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Background Analysis of fluid metabolites has the potential to provide insight into the neuropathophysiology of injury in patients with traumatic brain injury (TBI). Objective Using a 1H nuclear magnetic resonance (NMR)-based quantitative metabolic profiling approach, this study determined (1) if urinary metabolites change during recovery in patients with mild to severe TBI; (2) whether changes in urinary metabolites correlate to injury severity; (3) whether biological pathway analysis reflects mechanisms that mediate neural damage/repair throughout TBI recovery. Methods Urine samples were collected within 7 days and at 6-months post-injury in male participants (n = 8) with mild-severe TBI. Samples were analyzed with NMR-based quantitative spectroscopy for metabolomic profiles and analyzed with multivariate statistical and machine learning-based analyses. Results Lower levels of homovanillate (R = −0.74, p ≤ 0.001), L-methionine (R = −0.78, p, Highlights • NMR-based metabolomics of urine can identify metabolic fingerprints associated with functional recovery following TBI. • Metabolic profiles in urine correlate to injury severity. • Biological pathway analysis reflects mechanisms that mediate neural damage and repair processes throughout recovery. • Metabolomics provides insight into the neuropathophysiology of injury in TBI patients.

Huntington&rsquo, s disease is a rare neurodegenerative disease caused by a cytosine&ndash, adenine&ndash, guanine (CAG) trinucleotide expansion in the Huntingtin (HTT) gene. Although Huntington&rsquo, s disease (HD) is well studied, the pathophysiological mechanisms, genes and metabolites involved in HD remain poorly understood. Systems bioinformatics can reveal synergistic relationships among different omics levels and enables the integration of biological data. It allows for the overall understanding of biological mechanisms, pathways, genes and metabolites involved in HD. The purpose of this study was to identify the differentially expressed genes (DEGs), pathways and metabolites as well as observe how these biological terms differ between the pre-symptomatic and symptomatic HD stages. A publicly available dataset from the Gene Expression Omnibus (GEO) was analyzed to obtain the DEGs for each HD stage, and gene co-expression networks were obtained for each HD stage. Network rewiring, highlights the nodes that change most their connectivity with their neighbors and infers their possible implication in the transition between different states. The CACNA1I gene was the mostly highly rewired node among pre-symptomatic and symptomatic HD network. Furthermore, we identified AF198444 to be common between the rewired genes and DEGs of symptomatic HD. CNTN6, DEK, LTN1, MST4, ZFYVE16, CEP135, DCAKD, MAP4K3, NUPL1 and RBM15 between the DEGs of pre-symptomatic and DEGs of symptomatic HD and CACNA1I, DNAJB14, EPS8L3, HSDL2, SNRPD3, SOX12, ACLY, ATF2, BAG5, ERBB4, FOCAD, GRAMD1C, LIN7C, MIR22, MTHFR, NABP1, NRG2, OTC, PRAMEF12, SLC30A10, STAG2 and Y16709 between the rewired genes and DEGs of pre-symptomatic HD. The proteins encoded by these genes are involved in various biological pathways such as phosphatidylinositol-4,5-bisphosphate 3-kinase activity, cAMP response element-binding protein binding, protein tyrosine kinase activity, voltage-gated calcium channel activity, ubiquitin protein ligase activity, adenosine triphosphate (ATP) binding, and protein serine/threonine kinase. Additionally, prominent molecular pathways for each HD stage were then obtained, and metabolites related to each pathway for both disease stages were identified. The transforming growth factor beta (TGF-&beta, ) signaling (pre-symptomatic and symptomatic stages of the disease), calcium (Ca2+) signaling (pre-symptomatic), dopaminergic synapse pathway (symptomatic HD patients) and Hippo signaling (pre-symptomatic) pathways were identified. The in silico metabolites we identified include Ca2+, inositol 1,4,5-trisphosphate, sphingosine 1-phosphate, dopamine, homovanillate and L-tyrosine. The genes, pathways and metabolites identified for each HD stage can provide a better understanding of the mechanisms that become altered in each disease stage. Our results can guide the development of therapies that may target the altered genes and metabolites of the perturbed pathways, leading to an improvement in clinical symptoms and hopefully a delay in the age of onset.

Chamaecyparis obtusa and Chamaecyparis pisifera are cypress species native to and widely distributed in Japan and South Korea that belong to the Cupressaceae family. Various pharmacological properties that have been identified in these species could be useful in the development of medicinal products, and the ability to differentiate these two species has become essential due to their different specific pharmacological properties. Metabolic analysis using 1H nuclear magnetic resonance spectroscopy coupled with multivariate statistical techniques was performed to elucidate the differences between C. obtusa and C. pisifera leaves. An optimized partial least-squares discriminant analysis (PLS-DA) model for their discrimination was obtained by selecting variables based on a cutoff for their importance in the projection value of 1.1. The following 24 compounds were profiled and compared between C. obtusa and C. pisifera leaves: 2-hydroxyisobutyrate, acetate, catechin, choline, glucose, betaine, malonate, 4-hydroxyphenylacetate, homovanillate, fructose, 5-hydroxyindoleacetate, glycine, sucrose, glycolate, tartrate, 3,4-dihydroxymandelate, uracil, myricetin, fumarate, 4-hydroxybenzoate, protocatechuate, cinnamate, hypoxanthine, and formate. The key compounds contributing to the discrimination of the two leaf types were found to be acetate, homovanillate, protocatechuate, sucrose, catechin, formate, 2-hydroxyisobutyrate, and hypoxanthine.

Background and objectives Causes of CKD differ in prognosis and treatment. Metabolomic indicators of CKD cause may provide clues regarding the different physiologic processes underlying CKD development and progression. Design, setting, participants & measurements Metabolites were quantified from serum samples of participants in the Modification of Diet in Renal Disease (MDRD) Study, a randomized controlled trial of dietary protein restriction and BP control, using untargeted reverse phase ultraperformance liquid chromatography tandem mass spectrometry quantification. Known, nondrug metabolites (n=687) were log-transformed and analyzed to discover associations with CKD cause (polycystic kidney disease, glomerular disease, and other cause). Discovery was performed in Study B, a substudy of MDRD with low GFR (n=166), and replication was performed in Study A, a substudy of MDRD with higher GFR (n=423). Results Overall in MDRD, average participant age was 51 years and 61% were men. In the discovery study (Study B), 29% of participants had polycystic kidney disease, 28% had glomerular disease, and 43% had CKD of another cause; in the replication study (Study A), the percentages were 28%, 24%, and 48%, respectively. In the discovery analysis, adjusted for demographics, randomization group, body mass index, hypertensive medications, measured GFR, log-transformed proteinuria, and estimated protein intake, seven metabolites (16-hydroxypalmitate, kynurenate, homovanillate sulfate, N2,N2-dimethylguanosine, hippurate, homocitrulline, and 1,5-anhydroglucitol) were associated with CKD cause after correction for multiple comparisons (P Conclusions Metabolomic profiling identified several metabolites strongly associated with cause of CKD.

This review outlines information about the Gram-negative, aerobic bacterium Variovorax paradoxus. The genomes of these species have G+C contents of 66.5-69.4 mol%, and the cells form yellow colonies. Some strains of V. paradoxus are facultative lithoautotrophic, others are chemoorganotrophic. Many of them are associated with important catabolic processes including the degradation of toxic and/or complex chemical compounds. The degradation pathways or other skills related to the following compounds, respectively, are described in this review: sulfolane, 3-sulfolene, 2-mercaptosuccinic acid, 3,3'-thiodipropionic acid, aromatic sulfonates, alkanesulfonates, amino acids and other sulfur sources, polychlorinated biphenyls, dimethyl terephthalate, linuron, 2,4-dinitrotoluene, homovanillate, veratraldehyde, 2,4-dichlorophenoxyacetic acid, anthracene, poly(3-hydroxybutyrate), chitin, cellulose, humic acids, metal-EDTA complexes, yttrium, rare earth elements, As(III), trichloroethylene, capsaicin, 3-nitrotyrosine, acyl-homoserine lactones, 1-aminocyclopropane-1-carboxylate, methyl tert-butyl ether, geosmin, and 2-methylisoborneol. Strains of V. paradoxus are also engaged in mutually beneficial interactions with other plant and bacterial species in various ecosystems. This species comprises probably promising strains for bioremediation and other biotechnical applications. Lately, the complete genomes of strains S110 and EPS have been sequenced for further investigations.

Objective:To determine whether a Parkinson disease (PD)-specific biochemical signature might be found in the total body metabolic milieu or in the CSF compartment, especially since this disorder has systemic manifestations beyond the progressive loss of dopaminergic nigrostriatal neurons.Methods:Our goal was to discover biomarkers of PD progression. Using ultra-high-performance liquid chromatography linked to gas chromatography and tandem mass spectrometry, we measured concentrations of small-molecule (≤1.5 kDa) constituents of plasma and CSF from 49 unmedicated, mildly affected patients with PD (mean age 61.4 years; mean duration of PD 11.4 months). Specimens were collected twice (baseline and final) at intervals up to 24 months. During this time, mean Unified Parkinson’s Disease Rating Scale (UPDRS) parts 2 + 3 scores increased 47% (from 28.8 to 42.2). Measured compounds underwent unbiased univariate and multivariate analyses, including fitting data into multiple linear regression with variable selection using least absolute shrinkage and selection operator (LASSO).Results:Of 575 identified plasma and 383 CSF biochemicals, LASSO led to selection of 15 baseline plasma constituents with high positive correlation (0.87, p = 2.2e−16) to baseline-to-final change in UPDRS parts 2 + 3 scores. Three of the compounds had xanthine structures, and 4 were either medium- or long-chain fatty acids. For the 15 LASSO-selected biomarkers, pathway enrichment software found no overrepresentation among metabolic pathways. CSF concentrations of the dopamine metabolite homovanillate showed little change between baseline and final collections and minimal correlation with worsening UPDRS parts 2 + 3 scores (0.29, p = 0.041).Conclusions:Metabolomic profiling of plasma yielded strong prediction of PD progression and offered biomarkers that may provide new insights into PD pathogenesis.

Acute kidney injury (AKI) staging has been developed in the adult and pediatric populations, but these do not yet exist for the neonatal population. Metabolomics was utilized to uncover biomarkers of normal and AKI-associated renal function in preterm infants. The study comprised 20 preterm infants with an AKI diagnosis who were matched by gestational age and gender to 20 infants without an AKI diagnosis. Urine samples from pre-term newborn infants collected on day 2 of life were analyzed using broad-spectrum nuclear magnetic resonance (NMR) metabolomics. Multivariate analysis methods were used to identify metabolite profiles that differentiated AKI and no AKI, and to identify a metabolomics profile correlating with gestational age in infants with and without AKI. There was a clear distinction between the AKI and no-AKI profiles. Two previously identified biomarkers of AKI, hippurate and homovanillate, differentiated AKI from no-AKI profiles. Pathway analysis revealed similarities to cholinergic neurons, prenatal nicotine exposure on pancreatic β cells, and amitraz-induced inhibition of insulin secretion. Additionally, a pH difference was noted. Both pH and the metabolites were found to be associated with AKI; however, only the metabotype was a significant predictor of AKI. Pathways for the no-AKI group that correlated uniquely with gestational age included aminoacyl-t-RNA biosynthesis, whereas pathways in the AKI group yielded potential metabolite changes in pyruvate metabolism. Metabolomics was able to differentiate the urinary profiles of neonates with and without an AKI diagnosis and metabolic developmental profiles correlated with gestational age. Further studies in larger cohorts are needed to validate these results.

The essential oil from air dried buds and roots of Calligonum polygonoides Linn., has been extracted from dry steam distillation and analysed for chemical composition by gas chromatography-mass spectrometry. In total, 27 and 10 compounds were analysed qualitatively and quantitatively, accounting for 68.42% and 82.12% total contents of the essential oils of buds and roots, respectively. It contains a complex mixture of terpenoids, hydrocarbons, phenolic compounds, acid derivatives and ketones. The main component of essential oil was ethyl homovanillate (11.79%) in buds and drimenol (29.42%) in roots.

In the present study, the effects of the novel vanilloid agonist, 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), on oxygen consumption (VO2) and vascular resistance (perfusion pressure, PP) were investigated in the constant flow, perfused rat hindlimb. The acute desensitizing properties of this novel synthetic agent were also examined. Maximum stimulation of VO2 was produced by 0.2 μM PPAHV (ΔVO2, 0.83±0.06 μmol g−1 h−1) and was accompanied by mild vasoconstriction (increase in PP; 8.0±1.1 mmHg). The highest concentration of PPAHV tested (2 μM) caused inhibition of VO2 (ΔVO2, −2.73±0.51 μmol g−1 h−1) and strong vasoconstriction (ΔPP, 42.0±1.2 mmHg). Capsazepine (10 μM) caused a parallel shift to the right of both VO2 and PP concentration-response curves for PPAHV (pKb=5.00), indicative of competitive binding to vanilloid receptors. The stimulation of VO2 produced by 0.2 μM PPAHV decreased, but was not completely abolished, after repeated infusion of PPAHV (change in VO2, first infusion, 0.66±0.18 μmol g−1 h−1; sixth infusion, 0.29±0.08 μmol g−1 h−1, P

Our objective was to characterize the mechanism of action of intrastriatal infusion of domoic acid on extracellular dopamine levels, using in vivo dialysis in conscious and freely moving rats. The local infusion of domoic acid (500 microM) caused an increase (567.9+/-142.5%, versus basal) in dopamine extracellular levels associated with a decrease in its metabolites: dihydroxyphenylacetate (DOPAC) and homovanillate (HVA) (47.3+/-4.4% and 33.8+/-4.2%, respectively, compared to basal). Infusion of the amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/kainate) receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX; 200 microM) reversed the effect of domoic acid infusion on striatal dopamine levels. However, the infusion of the selective non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, dizocilpine (MK-801; 50 microM), did not change significantly the effect of domoic acid on dopamine extracellular levels. In conclusion, based on results with a microdialysis technique, we suggest that domoic acid may act through AMPA/kainate receptors in striatum.

The microdialysis technique was used to examine the effect of the neurotoxin domoate, an analog of glutamic acid, on striatal dopamine activity. Our results show that the intracerebral administration of different concentrations of domoate (100 and 500 μM) produced increases in the extracellular levels of dopamine associated to decreases in the extracellular levels of its metabolites dihydroxyphenylacetate and homovanillate from rat striatum. These changes seem to be related according to a time sequence, indicating a possible effect on the metabolism of dopamine. Changes were also observed in locomotor activity (cycling behavior, sniffing around and chewing) in rats during the domoate infusion. The physiological mechanism by which domoate increased dopamine release remains to be worked out.

J.E. TURNER AND N. ALLISON. 1995. A newly-isolated strain of Pseudomonas putida (HVA-1) utilized homovanillic acid as sole carbon and energy source. Homovanillate-grown bacteria oxidized homovanillate and homoprotocatechuate but monohydroxylated and other methoxylated phenylacetic acids were oxidized poorly; methoxy-substituted benzoates were not oxidized. Extracts of homovanillate-grown cells contained homoprotocatechuate 2,3-dioxygenase but the primary homovanillate-degrading enzyme could not be detected. No other methoxylated phenylacetic acid supported growth of the organism but vanillate was utilized as a carbon and energy source. When homovanillate-grown cells were used to inoculate media containing vanillate a 26 h lag period occurred before growth commenced. Vanillate-grown bacteria oxidized vanillate and protocatechuate but no significant oxygen uptake was obtained with homovanillate and other phenylacetic acid derivatives. Analysis of pathway intermediates revealed that homovanillate-grown bacteria produced homoprotocatechuate, formaldehyde and the ring-cleavage product 5-carboxymethyl 2-hydroxymuconic semialdehyde (CHMS) when incubated with homovanillate but monohydroxylated or monomethoxylated phenylacetic acids were not detected. These results suggest that homovanillate is degraded directly to the ring-cleavage substrate homoprotocatechuate by an unstable but highly specific demethylase and then undergoes extradiol cleavage to CHMS. It would also appear that the uptake/degradatory pathways for homovanillate and vanillate in this organism are entirely separate and independently controlled. If stabilization of the homovanillate demethylase can be achieved, there is potential for exploiting the substrate specificity of this enzyme in both medical diagnosis and in the paper industry.

In vivo microdialysis was used to study the effects of systemically administered phencyclidine (PCP, 10 mg/kg) on the extracellular levels of dopamine, dihydroxyphenylacetate (DOPAC), homovanillate (HVA), 5-hydroxy-indolacetate (5-HIAA), gamma-aminobutyrate (GABA), glutamate, and aspartate in the rat dorsolateral striatum. In order to demarcate the effects of anesthesia, tissue trauma and gliosis, the effect of PCP was studied in both anesthetized rats with long and short probe implantation periods and in conscious rats with a long probe implantation period. PCP significantly increased the extracellular levels of dopamine in all experimental groups, though the post-implantation interval and anesthesia modulated the degree of increase. PCP increased 5-HIAA levels in both conscious and anesthetized rats after a long post-implantation period and HVA only in anesthetized rats after a long post-implantation period. Glutamate, aspartate, and DOPAC were not affected by PCP challenge but our study indicated for the first time that systemic PCP elevates extracellular GABA in conscious rats.

Motivation: For the early detection of cancer, highly sensitive and specific biomarkers are needed. Particularly, biomarkers in bio-fluids are relatively more useful because those can be used for non-biopsy tests. Although the altered metabolic activities of cancer cells have been observed in many studies, little is known about metabolic biomarkers for cancer screening. In this study, a systematic method is proposed for identifying metabolic biomarkers in urine samples by selecting candidate biomarkers from altered genome-wide gene expression signatures of cancer cells. Biomarkers identified by the present study have increased coherence and robustness because the significances of biomarkers are validated in both gene expression profiles and metabolic profiles. Results: The proposed method was applied to the gene expression profiles and urine samples of 50 breast cancer patients and 50 normal persons. Nine altered metabolic pathways were identified from the breast cancer gene expression signatures. Among these altered metabolic pathways, four metabolic biomarkers (Homovanillate, 4-hydroxyphenylacetate, 5-hydroxyindoleacetate and urea) were identified to be different in cancer and normal subjects (p Contact: dhlee@biosoft.kaist.ac.kr Supplementary information: Supplementary data are available at Bioinformatics online.

Cow's milk allergy (CMA) is the most common form of food allergy affecting 2.5% of children, but the diagnosis is often difficult. Both intestinal microbiota and barrier function seem to be disturbed in patients with food allergies, and administration of probiotics has been shown to normalize intestinal microbiota and alleviate symptoms. We hypothesized that the differences in intestinal metabolic activity and permeability could lead to detectable changes in the end-products of metabolism in patients with CMA. This could offer new diagnostic possibilities. The urinary concentrations of 37 organic acids were studied by a mass spectrometry-based method in 35 infants aged under 1 yr with atopic eczema, 16 of them having CMA diagnosed with a double-blind placebo-controlled food challenge test. The control group consisted of the remaining 19 infants with only atopic eczema. In a second study, Lactobacillus rhamnosus GG (LGG) or placebo was administered to the infants with CMA for 4 wk and the urinary organic acids were analysed again. CMA patients and patients with only atopic eczema had statistically significant differences in urinary concentrations of hydroxybutyrate (p

The effects of acute and chronic stress on the production of systemic metabolites were investigated in male Sprague-Dawley (SD) rats. Metabolites excreted in urine were analyzed using GC/MS in conjunction with multivariate and univariate statistical techniques. SD rats were subjected to two kinds of acute stress and chronic unpredictable mild stress, respectively. Metabolic analysis demonstrated that urinary expression of a number of metabolites including glutamate, glutamine, homovanillate, proline, succinate, citrate, and tyrosine altered in the acute stress model in the same way as in the chronic model, while pimelate and hippurate changed in the opposite trend. The results suggested that the stress induced metabolic perturbations were reversible and nonspecific. Metabolic response to chronic combined stress revealed biochemical clues to depression-like symptoms validated by behavior and physiologic results. This study provides a noninvasive and dynamic analytical strategy for the characterization of endogenous metabolic perturbations induced by external stress.

PPAHV (Phorbol 12-phenylacetate 12-acetate 20-homovanillate) was synthesized as part of an effort to define structural motifs which render the ultrapotent …

We previously described a series of N-(3-acyloxy-2-benzylpropyl) homovanillate and N'-(4-hydroxy-3-methoxybenzyl) thiourea derivatives that were potent VR1 agonists with high-affinities and excellent analgesic profiles. The design of these simplified RTX analogues was based on our RTX-derived pharmacophore model which incorporates the 4-hydroxy-3-methoxyphenyl (A-region), C(20)-ester (B-region), orthophenyl (C1-region) and C(3)-keto (C2-region) groups of RTX. For the purpose of optimizing the spatial arrangement of the four principal pharmacophores on the lead agonists (1-4), we have modified the distances in the parent C-region, 3-acyloxy-2-benzylpropyl groups, by lengthening or shortening one carbon to vary the distances between the pharmacophores. We find that two of the amides, 4 and 19, possess EC(50) values1 nM for induction of calcium influx in the VR1-CHO cells. As observed previously, the structure-activity relations for inhibition of RTX binding to VR1 and for induction of calcium uptake were distinct, presumably reflecting both intrinsic and methodological factors. In order to find the active conformation of VR1 ligands, the energy-minimized conformations of seven selected agonists were determined and the positions of their four pharmacophores were matched with those of five low energy RTX conformations. The rms values for the overlaps in the pharmacophores were calculated and correlated with the measured binding affinities (K(i)) and calcium influx (EC(50)) values. The binding affinities of the agonists correlated best with the RMS values derived from RTX conformation E (r(2)=0.92), predicting a model of the active conformation of RTX and related vanilloids for binding to VR1. Poorer correlation was obtained between any of the conformations and the EC(50) values for calcium influx.

In vivo microdialysis was used to study the effects of restraint stress (30 min) and amphetamine (AMPH) (5 mg/kg, i.p.) in awake adult male rats with neonatal ventral hippocampal (VH) damage. Extracellular levels of dopamine (DA), dihydrophenylacetate (DOPAC), homovanillate (HVA) and 5-hydroxyindolacetate (5-HIAA) were measured in the nucleus accumbens (NA). There were no differences in the baseline levels of DA, DOPAC, HVA or 5-HIAA in the lesioned as compared to the sham rats. Release from restraint resulted in increased extracellular levels of DA in the sham but not in the lesioned animals. AMPH increased DA release in both sham operated and lesioned animals, but this increase was significantly attenuated in the lesioned rats. Our data suggest that this developmental lesion alters function of the dopaminergic system in response to environmental and pharmacological challenge.

Capsaicin, the vanilloid responsible for the pungent taste of hot peppers, binds to receptors found primarily in polymodal nociceptors. Capsaicin initially stimulates polymodal nociceptors and subsequently inhibits them from responding to a variety of stimuli. This property makes it useful clinically as an analgesic and anti-inflammatory compound. There is mounting, albeit indirect, evidence for the existence of several subtypes of vanilloid receptors. One such piece of evidence comes from studying analogues of capsaicin, such as phorbol 12-phenylacetate 13 acetate 20-homovanillate. This compound binds to (capsaicin) vanilloid receptors on sensory neurons, but unlike capsaicin it is non-pungent and does not produce hypothermia. To determine how sensory neurons respond to phorbol 12-phenylacetate 13 acetate 20-homovanillate, and to compare these responses with those evoked by capsaicin, whole-cell patch-clamp measurements were performed on cultured rat trigeminal ganglion neurons. It was found that 63% of the neurons held at -60 mV were activated by 3 microM, phorbol 12-phenylacetate 13 acetate 20-homovanillate, and 87% of these were also activated by 1 microM capsaicin. In a given neuron, phorbol 12-phenylacetate 13 acetate 20-homovanillate, like capsaicin, could activate kinetically distinct inward currents. The current-voltage curves characterizing phorbol 12-phenylacetate 13 acetate 20-homovanillate responses were asymmetric and had reversal potentials at -5.8 +/- 6.0 mV and 10.4 +/- 4 mV. The averaged dose-response curves for phorbol 12-phenylacetate 13 acetate 20-homovanillate were fit to the Hill equation and had binding constants (K(1/2)s) of 2.73 microM and 0.96 microM and Hill coefficients (ns) of approximately 1 for a rapidly- and slowly-activating current, respectively. These parameters are consistent with those obtained from binding experiments and calcium-influx experiments on sensory nerves. Repeated applications of phorbol 12-phenylacetate 13 acetate 20-homovanillate every 3 min caused a complete reduction in the rapidly-activating currents leaving only a reduced slowly-activating current. This provides strong evidence for the independence of these currents and the existence of subtypes of vanilloid receptors. Additional evidence for the existence of receptor subtypes is that 10 microM capsazepine, a specific and competitive inhibitor of capsaicin-evoked responses, did not inhibit the phorbol 12-phenylacetate 13 acetate 20-homovanillate-induced currents in some neurons and partially inhibited them in other neurons. Thus, there are capsazepine-sensitive and capsazepine-insensitive subtypes of vanilloid receptors. In summary, we have obtained electrophysiological and pharmacological evidence for distinct subtypes of vanilloid receptors.

Capsaicin binds to a specific recognition site, referred to as the vanilloid receptor, which it shares with the natural, ultrapotent agonist resiniferatoxin and with the competitive antagonist capsazepine. Upon binding to its receptor, capsaicin opens a cation channel leading to Ca2+ influx. The binding of capsaicin or resiniferatoxin by the vanilloid receptor follows a sigmoidal saturation curve, indicative of positive cooperativity. The biological significance of this positive cooperative behaviour is unknown, as is the mechanism responsible for it. We have developed a novel ligand, phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), which binds to cultured rat sensory neurons (with a Ki of 3.1 +/- 0.4 microM), and induces Ca2+ uptake by them (with an ED50 of 1.8 +/- 0.3 microM) with similar affinities and in a non-cooperative manner (Hill coefficients are 0.99 and 1.06 for binding and Ca2+ uptake, respectively). The behaviour of PPAHV thus contrasts with resiniferatoxin or capsaicin not only in the lack of cooperativity but also in the relative potencies for resiniferatoxin binding versus Ca2+ uptake (resiniferatoxin is less potent and capsaicin is more potent for induction of Ca2+ uptake than for binding). In further experiments in which the concentration of [3H]resiniferatoxin was varied, 1 microM PPAHV likewise reduced the cooperativity index that characterizes resiniferatoxin binding to rat spinal cord membranes from 2.3 +/- 0.1 to 1.1 +/- 0.2; in parallel experiments, neither capsaicin nor capsazepine (both at a concentration of 2 microM) affected binding cooperativity. Moreover, PPAHV (1 microM) turned the bi-phasic dissociation curve of resiniferatoxin into a monophasic curve, eliminating the second, slow-dissociation phase. The present results suggest that positive cooperativity is a ligand-induced feature rather than an inherent property of vanilloid receptors. A comparison of the spectrum of biological activity of ligands which bind to vanilloid receptors with different degrees of cooperativity may provide an approach to explore the functional significance of this binding behaviour.

A newly isolated strain of Variovorax paradoxus could grow on homovanillate and several monohydroxylated phenylacetic acids. During growth on homovanillate, the organism formed separate NAD(P)H-dependent hydroxylases with activity towards 4-hydroxyphenylacetic acid and homovanillate. Homovanillate hydroxylase catalysed a typical monooxygenase reaction and had little activity towards 4-hydroxyphenylacetic acid. GC-MS and TLC analysis suggested that homovanillate was 1-hydroxylated to yield a dihydroxymonomethoxyphenylacetic acid which served as a substrate for homogentisate 1,2-dioxygenase. Methanol, but not formaldehyde, was released either during ring-cleavage or subsequent metabolism of the ring-cleavage product.

The dose-dependent effects of systemically and locally administered phencyclidine (PCP) on the extracellular levels of dopamine, dihydroxyphenylacetate (DOPAC), homovanillate (HVA), 5-hydroxyindolacetate (5-HIAA), gamma-aminobutyrate (GABA), glutamate and aspartate in the dorsolateral striatum of anaesthetized rats were studied by in vivo microdialysis. Both local (1, 5, 50 and 100 microM) and systemic (2 and 10 mg kg-1 i.p.) PCP caused a dose-dependent increase in the extracellular levels of dopamine. The lowest PCP doses caused only a moderate but long-lasting increase in the extracellular levels of dopamine, while the highest PCP doses caused a massive but transient increase followed by a rebound decrease. The low doses of both systemic and local PCP tended to increase the levels of DOPAC, while those of HVA were not changed. The extracellular levels of 5-HIAA were increased only by the lowest (1 microM) locally administered dose of PCP. GABA levels were increased when PCP was administered locally at two doses. None of the treatments affected the extracellular levels of glutamate and aspartate. The results show that the effects of local and systemic PCP administration are dissimilar on the extracellular levels of 5-HIAA and GABA and thus provide new information on the neurochemical effects of PCP.

In order to investigate the relationship between visual dysfunction and retinal DA metabolism in zitter rats and spontaneously epileptic rats (SER), we measured the amounts of retinal dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillate (HVA). DA, DOPAC and HVA contents were markedly decreased in zitter rats as compared with the controls, Kyo:Wistar or Sprague-Dawley rats. In contrast, in SER, retinal DA and its metabolite contents were not significantly different from those of Kyo:Wistar rats. SER showed higher levels of DA and its metabolites in comparison with Sprague-Dawley rats. Thus, it is suggested that the debilitation of retinal DA synthesis is at least partly related to the visual dysfunction observed in zitter rats, but not that of SER.

Aspartic acid, 5-hydroxyindoleacetic acid, glutamic acid, homovanillic acid and 3-methoxy-4-hydroxyphenylethylene glycol was determined in samples of ventricular fluid from 82 subjects. Laminar distribution of the total number (Bmax value) of serotonin 1A receptors was determined on seven neurosurgical samples of neocortex. Apart from an association in a small subgroup of subjects between homovanillate concentration and corticosteroid medication, no complicating influences of treatment preceding operation were found. The content of the serotonin metabolite alone was significantly reduced in intractable depressive illness (bipolar and major depressive disorders) compared with neurological conditions subdivided into Alzheimer's disease, other dementias and other conditions. There was no other significant difference between these groups for the compounds measured. The total number of serotonin 1A receptors was highest in the superficial layers, being considerably higher than in the rat, irrespective of cortical layer. This part of the study indicated that these receptors are important for regulating activity of human corticocortical glutamatergic neurons. The results are discussed in relation to treating depression with serotonergic agents and targeting corticocortical glutamatergic neurons as well as acetylcholine in Alzheimer's disease.