Changes in the size of isolated chromaffin granules in ATP-evoked catecholamine release

The average size of chromaffin granules isolated from bovine adrenal medullae was analyzed by a quasi-elastic laser light scattering method. The granule diameter increased by a factor of 1.3 by addition of Mg-ATP in the medium. The ATP effect was completely suppressed in the presence of an anion transport blocker (SITS), and partly depressed by a proton transport blocker (DCCD).     

Affinity purified tetanus toxin binds to isolated chromaffin granules and inhibits catecholamine release in digitonin-permeabilized chromaffin cells

Tetanus toxin, a potent neurotoxin which blocks neurotransmitter release in the CNS, also inhibits Ca2+-induced catecholamine release from digitonin-permeabilized, but not from intact bovine chromaffin cells. In searching for intracellular targets for the toxin we studied the binding of affinity-purified tetanus toxin to bovine adrenal chromaffin granules. Tetanus toxin bound in a neuraminidase-sensitive fashion to intact granules and to isolated granule membranes, as assayed biochemically and visualized by electron microscopic techniques. The binding characteristics of the toxin to chromaffin granule membranes are very similar to the binding of tetanus toxin to brain synaptosomal membranes. We suggest that the toxin-binding site is a glycoconjugate of the G1b type (a polysialoganglioside or a glycoprotein-proteoglycan) which is localized on the cytoplasmic face of the granule membrane and might directly be involved in exocytotic membrane fusion.     

Ion permeability of isolated chromaffin granules.

The passive ion permeability, regulation of volume, and internal pH of isolated bovine chromaffin granules were studied by radiochemical, potentiometric, gravimetric, and spectrophotometric techniques. Chromaffin granules behave as perfect osmometers between 340 and 1,000 mosM in choline chloride, NaCl, and KCl as measured by changes in absorbance at 430 nm or from intragranular water measurements using 3H2O and [14C]polydextran. By suspending chromaffin granules in iso-osmotic media of various metal ions and selectively increasing the permeability to either the cation or the anion by intrinsically permeable ions or specific ionophores, it was possible to determine by turbidity and potentiometric measurements the permeability to the counterion. These measurements indicate that the chromaffin granule is impermeable to the cations tested (Na+, K+, and H+). Limited H+ permeability across the chromaffin granule membrane was also shown by means of the time course of pH re-equilibration after pulsed pH changes in the surrounding media. The measurement of [14C]methylamine distribution indicates that a significant deltapH exists across the membrane, inside acidic, which at an external value of 6.85 has a value of 1.16. The deltapH is relatively insensitive to changes in the composition of the external media and can be enhanced or collapsed by the addition of ionophores and uncouplers. Measurement at various values of external pH indicates an internal pH of 5.5. Use of the ionophore A23187 indicates that Ca++ and Mg++ can be accumulated against an apparent concentration gradient with calcium uptake exceeding 50 nmol/mg of protein at saturation. These measurements also show that Ca++ and Mg++ are impermeable. Measurement of catecholamine release under conditions where intravesicular calcium accumulation is maximal indicates that catecholamine release does not occur. The physiological significance of the high impermeability to ions and the existence of a large deltapH are discussed in terms of regulation of uptake, storage, and release of catecholamines in chromaffin granules.     

Solubilization and reconstitution of the catecholamine transporter from bovine chromaffin granules.

The catecholamine transporter from bovine chromaffin granules has been solubilized by using low concentrations of sodium cholate in the presence of phospholipids. The functional solubilized protein has been incorporated into liposomes after removal of the detergent either by gel filtration or by dialysis. Reserpine-sensitive accumulation against a concentration gradient is achieved by artifically imposing a pH gradient across the membrane. In the reconstituted system adenosine 5'-triphosphate (ATP) serves as an energy source only at higher detergent concentrations. The proton-translocating adenosine triphosphatase (ATPase) is solubilized in parallel with the increasing efficiency of ATP as an energy source. Several criteria are proposed to distinguish between carrier-mediated (reserpine sensitive) and unmediated transport in the reconstituted system. The reserpine-sensitive process shows affinity and ss presented in this communication provide further support for the contention that concentrative uptake in biogenic amine storage vesicles is driven by a transmembrane pH gradient, which, in the native system, is generated by a proton-translocating ATPase. Moreover, the assays described provide a tool for the isolation and purification of the transport protein.     

Catecholamine transport and energy-linked function of chromaffin granules isolated from a human pheochromocytoma

The structure and function of chromaffin granulles of human phoechromocytoma was extensively investigated in a highly purified granule fraction obtained from a single specimen of human pheochromocytoma tissue. Pheochromocytoma chromaffin granules were analyzed for catecholamine, ATP, enkephalin, phospholipid, cytochrome and ion content. Using a variety of techniques it was found that the membrane of these granules is highly impermeable to Na⁺, K⁺, and H⁺, and that the intragranular pH was maintained at 5.1 irrespective of suspending media. The presence of MgATP induces a transmembrane potential (ΔΨ) across the membrane of these granules which is positive inside and which corresponds to 90 mV. Both ΔpH and ΔΨ are coupled to biogenic amine accumulation into the granules in a process which is reserpine sensitive. These properties are compared with those of chromaffin granules isolated from normal human tissue or from other animal species and are discussed in terms of possible explanation at a biochemical or subcellular level of the clinical manifestation of the pheochromocytoma.     

Protonmotive force in giant mitochondria

The accompanying communication [Eur. J. Biochem. 141 (1984) 1-4] indicates that the microinjection of the pH fluorescent indicator pyranine (8-hydroxy-1,2,6- pyrenesulfonate ) into giant mitochondria or mitoplasts does not affect their ability to carry out oxidative phosphorylation. The dye can therefore be used as a quantitative indicator of internal mitochondrial pH. We found that activation of metabolism in rotenone-inhibited giant mitochondria by the addition of succinate produces an internal pH change corresponding to a pH shift of 0.3 to the alkaline range, approximately the same value found previously for conventional rat liver mitochondria.     

Protonmotive force and motility of Bacillus subtilis.

Motility of Bacillus subtilis was inhibited within a few minutes by a combination of valinomycin and a high concentration of potassium ions in the medium at neutral pH. Motility was restored by lowering the concentration of valinomycin or potassium ions. The valinomycin concentration necessary for motility inhibition was determined at various concentrations of potassium ions and various pH's. At pH 7.5, valinomycin of any concentration did not inhibit the motility, when the potassium ion concentration was lower than 9 mM. In the presence of 230 mM potassium ion, the motility inhibition by valinomycin was not detected at pH lower than 6.1. These results are easily explained by the idea that the motility of B. subtilis is supported by the electrochemical potential difference of the proton across the membrane, or the protonmotive force. The electrochemical potential difference necessary for motility was estimated to be about -90 mV.     

Exocytosis of catecholamine (CA)-containing and CA-free granules in chromaffin cells

Recent evidence suggests that endocytosis in neuroendocrine cells and neurons can be tightly coupled to exocytosis, allowing rapid retrieval from the plasma membrane of fused vesicles for future use. This can be a much faster mechanism for membrane recycling than classical clathrin-mediated endocytosis. During a fast exo-endocytotic cycle, the vesicle membrane does not fully collapse into the plasma membrane; nevertheless, it releases the vesicular contents through the fusion pore. Once the vesicle is depleted of transmitter, its membrane is recovered without renouncing its identity. In this report, we show that chromaffin cells contain catecholamine-free granules that retain their ability to fuse with the plasma membrane. These catecholamine-free granules represent 7% of the total population of fused vesicles, but they contributed to 47% of the fusion events when the cells were treated with reserpine for several hours. We propose that rat chromaffin granules that transiently fuse with the plasma membrane preserve their exocytotic machinery, allowing another round of exocytosis.     

Protonmotive force and bacterial sensing

The role of the proton gradient and external pH in the motility and chemotaxis of Bacillus subtilis was investigated. Presence of a substantial proton gradient is not necessary for motility or chemotaxis, as long as the electrical potential is sufficient to maintain motility. Changes in the proton gradient do, however, lead to changes in swimming behavior, and these changes are mediated by two processes. One is sensitive to external pH and probably operates through a pH receptor. The second is sensitive to changes in the proton gradient. When the level of the protonmotive force is high enough to maintain motiligy, changes in the components of the protonmotive force are sensed by the bacteria and lead to behavioral changes, but changes in the protonmotive force are not necessary for chemotaxis.     

Protonmotive force in yeasts--pH, buffer and species dependence.

Using yeast species Saccharomyces cerevisiae K, Rhodotorula gracilis, and Lodderomyces elongisporus, their intracellular pH value and their membrane potential were estimated at pH 3.5-7.5 in four different buffers: triethanolamine--phthalic acid (TEPA), citric acid--trisodium citrate (CASC), acetic acid--NaOH (AANA) and MES. The pHin followed the same pattern in all buffers, with rather constant values below pHout = 5 and again above pHout = 7. The membrane potential decreased regularly with decreasing pHout. The apparent protonmotive force increased with decreasing pHout. It is seen that for all the yeast species pHin and pHout are the same between 5.0 and 6.0 which is thus the pH range of choice for various transport measurements. Of the four buffers, TEPA gives smoothest pHin dependences on pHout and, being metabolically inert, is the one to be recommended.     

Asbestos-elicited catecholamine secretion from isolated bovine adrenal chromaffin cells

Standard (UICC) chrysotile B asbestos fibres caused rapid (within minutes) 5-to-8-fold stimulations of catecholamine secretion from isolated bovine adrenal chromaffin cells without affecting their viability (97%). The stimulation of catecholamine secretion by asbestos was selective to chrysotile type fibres, half-maximal stimulation by standard chrysotile B, chrysotile A, crocidolite, amosite and silica fibres being observed at 7, 73, 160, 250 and ? 500 μg per ml, respectively. The secretory effect of chrysotile B was additive to that of acetylcholine and blocked by either the divalent cations, Co²⁺, Ni²⁺ and Mg²⁺ or the ion chelators, EGTA and EDTA. Conversely, neither verapamil, methoxyverapamil, or removal of extracellular calcium affected the asbestos-evoked catecholamine secretion. These data indicate that the selective stimulatory effect of chrysotile type asbestos on adrenal chromaffin cells can be mediated by membrane or intracellular calcium and raise the question of the possible involvement of catecholamines in the pathogenesis of asbestos related diseases.     

ATP-stimulated transmitter release and cyclic amp synthesis in isolated chromaffin granules

ATP stimulates chromaffin granules from the bovine adrenal medulla to release epinephrine and specific soluble proteins. ATP analogs substituted in the β-γ-position with either nitrogen or carbon were also found to be effective at inducing release from isolated chromaffin granules. However, an ATP analog substituted at the α-β position with carbon was strongly inhibitory. Cyclic AMP was also found to be synthesized by isolated chromaffin granules under release conditions. ATP analogs were effective as substrates for adenylate cyclase in the same order as their efficiency for inducing release from vesicles. Hydrolysis at the β-γ linkage of ATP therefore is probably not necessary for release; however, hydrolysis at the α-β position may be important in the release process. Cyclic AMP may be produced and play a regulatory role in this event.     

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.     

Temperature-induced lysis of chromaffin granules provides evidence against the two-pool hypothesis of catecholamine storage

The temperature-dependent release of core constituents from isolated chromaffin granules in isotonic sucrose has been a controversial and puzzling phenomenon that has been interpreted either as selective catecholamine efflux from different catecholamine pools or as temperature-dependent lysis. We have analysed the kinetics, temperature dependence and physical basis of this process. Our results demonstrate that, upon increasing the ambient temperature, chromaffin granules show a shift in their osmotic fragility to higher osmolarities, which is linearly dependent on temperature and leads to measurable lysis in 0.26 M buffered sucrose at temperatures above 12 degrees C. It is possible to demonstrate both protein and dopamine beta-hydroxylase release when lysis as a function of temperature is measured in 0.26 M buffered sucrose. Real time measurements of the lysis kinetics were recorded on cassettes and analysed by a computer program for exponential decay kinetics. It is shown that the temperature-dependent lysis proceeds in two separate phases, the fast one of which is associated with temperature-dependent shift in the osmotic fragility curve. It has no characteristics of any exponential decay kinetics. The slow phase, when followed over several hours, leads to complete lysis of the granules in a sigmoidal time course at 30 degrees C. We conclude from the absence of exponentiality that there is no basis on which to assume the existence of different catecholamine pools. The fast phase of temperature-dependent lysis can be best explained as a simple temperature-dependent increase of the granule core solution's osmotic pressure, while the slow phase is probably caused by sucrose permeation into the granules. On the basis of these results, we warn against any efflux experiments measuring the temperature-dependent transmitter release from secretory vesicles with highly concentrated core solutions.     

Molecular pharmacology of the catecholamine transporter of chromaffin granules from the bovine adrenal medulla

Tetrabenazine (TBZ) and reserpine are two inhibitors of the catecholamine uptake system of the chromaffin granule membrane. They are structural analogs of the substrates dopamine and serotonin and they inhibit the monoamine transporter, which catalyzes a H+/neutral amine antiport. [3H]Dihydrotetrabenazine ([3H]TBZOH) is bound by chromaffin granule membranes on one class of site (T sites, KD = 3 nM); [3H]reserpine is bound on T sites and a second class of site (R1 sites, KD = 0.7 nM). The two sites are involved in monoamine translocation. The substrates displace the ligands with different efficiency: noradrenaline (Km = 10 microM) displaces reserpine efficiently (EC50 = 30 microM), but TBZOH poorly (EC50 = 2000 microM); m-iodobenzylguanidine, which has recently been shown to be a substrate of the monoamine uptake system (Km = 5 microM), displaces TBZOH efficiently (EC50 = 25 microM), but reserpine inefficiently (EC50 = 300 microM). Since both substrates are translocated by the same transporter, this result confirms the existence of two sites with different properties. T sites are characterized by a linear relationship between the reciprocal of the dissociation constants of various drugs displacing [3H]TBZOH and their partition coefficient in octanol/H2O mixtures. This relationship, which indicates a hydrophobic environment of T sites, does not exist for R1 sites. T sites have been identified by covalent labeling with a derivative of TBZ coupled to an arylazido group. The labeled sites are borne by a 65,000 dalton protein. The kinetics of reserpine binding are accelerated in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

The ionogenic nature of the secretory-granule membrane. Electrokinetic properties of isolated chromaffin granules

1. Chromaffin granules isolated from the bovine adrenal medulla possess an electrophoretic mobility of -1.12mum.s(-1).cm.V(-1), corresponding to a surface zeta potential of -14.4mV and surface charge density of 1.38x10(-6)C.cm(-2). 2. The mobility of chromaffin granules is pH-dependent, indicating an amphoteric surface with an isoelectric point at pH3.0 and acidic groups with a pK(a) of 3.11. 3. Addition of bi- and ter-valent cations decreased the mobility of chromaffin granules in a dose-dependent fashion with a relative potency of La(3+)>Mn(2+)>Ca(2+) >Sr(2+)>Mg(2+)>Ba(2+). 4. Treatment with neuraminidase decreased the mobility of erythrocytes by 84%, whereas chromaffin-granule mobility was decreased by only 14%. This correlates well with the small complement of neuraminic acid present in the granule membrane. 5. The nature, origin and significance of the anionic surface charge of the chromaffin granule is discussed. It is concluded that the net negative charge at the surface of shear derives chiefly from a single type of chemical group, namely -CO(2) (-), contributed by the alpha-carboxyl group of constituent proteins, the phospholipid phosphatidylserine and, to a lesser extent, the sialic acid component of glycoproteins.     

Aggregation and dispersity of isolated chromaffin granules studied by intensity fluctuation spectroscopy

The aggregation and dispersity of isolated bovine adrenal secretory vesicles (chromaffin granules) were studied by intensity fluctuation spectroscopy. The degree of dispersity and the Z-average translational diffusion coefficients were calculated from the autocorrelation functions of the intensity fluctuations in lase light scattered from the granules in solution. Granules purified by sedimentation through 0.3 M sucrose/Ficoll/²H₂O showed greater dispersity than granules purified by sedimentation through 1.6 M sucrose. By monitoring the scattered light intensity and the diffusion coefficients of the granules, many of the difficulties encountered in the interpretation of absorbance measurements were avoided. Measurements over a range of granule concentrations in sucrose solutions (10 mM HEPES, pH 7.0), indicated that aggregation of the granules occurred at concentrations above 150 μg protein/ml. At low granule concentrations (15–30 μg protein/ml) Ca²⁺-induced aggregation was detected at a threshold of 2–10 mM calcium.     

Protonmotive force and photophosphorylation in single swollen thylakoid vesicles

Swollen vesicles generally 40 micron in diameter were prepared from spinach chloroplasts. These vesicles appear to originate from thylakoids. The present study reports results obtained with individual vesicles using micromanipulative procedures. The electric potential across the membrane was measured with microelectrodes and the pH of the internal space was calculated from the fluorescence of the pH indicator pyranine. The individual vesicles photophosphorylate as measured with luciferin-luciferase. Impalement with microelectrodes did not affect the ability of individual vesicles to photophosphorylate. However, there was no significant membrane potential either with continuous illumination or light flashes. In contrast, we found a delta pH of 3.7 under photophosphorylative conditions and the incubation with the appropriate buffers blocked photophosphorylation presumably by preventing formation of a pH gradient. We propose that, in these vesicles, the membrane potential plays no role in photophosphorylation, whereas a pH gradient is obligatory.