olvanil and iodoresiniferatoxin

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

Olvanil ((N-vanillyl)-9-oleamide), a non-pungent transient receptor potential vanilloid type 1 agonist, desensitizes nociceptors and alleviates pain. But its molecular targets and signaling mechanisms are little known. Calcium influx through voltage-activated Ca(2+) channels plays an important role in neurotransmitter release and synaptic transmission. Here we determined the effect of olvanil on voltage-activated Ca(2+) channel currents and the signaling pathways in primary sensory neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated rat dorsal root ganglion neurons. Olvanil (1 microM) elicited a delayed but sustained inward current, and caused a profound inhibition (approximately 60%) of N-, P/Q-, L-, and R-type voltage-activated Ca(2+) channel current. Pretreatment with a specific transient receptor potential vanilloid type 1 antagonist or intracellular application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid abolished the inhibitory effect of olvanil on voltage-activated Ca(2+) channel current. Calmodulin antagonists (ophiobolin-A and calmodulin inhibitory peptide) largely blocked the effect of olvanil and capsaicin on voltage-activated Ca(2+) channel current. Furthermore, calcineurin (protein phosphatase 2B) inhibitors (deltamethrin and FK-506) eliminated the effect of olvanil on voltage-activated Ca(2+) channel current. Notably, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin antagonists, and calcineurin inhibitors each alone significantly increased the amplitude of voltage-activated Ca(2+) channel current. In addition, double immunofluorescence labeling revealed that olvanil induced a rapid internalization of Ca(V)2.2 immunoreactivity from the membrane surface of dorsal root ganglion neurons. Collectively, this study suggests that stimulation of non-pungent transient receptor potential vanilloid type 1 inhibits voltage-activated Ca(2+) channels through a biochemical pathway involving intracellular Ca(2+)-calmodulin and calcineurin in nociceptive neurons. This new information is important for our understanding of the signaling mechanisms of desensitization of nociceptors by transient receptor potential vanilloid type 1 analogues and the feedback regulation of intracellular Ca(2+) and voltage-activated Ca(2+) channels in nociceptive sensory neurons.

Topics: Animals; Aryldialkylphosphatase; Calcium Channels; Calcium Channels, N-Type; Capsaicin; Chelating Agents; Diterpenes; Dose-Response Relationship, Radiation; Drug Interactions; Egtazic Acid; Electric Stimulation; Enzyme Inhibitors; Ganglia, Spinal; Immunohistochemistry; Male; Membrane Potentials; Neural Inhibition; Neurons, Afferent; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Signal Transduction; TRPV Cation Channels

Certain fatty acid amides such as anandamide (AEA) and olvanil are agonists for the transient receptor potential, vanilloid-1 (TRPV1) receptor, but have been found to activate TRPV1-containing C-fibers in some tissues but not others. We used extracellular recording and whole-cell patch clamp techniques to investigate the effect of olvanil and AEA on different types of vagal C-fibers innervating the same tissue, namely jugular and nodose vagal C-fibers in guinea pig lungs. A 30 s exposure to AEA and olvanil caused action potential discharge in all nodose C-fiber innervating lung but failed to activate jugular C-fibers innervating lung and airways. The activation of nodose C-fibers was blocked by the TRPV1 antagonist iodo-resiniferatoxin. In whole-cell patch clamp recordings of dissociated nodose and jugular capsaicin-sensitive neurons labeled from lungs and airways, olvanil induced large TRPV1-dependent inward currents in cell bodies of both nodose and jugular ganglion neurons. Prolonged exposure (up to 5 min) to olvanil caused action potential discharge in jugular C-fiber innervating lung but the onset latency was four times longer in jugular than in nodose C-fibers. The onsets of capsaicin response in nodose and jugular C-fibers were not different. Decreasing the tissue temperature to 25 degrees C increased the onset latency of olvanil-induced activation of nodose C-fibers 2-3-fold, but did not effect the latency of the capsaicin response. Capsaicin, olvanil, and AEA stimulate jugular C-fibers leading to tachykinergic contractions of isolated bronchi. The time to reach half-maximum is more than four times longer for olvanil and AEA, as compared to capsaicin in evoking contractions. We conclude that brief exposure to certain fatty acid amides, such as AEA and olvanil activate nodose but not jugular C-fiber terminals in the lungs. We hypothesize that this is because the nodose C-fiber terminals are equipped with a temperature-dependent mechanism for effectively and rapidly transporting the TRPV1 agonists so that they gain access to the intracellular binding sites on TRPV1. This transport mechanism may be differently expressed in two distinct subtypes of pulmonary C-fiber terminals innervating the same tissue.

Topics: Action Potentials; Animals; Arachidonic Acids; Bronchi; Capsaicin; Diterpenes; Endocannabinoids; Glomus Jugulare; Guinea Pigs; In Vitro Techniques; Lung; Male; Nerve Fibers, Unmyelinated; Nodose Ganglion; Polyunsaturated Alkamides; Temperature; Time Factors; Trachea; TRPV Cation Channels; Vagus Nerve

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