The mechanisms of inflammatory pain need to be identified in order to find new superior treatments. Protease-activated receptors 2 (PAR2) and transient receptor potential vanilloid 1 (TRPV1) are highly co-expressed in dorsal root ganglion neurons and implicated in pain development. Here, we examined the role of spinal PAR2 in hyperalgesia and the modulation of synaptic transmission in carrageenan-induced peripheral inflammation, using intrathecal (i.t.) treatment in the behavioral experiments and recordings of spontaneous, miniature and dorsal root stimulation-evoked excitatory postsynaptic currents (sEPSCs, mEPSCs and eEPSCs) in spinal cord slices. Intrathecal PAR2-activating peptide (AP) administration aggravated the carrageenan-induced thermal hyperalgesia, and this was prevented by a TRPV1 antagonist (SB 366791) and staurosporine i.t. pretreatment. Additionally, the frequency of the mEPSC and sEPSC and the amplitude of the eEPSC recorded from the superficial dorsal horn neurons were enhanced after acute PAR2 AP application, while prevented with SB 366791 or staurosporine pretreatment. PAR2 antagonist application reduced the thermal hyperalgesia and decreased the frequency of mEPSC and sEPSC and the amplitude of eEPSC. Our findings highlight the contribution of spinal PAR2 activation to carrageenan-induced hyperalgesia and the importance of dorsal horn PAR2 and TRPV1 receptor interactions in the modulation of nociceptive synaptic transmission.

• MeSH
• anilidy farmakologie   MeSH
• buňky zadních rohů míšních účinky léků   metabolismus   fyziologie   MeSH
• cinnamáty farmakologie   MeSH
• excitační postsynaptické potenciály MeSH
• hyperalgezie etiologie   metabolismus   patofyziologie   MeSH
• karagenan farmakologie   toxicita   MeSH
• kationtové kanály TRPV antagonisté a inhibitory   metabolismus   MeSH
• krysa rodu rattus MeSH
• miniaturní postsynaptické potenciály MeSH
• nocicepce MeSH
• potkani Wistar MeSH
• receptor PAR-2 metabolismus   MeSH
• staurosporin farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca2+ concentration ([Ca2+]i) remain unknown. We analyzed mechanisms regulating resting [Ca2+]i in dissociated rat melanotrophs by Ca2+-imaging and patch-clamp techniques. Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca2+ channels (VGCCs) as well as removal of extracellular Ca2+ resulted in a rapid and reversible decrease in [Ca2+]i, indicating constitutive Ca2+ influx through L-type VGCCs. Reduction of extracellular Na+ concentration (replacement with NMDG+) similarly decreased resting [Ca2+]i. When cells were champed at -80 mV, decrease in the extracellular Na+ resulted in a positive shift of the holding current. In cell-attached voltage-clamp and whole-cell current-clamp configurations, the reduction of extracellular Na+ caused hyperpolarisation. The holding current shifted in negative direction when extracellular K+ concentration was increased from 5 mM to 50 mM in the presence of K+ channel blockers, Ba2+ and TEA, indicating cation nature of persistent conductance. RT-PCR analyses of pars intermedia tissues detected mRNAs of TRPV1, TRPV4, TRPC6, and TRPM3-5. The TRPV channel blocker, ruthenium red, shifted the holding current in positive direction, and significantly decreased the resting [Ca2+]i. These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca2+]i in rat melanotrophs.

• MeSH
• kationtové kanály TRPV antagonisté a inhibitory   metabolismus   MeSH
• krysa rodu rattus MeSH
• melanotropní buňky metabolismus   MeSH
• metoda terčíkového zámku MeSH
• potkani Wistar MeSH
• rutheniová červeň farmakologie   MeSH
• sodík metabolismus   MeSH
• vápník metabolismus   MeSH
• vápníkové kanály - typ L metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Transient receptor potential vanilloid type 1 (TRPV1) receptors are important in the development of different pathological chronic pain states. Here we examined the role of spinal cord TRPV1 receptors in the mechanisms leading to activation of dorsal horn neurons after paclitaxel (PAC) treatment. PAC is a widely used chemotherapeutic drug that often leads to development of painful neuropathy. Immunohistochemical analysis of c-Fos protein expression in dorsal horn neurons was used as a marker of neuronal activation. Rat spinal cord slices were processed for in vitro incubation with PAC (100 nM) and TRPV1 receptor antagonists (SB366791 and AMG9810; 10 microM). PAC treatment induced significant upregulation of c-Fos nuclear expression in superficial dorsal horn neurons that was diminished by TRPV1 receptor antagonists pre-incubation. These results further substantiated the role of spinal TRPV1 receptors in the development of paclitaxel-induced neuropathic pain and contribute to better understanding of the pathological mechanisms involved.

• MeSH
• antitumorózní látky fytogenní farmakologie   MeSH
• buňky zadních rohů míšních účinky léků   metabolismus   MeSH
• exprese genu MeSH
• kationtové kanály TRPV antagonisté a inhibitory   fyziologie   MeSH
• krysa rodu rattus MeSH
• paclitaxel farmakologie   MeSH
• potkani Wistar MeSH
• protoonkogenní proteiny c-fos agonisté   biosyntéza   genetika   MeSH
• zadní rohy míšní účinky léků   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Protease-activated receptors 2 (PAR2) and transient receptor potential vanilloid 1 (TRPV1) receptors in the peripheral nerve endings are implicated in the development of increased sensitivity to mechanical and thermal stimuli, especially during inflammatory states. Both PAR2 and TRPV1 receptors are co-expressed in nociceptive dorsal root ganglion (DRG) neurons on their peripheral endings and also on presynaptic endings in the spinal cord dorsal horn. However, the modulation of nociceptive synaptic transmission in the superficial dorsal horn after activation of PAR2 and their functional coupling with TRPV1 is not clear. To investigate the role of spinal PAR2 activation on nociceptive modulation, intrathecal drug application was used in behavioural experiments and patch-clamp recordings of spontaneous, miniature and dorsal root stimulation-evoked excitatory postsynaptic currents (sEPSCs, mEPSCs, eEPSCs) were performed on superficial dorsal horn neurons in acute rat spinal cord slices. Intrathecal application of PAR2 activating peptide SLIGKV-NH2 induced thermal hyperalgesia, which was prevented by pretreatment with TRPV1 antagonist SB 366791 and was reduced by protein kinases inhibitor staurosporine. Patch-clamp experiments revealed robust decrease of mEPSC frequency (62.8 ± 4.9%), increase of sEPSC frequency (127.0 ± 5.9%) and eEPSC amplitude (126.9 ± 12.0%) in dorsal horn neurons after acute SLIGKV-NH2 application. All these EPSC changes, induced by PAR2 activation, were prevented by SB 366791 and staurosporine pretreatment. Our results demonstrate an important role of spinal PAR2 receptors in modulation of nociceptive transmission in the spinal cord dorsal horn at least partially mediated by activation of presynaptic TRPV1 receptors. The functional coupling between the PAR2 and TRPV1 receptors on the central branches of DRG neurons may be important especially during different pathological states when it may enhance pain perception.

• MeSH
• alergie metabolismus   patologie   MeSH
• anilidy farmakologie   MeSH
• buňky zadních rohů míšních účinky léků   fyziologie   MeSH
• chování zvířat účinky léků   MeSH
• cinnamáty farmakologie   MeSH
• excitační postsynaptické potenciály účinky léků   MeSH
• hyperalgezie etiologie   prevence a kontrola   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• kationtové kanály TRPV antagonisté a inhibitory   metabolismus   MeSH
• krysa rodu rattus MeSH
• metoda terčíkového zámku MeSH
• mícha metabolismus   MeSH
• nervový přenos fyziologie   MeSH
• oligopeptidy farmakologie   MeSH
• potkani Wistar MeSH
• receptor PAR-2 agonisté   metabolismus   MeSH
• staurosporin farmakologie   MeSH
• techniky in vitro MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• analgetika farmakologie   terapeutické užití   MeSH
• bolest farmakoterapie   patofyziologie   MeSH
• kapsaicin farmakologie   terapeutické užití   MeSH
• kationtové kanály TRPV antagonisté a inhibitory   farmakologie   MeSH
• klinická studie jako téma MeSH
• lidé MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Peripheral neuropathy is dose limiting in paclitaxel cancer chemotherapy and can result in both acute pain during treatment and chronic persistent pain in cancer survivors. The hypothesis tested was that paclitaxel produces these adverse effects at least in part by sensitizing transient receptor potential vanilloid subtype 1 (TRPV1) through Toll-like receptor 4 (TLR4) signaling. The data show that paclitaxel-induced behavioral hypersensitivity is prevented and reversed by spinal administration of a TRPV1 antagonist. The number of TRPV1(+) neurons is increased in the dorsal root ganglia (DRG) in paclitaxel-treated rats and is colocalized with TLR4 in rat and human DRG neurons. Cotreatment of rats with lipopolysaccharide from the photosynthetic bacterium Rhodobacter sphaeroides (LPS-RS), a TLR4 inhibitor, prevents the increase in numbers of TRPV1(+) neurons by paclitaxel treatment. Perfusion of paclitaxel or the archetypal TLR4 agonist LPS activated both rat DRG and spinal neurons directly and produced acute sensitization of TRPV1 in both groups of cells via a TLR4-mediated mechanism. Paclitaxel and LPS sensitize TRPV1 in HEK293 cells stably expressing human TLR4 and transiently expressing human TRPV1. These physiological effects also are prevented by LPS-RS. Finally, paclitaxel activates and sensitizes TRPV1 responses directly in dissociated human DRG neurons. In summary, TLR4 was activated by paclitaxel and led to sensitization of TRPV1. This mechanism could contribute to paclitaxel-induced acute pain and chronic painful neuropathy. Significance statement: In this original work, it is shown for the first time that paclitaxel activates peripheral sensory and spinal neurons directly and sensitizes these cells to transient receptor potential vanilloid subtype 1 (TRPV1)-mediated capsaicin responses via Toll-like receptor 4 (TLR4) in multiple species. A direct functional interaction between TLR4 and TRPV1 is shown in rat and human dorsal root ganglion neurons, TLR4/TRPV1-coexpressing HEK293 cells, and in both rat and mouse spinal cord slices. Moreover, this is the first study to show that this interaction plays an important role in the generation of behavioral hypersensitivity in paclitaxel-related neuropathy. The key translational implications are that TLR4 and TRPV1 antagonists may be useful in the prevention and treatment of chemotherapy-induced peripheral neuropathy in humans.

• MeSH
• antitumorózní látky fytogenní antagonisté a inhibitory   farmakologie   MeSH
• excitační postsynaptické potenciály účinky léků   MeSH
• HEK293 buňky MeSH
• hyperalgezie chemicky indukované   patofyziologie   MeSH
• kationtové kanály TRPV antagonisté a inhibitory   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• měření bolesti účinky léků   MeSH
• metoda terčíkového zámku MeSH
• mícha účinky léků   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nervové receptory účinky léků   MeSH
• paclitaxel antagonisté a inhibitory   farmakologie   MeSH
• potkani Sprague-Dawley MeSH
• signální transdukce účinky léků   MeSH
• spinální ganglia cytologie   účinky léků   MeSH
• toll-like receptor 4 antagonisté a inhibitory   účinky léků   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

In recent years, several studies have explored the involvement of the deregulation of the hypothalamus-pituitary-adrenal (HPA) axis in the pathophysiology of stress-related disorders. HPA hyper-activation as a consequence of acute/chronic stress has been found to play a major role in the neurobiological changes that are responsible for the onset of such states. Currently available medications for depression, one of the most relevant stress-related disorders, present several limitations, including a time lag for treatment response and low rates of efficacy. N-Arachidonoylserotonin (AA-5-HT), a dual blocker at fatty acid amide hydrolase (FAAH, the enzyme responsible for the inactivation of the endocannabinoid anandamide) and transient receptor potential vanilloid type-1 channel (TRPV1), produces anxiolytic-like effects in mice. The present study was designed to assess the capability of AA-5-HT to reverse the behavioral despair following exposure to stress in rats and the role of the HPA-axis. Behavioral tasks were performed, and corticosterone and endocannabinoid (anandamide and 2-arachidonoylglycerol) levels were measured in selected brain areas critically involved in the pathophysiology of stress-related disorders (medial PFC and hippocampus) under basal and stress conditions, and in response to treatment with AA-5-HT. Our data show that AA-5-HT reverses the rat behavioral despair in the forced swim test under stress conditions, and this effect is associated with the normalization of the HPA-axis deregulation that follows stress application and only in part with elevation of anandamide levels. Blockade of FAAH and TRPV1 may thus represent a novel target to design novel therapeutic strategies for the treatment of stress-related disorders.

• MeSH
• amidohydrolasy antagonisté a inhibitory   genetika   metabolismus   MeSH
• chování zvířat účinky léků   MeSH
• endokanabinoidy metabolismus   MeSH
• fyzické omezení MeSH
• glyceridy metabolismus   MeSH
• kationtové kanály TRPV antagonisté a inhibitory   genetika   metabolismus   MeSH
• kortikosteron krev   MeSH
• krysa rodu rattus MeSH
• kyseliny arachidonové metabolismus   farmakologie   terapeutické užití   MeSH
• mozek účinky léků   metabolismus   MeSH
• mozkový neurotrofický faktor genetika   metabolismus   MeSH
• plavání MeSH
• polynenasycené alkamidy metabolismus   MeSH
• potkani Wistar MeSH
• psychický stres krev   farmakoterapie   metabolismus   MeSH
• serotonin analogy a deriváty   farmakologie   terapeutické užití   MeSH
• systém hypofýza - nadledviny MeSH
• systém hypotalamus-hypofýza MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Acute postoperative pain is one of the frequent reasons for pain treatment. However, the exact mechanisms of its development are still not completely clear. Transient receptor potential vanilloid 1 (TRPV1) receptors are involved in nociceptive signaling in various hypersensitive states. Here we have investigated the contribution of TRPV1 receptors expressed on cutaneous peripheral nociceptive fibers and in the spinal cord on the development and maintenance of hypersensitivity to thermal and mechanical stimuli following surgical incision. A rat plantar incision model was used to test paw withdrawal responses to thermal and mechanical stimuli. The effect of the TRPV1 receptor antagonist SB366791 was investigated 1) by intrathecal injection 15 min before incision and 2) intradermal injection before (30 min) and immediately after the surgery. Vehicle-injected rats and naïve animals treated identically were used as controls. RESULTS: Plantar incision induced mechanical allodynia and hyperalgesia and thermal hyperalgesia. A single intrathecal administration of SB366791 significantly reduced postincisional thermal hyperalgesia and also attenuated mechanical allodynia, while mechanical hyperalgesia remained unaffected. Local intradermal SB366791 treatment reduced thermal hyperalgesia and mechanical allodynia without affecting mechanical hyperalgesia. CONCLUSIONS: Our experiments suggest that both peripheral and spinal cord TRPV1 receptors are involved in increased cutaneous sensitivity following surgical incision. The analgesic effect of the TRPV1 receptor antagonist was especially evident in the reduction of thermal hyperalgesia. The activation of TRPV1 receptors represents an important mechanism in the development of postoperative hypersensitivity.

• MeSH
• anilidy farmakologie   terapeutické užití   MeSH
• časové faktory MeSH
• cinnamáty farmakologie   terapeutické užití   MeSH
• fyzikální stimulace škodlivé účinky   MeSH
• hmat MeSH
• hyperalgezie farmakoterapie   etiologie   MeSH
• kationtové kanály TRPV antagonisté a inhibitory   metabolismus   MeSH
• krysa rodu rattus MeSH
• měření bolesti účinky léků   MeSH
• mícha účinky léků   metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• pooperační bolest komplikace   farmakoterapie   MeSH
• potkani Wistar MeSH
• práh bolesti účinky léků   MeSH
• reakční čas účinky léků   MeSH
• vysoká teplota MeSH
• způsoby aplikace léků MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Modulation of nociceptive synaptic transmission in the spinal cord is implicated in the development and maintenance of several pathological pain states. The chemokine CCL2 (C-C motif ligand 2) was shown to be an important factor in the development of neuropathic pain after peripheral nerve injury. In our experiments we have studied the effect of CCL2 application and TRPV1 (transient receptor potential vanilloid 1) receptor activation on nociceptive signaling and the modulation of synaptic transmission. Intrathecal drug application in behavioral experiments and patch-clamp recordings of spontaneous, miniature and dorsal root stimulation-evoked excitatory postsynaptic currents (sEPSCs, mEPSCs, eEPSCs) from superficial dorsal horn neurons in acute rat spinal cord slices were used. The intrathecal application of CCL2 induced thermal hyperalgesia and mechanical allodynia, while pretreatment with the TRPV1 receptor antagonist SB366791 diminished the thermal but not the mechanical hypersensitivity. Patch-clamp experiments showed an increase of sEPSC and mEPSC (124.5 ± 12.8% and 161.2 ± 17.3%, respectively) frequency in dorsal horn neurons after acute CCL2 application. This CCL2-induced increase was prevented by SB366791 pretreatment (89.4 ± 6.0%, 107.5 ± 14.2%). CCL2 application increased the amplitude of eEPSCs (188.1 ± 32.1%); this increase was significantly lower in experiments with SB366791 pretreatment (120.8 ± 17.2%). Our results demonstrate that the activation of spinal TRPV1 receptors plays an important role in the modulation of nociceptive signaling induced by CCL2 application. The mechanisms of cooperation between the CCL2 activated receptors and TRPV1 receptors on the central branches of primary afferent fibers may be especially important during different pathological pain states and need to be further investigated.

• MeSH
• anilidy terapeutické užití   MeSH
• buňky zadních rohů míšních účinky léků   fyziologie   MeSH
• časové faktory MeSH
• chemokin CCL2 toxicita   MeSH
• cinnamáty terapeutické užití   MeSH
• excitační postsynaptické potenciály účinky léků   MeSH
• hyperalgezie chemicky indukované   farmakoterapie   metabolismus   MeSH
• kapsaicin farmakologie   MeSH
• kationtové kanály TRPV antagonisté a inhibitory   metabolismus   MeSH
• krysa rodu rattus MeSH
• látky ovlivňující senzorický systém farmakologie   MeSH
• MAP kinasa-kinasa-kinasa 3 metabolismus   MeSH
• metoda terčíkového zámku MeSH
• mícha cytologie   účinky léků   metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• novorozená zvířata MeSH
• potkani Wistar MeSH
• práh bolesti účinky léků   MeSH
• techniky in vitro MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: The recent discovery that camphor activates and strongly desensitizes the capsaicin-sensitive and noxious heat-sensitive channel transient receptor potential vanilloid subfamily member 1 (TRPV1) has provided new insights and opened up new research paths toward understanding why this naturally occurring monoterpene is widely used in human medicine for its local counter-irritant, antipruritic, and anesthetic properties. However, the molecular basis for camphor sensitivity remains mostly unknown. The authors attempt to explore the nature of the activation pathways evoked by camphor and narrow down a putative interaction site at TRPV1. METHODS: The authors transiently expressed wild-type or specifically mutated recombinant TRPV1 channels in human embryonic kidney cells HEK293T and recorded cation currents with the whole cell, patch clamp technique. To monitor changes in the spatial distribution of phosphatidylinositol 4,5-bisphosphate, they used fluorescence resonance energy transfer measurements from cells transfected with the fluorescent protein-tagged pleckstrin homology domains of phospholipase C. RESULTS: The results revealed that camphor modulates TRPV1 channel through the outer pore helix domain by affecting its overall gating equilibrium. In addition, camphor, which generally is known to decrease the fluidity of cell plasma membranes, may also regulate the activity of TRPV1 by inducing changes in the spatial distribution of phosphatidylinositol-4,5-bisphosphate on the inner leaflet of the plasma membrane. CONCLUSIONS: The findings of this study provide novel insights into the structural basis for the modulation of TRPV1 channel by camphor and may provide an explanation for the mechanism by which camphor modulates thermal sensation in vivo.

• MeSH
• gating iontového kanálu účinky léků   fyziologie   MeSH
• HEK293 buňky MeSH
• jaderný pór účinky léků   metabolismus   MeSH
• kafr chemie   farmakologie   MeSH
• kationtové kanály TRPV antagonisté a inhibitory   chemie   fyziologie   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• terciární struktura proteinů účinky léků   fyziologie   MeSH
• vazebná místa účinky léků   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH