Sally E. Carty

The internal pH of mast cell granules.

Mast cells, which are found in all anatomical structures of the body except the blood and lymph, possess one of the largest contents of the biogenic amines histamine and serotonin (S-hydroxytryptamine) found within mammalian cells [ 1,2]. These amines, along with large amounts of the anionjc mucopolysaccharide heparin and proteolytic enzymes, are stored in high concentration within the osmophilic storage granules of the mast cell [3,4]. Varying in size from 300-800 nm, the spherical mast cell granules can be easily viewed by light microscopy [5]. According to [4] histamine is synthesized from histidine in the cytosol of the mast cell via the enzyme histidine decarboxylase, however fluorescence microscopy has established that histamine is localized in high concentration only in the granules [5]. Although this evidence suggests active ~stamine accumulation into mast cell granules against an apparent concentration gradient, the precise mechanism by which histamine and serotonin are transported and stored is unknown. Considerable similarity exists between the mast cell granule, the chromaffm granule, and the platelet dense granule with respect to the existence of a high content of basic biogenic amines, the presence of large i& acidic molecules implicated in the storage complex, the presence of associated peptide hormones, and the release by a stimulus coupling mechanism [ 1,671. Chromaffin granules and platelet granules, which store large quantities of catecholamines and serotonin, respectively, are remarkable in that they both maintain a distinctly acidic intragranular pH of 5.5 even after isolation and resuspension at neutral pH [6-81. There is good evidence that this intravesicular acidic pH is generated by a H+-translocating ATPase within the granule membrane; the

The isolation of intact adrenal chromaffin granules using isotonic percoll density gradients

An improved method for the isolation of intact adrenal chromaffin granules under isotonic conditions, using a Percoll density gradient, is presented. After dissection, homogenization, and differential centrifugation, the crude granule homogenate was layered onto a gradient medium previously centrifuged at 20,200g × 5 min, consisting of 30% (vv) Percoll, 0.27 m sucrose, and 10 mm Tris-maleate, pH 7.0. After centrifugation for 40 min at 8650g in a standard preparative centrifuge, the chromaffin granules were found to band in the lowest fraction, where 45% of the catecholamines and 60% of the ATP could be recovered. With respect to other published methods, the percentage of lysosomal and mitochondrial contamination compared favorably. In addition, granules isolated by the Percoll gradient method were found to have at least 42 and 14% higher ATP and catecholamines, respectively, per milligram of protein. It is suggested that this method offers the advantages of ease of preparation, purity, and cost efficiency when compared with previously published techniques.

Mechanisms of accumulation of tyramine, metaraminol, and isoproterenol in isolated chromaffin granules and ghosts

The effects of the transmembrane pH gradient (delta pH) and the transmembrane potential gradient (delta psi) on the uptake of several sympathomimetic amines were investigated, using bovine adrenal chromaffin granules isolated in isotonic sucrose. As previously described [R. Johnson and A. Scarpa, J. Biol. Chem. 254 3750 (1979)], freshly isolated chromaffin granules maintain an intragranular pH of 5.5 as measured by [14C] methylamine distribution and, in the presence of ATP, generate a delta psi of 80 mV, positive inside, as measured by [14C] methylamine distribution. When tyramine, metaraminol, and isoproterenol (1-50 mM) were added to well-buffered suspensions of granules at pH 7.0, a dose-related alkalinization of the granule interior was observed. Study of the time-resolved influx of the same amines labeled radiochemically (5-21 microM) revealed that all the amines were accumulated against an apparent concentration gradient. However, while accumulation of [14C] serotonin and [3H] isoproterenol was totally inhibited by reserpine, [14C] tryramine accumulation was inhibited by only 60% and [14C[ metaraminol uptake was unaffected. The ATP-dependent generation of a delta psi produced a stimulation of amine uptake in the order: serotonin greater than isoproterenol greater than tyramine; metaraminol accumulation was not enhanced by ATP addition. The relationship between the electrochemical proton gradient (delta micro H+) and the electrochemical gradient for each of the sympathomimetic amines (delta micro A) was investigated utilizing chromaffin ghosts devoid of endogenous matrix gradients or components. All amines were accumulated in the presence of delta pH alone. In the presence of delta psi alone, [14C] serotonin, (14C] tyramine, and [3H] isoproterenol were accumulated, but no [3H] metaraminol uptake was demonstrable. The results indicate that serotonin and isoproterenol accumulated in isolated chromaffin granules and ghosts via a reserpine-sensitive mechanism, driven by the magnitude of the electrochemical proton gradient. Conversely, metaraminol permeated the membrane of the chromaffin granule through the apolar lipid phase and distributed according to the delta pH alone. Tyramine uptake proceeded by both mechanisms. The implications of the mechanism of accumulation of these potent physiologic and pharmacologic agents for their in vivo action are discussed.

Phospholipid composition of some amine storage granules

Several mammalian tissues or cells contain subcellular organelles which accumulate and store biogenic amines, e.g., adrenal medulla chromaffm granules, platelet dense granules,mast cell granules, and anterior pituitary granules. The similarity among these granules is not restricted to a high content of biogenic amines, but extends to the presence of ATP, peptide hormones, and large M, acidic molecules implicated in the storage complex aswell;also to the extremely low membrane permeability to ions, to the maintenance of a distinctly acidic intragranular pH of 5.5-6.1, and to the release of amines by a calcium-dependent stimulus coupling mechanism [l-6]. Adrenal chromaffm granules are known to be characterized by an unusually high amount (up to 17 mol%) of lysophosphatidylcholine in their membranes [7-131, and this observation has led to much speculation as to the role of lysophospholipid in secretory granule membranes. The measurements have also generated controversy over whether the high lysophosphatidylcholine content is a manifestation of post-mortem artifacts. In view of this controversy, recently fueled by several reports [ 14,151, we have carried out a comparative study of the phospholipid composition of several amine storage organelles. This comparison, which utilized similar isolation procedures and one standardized analytical technique for phospholipid measurement, included bovine chromaffm granules purified through different density gradients, chromaffin granules isolated from a human pheochromocytoma (an adrenal medullary tumor), serotonin dense granules from pig platelets, bovine anterior pituitary granules,and intact rat peritoneal mast cells. The results indicate that the phospholipid composition of bovine chromaffm granules is independent

H+-Translocating ATPase and Other Membrane Enzymes Involved in the Accumulation and Storage of Biological Amines in Chromaffin Granules

Within the adrenal medulla, catecholamines accumulate into and are stored within a highly specialized subcellular organelle, the chromaffin granule. Since their isolation almost 30 years ago, an interdisciplinary approach from pharmacologists, transport and cellular physiologists, electron microscopists, and anatomists has helped to delineate the salient aspects of the structure, composition, and function of these granules. One of the reasons for such profound interest is that the adrenal chromaffin granule has a common embryologic origin with the adrenergic neurotransmitter granules of the central and peripheral nervous system, suggesting that the two amine storage organelles may have functional properties in common.

Accumulation of amantadine by isolated chromaffin granules

Abstract The accumulation of amantadine and the effect of this pharmacologic amine upon the magnitude of the transmembrane proton gradient (ΔpH) and of the transmembrane potential gradient (ΔΨ) were investigated in bovine adrenal chromaffin granules isolated in isotonic sucrose. Freshly isolated chromaffin granules have an intragranular pH of 5.5, as measured by [ 14C ]methylamine distribution [R. G. Johnson and A. Scarpa, J. gen. Physiol. 68 , 601 (1976)]. The addition of amantadine (1–50 mM) to well-buffered suspensions of granules at pH 7.0 resulted in a dose-related alkalinization of the granule interior. Similar results were obtained with equivalent external concentrations of ammonia. When the time-resolved influx of labeled amines into the granules was studied radiochemically, using low external amine concentrations, the accumulation of [ 3 H]amantadine was quite similar to that of [ 14 C]methylamine with regard to rate and extent over a wide range of magnitudes of the transmembrane proton gradient. However, unlike biogenic amine accumulation into the chromaffin granule, which is driven by both transmembrane proton and potential gradients, the accumulation of [ 3 H] amantadine was not stimulated by the existence of a transmembrane potential, nor was it inhibited by reserpine. Moreover, low concentrations of amantadine did not competitively inhibit biogenic amine accumulation in the isolated granules. These results indicate that amantadine can distribute across the membrane of chromaffin granules according to the magnitude of the endogenous ΔpH, and suggest that in vivo amantadine may be concentrated and stored as a pharmacologic agent in amine containing granules.

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

The structure and function of chromaffin granules of human pheochromocytoma 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 (delta psi) across the membrane of these granules which is positive inside and which corresponds to 90 mV. Both delta pH and delta psi 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.

Kinetic and quantitative measurements of catecholamine transport in chromaffin ghosts using a glassy carbon electrode

Abstract A method for the on-line kinetic measurement of net catecholamine uptake and release in ghosts derived from bovine chromaffin granules is described. Changes in free catecholamine concentration in 1 to 2 ml of media containing chromaffin ghosts were continuously measured through the amperometric detection of their oxidation products through a glassy carbon electrode set at 0.5-V potential vs a reference electrode. Parallel measurements of catecholamine uptake and release in the ghosts under various metabolic conditions show a good quantitative agreement between the values obtained with the electrode and those obtained through high-performance liquid chromatography after separation of the ghosts from the medium. Initial velocities of ATP-dependent uptake of epinephrine, norepinephrine, dopamine, and isoproternol by the ghosts are shown. This method permits, for the first time, quantitation of unidirectional movement of catecholamines in the presence of minute quantities of biological samples. The advantages, limitations, and suitability of this method to measure catecholamine transport in other systems are discussed.

Net uptake of catecholamines into isolated chromaffin granules demonstrated by a novel polarographic technique

The recognition of the importance of chromaffin granules both in the control of adrenal catecholamine homeostasis and as an excellent model for the broader investigation of biogenic amine transport has led to the development of procedures for the routine isolation of chromaffin granules in high yield and purity. Biochemical analysis of the composition of granules isolated in vitro indicates that they contain large quantities of catecholamines 01 pmol/mg protein) [ 1,2]. The mechanism by which the granule accumulates and stores catecholamines against an apparent concentration gradient has recently become an increasingly active area of biologic investigation, and a molecular model for the active accumulation of catecholamines has been proposed. The model is based upon the documented existence of a H+-translocating ATPase within the chromaffin granule membrane, the generation of a transmembrane pH gradient (ApH, inside acidic), and transmembrane potential (A\k, inside positive), and the coupling of the resulting APB+ to a catecholamine carrier [3-l 31. of catecholamine transport, the intrinsic limitations of the radiochemical amine distribution methodology have greatly limited further characterization of the precise molecular mechanism of coupling of catecholamine transport to the A,~H+. In addition, despite the rather sophisticated analysis of kinetic catecholamine fluxes precent in the literature, the objection has been raised that a basic criterion for demonstration and study of catecholamine transport, that of net accumulation into granules or ghosts, has yet to be demonstrated [ 16,171. This study presents two techniques which overcome many of the limitations of the methodologies available for measurement of catecholamine accumulation.