Physical studies of the chromaffin granule and its membrane

Chromaffin granules, the catecholamine storage vesicles abundant in the chromaffin cells of the adrenal medulla, are immunologically identical to the adrenergic vesicles of the sympathetic nervous system. As a consequence of their common embryological origin, the chromaffin cell serves as a useful model system in which to investigate several aspects of nerve action. Chromaffin granules contain large concentrations of catecholamines and of ATP, acidic proteins termed the chromogranins, and the divalent metal ions, calcium and magnesium, The membrane of the chromaffin granule contains approximately 30% protein by weight. These proteins include a Mg2+-activated ATPase, an electron transfer chain with an NADH:(acceptor) oxidoreductase activity and the enzyme dopamine-β-hydroxylase.The membrane has an unusually large content of lysolecithin, approximately 16% of the total phospholipid.
The adrenal medulla is innervated directly by a branch of the greater splanchnic nerve. Acetylcholine, released at the synapses, potentiates an influx of calcium ions into the chromaffin cell. This results in the migration of the granules through the cytoplasm to the cell plasma membrane where the two membranes fuse, expelling the total contents of the granules into the interstitial space. The lysolecithin present in the membrane has been implicated in this process of fusion.
There are many problems associated with the chromaffin granule system. It it not known how such large quantities of catecholamines, nucleotides and proteins can be assimilated and then maintained within the granule against the very large osmotic and concentration gradients. The roles of the membrane proteins, in vivo, have not been determined (apart from that of dopamine-β-hydroxylase). Roles have been proposed for the ATPase involving it in catecholamine synthesis and uptake, ATP synthesis, interaction with microtubules in the transcytoplasmic migration and also interaction with, the plasma membrane in exocytosis itself. Apart from the knowledge that the secretory stimulus is a calcium influx and that exocytosis involves membrane fusion, virtually nothing has been determined about the intermediate processes.
The aim of this thesis has been threefold; to determine the role of the ATPase, to discover the nature of the granule storage complex and to investigate the structure of the granule membrane in view of the proposed properties and the function of the membrane in exocytosis.
The studies on the ATPase are described in Chapter Two. It was found that the activity of the ATPase of both intact granules and granule membranes is uncoupler sensitive i.e. the activity is greatly enhanced in the presence of conventional uncouplers of the mitochondrial ATPase activity and the electron transfer chain. The uncouplers which affected the chroiuaffin granule ATPase include 5-chloro,3-t-butyl, 2'-chloro, 4'-nitrosalicylanilide (S-13) and bis-hexafluoro-acetone (1799). The ATPase activity of intact granules was enhanced optimally four-fold, whilst, that of the granule membrane was enhanced only 50%. The uncoupler sensitivity implies, according to the chemiosmotic hypothesis of oxidative phosphorylation, that the ATPase is able to generate a cation or proton gradient across the granule membrane. Neither the coupled nor the uncoupled ATPase activities were inhibited by oligomycin or aurovertin, but both were greatly inhibited by dicyclohexyl carbodiimide (DCCD) which is a potent inhibitor of the mitochondrial ATPase, and is thought to block proton channels. The inhibition by DCCD was only partially restored on solubilisation of the granule membrane although the uncoupling by S-13 was completely abolished. This indicates that the uncoupling phenomenon is dependent on the integrity of the membrane whilst DCCD inhibition is due, in part, to its direct interaction with the enzyme or tightly bound protein factor. It was also shown that the chromaffin granule membrane can generate an 'ANS Response' in the presence of ATP-Mg2+ i.e. the fluorescence of the probe, 1-anilinonaphthalene-8-sulphonate (ANS) interacting with the granule membrane was greatly enhanced on addition of ATP-Mg2+. This effect is also observed with submitochondrial particles, and is believed to be a reflection of the generation of a transmembrane potential. The enhancement was abolished when uncoupler molecules were added and was prevented by preincubation of the membrane with DCCD.
The pH dependence of the ATPase activity and the ANS response were investigated in the presence and absence of uncoupler. The pH optima of the coupled and uncoupled ATPase activities were very different. The coupled ATPase did not show an optimum; the activity remained constant between 6.0 and 8.0. The uncoupled activity was maximal at pH 6.4, as was the fluorescence enhancement of the ANS response. The 'reversal' of the ANS fluorescence enhancement by uncoupler had a linear dependence on pH at pHs greater than 6.0, but at pHs less than approximately 5.5, there was further enhancement.
The uptake of catecholamines by intact granules and by granule membrane vesicles, which is dependent on the hydrolysis of ATP, was abolished in the presence of uncouplers, even although the ATPase was up to four times as active. The uptake of these biogenic amines is, therefore, not a direct result of the ATPase activity, but is most probably a consequence of a potential energy gradient generated by the ATP hydrolysis. It is this gradient that is dissipated by uncouplers. The uptake is also inhibited by DCCD. These results indicate that the ATPase of the chromaffin granule membrane has similar properties to that of submitochondrial particles, where the ATP-dependent uptake of cations is abolished in the presence of uncouplers. It has not been possible to determine whether the electron transfer chain of the granule membrane is coupled to the ATPase, since the rates of NADH:(acceptor) oxidcreductase activity are very low.
Electron spin resonance (ESR) studies of the chromaffin granule membrane are described in Chapter Three. The structure of the membrane has been probed using a variety of spin labels. Both the effects of temperature and high levels of calcium have been studied. The results from three positional isomers of the stearic acid spin label demonstrate that a substantial part of the membrane lipid is in a bilayer structure which undergoes a structural transition at 32°-38°C, characterised by an increase in the population of gauche isomers in the lipid chains. A possible mechanism for this transition would be preferential segregation of cholesterol. The covalently bound iodoacetamide spin label revealed a transition within the protein component of the membrane, or its immediate lipid environment, at approximately 32°C. This transition corresponds to an increased degree of motional freedom of the spin label. The lipid soluble TEMPO spin label exhibits a break at 34°C in the temperature dependence of its partitioning into the membrane. This could correspond to the onset of a lateral phase separation in the membrane lipid, again possibly involving a redistribution of cholesterol. Calcium abolishes, diminishes or shifts the transition observed by TEMPO, and decreases the amplitude of motion of the stearic acid spin labels. This also may be due to a redistribution of lysolecithin and cholesterol. The temperatures of the structural transitions agree well with those observed in the enzymic activity of the membrane ATPase and the NADH oxidase functions, and also with the results of fluorescent probe studies. It is possible that triggering of the transition either with calcium or some other stimulus may play a role in catecholamine release and membrane fusion.
Nuclear magnetic resonance (NMR) studies have been made of intact adrenal glands, chromaffin granules and granule membranes (Chapter Four). 31Phosphorus NMR has been used as a sensitive non-destructive probe, permitting comparison of the structure of the contents of isolated granules with those of granules in intact glands, by measurement of the frequencies and line widths of the ATP resonances. Adrenal glands and chromaffin granules from pigs, sheep and cows have been examined, and it has been shown that the preparative procedure does not affect the contents of the granules, as assessed by these criteria. The nature of the granule complex has been studied by making pH titrations of the ATP resonances in simulated contents at varying degrees of complexity. It has been concluded from these studies that the internal pH of the bovine chromaffin granule is 5.45 ± 0.2. This is in excellent agreement with the findings described in Chapter Two. The simulation studies show that the reported contents themselves do not account for the broadening of the resonances observed in granule spectra, and that it is likely that the additional broadening is a result of the presence of low concentrations of paramagnetic ions. The total simulated complex, containing chromogranins and phospholipids in addition to ATP, catecholamines and divalent metal ions, is completely soluble at room temperature. This is a good indication that in vivo the storage complex does not exist as a crystalline or paracrystalline structure but as a very viscous solution. 1H and 13C NMR studies, in addition to 31P, show that the chemical shifts present in spectra of intact granules and of ATP and adrenaline at 122mM and 550mM respectively are due to general ring current shifting, as a consequence of the large concentration of aromatic rings present in the solutions. There is no evidence for specific complex formation of more than a transitory nature. This is supported by preliminary relaxation studies.
The 1H NMR spectrum of purified granule membranes shows no detail of lipid structure but resonances attributable to protein components, similar to the chromogranins, have been observed. 31P NMR studies of the chromaffin granule membrane indicate that the spectrum is similar to that expected from a consideration of chemical shift anisotropy effects. A temperature study was made of this membrane. Unfortunately the spectra are rather poor, but changes in the spectrum can be observed between 30° and 40°C. This is in agreement with the ESR, fluorescence and enzyme activity studies.
Finally, the chromaffin granule mwcbrane was examined by X-ray diffraction (Chapter Five). The intention was to obtain an electron density profile of the membrane, and to investigate the proposed existence of a protein lattice in the plane of the membrane. It was not possible to obtain this electron density profile since it was discovered that the membrane was not stable to the dehydration necessary to produce orientation. Electron microscopy of ultracentrifuged, partially dehydrated membrane pellets revealed the presence of large multilamellar structures which produce a sharp optical diffraction pattern,spacing approximately 46Å . X-ray diffraction of this pellet resulted in a strong lamellar diffraction, with a spacing of approximately 55Å, extending to the sixth or seventh order. No other diffracting species was present in the electron micrographs, but the spacings obtained from optical and X-ray diffraction are not in complete agreement. The reason for this has not been resolved. Preliminary studies at high humidity indicate that it may be possible to produce membrane vesicles that are sufficiently well oriented to give lamellar diffraction to the necessary resolution. At no time did the X-ray diffraction patterns show the presence of equatorial reflections which could be attributed to a protein lattice. This agrees with freeze-fracture studies carried out in this laboratory. The chromaffin granule membrane lipid extract was examined and the diffraction patterns at a range of humidities obtained. Although there were several minor diffraction series present, a major lamellar diffraction always occured. From the spacings and the intensities of these lamellar diffractions, a one-dimensional Fourier was synthesised, and an electron density profile of the lipid bilayer was obtained. The electron density maxima of this profile are 45Å apart, the same distance as that found with extracted mitochondrial lipids. This spacing is attributed to the presence of cholesterol in the bilayer. When chromaffin granules were irradiated there was no indication of an ordered diffraction of a crystalline nature, supporting the proposal that the storage complex is in a soluble, disordered state.
In conclusion, this study has proposed that the ATPase of the chromaffin granule membrane is energy-coupled and may be similar to the mitochondrial and related ATPases. It is capable of generating a potential gradient across the membrane, which can drive other functions including catecholamine uptake. The internal pH of the chromaffin granule is around 5.5, which means that there should be a pH gradient across the membrane of greater than one pH unit. There also appears to be a phase transition in the granule membrane, at approximately 34°C, which may involve the partitioning of cholesterol and the consequent formation of lysolecithin-enriched areas.