TRPV4-Mediated Calcium Influx into Human Bronchial Epithelia upon Exposure to Diesel Exhaust Particles

BACKGROUND: Human respiratory epithelia function in airway mucociliary clearance and barrier function and have recently been implicated in sensory functions. OBJECTIVE: We investigated a link between chronic obstructive pulmonary disease (COPD) pathogenesis and molecular mechanisms underlying Ca2+ influx into human airway epithelia elicited by diesel exhaust particles (DEP). METHODS AND RESULTS: Using primary cultures of human respiratory epithelial (HRE) cells, we determined that these cells possess proteolytic signaling machinery, whereby proteinase-activated receptor-2 (PAR-2) activates Ca2+-permeable TRPV4, which leads to activation of human respiratory disease-enhancing matrix metalloproteinase-1 (MMP-1), a signaling cascade initiated by diesel exhaust particles (DEP), a globally relevant air pollutant. Moreover, we observed ciliary expression of PAR-2, TRPV4, and phospholipase-Cβ3 in human airway epithelia and their DEP-enhanced protein-protein complex formation. We also found that the chronic obstructive pulmonary disease (COPD)-predisposing TRPV4P19S variant enhances Ca2+ influx and MMP 1 activation, providing mechanistic linkage between man-made air pollution and human airway disease. CONCLUSION: DEP evoked protracted Ca2+ influx via TRPV4, enhanced by the COPD-predisposing human genetic polymorphism TRPV4P19S. This mechanism reprograms maladaptive inflammatory and extracellular-matrix-remodeling responses in human airways. The novel concept of air pollution-responsive ciliary signal transduction from PAR-2 to TRPV4 in human respiratory epithelia will accelerate rationally targeted therapies, possibly via the inhalatory route

Small molecule dual-inhibitors of TRPV4 and TRPA1 for attenuation of inflammation and pain

TRPV4 ion channels represent osmo-mechano-TRP channels with pleiotropic function and wide-spread expression. One of the critical functions of TRPV4 in this spectrum is its involvement in pain and inflammation. However, few small-molecule inhibitors of TRPV4 are available. Here we developed TRPV4-inhibitory molecules based on modifications of a known TRPV4-selective tool-compound, GSK205. We not only increased TRPV4-inhibitory potency, but surprisingly also generated two compounds that potently co-inhibit TRPA1, known to function as chemical sensor of noxious and irritant signaling. We demonstrate TRPV4 inhibition by these compounds in primary cells with known TRPV4 expression - articular chondrocytes and astrocytes. Importantly, our novel compounds attenuate pain behavior in a trigeminal irritant pain model that is known to rely on TRPV4 and TRPA1. Furthermore, our novel dual-channel blocker inhibited inflammation and pain-associated behavior in a model of acute pancreatitis - known to also rely on TRPV4 and TRPA1. Our results illustrate proof of a novel concept inherent in our prototype compounds of a drug that targets two functionally-related TRP channels, and thus can be used to combat isoforms of pain and inflammation in-vivo that involve more than one TRP channel. This approach could provide a novel paradigm for treating other relevant health conditions

Selective Cholinergic Depletion in Medial Septum Leads to Impaired Long Term Potentiation and Glutamatergic Synaptic Currents in the Hippocampus

Cholinergic depletion in the medial septum (MS) is associated with impaired hippocampal-dependent learning and memory. Here we investigated whether long term potentiation (LTP) and synaptic currents, mediated by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors in the CA1 hippocampal region, are affected following cholinergic lesions of the MS. Stereotaxic intra-medioseptal infusions of a selective immunotoxin, 192-saporin, against cholinergic neurons or sterile saline were made in adult rats. Four days after infusions, hippocampal slices were made and LTP, whole cell, and single channel (AMPA or NMDA receptor) currents were recorded. Results demonstrated impairment in the induction and expression of LTP in lesioned rats. Lesioned rats also showed decreases in synaptic currents from CA1 pyramidal cells and synaptosomal single channels of AMPA and NMDA receptors. Our results suggest that MS cholinergic afferents modulate LTP and glutamatergic currents in the CA1 region of the hippocampus, providing a potential synaptic mechanism for the learning and memory deficits observed in the rodent model of selective MS cholinergic lesioning

TRPV4 is necessary for trigeminal irritant pain and functions as a cellular formalin receptor.

Detection of external irritants by head nociceptor neurons has deep evolutionary roots. Irritant-induced aversive behavior is a popular pain model in laboratory animals. It is used widely in the formalin model, where formaldehyde is injected into the rodent paw, eliciting quantifiable nocifensive behavior that has a direct, tissue-injury-evoked phase, and a subsequent tonic phase caused by neural maladaptation. The formalin model has elucidated many antipain compounds and pain-modulating signaling pathways. We have adopted this model to trigeminally innervated territories in mice. In addition, we examined the involvement of TRPV4 channels in formalin-evoked trigeminal pain behavior because TRPV4 is abundantly expressed in trigeminal ganglion (TG) sensory neurons, and because we have recently defined TRPV4's role in response to airborne irritants and in a model for temporomandibular joint pain. We found TRPV4 to be important for trigeminal nocifensive behavior evoked by formalin whisker pad injections. This conclusion is supported by studies with Trpv4(-/-) mice and TRPV4-specific antagonists. Our results imply TRPV4 in MEK-ERK activation in TG sensory neurons. Furthermore, cellular studies in primary TG neurons and in heterologous TRPV4-expressing cells suggest that TRPV4 can be activated directly by formalin to gate Ca(2+). Using TRPA1-blocker and Trpa1(-/-) mice, we found that both TRP channels co-contribute to the formalin trigeminal pain response. These results imply TRPV4 as an important signaling molecule in irritation-evoked trigeminal pain. TRPV4-antagonistic therapies can therefore be envisioned as novel analgesics, possibly for specific targeting of trigeminal pain disorders, such as migraine, headaches, temporomandibular joint, facial, and dental pain, and irritation of trigeminally innervated surface epithelia

Cholinergic depletion in the MS leads to alterations in NMDA elicited synaptic single channel currents.

<p>Sample traces and respective current amplitude histograms of (A) non-infused control rats, (B) saline infused rats, and (C) 192-saporin infused rats show that channel open peak (right peaks in each histogram) in the 192-saporin data is reduced demonstrating a decrease in open probability (Calibration: each 4 pA, 200 ms). Dwell open time histograms of NMDA currents were best fitted with two terms for (D) non-infused, (E) saline infused, and (F) 192 saporin infused data. The NMDA elicited currents were confirmed by addition of APV to the extracellular solution to completely block these currents (Data not shown).</p

Inhibition of MS cholinergic pathway decreases the AMPA receptor activity in the CA1 pyramidal neurons of the hippocampus.

<p>(A) Representatives of AMPA receptor mediated sEPSC traces in controls and 192-saporin infused rats recorded at −65 mV membrane potential; Calibration: 40 pA, 1 s. The adjacent average traces depict the reduction in the amplitude in the MS cholinergic lesioned rats compared to control rats, which show no difference in amplitude; Calibration: 15 pA, 30 ms. (B) Cumulative fraction plot of sEPSC amplitude from the composite data shows the shift of 192-saporin curve to the left from the controls, indicating reductions in amplitude. (C) Cumulative fraction plots of sEPSC interevent intervals exhibiting increased values for 192-saporin infused rats suggesting decreased frequency. (D) Representative traces of AMPA receptor mediated mEPSCs for the three groups (Calibration: 10 pA, 1 s) and the mEPSCs (Calibration: 10 pA, 30 ms) to show reduced amplitude in the 192-saporin infused rats. (E) Cumulative fraction plots for amplitude and (F) interevent interval exhibiting reduced amplitude and frequency.</p

Impairment of LTP in 192-saporin infused rats.

<p>(A) Summary data for experiments in which LTP was induced by TBS and measured at 55–60 min after TBS. LTP in 192-saporin infuced rats (gray circles) was reduced compared to the saline infused controls (black circles). (B) Sample traces depicting LTP in saline infused control rats and lack of LTP in 192-saporin infused rats. Traces with gray lines represent those collected prior to TBS, during baseline recording, and the traces with black lines represent those taken 55–60 min after TBS. Calibration: 1 mV, 20 ms. (c) Bar chart showing drastic reduction of LTP in 192-saporin infused rats (gray) compared to the saline infused controls (black).</p

Burst analysis of AMPA and NMDA receptor single channel currents in synaptosomes.

<p>*<i>P</i><0.05,</p>#<p><i>P</i><0.01.</p

Reduction in the TBS facilitation in 192-saporin infused rats.

<p>(A) TBS stimulation protocol and samples of TBS induced traces from saline infused control and 192-saporin infused rats. (B) Bar chart exhibiting reduction of facilitation within the first TBS in 192-saporin infused rats (gray) compared to the controls (black); Calibration: 4 mV, 200 ms. (C) Facilitation of between TBS potentiation was impaired in 192-saporin infused rats (gray bars) compared to the saline infused controls (black bars) as shown by the sample traces and the bar chart. Sample traces are the fEPSPs in response to the first pulse in the first TBS. Calibration: 2 mV, 20 ms.</p

Infusion of 192-saporin results in lesioning of cholinergic neurons.

<p>Photomicrographs (50× magnification) of MS region immunostained with anti-ChAT antibody in (A) a saline infused rat and (B) in a rat infused with 192-saporin.</p