Allan S. Schneider

Rapid rise in cyclic GMP accompanies catecholamine secretion in suspensions of isolated adrenal chromaffin cells

Acetylcholine stimulates a five-fold increase in cyclic GMP within seconds of addition to suspensions of isolated adrenal chromaffin cells. The rapid kinetics and dose-response curve for cyclic GMP accumulation are compared with corresponding results for catecholamine secretion and a possible role of cyclic GMP in stimulus-secretion coupling is considered.

Stimulus-secretion coupling in isolated adrenal chromaffin cells: calcium channel activation and possible role of cytoskeletal elements.

Abstract: The catecholamine secretory function of a preparation of isolated bovine adrenal chromaffin cells has been further characterized under conditions designed to elucidate the mechanism of calcium channel activation and the possible role of cytoskeletal elements in stimulus‐secretion coupling. Three related sets of data were obtained: (1) Differences in kinetics, Ca dependence, strength, and additivity of the secretory response to acetylcholine (ACh) versus excess K; (2) the effects on secretion of the Ca channel‐blocking agents, Ni, Mg, and verapamil; and (3) the Ca dependence of vinblastine action on ACh‐ and K‐evoked secretion. The results suggest that a major portion of the Ca influx required for catecholamine release enters the cell via voltage‐dependent Ca channels with some additional Ca influx via the ACh receptor channel. Comparison of the present secretion data with corresponding known electrophysiological properties of isolated chromaffin cells provides added evidence for a role of chromaffin cell action potentials in regulation of Ca influx and the secretory response. Elevated Ca concentrations enhanced K‐evoked secretion to levels comparable to that of ACh but did not induce a vinblastine block of K‐evoked release. This provides further evidence against a role of microtubules in the common exocytosis event per se. However, a role of cytoskeletal elements in directing the movement of secretory granules, or an action of vinblastine at cholinergic receptors, remain distinct possibilities.

Mobility of water bound to biological membranes. A proton NMR relaxation study.

Water proton nuclear magnetic resonance relaxation measurements have been obtained for aqueous suspensions of red cell membranes. These data support a model in which water molecules are exchanging rapidly between a bound phase with restricted motions and a free phase with dynamic properties similar to liquid water. From this model and these data, estimates are obtained for the relaxation time for bound phase water. Possible relaxation mechanisms for bound phase water are discussed and some support is found for an intermolecular interaction modulated by translational motions characterized by a diffusion constant of 10(-9) cm2/s.

Cholinergic stimulants and excess potassium ion increase the fluidity of plasma membranes isolated from adrenal chromaffin cells.

Chromaffin cell membranes from the bovine adrenal medulla were labelled with the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene, and the fluorescence polarization (P) of the membrane suspensions was measured as a function of temperature. The P versus t profiles, between 20 and 37 degrees C, showed two linear regions separated by a break in the vicinity of 30 degrees C, reflecting a change in the phase behaviour of the constitutent lipids. Decreases in P values at higher temperature indicated progressive fluidization of the lipid bilayer. Previous incubation with either acetylcholine (0.5 mM) or nicotine (50 microM) produced further fluidization, the extent of which depended on the presence of added Ca2+ (2.2 mM). Thus, the flow activation energy, delta E, between approx. 30 and 37 degrees C was 9.1 kcal/mol for acetylcholine and 8.8 kcal/mol for acetylcholine plus Ca2+, as compared to 7.9 kcal/mol in the absence of acetylcholine and Ca2+. In the presence of nicotine, delta E was 11.4 kcal/mol when Ca2+ was absent and 9.5 kcal/mol when it was present. The cholinergic blocker, hexamethonium (0.5 mM), abolished the acetylcholine- or nicotine-induced changes. 65 mM K+ produced a similar fluidization, which was reversed by addition of Ca2+. An additive effect was observed when the membranes were incubated with both nicotine and K+, with delta E = 16.6 kcal/mol in the presence of Cas2+. These results indicate a receptor-mediated modulation of the lipid distribution between rigid and fluid regions in the membrane, which could be of importance for stimulated catecholamine secretion in the intact cell.

Acetylcholine-induced in vitro fusion between cell membrane vesicles and chromaffin granules from the bovine adrenal medulla

Catecholamine (CA) secretion from the adrenal medullary chromaffin cell (CC) occurs by means of exocytotic membrane fusion [l]. In studies at the plasma membrane fusion with the chromaffm granule (CG) and subsequent efflux of CA and associated granule components, it is useful to have an in vitro system in which such a process can be demonstrated. The ‘leaky’ CC has been proposed for this purpose E21. Here we report data indicating that fusion between isolated CC plasma membranes and intact CG in suspension can be induced by acetylcholine (ACh), the natural cholinergic agonist of the adrenal medulla, The method is based on our ob~rvation that the fluorescence of 6~arboxy-~uoresce~e (CF) is quenched by relatively high CA concentrations. Emission quenching of the parent compound, fluoresceine, by a variety of phenolic compounds, was reported in [3]. Our approach is different to previous applications of CF where increases in fluorescence occu~~g when liposomecontained CF was transferred into intact lymphocytes and other cells were recorded, with a consequent ‘relief of self-quenching’ [4-61. Changes in CF emission yield, in a suspension containing intact CG and CC plasma membrane vesicles loaded with CF, can occur for different reasons. In the absence of fusion between the two different types of vesicles, the fluorescence intensity of the suspension will remain constant if the CF-loaded plasma membrane vesicles are tight. If they are leaky, a progressive increase in fluorescence will occur, owing to CF release into the medium. In the event