Mariana L. Papouchado

Highly-sensitive and specific enzyme-linked immunosorbent assays for GAD65 autoantibodies using a thioredoxin-GAD65 fusion antigen

Autoantibodies against glutamic acid decarboxylase (GAD65) are present in the sera of most patients with recently diagnosed insulin-dependent diabetes mellitus (IDDM). These antibodies appear years before the clinical symptoms, and they are considered to be early markers of the disease. To detect GAD65 autoantibodies (GADA), we developed new enzyme-linked immunosorbent assays (ELISA) with a fusion protein thioredoxin-GAD65 (Trx-GAD65) produced in E. coli as the antigen. These assays were compared with the reference radiobinding assay (RBA). Since most GADA are directed against native epitopes, and adsorption of GAD65 to plastic may cause disruption of its native conformation, the new assays rely on the following immobilization procedures: (a) capture ELISA (c-ELISA) with Trx-GAD65 (protocol A) or biotin-Trx-GAD (protocol B) indirectly immobilized by a non-adsorptive process; (b) ELISA with antigen-antibody preincubation in solution (p-ELISA) in which GADA were reacted first with Trx-GAD65 (protocol C) or biotin-Trx-GAD (protocol D) and the free antigen was determined by conventional ELISA. The results obtained with 42 newly diagnosed IDDM patients and 30 normal individuals were as follows: RBA had 79% sensitivity (percentage of IDDM patients detected) and 97% specificity (100% minus the percentage of false positives). c-ELISA showed low sensitivity (36 and 50%, respectively for protocols A and B), and high specificity (100 and 97%, respectively). p-ELISA were highly-sensitive (74 and 79%, respectively) and specific (97 and 93% for protocols C and D, respectively). Thus, protocols C and D had a performance similar to the reference method. The results reported here provide the basis for simple, highly-sensitive, specific, and widely-applicable tests for GADA that eliminate many of the drawbacks of the radioactive methods.

Effects of atrial natriuretic peptide and angiotensin III on the uptake and intracellular distribution of norepinephrine in medulla oblongata of the rat

1. Ten micromoles angiotensin III decreased total 3H-norepinephrine uptake in medulla oblongata of the rat and 100 nM atrial natriuretic peptide increased it. These were the threshold concentrations for the peptides to modify the uptake of the amine. 2. A threshold concentrations (1 nM) of atrial natriuretic peptide reversed the effects produced by 10 microM angiotensin III on total 3H-norepinephrine uptake, but subthreshold angiotensin III concentrations failed to alter the effects produced by 100 nM atrial natriuretic peptide. 3. Angiotensin III, as well as atrial natriuretic peptide, modified only neuronal norepinephrine uptake and did not alter non-neuronal norepinephrine uptake. 4. Angiotensin III and atrial natriuretic peptide did not modify the intracellular distribution of norepinephrine in medulla oblongata.

Modulation of the rat adrenal medulla norepinephrine secretion in a sodium-free medium by atrial natriuretic factor

The effects of atrial natriuretic factor (ANF) on [3H]norepinephrine ([3H]NE) release evoked by a sodium-free medium (SFM) were studied. Experiments were performed in rat adrenal medulla slices incubated in vitro. Results showed that [3H]NE release evoked by the omission of Na+ was decreased by 10 nM ANF. In addition, when the Ca2+ was omitted from the SFM, NE output was partially diminished. Nevertheless, if ANF was added to SFM/Ca(2+)-free medium (CFM), NE secretion showed no modifications compared with SFM/CFM. Present results raise the hypothesis that two mechanisms could be involved in NE output evoked by a SFM in the rat adrenal medulla: one independent of and the other dependent on the extracellular calcium. Moreover, ANF only diminished NE secretion evoked by SFM dependent on extracellular calcium and did not modify calcium-independent NE release.