CATECHOLAMINES REDUCE TRANSIENT RECEPTOR POTENTIAL VANILLOID TYPE 1 DESENSITIZATION IN CULTURED DORSAL ROOT GANGLIA NEURONS

TRPV1 (transient receptor potential vanilloid type 1) is a non-selective cation channel, highly expressed in a subset of primary somatosensory neurons located in the dorsal root and trigeminal ganglia, acting as a polymodal nociceptor. It is synergistically activated by multiple noxious stimuli such as capsaicin, extracellular acidity (protons) or noxious heat (1-3), and also by other endogenous or exogenous agonists (4, 5).

A large body of experimental evidence suggests that the sympathetic nervous system is involved in increasing dorsal root ganglia (DRG) neuronal activity to promote pain. Spinal nerve ligation in rats induces a sympathetic postganglionic nerve fiber sprouting in the related DRG (6), which, by releasing norepinephrine promotes mechanical hyperalgesia (7) and augments ectopic discharges associated with neuropathic pain (8, 9). N-type Cα2+ channels or TTX (tetrodotoxin)-resistant Na+ channels activation might be responsible for such an increased sensitivity (7, 8), but TRPV1 channels could be involved as well. In vivo studies have shown that intradermal injection of capsaicin requires integrity of sympathetic nerve fibers in order to be able to increase C-nociceptor sensitivity and to promote the release of proinflammatory neuropeptides from the sensory nerve endings as part of the neurogenic inflammatory reaction (10-13). In addition, norepinephrine can increase the duration of painful sensation caused by local administration of capsaicin in experimental animals (14) and it was correlated with amplification of the inflammatory response induced by topical administration of capsaicin in humans (15). In vitro studies have shown that sympathetic nervous system sensitizes TRPV1 receptors in DRG neurons innervating colon by norepinephrine released from sympathetic efferents acting via adrenergic receptors in a cystathionine β-synthetase-dependent manner (16) or that it can upregulate TRPV1 expression by ATP released from sympathetic efferents via a PKC (protein kinase C) cascade (17). However, there are no data showing if reduced desensitization might also contribute to increased excitability in DRG neurons.

Primary sensory neurons normally express α1 (18-20), α2 (21, 22) and β2 adrenergic receptors (23), the expression of which is altered after injuries of peripheral nerve fibers or inflammatory processes (24-26). Of all of them, α1 adrenergic receptors seemed to be more involved in modulating capsaicin induced-response:

1) skin inflammatory responses induced by capsaicin depend on them (11, 12, 27); 2) preincubation with α1 blocker prazosin, but not with α2 blocker yohimbine blocks the capsaicin-evoked substance P (SP) release in E15 embryonic rat primary sensory neurons (28); 3) selective α1 agonist phenylephrine increases capsaicin-evoked SP release from primary cultured DRG neurons (29); 4) incubation with α1 adrenergic receptor agonists causes depolarization and increased neuronal excitability in primary cultures of DRG neurons (30), and (5) administration of the selective α2 adrenoreceptor agonist clonidine for 4 days decreases capsaicin-evoked SP release, but not basal SP release (31).

Considering these data, in the present study we aimed to evaluate the effects of epinephrine and norepinehrine on TRPV1 desensitization induced by repeated applications of capsaicin in dorsal root ganglia cultured neurons and to assess the involvement of the major α1, α2 and β adrenergic receptors subtypes in mediating this effect.

MATERIALS AND METHODS

Animals

For this study, 38 adult male Wistar rats (100 – 150 g) from the animal facility of the ‘Ion Cantacuzino’ National Institute, Bucharest, Romania were used.

All procedures were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 86-23) revised in 1996 and with Directive 2010/63/EU revising Directive 86/609/EEC on the protection of animals used for scientific purposes and were approved by the Bioethics Committee of the Faculty of Biology, University of Bucharest.

Dorsal root ganglia primary cultures

Rats were killed by inhalation of 100% CO2 followed by decapitation. DRG from all spinal segments were removed and dissociated in 0.5 mg/ml collagenase IA and 2 mg/ml dispase (Gibco, 17105041) for 1 hour at 37°C, then plated on 13-mm glass coverslips pretreated with poly-D-lysine (0.1 mg/ml for 30 min) and cultured in a NGF-free 1:1 mixture of 7.4 mM glucose DMEM and Hams’s F10 medium with 10% horse serum, 0.5% Penicillin/Streptomycin and 1% L-glutamine (Thermo Fisher Scientific, α1286001) at 37°C, in 5% CO2 in air. If not otherwise specified, all reagents were from Sigma. Experiments were conducted at room temperature (25°C), 24 hours after plating the cells.

Intracellular Cα2+ imaging

DRG neurons cultured on coverslips were incubated for 30 min at 37°C in standard extracellular solution (see Solutions below) containing 2 µM Calcium Green-1 AM and 0.02% Pluronic F-127 (both from Thermo Fisher Scientific, Waltham, MA, USA), washed and left to recover for 30 min before use. Coverslips were mounted in a Teflon chamber (RC-40HP, Harvard Apparatus, Holliston, MA, USA) on the stage of an Eclipse TE300 inverted microscope (Nikon, Tokyo, Japan) and left for 5 minutes to adapt to the extracellular solution flow at 25°C. Neurons were illuminated with an Optoscan monochromator (Cairn Instruments, Faversham, UK) and the fluorescence changes were captured with a 12-bit CCD SensiCam camera (PCO, Kelheim, Germany). Data were recorded and analyzed using Axon Imaging Workbench 4.0 (Indec Biosystems, Mountain View, CA, USA). After background subtraction, data were quantified as ΔF / F0 for each recorded cell, representing the ratio between the maximum fluorescence change during the stimulus and the baseline fluorescence before the stimulus.

The stimulation protocol included 5 consecutive applications of 0.3 µM capsaicin for 20 s spaced at 4-min intervals, with a final application of 50 mM KCl to check if the cells were viable neurons. To establish capsaicin sensitivity, only the responses to the 1st capsaicin application were used to avoid tachyphylaxis and desensitization known to occur at repetitive capsaicin application (32). A histogram with all the analyzed cells in control conditions, after the 1st capsaicin application (n = 193, see also Table 1, 1st row) was fitted with a two-peak envelope of Gaussian functions and the cutoff value was taken as the mean ± 2 SD of the Gaussian peak centered closest to zero (Fig. 1). The cells which responded to capsaicin application with a ΔF / F0 ≥ 0.08 were considered capsaicin-sensitive (n = 136). Out of these cells were selected for further analysis only the cells that had ΔF / F0 ≥ 0.08 for each of the first 3 capsaicin applications (n = 49, see also Table 1, 2nd row). As above, the histogram for this population was based only on the responses to the 1st capsaicin application (Fig. 1).

Fig. 1. Histogram of all DRG neurons that responded to capsaicin application, based on which the capsaicin sensitivity was established at ΔF / F0 ≥ 0.08. Included is also the histogram of the DRG neurons which were further selected for analysis.

Table 1. Mean ΔF / F0 values for capsaicin applications in control experiments.

In the selected cells, the 4th capsaicin application was preceded by a 1-min application of either 1 µM epinephrine, 10 µM norepinephrine or a combination of 1 µM epinephrine or 10 µM norepinephrine plus 10 µM phentolamine / 10 µM prazosin / 10 µM propranolol. The adrenergic agents were applied during the 4th capsaicin application as well. The 5th capsaicin application was made to test the persistence of cathecolamines’ effect. For the 4th and 5th capsaicin application, the ΔF / F0 ≥ 0.08 criteria was also applied.

By this protocol we wanted to eliminate the cells which have undergone excessive desensitization and had responses below the threshold after the 2nd application of capsaicin, and test the effects of cathecolamines on the population of cells which, even though continued to show desensitization, had responses above the threshold after the 2nd application of capsaicin.

Solutions

Standard extracellular Ringer solution contained (in mM): NaCl 140, KCl 4, MgCl2 1, CaCl2 2, HEPES 10, NaOH 4.54, pH 7.40 at 25°C, freshly supplemented with D-glucose 7.4 mM. Drugs were freshly added from stock solutions prepared as follows: 1 mM capsaicin in ethanol, 10 mM (±)-epinephrine (Sigma, E4642) in 1M HCl, 10 mM DL-norepinephrine hydrochloride (Sigma, A7256), 10 mM (±)-propranolol hydrochloride (Sigma, P0884), 10 mM phentolamine hydrochloride (Sigma, P7547), and 10 mM prazosin hydrochloride (Sigma, P7791) in deionized water.

Data analysis

Analysis was performed using Axon Imaging Workbench 4.0 (Indec Biosystems, Mountain View, CA, USA), Microsoft Excel 2007 (Microsoft Inc., USA), Prism 5.01 (GraphPad Software Inc., USA) software and a macro analysis routine written by Liviu Soltuzu for Microsoft Excel 2007. All data were reported as means ± S.E.M.; statistical significance was tested using two-tailed Mann-Whitney test and a value of P < 0.05 was set as level of significance.

RESULTS

To investigate the effect of adrenergic stimulation on TRPV1 desensitization, we examined the effect of 1 µM epinephrine and 10 µM norepinephrine applied for 1 min before and during the 4th capsaicin application in a population of cells selected according to the criteria mentioned above. The fluorescence change between the 4th capsaicin application and the 3rd one showed a significant increase after both epinephrine and norepinephrine compared to control conditions, suggesting a strong reduction of TRPV1 desensitization by both catecholamines (4th to 3rd capsaicin ΔF/F0 ratio (%) in control conditions was 83.97 ± 6.1%, n = 49, after epinephrine it was 130.6 ± 11.01%, n = 68, P < 0.05, and after norepinephrine 130.6 ± 7.6%, n = 37, P < 0.05, Fig. 2a-2c and Table 2). Surprisingly, the mean for both catecholamines was equal even though the population size was different, suggesting higher ratio values in the norepinephrine population than in the epinephrine population. In contrast, the fluorescence change between the 5th capsaicin application and the 3rd one showed no difference after both epinephrine and norepinephrine compared to control conditions, suggesting that the reduced desensitization of TRPV1 receptors was short-lasting (5th to 3rd capsaicin ΔF / F0 ratio (%) in control conditions was 95.83 ± 11.67%, n = 49, after epinephrine it was 72.07 ± 5.54%, n = 68, P > 0.05, and after norepinephrine 91.22 ± 8.23 %, n = 37, P > 0.05, Fig. 2d).

Table 2. Number of measured and selected cells in all conditions.

Fig. 2. Epinephrine and norepinephrine transiently reduce TRPV1 desensitization. Representative traces of 5 consecutive applications of 0.3 µM capsaicin, with 60-s application of either epinephrine (a) or norepinephrine (b) before and during the 4th capsaicin application. The insert shows magnified responses to the 4th compared to the preceding (3rd) capsaicin application in the presence of catecholamines versus control conditions. Traces in (a) and (b) were processed by subtraction of a linear decay in ΔF / F0 due to photobleaching. Mean ΔF / F0 ratio during 4th versus 3rd capsaicin application (c) and 5th versus 3rd capsaicin application (d) with catecholamines and in control conditions (mean ± SEM). *** P < 0.001. Cps - capsaicin; Epi - epinephrine; NorEpi - norepinephrine.

To confirm that catecholamines have indeed an effect on TRPV1 receptors, we performed experiments in which we tested if pre-incubation with epinephrine sensitizes TRPV1 receptors. Previously it has been shown that 5 min incubation with 10 µM norepinephrine sensitizes TRPV1 receptors from T13-L2 DRG neurons which specifically innervate colon (16). In our case, 80 seconds incubation with 1 µM epinephrine applied before and during 0.3 µM capsaicin application, was able to sensitize TRPV1 response (Fig. 3a), even though the culture included DRG from all spinal segments. More specifically, epinephrine pre-incubation led to a ~13% increase in the response to capsaicin: control ΔF / F0 = 0.509 ± 0.031, n = 166; epinephrine pre-incubation ΔF / F0 = 0.574 ± 0.027 n = 111, P < 0.05, Fig. 3b. Our data were lower than previously reported results, most likely due to the fact that we used DRG coming from more spinal levels which could induce additional heterogeneity in TRPV1 response to catecholamines.

Fig. 3. Pre incubation with epinephrine sensitizes TRPV1 receptors. (a) Representative traces for the first response to capsaicin in control conditions or after 80 seconds pre-incubation with 1 µM epinephrine. (b) Mean ΔF / F0 ratio after incubation with epinephrine, compared to 0.3 µM capsaicin application.

In order to exclude a possible Cα2+ influx due to catecholamines, we performed a set of experiments in which 80 seconds application of 1 µM epinephrine or 10 µM norepinephrine were followed at 4-min intervals by 0.3 µM capsaicin and 50 mM KCl applications. As shown in Fig. 4, neither epinephrine nor norepinephrine did not induce by themselves an increase in [Cα2+]i compared to an application of Ringer solution of similar length: mean ΔF / F0 for epinephrine was 0.04 ± 0.01, n = 53 and for Ringer it was 0.025 ± 0.005, n = 53; the two values were not significantly different (P > 0.05, Fig. 4a and 4b). Similarly, mean ΔF / F0 for norepinephrine was 0.07 ± 0.01, n = 51 and for Ringer it was 0.06 ± 0.01, n = 51; the two values were not significantly different (P > 0.05, Fig. 4c and 4d). In both cases, the [Cα2+]i increase induced by capsaicin was significantly above the pre-established threshold, i.e mean ΔF / F0 in epinephrine experiments was 0.79 ± 0.01, n = 53, and in norepinephrine experiments it was 0.69 ± 0.06, n = 51, Fig. 4a-4d.

Fig. 4. Epinephrine and norepinephrine alone do not induce a calcium influx. (a) Mean ΔF / F0 ratio after epinephrine alone, compared to Ringer and 0.3 µM capsaicin and a representative trace (b). (c) Mean ΔF / F0 ratio after norepinephrine alone, compared to Ringer and 0.3 µM capsaicin and a representative trace (d). Traces in (b) and (d) were pre-processed by removal of photobleaching effects.

To further examine if the effect of catecholamines on TRPV1 desensitization occured via a and/or b adrenergic receptor stimulation, we tested the effect of epinephrine and norepinephrine on the 4th capsaicin response in the presence of 10 µM phentolamine (a non-selective a adrenergic antagonist), 10 µM prazosin (an α1 receptor antagonist) and 10 µM propranolol (a non-selective β antagonist). Phentolamine and prazosin significantly reduced the effects of both epinephrine and norepinephrine: 4th to 3rd capsaicin ΔF / F0 ratio (%) after epinephrine and phentolamine was 58.18 ± 8.28%, n = 32, and after epinephrine and prazosin it was 77.13 ± 7.82%, n = 59; these values were significantly smaller than after epinephrine alone (130.6 ± 11.01%, n = 68, P < 0.05, Fig. 5a, 5b and Table 2). In a similar manner, 4th to 3rd capsaicin ΔF / F0 ratio (%) after norepinephrine and phentolamine was 102.2 ± 11.21%, n = 40, and after norepinephrine and prazosin it was 57.46 ± 7.63%, n = 42; these values were significantly smaller than after norepinephrine alone (130.6 ± 7.6%, n = 37, P < 0.05, Fig. 5c, 5d, and Table 2). On the other side, propranolol reduced in a significant manner only the epinephrine action, and had no impact on the norepinephrine sensitizing effect: 4th to 3rd capsaicin ΔF / F0 ratio (%) after epinephrine and propranolol was 96.75 ± 9.98%, n = 46; the value was significantly smaller than after epinephrine alone, P < 0.05, Fig. 5a and 5b, while 4th to 3rd capsaicin ΔF / F0 ratio (%) after norepinephrine and propranolol was 163.3 ± 13.74%, n = 52; this value showed no significant effect compared to norepinephrine alone (P > 0.05, Fig. 5c, 5d and Table 2).

Fig. 5. Adrenergic antagonists specifically block catecho-lamines effects. (a) Mean ΔF / F0 ratio of epinephrine alone or co-applied with phentolamine, prazosin, or propranolol (10 µM each). (b) Representative traces (3rd and 4th capsaicin application) of the effects of epinephrine alone or combined with alpha- or beta-adrenergic inhibitors. (c) Mean ΔF / F0 ratio of norepinephrine alone or co-applied with phentolamine, prazosin, or propranolol (10 µM each). (d) Representative traces (3rd and 4th capsaicin application) of the effects of norepinephrine alone or combined with alpha- or beta-adrenergic inhibitors. Values are represented as mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001. Cps - capsaicin; Epi - epinephrine; NorEpi - norepinephrine.

DISCUSSION

The present study showed that epinephrine and norepinephrine reduce in a transient manner TRPV1 desensitization induced by repeated applications of capsaicin in cultured DRG neurons. Both catecholamines showed similar effects, which were mediated by α1, α2 and ± β2 receptors.

A well-known characteristic of TRPV1 functioning is that its response can be sensitized or desensitized. The sensitization which can be mediated via phosphorylation by PKC (33), particularly the minor e isoform, PKA (34), CaMKII (35) or tyrosine kinases like the JAK/STAT pathway or a non-receptor c-Src kinase (36), is associated with higher or longer responses to specific agonists, which subsequently depolarize the neurons and increase excitability. Desensitization, which can be mediated via protein phosphatases dephosphorylation (37), by calmodulin interaction with TRPV1 N-terminal residues in a Ca-dependent manner (38, 39) or by IP3-induced calcium release from intracellular stores (40), is associated with reduced responses to consecutive applications of capsaicin, and therefore reduced excitability.

Reduced desensitization is also associated with increased excitability, because under this condition TRPV1 receptors are able to still open to new/repetitive stimuli, and thus allow depolarization. Reduced desensitization was associated with increased functioning of TRPV1 receptors after 4 h incubation with 1.5 µM CXCL1 chemokine (41) or after overnight exposure to hypoxia/hyperglycemia specific to diabetic conditions (42). In our experiments we also noticed a reduced desensitization when either catecholamine was applied before the 4th capsaicin application, which lasted only for few minutes and did not affect the 5th capsaicin application as well. These data show that reduced desensitization, besides increased sensitization which was shown before (16) and it was reconfirmed in our experiments, is an additional mechanism that could account for increased pain sensitivity to capsaicin under adrenergic stimulation. In the experiments testing the effect on the rate of desensitization or reconfirming sensitization we applied catecholamines both before and during capsaicin application. Since catecholamines have no effect by themselves on the [Cα2+]i (Fig. 4), their effects on TRPV1 response is most likely due to events that occur before capsaicin application.

Of the adrenergic receptors, α1 and α2 adrenoreceptors have equal affinity for epinephrine and norepinephrine, while β2 receptors have a significantly greater affinity for epinephrine than for norepinephrine (43). In our study, the experiments with antagonists suggested a more important contribution of a adrenergic receptors in mediating the effects on TRPV1 desensitization. Phentolamine, a nonselective α-adrenergic antagonist and prazosin, an α1 receptor antagonist, significantly inhibited both epinephrine and norepinephrine effects. The significant difference (P > 0.05) between the effect of phentolamine on epinephrine and on norepinephrine despite the fact that the two catecholamines can equally bind to a receptors, could only be explained by the fact that this drug is more effective in antagonizing responses to circulating epinephrine and/or norepinephrine than in antagonizing responses to mediator released at the adrenergic nerve ending (44). Since in normal conditions circulating norepinephrine is far less than epinephrine (i.e. 20% of the total adrenal secretion of catecholamines (43), this effect might suggest that on adrenergic receptors in DRG neurons phentolamine antagonizes in a competitive manner preferentially epinephrine binding, and less strongly, although still statistically significant, norepinephrine binding. In the case of prazosin there was no statistical difference between the effects on epinephrine and on norepinephrine (P < 0.05), suggesting an equal competitive antagonism for both epinephrine and norepinephrine. On the other hand, propranolol, a nonselective β-adrenergic blocker, significantly inhibited only the effect of epinephrine, although slightly less than α adrenergic antagonists. Our data are in agreement with the known reduced affinity of β2 receptors for norepinephrine, but is in contrast with experimental data in which visceral hypersensitivity associated with irritable bowel syndrome is mediated by β2-adrenergic receptors mainly activated by norepinephrine (16). However, in our case we used DRG neurons from all spinal levels, while in the above mentioned study they used only T13-L2 DRG neurons which specifically innervate the colon. It is possible that this β2 population which was more sensitive to norepinephrine was too small to influence the mean response in the whole population of neurons that was analyzed in our experiments.

Extended activation of TRPV1 receptors due to catecholamines-induced decreased desensitization may initiate a cascade of events that could potentiate even more the pain sensitivity. One such mechanism is the activation of other receptors, like TRPα1, which are known to be highly co-expressed with TRPV1 in a subset of small- to medium-diameter peripheral sensory neurons (45) and to be dependent of TRPV1 for the enhancement of adrenaline secretion from the adrenal gland by its specific agonists (46). The adrenaline thus secreted may act back on DRG neurons in a positive feedback loop that may further activate TRPV1 as shown above, increasing pain sensitivity. Even more, co-expression of TRPV1 and TRPα1 at the epidermal keratinocytes level contribute to acute pain in herpes zoster or diabetes (47, 48) and may facilitate as well releasing of proinflammatory neuropeptides from the sensory nerve endings (12) or increasing the epidermal blood flow as part of the neurogenic inflammatory reaction (49). Therefore, by better understanding the functioning of TRPV1 receptors and by putting it in a broader physiological context in which these receptors are not independent but cooperate with other receptors or ionic channels, we can get a more clear picture of the pathophysiological mechanism of pain associated with different diseases, and in particular skin diseases.

Overall, our data suggest that the molecular mechanism through which sympathetic nervous system could induce increased DRG neuronal activity to promote pain, besides sensitization or upregulation of TRPV1 receptors, is also by reducing TRPV1 desensitization. In addition to the already known data that α1 receptors are mainly responsible for mediating cathecolamines’ effects on TRPV1 capsaicin-induced response, we showed that α2 and β2 receptors also have a contribution, as suggested by the phentolamine studies which antagonizes α2 receptors as well, and by the propranolol studies which were more specific for antagonizing epinephrine binding than for norepinephrine binding. This study completes the general information about TRPV1 sensitization via adrenergic stimulation and may open perspectives for novel pharmacological approaches in skin inflammatory disorders and pain therapy.

Acknowledgements: The authors express their gratitude to Prof. Leon Zagrean, Alexandru Babes, Cristian Neacsu, Adriana Georgescu, Livia Petrescu for support and to Cornelia Dragomir and Geanina Haralambie for expert technical assistance. This research was funded by the Romanian Government via UEFISCDI (Executive Unit for Higher Education, Research, Development and Innovation Funding) grants 117/2011 awarded to V.R., 33891/2014 awarded to C.C., and PN2 80/2012 awarded to Prof. dr. Aurel Popa-Wagner.

A. Filippi and C. Caruntu the authors contributed equally to this publication.

Conflict of interests: None declared.

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