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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. [ABSTRACT FROM AUTHOR]

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 (H2PO4- ; 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. [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]

The contribution of low-frequency and damaging genetic variants associated with platelet function to ischemic stroke (IS) susceptibility remains unknown. We employed a deep re-sequencing approach in Polish patients in order to investigate the contribution of rare variants (minor allele frequency, MAF < 1%) to the IS genetic susceptibility in this population. The genes selected for re-sequencing consisted of 26 genes coding for proteins associated with the surface membrane of platelets. Targeted pooled re-sequencing (Illumina HiSeq 2500) was performed on genomic DNA of 500 cases (patients with history of clinically proven diagnosis of large-vessel IS) and 500 controls. After quality control and prioritization based on allele frequency and damaging probability, follow-up individual genotyping of deleterious rare variants was performed in patients from the original cohort. Gene-based analyses identified an association between IS and 6 rare functional and damaging variants in the purinergic genes (P2RY1 and P2RY12 locus). The predicted properties of the most damaging rare variants in P2RY1 and P2RY12 were confirmed by using mouse fibroblast cell cultures transfected with plasmid constructs containing cDNA of mutated variants (FLIPR on FlexStation3). This study identified a putative role for rare variants in P2RY1 and P2RY12 genes involved in platelet reactivity on large-vessel IS susceptibility in a Polish population. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

00974.2015.--Mechanical and metabolic stimuli arising from contracting muscles evoke the exercise pressor reflex. This reflex is greater in a rat model of simulated peripheral arterial disease in which a femoral artery is chronically ligated than it is in rats with freely perfused femoral arteries. The role played by the mechanically sensitive component of the exaggerated exercise pressor reflex in ligated rats is unknown. We tested the hypothesis that the mechano-gated channel inhibitor GsMTx4, a relatively selective inhibitor of mechano-gated Piezo channels, reduces the exercise pressor reflex in decerebrate rats with ligated femoral arteries. Injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the pressor response to Achilles 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 (purely metabolic stimuli). Moreover, injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced both the integrated pressor area (control 535 ± 21, GsMTx4 218 ± 24 mmHg·s; P < 0.01), peak pressor (control 29 ± 2, GsMTx4 14 ± 3 mmHg; P < 0.01), and renal sympathetic nerve responses to electrically induced intermittent hindlimb muscle contraction (a mixed mechanical and metabolic stimulus). The reduction of the integrated pressor area during contraction caused by GsMTx4 was greater in rats with ligated femoral arteries than it was in rats with freely perfused femoral arteries. We conclude that the mechanically sensitive component of the reflex contributes to the exaggerated exercise pressor reflex during intermittent hindlimb muscle contractions in rats with ligated femoral arteries. [ABSTRACT FROM AUTHOR]

The mechanisms by which G proteins modulate voltage-gated Ca2+ channel currents (CaV), particularly CaV2.2 and CaV2.3, are voltage dependent (VD) or voltage independent (VI). VD pathways are typically mediated by Gαi/o and GαS subfamilies. On the other hand, VI inhibition modulation is coupled to the Gαq subfamily and signaling pathways downstream of phospholipase C stimulation. In most studies, this latter pathway has been shown to be linked to Gαq and/or Gα11 protein subunits. However, there are no studies that have examined whether natively expressed Gα14 subunits (Gαq subfamily member) couple G protein-coupled receptors (GPCR) with CaV2.2 channels. We report that Gα14 subunits functionally couple the substance P (SP)/neurokinin-1 (NK-1) receptor pathway to CaV2.2 channels in acutely dissociated rat celiac-superior mesenteric ganglion (CSMG) neurons. Exposure of CSMG neurons to SP blocked the CaV2.2 currents in a predominantly VD manner that was pertussis toxin and cholera toxin resistant, as well as Gαq/11 independent. However, silencing Gα14 subunits significantly attenuated the SP-mediated Ca2+ current block. In another set of experiments, exposure of CSMG neurons to SP led to the inhibition of KCNQ K+ M-currents. The SP-mediated M-current block was significantly reduced in neurons transfected with Gα14 small-interference RNA. Finally, overexpression of the GTP-bound Gαq/11 binding protein RGS2 did not alter the block of M-currents by SP but significantly abolished the oxotremorine methiodide-mediated M-current inhibition. Taken together, these results provide evidence of a new Gα14-coupled signaling pathway that modulates CaV2.2 and M-currents via SP-stimulated NK-1 receptors in CSMG neurons. [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]

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. [ABSTRACT FROM AUTHOR]

Objective: Sepsis is a condition associated with a dysregulated inflammatory response to infection with significant morbidity. Recent advances have elucidated the vital role that the short chain fatty acid glycoprotein receptor 43 (FFA2/GPR43) plays in inflammatory and immunomodulatory pathways. We hypothesized that elevated whole blood GPR43 RNA expression would be associated with increased 30-day survival in patients admitted with sepsis. Patients (n = 93) admitted to the intensive care unit with the diagnosis of sepsis underwent quantitative real time PCR within 48 h of intensive care unit admission. Clinical and demographical parameters were retrospectively extracted from the chart and compared to quantitative measurements of GPR43 RNA expression. Results: Utilizing logistic regression, we found that the odds of mortality decreased for every one-unit increase in GPR43 RNA expression for patients that survived to 30 days [OR = 0.71; 95% CI (0.50, 0.99) p = 0.049]. Using linear regression, we determined that the increase in whole blood GPR43 expression was not associated with whole blood white cell count [r = 0.04; 95% CI (-0.16, 0.24); p = 0.70] or body mass index [r = - 0.07; 95% CI (- 0.23, 0.18); p = 0.81]. We conclude that the GPR43 receptor plays an integral role in survival during and after sepsis. [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]

Septic shock, the most severe complication associated with sepsis, is manifested by tissue hypoperfusion due, in part, to cardiovascular and autonomic dysfunction. In many cases, the splanchnic circulation becomes vasoplegic. The celiac-superior mesenteric ganglion (CSMG) sympathetic neurons provide the main autonomic input to these vessels. We used the cecal ligation puncture (CLP) model, which closely mimics the hemodynamic and metabolic disturbances observed in septic patients, to examine the properties and modulation of Ca2+ channels by G protein-coupled receptors in acutely dissociated rat CSMG neurons. Voltage-clamp studies 48 hr post-sepsis revealed that the Ca2+ current density in CMSG neurons from septic rats was significantly lower than those isolated from sham control rats. This reduction coincided with a significant increase in membrane surface area and a negligible increase in Ca2+ current amplitude. Possible explanations for these findings include either cell swelling or neurite outgrowth enhancement of CSMG neurons from septic rats. Additionally, a significant rightward shift of the concentration-response relationship for the norepinephrine (NE)-mediated Ca2+ current inhibition was observed in CSMG neurons from septic rats. Testing for the presence of opioid receptor subtypes in CSMG neurons, showed that mu opioid receptors were present in ~70% of CSMG, while NOP opioid receptors were found in all CSMG neurons tested. The pharmacological profile for both opioid receptor subtypes was not significantly affected by sepsis. Further, the Ca2+ current modulation by propionate, an agonist for the free fatty acid receptors GPR41 and GPR43, was not altered by sepsis. Overall, our findings suggest that CSMG function is affected by sepsis via changes in cell size and α2-adrenergic receptor-mediated Ca2+ channel modulation. [ABSTRACT FROM AUTHOR]

The exercise pressor reflex, a crucial component of the cardiovascular response under physiological and pathophysiological states, is activated via metabolic and mechanical mediators that originate from contracting muscles and stimulate group III and IV afferents. We reported previously that stimulation of mu opioid receptors (MOR), expressed in both afferents, led to a significant attenuation of the reflex in rats whose femoral arteries had been occluded for 72 h. The present study examined the effect of arterial occlusion on the signaling components involved in the opioid-mediated modulation of Ca2+ channels in rat dorsal root ganglion neurons innervating the triceps surae muscles. We focused on neurons that were transfected with cDNA coding for enhanced green fluorescent protein whose expression is driven by the voltagegated Na+ channel 1.8 (NaV1.8) promoter region, a channel expressed primarily in nociceptive neurons. With the use of a small interference RNA approach, our results show that the pertussis toxin-sensitive Gαi3 subunit couples MOR with Ca2+ channels. We observed a significant leftward shift of the MOR agonist [D-Ala2-N-Me-Phe4- Glycol5]-enkephalin concentration-response relationship in neurons isolated from rats with occluded arteries compared with those that were perfused freely. Femoral occlusion did not affect Ca2+ channel density or the fraction of the main Ca2+ channel subtype. Furthermore, Western blotting analysis indicated that the leftward shift did not result from either increased Gαi3 or MOR expression. Finally, all neurons from both groups exhibited an inward current following exposure of the transient potential receptor vanilloid 1 (TRPV1) agonist, 8-methyl-N-vanillyl-6-nonenamide. These findings suggest that sensory neurons mediating the exercise pressor reflex express NaV1.8 and TRPV1 channels, and femoral occlusion alters the MOR pharmacological profile. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]