Combining the lack of chromogranins with chronic L-DOPA treatment affects motor activity in mice.

We have tested whether the lack of chromogranins (Cgs) A and B could provoke CNS disorders when combined with an excess of dopamine. We chronically treated (over 6 months) mice lacking both chromogranins A and B (Cgs-KO) with a low oral dosage of L-DOPA/benserazide (10/2.5 mg/kg). Motor performance in the rota-rod test, open field activity, and metabolic cages indicated a progressive impairment in motor coordination in these mice, and an increase in rearing behavior, which was accompanied by an increase in DA within the substantia nigra. We conclude that mild chronic L-DOPA treatment does not produce nigro-striatal toxicity that could be associated with parkinsonism, neither in control nor Cgs-KO mice. Rather, Cgs-KO mice exhibit behaviors compatible with an amphetamine-like effect, probably caused by the excess of catecholamines in the CNS.

Adrenergic chromaffin cells are adrenergic even in the absence of epinephrine.

Adrenal chromaffin cells release epinephrine (EPI) and norepinephrine (NE) into the bloodstream as part of the homeostatic response to situations like stress. Here we utilized EPI-deficient mice generated by knocking out (KO) the phenylethanolamine N-methyltransferase (Pnmt) gene. These Pnmt-KO mice were bred to homozygosis but displayed no major phenotype. The lack of EPI was partially compensated by an increase in NE, suggesting that EPI storage was optimized in adrenergic cells. Electron microscopy showed that despite the lack of EPI, chromaffin granules retain their shape and general appearance. This indicate that granules from adrenergic or noradrenergic cells preserve their characteristics even though they contain only NE. Acute insulin injection largely reduced the EPI content in wild-type animals, with a minimal reduction in NE, whereas there was only a partial reduction in NE content in Pnmt-KO mice. The analysis of exocytosis by amperometry revealed a reduction in the quantum size (-30%) and Imax (-21%) of granules in KO cells relative to the wild-type granules, indicating a lower affinity of NE for the granule matrix of adrenergic cells. As amperometry cannot distinguish between adrenergic or noradrenergic cells, it would suggest even a larger reduction in the affinity for the matrix. Therefore, our results demonstrate that adrenergic cells retain their structural characteristics despite the almost complete absence of EPI. Furthermore, the chromaffin granule matrix from adrenergic cells is optimized to accumulate EPI, with NE being a poor substitute. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

The crucial role of chromogranins in storage and exocytosis revealed using chromaffin cells from chromogranin A null mouse.

Chromogranins (Cgs) are the major soluble proteins of dense-core secretory vesicles. Chromaffin cells from Chga null mice [chromogranin A knock-out (CgA-KO)] exhibited approximately 30% reduction in the content and in the release of catecholamines compared with wild type. This was because of a lower secretion per single exocytotic event, rather than to a lower frequency of exocytotic events. Cell incubation with L-DOPA produced an increase in the vesicular amine content of wild-type, but not CgA-KO vesicles. In contrast, intracellular electrochemistry showed that L-DOPA produced a significantly larger increase in cytosolic amines in CgA-KO cells than in the wild type. These data indicate that the mechanisms for vesicular accumulation in CgA-KO cells were fully saturated. Patch-amperometry recordings showed a delayed initiation of the amperometric signal after vesicle fusion, whereas no changes were observed in vesicle size or fusion pore kinetics despite the smaller amine content. We conclude that intravesicular proteins are highly efficient systems directly implicated in transmitter accumulation and in the control of neurosecretion.