Fluorescent Ca2+ probes and digital photo-sectioning techniques were used to directly study the dynamics of Ca2+ in isolated mast cell granules of normal (CB/J) and beige (Bg(j)/Bg(j)) mice. The resting intraluminal free Ca2+ concentration ([Ca2+]L) is 25 +/- 4.2 microM (mean +/- SD, n = 68). Exposure to 3 microM inositol 1,4,5-trisphosphate (InsP3) induced periodic oscillations of luminal Ca2+ ([Ca2+]L) of approximately 10 microM amplitude and a period around 8-10 s. The [Ca2+]L oscillations were accompanied by a corresponding oscillatory release of [Ca2+]L to the extraluminal space. Control experiments using ruthenium red (2 microM) and thapsigargin (100 nM) ruled out artifacts derived from the eventual presence of mitochondria or endoplasmic reticulum in the isolated granule preparation. Oscillations of [Ca2+]L and Ca2+ release result from a Ca2+/K+ exchange process whereby bound Ca is displaced from the heparin polyanionic matrix by inflow of K+ into the granular lumen via an apamin-sensitive Ca2+-sensitive K+ channel (ASK(Ca)), whereas Ca2+ release takes place via an InsP3-receptor-Ca2+ (InsP3-R) channel. These results are consistent with previous observations of [Ca2+]L oscillations and release in/from the endoplasmic reticulum and mucin granules, and suggest that a highly conserved common mechanism might be responsible for [Ca2+]L oscillations and quantal periodic Ca2+ release in/from intracellular Ca2+ storage compartments.

Materials released by secretory cells are stored inside intracellular membrane‐bound vesicles. These moieties are not freely diffusible in the vesicle but remain immobilized in a Ca2+‐crosslinked condensed‐phase polyanionic polymer matrix. During exocytosis a Na+/Ca2+ ion exchange process triggers a volume phase transition resulting in massive swelling and release of the materials to the extracellular space. Here we formulate a simple model to assess Ca2+‐ion binding from the swelling kinetics of polymer networks. We found the diffusivity of the networks (D) exhibits a power‐law dependency on the Ca2+ concentration where D ∝ [Ca2+]−2/3. The model yields an estimate of charge density and ionic affinity of the polymer chains. Studies of post‐exocytic swelling kinetics in airway mucin granules, mast cell granules and granules from the microalga (Phaeocystis globosa) were used to validate predictions from our model. These results suggest that independent of the cell type, from animal to plant cells, a single polyelectrolyte interaction mechanism appear to be responsible for product release in exocytosis.

No abstract

SummaryThe smaller isoform of glutamate decarboxylase (GAD65) is a major autoantigen in type 1 diabetes (TID). Its hydrophobic character requires detergent to keep the protein in solution, which complicates studies of antigen processing and presentation. In this study an attempt was made to replace detergent with human serum albumin (HSA) for in vitro antigen presentation. Different preparations of recombinant human GAD65 complexed with HSA were incubated with Priess B cells (HLA DRB1*0401) and antigen presentation was tested with HLA DRB1*0401-restricted and epitopespecific T33.1 (GAD65 epitope 274-286) and T35 (GAD65 epitope 115-127) T cell hybridomas. Specific epitope recognition by T33.1 (274-286) and T35 (115-127) cells varied between the different GAD65/HSA preparations, and a reverse pattern of antigen presentation were detected by the two hybridoma. The HSA-specific T-cell hybridoma 17.9 response to the different GAD65/HSA preparations followed the same pattern as that observed for the T33.1 cells. The content of immunoreactive GAD65 measured with four GAD65 antibodies indicated that the lowest GAD65 concentration resulted in the highest 274-286, but the lowest 115-127 presentation. This suggests that HSA-GAD65 complexes qualitatively affect the epitope specificity of GAD65 presentation. HSA may enhance the 274-286 epitope presentation, while suppressing the 115-127 epitope.Keywords antigen presentation; GAD65; Human serum albumin; T cell assay; Type 1 diabetes; T cells; Diabetes; Autoimmunity; Antigen Presentation/Processing IntroductionType 1 diabetes mellitus (TID) is an autoimmune disease that results in the specific destruction of the pancreatic islet beta cells. The smaller isoform of glutamate decarboxylase (GAD65) is implicated as a major contributing autoantigen in the pathogenesis of beta cell destruction [1,2]. Autoantibodies directed to GAD65 (GAD65Ab) are present in the majority of new onset T1D patients [3,4] and GAD65-specific T cells have been identified in T1D patients and in spontaneously diabetic NOD mice and BB rats (for review see [5,6] [9]. The establishment of a standard T cell assay remains critical for the understanding of GAD65 antigen uptake, processing and presentation.GAD65, which catalyzes the generation of the neurotransmitter GABA, is associated with intracellular membranes [10,11]. In vitro, the highly hydrophobic GAD65 requires detergents to remain in solution [12]. However, because detergents are extremely cytotoxic, it is critical that the detergent is removed prior to incubation with antigen-presenting cells (APC) and responder T cells.In the present study, we tested the well known property of human serum albumin (HSA) to maintain proteins of hydrophobic character in solution (for review see [13,14]). The aim of our study was to test whether GAD65 complexed with HSA affected antigen presentation by human Priess B cells to two GAD65-and one HSA-specific specific HLA-matched T cell hybridoma as readouts of antigen presentation. The specific epitope response of the two ...