Phenylalanine as substrate for tyrosine hydroxylase in bovine adrenal chromaffin cells.
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ABSTRACT:
SUBMITTER: Fukami MH
PROVIDER: S-EPMC1131466 | biostudies-other | 1990 Jun
REPOSITORIES: biostudies-other
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Project description:EPHB6 is a member of the erythropoietin-producing hepatocellular kinase (EPH) family and a receptor tyrosine kinase with a dead kinase domain. It is involved in blood pressure regulation and adrenal gland catecholamine (CAT) secretion, but several facets of EPHB6-mediated CAT regulation are unclear. In this study, using biochemical, quantitative RT-PCR, immunoblotting, and gene microarray assays, we found that EPHB6 up-regulates CAT biosynthesis in adrenal gland chromaffin cells (AGCCs). We observed that epinephrine content is reduced in the AGCCs from male Ephb6-KO mice, caused by decreased expression of tyrosine hydroxylase, the rate-limiting enzyme in CAT biosynthesis. We demonstrate that the signaling pathway from EPHB6 to tyrosine hydroxylase expression in AGCCs involves Rac family small GTPase 1 (RAC1), MAP kinase kinase 7 (MKK7), c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, activator protein 1 (AP1), and early growth response 1 (EGR1). On the other hand, signaling via extracellular signal-regulated kinase (ERK1/2), p38 mitogen-activated protein kinase, and ELK1, ETS transcription factor (ELK1) was not affected by EPHB6 deletion. We further report that EPHB6's effect on AGCCs was via reverse signaling through ephrin B1 and that EPHB6 acted in concert with the nongenomic effect of testosterone to control CAT biosynthesis. Our findings elucidate the mechanisms by which EPHB6 modulates CAT biosynthesis and identify potential therapeutic targets for diseases, such as hypertension, caused by dysfunctional CAT biosynthesis.
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Project description:The erythropoietin-producing human hepatocellular receptor EPH receptor B6 (EPHB6) is a receptor tyrosine kinase that has been shown previously to control catecholamine synthesis in the adrenal gland chromaffin cells (AGCCs) in a testosterone-dependent fashion. EPHB6 also has a role in regulating blood pressure, but several facets of this regulation remain unclear. Using amperometry recordings, we now found that catecholamine secretion by AGCCs is compromised in the absence of EPHB6. AGCCs from male knockout (KO) mice displayed reduced cortical F-actin disassembly, accompanied by decreased catecholamine secretion through exocytosis. This phenotype was not observed in AGCCs from female KO mice, suggesting that testosterone, but not estrogen, contributes to this phenotype. Of note, reverse signaling from EPHB6 to ephrin B1 (EFNB1) and a 7-amino acid-long segment in the EFNB1 intracellular tail were essential for the regulation of catecholamine secretion. Further downstream, the Ras homolog family member A (RHOA) and FYN proto-oncogene Src family tyrosine kinase (FYN)-proto-oncogene c-ABL-microtubule-associated monooxygenase calponin and LIM domain containing 1 (MICAL-1) pathways mediated the signaling from EFNB1 to the defective F-actin disassembly. We discuss the implications of EPHB6's effect on catecholamine exocytosis and secretion for blood pressure regulation.
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Project description:Phenylalanine hydroxylase (PheH) is an iron(II)-dependent enzyme that catalyzes the hydroxylation of aromatic amino acid l-phenylalanine (L-Phe) to l-tyrosine (L-Tyr). The enzymatic modification has been demonstrated to be highly regiospecific, forming proteinogenic para-Tyr (p-Tyr) exclusively. Here we biochemically characterized the first example of a phenylalanine 3-hydroxylase (Phe3H) that catalyzes the synthesis of meta-Tyr (m-Tyr) from Phe. Subsequent mutagenesis studies revealed that two residues in the active site of Phe3H (Cys187 and Thr202) contribute to C-3 rather than C-4 hydroxylation of the phenyl ring. This work sets the stage for the mechanistic and structural study of regiospecific control of the substrate hydroxylation by PheH.
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Project description:We studied the participation of adrenal medulla (AM) chromaffin cells in hypercapnic chemotransduction. Using amperometric recordings, we measured catecholamine (CAT) secretion from cells in AM slices of neonatal and adult rats perfused with solutions bubbled with different concentrations of CO2. The secretory activity augmented from 1.74 +/- 0.19 pC/min at 5% CO2 to 6.36 +/- 0.77 pC/min at 10% CO2. This response to CO2 was dose dependent and appeared without changes in extracellular pH, although it was paralleled by a drop in intracellular pH. Responsiveness to hypercapnia was higher in neonatal than in adult slices. The secretory response to hypercapnia required extracellular Ca2+ influx. Both the CO2-induced internal pH drop and increase in CAT secretion were markedly diminished by methazolamide (2 microm), a membrane-permeant carbonic anhydrase (CA) inhibitor. We detected the presence of two CA isoforms (CAI and CAII) in neonatal AM slices by in situ hybridization and real-time PCR. The expression of these enzymes decreased in adult AM together with the disappearance of responsiveness to CO2. In patch-clamped chromaffin cells, hypercapnia elicited a depolarizing receptor potential, which led to action potential firing, extracellular Ca2+ influx, and CAT secretion. This receptor potential (inhibited by methazolamide) was primarily attributable to activation of a resting cationic conductance. In addition, voltage-gated K+ current amplitude was also decreased by high CO2. The CO2-sensing properties of chromaffin cells may be of physiologic relevance, particularly for the adaptation of neonates to extrauterine life, before complete maturation of peripheral and central chemoreceptors.
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Project description:Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes oxidation of phenylalanine to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH has a regulatory domain in which binding of the substrate leads to allosteric activation of the enzyme. However, the existence of PAH regulation in evolutionarily distant organisms, for example some bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum, a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site 15.7 Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 μM for phenylalanine. Under the same conditions, ITC revealed no detectable binding for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of amino acid residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) led to impaired binding, consistent with the presence of distal site binding in solution. Although kinetic analysis revealed that the distal site mutants suffer discernible loss of their catalytic activity, X-ray crystallographic analysis of Y155A and F258A, the two mutants with the most noticeable decrease in activity, revealed no discernible change in the structure of their active sites, suggesting that the effect of distal binding may result from protein dynamics in solution.
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Project description:The aromatic amino acid hydroxylases tyrosine hydroxylase (TyrH) and phenylalanine hydroxylase (PheH) have essentially identical active sites; however, PheH is nearly incapable of hydroxylating tyrosine, while TyrH can readily hydroxylate both tyrosine and phenylalanine. Previous studies have indicated that Asp425 of TyrH is important in determining the substrate specificity of that enzyme [Daubner, S. C., Melendez, J., and Fitzpatrick, P. F. (2000) Biochemistry 39, 9652-9661]. Alanine-scanning mutagenesis of amino acids 423-427, a mobile loop containing Asp425, shows that only mutagenesis of Asp425 alters the activity of the enzyme significantly. Saturation mutagenesis of Asp425 results in large (up to 10(4)) decreases in the V(max) and V(max)/K(tyr) values for tyrosine hydroxylation, but only small decreases or even increases in the V(max) and V(max)/K(phe) values for phenylalanine hydroxylation. The decrease in the tyrosine hydroxylation activity of the mutant proteins is due to an uncoupling of tetrahydropterin oxidation from amino acid hydroxylation with tyrosine as the amino acid substrate. In contrast, with the exception of the D425W mutant, the extent of coupling of tetrahydropterin oxidation and amino acid hydroxylation is unaffected or increases with phenylalanine as the amino acid substrate. The decrease in the V(max) value with tyrosine as the substrate shows a negative correlation with the hydrophobicity of the amino acid residue at position 425. The results are consistent with a critical role of Asp425 being to prevent a hydrophobic interaction that results in a restricted active site in which hydroxylation of tyrosine does not occur.
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Project description:The pro-inflammatory cytokines, tumor necrosis factor-?, and interleukin-1?/? modulate catecholamine secretion, and long-term gene regulation, in chromaffin cells of the adrenal medulla. Since interleukin-6 (IL6) also plays a key integrative role during inflammation, we have examined its ability to affect both tyrosine hydroxylase activity and adrenomedullary gene transcription in cultured bovine chromaffin cells. IL6 caused acute tyrosine/threonine phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), and serine/tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3). Consistent with ERK1/2 activation, IL6 rapidly increased tyrosine hydroxylase phosphorylation (serine-31) and activity, as well as up-regulated genes, encoding secreted proteins including galanin, vasoactive intestinal peptide, gastrin-releasing peptide, and parathyroid hormone-like hormone. The effects of IL6 on the entire bovine chromaffin cell transcriptome were compared to those generated by G-protein-coupled receptor (GPCR) agonists (histamine and pituitary adenylate cyclase-activating polypeptide) and the cytokine receptor agonists (interferon-? and tumor necrosis factor-?). Of 90 genes up-regulated by IL6, only 16 are known targets of IL6 in the immune system. Those remaining likely represent a combination of novel IL6/STAT3 targets, ERK1/2 targets and, potentially, IL6-dependent genes activated by IL6-induced transcription factors, such as hypoxia-inducible factor 1?. Notably, genes induced by IL6 include both neuroendocrine-specific genes activated by GPCR agonists, and transcripts also activated by the cytokines. These results suggest an integrative role for IL6 in the fine-tuning of the chromaffin cell response to a wide range of physiological and paraphysiological stressors, particularly when immune and endocrine stimuli converge.
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Project description:Nicotinic acetylcholine receptors (nAChRs) that contain ?6 and ?4 subunits have been demonstrated functionally in human adrenal chromaffin cells, rat dorsal root ganglion neurons, and on noradrenergic terminals in the hippocampus of adolescent mice. In human adrenal chromaffin cells, ?6?4* nAChRs (the asterisk denotes the possible presence of additional subunits) are the predominant subtype whereas in rodents, the predominant nAChR is the ?3?4* subtype. Here we present molecular and pharmacological evidence that chromaffin cells from monkey (Macaca mulatta) also express ?6?4* receptors. PCR was used to show the presence of transcripts for ?6 and ?4 subunits and pharmacological characterization was performed using patch-clamp electrophysiology in combination with ?-conotoxins that target the ?6?4* subtype. Acetylcholine-evoked currents were sensitive to inhibition by BuIA[T5A,P6O] and MII[H9A,L15A]; ?-conotoxins that inhibit ?6-containing nAChRs. Two additional agonists were used to probe for the expression of ?7 and ?2-containing nAChRs. Cells with currents evoked by acetylcholine were relatively unresponsive to the ?7-selctive agonist choline but responded to the agonist 5-I-A-85380. These studies provide further insights into the properties of natively expressed ?6?4* nAChRs.
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Project description:Adrenal glands are the major organs releasing catecholamines and regulating our stress response. The mechanisms balancing generation of adrenergic chromaffin cells and protecting against neuroblastoma tumors are still enigmatic. Here we revealed that serotonin (5HT) controls the numbers of chromaffin cells by acting upon their immediate progenitor "bridge" cells via 5-hydroxytryptamine receptor 3A (HTR3A), and the aggressive HTR3Ahigh human neuroblastoma cell lines reduce proliferation in response to HTR3A-specific agonists. In embryos (in vivo), the physiological increase of 5HT caused a prolongation of the cell cycle in "bridge" progenitors leading to a smaller chromaffin population and changing the balance of hormones and behavioral patterns in adulthood. These behavioral effects and smaller adrenals were mirrored in the progeny of pregnant female mice subjected to experimental stress, suggesting a maternal-fetal link that controls developmental adaptations. Finally, these results corresponded to a size-distribution of adrenals found in wild rodents with different coping strategies.
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Project description:To identify the Na+/Ca2+ exchanger expressed in bovine chromaffin cells, the ncx gene was cloned from a bovine chromaffin cell cDNA library. Five partial clones were obtained and their nucleotide sequences showed that there were at least three isoforms containing different intracellular loops. The 3'-untranslated region was the same in all the clones. To examine the Na+/Ca2+ exchange activity of the clones, full-length ncx1 genes were constructed by replacing the corresponding region of bovine cardiac ncx1 clone p17 with the different regions from two bovine chromaffin cell clones; these were designated p17c and p17h. p17h, but not p17c, showed Na+/Ca2+ exchange activity when expressed in Chinese hamster ovary cells and Xenopus oocytes. The expressed exchange activity of p17 was inhibited by 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP) but was not affected by PMA. However, the activity of p17h was inhibited by PMA but enhanced by 8-Br-cAMP. The agents that changed the activity of protein kinase C and cAMP-dependent protein kinase modulated the endogenous Na+/Ca2+ exchange current of chromaffin cells in a manner similar to that of p17h. Our results suggest that the p17h clone is the major isoform of the exchanger in chromaffin cells and is similar to the major ncx1 isoform in kidney. The exchange activity could be regulated by phosphorylation, and the variable region in the intracellular loop is important for the different effects of phosphorylation on the different isoforms.
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