A key issue in signal transduction is how signaling pathways common to many systems-so-called canonical signaling cassettes-integrate signals from molecules having a wide spectrum of activities, such as hormones and neurotrophins, to deliver distinct biological outcomes. The neuroendocrine cell line PC12, derived from rat pheochromocytoma, provides an example of how one canonical signaling cassette-the Raf --> mitogen-activated protein kinase kinase (MEK) --> extracellular signal-regulated kinase (ERK) pathway-can promote distinct outcomes, which in this case include neuritogenesis, gene induction, and proliferation. Two growth hormones, epidermal growth factor (EGF) and nerve growth factor (NGF), use the same pathway to cause PC12 proliferation and differentiation, respectively. In addition, pituitary adenylate cyclase-activating polypeptide (PACAP), a neurotransmitter that also causes differentiation, uses the same canonical cassette as NGF but in a different way. The Connections Map for PC12 Cell Differentiation brings into focus the complex array of specific cellular responses that rely on canonical signal transduction systems.

We present evidence that regulation of dense-core secretory granule biogenesis and hormone secretion in endocrine cells is dependent on chromogranin A (CGA). Downregulation of CGA expression in a neuroendocrine cell line, PC12, by antisense RNAs led to profound loss of dense-core secretory granules, impairment of regulated secretion of a transfected prohormone, and reduction of secretory granule proteins. Transfection of bovine CGA into a CGA-deficient PC12 clone rescued the regulated secretory phenotype. Stable transfection of CGA into a CGA-deficient pituitary cell line, 6T3, lacking a regulated secretory pathway, restored regulated secretion. Overexpression of CGA induced dense-core granules, immunoreactive for CGA, in nonendocrine fibroblast CV-1 cells. We conclude that CGA is an "on/off" switch that alone is sufficient to drive dense-core secretory granule biogenesis and hormone sequestration in endocrine cells.

A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1-mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2-mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function.Storage of monoamines in secretory organelles of neurons, endocrine/paracrine cells, basophils, blood platelets, and mast cells is critical for their regulated, physiological secretion. Monoamine accumulation from the cytoplasm into storage organelles is mediated by vesicular monoamine transporters (VMATs) with an absolute dependence on a vacuolar ATPase-generated proton gradient to transport the cationic amine substrates into the storage organelle in exchange for protons (1).Recently, we cloned a VMAT (formerly named MAT) from a rat basophilic leukemia cell line (RBL-2H3) by functional expression of T7 promoter-driven cDNA sublibraries in CV-1 fibroblasts infected with T7 polymerase-expressing recombinant vaccinia virus (2). Permeabilization of the plasma membrane with digitonin provided the first demonstration that monoamine substrates could be directly accumulated by an intracellular compartment of nonneuroendocrine cells expressing this transporter in an ATP-dependent fashion that was sensitive to the specific inhibitors reserpine and tetrabenazine (TBZ). The mRNA for this transpor...

A cDNA for a rat vesicular monoamine transporter, desgatd MAT, was isolated by expression cloning in a mammalian cell line (CV-1). (1-4). The biogenic amine Na+-dependent transporters (reuptake transporters) located at the plasma membrane are responsible for transport of the released monoamines back into the cytoplasm, where they may be repackaged by the vesicular transporter into storage organelles or degraded by monoamine oxidases (5, 6).Several distinguishing features of the MAT are as follows: (i) broad selectivity for 5-hydroxytryptamine (serotonin) (5HT), dopamine (DA), and norepinephrine (NE) uptake, (ii) specific inhibition of transport by reserpine (RES) and tetrabenazine (TBZ), and (iii) transmembrane H+-electrochemical dependence of monoamine accumulation (1-4) .