Role of bradykinin and prostaglandin EP4 receptors in regulating TRPV1 channel sensitization in rat dorsal root ganglion neurons.

Transient receptor potential vanilloid type-1 (TRPV1) channels play key roles in chronic pain conditions and are modulated by different inflammatory mediators to elicit heat sensitisation. Bradykinin is a 9-amino acid peptide chain that promotes inflammation. The aim of present study is to investigate how bradykinin and prostaglandin receptors (EP3 and EP4 ) modulate the sensitisation of TRPV1-mediated responses. Calcium imaging studies of rat dorsal root ganglion (DRG) neurons were employed to investigate the desensitizing responses of TRPV1 ion channels by capsaicin, and the re-sensitization of TRPV1 by bradykinin, then to explore the role EP3 and EP4 receptors in mediating these bradykinin-dependent effects. Immunocytochemistry was used to study the co-expression and distribution of EP4, TRPV1, COX-1 and B2 in rat DRG neurons. Desensitization was seen upon repeated capsaicin application, we show that bradykinin-mediated sensitization of capsaicin-evoked calcium responses in rat DRG neurons occurs is dependent on COX-1 activity and utilizes a pathway that involves EP4 but not EP3 receptors. Immunocytochemical techniques revealed that EP4, TRPV1, COX-1 and B2 proteins are expressed mainly in small diameter (<1000 µm2 ) cell bodies of rat DRG neurons which are typically nociceptors. The present study provides suggestive evidence for a potential signalling pathway through which bradykinin may regulate TRPV1 ion channel function via EP4 receptors. In addition to confirming existing knowledge, the anatomical distribution and colocalization of these proteins in DRG neurons as revealed by this study offer valuable insight.

TRPA1-Mediated Src Family Kinases Activity Facilitates Cortical Spreading Depression Susceptibility and Trigeminovascular System Sensitization.

Transient receptor potential ankyrin 1 (TRPA1) plays a role in migraine and is proposed as a promising target for migraine therapy. However, TRPA1-induced signaling in migraine pathogenesis is poorly understood. In this study, we explored the hypothesis that Src family kinases (SFKs) transmit TRPA1 signaling in regulating cortical spreading depression (CSD), calcitonin gene-related peptide (CGRP) release and neuroinflammation. CSD was monitored in mouse brain slices via intrinsic optical imaging, and in rats using electrophysiology. CGRP level and IL-1ß gene expression in mouse trigeminal ganglia (TG) was detected using Enzyme-linked Immunosorbent Assay and Quantitative Polymerase Chain Reaction respectively. The results showed a SFKs activator, pYEEI (EPQY(PO3H2)EEEIPIYL), reversed the reduced cortical susceptibility to CSD by an anti-TRPA1 antibody in mouse brain slices. Additionally, the increased cytosolic phosphorylated SFKs at Y416 induced by CSD in rat ipsilateral cerebral cortices was attenuated by pretreatment of the anti-TRPA1 antibody perfused into contralateral ventricles. In mouse TG, a SFKs inhibitor, saracatinib, restored the CGRP release and IL-1ß mRNA level increased by a TRPA1 activator, umbellulone. Moreover, umbellulone promoted SFKs phosphorylation, which was reduced by a PKA inhibitor, PKI (14-22) Amide. These data reveal a novel mechanism of migraine pathogenesis by which TRPA1 transmits signaling to SFKs via PKA facilitating CSD susceptibility and trigeminovascular system sensitization.

ROS/TRPA1/CGRP signaling mediates cortical spreading depression.

OBJECTIVES: The transient receptor potential ankyrin A 1 (TRPA1) channel and calcitonin gene-related peptide (CGRP) are targets for migraine prophylaxis. This study aimed to understand their mechanisms in migraine by investigating the role of TRPA1 in cortical spreading depression (CSD) in vivo and exploring how reactive oxygen species (ROS)/TRPA1/CGRP interplay in regulating cortical susceptibility to CSD. METHODS: Immunohistochemistry was used for detecting TRPA1 expression. CSD was induced by K+ on the cerebral cortex, monitored using electrophysiology in rats, and intrinsic optical imaging in mouse brain slices, respectively. Drugs were perfused into contralateral ventricle of rats. Lipid peroxidation (malondialdehyde, MDA) analysis was used for indicating ROS level. RESULTS: TRPA1 was expressed in cortical neurons and astrocytes of rats and mice. TRPA1 deactivation by an anti-TRPA1 antibody reduced cortical susceptibility to CSD in rats and decreased ipsilateral MDA level induced by CSD. In mouse brain slices, H2O2 facilitated submaximal CSD induction, which disappeared by the antioxidant, tempol and the TRPA1 antagonist, A-967079; Consistently, TRPA1 activation reversed prolonged CSD latency and reduced magnitude by the antioxidant. Further, blockade of CGRP prolonged CSD latency, which was reversed by H2O2 and the TRPA1 agonist, allyl-isothiocyanate, respectively. CONCLUSIONS: ROS/TRPA1/CGRP signaling plays a critical role in regulating cortical susceptibility to CSD. Inhibition ROS and deactivation of TRPA1 channels may have therapeutic benefits in preventing stress-triggered migraine via CGRP.

Electrophysiological effects of nicotinic and electrical stimulation of intrinsic cardiac ganglia in the absence of extrinsic autonomic nerves in the rabbit heart.

BACKGROUND: The intrinsic cardiac nervous system is a rich network of cardiac nerves that converge to form distinct ganglia and extend across the heart and is capable of influencing cardiac function. OBJECTIVE: The goals of this study were to provide a complete picture of the neurotransmitter/neuromodulator profile of the rabbit intrinsic cardiac nervous system and to determine the influence of spatially divergent ganglia on cardiac electrophysiology. METHODS: Nicotinic or electrical stimulation was applied at discrete sites of the intrinsic cardiac nerve plexus in the Langendorff-perfused rabbit heart. Functional effects on sinus rate and atrioventricular conduction were measured. Immunohistochemistry for choline acetyltransferase (ChAT), tyrosine hydroxylase, and/or neuronal nitric oxide synthase (nNOS) was performed using whole mount preparations. RESULTS: Stimulation within all ganglia produced either bradycardia, tachycardia, or a biphasic brady-tachycardia. Electrical stimulation of the right atrial and right neuronal cluster regions produced the largest chronotropic responses. Significant prolongation of atrioventricular conduction was predominant at the pulmonary vein-caudal vein region. Neurons immunoreactive (IR) only for ChAT, tyrosine hydroxylase, or nNOS were consistently located within the limits of the hilum and at the roots of the right cranial and right pulmonary veins. ChAT-IR neurons were most abundant (1946 ± 668 neurons). Neurons IR only for nNOS were distributed within ganglia. CONCLUSION: Stimulation of intrinsic ganglia, shown to be of phenotypic complexity but predominantly of cholinergic nature, indicates that clusters of neurons are capable of independent selective effects on cardiac electrophysiology, therefore providing a potential therapeutic target for the prevention and treatment of cardiac disease.

Protection from noise-induced hearing loss by Kv2.2 potassium currents in the central medial olivocochlear system.

The central auditory brainstem provides an efferent projection known as the medial olivocochlear (MOC) system, which regulates the cochlear amplifier and mediates protection on exposure to loud sound. It arises from neurons of the ventral nucleus of the trapezoid body (VNTB), so control of neuronal excitability in this pathway has profound effects on hearing. The VNTB and the medial nucleus of the trapezoid body are the only sites of expression for the Kv2.2 voltage-gated potassium channel in the auditory brainstem, consistent with a specialized function of these channels. In the absence of unambiguous antagonists, we used recombinant and transgenic methods to examine how Kv2.2 contributes to MOC efferent function. Viral gene transfer of dominant-negative Kv2.2 in wild-type mice suppressed outward K(+) currents, increasing action potential (AP) half-width and reducing repetitive firing. Similarly, VNTB neurons from Kv2.2 knock-out mice (Kv2.2KO) also showed increased AP duration. Control experiments established that Kv2.2 was not expressed in the cochlea, so any changes in auditory function in the Kv2.2KO mouse must be of central origin. Further, in vivo recordings of auditory brainstem responses revealed that these Kv2.2KO mice were more susceptible to noise-induced hearing loss. We conclude that Kv2.2 regulates neuronal excitability in these brainstem nuclei by maintaining short APs and enhancing high-frequency firing. This safeguards efferent MOC firing during high-intensity sounds and is crucial in the mediation of protection after auditory overexposure.

Neuroprotection mediated by the EP4 receptor avoids the detrimental side effects of COX-2 inhibitors following ischaemic injury.

Although COX-2 inhibition in animal models of ischaemia has shown neuroprotection, clinical trials revealed long term side effects with COX-2 inhibitors. A more focussed approach is necessary to retain the therapeutic effects of prostaglandins. This study investigated the role of the PGE(2) EP(4) receptor using both in vitro and in vivo models of ischaemia. To demonstrate whether targeting the EP(4) receptor is as neuroprotective as COX-2 inhibition, simultaneous experiments were carried out using a selective COX-2 inhibitor. Organotypic hippocampal sliced cultures, exposed to 2 h of oxygen glucose deprivation, were treated with; DMSO only, COX-2 inhibitor (NS-398), EP(4) agonist (L-902688) or EP(4) antagonist (GW627368X) and cell death was assessed. The EP(4) agonist and the COX-2 inhibitor significantly reduced cell death following in vitro ischaemia, whereas treatment with the EP(4) antagonist significantly increased cell death in hippocampal cultures. Following a 1 h occlusion of middle cerebral artery, mice were treated with the COX-2 inhibitor (10 mg kg, I.P), EP(4) agonist (0.75 µg/kg, I.P) or vehicle (I.P), at the onset of reperfusion and again at 24 h post stroke. The COX-2 inhibitor and EP(4) agonist treated animals showed a significant reduction in infarct volume (P < .05) at 48 h post stroke compared to the vehicle treated group. These results show that selective activation of the EP(4) receptor following acute ischaemic damage is neuroprotective, and support the concept of targeting protective prostaglandin receptor signalling as a potential therapeutic target for cerebral stroke.

Investigation of the mechanisms of progesterone protection following oxygen-glucose deprivation in organotypic hippocampal slice cultures.

This study aimed to test the hypothesis that progesterone is neuroprotective against oxygen-glucose deprivation (OGD) through its conversion to the active metabolite allopregnanolone (AlloP) and the potentiation of GABA(A) receptors. Organotypic hippocampal cultures were exposed to 2h of OGD and the resulting cell death was quantified 24h later using combined propidium iodide and Hoechst immunostaining. Initially, we confirmed, that both progesterone and AlloP were protective in terms of reducing cell death following OGD in hippocampal cultures and for both, the optimal level of protection was observed at a concentration of 0.1µM. However, the protective effect of progesterone was absent in the presence of finasteride (10µM) which inhibits the metabolism of progesterone to active metabolites, including AlloP. In addition, the concurrent application of picrotoxin (100µM), a potent GABA(A) receptor antagonist, prevented the protection previously seen by either progesterone or AlloP alone. These results indicate that progesterone protects hippocampal cultures from cell death following OGD largely due to its conversion to AlloP and that GABA(A) receptors are important mediators of the protective effects of both progesterone and AlloP.

Ototrauma induces sodium channel plasticity in auditory afferent neurons.

Exposure to intense sound can cause damage to the delicate sensory and neuronal components of the cochlea leading to hearing loss. Such damage often causes the dendrites of the spiral ganglion neurons (SGN), the neurons that provide the afferent innervation of the hair cells, to swell and degenerate thus damaging the synapse. In models of neuropathic pain, axotomy, another form of afferent nerve damage, is accompanied by altered voltage-gated sodium channel (VGSC) expression, leading to neuronal hyperactivity. In this study, adult Wistar rats were exposed to noise which produced a mild, 20 dB hearing threshold elevation and their VGSC expression was investigated. Quantitative PCR showed decreased Na(V)1.1 and Na(V)1.6 mRNA expression in the SGN following noise exposure (29% and 56% decrease respectively) while Na(V)1.7 mRNA expression increased by approximately 20% when compared to control rats. Immunohistochemistry extended these findings, revealing increased staining for Na(V)1.1 along the SGN dendrites and Na(V)1.7 in the cell bodies after noise. These results provide the first evidence for selective changes in VGSC expression following moderate noise-induced hearing loss and could contribute to elevated hearing thresholds and to the generation of perceptual anomalies commonly associated with cochlear damage, such as tinnitus and hyperacusis.

Bradykinin-induced nociceptor sensitisation to heat depends on cox-1 and cox-2 in isolated rat skin.

Bradykinin is an important inflammatory mediator that can either activate and/or sensitise nociceptors to heat stimuli applied to the skin. Several studies have suggested that prostaglandins and thus the cyclooxygenase (cox) enzymes are important in the sensitisation process but little is known about the relative involvement of the two cox isoforms, cox-1 and cox-2. Extracellular recordings were made from C-mechanoheat-sensitive fibres in isolated rat skin-saphenous nerve preparations. Bradykinin-mediated sensitisation of heat responses in these afferents was significantly attenuated by the selective cox-1 inhibitor, SC-560, and by the selective cox-2 inhibitor, NS-398. In the same experiments, bradykinin-mediated induction of ongoing activity was reduced by SC-560 but not NS-398. In a second series of experiments, bradykinin-stimulated synthesis and release of prostaglandin E2 (PGE2) was measured in isolated skin-nerve preparations. Although the basal release of PGE2 appeared unaffected by either drug, bradykinin-stimulated PGE2 release from the skin was inhibited by both SC-560 and NS-398. Immunocytochemical evaluation revealed cox-1 immunostaining was present in large cutaneous nerve branches, small intradermal nerve bundles as well as nerve endings within the skin. Cox-1 labelling was also present in non-neuronal cell types such as mast cells. Cox-2 immunoreactivity was weak but where present was located in small nerve bundles, smaller intradermal nerve bundles and nerve endings. This study shows that both cox isoforms are present in skin and that they have an important role in mediating bradykinin-evoked heat sensitisation of C-MH sensitive fibres through cox-1 and cox-2 dependent prostaglandin synthesis.

Statin-sensitive endocytosis of albumin by glomerular podocytes.

Glomerular podocytes are critical regulators of glomerular permeability via the slit diaphragm and may play a role in cleaning the glomerular filter. Whether podocytes are able to endocytose proteins is uncertain. We studied protein endocytosis in conditionally immortalized mouse and human podocytes using FITC-albumin by direct quantitative assay and by fluorescence microscopy and electron microscopy in mouse podocytes. Furthermore, in vivo uptake was studied in human, rat, and mouse podocytes. Both mouse and human podocytes displayed specific one-site binding for FITC-albumin with K(d) of 0.91 or 0.44 mg/ml and B(max) of 3.15 or 0.81 microg/mg cell protein, respectively. In addition, they showed avid endocytosis of FITC-albumin with K(m) of 9.48 or 4.5 mg/ml and V(max) of 474.3 or 97.4 microg.mg cell protein(-1).h(-1), respectively. Immunoglobulin and transferrin were inefficient competitors of this process, indicating some specificity for albumin. Accumulation of endocytosed albumin could be demonstrated in intracellular vesicles by fluorescence confocal microscopy and electron microscopy. Endocytosis was sensitive to pretreatment with simvastatin. In vivo accumulation of albumin was found in all three species but was most pronounced in the rat. We conclude that podocytes are able to endocytose protein in a statin-sensitive manner. This function is likely to be highly significant in health and disease. In addition, protein endocytosis by podocytes may represent a useful, measurable phenotypic characteristic against which potentially injurious or beneficial interventions can be assessed.

Activation of sensory neurons in the arthritic joint.

Joints are richly innervated with a range of sensory nerve fibres that convey information to the central nervous system about forces exerted on articular tissues by both low and high threshold mechanical stimuli. High threshold nociceptive afferents terminate primarily in the synovium and periosteum, and normally respond only to movement of the joint beyond the working limits. Following joint damage, two factors combine to alter the mechanical sensitivity of articular nociceptors. Firstly, physical changes (joint effusion and tissue oedema) alter the resting and movement-induced forces exerted on the joint tissues and secondly, inflammatory mediators released within the damaged tissue sensitize articular nociceptive afferents by binding to receptors on the nerve endings. These factors result in a reduction of the mechanical threshold for activation of articular nociceptors such that manipulation of the joint within the normal range is easily sufficient to activate them. Acute and chronic animal models of joint inflammation have been used to study the mechanisms of articular nociceptor sensitization and a number of inflammatory mediators and their receptors have been implicated. The focus of this paper will be to introduce some of the important issues involved in the sensitization of nociceptive articular afferents.

Immunocytochemical identification of G-protein-coupled receptor expression and localization.

Immunocytochemistry exploits the incomparable specificity of the antibody-antigen interaction to form the basis of a flexible approach to the study of expression and localization of proteins both in model systems and their physiological context. This chapter details the theory and practice of the technique as well as lists the materials required. A general protocol is proposed, which can be adapted to suit the needs of individual investigators using the suggestions outlined in the Notes. The use of frozen tissue sections and cultured cells is described. Finally, the most common causes for failure of the technique are presented, along with likely solutions.

Nuclear immunostaining in rat neuronal cells using two anti-Kir2.2 ion channel polyclonal antibodies.

The inwardly rectifying potassium ion channel Kir2.2 has recently been demonstrated to have nuclear and plasma membrane subcellular localization. Nuclear expression of Kir2.2 is controversial, as a functional role for Kir2.0 potassium channels in the nucleus has not been investigated. However, in this report we have demonstrated Kir2.2 nuclear localization in sections of rat hindbrain and dorsal root ganglia tissue, using two anti- Kir2.2 polyclonal antisera with different epitope specificities. These data confirm nuclear localization and are suggestive of new functions of Kir2.0 potassium ion channels in the nucleus.

The Effects of NMDA Antagonists on Neuronal Activity in Cat Spinal Cord Evoked by Acute Inflammation in the Knee Joint.

In alpha-chloralose-anaesthetized, spinalized cats we examined the effects of NMDA antagonists on the discharges of 71 spinal neurons which had afferent input from the knee joint. These neurons were rendered hyperexcitable by acute arthritis in the knee induced by kaolin and carrageenan. They were located in the deep dorsal and ventral horn and some of them had ascending axons. The N-methyl-d-aspartate (NMDA) antagonists ketamine and d-2-amino-5-phosphonovalerate (AP5), were administered ionophoretically, and ketamine was also administered intravenously. In some of the experiments the antagonists were tested against the agonists NMDA and quisqualate. The effects of the NMDA antagonists consisted of a significant reduction in the resting activity of neurons and/or the responses of the same neurons to mechanical stimulation of the inflamed knee. Intravenous ketamine was most effective in suppressing the resting and mechanically evoked activity in 25 of 26 neurons tested. Ionophoretically applied ketamine had a suppressive effect in 11 of 21 neurons, and AP5 decreased activity in 17 of 24 cells. The reduction in the resting and/or the mechanically evoked discharges was achieved with doses of the antagonists which suppressed the responses to NMDA but not those to quisqualate. These results suggest that NMDA receptors are involved in the enhanced responses and basal activity of spinal neurons induced by inflammation in the periphery.