iodoresiniferatoxin and resiniferatoxin

As canine mammary tumours (CMT) and human breast cancer share clinical and prognostic features, the former have been proposed as a model to study carcinogenesis and improved therapeutic treatment in human breast cancer. In recent years, it has been shown that transient receptor potential vanilloid 1 (TRPV1) is expressed in different neoplastic tissues and its activation has been associated with regulation of cancer growth and progression. The aim of the present research was to demonstrate the presence of TRPV1 in human and canine mammary cancer cells, MCF-7 and CF.41, respectively, and to study the role of TRPV1 in regulating cell proliferation. The images obtained by Western blot showed a signal at 100 kDa corresponding to the molecular weight of TRPV1 receptor. All tested TRPV1 agonists and antagonists caused a significant decrease (P < 0.05) of cell growth rate in MCF-7 cells. By contrast, in CF.41 cells capsaicin and capsazepine induced a significant increase (P < 0.05) in cell proliferation, whereas resiniferatoxin (RTX) and 5-iodo-resiniferatoxin (5-I-RTX) had no influence on CF.41 cell proliferation. Further studies are needed to elucidate the underlying molecular mechanism responsible for the different effects evoked by TRPV1 activation in MCF-7 and CF.41 cells.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Capsaicin; Cell Line, Tumor; Cell Proliferation; Diterpenes; Dogs; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Animal; MCF-7 Cells; TRPV Cation Channels

The chiral isomers of the two potent simplified RTX-based vanilloids, compounds 2 and 3, were synthesized employing highly enantioselective PTC alkylation and evaluated as hTRPV1 ligands. The analysis indicated that the R-isomer was the eutomer in binding affinity and functional activity. The agonism of compound 2R was comparable to that of RTX. Docking analysis of the chiral isomers of 3 suggested the basis for its stereospecific activity and the binding mode of 3R.

Topics: Diterpenes; Dose-Response Relationship, Drug; Ligands; Models, Molecular; Molecular Structure; Stereoisomerism; Structure-Activity Relationship; TRPV Cation Channels

Bladder cancer (BC) is the fifth most common non-cutaneous malignancy and the most common form of BC in Western countries is transitional cell carcinoma. Resiniferatoxin (RTX) has found therapeutic usefulness for the treatment of bladder dysfunction but no data are available on its use as chemotherapeutic agent. The aim of this work is to evaluate the use of RTX as new anti-cancer drug in BC therapy. The effects of RTX on cell viability and cell death were evaluated on T24 and 5637 BC cell lines by MTT assay, cell cycle analysis, Annexin-V/PI staining and agarose gel electrophoresis of DNA. Mitochondrial depolarization and ROS production were assessed by flow cytometry. ADP/ATP ratio was measured by bioluminescence and caspase 3 cleavage by Western blot. For in vivo experiments, athymic nude mice, xenografted with T24 cells, received subcutaneous administrations of RTX. Tumor volumes were measured and immunohistochemistry was performed on tumor sections. Our data demonstrated that RTX influences cell cycle and induces necrotic cell death of BC cells by altering mitochondrial function, leading to depolarization, increase in ADP/ATP ratio and ROS production. Moreover, RTX is able to reduce tumor growth in a xenograft mouse model. Overall, we demonstrated that RTX induces necrotic cell death of BC cells and reduces tumor growth in a xenograft mouse model of BC, suggesting RTX as a new potential anti-cancer drug in BC chemotherapy.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Antineoplastic Agents; Carcinoma, Transitional Cell; Cell Cycle Checkpoints; Cell Line, Tumor; Diterpenes; Homeostasis; Humans; Male; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Mitochondria; Necrosis; Oxidation-Reduction; Reactive Oxygen Species; TRPV Cation Channels; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

A series of carbonate analogues of 5'-halogenated RTX have been investigated in order to examine the effect of the carbonate group as a linker and the role of halogens in the reversal of activity from agonism to antagonism for rat and human TRPV1 heterologously expressed in Chinese hamster ovary cells. The carbonate analogues showed similar activities to the corresponding RTX derivatives in rat TRPV1 but lower potency in human TRPV1. 5-Halogenation converted the agonists to partial agonists or full antagonists and the extent of antagonism reflected the order of I>Br>Cl>F, with a somewhat greater extent of antagonism for the derivatives of the 4-amino RTX surrogates compared to the corresponding derivatives of RTX itself. The carbonate analogues of I-RTX (60) and 5-bromo-4-amino-RTX (66) were potent and full antagonists with Ki(ant)=2.23 and 2.46 nM, respectively, for rat TRPV1, which were ca. 5-fold more potent than I-RTX (2) under our conditions. The conformational analysis of the I-RTX-carbonate (60) indicated that its bent conformation was similar to that of I-RTX, consistent with compound 60 and I-RTX showing comparable potent antagonism.

Topics: Animals; Carbonates; CHO Cells; Cricetinae; Cricetulus; Diterpenes; Halogens; Humans; Ligands; Molecular Conformation; Protein Binding; Rats; TRPV Cation Channels

The side chain benzylic methylene is a critical element for the vanilloid activity of resiniferatoxin (2a, RTX), and introduction of branching, oxygen functions, or isosteric substitution at this center proved detrimental, with a decrease of potency of 2-3 orders of magnitude compared to the natural product. Conversely, only a modest erosion of activity was observed upon alpha-methylation and alpha-methylenation of the side chain. Surprisingly, introduction of an iodine atom in the guaiacyl moiety of the oxygen isoster 2h led to an unexpected and remarkable (>1000-fold) increase of potency, affording 2i, a compound that outperforms RTX in terms of vanilloid agonism and represents the first one-digit picomolar ligand of a TRP channel discovered to date.

Topics: Cell Line; Diterpenes; Humans; Iodine; Methylation; Structure-Activity Relationship; TRPV Cation Channels

The transient receptor potential vanilloid-1 (TRPV1) receptor is involved in peripheral and spinal nociceptive processing and is a therapeutic target for pain. We have shown previously that TRPV1 in the ventrolateral periaqueductal gray (VL-PAG) tonically contributes to brain stem descending antinociception by stimulating glutamate release into the rostral ventromedial medulla and off neuron activity. Because both opioid and vanilloid systems integrate and transduce pain sensation in these pathways, we studied the potential interaction between TRPV1 and mu-opioid receptors in the VL-PAG-rostral ventromedial medulla (RVM) system. We found that the TRPV1 agonist, capsaicin, and the mu-receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin, when coadministered into the ventrolateral-PAG at doses nonanalgesic per se, produce 1) antinociception in tests of thermal nociception; 2) stimulation of glutamate release into the RVM; and 3) inhibition of on neuron activity in the RVM. These effects were all antagonized by the TRPV1 and opioid receptor antagonists 5'-iodo-resiniferatoxin and naloxone, respectively, thus suggesting the existence of a TRPV1-mu-opioid interaction in the VL-PAG-RVM system. By using double immunofluorescence techniques, we found that TRPV1 and mu-opioid receptors are coexpressed in several neurons of the VL-PAG. These findings suggest that mu-receptor activation not only acts on inhibitory neurons to disinhibit PAG output neurons but also interacts with TRPV1 activation at increasing glutamate release into the RVM, possibly by acting directly on PAG output neurons projecting to the RVM.

Topics: Action Potentials; Animals; Capsaicin; Diterpenes; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Glutamic Acid; Hyperalgesia; Male; Medulla Oblongata; Microinjections; Naloxone; Pain Measurement; Pain Threshold; Periaqueductal Gray; Rats; Rats, Wistar; Reaction Time; Receptors, Opioid, mu; TRPV Cation Channels

Long-term depression (LTD) in the rodent superior colliculus (SC) is regarded as a model of synaptic refinement because it can be induced during development but not in adults. We investigated the role of transient receptor potential vanilloid type-1 (TRPV1) channels in this type of synaptic plasticity. Experiments were carried out in pigmented mice aged between postnatal day 8 (P8) and 42 (P42) and in adult mice. Retinal axons to the SC were labelled by injection of cholera toxin-beta (CTbeta) into the eye. Immunohistochemical staining for CTbeta, TRPV1 and markers of glutamatergic and GABAergic cells and fibres (VGLUT1 and VGAT or GAD65, respectively) was performed by using multiple immunofluorescence. This showed that both glutamatergic retinal afferents to, and some GABAergic neurones in, the superficial SC are TRPV1 positive in juvenile but not adult mice. Field potential recordings were made from the superficial grey layer in parasagittal SC slices, and LTD (76 +/- 8% of control responses) was induced with a 50 Hz, 20 s tetanus. Activation of TRPV1 with resiniferatoxin also reduced field potential amplitude to 84 +/- 8% of control values. Blockade of TRPV1 with the selective antagonist 5'-iodo-resiniferatoxin prevented the induction of LTD (98 +/- 4% of control values), but did not cause its reversal if LTD was already established. N-acylphosphatidylethanolamine-specific phospholipase D and 12-lipoxygenase, two proposed endovanilloid biosynthesizing enzymes, were co-expressed with TRPV1 in the SC at P14 and P28. These results suggest that TRPV1 modulates retinocollicular responses in the developing SC and is activated during tetanic stimulation by endovanilloid ligands to participate in the induction of LTD.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Age Factors; Aging; Animals; Arachidonate 12-Lipoxygenase; Diterpenes; Ethanolamines; Excitatory Postsynaptic Potentials; Glutamate Decarboxylase; In Vitro Techniques; Ligands; Long-Term Synaptic Depression; Male; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Neurons; Phospholipase D; Staining and Labeling; Superior Colliculi; Synaptic Transmission; Time Factors; TRPV Cation Channels; Vesicular Glutamate Transport Protein 1; Vesicular Inhibitory Amino Acid Transport Proteins

The transient receptor potential (TRP) vanilloid receptor subtype 1 (TRPV1) is a ligand-gated, Ca(2+)-permeable ion channel in the TRP superfamily of channels. We report the establishment of the first neuronal model expressing recombinant human TRPV1 (SH-SY5Y(hTRPV1)). SH-SY5Y human neuroblastoma cells were stably transfected with hTRPV1 using the Amaxa Biosystem (hTRPV1 in pIREShyg2 with hygromycin selection). Capsaicin, olvanil, resiniferatoxin and the endocannabinoid anandamide increased [Ca(2+)](i) with potency (EC(50)) values of 2.9 nmol/L, 34.7 nmol/L, 0.9 nmol/L and 4.6 micromol/L, respectively. The putative endovanilloid N-arachidonoyl-dopamine increased [Ca(2+)](i) but this response did not reach a maximum. Capsaicin, anandamide, resiniferatoxin and olvanil mediated increases in [Ca(2+)](i) were inhibited by the TRPV1 antagonists capsazepine and iodo-resiniferatoxin with potencies (K(B)) of approximately 70 nmol/L and 2 nmol/L, respectively. Capsaicin stimulated the release of pre-labelled [(3)H]noradrenaline from monolayers of SH-SY5Y(hTRPV1) cells with an EC(50) of 0.6 nmol/L indicating amplification between [Ca(2+)](i) and release. In a perfusion system, we simultaneously measured [(3)H]noradrenaline release and [Ca(2+)](i) and observed that increased [Ca(2+)](i) preceded transmitter release. Capsaicin treatment also produced a cytotoxic response (EC(50) 155 nmol/L) that was antagonist-sensitive and mirrored the [Ca(2+)](I) response. This model displays pharmacology consistent with TRPV1 heterologously expressed in standard non-neuronal cells and native neuronal cultures. The advantage of SH-SY5Y(hTRPV1) is the ability of hTRPV1 to couple to neuronal biochemical machinery and produce large quantities of cells.

Topics: Arachidonic Acids; Calcium; Calcium Signaling; Capsaicin; Cell Culture Techniques; Cell Death; Cell Line, Tumor; Cell Proliferation; Diterpenes; Dopamine; Endocannabinoids; Humans; Models, Biological; Neuroblastoma; Neurons; Norepinephrine; Polyunsaturated Alkamides; Recombinant Proteins; Synaptic Transmission; Transfection; TRPV Cation Channels; Up-Regulation

Capsaicin (vanilloid) sensitivity has long served as the functional signature of a subset of nociceptive sensory neurons. Mutagenesis studies have revealed seemingly distinct regions involved in mediating ligand binding and channel activation at the capsaicin binding site. Residue 547 (transmembrane region 4) mediates significant species differences in resiniferatoxin (RTX) sensitivity, and the Ser(512) residue is critical in discriminating between pH and capsaicin gating. In the present study, the pharmacological profiles of a variety of ligands were studied to investigate cross-talk between these two regions. Exchange of residue 547 between species mediated a difference in capsaicin and RTX-dependent gating. Likewise, the potency of iodoresiniferatoxin (I-RTX) and a novel transient receptor potential vanilloid 1 antagonist were also altered. Experiments using the S512Y mutant channel have confirmed the importance of residue 512 for functional interaction of capsaicin and our novel antagonist. In this study, we were surprised to find that the mutation S512Y converted the activity of the antagonist I-RTX into an intrinsic agonist, albeit with a lower potency than its parent compound, RTX. Recent studies have proposed a novel model for the receptor, based on the X-ray crystal structure of the voltage-dependent potassium channel, in which both the 512 and 547 amino acid residues are in close proximity. Our data support the model whereby intracellular ligand interaction occurs within an S3-S4 "sensor" domain, enabling binding of ligands to be transduced to functional gating of the channel. The binding pocket also seems to be exquisitely sensitive to residue-specific interaction with ligands, because subtle changes in either ligand or channel structure can have profound effects on channel activity.

Topics: Amino Acids; Animals; Binding Sites; Cricetinae; Cricetulus; Diterpenes; Dose-Response Relationship, Drug; Electrophysiology; Humans; Inhibitory Concentration 50; Ligands; Mutant Proteins; Rats; TRPV Cation Channels

The present study examined the expression of transient receptor potential vanilloid subtype 1 (TRPV1) in microglia, and its association with microglial cell death. In vitro cell cultures, RT-PCR, Western blot analysis, and immunocytochemical staining experiments revealed that rat microglia and a human microglia cell line (HMO6) showed TRPV1 expression. Furthermore, exposure of these cells to TRPV1 agonists, capsaicin (CAP) and resiniferatoxin (RTX), triggered cell death. This effect was ameliorated by the TRPV1 antagonists, capsazepine and iodo-resiniferatoxin (I-RTX), suggesting that TRPV1 is directly involved. Further examinations revealed that TRPV1-induced toxicity was accompanied by increases in intracellular Ca(2+), and mitochondrial damage; these effects were inhibited by capsazepine, I-RTX, and the intracellular Ca(2+) chelator BAPTA-AM. Treatment of cells with CAP or RTX led to increased mitochondrial cytochrome c release and enhanced immunoreactivity to cleaved caspase-3. In contrast, the caspase-3 inhibitor z-DEVD-fmk protected microglia from CAP- or RTX-induced toxicity. In vivo, we also found that intranigral injection of CAP or 12-hydroperoxyeicosatetraenoic acid, an endogenous agonist of TRPV1, into the rat brain produced microglial damage via TRPV1 in the substantia nigra, as visualized by immunocytochemistry. To our knowledge, this study is the first to demonstrate that microglia express TRPV1, and that activation of this receptor may contribute to microglial damage via Ca(2+) signaling and mitochondrial disruption.

Topics: Animals; Blotting, Western; Calcium; Capsaicin; Cell Death; Cell Line; Cytochromes c; Diterpenes; Enzyme Inhibitors; Humans; Immunohistochemistry; In Situ Nick-End Labeling; In Vitro Techniques; Microglia; Mitochondria; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Substantia Nigra; TRPV Cation Channels

Transient receptor potential vanilloid receptor-1 (TRPV1) is a sensory neuron-specific cation channel capable of integrating various noxious chemical and physical stimuli. The dog orthologue of TRPV1 was cloned using cDNA from nodose ganglia and heterologously expressed in HEK293(OFF) cells. At the amino acid level, dTRPV1 displays 85-89% sequence identity to other TRPV1 orthologues. Molecular pharmacological characterization of HEK293(OFF) cells expressing TRPV1 was assessed using a fluorescence imaging plate reader (FLIPR)-based calcium imaging assay. Dog TRPV1 was activated by various known TRPV1 agonists in a concentration-dependent manner: Ag23 = resiniferatoxin > olvanil approximately arvanil > capsaicin > phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) > N-oleoyldopamine (OLDA). In addition, select TRPV1 antagonists (capsazepine, I-resiniferatoxin and N-(-4-tertiarybutylphenyl)-4-(3-cholorpyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC)) were able to block the response of dTRPV1 to capsaicin. Furthermore, the dog TRPV1 lacked a conserved protein kinase A (PKA) phosphorylation site (117) found in other cloned orthologues, which may have physiological consequences on dog TRPV1 function. Taken together, these data constitute the first study of the cloning, expression and pharmacological characterization of dog TRPV1.

Topics: Amino Acid Sequence; Animals; Biological Transport; Calcium; Capsaicin; Cell Line; Cloning, Molecular; Diterpenes; DNA, Complementary; Dogs; Dopamine; Dose-Response Relationship, Drug; Fluorometry; Genetic Vectors; Genotype; Humans; Molecular Sequence Data; Mutation, Missense; Phorbol Esters; Phylogeny; Pyrazines; Pyridines; Receptors, Drug; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Transfection

Transient receptor potential (TRP) receptors are, typically, calcium-permeant cation channels that transduce environmental stimuli. Both kidney epithelial and inner ear sensory cells express TRPV1, are mechanosensors and accumulate the aminoglycoside antibiotic gentamicin. Recently, we showed that Texas Red-conjugated gentamicin (GTTR) enters kidney cells via an endosome-independent pathway. Here, we used GTTR to investigate this non-endocytotic mechanism of gentamicin uptake. In serum-free buffers, GTTR penetrated MDCK cells within 30 s and uptake was modulated by extracellular, multivalent cations (Ca2+, La3+, Gd3+) or protons. We verified the La3+ modulation of GTTR uptake using immunocytochemical detection of unconjugated gentamicin. Membrane depolarization, induced by high extracellular K+ or valinomycin, also reduced GTTR uptake, suggesting electrophoretic permeation through ion channels. GTTR uptake was enhanced by the TRPV1 agonists, resiniferatoxin and anandamide, in Ca2+-free media. Competitive antagonists of the TRPV1 cation current, iodo-resiniferatoxin and SB366791, also enhanced GTTR uptake independently of Ca2+, reinforcing these antagonists' potential as latent agonists in specific situations. Ruthenium Red blocked GTTR uptake in the presence or absence of these TRPV1-agonists and antagonists. In addition, GTTR uptake was blocked by RTX in the presence of more physiological levels (2 mM) of Ca2+. Thus gentamicin enters cells via cation channels, and gentamicin uptake can be modulated by regulators of the TRPV1 channel.

Topics: Anilides; Animals; Anti-Bacterial Agents; Arachidonic Acids; Calcium; Calcium Channel Blockers; Cell Line; Cinnamates; Diterpenes; Dogs; Endocannabinoids; Fluorescent Dyes; Gadolinium; Gentamicins; Hydrogen-Ion Concentration; Indicators and Reagents; Ionophores; Kidney Tubules, Distal; Lanthanum; Membrane Potentials; Polyunsaturated Alkamides; Ruthenium Red; TRPV Cation Channels; Valinomycin; Xanthenes

Transient receptor potential vanilloid 1 (TRPV1) is a capsaicin- and heat-gated ion channel required for normal in vivo responses to these painful stimuli. However, growing evidence suggests that TRPV1 also participates in thermoregulation. Therefore, we examined the effects of a selective TRPV1 antagonist, 5-iodoresiniferatoxin (I-RTX), on mouse body temperature. Surprisingly, s.c. administration of I-RTX (0.1-1 micromol/kg) evoked a hypothermic response similar to that evoked by capsaicin (9.8 micromol/kg) in naive wild-type mice, but not in mice pretreated with resiniferatoxin, a potent TRPV1 agonist, or in naive TRPV1-null mice. In response to I-RTX in vitro, HEK293 cells expressing rat TRPV1 exhibited increases in intracellular Ca(2+) (biphasic, EC(50) = 56.7 nM and 9.9 microM) that depended on Ca(2+) influx and outwardly rectifying, capsazepine-sensitive currents that were smaller than those evoked by 1 microM capsaicin. Thus, I-RTX induces TRPV1-dependent hypothermia in vivo and is a partial TRPV1 agonist in vitro.

Topics: Animals; Body Temperature; Calcium; Capsaicin; Diterpenes; Humans; Hypothermia; Ion Channels; Mice; Mice, Inbred C57BL; TRPV Cation Channels

The Transient Receptor Potential cation channel V1 (TRPV1) is expressed in peripheral nociceptive neurons and is subject to polymodal activation via various agents including capsaicin, noxious heat, low extracellular pH, and direct phosphorylation by protein kinase C (PKC). We have cloned and heterologously expressed mouse TRPV1 (mTRPV1) and characterized its function utilizing FLIPR-based calcium imaging to measure functional responses to various small molecule agonists, low pH and direct phosphorylation via PKC. The various TRPV1 agonists activated mTRPV1 with a rank order of agonist potency of (resiniferatoxin (RTX) = arvanil > capsaicin = olvanil > OLDA > PPAHV) (EC50 values of 0.15+/-0.04 nM, 0.27+/-0.07 nM, 9.1+/-1.2 nM, 3.7+/-0.3 nM, 258+/-105 nM, and 667+/-151 nM, respectively). Additionally, mTRPV1 was activated by either low pH or with addition of the PKC activator phorbol 12-myristate 13-acetate (PMA). The TRPV1 antagonists iodinated-resiniferatoxin (I-RTX) or BCTC were both able to block capsaicin, pH and PKC-induced responses of mTRPV1 (IC50 (I-RTX) = 0.35+/-0.12 nM, 1.9+/-0.7 nM, and 0.80+/-0.68 nM, IC50 (BCTC) = 1.3+/-0.36 nM, 0.59+/-0.16 nM, and 0.37+/-0.15 nM, respectively). However, the antagonist capsazepine was only able to inhibit a capsaicin-evoked response of mTRPV1 with an IC50 of 1426+/-316 nM. Comparable results were achieved with rat TRPV1, while capsazepine blocked all modes of human TRPV1 activation. Thus, the mTRPV1 cation channel has a molecular pharmacological profile more akin to rat TRPV1 than either human or guinea pig TRPV1 and the molecular pharmacology suggests that capsazepine may be an ineffective TRPV1 antagonist for in vivo models of inflammatory pain in the mouse.

Topics: Amino Acid Sequence; Animals; Calcium; Capsaicin; Cell Line; Cloning, Molecular; Cyclic AMP-Dependent Protein Kinases; Diterpenes; Enzyme Activation; Guinea Pigs; Humans; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Intracellular Space; Ion Channels; Mice; Phorbol Esters; Phosphorylation; Rabbits; Rats; Receptors, Drug; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection; TRPV Cation Channels