Journal Club 2017.10.27

Contribution of TRPC3 to store-operated calcium entry and inflammatory transductions in primary nociceptors.

Alkhani H, Ase AR, Grant R, O’Donnell D, Groschner K, Séguéla P1

Abstract

BACKGROUND:

Prolonged intracellular calcium elevation contributes to sensitization of nociceptors and chronic pain in inflammatory conditions. The underlying molecular mechanisms remain unknown but store-operated calcium entry (SOCE) components participate in calcium homeostasis, potentially playing a significant role in chronic pain pathologies. Most G protein-coupled receptors activated by inflammatory mediators trigger calcium-dependent signaling pathways and stimulate SOCE in primary afferents. The aim of the present study was to investigate the role of TRPC3, a calcium-permeable non-selective cation channel coupled to phospholipase C and highly expressed in DRG, as a link between activation of pro-inflammatory metabotropic receptors and SOCE in nociceptive pathways.

RESULTS:

Using in situ hybridization, we determined that TRPC3 and TRPC1 constitute the major TRPC subunits expressed in adult rat DRG. TRPC3 was found localized exclusively in small and medium diameter sensory neurons. Heterologous overexpression of TRPC3channel subunits in cultured primary DRG neurons evoked a significant increase of Gd3+-sensitive SOCE following thapsigargin-induced calcium store depletion. Conversely, using the same calcium add-back protocol, knockdown of endogenous TRPC3 with shRNA-mediated interference or pharmacological inhibition with the selective TRPC3 antagonist Pyr10 induced a substantial decrease of SOCE, indicating a significant role of TRPC3 in SOCE in DRG nociceptors. Activation of P2Y2 purinoceptors or PAR2 protease receptors triggered a strong increase in intracellular calcium in conditions of TRPC3 overexpression. Additionally, knockdown of native TRPC3 or its selective pharmacological blockade suppressed UTP- or PAR2 agonist-evoked calcium responses as well as sensitization of DRG neurons. These data show a robust link between activation of pro-inflammatory receptors and calcium homeostasis through TRPC3-containing channels operating both in receptor- and store-operated mode.

CONCLUSIONS:

Our findings highlight a major contribution of TRPC3 to neuronal calcium homeostasis in somatosensory pathways based on the unique ability of these cation channels to engage in both SOCE and receptor-operated calcium influx. This is the first evidence for TRPC3 as a SOCE component in DRG neurons. The flexible role of TRPC3 in calcium signaling as well as its functional coupling to pro-inflammatory metabotropic receptors involved in peripheral sensitization makes it a potential target for therapeutic strategies in chronic pain conditions.

Contribution of TRPC3 to SOCE and inflammatory transductions in primary nociceptors

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17.10.20

Involvement of TRPV4 in Serotonin-Evoked Scratching.

Akiyama T1, Ivanov M2, Nagamine M2, Davoodi A2, Carstens MI2, Ikoma A3, Cevikbas F3, Kempkes C3, Buddenkotte J4, Steinhoff M5, Carstens E6.
Abstract

Several thermosensitive transient receptor potential channels (transient receptor potential vanilloid type-1, -3; transient receptor potential cation channel, subfamily A, member 1) have been implicated in itch. In contrast, the role of transient receptor potential vanilloid type-4 (TRPV4) in itch is unknown. Therefore, we investigated if TRPV4, a temperature-sensitive cation channel, plays an important role in acute itch in mice. Four different pruritogens, including serotonin (5-hydroxytryptamine [5-HT]), histamine, SLIGRL (protease-activated receptors 2/mas-related G-protein-coupled receptor C11 agonist), and chloroquine (mas-related G-protein-coupled receptor A3 agonist), were intradermally injected into mice and itch-related scratching behavior was assessed. TRPV4 knockout mice exhibited significantly fewer 5-HT-evoked scratching bouts compared with wild-type mice. Notably, no differences between TRPV4 knockout and wild-type mice were observed in the number of scratch bouts elicited by SLIGRL and histamine. Pretreatment with a TRPV4 antagonist significantly attenuated 5-HT-evoked scratching in vivo. Using calcium imaging in cultured primary murine dorsal root ganglion neurons, the response of neurons after 5-HT application, but not other pruritogens, was significantly lower in TRPV4 knockout compared with wild-type mice. A TRPV4 antagonist significantly suppressed 5-HT-evoked responses in dorsal root ganglion cells from wild-type mice. Approximately 90% of 5-HT-sensitive dorsal root ganglion neurons were immunoreactive for an antibody to TRPV4, as assessed by calcium imaging. These results indicate that 5-HT-induced itch is linked to TRPV4.

10.20. Involvement of TRPV4 in serotonin-evoked scratching

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Journal Club 2017. 10. 13.

synergistic antipruritic effects of gamma aminobutyric acid A and B agonists ina a mouse model of atopic dermatitis

Ferda Cevikbas, PhD,a,b* Joao M. Braz, PhD,a* Xidao Wang, PhD,a Carlos Solorzano, PhD,a Mathias Sulk, MD,b,c
Timo Buhl, MD,b,d Martin Steinhoff, PhD,b,e,f,g,h and Allan I. Basbaum, PhDa

 

Atopic dermatitis (AD), an inflammatory, relapsing chronic
pruritic skin disease, is an often intractable form of chronic itch
that negatively impacts the quality of life of millions of patients.1
Unfortunately, because chronic itch conditions have very
different etiologies, most treatments have poor outcomes and
are accompanied by unacceptable adverse side effects, notably
sedation.2 Clearly, a better understanding of the pathophysiology
of these chronic itch conditions is critical to designing successful
therapeutic strategies.
Studies of the etiology of chronic itch3 generally focus on
changes in skin and immune dysfunction. However, there is
now considerable evidence for a contribution of primary afferent
pruritoceptors that transmit itch messages to spinal cord and
brainstem circuits engaged by and that regulate these messages.4
Of particular interest are studies demonstrating commonalities in
the mechanisms underlying nerve injury-induced neuropathic
pain and itch and the possibility that comparable approaches
may be appropriate for their management.5
Although there is evidence for specificity in the transmission of
itch and pain messages at the level of the primary afferent
nociceptor and pruritoceptor,6,7 both pain and itch are under
spinal cord inhibitory interneuron-mediated control. For example,
loss of spinal cord gamma aminobutyric acid (GABA) or glycinergic
function is a major contributor to the spontaneous pain and hypersensitivity
that develops following nerve injury.8-10 Moreover,
persistent scratching, a manifestation of chronic itch, occurs in the
Bhlhb5 mutant mouse, in which there is dramatic loss of dorsal
horn GABAergic inhibitory interneurons.11 Ablation of glycinergic
interneurons also induces excessive scratching and pain.12
And in a model of dry skin–induced scratching in the mouse,
GABA and glycine receptor antagonists can block scratchinginduced
inhibition of firing in superficial dorsal horn neurons.13
Finally, in patients, acute withdrawal of intrathecal baclofen, a
GABA-B receptor agonist, can induce pruritus.14
Given the evidence for a potential contribution of GABA
agonists in the management of pruritus, it is surprising that there
are no studies that assessed their utility in preclinical or clinical
conditions. Here, we demonstrate that both GABA-A and

GABA-B agonists are not only effective in models of acute itch,
but we also show that systemic administration of very low doses
of these agonists has synergistic antipruritic effects in IL-31
overexpressing transgenic mouse, a model of AD15 that is refractory
to antihistamines1,16 and thus particularly difficult to
manage. Most importantly, the antipruritic synergy could be produced
without concomitant sedation. Finally, we show that sustaining
high levels of GABA inhibition can be achieved using
intraspinal transplantation of cortical GABAergic interneuron
precursor cells. The transplants not only attenuated spontaneous
scratching but also dramatically reduced skin lesions in the IL-
31 overexpressing transgenic mouse (IL-31Tg) mice.

 

 

 

 

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