Characteristics and determinants of osmotic lysis in chromaffin granules

(1) Using isolated bovine chromaffin granules, we demonstrate that osmotic lysis is not a random process and establish the osmotic pressure dependence of osmotic lysis in chromaffin granules, the so-called osmotic fragility curve. (2) We show by measuring the release of constituents of the granule core and correlating these with changes in spectroscopic parameters (turbidity and endogenous catecholamine fluorescence), that the latter can be safely used to measure lysis. (3) Within a particular granule population, noradrenaline granules lyse at higher osmolarities than adrenaline granules, suggesting a higher core osmolarity of the noradrenaline granules. (4) The size distribution of chromaffin granules as a function of lysis was determined by the use of whole mount electron microscopy. It is shown that the mean size of chromaffin granules decreases as a function of lysis. (5) On the basis of theoretical considerations three alternative models of the sequence of osmotic lysis in chromaffin granules are proposed. The experimental results best support a model which postulates that during partial osmotic lysis, granule membranes reseal into smaller vesicles after graded release of contents. The osmotic fragility would represent several cycles of lysis and resealing and would not be a reflection of the distribution of osmotic pressures in the granule population.     

Catecholamine content of chromaffin granule ‘ghosts’ isolated from bovine adrenal glands

Studies on the mechanism of catecholamine transport into chromaffin granules is complicated by the release of endogenous catecholamines. To overcome this problem chromaffin granule ghosts have been prepared by many investigators by osmotic lysis of the granules which results in a loss of over 90% of the endogenous catecholamine. However, in the studies reported here, the resulting ghosts still contained 36 ± 3.9 nmol epinephrine/mg of protein if they were lysed by passage through a Sephadex G-50 column preequilibrated with hypoosmtic media. This residual catecholamine was foun the slowly diffuse out of the ghosts in a temperature-dependent process at a rate sufficient to interfere with kinetic analysis of catecholamine transport. Attempts to remove the endogenous catecholamine from the ghosts indicated that most of it could not be removed by further osmotic shock or freeze-thaw treatments, but that over 85% of it was released from the granules by incubating them at 30°C for 90 min or by dialysis with a 35 and 86% loss of rate of catecholamine transport into the ghosts, respectively. If the endogenous catecholamine was removed from chromaffin granule ghosts by preincubating them for 90 min at 30°C, these resulting ghosts transported catecholamine with a linear Lineweaver-Burk plot indicating a K_m of 12±2 μM. In addition, the resulting ghosts did not leak catecholamines over a 10 min period at 30°C, and the transport of catecholamines was blocked by reserpine and enhanced with increasing pH from 6.0 to 8.5.     

An osmotic mechanism for exocytosis from dissociated chromaffin cells

Dissociated chromaffin cells from bovine adrenal medulla were stimulated to secrete epinephrine and dopamine beta-hydroxylase with a variety of secretagogues in a study designed to test the hypothesis that the chemiosmotic lysis reaction of isolated chromaffin granules might in some way be related to the mechanism of release during exocytosis. Increasing the osmotic strength of the incubation medium with either NaCl or sucrose led to suppression of secretion of epinephrine from the cells regardless of whether secretion was induced with veratridine or acetylcholine. Suppression of secretion was approximately exponential with respect to osmotic strength. Epinephrine secretion occurred only if the medium contained a permeant anion such as chloride, and secretion induced by veratridine was suppressed when Na isethionate replaced NaCl in the medium. In an extensive study with different monovalent anions veratridine supported epinephrine secretion according to the following activity series: Br-, I-, NO3- greater than methylsulfate, SCN- greater than Cl greater than acetate much greater than isethionate. A similar series, except for the potency of NO3-, was observed with A23187 as agonist. In general, the anion series for granule lysis was analogous. However, there was a poor quantitative correlation between the anion dependence of chemiosmotic granule lysis and the anion dependence of cell secretion. Anion transport inhibitors such as probenecid and pyridoxal phosphate also inhibited secretion while the stilbene disulfonates were inactive. The ineffectiveness of the stilbene disulfonates further distinguished chemiosmotic granule lysis from cell secretion. Secretion of catecholamines, induced by veratridine or nicotine, a cholinergic agonist, was suppressed when NaCl in the medium was replaced by isosmotic sucrose and unexpectedly low levels of dopamine beta-hydroxylase were observed in some cases. In sum, these properties of secreting chromaffin cells resembled some properties of isolated chromaffin granules incubated in ATP and Cl-, but were different in a number of instances. We, therefore, have interpreted our data to indicate that while some mechanistic relationships may indeed exist between the release event in exocytosis from chromaffin cells and the chemiosmotic lysis reaction characteristic of isolated chromaffin granules, an understanding of the energetics of exocytosis awaits the discovery of reasons for the quantitative differences between the two systems.     

Tensile Strength of the Chromaffin Granule Membrane

Catecholamine release from chromaffin granules, suspended in sucrose solutions of various osmotic strengths, was determined at different temperatures between 2° and 44°C. Dynamic measurements showed that steady state is achieved within 15 min of incubation at all temperatures. The effect of temperature on the release was established in terms of the median granular fragility (MGF) defined as the concentration of sucrose solution causing 50% lysis. The MGF was determined as the inflection point of the Gaussian distribution of granular fragility. The MGF was found to decrease with fall in temperature implying a corresponding increase of the tensile strength of the vesicle membrane. Critical resultant forces at lysis were calculated and found to vary from 8.2 dyn/cm at 2°C to 4.2 dyn/cm at 44°C. These compare well with tensions at lysis found earlier for erythrocytes.     

Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.     

Effects of changes in osmolality on the stability and function of cultured chromaffin cells and the possible role of osmotic forces in exocytosis

Recent evidence indicates that osmotic forces may play a role in exocytosis. To examine this possibility and to investigate the osmotic properties of storage granules within cells, we investigated the effects of changes of osmolality on stability and function of cultured bovine chromaffin cells. Cell volume measurements indicated that the cells behaved as osmometers and that the intracellular osmolality rapidly equilibrated with the osmolality of the extracellular medium. Hyperosmotic solutions strongly inhibited nicotinic agonist-stimulated secretion but did not alter nicotinic agonist-stimulated Ca(2+) uptake. Hyperosmotic solutions also strongly inhibited elevated potassium- stimulated secretion but only weakly inhibited elevated K(+)-stimulated Ca(2+) uptake. Thus, hyperosmotic solutions inhibited secretion at a step after calcium entry. Cells exposed to 165 mOs(1) solutions did not lyse and retained their capacity to store and secrete catecholamine upon stimulation. Significant intracellular lysis of chromaffin granules occurred within cells exposed to lower osmolalities. In contrast, 75 percent of the catecholamine was released from granules from cultured cells or from fresh adrenal medulla incubated in vitro at 210 mOs. The data provide evidence for a role for osmotic forces in exocytosis and suggest that if osmotic stress of the granule occurs during exocytosis, then water influx into chromaffin granules increases granule volume by at least 70 percent. The results also indicate that the osmotic properties of the granules are altered upon homogenization and subcellular fractionation of the cells.     

Steady-state catecholamine distribution in chromaffin granule preparations: a test of the pump-leak hypothesis of general anesthesia

The molecular mechanism of general anesthesia is not understood. Possible modes of action include binding at a protein site, such as a receptor or channel, or physical effects on membrane lipid properties. The pump-leak hypothesis suggests that anesthetics perturb the bilayer of synaptic vesicles, thereby increasing ionic permeability. This results in decay of proton gradients required for transport and accumulation of neurotransmitters. The subsequent loss of neurotransmitters from synaptic vesicles reduces the efficiency of synaptic transmission and results in the anesthetized state. We have determined the effects of general anesthetics on certain parameters of enzyme activity and membrane permeability relevant to the pump-leak hypothesis. We used chromaffin granules as a convenient model system and focused on clinically relevant anesthetic concentrations (ED50), quantitative measurements of permeability changes, and the kinetics of gradient decay. General anesthetics at ED50 have little or no effect on the proton-transport ATPase activity, but do cause modest increments in proton permeability that change the catecholamine distribution in actively pumping chromaffin granule preparations. We found that pH gradients do not collapse entirely under these conditions and that only a fraction of total catecholamine is lost from the chromaffin granules. When total collapse is induced by other means, efflux of catecholamines occurs with a half-time near 30 min. These results suggest that if the pump-leak hypothesis is valid, then very small losses of catecholamines must be sufficient to induce anesthesia. We conclude that the weight of evidence favors other mechanisms, notably direct binding of anesthetics to sensitive proteins.     

Granule matrix property and rapid "kiss-and-run" exocytosis contribute to the different kinetics of catecholamine release from carotid glomus and adrenal chromaffin cells at matched quantal size

Catecholamine-containing small dense core granules (SDCGs, vesicular diameter of ~100 nm) are prominent in carotid glomus (chemosensory) cells and some neurons, but the release kinetics from individual SDCGs has not been studied in detail. In this study, we compared the amperometric signals from glomus cells with those from adrenal chromaffin cells, which also secrete catecholamine but via large dense core granules (LDCGs, vesicular diameter of ~200-250 nm). When exocytosis was triggered by whole-cell dialysis (which raised the concentration of intracellular Ca(2+) ([Ca(2+)](i)) to ~0.5 μmol/L), the proportion of the type of signal that represents a flickering fusion pore was 9-fold higher for glomus cells. Yet, at the same range of quantal size (Q, the total amount of catecholamine that can be released from a granule), the kinetics of every phase of the amperometric spike signals from glomus cells was faster. Our data indicate that the last phenomenon involved at least 2 mechanisms: (i) the granule matrix of glomus cells can supply a higher concentration of free catecholamine during exocytosis; (ii) a modest elevation of [Ca(2+)](i) triggers a form of rapid "kiss-and-run" exocytosis, which is very prevalent among glomus SDCGs and leads to incomplete release of their catecholamine content (and underestimation of their Q value).     

Hyperosmotic relaxation lysis of chromaffin granules is caused by interactions between the granular membrane and intragranular vesicles

Bovine chromaffin granules undergo irreversible structural changes during osmotic shrinkage in hypertonic sucrose and salt solutions, such that, on reexposure to isoosmotic conditions they do not regain their original morphology, but undergo lysis ('hyperosmotic relaxation lysis'). Irreversible alterations of granules were induced by hypertonic incubations lasting for as little as 1 min. Fluorescence and EPR membrane labelling experiments showed that hypertonicity did not induce membrane loss for instance by inwardly or outwardly directed pinching off of membrane material. The mean sizes of chromaffin granules as a function of increasing and subsequently decreasing osmotic pressure were measured by photon correlation spectroscopy; there was no significant difference in sizes of hyperosmotically pretreated granules as compared with controls. Freeze-fracture electron micrographs showed the formation of 'twins' and 'triplets' under hypertonic conditions. They also revealed intragranular vesicles of 50-200 nm in diameter in both hypertonically and isotonically suspended granules. 'Twin' and 'triplet' granules were formed by the attachment of intragranular vesicles to the granule membranes. We suggest that hyperosmotic relaxation lysis is caused by the fact that this adhesion partly prevents the granule membrane from reexpanding, thus, leading to its rupture.     

Synapsin II negatively regulates catecholamine release

We have assessed the role of synapsins in catecholamine release by comparing the properties of exocytosis in adrenal chromaffin cells from wild-type and synapsin triple knock-out (TKO) mice. Brief depolarizations led to a greater amount of catecholamine release in chromaffin cells from TKO mice in comparison to chromaffin cells from wild-type mice. This increase in catecholamine release was due to an increased number of exocytotic events, while the properties of individual quanta of released catecholamine were unchanged. Barium ions produced similar amounts of catecholamine release from TKO and wild-type chromaffin cells, suggesting that the reserve pool of chromaffin granules is unchanged following loss of synapsins. Because expression of synapsin IIa in TKO chromaffin cells rescued the defect in depolarization-induced exocytosis, the TKO phenotype apparently results from loss of synapsin IIa. We conclude that synapsin IIa serves as a negative regulator of catecholamine release and that this protein influences exocytosis from a readily releasable pool of chromaffin granules. Further, because these defects in catecholamine release are different from those observed for glutamate and GABA release in TKO mice, we conclude that the functions of synapsins differ for vesicles containing different types of neurotransmitters.     

On the interaction between chromaffin granule membranes and intragranular vesicles--theory and analysis of freeze-fracture micrographs

We present a model for the calculation of intragranular vesicle adhesion energy in a two-vesicle system consisting of an external secretory vesicle (chromaffin granule) and an intragranular vesicle (IGV) that adheres from the inside to the granule membrane. The geometrical parameters characterizing the granule-IGV systems were derived from freeze-fracture electron micrographs. Adhesion is brought about by incubation of the granules in hyperosmolar sucrose solutions. It is accompanied by a deformation of the granule because the intragranular vesicle bulges it outwards, and by segregation of intramembraneous particles from the adherent part of the granule membrane. Adhesion prevents the deformed granules from osmotic reexpansion and, therefore, causes hyperosmotic relaxation lysis. We estimated specific adhesion energy at -3 erg/cm2, a value which is 10 - 1000 times larger than the energy of van der Waals interaction between membranes. This large interaction energy probably results from changes of the granule core induced by dehydration. A minimization of the interface between the granule core and adjacent membranes could exclude intragranular vesicles from the core and squeeze them towards the granule membrane. This might induce a new kind of interaction between both membranes, which is irreversible and causes lysis upon osmotic relaxation.     

Sodium-dependent calcium efflux from adrenal chromaffin cells following exocytosis. Possible role of secretory vesicle membranes.

Although cytosolic Ca2+ transients are known to influence the magnitude and duration of hormone and neurotransmitter release, the processes regulating the decay of such transients after cell stimulation are not well understood. Na(+)-dependent Ca2+ efflux across the secretory vesicle membrane, following its incorporation into the plasma membrane, may play a significant role in Ca2+ efflux after stimulation of secretion. We have measured an enhanced 45Ca2+ efflux from cultured bovine adrenal chromaffin cells following cell stimulation with depolarizing medium (75 mM K+) or nicotine (10 microM). Such stimulation also causes Ca2+ uptake via voltage-gated Ca2+ channels and secretion of catecholamines. Na+ replacement with any of several substitutes (N-methyl-glucamine, Li+, choline, or sucrose) during cell stimulation inhibited the enhanced 45Ca2+ efflux, indicating and Na(+)-dependent Ca2+ efflux process. Na+ deprivation did not inhibit 45Ca2+ uptake or catecholamine secretion evoked by elevated K+. Suppression of exocytotic incorporation of secretory vesicle membranes into the plasma membrane with hypertonic medium (620 mOsm) or by lowering temperature to 12 degrees C inhibited K(+)-stimulated 45Ca2+ efflux in Na(+)-containing medium but did not inhibit the stimulated 45Ca2+ uptake. Enhancement of exocytotic secretion with pertussis toxin resulted in an enhanced 45Ca2+ efflux without affecting calcium uptake. The combined results suggest that Na(+)-dependent Ca2+ efflux across secretory vesicle membranes, following their incorporation into the plasma membrane during exocytosis, plays a significant role in regulating calcium efflux and the decay of cytosolic Ca2+ in adrenal chromaffin cells and possibly in related secretory cells.     

Two functionally distinct forms of guanosine cyclic 3',5'-phosphate stimulated cation channels in a bovine rod photoreceptor disk preparation

Cyclic nucleotide stimulated efflux of 22Na+ and 45Ca2+ from a purified bovine rod outer segment disk preparation was measured on the 25-100-ms time scale by a novel rapid superfusion method. Activation of cation efflux by 8-bromoguanosine cyclic 3',5'-phosphate (8-Br-cGMP) was maximal within 25 ms. Over a wide range of concentrations of 8-Br-cGMP, the kinetics of termination of efflux precisely conformed to the sum of two exponential decay processes: a rapid phase (decay constant of 200 ms) and a slower phase (decay constant of 1.6 s). The kinetics of the biphasic decay of efflux cannot be explained by depletion of a pool of releasable 22Na but appear to reflect an intrinsic process for inactivation of the channels. 8-Br-cGMP-stimulated release of actively accumulated 45Ca exhibited identical biphasic decay kinetics. The maximum rate of Ca release [5 nmol.(mg of disk protein)-1.min-1] may be sufficient to produce a 1 microM change in local cytoplasmic [Ca] within 20 ms. The Ca:Na selectivity ratio is approximately 0.5:1 for both decay phases. 8-Br-cGMP demonstrated a lower potency (EC50 of 8.4 microM vs 2.8 microM) but a higher degree of cooperativity in its activation of the rapid vs the slower decay phase of 22Na efflux. The slower phase of decay was selectively inhibited by 25 microM l-cis-diltiazem, a relatively weak inhibitor of the rapid decay phase. Sodium ion (5-10 mM) selectively inhibited the rapid decay phase of 8-Br-cGMP-stimulated 45Ca release. These two kinetically and pharmacologically distinct phases of decay are hypothesized to represent two functionally distinct forms of cGMP-stimulated cation channels.     

cis-Unsaturated fatty acids induce the fusion of chromaffin granules aggregated by synexin

When isolated chromaffin granules were aggregated by synexin (a Ca2+-binding protein present in chromaffin and other secretory tissues) and then exposed to cis-unsaturated fatty acids at 37 degrees C, they fused together to form large vesicles. The fusion was monitored by phase and electron microscopy and by turbidity measurements on the granule suspension. Arachidonic acid was the most effective fusogen, whereas trans-unsaturated fatty acids, saturated fatty acids, detergents or lysolecithin were inactive. During fusion some of the epinephrine of the granules was released but the soluble core proteins remained trapped in the resulting vesicles. These vesicles swelled to enclose the maximum volume. Although this swelling could be inhibited by increasing the osmotic strength of the medium, it did not appear to depend on the chemiosmotic properties of the granule membranes as it was not influenced by ATP, a proton ionophore, or an anion transport inhibitor. The regulators of this in vitro fusion--Ca2+, synexin, and free, cis-unsaturated fatty acids--may be present in the cytoplasm of the chromaffin cell when it is stimulated to release epinephrine and granule proteins by exocytosis. Therefore, this fusion event may be the same that occurs between chromaffin granules undergoing compound exocytosis.     

Composition of the aqueous phase of chromaffin granules

Nuclear magnetic resonance spectroscopy has been used to determine the composition of the aqueous phase of bovine chromaffin granules. Relative concentrations of catecholamines (epinephrine plus norepinephrine), ATP and chromogranins have been measured from integrated intensities in the proton spectra using computer simulation techniques. Most or all of the catecholamines (97 ± 8%) are present in the aqueous phase and contribute to the high resolution spectrum. The catecholamine: ATP molar ratio (4.41 ± 0.45) determined by NMR is close to the value (4.45) derived from biochemical assay indicating that most or all of the ATP is present with catecholamine in the aqueous phase. Catecholamine: protein ratios show that approximately 45% of the soluble protein freed by lysis is not NMR visible. Intensity from this fraction does not appear under highly denaturing conditions (8 M urea) but reappears after hydrolysis. This behavior is similar to that of recently isolated soluble lipoprotein complexes. Variations in the NMR spectra associated with (1) different preparative procedures; (2) different suspension media, and (3) increasing osmolality are described. The fact that high concentrations of epinephrine and ATP (approximately 700 mM total) are dissolved in the aqueous phase implies that solution phase interactions at least partially ionic in nature are responsible for the low internal osmolality of chromaffin granules in vivo. Ordered phases containing a substantial fraction of the total catecholamine in an osmotically inactive form are not present.     

Ascorbic acid and catecholamine release from digitonin-treated chromaffin cells

The subcellular localization of catecholamines and ascorbic acid in cultured bovine adrenal chromaffin cells was studied by permeabilizing the cells with digitonin, a steroid glycoside. Catecholamine release from permeabilized chromaffin cells was dependent on the free calcium concentration and the temperature of the incubation mixture. By contrast, [14C]ascorbic acid, preloaded into the cells, was released by digitonin treatment in a manner independent of the concentration of free calcium and with only moderate regard to the incubation temperature. The sensitivity of ascorbic acid release to digitonin treatment was identical to that of calcium-dependent catecholamine release. These results thus suggest that ascorbic acid preloaded into the cells may directly efflux from the cell cytoplasm as a result of the permeabilization of the plasma membrane. Dimethylepinephrine, a permanently positively charged catecholamine analog which is known to be excluded from vesicular fractions, was also released by digitonin treatment in a manner independent of calcium. The time course of dimethylepinephrine release was very similar to that of ascorbic acid release. Thus, newly accumulated ascorbic acid in chromaffin cells may be localized to a free pool in the cell cytoplasm rather than in a vesicular compartment.     

Composition of the aqueous phase of chromaffin granules.

Nuclear magnetic resonance spectroscopy has been used to determine the composition of the aqueous phase of bovine chromaffin granules. Relative concentrations of catecholamines (epinephrine plus norepinephrine), ATP and chromogranins have been measured from integrated intensities in the proton spectra using computer simulation techniques. Most or all of the catecholamines (97 +/- 8%) are present in the aqueous phase and contribute to the high resolution spectrum. The catecholamine:ATP molar ratio (4.41 +/- 0.45) determined by NMR is close to the value (4.45) derived from biochemical assay indicating that most or all of the ATP is present with catecholamine in the aqueous phase. Catecholamine:protein ratios show that approximately 45% of the soluble protein freed by lysis is not NMR visible. Intensity from this fraction does not appear under highly denaturing conditions (8 M urea) but reappears after hydrolysis. This behavior is similar to that of recently isolated soluble lipoprotein complexes. Variations in the NMR spectra associated with (1) different preparative procedures; (2) different suspension media, and (3) increasing osmolality are described. The fact that high concentrations of epinephrine and ATP (approximately 700 mM total) are dissolved in the aqueous phase implies that solution phase interactions at least partially ionic in nature are responsible for the low internal osmolality of chromaffin granules in vivo. Ordered phases containing a substantial fraction of the total catecholamine in an osmotically inactive form are not present.     

Effects of mono-ADP-ribosylation on cytoskeletal actin in chromaffin cells and their release of catecholamine.

To better understand the physiological role of mono-ADP-ribosylation in animals, we examined its role in chromaffin cells. Monoclonal antibodies against rat brain ADP-ribosylhydrolase were prepared, one of which (9E7) completely inhibited the enzyme's activity with ADP-ribosylated actin as the substrate. After actin monomers were polymerized by the addition of Mg2+, mono-ADP-ribosylation induced actin depolymerization. After mono-ADP-ribosylation, the actin monomers did not polymerize by the addition of Mg2+. Polymerized actin cosedimented with chromaffin granules but mono-ADP-ribosylated actin did not. After ADP-ribosylhydrolase on the membrane of chromaffin granules was incubated with 9E7, mono-ADP-ribosylated actin did not cosediment with chromaffin granules. When chromaffin cells permeabilized with saponin were incubated with NAD and 9E7, actin and rho protein was mono-ADP-ribosylated and stimulated catecholamine release from the cells. In histochemical experiments, catecholamine and actin filaments disappeared when the permeabilized chromaffin cells were treated with NAD and 9E7. These findings indicate that mono-ADP-ribosylation breaks the actin barrier in order to move granules during exocytosis, and ADP-ribosylactin hydrolase may keep the granules within the actin barrier.     

Isolation and characterization of noradrenalin storage granules of bovine adrenal medulla.

A method is described for the preparation of (1) the heavy population of bovine adrenal chromaffin granules (SH (average sedimentation coefficient) = 12 400 S in 0.25 M sucrose) essentially free from contamination with mitochondria and other organelles, and (2) a subpopulation of this heavy population which is highly enriched in noradrenalin (greater than or approximately 95% of the total catecholamine is noradrenalin). The method is based on isopycnic gradient centrifugation using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.5 M sucrose medium. The isolated population of noradrenalin granules appeared highly electron dense in transmission electron microscopy and revealed a rather narrow size distribution. The specific content of amine and adenine nucleotides (with reference to total granule protein) was markedly higher than for the total population of heavy chromaffin granules. The molar ratio of amines to adenine nucleotides was, however, lower in the noradrenalin granules, i.e. 4.8 vs. 11.9.