Brain serotonin determines maternal behavior and offspring survival.

Maternal care is an indispensable component of offspring survival and development in all mammals and necessary for reproductive success. Although brain areas regulating maternal behaviors are innervated by serotonergic afferents, very little is known about the role of this neurotransmitter in these behaviors. To evaluate the contribution of serotonin to maternal care, we used mice with a null mutation in the gene for tryptophan hydroxylase-2 (TPH2), which results in a genetic depletion of brain serotonin, and tested them in a wide range of maternal behavior paradigms. We found that litters born to and reared by TPH2(-/-) mothers showed decreased survival, lower weaning weights and increased cannibalization. In addition, TPH2(-/-) mothers performed poorly in pup retrieval, huddling, nest construction and high-arched back nursing. Aggression in TPH2(-/-) dams was not triggered by lactation and was steadily high. Survival and weaning weight deficits of TPH2(-/-) pups were rescued by cross-fostering and in litters of mixed genotype (TPH2(-/-) and TPH2(-/+) ). However, the maternal behaviors of TPH2(-/-) dams did not improve when rearing either TPH2(+/+) pups or mixed-genotype litters. In addition, TPH2(-/-) pups significantly worsened the behavior of TPH2(+/+) dams with respect to cannibalism, weaning weight and latency to attack. Olfactory and auditory functions of TPH2(-/-) females or anxiety-like behaviors did not account for these maternal alterations as they were equal to their TPH2(+/+) counterparts. These findings illustrate a profound influence of brain serotonin on virtually all elements of maternal behavior and establish that TPH2(-/-) pups can engender maladaptive mothering in dams of both genotypes.

The existence of a local 5-hydroxytryptaminergic system in peripheral arteries.

BACKGROUND AND PURPOSE: 5-HT is a vasoconstrictor exhibiting enhanced effects in systemic arteries from subjects with cardiovascular disease. The effect of endogenous 5-HT on arteries is controversial, because the concentration of free circulating 5-HT is low and a 5-hydroxytryptaminergic system has not been identified in peripheral arteries. We hypothesized that a local 5-hydroxytryptaminergic system (including 5-HT synthesis, metabolism, uptake and release) with physiological function exists in peripheral arteries. EXPERIMENTAL APPROACH: The presence of key components of a 5-hydroxytryptaminergic system in rat aorta and superior mesenteric artery was examined using western blot analyses, immunohistochemistry and immunocytochemistry. The function of the rate-limiting enzyme in 5-HT biosynthesis, tryptophan hydroxylase (TPH), and 5-HT transporter was tested by measuring enzyme activity and 5-HT uptake, respectively. Isometric contraction of arterial strips was used to demonstrate the function of released endogenous 5-HT in arterial tissues. KEY RESULTS: mRNA for TPH-1 was present in arteries, with low levels of TPH protein and TPH activity. Expression and function of MAO A (5-HT metabolizing enzyme) was supported by immunohistochemistry, western analyses and the elevation of concentrations of 5-hydroxyindoleacetic acid (5-HT metabolite) after exposure to exogenous 5-HT. The 5-HT transporter was localized to the plasma membrane of freshly isolated aortic smooth muscle cells. Peripheral arteries actively took up 5-HT in a time-dependent and 5-HT transporter-dependent manner. The 5-HT transporter substrate, (+)-fenfluramine, released endogenous 5-HT from peripheral arteries, which potentiated noradrenaline-induced arterial contraction. CONCLUSIONS AND IMPLICATIONS: This study revealed the existence of a local 5-hydroxytryptaminergic system in peripheral arteries.

Indoor mold, toxigenic fungi, and Stachybotrys chartarum: infectious disease perspective.

Damp buildings often have a moldy smell or obvious mold growth; some molds are human pathogens. This has caused concern regarding health effects of moldy indoor environments and has resulted in many studies of moisture- and mold-damaged buildings. Recently, there have been reports of severe illness as a result of indoor mold exposure, particularly due to Stachybotrys chartarum. While many authors describe a direct relationship between fungal contamination and illness, close examination of the literature reveals a confusing picture. Here, we review the evidence regarding indoor mold exposure and mycotoxicosis, with an emphasis on S. chartarum. We also examine possible end-organ effects, including pulmonary, immunologic, neurologic, and oncologic disorders. We discuss the Cleveland infant idiopathic pulmonary hemorrhage reports in detail, since they provided important impetus for concerns about Stachybotrys. Some valid concerns exist regarding the relationship between indoor mold exposure and human disease. Review of the literature reveals certain fungus-disease associations in humans, including ergotism (Claviceps species), alimentary toxic aleukia (Fusarium), and liver disease (Aspergillys). While many papers suggest a similar relationship between Stachybotrys and human disease, the studies nearly uniformly suffer from significant methodological flaws, making their findings inconclusive. As a result, we have not found well-substantiated supportive evidence of serious illness due to Stachybotrys exposure in the contemporary environment. To address issues of indoor mold-related illness, there is an urgent need for studies using objective markers of illness, relevant animal models, proper epidemiologic techniques, and examination of confounding factors.

Uses and limitations of the XTT assay in studies of Candida growth and metabolism.

Colorimetric tetrazolium assays are used increasingly in studies of fungi, often in the absence of standardization or correlation with other methods. We examined species- and strain-related tetrazolium metabolism in Candida albicans and Candida parapsilosis by using XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide] and WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphonyl)-2H-tetrazolium] and found marked variations. Also, significant signal was often missed in the absence of dimethyl sulfoxide extraction.

Voriconazole -- better chances for patients with invasive mycoses.

The past two decades have witnessed an increase in serious fungal infections, without corresponding growth in available antifungal agents. Voriconazole (VRC) is a novel triazole antifungal, recently approved in Europe for treatment of serious infections caused by Aspergillus, Fusarium, Scedosporium, and resistant Candida species. Voriconazole has in vitro activity against yeasts and yeast-like fungi similar, or superior to, fluconazole (FLC), itraconazole (ITC) and amphotericin B (AMB). Candida albicans is generally the most susceptible yeast (VRC MIC subset90 of 0.06 microg/ml); C. krusei often has low MICs even in the face of FLU/ITC resistance. Voriconazole has demonstrated comparable, or better, in vitro activity than ITC and AMB against Aspergillus (mean MICs 0.19-0.58 microg/ml), Ascomycetes, Bipolaris, Fusarium, Blastomyces dermatitidis, Coccidioides immitis, dermatophytes, Histoplasma capsulatum, Malassezia, and Scedosporium angiospermum (P. boydii). The drug possesses potent fungicidal activity against moulds including Aspergillus, Scedosporium, and Fusarium. Fungicidal activity is likely due to the high affinity of VRC for fungal 14-alpha-demethylase, a concept supported by ultrastructural and biochemical analysis. Animal studies confirmed the activity of VRC against infections including pulmonary and invasive aspergillosis (IA); A. fumigatus endocarditis; fusariosis; pulmonary cryptococcosis; and invasive candidiasis. Most importantly, well-designed human clinical trials have confirmed the efficacy of VRC in the treatment of candidal esophagitis, IA, and febrile neutropenia. Smaller studies and case reports have shown VRC is useful for salvage therapy of IA, cerebral aspergillosis, Scedosporium, and other fungal infections. Clinical testing has shown VRC is safe and well tolerated; the most common side effect is benign, self-limited visual disturbance.

Antifungal susceptibility of Candida biofilms: unique efficacy of amphotericin B lipid formulations and echinocandins.

Biofilms, likely the predominant mode of device-related microbial infection, exhibit resistance to antimicrobial agents. Evidence suggests that Candida biofilms have dramatically reduced susceptibility to antifungal drugs. We examined antifungal susceptibilities of Candida albicans and Candida parapsilosis biofilms grown on a bioprosthetic model. In addition to conventional agents, we determined if new antifungal agents (triazoles, amphotericin B lipid formulations, and echinocandins) have activities against Candida biofilms. We also explored effects of preincubation of C. albicans cells with subinhibitory concentrations (sub-MICs) of drugs to see if they could modify subsequent biofilm formation. Finally, we used confocal scanning laser microscopy (CSLM) to image planktonic- and biofilm-exposed blastospores to examine drug effects on cell structure. Candida biofilms were formed on silicone elastomer and quantified by tetrazolium and dry weight (DW) assays. Susceptibility testing of fluconazole, nystatin, chlorhexidine, terbenafine, amphotericin B (AMB), and the triazoles voriconazole (VRC) and ravuconazole revealed resistance in all Candida isolates examined when grown as biofilms, compared to planktonic forms. In contrast, lipid formulations of AMB (liposomal AMB and AMB lipid complex [ABLC]) and echinocandins (caspofungin [Casp] and micafungin) showed activity against Candida biofilms. Preincubation of C. albicans cells with sub-MIC levels of antifungals decreased the ability of cells to subsequently form biofilm (measured by DW; P < 0.0005). CSLM analysis of planktonic and biofilm-associated blastospores showed treatment with VRC, Casp, and ABLC resulted in morphological alterations, which differed with each agent. In conclusion, our data show that Candida biofilms show unique susceptibilities to echinocandins and AMB lipid formulations.

Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces.

Little is known about fungal biofilms, which may cause infection and antibiotic resistance. In this study, biofilm formation by different Candida species, particularly Candida albicans and C. parapsilosis, was evaluated by using a clinically relevant model of Candida biofilm on medical devices. Candida biofilms were allowed to form on silicone elastomer and were quantified by tetrazolium (XTT) and dry weight (DW) assays. Formed biofilm was visualized by using fluorescence microscopy and confocal scanning laser microscopy with Calcofluor White (Sigma Chemical Co., St. Louis, Mo.), concanavalin A-Alexafluor 488 (Molecular Probes, Eugene, Oreg.), and FUN-1 (Molecular Probes) dyes. Although minimal variations in biofilm production among invasive C. albicans isolates were seen, significant differences between invasive and noninvasive isolates (P < 0.001) were noted. C. albicans isolates produced more biofilm than C. parapsilosis, C. glabrata, and C. tropicalis isolates, as determined by DW assays (P was <0.001 for all comparisons) and microscopy. Interestingly, noninvasive isolates demonstrated a higher level of XTT activity than invasive isolates. On microscopy, C. albicans biofilms had a morphology different from that of other species, consisting of a basal blastospore layer with a dense overlying matrix composed of exopolysaccharides and hyphae. In contrast, C. parapsilosis biofilms had less volume than C. albicans biofilms and were comprised exclusively of clumped blastospores. Unlike planktonically grown cells, Candida biofilms rapidly (within 6 h) developed fluconazole resistance (MIC, >128 microg/ml). Importantly, XTT and FUN-1 activity showed biofilm cells to be metabolically active. In conclusion, our data show that C. albicans produces quantitatively larger and qualitatively more complex biofilms than other species, in particular, C. parapsilosis.

Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance.

Biofilms are a protected niche for microorganisms, where they are safe from antibiotic treatment and can create a source of persistent infection. Using two clinically relevant Candida albicans biofilm models formed on bioprosthetic materials, we demonstrated that biofilm formation proceeds through three distinct developmental phases. These growth phases transform adherent blastospores to well-defined cellular communities encased in a polysaccharide matrix. Fluorescence and confocal scanning laser microscopy revealed that C. albicans biofilms have a highly heterogeneous architecture composed of cellular and noncellular elements. In both models, antifungal resistance of biofilm-grown cells increased in conjunction with biofilm formation. The expression of agglutinin-like (ALS) genes, which encode a family of proteins implicated in adhesion to host surfaces, was differentially regulated between planktonic and biofilm-grown cells. The ability of C. albicans to form biofilms contrasts sharply with that of Saccharomyces cerevisiae, which adhered to bioprosthetic surfaces but failed to form a mature biofilm. The studies described here form the basis for investigations into the molecular mechanisms of Candida biofilm biology and antifungal resistance and provide the means to design novel therapies for biofilm-based infections.

Tetrahydrobiopterin enhances apoptotic PC12 cell death following withdrawal of trophic support.

(6R)-Tetrahydro-l-biopterin (BH(4)) is the rate-limiting cofactor in the production of catecholamine and indoleamine neurotransmitters and is also essential for the synthesis of nitric oxide by nitric-oxide synthase. We have previously reported that BH(4) administration induces PC12 cell proliferation and that nerve growth factor- or epidermal growth factor-induced PC12 cell proliferation requires the elevation of intracellular BH(4) levels. We show here that BH(4) accelerates apoptosis in undifferentiated PC12 cells deprived of serum and in differentiated neuron-like PC12 cells after nerve growth factor withdrawal. Increased production of catecholamines or nitric oxide cannot account for the enhancement of apoptosis by BH(4). Furthermore, increased calcium influx by exogenous BH(4) administration is not involved in the BH(4) proapoptotic effect. Our data also argue against the possibility that increased oxidative stress, due to BH(4) autoxidation, is responsible for the observed BH(4) effects. Instead, they are consistent with the hypothesis that BH(4) induces apoptosis by increasing cell cycle progression. Elevation of intracellular BH(4) during serum withdrawal increased c-Myc (and especially Myc S) expression earlier than serum withdrawal alone. Furthermore, N-acetylcysteine and the cyclin-dependent kinase inhibitor olomoucine ameliorated the BH(4) proapoptotic effect. These data suggest that BH(4) affects c-Myc expression and cell cycle-dependent events, possibly accounting for its effects on promoting cell cycle progression or apoptosis.

Molecular footprints of neurotoxic amphetamine action.

Methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy) are amphetamine analogs with high abuse potential. These drugs also cause damage to dopamine and serotonin nerve terminals in vivo. The mechanisms by which these drugs cause neurotoxicity are not known, but a great deal of attention has been focused on reactive oxygen species (ROS) and reactive nitrogen species (RNS) as mediators of this toxicity. ROS and RNS have very short biological half-lives in vivo, and it is virtually impossible to measure them in brain directly. However, ROS and RNS are also characterized by their extreme reactivity with proteins and nucleotides. Tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH), the initial and rate limiting enzymes in the synthesis of serotonin and dopamine, respectively, are identified targets for the actions of METH and MDMA. Using recombinant forms of these proteins, we have found that nitric oxide, catechol-quinones, and peroxynitrite, all of which are potentially produced by the neurotoxic amphetamines, covalently modify both TPH and TH. The ROS and RNS cause reductions in catalytic function of these enzymes in a manner that is consistent with the effects of METH and MDMNA in vivo. Protein-bound ROS or RNS may serve as molecular footprints of neurotoxic amphetamine action.

L-DOPA-quinone inactivates tryptophan hydroxylase and converts the enzyme to a redox-cycling quinoprotein.

Tryptophan hydroxylase, the initial and rate limiting enzyme in the biosynthesis of serotonin (5-HT), is inactivated by the quinone of L-DOPA. L-DOPA itself has no effect on enzyme activity. The inactivation of tryptophan hydroxylase could be prevented by glutathione (GSH), dithiothreitol, cysteine, and ascorbic acid but not by scavengers of hydrogen peroxide (catalase), hydroxyl radical (DMSO), or superoxide (superoxide dismutase). All cysteinyl residues within tryptophan hydroxylase are modified after treatment with L-DOPA-quinone as revealed by loss of DTNB-reactivity and formation of cysteinyl-DOPA residues. L-DOPA-quinone also converts tryptophan hydroxylase to a redox-cycling quinoprotein. These results suggest a possible mechanism of 5-HT neuronal damage in Parkinson's Disease by a redox-cycling quinoprotein.

Peroxynitrite inactivation of tyrosine hydroxylase: mediation by sulfhydryl oxidation, not tyrosine nitration.

Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme in the biosynthesis of dopamine (DA). TH activity is significantly diminished in Parkinson's disease (PD) and by the neurotoxic amphetamines, thereby accentuating the reductions in DA associated with these conditions. Reactive oxygen and nitrogen species have been implicated in the damage to DA neurons seen in PD and in reaction to amphetamine drugs of abuse, so we investigated the hypothesis that peroxynitrite (ONOO(-)) could interfere with TH catalytic function. ONOO(-) caused a concentration-dependent inactivation of TH. The inactivation was associated with tyrosine nitration (maximum of four tyrosine residues nitrated per TH monomer) and extensive sulfhydryl oxidation. Tetranitromethane, which causes sulfhydryl oxidation at pH 6 and 8 but which nitrates tyrosines only at pH 8, inactivated TH equally at either pH. Bicarbonate protected TH from ONOO(-)-induced inactivation and sulfhydryl oxidation but increased significantly tyrosine nitration. PNU-101033 blocked ONOO(-)-induced tyrosine nitration in TH but could not prevent enzyme inactivation or sulfhydryl oxidation. Together, these results indicate that the inactivation of TH by ONOO(-) is mediated by sulfhydryl oxidation. The coincident nitration of tyrosine residues appears to exert little influence over TH catalytic function.

Peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Coincident nitration of enzyme tyrosyl residues has minimal impact on catalytic activity.

Tryptophan hydroxylase, the initial and rate-limiting enzyme in serotonin biosynthesis, is inactivated by peroxynitrite in a concentration-dependent manner. This effect is prevented by molecules that react directly with peroxynitrite such as dithiothreitol, cysteine, glutathione, methionine, tryptophan, and uric acid but not by scavengers of superoxide (superoxide dismutase), hydroxyl radical (Me(2)SO, mannitol), and hydrogen peroxide (catalase). Assuming simple competition kinetics between peroxynitrite scavengers and the enzyme, a second-order rate constant of 3.4 x 10(4) M(-1) s(-1) at 25 degrees C and pH 7.4 was estimated. The peroxynitrite-induced loss of enzyme activity was accompanied by a concentration-dependent oxidation of protein sulfhydryl groups. Peroxynitrite-modified tryptophan hydroxylase was resistant to reduction by arsenite, borohydride, and dithiothreitol, suggesting that sulfhydryls were oxidized beyond sulfenic acid. Peroxynitrite also caused the nitration of tyrosyl residues in tryptophan hydroxylase, with a maximal modification of 3.8 tyrosines/monomer. Sodium bicarbonate protected tryptophan hydroxylase from peroxynitrite-induced inactivation and lessened the extent of sulfhydryl oxidation while causing a 2-fold increase in tyrosine nitration. Tetranitromethane, which oxidizes sulfhydryls at pH 6 or 8, but which nitrates tyrosyl residues at pH 8 only, inhibited tryptophan hydroxylase equally at either pH. Acetylation of tyrosyl residues with N-acetylimidazole did not alter tryptophan hydroxylase activity. These data suggest that peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Modification of tyrosyl residues by peroxynitrite plays a relatively minor role in the inhibition of tryptophan hydroxylase catalytic activity.

Tyrosine hydroxylase is inactivated by catechol-quinones and converted to a redox-cycling quinoprotein: possible relevance to Parkinson's disease.

Quinone derivatives of DOPA, dopamine, and N-acetyldopamine inactivate tyrosine hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the catecholamine neurotransmitters. The parent catechols are inert in this capacity. The effects of the catecholquinones on tyrosine hydroxylase are prevented by antioxidants and reducing reagents but not by scavengers of hydrogen peroxide, hydroxyl radicals, or superoxide radicals. Quinone modification of tyrosine hydroxylase modifies enzyme sulfhydryl groups and results in the formation of cysteinyl-catechols within the enzyme. Catecholquinones convert tyrosine hydroxylase to a redox-cycling quinoprotein. Quinotyrosine hydroxylase causes the reduction of the transition metals iron and copper and may therefore contribute to Fenton-like reactions and oxidative stress in neurons. The discovery that a phenotypic marker for catecholamine neurons can be converted into a redox-active species is highly relevant for neurodegenerative conditions such as Parkinson's disease.

Tryptophan hydroxylase regulation. Drug-induced modifications that alter serotonin neuronal function.

Tryptophan hydroxylase is the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin. A variety of drugs are known to diminish the function of this enzyme, and possibly cause damage to serotonin neurons. These include the substituted amphetamines methamphetamine and 3,4-methylenedioxy-methamphetamine, as well as L-DOPA, the most common therapy for Parkinsons Disease. In view of the important role for dopamine in the effects of these drugs on tryptophan hydroxylase and on serotonin neurons, we tested whether dopamine could alter the activity of this important enzyme. We found that dopamine-derived quinones, but not dopamine, inactivate tryptophan hydroxylase and convert the protein to a redox-cycling quinoprotein. This posttranslational modification of tryptophan hydroxylase could play a role in the drug-induced reduction in serotonin synthesis.

Dopamine inactivates tryptophan hydroxylase and forms a redox-cycling quinoprotein: possible endogenous toxin to serotonin neurons.

Exposure of tryptophan hydroxylase (TPH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, to dopamine under mild oxidizing conditions (iron + H2O2) or in the presence of tyrosinase results in a concentration-dependent inactivation of the enzyme. Dopamine, iron, H2O2, or tyrosinase alone does not alter TPH activity. Similarly, N-acetyldopamine oxidized with one equivalent of sodium periodate causes a concentration-dependent inactivation of TPH as well. TPH is protected from dopamine-induced inactivation by reduced glutathione, ascorbic acid, and dithiothreitol but not by the radical scavengers DMSO, mannitol, or superoxide dismutase. Parallel studies with [3H]dopamine reveal a high negative correlation between inhibition of catalysis and incorporation of tritium into the enzyme. Those reducing agents and antioxidants that protect TPH from inactivation are effective in preventing the labeling of TPH by [3H]dopamine. Acid hydrolysis and HPLC with electrochemical detection (HPLC-EC) analysis of inactivated TPH revealed the formation of cysteinyl-dopamine residues within the enzyme. Exposure of dopamine-modified TPH to redox-cycling staining after SDS-PAGE confirmed the formation of a quinoprotein. These results indicate that dopamine-quinones covalently modify cysteinyl residues in TPH, leading directly to the loss of catalytic activity, and establish that TPH could be a target for dopamine-quinones in vivo after drugs (e.g., neurotoxic amphetamines) that cause dopamine-dependent inactivation of TPH. Redox cycling of a TPH-quinoprotein could also participate in the serotonin neuronal toxicity caused by these same drugs.

Vasoactive intestinal peptide induces both tyrosine hydroxylase activity and tetrahydrobiopterin biosynthesis in PC12 cells.

Vasoactive intestinal peptide plays an important role in the trans-synaptic activation of tyrosine hydroxylase in sympathoadrenal tissues in response to physiological stress. Since tyrosine hydroxylase is thought to be subsaturated with its cofactor, tetrahydrobiopterin, we tested the hypothesis that up-regulation of tyrosine hydroxylase gene expression following vasoactive intestinal peptide treatment is accompanied by a concomitant elevation of intracellular tetrahydrobiopterin biosynthesis. We also investigated the second messenger systems involved in vasoactive intestinal peptide's effects on tetrahydrobiopterin metabolism. Our results demonstrate that treatment of PC12 cells for 24 h with vasoactive intestinal peptide induced intracellular tetrahydrobiopterin levels 3.5-fold. This increase was due to increased expression of the gene encoding GTP cyclohydrolase, the initial and rate-limiting enzyme in tetrahydrobiopterin biosynthesis, which was blocked by the transcriptional inhibitor, actinomycin D. Activation of tyrosine hydroxylase and GTP cyclohydrolase by vasoactive intestinal peptide was mediated by cyclic-AMP. Furthermore, stimulation of cyclic-AMP-mediated responses or protein kinase C activity induced the maximal in vitro activities of both tyrosine hydroxylase and GTP cyclohydrolase; the responses were additive when both treatments were combined. Induction of sphingolipid metabolism had no effect on the activation of tyrosine hydroxylase, while it induced GTP cyclohydrolase in a protein kinase C-independent manner. Our results support the hypothesis that intracellular tetrahydrobiopterin levels are tightly linked to tyrosine hydroxylation and that tetrahydrobiopterin bioavailability modulates catecholamine synthesis.

Tetrahydrobiopterin as a mediator of PC12 cell proliferation induced by EGF and NGF.

Epidermal growth factor and nerve growth factor increased the proliferation of rat phaeochromocytoma PC12 cells through obligatory elevation of intracellular (6R)-tetrahydrobiopterin (BH4). Epidermal growth factor and nerve growth factor increased intracellular BH4 by inducing GTP-cyclohydrolase, the rate-limiting enzyme in BH4 biosynthesis. Specific inhibitors of BH4 biosynthesis prevented growth factor-induced increases in BH4 levels and proliferation. The induction of GTP cyclohydrolase, BH4 and cellular proliferation by nerve growth factor was mediated by cAMP. Elevation of BH4 biosynthesis occurred downstream from cAMP in the cascade used by nerve growth factor to increase proliferation. Thus, intracellular BH4 is an essential mediator of the proliferative effects of epidermal growth factor and nerve growth factor in PC12 cells.

Molecular mechanism of the inactivation of tryptophan hydroxylase by nitric oxide: attack on critical sulfhydryls that spare the enzyme iron center.

Tryptophan hydroxylase (TPH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin (5-HT), is irreversibly inactivated by nitric oxide (NO). We have expressed brain TPH as a recombinant glutathione-S-transferase fusion protein and delineated the catalytic domain of the enzyme as the region spanning amino acids 99-444. Highly purified TPH catalytic core, like the native enzyme from brain, is inactivated by NO in a concentration-dependent manner. Removal of iron from TPH produces an apoenzyme with low activity that can be reconverted to its highly active holo-form by the addition of ferrous iron. Apo-TPH exposed to NO cannot be reactivated by iron. Treatment of holo-TPH (iron-loaded) with the disulfide 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) causes an inactivation of TPH that is readily reversed by dithiothreitol (DTT). DTNB-treated TPH [sulfhydryl (SH)-protected] exposed to NO is returned to full activity by thiol reduction with DTT. The inactivation of native TPH by NO cannot be reversed by either iron or DTT. These data indicate that NO inactivates TPH by selective action on critical SH groups (i.e., cysteine residues) while sparing catalytic iron sites within the enzyme. The results are interpreted with reference to the substituted amphetamines, which are neurotoxic to 5-HT neurons, that inactivate TPH in vivo and are now known to produce NO and other reactive oxygen species in vivo.