Paradoxical Potentiation of the Exercise Pressor Reflex by Endomorphin 2 in the Presence of Naloxone.

When contracting muscles are freely perfused, the acid-sensing ion channel 3 (ASIC3) on group IV afferents plays a minor role in evoking the exercise pressor reflex. We recently showed in dorsal ganglion neurons innervating the gastrocnemius muscles that two mu opioid receptor agonists, endomorphin 2 and oxycodone, potentiated the sustained inward ASIC3 current evoked by acidic solutions. This finding prompted us to determine if endomorphin 2 and oxycodone, infused into the arterial supply of freely perfused muscles, potentiated the exercise pressor reflex. We found that infusion of endomorphin 2 and naloxone in decerebrated rats potentiated the pressor responses to contraction. The endomorphin 2-induced potentiation of the pressor responses to contraction were prevented by infusion of APETx2, an ASIC3 antagonist. Specifically, the peak pressor response to contraction averaged 19.3 ± 5.6 mmHg for control, 27.2 ± 8.1 mmHg after naloxone and endomorphin 2 infusion, and 20 ± 8 mmHg after APETx2 and endomorphin 2 infusion (n=10). Infusion of endomorphin and naloxone did not potentiate the pressor responses in ASIC3 knockout rats (n=6). Oxycodone infusion significantly increased the exercise pressor reflex over its control level, but subsequent APETx2 infusion failed to restore the increase to its control level. The peak pressor response averaged 23.1 ± 8.6 mmHg for control, 33.2 ± 11 mmHg after naloxone and oxycodone were infused, and 27 ± 8.6 mmHg after APETx2 and oxycodone were infused (n=9). Our data suggest that after opioid receptor blockade, ASIC3 stimulation by endomorphin 2 potentiated the exercise pressor reflex.

Inhibition and potentiation of the exercise pressor reflex by pharmacological modulation of TRPC6 in male rats.

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. KEY POINTS: 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.

Coexpressed δ-, µ-, and κ-Opioid Receptors Modulate Voltage-Gated Ca2+ Channels in Gastric-Projecting Vagal Afferent Neurons.

Opioid analgesics are frequently associated with gastrointestinal side effects, including constipation, nausea, dysphagia, and reduced gastric motility. Though it has been shown that stimulation of opioid receptors expressed in enteric motor neurons contributes to opioid-induced constipation, it remains unclear whether activation of opioid receptors in gastric-projecting nodose ganglia neurons contributes to the reduction in gastric motility and emptying associated with opioid use. In the present study, whole-cell patch-clamp recordings were performed to determine the mechanism underlying opioid receptor-mediated modulation of Ca2+ currents in acutely isolated gastric vagal afferent neurons. Our results demonstrate that CaV2.2 channels provide the majority (71% ± 16%) of Ca2+ currents in gastric vagal afferent neurons. Furthermore, we found that application of oxycodone, U-50488, or deltorphin II on gastric nodose ganglia neurons inhibited Ca2+ currents through a voltage-dependent mechanism by coupling to the Gα i/o family of heterotrimeric G-proteins. Because previous studies have demonstrated that the nodose ganglia expresses low levels of δ-opioid receptors, we also determined the deltorphin II concentration-response relationship and assessed deltorphin-mediated Ca2+ current inhibition following exposure to the δ-opioid receptor antagonist ICI 174,864 (0.3 µM). The peak mean Ca2+ current inhibition following deltorphin II application was 47% ± 24% (EC50 = 302.6 nM), and exposure to ICI 174,864 blocked deltorphin II-mediated Ca2+ current inhibition (4% ± 4% versus 37% ± 20%). Together, our results suggest that analgesics targeting any opioid receptor subtype can modulate gastric vagal circuits. SIGNIFICANCE STATEMENT: This study demonstrated that in gastric nodose ganglia neurons, agonists targeting all three classical opioid receptor subtypes (µ, δ, and κ) inhibit voltage-gated Ca2+ channels in a voltage-dependent mechanism by coupling to Gαi/o. These findings suggest that analgesics targeting any opioid receptor subtype would modulate gastric vagal circuits responsible for regulating gastric reflexes.

Heterologous expression of the human wild-type and variant NaV 1.8 (A1073V) in rat sensory neurons.

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 NaV 1.8 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 NaV 1.8 variant in rat sensory neurons would exhibit different biophysical characteristics than previously observed in sympathetic neurons. METHODS: Endogenous NaV 1.8 channels were first silenced in DRG neurons and then either 1073A or 1073V human NaV 1.8 cDNA constructs were transfected. NaV 1.8 currents were recorded with the whole-cell patch-clamp technique. KEY RESULTS: The results indicate that 1073A and 1073V NaV 1.8 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.

Functional knockout of the TRPV1 channel has no effect on the exercise pressor reflex in rats.

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.

Dissimilar effects of stereoisomers and racemic hydroxychloroquine on Ca2+ oscillations in human induced pluripotent stem cell-derived cardiomyocytes.

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.

A 10-mg dose of amiloride increases time to failure during blood-flow-restricted plantar flexion in healthy adults without influencing blood pressure.

Amiloride has been shown to inhibit acid-sensing ion channels (ASICs), which contribute to ischemia-related muscle pain during exercise. The purpose of this study was to determine if a single oral dose of amiloride would improve exercise tolerance and attenuate blood pressure during blood-flow-restricted (BFR) exercise in healthy adults. Ten subjects (4 females) performed isometric plantar flexion exercise with BFR (30% maximal voluntary contraction) after ingesting either a 10-mg dose of amiloride or a volume-matched placebo (random order). Time to failure, time-tension index (TTI), and perceived pain (visual analog scale) were compared between the amiloride and placebo trials. Mean blood pressure, heart rate, blood pressure index (BPI), and BPI normalized to TTI (BPInorm) were also compared between trials using both time-matched (TM50 and TM100) and effort-matched (T50 and T100) comparisons. Time to failure (+69.4 ± 63.2 s, P < 0.01) and TTI (+1,441 ± 633 kg·s, P = 0.02) were both significantly increased in the amiloride trial compared with placebo, despite no increase in pain (+0.4 ± 1.7 cm, P = 0.46). In contrast, amiloride had no significant influence on the mean blood pressure or heart rate responses, nor were there any significant differences in BPI or BPInorm between trials when matched for time (all P ≥ 0.13). When matched for effort, BPI was significantly greater in the amiloride trial (+5,300 ± 1,798 mmHg·s, P = 0.01), likely owing to an increase in total exercise duration. In conclusion, a 10-mg oral dose of amiloride appears to significantly improve the tolerance to BFR exercise in healthy adults without influencing blood pressure responses.

In Vivo Transfection of Rat Salivary Glands With Fluorescently Tagged Aquaporin-5 Channel DNA.

Background  The acinar cells of salivary glands are responsible for most saliva production and are, unfortunately, h--ighly radiosensitive. As such, dry mouth or xerostomia is an adverse effect experienced by half of head and neck cancer patients treated with radiation. We evaluate a novel method of gene transfection of aquaporin channels to rat salivary glands. Materials and methods A green fluorescent protein (GFP)-tagged human Aquaporin-5 (AQP5) cDNA sequence cloned into a pCMV6-AC-GFP vector was complexed with lipofectamine 2000. One submandibular gland of the anesthetized rats was injected with the complexed cDNA and lipid solution under ultrasound guidance, while the opposite gland was injected with the vehicle control. The animals were sacrificed between 24 to 48 hours post-injection. The salivary glands were removed and evaluated via fluorescence imaging. Western blot assays were also performed to determine AQP5 cDNA expression. Results  In the experiments, the submandibular glands were identified and injected under ultrasound guidance. Four control glands and eight experimental glands were evaluated. The cDNA was expressed successfully and variably within the experimental glands, noting greater intensity along the cell surface consistent with appropriate trafficking of the AQP5 channel. Western blot analysis demonstrated variable expression in the experimental sample with no expression in the control sample. Several glands across the groups showed mild to moderate interstitial edema or inflammation. Conclusion  In this study, we demonstrate an alternative in vivo transfection method via lipofection and demonstrate the successful expression of the AQP5 channel in rat salivary gland tissue.

Resistin production does not affect outcomes in a mouse model of acute surgical sepsis.

INTRODUCTION: Because of the strong correlation between the blood concentration of circulating resistin and the illness severity of septic patients, resistin has been proposed as a mediator of sepsis pathophysiology. In vitro data indicate that human resistin directly impairs neutrophil migration and intracellular bacterial killing, although the significance of these findings in vivo remain unclear. OBJECTIVE: The objectives of the present study were: (1) to validate the expression of human resistin in a clinically relevant, murine model of surgical sepsis, (2) to assess how sepsis-induced changes in resistin correlate with markers of infection and organ dysfunction, and (3) to investigate whether the expression of human resistin alters immune function or disease outcomes in vivo. METHODS: 107 male, C57BL/6 mice transgenic for the human resistin gene and its promoter elements (Retn+/-/-, or Retn+) were generated on a Retn-/- (mouse resistin knockout, or Rko) background. Outcomes were compared between age-matched transgenic and knockout mice. Acute sepsis was defined as the initial 24 h following cecal ligation and puncture (CLP). Physiologic and laboratory parameters correlating to the human Sequential Organ Failure Assessment (SOFA) Score were measured in mice, and innate immune cell number/function in the blood and peritoneal cavity were assessed. RESULTS: CLP significantly increased circulating levels of human resistin. The severity of sepsis-induced leukopenia was comparable between Retn+ and Rko mice. Resistin was associated with increased production of neutrophil reactive oxygen species, a decrease in circulating neutrophils at 6 h and an increase in peritoneal Ly6Chi monocytes at 6 h and 24 h post-sepsis. However, intraperitoneal bacterial growth, organ dysfunction and mouse survival did not differ with resistin production in septic mice. SIGNIFICANCE: Ex vivo resistin-induced impairment of neutrophil function do not appear to translate to increased sepsis severity or poorer outcomes in vivo following CLP.

Serotonin-Mediated Activation of Serotonin Receptor Type 1 Oppositely Modulates Voltage-Gated Calcium Channel Currents in Rat Sensory Neurons Innervating Hindlimb Muscle.

Serotonin (5-HT) is a multifaceted neurotransmitter that has been described to play a role as a peripheral inflammatory mediator when released in ischemic or injured muscle. Dorsal root ganglia (DRG) neurons are key sensors of noxious stimuli that are released under inflammatory conditions or mechanical stress. Little information is available on the specific 5-HT receptor subtypes expressed in primary afferents that help regulate reflex pressor responses. In the present study, the whole-cell patch-clamp technique was employed to examine the modulation of voltage-gated calcium channel (CaV) 2.2 currents by 5-HT and to identify the 5-HT receptor subtype(s) mediating this response in acutely dissociated rat DRG neurons innervating triceps surae muscle. Our results indicate that exposure of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled DRG neurons to 5-HT can exert three modulatory effects on CaV currents: high inhibition, low inhibition, and enhancement. Both 5-HT-mediated inhibition responses were blocked after pretreatment with pertussis toxin (PTX), indicating that 5-HT receptors are coupled to CaV2.2 via Gα i/o protein subunits. Application of selective serotonin receptor type 1 (5-HT1) agonists revealed that modulation of CaV2.2 currents occurs primarily after 5-HT1A receptor subtype stimulation and minimally from 5-HT1D activation. Finally, the intrathecal administration of the selective 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), significantly (P < 0.05) decreased the pressor response induced by intra-arterial administration of lactic acid. This suggests that 5-HT1A receptors are expressed presynaptically on primary afferent neurons innervating triceps surae muscle. Our findings indicate that preferential stimulation of 5-HT1 receptors, expressed on thin fiber muscle afferents, serves to regulate the reflex pressor response to metabolic stimuli. SIGNIFICANCE STATEMENT: The monoamine serotonin (5-HT), released under ischemic conditions, can contribute to the development of inflammation that negatively affects the exercise pressor reflex. The 5-HT receptor subtype and signaling pathway that underlies calcium channel modulation in dorsal root ganglia afferents, innervating hindlimb muscles, are unknown. We show that 5-HT can either block (primarily via serotonin receptor type 1 (5-HT1)A subtypes) or enhance voltage-gated calcium channel (CaV2.2) currents. Our findings suggest 5-HT exhibits receptor subtype selectivity, providing a complexity of cellular responses.

Spinal cord injury-mediated changes in electrophysiological properties of rat gastric nodose ganglion neurons.

In preclinical rodent models, spinal cord injury (SCI) manifests as gastric vagal afferent dysfunction both acutely and chronically. However, the mechanism that underlies this dysfunction remains unknown. In the current study, we examined the effect of SCI on gastric nodose ganglia (NG) neuron excitability and on voltage-gated Na+ (NaV) channels expression and function in rats after an acute (i.e. 3-days) and chronic (i.e. 3-weeks) period. Rats randomly received either T3-SCI or sham control surgery 3-days or 3-weeks prior to experimentation as well as injections of 3% DiI solution into the stomach to identify gastric NG neurons. Single cell qRT-PCR was performed on acutely dissociated DiI-labeled NG neurons to measure NaV1.7, NaV1.8 and NaV1.9 expression levels. The results indicate that all 3 channel subtypes decreased. Current- and voltage-clamp whole-cell patch-clamp recordings were performed on acutely dissociated DiI-labeled NG neurons to measure active and passive properties of C- and A-fibers as well as the biophysical characteristics of NaV1.8 channels in gastric NG neurons. Acute and chronic SCI did not demonstrate deleterious effects on either passive properties of dissociated gastric NG neurons or biophysical properties of NaV1.8. These findings suggest that although NaV gene expression levels change following SCI, NaV1.8 function is not altered. The disruption throughout the entirety of the vagal afferent neuron has yet to be investigated.

Lipid-Encapsulated Silica Nanobowls as an Efficient and Versatile DNA Delivery System.

Nonmesoporous Janus silica nanobowls (NBs) are unique in that they possess two different nonporous surfaces per particle for loading biological molecules and can thus be designed with multifunctional properties. Although silica NBs have been successfully employed for both targeted therapeutic and diagnostic applications, their ability to deliver DNA has not yet been fully explored. The purpose of this study was to design and develop an in vitro transfection agent that would exploit the distinct characteristics of the silica NB. First, we determined that the NB surface can be linked to either supercoiled cDNA plasmids or vectorless, linear cDNA constructs. Additionally, the linearized cDNA can be functionalized and chemisorbed on NBs to obtain a controlled release. Second, the successful transfection of cells studied was dependent on lipid coating of the NB (LNBs). Although both NBs and LNBs were capable of undergoing endocytosis, NBs appeared to remain within vesicles as shown by transmission electron microscopy (TEM). Third, fluorescence microscopy and Western blotting assays revealed that transfection of four different cell lines and acutely isolated rat sensory neurons with LNBs loaded with either linear or supercoiled cDNA constructs coding for the fluorescent protein, clover and tdTomato, resulted in protein expression. Fourth, two separate opioid receptor-ion channel signaling pathways were functionally reconstituted in HEK cells transfected with LNBs loaded with three separate cDNA constructs. Overall, these results lay the foundation for the use and further development of LNBs as in vitro transfection agents.

Impact of the NaV1.8 variant, A1073V, on post-sigmoidectomy pain and electrophysiological function in rat sympathetic neurons.

NaV1.8 channels play a crucial role in regulating the action potential in nociceptive neurons. A single nucleotide polymorphism in the human NaV1.8 gene SCN10A, A1073V (rs6795970, G>A), has been linked to the diminution of mechanical pain sensation as well as cardiac conduction abnormalities. Furthermore, studies have suggested that this polymorphism may result in a "loss-of-function" phenotype. In the present study, we performed genomic analysis of A1073V polymorphism presence in a cohort of patients undergoing sigmoid colectomy who provided information regarding perioperative pain and analgesic use. Homozygous carriers reported significantly reduced severity in postoperative abdominal pain compared with heterozygous and wild-type carriers. Homozygotes also trended toward using less analgesic/opiates during the postoperative period. We also heterologously expressed the wild-type and A1073V variant in rat superior cervical ganglion neurons. Electrophysiological testing demonstrated that the mutant NaV1.8 channels activated at more depolarized potentials compared with wild-type channels. Our study revealed that postoperative abdominal pain is diminished in homozygous carriers of A1073V and that this is likely due to reduced transmission of action potentials in nociceptive neurons. Our findings reinforce the importance of NaV1.8 and the A1073V polymorphism to pain perception. This information could be used to develop new predictive tools to optimize patient pain experience and analgesic use in the perioperative setting.NEW & NOTEWORTHY We present evidence that in a cohort of patients undergoing sigmoid colectomy, those homozygous for the NaV1.8 polymorphism (rs6795970) reported significantly lower abdominal pain scores than individuals with the homozygous wild-type or heterozygous genotype. In vitro electrophysiological recordings also suggest that the mutant NaV1.8 channel activates at more depolarizing potentials than the wild-type Na+ channel, characteristic of hypoactivity. This is the first report linking the rs6795970 mutation with postoperative abdominal pain in humans.

Sporadic Erythromelalgia Associated with a Homozygous Carrier of Common Missense Polymorphism in SCN9A Gene Coding for NaV1.7 Voltage-gated Sodium Channel.

A 68-year-old female with a history of sporadic type and presumably secondary erythromelalgia with chronic intractable pain presented for foot surgery. The procedure was performed with combined general anesthesia and regional anesthesia consisting of the placement of a popliteal pain catheter for postoperative pain management. Subsequent whole-genome sequencing revealed that the patient was a homozygous carrier of the common missense mutation in the SCN9A gene coding for voltage-gated sodium channel (NaV1.7) - dbSNP rs6746030 (R1150W). The occurrence of this single nucleotide polymorphism (SNP) was previously suggested not to be associated with erythromelalgia but rather thought to be part of quantitative changes in the pain threshold in different cohorts of patients. The placement of the pain catheter, although controversial in patients with erythromelalgia, provided effective postoperative pain relief without any side effects.

Effect of knockout of the ASIC3 on cardiovascular reflexes arising from hindlimb muscle in decerebrated rats.

The exercise pressor reflex is initiated by the contraction-induced activation of group III and IV muscle afferents. The reflex is manifested by increases in arterial blood pressure and cardiac output, which, in turn, are generated by increases in the sympathetic outflow to the heart and vasculature and decreases in the vagal outflow to the heart. In previous experiments, we used a pharmacological approach to assess the role played by the acid-sensing ion channel 3 (ASIC3) on group III and IV afferents in evoking the exercise pressor reflex. In the present experiments, we used an alternative approach, namely functional knockout (KO) of the ASIC3 gene, to confirm and extend our previous finding that pharmacological blockade of the ASIC3 had only a small impact on the expression of the exercise pressor reflex when the arterial supply to the contracting hindlimb muscles of rats was patent. Using this alternative approach, we compared the magnitude of the exercise pressor reflex evoked in ASIC3 KO rats with that evoked in their wild-type (WT) counterparts. We found both WT and ASIC3 KO rats displayed similar pressor responses to static contraction (WT, n = 10, +12 ± 2 mmHg; KO, n = 9, +11 ± 2 mmHg) and calcaneal tendon stretch (WT, n = 9, +13 ± 2 mmHg; KO, n = 7, +11 ± 2 mmHg). Likewise, both WT and ASIC3 KO displayed similar pressor responses to intra-arterial injection of 12 mM lactic acid (WT, n = 9, +14 ± 3 mmHg; KO, n = 8, +18 ± 5 mmHg), 24 mM lactic acid (WT, n = 9,+24 ± 2 mmHg; KO, n = 8, +20 ± 5 mmHg), capsaicin (WT, n = 9,+27 ± 5 mmHg; KO, n = 10, +29 ± 5 mmHg), and diprotonated phosphate ([Formula: see text]; WT, n = 6,+22 ± 3 mmHg; KO, n = 6, +32 ± 6 mmHg). We conclude that redundant receptors are responsible for evoking the pressor reflexes arising from group III and IV afferents.

Resistin directly inhibits bacterial killing in neutrophils.

BACKGROUND: Sepsis-induced immunosuppression is a key factor contributing to the morbidity and mortality of critically ill patients, and polymorphonuclear neutrophil dysfunction is believed to be a hallmark of this immunosuppression. Circulating myeloid cells produce the cytokine resistin (RETN), which has been associated with poor outcomes in sepsis/septic shock and can directly inhibit neutrophil function. We previously demonstrated that resistin caused a dose-dependent impairment in neutrophil migration, reactive oxygen species production, and bacterial clearance in neutrophil cell lines. However, the relative antimicrobial responses of other innate immune cells to Gram-positive and Gram-negative infections in the presence of elevated levels of resistin have not been evaluated. We hypothesized that resistin directly contributes to sepsis-induced immunosuppression by selectively targeting the neutrophil component of the innate cellular immune system. Thus, the goal of the present study was to compare the effect of resistin on bacterial killing using monocultures or co-cultures of monocyte and neutrophil cell lines, as well as to extend our findings to primary immune cells. RESULTS: Our results indicate that human resistin impairs the ability of neutrophils to kill the Gram-negative bacterium Pseudomonas aeruginosa and the Gram-positive bacterium Staphylococcus aureus. In contrast, with the exception of macrophages incubated with P. aeruginosa, resistin did not affect the ability of macrophages or monocytes to kill either Gram-positive or Gram-negative organisms. Furthermore, co-incubation of neutrophils with increasing proportions of monocytes did not enhance bacterial killing. Resistin blocked bactericidal activity through partial reduction of F-actin polymerization and suppression of the oxidative burst in neutrophils. CONCLUSIONS: Our studies indicate that resistin selectively impairs neutrophil bacterial killing. These findings further support the notion that resistin can mimic cell type-dependent immunosuppressive effects. This is consistent with its putative role in the pathogenesis of bacterial sepsis.

Purinergic receptor expression and function in rat vagal sensory neurons innervating the stomach.

The nodose ganglion (NG) is the main parasympathetic ganglion conveying sensory signals to the CNS from numerous visceral organs including digestive signals such as gastric distension or the release the gastrointestinal peptides. The response characteristics of NG neurons to ATP and ADP and pharmacological interrogation of purinergic receptor subtypes have been previously investigated but often in NG cells of undetermined visceral origin. In this study, we confirmed the presence of P2X3 and P2Y1 receptors and characterized P2X and P2Y responses in gastric-innervating NG neurons. Application of ATP-evoked large inward currents and cytosolic Ca2+ increases in gastric-innervating NG neurons. Despite the expression of P2Y1 receptors, ADP elicited only minor modulation of voltage-gated Ca2+ channels. Considering the sensitivity of NG neurons to comorbidities associated with disease or neural injury, purinergic modulation of gastric NG neurons in disease- or injury-states is worthy of further investigation.

In a Model of Neuroinflammation Designed to Mimic Delirium, Quetiapine Reduces Cortisol Secretion and Preserves Reversal Learning in the Attentional Set Shifting Task.

Quetiapine, an atypical antipsychotic medication has lacked pre-clinical validation for its purported benefits in the treatment of delirium. This laboratory investigation examined the effects of quetiapine on the attentional set shifting task (ASST), a measure of cognitive flexibility and executive functioning, in a rodent model of lipopolysaccharide (LPS) mediated neuroinflammation. 19 Sprague Dawley female rats were randomly selected to receive intraperitoneal placebo (N = 5), LPS and placebo (N = 7) or LPS and quetiapine (n = 7) and performed the ASST. We measured trials to criterion, errors, non-locomotion episodes and latency to criterion, serum cortisol and tumor necrosis factor alpha (TNF-α) levels. TNF-α levels were not different between groups at 24 h. Cortisol levels in the LPS + Quetiapine group were reduced compared to LPS + Placebo (P < 0.001) and did not differ from the placebo group (P = 0.15). Analysis between LPS + Quetiapine and LPS + Placebo treated rats demonstrated improvement in the compound discrimination reversal (CD Rev1) (P = 0.016) and the intra-dimensional reversal (ID Rev2) (P = 0.007) discriminations on trials to criterion. LPS + Quetiapine treated rats had fewer errors than LPS + Placebo treated animals in the compound discrimination (CD) (P = 0.007), CD Rev1 (P = 0.005), ID Rev2 (P < 0.001) discriminations. There was no difference in non-locomotion frequency or latency to criterion between the three groups in all discriminations (P > 0.0167). We demonstrated preserved reversal learning, no effect on attentional set shifting and normalized cortisol levels in quetiapine-treated rats in this neuroinflammatory model of delirium. This suggests that quetiapine's beneficial effects in delirium may be related to the preservation of reversal learning and potential downstream effects related to reduction in cortisol production. Graphical Abstract.

Increased burden of rare deleterious variants of the KCNQ1 gene in patients with large­vessel ischemic stroke.

The impact of rare and damaging variants in genes associated with platelet function in large­vessel ischemic stroke (LVIS) remains unknown. The aim of this study was to investigate the contribution of some of these variants to the genetic susceptibility to LVIS in Polish patients using a deep re­sequencing of 54 selected genes, coding for proteins associated with altered platelet function. Targeted pooled re­sequencing (Illumina HiSeq 2500) was performed on genomic DNA of 500 cases (patients with history of clinically proven diagnosis of LVIS) and 500 age­, smoking status­, and sex­matched controls (no history of any type of stroke), and from the same population as patients with LVIS. After quality control and prioritization based on allele frequency and damaging probability, individual genotyping of all deleterious rare variants was performed in patients from the original cohort, and stratified to concomitant cardiac conditions differing between the study and stroke groups. We demonstrated a statistically significant increase in the number of rare and potentially damaging variants in some of the investigated genes in the LVIS pool (an increase in the genomic variants burden). Furthermore, we identified an association between LVIS and 6 rare functional and damaging variants in the Kv7.1 potassium channel gene (KCNQ1). The predicted functional properties (partial loss­of function) for the three most damaging variants in KCNQ1 coding locus were further confirmed in vitro by analyzing the membrane potential changes in cell lines co­transfected heterogeneously with human muscarinic type 1 receptor and wild­type or mutated KCNQ1 cDNA constructs using fluorescence imaging plate reader. The study demonstrated an increased rare variants burden for 54 genes associated with platelet function, and identified a putative role for rare damaging variants in the KCNQ1 gene on LVIS susceptibility in the Polish population.

Opioid-Mediated Modulation of Acid-Sensing Ion Channel Currents in Adult Rat Sensory Neurons.

Muscle ischemia, associated with peripheral artery disease (PAD), leads to the release of proinflammatory mediators that decrease extracellular pH and trigger the activation of proton-activated acid-sensing ion channels (ASIC). Claudication pain, linked with low blood flow, can be partially relieved by endogenous opioid peptide release. However, we previously reported that sustained ASIC currents in dorsal root ganglion (DRG) neurons were enhanced by naturally occurring endomorphin-1 and -2 opioid peptides, indicating a role of opioid involvement in hyperalgesia. The present study examined whether clinically employed synthetic (fentanyl, remifentanil) and the semisynthetic opioid (oxycodone) would also potentiate sustained ASIC currents, which arise from ASIC3 channel isoforms. Here, we show that exposure of each opioid to DRG neurons resulted in potentiation of the sustained ASIC currents. On the other hand, the potentiation was not observed in DRG neurons from ASIC3 knockout rats. Further, the enhancement of the ASIC currents was resistant to pertussis toxin treatment, suggesting that Gα i/Gα o G-proteins are not involved. Additionally, the potentiation of sustained ASIC currents was greater in DRG neurons isolated from rats with ligated femoral arteries (a model of PAD). The effect of all three opioids on the transient ASIC peak current was mixed (increase, decrease, no effect). The inhibitory action appears to be mediated by the presence of ASIC1 isoform, while the potentiating effect is primarily due to ASIC3 isoform expression. These findings reveal that, under certain conditions, these three opioids can increase ASIC channel activity, possibly giving rise to opioid-induced hyperalgesia.