Effect of Prolonged Exposure to Short Daylight and a Tryptophan Hydroxylase Inhibitor on the Behavior and Brain Serotonin System in Danio rerio.

We studied the effect of reduced tryptophan hydroxylase (TPH) activity and short daylight exposure on the behavior and the 5-HT system of the brain in D. rerio. Male and female D. rerio were exposed for 30 days to standard (12:12 h light:dark) and short (4:20 h light:dark) photoperiods in the presence or absence of TPH inhibitor (p-chlorophenylalanine, pCPA, 5 mg/liter). On day 31, the fish behavior in the "novel tank diving" test, their sex and body weight were determined, and the levels of pCPA, 5-HT, and its metabolite 5-HIAA were measured by HPLC; the levels of the key genes encoding metabolism enzymes (Tph1a, Tph1b, Tph2, and Mao) and receptors of 5-HT (Htr1aa, Htr2aa) were assessed by real-time PCR with reverse transcription. The short daylight exposure caused masculinization of females, reduced body weight, and motor activity in the "novel tank diving" test, but did not affect the 5-HT system of the brain. Long-term pCPA treatment had no effect on sex and body weight, significantly reduced the 5-HIAA level, but increased Tph1a and Tph2 gene expression in the brain. No effects of the interaction of short daylight and pCPA exposure on the sex, body weight, behavior, and 5-HT system of the brain were found. Thus, a moderate decrease in TPH activity cannot potentiate the negative effects of short daylight exposure on the sex, body weight, behavior, and 5-HT system of D. rerio.

Efficacy of electroacupuncture stimulating Shenmen (HT7), Baihui (GV20), Sanyinjiao (SP6) on spatial learning and memory deficits in rats with insomnia induced by para-chlorophenylalanine: a single acupoint combined acupoints.

OBJECTIVE: To investiage the effect of electroacupuncture (EA) at a single acupoint of Shenmen (HT7), Baihui (GV20), Sanyinjiao (SP6) and at combined acupoints of Shenmen (HT7) and Baihui (GV20) and Sanyinjiao (SP6) on the PKA/CREB and BDNF/TrkB signaling, as well as neuroapoptosis and neurogenesis in hippocampus and elucidate the underlying mechanism of single and combined acupoints on ameliorating spatial learning and memory deficits in a rat model of primary insomnia. METHODS: Primary insomnia was modeled by intraperitoneal injection of para-chlorophenylalanine (PCPA) once daily for 2 d. EA was applied at Shenmen (HT7), Baihui (GV20), Sanyinjiao (SP6), or Shenmen (HT7) + Baihui (GV20) + Sanyinjiao (SP6) (combined) for 30 min daily for 4 d. Spatial learning and memory function was evaluated by the Morris water maze (MWM) test. Protein expressions of hippocampal cAMP-dependent protein kinase (PKA)-Cß, phosphorylated cAMP-responsive element-binding protein (p-CREB), brain-derived neurotrophic factor (BDNF), and tyrosine kinase receptor B (TrkB) were evaluated by Western blotting. Neuronal apoptosis in the hippocampus was detected with the transferase-mediated dUTP-X nick end labeling assay. Endogenous neurogenesis was examined with bromodeoxyuridine staining. The MWM test and hippocampal p-CREB, BDNF, and TrkB protein levels in the combined acupoints group were evaluated after the administration of a PKA-selective inhibitor (H89). RESULTS: Spatial learning and memory were significantly impaired in rats with insomnia. The spatial learning deficits were ameliorated in the Shenmen (HT7), Baihui (GV20), Sanyinjiao (SP6), and combined groups; this improvement was significantly greater in the combined group than the single acupoint groups. The spatial memory impairment was improved in the combined, Baihui (GV20), and Shenmen (HT7) groups, but not the Sanyinjiao (SP6) group. The expressions of PKA-Cß, p-CREB, BDNF, and TrkB were decreased in rats with insomnia. All these proteins were significantly upregulated in the combined group. PKA/p-CREB protein levels were elevated in the Baihui (GV20) and Shenmen (HT7) groups, whereas BDNF/TrkB expression was upregulated in the Sanyinjiao (SP6) group. The staining results showed significant attenuation of hippocampal cell apoptosis and increased numbers of proliferating cells in the combined group, whereas the single acupoint groups only showed decreased numbers of apoptotic cells. In the combined group, the PKA inhibitor reversed the improvement of spatial memory and upregulation of p-CREB expression caused by EA, but did not affect its activation of BDNF/TrkB signaling. CONCLUSIONS: EA at the single acupoints Baihui (GV20), Shenmen (HT7), or Sanyinjiao (SP6) had an ameliorating effect on the spatial learning and memory deficits induced by insomnia. EA at combined acupoints exerted a synergistic effect on the improvements in spatial learning and memory impairment in rats with insomnia by upregulating the hippocampal PKA/CREB and BDNF/TrkB signaling, facilitating neurogenesis, and inhibiting neuronal apoptosis. These findings indicate that EA at combined acupoints [(Baihui (GV20), Shenmen (HT7), and Sanyinjiao (SP6)] achieves a more pronounced regulation of hippocampal neuroplasticity than EA at single acupoints, which may partly explain the underlying mechanisms by which EA at combined acupoints exerts a better ameliorative effect on the cognitive dysfunction caused by insomnia.

DNA damage induced by phenylalanine and its analogue p-chlorophenylalanine in blood and brain of rats subjected to a model of hyperphenylalaninemia.

Phenylketonuria (PKU) is a disease caused by a deficiency of phenylalanine hydroxylase (PAH), resulting in an accumulation of phenylalanine (Phe) in the brain tissue, cerebrospinal fluid, and other tissues of PKU patients. Considering that high levels of Phe are associated with neurological dysfunction and that the mechanisms underlying the neurotoxicity in PKU remain poorly understood, the main objective of this study was to investigate the in vivo and in vitro effects of Phe on DNA damage, as determined by the alkaline comet assay. The results showed that, compared to control group, the levels of DNA migration were significantly greater after acute administration of Phe, p-chlorophenylalanine (p-Cl-Phe, an inhibitor of PAH), or a combination thereof in cerebral cortex and blood, indicating DNA damage. These treatments also provoked increase of carbonyl content. Additionally, when Phe or p-Cl-Phe was present in the incubation medium, we observed an increase in the frequency and index of DNA damage in the cerebral cortex and blood, without affecting lactate dehydrogenase (LDH) release. Our in vitro and in vivo findings indicate that DNA damage occurs in the cerebral cortex and blood of rats receiving Phe, suggesting that this mechanism could be, at least in part, responsible for the neurological dysfunction in PKU patients.

Visceral hypersensitivity induced by activation of transient receptor potential vanilloid type 1 is mediated through the serotonin pathway in rat colon.

This study aimed to clarify the relationship between TRPV1 activation-induced visceral pain and the serotonin pathway in the colon of rats. The effects of para-chlorophenylalanine (pCPA) on visceral pain threshold pressure were assessed in capsaicin -induced visceral pain of rats. The expression of TRPV1 in the colon was examined by immunohistochemistry and Western blot analysis, and TRPV1 excitability in dorsal root ganglion (DRG) neurons was examined by whole-cell patch-clamp recording in pCPA-treated rats. Calcineurin and Ca(2+)-calmodulin-dependent kinase II (CaMKII), the important proteins in maintaining TRPV1 function in the colon, were also tested by Western blot analysis and immunofluorescence staining. Results showed that pCPA significantly increased the capsaicin-induced visceral pain threshold by 2.3-fold, and the enhanced visceral pain threshold corresponded with decreased 5-HT content (58% depleted) and enterochromaffin cell number (80% reduced). The reduced excitability of TRPV1 in DRG neurons, instead of changed TRPV1 expression, is responsible for the enhanced visceral pain threshold in 5-HT-depleted rats, and the mechanism may be related to the decreased expression of pCaMKII. These results indicate that visceral hypersensitivity induced by TRPV1 activation is modulated through 5-HT pathways and the attenuated function of TRPV1 and decreased protein expression of pCaMKII may play an important role in capsaicin-induced TRPV1 desensitization under 5-HT-depleted condition. The important role of TRPV1 and 5-HT in generating and maintaining visceral hypersensitivity may provide insights for the treatment of visceral hypersensitivity.

Pharmacological blockage of serotonin biosynthesis and circadian changes in oxaliplatin toxicity in rats.

This study investigates if the serotoninergic system plays a role in chronotoxic effects of the anticancer agent oxaliplatin (l-OHP). Four groups of female rats (120 in total) synchronized with light-dark (12 h:12 h) were treated with: (i) saline, (ii) para-chlorophenylalanine (pCPA, an inhibitor of serotonin biosynthesis: 300 mg/kg/d, i.p. for two consecutive days), (iii) l-OHP (23 mg/kg, i.v.) at three different dosing times, or (iv) both pCPA and l-OHP. The results show pCPA (ii) obliterates the circadian rhythm in plasma ACTH but not in corticosterone or leukocytes, and (iii) l-OHP exerts circadian time-dependent toxic effects (body weight loss, leukopenia, and intestinal lesions) with greatest toxicity coinciding with treatment at the end of the nocturnal activity span (P < 0.05). In rats whose serotonin biosynthesis was blocked (iv), the circadian rhythms in the toxic effects of l-OHP and in ACTH were obliterated, while the rhythms in corticosterone and leukocytes persisted.

A mechanism for hydroxylation by tyrosine hydroxylase based on partitioning of substituted phenylalanines.

The iron-containing enzyme tyrosine hydroxylase catalyzes the hydroxylation of tyrosine to dihydroxyphenylalanine. A series of 4-X-substituted (X = H, F, Br, Cl, CH3, or CH3O) phenylalanines have been characterized as substrates to gain insight into the mechanism of hydroxylation. Multiple hydroxylated products were formed in most cases. As the size of the substituent at the 4-position increased, the site of hydroxylation switched from the 4- to the 3-position of the aromatic ring. The total amount of product formed with each amino acid showed a very good correlation with the sigma parameter of the substituent, with rho values of -4.3 +/- 0.7 or -5.6 +/- 0.8 when tetrahydrobiopterin or 6-methyltetrahydropterin, respectively, was used as cosubstrate. These values are consistent with a highly electron deficient transition state for hydroxylation. Oxygen addition at the 4-position resulted in either elimination of the substituent to form tyrosine or an NIH shift to form the respective 3-X-tyrosine. The relative amount of the product due to an NIH shift decreased in the order Br > CH3 > Cl >> F approximately CH3O approximately 0. A chemical mechanism for hydroxylation by tyrosine hydroxylase is presented to account for product formation from the various 4-substituted phenylalanines.

Relaxing the substrate specificity of an aminoacyl-tRNA synthetase allows in vitro and in vivo synthesis of proteins containing unnatural amino acids.

It has previously been demonstrated that the unnatural amino acid p-Cl-phenylalanine can be attached to tRNA(Phe) by a modified phenylalanyl-tRNA synthetase with relaxed amino acid substrate specificity. We show that this modification to the translational machinery of Escherichia coli is the only requirement for the incorporation of either p-Cl- or p-Br-phenylalanine into full-length luciferase in vitro. The incorporation of p-Cl-phenylalanine was also demonstrated in vivo using a suitably modified host strain. These results represent the first description of the incorporation into a protein in vivo of an unnatural amino acid which is normally rejected by the cellular translational machinery.

pKSS--a second-generation general purpose cloning vector for efficient positive selection of recombinant clones.

A new small plasmid vector (pKSS) for the direct selection of insert-containing plasmid clones is presented. The selection strategy is based on the acquired sensitivity of Escherichia coli cells to p-chloro-phenylalanine (p-Cl-Phe) if they carry a pheS allele encoding a phenylalanyl-tRNA synthetase alpha subunit with relaxed substrate specificity. This pheS allele is present on pKSS. Insertion into, or replacement of, the plasmidial pheS gene by a cloned fragment enables transformed pheS wild-type cells to survive on agar plates containing p-Cl-Phe plus ampicillin. This host strain-independent positive selection of recombinant clones proved to be highly efficient (> 99%) and did not require purification of the vector fragment prior to cloning. The high-copy-number vector pKSS offers a multitude of restriction sites and all of the features for analysis of cloned fragments that stem from the cloning vector pBluescript (Stratagene, La Jolla, CA, USA). Thus, pKSS represents a valuable alternative to previously reported positive-selection vectors; it should prove particularly useful for cloning when expecting a high fraction of cells transformed with non-recombinant vector, and for construction of DNA libraries.

5-Hydroxytryptamine in the phrenic nerve diaphragm: evidence for its existence and release.

Evidence is presented for the existence of 5-hydroxytryptamine (5-HT) within the phrenic nerve of the rat and its release following electrical stimulation. Contents of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in the phrenic nerve and the indoleamine released into the bathing fluid were estimated fluorimetrically after isolation on Sephadex G-10 and/or solvent-solvent extraction. Bioassays of 5-HT were done on rat fundus strip. The phrenic nerve and the end-plate zone contains high levels of 5-HT (1.9 micrograms/g wet weight) and 5-HIAA (1.5 micrograms/g wet weight). The resting release of around 1 ng 5-HT/diaphragm/min was enhanced by 50% (1.5 ng 5-HT/diaphragm/min) upon supramaximal (2-4 V) electrical stimulation of 5 Hz. Phrenic nerve diaphragm prepared from the denervated and p-chlorophenylalanine (300 mg/kg/day i.p. for 3 days) treated rats failed to release 5-HT confirming the neuronal origin and the identity of the indoleamine respectively. Furthermore, methysergide, an antagonist of 5-HT in rat fundus strip, blocked the response obtained by the sample on it. A modulatory role of 5-HT in the phrenic nerve diaphragm of the rat is envisaged from the present study.

Studies on the mechanisms by which clenbuterol, a beta-adrenoceptor agonist, enhances 5-HT-mediated behaviour and increases metabolism of 5-HT in the brain of the rat.

The head twitch response in mice produced by injection of 5-hydroxytryptophan (100 mg/kg i.p.) and carbidopa (25 mg/kg i.p.) was enhanced by administration of clenbuterol (0.5 mg/kg i.p.), a beta-adrenoceptor agonist. Clenbuterol also enhanced the hyperactivity syndrome in rats produced by quipazine (25 mg/kg i.p.), a 5-hydroxytryptamine (5-HT) agonist. This enhancement was not prevented by depletion of 5-HT in brain with p-chlorophenylalanine or after pretreatment with prazosin. The behavioural responses of the rats to administration of the alpha 2-adrenoceptor agonist, clonidine, was unaltered by acute or longer-term administration of clenbuterol. Following chronic administration of clenbuterol (5 mg/kg daily for 14 days), a procedure resulting in down-regulation of central beta-adrenoceptors, a larger dose of clenbuterol was necessary to enhance the quipazine-induced hyperactivity, suggesting that the mechanism of enhancement involved central post-synaptic beta-adrenoceptors. Further evidence for this conclusion was that a lesion of central noradrenaline pathways produced by 6-hydroxydopamine did not abolish the clenbuterol-induced enhancement of the quipazine-mediated behaviour. The binding characteristics of 5-HT2-receptors were unchanged by acute or chronic administration of clenbuterol. Clenbuterol (5 mg/kg) increased the percentage of plasma free (non-albumin bound) tryptophan, plasma free fatty acid concentration and the concentration of tryptophan and 5-hydroxyindoleacetic acid (5-HIAA) in the brain. The increase in 5-HT turnover in brain was prevented by pretreatment with the beta 1-adrenoceptor antagonist atenolol, which enters the brain poorly. It is therefore suggested that the clenbuterol-induced increase in 5-HT metabolism results from the increase in the concentration of plasma free fatty acid which increases plasma free tryptophan and thus increases the concentration of tryptophan in brain and 5-HT synthesis in brain. The clenbuterol-induced enhancement of 5-HT-mediated behaviour is therefore not associated with its effect on 5-HT metabolism. The data are discussed in relation to that obtained after administration of antidepressant drugs.

Placental transfer of aromatic amino acids in non-human primates.

Macaca fascicularis (crab-eating monkeys) underwent an operative procedure at 120 to 130 days of pregnancy that allowed fetal blood sampling. During subsequent experiments L-phenylalanine and p-chlorophenylalanine were injected into the maternal circulation. Blood obtained from mother and fetus revealed that phenylalanine is actively transported across the placenta and hence is markedly increased in the fetus if the maternal blood phenylalanine concentration is below the "saturation" level of 1.82 mM to 2.12 mM. The results of these studies which provide a better understanding of placental transport mechanisms of aromatic amino acids will be of assistance in future management of pregnant phenylketonuric females.

Placental transfer of phenylalanine in Macaca mulatta and Macaca fascicularis.

We investigated placental transport mechanisms of phenylalanine in Macaca mulatta and Macaca fascicularis. In the beginning of the third trimester we administered i.v. phenylalanine and p-chlorophenylalanine to pregnant animals. Initial higher phenylalanine concentrations were observed followed by a rapid decrease in both rhesus mothers and fetuses when compared with phenylalanine levels in fascicularis mothers and fetuses. In general, however, placental transfer mechanisms of phenylalanine did not differ significantly between the two species.

Development of a refractory stage in a dog model for phenylketonuria.

Dogs were fed a continuous diet of phenylalanine (Phe) daily and para-chloro-phenylalanine (p-CPhe), an inhibitor of phenylalanine hydroxylase (PH-ase) every second day. It was reported that such a diet produces a sustained hyperphenylalanemia in rats. We have found, however, that in dogs an initial rise in circulating Phe is followed, after a period of time, by a return to normal levels in spite of diet maintenance. PH-ase ws measured in liver samples obtained from dogs before the start of the above diet and at the time when Phe levels returned to normal. It was found that the post-feeding liver sample had 57%-100% less activity than the normal sample. Accordingly, decline in Phe levels cannot be attributed to an increase in PH-ase activity. Experiments are being initiated using labelled Phe, to investigate whether chronic feeding of Phe and p-CPhe resulted in an intestinal block to Phe absorption, which may explain our experimental findings.

Amino acid analogues: uptake, pool formation and incorporation of phenylalanine and two halogenated derivatives in cultured mammalian cells.

HeLa cells take up Phe and two of its ring halogenated derivatives (pFPhe and pClPhe) with rpaidity, concentrating them against the external medium both at 4 and 37 degrees C. The majority of amino acid (greater than 90%) is accumulated without energy expenditures at 4 degrees C, and can be quickly discharged by normal cell washing procedures in saline. At 37 degrees C the freely-diffusible (FDP) pool is accompanied by another which develops more slowly and cannot diffuse out freely during washings with saline but is extractable with trichloracetic acid (the slowly-diffusible pool, SDP, or more conventionally, the acid-soluble pool). Both of the analogues produced larger pools of the latter type than Phe itself from external concentrations ranging from 10(-5) to 10(-3) M. The incorporation of pFPhe into proteins over these same concentrations ranged from 30 to 90--95% of Phe incorporation, whereas pClPhe showed negligible incorporation. From these and similar analyses it can be concluded that amino acid pools form largely independently of protein synthesis, but bear a close relationship with the external amino acid concentration. The fraction of total uptake into cellular pools entering the SDP was relatively constant over a wide range of external concentrations. pFPhe incorporation into cellular proteins produced the same labelling distribution of Phe. It appears to ener all proteins, the vast majority of which have similar half-lives and turnover rates to Phe proteins. In competition, little or no interference was experienced between the analogue and Phe in uptake and pool formation until excessive amounts of one or the other were present (50--100x). By contrast, incorporation of pFPhe into protein was markedly reduced by the presence of Phe. However, the development of normal or large pools of pFPhe or Phe in cells prior to 3H-Phe incorporation did not affect the linear incorporation pattern of the radioisotope into protein. The relationship of pools to protein synthesis is discussed, and it is concluded that, although the SDP could contain potential precursor molecules for protein synthesis, it does not usually act as the direct supplier of amino acid for protein synthesis. Alternative explanations for precursor supply are discussed.

Impairment of olfactory perception in male rats by treatment with p-chlorophenylalanine and hydrocortisone.

p-Chlorophenylalanine treatment eliminated the normal male rat preference for the female preputial gland odor. Oral administration of hydrocortisone had a similar effect on dimethyl sulfite preference, which slowly reappeared after the discontinuance of the treatment. These changes of behavior are discussed in relation to possible serotonin alterations resulting from the treatments.

Exocrine pancreas under experimental conditions. I. Formation of paracrystalline inclusions in vivo.

After the application of parachlorophenylalanine (pCPA), an amino acid analogue, paracrystalline inclusions are observed in the exocrine pancreas of the rat. The formation of the paracrystalline structures varies according to the dose and the time of examination. Although the first alterations can be seen in the Golgi apparatus and the condensing vacuoles, the main localization of these structures is within the cisternae of the RER. At the same time as degenerative changes occur in the cells, involving autophagic and heterophagic processes, regeneration also takes place. With the freeze-fracturing method, the paracrystalline inclusions are interpreted as lamellae or plates of probably altered secretory proteins in extremely extended RER-cisternae. The fracture surfaces of the paracrystals show a periodicity of about 80 A running diagnonally to the main axis of the paracrystalline structures, which are mainly oriented from the basal parts of the exocrine pancreatic cells to the cell apices. The mechanism of paracrystalline formation is discussed on the basis of the morphologic results. It could be shown that after pCPA administration the amylase content is decreased concomittantly with degranulation. pCPA seems not to be incorporated into secretory proteins; high intracellular concentrations, however, are required to induce the formation of the paracrystalline structures. This morphological study is the basis for other studies dealing with secretion and intracellular transport in the pancreatic acinar cell under experimental conditions.