|
|
Studies on molecular interactions of the mu-opioid and TRPV1 receptors
Melkes, Barbora ; Novotný, Jiří (advisor) ; Blahoš, Jaroslav (referee) ; Krůšek, Jan (referee)
In this work, we investigated the behavior of the -opioid receptor (MOR) and the transient receptor potential vanilloid 1 (TRPV1) ion channel in the plasma membrane and their mutual communication. Both these receptors are implicated in pain perception and analgesia. We observed that the lateral mobility of MOR was strongly affected by different biased opioid agonists. DAMGO and endomorphin-2 display opposite bias towards MOR. According to our results, they also have the opposite effects on the mobility of MOR. Morphine induced only small changes in the mobility of MOR. Moreover, cholesterol depletion and blockage of G protein signaling by pertussis toxin (PTX) affected the ability of different MOR agonists to alter MOR mobility in a unique manner. The effects of DAMGO and endomorphin-2 were compromised under these conditions. On the other hand, we observed increased movement of MOR after the addition of morphine. PTX alone did not affect receptor movement, but it completely disrupted the effect of cholesterol depletion on morphine induced changes the mobility of MOR. Next we studied the mobility of TRPV1. The TRPV1 agonist capsaicin changed the lateral mobility of TRPV1. Surprisingly, after adding the MOR antagonist naloxone, the apparent diffusion coefficient of TRPV1 but to a lower extent than...
|
|
|
Mechanisms of Activation and Modulation of Ion Channels Specific for Nociceptive Neurones
Touška, Filip ; Vlachová, Viktorie (advisor) ; Paleček, Jiří (referee) ; Tureček, Rostislav (referee)
Human body detects potentially damaging stimuli by specialized sensory nerve endings in the skin, the nociceptors. Their membranes are equipped with ion channels, molecular sensors, coding the outside stimuli into the trains of action potentials and conducting them to the higher brain centers. The most prominent group of transduction ion channels is the transient receptor potential (TRP) channel family followed by ion channels responsible for generation and conduction of action potentials from the periphery to the brain, the voltage-gated sodium channels (VGSCs). Understanding the mechanisms how particular stimulus is encoded and processed is of particular importance to find therapeutics for various types of pain conditions. We characterized the properties of VGSC subtypes NaV1.9 and NaV1.8 at high temperatures. We showed that NaV1.9 undergo large increase in current with increasing temperatures and significantly contribute to the action potential generation in dorsal root ganglion (DRG) neurons. Ciguatoxins (CTXs) are sodium channels activator toxins causing ciguatera fish poisoning, a disease manifested by sensory and neurological disturbances. We elucidated the mechanism of CTX- induced cold allodynia, a pathological phenomenon where normally innocuous cool temperatures are perceived as pain. We...
|
|
|
The role of nociceptive synaptic transmission modulation at the spinal cord level in different pain states
Adámek, Pavel
Pain is a common symptom of many clinical syndromes and diseases. In particular, the treatment of neuropathic pain represents a serious public health issue because currently available analgesia is ineffective in many cases or it has adverse effects. Treatment of pain-related suffering requires knowledge of how pain signals are initially generated and subsequently transmitted by the nervous system. A nociceptive system plays a key role in this process of encoding and transmission of pain signals. Modulation of the nociceptive synaptic transmission in the spinal cord dorsal horn represents an important mechanism in the development and maintenance of different pathological pain states. This doctoral thesis has aimed to investigate and clarify some of the mechanisms involved in the modulation of the spinal nociceptive processing in different pain states. The main attention was paid to study the following issues: (I.) Which is the role of Transient Receptor Potential Vanilloid type 1 channels (TRPV1), Toll-Like Receptors 4 (TLR4), and phosphatidylinositol 3-kinase (PI3K) in the development of neuropathic pain induced by paclitaxel (PAC) chemotherapy in acute in vitro, and subchronic in vivo murine model of PAC-induced peripheral neuropathy (PIPN)? (II.) How is affected spinal inhibitory synaptic control...
|
|
|
Modulation of synaptic transmission in the development of painful states
Slepička, Jakub ; Paleček, Jiří (advisor) ; Hejnová, Lucie (referee)
My thesis introduces the topic of nociceptive signalisation and processes involved in the formation and spreading of neuropathic pain. This study focuses on the mechanisms of nociceptive synaptic transmission mechanisms in the level of spinal dorsal horn and its modulation by paclitaxel, a chemotherapeutic drug inducing neuropathic changes. The attention is put especially on the possibility of glial activity participation in paclitaxel side effects. This idea stems from the existing hypothesis of the functional connection between TLR4 and TRPV1 receptor activity. TRPV1 is well known for its participation in chemical, thermal and nociceptive sensory transmission. Minocycline antibiotic is considered as an inhibitor of microglial activation therefore it was used for blocking neuroinflammation. The experimental part is comparing an impact of substances applied to the model of tachyphylaxis used for monitoring of nociceptive transmission changes according to decreasing activity of TRPV1 receptors. Electrophysiological recording of miniature excitatory postsynaptic currents from neurons in the Rexed laminae I. and II. of spinal dorsal horn was used. The results of my measurements show that minocycline is able to suppress acute effects of paclitaxel application in vitro if the spinal slice is incubated...
|
|
|
The role of nociceptive synaptic transmission modulation at the spinal cord level in different pain states
Adámek, Pavel ; Paleček, Jiří (advisor) ; Vaculín, Šimon (referee) ; Vlachová, Viktorie (referee)
Pain is a common symptom of many clinical syndromes and diseases. In particular, the treatment of neuropathic pain represents a serious public health issue because currently available analgesia is ineffective in many cases or it has adverse effects. Treatment of pain-related suffering requires knowledge of how pain signals are initially generated and subsequently transmitted by the nervous system. A nociceptive system plays a key role in this process of encoding and transmission of pain signals. Modulation of the nociceptive synaptic transmission in the spinal cord dorsal horn represents an important mechanism in the development and maintenance of different pathological pain states. This doctoral thesis has aimed to investigate and clarify some of the mechanisms involved in the modulation of the spinal nociceptive processing in different pain states. The main attention was paid to study the following issues: (I.) Which is the role of Transient Receptor Potential Vanilloid type 1 channels (TRPV1), Toll-Like Receptors 4 (TLR4), and phosphatidylinositol 3-kinase (PI3K) in the development of neuropathic pain induced by paclitaxel (PAC) chemotherapy in acute in vitro, and subchronic in vivo murine model of PAC-induced peripheral neuropathy (PIPN)? (II.) How is affected spinal inhibitory synaptic control...
|
|
|
Modulation of nociceptive synaptic transmission
Nerandžič, Vladimír ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee) ; Hejnová, Lucie (referee)
Modulation of synaptic transmission in the spinal cord dorsal horn plays an important role in development and maintenance of pathological pain states. The indisputable part of this modulation is conducted via activity of the transient receptor potential cation channel subfamily V member 1 (TRPV1) and the cannabinoid receptor 1 (CB1), expressed on presynaptic endings of primary afferents in the superficial spinal cord dorsal horn. Under physiological conditions, activation of TRPV1 receptors is pronociceptive while CB1 receptor activation leads to attenuation of nociceptive signalling. However, both receptors share also one endogenous agonist anandamide (AEA) that may be produced from N-arachidonoyl phosphatidylethanolamine (20:4-NAPE). Main objective of this thesis focuses on the effect of 20:4-NAPE on nociceptive synaptic transmission in spinal cord slices under naïve and inflammatory conditions and consequent on the possible interaction of TRPV1 and CB1 receptors. First, 20:4-NAPE application induced significant release of anandamide from spinal cord slices under in vitro conditions. Next, patch- clamp recordings of excitatory postsynaptic currents (mEPSC and sEPSC) from superficial dorsal horn (DH) neurons in acute spinal cord slices were used. 20:4-NAPE application under the physiological...
|
|
|
The role of spinal TRPV1 receptors in nociceptive signalling and the modulatory effect of chemokine CCL2 and µ-opioid receptor agonists
Šulcová, Dominika ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee)
The first nociceptive synapse in the spinal cord dorsal horn represents an important site, where nociceptive synaptic transmission can be modulated under pathological conditions. One of the modulatory mechanism involves activation of the transient receptor potential vanilloid 1 (TRPV1) that is expressed on central terminals of primary nociceptive neurons, where it regulates release of neurotransmitters and neuromodulators. Previous studies suggested that changes in TRPV1 activity may be related to effects of chemokine CCL2 (C-C motif ligand 2) and may be also involved in synaptic transmission modulation after µ-opioid receptors (MOP-R) activation. Because CCL2 receptors CCR2 often co-localize with TRPV1 and MOP-R, the goal of this work was to studypossible interactions of these receptors on the pre-synaptic endings of primaryafferents in the spinal cord dorsal horn and their role in nociceptive signalling under pathological conditions. The presented thesis focused on the effect of CCL2 during peripheral neuropathy and its interference with µ-opioid receptor activation. To studysynaptic transmission at the spinal cord level, patch-clamp recordings of excitatory post-synaptic currents (EPSC) in superficial spinal cord dorsal horn neurons in acute lumbar spinal cord slices from rats was used....
|
|
|
The role of TLR-4 receptors for modulation of nociceptive signalling.
Slepička, Jakub ; Paleček, Jiří (advisor) ; Zímová, Lucie (referee)
The ability to feel pain is subjective, but a crucial factor allowing us to maintain life with minimal body impairment. However, if the pain crosses the physiological signalling function it becomes a life restrictive symptom that is often difficult to treat. Development of pain and its change into pathological chronic symptom and the role of different molecular and cellular mechanisms in this process are best to be studied during its development, nociception. In this work the basic characteristics of nociceptive signalling, with focus on neuropathy, a state originating from nervous system damage is given. The exact mechanisms of neuropathic pain development are not known, which is one of the reasons why these painful states are difficult to treat. Neuroinflammation was lately shown to play an important part in the neuropathy development. In this context a review of Toll-like receptors is given. These receptors have a fundamental role in the innate immunity and their role in the nervous system was documented recently. In this review a focus is given on TLR-4 subtype that was shown to be involved in a modulation of nociceptive signalling. The basic features of this receptor and information about its involvement in neuropathic pain development are given. Studying the role of TLR-4 in different models...
|
|
|
The role of TRPV1 receptors in chemokine CCL2 induced modulation of nociceptive synaptic transmission at spinal cord level
Adámek, Pavel ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee)
Modulation of nociceptive synaptic transmission in the spinal cord dorsal horn is a significant mechanism in the development and maintenance of different pathological pain states. Accumulating evidence indicates that the TRPV1 (transient receptor potential vanilloid 1) receptor and chemokine CCL2 (C-C motif ligand 2) may play a critical role in this process. The aim of this diploma thesis was to investigate the CCL2 induced modulation of nociceptive synaptic transmission in the dorsal horn of spinal cord and the role of the TRPV1 receptors. To investigate this aim patch-clamp recordings of spontaneous and miniature excitatory postsynaptic currents (sEPSC, mEPSC) from superficial dorsal horn neurons in acute rat lumbar spinal cord slices were used. After acute application of CCL2 on the slice preparation from naïve animals, a frequency increase of both sEPSC and mEPSC was present. This CCL2 induced increase in both sEPSC and mEPSC frequency was prevented by the TRPV1 receptor antagonist SB366791 application. No changes were observed in the amplitudes of sEPSC or mEPSC after application of the CCL2, SB366791, or co-application of CCL2 and SB366791. This suggests that the observed changes were mediated predominantly by presynaptic mechanisms. The preliminary results indicate that after chronic constriction...
|
|
|
Mechanisms of activation and modulation of vanilloid TRP channels
Boukalová, Štěpána ; Vlachová, Viktorie (advisor) ; Hock, Miroslav (referee) ; Zemková, Hana (referee)
Štěpána Boukalová Mechanisms of activation and modulation of vanilloid TRP channels TRPV1 and TRPV3 are thermosensitive ion channels from the vanilloid subfamily of TRP receptors. TRPV1, which is primarily expressed in nociceptive sensory neurons, is an important transducer of painful stimuli and is also involved in the detection of noxious heat. TRPV3 is expressed mainly in the skin where it regulates proliferation and differentiation of keratinocytes. Similarly to voltage-dependent potassium (Kv) channels, TRP receptors are comprised of four subunits, each with six transmembrane segments (S1-S6). Using mutational approach, we tried to elucidate the role of S1 in TRPV1 functioning. Our results indicate that the extracellular portion of S1 plays a crucial role in TRPV1 gating. TRPV1 channels with a conservative mutation of positively charged residue in this region (R455K substitution) were overactive. However, they were neither activated nor potentiated by low pH; on the contrary, protons stabilized the closed conformation of this mutant channel. Very similar phenotypic properties were found in other TRPV1 mutants with substitution in S4/S5-S5 region and in the pore helix. In Kv channels, extracelular portion of S1 forms a small contact surface with the pore helix, which allows efficient transmission of...
|
guest ::
