Krebs Solution Research Articles

Synthesis, crystal structures and biological activities of halogeno-(O-alkylphenylcarbamothioate)bis(triarylphosphine)copper(I) complexes

The aim of this work was to synthesize and characterize a new series of copper(I) complexes with thiocarbamate ligands and to evaluate their biological activity. Treatment of CuX salts (X = Cl, Br, I) with O-alkyl N-phenylcarbamothioates in the presence of two equivalents of PAr3 (Ar = Ph, p-C6H4OMe) affords a series of mononuclear thione complexes [(Ar3P)2Cu{ROC(=S)N(H)Ph}X)] C1-C10. According to X-ray diffraction analyses, all complexes adopt a distorted tetrahedral geometry and feature intramolecular NH···Hal bonding, giving rise to six-membered cycles. Like in the case of the literature-known chloro‑compounds C1 and C2 (d(NH···Cl) ∼ 2.29 Å), the bromo‑derivatives C3–C6 exhibit strong NH···Hal distances varying from 2.48 to 2.63 Å. This intramolecular bonding, which is weaker for the iodo complexes C7-C10 (∼ 2.75 Å), has also been studied by Hirshfeld surface analysis of C1, C3, C4 and C6. The crystal structure of two 1D-polymeric intermediates occurring during the synthesis of C7 and C9, namely [{Cu(μ2-I)2Cu}(μ2-L)2]nCP1 and CP2, has also been elucidated. The complexes display weak luminescence in solution centered on the triarylphosphine ligands. In contrast, in the solid state a high-energy band around 350–450 nm centered on the thioamidate and triarylphosphine ligands is observed, along with a second low energy band typical for MLCT/XLCT states around 450–550 nm. The screening of the biological activity of these complexes reveals the absence of any significant antibacterial capacity. However, the presence of an antioxidant activity with a maximum activity for complexes C3 and C4 has been evidenced using the colorimetric DDPH method. This has been probed in vivo on Wistar rats for the preservation of hepatocytes integrity after liver hypothermic conservation (4 °C) for 24 h in Krebs solution with normal cellular state by addition of [(Ph3P)2Cu{EtOC(=S)N(HPHh}I] C8.

A275 MECHANOSENSITIVE KCA2 + 1.1 CHANNEL MEDIATES INTRALUMINAL DISTENSION-INDUCED CALCIUM INFLUX IN ENTERIC NEURONS

Abstract Background The enteric nervous system (ENS) is the extensive neural network within the wall of the gastrointestinal (GI) tract that is essential for the control of gut function. However, the mechanisms whereby the ENS responds to the mechanical forces associated with distension are not well understood, and this has implications for gut function in health and disease. Purpose This study aims to determine the mechanisms underlying the responsiveness of enteric neural circuits in the myenteric plexus to intraluminal distension. Method Intact segments of the colon from mice expressing a genetically encoded fluorescent calcium reporter specifically in enteric neurons (Wnt-1-GCaMP6 mice) or cholinergic neurons (Chat-GCaMP6 mice) were bathed and luminally perfused with Krebs solution in a custom-designed chamber (35-37°C, 95% O2 ,5% CO2, pH 7.4). Intracellular Ca2+ fluorescence in enteric neurons was visualized by live-cell confocal microscopy and was analyzed using Imaris™ software. Intraluminal distension was accomplished either by increasing luminal perfusion pressure or using colonic preparations containing natural fecal pellets. The degree of intraluminal distension produced by perfusion was defined by the relative distance between identified neurons in preparation and is expressed as a percentage increase in the distance between them. Distension was studied over a range from 0-75%. The degree of distension produced by a fecal pellet in the distal colon is approximately 75%. Result(s) Intraluminal distension significantly increased intracellular Ca2+ fluorescence in most (>85%) of neurons of the myenteric plexus. When the colon was distended, the response to depolarization by KCl, a general cell depolarization activator, and DMPP, a nicotinic receptor agonist in Wnt1-cre GCaMP6 mice and Chat GCaMP6 mice, respectively was substantially inhibited. To identify how distention induced an increase in intracellular Ca2+ fluorescence and inhibited depolarization, pharmacological inhibitors of mechanosensitive ion channels were used. The nonspecific mechanosensitive channel inhibitor, gadolinium (50 mM) and the Kca1.1 channel inhibitor, paxilline (10 mM), both significantly reduced the luminal distension-induced increase in intracellular Ca2+ fluorescence. The effect of luminal distension-inhibited cell depolarization by KCl recovered in the presence of paxilline. This was not observed in the Ca2+ responses evoked by DMPP, suggesting that other mechanisms may play a role in the inhibition of neuronal responses in cholinergic enteric neurons. Conclusion(s) The majority of neurons of the colonic myenteric plexus are mechanosensitive and show an increase in intracellular Ca2+ when the gut is distended. Myenteric neurons do not respond to nicotinic receptor activation when the gut is distended through a mechanism that is dependent on the activity of Kca1.1 channel. Supported by CIHR Disclosure of Interest None Declared

Screening of Volatile Constituents of N. sativa on Calcium Depleted Heart Model

The number of deaths from cardiovascular disorders is rising every year. Nigella sativa, one of the accessible natural plants, has a broad range of pharmacological effects. The Nigella sativa seeds were removed, cleaned, and preserved in order to research the effects of the plant on the cardiovascular system. After being ground into a fine powder, the seeds were used to extract the volatile oil from the seeds using the steam distillation process. The hypodynamic model was used after the rat's heart was removed. Using the standard kreb's hanselet salt solution to mount the isolated heart, a typical graph was produced. As the calcium concentration was reduced, the graph's negative tropic activity became apparent. When N. sativa was administered it has produced additional negative tropic effect on hypodynamic heart. At 0.1 ml produced negative tropic effect and keeps on decreasing as the dose increases by 0.2, 0.4, 0.8ml in dose dependent manner.

Physiological Effects of Condensed Tannins from Black Currant (Ribes nigrum L.) on Isolated Rat Duodenal Contraction

Condensed tannins (CTs) extracted from various plants have been shown to possess antioxidant, antidiabetic, anthelmintic, anti-palatable and anti-diarrhea activity. Black currant (Ribes nigrum L.), a native plant of northern Europe and Asia, is rich in phenolic compounds, including CTs. Among the biological activities of CTs, their astringent property is likely to affect gastrointestinal motility. This study aimed to investigate the physiological effect of CTs from black currant (R. nigrum L.) leaves on isolated rat duodenal contraction. Duodenal segments were fixed in organ baths containing carbogen aerated Krebs solution at the resting tension of 0.7 - 0.8 g. The frequency, amplitude, and tone of duodenal contraction were recorded. Either CTs or acetylcholine (ACh) were cumulatively added into the bath at the concentration of between 0.001 - 10 µg/mL and 10–8 - 10–4 M, respectively. The mechanisms of CTs and ACh actions were studied using muscarinic receptor antagonist (atropine, 1.55×10–5 M) and calcium channel blocker (verapamil, 10–6 M). It is found that CTs at the concentration between 0.001 - 10 µg/mL had no direct effect on duodenal frequency, amplitude, and tone of contraction, whereas ACh showed a significant increase in tonic contraction, was suppressed by atropine. Interestingly, in the presence of atropine and verapamil, CTs showed a further significant decrease in the amplitude of duodenal contraction compared to the effect of these 2 blockers alone. It is concluded that CTs would synergize the activity of the muscarinic receptor antagonist and the calcium channel blocker at duodenal enteric neurons or smooth muscle membrane. However, the use of CTs from black currant (R. nigrum L.) leaves to treat gastro-intestinal disorders while having muscarinic receptor antagonist or calcium channel blocker need cautions.
 HIGHLIGHTS
 
 Black currant (Ribes nigrum L.), a native plant of northern Europe and Asia, is rich in phenolic compounds including condensed tannins (CTs) and their astringent property is likely to affect gastrointestinal motility
 CTs isolated from leaves of black currant (R. nigrum L.) at the physiological doses (0.001 - 10.0 µg/mL) have no direct effect on isolated rat duodenal contraction
 CTs may possess the synergistic or additive effect with either atropine, a nonselective muscarinic receptor, or verapamil, a calcium channel blocker on duodenal amplitude of contraction
 
 GRAPHICAL ABSTRACT

Inorganic phosphate accelerates cardiac myofilament relaxation in response to lengthening.

Myocardial relaxation in late systole is enhanced by increasing velocities of lengthening. Given that inorganic phosphate (Pi) can rebind to the force-producing myosin enzyme prior to MgADP release and hasten crossbridge detachment, we hypothesized that myocardial relaxation in late systole would be further enhanced by lengthening in the presence of Pi. Wistar rat left ventricular papillary muscles were attached to platinum clips, placed between a force transducer and a length motor at room temperature, and bathed in Krebs solution with 1.8 mM Ca2+ and varying Pi of 0, 1, 2, and 5 mM. Tension transients were elicited by electrical stimulation at 1 Hz. Peak tension was significantly enhanced by Pi: 0.593 ± 0.088 mN mm−2 at 0 mM Pi and 0.817 ± 0.159 mN mm−2 at 5 mM Pi (mean ± SEM, p < 0.01 by ANCOVA). All temporal characteristics of the force transient were significantly shortened with increasing Pi, e.g., time-to-50% recovery was shortened from 305 ± 14 ms at 0 mM Pi to 256 ± 10 ms at 5 mM Pi (p < 0.01). A 1% lengthening stretch with varying duration of 10–200 ms was applied at time-to-50% recovery during the descending phase of the force transient. Matching lengthening stretches were also applied when the muscle was not stimulated, thus providing a control for the passive viscoelastic response. After subtracting the passive from the active force response, the resulting myofilament response demonstrated features of faster myofilament relaxation in response to the stretch. For example, time-to-70% relaxation with 100 ms lengthening duration was shortened by 8.8 ± 6.8 ms at 0 Pi, 19.6 ± 4.8* ms at 1 mM Pi, 31.0 ± 5.6* ms at 2 Pi, and 25.6 ± 5.3* ms at 5 mM Pi (*p < 0.01 compared to no change). Using skinned myocardium, half maximally calcium-activated myofilaments underwent a 1% quick stretch, and the tension response was subjected to analysis for sensitivity of myosin detachment rate to stretch, g 1, at various Pi concentrations. The parameter g 1 was enhanced from 15.39 ± 0.35 at 0 Pi to 22.74 ± 1.31 s−1/nm at 8 Pi (p < 0.01). Our findings suggest that increasing Pi at the myofilaments enhances lengthening-induced relaxation by elevating the sensitivity of myosin crossbridge detachment due to lengthening and thus speed the transition from late-systole to early-diastole.

Abstract P080: Il-18 Potentiates Contractile Responses To Phenylephrine And Inhibits Relaxation Responses To Acetylcholine In Resistance Arteries From Wistar Rats

Background: The etiology of essential hypertension remains unclear, and its mechanisms are complex. Indeed, low-grade inflammation and activation of the immune system have been implicated in hypertension. Clinical and population studies have consistently found increased circulating levels of interleukin 18 (IL-18) in patients with hypertension. Hypothesis: This study tests the hypothesis that IL-18 will enhance contractile responses to phenylephrine and impair relaxation responses to acetylcholine in isolated arterial rings from rats. Methods: Vascular function was performed on isolated pudendal arteries from male Wistar rats using wire myographs, kept in Krebs solution at 37°C, constantly aerated with 95%O 2 /5%CO 2 . Concentration-response curves to phenylephrine (PE; 10 -9 -3x10 -5 M), and acetylcholine (ACh; 10 -9 - 3x10 -5 M) were performed in the absence or in the presence of IL-18 (20ng/ml) for 1 hour and 6 hours respectively. Ten minutes prior to the incubation with IL-18, some arterial rings were incubated separately with the mitogen-activated protein kinase (MKK / MEK) inhibitor, PD98059 (10 -5 M). Concentration-response curves were analyzed using non-linear regression analysis. Data are presented as mean ± S.E.M. Statistical significance set at p&lt;0.05. Data analyzed with GraphPad Prism 9.2.0. Results: In the pudendal arterial rings, the potency of PE was increased in the presence of IL-18 (pEC 50 6.510 ± 0.3987 vs 6.022 ± 0.2738 for control; p = 0.0268), Incubation with IL-18 plus PD98059 restored the contraction to PE to control levels. (pEC 50 6.105 ± 0.4007 vs 6.170 ± 0.2847 for control; p = 0.7120). Relaxation responses to Ach were impaired by incubation with IL-18 for 6 hours (pEC 50 5.942 ± 0.1575 vs 7.341 ± 0.1796 for control; p=0.0042) Conclusion: Our results show that IL-18 enhances the contractile responses to PE in pudendal arteries from Wistar rats by activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Thus, IL-18 may contribute to the increased vascular resistance characteristic of hypertension.

Abstract P140: Indanyloxyacetic Acid-94 Decreases Contraction In Uterine Arteries In Response To Phenylephrine And Decreases Response To Phenylephrine After Ca 2+ Replacement

Background: Chloride Intracellular Channels (CLICs) are a family of transmembrane proteins that can be present on the plasma membrane or the membranes of a number of organelles. These channels are redox and pH sensitive and present a unique target to understanding the vascular response to reactive oxygen species (ROS). By regulating oxidative stress, these channels may play a role in the pathology of vascular dysfunction. Uterine arteries were chosen as the focus of this project because of the lack of research involving its vascular function and role in disease. Hypothesis: This project hypothesizes that IAA94 will decrease vasoconstriction in response phenylephrine and enhance the relaxation response to acetylcholine in an endothelial-dependent manner. Methods: Vascular reactivity tests on wire myographs were performed in endothelium intact (E+) and denuded (E-) uterine arteries from Wistar rats. Rings were kept in Krebs solution at 37°C and constantly aerated with 95%O 2 /5%CO 2 . Concentration-response curves to phenylephrine (PE; 10 -9 -3x10 -5 M), and acetylcholine (ACH; 10 -9 - 3x10 -5 M) were performed in the absence or in the presence of IAA94 (50 uM). Vascular reactivity to 3*10 -6 M PE was also performed in Ca 2+ free Krebs solution with washout of intracellular Ca 2+ and replenishing of external Ca 2+ . A concentration response curve was completed to potassium chloride (KCL; 1mM - 100mM). Concentration-response curves were analyzed using non-linear regression analysis. Data are presented as mean ± S.E.M. Statistical significance set at p&lt;0.05. Data analyzed with GraphPad Prism 9.2.0. Results: In the E+ rings, IAA -94 inhibited contractile response to PE (pEC 50 : control 6.684 ± 0.1433 vs. IAA94 treated 5.616 ± 0.89, p&lt;0.0001). The E- had decreased inhibitory effects of IAA-94 on contractile response to PE (pEC 50 : control 5.832 ± 0.19 vs. IAA94 treated 5.32 ± 0.21, p= 0.2034). IAA94 treatment did not significantly change the contraction to KCL, while the IAA94 treatment did significantly decrease the rings response to PE after Ca 2+ washout and replenishment. Conclusion: The data show that IAA94 decreases the contractile response to PE in an endothelial dependent manner that is separate from the K + flux from KCL-induced contraction.

Abstract P335: Contribution Of The Gut To The Salt-sensitive Phenotype Of Dahl S Rats Is Reflected In Increased Sodium Absorption In The Distal Colon

Salt-sensitive hypertension in Dahl sensitive (Dahl S) rats is primarily attributed to sodium mishandling associated with kidney dysfunction, and very few studies interrogated sodium absorption in the gut of Dahl S rats and its potential contribution to the salt-sensitive phenotype. To assess this, we compared sodium channel (ENaC)-driven sodium (Na + ) currents, as a measure of Na + transport across the distal colon, the primary sodium absorption site in the GI tract, between Dahl S and Dahl salt-resistant (Dahl R) rats ex vivo . In parallel, we evaluated fecal sodium levels to assess overall fecal sodium loss in Dahl S and R rats. Male adult (12-14wo) Dahl S and R were administered a low sodium diet (0.3% NaCl, Teklad) ad libitum from weaning. Prior to the experiment, food intake/rat was measured every day for three consecutive days. On the day of experiment, rats were euthanized by an overdose of isoflurane. Distal colons were dissected and transferred to the Ussing chamber with oxygenated warm Krebs solution containing d-glucose. Increasing concentrations of the ENaC blocker amiloride (0.01-1000uM) were applied to the Ussing chamber on the apical side of the distal colon in 10-minute intervals. Maximum decreases in total ionic current across the distal colon were measured in real time following addition of amiloride to quantify ENaC Na + current across the distal colon. Fecal samples were homogenized in distilled water, and sodium levels per gram of fecal mass were determined by flame photometry. Despite no differences in food intake, we observed a dose-dependent 2 to 4-fold higher responses to amiloride in the distal colon of Dahl S compared to Dahl R rats, suggesting elevated Na + current in the Dahl S. Conversely, a ~2-fold lower Na + concentration was detected in the feces of Dahl S compared to Dahl R rats. In conclusion, elevated ENaC-driven Na + current in the distal colon, coupled with reduced fecal Na + concentration, suggest higher absorption/transport of Na + across the distal colon of Dahl S compared to Dahl R rats, which was independent of salt intake. Considering that the circulating Na + levels are reportedly maintained in the Dahl S by the overworked kidney, elevated colonic sodium absorption may contribute to kidney disease and salt-sensitive phenotype in Dahl S.

Effect of metaclopramide on urinary bladder smooth muscle contraction-relaxation mechanism

Objectives: Metoclopramide, a dopamine receptor antagonist, a benzamide, is a commonly used antiemetic drug in many diseases with nausea and vomiting. The aim of this study is to examine the effects of metoclopramide, which is also used in urinary system infections, on the urinary bladder contraction-relaxation mechanism. Methods: In the study, bladder tissue strips obtained from adult female Wistar rats in diestrus were placed in an isolated organ bath containing Krebs solution. The effect of metoclopramide at concentrations of 10 µM and 20 µM on sections where spontaneous contractions were observed under 1.5 g of tension was investigated. Results: Metaclopramide caused a statistically significant increase in the area under the contraction curve (AUC) and peak to peak (p-p) parameters of spontaneous bladder contractions at concentrations of 10 and 20 μM (p &amp;lt; 0.01). Conclusions: Metaclopramide has an activator effect on spontaneous bladder contractions. This effect should be taken into consideration in clinical use, especially when used in urinary system infections.

Effects of lysophosphatidic acid on sling and clasp fibers of the human lower esophageal sphincter

Background This study aims to explore the role of lysophosphatidic acid receptors in the regulation mechanisms of contraction and relaxation of human lower esophageal sphincter. Methods Between July 2015 and March 2016, muscle strips were collected from a total of 30 patients (19 males, 11 females; mean age: 62±9.9 years; range, 52 to 68 years) who underwent an esophagectomy for mid-third esophageal carcinomas. The specimens were maintained in oxygenated Krebs solution. Muscle tension measurement technique in vitro was used to examine the effects of non-selective lysophosphatidic acid receptors agonists and antagonists, as well as selective lysophosphatidic acid receptors agonists on the clasp and sling fibers of human lower esophageal sphincter. Results The non-selective dopamine receptor agonist lysophosphatidic acid induced the contraction of the clasp and sling fibers of the human lower esophageal sphincter. The response induced by nonselective lysophosphatidic acid receptor agonist was inhibited completely by non-selective lysophosphatidic acid receptor antagonist. The selective lysophosphatidic acid 1 and 2 receptor agonist and the selective lysophosphatidic acid 3 receptor agonist induced a concentration-dependent contractile response of the clasp and sling fibers of the human lower esophageal sphincter. There was no significant difference in contraction rates between the clasp and sling fibers (p>0.05). Conclusion This study indicates that lysophosphatidic acid regulates the lower esophageal sphincter is through its receptor; the lysophosphatidic acid receptors may be involved in the contractile response of the human lower esophageal sphincter.

Vibegron inhibits enhanced spontaneous contractions induced by anoxia/reoxygenation in isolated whole bladder from rats

It has been recently proposed that repeated bladder ischemia/reperfusion induced by chronic pelvic ischemia may lead to detrusor overactivity, followed by lower urinary tract symptoms. Vibegron is a selective β3-adrenoceptor agonist approved for the treatment of overactive bladder. Several studies have tested β3-adrenoceptor agonists using animal models with detrusor overactivity related to bladder ischemia/reperfusion. However, whether β3-adrenoceptor agonists directly affect ischemia/reperfusion-evoked detrusor overactivity is unclear. Therefore, we examined whether bladder anoxia/reoxygenation could enhance spontaneous bladder contractions (SBCs) and investigated the effect of vibegron on enhanced SBCs. Isolated whole bladders from rats were incubated with Krebs solution aerated with 95% N2 + 5% CO2 for 5 h (anoxia). Subsequently, the bathing solution was replaced with an oxygen-saturated solution (reoxygenation). Anoxia/reoxygenation caused enhancement of the amplitude but not the frequency of SBC compared with that before reoxygenation. Vibegron (0.3–30 μM) inhibited this increase in SBC amplitude, but not the frequency, in a dose-dependent manner. The inhibitory effect of vibegron was not affected by pretreatment with the adenylyl cyclase inhibitor SQ22536 (100 μM) or protein kinase A inhibitor KT5720 (1 μM) and was not accompanied by considerable changes in cyclic adenosine monophosphate (cAMP) content in the bladder. In contrast, the large conductance potassium channel inhibitor iberiotoxin (100 nM) suppressed the inhibitory effect of vibegron. These results suggest that bladder ischemia/reperfusion induces SBC enhancement and vibegron directly inhibits detrusor overactivity via the large conductance potassium channel, which involves β3-adrenoceptor, rather than the cAMP signaling pathway.

Interleukin‐18 Enhances Contractile Responses to Phenylephrine in Pudendal Artery from Wistar Rats

BackgroundHypertension is a major risk factor for the development of cardiovascular diseases and is a leading cause of mortality. The etiology of essential hypertension remains unclear, and its mechanisms are complex. Inflammation is a complex phenomenon, comprises the first response of the immune system in the presence of harmful stimuli and is considered to be a major cause of increased blood pressure. Indeed, low‐grade inflammation and activation of the immune system have been implicated in hypertension. Cytokine levels are elevated in hypertension and have been reported to be implicated in the associated disturbances in vascular reactivity reported in this disease. Clinical and population studies have consistently found increased circulating levels of interleukin 18 (IL‐18) in patients with hypertension.HypothesisThis study tests the hypothesis that IL‐18 will enhance contractile responses to phenylephrine in isolated arterial rings from rats.MethodsVascular function was performed on isolated pudendal arteries from male Wistar rats using wire myographs, kept in Krebs solution at 37°C, constantly aerated with 95%O2/5%CO2. Concentration‐response curves to phenylephrine (PE; 10‐9‐3x10‐5M), Acetylcholine (ACH; 10‐9 – 3x10‐5M) were performed in the absence or in the presence of IL‐18 (20ng/ml) for 1 hour. 10 minutes prior to the incubation with IL‐18, some arterial rings were incubated separately with the following agents: NADPH oxidase 2 inhibitor, gp91 ds‐tat (10‐5M); NADPH oxidase 1 and 4 inhibitor, GKT137831(10‐6M); and mitogen‐activated protein kinase (MKK / MEK) inhibitor, PD98059 (10‐5M) Concentration‐response curves were analyzed using non‐linear regression analysis. Data are presented as mean ± S.E.M. Statistical significance set at p&lt;0.05. Data was analyzed with GraphPad Prism 9.2.0.ResultsIn the Wistar pudendal arterial rings, the potency of PE was increased in the presence of IL‐18 (pEC506.510 ± 0.3987 vs 6.022 ± 0.2738 for control; p = 0.0268) A, IL‐18 plus gp91 ds‐tat (pEC506.643 ± 0.2215 vs 6.264 ± 0.2471 for control; p = 0.0058) B and IL‐18 plus GKT137831 (pEC506.700 ± 0.2937 vs 6.272 ± 0.2963 for control; p = 0.0003) C. Incubation with IL‐18 plus PD98059 restored the contraction to PE to control levels. (pEC506.105 ± 0.4007 vs 6.170 ± 0.2847 for control; p = 0.7120) D. The relaxation response to ACh was not changed by IL‐18 after incubation for 1 hr.ConclusionOur results show that IL‐18 enhances the contractile responses to PE in pudendal arteries from Wistar rats by activation of the Mitogen‐Activated Protein Kinase/Extracellular Signal‐Regulated Kinase Pathway.

Chloride Intracellular Channel Protein 1 (CLIC1) Inhibitor (IAA94) Decreases Contractile Force in Rat Uterine Arteries

ObjectiveThe objective of this study was to determine the effects of the Chloride Intracellular Channel Protein 1 (CLIC1) inhibitor, IAA94, on uterine arteries in rats.HypothesisThis study tested the hypothesis that the inhibition of CLIC1 with IAA94 decreases contractility of the uterine artery.MethodsUterine arteries were harvested from 12‐week‐old female Wistar Rats. The arterial rings were mounted in a wire myograph and kept at 37ºC in Krebs solution, constantly aerated with a 95% O2+ 5% CO2 gas mix. Concentration‐response curves to Phenylephrine (PE, ‐9M to ‐4.5M). After washout, the arterial rings were incubated with the CLIC1 inhibitor (IAA94, 50 µM) for 30 minutes and then, concentration‐responses curves to PE were repeated. The curves were analyzed using nonlinear regression analysis and pEC50 and Emax were calculated with an N=8 for both groups. Data was analyzed by GraphPad Prism 9.2.0., and is presented as Mean ± SEM (n=8).Summary of ResultsIn the Wistar uterine arterial rings, the potency of PE was decreased in the presence of the CLIC1 inhibitor IAA94 (pEC50: 5.64 ± 0.28 vs. 6.59 ± 0.30 for IAA94 vs control, respectively; p &lt;0.0001). Similarly, Emax to PE was decreased after incubation with IAA94 (105 ± 2% vs 128 ± 5%, for IAA94 vs control, respectively; p &lt;0.001). These data show that the inhibition of CLIC1 plays a role in the contraction induced by PE. Further experiments are necessary to understand the mechanisms underlying this effect.ConclusionThe addition of the CLIC1 inhibitor, IAA94, elicits a decreased contractile response to PE. CLIC1 has been identified to be dysregulated in certain cancer types and aging. There are numerous overlaps between cell senescence/cancer morphology and the pathophysiology of preeclampsia. These preliminary experiments shed light on the uterine artery’s reactivity to a CLIC1 inhibitor and would be an interesting topic to pursue in pregnant rats in both SHR and Wistar strains. With hypertensive patients, we see an increase in peripheral contractility and a decrease in responsiveness to vasodilators. CLIC1 could be a possible target in vasoconstrictive pathways, especially those that occur during pregnancy and in preeclamptic patients.

A method for evaluating drug penetration and absorption through isolated buccal mucosa with highly accuracy and reproducibility.

The purpose of the project is to establish a standardized operation method of the in vitro permeability model to maximize mucosal integrity and viability. The model drug lidocaine permeability, 20 kDa fluorescein isothiocyanate-dextran, H&E staining, and mucosal viability were used as evaluation indicators. Firstly, the buccal mucosae of rats, rabbits, dogs, porcine, and humans were analyzed by H&E staining and morphometric analysis to compare the differences. Then, we studied a series of operation methods of isolated mucosa. The buccal mucosae were found to retain their integrity in Kreb's bicarbonate ringer solution at 4 °C for 36 h. Under the long-term storage method with program cooling, freezing at -80 °C, thawing at 37 °C, and using cryoprotectants of 20% glycerol and 20% trehalose, mucosal integrity and biological viability can be maintained for 21 days. The heat separation method was used to prepare a permeability model with a mucosal thickness of 500 μm, which was considered to be the optimal operation. In summary, this study provided an experimental basis for the selection and operation of in vitro penetration models, standardized the research process of isolated mucosa, and improved the accuracy of permeability studies.

A243 HISTAMINE H1-RECEPTOR ANTAGONISTS SUPPRESS THE HYPEREXCITABILITY OF NOCICEPTIVE NEURONS EXPOSED TO IBS-C PATIENT STOOL SUPERNATANTS

Abstract Background Abdominal pain is the primary cause of morbidity in many chronic GI disorders such as IBS, but the molecular mechanisms contributing to pain signaling are unclear. Stool supernatants from patients with diarrhea-predominant IBS increase the excitability of DRG neurons compared to supernatants from healthy controls, suggesting that mediators in the stool may sensitize nociceptors. Additionally, histamine has been implicated as a mediator of hypersensitivity in IBS patients. However, it is unclear if stool supernatants from constipation-predominant IBS (IBS-C) patients affect the excitability of DRG neurons. Furthermore, it is unknown if specific neuro-mediators in stool such as histamine impact the excitability of nociceptive neurons in this subgroup of IBS patients. Aims To evaluate whether IBS-C stool supernatants induce nociceptive signaling in DRG neurons compared to healthy control (HC) stool supernatants. If so, to evaluate the role of histamine 1 (H1) receptors in DRG neuron nociceptive signaling initiated by mediators in IBS-C stool supernatants. Methods IBS-C (n=5) and HC (n=2) patient stool was collected, filtered, and dissolved with Krebs solution in a 1:8 (g/v) dilution. Dorsal root ganglion (DRG) neurons from C57BL/6 mice were incubated with HC or IBS-C stool supernatant for 30 minutes. To evaluate whether histamine in stool supernatants can sensitize H1 receptors on nociceptors, DRG neurons were pre-incubated with the H1-receptor antagonist pyrilamine (1μM, 30 min) before stool supernatants. Changes in DRG neuronal excitability were recorded using perforated patch-clamp techniques to measure the rheobase (minimum input current needed to elicit an action potential) and the resting membrane potential (RMP). Results In neurons incubated with IBS-C stool supernatants (n=28) the rheobase decreased (63%) compared to healthy controls (78 ± 13.7 pA; n=6). This effect was reversed in DRG neurons pre-incubated with the H1 receptor antagonist, pyrilamine, (n=26) (77 ± 5.9 pA, p&amp;lt;0.001) compared to neurons incubated with IBS-C supernatant alone (50 ± 5.13 pA; n=28). No changes were found in the RMP. The data were analyzed with the non-parametric Kruskal-Wallis test. Conclusions IBS-C stool supernatants increase the excitability of DRG neurons compared to HC. Furthermore, the H1-receptor antagonist pyrilamine inhibits the neuronal hyperexcitability evoked by mediators in IBS-C patient stool. These findings suggest that the neuroactive metabolite histamine may contribute to visceral pain experienced by patients with IBS-C. Further studies are needed to examine whether similar signaling to nociceptive neurons by stool supernatants occurs in other subtypes of IBS. Funding Agencies CAG, CIHR

A242 STOOL FROM IBS-D PATIENT WITH A HISTORY OF A DYSBIOTIC AND NON-DYSBIOTIC ONSET MODULATE NEURONAL EXCITABILITY VIA DIFFERENT MECHANISMS

Abstract Background We have shown that Irritable Bowel Syndrome diarrhea-predominant (IBS-D) patients with a history of a dysbiotic-like onset have distinct stool metabolomic profiles versus those with a non-dysbiotic-like onset. IBS stool supernatants can sensitize mouse colonic afferent nerves via both histamine and proteases. However, it is unknown if stool supernatants from the two IBS-D subgroups modulate the excitability of nociceptive neurons via different neuroactive mediators Aims To evaluate whether there are differences in neuroactive mediators within stool supernatants from subgroups of IBS-D patients that can modulate the excitability of nociceptive neurons. Methods Stool samples from healthy control (HC) (N=5) and IBS-D patients with a dysbiotic (N=7) or non-dysbiotic-like (N=7) onset, was homogenized with Krebs solution and filtered. Proteolytic activity was assessed using casein as a substrate with and without protease inhibitors. Histamine was quantified by ELISA. DRG neurons from C57BL/6 mice were incubated overnight or acutely (30 min) with stool supernatants. Some neurons were pre-incubated with PAR2 (GB83, 10 μM) or H1R (pyrilamine, 1μM) antagonists prior to supernatant incubation. Changes in neuronal excitability were assessed with perforated patch-clamp by measuring the rheobase (current that elicits an action potential). Results Proteolytic activity in dysbiotic-like (57.9 U/μg, p&amp;lt;0.05) but not non-dysbiotic like (37.4 U/μg) stool supernatant was increased compared to HC (25.2 U/μg). Serine inhibitor decreased proteolytic activity of dysbiotic (46.6%, p&amp;lt;0.05) and non-dysbiotic (34.2%, p&amp;lt;0.01) supernatant whereas cysteine, aspartic and metalloproteases inhibitors had no effect. Histamine was increased 78% (p&amp;lt;0.05) in IBS-D compared to HC. No differences in proteolytic activity and histamine concentration between IBS-D subtypes were found. In patch-clamp recordings, overnight incubation with dysbiotic (19%, p&amp;lt;0.05) and non-dysbiotic (22%, p&amp;lt; 0.01) stool supernatant decreased rheobase of DRG neurons compared to HC. No difference in rheobase was observed between IBS-D subtypes. GB83 blocked the overnight actions of supernatants from both subgroups, but pyrilamine had no effect. In contrast, following acute incubation, pyrilamine blocked the hyperexcitability evoked by dysbiotic like supernatant (60pA vs 48pA, p&amp;lt;0.001) but had no effect on non-dysbiotic like supernatants. Conclusions Proteases in stool supernatants from IBS-D patients increase neuronal excitability but only those with a history of dysbiotic like onset acutely increase neuronal excitability through H1 receptors. These findings suggest that stool supernatants from subgroups of IBS-D may modulate nociceptor excitability via different mechanisms. Funding Agencies CIHRAmerican Neurogastroenterology and Motility Society

Hydroxychloroquine induces endothelium-dependent and endothelium-independent relaxation of rat aorta.

Hydroxychloroquine (HCQ) is an antimalarial that is widely used in the management of rheumatoid arthritis and other autoimmune diseases. In this study, we aimed to examine the vascular effects of HCQ on rat aorta (RA). The RA rings were suspended in isolated organ baths and tension was recorded isometrically. HCQ-induced relaxations were tested in the presence of the nitric oxide synthase inhibitor, nitro-L-arginine methyl ester (L-NAME, 100 mM); the cyclooxygenase enzyme inhibitor, indomethacin (10 mM); the calcium (Ca2+) ion channel blocker, nilvadipine (10 μM); and the K+ ion channel inhibitors, tetraethylammonium (1 mM), glibenclamide (10 mM), 4-aminopyridine (1 mM), and barium chloride (30 mM). The effect of HCQ on Ca2+ channels was examined using Ca2+-free Krebs solution, and adding calcium chloride (CaCl2 , 10-5- 10-2 M) cumulatively to baths incubated with HCQ. Removing the endothelium resulted in less relaxation of RA rings compared to endothelium-intact rings (p < 0.05). The effect of endothelium was supported by using L-NAME where HCQ produced-vasorelaxation was decreased (p < 0.05). The contraction of vascular rings was inhibited to a significant degree following the addition of CaCl2 , PE, or KCl on HCQ-incubated RA rings (p < 0.05). The incubation of the RA rings with the Ca2+ channel blocker, the K+ channel blockers, and the COX inhibitor, indomethacin did not significantly affect vascular relaxation induced by HCQ. HCQ produced relaxation of RA rings. The relaxation mechanism differs according to the concentration of HCQ. At con-centrations of 10-6 and 10-5 M, the relaxation is endothelium-dependent and mediated by NO. We strongly suggest that Ca2+ channel inhibition is involved at concentrations of 10-5 and 10-4 M, as well as NO.

Roles of Endothelial Motilin Receptor and Its Signal Transduction Pathway in Motilin-Induced Left Gastric Artery Relaxation in Dogs.

Background: Motilin increases left gastric artery (LGA) blood flow in dogs via the endothelial motilin receptor (MLNR). This article investigates the signaling pathways of endothelial MLNR.Methods: Motilin-induced relaxation of LGA rings was assessed using wire myography. Nitric oxide (NO), and cyclic guanosine monophosphate (cGMP) levels were measured using an NO assay kit and cGMP ELISA kit, respectively.Results: Motilin concentration-dependently (EC50=9.1±1.2×10−8M) relaxed LGA rings precontracted with U46619 (thromboxane A2 receptor agonist). GM-109 (MLNR antagonist) significantly inhibited motilin-induced LGA relaxation and the production of NO and cGMP. N-ethylmaleimide (NEM; G-protein antagonist), U73122 [phospholipase C (PLC) inhibitor], and 2-aminoethyl diphenylborinate [2-APB; inositol trisphosphate (IP3) blocker] partially or completely blocked vasorelaxation. In contrast, chelerythrine [protein kinase C (PKC) inhibitor] and H89 [protein kinase A (PKA) inhibitor] had no such effect. Low-calcium or calcium-free Krebs solutions also reduced vasorelaxation. N-nitro-L-arginine methyl ester [L-NAME; nitric oxide synthase (NOS) inhibitor] and ODQ [soluble guanylyl cyclase (sGC) inhibitor] completely abolished vasodilation and synthesis of NO and cGMP. Indomethacin (cyclooxygenase inhibitor), 18α-glycyrrhetinic acid [18α-GA; myoendothelial gap junction (MEGJ) inhibitor], and K+ channel inhibition through high K+ concentrations or tetraethylammonium (TEA-Cl; KCa channel blocker) partially decreased vasorelaxation, whereas glibenclamide (KATP channel blocker) had no such effect.Conclusion: The current study suggests that motilin-induced LGA relaxation is dependent on endothelial MLNR through the G protein-PLC-IP3 pathway and Ca2+ influx. The NOS-NO-sGC-cGMP pathway, prostacyclin, MEGJ, and K+ channels (especially KCa) are involved in endothelial-dependent relaxation of vascular smooth muscle (VSM) cells.

Novel intrinsic neurogenic and myogenic mechanisms underlying the formation of faecal pellets along the large intestine of guinea-pigs.

Soft faecal material is transformed into discrete, pellet-shaped faeces at the colonic flexure. Here, analysis of water content in natural faecal material revealed a decline from cecum to rectum without significant changes at the flexure. Thus, pellet formation is not explained by changes in viscosity alone. We then used video imaging of colonic wall movements with electromyography in isolated preparations containing guinea-pig proximal colon, colonic flexure and distal colon. To investigate the pellet formation process, the colonic segments were infused with artificial contents (Krebs solution and 4-6% methylcellulose) to simulate physiological faecal content flow. Remarkably, pellet formation took place in vitro, without extrinsic neural inputs. Infusion evoked slowly propagating neurogenic contractions, the proximal colon migrating motor complexes (∼0.6cpm), which initiated pellet formation at the flexure. Lesion of the flexure, but not the proximal colon, disrupted the formation of normal individual pellets. In addition, a distinct myogenic mechanism was identified, whereby slow phasic contractions (∼1.9cpm) initiated at the flexure and propagated short distances retrogradely into the proximal colon and antegradely into the distal colon. There were no detectable changes in the density or distribution of pacemaker-type interstitial cells of Cajal across the flexure. The findings provide new insights into how solid faecal content is generated, suggesting the major mechanisms underlying faecal pellet formation involve the unique interaction at the colonic flexure between antegrade proximal colon migrating motor complexes, organized by enteric neurons, and retrograde myogenic slow phasic contractions. Additional, as yet unidentified extrinsic and/or humoral influences appear to contribute to processing of faecal content in vivo. KEY POINTS: In herbivores, including guinea-pigs, clearly defined faecal pellets are formed at a distinct location along the large intestine (colonic flexure). The mechanism underlying the formation of these faecal pellets at this region has remained unknown. We reveal a progressive and gradual reduction in water content of faecal content along the bowel. Hence, the distinct transition from amorphous to pellet shaped faecal content could not be explained by a dramatic increase in water reabsorption from a specific site. We discovered patterns of anterograde neurogenic and retrograde myogenic motor activity that facilitate the formation of faecal pellets. The formation of 'pellet-like' boluses at the colonic flexure involves interaction of an antegrade migrating motor complex in the proximal colon and retrograde myogenic slow phasic contractions that emerge from the colonic flexure. The findings uncover intrinsic mechanisms responsible for the formation of discrete faecal scybala in the large intestine of a vertebrate.

The residence of synaptically released dopamine on D2 autoreceptors.

SUMMARYNeuromodulation mediated by synaptically released endogenous transmitters acting in G-protein-coupled receptors (GPCRs) is slow primarily because of multistep downstream signaling. What is less well understood is the spatial and temporal kinetics of transmitter and receptor interaction. The present work uses the combination of the dopamine sensor, dLight, to detect the spatial release and diffusion of dopamine and a caged form of a D2-dopamine receptor antagonist, CyHQ-sulpiride, to rapidly block the D2 autoreceptors. Photoactivation of the CyHQ-sulpiride blocks receptors in milliseconds such that the time course of dopamine/receptor interaction is mapped onto the downstream signaling. The results show that highly localized release, but not dopamine diffusion, defines the time course of the functional interaction between dopamine and D2 autoreceptors, which determines downstream inhibition.