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Pre-clinical models indicate that Amiloride (AMD) reduces baroreflex sensitivity and perturbs homeostatic blood pressure (BP) regulation. However, it remains unclear whether these findings translate to humans. This study investigated whether oral administration of AMD reduces spontaneous cardiac and sympathetic baroreflex sensitivity and perturbs BP regulation in healthy young humans. Heart rate (HR; electrocardiography), beat-to-beat BP (photoplethysmography), and muscle sympathetic activity (MSNA, microneurography) were continuously measured in 10 young subjects (4 females) during rest across two randomized experimental visits: (1) after 3 hours of oral administration of placebo (PLA - 10 mg of methylcellulose within a gelatin capsule) and (2) after 3 hours of oral administration of AMD (10 mg). Visits were separated for at least 48 hours. We calculated the standard deviation and other indices of BP variability. Spontaneous cardiac baroreflex was assessed via the sequence technique and cardiac autonomic modulation through time- and frequency-domain HR variability. The sensitivity (gain) of the sympathetic baroreflex was determined via weighted linear regression analysis between MSNA and diastolic BP. AMD did not affect HR, BP, and MSNA compared to PLA. Indexes of cardiac autonomic modulation (time- and frequency-domain HR variability) and BP variability were also unchanged after AMD ingestion. Likewise, AMD did not modify the gain of both spontaneous cardiac and sympathetic arterial baroreflex. A single oral dose of AMD does not affect spontaneous arterial baroreflex sensitivity and BP variability in healthy young adults.
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The orexigenic gut peptide ghrelin is an endogenous ligand for the growth hormone secretagogue receptor type 1a (GHSR1a). Systemic ghrelin administration has previously been shown to increase gastric motility and emptying. While these effects are known to be mediated by the vagus nerve, the cellular mechanism underlying these effects remains unclear. Therefore, the purpose of the present study was to investigate the signaling mechanism by which GHSR1a inhibits voltage-gated Ca2+ channels in isolated rat gastric vagal afferent neurons using whole-cell patch-clamp electrophysiology. The ghrelin pharmacological profile indicated that Ca2+ currents were inhibited with a log (Ic50)=-2.10 {plus minus} 0.44 and a maximal inhibition of 42.8 {plus minus} 5.0%. Exposure to the GHSR1a receptor antagonist (D-Lys3)-GHRP-6 reduced ghrelin-mediated Ca2+ channel inhibition (29.4 {plus minus} 16.7% vs 1.9 {plus minus} 2.5%, n=6, p=0.0064). Interestingly, we observed that activation of GHSR1a inhibited Ca2+ currents through both voltage-dependent and voltage-independent pathways. We also treated the gastric neurons with either pertussis toxin (PTX) or YM-254890 to examine whether the Ca2+ current inhibition was mediated by Gαi/o or Gαq/11 family of subunits. Treatment with both PTX (Ca2+ current inhibition=15.7 {plus minus} 10.6%, n=8, p=0.0327) and YM-254890 (15.2 {plus minus} 11.9%, n=8, p=0.0269) blocked ghrelin's effects on Ca2+ currents, as compared to control neurons (34.3 {plus minus} 18.9%, n=8). These results indicate GHSR1a can couple to both Gαi/o and Gαq/11 in gastric vagal afferent neurons. Overall, our findings suggest GHSR1a-mediated inhibition of Ca2+ currents occurs through two distinct pathways, offering necessary insights into the cellular mechanisms underlying ghrelin's regulation of gastric vagal afferents. Significance Statement This study demonstrated that in gastric vagal afferent neurons, activation of GHSR1a by ghrelin inhibits voltage-gated Ca2+ channels through both voltage-dependent and voltage-independent signaling pathways. These results provide necessary insight into the cellular mechanism underlying ghrelin regulation of gastric vagal afferent activity, which may benefit future studies investigating ghrelin mimetics to treat gastric motility disorders.
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Pain is a major issue in healthcare throughout the world. It remains one of the major clinical issues of our time because it is a common sequela of numerous conditions, has a tremendous impact on individual quality of life, and is one of the top drivers of cost in medicine, due to its influence on healthcare expenditures and lost productivity in those affected by it. Patients and healthcare providers remain desperate to find new, safer and more effective analgesics. Growing evidence indicates that the voltage-gated sodium channel Nav1.8 plays a critical role in transmission of pain-related signals throughout the body. For that reason, this channel appears to have strong potential to help develop novel, more selective, safer, and efficacious analgesics. However, many questions related to the physiology, function, and clinical utility of Nav1.8 remain to be answered. In this article, we discuss the latest studies evaluating the role of Nav1.8 in pain, with a particular focus on visceral pain, as well as the steps taken thus far to evaluate its potential as an analgesic target. We also review the limitations of currently available studies related to this topic, and describe the next scientific steps that have already been undertaken, or that will need to be pursued, to fully unlock the capabilities of this potential therapeutic target.
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INTRODUCTION: The pain associated with lower extremity arterial disease is difficult to treat, even with lower extremity revascularization. We sought to evaluate in-hospital and post-operative opioid usage in patients with different disease severities and treatments for lower extremity vascular disease. METHODS: A retrospective review was performed for all hospital encounters for patients with an International Classification of Diseases (ICD) code consistent with lower extremity arterial disease admitted to a single center between January 2018 and March 2023. Cases included patients admitted to the hospital with a primary diagnosis of lower extremity arterial disease. These patients were subdivided based on disease severity, treatment type, and comorbid diagnosis of diabetes mellitus. The analysis focused on in-hospital opioid use frequency and dosage among these patients. The control group (CON) included encounters for patients admitted with a secondary diagnosis of lower extremity atherosclerotic disease. A total of 438 patients represented by all the analyzed encounters were then reviewed for the number and type of vascular procedures performed as well as opioid use in the outpatient setting for one year. RESULTS: Critical limb ischemia (CLI) encounters were more likely to use opioids as compared to the CON and peripheral arterial disease (PAD) without rest pain, ulcer or gangrene groups (CLI 67.9% (95% CI: 63.6%-71.6%) versus CON 52.1% (95% CI: 48.5%-55.7%), p < 0.001 and CLI 67.9% (95% CI: 63.6%-71.6%) versus PAD 50.2% (95% CI: 42.6%-57.4%), p < 0.001). Opioid use was also more common in encounters for gangrene and groups treated with revascularization (REVASC) and amputation (AMP) as compared to CON (gangrene 74.5% (95% CI: 68.5%-82.1%) versus CON 52.1% (95% CI: 48.5%-55.7%), p < 0.01; REVASC 58.3% (95% CI: 57.3%-66.4%) versus CON 52.1% (95% CI: 48.5%-55.7%), p =0.01; and AMP 72.3% (95% CI: 62.1%-74.0%) versus CON 52.1% (95% CI: 48.5%-55.7%), p < 0.01). Significantly increased oral opioid doses per day (MME/day) were not noted for any of the investigated groups as compared to the CON. In the outpatient setting, 186 (42.5% (95% CI: 37.2%-46.4%)) patients were using opioids one month after the most recent vascular intervention. By one year, 31 (7.1% (95% CI: 1.30%-7.70%)) patients were still using opioids. No differences in opioid usage were noted for patients undergoing single versus multiple vascular interventions at one year. Patients undergoing certain vascular surgery procedures were more likely to be using opioids at one year. CONCLUSION: Patients with CLI and gangrene as well as those undergoing vascular treatment have a greater frequency of opioid use during hospital encounters as compared to those patients with less severe disease and undergoing conservative management, respectively. However, these findings do not equate to higher doses of opioids used during hospitalization. Patients undergoing multiple vascular procedures are not more likely to be using opioids long-term (at one year) as compared to those patients treated with single vascular procedures.
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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. KEY POINTS: 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.
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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 isolated dorsal root ganglion neurons innervating the gastrocnemius muscles that two mu opioid receptor agonists, namely endomorphin 2 and oxycodone, potentiated the sustained inward ASIC3 current evoked by acidic solutions. This in vitro finding prompted us to determine whether endomorphin 2 and oxycodone, when infused into the arterial supply of freely perfused contracting hindlimb muscles, potentiated the exercise pressor reflex. We found that infusion of endomorphin 2 and naloxone in decerebrated rats potentiated the pressor responses to contraction of the triceps surae muscles. The endomorphin 2-induced potentiation of the pressor responses to contraction was prevented by infusion of APETx2, an ASIC3 antagonist. Specifically, the peak pressor response to contraction averaged 19.3 ± 5.6 mmHg for control (n = 10), 27.2 ± 8.1 mmHg after naloxone and endomorphin 2 infusion (n = 10), and 20 ± 8 mmHg after APETx2 and endomorphin 2 infusion (n = 10). Infusion of endomorphin 2 and naloxone did not potentiate the pressor responses to contraction in ASIC3 knockout rats (n = 6). Partly similar findings were observed when oxycodone was substituted for endomorphin 2. 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 (n = 9). The peak pressor response averaged 23.1 ± 8.6 mmHg for control (n = 9), 33.2 ± 11 mmHg after naloxone and oxycodone were infused (n = 9), and 27 ± 8.6 mmHg after APETx2 and oxycodone were infused (n = 9). Our data suggest that after opioid receptor blockade, ASIC3 stimulation by the endogenous mu opioid, endomorphin 2, potentiated the exercise pressor reflex.NEW & NOTEWORTHY This paper provides the first in vivo evidence that endomorphin 2, an endogenous opioid peptide, can paradoxically increase the magnitude of the exercise pressor reflex by an ASIC3-dependent mechanism even when the contracting muscles are freely perfused.
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Canais Iônicos Sensíveis a Ácido , Contração Muscular , Músculo Esquelético , Naloxona , Oligopeptídeos , Receptores Opioides mu , Reflexo , Animais , Masculino , Ratos , Canais Iônicos Sensíveis a Ácido/metabolismo , Analgésicos Opioides/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Oligopeptídeos/farmacologia , Oxicodona/farmacologia , Oxicodona/administração & dosagem , Condicionamento Físico Animal/fisiologia , Ratos Sprague-Dawley , Receptores Opioides mu/metabolismo , Reflexo/efeitos dos fármacos , Reflexo/fisiologiaRESUMO
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.
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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 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.
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Doenças Inflamatórias Intestinais , Células Receptoras Sensoriais , Animais , Humanos , Ratos , Doenças Inflamatórias Intestinais/metabolismo , Dor/metabolismo , Células Receptoras Sensoriais/metabolismoRESUMO
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.
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Analgésicos Opioides , Receptores Opioides kappa , Humanos , Analgésicos Opioides/farmacologia , Receptores Opioides mu/fisiologia , Constipação Intestinal , Neurônios Aferentes , Receptores Opioides , Analgésicos/farmacologiaRESUMO
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.
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Capsaicina , Canais de Potencial de Receptor Transitório , Animais , Ratos , Pressão Sanguínea , Capsaicina/farmacologia , Artéria Femoral/metabolismo , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Ratos Sprague-Dawley , Reflexo/fisiologia , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
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.
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Hidroxicloroquina , Células-Tronco Pluripotentes Induzidas , Humanos , Hidroxicloroquina/farmacologia , Estereoisomerismo , Miócitos Cardíacos , SulfatosRESUMO
Spatially extended aggregates or clusters of dopants are ubiquitous in a plethora of granular superconducting systems, such as Al-dopedMgB2and N-dopedMo2N, forming a droplet network that is very important to their characterization and to the description of their superconducting properties. At the same time, one of the most studied classes of unconventional superconducting materials are the high-temperature superconductors, where special attention is given to the hole-doped cuprates, where the carrier concentration is controlled by the amount of extra interstitial oxygen dopants. In this context, the formation of spatially inhomogeneous aggregates of interstitial dopant oxygen atoms, in the form of nanosized superpuddles, is not only relevant, but also a subject of intense recent experimental and theoretical surveys. Following these efforts, in this work we investigate the consequences of the presence of networks of inhomogeneously distributed dopant superpuddles on the superconducting state. Starting from the inhomogeneous extended disordered background brought by the network of superpuddles, we demonstrate, with the aid of an effective interaction between electrons mediated by the local vibrational degrees of freedom of each puddle, that the Cooper pairs arising from an attractive interaction in an inhomogeneous medium have a finite center-of-mass (CM) momentum,p, that breaks up the Cooper channel. Furthermore, we derive an analytical expression for the amplitude of the superconducting gap,Δk, in terms of disorder and finite CM momentum and show that amplitude fluctuations are induced in the superconducting state by the presence of the superpuddles, where both the gap and the critical temperature are reduced by disorder and finite momentum pairs. Finally, we discuss our findings in the context of synchronized networks of superconducting oxygen nano-puddles in cuprates and in other granular superconducting systems.
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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.
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Amilorida , Treinamento Resistido , Adulto , Feminino , Humanos , Masculino , Amilorida/farmacologia , Pressão Sanguínea/fisiologia , Frequência Cardíaca/fisiologia , Hemodinâmica , Fluxo Sanguíneo Regional/fisiologiaRESUMO
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.
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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.
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Resistina , Sepse , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Insuficiência de Múltiplos Órgãos/metabolismo , Neutrófilos/metabolismo , Resistina/genética , Resistina/metabolismoRESUMO
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.
Assuntos
Receptor 5-HT1A de Serotonina , Serotonina , Animais , Canais de Cálcio/metabolismo , Membro Posterior/metabolismo , Músculos/metabolismo , Ratos , Receptor 5-HT1A de Serotonina/metabolismo , Receptores de Serotonina/metabolismo , Células Receptoras Sensoriais/metabolismo , Serotonina/metabolismo , Serotonina/farmacologiaRESUMO
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.
Assuntos
Potenciais de Ação/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.8/fisiologia , Gânglio Nodoso/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Masculino , Neurônios/fisiologia , Ratos , Ratos WistarRESUMO
INTRODUCTION AND HYPOTHESIS: We sought to develop a Spanish translation of the Female Genitourinary Pain Index (GUPI) and to validate this instrument in US Latina women. METHODS: Translation back-translation was performed to create the initial Spanish version. Bilingual women with pelvic and/or genitourinary pain were recruited from clinical sites and social media. Participants reported demographics and completed the Female GUPI in both English and Spanish. Agreement was assessed for each item, subscale and total score. Additionally, we performed cognitive debriefing interviews to further test face validity. A consensus group of bilingual physicians and healthcare personnel utilized comments from the interviews to create a final Spanish version. RESULTS: Thirty-four participants completed the questionnaire. Their average age was 33 years, 80% reported attending some college, and 20% reported an undergraduate degree or higher. Most were born in mainland USA (57%) or Mexico (27%). Agreement for the pain, urinary and quality of life subscales between the English and Spanish versions of the measure were excellent (0.91, 0.89 and 0.92, respectively) with 0.96 agreement for the measure as a whole. Despite favorable psychometrics, preferences for alternate wording were reported over 50 times. Based on that feedback, a consensus group was formed, which recommended changes to 13 of the 15 items, 3 of which required complete rewriting. CONCLUSIONS: The Spanish Female GUPI is strongly correlated with the English original; however, participants reported the language was overly complex. Translation and validation should include review of the measure and feedback by the target audience for optimal clarity and readability.