Immunocytochemical localization of aromaticl-amino acid decarboxylase and catechol-O-methyltransferase in blood vessel wall of the human dental pulp

Relatively large amounts of DOPA as compared with the concentration of norepinephrine are found in human dental pulp. AADC and COMT are localized in blood vessel walls of human dental pulp. This localization suggests a functional relationship between COMT and AADC with regard to the metabolism of DOPA.     

Microdialysis with radiometric monitoring of -[β-11C]DOPA to assess dopaminergic metabolism: effect of inhibitors of -amino acid decarboxylase,monoamine oxidase,and catechol-O-methyltransferase on rat striatal dialysate

The catecholamine, dopamine (DA), is synthesized from 3,4-dihydroxy--phenylalanine (-DOPA) by aromatic -amino acid decarboxylase (AADC). Dopamine metabolism is regulated by monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT). To measure dopaminergic metabolism, we used microdialysis with radiometric detection to monitor -[β-¹¹C]DOPA metabolites in the extracellular space of the rat striatum. We also evaluated the effects of AADC, MAO, and COMT inhibitors on metabolite profiles. The major early species measured after administration of -[β-¹¹C]DOPA were [¹¹C]3,4-dihydroxyphenylacetic acid ([¹¹C]DOPAC) and [¹¹C]homovanillic acid ([¹¹C]HVA) in a 1:1 ratio, which shifted toward [¹¹C]HVA with time. An AADC inhibitor increased the uptake of -[β-¹¹C]DOPA and -3-O-methyl-[¹¹C]DOPA and delayed the accumulation of [¹¹C]DOPAC and [¹¹C]HVA. The MAO and COMT inhibitors increased the production of [¹¹C]3-methoxytyramine and [¹¹C]DOPAC, respectively. These results reflect the -DOPA metabolic pathway, suggesting that this method may be useful for assessing dopaminergic metabolism.     

Use of catechol‐O‐methyltransferase inhibition to minimize L‐3,4‐dihydroxyphenylalanine‐induced dyskinesia in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐lesioned macaque

L‐3,4‐dihydroxyphenylalanine (L‐DOPA)‐induced dyskinesia is a complication of dopaminergic treatment in Parkinson's disease. Lowering the L‐DOPA dose reduces dyskinesia but also reduces the antiparkinsonian benefit. A therapy that could enhance the antiparkinsonian action of low‐dose L‐DOPA (LDl) without exacerbating dyskinesia would thus be of considerable therapeutic benefit. This study assessed whether catechol‐O‐methyltransferase (COMT) inhibition, as an add‐on to LDl, might be a means to achieve this goal. Cynomolgus macaques were administered 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine. Dyskinesia was established by chronic treatment with L‐DOPA. Two doses of L‐DOPA were identified – high‐dose L‐DOPA (LDh), which provided good antiparkinsonian benefit but was compromised by disabling dyskinesia, and LDl, which was sub‐threshold for providing significant antiparkinsonian benefit, without dyskinesia. LDh and LDl were administered in acute challenges in combination with vehicle and, for LDl, with the COMT inhibitor entacapone (5, 15 and 45 mg/kg). The duration of antiparkinsonian benefit (ON‐time), parkinsonism and dyskinesia were determined. The ON‐time after LDh was ～170 min and the ON‐time after LDl alone (～98 min) was not significantly different to vehicle (～37 min). In combination with LDl, entacapone significantly increased the ON‐time (5, 15 and 45 mg/kg being ～123, ～148 and ～180 min, respectively). The ON‐time after LDl/entacapone 45 mg/kg was not different to that after LDh. However, whereas the percentage ON‐time that was compromised by disabling dyskinesia was ～56% with LDh, it was only ～31% with LDl/entacapone 45 mg/kg. In addition to the well‐recognized action of COMT inhibition to reduce wearing‐OFF, the data presented suggest that COMT inhibition in combination with low doses of L‐DOPA has potential as a strategy to alleviate dyskinesia.     

The O-methylated derivative of -DOPA, 3-O-methyl- -DOPA, fails to inhibit neuronal and non-neuronal aromatic -amino acid decarboxylase

The present study examined whether the O-methylated derivative of -DOPA, 3-O-methyl- -DOPA (3-OM- -DOPA), inhibits neuronal (brain) and non-neuronal (liver and kidney) aromatic -amino acid decarboxylase (AADC) activity. The incubation of brain, liver and kidney homogenates with 3-OM- -DOPA (5 mM) did not result in the formation of 3-methoxytyramine, the compound expected to result from the decarboxylation of 3-OM- -DOPA. Incubation of tissue homogenates with -DOPA resulted in a concentration-dependent formation of dopamine, revealing K_m values (in mM) of similar magnitude for brain (0.8), liver (1.6) and kidney (1.0). Both benserazide and -5-hydroxytryptophan ( -5-HTP) were found to produce concentration dependent decreases in AADC activity with K_i values in the μM range. By contrast, 3-OM- -DOPA did not reduce the activity of either neuronal AADC (brain) or non-neuronal AADC (liver and kidney). The administration of benserazide in vivo (1, 3 and 10 mg/kg) produced marked reductions in AADC activity in both liver and kidney, but had no effect upon brain AADC. The effect of increasing the dose of benserazide up to 30 mg/kg (p.o.) was an almost complete inhibition (>95% reduction) in liver and kidney AADC activity accompanied by a marked decrease (49% reduction) in brain AADC activity. By contrast, the administration of 30 mg/kg (p.o.) 3-OM- -DOPA, which generates levels in brain, liver and kidney six-fold those in -DOPA-treated rats, was found to change neither neuronal nor non-neuronal AADC activity. In conclusion, 3-OM- -DOPA fails to interact with neuronal and non-neuronal AADC, either as substrate or inhibitor.     

Central action of benserazide after COMT inhibition demonstrated in vivo by PET

Summary Positron emission tomography (PET) following intravenous administration of -[¹¹ C]-L-DOPA provides a method of assessing regional cerebral uptake and utilization of levodopa. Cerebral levodopa kinetics in the rhesus monkey were investigated after the inhibition of catechol-O-methyltransferase (COMT) with RO 40-7592, and after coadministration of the peripheral aromatic L-amino acid decarboxylase (AADC) inhibitors benserazide and carbidopa. Pretreatment with RO 40-7592 (10 mg/kg), benserazide (10 mg/kg) or carbidopa (3.5 mg/kg) did not change striatal k₃, which mainly reflects the ability for the brain tissue to convert [¹¹C]-L-DOPA to [¹¹C]-dopamine, although the brain's uptake of radioactivity increased substantially after pretreatment with the AADC inhibitors. When benserazide was coadministered with RO 40-7592 (10 mg/kg) a dose-dependent decrease in striatal k₃ was measured with an apparent ED₅₀ of 3 mg/kg. No such effect was indicated after pretreatment with the combination of RO 40-7592 (10 mg/kg) and carbidopa (3.5 mg/kg). The possible negative interactions of coadministration with COMT inhibitors and predominantly peripherally acting AADC inhibitors must be considered when used in the therapy of Parkinson's disease.A contribution from the Uppsala University PET centre     

Effects of scoparone on dopamine biosynthesis and L‐DOPA‐induced cytotoxicity in PC12 cells

The effects of scoparone on dopamine biosynthesis and L‐DOPA‐induced cytotoxicity in PC12 cells were investigated. PC12 cells treated with scoparone at concentrations of 100–200 μM showed a 128–136% increase in dopamine levels over the course of 24 hr. Scoparone significantly increased the secretion of dopamine into the culture medium. Under the same conditions, the activities of tyrosine hydroxylase (TH) and aromatic L‐amino acid decarboxylase (AADC) were enhanced by treatment with 200 μM scoparone for 6–48 hr, but the activity of TH was regulated for a longer period than that of AADC. The intracellular levels of cyclic AMP and Ca²⁺ were increased by treatment with 200 μM scoparone. The levels of TH mRNA and the phosphorylation of cyclic AMP‐response element‐binding protein (CREB) were also significantly increased by treatment with 200 μM scoparone. In addition, scoparone at a concentration of 200 μM stimulated the activities of cyclic AMP‐dependent protein kinase (PKA), protein kinase C (PKC), and Ca²⁺/calmodulin kinase II (CaMK II). Finally, pretreatment with 200 μM scoparone reduced the cytotoxicity induced by L‐DOPA (20–100 μM) at 24 hr. These results suggest that scoparone enhances dopamine biosynthesis by regulating TH activity and TH gene expression, which is mediated by the PKA, CREB, PKC, and CaMK II pathways, and protects cells from L‐DOPA‐induced cytotoxicity by inducing cyclic AMP‐PKA systems in PC12 cells. © 2009 Wiley‐Liss, Inc.     

The discovery of the pressor effect of DOPS and its blunting by decarboxylase inhibitors

In the 1950s it was found that an artificial aminoacid, 3,4-threo-dihydroxyphenylserine (DOPS), was converted to norepinephrine (NE) in a single step by the enzyme L-aromatic amino acid decarboxylase (AADC), bypassing the need for the rate limiting enzyme dopamine beta hydroxylase. Trying to replicate the success of dihydroxyphenylalanine (DOPA) in the treatment of Parkinson disease, treatment with DOPS was attempted in patients with autonomic failure who have impaired NE release. DOPS improved orthostatic hypotension in patients with familial amyloid polyneuropathy, congenital deficiency of dopamine beta hydroxylase, pure autonomic failure and multiple system atrophy. DOPS pressor effect is due to its conversion to NE outside the central nervous system because concomitant administration of carbidopa, an inhibitor of AADC that does not cross the blood-brain barrier, blunted both the increase in plasma NE and the pressor response. DOPS pressor response is not dependent on intact sympathetic terminals because its conversion to NE also occurs in non-neuronal tissues.     

Immunocytochemical localization of serotonin, monoamine oxidase and assessment of monoamine oxidase activity in human dental pulp

In the present study serotonin (5-hydroxytryptamine, 5-HT) and monoamine oxidase (MAO) were both found localized in the blood vessel walls of human dental pulp. Our discovery of MAO activity in human dental pulp suggests a functional relationship between serotonin and MAO in this region.     

Dental pulp can be a good candidate for nerve grafting in a xeno-graft model

Dental pulp is discarded after extirpation of dental pulp and after tooth extraction. However, it contains nerve tissue abundantly and could be used more effectively. This study was designed to examine whether a dental pulp could be a candidate of donor for nerve grafting in xenografting model. The dental pulp was obtained from a human vital extracted tooth for orthodontic treatment, and treated with freezing and thawing method for reducing antigenicity. The treated sample was inserted into chitosan mesh tube for easy suturing, and then the complex was implanted into transected sciatic nerve in Sprague-Dawley (SD) rats (dental pulp group). As controls, chitosan tubes with and without sciatic nerve harvested from another SD rats were implanted (isograft group and tube group, respectively). As early as 4 weeks after grafting, regenerating axons accompanied by host Schwann cells were found to grout out through basal laminae by electron microscopy. The intact structure of basal laminae at this period suggested that they were derived from the original structure of donor graft. Twelve weeks after grafting, sporadic axonal regeneration was confirmed by light microscopy in the dental pulp group. Thirty-two weeks after implantation, aggregation of axons was observed in this group and matched that in isograft group. The average diameter of axons in dental pulp group was comparable to that in isograft group, whereas number of minifascicles and axon proportion were smaller. It was suggested that some delay occurred in dental pulp group because of the phagocytosis and absorption of tissue debris components remained after the freezing and thawing treatment. These findings clearly demonstrate that even dental pulp can act as conduits for regenerating axons.     

R(+)-8-OH-DPAT, a selective 5-HT1A receptor agonist, attenuated amphetamine-induced dopamine synthesis in rat striatum, but not nucleus accumbens or medial prefrontal cortex

R(+)-8-OH-DPAT (0.05, but not 0.025, 0.1, 1 mg/kg), a 5-HT_1A receptor agonist, decreased l-3,4-dihydroxyphenylalanine (DOPA) accumulation in rat striatum following NSD-1015, an l-aromatic amino acid decarboxylase inhibitor. Amphetamine (1 mg/kg) increased striatal DOPA accumulation, an effect attenuated by R(+)-8-OH-DPAT (0.05 mg/kg). However, both amphetamine (1 mg/kg) and R(+)-8-OH-DPAT (0.05 mg/kg) decreased cortical DOPA accumulation; there were no additional decreases from their combination. Neither amphetamine (1 mg/kg), R(+)-8-OH-DPAT (0.05 mg/kg), or the combination, significantly affected DOPA accumulation in the nucleus accumbens. The significance of and possible mechanisms for these findings are discussed.     

Short-photoperiod exposure reduces l-aromatic-amino-acid decarboxylase immunostaining in the arcuate nucleus and median eminence of male Syrian hamsters

In male hamsters, exposure to short photoperiod (SD) results in a decrease in median eminence and posterior pituitary dopamine (DA) concentrations. To determine if the SD-induced decline in DA is due to a decrease in the number of neurons synthesizing DA, immunocytochemistry was used to identify cells containing tyrosine hydroxylase (TH) and l-aromatic amino acid decarboxylase (AADC) in animals housed under long photoperiod (LD) or SD. Immunopositive cells were counted in the arcuate and caudal periventricular nuclei, regions where the DA neurons that project to the median eminence and posterior pituitary are located. AADC immunopositive (AADC + ) cells were also counted in the median eminence. Photoperiod did not affect the number of TH immunopositive (TH + ) or AADC + cells in the caudal periventricular nuclei. In the arcuate nuclei, SD exposure did not affect the number of TH + neurons, but produced a significant decline in the number of AADC + cells. The number of AADC + cells was also reduced in the median eminence of SD-exposed animals. This decline in AADC + cells may play a role in SD-induced changes in hypothalamic regulation of anterior pituitary hormone release, including the decline in median eminence DA concentrations.     

Glial cell line-derived neurotrophic factor (GDNF) from adult rat tooth serves a distinct population of large-sized trigeminal neurons

Glial cell line-derived neurotrophic factor (GDNF) mediates trophic effects for specific classes of sensory neurons. The adult tooth pulp is a well-defined target of sensory trigeminal innervation. Here we investigated potential roles of GDNF in the regulation of adult trigeminal neurons and the dental pulp nerve supply of the rat maxillary first molar. Western blot analysis and radioactive 35S-UTP in situ hybridization revealed that GDNF in the dental pulp and its mRNAs were localized with Ngf in the coronal pulp periphery, in particular in the highly innervated subodontoblast layer. Retrograde neuronal transport of iodinated GDNF and Fluorogold (FG) from the dental pulp indicated that GDNF was transported in about one third of all the trigeminal dental neurons. Of the GDNF-labelled neurons, nearly all (97%) were large-sized (> or =35 microm in diameter). Analysis of FG-labelled neurons revealed that, of the trigeminal neurons supporting the adult dental pulp, approximately 20% were small-sized, lacked isolectin B4 binding and did not transport GDNF. Of the large-sized dental trigeminal neurons approximately 40% transported GDNF. About 90% of the GDNF-accumulating neurons were negative for the high-temperature nociceptive marker VRL-1. Our results show that a subclass of large adult trigeminal neurons are potentially dependent on dental pulp-derived GDNF while small dental trigeminal neurons seems not to require GDNF. This suggests that GDNF may function as a neurotrophic factor for subsets of nerves in the tooth, which apparently mediate mechanosensitive stimuli. As in dorsal root ganglia both small- and large-sized neurons are known to be GDNF-dependent; these data provide molecular evidence that the sensory supply in the adult tooth differs, in some aspects, from the cutaneous sensory system.     

Sensorimotor gating of schizophrenia patients depends on Catechol O-methyltransferase Val158Met polymorphism

It has been recently shown that Catechol O-methyltransferase (COMT) Val(158)Met polymorphism strongly influences prepulse inhibition (PPI) of the acoustic startle response (ASR) in healthy human volunteers. Given that schizophrenia patients exhibit impairment in PPI and that COMT is a putative susceptibility gene for schizophrenia, we investigated the impact of the COMT Val(158)Met polymorphisms on PPI in schizophrenic inpatients. We analyzed COMT Val(158)Met polymorphisms and assessed startle reactivity, habituation, and PPI of ASR in 68 Caucasian schizophrenia inpatients. Clinical symptoms were measured with the Positive and Negative Syndrome Scale (PANSS). Patients carrying the Val(158)Met Met/Met allele showed elevated PPI levels whereas startle reactivity and habituation did not differ from the other two genotypes. These preliminary results imply that PPI is influenced by COMT Val(158)Met genotype in schizophrenia as well. In concert with other findings, our data suggest that PPI is a polygenic trait.     

Therapeutic effects of dental pulp stem cells on vascular dementia in rat models

Dental pulp stem cells are a type of adult stem cells with strong proliferative ability and multi-differentiation potential. There are no studies on treatment of vascular dementia with dental pulp stem cells. In the present study, rat models of vascular dementia were established by two-vessel occlusion, and 30 days later, rats were injected with 2 × 10⁷ dental pulp stem cells via the tail vein. At 70 days after vascular dementia induction, dental pulp stem cells had migrated to the brain tissue of rat vascular dementia models and differentiated into neuron-like cells. At the same time, doublecortin, neurofilament 200, and NeuN mRNA and protein expression levels in the brain tissue were increased, and glial fibrillary acidic protein mRNA and protein expression levels were decreased. Behavioral testing also revealed that dental pulp stem cell transplantation improved the cognitive function of rat vascular dementia models. These findings suggest that dental pulp stem cell transplantation is effective in treating vascular dementia possibly through a paracrine mechanism. The study was approved by the Animal Ethics Committee of Harbin Medical University (approval No. KY2017-132) in 2017.

Chinese Library Classification No. R456; R741; Q983+.8     

The central catechol-O-methyltransferase inhibitor tolcapone increases striatal hydroxyl radical production in l-DOPA/carbidopa treated rats

Summary. Inhibition of catechol catechol-O-methyltransferase (COMT) in the brains of subjects treated with l-DOPA (l-3,4-dihydroxylphenylalanine) and an aromatic amino acid decarboxylase (AADC) inhibitor is suggested to cause an increase of l-DOPA, which might lead to oxidative damage through enhanced formation of free radicals. To investigate this hypothesis, the acute effects of two doses of the systemically administered COMT inhibitors entacapone (peripheral) and tolcapone (peripheral and central) on the extracellular formation of hydroxyl radicals in vivo following treatment with l-DOPA and the AADC inhibitor carbidopa were examined. The formation of extracellular hydroxyl radicals were determined by the measurement of 2,3-dihydroxybenzoic acid (2,3-DHBA), a reaction product of hydroxyl radicals with sodium salicylate, using microdialysis in the striatum of anesthetised rats. The COMT inhibitors were administered together with 50 mg/kg i.p. carbidopa as 5% gum arabic suspensions intraperitoneally (i.p.) at doses of 0, 1.0, and 10 mg/kg body weight to a total of 36 male HAN-Wistars rats. l-DOPA was injected i.p. 40 min after drugs of interest. Microdialysis samples were collected every 20 min for 400 min at a perfusion rate of 1 μl/min. Systemically administered 10 mg/kg tolcapone, but not entacapone, induced an increase in hydroxyl radical formation in the striatum of anesthetised rats following treatment with l-DOPA/carbidopa. The increase in hydroxyl radical formation was reflected by higher extracellular concentrations of the hydroxylate product of salicylate, 2,3-DHBA, peaking at 192% of baseline at the end of the observation period. Similar results were also found using the AUC (area under the curve) value estimated for the observation period. We conclude that the increase in hydroxyl radical formation is likely to result from an increased rate of monoamine oxidase-mediated and non-enzymatic (autoxidation) dopamine metabolism following increased central availability caused by reduction in COMT-mediated metabolism. We cannot, however, exclude the possibility that hydroxyl radicals are produced by tolcapone as a result of uncoupling mitochondrial oxidative phosphorylation. Received June 30, 2000; accepted August 17, 2000     

Characterization of Non-Nitrocatechol Pan and Isoform Specific Catechol-O-methyltransferase Inhibitors and Substrates

Reduced dopamine neurotransmission in the prefrontal cortex has been implicated as causal for the negative symptoms and cognitive deficit associated with schizophrenia; thus, a compound which selectively enhances dopamine neurotransmission in the prefrontal cortex may have therapeutic potential. Inhibition of catechol-O-methyltransferase (COMT, EC 2.1.1.6) offers a unique advantage, since this enzyme is the primary mechanism for the elimination of dopamine in cortical areas. Since membrane bound COMT (MB-COMT) is the predominant isoform in human brain, a high throughput screen (HTS) to identify novel MB-COMT specific inhibitors was completed. Subsequent optimization led to the identification of novel, non-nitrocatechol COMT inhibitors, some of which interact specifically with MB-COMT. Compounds were characterized for in vitro efficacy versus human and rat MB and soluble (S)-COMT. Select compounds were administered to male Wistar rats, and ex vivo COMT activity, compound levels in plasma and cerebrospinal fluid (CSF), and CSF dopamine metabolite levels were determined as measures of preclinical efficacy. Finally, novel non-nitrocatechol COMT inhibitors displayed less potent uncoupling of the mitochondrial membrane potential (MMP) compared to tolcapone as well as nonhepatotoxic entacapone, thus mitigating the risk of hepatotoxicity.     

L-dopa therapy for Parkinson's disease: Past,present, and future

Dopamine (DA) supplementation therapy by L-dopa for Parkinson's disease (PD) was established around 1970. The dose of L-dopa can be reduced by the combined administration of inhibitors of peripheral L-amino acid decarboxylase (AADC), catechol O-methyltransferase (COMT), or monoamine oxidase B (MAO B). DA in the striatum may be produced from exogenously administered L-dopa by various AADC-containing cells, such as serotonin neurons. The long-term administration of L-dopa in PD patients may produce L-dopa–induced dyskinesia (LID), which may be due to chronic overstimulation of supersensitive DA D1 receptors. L-dopa may be used in combination with various new strategies such as gene therapy or transplantation in the future.