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.     

In Adrenal Medulla Synaptophysin (Protein p38) Is Present in Chromaffin Granules and in a Special Vesicle Population

We have analyzed the properties and subcellular localization of synaptophysin (protein p38) in bovine adrenal medulla. In one-dimensional immunoblotting the adrenal antigen appears identical to synaptophysin of rat synaptic vesicles. In two-dimensional immunoblotting it migrates as a heterogeneous band varying in pI from 4.5 to 5.8. Subcellular fractionation by various sucrose gradients revealed that synaptophysin was present in two different cell particles. More than half of the antigens present in adrenal medulla were confined to special membranes that sedimented both with the "large granules" and with microsomal elements. These membranes could be removed from the large granule sediment by washing. In gradients it equilibrated in regions of low sucrose density. These membranes did not contain any markers for chromaffin granules. Less than half of the amount of synaptophysin present in adrenal medulla copurified with chromaffin granules. Despite several variations in the fractionation scheme synaptophysin could not be removed from chromaffin granules. After washing of granule membranes with alkaline solution synaptophysin still cosedimented in gradients with typical granule markers. The concentration of synaptophysin in membranes of chromaffin granules is low (less than 10%) when compared with synaptic vesicles. It is concluded that in adrenal medulla synaptophysin is present in special membranes, probably in high concentration, and in membranes of chromaffin granules, either in a low concentration in all or in a higher concentration in some of them.     

Adrenal Chromaffin Cell Calmodulin: Its Subcellular Distribution and Binding to Chromaffin Granule Membrane Proteins

Bovine adrenal medullae were homogenized in the presence or in the absence of EGTA and different subcellular fractions were prepared by differential and density gradient centrifugations. In the presence of the chelating agent, 69% of the total calmodulin, measured by radioimmunoassay, was present in the cytosol; the rest was bound to different membrane-containing fractions (nuclei, microsomal, and crude granule fraction). When the chelating agent was omitted, 43% of the calmodulin was present in the cytosol, the remaining calmodulin being membrane-bound. Further resolution of the crude granule fraction by sucrose density centrifugation demonstrated that the distribution of calmodulin in the density gradient was similar to the distribution of chromaffin granules rather than to that of mitochondria, Golgi elements, and lysosomes. In this case, there was also more calmodulin bound to chromaffin granules when EGTA was omitted from the density gradient. Experiments with 125I-calmodulin indicated the presence of high-affinity binding sites (KD = 1.3 X 10(-8) M; Bmax = 30 pmol/mg protein) for calmodulin in chromaffin granule membranes. Further, photoaffinity crosslinking experiments with 125I-calmodulin followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography indicated the presence of three calmodulin-binding polypeptide complexes (84,000; 41,000; and 38,000 daltons) in chromaffin granule membranes. These polypeptides were not labelled when either Ca2+ was omitted or an excess of nonradioactive calmodulin was present in the photolysis buffer, indicating the Ca2+ dependency and the specificity of the interaction. On the basis of the results described, it is suggested that the cellular levels of Ca2+ control the cellular distribution of calmodulin and its binding to specific chromaffin granule membrane proteins. Further, it is also suggested that the interactions between calmodulin and granule proteins might play a role in stimulus-secretion coupling.     

Noncholinergic Transmitter(s) Maintains Secretion of Catecholamines from Rat Adrenal Medulla for Several Hours of Continuous Stimulation of Splanchnic Neurons

The effect of continuous stimulation of splanchnic nerves at 1, 3, and 10 Hz on the secretion of catecholamines from the isolated rat adrenal gland was examined. Secretion evoked at 10 Hz declined over 60% in 1 h, and by the end of 4 h the secretion was only 10% of the initial value. The secretion evoked at 3 Hz was unchanged in the first hour, but showed a gradual decline in subsequent hours. In contrast, secretion evoked at 1 Hz was well maintained for several hours. Even after 6 h of continuous stimulation, the decline was only about 35%. Atropine plus hexamethonium reduced the secretion evoked at 10 Hz by over 80%, but that evoked at 1 Hz was reduced by about 35%; addition of naloxone reduced it to 75%. When the secretion declined to very low levels after continuous stimulation at 10 Hz for 100 min, a change in frequency to 3 Hz or 1 Hz caused a sharp rebound in the secretory response. Returning the frequency back to 10 Hz led to a sharp drop in the secretion, whereas reducing the frequency to 1 or 3 Hz once again increased the secretion. The rebound in the secretory response after switchover of frequencies was observed in the presence of atropine plus hexamethonium, but was abolished by naloxone. Extensive stimulations, which caused large amounts of catecholamine secretions at each frequency, were not associated with any loss in tissue catecholamine contents. The major conclusion is that secretion of catecholamines is maintained uninterrupted for several hours when splanchnic nerves are stimulated at low frequency.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subcellular Distribution Studies of Diadenosine Polyphosphates—Ap4A and Ap5A—in Bovine Adrenal Medulla: Presence in Chromaffin Granules

Diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) have been identified in bovine adrenal medullary tissue using an HPLC method. The values obtained were 0.1 +/- 0.05 mumol/g of tissue for both compounds. The subcellular fraction where Ap4A and Ap5A were present in the highest concentration was chromaffin granules: 32 nmol/mg of protein for both compounds (approximately 6 mM intragranularly). This value was 30 times higher than in the cytosolic fraction. Enzymatic degradation of Ap4A and Ap5A, isolated from chromaffin granules, with phosphodiesterase produces AMP as the final product. The Ap4A and Ap5A obtained from this tissue were potent inhibitors of adenosine kinase. Their Ki values relative to adenosine were 0.3 and 2 microM for Ap4A and Ap5A, respectively. The cytosolic fraction also contains enzymatic activities that degrade Ap4A as well as Ap5A. These activities were measured by an HPLC method; the observed Km values were 10.5 +/- 0.5 and 13 +/- 1 microM for Ap4A and Ap5A, respectively.     

Tyrosine Kinases Are Required for Catecholamine Secretion and Mitogen-Activated Protein Kinase Activation in Bovine Adrenal Chromaffin Cells

Abstract: Nicotine-induced catecholamine secretion in bovine adrenomedullary chromaffin cells is accompanied by rapid tyrosine phosphorylation of multiple cellular proteins, most notably the mitogen-activated protein kinases (MAPKs). The requirement for activation of tyrosine kinases and MAPKs in chromaffin cell exocytosis was investigated using a panel of tyrosine kinase inhibitors. Genistein and tyrphostin 23, two compounds that inhibit tyrosine kinases by distinct mechanisms, were found to inhibit secretion by >90% in cells stimulated by nicotine, 55 m M KCI, or the Ca²⁺ ionophore A23187. Inhibition of secretion induced by all three secretagogues correlated with a block in both protein tyrosine phosphorylation and activation of the MAPKs and their activators (MEKs) in situ. However, neither genistein nor tyrphostin 23 inhibited the activities of the MAPKs or MEKs in vitro. These results indicate that the target(s) of inhibition lie down-stream of Ca²⁺ influx and upstream of MEK activation. This Ca²⁺-activated tyrosine kinase activity could not be accounted for entirely by c-Src or Fyn (two nonreceptor tyrosine kinases that are expressed abundantly in chromaffin cells), because their in vitro kinase activities were not inhibited by tyrphostin 23 and only partially inhibited by genistein. These results demonstrate that an unidentified Ca²⁺-activated tyrosine kinase(s) is required for MAPK activation and exocytosis in chromaffin cells and suggest that MAPK participates in the regulation of secretion.     

Dopamine β-Hydroxylase and Other Glycoproteins from the Soluble Content and the Membranes of Adrenal Chromaffin Granules: Isolation and Carbohydrate Analysis

Abstract: Chromogranin A and two other proteins (A₁ and A₂) of the soluble proteins of bovine chromaffin granules were isolated by extraction from polyacrylamide gels after electrophoresis. The carbohydrate content of these proteins was 5%, with galactose, N -acetylgalactosamine, and sialic acid as the main sugars. Membranes of chromaffin granules were solubilized with sodium dodecyl sulphate (SDS) and three glycoproteins were isolated by sequential affinity chromatography on Concanavalin A (Con A) and wheat germ lectin (WGL) Sepharose columns. Two glycoproteins, designated GP II and III, were found to have a high carbohydrate content of about 30%. Mannose, galactose, N -acetylgalactosamine, and sialic acid were the main sugars. In addition membrane-bound dopamine β-hydroxylase was isolated by this procedure. No significant differences between the carbohydrate composition of the membrane-bound and the soluble enzyme were revealed. It was shown that all four subunits of dopamine β-hydroxylase possess carbohydrate chains with an affinity for Con A. The isolation methods established in this study will be useful for immunological studies on these glycoproteins.     

Differential In Vivo Regulation of mRNA Encoding the Norepinephrine Transporter and Tyrosine Hydroxylase in Rat Adrenal Medulla and Locus Ceruleus

Abstract: To investigate the regulation of norepinephrine transporter mRNA in vivo, we analyzed the effects of reserpine on its expression in the rat adrenal medulla and locus ceruleus. First, PCR was used to clone a 0.5-kb rat cDNA fragment that exhibits 87% nucleotide identity to the corresponding human norepinephrine transporter cDNA sequence. In situ, the cDNA hybridizes specifically within norepinephrine-secreting cells, but in neither dopamine nor serotonin neurons, suggesting strongly it is a partial rat norepinephrine transporter cDNA. Reserpine, 10 mg/kg administered 24 h premortem, decreased steady-state levels of norepinephrine transporter mRNA in the adrenal medulla by ∼65% and in the locus ceruleus by ∼25%, as determined by quantitative in situ hybridization. Northern analysis confirmed the results of the in situ hybridization analysis in the adrenal medulla but did not detect the smaller changes observed in the locus ceruleus. Both analyses showed that reserpine increased tyrosine hydroxylase expression in the adrenal medulla and locus ceruleus. These results suggest that noradrenergic neurons and adrenal chromaffin cells can coordinate opposing changes in systems mediating catecholamine uptake and synthesis, to compensate for catecholamine depletion.     

Arachidonic Acid and Other Unsaturated Fatty Acids Alter Membrane Potential in PC12 and Bovine Adrenal Chromaffin Cells

Abstract: The action of arachidonic acid and other fatty acids on membrane potential in PC 12 and bovine chromaffin cells was investigated using a membrane potential-sensitive fluorescent dye. Arachidonic acid (1–40 μ M ) provoked dose-dependent membrane hyperpolarization, thereby reducing hyperpolarization induced by the K⁺-selective ionophore valinomycin. Other cis-unsaturated fatty acids, but not lipoxygenase products or the saturated fatty acid palmitic acid, also affected membrane potential. Tetraethylammonium blocked the arachidonic acid-induced hyperpolarization. These data suggest that cis-unsaturated fatty acids alter membrane potential in PC 12 and bovine chromaffin cells by modulating K⁺ conductances. Valinomycin-generated hyperpolarization had no effect on agonist-induced Ca²⁺ influx into bovine chromaffin cells, whereas preincubation with arachidonic acid and other cis-unsaturated fatty acids blocked Ca²⁺ influx and secretion. We propose a model where internally generated fatty acids act as a feed-back to desensitize the stimulated cell via inhibition of receptor-dependent Ca²⁺ influx and induction of membrane hyperpolarization.     

Characterization of Insulin-Like Growth Factor-I Receptors in PC 12 Pheochromocytoma Cells and Bovine Adrenal Medulla

Competitive binding studies indicated that PC12 cells have receptors for insulin-like growth factor-I (IGF-I). There are approximately 11,000 +/- 1,500 IGF-I receptors/cell; these receptors have an apparent KD for IGF-I of 7.2 +/- 0.6 nM. Covalent cross-linking of 125I-IGF-I to PC12 cells labeled a 125,000-130,000-Mr protein, presumably the alpha-subunit of the IGF-I receptor. Although PC12 cells also have insulin receptors, the 125I-IGF-I appeared to be cross-linked to IGF-I receptors, because 100 nM IGF-I competed for labeling but 100 nM insulin did not. Bovine chromaffin cells also have IGF-I receptors. The protein tyrosyl kinase activity of IGF-I receptors from bovine adrenal medulla and PC12 cells was examined after purification of the receptors by wheat germ agglutinin-Sepharose chromatography. IGF-I (10 nM) stimulated autophosphorylation of the beta-subunits of the IGF-I receptors from both preparations; the beta-subunits from both sources had Mr values of approximately 97,000. IGF-I also stimulated phosphorylation of the synthetic substrate poly(Glu:Tyr)4:1 by both receptor preparations. IGF-I (IC50 of approximately 0.2 nM) was much more potent than insulin at stimulating phosphorylation of poly(Glu:Tyr) by the bovine adrenal medulla preparation. A maximal concentration of IGF-I (10 nM) increased phosphorylation approximately threefold. IGF-I was slightly more effective than insulin at stimulating the phosphorylation of poly(Glu:Tyr) by the PC12 cell receptor preparation, but neither ligand produced a maximal effect at concentrations up to 100 nM. This result probably reflects the presence of comparable numbers of IGF-I and insulin receptors on PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulatory Role of the GTP-Binding Protein, Go, in the Mechanism of Exocytosis in Adrenal Chromaffin Cells

To elucidate the possible involvement of GTP-binding proteins (G proteins) in the mechanism of exocytosis, we studied effects of pertussis toxin (PTX), guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), and antibodies against the G proteins (Gi and G(o)) on the secretory function of bovine adrenal chromaffin cells. Pretreatment of chromaffin cells with PTX resulted in an increase in acetylcholine-evoked catecholamine release. High K(+)-, histamine-, or gamma-aminobutyric acid-evoked catecholamine release was also potentiated by PTX pretreatment. The concentration of extracellular Ca2+ required for maximal release by 10(-4) M acetylcholine was decreased significantly in PTX-treated cells. In digitonin-permeabilized cells, PTX pretreatment resulted in a decrease of the half-maximal concentration (Km) of Ca2+ required for exocytosis with no significant change in the maximal stimulation (Vmax). Exposure of permeabilized cells to GTP-gamma-S (a nonhydrolyzable GTP analogue) inhibited Ca(2+)-dependent exocytosis by reducing the affinity for Ca2+. The effects of PTX pretreatment were mimicked by treatment of permeabilized cells with polyclonal antibodies selective for the alpha subunit of the PTX-sensitive G protein, G(o). Treatment with similar antibodies against the alpha subunit of Gi had no effect. These findings suggest that G(o) directly controls the Ca(2+)-triggered process in the machinery of exocytosis by lowering the affinity of the unknown target for Ca2+.     

Restoration of Catecholamine Content of Previously Depleted Adrenal Medulla In Vitro: Importance of Synthesis in Maintaining the Catecholamine Stores

The functional integrity of adrenal chromaffin storage vesicles was studied in the perfused rat adrenal gland subjected to intense exocytosis. Continuous perfusion with 55 mM K+-Krebs solution produced a large and uninterrupted secretion of catecholamines. Total amounts secreted within 45 min were 4.66 micrograms and represented almost 30% of the total tissue catecholamine content. If perfusion with excess K+ was extended to 90 min, the secretion increased further to 5.76 micrograms. Despite such a large secretory response, the catecholamine content of the K+-stimulated adrenal medulla was comparable to that of unstimulated control, suggesting an enhanced resynthesis to maintain the normal levels. Pretreatment of rats with alpha-methyl-p-tyrosine, and including this agent in the perfusion medium during stimulation with K+, caused a marked reduction in catecholamine content. The degree of depletion depended on the extent of stimulation with K+ (45% in 45 min and 60% in 90 min). Although depleted catecholamine stores did not show spontaneous recovery in 2 h, inclusion of tyrosine, L-3,4-dihydroxyphenylalanine or dopamine (but not epinephrine or norepinephrine) completely restored the catecholamine content of previously depleted adrenal medulla. Repletion achieved by tyrosine was time dependent (evident in 30 min and maximum in 2 h) and blocked by alpha-methyl-p-tyrosine but not by calcium deprivation. The ratio of epinephrine to norepinephrine remained constant during various stages of the experiment, suggesting both types of vesicles were equally affected by different treatments. The secretory response (10 Hz for 30 s) was unaffected even though tissue catecholamine stores were significantly depleted (50%). In summary, we have demonstrated that catecholamine content of the isolated perfused adrenal gland can be reduced by stimulation of exocytotic secretion in the presence of tyrosine hydroxylase inhibitor. Since the depleted stores can be fully refilled by synthesis of catecholamines from its precursors, it is suggested that chromaffin vesicles may be reutilized for the purpose of synthesis, storage, and secretion of adrenal medullary hormones.     

Subcellular Site of Biosynthesis of the Catecholamine Biosynthetic Enzymes in Bovine Adrenal Medulla

The subcellular site of biosynthesis of the catecholamine biosynthetic enzymes was examined. Free and membrane-bound polysomes were prepared from bovine adrenal medulla and mRNA was isolated from these polysomes. Both were active in directing cell-free translations. Immunoprecipitation of cell-free products with specific antisera localized the biosynthesis of the subunits of tyrosine hydroxylase (TH) (apparent Mr = 61,000) and of phenylethanolamine N-methyltransferase (PNMT) (apparent Mr = 32,000) on free polysomes, compared with biosynthesis of subunits of dopamine beta-hydroxylase (DBH) (apparent Mr = 67,000) on membrane-bound polysomes. Cross-reactivity between translation products was observed. Antibodies for DBH recognized a polypeptide with electrophoretic mobility identical to newly synthesized PNMT. However increasing concentrations of antibodies to DBH recognized at most 1/20 of the PNMT formed. The results of this study show the subcellular distribution of the catecholamine synthesizing enzymes is determined by their site of biosynthesis.     

α1-Antichymotrypsin-Like Proteins I and II Purified from Bovine Adrenal Medulla Are Enriched in Chromaffin Granules and Inhibit the Proenkephalin Processing Enzyme "Prohormone Thiol Protease"

Proteolytic processing of inactive proenkephalin and proneuropeptides is essential for the production of biologically active enkephalins and many neuropeptides. The incomplete processing of proenkephalin in adrenal medulla suggests that endogenous protease inhibitors may inhibit proenkephalin processing enzymes. This study demonstrates the isolation and characterization of two isoforms of adrenal medullary alpha1-antichymotrypsin (ACT), referred to as ACT-like proteins I and II, which are colocalized with enkephalin in chromaffin granules and which inhibit the proenkephalin processing enzyme known as prohormone thiol protease (PTP). Subcellular fractionation demonstrated enrichment of 56- and 60-kDa ACT-like proteins I and II, respectively, to enkephalin-containing chromaffin granules (secretory vesicles). Immunofluorescence cytochemistry of chromaffin cells indicated a discrete, punctate pattern of ACT immunostaining that resembles that of [Met]enkephalin that is stored in secretory vesicles. Chromatography of adrenal medullary extracts through DEAE-Sepharose and chromatofocusing resulted in the separation of ACT-like proteins I and II that possess different isoelectric points of 5.5 and 4.0, respectively. The 56-kDa ACT-like protein I was purified to apparent homogeneity by Sephacryl S200 chromatography; the 60-kDa ACT-like protein II was isolated by butyl-Sepharose, Sephacryl S200, and concanavalin A-Sepharose columns. The proenkephalin processing enzyme PTP was potently inhibited by ACT-like protein I, with a K(i,app) of 35 nM, but ACT-like protein II was less effective. ACT-like proteins I and II had little effect on chymotrypsin. These results demonstrate the biochemical identification of two secretory vesicle ACT-like proteins that differentially inhibit PTP. The colocalization of the ACT-like proteins and PTP within chromaffin granules indicates that they could interact in vivo. Results from this study suggest that these ACT-like proteins may be considered as candidate inhibitors of PTP, which could provide a mechanism for limited proenkephalin processing in adrenal medulla.     

Characterization of Catecholamine-Storage Organelles in Transplantable Phaeochromocytoma and Adrenal Glands of Rats

Abstract: The properties of the catecholamine-storing organelles from transplantable rat phaeochromocytoma and rat adrenal glands were compared by density gradient centrifugation. It was shown that tumour granules are more heterogeneous and less dense than adrenal granules. Both granule preparations can take up catecholamines and nucleotides by a process driven by an electrochemical proton gradient. Dopamine β-hydroxylase and glycoprotein III were analysed by immunological techniques. Glycoprotein III was shown to be a specific component of chromaffin granules. Tumour tissue (average weight 700 mg) contains amounts of these antigens comparable to those in 210 adrenals. The biosynthesis of granules in the tumour apparently occurs at a low rate, making turnover studies difficult. The transplantable rat phaeochromocytoma is very useful for studies on the uptake properties and the immunological characteristics of rat catecholamine storage granules because one tumour provides an amount of material that could otherwise be obtained only from a large number of adrenal glands.     

Exocytosis Coupled to Mobilization of Intracellular Calcium by Muscarine and Caffeine in Rat Chromaffin Cells

Abstract: We used cultured rat chromaffin cells to test the hypothesis that Ca²⁺ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca²⁺ sources: (a) Ca²⁺ surrounding single cells was transiently displaced by applying agonist with or without Ca²⁺ from an ejection pipette. (b) Intracellular stores of Ca²⁺ were depleted by soaking cells in Ca²⁺-free plus 1 mM EGTA solution before transient exposure to agonist plus Ca²⁺. Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured by indo-1 fluorescence. KCl (35 mM) and nicotine (10 µM) caused an immediate increase in [Ca²⁺]_i and secretion in cells with or without internal Ca²⁺ stores, but only when applied with Ca²⁺ in the ejection pipette. Caffeine (10 mM) and muscarine (30 µM) evoked exocytosis whether or not Ca²⁺ was included in the pipette, but neither produced responses in cells depleted of internal Ca²⁺ stores. Pretreatment with ryanodine (0.1 µM) inhibited caffeine- but not muscarine-stimulated responses. Elevated [Ca²⁺]_i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 ± 0.38 s) or muscarine (2.2 ± 0.25 s). However, the duration of caffeine-evoked exocytosis (7.1 ± 0.8 s) was significantly shorter than that evoked by muscarine (33.1 ± 3.5 s). The duration of caffeine-evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An ～20-s refractory period was found between repeated caffeine-evoked exocytotic bursts even though [Ca²⁺]_i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca²⁺ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca²⁺. These results and conclusions are consistent with our original observations in the perfused adrenal gland.     

Differential Expression of SNAP-25 Isoforms and SNAP-23 in the Adrenal Gland

Abstract : In the rat adrenal gland, we previously observed that SNAP-25 is not restricted to the plasmalemma in noradrenergic cells as it is in adrenergic cells, and hypothesized that SNAP-25 isoform expression is different in the two phenotypes. Expression of SNAP-25 isoforms and SNAP-23 was examined by immunoblotting, immunofluorescence, and RT-PCR. Amplifications of SNAP-25 mRNAs were combined with Southern hybridization, restriction enzyme analysis, and sequencing of cloned PCR products to compare SNAP-25 isoform expression in rat and bovine adrenal glands. SNAP-25 and SNAP-23 mRNA and protein are expressed in the glands ; SNAP-23 is enriched in the adrenal cortex, whereas SNAP-25 is restricted to the adrenal medulla. Furthermore, high levels of SNAP-25 and low levels of SNAP-23 are observed in the PC12 cells, whereas both SNAP-25 and SNAP-23 are expressed in adrenal medullary cultures. In all extracts, the SNAP-23 mRNA corresponded to SNAP-23a. SNAP-25a is the major form expressed in rat adrenal glands (75%), as it is in PC12 cells (80%), but both SNAP-25a and SNAP-25b (40% vs. 60%) are expressed in bovine adrenal medulla in situ and in culture. In addition, an enriched population of adrenergic cells (93%) expressed a higher level of SNAP-25b (70%), suggesting that this isoform may not be restricted to fast neurotransmission.     

Characterization and Distribution of Transferrin Receptors in the Rat Brain

The mechanism of calcium transport across the plasma membrane of chromaffin cells was studied using plasma membrane vesicles prepared from cells of adrenal medulla. Purification of the plasma membrane was about 30-fold, based on the alpha-bungarotoxin binding activity. The isolated membrane vesicles have both Na+/Ca2+ exchange and calcium pump activities. The Na+/Ca2+ exchange activity increased with the free calcium concentration and was not saturated at 1 mM, the highest concentration tried. The K1/2 of the calcium pump for calcium is 0.06 microM. Part of the Na+/Ca2+ exchange activity was inhibited by preincubation of the membrane vesicles with veratridine and the effect of veratridine was reversed by tetrodotoxin. The calcium taken up by the calcium pump was released by 0.005% saponin, but was not affected by oxalate. The calcium taken up by the calcium pump was released by exchanging with the external sodium, which suggests that the two calcium transport systems are located on the same population of membrane vesicles. The above evidence indicates that both calcium transport activities are located on the plasma membrane and not on contaminating organelle membranes. The significance of the two calcium transport systems in regulation of cytosolic calcium concentration of chromaffin cells is discussed.     

Proadrenomedullin N-Terminal 20 Peptide (PAMP), an Endogenous Anticholinergic Peptide: Its Exocytotic Secretion and Inhibition of Catecholamine Secretion in Adrenal Medulla

Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca²⁺-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC₅₀ = 50.1 µ M ) and catecholamines (EC₅₀ = 63.0 µ M ), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH₂ inhibited carbachol-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀ = 2.5 µ M ) in a noncompetitive manner and thereby reduced carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels (IC₅₀ = 1.0 µ M ) and catecholamine secretion (IC₅₀ = 1.6 µ M ). It did not alter high K⁺-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels or veratridine-induced ²²Na⁺ influx via voltage-dependent Na⁺ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca²⁺-dependent exocytosis in response to nicotinic receptor stimulation.     

Fine structure and cytochemistry of specific granules in the lamprey atrium

Summary Fine structural and cytochemical studies were performed to examine the nature of three types of specific granules found in the atrium of lamprey; specific granules of the atrial muscle cell (ASG), interstitial cell granules (ICG) and endocardial endothelial granules (ESG).Ultrastructurally, ASG and ICG appeared quite similar in size, shape and electron opacity, while ESG were much larger and less dense in opacity than the other two.None of the granules showed positive DAB reaction or acid phosphatase reaction. Only ICG revealed positive chromaffin reaction, which agreed with formaldehyde induced green fluorescence along the atrial lumen. Phosphotungstic acid at low pH stained ICG and ASG strongly positive, and ESG weakly positive. Pronase treatment in Epon sections for 24h digested ASG alone, whereas in glycol-methacrylate embedded sections, ESG were digested first, ASG were digested thoroughly after 30 min, but ICG were not digested completely after 90 min.From these results it can be concluded that the three types of specific granules have different constituents. ESG consist of protein with some polysaccharides; ASG are composed of protein carbohydrate complexes and lack catecholamines; ICG contain catecholamine as well as protein carbohydrate complexes.This work was supported by a grant from the Ministry of Education, JapanThe authors would like to express their gratitude to K. Wasano, M.D. for his technical assistance in fluorescence microscopy