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B-type natriuretic peptide-induced delayed modulation of TRPV1 and P2X3 receptors of mouse trigeminal sensory neurons.

Vilotti S, Marchenkova A, Ntamati N, Nistri A - PLoS ONE (2013)

Bottom Line: Nonetheless, 24 h application of BNP depressed TRPV1 mediated currents (an effect blocked by the NPR-A antagonist anantin) without changing responses to α,β-meATP or GABA.Anantin alone decreased basal cGMP production and enhanced control α,β-meATP-evoked responses, implying constitutive regulation of P2X3 receptors by ambient BNP.These data suggest a slow modulatory action by BNP on TRPV1 and P2X3 receptors outlining the role of this peptide as a negative regulator of trigeminal sensory neuron excitability to nociceptive stimuli.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Department, International School for Advanced Studies (SISSA), Trieste, Italy.

ABSTRACT
Important pain transducers of noxious stimuli are small- and medium-diameter sensory neurons that express transient receptor vanilloid-1 (TRPV1) channels and/or adenosine triphosphate (ATP)-gated P2X3 receptors whose activity is upregulated by endogenous neuropeptides in acute and chronic pain models. Little is known about the role of endogenous modulators in restraining the expression and function of TRPV1 and P2X3 receptors. In dorsal root ganglia, evidence supports the involvement of the natriuretic peptide system in the modulation of nociceptive transmission especially via the B-type natriuretic peptide (BNP) that activates the natriuretic peptide receptor-A (NPR-A) to downregulate sensory neuron excitability. Since the role of BNP in trigeminal ganglia (TG) is unclear, we investigated the expression of BNP in mouse TG in situ or in primary cultures and its effect on P2X3 and TRPV1 receptors of patch-clamped cultured neurons. Against scant expression of BNP, almost all neurons expressed NPR-A at membrane level. While BNP rapidly increased cGMP production and Akt kinase phosphorylation, there was no early change in passive neuronal properties or responses to capsaicin, α,β-meATP or GABA. Nonetheless, 24 h application of BNP depressed TRPV1 mediated currents (an effect blocked by the NPR-A antagonist anantin) without changing responses to α,β-meATP or GABA. Anantin alone decreased basal cGMP production and enhanced control α,β-meATP-evoked responses, implying constitutive regulation of P2X3 receptors by ambient BNP. These data suggest a slow modulatory action by BNP on TRPV1 and P2X3 receptors outlining the role of this peptide as a negative regulator of trigeminal sensory neuron excitability to nociceptive stimuli.

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cGMP production by mouse TG cultures.A, ELISA-based quantification of intracellular cGMP in primary cultures from P12 mice. Cultures were treated with BNP (100 or 500 ng/ml for 5, 10 or 30 min) and/or anantin (100 and 500 nM, 30 min), or treated with vehicle (indicated as 0). Note large rise in cGMP that declines at 60 min; anantin alone (500 nM) decreases basal cGMP. Data were normalized to the total protein content in each sample and are presented as mean value ± SEM (n=3). *p≤0.05. B (top), example of western blot of Akt phosphorylation induced by BNP (500 ng/ml) applied alone or together with anantin (100 and 500 nM) for various times as indicated. Bottom histograms quantify these data from at least 3 experiments, demonstrating early and transient increment in P-Akt expression.
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pone-0081138-g003: cGMP production by mouse TG cultures.A, ELISA-based quantification of intracellular cGMP in primary cultures from P12 mice. Cultures were treated with BNP (100 or 500 ng/ml for 5, 10 or 30 min) and/or anantin (100 and 500 nM, 30 min), or treated with vehicle (indicated as 0). Note large rise in cGMP that declines at 60 min; anantin alone (500 nM) decreases basal cGMP. Data were normalized to the total protein content in each sample and are presented as mean value ± SEM (n=3). *p≤0.05. B (top), example of western blot of Akt phosphorylation induced by BNP (500 ng/ml) applied alone or together with anantin (100 and 500 nM) for various times as indicated. Bottom histograms quantify these data from at least 3 experiments, demonstrating early and transient increment in P-Akt expression.

Mentions: We sought to understand whether the large expression of membrane bound NPR-A was actually indicative of a functional receptor system. To this end, since activation of NPR-A by ANP and BNP has been shown to increase intracellular cGMP production [40], we investigated if application of BNP to mouse TG cultures could increase intracellular cGMP production, as determined by ELISA. Figure 3 A shows that application of 100 or 500 ng/ml BNP (compared to the vehicle control treatment) increase the mean cGMP levels in a concentration- and time-related fashion. In fact, the cGMP rise was already detectable after 5 min and reached its peak after 30 min with a late (1 h) decline. The greatest increment was observed after 30 min application of 500 ng/ml BNP. Even 100 ng/ml BNP was sufficient to activate the NPR-A mediated pathways by significantly increasing cGMP by almost 2-fold compared to basal levels. This effect was prevented by the application of 500 nM anantin, a specific antagonist of NPR-A [41,42], that completely inhibited the elevation of cGMP levels. A lower anantin concentration (100 nM) failed to produce significant antagonism of BNP-elicited rise in cGMP (Figure 3 A). It is noteworthy that anantin (500 nM) per se significantly decreased the basal level of cGMP.


B-type natriuretic peptide-induced delayed modulation of TRPV1 and P2X3 receptors of mouse trigeminal sensory neurons.

Vilotti S, Marchenkova A, Ntamati N, Nistri A - PLoS ONE (2013)

cGMP production by mouse TG cultures.A, ELISA-based quantification of intracellular cGMP in primary cultures from P12 mice. Cultures were treated with BNP (100 or 500 ng/ml for 5, 10 or 30 min) and/or anantin (100 and 500 nM, 30 min), or treated with vehicle (indicated as 0). Note large rise in cGMP that declines at 60 min; anantin alone (500 nM) decreases basal cGMP. Data were normalized to the total protein content in each sample and are presented as mean value ± SEM (n=3). *p≤0.05. B (top), example of western blot of Akt phosphorylation induced by BNP (500 ng/ml) applied alone or together with anantin (100 and 500 nM) for various times as indicated. Bottom histograms quantify these data from at least 3 experiments, demonstrating early and transient increment in P-Akt expression.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842315&req=5

pone-0081138-g003: cGMP production by mouse TG cultures.A, ELISA-based quantification of intracellular cGMP in primary cultures from P12 mice. Cultures were treated with BNP (100 or 500 ng/ml for 5, 10 or 30 min) and/or anantin (100 and 500 nM, 30 min), or treated with vehicle (indicated as 0). Note large rise in cGMP that declines at 60 min; anantin alone (500 nM) decreases basal cGMP. Data were normalized to the total protein content in each sample and are presented as mean value ± SEM (n=3). *p≤0.05. B (top), example of western blot of Akt phosphorylation induced by BNP (500 ng/ml) applied alone or together with anantin (100 and 500 nM) for various times as indicated. Bottom histograms quantify these data from at least 3 experiments, demonstrating early and transient increment in P-Akt expression.
Mentions: We sought to understand whether the large expression of membrane bound NPR-A was actually indicative of a functional receptor system. To this end, since activation of NPR-A by ANP and BNP has been shown to increase intracellular cGMP production [40], we investigated if application of BNP to mouse TG cultures could increase intracellular cGMP production, as determined by ELISA. Figure 3 A shows that application of 100 or 500 ng/ml BNP (compared to the vehicle control treatment) increase the mean cGMP levels in a concentration- and time-related fashion. In fact, the cGMP rise was already detectable after 5 min and reached its peak after 30 min with a late (1 h) decline. The greatest increment was observed after 30 min application of 500 ng/ml BNP. Even 100 ng/ml BNP was sufficient to activate the NPR-A mediated pathways by significantly increasing cGMP by almost 2-fold compared to basal levels. This effect was prevented by the application of 500 nM anantin, a specific antagonist of NPR-A [41,42], that completely inhibited the elevation of cGMP levels. A lower anantin concentration (100 nM) failed to produce significant antagonism of BNP-elicited rise in cGMP (Figure 3 A). It is noteworthy that anantin (500 nM) per se significantly decreased the basal level of cGMP.

Bottom Line: Nonetheless, 24 h application of BNP depressed TRPV1 mediated currents (an effect blocked by the NPR-A antagonist anantin) without changing responses to α,β-meATP or GABA.Anantin alone decreased basal cGMP production and enhanced control α,β-meATP-evoked responses, implying constitutive regulation of P2X3 receptors by ambient BNP.These data suggest a slow modulatory action by BNP on TRPV1 and P2X3 receptors outlining the role of this peptide as a negative regulator of trigeminal sensory neuron excitability to nociceptive stimuli.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Department, International School for Advanced Studies (SISSA), Trieste, Italy.

ABSTRACT
Important pain transducers of noxious stimuli are small- and medium-diameter sensory neurons that express transient receptor vanilloid-1 (TRPV1) channels and/or adenosine triphosphate (ATP)-gated P2X3 receptors whose activity is upregulated by endogenous neuropeptides in acute and chronic pain models. Little is known about the role of endogenous modulators in restraining the expression and function of TRPV1 and P2X3 receptors. In dorsal root ganglia, evidence supports the involvement of the natriuretic peptide system in the modulation of nociceptive transmission especially via the B-type natriuretic peptide (BNP) that activates the natriuretic peptide receptor-A (NPR-A) to downregulate sensory neuron excitability. Since the role of BNP in trigeminal ganglia (TG) is unclear, we investigated the expression of BNP in mouse TG in situ or in primary cultures and its effect on P2X3 and TRPV1 receptors of patch-clamped cultured neurons. Against scant expression of BNP, almost all neurons expressed NPR-A at membrane level. While BNP rapidly increased cGMP production and Akt kinase phosphorylation, there was no early change in passive neuronal properties or responses to capsaicin, α,β-meATP or GABA. Nonetheless, 24 h application of BNP depressed TRPV1 mediated currents (an effect blocked by the NPR-A antagonist anantin) without changing responses to α,β-meATP or GABA. Anantin alone decreased basal cGMP production and enhanced control α,β-meATP-evoked responses, implying constitutive regulation of P2X3 receptors by ambient BNP. These data suggest a slow modulatory action by BNP on TRPV1 and P2X3 receptors outlining the role of this peptide as a negative regulator of trigeminal sensory neuron excitability to nociceptive stimuli.

Show MeSH
Related in: MedlinePlus