Chromaffin cells vesicles

In this lecture we will be concerned by exocytosis of neurotransmitters by chromaffin cells. These cells, located above kidneys, produce the adrenaline burst which induces fast body reactions they are used in neurosciences as standard models for the study of exocytosis by catecholaminergic neurons. Prior to exocytosis, adrenaline is contained at highly concentrated solutions into a polyelectrolyte gel matrix packed into small vesicles present in the cell cytoplasm and brought by the cytoskeleton near the cell outer membrane. Stimulation of the cell by divalent ions induces the fusion of the vesicles membrane with that of the cell and hence the release of the intravesicular content into the outer-cytoplasmic region. 

Together with dopamine, adrenaline and noradrenaline belong to the endogenous catecholamines that are synthesized from the precursor amino acid tyrosine (Fig. 1). In the first biosynthetic step, tyrosine hydroxylase generates l-DOPA which is further converted to dopamine by the aromatic L-amino acid decarboxylase ( Dopa decarboxylase). Dopamine is transported from the cytosol into synaptic vesicles by a vesicular monoamine transporter. In sympathetic nerves, vesicular dopamine (3-hydroxylase generates the neurotransmitter noradrenaline. In chromaffin cells of the adrenal medulla, approximately 80% of the noradrenaline is further converted into adrenaline by the enzyme phenylethanolamine-A-methyltransferase. 

At least two classes of regulated secretion can be defined [54]. The standard regulated secretion pathway is common to all secretory cells (i.e. adrenal chromaffin cells, pancreatic beta cells, etc.) and works on a time scale of minutes or even longer in terms of both secretory response to a stimulus and reuptake of membranes after secretion. The second, much faster, neuron-specific form of regulated secretion is release of neurotransmitters at the synapse. Release of neurotransmitters may occur within fractions of a second after a stimulus and reuptake is on the order of seconds. Indeed, synaptic vesicles may be recycled and ready for another round of neurotransmitter release within 1-2 minutes [64]. These two classes of regulated secretion will be discussed separately after a consideration of secretory vesicle biogenesis. 

The SNAREs involved in the fusion of synaptic vesicles and of secretory granules in neuroendocrine cells, referred to as neuronal SNAREs, have been intensely studied and serve as a paradigm for all SNAREs. They include syntaxin 1A and SNAP-25 at the presynaptic membrane and synaptobrevin 2 (also referred to as VAMP 2) at the vesicle membrane. Their importance for synaptic neurotransmission is documented by the fact that the block in neurotransmitter release caused by botulinum and tetanus neurotoxins is due to proteolysis of the neuronal SNAREs (Schiavo et al. 2000). Genetic deletion of these SNAREs confirmed their essential role in the last steps of neurotransmitter release. Intriguingly, analysis of chromaffin cells from KO mice lacking synaptobrevin or SNAP-25 showed that these proteins can be at least partially substituted by SNAP-23 and cellubrevin, respectively (Sorensen et al. 2003 Borisovska et al. 2005), i.e., the corresponding SNAREs involved in constitutive exocytosis. 

Artalejo CR, Elhamdani A, Palfrey HC (2002) Sustained stimulation shifts the mechanism of endocytosis from dynamin-1-dependent rapid endocytosis to clathrin- and dynamin-2-mediated slow endocytosis in chromaffin cells. Proc Natl Acad Sci USA 99 6358-63 Becher A, Drenckhahn A, Pahner I, Margittai M, Jahn R, Ahnert-Hilger G (1999) The synaptophysin-synaptobrevin complex a hallmark of synaptic vesicle maturation. J Neurosd 19 1922-31... 

Miller RJ (1998) Presynaptic receptors. Annu Rev Pharmacol Toxicol 38 201-27 Milosevic I, Sorensen JB, Lang T et al (2005) Plasmalemmal phosphatidylinositol-4,5-bisphosphate level regulates the releasable vesicle pool size in chromaffin cells. J Neurosci 25 2557-65... 

Storage of norepinephrine in vesicles Dopamine is transported into synaptic vesicles by an amine transporter system that is also involved in the re-uptake of preformed norepinephrine. This carrier system is blocked by reserpine (see p. 78). Dopamine is hydroxylated to form norepinephrine by the enzyme, dopamine 3-hydroxylase. Synaptic vesicles contain dopamine or norepinephrine plus adenosine triphosphate and the 3-hydroxylase. Not all of the norepinephrine is packaged in vesicles some exists in a cytoplasmic pool that can be displaced. In the adrenal medulla, norepinephrine is methylated to yield epinephrine both are stored in chromaffin cells. On stimulation, the adrenal medulla releases about 85% epinephrine and 15% norepinephrine. 

Amatore C, Arbault S, Bonifas I, Bouret Y, Erard M, et al. 2005. Correlation between vesicle quantal size and fusion pore chromaffin cell exocytosis. Biophys J 88 4411-4420. 

Tian J-H, Wu Z-X, Unzicker M, Lu L, Cai Q, et al. 2005. The role of snapin in neurosecretion Snapin knock-out mice exhibit impaired calcium-dependent exocytosis of large dense-core vesicles in chromaffin cells. J Neurosci 25 ... 

Studies of the cleavage specificity of cathepsin L demonstrated that it prefers to cleave on the NH2-terminal side of dibasic residue processing sites of enkephalin-containing peptide substrates BAM-22P and Peptide F (22) and to cleave at the N-terminal sides of dibasic residues within peptide-MCA substrates (32). The cleavage specificity of cathepsin L results in enkephalin intermediate peptides with NH2-terminal basic residue extensions, which are then removed by Arg/Lys aminopeptidase. Secretory vesicles from adrenal medullary chromaffin cells (33) and from pituitary (34) contain Arg/Lys aminopeptidase activity for neuropeptide production. 

Figure 5 Proteomics reveals functional secretory vesicle protein systems for neuropeptide biosynthesis, storage, and secretion. Chromaffin secretory vesicles (also known as chromaffin granules) were isolated and subjected to proteomic analyses of proteins in the soluble and membrane components of the vesicles. Protein systems in secretory vesicle function consisted of those for 1) production of hormones, neurotransmitters, and neuromodulatory factors, 2) generating selected internal vesicular conditions for reducing condition, acidic pH conditions maintained by ATPases, and chaperones for protein folding, and 3) vesicular trafficking mechanisms to allow the mobilization of secretory vesicles for exocytosis, which uses proteins for nucleotide-binding, calcium regulation, and vesicle exocytosis. These protein systems are coordinated to allow the secretory vesicle to synthesize and release neuropeptides for cell-cell communication in the control of neuroendocrine functions.

Hook V, Toneff T, Bogyo M, Greenbaum D, Medzihradszky KF, Neveu J, Lane W, Hook G, Reisine T. Inhibition of cathepsin B reduces -amyloid production in regulated secretory vesicles of neuronal chromaffin cells evidence for cathepsin B as a candidate -secretase of Alzheimer s disease. Biol. Chem. 2005 386 931-940. 

Hayashi T, Yamasaki S, Nauenburg S, Binz T, Niemann H (1995) Disassembly of the reconstituted synaptic vesicle membrane fusion complex in vitro. In EMBOJ. 14 2317-25 Melting TB, Zwisler O (1977) Structure of tetanus toxin. In J. Biol. Chem. 252 187-93 Hohne-Zell B, Ecker A, Weller U, Gratzl M (1994) Synaptobrevin cleavage by tetanus toxin light chain is linked to inhibition of exocytosis in chromaffin cells. In FEBS Lett. 355 131 -4... 

SollnerTH, Bennett MK, Whiteheart SW, Scheller RH, Rothman JE (1993 b) A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. In Cell 75 409-18 Stecher B, Weller U, Habermann E, Gratzl M, Anhert-Hilger G (1989) The light chain but not the heavy chain of botulinum A toxin inhibits exocytosis from per-meabilized adrenal chromaffin cells. In FEBS Lett. 255 391 -4... 

---