Molecular Mechanism Of Pruritus Induced By Endothelin-1

Endothelin-1(ET-1) is secreted by a variety of cell types, including keratinocytes, vascular smooth muscle cells, leukocytes, cardiomyocytes and mesangial cells, and some tumor cell lines since its first isolation from the cultures of porcine aortic endothelial cell cultures. It is the most potent vasoconstrictor known. The signaling of ET-1is mediated via two main membrane G-protein-coupled receptor (GPCR) subtypes, ETA and ETB receptors. The expression of ETA receptor is largely confined to the vascular endothelium, as well as neurons and mast cells, while ETB receptor is mainly expressed by keratinocytes, smooth muscle cells and vascular endothelium.ET-1enhances capsaicin-evoked increases of intracellular calcium levels in freshly dissociated DRG neurons in a protein kinase C (PKC) dependent manner, and potentiation of capsaicin-induced currents. Local administration of ET-1induces nociceptive behaviors in animals and causes pain in humans. ET-1has been demonstrated to elicit pruritus in mice and humans also. How to cope with the messages of itch and pain inuuced by the same agent, ET-1. Little is known about the molecular mechanisms of the pruritogenic action of ET-1. Gastrin-releasing peptide receptor (GRPR) is an itch-specific molecule in the spinal cord [28]. Mice selectively ablated lamina I neurons expressing GRPR in the spinal cord showed profound scratching deficits in response to all of the itching (pruritogenic) stimuli (including ET-1) tested [29]. It is suggested that different neurotransmitters are released by the primary neurons to excite the pain-or itch-specicif second neurons in the spinal. The current study was designed to elucidate the molecular signals of itch induced by ET-1.MethodsAnimalsMale C57BL/6J mice, weighing20-22g, were purchased from the Center for Laboratory Animals, Sun Yat-Shen University. The experimental procedures and the animal use and care protocols were approved by the Committee on Ethical Use of Animals of Guangdong Academy of Medical Sciences. All efforts were made to minimize animal suffering and to reduce the number of animals used.Pruritus model of neck injectionOne day after shaving the rostral part of the back of the neck, mice were placed into a small plastic chamber30min before the experiment. For drug administration, mice were briefly removed from the chamber, and each test drug was intradermally injected with a30-gauge needle. Then the mice were returned to the chamber, and the hind limb scratching directed towards the shaved area at the back of the neck was observed and recorded for30min. One scratch was defined as a lift of the hind limb towards the injection site and then a reposition of the limb back to the floor, regardless of the scratching strokes that took place between the two movements.Cheek injection modelOne day after shaving the cheek, mice were placed into a small plastic chamber30min before the experiment. For drug administration, mice were briefly removed from the chamber, and each test drug was intradermally injected with a30-gauge needle.A volume of10μl was injected intradermally into the cheek. Then the mice were returned to the chamber, number of hindpaw scratches directed to the injected area as an indicator of itch and number of ipsilateral forelimb wipes directed to the injected area as an indicator of pain were recorded for a period of30min after the injection. Paw withdrawal latencyMice were placed into a small plastic chamber on glass table with the positioned in an heat stimulation apparatus for radiant heat stimulation on the plantar surface. Mice were placed into the small plastic chamber for1h one day before the experiment. Intensity of heat stimulus was adjusted for paw withdrawal latency of8-12s in normal animals. A cut-off time was set at20s to avoid injury to the hindpaw.Depletion of TRPV1neurons in neonatal miceMale neonatal mice (aged2days) were anesthetized with sevoflurane and injected subcutaneously with capsaicin (50mg/kg) or the vehicle (10%ethanol,10%Tween-80and80%PBS) as described previously. Animals were included in the study6weeks after the injection of capsaicin or the vehicle. The effects of capsaicin were expected to cause depletion of TRPV1neurons. It was verified by the eye-wiping test. For this test,0.01%capsaicin at the volume of20μl was sprayed into the eye and the number of wiping movements that occurred within1min was counted. The animal was considered to be desensitized to TRPV1by neonatal capsaicin treatment when the animal wiped its eyes no more than five times.Cell culture of dorsal root ganglionAdult male C57BL/6J mice were anesthetized with sevoflurane and the skin of the back were disinfected. The spinal were removed and transfered into cold L15medium and placed on ice. DRGs were removed bilaterally from lumbar and thoracic spinal levels under sterile conditions and incubated with0.25%collagenase type IV at37℃for60min with gentle agitation. Then, DRGs were centrifuged at1000rpm for5min in4℃after mechanically dissociated with pipette. Cells were resuspended with Dulbecco’s modified Eagle’s medium containing nerve growth factor (50ng/ml, Invitrogen),10%fetal bovine serum (Invitrogen), penicillin (100units/ml, In-vitrogen) and streptomycin (100g/ml, Invitrogen) and plated onto glass coverslips or plates precoated with poly-L-lysine and cultured in incubator containing5%CO2at37℃. Membrane translocation of PKCsCells were plated on10-mm diameter glass coverslips and were washed with0.01M PBS and then exposed to0.01M PBS or10nM ET-1for60s in incubator containing5%CO2at37℃, and immediately fixed with4%paraformaldehyde for20min, rinsed using PBS, and then incubated with10%normal goat for1h. Fixed slips were incubated with primary antibodies in0.2%Triton for16h at4℃. Then slips were incubated with second antibodies conjugated to Alexa Fluor488or Alexa Fluor594.Measurement of cAMPCells were plated in24wells plate and washed with PBS before test, and preincubated at37℃for10min in the presence of the phosphodiesterase inhibitor3-isobutyl-l-methylxanthine (2.5mM) in PBS containing0.1%glucose. Cells were incubated for an additional60s at37℃with or without ET-1(10nM in2.5mM IBMX in PBS). Then PBS was removed, and250μl of HCl(0.1M) with0.8%Triton X-100(Sigma-Aldrich) was added to the plates. After10-min incubation at room temperature, the lysate was removed from the plates and centrifuged for2min. The supernatant was collected and100u1of it was measureded for cAMP concentration using a monoclonal cAMP enzyme immunoassay kit.All reagents and plate are prepared at room temperature20min before test. Sample of cell extracts and standard cAMP are added to96-well plate coated with goat-anti-mouse serum. cAMP will competitively bind to the monoclonal anti-cAMP antibody in the presence of fixed amounts of cAMP-conjugated horse-radish peroxidase. Standard cAMP are used to generate the calculation curve. After a short incubation, the excess reagents are washed away and substrate (3,3’,5,5’-tetramethylbenzidine, TMB) is added. The multiwell plates are then read on a microplate reader at450nm. The intensity of the yellow color is inversely proportional to the concentration of cAMP in samples. The measured optical density is used to calculate the concentration of cAMP in samples based on the curve from the cAMP standards.The production of cAMP was normalized to the protein concentration in each well (pmol/mg protein). Protein content was determined using a bicinchoninic acid protein assay kit according to the manufacturer’s instructions and determined at562nm.Results1ET-1induces scratches through activation of ETA receptorIntradermal injection of ET-1to the back evoked the number of scratches in a dose-dependent manner, with a significant increase starting from1pmol (in100u1PBS) of ET-1. It induced an average of360bouts of scratches at the dose of1000pmol. At100pmol, ET-1induced a moderate number of scratches, within the linear range of ET-1effects. Therefore, the dose of100pmol (in100μl PBS) was selected for subsequent experiments.Intradermal injection of ET-1to the cheek an average of18bouts of wipes at the dose of100pmol (in10μ1PBS). No wipes was evoked when the dose of ET-1lower than10pmol. Cheek injection of ET-1dose-dependently evoked scratching response when the dose of ET-1higher than10pmol (in10μl PBS).Co-injection of BQ-123(selective antagonists for ETA) with ET-1at100nmol significantly reduced the number of scratches, while BQ-788(selective antagonists for ETB receptor) at30nmol increased the number of scratches induced by ET-1. These results show that ETA receptor mediates the pruritogenic effects, and the ETB receptor exerts anti-pruritic effects.2PKC mediates pruritogenic effects of ET-1Co-injection of Bisindolylmaleimide I (PKC inhibitor) at the dose of2u g or10μg reduced the scratching response induced by ET-1. Membrane translocation of PKCs were found in cultured dorsal root ganglion neurons after ET-1(10nM for60s) stimulation.Scratching response was also induced by the PKC activator phorbol-12-myristate-13-acetate (PMA) at the dose above100pmol. Intraplantar injection of PMA at the dose of1pmol (in10μl PBS) reduced the paw withdraw latency from heat stimulation about that induced by ET-1at the dose of100pmol (in10μl PBS). Thermal hyperalgesia induced by PMA and ET-1were inhibited by premedication with Bisindolylmaleimide I.3cAMP-PKC mediates pruritogenic effects of ET-1PKC is downstream of phospholipaseC (PLC). Co-injection of U73122(PLC inhibitor) with ET-1increased the scratching number. PKC activated by cAMP, a non-classical pathway was evaluated. The concentration of cyclic adenosine monophosphate (cAMP) in dorsal root ganglion ganglion neurons were increased after ET-1(lOnM for60s)stimulation. Co-injection of SQ22536(adenylyl cyclase inhibitor) reduced the number of scratches in a dose-dependent manner. Activation of AC causes increases of intracellular cAMP concentration, protein kinase A (PKA)is activated accordingly. Co-injection of H89(PKA inhibitor) didn’t affect the response to ET-1.The effects of high concentration of U73122on scratching response induced by ET-1and compound48/80(C48/80) was evaluated to exclude the non-specific effects of U73122on PLC which might affect the results. Histamine is released by mast cell in culture upon the stimulation of C48/80. U73122at the concentration of10μM is sufficient to block the release of histamine evoked by C48/80. Scratching response was induced by intradermal injection of C48/80, which is inhibited by co-injection with U73122(10μM,1000pmol). Co-injection of U73122(10μM,1000pmol) with ET-1increased the scratching number induced by ET-1. Premedication with D609(phosphatidylcholine specific phospholipase C inhibitor,500nmol, intradermal injection) at the same site before administration of ET-1reduced the number of scratches, while it didn’t affect the scratching response induced by C48/80..4Role of TRPV1,H1R,TRP and TRPA1in pruritus inuced by ET-1Intraperitoneal mepyramine (H1R antagonist,40mg/kg), capsazepine (TRPV1antagonist,4mg/kg) didn’t affect the response to ET-1. Scratching response was inhibited in capsaicin treated-mice in neonatal period. Co-injection of ruthenium red (RR,5nmol) and AP18(100nmol) with ET-1increased the scratching number. TRPA1is involved in the pain-like behavior induced by ET-1. Co-injection of RR or AP18with ET-1inhibited the pain-like behavior induced by ET-1. To examined the effect of RR and AP18on pruritus caused by ET-1could be attributed to pain relief, morphine (5mg/kg) was injected subcutaneously15min before the administration of ET-1. Morphine had no effect on the number of scratches caused by ET-1. This provided evidence that ET-1induced itch is not related to the pain.5Antipruritic effect of ETBPretreatment with systemically administered naloxone0.5mg/kg significantly reduced the number of scratches induced by ET-1. Co-injection of naloxone2nmol or0.5mg/kg augmented the effect of ET-1. Thus, local, but not systemic naloxone, prevented the antipruritic effect induced by activation of the ETB receptor, suggesting the involvement of peripheral opioid receptors in pruritis. We next examined the effects of CTOP, nor-BNI and naltrindole (μ-, κ-and8-opioid receptor antagonists, respectively) on the scratching response induced by ET-1. Intradermal injections of nor-BNI evoked dose-dependent scratching bouts. Five nmol of nor-BNI was selected because it did not cause a noticeable scratching response. Co-injection of nor-BNI significantly increased the number of scratches induced by ET-1, CTOP and naltrindole did not alter the scratching response to ET-1.Nitric oxide (NO) is involved in the antinociception effects of KOR agonist. There are enothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS). Inhibitor of nNOS abolished the analgesia effects of KOR agonist. Co-injection of L-NAME (non-selective NOS inhibitor,200nmol) or together with selective inhibitors for eNOS (L-NIO,200nmol), with ET-1increased the scratching number. Co-injection of AMT (iNOS inhibitor,5nmol) and nNOS (Nco-Propyl-L-arginine,20nmol) did not alter the scratching response to ET-1. This resluts suggest that keratinocyte mediates the antipruitic effects of KOR agonist as neuron expresses mainly nNOS, while keratinocyte expresses mainly eNOS,Conclusions1ET-1induces pruritus through ETA/AC/PKC pathway.2PKC activated by cAMP is a non-classical pathway. The cAMP-to-PKC signaling is mediated through the cAMP-activated guanine exchange factor Epac, which signals downstream to PI-PLC and PLD, both are necessary for activation of PKC in vitro and in vivo. However, the PKC signaling did not seem to descend from the PI-PLC pathway in our study, since blocking PI-PLC by U73122produced an opposite effect from blocking PKC. Our results reveal a new pathway for the cAMP-to-PKC signaling that is PI-PLC-independent, possibly via PC-PLC pathway. 3ETB exerts antipruritic effects, and peripheral KOR mediate the antipruritic effects through eNOS. Nitric oxide released from keratinocytes are involved in the antipruritic effects possibly.