Alexander J. Davies

TRPV1 in GABAergic Interneurons Mediates Neuropathic Mechanical Allodynia and Disinhibition of the Nociceptive Circuitry in the Spinal Cord

Neuropathic pain and allodynia may arise from sensitization of central circuits. We report a mechanism of disinhibition-based central sensitization resulting from long-term depression (LTD) of GABAergic interneurons as a consequence of TRPV1 activation in the spinal cord. Intrathecal administration of TRPV1 agonists led to mechanical allodynia that was not dependent on peripheral TRPV1 neurons. TRPV1 was functionally expressed in GABAergic spinal interneurons and activation of spinal TRPV1 resulted in LTD of excitatory inputs and a reduction of inhibitory signaling to spinothalamic tract (STT) projection neurons. Mechanical hypersensitivity after peripheral nerve injury was attenuated in TRPV1(-/-) mice but not in mice lacking TRPV1-expressing peripheral neurons. Mechanical pain was reversed by a spinally applied TRPV1 antagonist while avoiding the hyperthermic side effect of systemic treatment. Our results demonstrate that spinal TRPV1 plays a critical role as a synaptic regulator and suggest the utility of central nervous system-specific TRPV1 antagonists for treating neuropathic pain.

Eugenol reverses mechanical allodynia after peripheral nerve injury by inhibiting hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.

Summary Eugenol reverses mechanical allodynia at relatively lower concentrations than that which effectively blocks thermal hyperalgesia in the trigeminal system, acting via the inhibition of Ih currents. ABSTRACT Mechanical allodynia is a common symptom found in neuropathic patients. Hyperpolarization‐activated cyclic nucleotide‐gated channels and their current, Ih, have been suggested to play an important role in neuropathic pain, especially in mechanical allodynia and spontaneous pain, by involvement in spontaneous ectopic discharges after peripheral nerve injury. Thus, Ih blockers may hold therapeutic potential for the intervention of mechanical allodynia under diverse neuropathic conditions. Here we show that eugenol blocks Ih and abolishes mechanical allodynia in the trigeminal system. Eugenol produced robust inhibition of Ih with IC50 of 157 μM in trigeminal ganglion (TG) neurons, which is lower than the dose of eugenol that inhibits voltage‐gated Na channels. Eugenol‐induced Ih inhibition was not mediated by Gi/o‐protein activation, but was gradually diminished by an increase in intracellular cAMP concentration. Eugenol also inhibited Ih from injured TG neurons which were identified by retrograde labeling with DiI and reversed mechanical allodynia in the orofacial area after chronic constriction injury of infraorbital nerve. We propose that eugenol could be potentially useful for reversing mechanical allodynia in neuropathic pain patients.

Intracellular Acidification Is Associated with Changes in Free Cytosolic Calcium and Inhibition of Action Potentials in Rat Trigeminal Ganglion

The effect of intracellular acidification and subsequent pH recovery in sensory neurons has not been well characterized. We have studied the mechanisms underlying Ca2+-induced acidification and subsequent recovery of intracellular pH (pHi) in rat trigeminal ganglion neurons and report their effects on neuronal excitability. Glutamate (500 μm) and capsaicin (1 μm) increased intracellular Ca2+ concentration ([Ca2+]i) with a following decrease in pHi. The recovery of [Ca2+]i to the prestimulus level was inhibited by LaCl3 (1 mm) and o-vanadate (10 mm), a plasma membrane Ca2+/ATPase (PMCA) inhibitor. Removal of extracellular Ca2+ also completely inhibited the acidification induced by capsaicin. TRPV1 was expressed only in small and medium sized trigeminal ganglion neurons. mRNAs for Na+/H+ exchanger type 1 (NHE1), pancreatic Na+-HCO3− cotransporter type 1 (pNBC1), NBC3, NBC4, and PMCA types 1–3 were detected by RT-PCR. pHi recovery was significantly inhibited by pretreatment with NHE1 or pNBC1 siRNA. We found that the frequency of action potentials (APs) was dependent on pHi. Application of the NHE1 inhibitor 5′-(N-ethyl-N-isopropyl) amiloride (5 μm) or the pNBC1 inhibitor 4′,4′-di-isothiocyanostilbene-2′,2′-sulfonic acid (500 μm) delayed pHi recovery and decreased AP frequency. Simultaneous application of 5′-(N-ethyl-N-isopropyl) amiloride and 4′,4′-di-isothiocyanostilbene-2′,2′-sulfonic acid almost completely inhibited APs. In summary, our results demonstrate that the rise in [Ca2+]i in sensory neurons by glutamate and capsaicin causes intracellular acidification by activation of PMCA type 3, that the pHi recovery from acidification is mediated by membrane transporters NHE1 and pNBC1 specifically, and that the activity of these transporters has direct consequences for neuronal excitability.

Chaperone stress 70 protein (STCH) binds and regulates two acid/base transporters NBCe1-B and NHE1

Background: Regulation of intracellular pH is critical for cellular homeostasis. Results: Stress 70 protein chaperone (STCH) interacts with pH regulating transporters NBCe1-B and NHE1 and modulates their functional expression. Conclusion: STCH chaperone dynamically regulates intracellular pH through site-specific interactions with ion transporters. Significance: These novel STCH interactions provide a mechanism for intracellular pH regulation in response to homeostatic stress. Regulation of intracellular pH is critical for the maintenance of cell homeostasis in response to stress. We used yeast two-hybrid screening to identify novel interacting partners of the pH-regulating transporter NBCe1-B. We identified Hsp70-like stress 70 protein chaperone (STCH) as interacting with NBCe1-B at the N-terminal (amino acids 96–440) region. Co-injection of STCH and NBCe1-B cRNA into Xenopus oocytes significantly increased surface expression of NBCe1-B and enhanced bicarbonate conductance compared with NBCe1-B cRNA alone. STCH siRNA decreased the rate of Na+-dependent pHi recovery from NH4+ pulse-induced acidification in an HSG (human submandibular gland ductal) cell line. We observed that in addition to NBCe1-B, Na+/H+ exchanger (NHE)-dependent pHi recovery was also impaired by STCH siRNA and further confirmed the interaction of STCH with NHE1 but not plasma membrane Ca2+ ATPase. Both NBCe1-B and NHE1 interactions were dependent on a specific 45-amino acid region of STCH. In conclusion, we identify a novel role of STCH in the regulation of pHi through site-specific interactions with NBCe1-B and NHE1 and subsequent modulation of membrane transporter expression. We propose STCH may play a role in pHi regulation at times of cellular stress by enhancing the recovery from intracellular acidification.

Painful Neuron-Microglia Interactions in the Trigeminal Sensory System

The trigeminal sensory system is unique in its innervation of structures specific to the orofacial area. Nocicep- tive trigeminal afferents are known to synapse with second-order neurons in the trigeminal subnucleus caudalis (Sp5C) in the brain stem. The activity of neurons within the Sp5C is responsible for the relay of nociceptive signals to higher brain centers. Recent evidence suggests that central sensitization may be fundamental to many trigeminal-specific painful neu- ropathies, including trigeminal neuralgia and migraine. Glia within the Sp5C are emerging as prime suspects in trigeminal central sensitization. In particular, microglial activation has been implicated in the development of neuropathic pain. It is possible that activated microglia release factors that alter the activity of second-order neurons or the synaptic activity of peripheral terminals within the Sp5C. Microglial activation has been characterized by changes in morphology, expression of membrane receptors and ion chan- nels, as well as alterations to cytokine and chemokine release. In addition, microglia have been studied in brain slice and dissociated culture where activation is characterized by changes to P2X receptor and potassium channel membrane cur- rents. However, little is known about resting and activated microglial membrane properties in the Sp5C and, furthermore, how these properties are affected following trigeminal nerve injury. This review summarizes the anatomical and patho- physiological importance of the Sp5C and focuses on recent studies on neurons and microglia in the trigeminal sensory system. The final part of the review aims to link important aspects of microglial membrane physiology with their potential role in chronic trigeminal pain conditions.

Attenuation of natural killer cell functions by capsaicin through a direct and TRPV1- independent mechanism

The assessment of the biological activity of capsaicin, the compound responsible for the spicy flavor of chili pepper, produced controversial results, showing either carcinogenicity or cancer prevention. The innate immune system plays a pivotal role in cancer pathology and prevention; yet, the effect of capsaicin on natural killer (NK) cells, which function in cancer surveillance, is unclear. This study found that capsaicin inhibited NK cell-mediated cytotoxicity and cytokine production (interferon-γ and tumor necrosis factor-α). Capsaicin impaired the cytotoxicity of NK cells, thereby inhibiting lysis of standard target cells and gastric cancer cells by modulating calcium mobilization in NK cells. Capsaicin also induced apoptosis in gastric cancer cells, but that effect required higher concentrations and longer exposure times than those required to trigger NK cell dysfunction. Furthermore, capsaicin inhibited the cytotoxicity of isolated NK cells and of an NK cell line, suggesting a direct effect on NK cells. Antagonists of transient receptor potential vanilloid subfamily member 1 (TRPV1), a cognate capsaicin receptor, or deficiency in TRPV1 expression failed to prevent the defects induced by capsaicin in NK cells expressing functional TRPV1. Thus, the mechanism of action of capsaicin on NK cells is largely independent of TRPV1. Taken together, capsaicin may have chemotherapeutic potential but may impair NK cell function, which plays a central role in tumor surveillance.

Acute inflammation reveals GABAA receptor-mediated nociception in mouse dorsal root ganglion neurons via PGE2 receptor 4 signaling

Gamma‐aminobutyric acid (GABA) depolarizes dorsal root ganglia (DRG) primary afferent neurons through activation of Cl− permeable GABAA receptors but the physiologic role of GABAA receptors in the peripheral terminals of DRG neurons remains unclear. In this study, we investigated the role of peripheral GABAA receptors in nociception using a mouse model of acute inflammation. In vivo, peripheral administration of the selective GABAA receptor agonist muscimol evoked spontaneous licking behavior, as well as spinal wide dynamic range (WDR) neuron firing, after pre‐conditioning with formalin but had no effect in saline‐treated mice. GABAA receptor‐mediated pain behavior after acute formalin treatment was abolished by the GABAA receptor blocker picrotoxin and cyclooxygenase inhibitor indomethacin. In addition, treatment with prostaglandin E2 (PGE2) was sufficient to reveal muscimol‐induced licking behavior. In vitro, GABA induced sub‐threshold depolarization in DRG neurons through GABAA receptor activation. Both formalin and PGE2 potentiated GABA‐induced Ca2+ transients and membrane depolarization in capsaicin‐sensitive nociceptive DRG neurons; these effects were blocked by the prostaglandin E2 receptor 4 (EP4) antagonist AH23848 (10 μmol/L). Furthermore, potentiation of GABA responses by PGE2 was prevented by the selective Nav1.8 antagonist A887826 (100 nmol/L). Although the function of the Na+‐K+‐2Cl‐ co‐transporter NKCC1 was required to maintain the Cl‐ ion gradient in isolated DRG neurons, NKCC1 was not required for GABAA receptor‐mediated nociceptive behavior after acute inflammation. Taken together, these results demonstrate that GABAA receptors may contribute to the excitation of peripheral sensory neurons in inflammation through a combined effect involving PGE2‐EP4 signaling and Na+ channel sensitization.

Pain Fiber Anesthetic Reduces Brainstem Fos after Tooth Extraction

We recently demonstrated that pain-sensing neurons in the trigeminal system can be selectively anesthetized by co-application of QX-314 with the TRPV1 receptor agonist, capsaicin (QX cocktail). Here we examined whether this new anesthetic strategy can block the neuronal changes in the brainstem following molar tooth extraction in the rat. Adult male Sprague-Dawley rats received infiltration injection of anesthetic 10 min prior to lower molar tooth extraction. Neuronal activation was determined by immunohistochemistry for the proto-oncogene protein c-Fos in transverse sections of the trigeminal subnucleus caudalis (Sp5C). After tooth extraction, c-Fos-like immunoreactivity (Fos-LI) detected in the dorsomedial region of bilateral Sp5C was highest at 2 hrs (p < .01 vs. naïve ipsilateral) and declined to pre-injury levels by 8 hrs. Pre-administration of the QX cocktail significantly reduced to sham levels Fos-LI examined 2 hrs after tooth extraction; reduced Fos-LI was also observed with the conventional local anesthetic lidocaine. Pulpal anesthesia by infiltration injection was confirmed by inhibition of the jaw-opening reflex in response to electrical tooth pulp stimulation. Our results suggest that the QX cocktail anesthetic is effective in reducing neuronal activation following tooth extraction. Thus, a selective pain fiber ‘nociceptive anesthetic’ strategy may provide an effective local anesthetic option for dental patients in the clinic.