors of adenosine kinase. Their Ki values relative to adenosine were 0.3 and 2 μM for Ap4A and Ap5A, respectively. The cytosolic fraction also contains enzymatic activities that degrade Ap4A as well as Ap5A. These activities were measured by an HPLC method; the observed Km values were 10.5 ± 0.5 and 13 ± 1 μM for Ap4A and Ap5A, respectively.

Cannabinoid Type 2 Receptor Activation Downregulates Stroke-Induced Classic and Alternative Brain Macrophage/Microglial Activation Concomitant to Neuroprotection

Background and Purpose— Ischemic stroke continues to be one of the main causes of death worldwide. Inflammation accounts for a large part of damage in this pathology. The cannabinoid type 2 receptor (CB2R) has been proposed to have neuroprotective properties in neurological diseases. Therefore, our aim was to determine the effects of the activation of CB2R on infarct outcome and on ischemia-induced brain expression of classic and alternative markers of macrophage/microglial activation. Methods— Swiss wild-type and CB2R knockout male mice were subjected to a permanent middle cerebral artery occlusion. Mice were treated with either a CB2R agonist (JWH-133), with or without a CB2R antagonist (SR144528) or vehicle. Infarct outcome was determined by measuring infarct volume and neurological outcome. An additional group of animals was used to assess mRNA and protein expression of CB2R, interleukin (IL)-1&bgr;, IL-6, tumor necrosis factor &agr; (TNF-&agr;), monocyte chemoattractant protein–1 (MCP-1), macrophage inflammatory peptide (MIP) –1&agr;, RANTES, inducible nitric oxide synthase (iNOS), cyclooxygenase-2, IL-4, IL-10, transforming growth factor &bgr; (TGF-&bgr;), arginase I, and Ym1. Results— Administration of JWH-133 significantly improved infarct outcome, as shown by a reduction in brain infarction and neurological impairment. This effect was reversed by the CB2R antagonist and was absent in CB2R knockout mice. Concomitantly, administration of JWH-133 led to a lower intensity of Iba1+ microglia/macrophages and a decrease in middle cerebral artery occlusion–induced gene expression of both classic (IL-6, TNF-&agr;, MCP-1, MIP-1&agr;, RANTES, and iNOS) and alternative mediators/markers (IL-10, TGF-&bgr;, and Ym1) of microglial/macrophage activation after permanent middle cerebral artery occlusion. Conclusions— The inhibitory effect of CB2R on the activation of different subpopulations of microglia/macrophages may account for the protective effect of the selective CB2R agonist JWH-133 after stroke.

Characterization and quantification of diadenosine hexaphosphate in chromaffin cells: Granular storage and secretagogue-induced release

The presence of diadenosine hexaphosphate (Ap6A) in chromaffin cells is described. The characterization of Ap6A has been accomplished by HPLC techniques, using three different elution conditions, rechromatography, and coelution with standards. Treatment with phosphodiesterase from Crotalus durissus produced AMP and adenosine pentaphosphate. The HPLC techniques described allowed the quantification of Ap6A in the picomole range. Chromaffin granules store Ap6A in a quantity of 48.5 +/- 9.7 nmol/mg protein, with a molar ratio ATP/Ap6A of 27. In chromaffin cells the Ap6A value was 1.46 +/- 0.32 nmol/10(6) cells. Diadenosine hexaphosphate was released from chromaffin cells by the action of carbachol and a value of 64 +/- 15 pmol/10(6) cells was obtained, which represents 4-5% of the total cellular content.

Carbachol induced release of diadenosine polyphosphates -Ap4A and Ap5A- from perfused bovine adrenal medulla and isolated chromaffin cells

The diadenosine polyphosphates--Ap4A and Ap5A--were released from perfused bovine adrenal glands and recently isolated chromaffin cells by the action of carbachol. The H.P.L.C. technique reported here allowed the quantification of pmol amounts of these compounds present in biological samples from the perfusion media after stimulation. Both compounds (Ap4A and Ap5A) were identified by the retention time in H.P.L.C. chromatography, co-elution with standards, re-chromatography and destruction by the phosphodiesterase action. Bovine adrenal glands stimulated with 100 microM carbachol released 0.47 +/- 0.12 nmol/gland of Ap4A and 1.11 +/- 0.26 nmol/gland of Ap5A. Isolated bovine chromaffin cells after 100 microM carbachol, as secretagogue, released 11.1 +/- 0.8 pmol/10(6) cells of Ap4A and 15.8 +/- 1.1 pmol/10(6) cells of Ap5A. The ratio of these compounds with respect to the exocytotically released ATP and catecholamines was in the same order as that found in isolated chromaffin granules.

Comparative effects of several nitric oxide donors on intracellular cyclic GMP levels in bovine chromaffin cells: correlation with nitric oxide production.

Sodium nitroprusside, S‐nitroso‐N‐acetyl‐D,L‐penicillamine, Spermine NONOate and DEA NONOate raised cyclic GMP levels in bovine chromaffin cells in a time and concentration dependent manner with different potencies, the most potent being DEA/NO with an EC50 value of 0.38±0.02 μM. Measurements of NO released from these donors revealed that DEA/NO decomposed with a half‐life (t1/2) of 3.9±0.2 min. The t1/2 for SPER/NO was 37±3 min. SNAP decomposed more slowly (t1/2=37±4 h) and after 60 min the amount of NO produced corresponded to less than 2% of the total SNAP present. The rate of NO production from SNAP was increased by the presence of glutathione. For DEA/NO and SPER/NO there was a clear correlation between nitric oxide production and cyclic GMP increases. Their threshold concentrations were 0.05 μM and maximal effective concentration between 2.5 and 5 μM. For SNAP, threshold activation was seen at 1 μM, whereas full activation required a higher concentration (500–750 μM). The dose‐response for SNAP increases in cyclic GMP was shifted nearly two orders of magnitude lower in the presence of glutathione. At higher concentrations an inhibition of cyclic GMP accumulation was found. This effect was not observed with either the nitric oxide‐deficient SNAP analogue or other NO donors. Although NO‐donors are likely to be valuable for studying NO functions, their effective concentrations and the amount of NO released by them are very different and should be assessed in each system to ensure that physiological concentrations of NO are used.

Effect of diadenosine polyphosphates on catecholamine secretion from isolated chromaffin cells

1 The action of several diadenosine polyphosphates (AP3A, AP4A and AP5A) on basal, and on nicotine‐and high K+‐evoked, catecholamine (CA) release has been investigated. Each of the three diadenosine polyphosphates weakly but significantly increased basal CA secretion. This enhancement represented about 10% of the response evoked by 2 μm nicotine. 2 The evoked secretory response to diadenosine polyphosphates had an absolute requirement for extracellular Ca2+. 3 In contrast, these compounds had an inhibitory action on nicotine‐evoked release. This response was concentration‐dependent, EC50 values being 3.2 ± 0.4 μm, 4.0 ± 1.6 μm and 19.3 ± 4.0 μm for AP3A, AP4A, and AP5A, respectively. The lower the concentration of nicotine used to evoke secretion, the higher the inhibitory power of these compounds. 4 The CA secretion evoked by K+‐rich solutions was further enhanced by AP3A and AP5A, whereas AP4A inhibited it. The possible physiological role of these dual actions is discussed.

Characterization of diadenosine tetraphosphate (Ap4A) binding sites in cultured chromaffin cells: evidence for a P2y site.

1 Diadenosine tetraphosphate (Ap4A) a dinucleotide, which is stored in secretory granules, presents two types of high affinity binding sites in chromaffin cells. A Kd value of 8 ± 0.65 × 10−11 m and Bmax value of 5420 ± 450 sites per cell were obtained for the high affinity binding site. A Kd value of 5.6 ± 0.53 × 10−9 m and a Bmax value close to 70,000 sites per cell were obtained for the second binding site with high affinity. 2 The diadenosine polyphosphates, Ap3A, Ap4A, Ap5A and Ap6A, displaced [3H]‐Ap4A from the two binding sites, the Ki values being 1.0 nm, 0.013 nm, 0.013 nm and 0.013 nm for the very high affinity binding site and 0.5 μm, 0.13 μm, 0.062 μm and 0.75 μm for the second binding site. 3 The ATP analogues displaced [3H]‐Ap4A with the potency order of the P2y receptors, adenosine 5′‐O‐(2 thiodiphosphate) (ADP‐β‐S) > 5′‐adenylyl imidodiphosphate (AMP‐PNP) > α,β‐methylene ATP (α,β‐MeATP), in both binding sites. The Ki, values were respectively 0.075 nm, 0.2 nm and 0.75 nm for the very high affinity binding site and 0.125 μm, 0.5 μm and 0.9 μm for the second binding site.

Functional coupling of nitric oxide synthase and soluble guanylyl cyclase in controlling catecholamine secretion from bovine chromaffin cells

This study was designed to evaluate whether the enzymes of the nitric oxide/cyclic-GMP pathway, nitric oxide synthase and soluble guanylyl cyclase, are functionally coupled in controlling catecholamine secretion in primary cultures of bovine chromaffin cells. In immunocytochemical studies, 80-85% of the tyrosine hydroxylase-positive chromaffin cells also possessed phenylethanolamine-N-methyltransferase, f1p4cating their capability to synthesize epinephrine. Immunoreactivity for neuronal-type nitric oxide synthase was found in over 90% of all chromaffin cells. Reverse transcription-polymerase chain reaction also demonstrated neuronal-type nitric oxide synthase messenger RNA. Immunoreactivity for soluble guanylyl cyclase was detectable in over 95% of chromaffin cells. Double-labeling immunofluorescence studies co-localized neuronal-type nitric oxide synthase and soluble guanylyl cyclase with tyrosine hydroxylase and phenylethanolamine-N-methyltransferase in the majority of chromaffin cells. Chromaffin cells possessed basal nitric oxide synthase activity which could be stimulated by acetylcholine and inhibited by NG-nitro-L-arginine methyl ester. Activation of soluble guanylyl cyclase by endogenously synthesized nitric oxide or the nitric oxide donor compound sodium nitroprusside was blocked by the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Catecholamine release and the increase in cytosolic Ca2+ concentration evoked by acetylcholine were enhanced by inhibitors of the endogenous nitric oxide/cyclic-GMP pathway such as NG-nitro-L-arginine methyl ester, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and the protein kinase G inhibitor Rp-8-pCPT-cGMPS. These data indicate that chromaffin cells possess an autocrine nitric oxide/cyclic-GMP pathway tonically controlling the inhibition of catecholamine release.

Pre-synaptic GABAB receptors inhibit glutamate release through GIRK channels in rat cerebral cortex

Neuronal G protein‐gated inwardly rectifying potassium (GIRK) channels mediate the slow inhibitory effects of many neurotransmitters post‐synaptically. However, no evidence exists that supports that GIRK channels play any role in the inhibition of glutamate release by GABAB receptors. In this study, we show for the first time that GABAB receptors operate through two mechanisms in nerve terminals from the cerebral cortex. As shown previously, GABAB receptors reduces glutamate release and the Ca2+ influx mediated by N‐type Ca2+ channels in a mode insensitive to the GIRK channel blocker tertiapin‐Q and consistent with direct inhibition of this voltage‐gated Ca2+ channel. However, by means of weak stimulation protocols, we reveal that GABAB receptors also reduce glutamate release mediated by P/Q‐type Ca2+ channels, and that these responses are reversed by the GIRK channel blocker tertiapin‐Q. Consistent with the functional interaction between GABAB receptors and GIRK channels at nerve terminals we demonstrate by immunogold electron immunohistochemistry that pre‐synaptic boutons of asymmetric synapses co‐express GABAB receptors and GIRK channels, thus suggesting that the functional interaction of these two proteins, found at the post‐synaptic level, also occurs at glutamatergic nerve terminals.