Sensory neuron-specific GPCRs Mrgprs are itch receptors mediating chloroquine-induced pruritus methods
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Cultures of dissociated DRG neurons
DRG from all spinal levels of 4-week old mice or rats were collected in cold DH10 medium and treated with enzyme solution at 37°C. Following trituration and centrifugation, cells were resuspended in DH10, plated on glass cover slips coated with poly-D-lysine and laminin, cultured in an incubator at 37°C and used within 24 hours. Details are available (see ).
Mrgpr-clusterΔ -/- mice exhibit severe reduction in CQ-induced scratching
In addition to pain, we evaluated chemically induced itch responses in Mrgpr-clusterΔ -/- mice. No significant difference was found between Mrgpr-clusterΔ -/- and WT mice in the total number of scratching bouts induced by histamine over a period of 30 min ( ). Consistent with this result, WT and mutant mice also showed similar responses to compound 48/80, a drug that elicits mast cell degranulation and induces histamine-dependent itch (; ) ( ). These results suggest that Mrgprs are not involved in histamine-dependent itch.
Mrgpr-clusterΔ -/- mice exhibit severe reduction in CQ-induced scratching
Strikingly, itch induced by CQ was strongly reduced in Mrgpr-clusterΔ -/- mice. shows the time course of scratching bouts at 5 min intervals after CQ injection. Typically, the first bout was observed within 1 to 2 min after injection, and scratching peaked within 15 min in WT mice. In contrast, Mrgpr-clusterΔ -/- mice showed a delayed occurrence of the first scratching behavior (WT 57.8 ± 25.4 sec versus KO 280.5 ± 35.0 sec; P=0.0004). The total number of scratching bouts induced by CQ were 278 ± 21 in WT mice and 104 ± 13 in Mrgpr-clusterΔ -/- mice ( ). Interestingly, injection of a CQ precursor, quinoline, did not evoke any scratching behavior (see below for quinoline's structure and inability of activating Mrgprs) in both WT and mutant mice ( ). However, immediately after quinoline treatment, CQ injection...
CQ directly excites DRG neurons in an Mrgpr-dependent manner
Since CQ-induced itch is not considered an allergic reaction, we hypothesized given our results that this type of itch results from a direct activation of DRG neurons by the drug. If so, then the behavioral deficit seen in mutant mice would be attributed to a loss of CQ responsiveness in primary sensory neurons. Indeed, 1 mM CQ treatment of cultured DRG neurons evoked a robust intracellular calcium ([Ca 2+ ] i ) increase in ~4-5% of the cells from WT mice. In contrast, none of the neurons in cultures derived from Mrgpr-deficient mice exhibited any significant response to CQ ( ). These data indicate that the CQ-evoked [Ca 2+ ] i increases seen in WT DRG cultures reflect specific activation of a subset of cells, and that this activation is Mrgpr-dependent. In contrast, the percentage of DRG neurons responding to histamine was identical between WT and mutant cultures, consistent wi...
CQ specifically activates mouse MrgprA3 and human MrgprX1
In order to determine the lowest concentrations of CQ capable of activating MrgprA3 and X1, we performed dose-response experiments in HEK293 cells. These experiments indicated that the receptors could be activated by the drug at micromolar concentrations with the mouse receptor showing 10-fold higher sensitivity than the human receptor ( ). EC 50 s for MrgprA3 and MrgprX1 are 27.55 ± 2.03 and 297.68 ±2.10 µM, respectively (see Discussion regarding the low potency of CQ on MrgprX1). Besides CQ, we also determined the sensitivities of these receptors to other structurally related compounds (i.e. quinoline, quinine and serotonin) ( ). Quinoline is used as an intermediate in the production of various compounds including CQ. Despite the presence of a bicyclic structure, quinoline completely failed to activate MrgprA3 and MrgprX1 suggesting that the side chain in CQ is also n...
MrgprA3 and MrgprX1 rescue the phenotypes of Mrgpr-deficient neurons
We next asked whether MrgprA3 or MrgprX1 can rescue the phenotypes of DRG neurons from Mrgpr-clusterΔ -/- mice. To answer this question, we electroporated the Mrgpr expression constructs used in the heterologous studies into dissociated adult DRG neurons from these mice. After 24 hour in culture, expression and membrane localization of the transfected Mrgprs in the mutant neurons could be readily visualized by GFP ( ). Strikingly, all MrgprA3-expressing mutant neurons generated numerous APs in response to CQ treatment ( ) whereas neighboring GFP-negative neurons remained silent (n=6, not shown). The number of APs generated in the GFP-positive neurons was comparable to that produced by CQ treatment of WT DRG neurons, indicating a nearly complete rescue by MrgprA3. Similar results were obtained for Mrgpr-deficient neurons electroporated with MrgprX1 ( ). In contrast, fe...
Behavioral studies
All behavioral tests were performed with an experimenter blind to genotype. The mice were 2 to 3 month-old males (20-30 g) that had been backcrossed to C57Bl/6 mice for at least ten generations. Rat studies were done using four week old CD® animals (Charles River Laboratories). All experiments were performed under the protocol approved by the Animal Care and Use Committee of Johns Hopkins University School of Medicine. Pruritic compounds (i.e. histamine, compound 48/80, and CQ) were subcutaneously injected into the nape of the neck after acclimatization and scratching behavior was observed for 30 min. A bout of scratching was defined as continuous scratch movements with hind paws directed at the area around the injection site. Scratching behavior was quantified by recording the number of scratching bouts at 5 min intervals over the 30 min observation period. Details for oth...
Calcium imaging
Neurons or HEK293 cells were loaded with fura 2-acetomethoxy ester (Molecular Probes) for 30 min in the dark at room temperature or 45 min at 37°C, respectively. After washing, cells were imaged at 340 and 380 nm excitation to detect intracellular free calcium. Calcium imaging assays were performed with an experimenter blind to genotype. Each experiment was done at least three times and at least 100 cells (neurons or HEK293 cells) were analyzed each time.
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DRG from all spinal levels of 4-week old mice or rats were collected in cold DH10 medium and treated with enzyme solution at 37°C. Following trituration and centrifugation, cells were resuspended in DH10, plated on glass cover slips coated with poly-D-lysine and laminin, cultured in an incubator at 37°C and used within 24 hours. Details are available (see ).
In addition to pain, we evaluated chemically induced itch responses in Mrgpr-clusterΔ -/- mice. No significant difference was found between Mrgpr-clusterΔ -/- and WT mice in the total number of scratching bouts induced by histamine over a period of 30 min ( ). Consistent with this result, WT and mutant mice also showed similar responses to compound 48/80, a drug that elicits mast cell degranulation and induces histamine-dependent itch (; ) ( ). These results suggest that Mrgprs are not involved in histamine-dependent itch.
Strikingly, itch induced by CQ was strongly reduced in Mrgpr-clusterΔ -/- mice. shows the time course of scratching bouts at 5 min intervals after CQ injection. Typically, the first bout was observed within 1 to 2 min after injection, and scratching peaked within 15 min in WT mice. In contrast, Mrgpr-clusterΔ -/- mice showed a delayed occurrence of the first scratching behavior (WT 57.8 ± 25.4 sec versus KO 280.5 ± 35.0 sec; P=0.0004). The total number of scratching bouts induced by CQ were 278 ± 21 in WT mice and 104 ± 13 in Mrgpr-clusterΔ -/- mice ( ). Interestingly, injection of a CQ precursor, quinoline, did not evoke any scratching behavior (see below for quinoline's structure and inability of activating Mrgprs) in both WT and mutant mice ( ). However, immediately after quinoline treatment, CQ injection at the same location induced robust scratching behavior in WT mice; and the number of scratches induced by this treatment was again severely reduced in Mrgpr-clusterΔ -/- mice ( ). These results suggest that CQ-induced itch, but not histaminergic itch, is affected in the cluster del...
Since CQ-induced itch is not considered an allergic reaction, we hypothesized given our results that this type of itch results from a direct activation of DRG neurons by the drug. If so, then the behavioral deficit seen in mutant mice would be attributed to a loss of CQ responsiveness in primary sensory neurons. Indeed, 1 mM CQ treatment of cultured DRG neurons evoked a robust intracellular calcium ([Ca 2+ ] i ) increase in ~4-5% of the cells from WT mice. In contrast, none of the neurons in cultures derived from Mrgpr-deficient mice exhibited any significant response to CQ ( ). These data indicate that the CQ-evoked [Ca 2+ ] i increases seen in WT DRG cultures reflect specific activation of a subset of cells, and that this activation is Mrgpr-dependent. In contrast, the percentage of DRG neurons responding to histamine was identical between WT and mutant cultures, consistent with the behavioral data and providing further evidence that histamine-induced itch is unaffected in Mrgpr-clusterΔ -/- mice ( ).
In order to determine the lowest concentrations of CQ capable of activating MrgprA3 and X1, we performed dose-response experiments in HEK293 cells. These experiments indicated that the receptors could be activated by the drug at micromolar concentrations with the mouse receptor showing 10-fold higher sensitivity than the human receptor ( ). EC 50 s for MrgprA3 and MrgprX1 are 27.55 ± 2.03 and 297.68 ±2.10 µM, respectively (see Discussion regarding the low potency of CQ on MrgprX1). Besides CQ, we also determined the sensitivities of these receptors to other structurally related compounds (i.e. quinoline, quinine and serotonin) ( ). Quinoline is used as an intermediate in the production of various compounds including CQ. Despite the presence of a bicyclic structure, quinoline completely failed to activate MrgprA3 and MrgprX1 suggesting that the side chain in CQ is also necessary for activation ( ). Consistently, quinoline does not induce any scratching behavior in mice ( ). Serotonin also has a bicyclic structure. But unlike CQ or quinoline, its bicyclic structure consists of a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring ( )....
We next asked whether MrgprA3 or MrgprX1 can rescue the phenotypes of DRG neurons from Mrgpr-clusterΔ -/- mice. To answer this question, we electroporated the Mrgpr expression constructs used in the heterologous studies into dissociated adult DRG neurons from these mice. After 24 hour in culture, expression and membrane localization of the transfected Mrgprs in the mutant neurons could be readily visualized by GFP ( ). Strikingly, all MrgprA3-expressing mutant neurons generated numerous APs in response to CQ treatment ( ) whereas neighboring GFP-negative neurons remained silent (n=6, not shown). The number of APs generated in the GFP-positive neurons was comparable to that produced by CQ treatment of WT DRG neurons, indicating a nearly complete rescue by MrgprA3. Similar results were obtained for Mrgpr-deficient neurons electroporated with MrgprX1 ( ). In contrast, fewer than half of the MrgprA1-electroporated neurons elicited a few APs in response to CQ ( ). Rescue by MrgprA3 and MrgprX1 was also seen using calcium imaging, with an increase in [Ca 2+ ] i induced by CQ ( ). Together these results strongly suggest that mouse MrgprA3 and human MrgprX1 are the majo...
All behavioral tests were performed with an experimenter blind to genotype. The mice were 2 to 3 month-old males (20-30 g) that had been backcrossed to C57Bl/6 mice for at least ten generations. Rat studies were done using four week old CD® animals (Charles River Laboratories). All experiments were performed under the protocol approved by the Animal Care and Use Committee of Johns Hopkins University School of Medicine. Pruritic compounds (i.e. histamine, compound 48/80, and CQ) were subcutaneously injected into the nape of the neck after acclimatization and scratching behavior was observed for 30 min. A bout of scratching was defined as continuous scratch movements with hind paws directed at the area around the injection site. Scratching behavior was quantified by recording the number of scratching bouts at 5 min intervals over the 30 min observation period. Details for other behavior assays are available in the.
Neurons or HEK293 cells were loaded with fura 2-acetomethoxy ester (Molecular Probes) for 30 min in the dark at room temperature or 45 min at 37°C, respectively. After washing, cells were imaged at 340 and 380 nm excitation to detect intracellular free calcium. Calcium imaging assays were performed with an experimenter blind to genotype. Each experiment was done at least three times and at least 100 cells (neurons or HEK293 cells) were analyzed each time.
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