Depolarization-stimulated catecholamine biosynthesis: involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ.

Depolarizing stimuli increase catecholamine (CA) biosynthesis, tyrosine hydroxylase (TH) activity, and TH phosphorylation at Ser19, Ser31, and Ser40 in a Ca(2+)-dependent manner. However, the identities of the protein kinases that phosphorylate TH under depolarizing conditions are not known. Furthermore, although increases in Ser31 or Ser40 phosphorylation increase TH activity in vitro, the relative influence of phosphorylation at these sites on CA biosynthesis under depolarizing conditions is not known. We investigated the participation of extracellular signal-regulated protein kinase (ERK) and cAMP-dependent protein kinase (PKA) in elevated K(+)-stimulated TH phosphorylation in PC12 cells using an ERK pathway inhibitor, PD98059, and PKA-deficient PC12 cells (A126-B1). In the same paradigm, we measured CA biosynthesis. TH phosphorylation stoichiometry (PS) was determined by quantitative blot-immunolabeling using site- and phosphorylation state-specific antibodies. Treatment with elevated K(+) (+ 58 mM) for 5 min increased TH PS at each site in a Ca(2+)-dependent manner. Pretreatment with PD98059 prevented elevated K(+)-stimulated increases in ERK phosphorylation and Ser31 PS. In A126-B1 cells, Ser40 PS was not significantly increased by forskolin, and elevated K(+)-stimulated Ser40 PS was three- to five-fold less than that in PC12 cells. In both cell lines, CA biosynthesis was increased 1.5-fold after treatment with elevated K(+) and was prevented by pretreatment with PD98059. These results suggest that ERK phosphorylates TH at Ser31 and that PKA phosphorylates TH at Ser40 under depolarizing conditions. They also suggest that the increases in CA biosynthesis under depolarizing conditions are associated with the ERK-mediated increases in Ser31 PS.

The transient nicotinic stimulation of tyrosine hydroxylase gene transcription in bovine adrenal chromaffin cells is independent of c-fos gene activation.

The nicotinic agonist dimethylphenylpiperazinium (DMPP) transiently stimulates tyrosine hydroxylase (TH) gene transcription in cultured bovine adrenal chromaffin cells (Craviso et al., J. Neurochem., 59 (1992) 2285-2296). The present studies examined the mechanism of this stimulation, exploring the hypothesis that c-fos- and/or cyclic AMP-related mechanisms are involved. As determined by nuclear run-on assay, exposure of chromaffin cells to DMPP (1 microM) induced c-fos and TH gene transcription fivefold and twofold, respectively. Nitrendipine (20 microM) blocked both responses, indicating a similar dependency of each on extracellular calcium. Both c-fos and TH gene transcription rates were also elevated by entry of calcium due to the presence of the calcium ionophore A23187 (5 microM). Comparison of the time dependence of the DMPP stimulation of c-fos and TH gene transcription revealed similar time courses. Both were rapid and transient, peaking within 10-30 min of nicotinic receptor occupancy and returning to control values by 1 h. This simultaneous activation of the TH and c-fos genes indicates that Fos induction cannot be responsible for the stimulation of TH gene transcription. This conclusion was further supported by a failure of anisomycin (100 microM) pretreatment of chromaffin cells, which blocked protein synthesis 99%, to have any effect on either the rapid stimulation of TH gene transcription or the length of time that the TH gene was activated by DMPP. Thus, neither Fos nor other high turnover-rate transcription factors appear to be responsible for the stimulation, or return to control level, of TH gene activity following nicotinic stimulation of chromaffin cells. In other experiments, treating chromaffin cells with a combination of maximally effective concentrations of DMPP and forskolin was found to produce no greater stimulation of TH gene transcription than either agent alone, suggesting that DMPP acts through the same mechanism as forskolin. Taken together, these results support the conclusion that the mechanism of TH gene activation in chromaffin cells by DMPP involves a cyclic AMP-dependent process and not the induction of transcription factors such as Fos.

Regulation of Raf-1 kinase activity by the 14-3-3 family of proteins.

We have identified the beta (beta) isoform of the 14-3-3 family of proteins as an activator of the Raf-1 protein kinase. 14-3-3 was isolated in a yeast two-hybrid screen for Raf-1 kinase domain binding proteins. Purified bovine brain 14-3-3 interacted specifically with both c-Raf-1 and the isolated Raf-1 kinase domain. Association was sensitive to the activation status of Raf-1; 14-3-3 bound to unactivated Raf-1, but not Raf-1 activated by protein kinase C alpha or Ras and Lck. The significance of these interactions under physiological conditions was demonstrated by co-immunoprecipitation of Raf-1 and 14-3-3 from extracts of quiescent, but not mitogen-stimulated, NIH 3T3 cells. 14-3-3 was not a preferred Raf-1 substrate in vitro and did not significantly affect Raf-1 kinase activity in a purified system. However, in cell-free extracts 14-3-3 acted as a Ras-independent activator of both c-Raf-1 and the Raf-1 kinase domain. The same results were obtained in vivo using transfection assays; 14-3-3 enhanced both c-Raf-1- and Raf-1 kinase domain-stimulated expression of AP-1- and NF-kappa B-dependent reporter genes and accelerated Raf-1 kinase domain-triggered differentiation of PC12 cells. We conclude that 14-3-3 is a latent co-activator bound to unactivated Raf-1 in quiescent cells and mediates mitogen-triggered but Ras-independent regulatory effects aimed directly at the kinase domain.

Activation of protein kinase C by purified bovine brain 14-3-3: comparison with tyrosine hydroxylase activation.

In the course of the purification of 14-3-3 protein (14-3-3) we found that 14-3-3 isolated from bovine forebrain activates protein kinase C (PKC), rather than the previously reported protein kinase C inhibitory activity (KCIP). We have characterized the 14-3-3 activation of PKC. The physical properties of purified PKC activator are the same as those previously reported for 14-3-3 and KCIP; i.e., (1) it is composed of subunits of molecular weight 32,000, 30,000, and 29,000; (2) it is homogeneous with respect to molecular weight, as judged by native gradient-gel electrophoresis, with a molecular weight of 53,000; and (3) it is composed of at least six isoforms when analyzed by reverse-phase HPLC. The concentration dependence of PKC activation by 14-3-3 is in the same range as that shown previously for KCIP inhibition of PKC, and as that required for 14-3-3 activation of tyrosine hydroxylase; a maximal stimulation of two- to three-fold occurs at 40-100 micrograms/ml. 14-3-3's activation of PKC is sensitive to alpha-chymotrypsin digestion but is not heat labile. Activation is specific to PKC; at least two other protein kinases, cyclic AMP- and calcium/calmodulin-dependent protein kinases, are not activated. The activation of PKC by 14-3-3 is independent of phosphatidylserine and calcium and, as such, is an alternative mechanism for the activation of PKC that obviates its translocation to membranes.

Nicotinic cholinergic regulation of tyrosine hydroxylase gene expression and catecholamine synthesis in isolated bovine adrenal chromaffin cells.

Isolated bovine adrenal chromaffin cells were used to study the nicotinic regulation of tyrosine hydroxylase (TH) gene expression. Continuous exposure of the cells to carbachol or the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) produces a time- and concentration-dependent increase in TH enzyme activity, whereas muscarine has no effect. DMPP at 1 microM (EC50 = 0.3 microM) elicits a two- to threefold elevation of both TH activity and TH immunoreactive protein level after 3-5 days in the presence of 2.5 mM calcium; the increase in enzyme levels is significantly less at lower extracellular calcium levels. The rate of hydroxylation of tyrosine to dopamine (DA) in intact cells, an index of endogenous TH activity, increases in parallel with the rise in TH levels. The TH mRNA level is elevated before the increase in protein levels. As determined by nuclear run-on assays, TH gene transcription is stimulated two- to threefold within 30 min of addition of 1 microM DMPP to the cells; transcription returns to basal levels by 2 h. Nitrendipine (20 microM) blocks the stimulation of transcription by DMPP. Pretreatment of the cells with cycloheximide (5 microM) does not prevent the DMPP stimulation of transcription. Forskolin (10 microM) also increases TH transcription (fourfold in 15 min) by a mechanism that is not blocked by cycloheximide. These results show that nicotinic receptor stimulation increases TH mRNA synthesis, TH protein levels, and TH activity in a calcium-dependent manner. Furthermore, the nicotinic influence on TH gene expression does not appear to require the synthesis of a protein factor for its effects. That in situ DA synthesis rates are elevated consequent to the rise in TH levels demonstrates that TH induction serves as a mechanism for enhancing the catecholamine-synthesizing capacity of the chromaffin cell on a long-term basis.

Vasoactive intestinal peptide stimulates catecholamine biosynthesis in isolated adrenal chromaffin cells: evidence for a cyclic AMP-dependent phosphorylation and activation of tyrosine hydroxylase.

Vasoactive intestinal peptide (VIP) increased catecholamine biosynthesis in bovine adrenal chromaffin cells by 50-200%. Six related peptides produced no effects. In addition, VIP increased tyrosine hydroxylase (TH) activity measured in gel-filtered supernatants prepared from homogenates of treated cells. The hypothesis that cyclic AMP is the second messenger involved in these effects of VIP was also evaluated. VIP led to an elevation of cyclic AMP levels, and this increase occurred over a similar concentration range and time course as the activation of TH and the increase in catecholamine biosynthesis. Each measure reached maximal levels at 10-20 microM VIP within 1 min and remained elevated for at least 16 min. These changes produced by VIP were paralleled by enhanced phosphorylation of TH, and this phosphorylation occurred on a single tryptic peptide that was the same peptide whose phosphorylation has been previously shown to be stimulated by forskolin. In contrast to VIP and forskolin, 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester known to activate protein kinase C, increased the phosphorylation on a total of three tryptic peptides of TH. Our results indicate that VIP stimulates catecholamine biosynthesis in chromaffin cells through the phosphorylation and activation of TH and support the conclusion that a cyclic AMP-dependent phosphorylation of TH is responsible for these effects.

Phosphoglyceride biosynthesis in bovine adrenal chromaffin cells.

Cultured adrenal chromaffin cells, representing a virtually homogeneous population of neuronal elements, have been utilized to examine the final enzymes in the formation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), namely, choline phosphotransferase, ethanolaminephosphotransferase, and the N-methyltransferases in the sequential methylation of PE to PC. Each enzyme has been characterized extensively in terms of substrate requirements, pH optima, detergent and cation effects, and response to inhibitors revealing properties very similar to those in other neural preparations. The respective activities are stable for up to two weeks of adrenal chromaffin cell culture suggesting that this system is a suitable model for examining the relative roles and the regulation of each pathway in PC formation.

In vitro phosphorylation of bovine adrenal chromaffin cell tyrosine hydroxylase by endogenous protein kinases.

Under phosphorylating conditions, addition of Ca2+ or cyclic AMP to the 100,000 g supernatant of purified bovine adrenal chromaffin cells increases both the incorporation of 32P into tyrosine hydroxylase and the activity of the enzyme. Combining maximally effective concentrations of each of these stimulating agents produces an additive increase in both the level of 32P incorporation into tyrosine hydroxylase and the degree of activation of the enzyme. The increased phosphorylation by Ca2+ is due to stimulation of endogenous Ca2+-dependent protein kinase activity and not inhibition of phosphoprotein phosphatases. When the chromaffin cell supernatant is subjected to diethylaminoethyl (DEAE) chromatography to remove calmodulin and phospholipids, tyrosine hydroxylase is no longer phosphorylated or activated by Ca2+; on the other hand, phosphorylation and activation of tyrosine hydroxylase by cyclic AMP are not affected. Subsequent replacement of either Ca2+ plus calmodulin or Ca2+ plus phosphatidylserine to the DEAE-fractionated cell supernatant restores the phosphorylation, but not activation of the enzyme. Reverse-phase HPLC peptide mapping of tryptic digests of tyrosine hydroxylase from the 100,000 g supernatant shows that the Ca2+-dependent phosphorylation occurs on three phosphopeptides, whereas the cyclic AMP-dependent phosphorylation occurs on one of these peptides. In the DEAE preparation, either cyclic AMP alone or Ca2+ in the presence of phosphatidylserine stimulates the phosphorylation of only a single phosphopeptide peak, the same peptide phosphorylated by cyclic AMP in the crude supernatant. In contrast, Ca2+ in the presence of calmodulin stimulates the phosphorylation of three peptides having reverse-phase HPLC retention times that are identical to peptides phosphorylated by Ca2+ addition to the crude unfractionated 100,000 g supernatant. Rechromatography of the peaks from each of the in vitro phosphorylations, either in combination with each other or in combination with each of the seven peaks generated from phosphorylation of tyrosine hydroxylase in situ, established that cyclic AMP, Ca2+/phosphatidylserine, and Ca2+/calmodulin all stimulate the phosphorylation of the same reverse-phase HPLC peptide: in situ peptide 6. Ca2+/calmodulin stimulates the phosphorylation of in situ peptides 3 and 5 as well. Thus, tyrosine hydroxylase can be phosphorylated in vitro by protein kinases endogenous to the chromaffin cell. Phosphorylation occurs on a maximum of three of the seven in situ phosphorylated sites, and all three of these sites can be phosphorylated by a Ca2+/calmodulin-dependent protein kinase.

Phosphorylation of bovine adrenal chromaffin cell tyrosine hydroxylase. Temporal correlation of acetylcholine's effect on site phosphorylation, enzyme activation, and catecholamine synthesis.

Tryptic peptide fragments of tyrosine hydroxylase isolated from 32PO4-prelabeled bovine adrenal chromaffin cells are resolved into seven phosphopeptides by reverse phase-high performance liquid chromatography. All seven of the peptides are phosphorylated on serine residues. Three of these putative phosphorylation sites, peptides 3, 5, and 6, are rapidly phosphorylated (5-fold in 15 s) by both acetylcholine stimulation and potassium depolarization of the cells, and this phosphorylation is accompanied by a similarly rapid activation of the enzyme. Both phosphorylation and activation are transient and do not account for the prolonged increase in catecholamine biosynthesis produced by these stimuli. Peptides 4 and 7 show a much slower and sustained increase in phosphorylation (3-fold in 4 min) in response to acetylcholine and potassium. Phosphorylation of these peptides correlates with the sustained increase in catecholamine biosynthesis rather than enzyme activation. Peptides 1 and 2 are not stimulated by any agonist yet employed and thus show no relation to enzyme activation or catecholamine biosynthesis. Phosphorylation of all five peptides by acetylcholine or potassium is calcium-dependent. In contrast to the stimulation of phosphorylation of tyrosine hydroxylase on multiple sites, forskolin stimulates the phosphorylation of only peptide 6, and this is accompanied by a coordinated activation of tyrosine hydroxylase and increased catecholamine biosynthesis. These findings show that the phosphorylation of tyrosine hydroxylase in intact cells is more complex than predicted from in vitro results, that at least two protein kinases are involved in the secretagogue-induced phosphorylation of tyrosine hydroxylase, and that the regulation of catecholamine biosynthesis, in response to phosphorylation, appears to involve both tyrosine hydroxylase activation and other mechanisms.

In situ phosphorylation of tyrosine hydroxylase in chromaffin cells: Localization to soluble compartments.

The present studies investigated the subcellular distribution of acetylcholine's effects upon the phosphorylation of tyrosine hydroxylase in isolated purified bovine adrenal chromaffin cells. After labeling the intact chromaffin cells with (32)P(i), over 90% of the [(32)P]tyrosine hydroxylase was found in soluble fractions. Stimulation of the cells with acetylcholine, the natural secretagogue of chromaffin cells, increased the phosphorylation of tyrosine hydroxylase and over 90% of the increase was associated with soluble tyrosine hydroxylase. Homogenates and subcellular fractions from chromaffin cells were also prepared and phosphorylated in vitro in an attempt to optimize detection of tyrosine hydroxylase phosphorylation. In chromaffin cell homogenates, both 8-bromo-cyclic AMP and calcium increased (32)P incorporation into tyrosine hydroxylase, and again over 90% of the increase was observed in soluble fractions. In the particulate fraction, phosphorylation of a band which comigrated with tyrosine hydroxylase in electrophoresis was occasionally detected but only with very long autoradiographic exposures. Tyrosine hydroxylase enzymatic activity in the isolated purified chromaffin cells was also found to be associated predominantly (approx 90%) with soluble fractions. In contrast, a large portion (40-50%) of the tyrosine hydroxylase activity from crude bovine adrenal medullae was associated with the particulate fraction. The data indicate that although tyrosine hydroxylase (and possibly kinases) can associate with particulate fractions when isolated from crude bovine adrenal medullae, the enzyme is predominantly soluble when isolated from the isolated cells. Further, the effects of acetylcholine on the isolated chromaffin cells are predominantly associated with this soluble tyrosine hydroxylase and its attendant kinases.

Regional analysis of neostriatal cholinergic and dopaminergic receptor binding and tyrosine hydroxylase activity as a function of aging.

Tyrosine hydroxylase (TH) activity, as well as dopaminergic and cholinergic muscarinic receptor binding were measured in discrete regions of the neostriatum of rats 7, 17, and 27 months old. Activity of TH was highest in the rostral neostriatum as compared to the caudal region. Age-related differences in TH were detected in only one region. The density of dopamine receptors was also highest in the rostral neostriatum of all age groups, but there were no significant age-related differences. The distribution of cholinergic muscarinic receptor binding was similar to that of TH and dopaminergic receptors, and showed age-related changes in discrete regions. These results, when considered with previous data, suggest that in selected striatal regions in the Sprague Dawley rat the cholinergic system is more vulnerable than the dopaminergic system to aging effects.

The localization of tyrosine hydroxylase-like immunoreactivity in the central nervous system: methodological considerations.

The ideal methodologies for the localization of tyrosine hydroxylase-like immunoreactivity were investigated using the quantitative capabilities of the Leitz MPV-3 microspectrofluorometer to determine the best protocol. The following method was found to give the best results. The animals were perfused with the two-stage procedure [1] consisting of an initial perfusion with 4% paraformaldehyde at pH 6.5 and a second perfusion of 4% paraformaldehyde at pH 11. The brains were sectioned on a cryostat and the loose sections were placed in the primary antibody for 80 hours (the time of saturation of the reaction). The primary antibody solution contained 0.1-0.5% lambda-carregeenan and 0.3% Triton-X 100 in phosphate buffered saline. The sections were rinsed and placed in a 0.1-0.3% carregeenan solution with 0.1% Triton-X 100 and containing the secondary antibody. The sections were mounted onto chrome alum subbed slides and allowed to dry. The sections were coverslipped with buffered glycerine (pH 8.6) containing 2 mg/ml paraphenylene diamine as a mounting medium. This medium provided excellent protection from fading but certain subsequent enzyme staining (notably acetylcholinesterase) required the use of buffered glycerine alone. Various counterstains were evaluated for their compatability with the FITC fluorescence. A detailed methodology is presented.

Bovine adrenal chromaffin cells: high-yield purification and viability in suspension culture.

A method for purifying chromaffin cells from adult, bovine, adrenal medullae and the techniques for maintaining the cells in suspension culture for at least 14 days are presented. Perfusion of medullae with a collagenase-containing medium produced a cell fraction that contained, in addition to chromaffin cells, a significant percentage of non-chromaffin cells. These cells were found to attach more rapidly than chromaffin cells to glass and tissue-culture plasticware. Using this property, we devised a selective plating procedure that yielded approximately 1-2 x 10(8) chromaffin cells per adrenal medulla at a purity of 95% or higher. On the basis of catecholamine levels and enzyme activities, suspension (as opposed to monolayer) cultures were chosen to further investigate their potential as a model system for the regulation of adrenergic function. In contrast to chromaffin cells cultured in monolayer, chromaffin cells in suspension had a more rounded appearance and formed multicellular aggregates with time in culture. Very few neurite-like structures, commonly observed in monolayer cultures, were present in the suspension cultures. Also, inhibitors of mitosis were not necessary to prevent overgrowth by non-chromaffin cells as there was little or no cell division in the suspension cultures. Catecholamine levels were relatively stable for at least 2 weeks, although a gradual decline in epinephrine occurred after day 5. Unlike other enzymes involved in catecholamine metabolism, phenylethanolamine N-methyl transferase activity declined significantly with time in culture in parallel to the gradual loss of epinephrine. In addition, both oxygen consumption and amino acid incorporation into proteins were relatively stable. Thus, the primary suspension cultures of adult, bovine chromaffin cells seem to offer several advantages for studying long-term regulation of chromaffin cell function and provide a stable source of adrenergic cells for examining short-term regulatory processes.

Multiple site phosphorylation of tyrosine hydroxylase. Differential regulation in situ by a 8-bromo-cAMP and acetylcholine.

Suspension cultures of purified bovine adrenal chromaffin cells incorporated 32P from exogenous 32Pi into a protein of approximately M4 = 60,000 (isolated by discontinuous, sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis). Phosphorylated tyrosine hydroxylase, purified from chromaffin cell supernatants by immunoprecipitation, co-migrated with the Mr = 60,000 band. Tryptic fragments prepared fom either the Mr congruent to 60,000 band or the immunoprecipitated tyrosine hydroxylase band were analyzed after separation with two-dimensional electrophoresis/chromatography. Two distinct 32P-peptides were present in either sample. After a 2-3-min lag period. 32P incorporation into both peptides was relatively linear with time for at least 20 min. In the presence of calcium, exogenous acetylcholine (100 microM) increased 32P incorporation into both of the 32P-labeled tryptic peptides whereas 8-bromo-cAMP (1 mM) increased 32P incorporation into only one of the two. Ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and MnCl2 inhibited the acetylcholine-induced phosphorylation of both tryptic peptides. Thus, tyrosine hydroxylase is phosphorylated in situ at more than one site, and the phosphorylation of these sites is affected differently by acetylcholine and 8-bromo-cAMP. The data imply that kinase activity other than (or in addition to) cAMP-dependent protein kinase activity attends tyrosine hydroxylase in the intact chromaffin cells and that multiple kinase activities may be involved in the short term regulation of catecholamine biosynthesis by afferent activity.

Activating antibodies to tyrosine hydroxylase.

Crude immunoglobulin fractions from a sheep, immunized with rat pheochromocytoma tyrosine hydroxylase, increased the catalytic activity of tyrosine hydroxylase in vitro up to 20-fold. Kinetic analysis of the activation revealed both a decrease in Km(app) for pterin cofactor and an increase in Vmax(app). No change Km(app) for tyrosine was observed. Activation was maximal at the minimum antibody/enzyme ratio required to remove tyrosine hydroxylase activity from solution with protein A immunoadsorbent. Chromatography of the crude immunoglobulins on either DEAE-Sephacel or protein A-Sepharose separated two immunoglobulin fractions. In both cases, the activating influence and the anti-tyrosine hydroxylase activity bound to the matrices and were eluted together. As supported by data from a solid phase radioimmunoassay using rabbit anti-bovine IgG1, this chromatographic data was consistent with the activating activity and the anti-tyrosine hydroxylase activity both being sheep IgG1 immunoglobulins. Preadsorption of the active immunoglobulin fraction from DEAE-Sephacel chromatography (with protein A immunoadsorbent precoated with rabbit anti-sheep IgG) eliminated the activating influence. Moreover, after incubation with the active fraction, tyrosine hydroxylase was bound to protein A-Sepharose and the activity recovered was severalfold higher than the initial activity. These data clearly demonstrate the involvement of antibody in the activation of the enzyme and suggest that the anti-tyrosine hydroxylase activity and the activating activity may derive from one and the same set of interactions.

Morphine induced increases in the incorporation of 3H-thymidine into brain striatal DNA.

3H-thymidine uptake into DNA fractions of rat brain regions was measured following in vivo administration of (methyl-3H)-thymidine and morphine. Acute morphine administration (10 mg/kg; 30 min prior to 3H-thymidine) increased incorporation of 3H-thymidine into DNA of rat striatum. This effect was antagonized by naloxone (1 mg/kg). Further, the observed change in incorporation of 3H-thymidine into DNA in striatum could not be accounted for by differences in the local availability of the label in morphinized rats. An autoradiographic study revealed that the 3H-thymidine was localized in nuclei in cells of the sub-ependymal layer lining the lateral ventricles, an area of glial cell proliferation in adult rats. No change in 3H-thymidine incorporation into DNA was observed in any area of the brain in morphine-addicted rats or in rats undergoing naloxone-precipitated withdrawal. The results indicate that opiates may induce permanent anatomical changes in the brain, including alterations of neuroglial interactions.