ATP release mechanisms of endothelial cell-mediated stimulus-dependent hyperalgesia

Abstract

Endothelin-1 acts on endothelial cells to enhance mechanical stimulation-induced release of ATP, which in turn can act on sensory neurons innervating blood vessels to contribute to vascular pain, a phenomenon we have referred to as stimulus-dependent hyperalgesia (SDH). In the present study we evaluated the role of the major classes of ATP release mechanisms to SDH: vesicular exocytosis, plasma membrane associated ATP synthase, ATP-Binding Cassette (ABC) transporters, and ion channels. Inhibitors of vesicular exocytosis (i.e., monensin, brefeldin A and bafilomycin), plasma membrane associated ATPase (i.e., oligomycin and pigment epithelium-derived factor-derived peptide 34-mer) and connexin ion channels (carbenoxolone and flufenamic acid), but not ABC transporters (i.e., dipyridamole, nicardipine or CFTR_inh-172) attenuated stimulus-dependent hyperalgesia. These studies support a role of ATP in SDH, and suggest novel targets for the treatment of vascular pain syndromes.

Introduction

We have recently discovered a novel mechanism by which endothelial cells, which line the lumen of blood vessels, contribute to peripheral vascular pain mechanisms ^29–32. We demonstrated two separate mechanisms by which endothelin-1 (ET-1) causes hyperalgesia; intradermal administration of ET-1 decreases nociceptive threshold to a similar magnitude when measured for the first time at 15 or 30 min after administration, however, when readings are taken every 5 min, in the interval between 15 and 30 min, there is a progressive decrease in nociceptive threshold, i.e., the mechanical stimulation of the testing itself enhanced ET-1 hyperalgesia ³⁰. This phenomenon, referred to as stimulus-dependent hyperalgesia (SDH), is elicited by two prominent vasoactive compounds, ET-1 and epinephrine, acting at their cognate receptors on endothelial cells to produce a state in which mechanical stimulation now produces enhanced release of ATP that, in turn, acts at P2X3 receptors on sensory neurons ⁹. One key feature of SDH that remains to be elucidated is the mechanism mediating ATP release, in response to the mechanical stimulus in the ET-1 and epinephrine sensitized endothelial cell.

There are several mechanisms that have been described by which ATP is released from cells, including: 1) vesicular exocytosis (ATP is found in high concentration in synaptic vesicles where it is thought to function as a co-transmitter ⁴), 2) plasma membrane associated ATP synthase (this enzyme is found in the plasma membrane as well as mitochondrial membrane ^{8, 10}), 3) ATP-binding cassette (ABC) transporters ^{41, 50}, and 4) various ion channels ^{23, 25, 41, 55} (e.g., connexin hemichannels, pannexin channels, maxi-anion channels, volume regulated anion channels and P2X7 receptors). Several of these mechanisms have already been demonstrated to be present in endothelial cells ^{40, 41} and, importantly from a practical standpoint, there is a relatively well-developed pharmacology available for the study of these mechanisms. Therefore, in the present study we evaluated the role of these ATP release mechanisms in ET-1 induced SDH.

Materials and Methods

Animals

Experiments were performed on male Sprague Dawley rats (200–250 g; Charles River). Animals were housed three per cage, under a 12 h light/dark cycle, in a temperature- and humidity-controlled environment. Food and water were available ad libitum. All behavioral nociceptive testing was performed between 10:00 A.M. and 4:00 P.M. All experimental protocols were approved by the University of California, San Francisco Committee on Animal Research and conformed to National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used and their suffering.

Nociceptive testing

Rats were acclimatized to the experimental environment and behavioral testing procedures, before performing experiments. To acclimatize rats to the testing environment, they were brought to the experimental area in their home cage and left in their cage for 15–30 min, after which they were placed in a restrainer (cylindrical transparent acrylic tubes that have side openings to allow extension of the hind limbs from the restrainer, for nociceptive testing). Rats were left undisturbed in the restrainer for another 15–30 min before nociceptive testing was started.

The nociceptive flexion reflex was quantified with an Ugo Basile Analgesymeter (Stoelting^®), which applies a linearly increasing mechanical force to the dorsum of the rat’s hind paw. Nociceptive threshold was defined as the force, in grams, at which the rat withdrew its paw; the experimenter was not blinded to treatment group. Baseline nociceptive threshold, was defined as the mean of three readings taken at 5-min intervals, determined prior to all experiments. Hyperalgesia was defined as a decrease in mechanical nociceptive threshold, here presented as percent reduction from baseline [% reduction in threshold = (pretreatment threshold - post-treatment threshold)/(pretreatment threshold)] × 100. Each paw was treated as an independent measure; both paws of the same rat received the same treatment ^{2, 3, 36}. Each experiment was performed on separate groups of rats. Inhibitors were injected intradermally into the dorsum of both hind paws at the site of nociceptive testing (to determine whether the inhibitors alone affected nociceptive threshold), 15 min before the administration of ET-1 and paw-withdrawal thresholds compared, before and after drug treatment. The doses used in this study are based on a very large number of studies that have used 1 μg as a screening dose for intradermal drugs ^{14, 29–33}, all inhibitors were administered at concentrations known to inhibit their ATP release mechanisms ^{1, 13, 16, 17, 48, 62}.

Drugs

Vesicular exocytosis mechanisms

The role of exocytosis (e.g., vesicular exocytosis) mechanisms was studied by administering monensin (Sigma Chemical Co., St. Louis, MO), which interferes with vesicle formation from the Golgi formation ^{4, 39}, bafilomycin A1 (VWR International, Brisbane CA), an inhibitor of vacuolar H⁺-ATPases that produce proton gradients in endoplasmic reticulum thereby reducing the driving force for uptake of ATP into the vesicle ²⁰ and brefeldin A (Sigma Chemical Co) which blocks the activation of a subset of ADP-ribosylation factors and thereby inhibiting vesicle trafficking ³⁸.

ATP-binding cassette (ABC) transporters

ATP-binding cassette (ABC) transporters have been implicated in ATP release, including the multidrug resistance gene product, MDR1 (also known as P-glycoprotein, ABCB1 ⁵⁹), and the cystic fibrosis transmembrane conductance regulator CFTR (ABCC7) ⁵⁸. We tested for a contribution of ABC transporters to SDH by administering ABC transporter inhibitors dipyridamole (Sigma Chemical Co.), an inhibitor of MDR1/P-glycoprotein/BCRP ⁵², and nicardipine (Santa Cruz Biotechnology, Paso Robles, CA), a dihydropyridine in different chemical class from dipyridamole, that is another potent inhibitor of MDR1/P-glycoprotein ⁵², as well as breast cancer resistance protein ⁶⁷. We also tested the highly selective inhibitor of CFTR, CFTR_inh-172 (Santa Cruz Biotechnology), a thiazolidinone compound that acts directly on the CFTR ^{57, 58}.

Ion channels

Finally, the role of ion channels (e.g., connexin hemichannels, pannexin channels, maxi-anion channels, and volume regulated anion channels) has been commonly studied by administering the gap junction inhibitor flufenamic acid (Sigma Chemical Company), an inhibitor of Connexin 43 ²², and carbenoxolone (Sigma Chemical Company), a non-specific gap junction blocker ¹².

Plasma membrane ATP synthase

In addition to its synthesis by mitochondria, ATP is also synthesized by a plasma membrane ATP synthase. The role of plasma membrane ATP synthase was evaluated by administering the ATP synthesis inhibitor oligomycin (Sigma Chemical Co.) ⁴⁶ and PEDF (pigment epithelium-derived factor peptide 34-mer, Phoenix Pharmaceutical, Brisbane, CA) a ligand of cell-surface F1 ATP synthase, that only inhibits extracellular ATP synthesis ^{13, 47}, to rule out the mitochondrion as the site of ATP synthase inhibition.

All drugs were administered intradermally (i.d.) in a volume of 5 μl using a 30-gauge hypodermic needle attached to a micro-syringe (Hamilton, Reno, NV) by PE-10 polyethylene tubing. All inhibitors were administered 15 min prior to ET-1 (Fisher Scientific, Houston, TX) and nociceptive thresholds measured (four times), at 15, 20, 25 and 30 min post ET-1. The per se effect of all the inhibitors were separately evaluated and none had a significant effect on paw-withdrawal threshold of the naïve rats (data not shown). Monensin¹, oligomycin ⁶², PEDF ¹³, carbenoxolone ¹⁶, flufenamic acid ¹⁷, brefeldin A and bafilomycin ⁴⁸ were given at concentrations that have been shown to inhibit ATP release or degradation.

Statistics

The dependent variable in experiments evaluating cutaneous nociceptive threshold was change in paw withdrawal threshold from the pretreatment baseline threshold. Group data are represented as mean ± SEM. Statistical significance was determined by one- or two-way repeated-measures ANOVA, followed by Dunnet’s post hoc test. p < 0.05 was considered statistically significant.

Results

Vesicular exocytosis

We administered three inhibitors of vesicular release mechanisms, monensin, brefeldin A and bafilomycin, to evaluate the role of this release mechanism in endothelial cell mediated ET-1-induced SDH. Rats received either vehicle (0.9% sodium chloride), monensin, bafilomycin or brefeldin A, 15 min before ET-1 administration. Nociceptive threshold was evaluated every 5 min beginning 15 min after ET-1, the standard protocol for detecting SDH ^{30, 31}. In rats pretreated with vehicle, ET-1 hyperalgesia increased with each subsequent test of mechanical threshold, indicating the presence of SDH, as previously described ³⁰. However, in rats pretreated with either monensin, brefeldin A or with bafilomycin, this enhancement of hyperalgesia by mechanical stimulation was abolished (Figure 1). Monensin also affects ET-1 hyperalgesia, a phenomenon we have previously observed with β₂-adrenergic and 5HT_1B/D receptor antagonists ³¹, presumably due to action on the nociceptor terminal.

Figure 1. Effect of bafilomycin (vacuolar H-ATPase inhibitor), monensin (inhibitor of vesicle formation) and brefeldin A (inhibitor of vesicle transport) on ET-1 induced mechanical hyperalgesia and stimulus-dependent hyperalgesia (SDH).

15 min before ET-1, rats received vehicle (5 μl), bafilomycin, monensin or brefeldin A (each 1 μg in 5 μl/paw). Paw withdrawal thresholds were measured 15, 20, 25 & 30 min after ET-1 administration. Bafilomycin, monensin and brefeldin A each significantly inhibited ET-1–induced SDH compared to vehicle treated controls, and monensin also significantly inhibited ET-1 hyperalgesia (*P < 0.001, two-way repeated measures ANOVA, followed by Bonferroni post test, N = 6).

ATP-binding cassette (ABC) transporters

We administered inhibitors of three ATP-binding cassette (ABC) transporters, dipyridamole, nicardipine and CFTR_inh-172, to evaluate the role of ABC transporters in endothelial cell mediated SDH. Rats received vehicle (0.9% sodium chloride, or 10% DMSO in 0.9% saline for CFTR_inh-172), dipyridamole, nicardipine or CFTR_inh-172 15 min before ET-1. Nociceptive threshold was evaluated every 5 min beginning 15 min after ET-1. Neither dipyridamole, nicardipine nor CFTR_inh-172 affected the development of SDH, but CFTR_inh-172 significantly attenuated ET-1–induced hyperalgesia (Figure 2).

Figure 2. Effect of dipyridamole, nicardipine and CFTR_inh-172 (ABC transport inhibitors) on ET-1 induced mechanical hyperalgesia and SDH.

15 min before ET-1, rats received vehicle (5 μl), dipyridamole, nicardipine or CFTR_inh-172 (all 1 μg in 5 μl/paw). Paw withdrawal thresholds were measured 15, 20, 25 & 30 min after ET-1 administration. Neither dipyridamole, nicardipine nor CFTR_inh-172 affected ET-1 SDH (P=N.S., two-way repeated measures ANOVA, N = 6), however CFTR_inh-172 significantly attenuated ET-1 hyperalgesia (2-way ANOVA with Dunnet’s post hoc test, *P<0.05). Note that the ET-1 alone data is the same group as in Figure 1.

Ion channels

We administered two ion channel inhibitors, flufenamic acid (a voltage gated sodium channel blocker) and carbenoxolone (an interneuronal gap junction blocker), to evaluate the role of ion channels in endothelial cell mediated SDH. Rats received vehicle (0.9% sodium chloride), flufenamic acid or carbenoxolone 15 min before ET-1. Nociceptive threshold was evaluated every 5 min beginning 15 min after ET-1. Flufenamic acid and carbenoxolone pretreatment completely prevented the development of SDH, and flufenamic acid attenuated ET-1 hyperalgesia (Figure 3).

Figure 3. Effect of flufenamic acid (voltage gated sodium channel blocker) and carbenoxolone (interneuronal gap junction blocker) on ET-1 induced mechanical hyperalgesia and SDH.

15 min before ET-1, rats received vehicle (5 μl), flufenamic acid or carbenoxolone (both 1 μg in 5 μl/paw). Paw withdrawal thresholds were measured 15, 20, 25 & 30 min after ET-1 administration. Both flufenamic acid and carbenoxolone significantly inhibited SDH; flufenamic acid, but not carbenoxolone, significantly attenuated ET-1 induced hyperalgesia (*P < 0.001, two-way repeated measures ANOVA, followed by Bonferroni post test, N = 6). Note that the ET-1 alone data is the same group as in Figure 1.

Plasma membrane associated ATP synthase

Finally, we administered two inhibitors of plasma membrane associated ATP synthase, oligomycin and PEDF, to evaluate the role of ATP synthase in endothelial cell mediated SDH. Rats received either vehicle (0.9% sodium chloride or 10% DMSO in 0.9% saline for oligomycin), oligomycin or PEDF 15 min before ET-1. Nociceptive threshold was evaluated every 5 min beginning 15 min after ET-1. Both oligomycin and PEDF pretreatment prevented both ET-1 hyperalgesia and the development of ET-1–induced SDH (Figure 4).

Figure 4. Effect of oligomycin A (ATP synthase inhibitor) or PEDF-34-mer (extracellular ATP synthase inhibitor) on ET-1 induced mechanical hyperalgesia and SDH.

15 min before ET-1, rats received vehicle (5 μl), oligomycin or PEDF-34-mer (both 1 μg in 5 μl/paw). Paw withdrawal thresholds were measured 15, 20, 25 & 30 min after ET-1 administration. Both oligomycin and PEDF-34-mer significantly inhibited SDH as well as ET-1 hyperalgesia compared to vehicle treated controls (*P < 0.05, two-way repeated measures ANOVA, followed by Bonferroni post test, N = 6).

Discussion

The intradermal administration of ET-1 produces mechanical hyperalgesia, which is further enhanced by repeated testing with a threshold nociceptive intensity mechanical stimulus, a phenomenon that we have termed stimulus dependent hyperalgesia (SDH) ³⁰. While ET-1 hyperalgesia is mediated by its action at ET receptors on the primary afferent nociceptors ²⁹, we hypothesized that SDH is mediated via ET receptors on endothelial cells. Since a role of the endothelial cell in vascular pain has been postulated ^{6, 44, 51, 56}, we used a method employed in the cardiovascular and renal vascular literature to impair endothelial function: administration of octoxynol-9 to the luminal side of blood vessels, in vitro ^{11, 26} and in vivo ⁵⁴. We observed that this method of impairing endothelial function eliminates ET-1–induced SDH while leaving ET-1 hyperalgesia undiminished ^{29, 31}.

A signal feature of SDH is that it is produced by repeated mechanical stimulation. Since endothelial cells are activated by mechanical stimuli to release ATP ^{4, 6, 43, 65}, and ATP acts on P2X3 receptors on nociceptors to mediate pain, we hypothesized that ET-1 acts on endothelial cells to enhance mechanical stimulation-induced ATP release, giving rise to SDH. In this study, we examined the contribution of ATP release mechanisms to SDH.

We observed that inhibitors of ATP release via vesicular mechanisms (bafilomycin A, monensin, brefeldin A), and ion channel-dependent mechanisms (flufenamic acid, carbenoxolone) significantly attenuate SDH. We also observed that inhibition of ATP synthase activity with oligomycin A, an inhibitor of the ATP synthase, as well as by PEDF-34-mer, which is specific for ecto F1-ATP synthase on the plasma membrane ⁴⁷. However, we did not observe attenuation in ET-1–induced SDH with dipyridamole, a potent inhibitor of the ABC transporters, including P-glycoprotein (ABCB1) ⁶⁰, multidrug resistance-associated protein (ABCC1) ²⁸, and breast cancer resistance protein (ABCG2) ⁶⁴, or by nicardipine (a dihydropyridine, different chemical class from dipyridamole) another potent inhibitor of P-glycoprotein ⁵² and breast cancer resistance protein ⁶⁷. CFTR_inh-172, a potent and highly selective antagonist for the CFTR transport mechanism, also failed to attenuate ET-1 induced SDH. However, CFTR_inh-172 attenuation of ET-1 hyperalgesia may be related to the role CFTR plays in mediating the release of ATP from dorsal root ganglia (DRG) ³⁴; presence of CFTR_inh-172 may decrease the release of ATP from DRG in response to increased extracellular levels of ATP ⁴².

Since the drugs used in this study have actions other than inhibition of a specific ATP release mechanism, we used more than one class of drug for each release mechanism (i.e. bafilomycin A, monensin and brefeldin A for vesicular release, and flufenamic acid and carbenoxolone for ion channel-dependent release). This approach is used routinely in the endothelial cell literature to establish the role of these mechanisms in ATP release ^{4, 15, 17, 24, 40, 41, 63}. Of note in this regard, specifically identified following high throughput screening of 50,000 molecules, CFTR_inh-172 is a potent and highly selective antagonist for the CFTR transport mechanism ³⁵. Our observations implicate multiple ATP release mechanisms in response to mechanical stimulation, as seen in other cell types ^{20, 39, 61}.

While release of ATP from endothelial cells in response to mechanical stress is well-established ^{4, 5, 40, 43, 65}, the specific mechanisms mediating release are still under investigation ⁴¹. Our observation that monensin, bafilomycin and brefeldin A inhibit ET-1–induced SDH support the hypothesis that vesicular release mechanism, presumably ATP release based on our previous P2X3 antagonist studies ³¹, is involved in the expression of SDH. This hypothesis is consistent with histological evidence that ATP is present in vesicles in endothelial cells, and release of ATP from cultured endothelial cells exposed to a shear stress is blocked by monensin ⁴. Of note, it is also possible that the compounds used in this study to inhibit ATP release, may have action on cell types in the periphery other than the endothelial cell. For example, gene products of glial cells, such as Schwann cells, have been hypothesized to play a role in nerve injury and sensory nerve function ^{7, 19}, while a role for immune cells (e.g. neutrophils, macrophages, T cells) in acute and chronic inflammatory and neuropathic pain is well-established ^{21, 45, 53}. Other cell types, such as keratinocytes ^{18, 37} and mast cells ^{27, 49} are also implicated in peripheral nociceptive mechanisms. It is likely that actions of mediators, released from these cell types, on nociceptors contribute to regulating nociceptor function in chronic pain.

We also observed that inhibition of ATP synthesis by oligomycin or PEDF-derived peptide 34-mer (which is selective for extracellular ATPase), prevented both ET-1 induced hyperalgesia as well as SDH. While it might be assumed that inhibition of extracellular ATP synthesis would be independent of mechanical stimulation-induced release of ATP, it has previously been shown that inhibition of cell surface ATP synthase with angiostatin, piceatannol, or anti-ATP synthase antibody (none of which cross the plasma membrane, and so have no effect on mitochondrial ATP synthase activity) markedly decrease mechanical stimulation-induced ATP release ⁶⁶.

Taken together with our previous observations that ET-1 induced SDH is attenuated by a selective antagonist, for the P2X3 receptor, A-317491, present on the nociceptor and activated by ATP ²⁹, our data provide additional evidence for mechanical stimulation-induced release of ATP from endothelial cells via vesicular and ion channel-dependent mechanisms mediating SDH.

Perspective.

Endothelin-1 acts on endothelial cells to produce mechanical stimulation-induced hyperalgesia. Inhibitors of three different ATP release mechanisms attenuated this stimulus-dependent hyperalgesia. These studies provide support for a role of ATP in stimulus-dependent hyperalgesia, and suggest novel targets for the treatment of vascular pain syndromes.