Impact of Central and Peripheral TRPV1 and ROS Levels on Proinflammatory Mediators and Nociceptive Behavior

Background: Transient receptor potential vanilloid 1 (TRPV1) channels are important membrane sensors on peripheral nerve endings and on supportive non-neuronal synoviocytes in the knee joint. TRPV 1 ion channels respond with activation of calcium and sodium fluxes to pH, thermal, chemical, osmotic, mechanical and other stimuli abundant in inflamed joints. In the present study, the kaolin/carrageenan (k/c) induced knee joint arthritis model in rats, as well as primary and clonal human synoviocyte cultures were used to understand the reciprocal interactions between reactive nitroxidative species (ROS) and functional TRPV1 channels. ROS generation was monitored with ROS sensitive dyes using live cell imaging in vitro and in spinal tissue histology, as well as with measurement of ROS metabolites in culture media using HPLC. Results: Functional responses in the experimental arthritis model, including increased nociceptive responses (thermal and mechanical hyperalgesia and allodynia), knee joint temperature reflecting local blood flow, and spinal cord ROS elevations were reduced by the ROS scavenger PBN after intraperitoneal pretreatment. Increases in TRPV1 and ROS, generated by synoviocytes in vitro, were reciprocally blocked by TRPV1 antagonists and the ROS scavenger. Further evidence is presented that synoviocyte responses to ROS and TRPV1 activation include increases in TNFalpha and COX-2, both measured as an indicator of the inflammation in vitro. Cconclusions: The results demonstrate that contributions of ROS to pronociceptive responses and neurogenic inflammation are mediated both centrally and peripherally. Responses are mediated by TRPV1 locally in the knee joint by synoviocytes, as well as by ROS-induced sensitization in the spinal cord. These findings and those of others reported in the literature indicate reciprocal interactions between TRPV1 and ROS play critical roles in the pathological and nociceptive responses active during arthritic inflammation

Activation of endothelial transient receptor potential C3 channel is required for small conductance calcium-activated potassium channel activation and sustained endothelial hyperpolarization and vasodilation of cerebral artery

BACKGROUND: Transient receptor potential C3 (TRPC3) has been demonstrated to be involved in the regulation of vascular tone through endothelial cell (EC) hyperpolarization and endothelium-dependent hyperpolarization-mediated vasodilation. However, the mechanism by which TRPC3 regulates these processes remains unresolved. We tested the hypothesis that endothelial receptor stimulation triggers rapid TRPC3 trafficking to the plasma membrane, where it provides the source of Ca(2+) influx for small conductance calcium-activated K(+) (SKCa) channel activation and sustained EC hyperpolarization. METHODS AND RESULTS: Pressurized artery studies were performed with isolated mouse posterior cerebral artery. Treatment with a selective TRPC3 blocker (Pyr3) produced significant attenuation of endothelium-dependent hyperpolarization-mediated vasodilation and endothelial Ca(2+) response (EC-specific Ca(2+) biosensor) to intraluminal ATP. Pyr3 treatment also resulted in a reduced ATP-stimulated global Ca(2+) and Ca(2+) influx in primary cultures of cerebral endothelial cells. Patch-clamp studies with freshly isolated cerebral ECs demonstrated 2 components of EC hyperpolarization and K(+) current activation in response to ATP. The early phase was dependent on intermediate conductance calcium-activated K(+) channel activation, whereas the later sustained phase relied on SKC a channel activation. The SKC a channel-dependent phase was completely blocked with TRPC3 channel inhibition or in ECs of TRPC3 knockout mice and correlated with increased trafficking of TRPC3 (but not SKC a channel) to the plasma membrane. CONCLUSIONS: We propose that TRPC3 dynamically regulates SKC a channel activation through receptor-dependent trafficking to the plasma membrane, where it provides the source of Ca(2+) influx for sustained SKC a channel activation, EC hyperpolarization, and endothelium-dependent hyperpolarization-mediated vasodilation.Fil: Kochukov, Mikhail Y.. Baylor College of Medicine; Estados UnidosFil: Balasubramanian, Adithya. Baylor College of Medicine; Estados UnidosFil: Abramowitz, Joel. National Institute of Environmental Health Sciences Research; Estados UnidosFil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences Research; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marrelli, Sean P.. Baylor College of Medicine; Estados Unido

Activation of endothelial transient receptor potential C3 channel is required for small conductance calcium-activated potassium channel activation and sustained endothelial hyperpolarization and vasodilation of cerebral artery

BACKGROUND: Transient receptor potential C3 (TRPC3) has been demonstrated to be involved in the regulation of vascular tone through endothelial cell (EC) hyperpolarization and endothelium-dependent hyperpolarization-mediated vasodilation. However, the mechanism by which TRPC3 regulates these processes remains unresolved. We tested the hypothesis that endothelial receptor stimulation triggers rapid TRPC3 trafficking to the plasma membrane, where it provides the source of Ca(2+) influx for small conductance calcium-activated K(+) (SKCa) channel activation and sustained EC hyperpolarization. METHODS AND RESULTS: Pressurized artery studies were performed with isolated mouse posterior cerebral artery. Treatment with a selective TRPC3 blocker (Pyr3) produced significant attenuation of endothelium-dependent hyperpolarization-mediated vasodilation and endothelial Ca(2+) response (EC-specific Ca(2+) biosensor) to intraluminal ATP. Pyr3 treatment also resulted in a reduced ATP-stimulated global Ca(2+) and Ca(2+) influx in primary cultures of cerebral endothelial cells. Patch-clamp studies with freshly isolated cerebral ECs demonstrated 2 components of EC hyperpolarization and K(+) current activation in response to ATP. The early phase was dependent on intermediate conductance calcium-activated K(+) channel activation, whereas the later sustained phase relied on SKC a channel activation. The SKC a channel-dependent phase was completely blocked with TRPC3 channel inhibition or in ECs of TRPC3 knockout mice and correlated with increased trafficking of TRPC3 (but not SKC a channel) to the plasma membrane. CONCLUSIONS: We propose that TRPC3 dynamically regulates SKC a channel activation through receptor-dependent trafficking to the plasma membrane, where it provides the source of Ca(2+) influx for sustained SKC a channel activation, EC hyperpolarization, and endothelium-dependent hyperpolarization-mediated vasodilation.Fil: Kochukov, Mikhail Y.. Baylor College of Medicine; Estados UnidosFil: Balasubramanian, Adithya. Baylor College of Medicine; Estados UnidosFil: Abramowitz, Joel. National Institute of Environmental Health Sciences Research; Estados UnidosFil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences Research; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marrelli, Sean P.. Baylor College of Medicine; Estados Unido

Tumor necrosis factor-alpha (TNF-α) enhances functional thermal and chemical responses of TRP cation channels in human synoviocytes

<p>Abstract</p> <p>Background</p> <p>We have shown functional expression of several TRP channels on human synovial cells, proposing significance in known calcium dependent proliferative and secretory responses in joint inflammation. The present study further characterizes synoviocyte TRP expression and activation responses to thermal and osmotic stimuli after pre-treatment with proinflammatory mediator tumor necrosis factor alpha (TNF-α, EC50 1.3221 × 10^-10g/L).</p> <p>Results</p> <p>Fluorescent imaging of Fura-2 loaded human SW982 synoviocytes reveals immediate and delayed cytosolic calcium oscillations elicited by (1) TRPV1 agonists capsaicin and resiniferatoxin (20 – 40% of cells), (2) moderate and noxious temperature change, and (3) osmotic stress TRPV4 activation (11.5% of cells). TNF-alpha pre-treatment (1 ng/ml, 8 – 16 hr) significantly increases (doubles) capsaicin responsive cell numbers and [Ca2+]i spike frequency, as well as enhances average amplitude of temperature induced [Ca²⁺]_iresponses. With TNF-alpha pre-treatment for 8, 12, and 16 hr, activation with 36 or 45 degree bath solution induces bimodal [Ca²⁺]_iincrease (temperature controlled chamber). Initial temperature induced rapid transient spikes and subsequent slower rise reflect TRPV1 and TRPV4 channel activation, respectively. Only after prolonged TNF-alpha exposure (12 and 16 hr) is recruitment of synoviocytes observed with sensitized TRPV4 responses to hypoosmolarity (3–4 fold increase). TNF-alpha increases TRPV1 (8 hr peak) and TRPV4 (12 hr peak) immunostaining, mRNA and protein expression, with a TRPV1 shift to membrane fractions.</p> <p>Conclusion</p> <p>TNF-α provides differentially enhanced synoviocyte TRPV1 and TRPV4 expression and [Ca²⁺]_iresponse dependent on the TRP stimulus and time after exposure. Augmented relevance of TRPV1 and TRPV4 as inflammatory conditions persist would provide calcium mediated cell signaling required for pathophysiological responses of synoviocytes in inflammatory pain states.</p

Combinations of physiologic estrogens with xenoestrogens alter calcium and kinase responses, prolactin release, and membrane estrogen receptor trafficking in rat pituitary cells

<p>Abstract</p> <p>Background</p> <p>Xenoestrogens such as alkylphenols and the structurally related plastic byproduct bisphenol A have recently been shown to act potently via nongenomic signaling pathways and the membrane version of estrogen receptor-α. Though the responses to these compounds are typically measured individually, they usually contaminate organisms that already have endogenous estrogens present. Therefore, we used quantitative medium-throughput screening assays to measure the effects of physiologic estrogens in combination with these xenoestrogens.</p> <p>Methods</p> <p>We studied the effects of low concentrations of endogenous estrogens (estradiol, estriol, and estrone) at 10 pM (representing pre-development levels), and 1 nM (representing higher cycle-dependent and pregnancy levels) in combinations with the same levels of xenoestrogens in GH₃/B6/F10 pituitary cells. These levels of xenoestrogens represent extremely low contamination levels. We monitored calcium entry into cells using Fura-2 fluorescence imaging of single cells. Prolactin release was measured by radio-immunoassay. Extracellular-regulated kinase (1 and 2) phospho-activations and the levels of three estrogen receptors in the cell membrane (ERα, ERβ, and GPER) were measured using a quantitative plate immunoassay of fixed cells either permeabilized or nonpermeabilized (respectively).</p> <p>Results</p> <p>All xenoestrogens caused responses at these concentrations, and had disruptive effects on the actions of physiologic estrogens. Xenoestrogens reduced the % of cells that responded to estradiol via calcium channel opening. They also inhibited the activation (phosphorylation) of extracellular-regulated kinases at some concentrations. They either inhibited or enhanced rapid prolactin release, depending upon concentration. These latter two dose-responses were nonmonotonic, a characteristic of nongenomic estrogenic responses.</p> <p>Conclusions</p> <p>Responses mediated by endogenous estrogens representing different life stages are vulnerable to very low concentrations of these structurally related xenoestrogens. Because of their non-classical dose-responses, they must be studied in detail to pinpoint effective concentrations and the directions of response changes.</p

Progressive Changes in a Distributed Neural Circuit Underlie Breathing Abnormalities in Mice Lacking MeCP2

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in Methyl-CpG-binding protein 2 (MECP2). Severe breathing abnormalities are common in RTT and are reproduced in mouse models of RTT. Previously, we found that removing MeCP2 from the brainstem and spinal cord in mice caused early lethality and abnormal breathing. To determine whether loss of MeCP2 in functional components of the respiratory network causes specific breathing disorders, we used the Cre/LoxP system to differentially manipulate MeCP2 expression throughout the brainstem respiratory network, specifically within HoxA4-derived tissues, which include breathing control circuitry within the nucleus tractus solitarius and the caudal part of ventral respiratory column but do not include more rostral parts of the breathing control circuitry. To determine whether respiratory phenotypes manifested in animals with MeCP2 removed from specific pons medullary respiratory circuits, we performed whole-body plethysmography and electrophysiological recordings from in vitro brainstem slices from mice lacking MeCP2 in different circuits. Our results indicate that MeCP2 expression in the medullary respiratory network is sufficient for normal respiratory rhythm and preventing apnea. However, MeCP2 expression within components of the breathing circuitry rostral to the HoxA4 domain are neither sufficient to prevent the hyperventilation nor abnormal hypoxic ventilatory response. Surprisingly, we found that MeCP2 expression in the HoxA4 domain alone is critical for survival. Our study reveals that MeCP2 is differentially required in select respiratory components for different aspects of respiratory functions, and collectively for the integrity of this network functions to maintain proper respiration. SIGNIFICANCE STATEMENT Breathing abnormalities are a significant clinical feature in Rett syndrome and are robustly reproduced in the mouse models of this disease. Previous work has established that alterations in the function of MeCP2, the protein encoded by the gene mutated in Rett syndrome, within the hindbrain are critical for control of normal breathing. Here we show that MeCP2 function plays distinct roles in specific brainstem regions in the genesis of various aspects of abnormal breathing. This provides insight into the pathogenesis of these breathing abnormalities in Rett syndrome, which could be used to target treatments to improve these symptoms. Furthermore, it provides further knowledge about the fundamental neural circuits that control breathing

Progressive Changes in a Distributed Neural Circuit Underlie Breathing Abnormalities in Mice Lacking MeCP2.

UnlabelledRett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in Methyl-CpG-binding protein 2 (MECP2). Severe breathing abnormalities are common in RTT and are reproduced in mouse models of RTT. Previously, we found that removing MeCP2 from the brainstem and spinal cord in mice caused early lethality and abnormal breathing. To determine whether loss of MeCP2 in functional components of the respiratory network causes specific breathing disorders, we used the Cre/LoxP system to differentially manipulate MeCP2 expression throughout the brainstem respiratory network, specifically within HoxA4-derived tissues, which include breathing control circuitry within the nucleus tractus solitarius and the caudal part of ventral respiratory column but do not include more rostral parts of the breathing control circuitry. To determine whether respiratory phenotypes manifested in animals with MeCP2 removed from specific pons medullary respiratory circuits, we performed whole-body plethysmography and electrophysiological recordings from in vitro brainstem slices from mice lacking MeCP2 in different circuits. Our results indicate that MeCP2 expression in the medullary respiratory network is sufficient for normal respiratory rhythm and preventing apnea. However, MeCP2 expression within components of the breathing circuitry rostral to the HoxA4 domain are neither sufficient to prevent the hyperventilation nor abnormal hypoxic ventilatory response. Surprisingly, we found that MeCP2 expression in the HoxA4 domain alone is critical for survival. Our study reveals that MeCP2 is differentially required in select respiratory components for different aspects of respiratory functions, and collectively for the integrity of this network functions to maintain proper respiration.Significance statementBreathing abnormalities are a significant clinical feature in Rett syndrome and are robustly reproduced in the mouse models of this disease. Previous work has established that alterations in the function of MeCP2, the protein encoded by the gene mutated in Rett syndrome, within the hindbrain are critical for control of normal breathing. Here we show that MeCP2 function plays distinct roles in specific brainstem regions in the genesis of various aspects of abnormal breathing. This provides insight into the pathogenesis of these breathing abnormalities in Rett syndrome, which could be used to target treatments to improve these symptoms. Furthermore, it provides further knowledge about the fundamental neural circuits that control breathing

Nongenomic Signaling Pathways of Estrogen Toxicity

Xenoestrogens can affect the healthy functioning of a variety of tissues by acting as potent estrogens via nongenomic signaling pathways or by interfering with those actions of multiple physiological estrogens. Collectively, our and other studies have compared a wide range of estrogenic compounds, including some closely structurally related subgroups. The estrogens that have been studied include environmental contaminants of different subclasses, dietary estrogens, and several prominent physiological metabolites. By comparing the nongenomic signaling and functional responses to these compounds, we have begun to address the structural requirements for their actions through membrane estrogen receptors in the pituitary, in comparison to other tissues, and to gain insights into their typical non-monotonic dose-response behavior. Their multiple inputs into cellular signaling begin processes that eventually integrate at the level of mitogen-activated protein kinase activities to coordinately regulate broad cellular destinies, such as proliferation, apoptosis, or differentiation