Temporal and spatial summation of laser heat stimuli in cultured nociceptive neurons of the rat.

We studied the efficacy of a near-infrared laser (1475 nm) to activate rat dorsal root ganglion (DRG) neurons with short punctate radiant heat pulses (55 µm diameter) and investigated temporal and spatial summation properties for the transduction process for noxious heat at a subcellular level. Strength-duration curves (10-80 ms range) indicated a minimum power of 30.2mW for the induction of laser-induced calcium transients and a chronaxia of 13.9 ms. However, threshold energy increased with increasing stimulus duration suggesting substantial radial cooling of the laser spot. Increasing stimulus duration demonstrated suprathreshold intensity coding of calcium transients with less than linear gains (Stevens exponents 0.29/35mW, 0.38/60mW, 0.46/70mW). The competitive TRPV1 antagonist capsazepine blocked responses to short near-threshold stimuli and significantly reduced responses to longer duration suprathreshold heat. Heating 1/3 of the soma of a neuron was sufficient to induce calcium transients significantly above baseline (p < 0.05), but maximum amplitude was only achieved by centering the laser over the entire neuron. Heat-induced calcium increase was highest in heated cell parts but rapidly reached unstimulated areas reminiscent of spreading depolarization and opening of voltage-gated calcium channels. Full intracellular equilibrium took about 3 s, consistent with a diffusion process. In summary, we investigated transduction mechanisms for noxious laser heat pulses in native sensory neurons at milliseconds temporal and subcellular spatial resolution and characterized strength duration properties, intensity coding, and spatial summation within single neurons. Thermal excitation of parts of a nociceptor spread via both membrane depolarization and intracellular calcium diffusion.

The capsaicin receptor TRPV1 is the first line defense protecting from acute non damaging heat: a translational approach.

BACKGROUND: Pain is the vital sense preventing tissue damage by harmful noxious stimuli. The capsaicin receptor TRPV1 is activated by noxious temperatures, however, acute heat pain is only marginally affected in mice after TRPV1 knockout but completely eliminated in mice lacking TRPV1 positive fibers. Exploring contribution of candidate signal transduction mechanisms to heat pain in humans needs translational models. METHODS: We used focused, non-damaging, short near-infrared laser heat stimuli (wavelength 1470/1475 nm) to study the involvement of TRPV1-expressing nerve fibers in the encoding of heat pain intensity. Human psychophysics (both sexes) were compared to calcium transients in native rat DRG neurons and heterologously expressing HEK293 cells. RESULTS: Heating of dermal and epidermal nerve fibers in humans with laser stimuli of ≥ 2.5 mJ (≥ 25 ms, 100 mW) induced pain that increased linearly as a function of stimulus intensity in double logarithmic space across two orders of magnitude and was completely abolished by desensitization using topical capsaicin. In DRG neurons and TRPV1-expressing HEK cells, heat sensitivity was restricted to capsaicin sensitive cells. Strength duration curves (2-10 ms range) and thresholds (DRGs 0.56 mJ, HEK cells 0.52 mJ) were nearly identical. Tachyphylaxis upon repetitive stimulation occurred in HEK cells (54%), DRGs (59%), and humans (25%). CONCLUSION: TRPV1-expressing nociceptors encode transient non-damaging heat pain in humans, thermal gating of TRPV1 is similar in HEK cells and DRG neurons, and TRPV1 tachyphylaxis is an important modulator of heat pain sensitivity. These findings suggest that TRPV1 expressed in dermal and epidermal populations of nociceptors serves as first line defense against heat injury.

Analyses of Synthetic N-Acyl Dopamine Derivatives Revealing Different Structural Requirements for Their Anti-inflammatory and Transient-Receptor-Potential-Channel-of-the-Vanilloid-Receptor-Subfamily-Subtype-1 (TRPV1)-Activating Properties.

We studied the chemical entities within N-octanoyl dopamine (NOD) responsible for the activation of transient-receptor-potential channels of the vanilloid-receptor subtype 1 (TRPV1) and inhibition of inflammation. The potency of NOD in activating TRPV1 was significantly higher compared with those of variants in which the ortho-dihydroxy groups were acetylated, one of the hydroxy groups was omitted ( N-octanoyl tyramine), or the ester functionality consisted of a bulky fatty acid ( N-pivaloyl dopamine). Shortening of the amide linker (ΔNOD) slightly increased its potency, which was further increased when the carbonyl and amide groups (ΔNODR) were interchanged. With the exception of ΔNOD, the presence of an intact catechol structure was obligatory for the inhibition of VCAM-1 and the induction of HO-1 expression. Because TRPV1 activation and the inhibition of inflammation by N-acyl dopamines require different structural entities, our findings provide a framework for the rational design of TRPV1 agonists with improved anti-inflammatory properties.

Radiofluorinated N-Octanoyl Dopamine ([18F]F-NOD) as a Tool To Study Tissue Distribution and Elimination of NOD in Vitro and in Vivo.

To mitigate pretransplantation injury in organs of potential donors, N-octanoyl dopamine (NOD) treatment might be considered as it does not affect hemodynamic parameters in braindead (BD) donors. To better assess optimal NOD concentrations for donor treatment, we report on the fast and facile radiofluorination of the NOD-derivative [18F]F-NOD [18F]5 for in vivo assessment of NOD's elimination kinetics by means of PET imaging. [18F]5 was synthesized in reproducibly high radiochemical yields and purity (>98%) as well as high specific activities (>20 GBq/µmol). Stability tests showed no decomposition of [18F]5 over a period of 120 min in rat plasma. In vitro, low cell association was found for [18F]5, indicating no active transport mechanism into cells. In vivo, [18F]5 exhibited a fast blood clearance and a predominant hepatobiliary elimination. As these data suggest that also NOD might be cleared fast, further pharmacokinetic evaluation is warranted.

N-octanoyl dopamine treatment exerts renoprotective properties in acute kidney injury but not in renal allograft recipients.

BACKGROUND: N-octanoyl dopamine (NOD) treatment improves renal function when applied to brain dead donors and in the setting of warm ischaemia-induced acute kidney injury (AKI). Because it also activates transient receptor potential vanilloid type 1 (TRPV1) channels, we first assessed if NOD conveys its renoprotective properties in warm ischaemia-induced AKI via TRPV1 and secondly, if renal transplant recipients also benefit from NOD treatment. METHODS: We induced warm renal ischaemia in Lewis, wild-type (WT) and TRPV1(-/-) Sprague-Dawley (sd) rats by clamping the left renal artery for 45 min. Transplantations were performed in allogeneic and syngeneic donor-recipient combinations (Fisher to Lewis and Lewis to Lewis) with a cold ischaemia time of 20 h. Treatment was instituted directly after restoration of organ perfusion. Renal function, histology and perfusion were assessed by serum creatinine, microscopy and magnetic resonance imaging (MRI) using arterial spin labelling (ASL). RESULTS: NOD treatment significantly improved renal function in Lewis rats after warm ischaemia-induced AKI. It was, however, not effective after prolonged cold ischaemia. The renoprotective properties of NOD were only observed in Lewis or WT, but not in TRPV1(-/-) sd rats. Renal inflammation was significantly abrogated by NOD. MRI-ASL showed a significantly lower cortical perfusion in ischaemic when compared with non-ischaemic kidneys. No overall differences were observed in renal perfusion between NOD- and NaCl-treated rats. CONCLUSIONS: NOD treatment reduces renal injury in warm ischaemia, but is not effective in renal transplant in our experimental animal models. The salutary effect of NOD appears to be TPRV1-dependent, not involving large changes in renal perfusion.

Acetylsalicylic acid enhances tachyphylaxis of repetitive capsaicin responses in TRPV1-GFP expressing HEK293 cells.

Since many years acetylsalicylic acid (ASA) is known for its antithrombotic, antiphlogistic and analgesic effects caused by irreversible acetylation of cyclooxygenase. ASA also inhibits capsaicin- and heat-induced responses in cultured dorsal root ganglia (DRG) neurons, suggesting TRPV1 (transient receptor potential channel of the vanilloid receptor family, subtype 1) to be an additional target of ASA. We now studied the effect of ASA on heterologously expressed rat TRPV1 using calcium microfluorimetry. Capsaicin dose-dependently increased intracellular calcium with an EC50 of 0.29 µM in rTRPV1 expressing HEK293 cells. During repetitive stimulation the second response to capsaicin was reduced (53.4 ± 8.3% compared to vehicle control; p<0.005; Student's unpaired t-test) by 1µM ASA, a concentration much below the one needed to inhibit cyclooxygenase (IC50 of 35 µM in thromboxane B2 production assay). In contrast, calcium transients induced by a single stimulus of 0.3 or 1 µM capsaicin were not significantly reduced by 0.3 or 1 µM ASA. These data suggest that ASA increases the tachyphylaxis of rTRPV1 channel activation. Mechanisms are unknown and may be direct by e.g. stabilization of the desensitized state or indirect via inhibition of intracellular signaling pathways e.g. of the mitogen-activated protein kinase family (MAPK/ERK).

N-octanoyl-dopamine is an agonist at the capsaicin receptor TRPV1 and mitigates ischemia-induced [corrected] acute kidney injury in rat.

Since stimulation of transient receptor potential channels of the vanilloid receptor subtype 1 (TRPV1) mitigates acute kidney injury (AKI) and endogenous N-acyl dopamine derivatives are able to activate TRPV1, we tested if synthetic N-octanoyl-dopamine (NOD) activates TRPV1 and if it improves AKI. These properties of NOD and its intrinsic anti-inflammatory character were compared with those of dopamine (DA). TRPV1 activation and anti-inflammatory properties of NOD and DA were tested using primary cell cultures in vitro. The influence of NOD and DA on AKI was tested in a prospective, randomized, controlled animal study with 42 inbred male Lewis rats (LEW, RT1), treated intravenously with equimolar concentrations of DA or NOD one hour before the onset of warm ischemia and immediately before clamp release. NOD, but not DA, activates TRPV1 channels in isolated dorsal root ganglion neurons (DRG) that innervate several tissues including kidney. In TNFα stimulated proximal tubular epithelial cells, inhibition of NFκB and subsequent inhibition of VCAM1 expression by NOD was significantly stronger than by DA. NOD improved renal function compared to DA and saline controls. Histology revealed protective effects of NOD on tubular epithelium at day 5 and a reduced number of monocytes in renal tissue of DA and NOD treated rats. Our data demonstrate that NOD but not DA activates TRPV1 and that NOD has superior anti-inflammatory properties in vitro. Although NOD mitigates deterioration in renal function after AKI, further studies are required to assess to what extend this is causally related to TRPV1 activation and/or desensitization.

'TRPing' synaptic ribbon function in the rat pineal gland: neuroendocrine regulation involves the capsaicin receptor TRPV1.

Synaptic ribbons (SRs) are presynaptic structures thought to regulate and facilitate multivesicular release. In the pineal gland, they display a circadian rhythm with higher levels at night paralleling melatonin synthesis. To gain more insight into the processes involved and the possible functions of these structures, a series of experiments were conducted in rodents. We studied the regional distribution of a molecular marker of pineal SRs, the kinesin motor KIF3A in the gland. Respective immunoreactivity was abundant in central regions of the gland where sympathetic fibers were less dense, and vice versa, revealing that intercellular communication between adjacent pinealocytes is enhanced under low sympathetic influence. KIF3A was found to be colocalized to the transient receptor potential channel of the vanilloid receptor family, subtype 1 (TRPV1). The TRPV1 agonist capsaicin increased melatonin secretion from perifused pineals in a dose-dependent manner that was blocked by the competitive TRPV1 antagonist capsazepine. No change in free intracellular calcium was observed in response to TRPV1 ligands applied to pinealocytes responding to norepinephrine, bradykinin and/or depolarization. These data clearly indicate that TRPV1 actively regulates pineal gland function.

Co-expression of heat sensitive vanilloid receptor subtypes in rat dorsal root ganglion neurons.

Expression of the heat sensitive cation channels TRPV1 and TRPV2 was investigated by immunofluorescence in rat dorsal root ganglion (DRG) neurons. TRPV1-positive neurons were more frequent and had smaller diameters than TRPV2-positive neurons (35.7% vs 7.3%; 22.3 microm vs 27.6 microm), but size distributions overlapped and significant co-expression was seen in 20.7% of TRPV2-positive neurons (1.7% of all). Expression patterns did not differ between tissue sections typically used in immunocytochemistry and dissociated DRG neurons typically used in electrophysiology. Rectangular temperature pulses revealed two patterns of heat-evoked inward currents in small DRG neurons: low-threshold rapidly activating and high-threshold slowly activating. Slowly activating heat-evoked currents have not been described previously, but correspond to the type I heat responses of primary nociceptive afferents, which have been suggested to be mediated by TRPV2.