In vivo birthdating by BAPTISM reveals that trigeminal sensory neuron diversity depends on early neurogenesis

Among sensory systems, the somatic sense is exceptional in its ability to detect a wide range of chemical, mechanical and thermal stimuli. How this sensory diversity is established during development remains largely elusive. We devised a method (BAPTISM) that uses the photoconvertible fluorescent protein Kaede to simultaneously analyze birthdate and cell fate in live zebrafish embryos. We found that trigeminal sensory ganglia are formed from early-born and late-born neurons. Early-born neurons give rise to multiple classes of sensory neurons that express different ion channels. By contrast, late-born neurons are restricted in their fate and do not form chemosensory neurons expressing the ion channel TrpA1b. Accordingly, larvae lacking early-born neurons do not respond to the TrpA1b agonist allyl isothiocyanate. These results indicate that the multimodal specification and function of trigeminal sensory ganglia depends on the timing of neurogenesis.     

TRPA1 mediates the antinociceptive properties of the constituent of Crocus sativus L., safranal

Safranal, contained in Crocus sativus L., exerts anti‐inflammatory and analgesic effects. However, the underlying mechanisms for such effects are poorly understood. We explored whether safranal targets the transient receptor potential ankyrin 1 (TRPA1) channel, which in nociceptors mediates pain signals. Safranal by binding to specific cysteine/lysine residues, stimulates TRPA1, but not the TRP vanilloid 1 and 4 channels (TRPV1 and TRPV4), evoking calcium responses and currents in human cells and rat and mouse dorsal root ganglion (DRG) neurons. Genetic deletion or pharmacological blockade of TRPA1 attenuated safranal‐evoked release of calcitonin gene‐related peptide (CGRP) from rat and mouse dorsal spinal cord, and acute nociception in mice. Safranal contracted rat urinary bladder isolated strips in a TRPA1‐dependent manner, behaving as a partial agonist. After exposure to safranal the ability of allyl isothiocyanate (TRPA1 agonist), but not that of capsaicin (TRPV1 agonist) or GSK1016790A (TRPV4 agonist), to evoke currents in DRG neurons, contraction of urinary bladder strips and CGRP release from spinal cord slices in rats, and acute nociception in mice underwent desensitization. As previously shown for other herbal extracts, including petasites or parthenolide, safranal might exert analgesic properties by partial agonism and selective desensitization of the TRPA1 channel.     

Regulation of beta-adrenergic receptors and calcium channel agonist binding sites in cultured human embryonal smooth muscle cells

Recent electrophysiological studies with cell membrane patches of cardiac myocytes and an electrically excitable cell line derived from rat pituitary tumor suggested that voltage activated calcium channels must be phosphorylated to respond to membrane depolarization (Armstrong and Eckert 1986; Trautwein and Kameyama 1986). In view of the "phosphorylation hypothesis" we investigated the adenylate-cyclase activity, the characteristics of beta-adrenergic and calcium channel agonist binding sites in control and desensitized (exposure to isoproterenol) human embryonal cells (HEC), and in fragmented membrane preparations of canine coronary smooth muscle. Our results suggest that down-regulation of the membrane-bound beta-adrenergic receptors, induced by isoproterenol in human embryonal cells and also in adult canine vascular tissue, results in physical translocation of beta-adrenergic binding sites into the light membrane fraction. This phenomenon is accompanied with an increased intracellular concentration of cAMP in and an increased binding of the calcium channel agonist (3H) BAYK 8644 to both HEC and canine smooth muscle membrane preparations. It could be concluded that phosphorylation of beta-adrenergic receptors regulates not only the beta subcellular distribution of the beta receptors but also the availability of calcium channel agonist binding sites in the cellular membrane.     

Identification of species-specific determinants of the action of the antagonist capsazepine and the agonist PPAHV on TRPV1

The vanilloid receptor 1 (VR1 or TRPV1) ion channel is activated by noxious heat, low pH and by a variety of vanilloid-related compounds. The antagonist, capsazepine is more effective at inhibiting the human TRPV1 response to pH 5.5 than the rat TRPV1 response to this stimulus. Mutation of rat TRPV1 at three positions in the S3 to S4 region, to the corresponding human amino acid residues I514M, V518L, and M547L decreased the IC(50) values for capsazepine inhibition of the pH 5.5 response from >10,000 nm to 924 +/- 241 nm in [Ca(2+)](i) assays and increased capsazepine inhibition of the capsaicin response to levels seen for human TRPV1. We have previously noted that phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) is a strong agonist of rat TRPV1 but not human TRPV1 in [Ca(2+)](i) assays (1). Mutation of methionine 547 in S4 of rat TRPV1 to leucine, found in human TRPV1 (M547L), reduced the ability of PPAHV to activate TRPV1 by approximately 20-fold. The reciprocal mutation of human TRPV1 (L547M) enabled the human receptor to respond to PPAHV. These mutations did not significantly affect the agonist activity of capsaicin, resiniferatoxin (RTX) or olvanil in [Ca(2+)](i) assays. Introducing the equivalent mutation into guinea pig TRPV1 (L549M) increased the agonist potency of PPAHV by > 10-fold in the [Ca(2+)](i) assay and increased the amplitude of the evoked current. The rat M547L mutation reduced the affinity of RTX binding. Thus, amino acids within the S2-S4 region are important sites of agonist and antagonist interaction with TRPV1.     

Inverse regulation of calcium channels and beta-adrenergic receptors in virus-transformed human embryonal cells.

Monolayer cultures of human embryonal smooth muscle cells (HEC) were used to study the heterologous regulation of membrane beta-adrenergic receptors and Ca2+ channels. The relationships between the activation of membrane bound alpha-1 and beta-adrenergic receptors, the cyclic nucleotide response and Ca2+ channel binding were studied in a cellular model of latent virus infection (Herpes simplex, Type-2) in a human embryonal cell line. In the early stage of infection (72 h), there was a significant increase in the cell cAMP content, followed by a decrease in the binding capacity of the beta-adrenergic ligand with an increased total number of the 1,4-dihydropyridine Ca2+ channel agonist (-)-S-(3H)BAYK 8644 binding sites on the cell membrane of infected cells. The increased numbers of Ca2+ agonist binding sites were accompanied by an increased cAMP content in the cells and an increased membrane ATP-ase activity. Down-regulation of (3H)DHA binding, and an increased capacity of Ca2+ agonist binding were found after prolonged exposure of HEC to isoprenaline (10(-5) mol.l-1). Stimulation of alpha-1 adrenergic receptors with phenylephrine (10(-6) mol.l-1) was accompanied with only slight but significant increase in (3H)DHA binding and with a significant reduction in the total number of Ca2+ channel agonist binding sites.     

Evolution of multi-enzyme complexes: the case of tryptophan synthase

The prototypical tryptophan synthase is a stable heterotetrameric alpha-betabeta-alpha complex. The constituting TrpA and TrpB1 subunits, which are encoded by neighboring genes in the trp operon, activate each other in a bi-directional manner. Recently, a novel class of TrpB2 proteins has been identified, whose members contain additional amino acids that might sterically prevent complex formation with TrpA. To test this hypothesis, we characterized the TrpA and TrpB proteins from Sulfolobus solfataricus. This hyperthermophilic archaeon does not contain a TrpB1 protein but instead contains two TrpB2 homologues that are encoded within (TrpB2i) and outside (TrpB2o) the trp operon. We find that TrpB2i and TrpA form a weak and transient complex during catalysis, with a uni-directional activation of TrpA by TrpB2i. In contrast, TrpB2o and TrpA do not form a detectable complex. These results suggest a model for the evolution of the tryptophan synthase in which TrpB2o, TrpB2i, and TrpB1 reflect the stepwise increase of TrpB affinity for TrpA and the refinement of functional subunit interaction, concomitant with the co-localization of the encoding genes in the trp operon.     

Development of novel azabenzofuran TRPA1 antagonists as in vivo tools

The transient receptor potential ankyrin 1 (TRPA1) channel is activated by noxious stimuli including chemical irritants and endogenous inflammatory mediators. Antagonists of this channel are currently being investigated for use as therapeutic agents for treating pain, airway disorders, and itch. A novel azabenzofuran series was developed that demonstrated in vitro inhibition of allyl isothiocyanate (AITC)-induced ⁴⁵Ca²⁺ uptake with nanomolar potencies against both human and rat TRPA1. From this series, compound 10 demonstrated in vivo target coverage in an AITC-induced flinching model in rats while providing unbound plasma concentrations up to 16-fold higher than the TRPA1 rat IC₅₀.     

A sphingosine 1-phosphate receptor 2 selective allosteric agonist

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.     

Newly Synthesized Oleylgingerol and Oleylshogaol Activate TRPV1 Ion Channels

The oleyl moiety in vanilloids is important in activating vanilloid receptor 1 (TRPV1), but there was no ingredient of ginger containing the oleyl moiety in the natural form. We synthesized oleylgingerol and oleylshogaol and then evaluated their potential to activate a rat TRPV1 channel. Oleylgingerol is a stronger TRPV1 agonist than natural gingerols, but oleylshogaol is a weaker agonist than natural shogaols. The difference in structure between oleylgingerol and oleylshogaol is only the hydroxy group at carbon-5. This hydroxy group might have an important role in activating a TRPV1 channel.     

Allosteric activation of the follicle-stimulating hormone (FSH) receptor by selective, nonpeptide agonists

The pituitary glycoprotein hormones, luteinizing hormone and follicle-stimulating hormone (FSH), act through their cognate receptors to initiate a series of coordinated physiological events that results in germ cell maturation. Given the importance of FSH in regulating folliculogenesis and fertility, the development of FSH mimetics has been sought to treat infertility. Currently, purified and recombinant human FSH are the only FSH receptor (FSH-R) agonists available for infertility treatment. By screening unbiased combinatorial chemistry libraries, using a cAMP-responsive luciferase reporter assay, we discovered thiazolidinone agonists (EC50's = 20 microm) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC50 = 2 nm) with full efficacy compared with FSH that was FSH-R-selective and -dependent. The compound mediated progesterone production in Y1 cells transfected with the human FSH-R (EC50 = 980 nm) and estradiol production from primary rat ovarian granulosa cells (EC50 = 10.5 nm). This and related compounds did not compete with FSH for binding to the FSH-R. Use of human FSH/thyroid-stimulating hormone (TSH) receptor chimeras suggested a novel mechanism for receptor activation through a binding site independent of the natural hormone binding site. This study is the first report of a high affinity small molecule agonist that activates a glycoprotein hormone receptor through an allosteric mechanism. The small molecule FSH receptor agonists described here could lead to an oral alternative to the current parenteral FSH treatments used clinically to induce ovarian stimulation for both in vivo and in vitro fertilization therapy.     

Assay and screening methods for bioactive substances based on cellular signaling pathways

Assay and screening methods for bioactive substances based on cellular signaling pathways are presented. Examples include: (1) intracellular protein phosphorylation and protein-protein interaction, (1-i) a new assay method for evaluating chemical selectivity of agonists for insulin signaling pathways based on agonist-induced phosphorylation of a target peptide, (1-ii) an SPR-based screening method for agonist selectivity for insulin signaling pathways based on the binding of phosphotyrosine to its specific binding protein, (1-iii) a fluorescent indicator for tyrosine phosphorylation-based insulin signaling pathways, and (1-iv) split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing; (2) a screening method for antigen-specific IgE using mast cells based on intracellular calcium signaling; (3) a screening method for substrates of multidrug resistance-associated protein (MRP); and (4) fluorescent indicators for cyclic GMP based on cyclic GMP-dependent protein kinase Ialpha and green fluorescent proteins.     

Activation properties of heterologously expressed mammalian TRPV2: evidence for species dependence

TRPV2 has been proposed as a potential pain target, in part due to its relatedness to the nociceptor TRPV1 and to its reported activation by noxious high temperatures (>52 degrees C). However, TRPV2 responses to heat as well as to the nonselective agonist 2-aminoethoxydiphenyl borate (2-APB) have not been universally reproduced in other laboratories, leading to debate about the activation properties of this channel. Here, we report the expression of rat, mouse, and human TRPV2 in HEK293 cells and the differential properties of their responses to heat and 2-APB. Expression of mouse or rat TRPV2 in HEK293 cells resulted in robust channel activation when induced by either temperature (>53 degrees C) or 2-APB. By contrast, expression of human TRPV2 did not lead to detectable activation by either of these stimuli. Human TRPV2 protein was expressed at levels comparable with those of rat TRPV2, exhibited similar surface localization and responded to a novelly identified TRPV2 agonist, Delta(9)-tetrahydrocannabinol, indicating that human TRPV2 is functionally expressed on the cell surface. Studies using deletion mutants and chimeras between rat and human TRPV2 indicated that both amino- and carboxyl-cytoplasmic termini of rat TRPV2 are important for responses to heat and 2-APB but can be supplied in trans to form an active channel. The present study not only confirms and extends previous reports demonstrating that rat and mouse TRPV2 respond to 2-APB and noxious heat but also indicates that further investigation will be required to elucidate TRPV2 activation and regulatory mechanisms.     

Catalytically impaired TrpA subunit of tryptophan synthase from Chlamydia trachomatis is an allosteric regulator of TrpB

Intracellular growth and pathogenesis of Chlamydia species is controlled by the availability of tryptophan, yet the complete biosynthetic pathway for l‐Trp is absent among members of the genus. Some representatives, however, preserve genes encoding tryptophan synthase, TrpAB – a bifunctional enzyme catalyzing the last two steps in l‐Trp synthesis. TrpA (subunit α) converts indole‐3‐glycerol phosphate into indole and glyceraldehyde‐3‐phosphate (α reaction). The former compound is subsequently used by TrpB (subunit β) to produce l‐Trp in the presence of l‐Ser and a pyridoxal 5′‐phosphate cofactor (β reaction). Previous studies have indicated that in Chlamydia, TrpA has lost its catalytic activity yet remains associated with TrpB to support the β reaction. Here, we provide detailed analysis of the TrpAB from C. trachomatis D/UW‐3/CX, confirming that accumulation of mutations in the active site of TrpA renders it enzymatically inactive, despite the conservation of the catalytic residues. We also show that TrpA remains a functional component of the TrpAB complex, increasing the activity of TrpB by four‐fold. The side chain of non‐conserved βArg267 functions as cation effector, potentially rendering the enzyme less susceptible to the solvent ion composition. The observed structural and functional changes detected herein were placed in a broader evolutionary and genomic context, allowing identification of these mutations in relation to their trp gene contexts in which they occur. Moreover, in agreement with the in vitro data, partial relaxation of purifying selection for TrpA, but not for TrpB, was detected, reinforcing a partial loss of TrpA functions during the course of evolution.     

Microscopic kinetics and energetics distinguish GABA(A) receptor agonists from antagonists

Although agonists and competitive antagonists presumably occupy overlapping binding sites on ligand-gated channels, these interactions cannot be identical because agonists cause channel opening whereas antagonists do not. One explanation is that only agonist binding performs enough work on the receptor to cause the conformational changes that lead to gating. This idea is supported by agonist binding rates at GABA(A) and nicotinic acetylcholine receptors that are slower than expected for a diffusion-limited process, suggesting that agonist binding involves an energy-requiring event. This hypothesis predicts that competitive antagonist binding should require less activation energy than agonist binding. To test this idea, we developed a novel deconvolution-based method to compare binding and unbinding kinetics of GABA(A) receptor agonists and antagonists in outside-out patches from rat hippocampal neurons. Agonist and antagonist unbinding rates were steeply correlated with affinity. Unlike the agonists, three of the four antagonists tested had binding rates that were fast, independent of affinity, and could be accounted for by diffusion- and dehydration-limited processes. In contrast, agonist binding involved additional energy-requiring steps, consistent with the idea that channel gating is initiated by agonist-triggered movements within the ligand binding site. Antagonist binding does not appear to produce such movements, and may in fact prevent them.     

Resiniferatoxin binds to the capsaicin receptor (TRPV1) near the extracellular side of the S4 transmembrane domain

The capsaicin receptor (TRPV1) is a nonselective cation channel that is activated in nociceptors by several painful stimuli, and hence TRPV1 antagonists could represent a novel class of analgesic compounds. Resiniferatoxin (RTX), a potent agonist of TRPV1, and iodoresiniferatoxin (I-RTX), a potent antagonist of TRPV1, both bind with higher affinity to the rat TRPV1 (rTRPV1) than the human (hTRPV1) isoform. To identify the structural features responsible for this difference in affinity, [(3)H]RTX binding to chimeras between hTRPV1 and rTRPV1 was characterized. The "sensor" region within the transmembrane domain (S1-S4) was found to determine [(3)H]RTX binding affinity. All 16 different residues in this region were systematically substituted in hTRPV1 with rTRPV1 residues. A single mutation in the S4 membrane domain of hTRPV1, L547M, caused a 30-fold increase in [(3)H]RTX affinity whereas the inverse mutation in rTRPV1, M547L, caused a 30-fold decrease in affinity for [(3)H]RTX, and several other agonists and antagonists were similarly affected by these mutations. TRPV1 channels with mutations at position 547 were expressed in oocytes, and the relative response to RTX followed a pattern similar to that seen with [(3)H]RTX binding. These data suggest a model where Met-547 in the S4 domain of TRPV1 forms a binding pocket with Tyr-511 in the S3 domain. This model places RTX near the sensor domain thought to move during the gating process and should help to guide further work designed to understand the gating mechanisms of TRPV1 channels based on comparisons between the agonist RTX and the related competitive antagonist I-RTX.     

Time-resolved quantitative analysis of CCK1 receptor-induced intracellular calcium increase

Cholecystokinin (CCK) is a gastrointestinal hormone, which regulates many physiological functions such as satiety by binding to the CCK receptor (CCKR). Molecules, which recognize this receptor can mimic or block CCK signaling and thereby influence CCKR-mediated processes. We have set up a quantitative heterologous assay with CHO cells over-expressing the rat CCK1 receptor to screen for such candidate molecules. Receptor activation, induced by agonist binding, is followed by an intracellular calcium increase, which was monitored using a fluorescent sensor dye. For quantification of the calcium increase, a population average technique using a fluorescence plate reader was optimized and subsequently compared with a single-cell approach using confocal microscopy. With both strategies, dose-response curves were generated for the natural agonist CCK-8S, the partial agonist JMV-180 as well as the antagonist lorglumide. Significant differences were found between the ligands and a strong correspondence was observed between both methods in terms of maximum response and median effect concentrations. Both highly sensitive methods proved complementary: whereas the plate reader assay allowed faster, high throughput screening, the confocal microscopy identified single-cell variations and revealed factors that reduce specificity and sensitivity.     

Characterization of human and rodent native and recombinant adenosine A2B receptors by radioligand binding studies

Adenosine A_2B receptors of native human and rodent cell lines were investigated using [³H]PSB-298 [(8-{4-[2-(2-hydroxyethylamino)-2-oxoethoxy]phenyl}-1-propylxanthine] in radioligand binding studies. [³H]PSB-298 showed saturable and reversible binding. It exhibited a K_D value of 60 ± 1 nM and limited capacity (B_max = 3.511 fmol per milligram protein) at recombinant human adenosine A_2B receptors expressed in human embryonic kidney cells (HEK-293). The addition of sodium chloride (100 mM) led to a threefold increase in the number of binding sites recognized by the radioligand. The curve of the agonist 5′-N-ethylcarboxamidoadenosine (NECA) was shifted to the right in the presence of NaCl, while the curve of the antagonist PSB-298 was shifted to the left, indicating that PSB-298 may be an inverse agonist at A_2B receptors. Adenosine A_2B receptors were shown to be the major adenosine A₂ receptor subtype on the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 cells. Binding studies at rat INS-1 cells (insulin secreting cell line) demonstrated that [³H]PSB-298 is a selective radioligand for adenosine A_2B binding sites in this cell line.