Arteries are finely tuned thermosensors regulating myogenic tone and blood flow.

In response to changing blood pressure, arteries adjust their caliber to control perfusion. This vital autoregulatory property, termed vascular myogenic tone, stabilizes downstream capillary pressure. We discovered that tissue temperature critically determines myogenic tone. Heating steeply activates tone in skeletal muscle, gut, brain and skin arteries with temperature coefficients ( Q 10 ) of ∼11-20. Further, arterial thermosensitivity is tuned to resting tissue temperatures, making myogenic tone sensitive to small thermal fluctuations. Interestingly, temperature and intraluminal pressure are sensed largely independently and integrated to trigger myogenic tone. We show that TRPV1 and TRPM4 mediate heat-induced tone in skeletal muscle arteries. Variations in tissue temperature are known to alter vascular conductance; remarkably, thermosensitive tone counterbalances this effect, thus protecting capillary integrity and fluid balance. In conclusion, thermosensitive myogenic tone is a fundamental homeostatic mechanism regulating tissue perfusion. One-Sentence Summary: Arterial blood pressure and temperature are integrated via thermosensitve ion channels to produce myogenic tone.

TRPV1 in arteries enables a rapid myogenic tone.

Arterioles maintain blow flow by adjusting their diameter in response to changes in local blood pressure. In this process called the myogenic response, a vascular smooth muscle mechanosensor controls tone predominantly through altering the membrane potential. In general, myogenic responses occur slowly (minutes). In the heart and skeletal muscle, however, tone is activated rapidly (tens of seconds) and terminated by brief (100 ms) arterial constrictions. Previously, we identified extensive expression of TRPV1 in the smooth muscle of arterioles supplying skeletal muscle, heart and fat. Here we reveal a critical role for TRPV1 in the rapid myogenic tone of these tissues. TRPV1 antagonists dilated skeletal muscle arterioles in vitro and in vivo, increased coronary flow in isolated hearts, and transiently decreased blood pressure. All of these pharmacologic effects were abolished by genetic disruption of TRPV1. Stretch of isolated vascular smooth muscle cells or raised intravascular pressure in arteries triggered Ca2+ signalling and vasoconstriction. The majority of these stretch-responses were TRPV1-mediated, with the remaining tone being inhibited by the TRPM4 antagonist, 9-phenantrol. Notably, tone developed more quickly in arteries from wild-type compared with TRPV1-null mice. Furthermore, the immediate vasodilation following brief constriction of arterioles depended on TRPV1, consistent with a rapid deactivation of TRPV1. Pharmacologic experiments revealed that membrane stretch activates phospholipase C/protein kinase C signalling combined with heat to activate TRPV1, and in turn, L-type Ca2+ channels. These results suggest a critical role, for TRPV1 in the dynamic regulation of myogenic tone and blood flow in the heart and skeletal muscle. KEY POINTS: We explored the physiological role of TRPV1 in vascular smooth muscle. TRPV1 antagonists dilated skeletal muscle arterioles both ex vivo and in vivo, increased coronary perfusion and decreased systemic blood pressure. Stretch of arteriolar myocytes and increases in intraluminal pressure in arteries triggered rapid Ca2+ signalling and vasoconstriction respectively. Pharmacologic and/or genetic disruption of TRPV1 significantly inhibited the magnitude and rate of these responses. Furthermore, disrupting TRPV1 blunted the rapid vasodilation evoked by arterial constriction. Pharmacological experiments identified key roles for phospholipase C and protein kinase C, combined with temperature, in TRPV1-dependent arterial tone. These results show that TRPV1 in arteriolar myocytes dynamically regulates myogenic tone and blood flow in the heart and skeletal muscle.

International Union of Basic and Clinical Pharmacology. CX. Classification of Receptors for 5-hydroxytryptamine; Pharmacology and Function.

5-HT receptors expressed throughout the human body are targets for established therapeutics and various drugs in development. Their diversity of structure and function reflects the important role 5-HT receptors play in physiologic and pathophysiological processes. The present review offers a framework for the official receptor nomenclature and a detailed understanding of each of the 14 5-HT receptor subtypes, their roles in the systems of the body, and, where appropriate, the (potential) utility of therapeutics targeting these receptors. SIGNIFICANCE STATEMENT: This review provides a comprehensive account of the classification and function of 5-hydroxytryptamine receptors, including how they are targeted for therapeutic benefit.

Countermeasures for Preventing and Treating Opioid Overdose.

The only medication available currently to prevent and treat opioid overdose (naloxone) was approved by the US Food and Drug Administration (FDA) nearly 50 years ago. Because of its pharmacokinetic and pharmacodynamic properties, naloxone has limited utility under some conditions and would not be effective to counteract mass casualties involving large-scale deployment of weaponized synthetic opioids. To address shortcomings of current medical countermeasures for opioid toxicity, a trans-agency scientific meeting was convened by the US National Institute of Allergy and Infectious Diseases/National Institutes of Health (NIAID/NIH) on August 6 and 7, 2019, to explore emerging alternative approaches for treating opioid overdose in the event of weaponization of synthetic opioids. The meeting was initiated by the Chemical Countermeasures Research Program (CCRP), was organized by NIAID, and was a collaboration with the National Institute on Drug Abuse/NIH (NIDA/NIH), the FDA, the Defense Threat Reduction Agency (DTRA), and the Biomedical Advanced Research and Development Authority (BARDA). This paper provides an overview of several presentations at that meeting that discussed emerging new approaches for treating opioid overdose, including the following: (1) intranasal nalmefene, a competitive, reversible opioid receptor antagonist with a longer duration of action than naloxone; (2) methocinnamox, a novel opioid receptor antagonist; (3) covalent naloxone nanoparticles; (4) serotonin (5-HT)1A receptor agonists; (5) fentanyl-binding cyclodextrin scaffolds; (6) detoxifying biomimetic "nanosponge" decoy receptors; and (7) antibody-based strategies. These approaches could also be applied to treat opioid use disorder.

TRPV1 expressed throughout the arterial circulation regulates vasoconstriction and blood pressure.

KEY POINTS: The functional roles of the capsaicin receptor, TRPV1, outside of sensory nerves are unclear. We mapped TRPV1 in the mouse circulation, revealing extensive expression in the smooth muscle of resistance arterioles supplying skeletal muscle, heart and adipose tissue.  Activation of TRPV1 in vascular myocytes constricted arteries, reduced coronary flow in isolated hearts and increased systemic blood pressure. These functional effects were retained after sensory nerve ablation, indicating specific signalling by arterial TRPV1.  TRPV1 mediated the vasoconstrictive and blood pressure responses to the endogenous inflammatory lipid lysophosphatidic acid.  These results show that TRPV1 in arteriolar myocytes modulates regional blood flow and systemic blood pressure, and suggest that TRPV1 may be a target of vasoactive inflammatory mediators. ABSTRACT: The capsaicin receptor, TRPV1, is a key ion channel involved in inflammatory pain signalling. Although mainly studied in sensory nerves, there are reports of TRPV1 expression in isolated segments of the vasculature, but whether the channel localizes to vascular endothelium or smooth muscle is controversial and the distribution and functional roles of TRPV1 in arteries remain unknown. We mapped functional TRPV1 expression throughout the mouse arterial circulation. Analysis of reporter mouse lines TRPV1PLAP-nlacZ and TRPV1-Cre:tdTomato combined with Ca2+ imaging revealed specific localization of TRPV1 to smooth muscle of terminal arterioles in the heart, adipose tissue and skeletal muscle. Capsaicin evoked inward currents (current density ∼10% of sensory neurons) and raised intracellular Ca2+ levels in arterial smooth muscle cells, constricted arterioles ex vivo and in vivo and increased systemic blood pressure in mice and rats. Further, capsaicin markedly and dose-dependently reduced coronary flow. Pharmacological and/or genetic disruption of TRPV1 abolished all these effects of capsaicin as well as vasoconstriction triggered by lysophosphatidic acid, a bioactive lipid generated by platelets and atherogenic plaques. Notably, ablation of sensory nerves did not affect the responses to capsaicin revealing a vascular smooth muscle-restricted signalling mechanism. Moreover, unlike in sensory nerves, TRPV1 function in arteries was resistant to activity-induced desensitization. Thus, TRPV1 activation in vascular myocytes enables a persistent depolarizing current, leading to constriction of coronary, skeletal muscle and adipose arterioles and a sustained increase in systemic blood pressure.

Inhibition of Ligand-Gated TRPA1 by General Anesthetics.

Several general anesthetics (GAs) produce pain or irritation upon administration, and this occurs predominantly through the activation of the nociceptive ion channel, transient receptor potential ankyrin type 1 (TRPA1). However, the effects of GAs on agonist-mediated TRPA1 activity are unclear. Here we show that a diverse range of noxious and non-noxious volatile anesthetics, at clinically relevant concentrations, inhibit ligand-activated TRPA1 currents. These effects are species-specific; GAs blocks rodent TRPA1 without affecting the Drosophila ortholog. Furthermore, propofol inhibits rodent but not human TRPA1. Analysis of chimeric TRPA1 proteins and mutagenesis combined reveals two amino acid residues located in the S5 domain, Ser876 and Thr877, that are critical for the inhibitory effects of isoflurane and propofol. Introduction of these residues into Drosophila TRPA1 confers anesthetic inhibition. Furthermore, several residues lining the presumptive binding pocket for noxious GAs are not required for the inhibitory effects of GAs. We conclude that anesthetics inhibit TRPA1 by interacting at a site distinct from the activation site. The inhibitory effects of GAs at TRPA1 may contribute to the diverse pharmacological action of these drugs. SIGNIFICANCE STATEMENT: We show that both noxious and non-noxious general anesthetics inhibit agonist-evoked transient receptor potential ankyrin type 1 (TRPA1) activity and identify critical amino acid residues located in the pore domain. Inhibition of TRPA1 may affect pain and vascular signaling during anesthesia.

Human Immunodeficiency Virus Promotes Mitochondrial Toxicity.

Combined antiretroviral therapies (cART) have had remarkable success in reducing morbidity and mortality among patients infected with human immunodeficiency virus (HIV). However, mild forms of HIV-associated neurocognitive disorders (HAND), characterized by loss of synapses, remain. cART may maintain an undetectable HIV RNA load but does not eliminate the expression of viral proteins such as trans-activator of transcription (Tat) and the envelope glycoprotein gp120 in the brain. These two viral proteins are known to promote synaptic simplifications by several mechanisms, including alteration of mitochondrial function and dynamics. In this review, we aim to outline the many targets and pathways used by viral proteins to alter mitochondria dynamics, which contribute to HIV-induced neurotoxicity. A better understanding of these pathways is crucial for the development of adjunct therapies for HAND.

Identification of a putative binding site critical for general anesthetic activation of TRPA1.

General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABAA receptors. In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. Here we exploit the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, we show that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876, Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, our combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels.

Intrinsic Photosensitivity Enhances Motility of T Lymphocytes.

Sunlight has important biological effects in human skin. Ultraviolet (UV) light striking the epidermis catalyzes the synthesis of Vitamin D and triggers melanin production. Although a causative element in skin cancers, sunlight is also associated with positive health outcomes including reduced incidences of autoimmune diseases and cancers. The mechanisms, however, by which light affects immune function remain unclear. Here we describe direct photon sensing in human and mouse T lymphocytes, a cell-type highly abundant in skin. Blue light irradiation at low doses (<300 mJ cm-2) triggers synthesis of hydrogen peroxide (H2O2) in T cells revealed by the genetically encoded reporter HyPerRed. In turn, H2O2 activates a Src kinase/phospholipase C-γ1 (PLC-γ1) signaling pathway and Ca2+ mobilization. Pharmacologic inhibition or genetic disruption of Lck kinase, PLC-γ1 or the T cell receptor complex inhibits light-evoked Ca2+ transients. Notably, both light and H2O2 enhance T-cell motility in a Lck-dependent manner. Thus, T lymphocytes possess intrinsic photosensitivity and this property may enhance their motility in skin.

Protease induced plasticity: matrix metalloproteinase-1 promotes neurostructural changes through activation of protease activated receptor 1.

Matrix metalloproteinases (MMPs) are a family of secreted endopeptidases expressed by neurons and glia. Regulated MMP activity contributes to physiological synaptic plasticity, while dysregulated activity can stimulate injury. Disentangling the role individual MMPs play in synaptic plasticity is difficult due to overlapping structure and function as well as cell-type specific expression. Here, we develop a novel system to investigate the selective overexpression of a single MMP driven by GFAP expressing cells in vivo. We show that MMP-1 induces cellular and behavioral phenotypes consistent with enhanced signaling through the G-protein coupled protease activated receptor 1 (PAR1). Application of exogenous MMP-1, in vitro, stimulates PAR1 dependent increases in intracellular Ca2+ concentration and dendritic arborization. Overexpression of MMP-1, in vivo, increases dendritic complexity and induces biochemical and behavioral endpoints consistent with increased GPCR signaling. These data are exciting because we demonstrate that an astrocyte-derived protease can influence neuronal plasticity through an extracellular matrix independent mechanism.

Sex-dependent expression of TRPV1 in bladder arterioles.

Transient receptor potential vanilloid type 1 (TRPV1) is a major nociceptive ion channel implicated in bladder physiology and/or pathophysiology. However, the precise expression of TRPV1 in neuronal vs. nonneuronal bladder cells is uncertain. Here we used reporter mouse lines (TRPV1-Cre:tdTomato and TRPV1PLAP-nlacZ) to map expression of TRPV1 in postnatal bladder. TRPV1 was not detected in the urothelium, however, we found marked expression of TRPV1 lineage in sensory nerves, and surprisingly, in arterial/arteriolar smooth muscle (ASM) cells. Tomato fluorescence was prominent in the vesical arteries and in small-diameter (15-40 µm) arterioles located in the suburothelial layer with a near equal distribution in bladder dome and base. Notably, arteriolar TRPV1 expression was greater in females than in males and increased in both sexes after 90 days of age, suggesting sex hormone and age dependency. Analysis of whole bladder and vesical artery TRPV1 mRNA revealed a similar sex and developmental dependence. Pharmacological experiments confirmed functional TRPV1 protein expression; capsaicin increased intracellular Ca2+ in ∼15% of ASM cells from wild-type female bladders, but we observed no responses to capsaicin in bladder arterioles isolated from TRPV1-null mice. Furthermore, capsaicin triggered arteriole constriction that was rapidly reversed by the TRPV1 antagonist, BCTC. These data show that predominantly in postpubertal female mice, bladder ASM cells express functional TRPV1 channels that may act to constrict arterioles. TRPV1 may therefore play an important role in regulating the microcirculation of the female bladder, and this effect may be of significance during inflammatory conditions.

Menthol Enhances the Desensitization of Human α3ß4 Nicotinic Acetylcholine Receptors.

The α3ß4 nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the peripheral and central nervous systems, including in airway sensory nerves. The nAChR subtype transduces the irritant effects of nicotine in tobacco smoke and, in certain brain areas, may be involved in nicotine addiction and/or withdrawal. Menthol, a widely used additive in cigarettes, is a potential analgesic and/or counterirritant at sensory nerves and may also influence nicotine's actions in the brain. We examined menthol's effects on recombinant human α3ß4 nAChRs and native nAChRs in mouse sensory neurons. Menthol markedly decreased nAChR activity as assessed by Ca(2+) imaging, (86)Rb(+) efflux, and voltage-clamp measurements. Coapplication of menthol with acetylcholine or nicotine increased desensitization, demonstrated by an increase in the rate and magnitude of the current decay and a reduction of the current integral. These effects increased with agonist concentration. Pretreatment with menthol followed by its washout did not affect agonist-induced desensitization, suggesting that menthol must be present during the application of agonist to augment desensitization. Notably, menthol acted in a voltage-independent manner and reduced the mean open time of single channels without affecting their conductance, arguing against a simple channel-blocking effect. Further, menthol slowed or prevented the recovery of nAChRs from desensitization, indicating that it probably stabilizes a desensitized state. Moreover, menthol at concentrations up to 1 mM did not compete for the orthosteric nAChR binding site labeled by [(3)H]epibatidine. Taken together, these data indicate that menthol promotes desensitization of α3ß4 nAChRs by an allosteric action.

Transient receptor potential channels and energy homeostasis.

Transient receptor potential (TRP) channels are members of an ancient class of ion channels that are present in most mammalian tissues. Consistent with their wide tissue distribution, TRPs are capable of influencing diverse physiological processes including adipocyte function, energy intake and energy expenditure. TRPs function as transduction channels downstream of G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases, and some can also be direct sensors of chemical irritants that influence food intake or regulate body temperature and thermogenesis. TRP agonists were shown to reduce body weight and adiposity, suggesting that they might be exploited as therapeutic targets. In this review I discuss the current knowledge of how TRP channels influence energy balance.

TRPA1 is a polyunsaturated fatty acid sensor in mammals.

Fatty acids can act as important signaling molecules regulating diverse physiological processes. Our understanding, however, of fatty acid signaling mechanisms and receptor targets remains incomplete. Here we show that Transient Receptor Potential Ankyrin 1 (TRPA1), a cation channel expressed in sensory neurons and gut tissues, functions as a sensor of polyunsaturated fatty acids (PUFAs) in vitro and in vivo. PUFAs, containing at least 18 carbon atoms and three unsaturated bonds, activate TRPA1 to excite primary sensory neurons and enteroendocrine cells. Moreover, behavioral aversion to PUFAs is absent in TRPA1-null mice. Further, sustained or repeated agonism with PUFAs leads to TRPA1 desensitization. PUFAs activate TRPA1 non-covalently and independently of known ligand binding domains located in the N-terminus and 5(th) transmembrane region. PUFA sensitivity is restricted to mammalian (rodent and human) TRPA1 channels, as the drosophila and zebrafish TRPA1 orthologs do not respond to DHA. We propose that PUFA-sensing by mammalian TRPA1 may regulate pain and gastrointestinal functions.

5-HT and the immune system.

The classical neurotransmitter, serotonin (5-HT), plays an important role outside of the central nervous system in immune signaling. Here I review recent studies describing 5-HT uptake in dendritic cells and B lymphocytes, 5-HT synthesis in T lymphocytes, and the role of specific 5-HT receptor subtypes in innate and adaptive immune cells. Furthermore, a recent paper describing the immune phenotype of 5-HT deficient mice is discussed.

TRPV1 and synaptic transmission.

Transient Receptor Potential Vanilloid Type 1 is a prominent "pain" receptor expressed in sensory afferent neurons. TRPV1 on peripheral nerve terminals detects a variety of noxious stimuli generated at sites of injury and inflammation, and in turn, drives the excitation and sensitization of C-fibers neurons. Significantly, TRPV1 is also located on the central terminals of sensory neurons projecting to the spinal cord and brainstem. These TRPV1 channels appear to stimulate the secretion of glutamate. Further, TRPV1 is expressed diffusely in the brain and there is emerging evidence for TRPV1 modulating transmission at various brain synapses. Here we discuss our current understanding of the potential roles for TRPV1 in synaptic transmission.

Store-operated Ca²+ signaling in dendritic cells occurs independently of STIM1.

SOCE via CRAC channels is a critical signaling event in immune cells. Recent studies have identified key proteins underlying this process; STIM is an ER Ca²+ sensor that interacts with Orai, an intrinsic, pore-forming protein of the CRAC channel. In heterologous expression systems, STIM1 regulates SOCE by interacting with Orai1, -2, and -3. In native tissues, however, the precise roles of STIM and Orai proteins are not well defined. Here, we have investigated the molecular components of SOCE signaling in mouse DCs. We show that DCs predominantly express STIM2 and only very low levels of STIM1 compared with T lymphocytes. Upon store depletion with Tg, STIM2 aggregates and interacts selectively with Orai2. In contrast, Tg fails to aggregate STIM1 or enhance STIM1-mediated interactions with Orai proteins. Consistent with this biochemical characterization, stimulation of DCs with the adhesion molecule ICAM-1 selectively recruits STIM2 and Orai2 to the IS. Together, these data demonstrate a novel, STIM2-dependent SOCE signaling pathway in DCs.

General anesthetics sensitize the capsaicin receptor transient receptor potential V1.

General anesthetics (GAs) are central nervous system depressants that render patients unresponsive to external stimuli. In contrast, many of these agents are also known to stimulate peripheral sensory nerves, raising the possibility that they may exacerbate tissue inflammation. We have found that pungent GAs excite sensory neurons by directly activating the transient receptor potential (TRP) A1 ion channel. Here, we show that GAs also sensitize the capsaicin receptor TRPV1, a key ion channel expressed in nociceptive neurons. Clinically relevant concentrations of isoflurane, sevoflurane, enflurane, and desflurane sensitize TRPV1 to capsaicin and protons and reduce the threshold for heat activation. Furthermore, isoflurane directly activates TRPV1 after stimulation of protein kinase C. Likewise, isoflurane excites TRPV1 and sensory neurons during concomitant application of bradykinin, a key inflammatory mediator formed during tissue injury. Thus, GAs can enhance the activation of TRPV1 that occurs during surgically induced tissue damage. These results support the hypothesis that some GAs, through direct actions at TRP channels, increase postsurgical pain and inflammation.

General anesthetics activate a nociceptive ion channel to enhance pain and inflammation.

General anesthetics (GAs) have transformed surgery through their actions to depress the central nervous system and blunt the perception of surgical insults. Counterintuitively, many of these agents activate peripheral nociceptive neurons. However, the underlying mechanisms and significance of these effects have not been explored. Here, we show that clinical concentrations of noxious i.v. and inhalation GAs excite sensory neurons by selectively activating TRPA1, a key ion channel in the pain pathway. Further, these GAs induce pain-related responses in mice that are abolished in TRPA1-null animals. Significantly, TRPA1-dependent neurogenic inflammation is greater in mice anesthetized with pungent compared with nonpungent anesthetics. Thus, our results show that TRPA1 is essential for sensing noxious GAs. The pronociceptive effects of GAs combined with surgical tissue damage could lead to a paradoxical increase in postoperative pain and inflammation.

TRPV1-null mice are protected from diet-induced obesity.

We explored a role for the capsaicin receptor, transient receptor potential channel vanilloid type 1 (TRPV1), in the regulation of feeding and body mass. On a 4.5% fat diet, wild-type and TRPV1-null mice gained equivalent body mass. On an 11% fat diet, however, TRPV1-null mice gained significantly less mass and adiposity; at 44 weeks the mean body weights of wild-type and TRPV1-null mice were approximately 51 and 34g, respectively. Both groups of mice consumed equivalent energy and absorbed similar amounts of lipids. TRPV1-null mice, however, exhibited a significantly greater thermogenic capacity. Interestingly, we found that 3T3-L1 preadipocytes expressed functional calcitonin gene-related peptide receptors. Thus, these data support a potential neurogenic mechanism by which TRPV1-sensitive sensory nerves may regulate energy and fat metabolism.