Tight regulation of cephalic blood circulation is critical under normal physiological conditions, and dysregulation of blood flow to the head occurs in pathophysiological situations such as stroke and migraine headache. The facial artery is an extracranial artery which is one of branches from the external carotid artery territory and its extracranial position indicates its importance in regulating head hemodynamics. Transient receptor potential vanniloid type 1 (TRPV1) is a cation channel permeable to Ca2+ and Na+. Intracellular Ca2+ increase causes vasoconstriction. A previous study indicated the presence of TRPV1 in smooth muscle cells in the facial artery. Protein kinase C (PKC) is found to sensitize TRPV1 channels in neurons. Our lab's preliminary data suggested PKC modulates TRPV1 in the middle meningeal artery. Serotonin (5-HT) is an endogenous vasoconstrictor, and the 5-HT2 receptor is a Gq-protein-coupled receptor that activates PKC. In the present study, we found that 5-HT caused facial artery constriction. Thus, we studied whether TRPV1 channel acting as a Ca2+ entry channel is involved in 5-HT induced facial artery constriction. We used a pressurized arteriography technique to examine the artery diameter. The results indicate that 1) TRPV1 antagonist blunted 30 nM 5-HT-induced mouse facial artery constriction. 5-HT constriction on the facial artery from TRPV1 knock out mice was significantly blunted compared to the constriction on the facial artery from wild type mice; 2) PKC, which is a downstream signaling molecule of 5-HT2 receptor, is involved in capsaicin (TRPV1 agonist)-induced facial artery constriction; 3) 5-HT-induced facial artery constriction is mediated mostly by activation of 5-HT1 and 5-HT2 receptors; 4) 5-HT2 but not 5-HT1 receptor is involved in 5-HT-induced facial artery constriction via opening of TRPV1 channels; 5) PKC may be involved in 5-HT-induced facial artery constriction; 6) The L-type-voltage-dependent Ca2+ channel is involved in 5-HT-induced facial artery constriction. We conclude that activation of TRPV1 channel contributes to serotonin-induced 5-HT2 receptor-mediated constriction of the mouse facial artery.

Neuropathic pain is a complex clinical syndrome characterized by increased sensitivity to thermal and/or mechanical stimuli that may or may not be accompanied by the phenomenon of spontaneous or aberrant pain sensations. <p>Over the past decade, the mechanisms underlying the behavioral manifestations of inflammatory neuropathic pain have become more clearly elucidated. These include the involvement of: 1) transient receptor potential vanilloid receptor 1 (TRPV1) in the generation of thermal hyperalgesia; 2) acid sensing ion channel 3 (ASIC3) in some aspects of the development/maintenance of mechanical hypersensitivity; 3) the tetrodotoxin resistant sodium channels Nav1.8 and Nav1.9 in both hyperalgesia and spontaneous pain; and 4) activation of the MAP Kinases p38 and ERK1/2 in the regulation of expression of the aforementioned molecules.<p>Interestingly, it is the pro-inflammatory neurotrophin nerve growth factor (NGF) that is the common link between all of these mediators of neuropathic pain. Increased availability of NGF under conditions of inflammation has been shown to drive increased expression/upregulation of TRPV1, ASIC3, Nav1.8 and Nav1.9, as well as phospho-p38 and phospho-ERK1/2.<p>Evidence presented here continues to support a role for neurotrophin-3 (NT-3) in antagonizing the effects of increased NGF on trkA signaling, neuropathic pain behaviors and some of the molecules associated with the generation of such behaviors.<p>More specifically, the work culminating in this thesis demonstrates a novel role for NT-3 in negative modulation of TRPV1, ASIC3, Nav1.8 and Nav1.9, as well as phospho-p38 expression in response to the chronic constriction injury model of neuropathic pain. Finally, initial insights into how this negative regulation of these nociceptive markers might occur is elucidated in studies demonstrating that NT-3 differentially affects levels of the key signaling molecule phospho-ERK in trkA-positive versus trkC-positive neurons in naïve dorsal root ganglia (DRG).

Neuropathic pain is a complex clinical syndrome characterized by increased sensitivity to thermal and/or mechanical stimuli that may or may not be accompanied by the phenomenon of spontaneous or aberrant pain sensations. <p>Over the past decade, the mechanisms underlying the behavioral manifestations of inflammatory neuropathic pain have become more clearly elucidated. These include the involvement of: 1) transient receptor potential vanilloid receptor 1 (TRPV1) in the generation of thermal hyperalgesia; 2) acid sensing ion channel 3 (ASIC3) in some aspects of the development/maintenance of mechanical hypersensitivity; 3) the tetrodotoxin resistant sodium channels Nav1.8 and Nav1.9 in both hyperalgesia and spontaneous pain; and 4) activation of the MAP Kinases p38 and ERK1/2 in the regulation of expression of the aforementioned molecules.<p>Interestingly, it is the pro-inflammatory neurotrophin nerve growth factor (NGF) that is the common link between all of these mediators of neuropathic pain. Increased availability of NGF under conditions of inflammation has been shown to drive increased expression/upregulation of TRPV1, ASIC3, Nav1.8 and Nav1.9, as well as phospho-p38 and phospho-ERK1/2.<p>Evidence presented here continues to support a role for neurotrophin-3 (NT-3) in antagonizing the effects of increased NGF on trkA signaling, neuropathic pain behaviors and some of the molecules associated with the generation of such behaviors.<p>More specifically, the work culminating in this thesis demonstrates a novel role for NT-3 in negative modulation of TRPV1, ASIC3, Nav1.8 and Nav1.9, as well as phospho-p38 expression in response to the chronic constriction injury model of neuropathic pain. Finally, initial insights into how this negative regulation of these nociceptive markers might occur is elucidated in studies demonstrating that NT-3 differentially affects levels of the key signaling molecule phospho-ERK in trkA-positive versus trkC-positive neurons in naïve dorsal root ganglia (DRG).

Airway hyperresponsivness (AHR) is one of most prominent pathophysiological features of asthma. Increasing evidence suggests that vagal bronchopulmonary afferents may be involved in the development of AHR. However, the underlying mechanisms are not clear. Therefore, the purpose of this dissertation was to investigate the effect of chronic airway inflammation induced by allergen sensitization on vagal bronchopulmonary afferents. The study was carried out in an animal model of allergic asthma. Brown-Norway rats were sensitized by intraperitoneal Ovalbumin (Ova) and exposed to aerosolized Ova 3 times/week for three weeks. Control rats received the vehicle. In vivo single-fiber recording technique was applied in this study. Our results showed that chronic Ova exposure caused an elevated baseline activity of pulmonary Cfibers, and a distinctly higher sensitivity of these afferents to chemical stimulants and lung inflation. After an acute Ova inhalation challenge, the increase in baseline activity and the excitability of pulmonary C-fibers were further augmented in sensitized rats, but not in control rats. In addition, sensitivity of pulmonary myelinated afferents to capsaicin was significantly elevated after chronic airway inflammation was induced by allergen. Furthermore, immunohistochemsitry data showed that, in nodose ganglia the proportion of transient receptor potential vanilloids type 1 channels (TRPV1)-expressing bronchopulmonary neurons was significantly higher in sensitized rats than in controls. This increase of TRPV1 expression was found mainly in neurofilament-positive neurons (myelinated neurons), but this effect was absent in jugular ganglia. In conclusion, allergen-induced airway inflammation caused a pronounced sensitizing effect on vagal pulmonary non-myelinated (C-fiber) afferents and elevated the sensitivity of vagal pulmonary myelinated afferents to capsaicin. The latter was accompanied by the upregulation of TRPV1 expression in these myelinated neurons.

The complement system is a principal component of innate immunity. Recent studies have underscored the importance of C5a and other complement components in inflammatory and neuropathic pain, although the underlying mechanisms are largely unknown. In particular, it is unclear how the complement system communicates with nociceptors and which ion channels and receptors are involved. Here we demonstrate that inflammatory thermal and mechanical hyperalgesia induced by complete Freund’s adjuvant were accompanied by C5a upregulation and were markedly reduced by C5a receptor (C5aR1) knockout (KO) or treatment with the C5aR1 antagonist PMX53. Direct administration of C5a into the mouse hindpaw produced strong thermal and mechanical hyperalgesia, an effect that was absent in TRPV1 KO mice, and was blocked by the TRPV1 antagonist AMG9810. Immunohistochemistry of mouse plantar skin showed prominent expression of C5aR1 in macrophages. Additionally, C5a evoked strong Ca2+ mobilization in macrophages. Macrophage depletion in transgenic macrophage Fas-induced apoptosis (MAFIA) mice abolished C5a-dependent thermal and mechanical hyperalgesia. Examination of inflammatory mediators following C5a injection revealed a rapid upregulation of nerve growth factor (NGF), a mediator known to sensitize TRPV1. Pre-injection of an NGF-neutralizing antibody or Trk inhibitor GNF-5837 prevented C5a-induced thermal hyperalgesia. Notably, NGF-induced thermal hyperalgesia was unaffected by macrophage depletion. Collectively, these results suggest that C5a induces thermal and mechanical hyperalgesia by triggering macrophage-dependent signaling that involves mobilization of NGF and NGF-dependent sensitization of TRPV1. Our findings highlight the importance of macrophage-to-neuron signaling in pain processing and identify C5a, NGF and TRPV1 as key players in this cross-cellular communication.

Pain is a major personal and community burden throughout the world with currently limited treatment options for persistent pain due to unacceptable side effects, dependence or frank inefficacy. It is necessary to understand the anatomical and molecular pathways leading to pain to better cope with the current challenge of treating it.

Piperine, the major alkaloid derived from black pepper corns, has played an important role in traditional medicine worldwide. Current research has demonstrated piperine to have several anti-inflammatory properties, however, little is known concerning the effect of piperine on B cells. Spleen-derived murine B cells were cultured in the presence or absence of piperine during T-dependent or T-independent activation. Piperine reversibly inhibited B cell proliferation in a dose-dependent manner. This was due to a G0/1-phase cell cycle arrest, and was associated with a reduction in phospho-ERK, phospho-AKT, and Cyclin D1, D2, and D3. Piperine also inhibited antibody and cytokine production. Furthermore, piperine treatment diminished B cell-mediated antigen presentation determined by measuring OT-II transgenic T cell proliferation in response to OVA, which was attributed to the decreased MHC-II ad co-stimulatory molecule expression observed. This in vitro study shows that piperine has potent immuno-suppressive effects on B cell activation and effector function.

No description available.

Transient receptor potential (TRP) channels play an important role in sensory and nonsensory functions. TRPVanilloid 1 and TRPVanilloid 4 are proposed to be involved in inflammation-induced pain. TRPV1 is extensively studied and it is specifically involved in inflammatory thermal hypersensitivity. Mechanical hypersensitivity is one of the significant components of nociception. Several receptors have been proposed to underlie mechanosensation. The molecular entities responsible for mechanosensation are not fully understood. In this study, I have characterized the properties of TRPV4, a putative mechanosensitive ion channel expressed in dorsal root ganglion (DRG) neurons and nonsensory tissues. First, I have investigated the expression and function of TRPV4 and TRPV1 in the DRG neuronal cell bodies as well as their central terminals and determined the modulation by protein kinase C (PKC). Both TRPV4 and TRPV1 are expressed in DRG and laminae I and II of the spinal dorsal horn (DH). Ca2+ fluorescence imaging and whole-cell patch-clamp experiments showed that both capsaicin-induced TRPV1 response and 4alpha-phorbol 12, 13-didecanoate (4alpha-PDD)-induced TRPV4 response were observed in a proportion of the same DRG neurons, suggesting their co-expression. Incubation of DRG neurons with phorbol 12, 13-dibutyrate (PDBu), a PKC activator, resulted in a significantly greater potentiation of TRPV4 currents than TRPV1 currents. In HEK cells heterologously expressing TRPV4, PDBu potentiated TRPV4-mediated single-channel current activity. In patch-clamped DH neurons, the application of 4alpha-PDD at the first sensory synapse increased the frequency but not the amplitude of the miniature excitatory postsynaptic currents (mEPSCs), suggesting a presynaptic locus of action. 4alpha-PDD-induced increase in the frequency of mEPSC was further facilitated by PDBu. These results suggest that TRPV4 in the central terminals modulates synaptic transmission and is regulated by PKC. Second, I have studied the mechanosensitivity of TRPV4 in cell-attached patches by applying direct mechanical force via the patch pipette. In TRPV4 expressing HEK cells, the application of negative pressure evoked single-channel current activity in a reversible manner and the channel activity was enhanced after incubation with PDBu. TRPV4 has been shown to be activated by hypotonicity. Here I show that negative pressure exaggerated hypotonicity-induced single-channel current activity. However, in similar experimental conditions, cells expressing TRPV1 did not respond to mechanical force. TRP channels are also expressed in non-sensory regions and the role of these channels is not fully understood. Both TRPV4 and TRPV1 are expressed in the hippocampus. Using whole-cell patch-clamp techniques, I have found that 4alpha-PDD increased the frequency, but not the amplitude of mEPSCs in cultured hippocampal neurons, suggesting a presynaptic site of action. Interestingly, the application of capsaicin had no effect on synaptic transmission in hippocampal neuronal cultures. Finally, I have investigated the expression and function of TRP channels in diabetes because TRP channels have been shown to be involved in peripheral neuropathy as well as vascular complications in diabetes. ROS production plays a critical role in the progress of diabetes. I propose that lower levels of ROS up-regulate the expression TRP channels in the early stages of diabetes, leading to hyperalgesia, and higher levels of ROS or chronic exposure to ROS down-regulate TRP channels in the late stages of diabetes, resulting in hypoalgesia. I have found that the expression of TRPV1 and phospho p38 (p-p38) MAPK was increased in DRG of streptozotocin (STZ)-injected diabetic and non-diabetic hyperalgesic mice. An increase in TRPV1 and p-p38 MAPK levels was induced by STZ or H2O2 treatment in stably TRPV1 expressing HEK cells, suggesting the involvement of STZ-ROS-p38MAPK pathway. TRPV4 has been reported to be involved in vasodilatation by shear stress in blood vessels. Here, I have demonstrated that TRPV4 is expressed in lymphatic endothelial cells (LECs). Treatment with low concentration of H2O2 enhanced the expression of TRPV4 at mRNA and protein levels in LECs, suggesting that mild levels of ROS up-regulate TRPV4 expression. In diabetes, beta cell dysfunction is responsible for decreased insulin release. TRPV4 is expressed in RINm5F (beta cell line), islets and pancreas. It has been shown that hypotonicity induced insulin release in beta cell lines, which was mediated by activation of stretch-activated channels, raising the possibility of the involvement of TRPV4, a mechanosensitive channel. Therefore, I have studied the functional role of TRPV4 in beta cells. Incubation with 4alpha-PDD enhanced insulin release in RINm5F cells, suggesting TRPV4 regulates insulin secretion from pancreatic beta cells. Since TRPV4 expression levels are decreased in diabetes, insulin secretion from beta cells may be impaired. In summary, TRPV1, a thermosensitive channel, and TRPV4, a mechanosensitive channel, contribute to thermal and mechanical hyperalgesia, respectively in the early stage of DPN through their up-regulation by ROS-p38 MAPK and insulin/IGF-1 pathways. Due to the mechanical sensitivity of TRPV4 channel, the up-regulation in the early stage and down-regulation in the late stage may be involved in the development of vascular complications and regulation of insulin release in diabetes.

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...