Subcellular Distribution of 65,000 Calmodulin-Binding Protein (p65) and Synaptophysin (p38) in Adrenal Medulla

Both neuronal and endocrine cells contain secretory vesicles that store and release neurotransmitters and peptides. Neuronal cells release their secretory material from both small synaptic vesicles and large dense-core vesicles (LDCVs), whereas endocrine cells release secretory products from LDCVs. Neuronal small synaptic vesicles are known to express three integral membrane proteins: 65,000 calmodulin-binding protein (65-CMBP) (p65), synaptophysin (p38), and SV2. A controversial question surrounding these three proteins is whether they are present in LDCV membranes of endocrine and neuronal cells. Sucrose density centrifugation of adrenal medulla was performed to study and compare the subcellular distribution of two of these small synaptic vesicle proteins (65-CMBP and synaptophysin). Subsequent immunoblotting and 125I-Protein A binding experiments performed on the fractions obtained from sucrose gradients showed that 65-CMBP was present in fractions corresponding to granule membranes and intact chromaffin granules. Similar immunoblotting and 125I-Protein A binding experiments with synaptophysin antibodies showed that this protein was also present in intact granules and granule membrane fractions. However, an additional membrane component, equilibrating near the upper portion of the sucrose gradient, also showed strong immunoreactivity with anti-synaptophysin and high 125I-Protein A binding activity. In addition, immunoblotting experiments on purified plasma and granule membranes demonstrated that 65-CMBP was a component of both membranes, whereas synaptophysin was only present in granule membranes. Thus, there appears to be a different subcellular localization between 65-CMBP and synaptophysin in the chromaffin cell.     

Synaptin/synaptophysin, p65 and SV2: their presence in adrenal chromaffin granules and sympathetic large dense core vesicles.

The subcellular distribution of three proteins of synaptic vesicles (synaptin/synaptophysin, p65 and SV2) was determined in bovine adrenal medulla and sympathetic nerve axons. In adrenals most p65 and SV2 is confined to chromaffin granules. Part of synaptin/synaptophysin is apparently also present in these organelles, but a considerable portion is found in a light vesicle which does not contain significant concentrations of typical markers of chromaffin granules (cytochrome b-561, dopamine beta-hydroxylase or the amine carrier). An analogous finding was obtained for sympathetic axons. The large dense core vesicles contain most p65 and also SV2 but only a smaller portion of synaptin/synaptophysin. A lighter vesicle containing this latter antigen and some SV2 has also been found. These results establish that in adrenal medulla and sympathetic axons three typical antigens of synaptic vesicles are not restricted to light vesicles. Apparently, a varying part of these antigens is found in chromaffin granules and large dense core vesicles. On the other hand, the light vesicles do not contain significant concentrations of functional antigens of chromaffin granules. Thus, the biogenesis of small presynaptic vesicles which contain all three antigens as well as functional components like the amine carrier is likely to involve considerable membrane sorting.     

Demonstration of immunochemical identity between the synaptic vesicle proteins synaptin and synaptophysin/p38

The synaptic vesicle proteins synaptin and synaptophysin/p38 were shown to be immunochemically identical. Western immunoblot analysis of Triton X-100 extracts from rat brain showed that polyclonal polyspecific anti-synaptin antibodies and monoclonal antibody SY38 against synaptophysin both reacted with a band of 38 kDa. In two-dimensional immunoblots of chromaffin granule membranes from bovine adrenal medulla anti-synaptin and anti-synaptophysin antibodies also recognized the same component. Finally, in a Western immunoblotting experiment SY38 reacted with an immuno-isolated synaptin antigen.     

Catecholamines Are Present in a Synaptic-Like Microvesicle-Enriched Fraction from Bovine Adrenal Medulla

Abstract: "Synaptic-like microvesicles" are present in all neuroendocrine cells and cell lines. Despite their resemblance to small synaptic vesicles of the CNS. a thorough biochemical characterization is lacking. Moreover, the subcellular distribution of synaptophysin, the most abundant integral membrane protein of small synaptic vesicles, in adrenal medulla is still controversial. Using gradient centrifugation. we were able to compare the distribution of several markers for small synaptic vesicles and chromaffin granules. Synaptophysin was found at a high density (1.16 g/ml), purifying away from dopamine β-hydroxylase and cytochrome b₅₆₁. Both noradrenaline and adrenaline showed a parallel distribution with synaptophysin, suggesting their presence in synaptic-like microvesicles. Experiments in the presence of tetrabenazine did not influence the catecholamine content. Additionally, tetrabenazine binding showed a consistent shoulder in the region of synaptophysin. [³H]-Noradrenaline uptake was blocked by tetrabenazine, but not by desipramine. Also chromogranin A parallels the distribution of synaptophysin: however, a localization in the Golgi cannot be ruled out. Synaptophysin was shown to undergo very fast phosphorylation, together with another triplet protein of ∼ 18 kDa. In contrast, the latter showed a rather bimodal distribution coinciding with synaptophysin and dopamine β-hydroxylase. Immunoelectron microscopy of synaptic-like microvesicle fractions showed an intense labeling for synaptophysin on 60-90-nm organelles. Whereas abundant gold labeling for cytochrome b₅₆₁ was found over the entire surface of chromaffin granules, synaptophysin labeling was encountered mostly on vesicles adsorbed to granules. We conclude that catecholamines might be stored in synaptic-like microvesicles of the chromaffin cell.     

Quantification of p38/synaptophysin in highly purified adrenal medullary chromaffin vesicles

Chromaffin vesicles were first purified by differential and density gradient centrifugation in isotonic (Percoll) gradients. In subsequent sucrose gradients p38/synaptophysin exhibited the same distribution as established marker substances of chromaffin vesicles. Quantification of immunoblots revealed that 750 ng p38/synaptophysin per mg of protein were present in the chromaffin vesicles recovered from the sucrose gradient. Thus the amount of p38/synaptophysin per mg protein of chromaffin vesicles is about 100 times lower than that observed in clear (synaptic) vesicles. However, because of the large difference in surface area and protein content, the amount of p38/synaptophysin per single vesicle is the same in both types of organelles.     

Low molecular mass GTP-binding proteins of adrenal chromaffin cells are present on the secretory granule

Adrenal medullary homogenates and chromaffin granule membranes were separated by SDS-polyacrylamide gel electrophoresis and GTP-binding proteins detected using [alpha-32P]GTP binding to nitrocellulose blots. Four GTP-binding polypeptides of 24, 22, 20 and 18 kDa were routinely found in medullary homogenates and all were also found in isolated chromaffin granule membranes. The GTP-binding polypeptides co-sedimented with granule membrane markers following separation on sucrose gradients. On the basis of trypsin sensitivity and resistance to extraction, the GTP-binding proteins appeared to be tightly bound to the cytoplasmic surface of the granules. One or more of the secretory granule GTP-binding proteins could be involved in exocytosis in adrenal chromaffin cells.     

Characterization of Proenkephalin-Cleaving Proteinases in Bovine Adrenal Chromaffin Granules Using [35S]Proenkephalin Copolymerized into Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Proteinases capable of cleaving proenkephalin into smaller peptides have been identified in bovine adrenal chromaffin granules using [35S]methionine-labeled recombinant rat proenkephalin as a selective substrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis proteinase radiozymography. This technique was used for the screening of subcellular fractions, general characterization of pH optima, and the mechanistic characterization of proteinases with both reversible and irreversible inhibitors. Two enzymes with approximate molecular masses of 76 and 30 kDa were shown to be localized to the highest-density fractions of chromaffin granules by sucrose density gradient fractionation. Both were enriched in a 1 M NaCl wash of purified chromaffin granule membranes, were active at high pH, and were characterized as serine proteinases based on inhibition by soybean trypsin inhibitor. The 30-kDa enzyme was also inhibited by diisopropyl fluorophosphate, D-Phe-Pro-Arg-CH2Cl, and D-Val-Phe-Lys-CH2Cl and appeared to be the previously described adrenal trypsin-like enzyme. A third enzyme, of 66 kDa, was also associated with the 1 M NaCl wash of purified chromaffin granule membranes but was not localized exclusively to chromaffin granules in sucrose gradients. This proteinase was found to be Ca2+ activated and inhibited by EDTA but not diisopropyl fluorophosphate, soybean trypsin inhibitor, p-chloromercuriphenylsulfonic acid, 1,10-phenanthroline, or pepstatin.     

Tyrosine hydroxylase in secretory granules from bovine adrenal medulla. Evidence for an integral membrane form

Intact secretory granules isolated from bovine adrenal medulla express tyrosine hydroxylase (TH) activity. Granule-associated TH sediments on continuous sucrose gradients with dopamine beta-hydroxylase, a marker for granule membranes, indicating that TH is associated with chromaffin granules. Membranes prepared from lysed granules retain TH, whereas granule contents are free of the enzyme. TH immunoreactivity was detected in granule membranes by immunoblot analysis using a polyclonal antiserum against TH. TH immunoreactivity cannot be removed from membranes by washes in high ionic strength buffers and is only partially removed from membranes by treatment with either urea or Na2CO3. TH can be removed from granule membranes by the detergents Nonidet P-40, Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Treatment of membranes with a phosphatidylinositol-specific phospholipase C did not remove TH, ruling out the possibility of a glycosyl phosphatidyl anchor. Fractionation of granule membranes by temperature-induced phase separation in Triton X-114 revealed that TH is recovered in phases in which integral (detergent phase) and hydrophobic (phospholipid phase) membrane proteins are typically found. By contrast, TH from adrenal cytosol fractionated exclusively into the aqueous phase along with other soluble proteins. Digestion of granules with various protease enzymes revealed that TH is resistant to degradation, suggesting that the enzyme is embedded within membranes. TH becomes phosphorylated when intact granules are exposed to the catalytic subunit of the cAMP-dependent protein kinase, indicating that at least the N-terminal region of TH is exposed on the cytoplasmic surface of granules. These results establish that a fraction of TH is an integral component of bovine granule membranes. The association of TH with granule membranes may play a role in coordinating TH activity and catecholamine release.     

Fractionation of synaptophysin-containing vesicles from rat brain and cultured PC12 pheochromocytoma cells

Synaptophysin is a transmembrane glycoprotein of neuroendocrine vesicles. Its content and distribution in subcellular fractions from cultured PC12 cells, rat brain and bovine adrenal medulla were determined by a sensitive dot immunoassay. Synaptophysin-containing fractions appeared as monodispersed populations similar to synaptic vesicles in density and size distribution. Membranes from synaptic vesicles contained approximately 100-times more synaptophysin than chromaffin granules. In conclusion, synaptophysin is located almost exclusively in vesicles of brain and PC12 cells which are distinct from dense core granules.     

Osmotic fragility of chromaffin granules prepared under isoosmotic or hyperosmotic conditions and localization of acetylcholinesterase

In chromaffin cells of the adrenal medulla, catecholamines are stored in secretory granules. Different methods have been described to purify chromaffin granules. In the present study, storage granules were prepared using isoosmotic self-generating Percoll gradients or hyperosmotic sucrose gradients, and a comparison of their physical properties in response to osmotic changes was made. Catecholamines, dopamine beta-hydroxylase activity and protein were detected both in the external medium and in the granule fraction according to the medium osmolality. Suspension turbidity was used as a measure of organelle integrity. Acetylcholinesterase activity was found to be associated with both isoosmotically and hyperosomotically prepared granules. The total acetylcholinesterase activity was determined after adding Triton X-100 to the assay medium. When adrenal medullary tissue was homogenized in buffers containing echothiopate, an inhibitor of acetylcholinesterase, only 15-20% of enzyme activity was inhibited, excluding the possibility that main granule acetylcholinesterase could be due to contamination by plasma membrane fragments, endoplasmic reticulum and Golgi membranes. When granules were suspended in hypoosmotic buffers, a soluble acetylcholinesterase form was released into the external medium, while an insoluble acetylcholinesterase form was still found associated with the membrane fraction. Soluble acetylcholinesterase was found to be released differently than soluble dopamine beta-hydroxylase, indicating that acetylcholinesterase may be associated with a more osmotically resistant granule population.