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Background: Silent inflammatory bowel disease (IBD) is a condition in which individuals with the active disease experience minor to no pain. Voltage‐gated Na+ (NaV) channels expressed in sensory neurons play a major role in pain perception. Previously, we reported that a NaV1.8 genetic polymorphism (A1073V, rs6795970) was more common in a cohort of silent IBD patients. The expression of this variant (1073V) in rat sympathetic neurons activated at more depolarized potentials when compared to the more common variant (1073A). In this study, we investigated whether expression of either NaV1.8 variant in rat sensory neurons would exhibit different biophysical characteristics than previously observed in sympathetic neurons. Methods: Endogenous NaV1.8 channels were first silenced in DRG neurons and then either 1073A or 1073V human NaV1.8 cDNA constructs were transfected. NaV1.8 currents were recorded with the whole‐cell patch‐clamp technique. Key Results: The results indicate that 1073A and 1073V NaV1.8 channels exhibited similar activation values. However, the slope factor (k) for activation determined for this same group of neurons decreased by 5 mV, suggesting an increase in voltage sensitivity. Comparison of inactivation parameters indicated that 1073V channels were shifted to more depolarized potentials than 1073A‐expressing neurons, imparting a proexcitatory characteristic. Conclusions and Inferences: These findings differ from previous observations in other expression models and underscore the challenges with heterologous expression systems. Therefore, the use of human sensory neurons derived from induced pluripotent stem cells may help address these inconsistencies and better determine the effect of the polymorphism present in IBD patients. [ABSTRACT FROM AUTHOR]

The role played by the transient receptor potential vanilloid 1 (TRPV1) channel on the thin fibre afferents evoking the exercise pressor reflex is controversial. To shed light on this controversy, we compared the exercise pressor reflex between newly developed TRPV1+/+, TRPV1+/− and TRPV1−/− rats. Carotid arterial injection of capsaicin (0.5 μg), evoked significant pressor responses in TRPV1+/+ and TRPV1+/− rats, but not in TRPV1−/− rats. In acutely isolated dorsal root ganglion neurons innervating the gastrocnemius muscles, capsaicin evoked inward currents in neurons isolated from TRPV1+/+ and TRPV1+/− rats but not in neurons isolated from TRPV1−/− rats. The reflex was evoked by stimulating the tibial nerve in decerebrated rats whose femoral artery was either freely perfused or occluded. We found no difference between the reflex in the three groups of rats regardless of the patency of the femoral artery. For example, the peak pressor responses to contraction in TRPV1+/+, TRPV1+/− and TRPV1−/− rats with patent femoral arteries averaged 17.1 ± 7.2, 18.9 ± 12.4 and 18.4 ± 8.6 mmHg, respectively. Stimulation of the tibial nerve after paralysis with pancuronium had no effect on arterial pressure, findings which indicated that the pressor responses to contraction were not caused by electrical stimulation of afferent tibial nerve axons. We also found that expression levels of acid‐sensing ion channel 1 and endoperoxide 4 receptor in the L4 and 5 dorsal root ganglia were not upregulated in the TRPV1−/− rats. We conclude that TRPV1 is not needed to evoke the exercise pressor reflex in rats whose contracting muscles have either a patent or an occluded arterial blood supply. Key points: A reflex arising in contracting skeletal muscle contributes to the increases in arterial blood pressure, cardiac output and breathing evoked by exercise.The sensory arm of the reflex comprises both mechanoreceptors and metaboreceptors, of which the latter signals that blood flow to exercising muscle is not meeting its metabolic demand.The nature of the channel on the metaboreceptor sensing a mismatch between supply and demand is controversial; some believe that it is the transient receptor potential vanilloid 1 (TRPV1) channel.Using genetically engineered rats in which the TRPV1 channel is rendered non‐functional, we have shown that it is not needed to evoke the metaboreflex. [ABSTRACT FROM AUTHOR]

All currently employed pharmaceutical formulations of hydroxychloroquine (HCQ) sulfate are a racemate, consisting of equal parts mixture of two stereoisomers: R(−)HCQ and S(+)HCQ sulfates. The aims of the current study were first, to obtain and characterize pure HCQ enantiomers. The separation and purification of free base HCQ enantiomers from the racemate form were performed using semi‐preparative chiral high‐performance liquid chromatography. Second, we compared the pharmacological properties of both optical isomers and racemic mixture on the intracellular Ca2+ oscillations employing an in vitro model of human cardiomyocytes derived from induced pluripotent stem cells (iPSCs). The results of the pharmacological investigations indicate that the racemic and pure stereoisomer forms of HCQ sulfate exhibited a dose‐dependent inhibition of spontaneous Ca2+ oscillations (as measured from their frequency and Ca2+ peak widths) in cardiomyocytes 5–45 min following exposure. In addition, the concentration‐response relationships for all three compounds indicated that the rank order of potency (IC50) was R(−)HCQ >racemic HCQ >S(+)HCQ for the frequency of the Ca2+ oscillations and width of Ca2+ peaks for all time points examined. These studies indicate that both R(−) and S(+) stereoisomers exhibit differing pharmacological actions on hiPSC cardiomyocytes, with the former effecting a greater potency on cell Ca2+ handling. [ABSTRACT FROM AUTHOR]

Key points We investigated the role played by lactate and hydrogen in evoking the exercise pressor reflex (EPR) in decerebrated rats whose hindlimb muscles were either freely perfused or ischaemic. Production of lactate and hydrogen by the contracting hindlimb muscles was manipulated by knocking out the myophosphorylase gene (pygm). In knockout rats (pygm−/−; n = 13) or wild‐type rats (pygm+/+; n = 13), the EPR was evoked by isometrically contracting the triceps surae muscles. Blood pressure, tension, blood flow, renal sympathetic nerve activity and blood lactate concentrations were measured. Intramuscular metabolites and pH changes induced by the contractions were quantified by 31P‐magnetic resonance spectroscopy (n = 5). In a subset of pygm−/− rats (n = 5), contractions were evoked with prior infusion of lactate (pH 6.0) in an attempt to restore the effect of lactate and hydrogen ions. Contraction of freely perfused muscles increased blood lactate and decreased muscle pH in pygm+/+ rats only. Despite these differences, the reflex pressor and sympathetic responses to freely perfused contraction did not differ between groups (P = 0.992). During ischaemia, contraction increased muscle lactate and hydrogen ion production in pygm+/+ rats (P < 0.0134), whereas it had no effect in pygm−/− rats (P > 0.783). Likewise, ischaemia exaggerated the reflex pressor, and sympathetic responses to contraction in pygm+/+ but not in pygm−/− rats. This exaggeration was restored when a solution of lactate (pH 6.0) was infused prior to the contraction in pygm−/− rats. We conclude that lactate and hydrogen accumulation in contracting myocytes play a key role in evoking the metabolic component of the EPR during ischaemic but not during freely perfused contractions. Conflicting results exist about the role played by lactate and hydrogen ions in evoking the exercise pressor reflex. Using CRISP‐Cas9, we rendered the myophosphorylase gene non‐functional to block the production of lactate and hydrogen ions. The exercise pressor reflex was evoked in decerebrated rats by statically contracting the triceps surae muscles with or without muscle ischaemia. Static contraction elevated the concentration of lactate and hydrogen ions in pygm+/+ but not in pygm−/− rats. Despite these differences, the exercise pressor reflex was not different between groups. Acute muscle ischaemia exaggerated the concentration of lactate and hydrogen ions in pygm+/+ but not in pygm−/− rats. Likewise, acute muscle ischaemia exaggerated the exercise pressor reflex in pygm+/+ but not in pygm−/− rats. We conclude that lactate and hydrogen play a key role in evoking the exercise pressor reflex during ischaemic but not during freely perfused contractions. [ABSTRACT FROM AUTHOR]

The stability of Earth's critical zone is intimately linked with erosion, weathering and vegetation type and density. Therefore, it affects global biogeochemical processes which in turn affect the global climate by absorbing and reflecting solar radiation, and by altering fluxes of heat, water vapour, carbon dioxide and other trace gases through various feedback mechanisms. However, there is a lack of knowledge about how Earth's critical zone processes have changed over time and their link with past monsoon variability, especially in Asia. The study of lake sediments, which contain a suite of inorganic elemental and isotopic proxies, may facilitate the understanding of the Earth's critical zone processes on millennial timescales. Here we reconstruct the history of erosion-weathering-vegetation interactions since ~14.7 ka using geochemical records from a radiocarbon-dated sediment core from Lake Gonghai in the monsoon-arid transitional zone of north China. Detrital (Al, Ti, K, Rb) and authigenic (Ca, Sr) elemental records reveal distinct, millennial-scale, late deglacial-Holocene erosion and weathering patterns and transitions with the former (latter) elements showing higher (lower) values in warm intervals and vice versa. Chemical Index of Alteration (CIA) molar, a humidity proxy, suggests low humidity during the late deglacial ~11.5-14.7 ka, high humidity during the early-mid Holocene ~11.5-3.2 ka and intermediate humidity during the late Holocene interval since ~3.2 ka. The results of cross-spectral analysis and comparison of our records with other climate reconstructions also suggest a pattern of orbitally-phased humidity changes in north China. Overall, our results provide evidence for the solar-forcing of Earth's surface processes in mid-latitude China under natural climatic conditions. [ABSTRACT FROM AUTHOR]

Key points Chronic limb ischaemia, characterized by inflammatory mediator release and a low extracellular pH, leads to acid-sensing ion channel (ASIC) activation and reflexively increases mean arterial pressure; endomorphin release is also increased under inflammatory conditions., We examined the modulation of ASIC currents by endomorphins in sensory neurons from rats with freely perfused and ligated femoral arteries: peripheral artery disease (PAD) model., Endomorphins potentiated sustained ASIC currents in both groups of dorsal root ganglion neurons, independent of mu opioid receptor stimulation or G protein activation., Intra-arterial administration of lactic acid (to simulate exercising muscle and evoke a pressor reflex), endomorphin-2 and naloxone resulted in a significantly greater pressor response than lactic acid alone, while administration of APETx2 inhibited endomorphin's enhancing effect in both groups., These results suggest a novel role for endomorphins in modulating ASIC function to effect lactic acid-mediated reflex increase in arterial pressure in patients with PAD., Abstract Chronic muscle ischaemia leads to accumulation of lactic acid and other inflammatory mediators with a subsequent drop in interstitial pH. Acid-sensing ion channels (ASICs), expressed in thin muscle afferents, sense the decrease in pH and evoke a pressor reflex known to increase mean arterial pressure. The naturally occurring endomorphins are also released by primary afferents under ischaemic conditions. We examined whether high affinity mu opioid receptor (MOR) agonists, endomorphin-1 (E-1) and -2 (E-2), modulate ASIC currents and the lactic acid-mediated pressor reflex. In rat dorsal root ganglion (DRG) neurons, exposure to E-2 in acidic solutions significantly potentiated ASIC currents when compared to acidic solutions alone. The potentiation was significantly greater in DRG neurons isolated from rats whose femoral arteries were ligated for 72 h. Sustained ASIC current potentiation was also observed in neurons pretreated with pertussis toxin, an uncoupler of G proteins and MOR. The endomorphin-mediated potentiation was a result of a leftward shift of the activation curve to higher pH values and a slight shift of the inactivation curve to lower pH values. Intra-arterial co-administration of lactic acid and E-2 led to a significantly greater pressor reflex than lactic acid alone in the presence of naloxone. Finally, E-2 effects were inhibited by pretreatment with the ASIC3 blocker APETx2 and enhanced by pretreatment with the ASIC1a blocker psalmotoxin-1. These findings have uncovered a novel role of endomorphins by which the opioids can enhance the lactic acid-mediated reflex increase in arterial pressure that is MOR stimulation-independent and APETx2-sensitive. [ABSTRACT FROM AUTHOR]

Key points Mechanical and metabolic stimuli from contracting muscles evoke reflex increases in blood pressure, heart rate and sympathetic nerve activity. Little is known, however, about the nature of the mechano-gated channels on the thin fibre muscle afferents that contribute to evoke this reflex, termed the exercise pressor reflex., We determined the effect of GsMTx4, an inhibitor of mechano-gated Piezo channels, on the exercise pressor reflex evoked by intermittent contraction of the triceps surae muscles in decerebrated, unanaesthetized rats., GsMTx4 reduced the pressor, cardioaccelerator and renal sympathetic nerve responses to intermittent contraction but did not reduce the pressor responses to femoral arterial injection of compounds that stimulate the metabolically-sensitive thin fibre muscle afferents., Expression levels of Piezo2 channels were greater than Piezo1 channels in rat dorsal root ganglia., Our findings suggest that mechanically-sensitive Piezo proteins contribute to the generation of the mechanical component of the exercise pressor reflex in rats., Abstract Mechanical and metabolic stimuli within contracting skeletal muscles evoke reflex autonomic and cardiovascular adjustments. In cats and rats, gadolinium has been used to investigate the role played by the mechanical component of this reflex, termed the exercise pressor reflex. Gadolinium, however, has poor selectivity for mechano-gated channels and exerts multiple off-target effects. We tested the hypothesis that GsMTX4, a more selective mechano-gated channel inhibitor than gadolinium and a particularly potent inhibitor of mechano-gated Piezo channels, reduced the exercise pressor reflex in decerebrate rats. Injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the peak pressor (control: 24 ± 5, GsMTx4: 12 ± 5 mmHg, P < 0.01), cardioaccelerator and renal sympathetic nerve responses to tendon stretch, a purely mechanical stimulus, but had no effect on the pressor responses to intra-arterial injection of α,β-methylene ATP or lactic acid. Moreover, injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the peak pressor (control: 24 ± 2, GsMTx4: 14 ± 3 mmHg, P < 0.01), cardioaccelerator and renal sympathetic nerve responses to electrically-induced intermittent hindlimb muscle contractions. By contrast, injection of 10 μg of GsMTx4 into the jugular vein had no effect on the pressor, cardioaccelerator, or renal sympathetic nerve responses to contraction. Quantitative RT-PCR and western blot analyses indicated that both Piezo1 and Piezo2 channel isoforms were natively expressed in rat dorsal root ganglia tissue. We conclude that GsMTx4 reduced the exercise pressor reflex in decerebrate rats and that the reduction was attributable, at least in part, to its effect on mechano-gated Piezo channels. [ABSTRACT FROM AUTHOR]

Key points In decerebrated rats, the exercise pressor reflex arising from a hindlimb whose femoral artery was occluded for 72 h was significantly higher than that arising from a hindlimb whose femoral artery was freely perfused., Blockade of endoperoxide 4 receptors, but not blockade of endoperoxide 3 receptors, prevented the exaggerated exercise pressor reflex in rats with ligated femoral arteries., Blockade of endoperoxide 3 or 4 receptors in rats with freely perfused femoral arteries had no effect on the exercise pressor reflex., Western immunoblots showed that ligation of the femoral artery for 72 h increased the endoperoxide 4 receptor protein in the L4 and L5 dorsal root ganglia over their freely perfused counterparts by 24% ( P < 0.05)., Abstract Ligating the femoral artery for 72 h in decerebrated rats exaggerates the exercise pressor reflex. The sensory arm of this reflex is comprised of group III and IV afferents, which can be either sensitized or stimulated by PGE2. In vitro studies showed that endoperoxide (EP) 3 and 4 receptors were responsible for the PGE2-induced sensitization of rat dorsal root ganglion cells. This in vitro finding prompted us to test the hypothesis that blockade of EP3 and/or EP4 receptors attenuated the exaggerated exercise pressor reflex in rats with ligated femoral arteries. We measured the cardiovascular responses to static hindlimb contraction or tendon stretch before and after femoral arterial injection of L798106 (an EP3 antagonist) or L161982 (an EP4 antagonist). The pressor and cardioaccelerator responses to either contraction or tendon stretch were not attenuated by L798106 in either the ligated or freely perfused rats. Likewise in five rats whose hindlimb muscles were freely perfused, the pressor and cardioaccelerator responses to either contraction or tendon stretch were not attenuated by L161982. In the six ligated rats, however, the pressor response to contraction was attenuated by L161982, averaging 37 ± 3 mmHg before, 18 ± 2 mmHg afterward ( P < 0.05). Western blotting analysis revealed that ligation of the femoral artery for 72 h increased the EP4 receptor protein in the L4 and L5 dorsal root ganglia over their freely perfused counterparts by 24% ( P < 0.05). We conclude that EP4 receptors, but not EP3 receptors, play an important role in the exaggerated exercise pressor reflex found in rats with ligated femoral arteries. [ABSTRACT FROM AUTHOR]

Key point Sympathetic stellate ganglion (SG) neurons that innervate cardiac muscle play an important role in regulating heart rate and contractility., In this study, we examined which Gβ protein subunit couples Ca2+ channels to opioid and adrenergic receptors expressed in SG neurons., We show that Gβ2 and Gβ4 are important signalling elements that maintain the transduction pathway of both receptor subtypes and Ca2+ channels, and how their expression levels are differentially compensated., Our results also indicate that Gβ1 subunits do not appear to play a role in either signalling pathway., The elucidation of specific G proteins that couple Ca2+ channels with opioid and adrenergic receptors will help us better understand how these receptors regulate synaptic transmission of SG neurons that innervate cardiac muscle., Abstract Cardiac function is regulated in part by the sympathetic branch of the autonomic nervous system via the stellate ganglion (SG) neurons. Neurotransmitters, such as noradrenaline (NA), and neuropeptides, including nociceptin (Noc), influence the excitability of SG neurons by modulating Ca2+ channel function following activation of the adrenergic and nociceptin/orphanin FQ peptide (NOP) opioid receptors, respectively. The regulation of Ca2+ channels is mediated by Gβγ, but the specific Gβ subunit that modulates the channels is not known. In the present study, small interference RNA (siRNA) was employed to silence the natively expressed Gβ proteins in rat SG tissue and to examine the coupling specificity of adrenergic and NOP opioid receptors to Ca2+ channels employing the whole-cell variant of the patch-clamp technique. Western blotting analysis showed that Gβ1, Gβ2 and Gβ4 are natively expressed. The knockdown of Gβ2 or Gβ4 led to a significant decrease of the NA- and Noc-mediated Ca2+ current inhibition, while Gβ1 silencing was without effect. However, sustaining low levels of Gβ2 resulted in an increased expression of Gβ4 and a concomitant compensation of both adrenergic and opioid signalling pathways modulating Ca2+ channels. Conversely, Gβ4-directed siRNA was not accompanied with a compensation of the signalling pathway. Finally, the combined silencing of Gβ2 and Gβ4 prevented any additional compensatory mechanisms. Overall, our studies suggest that in SG neurons, Gβ2 and Gβ4 normally maintain the coupling of Ca2+ channels with the receptors, with the latter subtype responsible for maintaining the integrity of both pathways. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi kinetoplastid membrane protein-11 (KMP-11) is able to induce protective immunity in mice. In humans, T-cell immunity during Chagas disease has been documented using parasite antigens allowing the identification of specific CD8+ T cells. However, little is known about the CD4+ T-cell response during the evolution of the disease. In this paper, the induction of a natural CD4+ T-cell response against the KMP-11 protein in T. cruzi infected humans was studied to assess whether this parasite-derived protein could be processed, presented and detected as a major histocompatibility complex class II restricted epitope. The results show that helper T cells from 5 out of 13 chagasic patients specifically produced interferon-γ after exposure to the KMP-11 antigen, whereas healthy donors and non-chagasic cardiopathic patients did not respond. This is the first description of T. cruzi KMP-11 protein recognition by CD4+ T cells in chronic chagasic patients.Immunology and Cell Biology (2009) 87, 149–153; doi:10.1038/icb.2008.76; published online 28 October 2008 [ABSTRACT FROM AUTHOR]

Key points We determined the role played by the transient receptor potential canonical 6 (TRPC6) channel in evoking the mechanical component of the exercise pressor reflex in male decerebrated Sprague–Dawley rats. TRPC6 channels were identified by quadruple‐labelled (DiI, TRPC6, neurofilament‐200 and peripherin) immunohistochemistry in dorsal root ganglion (DRG) cells innervating the triceps surae muscles (n = 12). The exercise pressor reflex was evoked by statically contracting the triceps surae muscles before and after injection of the TRPC6 antagonist BI‐749327 (n = 11; 12 μg kg−1) or SAR7334 (n = 11; 7 μg kg−1) or the TRPC6 positive modulator C20 (n = 11; 18 μg kg−1). Similar experiments were conducted while the muscles were passively stretched (n = 8–12), a manoeuvre that isolated the mechanical component of the reflex. Blood pressure, tension, renal sympathetic nerve activity (RSNA) and blood flow were recorded. Of the DRG cells innervating the triceps surae muscles, 85% stained positive for the TRPC6 antigen, and 45% of those cells co‐expressed neurofilament‐200. Both TRPC6 antagonists decreased the reflex pressor responses to static contraction (−32 to −42%; P < 0.05) and to passive stretch (−35 to −52%; P < 0.05), whereas C20 increased these responses (55–65%; P < 0.05). In addition, BI‐749327 decreased the peak and integrated RSNA responses to both static contraction (−39 to −43%; P < 0.05) and passive stretch (−56 to −62%; P < 0.05), whereas C20 increased the RSNA to passive stretch only. The onset latency of the decrease or increase in RSNA occurred within 2 s of the onset of the manoeuvres (P < 0.05). Collectively, our results show that TRPC6 plays a key role in evoking the mechanical component of the exercise pressor reflex. The exercise pressor reflex plays a key role in the sympathetic and haemodynamic responses to exercise. This reflex is composed of two components, namely the mechanoreflex and the metaboreflex. The receptors responsible for evoking the mechanoreflex are poorly documented. A good candidate for this function is the transient receptor potential canonical 6 (TRPC6) channel, which is activated by mechanical stimuli and expressed in dorsal root ganglia of rats. Using two TRPC6 antagonists and one positive modulator, we investigated the role played by TRPC6 in evoking the mechanoreflex in decerebrated rats. Blocking TRPC6 decreased the renal sympathetic and the pressor responses to both contraction and stretch, the latter being a manoeuvre that isolates the mechanoreflex. In contrast, the positive modulator increased the pressor reflex to contraction and stretch, in addition to the sympathetic response to stretch. Our results provide strong support for a role played by the TRPC6 channel in evoking the mechanoreflex. [ABSTRACT FROM AUTHOR]

Background: Abdominal pain is a frequent and persistent problem in the most common gastrointestinal disorders, including irritable bowel syndrome and inflammatory bowel disease. Pain adversely impacts quality of life, incurs significant healthcare expenditures, and remains a challenging issue to manage with few safe therapeutic options currently available. It is imperative that new methods are developed for identifying and treating this symptom. A variety of peripherally active neuroendocrine signaling elements have the capability to influence gastrointestinal pain perception. A large and growing body of evidence suggests that voltage‐gated sodium channels (VGSCs) play a critical role in the development and modulation of nociceptive signaling associated with the gut. Several VGSC isoforms demonstrate significant promise as potential targets for improved diagnosis and treatment of gut‐based disorders associated with hyper‐ and hyposensitivity to abdominal pain. Purpose: In this article, we critically review key investigations that have evaluated the potential role that VGSCs play in visceral nociception and discuss recent advances related to this topic. Specifically, we discuss the following: (a) what is known about the structure and basic function of VGSCs, (b) the role that each VGSC plays in gut nociception, particularly as it relates to human physiology, and (c) potential diagnostic and therapeutic uses of VGSCs to manage disorders associated with chronic abdominal pain. Voltage‐gated sodium channels (VGSCs) are critical for the transmission of nociceptive signals that are interpreted as pain, including from the gastrointestinal (GI) tract. Here, we review the basic function and pathophysiology of VGSCs in the context of gastrointestinal pain perception. [ABSTRACT FROM AUTHOR]