Rab3 Proteins Involved in Vesicle Biogenesis and Priming in Embryonic Mouse Chromaffin Cells

The four Rab3 paralogs A–D are involved in exocytosis, but their mechanisms of action are hard to study due to functional redundancy. Here, we used a quadruple Rab3 knockout (KO) (rab3a, rab3b, rab3c, rab3d null, here denoted as ABCD^?/?) mouse line to investigate Rab3 function in embryonic mouse adrenal chromaffin cells by electron microscopy and electrophysiological measurements. We show that in cells from ABCD^?/? animals large dense‐core vesicles (LDCVs) are less abundant, while the number of morphologically docked granules is normal. By capacitance measurements, we show that deletion of Rab3s reduces the size of the releasable vesicle pools but does not alter their fusion kinetics, consistent with an altered function in vesicle priming. The sustained release component has a sigmoid shape in ABCD^?/? cells when normalized to the releasable pool size, indicating that vesicle priming follows at a higher rate after an initial delay. Rescue experiments showed that short‐term (4–6 h) overexpression of Rab3A or Rab3C suffices to rescue vesicle priming and secretion, but it does not restore the number of secretory vesicles. We conclude that Rab3 proteins play two distinct stimulating roles for LDCV fusion in embryonic chromaffin cells, by facilitating vesicle biogenesis and stabilizing the primed vesicle state.     

Structure and Dynamics of a Two‐Helix SNARE Complex in Live Cells

SNAREs are clustered membrane proteins essential for intracellular fusion steps. During fusion, three to four SNAREs with a Q_a‐, Q_b‐, Q_c‐ and R‐SNARE‐motif form a complex. The core complex represents a Q_aQ_bQ_cR‐SNARE‐motif bundle, most certainly assembling in steps. However, to date it is unknown which intermediate SNARE complex observed in vitro also exists in vivo. Here we have applied comparative fluorescence recovery after photobleaching (FRAP)‐studies as a novel approach for studying in intact cells a SNARE interaction involved in synaptic vesicle fusion [catalyzed by syntaxin 1A (Q_a), SNAP25 (Q_b/Q_c) and synaptobrevin 2 (R)]. We find that the Q_b‐SNARE‐motif of SNAP25 interacts reversibly with clustered syntaxin. The interaction requires most of the alpha helical Q_b‐SNARE‐motif and depends on its position within the molecule. We conclude that a zippered Q_aQ_b‐SNARE complex represents a short‐lived SNARE intermediate in intact cells, most likely providing an initial molecular platform toward membrane fusion.     

S100A10‐Mediated Translocation of Annexin‐A2 to SNARE Proteins in Adrenergic Chromaffin Cells Undergoing Exocytosis

In neuroendocrine cells, annexin‐A2 is implicated as a promoter of monosialotetrahexosylganglioside (GM1)‐containing lipid microdomains that are required for calcium‐regulated exocytosis. As soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) require a specific lipid environment to mediate granule docking and fusion, we investigated whether annexin‐A2‐induced lipid microdomains might be linked to the SNAREs present at the plasma membrane. Stimulation of adrenergic chromaffin cells induces the translocation of cytosolic annexin‐A2 to the plasma membrane, where it colocalizes with SNAP‐25 and S100A10. Cross‐linking experiments performed in stimulated chromaffin cells indicate that annexin‐A2 directly interacts with S100A10 to form a tetramer at the plasma membrane. Here, we demonstrate that S100A10 can interact with vesicle‐associated membrane protein 2 (VAMP2) and show that VAMP2 is present at the plasma membrane in resting adrenergic chromaffin cells. Tetanus toxin that cleaves VAMP2 solubilizes S100A10 from the plasma membrane and inhibits the translocation of annexin‐A2 to the plasma membrane. Immunogold labelling of plasma membrane sheets combined with spatial point pattern analysis confirmed that S100A10 is present in VAMP2 microdomains at the plasma membrane and that annexin‐A2 is observed close to S100A10 and to syntaxin in stimulated chromaffin cells. In addition, these results showed that the formation of phosphatidylinositol (4,5)‐bisphosphate (PIP₂) microdomains colocalized with S100A10 in the vicinity of docked granules, suggesting a functional interplay between annexin‐A2‐mediated lipid microdomains and SNAREs during exocytosis.     

Secretory Vesicle Pools and Rate and Kinetics of Single Vesicle Exocytosis in Neurosecretory Cells

Secretory vesicles are localized in specific compartments within neurosecretory cells. Morphometric, cytochemical and electrophysiological techniques have allowed the definition of secretory vesicle compartments. These are different pools in which vesicles are in various states of releasability. The transit of vesicles between compartments is not random, but an event controlled and regulated by Ca²⁺ and the cortical F-actin network. Cortical F-actin disassembly, a Ca²⁺-dependent event, controls the transit of secretory vesicles from the reserve compartment to the release-ready vesicle pool. Furthermore, the recent development of new technical approaches (patch-clamp membrane capacitance, electrochemical detection of amines with carbon-fibre microelectrodes) has now permitted us to understand the kinetics of single vesicle exocytosis.     

Effects of Osmolality and Ionic Strength on Secretion from Adrenal Chromaffin Cells Permeabilized with Digitonin

Hyperosmotic solutions inhibit exocytosis of catecholamine from adrenal chromaffin cells at a step after Ca2+ entry into the cells. The possibility that the inhibition resulted from an inability of shrunken secretory granules to undergo exocytosis was investigated in cells with plasma membranes permeabilized by digitonin. The osmoticants and salts used in this study rapidly equilibrated across the plasma membrane and bathed the intracellular organelles. When sucrose was the osmoticant, secretion was not significantly inhibited unless the osmolality was raised above 1,000 mOs. When the osmolality was raised with the tetrasaccharide stachyose or a low-molecular-weight maltodextrin fraction (average size a tetrasaccharide), one-half maximal inhibition occurred at 900-1,000 mOs. Prior treatment of permeabilized cells with Ca2+ in hyperosmotic solution did not result in enhanced secretion when cells were restored to normal osmolality. Increased concentrations of potassium glutamate or sodium isethionate were more potent than carbohydrate in inhibiting secretion. Half-maximal inhibition occurred at 600-700 mOs or when the ionic strength was approximately doubled. The inhibition by elevated potassium glutamate also occurred when the osmolality was kept constant with sucrose. Increasing the ionic strength did not alter the Ca2+ sensitivity of the secretory response. Reducing the ionic strength by substituting sucrose for salt reduced the Ca2+ concentration required for half-maximal stimulated secretion from approximately 1.2 microM to 0.5 microM. Chromaffin granules, the secretory granules, are known to shrink in hyperosmotic solution. The experiments indicate that shrunken chromaffin granules can undergo exocytosis and suggest that in intact cells elevated ionic strength rather than chromaffin granule shrinkage contributes to the inhibition of secretion by hyperosmotic solutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical and Functional Studies of Cortical Vesicle Fusion: The SNARE Complex and Ca2+ Sensitivity

Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca²⁺ activity curve comparable to exocytosis (CV–PM fusion). Here we show that Sr²⁺ and Ba²⁺ also trigger CV–CV fusion, and agents affecting different steps of exocytotic fusion block Ca²⁺, Sr²⁺, and Ba²⁺-triggered CV–CV fusion. The maximal number of active fusion complexes per vesicle, <n\>_Max, was quantified by NEM inhibition of fusion, showing that CV–CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both <n\>_Max and the Ca²⁺ sensitivity of fusion complex activation were comparable to that determined for CV–PM fusion. Using Ca²⁺-induced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV–CV and CV–PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca²⁺, Sr²⁺, and Ba²⁺, the complex remains Ca²⁺- sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca²⁺ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca²⁺-triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca²⁺ sensitivity of fusion.     

Vesicle Shrinking and Enlargement Play Opposing Roles in the Release of Exocytotic Contents

1. Download : Download high-res image (176KB)
2. Download : Download full-size image

     

Ternary SNARE complexes are enriched in lipid rafts during mast cell exocytosis

Lipid rafts are membrane microdomains rich in cholesterol and glycosphingolipids that have been implicated in the regulation of intracellular protein trafficking. During exocytosis, a class of proteins termed SNAREs mediate secretory granule-plasma membrane fusion. To investigate the role of lipid rafts in secretory granule exocytosis, we examined the raft association of SNARE proteins and SNARE complexes in rat basophilic leukemia (RBL) mast cells. The SNARE protein SNAP-23 co-localized with a lipid raft marker and was present in detergent-insoluble lipid raft microdomains in RBL cells. By contrast, only small amounts (<20%) of the plasma membrane SNARE syntaxin 4 or the granule-associated SNARE vesicle-associated membrane protein (VAMP)-2 were present in these microdomains. Despite this, essentially all syntaxin 4 and most of VAMP-2 in these rafts were present in SNARE complexes containing SNAP-23, while essentially none of these complexes were present in nonraft membranes. Whereas SNAP-23 is membrane anchored by palmitoylation, the association of the transmembrane protein syntaxin 4 with lipid rafts was because of its binding to SNAP-23. After stimulating mast cells exocytosis, the amount of syntaxin 4 and VAMP-2 present in rafts increased twofold, and these proteins were now present in raft-associated phospho-SNAP-23/syntaxin 4/VAMP-2 complexes, revealing differential association of SNARE fusion complexes during the process of regulated exocytosis.     

Real-time imaging of plasma membrane deformations reveals pre-fusion membrane curvature changes and a role for dynamin in the regulation of fusion pore expansion

Assays for real-time investigation of exocytosis typically measure what is released from the granule. From this, inferences are made about the dynamics of membrane remodeling as fusion progresses from start to finish. We have recently undertaken a different approach to investigate the fusion process, by focusing not primarily on the granule, but rather its partner in exocytosis - the plasma membrane. We have been guided by the idea that biochemical interactions between the granule and plasma membranes before and during fusion, cause changes in membrane conformation. To enable study of membrane conformation, a novel imaging technique was developed combining polarized excitation of an oriented membrane probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI) with total internal reflection fluorescence microscopy (pTIRFM). Because this technique measures changes in membrane conformation (or deformations) directly, its usefulness persists even after granule cargo reporter (catecholamine, or protein), is no longer present. In this mini-review, we first summarize the workings of pTIRFM. We then discuss the application of the technique to investigate deformations in the membrane preceding fusion, and later, during fusion pore expansion. Finally, we discuss how expansion of the fusion pore may be regulated by the GTPase activity of dynamin.     

The SNARE protein vti1a functions in dense‐core vesicle biogenesis

The SNARE protein vti1a is proposed to drive fusion of intracellular organelles, but recent data also implicated vti1a in exocytosis. Here we show that vti1a is absent from mature secretory vesicles in adrenal chromaffin cells, but localizes to a compartment near the trans‐Golgi network, partially overlapping with syntaxin‐6. Exocytosis is impaired in vti1a null cells, partly due to fewer Ca²⁺‐channels at the plasma membrane, partly due to fewer vesicles of reduced size and synaptobrevin‐2 content. In contrast, release kinetics and Ca²⁺‐sensitivity remain unchanged, indicating that the final fusion reaction leading to transmitter release is unperturbed. Additional deletion of the closest related SNARE, vti1b, does not exacerbate the vti1a phenotype, and vti1b null cells show no secretion defects, indicating that vti1b does not participate in exocytosis. Long‐term re‐expression of vti1a (days) was necessary for restoration of secretory capacity, whereas strong short‐term expression (hours) was ineffective, consistent with vti1a involvement in an upstream step related to vesicle generation, rather than in fusion. We conclude that vti1a functions in vesicle generation and Ca²⁺‐channel trafficking, but is dispensable for transmitter release.     

Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.     

Characterization of Exocytotic Events From Single PC12 Cells: Amperometric Studies in Native PC12h, DA-Loaded PC12h and Bovine Adrenal Chromaffin Cells

Exocytotic events from rat pheochromocytoma (PC12) cells were characterized by amperometric analysis. For single-cell amperometric recordings, PC12h cells cultured onto poly-L-lysine corted glass-base dish were incubated with 1 mM dopamine (DA) for 60 min. Amperometric recordings, with a carbon fiber microelectrode (5 μm diameter), of catecholamine release from the individual cells were conducted under an inverted microscope at 25 ^∘C. To characterize a single exocytotic event that is detected as a single spike current, the spike number, spike parameters (rise time, middle width and area) and spike shape were analyzed. Exposure of DA-loaded PC12h cells to 60 mM KCl (1000 hps) for 5 min and for 4 s evoked a train of events with the event number of 114± 19 (spikes/response for 5 min) and 12± 3 (spikes/response for 15 s), respectively. We observed distinctive kinetics in the events (rise time = 0.83± 0.19 ms, middle width = 2.89± 0.62 ms, area = 62± 7.6 fC and the spikes with a “foot” = 15.4± 2.7% of total spikes). The number and mean height of the events were 3- to 4-fold higher than that in DA-unloaded cells, and the values of rise time and middle width in DA-loaded PC12h cells were approx. 5- and 10-fold less than those observed in cultured adrenal chromaffin cells. The successful application of amperometry to monitor DA released from secretory vesicles in DA-loaded PC12h cell suggest that this technique is applicable to characterize exocytotic events in neurons.     

Comparative Studies of Calcium Transients Induced by Acetylcholine in Rat Chromaffin Cells

We used an intracellular fluorescent probe, FURA-2M, to examine the responses of isolated rat chromaffin cells to applications of 1 mM acetylcholine (ACh). Our data showed two different populations of the cell responses to such stimulation. Responses of the first type demonstrated fast rise and decay phases of the Ca²⁺ transients and no significant decrease in their amplitude during repetitive stimulation of the cell with ACh. Cell responses of the second type showed remarkably slower rise and decay phases of the Ca²⁺ transients and a noticeable drop of the cell responses during repetitive ACh stimulation that could be recovered after KCl depolarization. We find no significant differences in the amplitudes of the transients in these two populations of the cells. We conclude that there is heterogeneity of the chromaffin cells according to their ACh receptors: the first subpopulation predominantly expresses ionotropic (nicotinic) receptors (n cells), whereas the second cell population has mainly metabotropic (muscarinic) ones (m cells), which are associated with Ca²⁺ release from the intracellular stores.     

Interactions between Presynaptic Calcium Channels and Proteins Implicated in Synaptic Vesicle Trafficking and Exocytosis

Monoclonal antibodies were generated by immunizing mice with chick brain synaptic membranes and screening for immunoprecipitation of solubilized conotoxin GVIA receptors (N-type calcium channels). Antibodies against two synaptic proteins (p35--syntaxin 1 and p58--synaptotagmin) were produced and used to purify and characterize a ternary complex containing N-type channels associated with these two proteins. These results provided the first evidence for a specific interaction between presynaptic calcium channels and SNARE proteins involved in synaptic vesicle docking and calcium-dependent exocytosis. Immunoprecipitation experiments supported the conclusion that syntaxin 1/SNAP-25/VAMP/synaptotagmin I or II complexes associate with N-type, P/Q-type, but not L-type calcium channels from rat brain nerve terminals. Immunofluorescent confocal microscopy at the frog neuromuscular junction was consistent with the co-localization of syntaxin 1, SNAP-25, and calcium channels, all of which are predominantly expressed at active zones of the presynaptic plasma membrane facing post-synaptic folds rich in acetylcholine receptors. The interaction of proteins implicated in calcium-dependent exocytosis with presynaptic calcium channels may locate the sensor(s) that trigger vesicle fusion within a microdomain of calcium entry.     

Ca2+-induced Ca2+ Release in Chromaffin Cells Seen from inside the ER with Targeted Aequorin

The presence and physiological role of Ca²⁺-induced Ca²⁺ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca²⁺] ([Ca²⁺]_c). Using targeted aequorin, we have directly monitored [Ca²⁺] changes inside the ER ([Ca²⁺]_ER) in bovine adrenal chromaffin cells. Ca²⁺ entry induced by cell depolarization triggered a transient Ca²⁺ release from the ER that was highly dependent on [Ca²⁺]_ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca²⁺ release was quantal in nature due to modulation by [Ca²⁺]_ER. Whereas caffeine released essentially all the Ca²⁺ from the ER, inositol 1,4,5-trisphosphate (InsP₃)- producing agonists released only 60–80%. Both InsP₃ and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca²⁺ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca²⁺]_c measurements showed that the wave of [Ca²⁺]_c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca²⁺ pool that can release Ca²⁺ both via InsP₃ receptors or CICR.     

Parallel but Separate Release of Catecholamines and Acetylcholinesterase from Stimulated Adrenal Chromaffin Cells in Culture

Abstract: A pharmacological study was made of the effects of veratridine and lasalocid on the release of catecholamines, acetylcholinesterase (AChE) and dopamine-β-hydroxylase (DBH) from cultures of isolated bovine adrenal chromaffin cells. Exposure of the cultures to veratridine resulted in concomitant release of catecholamines and AChE into the external medium in a dose-dependent and Ca^{2 +}-dependent manner. A Ca²⁺ iono-phore, lasalocid, also produced a dose-dependent and parallel release of both catecholamines and AChE. The release of the two components was accompanied by release of DBH. The present results provide pharmacological evidence for a parallel release of catecholamines, AChE, and DBH from cultured adrenal chromaffin cells, and the stoichiometry of the release evoked by different secretagogues suggests that AChE and catecholamines are released from different cellular compartments.     

Stimulation by ATP of Inositol Trisphosphate Accumulation and Calcium Mobilization in Cultured Adrenal Chromaffin Cells

Effects of ATP on accumulation of inositol phosphates and Ca2+ mobilization were investigated in cultured bovine adrenal chromaffin cells. When the cells were stimulated with 30 microM ATP, a rapid and transient rise in intracellular Ca2+ concentration was observed. At the same time, ATP rapidly increased accumulation of inositol phosphates. The concentration-response curve for the ATP-induced Ca2+ mobilization was similar to that for inositol trisphosphate (IP3) accumulation. ATP exerted its maximal effects at 30 microM for either IP3 accumulation or Ca2+ mobilization. The order of the efficacy of the agonists for IP3 accumulation and Ca2+ mobilization at 100 microM was ATP greater than ADP greater than AMP approximately adenosine, AMP (100 microM) and adenosine (300 microM) failed to induce IP3 accumulation and Ca2+ mobilization. Although 100 microM GTP and 100 microM UTP also induced IP3 accumulation and Ca2+ mobilization, their efficacy was less than that of ATP. CTP (100 microM) induced a slight IP3 accumulation, but it did not induce Ca2+ mobilization. Nifedipine (10 microM), a Ca2+ channel antagonist, and theophylline (100 microM), a P1-purinergic receptor antagonist, failed to inhibit the ATP-induced IP3 accumulation and Ca2+ mobilization. The above two cellular responses induced by ATP were also observed in the Ca2+-depleted medium. ATP induced a rapid and transient accumulation of 1,4,5-IP3 (5s), followed by a slower accumulation of 1,3,4-IP3. These results suggest that ATP induces the formation of 1,4,5-IP3 through the P2-purinergic receptor and consequently promotes Ca2+ mobilization from intracellular storage sites in cultured adrenal chromaffin cells.     

Cholinergic Receptor-Mediated Phosphorylation and Activation of Tyrosine Hydroxylase in Cultured Bovine Adrenal Chromaffin Cells

We have identified a 56-kilodalton protein in cultured bovine adrenal chromaffin cells that is phosphorylated when catecholamine secretion is stimulated. Immunodetection on Western blots from both one- and two-dimensional polyacrylamide gels indicated that this protein was tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. Two-dimensional polyacrylamide gel electrophoresis of proteins from unstimulated cells revealed small amounts of phosphorylated protein with a molecular weight of 56K and pI values of 6.37 and 6.27 which were subunits of tyrosine hydroxylase. Nicotinic stimulation of chromaffin cells caused the phosphorylation of three proteins of 56 kilodaltons with pI values of approximately 6.37, 6.27, and 6.15 which were tyrosine hydroxylase. The immunochemical analysis also revealed that there was unphosphorylated tyrosine hydroxylase 56 kilodaltons with a pI of 6.5 which may have decreased on nicotinic stimulation. The phosphorylation of tyrosine hydroxylase was associated with an increase in in situ conversion of [3H]tyrosine to [3H]dihydroxyphenylalanine ([3H]DOPA). Muscarinic stimulation also caused phosphorylation of tyrosine hydroxylase, but to a smaller extent than did nicotinic stimulation. The secretagogues, elevated K+ and Ba2+, stimulated phosphorylation of tyrosine hydroxylase and [3H]DOPA production. The effects of nicotinic stimulation and elevated K+ on tyrosine hydroxylase phosphorylation and [3H]DOPA production were Ca2+-dependent. Nicotinic agonists also raised cyclic AMP levels in chromaffin cells after 2 min. Dibutyryl cyclic AMP and forskolin, which have little effect on catecholamine secretion, also caused phosphorylation of tyrosine hydroxylase. These stimulators of cyclic AMP-dependent processes caused the appearance of two phosphorylated subunits of tyrosine hydroxylase with pI values of 6.37 and 6.27. There was also a small amount of phosphorylated subunit with a pI of 6.15. Both agents stimulated [3H]DOPA production. The experiments indicate that tyrosine hydroxylase is phosphorylated and activated when chromaffin cells are stimulated to secrete. The data suggest that the earliest phosphorylation of tyrosine hydroxylase induced by a nicotinic agonist occurs through stimulation of a Ca2+-dependent protein kinase. After 2 min phosphorylation by a cyclic AMP-dependent protein kinase may also occur. Phosphorylation of tyrosine hydroxylase is associated with an increase in in situ tyrosine hydroxylase activity.     

Differential Inhibition of Secretagogue-Stimulated Sodium Uptake in Adrenal Chromaffin Cells by Activation of D4 and D5 Dopamine Receptors

Abstract: Recent studies have demonstrated that D1-selective and D2-selective dopamine receptor agonists inhibit catecholamine secretion and Ca²⁺ uptake into bovine adrenal chromaffin cells by receptor subtypes that we have identified by PCR as D5, a member of the D1-like dopamine receptor subfamily, and D4, a member of the D2-like dopamine receptor subfamily. The purpose of this study was to determine whether activation of D5 or D4 receptors inhibits influx of Na⁺, which could explain inhibition of secretion and Ca²⁺ uptake by dopamine agonists. D1-selective agonists preferentially inhibited both dimethylphenylpiperazinium- (DMPP) and veratridine-stimulated ²²Na⁺ influx into chromaffin cells. The D1-selective agonists chloro-APB hydrobromide (CI-APB; 100 µ M ) and SKF-38393 (100 µ M ) inhibited DMPP-stimulated Na⁺ uptake by 87.5 ± 2.3 and 59.7 ± 4.5%, respectively, whereas the D2-selective agonist bromocriptine (100 µ M ) inhibited Na⁺ uptake by only 22.9 ± 5.0%. Veratridine-stimulated Na⁺ uptake was inhibited 95.1 ± 3.2 and 25.7 ± 4.7% by 100 µ M CI-APB or bromocriptine, respectively. The effect of CI-APB was concentration dependent. A similar IC₅₀ (∼18 µ M ) for inhibition of both DMPP- and veratridine-stimulated Na⁺ uptake was obtained. The addition of 8-bromo-cyclic AMP (1 m M ) had no effect on either DMPP- or veratridine-stimulated Na⁺ uptake. These observations suggest that D1-selective agonists are inhibiting secretagogue-stimulated Na⁺ uptake in a cyclic AMP-independent manner.