Real-World Outcomes of Patients Starting Intravenous and Transitioning to Subcutaneous Vedolizumab in Inflammatory Bowel Disease.

BACKGROUND: Real-world data on starting intravenous (IV) vedolizumab (VDZ) and transitioning to subcutaneous (SC) treatment in inflammatory bowel disease (IBD) are scarce. AIMS: To assess treatment outcomes of patients with IBD starting IV VDZ and switching to SC VDZ in routine clinical care. METHODS: Adult patients with IBD switching from IV to SC VDZ treatment between 1 March 2020 and 31 December 2021 were identified from the Swedish IBD quality register. The primary outcome was SC VDZ persistence. Secondary outcomes included clinical remission, changes in quality of life (QoL) according to EuroQual 5-Dimensions 5-Levels (EQ-5D-5L) and the Short-Health Scale (SHS) and inflammatory markers, including faecal Calprotectin (FCP). RESULTS: Altogether, 406 patients with IBD (Crohn's disease, n = 181; ulcerative colitis, n = 225) were identified. After a median follow-up of 30 months from starting IV VDZ treatment, the persistence rates were 98%(178/181) in Crohn's disease and 94% (211/225) in ulcerative colitis. Most patients (84%) transitioned during maintenance therapy, and the median follow-up from switch to SC VDZ was 10 months. Compared to baseline, statistically significant improvements were observed in all domains of the SHS, EQ-5D index value and visual analogue scale. Median (interquartile range) FCP concentrations (µg/g) decreased from 459 (185-1001) to 65 (26-227) in Crohn's disease (n = 45; p < 0.001) and from 646 (152-1450) to 49 (20-275) in ulcerative colitis (n = 58; p < 0.001). CONCLUSION: Initiating IV VDZ and switching to SC treatment was associated with high persistence rates and improvements in measures of QoL and FCP. These findings are reassuring for patients who start IV VDZ and switch to SC VDZ.

A pragmatic approach for mortality prediction after surgery in infective endocarditis: optimizing and refining EuroSCORE.

OBJECTIVE: To simplify and optimize the ability of EuroSCORE I and II to predict early mortality after surgery for infective endocarditis (IE). METHODS: Multicentre retrospective study (n = 775). Simplified scores, eliminating irrelevant variables, and new specific scores, adding specific IE variables, were created. The performance of the original, recalibrated and specific EuroSCOREs was assessed by Brier score, C-statistic and calibration plot in bootstrap samples. The Net Reclassification Index was quantified. RESULTS: Recalibrated scores including age, previous cardiac surgery, critical preoperative state, New York Heart Association >I, and emergent surgery (EuroSCORE I and II); renal failure and pulmonary hypertension (EuroSCORE I); and urgent surgery (EuroSCORE II) performed better than the original EuroSCOREs (Brier original and recalibrated: EuroSCORE I: 0.1770 and 0.1667; EuroSCORE II: 0.2307 and 0.1680). Performance improved with the addition of fistula, staphylococci and mitral location (EuroSCORE I and II) (Brier specific: EuroSCORE I 0.1587, EuroSCORE II 0.1592). Discrimination improved in specific models (C-statistic original, recalibrated and specific: EuroSCORE I: 0.7340, 0.7471 and 0.7728; EuroSCORE II: 0.7442, 0.7423 and 0.7700). Calibration improved in both EuroSCORE I models (intercept 0.295, slope 0.829 (original); intercept -0.094, slope 0.888 (recalibrated); intercept -0.059, slope 0.925 (specific)) but only in specific EuroSCORE II model (intercept 2.554, slope 1.114 (original); intercept -0.260, slope 0.703 (recalibrated); intercept -0.053, slope 0.930 (specific)). Net Reclassification Index was 5.1% and 20.3% for the specific EuroSCORE I and II. CONCLUSIONS: The use of simplified EuroSCORE I and EuroSCORE II models in IE with the addition of specific variables may lead to simpler and more accurate models.

Risk factors for exacerbation in chronic obstructive pulmonary disease: a prospective study.

BACKGROUND: Although acute exacerbations are key events in the progression of chronic obstructive pulmonary disease (COPD), their frequency and the factors associated with acute exacerbation are not fully known. OBJECTIVE: To determine the incidence and risk factors of very frequent exacerbations in COPD (⩾3 per year). PATIENTS AND METHODS: In a cohort study to analyse acute exacerbation and associated factors in 512 primary care patients during a 2-year follow-up, variables of interest were collected for each patient. Acute exacerbation was defined as an event that required antibiotics and/or systemic steroids (moderate) or hospital admission (severe). Odds ratios (OR) were used to determine factors associated with exacerbation. RESULTS: Incidence of exacerbation was 61.7% in the first year of follow-up and 63.9% in the second year. During the first year, the factors associated with very frequent exacerbation were previous hospital admission (OR 1.69), dyspnoea (moderate [OR 2.86] and severe-very severe [OR 5.83]) and the Charlson Index (OR 1.19); during the second year, associated factors were female sex (OR 4.17), history of previous hospital admissions (OR 2.90), smoking (smoker/ex-smoker) (OR 2.00) and forced vital capacity (OR 0.98). CONCLUSIONS: Incidence of exacerbation is high in COPD patients. Previous admission for exacerbation is a strong predictor and can identify patients at risk.

A major population of mucosal memory CD4+ T cells, coexpressing IL-18Rα and DR3, display innate lymphocyte functionality.

Mucosal tissues contain large numbers of memory CD4(+) T cells that, through T-cell receptor-dependent interactions with antigen-presenting cells, are believed to have a key role in barrier defense and maintenance of tissue integrity. Here we identify a major subset of memory CD4(+) T cells at barrier surfaces that coexpress interleukin-18 receptor alpha (IL-18Rα) and death receptor-3 (DR3), and display innate lymphocyte functionality. The cytokines IL-15 or the DR3 ligand tumor necrosis factor (TNF)-like cytokine 1A (TL1a) induced memory IL-18Rα(+)DR3(+)CD4(+) T cells to produce interferon-γ, TNF-α, IL-6, IL-5, IL-13, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-22 in the presence of IL-12/IL-18. TL1a synergized with IL-15 to enhance this response, while suppressing IL-15-induced IL-10 production. TL1a- and IL-15-mediated cytokine induction required the presence of IL-18, whereas induction of IL-5, IL-13, GM-CSF, and IL-22 was IL-12 independent. IL-18Rα(+)DR3(+)CD4(+) T cells with similar functionality were present in human skin, nasal polyps, and, in particular, the intestine, where in chronic inflammation they localized with IL-18-producing cells in lymphoid aggregates. Collectively, these results suggest that human memory IL-18Rα(+)DR3(+) CD4(+) T cells may contribute to antigen-independent innate responses at barrier surfaces.

Targeting T-cell migration in inflammatory bowel disease.

Crohn's disease and ulcerative colitis are chronic inflammatory disorders of the gastrointestinal tract and are collectively referred to as inflammatory bowel disease (IBD). IBD is a major cause of lifetime morbidity, has a severe impact on quality of life of patients (equivalent to that of rheumatoid arthritis, asthma, migraine or diabetes) and constitutes a substantial economic burden to the healthcare system. The introduction of anti-tumour necrosis factor (TNF) antibodies has dramatically improved the treatment of IBD, but approximately one-third of patients are nonresponders and another 30-50% will eventually lose the therapeutic effect or become intolerant to these antibodies. Thus, there is an urgent and unmet need for new therapies. The aetiologies of the different forms of IBD have not been fully elucidated but there is strong evidence implicating T cells and T-cell migration to the gut in initiating and perpetuating the intestinal inflammatory process and tissue destruction. In recent years, progress in basic science has shed light on the mechanisms regulating T-cell migration to the gut and new therapeutics targeting these pathways have been developed. It is interesting that some of the factors directing the localization of T cells to the gut have been shown to be relatively organ specific, potentially enabling new T-cell-targeted treatments to demonstrate improved safety whilst preserving therapeutic efficacy. Here, fundamental aspects of the gut immune system, the generation of tissue-tropic effector T cells and the mechanisms of T-cell trafficking to the gut mucosa will be reviewed. In addition, the role of these processes in IBD and how they have been exploited for the development of novel therapies for IBD will be discussed.

Ionic dependence of the velocity of release of ATP from permeabilized cholinergic synaptic vesicles.

Evidence is provided to show that synaptic vesicles have an internal matrix. Suspensions of cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata fish were permeabilized in solutions containing low concentrations of Na(+) or Ca(2+). The release of ATP from the vesicular matrix was 10 times more effective with Ca(2+) than with Na(+). We ascertained whether these two cations induced a different velocity of release of ATP from the matrix. The release of ATP was monitored with the chemiluminescent reaction of luciferin-luciferase. The light signal generated was the result of the kinetics of ATP release of the enzymatic reaction. To overcome the kinetics of the enzymatic reaction, the light records were deconvoluted. The actual kinetics of ATP release of vesicles containing Na(+) or Ca(2+) were coincident. To validate this result, comparison was made with ATP release from intact nerve terminals which were already deconvoluted. The results show that the real time course of release is longer than that obtained from synaptic vesicles. This was as expected given that the release of neurotransmitters is due to successive molecular steps of synaptic vesicle exocytosis.

ATP crossing the cell plasma membrane generates an ionic current in xenopus oocytes.

The presence of ATP within cells is well established. However, ATP also operates as an intercellular signal via specific purinoceptors. Furthermore, nonsecretory cells can release ATP under certain experimental conditions. To measure ATP release and membrane currents from a single cell simultaneously, we used Xenopus oocytes. We simultaneously recorded membrane currents and luminescence. Here, we show that ATP release can be triggered in Xenopus oocytes by hyperpolarizing pulses. ATP release (3.2 +/- 0.3 pmol/oocyte) generated a slow inward current (2.3 +/- 0.1 microA). During hyperpolarizing pulses, the permeability for ATP(4-) was more than 4000 times higher than that for Cl(-). The sensitivity to GdCl(3) (0. 2 mm) of hyperpolarization-induced ionic current, ATP release and E-ATPase activity suggests their dependence on stretch-activated ion channels. The pharmacological profile of the current inhibition coincides with the inhibition of ecto-ATPase activity. This enzyme is highly conserved among species, and in humans, it has been cloned and characterized as CD39. The translation, in Xenopus oocytes, of human CD39 mRNA encoding enhances the ATP-supported current, indicating that CD39 is directly or indirectly responsible for the electrodiffusion of ATP.

Constitutive expression of stromal derived factor-1 by mucosal epithelia and its role in HIV transmission and propagation.

HIV particles that use the chemokine receptor CXCR4 as a coreceptor for entry into cells (X4-HIV) inefficiently transmit infection across mucosal surfaces [1], despite their presence in seminal fluid and mucosal secretions from infected individuals [2] [3] [4]. In addition, although intestinal lymphocytes are susceptible to infection with either X4-HIV particles or particles that use the chemokine receptor CCR5 for viral entry (R5-HIV) during ex vivo culture [5], only systemic inoculation of R5-chimeric simian-HIV (S-HIV) results in a rapid loss of CD4(+) intestinal lymphocytes in macaques [6]. The mechanisms underlying the inefficient capacity of X4-HIV to transmit infection across mucosal surfaces and to infect intestinal lymphocytes in vivo have remained elusive. The CCR5 ligands RANTES, MIP-1alpha and MIP-1beta suppress infection by R5-HIV-1 particles via induction of CCR5 internalization, and individuals whose peripheral blood lymphocytes produce high levels of these chemokines are relatively resistant to infection [7] [8] [9]. Here, we show that the CXCR4 ligand stromal derived factor-1 (SDF-1) is constitutively expressed by mucosal epithelial cells at sites of HIV transmission and propagation. Furthermore, CXCR4 is selectively downmodulated on intestinal lymphocytes within the setting of prominent SDF-1 expression. We postulate that mucosally derived SDF-1 continuously downmodulates CXCR4 on resident HIV target cells, thereby reducing the transmission and propagation of X4-HIV at mucosal sites. Moreover, such a mechanism could contribute to the delayed emergence of X4 isolates, which predominantly occurs during the later stages of the HIV infection.

Mucosal T lymphocyte numbers are selectively reduced in integrin alpha E (CD103)-deficient mice.

The mucosal lymphocyte integrin alpha E(CD103)beta 7 is thought to be important for intraepithelial lymphocyte (IEL) localization or function. We cloned the murine integrin gene encoding alpha E, localized it to chromosome 11, and generated integrin alpha E-deficient mice. In alpha E-/- mice, intestinal and vaginal IEL numbers were reduced, consistent with the known binding of alpha E beta 7 to E-cadherin expressed on epithelial cells. However, it was surprising that lamina propria T lymphocyte numbers were diminished, as E-cadherin is not expressed in the lamina propria. In contrast, peribronchial, intrapulmonary, Peyer's patch, and splenic T lymphocyte numbers were not reduced in alpha E-deficient mice. Thus, alpha E beta 7 was important for generating or maintaining the gut and vaginal T lymphocytes located diffusely within the epithelium or lamina propria but not for generating the gut-associated organized lymphoid tissues. Finally, the impact of alpha E deficiency upon intestinal IEL numbers was greater at 3-4 wk of life than in younger animals, and affected the TCR alpha beta+ CD8+ T cells more than the gamma delta T cells or the TCR alpha beta+ CD4+CD8- population. These findings suggest that alpha E beta 7 is involved in the expansion/recruitment of TCR alpha beta+ CD8+ IEL following microbial colonization. Integrin alpha E-deficient mice will provide an important tool for studying the role of alpha E beta 7 and of alpha E beta 7-expressing mucosal T lymphocytes in vivo.

Synaptobrevin isoforms in secretory granules and synaptic-like microvesicles in anterior pituitary cells.

A set of synaptic proteins have been shown to be essential for the life cycle and exocytosis of synaptic vesicles at the nerve terminal. Recently, these proteins have also been identified in certain endocrine cells. Here we analysed the presence and location of some of these synaptic proteins in anterior pituitary cells. Immunoblotting data demonstrated that Rab3a, synaptotagmin, cellubrevin, synaptobrevin 2, syntaxin 1, SNAP-25 and synaptophysin were well represented in anterior pituitary cells as well as in the corticotroph cell line AtT-20. Cellubrevin was the most abundant synaptobrevin isoform present in pituitary cells. Moreover, both cellubrevin and synaptobrevin 2 took part of a protein complex involved in the fusion process in adenohypophyseal cells. Immunocytochemical and subcellular fractionation showed that cellubrevin, synaptobrevin 2, Rab3a and synaptotagmin were located in both secretory granules and synaptic-like microvesicles fractions. In contrast, SNAP-25 and syntaxin 1 were mainly associated with plasma membrane fractions. Therefore, these results suggest similar secretory mechanisms for synaptic vesicles and secretory organelles in both neuronal and endocrine cells.

Regulated secretion is impaired in AtT-20 endocrine cells stably transfected with botulinum neurotoxin type A light chain.

Botulinum neurotoxin type A (BoNT/A) inhibits neurotransmitter release by specific cleavage of SNAP-25, a synaptosome-associated protein also expressed in the ACTH secretory cell line AtT-20. Expression of light chain BoNT/A (L-BoNT/A) gene transfected into AtT-20 cells resulted in a cleaved form of SNAP-25 indistinguishable from that generated by bona fide BoNT/A. L-BoNT/A-transfected cells showed no difference in replication rate, viability, or phenotype, compared with control AtT-20 cells. In contrast, L-BoNT/A-transfected cells could not be induced to secrete ACTH upon stimulation by 8-bromo-cAMP or KCl. In addition, alpha-latrotoxin induced ACTH release from control cells, but not from L-BoNT/A-transfected cells. These experiments suggest an important role for SNAP-25 in regulated secretion from AtT-20 cells and underline the usefulness of this cell system as a tool for the study of the molecular mechanism of peptide hormone secretion.

Immunocytochemical analysis of the synaptic proteins SNAP-25 and Rab3A in human pituitary adenomas. Overexpression of SNAP-25 in the mammmosomatotroph lineages.

SNAP-25 and Rab3A were originally identified as synaptic proteins involved in neuronal membrane traffic. Recently, both proteins have been detected in several mammalian endocrine cell types and have been proposed as essential components of the exocytotic pathway in neuroendocrine cells. In this study, the expression of SNAP-25 and Rab3A was analysed in biopsied human anterior pituitary tumours (21 cases) by immunocytochemical methods. No differences in Rab3A immunoreactivity were observed between tumour and normal pituitary cells. Strong SNAP-25 immunoreactivity was detected in tumour cells of prolactinomas (n = 3). Several growth hormone (GH)/prolactin (PRL) tumours also displayed intense SNAP-25 immunolabelling (n = 3), whereas the remaining GH-secreting adenomas (n = 4) exhibited moderate to weak SNAP-25 immunoreactivity. In contrast, SNAP-25 near-background immunostaining was observed in tumour cells of adrenocorticotrophic hormone (ACTH)-secreting tumours (n = 4) and non-secreting tumours (n = 7), as well as in normal pituitary cells. Since SNAP-25 and Rab3A have been shown to be involved in exocytotic events in rodent endocrine cells, overexpression of SNAP-25 protein in PRL and GH/PRL tumour cells might be implicated in the mechanism of exocytosis of the neoplastic human mammosomatotroph lineages.

Expression of synaptosomal-associated protein SNAP-25 in endocrine anterior pituitary cells.

A growing body of evidence indicates that the fundamental molecular mechanism of exocytosis in the secretory pathway may be structurally similar in all eukaryotic cells. The synaptosomal-associated protein of 25 kDa (SNAP-25) is a plasma membrane protein involved in regulated exocytosis in neurons. In order to compare exocytotic components in neurons and endocrine cells, we have analyzed the expression of SNAP-25 in the rat anterior pituitary. Western blotting analysis documented the presence of SNAP-25 in anterior pituitary homogenates and cultured anterior pituitary cells. In addition to SNAP-25, other neuronal proteins involved in exocytosis (syntaxin, VAMP/synaptobrevin and Rab3A) were also detected in the anterior pituitary. The specific expression of SNAP-25 mRNA in anterior pituitary cells was also corroborated by Northern analysis. SNAP-25 immunoreactivity was located at the plasma membrane of endocrine anterior pituitary cells. Characteristically, patches of fine punctate deposits exhibited intense SNAP-25 immunoreactivity. Double-labeling immunocytochemistry revealed that SNAP-25 was mainly associated with gonadotroph cell populations. Furthermore, we demonstrate that in the anterior pituitary, SNAP-25 is selectively cleaved by clostridial neurotoxins. In conclusion, our results establish the presence of SNAP-25 in secretory anterior pituitary cells and suggest a potential role of this protein in the secretion of adenohypophysial hormone.

Calcium channel antagonist omega-conotoxin binds to intramembrane particles of isolated nerve terminals.

Voltage-sensitive calcium channels play a key role in evoked neurotransmitter release and their distribution in presynaptic membranes can be critical for fast signalling at chemical synapses. Using a biotinylated derivative of the neuronal calcium channel antagonist, omega-conotoxin, and a combination of colloidal gold labeling and freeze-fracture techniques, we have labeled calcium channels present at the membrane of nerve terminals isolated from the electric organ of Torpedo marmorata. The biotinylated blocker exerts an inhibitory action on the high potassium-evoked release of adenosine triphosphate as the native toxin does and its interaction with biological membranes is specific as shown in displacement experiments. This study shows that an antagonist specific for voltage-activated calcium channels binds to intramembrane particles in presynaptic membranes, reinforcing the idea that these particles, concentrated at neurotransmitter release sites, effectively represent calcium channels.

Carbohydrate patterns of the pure cholinergic synapse of Torpedo electric organ: a cytochemical and immunocytochemical electron microscopic approach.

Using post-embedding gold staining techniques, we investigated the ultrastructural distribution of terminal sugars and carbohydrate chains located at the pure cholinergic electric organ tissue of Torpedo marmorata. Neither alpha-N-acetylgalactosamine (GalNAc)-specific lectins (DBA, SBA, HPA) nor monoclonal antibodies (MAb) recognizing Tn (Gal-NAc alpha-O-Ser/Thr; MAb Cu-1) and sialyl-Tn epitopes (NeuAc alpha 2,6GalNAc alpha-O-Ser/Thr; MAb B72.3 and OSM-10) were capable of labeling any of the synaptic structures. The absence of gold labeling was likewise noted with UEA-I (L-fucose) and with PNA (T-antigen, Gal beta 1,3GalNAc alpha). After neuraminidase pre-treatment of ultra-thin sections, PNA labeling was rendered evident, indicating the presence of neuraminic acid-masked T-antigen. Certain synaptic vesicles were labeled for neuraminic acid (LFA) and for N-acetyllactosamine (DSA), whereas others were not labeled at all. Gold labeling with LFA, RCA-I (beta-galactose), and DSA in the membrane infoldings of the dorsal face of the electrocyte was visualized. As noted above, the PNA reaction was revealed only after pre-treatment with neuraminidase. Dorsal (non-synaptic) basal lamina were reactive with DSA, whereas the synaptic portion was likewise labeled with LFA and RCA-I. Finally, RCA-I labeling was noted in the Schwann cell nucleus. Comparisons between these results and those described at the neuromuscular junction were made.

Omega-conotoxin differentially blocks acetylcholine and adenosine triphosphate releases from Torpedo synaptosomes.

We have examined the effect of several blockers of voltage-sensitive calcium channels on the release of acetylcholine and ATP from synaptosomes isolated from Torpedo marmorata electric organ. Depolarization of these nerve terminals with high K(+)-containing solutions resulted in a calcium-dependent release of both molecules. Cadmium ions (10(-6) to 10(-3) M) inhibited similarly both releases whereas nickel ions (10(-4) M) in the external medium did not affect either neurotransmitter or nucleotide release. Both releases were completely resistant to the effect of 1,4-dihydropyridines (antagonists nimodipine, nifedipine and agonist Bay K 8644) and of a related compound (diltiazem) at concentrations up to 10(-5) M. These drugs failed to cause any effect even when synaptosomes were submaximally depolarized during incubation. Omega-conotoxin (10(-8) to 5 x 10(-5) M) showed a differential effect on acetylcholine and ATP releases. Nucleotide release was inhibited 90% at the highest concentration tested (50 microns) while acetylcholine release was only moderately decreased (30%). EC50 values for acetylcholine and ATP were of 167 and 2 microM respectively. The results suggest the implication of different types of calcium channels in the release of these molecules.

Opiates depress ACh and ATP release from cholinergic synaptosomes by blocking calcium uptake.

We have studied the effects of opiates on ATP and acetylcholine (ACh) release from cholinergic nerve terminals isolated from the electric organ of Torpedo marmorata. The release of ATP was inhibited by morphine and this action was reversed by naloxone. Morphine, [D-Met2-Pro5]enkephalinamide and [D-Ala2-Leu5]enkephalin also inhibited acetylcholine release. Naloxone prevented these inhibitory effects. The action of enkephalin on ACh release was less effective than that of morphine. The calcium uptake by nerve terminals of Torpedo electric organ was also inhibited by morphine, either under resting or depolarizing conditions, and this effect was reversed by naloxone. Using the quick freeze-fracture method, the structural changes induced by morphine in the presynaptic membrane were also studied. Morphine prevents the rearrangement of intramembrane particles (IMPs) at both freeze-fractured faces of the synaptosomal presynaptic membrane after depolarization. It is concluded that opiates depress the ATP and the ACh releases from cholinergic synaptosomes by inhibiting the calcium uptake by the nerve terminals and the rearrangement of the IMPs after potassium-induced depolarization. Furthermore, ACh release, but not ATP release, seems to be related with the rearrangement of IMPs in the presynaptic membrane.

The release of adenosine at the electric organ of Torpedo. A study using a continuous chemiluminescent method.

Acetylcholine and ATP are costored and coreleased during synaptic activity at the electric organ of Torpedo. It has been suggested that released ATP is converted to adenosine at the synaptic cleft, and in turn this nucleoside would depress the evoked release of acetylcholine. In the present communication we have used a chemiluminescent reaction that let us to monitor continuously the presence of adenosine in this preparation. The chemiluminescent reaction is based on the conversion of adenosine into uric acid and H2O2 by adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase enzymes. The hydrogen peroxide has been detected by peroxidase-luminol mixture. The reaction has a sensitivity on the picomol range and discerned between Adenosine, AMP, ADP, and ATP. We have developed this technique in the hope of understanding whether adenosine is released during synaptic activity or it comes from the released ATP. We have studied the release or formation of adenosine in fragments of the electric organ and in isolated cholinergic nerve terminals obtained from it. In both conditions we have followed the effect of potassium stimulation upon the detection of adenosine. Potassium stimulation increased the extracellular adenosine either in slices or the synaptosomal fraction of Torpedo electric organ. The presence of alpha, beta-methylene ADP, an inhibitor of 5'-nucleotidase, inhibits the detection of adenosine, suggesting that extracellular adenosine is a consequence of ectocellular dephosphorylation of released ATP.

Botulinum toxin type A blocks the morphological changes induced by chemical stimulation on the presynaptic membrane of Torpedo synaptosomes.

The action of botulinum neurotoxin on acetylcholine release, and on the structural changes at the presynaptic membrane associated with the transmitter release, was studied by using a subcellular fraction of cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ. Acetylcholine and ATP release were continuously monitored by chemiluminescent methods. To catch the membrane morphological changes, the quick-freezing method was applied. Our results show that botulinum neurotoxin inhibits the release of acetylcholine from these isolated nerve terminals in a dose-dependent manner, whereas ATP release is not affected. The maximal inhibition (70%) is achieved at neurotoxin concentrations as low as 125 pM with an incubation time of 6 min. This effect is not linked to an alteration of the integrity of the synaptosomes since, after poisoning by botulinum neurotoxin type A, they show a nonmodified occluded lactate dehydrogenase activity. Moreover, membrane potential is not altered by the toxin with respect to the control, either in resting condition or after potassium depolarization. In addition to acetylcholine release inhibition, botulinum neurotoxin blocks the rearrangement of the presynaptic intramembrane particles induced by potassium stimulation. The action of botulinum neurotoxin suggests that the intramembrane particle rearrangement is related to the acetylcholine secretion induced by potassium stimulation in synaptosomes isolated from the electric organ of Torpedo marmorata.