TRPA1 activation in a human sensory neuronal model: relevance to cough hypersensitivity?

The cough reflex becomes hyperresponsive in acute and chronic respiratory diseases, but understanding the underlying mechanism is hampered by difficulty accessing human tissue containing both nerve endings and neuronal cell bodies. We refined an adult stem cell sensory neuronal model to overcome the limited availability of human neurones and applied the model to study transient receptor potential ankyrin 1 (TRPA1) channel expression and activation.Human dental pulp stem cells (hDPSCs) were differentiated towards a neuronal phenotype, termed peripheral neuronal equivalents (PNEs). Using molecular and immunohistochemical techniques, together with Ca2+ microfluorimetry and whole cell patch clamping, we investigated roles for nerve growth factor (NGF) and the viral mimic poly I:C in TRPA1 activation.PNEs exhibited morphological, molecular and functional characteristics of sensory neurons and expressed functional TRPA1 channels. PNE treatment with NGF for 20 min generated significantly larger inward and outward currents compared to untreated PNEs in response to the TRPA1 agonist cinnamaldehyde (p<0.05). PNE treatment with poly I:C caused similar transient heightened responses to TRPA1 activation compared to untreated cells.Using the PNE neuronal model we observed both NGF and poly I:C mediated sensory neuronal hyperresponsiveness, representing potential neuro-inflammatory mechanisms associated with heightened nociceptive responses recognised in cough hypersensitivity syndrome.

Mucosal modulation of contractility in bladder strips from normal and overactive rat models and the effect of botulinum toxin A on overactive bladder strips.

AIMS: To investigate the local, regulatory role of the mucosa on bladder strip contractility from normal and overactive bladders and to examine the effect of botulinum toxin A (BoNT-A). METHODS: Bladder strips from spontaneously hyperactive rat (SHR) or normal rats (Sprague Dawley, SD) were dissected for myography as intact or mucosa-free preparations. Spontaneous, neurogenic and agonist-evoked contractions were investigated. SHR strips were incubated in BoNT-A (3 h) to assess effects on contractility. RESULTS: Spontaneous contraction amplitude, force-integral or frequency were not significantly different in SHR mucosa-free strips compared with intacts. In contrast, spontaneous contraction amplitude and force-integral were smaller in SD mucosa-free strips than in intacts; frequency was not affected by the mucosa. Frequency of spontaneous contractions in SHR strips was significantly greater than in SD strips. Neurogenic contractions in mucosa-free SHR and SD strips at higher frequencies were smaller than in intact strips. The mucosa did not affect carbachol-evoked contractions in intact versus mucosa-free strips from SHR or SD bladders. BoNT-A reduced spontaneous contractions in SHR intact strips; this trend was also observed in mucosa-free strips but was not significant. Neurogenic and carbachol-evoked contractions were reduced by BoNT-A in mucosa-free but not intact strips. Depolarisation-induced contractions were smaller in BoNT-A-treated mucosa-free strips. CONCLUSIONS: The mucosal layer positively modulates spontaneous contractions in strips from normal SD but not overactive SHR bladder strips. The novel finding of BoNT-A reduction of contractions in SHR mucosa-free strips indicates actions on the detrusor, independent of its classical action on neuronal SNARE complexes.

Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium.

Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca2+]i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25 mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months' streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the molecular and functional expression of TRPV4 channels in retinal microvascular endothelial cells. These changes may contribute to diabetes induced endothelial dysfunction and retinopathy.

GABAergic control of arteriolar diameter in the rat retina.

PURPOSE: To investigate the role of γ-aminobutryic acid (GABA) in the regulation of arteriolar diameter in the rat retina. METHODS: The actions of GABA on arteriolar diameter were examined using ex vivo retinal whole-mount preparations and isolated vessel segments. In most experiments, arterioles were partially preconstricted with endothelin (Et)-1. The expression levels of GABAA and GABAB receptors on isolated rat retinal Müller cells were assessed by immunohistochemistry. RESULTS: GABA (0.1-1 mM) evoked vasodilation or vasoconstriction of arterioles in whole-mount preparations. No such effects were observed with isolated vessel segments. In whole mount samples, the GABAA receptor agonist muscimol caused vasomotor responses in only a small proportion of vessels. In contrast, arteriolar responses to the GABAB receptor agonists baclofen and SKF97541 more closely resembled those observed with GABA. No responses were seen with the GABAC receptor agonist 5-methylimidazoleacetic acid. GABA-induced vasodilator responses were, for the most part, repeatable in the presence of the GABAA receptor antagonist bicuculline. These responses, however, were completely blocked in the presence of the GABAB receptor inhibitor 2-hydroxysaclofen. Strong immunolabeling for both GABAA and GABAB receptors was detected in isolated Müller cells. In the absence of Et-1-induced preconstriction, most vessels were unresponsive to bicuculline or 2-hydroxysaclofen. CONCLUSIONS: GABA exerts complex effects on arteriolar diameter in the rat retina. These actions appear largely dependent upon the activation of GABAB receptors in the retinal neuropile, possibly those located on perivascular Müller cells. Despite these findings, endogenous GABA appears to contribute little to the regulation of basal arteriolar diameter in the rat retina.

Identification of PDGFRα positive populations of interstitial cells in human and guinea pig bladders.

PURPOSE: The bladder wall comprises a complex array of cells, including urothelium, smooth muscle, nerves and interstitial cells. Interstitial cells have several subtypes based on site, morphology and differential expression of markers such as anti-vimentin and anti-KIT. We examined whether a subpopulation of interstitial cells immunopositive for PDGFRα exists in human and guinea pig bladders. MATERIALS AND METHODS: Human and guinea pig bladder tissues were processed for immunohistochemistry and examined by bright field or confocal microscopy. Whole mount tissues and paraffin sections were labeled with antibodies to PDGFRα, vimentin, KIT and PGP9.5. Protein expression was assessed by Western blot. RESULTS: PDGFRα(+) cells were present in human and guinea pig bladders. In the guinea pig PDGFRα(+) cells had a branched stellate morphology and formed networks in the lamina propria. In human and guinea pig detrusors PDGFRα(+) cells were elongated on the boundary of smooth muscle bundles or were seen as groups of stellate cells in the interbundle spaces. PDGFRα(+) cells were located close to nerves labeled by PGP9.5. Double labeling revealed that PDGFRα(+) cells were a subgroup of the vimentin(+) population. A significant proportion of PDGFRα(+) cells were also KIT(+). Bands corresponding to PDGFRα, KIT and vimentin proteins were detected on Western blot. CONCLUSIONS: To our knowledge this study is the first to identify PDGFRα(+)/KIT(+) cells in the bladder lamina propria and detrusor layers. These cells are a subgroup of the vimentin(+) population, showing the complexity of bladder interstitial cells. PDGFRα(+) cells are apparently structurally associated with intramural nerves, indicating integration with bladder control mechanisms.

The stretch-dependent potassium channel TREK-1 and its function in murine myometrium.

Smooth muscle of the uterus stays remarkably quiescent during normal pregnancy to allow sufficient time for development of the fetus. At present the mechanisms leading to uterine quiescence during pregnancy and how the suppression of activity is relieved at term are poorly understood. Myometrial excitability is governed by ion channels, and a major hypothesis regarding the regulation of contractility during pregnancy has been that expression of certain channels is regulated by hormonal influences. We have explored the expression and function of stretch-dependent K+ (SDK) channels, which are likely to be due to TREK channels, in murine myometrial tissues and myocytes using PCR, Western blots, patch clamp, intracellular microelectrode and isometric force measurements. TREK-1 is more highly expressed than TREK-2 in myometrium, and there was no detectable expression of TRAAK. Expression of TREK-1 transcripts and protein was regulated during pregnancy and delivery. SDK channels were activated in response to negative pressure applied to patches. SDK channels were insensitive to a broad-spectrum of K+ channel blockers, including tetraethylammonium and 4-aminopyridine, and insensitive to intracellular Ca2+. SDK channels were activated by stretch and arachidonic acid and inhibited by reagents that block TREK-1 channels, l-methionine and/or methioninol. Our data suggest that uterine excitability and contractility during pregnancy is regulated by the expression of SDK/TREK-1 channels. Up-regulation of these channels stabilizes membrane potential and controls contraction during pregnancy and down-regulation of these channels induces the onset of delivery.

Imaging of effector memory T cells during a delayed-type hypersensitivity reaction and suppression by Kv1.3 channel block.

Effector memory T (Tem) cells are essential mediators of autoimmune disease and delayed-type hypersensitivity (DTH), a convenient model for two-photon imaging of Tem cell participation in an inflammatory response. Shortly (3 hr) after entry into antigen-primed ear tissue, Tem cells stably attached to antigen-bearing antigen-presenting cells (APCs). After 24 hr, enlarged Tem cells were highly motile along collagen fibers and continued to migrate rapidly for 18 hr. Tem cells rely on voltage-gated Kv1.3 potassium channels to regulate calcium signaling. ShK-186, a specific Kv1.3 blocker, inhibited DTH and suppressed Tem cell enlargement and motility in inflamed tissue but had no effect on homing to or motility in lymph nodes of naive and central memory T (Tcm) cells. ShK-186 effectively treated disease in a rat model of multiple sclerosis. These results demonstrate a requirement for Kv1.3 channels in Tem cells during an inflammatory immune response in peripheral tissues. Targeting Kv1.3 allows for effector memory responses to be suppressed while central memory responses remain intact.

Nucleotide regulation of the voltage-dependent nonselective cation conductance in murine colonic myocytes.

ATP is proposed to be a major inhibitory neurotransmitter in the gastrointestinal (GI) tract, causing hyperpolarization and smooth muscle relaxation. ATP activates small-conductance Ca(2+)-activated K(+) channels that are involved in setting the resting membrane potential and causing inhibitory junction potentials. No reports are available examining the effects of ATP on voltage-dependent inward currents in GI smooth muscle cells. We previously reported two types of voltage-dependent inward currents in murine proximal colonic myocytes: a low-threshold voltage-activated, nonselective cation current (I(VNSCC)) and a relatively high-threshold voltage-activated (L-type) Ca(2+) current (I(L)). Here we have investigated the effects of ATP on these currents. External application of ATP (1 mM) did not affect I(VNSCC) or I(L) in dialyzed cells. ATP (1 mM) increased I(VNSCC) and decreased I(L) in the perforated whole-cell configuration. UTP and UDP (1 mM) were more potent than ATP on I(VNSCC). ADP decreased I(L) but had no effect on I(VNSCC). The order of effectiveness was UTP = UDP > ATP > ADP. These effects were not blocked by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS), but the phospholipase C inhibitor U-73122 reversed the effects of ATP on I(VNSCC). ATP stimulation of I(VNSCC) was also reversed by protein kinase C (PKC) inhibitors chelerythrine chloride or bisindolylmaleimide I. Phorbol 12,13-dibutyrate mimicked the effects of ATP. RT-PCR showed that P2Y(4) is expressed by murine colonic myocytes, and this receptor is relatively insensitive to PPADS. Our data suggest that ATP activates I(VNSCC) and depresses I(L) via binding of P2Y(4) receptors and stimulation of the phospholipase C/PKC pathway.

A pH-sensitive potassium conductance (TASK) and its function in the murine gastrointestinal tract.

The excitability of smooth muscles is regulated, in part, by background K+ conductances that determine resting membrane potential. However, the K+ conductances so far described in gastrointestinal (GI) muscles are not sufficient to explain the negative resting potentials of these cells. Here we describe expression of two-pore K+ channels of the TASK family in murine small and large intestinal muscles. TASK-2, cloned from murine intestinal muscles, resulted in a pH-sensitive, time-dependent, non-inactivating K+ conductance with slow activation kinetics. A similar conductance was found in native intestinal myocytes using whole-cell patch-clamp conditions. The pH-sensitive current was blocked by local anaesthetics. Lidocaine, bupivacaine and acidic pH depolarized circular muscle cells in intact muscles and decreased amplitude and frequency of slow waves. The effects of lidocaine were not blocked by tetraethylammonium chloride, 4-aminopyridine, glibenclamide, apamin or MK-499. However, depolarization by acidic pH was abolished by pre-treatment with lidocaine, suggesting that lidocaine-sensitive K+ channels were responsible for pH-sensitive changes in membrane potential. The kinetics of activation, sensitivity to pH, and pharmacology of the conductance in intestinal myocytes and the expression of TASK-1 and TASK-2 in these cells suggest that the pH-sensitive background conductance is encoded by TASK genes. This conductance appears to contribute significantly to resting potential and may regulate excitability of GI muscles.

Mechanism of active repolarization of inhibitory junction potential in murine colon.

Enteric inhibitory responses in gastrointestinal (GI) smooth muscles involve membrane hyperpolarization that transiently reduce the excitability of GI muscles. We examined the possibility that an active repolarization mechanism participates in the restoration of resting membrane potential after fast inhibitory junction potentials (IJPs) in the murine colon. Previously, we showed these cells express a voltage-dependent nonselective cation conductance (NSCC) that might participate in active repolarization of IJPs. Colonic smooth muscle cells were impaled with micro-electrodes and voltage responses to nerve-evoked IJPs, and locally applied ATP were recorded. Ba2+ (500 muM), a blocker of the NSCC, slowed the rate of repolarization of IJPs. We also tested the effects of Ba2+, Ni2+, and mibefradil, all blockers of the NSCC, on responses to locally applied ATP. Spritzes of ATP caused transient hyperpolarization, and the durations of these responses were significantly increased by the blockers of the NSCC. We considered whether NSCC blockers might affect ATP metabolism and found that Ni2+ decreased ATP breakdown in colonic muscles. Mibefradil had no effect on ATP metabolism. Because both Ni2+ and mibefradil had similar effects on prolonging responses to ATP, it appears that restoration of resting membrane potential after ATP spritzes is not primarily due to ATP metabolism. Neurally released enteric inhibitory transmitter and locally applied ATP resulted in transient hyperpolarizations of murine colonic muscles. Recovery of membrane potential after these responses appears to involve an active repolarization mechanism due to activation of the voltage-dependent NSCC expressed by these cells.

Contribution of Kv4 channels toward the A-type potassium current in murine colonic myocytes.

A rapidly inactivating K(+) current (A-type current; I(A)) present in murine colonic myocytes is important in maintaining physiological patterns of slow wave electrical activity. The kinetic profile of colonic I(A) resembles that of Kv4-derived currents. We examined the contribution of Kv4 alpha-subunits to I(A) in the murine colon using pharmacological, molecular and immunohistochemical approaches. The divalent cation Cd(2+) decreased peak I(A) and shifted the voltage dependence of activation and inactivation to more depolarized potentials. Similar results were observed with La(3+). Colonic I(A) was sensitive to low micromolar concentrations of flecainide (IC(50) = 11 microM). Quantitative PCR indicated that in colonic and jejunal tissue, Kv4.3 transcripts demonstrate greater relative abundance than transcripts encoding Kv4.1 or Kv4.2. Antibodies revealed greater Kv4.3-like immunoreactivity than Kv4.2-like immunoreactivity in colonic myocytes. Kv4-like immunoreactivity was less evident in jejunal myocytes. To address this finding, we examined the expression of K(+) channel-interacting proteins (KChIPs), which act as positive modulators of Kv4-mediated currents. Qualitative PCR identified transcripts encoding the four known members of the KChIP family in isolated colonic and jejunal myocytes. However, the relative abundance of KChIP transcript was 2.6-fold greater in colon tissue than in jejunum, as assessed by quantitative PCR, with KChIP1 showing predominance. This observation is in accordance with the amplitude of the A-type current present in these two tissues, where colonic myocytes possess densities twice that of jejunal myocytes. From this we conclude that Kv4.3, in association with KChIP1, is the major molecular determinant of I(A) in murine colonic myocytes.