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Bovine adrenal medullary cells, cultured on quartz plates, were superfused with buffer to which pulses of stimulant were added. Cytosolic Ca2" was measured by the fura-2 fluorescence method and the simultaneously released catecholamine was measured electrochemically. When stimulant concentrations were adjusted to given equivalent elevations of cytosolic Ca2+, secretion depended entirely on whether Ca2+ came from internal stores or from the extracellular medium. Calcium from internal stores did not support secretion under these conditions. This nonequivalence of the two sources of cytosolic Ca2+ points to important differences in the physiological roles of the two sources of calcium. Dimethylphenylpiperazinium (a cholinergic agonist) and elevated K+ MATERIALS AND METHODSCell Culture. Chromaffin cells were isolated from bovine adrenal medulla by collagenase digestions and purified by centrifugation on a Percoll gradient (6). The cells were cultured on quartz plates (1.25 x 4.5 cm), about 1 mm thick, that had been treated with fibronectin to promote cell adhesion. The cells were maintained up to 7 days in Eagle's minimal essential medium (GIBCO) containing 10% (vol/vol) heat-inactivated fetal calf serum (HyClone) plus antibiotics. Measurement of Fura-2 Fluorescence and CatecholamineRelease. Fura-2 fluorescence was used to measure cytosolic calcium (7). Chromaffin cells attached to quartz plates were incubated with 2-3 AuM fura 2 tetrakis(acetoxymethyl) ester in Eagle's minimal essential medium at 370C for 45-60 min.After incubation, cells were washed three times with Lockes solution (154 mM NaCl/5.6 mM KCI/2.2 mM CaCl2/10 mM glucose/5 mM Hepes, pH 7.3) before being placed in a flow chamber. The quartz plate with attached cells formed one side of the flow chamber of ==40 ,ul that was placed in the excitation beam of a Perkin-Elmer fluorescence spectrophotometer at an angle of 320 to the incident beam. Lockes solution flowed over the cells at a rate of 1 ml/min, at 29°C. The effluent from this flow chamber, in which fluorescence was measured, went immediately through capillary tubing to a Metrohm electrochemical detector to measure catecholamine released from the cells. In all experiments presented here, 6-sec pulses of the nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP), elevated K+ concentration, or other stimulants were injected into the flowing solution by a manual HPLC injection valve. There was a delay of 17 sec from the flow cell to the electrochemical detector. In the figures shown, the catecholamine curves have been made coincident with the fura-2 curve by using the known time delay factor. The dispersion ratios from the injection valve to the flow chamber and to the electrochemical detector are 0.56 and 0.37, respectively. In experiments not shown, we used digital imaging techniques to examine fura-2 fluorescence in individual cells. In no case did we see punctate or inhomogenious cytosolic fluorescence. The fura-2 seems to be uniformly distributed throughout the cytoplasm and not conce...

The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.exocytosis | purines | quantum size | secretory vesicles | VNUT V irtually most, and possibly all, types of secretory vesicles found in cells contain ATP, which often accumulates at high concentrations and, commonly, in conjunction with different types of neurotransmitters. However, the reason for this widespread distribution of ATP remains a mystery. Although ATP is present in all animal species, including primitive life forms like Giardia lamblia that lack Golgi complexes and mitochondria, the detection of ATP in the secretory vesicles of sympathetic neurons was considered to be the first example of cotransmission (1). However, given the ubiquitous accumulation of ATP in secretory vesicles, it might instead be considered that it is the other neurotransmitters that coincide with ATP, rather than the other way around (2). Indeed, perhaps ATP should be considered as the first molecule used as a transmitter in primitive forms of life.Astonishingly high concentrations of releasable species are stored inside secretory vesicles, far exceeding those in the cytosol (3, 4). For example, the catecholamine content of adrenal secretory granules (SGs), a type of large dense core secretory vesicles also known as chromaffin granules, was 0.8-1 M when measured directly in adrenal chromaffin cells using patch amperometry (5, 6). In addition, SGs from chromaffin cells contain ATP at ∼150 mM (7), calcium ∼40 mM (8), about 2 mM of granins, ascorbate, peptides, and other nucleotides, all in an acidic pH ∼5.5 environment.ATP possesses intrinsic chemical characteristics that make it relevant to the accumulation of soluble substances in secretory vesicles. The formation of weak complexes between monoamines and ATP, the two main soluble compounds in chromaffin granules, has been demonstrated in vitro by NMR (9), ultracentrifugation (10), infrared spectroscopy, and calorime...

The enzyme 3-phosphoglycerate kinase from yeast has been studied by observation of the proton nuclear magnetic resonance spectrum at 270 MHz using Fourier transform techniques. Difference spectroscopy was used to enhance the resolution and to identify specific ligand binding effects and conformational changes. Perturbations involving single protons of amino-acid residues could thus be detected despite the relatively high molecular weight of the protein (47000), particularly in the aromatic (6-9 ppm) and methylene (2-3 ppm) regions of the spectrum.3 . A pH titration enabled assignment of the C-2 and C-4 imidazole protons of each of three histidine residues remote from the active site, and the determination of their pK, values as 8.20 and 7.05 (two) respectively. The former histidine is deduced to lie buried within the polypeptide chain whilst the latter pair lie in practically identical environments on the surface of the enzyme exposed to solvent.2. Conformation changes were monitored on binding ADP and ATP both with and without Mg2'. A single site was observed for Mg . ADP, Mg . ATP, La . ATP and the uncomplexed nucleotides.3. Using paramagnetic difference spectroscopy, the line-broadening inhibitor Gd . ATP and the substrate Mn . ATP were found to induce identical perturbations of the enzyme resonances, showing that these species bind identically. The shift probes Pr . ATP and Eu . ATP could then be applied to map geometrically the enzyme active site. Three non-titratable (with pH) histidine resonances, three non-histidine aromatic resonances and one resonance corresponding to a methylene group of a polar amino acid were located vectorially relative to the ATP unit. No group is close enough to bind directly in the first coordination sphere of the metal. The conformation of lanthanide-ATP in the active site was effectively unchanged with respect to free lanthanide-ATP. 4. The conformation changes induced by the nucleotides were further resolved by analysis of the separate effects of adenosine and of lanthanide-pyrophosphate ; the latter caused no conformation change but bound specifically at the nucleotide phosphate binding site. Sulphate anions (which are often used as a medium in growing crystals for X-ray structure analysis) were shown also to compete for the phosphate groups of the nucleotides. 5. A conformation change was observed on binding 3-phospho-~-glycerate, and a study of the quaternary complexes E . ADP . P-glycerate . Gd and E . ATP . P-glycerate . Gd indicated that P-glycerate, although binding close to the nucleotides, did not significantly perturb the E . metal . nucleotide conformations. Abbreviations. NMR. nuclear magnetic resonance; PMR, proton magnetic resonance; Ln, lanthanide (Pr, Nd, ELI or Yb); EDTA. ethylenediamine tetraacetic acid, sodium salt; P-glycerate, 3-phospho-~-glyceric acid, sodium salt.

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