Bovine Chromaffin Granules Research Articles

Combining fluorescence and electrochemical imaging of chromaffin granule fusion with supported bilayers.

The main barrier to a full understanding of the detailed molecular conformational changes leading to fusion pore formation is the lack of a technique that allows to experimentally measure simultaneously and with (sub-)millisecond time resolution specific conformational changes in the fusion machinery and the formation of the fusion pore. To overcome this barrier we have developed microfabricated electrochemical detector (ECD) arrays patterned on microscope coverslips on which a chromaffin cell can be placed such that the cell surface in contact with the coverslip can simultaneously be imaged in total internal reflection fluorescence (TIRF) excitation mode while the released catecholamine molecules are oxidized by the ECDs producing amperometric oxidation current spikes. The amperometric spikes indicate not only the time of fusion with <1ms precision but also the location of the release event. Using SNAP25-based FRET probes we demonstrated a conformational change in the SNARE complex preceding fusion pore formation. However, the fluorescent protein (FP) based tags, are rather bulky and it is thus not clear what the conformational change exactly is. For a clear interpretation, the use of localized small labels will be needed. Labeling of specific residues requires the use of recombinant proteins in a reconstituted system. We therefore develop a technology where labeled recombinant SNARE proteins will be incorporated into a supported membrane that covers ECD arrays such that TIR-FRET imaging of conformational changes can be performed simultaneously with electrochemical detection of individual fusion events of dense core vesicles with the supported membrane. FRAP measurements show that a fluid membrane is indeed formed on the ECDs and docking and fusion of membrane labeled bovine chromaffin granules could be observed. Supported by NINDS (NIH) R21 NS118319.

Is the vesicular nucleotide transporter a molecular target of eicosapentaenoic acid?

Vesicular nucleotide transporter (VNUT), an active transporter for nucleotides in secretory vesicles, is responsible for the vesicular storage of ATP and plays an essential role in purinergic chemical transmission. Inhibition of VNUT decreases the concentration of ATP in the luminal space of secretory vesicles, followed by decreased vesicular ATP release, resulting in the blockade of purinergic chemical transmission. Very recently, Miyaji and colleagues reported that eicosapentaenoic acid (EPA) is a potent VNUT inhibitor and effective in treating neuropathic and inflammatory pain and insulin resistance through inhibition of vesicular storage and release of ATP. However, our validation study indicated that, in bovine adrenal chromaffin granule membrane vesicles, EPA inhibited the formation of an electrochemical gradient of protons across the membrane with the concentration of 50% inhibition (IC50) being 1.0μM without affecting concanamycin B-sensitive ATPase activity. Essentially, similar results were obtained with proteoliposomes containing purified vacuolar H+-ATPase. Consistent with these observations, EPA inhibited the ATP-dependent uptakes of ATP and dopamine by chromaffin granule membrane vesicles, with ID50 being 1.2 and 1.0μM, respectively. Furthermore, EPA inhibited ATP-dependent uptake of L-glutamate by mouse brain synaptic vesicles with ID50 being 0.35μM. These results indicate that EPA at sub-μM acts as a proton conductor and increases proton permeability across the membrane, regardless of the presence or absence of VNUT, thereby inhibiting non-specifically the vesicular storage of neurotransmitters. Thus, EPA may affect a broader range of chemical transmission than proposed.

Electron Transfer Reactions of Candidate Tumor Suppressor 101F6 Protein, a Cytochrome b561 Homologue, with Ascorbate and Monodehydroascorbate Radical

The candidate tumor suppressor 101F6 protein is a homologue of adrenal chromaffin granule cytochrome b561, which is involved in the electron transfer from cytosolic ascorbate to intravesicular monodehydroascorbate radical. Since the tumor suppressor activity of 101F6 was enhanced in the presence of ascorbate, it was suggested that 101F6 might utilize a similar transmembrane electron transfer reaction. Detailed kinetic analyses were conducted on the detergent-solubilized recombinant human 101F6 for its electron transfer reactions with ascorbate and monodehydroascorbate radical by stopped-flow and pulse radiolysis techniques. The reduction of oxidized 101F6 with ascorbate was found to be independent of pH in contrast to those observed for chromaffin granule and Zea mays cytochromes b561 in which both cytochromes exhibited very slow rates at pH 5.0 but faster at pH 6.0 and 7.0. The absence of the inhibition for the electron acceptance from ascorbate upon the treatment with diethyl pyrocarbonate suggested that 101F6 might not utilize a "concerted proton/electron transfer mechanism". The second-order rate constant for the electron donation from the ascorbate-reduced 101F6 to the pulse-generated monodehydroascorbate radical was found to be 5.0 × 10(7) M(-1)s(-1), about 2-fold faster than that of bovine chromaffin granule cytochrome b561 and about five times faster than that of Zea mays cytochrome b561, suggesting that human 101F6 is very effective for regenerating ascorbate from monodehydroascorbate radical in cells. Present observations suggest that 101F6 employs distinct electron transfer mechanisms on both sides of the membranes from those of other members of cytochrome b561 protein family.

Functional characterization of the recombinant human tumour suppressor 101F6 protein, a cytochrome b561 homologue

Candidate human tumour suppressor gene product, 101F6 protein, is a highly hydrophobic transmembrane protein and a member of cytochrome b(561) family. Purified 101F6 protein expressed in Pichia pastoris cells showed visible absorption spectra similar but distinct from those of cytochrome b(561). Haem content analysis indicated presence of two haems B per molecule. Midpoint potentials of the purified protein were found as +109 and +26 mV for two haems, slightly lower than those for bovine chromaffin granule or plant Zea mays cytochromes b(561). Electron paramagnetic resonance (EPR) spectra in oxidized state at 5 K showed only a highly anisotropic low-spin (HALS) signal at g(z) = 3.75. However, at 15 and 20 K, another HALS-type signal appeared at g(z) = 3.65 being overlapped with that of g(z) = 3.75. The rhombic EPR signal at g(z) = 3.16 previously seen in other cytochromes b(561) was not observed, suggesting distinct haem environments. Absence of the inhibition in the electron transfer from ascorbate by a treatment of 101F6 protein with diethylpyrocarbonate showed a remarkable contrast from those of other cytochromes b(561) where the 'concerted H(+)/e(-) transfer mechanism' at the cytosolic haem centre was blocked by specific Nε-carbethoxylation of haem-coordinating imidazole, suggesting that 101F6 protein might accept electrons via a mechanism distinct from other cytochromes b(561).

Controlling synaptotagmin activity by electrostatic screening

SummaryExocytosis of neurosecretory vesicles is mediated bythe SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins syntaxin-1, synaptobrevin, and SNAP-25, with synaptotagmin functioning as the major Ca2+-sensor for triggering membrane fusion. Here we show that bovine chromaffin granules readily fuse with large unilamellar liposomes in a SNARE-dependent manner. Fusion is enhanced by Ca2+ but only if the target liposomes contain PI(4,5)P2 and if polyphosphate anions such as nucleotides or pyrophosphate are present. Ca2+-dependent enhancement is mediated by endogenous synaptotagmin-1. Polyphosphates operate by an electrostatic mechanism that reverses an inactivating cis-association of synaptotagmin-1 with its own membrane whereas trans-binding is not affected. Hence, balancing trans- and cis-membrane interactions of synaptotagmin may be a crucial element in the pathway of Ca2+-dependent exocytosis.

Modulation of PC1/3 Activity by Self-Interaction and Substrate Binding

Prohormone convertase (PC)1/3 is a eukaryotic serine protease in the subtilase family that participates in the proteolytic maturation of prohormone and neuropeptide precursors such as proinsulin and proopiomelanocortin. Despite the important role of this enzyme in peptide synthesis, how PC1/3 activity is regulated is still poorly understood. Using ion exchange chromatography and two-dimensional gel electrophoresis we found that natural PC1/3 present in AtT-20 cells and bovine chromaffin granules, as well as recombinant PC1/3 secreted from overexpressing Chinese hamster ovary cells, exists as multiple ionic forms. Gel filtration and cross-linking studies revealed that protein oligomerization and aggregation contribute greatly to variability in surface charge. The most acidic forms of PC1/3 contained both inactive aggregates as well as oligomerized 87-kDa PC1/3 that exhibited stable activity which was partially latent and could be revealed by dilution. No such latency was observed for the more basic, 66/74-kDa forms of PC1/3. Fractions containing these species were stabilized by preincubation with micromolar concentrations of either fluorogenic substrate or peptides containing pairs of basic residues. In addition, the most active form of 87-kDa PC1/3, a probable homodimer, was activated by preincubation with these same peptides. Cleavage by PC1/3 is often the initiating step in the biosynthetic pathway for peptide hormones, implying that this is a natural step for regulation. Our data suggest that enzyme oligomerization and peptide stabilization represent important contributing factors for the control of PC1/3 activity within secretory granules.

Inositol 1,4,5-trisphosphate receptor in chromaffin secretory granules and its relation to chromogranins.

The inositol 1,4,5-trisphosphate (IP(3))-mediated intracellular Ca(2+) releases in secretory cells play vital roles in controlling not only the intracellular Ca(2+) concentrations but also the Ca(2+)-dependent exocytotic processes. Of intracellular organelles that release Ca(2+) in response to IP(3), secretory granules stand out as the most prominent organelle and are responsible for the majority of IP(3)-dependent Ca(2+) releases in the cytoplasm of chromaffin cells. Bovine chromaffin granules were the first granules that demonstrated the IP(3)-mediated Ca(2+) release as well as the presence of the IP(3) receptor (IP(3)R) in granule membranes. Secretory granules contain all three (type 1, 2, and 3) IP(3)R isoforms, and 58-69% of total cellular IP(3)R isoforms are expressed in bovine chromaffin granules. Moreover, secretory granules contain large amounts (2-4 mM) of chromogranins and secretogranins; chromogranins A and B, and secretogranin II being the major species. Chromogranins A and B, and secretogranin II are high-capacity, low-affinity Ca(2+) binding proteins, binding 30-93 mol of Ca(2+)/mol of protein with dissociation constants of 1.5-4.0 mM. Due to this high Ca(2+) storage properties of chromogranins secretory granules contain ~40 mM Ca(2+). Furthermore, chromogranins A and B directly interact with the IP(3)Rs and modulate the IP(3)R/Ca(2+) channels, i.e., increasing the open probability and the mean open time of the channels 8- to 16-fold and 9- to 42-fold, respectively. Coupled chromogranins change the IP(3)R/Ca(2+) channels to a more ordered, release-ready state, whereby making the IP(3)R/Ca(2+) channels significantly more sensitive to IP(3).

Commentary on Chapters ‘Clinical and Developmental Aspects’ and ‘Stress Responses of the Adrenal Medulla’

The division of the contributions to this volume into Chromogranins, Ion Channels, Secretion Mechanisms, Clinical and Developmental Aspects, and Stress Responses of the Adrenal Medulla is somewhat arbitrary: there is clearly much overlap among these domains of chromaffin cell research. This commentary covers both Clinical/ Developmental, and Stress Responses together, mainly to emphasize this overlap, and the translational importance of these two aspects of the chromaffin cell as a stress transducer. Genetics, development and stress were three topics of particular clinical relevance that were developed in depth through contributions presented at the 15th International Symposium for Chromaffin Cell Biology. The genetics of the chromaffin cell in normal physiology and disease were highlighted by contributions focusing on markers of chromaffin cell origin relevant to cancer including pheochromocytoma, and on catecholaminergic markers for hypertension and other cardiovascular diseases. Colon, pancreas, prostate and lung outrank the adrenal medulla as organs of medical concern in human cancer. Although pheochromocytoma, or cancer of adrenomedullary origin is rare, it is an important subject of study for several reasons. First, it arises from a limited and distinct set of cell types, and therefore is an attractive model for etiology of proliferative disease. Second, pheochromocytoma can be either metastatic, malignant or benign, and therefore can be used to search for markers to make this clinically crucial diagnosis. Murthy et al. propose carboxypeptidase E (CPE), the enkephalin prohormone processing enzyme first characterized in bovine chromaffin granules in 1982 (Hook et al., Nature 295:341–342, 1982), as such a marker. Meta-analysis of microarray expression data sets in the Gene Expression Omnibus (GEO) allowed Murthy and colleagues to identify CPE as a transcript frequently represented in the transcriptomes of metastatic cancers of both endocrine and epithelial origin. Thus, cervical, colorectal, renal, and bone (Ewing sarcoma), astrocytic, and oligodendroglial tumors or cells express higher levels of CPE mRNA than their non-cancerous cells and tissues of origin: in fact most of the corresponding cells of origin do not have appreciable concentrations of CPE. Lung, pituitary and pheochromocytoma tissue (i.e. neuroendocrine tumors) express higher CPE levels than their tissues of origin. Most significantly, metastatic pheochromocytoma expresses significantly higher CPE mRNA than benign. Should CPE prove a reliable prognostic marker for malignancy in pheochromocytoma and other neuroendocrine cancers, and perhaps even in non-endocrine cancers, its potential for translation to standard clinical practice would be high. Thouennon and Anouar report on expression of neuropeptides in pheochromocytoma that may be involved in trophic, proliferative and angiogenic paracrine/autocrine actions contributing to tumor growth. A strong correlation between NPY and PACAP expression was documented in 25 pheochromocytomas, and between RDC1, the adrenomedullin receptor, and VEGF, with angiogenic activity. These authors review the evidence for concerted expression This is a commentary to articles doi:10.1007/s10571-010-9535-7 10.1007/s10571-010-9567-z, 10.1007/s10571-010-9571-3, 10.1007/s10571-010-9578-9, 10.1007/s10571-010-9575-z, 10.1007/s10571-010-9582-0, 10.1007/s10571-010-9583-z, 10.1007/s10571-010-9592-y, 10.1007/s10571-010-9593-x, 10.1007/s10571-010-9594-9, 10.1007/s10571-010-9600-2, 10.1007/s10571-010-9606-9, 10.1007/s10571-010-9620-y.

Localization, Purification, and Functional Reconstitution of the P4-ATPase Atp8a2, a Phosphatidylserine Flippase in Photoreceptor Disc Membranes

P(4)-ATPases comprise a relatively new subfamily of P-type ATPases implicated in the energy-dependent translocation of aminophospholipids across cell membranes. In this study, we report on the localization and functional properties of Atp8a2, a member of the P(4)-ATPase subfamily that has not been studied previously. Reverse transcription-PCR revealed high expression of atp8a2 mRNA in the retina and testis. Within the retina, immunofluorescence microscopy and subcellular fractionation studies localized Atp8a2 to outer segment disc membranes of rod and cone photoreceptor cells. Atp8a2 purified from photoreceptor outer segments by immunoaffinity chromatography exhibited ATPase activity that was stimulated by phosphatidylserine and to a lesser degree phosphatidylethanolamine but not by phosphatidylcholine or other membrane lipids. Purified Atp8a2 was reconstituted into liposomes containing fluorescent-labeled phosphatidylserine to measure the ability of Atp8a2 to flip phosphatidylserine across the lipid bilayer. Fluorescence measurements showed that Atp8a2 flipped fluorescent-labeled phosphatidylserine from the inner leaflet of liposomes (equivalent to the exocytoplasmic leaflet of cell membranes) to the outer leaflet (equivalent to cytoplasmic leaflet) in an ATP-dependent manner. Our studies provide the first direct biochemical evidence that purified P(4)-ATPases can translocate aminophospholipids across membranes and further implicates Atp8a2 in the generation and maintenance of phosphatidylserine asymmetry in photoreceptor disc membranes.

Vesicular Monoamine Transporter Substrate/Inhibitor Activity of MPTP/MPP+ Derivatives: A Structure–Activity Study

The active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), N-methyl-4-phenylpyridinium (MPP(+)), selectively destroys the dopaminergic neurons and induces the symptoms of Parkinson's disease. Inhibition of mitochondrial complex I and/or the perturbation of dopamine metabolism through cellular and granular accumulation have been proposed as some of the major causes of neurotoxicity. In the present study we have synthesized and characterized a number of MPTP and MPP(+) derivatives that are suitable for the comparative neurotoxicity and complex I inhibition versus dopamine metabolism perturbation studies. Structure-activity studies with bovine chromaffin granule ghosts show that 3'-hydroxy-MPP(+) is one of the best known substrates for the vesicular monoamine transporter (VMAT). A series of compounds that combine the structural features of MPP(+) and a previously characterized VMAT inhibitor, 3-amino-2-phenyl-propene, have been identified as the most effective VMAT inhibitors. These derivatives have been used to define the structural requirements of the VMAT substrate and inhibitor activities.

Diphyllin, a Novel and Naturally Potent V-ATPase Inhibitor, Abrogates Acidification of the Osteoclastic Resorption Lacunae and Bone Resorption

Dissolution of the inorganic phase of bone by the osteoclasts mediated by V-ATPase and ClC-7 is a prerequisite for bone resorption. Inhibitors of osteoclastic V-ATPase or ClC-7 are novel approaches for inhibition of osteoclastic bone resorption. By testing natural compounds in acidification assays, diphyllin was identified. We characterized diphyllin with respect to the pharmacological effects on osteoclasts. Osteoclastic acidification of the resorption lacuna and bone resorption requires activity of both V-ATPase and the chloride channel ClC-7. Inhibition of these processes represents a novel approach for treatment of bone metabolic disorders. We identified diphyllin, a novel inhibitor of V-ATPase, and characterized this natural compound with respect to activity in human osteoclasts. Diphyllin was tested in the acid influx assay and V-ATPase assay using bovine chromaffin granules. Human osteoclasts were generated from CD14+ monocytes cultured with macrophage-colony stimulating factor (M-CSF) and RANKL. The effect of diphyllin on lysosomal acidification in human osteoclasts was studied using acridine orange. The effect of diphyllin on bone resorption by osteoclasts was measured as release of C-terminal cross-linked telopeptide of type I collagen (CTX-I) and calcium into the supernatants and by scoring pit area. Osteoclast number, TRACP activity, and cell viability were measured. Furthermore, the effect of diphyllin on bone nodule formation was tested using the mouse osteoblast cell line MC3T3-E1. In the acid influx assay, diphyllin potently inhibited the acid influx (IC50 = 0.6 nM). We found that diphyllin inhibited V-ATPase with an IC50 value of 17 nM, compared with 4 nM for bafilomycin A1. Moreover, diphyllin dose-dependently inhibited lysosomal acidification in human osteoclasts. Furthermore, we found that diphyllin inhibited human osteoclastic bone resorption measured by CTX-I (IC50 = 14 nM), calcium release, and pit area, despite increasing TRACP activity, numbers of osteoclasts, and cell viability. Finally, diphyllin showed no effect on bone formation in vitro, whereas bafilomycin A1 was toxic. We identified a natural compound that potently inhibits V-ATPase and thereby lysosomal acidification in osteoclasts, which leads to abrogation of bone resorption. Because recent studies indicate that inhibition of the osteoclastic acidification leads to inhibition of resorption without inhibiting formation, we speculate that diphyllin is a potential novel treatment for bone disorders involving excessive resorption.

Perturbation of Dopamine Metabolism by 3-Amino-2-(4′-halophenyl)propenes Leads to Increased Oxidative Stress and Apoptotic SH-SY5Y Cell Death

We have recently characterized a series of 3-amino-2-phenyl-propene (APP) derivatives as reversible inhibitors for the bovine adrenal chromaffin granule vesicular monoamine transporter (VMAT) that have been previously characterized as potent irreversible dopamine-beta-monooxygenase (DbetaM) and monoamine oxidase (MAO) inhibitors. Halogen substitution on the 4'-position of the aromatic ring gradually increases VMAT inhibition potency from 4'-F to 4'-I, parallel to the hydrophobicity of the halogen. We show that these derivatives are taken up into both neuronal and non-neuronal cells, and into resealed chromaffin granule ghosts efficiently through passive diffusion. Uptake rates increased according to the hydrophobicity of the 4'-substituent. More importantly, these derivatives are highly toxic to human neuroblastoma SH-SY5Y but not toxic to M-1, Hep G2, or human embryonic kidney 293 non-neuronal cells at similar concentrations. They drastically perturb dopamine (DA) uptake and metabolism in SH-SY5Y cells under sublethal conditions and are able to deplete both vesicular and cytosolic catecholamines in a manner similar to that of amphetamines. In addition, 4'-IAPP treatment significantly increases intracellular reactive oxygen species (ROS) and decreases glutathione (GSH) levels in SH-SY5Y cells, and cell death is significantly attenuated by the common antioxidants alpha-tocopherol, N-acetyl-l-cysteine and GSH, but not by the nonspecific caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. DNA fragmentation analysis further supports that cell death is probably due to a caspase-independent ROS-mediated apoptotic pathway. Based on these and other findings, we propose that drastic perturbation of DA metabolism in SH-SY5Y cells by 4'-halo APP derivatives causes increased oxidative stress, leading to apoptotic cell death.

An Arabidopsis cytochrome b561 with trans-membrane ferrireductase capability

Ascorbate-reducible cytochromes b561 (Cyts-b561) are a class of intrinsic trans-membrane proteins. Tonoplast Cyt-b561 (TCytb), one of the four Cyt-b561 isoforms in Arabidopsis was localized to the tonoplast. We demonstrate here that the optical spectra, EPR spectra and redox potentials of recombinant TCytb are similar to those of the well characterized bovine chromaffin granule Cyt-b561. We provide evidence for the reduction of ferric-chelates by the reduced TCytb. It is also shown that TCytb is capable of trans-membrane electron transport from intracellular ascorbate to extracellular ferric-chelates in yeast cells.

Purification and characterization of bovine adrenal cytochrome b561 expressed in insect and yeast cell systems

Bovine adrenal chromaffin granule cytochrome (cyt) b 561 is a transmembrane hemoprotein that plays a key role in transporting reducing equivalents from ascorbate to dopamine-β-hydroxylase for catecholamine synthesis. We have developed procedures for expression and purification of functional bovine adrenal cyt b 561 in insect and yeast cell systems. The bovine cyt b 561 coding sequence, with or without a hexahistidine-tag sequence at the C-terminus, was cloned into the pVL1392 transfer vector under the control of the polyhedrin promoter to generate recombinant baculovirus for protein expression in Sf9 insect cells (∼0.5 mg detergent-solubilized cyt b 561/L culture). For the yeast system, the cyt b 561 cDNA was modified with a hexahistidine-tag sequence at the C-terminus, and inserted into the pPICZB vector under the control of the alcohol oxidase promoter. The recombinant plasmid was transformed into Pichia pastoris GS115 competent cells to give methanol-inducible cyt b 561 expression (∼0.7 mg detergent-solubilized cyt b 561/L culture). Recombinant His-tagged cyt b 561 expressed in Sf9 or Pichia cells was readily solubilized from membrane fractions with dodecyl maltoside and purified to electrophoretic homogeneity by one-step chromatography on Ni–NTA affinity resin. The purified recombinant cytochrome from both systems had a heme to protein ratio close to two and was fully functional, as judged by comparison with the spectroscopic and kinetic parameters of the endogenous cytochrome from chromaffin granules. A novel procedure for isolation of chromaffin granule membranes was developed to utilize frozen adrenal glands instead of fresh tissue.

Characterization and location of post-translational modifications on chromogranin B from bovine adrenal medullary chromaffin granules.

Bovine chromoganin B (CGB)/secretogranin I, an acidic protein with a sequence of 626 residues and an isoelectric point of 5.2 is a major member of the chromogranin/secretogranin (CG/Sg) family. The difference between the theoretical molecular mass (76 kDa) and the value estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis results from post-translational modifications (glycosylation, phosphorylation and sulfation) and from the abundance of acidic residues (D 4.6%, and E 16.5%). Although the sequence of CGB is known, the structural analyses of the post-translational modifications have so far not been carried out. In the present study, using a combination of proteomic techniques including two-dimensional gel electrophoresis, Western blot, high-performance liquid chromatography purification, enzymatic digestion, sequencing, carbohydrate analysis, matrix-assisted laser desorption/ionization-time of flight and liquid chromatography mass spectrometry analysis, we have located 18 post-translational modifications on bovine CGB, isolated from adrenal medulla chromaffin granules. Furthermore, we have identified at the molecular level the presence of a mutation M/V on position 577 of natural CGB. All together these data reflect the complex structure of this protein marker of the neuroendocrine system.

Kinetic Evidence for Channeling of Dopamine between Monoamine Transporter and Membranous Dopamine-β-monooxygenase in Chromaffin Granule Ghosts

The nature of coupling between the uptake and dopamine-beta-monooxygenase (DbetaM) catalyzed hydroxylation of dopamine (DA) was studied in bovine chromaffin granule ghosts. Initial rate and transient kinetics of DA uptake and conversion were determined under a variety of conditions. The uptake kinetics of DA, norepinephrine (NE), and epinephrine demonstrate that DA is a better substrate than NE and epinephrine under optimal uptake conditions. The transient kinetics of DA accumulation and NE production under both optimal uptake and uptake and conversion conditions were zero-order with no detectable lag or burst periods. The mathematical analyses of the data show that a normal sequential uptake followed by the conversion process could not explain the observed kinetics, under any condition. On the other hand, all experimental data are in agreement with a mechanism in which DA is efficiently channeled from the vesicular monoamine transporter to membranous DbetaM for hydroxylation, prior to the release into the bulk medium of the ghost interior. The slow accumulation of DA under optimal conversion conditions appears to be caused by the slow leakage of DA from the channeling pathway to the ghost interior. Because DbetaM activity in intact granules is equally distributed between soluble and membranous forms of DbetaM, if an efficient channeling mechanism is operative in vivo, soluble DbetaM may not have access to the substrate, making the catalytic activity of soluble DbetaM physiologically insignificant, which is consistent with the increasing experimental evidence that membranous DbetaM may be the physiologically functional form.

The Yeast Vacuolar Proton-translocating ATPase Contains a Subunit Homologous to the Manduca sexta and Bovine e Subunits That Is Essential for Function

The yeast cwh36Delta mutant was identified in a screen for yeast mutants exhibiting a Vma(-) phenotype suggestive of loss of vacuolar proton-translocating ATPase (V-ATPase) activity. The mutation disrupts two genes, CWH36 and a recently identified open reading frame on the opposite strand, YCL005W-A. We demonstrate that disruption of YCL005W-A is entirely responsible for the Vma(-) growth phenotype of the cwh36Delta mutant. YCL005W-A encodes a homolog of proteins associated with the Manduca sexta and bovine chromaffin granule V-ATPase. The functional significance of these proteins for V-ATPase activity had not been tested, but we show that the protein encoded by YCL005W-A, which we call Vma9p, is essential for V-ATPase activity in yeast. Vma9p is localized to the vacuole but fails to reach the vacuole in a mutant lacking one of the integral membrane subunits of the V-ATPase. Vma9p is associated with the yeast V-ATPase complex in vacuolar membranes, as demonstrated by co-immunoprecipitation with known V-ATPase subunits and glycerol gradient fractionation of solubilized vacuolar membranes. Based on this evidence, we propose that Vma9p is a genuine subunit of the yeast V-ATPase and that e subunits may be a functionally essential part of all eukaryotic V-ATPases.

The Hippocampal Cholinergic Neurostimulating Peptide, the N-terminal Fragment of the Secreted Phosphatidylethanolamine-binding Protein, Possesses a New Biological Activity on Cardiac Physiology

Phosphatidylethanolamine-binding protein (PEBP), alternatively named Raf-1 kinase inhibitor protein, is the precursor of the hippocampal cholinergic neurostimulating peptide (HCNP) corresponding to its natural N-terminal fragment, previously described to be released by hippocampal neurons. PEBP is a soluble cytoplasmic protein, also associated with plasma and reticulum membranes of numerous cell types. In the present report, using biochemistry and cell biology techniques, we report for the first time the presence of PEBP in bovine chromaffin cell, a well described secretion model. We have examined its presence at the subcellular level and characterized this protein on both secretory granule membranes and intragranular matrix. In addition, its presence in bovine chromaffin cell and platelet exocytotic medium, as well as in serum, was reported showing that it is secreted. Like many other proteins that lack signal sequence, PEBP may be secreted through non-classic signal secretory mechanisms, which could be due to interactions with granule membrane lipids and lipid rafts. By two-dimensional liquid chromatography-tandem mass spectrometry, HCNP was detected among the intragranular matrix components. The observation that PEBP and HCNP were secreted with catecholamines into the circulation prompted us to investigate endocrine effects of this peptide on cardiovascular system. By using as bioassay an isolated and perfused frog (Rana esculenta) heart preparation, we show here that HCNP acts on the cardiac mechanical performance exerting a negative inotropism and counteracting the adrenergic stimulation of isoproterenol. All together, these data suggest that PEBP and HCNP might be considered as new endocrine factors involved in cardiac physiology.

Genome-wide Analysis of Iron-dependent Growth Reveals a Novel Yeast Gene Required for Vacuolar Acidification

We conducted a genome-wide screen in the budding yeast Saccharomyces cerevisiae of 4,792 homozygous diploid deletions to identify genes that function in iron metabolism. Strains unable to grow on iron-restricted medium contained deletions of genes that encode the structural components of the high affinity iron transport system (FET3, FTR1), the iron-sensing transcription factor AFT1 or genes required for the assembly of the transport system. We also identified genes that were not previously known to play a role in iron metabolism. Deletion of the gene CWH36 resulted in a severe growth defect on iron-limited medium, as well as increased sensitivity to Congo red and calcofluor white. Iron transport studies demonstrated that Deltacwh36 cells have an inability to copper load apoFet3p. Furthermore, Deltacwh36 cells demonstrated additional phenotypes including distorted vacuole morphology and altered kinetics of FM4-64 trafficking. We show that Deltacwh36 cells have a defect in vacuolar acidification through the use of the pH-sensitive dye LysoSensor Green DND-189. In Deltacwh36 cells, the vacuolar H+-ATPase is not assembled and there are reduced levels of at least one subunit of the V0 complex. The open reading frame responsible for the Deltacwh36 phenotypes is YCL005W-A. This gene contains two introns, has homologues in other Saccharomyces strains, and shows weak homology to a component of the vacuolar H+-ATPase found in organisms as diverse as insect and cow.

Heparin-binding epidermal growth factor-like growth factor: a component in chromaffin granules which promotes the survival of nigrostriatal dopaminergic neurones in vitro and in vivo

Chromaffin cells can restore function to the damaged nigrostriatal dopaminergic system in animal models of Parkinson's disease. It has been reported that a protein which is released from chromaffin granules can promote the survival of dopaminergic neurones in vitro and protect them against N-methylpyridinium ion toxicity. This neurotrophic effect has been found to be mediated by astroglial cells and blocked by inhibitors of the epidermal growth factor (EGF) receptor signal transduction pathway. Here we report the identification of bovine heparin-binding EGF-like growth factor (HB-EGF) in chromaffin granules and the cloning of the respective cDNA from bovine-derived adrenal gland. Protein extracts from bovine chromaffin granules were found to promote the survival of embryonic dopaminergic neurones in culture, to the same extent as recombinant human HB-EGF. Furthermore, the neurotrophic action of the chromaffin granule extract could be abolished by antiserum to recombinant human HB-EGF. We also show that intracerebral injection of recombinant human HB-EGF protected the nigrostriatal dopaminergic system in an in vivo adult rat model of Parkinson's disease. Intracerebral administration of this protein at the same time as a 6-hydroxydopamine lesion of the medial forebrain bundle was found to spare dopamine levels in the striatum and tyrosine hydroxylase-immunopositive neurones in the midbrain. This study has found that the main component in chromaffin granules responsible for their neurotrophic effect on dopaminergic neurones is HB-EGF. Furthermore, HB-EGF has significant protective effects on nigrostriatal dopaminergic neurones in vivo, making it a potential candidate for use in the treatment of Parkinson's disease.