Secretion of catecholamines from single bovine chromaffin cells in culture was elicited by brief pressure ejections from a micropipette containing nicotine, carbamoylcholine, or potassium ions or by mechanical stimulation. Release was monitored electrochemically with a carbon-fiber microelectrode placed adjacent to the cell. Cyclic voltammetry was used to identify secreted species, whereas constant potential amperometry was used for improved temporal resolution (millisecond range) of catecholamine detection. During secretion, brief current spikes were observed, which were shown to be due to detection of catecholamines by electrooxidation. The spikes have the physical characteristics of multimolecular packets of catecholamines released at random times and locations from the surface of the single cell. The half-width of the spikes was found to increase with an increase in cell-electrode spacing. The properties of the catecholamine spikes correlate well with expectations based on secretion from individual storage vesicles. Spikes do not occur in the absence of Ca2+ in the buffer, and the majority of spikes are found to be distributed between 0.2 and 2 picocoulombs, corresponding to 1-10 attomoles of catecholamine detected. The frequency of the spikes increases with the intensity of the stimulus, but the average quantity of catecholamine in each spike is independent of the stimulus. Thus, these measurements represent timeresolved observation of quantal secretion of catecholamines and provide direct evidence for the exocytotic hypothesis.

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 ϳ30% 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.

Catecholamine secretion has been measured with electrochemical techniques from isolated, single adrenal medullary chromaffin cells with carbon-fiber microelectrodes. The electrode tip, which is of similar dimensions to the cell, is placed adjacent to the cell to enable the measurement of local secretion. Secretion is caused by exposing the cell to nanoliter volumes of solution containing nicotinic receptor agonists or depolarizing agents. The identification of secreted substances is made with cyclic voltammetry at both bare electrodes and electrodes coated with a perfluorinated cation-exchange polymer. Catecholamine secretion is induced by nicotine (10-500 microM), carbamylcholine (1 mM), and K+ (60 mM). All agents that induce secretion lead to a broad envelope of secreted catecholamines on which sharp concentration spikes are superimposed. The concentration spikes can be monitored with a time resolution of tens of milliseconds when the electrodes are used in the amperometric mode. Release induced by nicotine and K+ is inhibited by Cd2+ (0.5 mM), and hexamethonium selectively blocks the nicotine-induced secretion. The actions of nicotine are found to continue for a longer period of time than those of the other secretagogues tested.

Neuiropeptide Y (NPY) is one of the most abundant peptide transmitters in the mammalian brain. In the periphery it is costored and coreleased with norepinephrine from sympathetic nerve terminals. However, the physiological functions of this peptide remain unclear because of the absence of specific high-affinity receptor antagonists. Three potent NPY receptor antagonists were synthesized and tested for their biological activity in in vitro, ex vivo, and in vivo functional assays. We describe here the effects of these antagonists inhibiting specific radiolabeled NPY binding at Y1 and Y2 receptors and antagonizing the effects of NPY in human erythroleukmia cell intracellular calcium mobilization, perfusion pressure in the isolated rat kidney, and mean arterial blood pressure in anesthetized rats.Neuropeptide Y (NPY) is a 36-amino acid peptide with an N-terminal tyrosine and a C-terminal tyrosine amide, first isolated from porcine brain by Tatemoto et al. in 1982 (1). NPY has been found to be an abundant mammalian neuropeptide, widely distributed throughout the central and peripheral nervous systems (2-4). On the basis of the pharmacological effects observed in experimental animals after central or peripheral administration of NPY, the peptide has tentatively been implicated in the regulation of a wide variety of biological functions such as vascular tone, feeding behavior, mood, and hormone secretion among others (for a review see ref. 5). At least two NPY receptor subtypes have been described based on the relative affinity of different NPY agonists: NPY-Y1 receptors require essentially the full NPY sequence of amino acids (see Fig. 1) for activation and have high affinity for the analog [Leu31,Pro34]NPY, whereas NPY-Y2 receptors can be activated by NPY and the shorter C-terminal fragment, NPY13-36, but have low affinity for [Leu31,Pro34]NPY (6,7). A third subtype (NPY-Y3) that recognizes all three of the above peptides but is insensitive to the NPY homolog, peptide YY, has been proposed (8, 9). Direct demonstration of a physiological and pathophysiological role for NPY has been hampered by the lack of specific, high-affinity NPY receptor antagonists. Receptor antagonists based on modified Cterminal fragments of NPY (10) Peptide Synthesis. Peptides were synthesized by the solidphase method. Compound 2 was obtained by oxidation of the reduced monomer and purification of the dimer by HPLC. Compound 3 was synthesized by using standard solid-phase synthesis. Compound 4 was synthesized by coupling BOC-Lglutamic acid fluorenylmethyl ester and a-Boc 3-FmOC-Ldiamino propionic acid in position 8 and 6, respectively. Dimerization was achieved on the resin by treatment with piperidine followed by a coupling reagent. Detailed synthesis is described in the compounds' patent publication (15).Binding Assays.[3H]NPY binding to rat brain membranes was done as described (16) except that incubations were terminated by filtration on a Brandel cell harvester through a Whatman GF/B filter, previously soaked overnight in 0.3% po...

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