Treatment Of Chromaffin Cells Research Articles

Commentary: Chromaffin Cell Plasticity in Stress, Inflammation, Development, and Disease

Chromaffin cells secrete catecholamine in response to acetylcholine stimulation released from splanchnic neurons. Various hormone and peptides co-released with catecholamine and acetylcholine have been shown to cause paracrine or autocrine regulation of stimulus–secretion coupling in chromaffin cells in normal conditions and during stress. In this meeting, several talks focused on this subject in three different sessions chaired by Youssef Anouar and Corey Smith and Kathrin Engisch. Several papers presented focused on the responses induced by pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide coreleased with acetylcholine from presynaptic terminals. Lee Eiden (USA) gave two talks: first, he reported a thorough microarray analysis of the transcriptomes induced by TNF and PACAP, respectively. Secondly, he reported the genes that were upregulated during restraint stress and hypoglycemia in adrenal medulla. Among them was galanin, a peptide implicated in the modulation of catecholamine release. The galanin transcript, as well as transcripts encoding catecholamine synthesizing enzymes (TH and PNMT) were upregulated by PACAP via a PKA-independent, ERK1/2dependent pathway, during stress. It appears that PACAP regulates catecholamine secretion at different levels. S.-A. Chan (USA), from Corey Smith’s group, reported that PACAP stimulates a phosphorylation-dependent recruitment of T-type Ca channels leading to an increase of catecholamine secretion. Eiden together with Smith have presented their work in a review on PACAP as a regulator of stimulus– secretion–synthesis coupling at the sympathoadrenal synapse during stress (below). Stephen Bunn (New Zealand) highlighted the importance of bidirectional signaling between the immune and neuroendocrine systems. He found that treatment of chromaffin cells with the cytokine IL-6 activated the ERK1/2 signal transduction pathway and induced significant changes in gene transcription. An extensive review of this work implicating the physiological significance of cytokine signaling on chromaffin cells during stress is presented by Bunn and Eiden (below). In addition to the classical model used for the study of the molecular mechanisms involved in stimulus-secretion coupling, chromaffin cells have also been used as a model for the study of cell survival and differentiation. Youssef Anouar (France) provided new evidence to support that PACAPregulated selenoprotien Texpression is involved in chromaffin cell survival during development. Vladimir Vukicevic (Germany) reported the isolation and characterization of the progenitors from adrenal medulla. He demonstrated that these cells can self-renew and differentiate into cells with characteristics of dopaminergic neurons. A review of the regulation of the progenitor state of chromaffin cells is presented below. Indeed, the presence of such progenitor/stem cells in the adrenal medulla is very exciting and raises the possibility of a potential source of dopaminergic neurons for treating neurodegenerative diseases such as Parkinson’s disease.

Up-Regulation of Cell Surface Insulin Receptor by Protein Kinase C-α in Adrenal Chromaffin Cells

Our previous study showed that treatment of cultured bovine adrenal chromaffin cells with phorbol 12, 13-dibutyrate (PDBu) or 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a rapid ( 15 h) translocation of both conventional (c) protein kinase C-α (PKC-α) and novel PKC-e (but not atypical PKC-ζ) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane association of only cPKC-α. In the present study, chronic (≥12 h) treatment of chromaffin cells with PDBu raised cell surface 125I-insulin binding without altering the KD value ; it developed in a concentration (EC50 = 1.9 nM)-and time (t1/2 = 14.6 h)-dependent manner, reaching its maximum 115% increase at 48 h. Either TPA (30 nM) or TMX (EC50 = 6.4 nM) also increased 125I-insulin binding by 97 or 88%, whereas the biologically inactive 4α-TPA had no effect. The increasing effect of PDBu (30 nM for 24 h) on 125I-insulin binding was significantly blocked, even when H7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treatment. Concurrent treatment with brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, abolished the PDBu-induced increment of 125I-insulin binding. Western blot analysis, using antibody against the β-subunit of the insulin receptor, showed that treatment with PDBu (30 nM) or TMX (EC50 = 2.3 nM) increased levels of insulin receptor precursor (~190 kDa ; t1/2 = 7.1 h) and insulin receptor β-subunit (t1/2 = 15.4 h), causing their almost maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by ~35% as soon as 3 h, producing its monophasic peak ~76% increases at 24 h. All of these increasing effects of PDBu and TMX on 125I-insulin binding and insulin receptor β-subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Go6976, a selective inhibitor of cPKC. These results suggest that long-term activation of cPKC-α up-regulates the density of the cell surface insulin receptor via transcriptional/translational events.

Catecholamine exocytosis is diminished in R6/2 Huntington’s disease model mice

In this work, the mechanisms responsible for dopamine (DA) release impairments observed previously in Huntington's disease model R6/2 mice were evaluated. Voltammetrically measured DA release evoked in striatal brain slices from 12-week old R6/2 mice by a single electrical stimulus pulse was only 19% of wild-type (WT) control mice. Iontophoresis experiments suggest that the concentration of released DA is not diluted by a larger striatal extracellular volume arising from brain atrophy, but, rather, that striatal dopaminergic terminals have a decreased capacity for DA release. This decreased capacity was not due to an altered requirement for extracellular Ca(2+), and, as in WT mice, the release in R6/2 mice required functioning vesicular transporters. Catecholamine secretion from individual vesicles was measured during exocytosis from adrenal chromaffin cells harvested from R6/2 and WT mice. While the number of exocytotic events was unchanged, the amounts released per vesicle were significantly diminished in R6/2 mice, indicating that vesicular catecholamines are present in decreased amounts. Treatment of chromaffin cells with 3-nitropropionic acid decreased the vesicular release amount from WT cells by 50%, mimicking the release observed from untreated R6/2 cells. Thus, catecholamine release from tissues isolated from R6/2 mice is diminished because of impaired vesicle loading.

Nitric oxide and peroxynitrite induce cellular death in bovine chromaffin cells: Evidence for a mixed necrotic and apoptotic mechanism with caspases activation

Treatment of chromaffin cells with nitric oxide (NO) donors (SNP and SNAP) and peroxynitrite produces a time- and dose-dependent necrotic and apoptotic cell death. Necrotic cell death was characterized by both an increase in lactate dehydrogenase and ATP release and changes in nuclei and cell morphology (as seen with fluorescence microscopy analysis with propidium iodide and Hoechst 33342). Apoptotic cell death was characterized by nuclear fragmentation and presence of apoptotic cell bodies, by a decrease in DNA content, and by an increase in DNA fragmentation. Treatment of chromaffin cells with lipopolysaccharide (LPS) or cytokines (interferon-gamma, tumor necrosis factor-alpha) resulted only in apoptotic cell death. Apoptotic effects of NO-inducing compounds were specifically reversed, depending on the stimuli, by the NO scavenger carboxy-PTIO (CPTio) or by the NOS inhibitors L-NMA and thiocitrulline. NO-induced apoptotic death in chromaffin cells was concomitant to a cell cycle arrest in G0G1 phase and a decrease in the number of chromaffin cells in the G2M and S phases of cell cycle. All NO-producing compounds were able to induce activation of caspase 3 and cytochrome c release, and specific inhibitors of caspase 3 and 9, such as Ac-DEVD-CHO (CPP32) and Ac-Z-LEHD-FMK, respectively, prevented NO-induced apoptosis in chromaffin cells. These results suggest that chromaffin cells could be good models for investigating the molecular basis of degeneration in diseases showing death of catecholaminergic neurons, phenomenon in which NO plays an important role.

Inhibition by Selenium Compounds of Catecholamine Secretion Due to Inhibition of Ca2+ Influx in Cultured Bovine Adrenal Chromaffin Cells

Selenium is an essential trace metal element, whereas large doses of selenium exert adverse effects to the human body. We examined the effects of selenium compounds, sodium selenite (Na2SeO3) and sodium selenate (Na2SeO4), on catecholamine secretion from cultured bovine adrenal chromaffin cells. Treatment of chromaffin cells with sodium selenite for 72, 48, and 24 h caused decreases in protein and catecholamine contents, in association with cell damage, at concentrations over 30, 300, and 300 microM, respectively. The cells treated with subtoxic conditions (<100 microM, 48 h) of sodium selenite were used for further experiments. Sodium selenite treatment for 48 h inhibited carbachol (CCh)-induced catecholamine secretion in a concentration-dependent and non-competitive manner, while it did not affect high K+- and veratridine-induced catecholamine secretion. Sodium selenite (100 microM) did not affect CCh- and veratridine-induced 22Na+ influx, while the compound inhibited 45Ca2+ influx induced only by CCh, but not high K+ and veratridine. Sodium selenate even at higher concentrations (1000 microM) did not affect any stimulus-induced catecholamine secretion and 45Ca2+ influx. Thus, sodium selenite may specifically exert adverse effects, such as inhibition of physiological stimulus-induced catecholamine secretion from adrenal chromaffin cells due to inhibition of Ca2+ influx.

Neuronal nitric oxide synthase modulates basal catecholamine secretion in bovine chromaffin cells.

The role of endogenously produced nitric oxide (NO) in the regulation of basal catecholamine (CA) secretion was studied in chromaffin cells. Treatment of chromaffin cells with nitric oxide synthase (NOS) inhibitors produced a dose-dependent increase in basal catecholamine secretion, which paralleled their ability to inhibit NOS activity. This inhibitory profile was similar to that found in neurons, suggesting the constitutive expression of neuronal NOS (nNOS) in these cells, which was confirmed by Western blot analysis. A study of the kinetics and pharmacology of nNOS activity expressed in chromaffin cells in culture indicated that NOS activity is calcium-dependent, increases with time, and is highly dependent on both intracellular concentrations of L-arginine (K(m) approximately 4 microM, V(max) = 908 +/- 60 pmol/hr x 10(6) cells) and transport of L-arginine into the cells (exhibiting two affinity constants of k(1) = 3.2 +/- 0.3 microM and k(2) = 126 +/- 5.5 microM). The effects of NOS inhibitors on CA secretion were mediated by the L-arginine-NO-cGMP pathway, insofar as exogenous L-arginine was able to partially block the increase in CA secretion evoked by them, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a specific inhibitor of guanylate cyclase, and zaprinast, an inhibitor of the cGMP phosphodiesterase, were able to increase and inhibit, respectively, basal CA secretion in a dose-dependent manner. These results suggest that chromaffin cells exhibit a tonic production of NO by nNOS that keeps the basal CA secretion at low levels, and this could be necessary for maintaining a normotensive state.

Lack of regulation of aromatic L-amino acid decarboxylase in intact bovine chromaffin cells.

Aromatic l-amino acid decarboxylase (AADC) is the second enzyme in the catecholamine biosynthetic pathway, and its activity is generally considered not to be limiting, and therefore not involved, in regulating flux through this pathway. Recent studies showing that its activity can be regulated in vivo and that the enzyme can be phosphorylated and activated in vitro have raised the possibility that AADC may play more than an obligatory role in catecholamine biosynthesis. In the present study, the phosphorylation and activity of AADC was evaluated relative to that of tyrosine hydroxylase (TH; the first and rate-limiting enzyme in the pathway) in intact bovine chromaffin cells. Treatment of chromaffin cells with elevated potassium, acetylcholine, phorbol dibutyrate, forskolin, or okadaic acid each increased 32P incorporation into TH (after metabolic labeling of ATP pools with 32P(i)) and TH activity. In contrast, as measured in matched samples, 32P incorporation into AADC was not detected and none of the treatments altered AADC activity. Thus, that AADC can be phosphorylated and activated in vitro has questionable physiological significance.

Beta-amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for beta-secretase activity in chromaffin vesicles.

A key factor in Alzheimer's disease (AD) is the beta-secretase activity that is required for the production of beta-amyloid (Abeta) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Abeta secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, beta-secretase activity in the regulated secretory pathway was examined for the production and secretion of Abeta in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Abeta(1-40) in APP-containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography. These vesicles also contain Abeta(1-42), measured by RIA. Significantly, regulated secretion of Abeta(1-40) from chromaffin cells represented the majority of secreted Abeta (> 95% of total secreted Abeta), compared with low levels of constitutively secreted Abeta(1-40). These results indicate the importance of Abeta production and secretion in the regulated secretory pathway as a major source of extracellular Abeta. Beta-secretase activity in isolated chromaffin vesicles was detected with the substrate Z-Val-Lys-Met-/MCA (methylcoumarinamide) that contains the beta-secretase cleavage site. Optimum beta-secretase activity in these vesicles required reducing conditions and acidic pH (pH 5-6), consistent with the in vivo intravesicular environment. Evidence for cysteine protease activity was shown by E64c inhibition of Z-Val-Lys-Met-MCA-cleaving activity, and E64c inhibition of Abeta(1-40) production in isolated chromaffin vesicles. Chromatography resolved the beta-secretase activity into two distinct proteolytic pathways consisting of: (i) direct cleavage of the beta-secretase site at Met-/Asp by two cysteine proteolytic activities represented by peaks Il-A and Il-B, and (ii) an aminopeptidase-dependent pathway represented by peak I cysteine protease activity that cleaves between Lys-/Met, followed by Met-aminopeptidase that would generate the beta-secretase cleavage site. Treatment of chromaffin cells in primary culture with the cysteine protease inhibitor E64d reduced the production of the beta-secretase product, a 12-14 kDa C-terminal APP fragment. In addition, BACE 1 and BACE 2 were detected in chromaffin vesicles; BACE 1 represented a small fraction of total beta-secretase activity in these vesicles. These results illustrate that multiple cysteine proteases, in combination with BACE 1, contribute to beta-secretase activity in the regulated secretory pathway. These results complement earlier findings for BACE 1 as beta3-secretase for Abeta production in the constitutive secretory pathway that provides basal secretion of Abeta into conditioned media. These findings suggest that drug inhibition of several proteases may be required for reducing Abeta levels as a potential therapeutic approach for AD.

Chromaffin Cell F-actin Disassembly and Potentiation of Catecholamine Release in Response to Protein Kinase C Activation by Phorbol Esters Is Mediated through Myristoylated Alanine-rich C Kinase Substrate Phosphorylation

The large majority of chromaffin vesicles are excluded from the plasma membrane by a cortical F-actin network. Treatment of chromaffin cells with phorbol 12-myristate 13-acetate produces disassembly of cortical F-actin, increasing the number of vesicles at release sites (Vitale, M. L., Seward, E. P., and Trifaró, J. M. (1995) Neuron 14, 353-363). Here, we provide evidence for involvement of myristoylated alanine-rich protein kinase C substrate (MARCKS), a protein kinase C substrate, in chromaffin cell secretion. MARCKS binds and cross-links F-actin, the latter is inhibited by protein kinase C-induced MARCKS phosphorylation. MARCKS was found in chromaffin cells by immunoblotting. MARCKS was also detected by immunoprecipitation. In intact or permeabilized cells MARCKS phosphorylation increased upon stimulation with 10(-7) m phorbol 12-myristate 13-acetate. This was accompanied by cortical F-actin disassembly and potentiation of secretion. MARCKS phosphorylation, cortical F-actin disassembly, and potentiation of Ca(2+)-evoked secretion were inhibited by a peptide (MARCKS phosphorylation site domain sequence (MPSD)) with amino acid sequence corresponding to MARCKS phosphorylation site. MPSD was phosphorylated in the process. A similar peptide (alanine-substituted phosphorylated site domain) with four serine residues of MPSD substituted by alanines was ineffective. These results provide the first evidence for MARCKS involvement in chromaffin cell secretion and suggest that regulation of cortical F-actin cross-linking might be involved in this process.

Up-regulation of cell surface insulin receptor by protein kinase C-alpha in adrenal chromaffin cells: involvement of transcriptional and translational events.

Our previous study showed that treatment of cultured bovine adrenal chromaffin cells with phorbol 12, 13-dibutyrate (PDBu) or 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a rapid (<15 min) and persistent (>15 h) translocation of both conventional (c) protein kinase C-alpha (PKC-alpha) and novel PKC-epsilon (but not atypical PKC-zeta) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane association of only cPKC-alpha. In the present study, chronic (>/=12 h) treatment of chromaffin cells with PDBu raised cell surface (125)I-insulin binding without altering the K(D) value; it developed in a concentration (EC(50) = 1.9 nM)-and time (t(1/2) = 14.6 h)-dependent manner, reaching its maximum 115% increase at 48 h. Either TPA (30 nM) or TMX (EC(50) = 6.4 nM) also increased (125)I-insulin binding by 97 or 88%, whereas the biologically inactive 4alpha-TPA had no effect. The increasing effect of PDBu (30 nM for 24 h) on (125)I-insulin binding was significantly blocked, even when H7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treatment. Concurrent treatment with brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, abolished the PDBu-induced increment of (125)I-insulin binding. Western blot analysis, using antibody against the beta-subunit of the insulin receptor, showed that treatment with PDBu (30 nM) or TMX (EC(50) = 2.3 nM) increased levels of insulin receptor precursor (approximately 190 kDa; t(1/2) = 7.1 h) and insulin receptor beta-subunit (t(1/2) = 15.4 h), causing their almost maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by approximately 35% as soon as 3 h, producing its monophasic peak approximately 76% increases at 24 h. All of these increasing effects of PDBu and TMX on (125)I-insulin binding and insulin receptor beta-subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Gö6976, a selective inhibitor of cPKC. These results suggest that long-term activation of cPKC-alpha up-regulates the density of the cell surface insulin receptor via transcriptional/translational events.

Neural regulation of phenylethanolamine N-methyltransferase (PNMT) gene expression in bovine chromaffin cells differs from other catecholamine enzyme genes.

Expression of the gene encoding the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) is regulated by hormonal and neural stimuli. Because the 5'-upstream regions of the PNMT do not contain sequences analogous to those demonstrated to convey neural regulation to the tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) catecholamine-synthesizing enzyme genes, functional and biochemical analyses have been utilized to characterize PNMT promoter responses to cholinergic and depolarizing agents. In primary cultures of bovine adrenal medullary chromaffin cells, reporter gene expression from transiently transfected 3- and 0.9-kb-containing PNMT promoter constructs is stimulated approximately twofold by nicotine and muscarine. Depolarizing concentrations of K+ produce fourfold increases in expression. These responses are not detected with constructs containing the proximal 0.3-kb promoter, indicating that the regions between -273 and -877 bp convey neural responsiveness for the PNMT gene in bovine chromaffin cells. Electrophoretic mobility shift assays (EMSAs) with oligonucleotides encoding these regions of the PNMT promoter revealed distinctions in migration of nuclear protein complexes formed following treatment of chromaffin cells with nicotine, muscarine, or 50 mM K+. Thus, the PNMT promoter between 0.3 and 0.9 kb contains sequences capable of responding to cholinergic and depolarization stimuli. Moreover, these treatments influence the interactions of specific nuclear proteins with this region of the PNMT promoter.

Neuropeptide Y Inhibits Chromaffin Cell Nicotinic Receptor-Stimulated Tyrosine Hydroxylase Activity through a Receptor-Linked G Protein-Mediated Process

Acetylcholine stimulation of bovine chromaffin cells results in increased norepinephrine and epinephrine secretion accompanied by a corresponding increase in synthesis. The addition of neuropeptide Y (NPY) to the culture medium prevents the increase in catecholamine synthesis but not secretion. Treatment of chromaffin cells with nicotine produces a concentration-dependent increase in tyrosine hydroxylase activity (IC50 = 1.2 microM) that is reduced if NPY is present during stimulation. Tyrosine hydroxylase activity decreases in a concentration-dependent fashion if increasing amounts of NPY are included in the culture medium, IC50 = 0.2 nM. Treatment with pertussis toxin completely prevents the effect of NPY. The rank order of potency for inhibition of tyrosine hydroxylase activity is NPY > or = [Leu31,Pro34]NPY > or = peptide YY > NPY2-36 > NPY13-36 > NPY18-36 > or = NPY26-36 >> NPY1-30, suggesting a NPY-Y1 receptor subtype. Examination of the effect of NPY on nicotine stimulation of chromaffin cell protein phosphorylation showed that NPY produces a concentration-dependent decrease in a 60-kDa protein, IC50 = 6.4 nM. The effect of NPY is pertussis toxin-sensitive. The rank order of potency is [Leu31,Pro34]NPY > or = NPY >> NPY18-36. Immunoprecipitation confirmed the identity of the 60-kDa protein as tyrosine hydroxylase.

Cyanide suppression of inwardly rectifying K+ channels in guinea pig chromaffin cells involves dephosphorylation.

Treatment of chromaffin cells with cyanide induced a gradual decrease in an inwardly rectifying K+ current (IIR), and washout of the mitochondrial inhibitor resulted in a rapid recovery of IIR. This diminution of IIR was reversed in a time-dependent manner by infusion of ATP or UTP, but not by that of GTP, ITP, or CTP. The restoration by ATP was not altered by addition to the pipette solution of 50 microM fluorescein 5-isothiocyanate, an inhibitor of various ATPases. A similar recovery of IIR occurred with injection of adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), but not of 5'-adenylylimidodiphosphate or alpha,beta-methyleneadenosine 5'-triphosphate. The ATP gamma S effect was biphasic, resulting in first a run-up of the current in ATP-depleted cells followed by a rundown of the current. This rundown was almost abolished by addition of guanosine 5'-O-(2-thiodiphosphate) to the ATP gamma S solution, suggesting the involvement of a G protein. Bath application of the protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine at 100 microM, but not N-(2-[methylamino]-ethyl)-5-isoquinolinesulfonamide, induced a reversible inhibition of IIR in the presence of pipette ATP, and the inhibition was diminished by 1 microM calyculin A, a phosphatase inhibitor. Bath application of 1 microM phorbol 12,13-dibutyrate did not affect IIR. It is concluded that cyanide suppresses inward rectifier K+ channel activity via dephosphorylation and that protein kinase C, adenosine 3',5'-cyclic monophosphate-dependent kinase, or guanosine 3',5'-cyclic monophosphate-dependent kinase is not involved in modulation of the channel.

Stimulation of “Prohormone Thiol Protease” (PTP) and [Met]Enkephalin by Forskolin.: BLOCKADE OF ELEVATED [Met]ENKEPHALIN BY A CYSTEINE PROTEASE INHIBITOR OF PTP

Proenkephalin and other prohormones require proteolytic processing at paired basic and monobasic residues for the biosynthesis of active neuropeptides. The novel "prohormone thiol protease" (PTP) has been proposed as a candidate proenkephalin processing enzyme for the production of [Met]enkephalin in chromaffin granules (Krieger, T. J., and Hook, V. Y. H. (1991) J. Biol. Chem. 266, 88376-8383). In this study, PTP was examined during elevation of cellular [Met]enkephalin by forskolin, a direct activator of adenylate cyclase that produces cAMP. Treatment of chromaffin cells with forskolin for 72 h increased enkephalin precursor cleaving activity (measured by following the conversion of the model substrate [35S-Met]preproenkephalin to trichloroacetic acid-soluble radioactivity) in isolated chromaffin granules by 170-180% over controls (100%). The increased activity was associated with the membrane fraction, rather than the soluble fraction, of chromaffin granules. The elevated activity was inhibited by E-64c, which is a potent inhibitor of PTP and cysteine proteases; however, the activity was not inhibited by serine or aspartic protease inhibitors. The elevated activity was identified as PTP based on immunoprecipitation by anti-PTP immunoglobulins. Stimulation of PTP synthesis was involved in the forskolin-induced increase in PTP activity, as demonstrated by a 10-fold increase in [35S]PTP pulse labeling in forskolin-treated chromaffin cells. Forskolin elevation of PTP protein levels within chromaffin granules was also detected in Western blots. Importantly, the forskolin-mediated rise in cellular [Met]enkephalin levels was completely blocked when cells were preincubated with the cysteine protease inhibitor Ep453, which is known to be converted by intracellular esterases to the more effective inhibitor E-64c (Buttle, D. J., Saklatvala, J., Tamai, M., and Barrett, A. J. (1992) Biochem. J. 281, 175-177). Both E-64c and Ep453 inhibit PTP, with E-64c being more potent (Azaryan, A. V., and Hook, V. Y. H. (1994b) Arch. Biochem. Biophys. 314, 171-177). These results demonstrate a role for PTP in proenkephalin processing in chromaffin cells and indicate that [Met] enkephalin formation and PTP are both regulated by cAMP.

Processing of proenkephalin in bovine chromaffin cells occurs in two phases

The processing of proenkephalin was studied in primary cultures of bovine adrenal medullary chromaffin cells by pulse-chase radiolabeling, immunopurification of proenkephalin and derivative peptides and quantitation following gel electrophoresis and Western blotting. Proenkephalin was processed with a t1/2 1 2 of approximately 1.1 h. Processing of proenkephalin-derived peptides of 15–25 kDa was essentially complete by 1 h. Treatment of chromaffin cells with brefeldin A to block the intracellular transport of proteins or with ammonium chloride to neutralize acidic intracellular compartments had only minor effects on the initial processing of proenkephalin. In contrast, both of these agents prevented a second, slower phase of proenkephalin processing. These studies suggest that proteolytic processing of proenkephalin in bovine adrenal medullary cells starts before transport to the trans-Golgi network and packaging into the chromaffin granules. A second phase of processing that requires an acidic environment occurs in or distal to the trans-Golgi network.

Pertussis toxin enhances proenkephalin synthesis in bovine chromaffin cells.

The synthesis of the neuropeptide precursor proenkephalin was measured in bovine adrenal chromaffin cells following radiolabeling with [35S]methionine. Treatment of chromaffin cells with pertussis toxin (100 ng/ml) approximately doubled proenkephalin synthesis without altering total protein synthesis. Pertussis toxin pretreatment also increased proenkephalin synthesis in chromaffin cells exposed to vasoactive intestinal peptide (VIP) and 3-isobutyl-1-methylxanthine (IBMX). Combinations of IBMX plus nicotine, VIP, or histamine also synergistically enhanced proenkephalin synthesis, with no further elevation when the cells were also pretreated with pertussis toxin. The action of forskolin, a direct activator of adenylate cyclase, on proenkephalin synthesis was similarly potentiated by pertussis toxin or IBMX, presumably reflecting the abilities of both the toxin and this phosphodiesterase inhibitor to enhance the cyclic AMP response to forskolin. In contrast, increased synthesis of proenkephalin in response to phorbol esters was not affected by pertussis toxin treatment. These results suggest that pertussis toxin potentiates proenkephalin synthesis primarily through inactivation of guanine nucleotide-binding proteins that inhibit adenylate cyclase, although other signaling pathways may also be involved.

Scinderin and chromaffin cell actin network dynamics during neurotransmitter release

It has been demonstrated that filamentous actin (F-A) is mainly localized in the cortical surface of the chromaffin cell. This F-A network acts as a barrier to the chromaffin granules impeding their contact with the plasma membrane. Stimulation of chromaffin cells with either nicotine or a depolarizing concentration of K + induces the disassembly of cortical F-A in focal areas underneath the plasma membrane. Sites of exocytosis are localised to these areas with low concentration of F-A. The cortical surface of the chromaffin cell also contains scinderin, a Ca 2+-dependent actin filament-severing protein recently isolated in our laboratory. Nicotine and high K + stimulation also induce redistribution of cortical scinderin. Both nicotine and high K +-induced scinderin redistribution and F-A disassembly are Ca 2+-dependent events which seem to precede neurotransmitter secretion. A possible target for protein kinase C in the modulation of secretion is the cortical F-A network. Treatment of chromaffin cells with phorbol esters prior to secretion induced scinderin redistribution, F-A disassembly and enhanced the initial rate of subsequent nicotine-evoked catecholamine release. The present results strongly indicate the involvement of the cortical cytoskeleton in the regulation of neurotransmitter release.

Thy-1 involvement in neurite outgrowth: Perturbation by antibodies, phospholipase C, and mutation

Thy-1 is a major cell surface protein anchored in the plasma membrane of neurons and lymphocytes by a covalent glycophosphotidylinositide linkage. Despite thorough characterization of the molecule's physicochemical properties, its biological function remains elusive. In this study we demonstrate that (i) monoclonal antibodies directed against Thy-1 are capable of enhancing neurite outgrowth from sympathetic neurons in culture, as well as stimulating the initiation of neurite sprouting from cultured adrenal chromaffin cells and PC12 cells. This effect is not observed with monovalent, Fab antibody fragments. Treatment with intact antibodies also results in the shedding of Thy-1 into the culture medium. (ii) Treatment of chromaffin cells with phosphotidylinositol-specific phospholipase C also results in an induction of neurite sprouting. The lipase effect can be blocked by preincubating the cells with monovalent anti-Thy-1 Fab fragments, indicating that the outgrowth stimulation is specifically due to removal of Thy-1. (iii) An entirely different approach to elucidating the function of Thy-1 involves mutagenesis of PC12 cells. Selection for Thy-1-deficient mutants revealed that cells lacking Thy-1 sprout neurites spontaneously at a very high frequency. A novel role for Thy-1 is proposed wherein the results of the mutant cell studies are compatible with the antibody and lipase data. Each of the perturbations can be viewed as releasing an inhibition that Thy-1 normally exerts on neurite outgrowth. We suggest that Thy-1 normally acts to stabilize neuronal membranes and processes, possibly through homophilic interactions.

Effects of pertussis toxin on the affinity of exocytosis for Ca 2+ in bovine adrenal chromaffin cells

Effects of pertussis toxin (islet-activating protein, IAP) on the secretory function of bovine adrenal chromaffin cells in culture were studied. Treatment of chromaffin cells with IAP resulted in an increase in both basal release of catecholamine and evoked-release by either acetylcholine (ACh) or high K +. In the dose-response curve for ACh-evoked release, IAP treatment produced an increase of the maximal response without affecting the half-maximal concentration of ACh. When the cells were permeabilized with digitonin after IAP-pretreatment, Ca 2+-dependent exocytosis was markedly increased where the affinity of exocytosis for Ca 2+ was augmented. These findings suggest that IAP-sensitive GTP-binding protein (or proteins) directory controls the Ca 2+-triggered process in the machinery of exocytosis by modulating the affinity for Ca 2+ of its unknown target.

Reserpine increases chromaffin cell enkephalin stores without a concomitant decrease in other proenkephalin-derived peptides.

Reserpine increases the levels of enkephalins in adrenal medullary chromaffin cells; however, the origin of the newly apparent pentapeptides has been the subject of debate, because no increase in the levels of proenkephalin mRNA has been observed. The present study was performed for determining if the reserpine-induced increase in context of enkephalins was derived from processing of preexisting fragments of proenkephalin. Bovine chromaffin cell enkephalins and larger enkephalin-containing peptides were separated by reversed-phase HPLC and identified by approximate molecular weight, elution with peptide standards, and enkephalin sequences contained. Treatment of the cells with reserpine increased the levels of enkephalins and of enkephalin-containing peptides of up to approximately 3 kilo-daltons without reducing the levels of larger enkephalin-containing peptides. Similar results were obtained with another catecholamine-depleting drug, tetrabenazine. In contrast, treatment of chromaffin cells with theophylline or forskolin increased the levels of both enkephalins and enkephalin-containing peptides of all sizes. The results suggest that new synthesis of proenkephalin is required for the effects of reserpine, although proenkephalin processing is also altered by this drug.