Obligatory role for endothelial heparan sulphate proteoglycans and caveolae internalization in catestatin-dependent eNOS activation.

The chromogranin-A peptide catestatin modulates a wide range of processes, such as cardiovascular functions, innate immunity, inflammation, and metabolism. We recently found that the cardiac antiadrenergic action of catestatin requires a PI3K-dependent NO release from endothelial cells, although the receptor involved is yet to be identified. In the present work, based on the cationic properties of catestatin, we tested the hypothesis of its interaction with membrane heparan sulphate proteoglycans, resulting in the activation of a caveolae-dependent endocytosis. Experiments were performed on bovine aortic endothelial cells. Endocytotic vesicles trafficking was quantified by confocal microscopy using a water-soluble membrane dye; catestatin colocalization with heparan sulphate proteoglycans and caveolin 1 internalization were studied by fluorimetric measurements in live cells. Modulation of the catestatin-dependent eNOS activation was assessed by immunofluorescence and immunoblot analysis. Our results demonstrate that catestatin (5 nM) colocalizes with heparan sulphate proteoglycans and induces a remarkable increase in the caveolae-dependent endocytosis and caveolin 1 internalization, which were significantly reduced by both heparinase and wortmannin. Moreover, catestatin was unable to induce Ser(1179) eNOS phosphorylation after pretreatments with heparinase and methyl-ß-cyclodextrin. Taken together, these results highlight the obligatory role for proteoglycans and caveolae internalization in the catestatin-dependent eNOS activation in endothelial cells.

Chromogranin A and vesicular monoamine transporter 2 immunolocalization in protein bodies of human locus coeruleus neurons.

Our previous histochemical and ultrastructural studies have identified, in human catecholamine neurons, abundant spherical acidophilic protein bodies (pb), which originate from regular mitochondria, retaining their double membrane. In locus coeruleus (LC) neurons, pb have somatodendritic distribution and are unequivocal storage vesicles for noradrenaline, as demonstrated by immunolocalization of Dopamine-ß-Hydroxylase. In the present study, in order to reinforce the identity of pb as monoamine storage sites in human LC, and to assess their potential of somatodendritic release, we studied the subcellular immunolocalization of chromogranin A (CgA) and vesicular monoamine transporter 2 (VMAT2), given the fact that their localization defines the vesicles capacity of filling with monoamine and hence exocytotic release. The data provided in the present study, demonstrate the novel ultrastructural immunolocalization of both CgA and VMAT2 in protein bodies, supporting their involvement in somatodendritic storage and release of noradrenaline in human LC. Since the molecular mechanism of LC somatodendritic exocytosis remains largely elusive, the present study may shed light to a better understanding of this mechanism.

Biogenesis of the secretory granule: chromogranin A coiled-coil structure results in unusual physical properties and suggests a mechanism for granule core condensation.

The secretory pro-hormone chromogranin A (CHGA) is densely packed into storage granules along with catecholamines, playing a catalytic role in granule biogenesis. 3-Dimensional structural data on CHGA are lacking. We found a superfamily structural homology for CHGA in the tropomyosin family of alpha-helical coiled-coils, even in mid-molecule regions where primary sequence identity is only modest. The assignment was confirmed by an independent algorithm, suggesting approximately 6-7 such domains spanning CHGA. We provide additional physiochemical evidence (chromatographic, spectral, microscopic) consistent with this unusual structure. Alpha-helical secondary structure (at up to approximately 45%) was confirmed by circular dichroism. CHGA molecular mass was estimated by MALDI-TOF mass spectrometry at approximately 50 kDa and by denaturing gel filtration at approximately 50-61 kDa, while its native Stokes radius was approximately 84.8 A, as compared to an expected approximately 30 A; the increase gave rise to an apparent native molecular weight of approximately 578 kDa, also consistent with the extended conformation of a coiled-coil. Small-angle X-ray scattering (SAXS) on CHGA in solution best fit an elongated cylindrical conformation in the monodisperse region with a radius of gyration of the rod cross-section (Rt) of approximately 52 A, compatible with a coiled-coil in the hydrated, aqueous state, or a multimeric coiled-coil. Electron microscopy with negative staining revealed an extended, filamentous CHGA structure with a diameter of approximately 94 +/- 4.5 A. Extended, coiled-coil conformation is likely to permit protein "packing" in the secretory granule at approximately 50% higher density than a globular/spherical conformation. Natural allelic variation in the catestatin region was predicted to disrupt the coiled-coil. Chromaffin granule ultrastructure revealed a approximately 108 +/- 6.3 A periodicity of electron density, suggesting nucleation of a binding complex by the CHGA core. Inhibition of CHGA expression, by siRNA, disrupted regulated secretory protein traffic by approximately 65%, while targeted ablation of the CHGA gene in the mouse reduced chromaffin granule cotransmitter concentrations by approximately 40-80%. These results suggest new roles for secretory protein tertiary structure in hormone and transmitter storage, with implications for secretory cargo condensation (or dense core "packing" structure) within the regulated pathway.