Fibrinolytic and factor XIII activity in subarachnoid hemorrhage Actividad fíbrinolítica y del factor XIII en la hemorragia subaracnoidea

The balance between fibrinolytic activity and coagulation mechanisms seems to play an important role in the rebleeding of a subarachnoid hemorrhage (SAH) due to aneurysmatic rupture. In the present paper we describe our findings in a group of patients (n10) with S AH. The plasmatic levels of fibrinogen and their degradation products (FDP), APTT, prothrombin activity and factor XIII were determined within 72 hours of initial bleeding, or of eventual rebleeding. Factor XIII activity in the first bleeding was 82.1 ±4%, while the levels of FDP were 3.8±lmg/ml. In patients presenting rebleeding (n4), Factor XIII activity was 67.3±4.5% the day it manifested, which is significantly less than the values previously observed (pEl balance entre la actividad fíbrinolítica y los mecanismos de la coagulación juegan un rol importante en el resangrado de una hemorragia subaracnoidea debido a ruptura aneurismástica. En el presente estúdio se describen nuestros hallazgos en un grupo de pacientes (n10) con hemorragia subaracnoidea (SAH). Los niveles plasmaticos de fibrinogeno y sus productos de degradation (FDP), la actividad de protrombina (APTT) y de factor XIII fueron determinados dentro de las 72 hs, dei sangrado inicial y ante el eventual resangrado. La actividad de factor XIII en el primer sangrado fue de 82,1±4%, mientras el nivel de FDP fue de 3,8±lmg/ml. En aquellos pacientes que resangraron (n4), la actividad de factor XIII fue de 67,3+4,5%, el dia del resangrado, el cual es significativamente menor que los valores previamente observados (p<0.01), mientras que los niveles de FDP fueron 4,1± 2mg/ml. La reduction de la actividad de factor XIII sugiere un importante rol de este en la establilidad del coágulo en el sitio de ruptura aneurismática, siendo posible atribuir un valor predicitivo a la reduction de su actividad

Facilitation of spontaneous acetylcholine release induced by activation of cAMP in rat neuromuscular junctions

Regulation of neurotransmitter release is thought to involve modulation of therelease probability by protein phosphorylation. Activation of the CAMP-proteinkinase A (PKA) pathway has been shown to facilitate synaptic transmission inmammalian neuromuscular synapses, although the relevant phosphorylation targetsare mostly unknown. We found that the inhibitor of the phosphodiesteraseaminophylline (1 m.M AMIN), the membrane-permeable analog of CAMP, 8-Br-CAMP (5 mM) and, the diect adenylate cyclase activator, forskolin (20 PM),induced an increase of miniature end-plate potentials (MEPPs) frequency in ratneuromuscular junctions. We investigated the possible involvement of the voltagedependentcalcium channels (VDCC), since these proteins are known to bephosphorylated by PKA. But this possibility was ruled out, since the increase inMEPPs frequency was not attenuated by the VDCC blocker Cd” (100 pM) and itwas observed when AMIN was studied on hyperosmotic response, which isindependent of [Ca2’10 and of Ca2’ infhrx through the VDCC. The lack of action ofAMIN on MEPPs frequency when [Ca’+]i was diminished by exposing thepreparations to zero Ca2’-EGTA solution (isotonic condition) or when nerveterminals were loaded with a permeant Ca2’ chelator (BAPTA-AM) (hypertoniccondition), indicate that cAMPmediated presynaptic facilitation is a function ofnerve terminal Ca2” concentration We also found that AMIN exerted a comparableincrease in MEPPs f%equency in control and high K” (10 and 15 mM), suggesting asingle mechanism of action for spontaneous and K+-induced secretion.Fil: Losavio, Adriana Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Muchnik, Salomon. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin

Effect of purines on calcium-independent acetylcholine release at the mouse neuromuscular junction

At the mouse neuromuscular junction, activation of adenosine A1 and P2Y receptors inhibits acetylcholine release by an effect on voltage dependent calcium channels related to spontaneous and evoked secretion. However, an effect of purines upon the neurotransmitter-releasing machinery downstream of Ca2+ influx cannot be ruled out. An excellent tool to study neurotransmitter exocytosis in a Ca2+-independent step is the hypertonic response. Intracellular recordings were performed on diaphragm fibers of CF1 mice to determine the action of the specific adenosine A1 receptor agonist 2-chloro-N6-cyclopentyl-adenosine (CCPA) and the P2Y12-13 agonist 2-methylthio-adenosine 5′-diphosphate (2-MeSADP) on the hypertonic response. Both purines significantly decreased such response (peak and area under the curve), and their effect was prevented by specific antagonists of A1 and P2Y12-13 receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and N-[2-(methylthioethyl)]-2-[3,3,3-trifluoropropyl]thio-5′-adenylic acid, monoanhydride with dichloromethylenebiphosphonic acid, tetrasodium salt (AR-C69931MX), respectively. Moreover, incubation of preparations only with the antagonists induced a higher response compared with controls, suggesting that endogenous ATP/ADP and adenosine are able to modulate the hypertonic response by activating their specific receptors. To search for the intracellular pathways involved in this effect, we studied the action of CCPA and 2-MeSADP in hypertonicity in the presence of inhibitors of several pathways. We found that the effect of CPPA was prevented by the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) while that of 2-MeSADP was occluded by the protein kinase C antagonist chelerythrine and W-7. On the other hand, the inhibitors of protein kinase A (N-(2[pbromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide, H-89) and phosphoinositide-3 kinase (PI3K) (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride, LY-294002) did not modify the modulatory action in hypertonicity of both purines. Our results provide evidence that activation of A1 and P2Y12-13 receptors by CCPA and 2-MeSADP inhibits ACh release from mammalian motor nerve terminals through an effect on a Ca2+-independent step in the cascade of the exocytotic process. Since presynaptic calcium channels are intimately associated with components of the synaptic vesicle docking and fusion processes, further experiments could clarify if the actions of purines on calcium channels and on secretory machinery are related.Fil: Veggetti, Mariela Iris. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Muchnik, Salomon. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Losavio, Adriana Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin

Excitatory effect of the A2A adenosine receptor agonist CGS-21680 on spontaneous and K+-evoked acetylcholine release at the mouse neuromuscular junction

The mechanism of action of the A2A adenosine receptor agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680) in the facilitation of spontaneous (isotonic and hypertonic condition) and K+-evoked acetylcholine (ACh) release was investigated in the mouse diaphragm muscles. At isotonic condition, the CGS-21680-induced excitatory effect on miniature end-plate potential (MEPP) frequency was not modified in the presence of CdCl2 and in a medium free of Ca2+ (0Ca2+-EGTA), but it was abolished after buffering the rise of intracellular Ca2+ with 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra(acetoxy-methyl) (BAPTA-AM) and when the Ca2+-ATPase inhibitor thapsigargin was used to deplete intracellular Ca2+ stores. CGS-21680 did not have a direct effect on the Ca2+-independent neurotransmitter-releasing machinery, since the modulatory effect on the hypertonic response was also occluded by BAPTA-AM and thapsigargin. CGS-21680 facilitation on K+-evoked ACh release was not altered by the P/Q-type voltage-dependent calcium channel (VDCC) blocker ω-Agatoxin IVA, but it was completely prevented by both, the L-type VDCC blocker nitrendipine (which is known to immobilize their gating charges), or thapsigargin, suggesting that the effects of CGS-21680 on L-type VDCC and thapsigargin-sensitive internal stores are associated. We found that the VDCC pore blocker Cd2+ (2 mM Ca2+ or 0Ca2+-EGTA) failed to affect the CGS-21680 effect in high K+ whereas nitrendipine in 0Ca2+-EGTA+Cd2+ occluded its action. The blockade of Ca2+ release from endoplasmic reticulum with ryanodine antagonized the facilitating effect of CGS-21680 in control and high K+ concentration. It is concluded that, at the mouse neuromuscular junction, activation of A2A receptors facilitates spontaneous and K+-evoked ACh release by an external Ca2+-independent mechanism but that involves mobilization of Ca2+ from internal stores: during spontaneous ACh release stimulating directly the ryanodine-sensitive stores and, at high K+, probably modulating the L-type VDCCs which may cause the opening of the ryanodine receptors that would be directly coupled to the channels. In both cases, Ca2+ released from the endoplasmic reticulum would be capable of activating the exocytotic machinery, thus producing facilitation of ACh release.Fil: Palma, Alejandra Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Neurofisiología; ArgentinaFil: Muchnik, Salomon. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Neurofisiología; ArgentinaFil: Losavio, Adriana Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Neurofisiología; Argentin

Effect of amyotrophic lateral sclerosis serum on calcium channels related to spontaneous acetylcholine release

Objectives: The aim of this work was to further investigate the effect of sera from sporadic amyotrophic lateral sclerosis (ALS) patients on miniature end-plate potentials (MEPP) frequency, by the mouse passive transfer model, and to study whether the transferred serum induces any change in the sensitivity of the L-type voltage-dependent calcium channels (VDCC) to its specific blocker Nitrendipine. Methods: A total of 35 CF1 mice were divided into 3 groups: (a) ALS group receiving sera from 15 patients that had been clinically and electromyographycally diagnosed as having sporadic ALS; (b) normal group receiving sera from 13 healthy volunteers and from 3 disease control patients, and (c) control group, which was kept untreated. Animals in groups (a) and (b) received daily intraperitoneal injection of 0.5-1ml of serum for 3 days, and 24h later the left hemidiaphragm was excised for electrophysiological recordings. Results: Analysis of MEPPs frequency recorded from ALS group showed that 3 of them induced an increase in spontaneous neurotransmitter release while in 4 a decrease was observed, suggesting that sera alter spontaneous secretion as result of an increased or decreased Ca2+ influx through the normally involved N-type or L-type VDCC, respectively. When the effect of Nitrendipine, an L-type VDCC blocker, was studied on ALS sera-injected mice, we found variable responses to the drug: only two mice showed control sensitivity to Nitrendipine, while in 7 its action was lower and surprisingly in 4 was greater than that without the drug. Conclusions: These results suggest that ALS sera contain factor(s) that are able to modify spontaneous neurotransmitter release by altering calcium current through L-type and N-type VDCC, and even inducing changes in the sensitivity to the L-type VDCC blocker.Fil: Muchnik, Salomon. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Losavio, Adriana Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: De Lorenzo, Silvana. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin

Role of adenosine in the early diagnosis of bipolar disorder

En trabajos previos encontramos que el suero de pacientes bipolares contiene una sustancia que, cuando es transferida pasivamente a ratones CF1, disminuye la liberación espontánea del neurotransmisor y ocluye el efecto inhibitorio de CCPA (agonista específico del receptor A1) en sinapsis neuromuscular. Esta acción no fue observada con el suero de pacientes esquizofrénicos, permitiendo realizar un diagnóstico diferencial entre ambas enfermedades. Para conocer la naturaleza de la sustancia activa, el suero de pacientes bipolares fue filtrado (cut off 3000) obteniéndose dos fracciones. La frecuencia de MEPP (fMEPP) registrada de diafragmas de ratones inyectados con la fracción de suero de alto PM (APM) estuvo dentro del rango control, mientras que la inyección con la fracción de suero de bajo PM (BPM) indujo una disminución de la fMEPP, y no permitió que CCPA ejerciera su efecto inhibitorio; esas acciones fueron similares a las producidas por el suero entero de pacientes bipolares. Debido a que el trastorno bipolar es una enfermedad asociada a la hiperactividad neuronal que implica un aumento de la secreción de neurotransmisor y ATP, evaluamos si la adenosina (metabolito activo del ATP), estaba involucrada en los resultados observados. Para ello estudiamos el efecto de la fracción de BPM + adenosina deaminasa (ADA, enzima que dregada adenosina a su metabolito inactivo inosina). Este procedimiento abolió la acción observada con la fracción de BPM sin ADA. Conclusiones: Estos resultados sugieren que la sustancia del suero de pacientes bipolares que induce disminución de la liberación espontánea de ACh en unión neuromuscular cuando es transferida pasivamente a los ratones, es compatible con adenosina (PM: 267.2). Esto fue demostrado por el hecho que los efectos biológicos no fueron observados cuando la fracción de BPM fue tratada con ADA. Por otro lado, se puede descartar que la sustancia involucrada sea una inmunoglobulina ya que estas tienen un PM mayor a 3000. La modulación observada con la fracción de BPM puede ser explicada por el incremento de la concentración de adenosina en el espacio sináptico como consecuencia de la hiperactividad neuronal. Este mecanismo no estaría involucrado en la esquizofrenia; por lo tanto, este estudio constituye una nueva herramienta diagnóstica para diferenciar el trastorno bipolar de la esquizofrenia.Fil: Veggetti, Mariela Iris. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: De Lorenzo, Silvana. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Losavio, Adriana Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Muchnik, Salomon. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin

Presynaptic inhibition of spontaneous acetylcholine release mediated by P2Y receptors at the mouse neuromuscular junction

At the neuromuscular junction, ATP is co-released with the neurotransmitter acetylcholine (ACh) and once in the synaptic space, it is degraded to the presynaptically active metabolite adenosine. Intracellular recordings were performed on diaphragm fibers of CF1 mice to determine the action of extracellular ATP (100 μM) and the slowly hydrolysable ATP analog 5′-adenylylimidodiphosphate lithium (βγ-imido ATP) (30 μM) on miniature end-plate potential (MEPP) frequency. We found that application of ATP and βγ-imido ATP decreased spontaneous secretion by 45.3% and 55.9% respectively. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective A1 adenosine receptor antagonist and α,β-methylene ADP sodium salt (αβ-MeADP), which is an inhibitor of ecto-5′-nucleotidase, did not prevent the inhibitory effect of ATP, demonstrating that the nucleotide is able to modulate spontaneous ACh release through a mechanism independent of the action of adenosine. Blockade of Ca2+ channels by both, Cd2+ or the combined application of nitrendipine and ω-conotoxin GVIA (ω-CgTx) (L-type and N-type Ca2+ channel antagonists, respectively) prevented the effect of βγ-imido ATP, indicating that the nucleotide modulates Ca2+ influx through the voltage-dependent Ca2+ channels related to spontaneous secretion. βγ-Imido ATP-induced modulation was antagonized by the non-specific P2 receptor antagonist suramin and the P2Y receptor antagonist 1-amino-4-[[4-[[4-chloro-6-[[3(or4)-sulfophenyl] amino]-1,3,5-triazin-2-yl]amino]-3-sulfophenyl] amino]-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic acid (reactive blue-2), but not by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt (PPADS), which has a preferential antagonist effect on P2X receptors. Pertussis toxin and N-ethylmaleimide (NEM), which are blockers of Gi/o proteins, prevented the action of the nucleotide, suggesting that the effect is mediated by P2Y receptors coupled to Gi/o proteins. The protein kinase C (PKC) antagonist chelerythrine and the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) occluded the effect of βγ-imido ATP, while the protein kinase A (PKA) antagonist KT-5720 and the inhibitor of the calcium/calmodulin-dependent protein kinase II (CAMKII) KN-62 failed to do so. βγ-Imido ATP did not affect 10, 15 and 20 mM K+-evoked release and application of reactive blue-2 before incubation in high K+ induced a higher asynchronous secretion. Thus, our results show that at mammalian neuromuscular junctions, ATP induces presynaptic inhibition of spontaneous ACh release due to the modulation of Ca2+ channels related to tonic secretion through the activation of P2Y receptors coupled to Gi/o proteins. We also demonstrated that at increasing degrees of membrane depolarization evoked by K+, endogenously released ATP induces presynaptic inhibition as a means of preventing excessive neurotransmitter secretion.Fil: de Lorenzo, María Soledad. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Veggetti, Mariela Iris. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Muchnik, Salomon. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Losavio, Adriana Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin

Differential effect of serum from bipolar versus schizophrenic patients on spontaneous acetylcholine release at mammalian neuromuscular junction

Objective: The diagnosis of bipolar disease frequently requires a long time since the age of onset, especially because the disease ismisdiagnosed with schizophrenia. The aim of the present work was to investigate whether sera from bipolar patients have an activesubstance that allows making a fast identification of the disease.Methods: Sera from healthy volunteers, euthymic and non-stabilized bipolar patients, and schizophrenic patients were passively transferredinto CF1 mice and after 2 day injections, MEPP frequency from diaphragm muscles was recorded. The same procedure was performedwith sera fraction of high and low MW (cut-off 3000).Results: Sera from non-stabilized bipolar patients induced a decreased MEPP frequency and occluded the presynaptic inhibitory effect ofthe specific adenosine A1 receptor agonist 2-chloro-N6-cyclopentyl-adenosine (CCPA) in the recipient mice, while in the euthymic bipolargroup spontaneous secretion reached control values although the action of CCPA was still prevented. Similar results were obtained withlow MW sera fraction from euthymic and non-stabilized bipolar patients. The addition of adenosine deaminase to the sera fraction preventedthe modification of spontaneous ACh release. In mice injected with sera from schizophrenic patients, MEPP frequency was withincontrol values and CCPA induced its typical inhibitory action.Conclusions: These results indicate that bipolar patients contain in their blood an active substance compatible with adenosine, which wasable to modify spontaneous ACh release in the recipient mice. This effect was not observed with sera from healthy volunteers and schizophrenicpatients. The increase of adenosine concentration may result from synaptic hyperactivity that presumably plays a role in thesymptoms of bipolar disorder and/or may derive from peripheral cells through a more general mechanism.Significance: The different results obtained with bipolar and schizophrenic sera raise the possibility that the passive transfer model couldbe used as a diagnostic test in the future.Fil: Veggetti, Mariela Iris. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: de Lorenzo, Silvana. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Cassone, Julieta. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Losavio, Adriana Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Muchnik, Salomon. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin