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Gi- and Gq-coupled ADP (P2Y) receptors act in opposition to modulate nociceptive signaling and inflammatory pain behavior.

Malin SA, Molliver DC - Mol Pain (2010)

Bottom Line: Agonists for these receptors inhibit nociceptive signaling in isolated neurons and reduce behavioral hyperalgesia in vivo.Anti-nociceptive actions of these receptors appear to be antagonized by the Gq-coupled ADP receptor, P2Y1, which is required for the full expression of inflammatory hyperalgesia.Taken together, our data suggest that Gi-coupled P2Y receptors are broadly expressed in nociceptors, inhibit nociceptive signaling in vivo, and represent potential targets for the development of novel analgesic drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept Medicine; Dept Neurobiology, University of Pittsburgh, Pittsburgh, PA, USA. sachamalin@gmail.com

ABSTRACT

Background: Investigations of nucleotide signaling in nociception to date have focused on actions of adenosine triphosphate (ATP). Both ATP-gated ion channels (P2X receptors) and G protein-coupled (P2Y) receptors contribute to nociceptive signaling in peripheral sensory neurons. In addition, several studies have implicated the Gq-coupled adenosine diphosphate (ADP) receptor P2Y1 in sensory transduction. In this study, we examined the expression and function of P2Y1 and the Gi-coupled receptors P2Y12, P2Y13 and P2Y14 in sensory neurons to determine their contribution to nociception.

Results: We detected mRNA and protein for ADP receptors P2Y12 and P2Y13 in mouse dorsal root ganglia (DRG). P2Y14, a homologous Gi-coupled nucleotide receptor, is also expressed in DRG. Immunohistochemical analysis of receptor distribution indicated that these receptors are widely expressed in nociceptive neurons. Using ratiometric calcium imaging, we found that ADP evokes increases in intracellular calcium in isolated DRG neurons and also produces a pertussis toxin-sensitive inhibition of depolarization-evoked calcium transients. The inhibitory effect of ADP was unaltered in the presence of the selective P2Y1 antagonist MRS2179 and in neurons isolated from P2Y1 knockout mice, whereas ADP-evoked calcium transients were greatly reduced. Analysis of behavioral responses to noxious heat before and after inflammatory injury (injection of complete Freund's adjuvant into the hindpaw) revealed that P2Y1 is required for the full expression of inflammatory hyperalgesia, whereas local injection of agonists for Gi-coupled P2Y receptors reduced hyperalgesia.

Conclusions: We report that Gi-coupled P2Y receptors are widely expressed in peripheral sensory neurons. Agonists for these receptors inhibit nociceptive signaling in isolated neurons and reduce behavioral hyperalgesia in vivo. Anti-nociceptive actions of these receptors appear to be antagonized by the Gq-coupled ADP receptor, P2Y1, which is required for the full expression of inflammatory hyperalgesia. We propose that nociceptor sensitivity is modulated by the integration of nucleotide signaling through Gq- and Gi-coupled P2Y receptors, and this balance is altered in response to inflammatory injury. Taken together, our data suggest that Gi-coupled P2Y receptors are broadly expressed in nociceptors, inhibit nociceptive signaling in vivo, and represent potential targets for the development of novel analgesic drugs.

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P2YGi receptor mRNA levels are regulated in response to inflammatory injury. A) Amplification of DRG cDNA by conventional RT-PCR using the P2YGi real-time PCR primers produced a single band for each receptor of the expected size (100-150 bp). B) Real-time PCR was used to analyze mRNA levels for each of the Gi-coupled P2Y receptors and the Gq-coupled P2Y1 at 1, 4 and 15 days after induction of inflammatory hyperalgesia by injection of complete Freund's adjuvant into the hindpaw. The shaded box indicates the period of significant heat hyperalgesia determined using the Hargreaves test. Data are normalized against baseline values. *p < 0.01, ‡p < 0.05, For PCR, n = 5 mice/time point. For behavior, n = 10 mice/time point.
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Figure 1: P2YGi receptor mRNA levels are regulated in response to inflammatory injury. A) Amplification of DRG cDNA by conventional RT-PCR using the P2YGi real-time PCR primers produced a single band for each receptor of the expected size (100-150 bp). B) Real-time PCR was used to analyze mRNA levels for each of the Gi-coupled P2Y receptors and the Gq-coupled P2Y1 at 1, 4 and 15 days after induction of inflammatory hyperalgesia by injection of complete Freund's adjuvant into the hindpaw. The shaded box indicates the period of significant heat hyperalgesia determined using the Hargreaves test. Data are normalized against baseline values. *p < 0.01, ‡p < 0.05, For PCR, n = 5 mice/time point. For behavior, n = 10 mice/time point.

Mentions: Expression of mRNA for all three of the Gi-coupled P2Y receptors was identified in mouse lumbar DRG by real-time PCR, with relative levels of expression of P2Y12 ~P2Y14 > P2Y13 (Table 1). To determine whether expression is altered in response to inflammatory injury, 20 μl complete Freund's adjuvant (CFA) was injected into the plantar surface of the hindpaw, and real-time PCR was used to quantify changes in mRNA abundance compared to uninjected baseline levels (Figure 1). The three P2YGi receptors were coordinately regulated: expression was initially reduced one day after CFA injection, but significantly upregulated at day 4. Expression of P2YGi receptor mRNA returned to baseline by day 15, by which time behavioral response thresholds to noxious heat had also returned to baseline levels.


Gi- and Gq-coupled ADP (P2Y) receptors act in opposition to modulate nociceptive signaling and inflammatory pain behavior.

Malin SA, Molliver DC - Mol Pain (2010)

P2YGi receptor mRNA levels are regulated in response to inflammatory injury. A) Amplification of DRG cDNA by conventional RT-PCR using the P2YGi real-time PCR primers produced a single band for each receptor of the expected size (100-150 bp). B) Real-time PCR was used to analyze mRNA levels for each of the Gi-coupled P2Y receptors and the Gq-coupled P2Y1 at 1, 4 and 15 days after induction of inflammatory hyperalgesia by injection of complete Freund's adjuvant into the hindpaw. The shaded box indicates the period of significant heat hyperalgesia determined using the Hargreaves test. Data are normalized against baseline values. *p < 0.01, ‡p < 0.05, For PCR, n = 5 mice/time point. For behavior, n = 10 mice/time point.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2865444&req=5

Figure 1: P2YGi receptor mRNA levels are regulated in response to inflammatory injury. A) Amplification of DRG cDNA by conventional RT-PCR using the P2YGi real-time PCR primers produced a single band for each receptor of the expected size (100-150 bp). B) Real-time PCR was used to analyze mRNA levels for each of the Gi-coupled P2Y receptors and the Gq-coupled P2Y1 at 1, 4 and 15 days after induction of inflammatory hyperalgesia by injection of complete Freund's adjuvant into the hindpaw. The shaded box indicates the period of significant heat hyperalgesia determined using the Hargreaves test. Data are normalized against baseline values. *p < 0.01, ‡p < 0.05, For PCR, n = 5 mice/time point. For behavior, n = 10 mice/time point.
Mentions: Expression of mRNA for all three of the Gi-coupled P2Y receptors was identified in mouse lumbar DRG by real-time PCR, with relative levels of expression of P2Y12 ~P2Y14 > P2Y13 (Table 1). To determine whether expression is altered in response to inflammatory injury, 20 μl complete Freund's adjuvant (CFA) was injected into the plantar surface of the hindpaw, and real-time PCR was used to quantify changes in mRNA abundance compared to uninjected baseline levels (Figure 1). The three P2YGi receptors were coordinately regulated: expression was initially reduced one day after CFA injection, but significantly upregulated at day 4. Expression of P2YGi receptor mRNA returned to baseline by day 15, by which time behavioral response thresholds to noxious heat had also returned to baseline levels.

Bottom Line: Agonists for these receptors inhibit nociceptive signaling in isolated neurons and reduce behavioral hyperalgesia in vivo.Anti-nociceptive actions of these receptors appear to be antagonized by the Gq-coupled ADP receptor, P2Y1, which is required for the full expression of inflammatory hyperalgesia.Taken together, our data suggest that Gi-coupled P2Y receptors are broadly expressed in nociceptors, inhibit nociceptive signaling in vivo, and represent potential targets for the development of novel analgesic drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept Medicine; Dept Neurobiology, University of Pittsburgh, Pittsburgh, PA, USA. sachamalin@gmail.com

ABSTRACT

Background: Investigations of nucleotide signaling in nociception to date have focused on actions of adenosine triphosphate (ATP). Both ATP-gated ion channels (P2X receptors) and G protein-coupled (P2Y) receptors contribute to nociceptive signaling in peripheral sensory neurons. In addition, several studies have implicated the Gq-coupled adenosine diphosphate (ADP) receptor P2Y1 in sensory transduction. In this study, we examined the expression and function of P2Y1 and the Gi-coupled receptors P2Y12, P2Y13 and P2Y14 in sensory neurons to determine their contribution to nociception.

Results: We detected mRNA and protein for ADP receptors P2Y12 and P2Y13 in mouse dorsal root ganglia (DRG). P2Y14, a homologous Gi-coupled nucleotide receptor, is also expressed in DRG. Immunohistochemical analysis of receptor distribution indicated that these receptors are widely expressed in nociceptive neurons. Using ratiometric calcium imaging, we found that ADP evokes increases in intracellular calcium in isolated DRG neurons and also produces a pertussis toxin-sensitive inhibition of depolarization-evoked calcium transients. The inhibitory effect of ADP was unaltered in the presence of the selective P2Y1 antagonist MRS2179 and in neurons isolated from P2Y1 knockout mice, whereas ADP-evoked calcium transients were greatly reduced. Analysis of behavioral responses to noxious heat before and after inflammatory injury (injection of complete Freund's adjuvant into the hindpaw) revealed that P2Y1 is required for the full expression of inflammatory hyperalgesia, whereas local injection of agonists for Gi-coupled P2Y receptors reduced hyperalgesia.

Conclusions: We report that Gi-coupled P2Y receptors are widely expressed in peripheral sensory neurons. Agonists for these receptors inhibit nociceptive signaling in isolated neurons and reduce behavioral hyperalgesia in vivo. Anti-nociceptive actions of these receptors appear to be antagonized by the Gq-coupled ADP receptor, P2Y1, which is required for the full expression of inflammatory hyperalgesia. We propose that nociceptor sensitivity is modulated by the integration of nucleotide signaling through Gq- and Gi-coupled P2Y receptors, and this balance is altered in response to inflammatory injury. Taken together, our data suggest that Gi-coupled P2Y receptors are broadly expressed in nociceptors, inhibit nociceptive signaling in vivo, and represent potential targets for the development of novel analgesic drugs.

Show MeSH
Related in: MedlinePlus