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Latexin, a carboxypeptidase A inhibitor, is expressed in rat peritoneal mast cells and is associated with granular structures distinct from secretory granules and lysosomes.

ABSTRACT: Latexin, a protein possessing inhibitory activity against rat carboxypeptidase A1 (CPA1) and CPA2, is expressed in a neuronal subset in the cerebral cortex and cells in other neural and non-neural tissues of rat. Although latexin also inhibits mast-cell CPA (MCCPA), the expression of latexin in rat mast cells has not previously been confirmed. In the present study we examined the expression and subcellular localization of latexin in rat peritoneal mast cells. Western blot and reverse-transcriptase-mediated PCR analyses showed that latexin was contained and expressed in the rat peritoneal mast cells. Immunocytochemically, latexin immunofluorescence was localized on granular structures distinct from MCCPA-, histamine- or cathepsin D-immunopositive granules. Immunoelectron microscopy revealed that latexin was associated with a minority population of granules. The latexin-associated granules were separated from MCCPA- or histamine-containing granules on a self-generating density gradient of polyvinylpyrrolidone-coated silica-gel particles (Percoll). Treatments with high ionic strength and heparinase released latexin from the granules, suggesting that latexin is non-covalently associated with a heparin-like component of the granules. MCCPA and histamine were released from the mast cells after non-immunological and immunological stimulation with compound 48/80, A23187 and anti-IgE antibody, whereas latexin was not released. These results show that latexin is synthesized in rat peritoneal mast cells and suggest that it is associated with a unique type of intracellular granules distinct from MCCPA- and histamine-containing secretory granules and lysosomes.

SUBMITTER: Uratani Y

PROVIDER: S-EPMC1220918 | biostudies-other | 2000 Mar

REPOSITORIES: biostudies-other

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Project description:We investigated the basis for previous observations that fluorescent-labeled neuropeptide Y (NPY) is usually released within 200 ms after fusion, whereas labeled tissue plasminogen activator (tPA) is often discharged over many seconds. We found that tPA and NPY are endogenously expressed in small and different subpopulations of bovine chromaffin cells in culture. We measured the mobility of these proteins (tagged with fluorophore) within the lumen of individual secretory granules in living chromaffin cells, and related their mobilities to postfusion release kinetics. A method was developed that is not limited by standard optical resolution, in which a bright flash of strongly decaying evanescent field (∼64 nm exponential decay constant) produced by total internal reflection (TIR) selectively bleaches cerulean-labeled protein proximal to the glass coverslip within individual granules. Fluorescence recovery occurred as unbleached protein from distal regions within the 300 nm granule diffused into the bleached proximal regions. The fractional bleaching of tPA-cerulean (tPA-cer) was greater when subsequently probed with TIR excitation than with epifluorescence, indicating that tPA-cer mobility was low. The almost equal NPY-cer bleaching when probed with TIR and epifluorescence indicated that NPY-cer equilibrated within the 300 ms bleach pulse, and therefore had a greater mobility than tPA-cer. TIR-fluorescence recovery after photobleaching revealed a significant recovery of tPA-cer (but not NPY-cer) fluorescence within several hundred milliseconds after bleaching. Numerical simulations, which take into account bleach duration, granule diameter, and the limited number of fluorophores in a granule, are consistent with tPA-cer being 100% mobile, with a diffusion coefficient of 2 × 10(-10) cm(2)/s (∼1/3000 of that for a protein of similar size in aqueous solution). However, the low diffusive mobility of tPA cannot alone explain its slow postfusion release. In the accompanying study, we suggest that, additionally, tPA itself stabilizes the fusion pore with dimensions that restrict its own exit.

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Latexin, a carboxypeptidase A inhibitor, is expressed in rat peritoneal mast cells and is associated with granular structures distinct from secretory granules and lysosomes.

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