Concentration-response Curves Research Articles

P35 Adaptation of a phenotypic screening assay to incorporate 384-well plates: opening the door to high-throughput screening

Abstract Introduction and aims High-throughput screening has significantly impacted drug discovery by increasing speed of experiments and reducing reagent and sample costs. We have previously developed several screening assays to measure myofibroblast inhibition via quantification of α-smooth muscle actin (α-SMA) expression, in a 96-well format. Currently, there are no drugs for the treatment of hypertrophic scarring, a major side-effect of burn injuries that occurs by myofibroblast transformation, leaving a huge gap in this sector. The aim for this project was to adapt our current assay for use in 384-well microplates. Methods Human primary dermal fibroblasts were transformed to myofibroblasts using 10 ng mL−1 transforming growth factor (TGF)-β1. α-SMA expression was measured using the In-Cell enzyme-linked immunosorbent assay method, with cell viability measured using DRAQ5 nuclear staining. Assay accuracy and reliability was determined by calculating the robust z-factor, with guidelines set to an average score over 0.5, with a minimum score per plate above 0.4. To further validate the assay, concentration-response curves (CRCs) for the drug vehicle [dimethyl sulfoxide (DMSO)] and our tool compound (SB-505124) were constructed at varying concentrations, in coincubation with TGF-β1. Results Optimization of well metrics was performed, producing an average robust z-factor of 0.52, confirming the assay Results are reliable and reproducible. The DMSO CRC produced no significant drop in cell viability at concentrations that are expected to be used in the screening of compounds. Our tool compound (SB-505124) produced a concentration-dependant curve for the inhibition of myofibroblast transformation. Conclusions A 384-well assay has been designed, tested and validated for the determination of myofibroblast transformation in human primary dermal fibroblasts. The assay has been shown to deliver an average robust z-factor of above 0.5, it contains a positive control and is compatible with compounds stored in up to 100% DMSO.

P06 A novel drug combination produces a synergistic effect when inhibiting myofibroblast transformation in an in vitro model of hypertrophic scarring

Abstract Introduction and aims Hypertrophic scars affect 91% of patients with burns and yet, there is currently no effective treatment for their prevention. Myofibroblasts are key cells in the pathogenesis of hypertrophic scars, with the inhibition of myofibroblast transformation having been suggested as an effective therapeutic strategy. We have previously screened 1954 drugs approved by the Food and Drug Administration to identify any that could inhibit myofibroblast transformation. Using this data, the aim of this study was to investigate the individual and combined antimyofibroblast effect of two drug classes. Methods Primary human fibroblasts were isolated from the scars of patients with burns, with myofibroblast transformation induced using transforming growth factor (TGF)-β1. Concentration-response curves for drug-class A and drug-class B were constructed at a range of concentrations (0.03–300 µmol L–1), with the In-Cell enzyme-linked immunosorbent assay method used to measure α-smooth muscle actin (α-SMA) expression. Combination experiments were carried out through construction of a concentration-response curve for drug-class A (0.03–100 µmol L–1) in the presence of 3 µmol L–1 of drug-class B. Results Both drug classes showed a concentration-dependent antimyofibroblast effect with an IC50 range of 12.2–120.1 µmol L–1 for drug-class A and 1.4–16.7 µmol L–1 for drug-class B. When given in combination the two classes of drugs produced a synergistic effect at all concentrations up to 10 µmol L–1. For example, an 18% reduction α-SMA expression was expected with 1 µmol L–1 of drug-class A and 3 µmol L–1 of drug-class B; however, a 24% reduction in α-SMA expression was observed instead. Conclusions This is the first study to identify this synergistic effect between these two drug classes, and therefore this novel drug combination could be developed for the prevention of hypertrophic scar formation.

The relaxation effect of endocannabinoid anandamide on isolated rat bladder and vas deferens tissues and possible mechanisms.

The endocannabinoid system plays important roles in various systems, including the genitourinary system; however, its mechanism of action is not fully understood. This study aimed to investigate the direct relaxant effects of anandamide and its possible mechanisms in isolated rat bladder and vas deferens tissues. Twenty-one adult male Wistar albino rats were used. Bladder and vas deferens (prostatic and epididymal portions) tissues were mounted in 10 mL of organ baths. Relaxation responses to anandamide were recorded at 3 and 10 μM concentrations. After the rest period, the procedures were repeated in the presence of cannabinoid (CB) and vanilloid receptor antagonists, various potassium channel blockers, cyclo-oxygenase, and nitric oxide synthase inhibitors. In different tissues to investigate the Ca2+-channel antagonistic effect of anandamide, concentration-response curves to CaCl2 were obtained in the absence and presence of anandamide. Anandamide caused a significant relaxation response in the bladder and epididymal vas deferens tissues, but not in the prostatic portion. The effect of anandamide was antagonized in the presence of the CB1 antagonist AM251 or the non-selective potassium channel blocker tetraethylammonium in bladder tissue. In the epididymal vas deferens, anandamide significantly inhibited the calcium contraction responses, especially at high concentrations. The CB2 antagonist AM630 reversed this inhibition. The results show that anandamide has a direct relaxant effect on the isolated rat bladder and epididymal vas deferens. Anandamide triggers different mechanisms in different types of tissues, and further studies are needed to elucidate the mechanism of action of anandamide.

Rapid-onset, short-duration induction of colorectal contractions in anesthetized, adult, male rats.

Rapid-onset, short-duration induction of colorectal contractions in anesthetized, adult, male rats.

Epithelium-derived 6-nitrodopamine modulates noradrenaline-induced contractions in human seminal vesicles

AimsTo evaluate the basal release of 6-nitrodopamine (6-ND) from human isolated seminal vesicles (HISV) and to characterize its action and origin. Main methodsLeft HISV obtained from patients undergoing prostatectomy surgery was suspended in a 3-mL organ bath containing warmed (37 °C) and gassed (95%O2:5%CO2) Krebs-Henseleit's solution (KHS) with ascorbic acid. An aliquot of 2 mL of the supernatant was used to quantify catecholamines by LC-MS/MS. For functional studies, concentration-responses curves to catecholamines were obtained, and pEC50 and Emax values were calculated. Detection of tyrosine hydroxylase and S100 protein were also carried out by both immunohistochemistry and fluorescence in-situ hybridization assays (FISH). Key findingsBasal release of 6-ND was higher than the other catecholamines (14.76 ± 14.54, 4.99 ± 6.92, 3.72 ± 4.35 and 5.13 ± 5.76 nM for 6-ND, noradrenaline, adrenaline, and dopamine, respectively). In contrast to the other catecholamines, the basal release of 6-ND was not affected by the sodium current (Nav) channel inhibitor tetrodotoxin (1 μM; 10.4 ± 8.9 and 10.4 ± 7.9 nM, before and after tetrodotoxin, respectively). All the catecholamines produced concentration-dependent HISV contractions (pEC50 4.1 ± 0.2, 4.9 ± 0.3, 5.0 ± 0.3, and 3.9 ± 0.8 for 6-ND, noradrenaline, adrenaline, and dopamine, respectively), but 6-ND was 10-times less potent than noradrenaline and adrenaline. However, preincubation with very low concentration of 6-ND (10−8 M, 30 min) produced significant leftward shifts of the concentration-response curves to noradrenaline. Immunohistochemical and FISH assays identified tyrosine hydroxylase in tissue epithelium of HISV strips. SignificanceEpithelium-derived 6-ND is the major catecholamine released from human isolated seminal vesicles and that modulates smooth muscle contractility by potentiating noradrenaline-induced contractions.

Menopause: An Early Obesogenic Condition Associated with Vascular Smooth Muscle Dysfunction

Background: For several years menopause was considered a natural event faced by women entering their 40’s. Evidence has now recognized that menopause causes a series of metabolic and vascular complications that negatively affect women later in life. Approximately, 65% of American menopausal women become overweight or obese which increases their risk of developing vascular events, such as hypertension. However, the mechanisms by which menopause leads to weight gain and vascular complications remain understudied. By using a preclinical model of menopause, our preliminary data shows that menopausal mice exhibit early weight gain and development of metabolic disorders. Therefore, we hypothesize that menopause causes an early onset of obesity and metabolic disorders that may precede the development of vascular complications. Methods: To address our hypothesis, eight-week-old female C57BL/6 mice were randomized into two experimental groups: Control/Sham Group (n=9) and Ovariectomized (OVX) group (n=8). The OVX group had their ovaries surgically removed and the Sham Group underwent surgery, but the ovaries were not removed. Metabolic parameters, including glucose metabolism and metabolic cages, and body weight were analyzed along the 20 weeks of the experimental protocol. At the terminal experiments, thoracic aortas were isolated and mounted in a wire myograph for vascular reactivity studies. Phenylephrine (PE) concentration response curves were utilized to assess vascular smooth muscle functionality. Results: After one week following the surgery, the OVX group showed a significant increase in body weight (19.4 ± 0.37g vs. 18.2 ± 0.40g controls, p<0.05), however, obesity was only confirmed in week 16 with the BMI (0.26 ± 0.003 vs. 0.24 ± 0.003 controls, p<0.05) and abdominal circumference (8.4 ± 0.14cm vs. 8.05 ± 0.06cm controls, p<0.05). Intriguingly, OVX group exhibited reduced food consumption (2.26 ± 0.11g vs. 3.36 ± 0.21g controls, p<0.05) water intake (3.11 ± 0.54 mL vs. 4.48 ± 0.18mL controls, p<0.05), fecal output (1.14 ± 0.07g vs. 2.10 ± 0.21g controls, p<0.05) and water intake (3.11 ± 0.54mL vs. 4.48 ± 0.18mL controls, p<0.05). Regarding glucose metabolism, the OVX group exhibited increased fasting glucose (125.4 ± 8.03mg/dL vs. 86.7 ± 2.67mg/dL controls, p<0.05), glucose intolerance (AUC: 39086.88 ± 2201.01 vs. 22939.56 ± 3187.55 controls, p<0.05), and increased triglycerides (116.0 ± 26.11mg/dL vs. 37.3 ± 4.23mg/dL controls, p<0.05). Vascular studies revealed a significant vascular smooth muscle dysfunction in the OVX group as evidenced by reduced aortic contractility to phenylephrine (EMax: 8.15 ± 1.1mN vs.12.3 ± 1.53mN controls, p<0.05). Conclusion: Our results reveals that menopause causes an early onset of weight gain that is not related to increased consumption of food but instead to an decreased metabolism. Menopausal mice developed central obesity, high fasting glucose, and high triglycerides, which taken together characterize metabolic syndrome that was accompanied by vascular smooth muscle dysfunction. NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Statistical physics investigation of the docking process of fruity odorants on Machilis hrabei MhOR5: New microscopic interpretations

In this paper, the putative phenomenon of adsorption involved in the insect olfactory perception of four fruity odorants on Machilishrabei olfactory receptor MhOR5 was studied at a molecular scale. Thus, the experimental concentration–response curves of propyl acetate, butyl acetate, isobutyl acetate, and prenyl acetate on MhOR5 were adjusted using the monolayer model of ideal gas established using the powerful statistical physics approach, which was selected as the most appropriate model among the others. The fitted model parameters were applied to stereographic and energetic analyze the four olfactory systems. Indeed, the modeling results indicated that the tested fruity key food odorants (KFOs) were docked as 2- or 3-aggregated molecules on one MhOR5 binding site via a nonparallel position (i.e., multi-molecular adsorption mechanism). Energetic parameters were used to characterize the adsorption process involved in the studied systems via the determination of the molar adsorption energies (ranging from 12.91 to 17.52 kJ/mol). Hence, the values of the molar adsorption energies are positive and inferior to 40 kJ/mol; this means that the putative adsorption mechanism presented an exothermic character and a physical type. The docking simulation results proved that the adsorption of propyl acetate, butyl acetate, isobutyl acetate, and prenyl acetate on MhOR5 may occur via physical interactions (weak interactions) like alkyl, pi-alkyl, van der Waals, carbon hydrogen bond, and conventional hydrogen bond. The statistical physics theory may also be utilized to quantitatively characterize Machilishrabei MhOR5 activated by propyl acetate, butyl acetate, isobutyl acetate, and prenyl acetate odorants via the estimation of the different site size distributions (SSDs) and site energy distributions (SEDs).

Evaluating anthelmintic activity through Caenorhabditis elegans egg hatching assay

The Caenorhabditis elegans egg hatching methodology is a valuable tool for assessing the anthelmintic activity of drugs and compounds and evaluating anthelmintic drug efficacy.Isolated eggs from gravid adults are exposed to different concentrations of selected drugs and the percentage of egg hatching is determined with respect to the control condition. The assay allows the construction of concentration-response curves and determination of EC50 or EC90 values for egg hatching inhibition. Also, it allows measurements of inhibition as a function of time of exposure.This approach addresses the urgent need for new anthelmintics, as resistance to current treatments poses a significant challenge in parasitic nematode infection. This resistance not only affects humans but also animals and plants, causing significant economic losses in livestock farming and agriculture.By using the free-living nematode C. elegans as a parasitic model organism, researchers can efficiently screen for potential treatments and assess drug combinations for synergistic effects. Importantly, this assay offers a cost-effective and accessible alternative to traditional methods, eliminating the need for specialized infrastructure, hosts, and trained animal maintenance personnel. Additionally, the methodology closely mimics natural conditions, providing insights into egg development and potential therapeutic targets.This method allows for evaluating the direct negative impact of drugs on egg hatching, which correlates with long-term anthelmintic effects, offering advantages in preventing or reducing the transmission and spread of worm infections by eggs.Overall, this approach represents a significant advancement for anthelmintic discovery, offering both practical applications and avenues for further scientific research.• The C. elegans egg hatching assay is a robust and effective method for assessing the anthelmintic potential of various drugs and compounds, allowing the generation of concentration-response curves.• By leveraging the free-living nematode C. elegans as a parasitic model organism, this method facilitates efficient screening of potential treatments and evaluation of drug combinations.• The method addresses the urgent need for new anthelmintics, offering a cost-effective and accessible alternative to traditional approaches.

OxLDL/LOX-1 Mediated Sex, Age, and Cell Dependent Alterations in Mouse Thoracic Aortic Vascular Reactivity, Stiffness, and Remodeling

Elevated oxidized low-density lipoprotein (oxLDL) is a risk factor and component that worsens cardiovascular disease states. OxLDL can elicit its detrimental action, via lectin-like oxLDL receptor 1 (LOX-1) and has been shown to disrupt vascular function. In this study, we determined whether oxLDL, via LOX-1, alters aortic vascular reactivity and determined if sex differences exist. Thoracic aortic endothelium-intact or -denuded ring segments were isolated from intact C57BL/6J female and male mice and incubated with oxLDL ex vivo (50ug/dL; 2h). Using wire myography, cumulative concentration-response curves to phenylephrine (PE) were generated to determine contractile responses. From these curves, the EC50 was determined and used to contract rings to assess acetylcholine (ACh) dependent relaxation. Calculated aortic stiffness and remodeling, as well as mRNA expression of vasoactive and pro-inflammatory mediators were assessed. BI-0115 (10μM; selective LOX-1 inhibitor) was used to determine LOX-1 dependence. In this study, we investigated the effects of oxidized low-density lipoprotein (oxLDL) via the LOX-1 receptor on murine thoracic aortic vasoreactivity, stiffness, and remodeling across age and sex. Our results demonstrate a differential sex, age, endothelial cell, and LOX-1 dependent alterations to the effcacy of PE-induced contractile responses and ACh-mediated vasorelaxation in the thoracic aortic rings following oxLDL exposure. Additionally, we observed a distinct sex and age effect on thoracic aortic stiffness following exposure to oxLDL. There was also a sex effect on calculated vessel diameter, as well as an age effect on oxLDL-mediated inward remodeling that was LOX-1 dependent. We also observed an oxLDL and age dependent increase in LOX-1, IL-6, endothelin-1 (ET-1), and ET-1 receptor alpha and beta mRNA expression. Thus, LOX-1 inhibition and the resulting attenuation of oxLDL/endothelial-mediated alterations in aortic function suggests that there are differential sex differences in the role of oxLDL/LOX-1 in the thoracic aorta of male and female mice. In conclusion, acute exposure to oxLDL led to altered vasoreactivity, endothelial dysfunction, and changes in aortic stiffness and remodeling. These effects were in-part age, sex, endothelial, and LOX-1 dependent. This study reveals potential complex interactions in oxLDL/LOX-1-mediated vascular responses that could serve as potential therapeutic intervention for vascular diseases such as atherosclerosis. University of Arizona Valley Research Partnership Grant VRP37 P2 (RJG), American Heart Association 19AIREA34480018 (RJG), UA VRP Grant VRP55 P1a (TSW). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Stronger vasoconstriction and weaker vasodilation in mesenteric arteries of Ossabaw than Göttingen minipigs before manifestation of metabolic syndrome

The metabolic syndrome predisposes and contributes to the development and progression of atherosclerosis. The minipig strain “Ossabaw” is characterized by a polygenic predisposition to develop metabolic syndrome and diffuse atherosclerosis in response to a hypercaloric atherogenic diet. The aim of this study was to investigate whether Ossabaw minipigs exhibit an altered vasomotor function even before they develop their diseased phenotype. Mesenteric arteries of adult Ossabaw (n=30) and Göttingen minipigs (n=40), a pig strain without genetic predisposition to a metabolic syndrome, were harvested postmortem, dissected, and mounted on a myograph for isometric force measurements. Maximal vasoconstriction to smooth muscle cell depolarization with potassium chloride (KClmax) was repeatedly induced (twice 0.6×10−1 mol/L and twice 1.2×10−1 mol/L). Cumulative concentration-response curves were determined in response to 1×10−9 -1×10−4 mol/L norepinephrine. Endothelium-dependent and -independent vasodilation were analyzed in response to carbachol and nitroprusside, respectively (1×10−9 - 1×10−4 mol/L, each) after pre-constriction by norepinephrine. The baseline diameter of mesenteric arteries from Ossabaw minipigs was smaller than that from Göttingen minipigs (329±20 vs. 435±11 μm), and mesenteric arteries of Ossabaw minipigs developed a higher baseline force of contraction than mesenteric arteries of Göttingen minipigs (6.2±0.7 vs. 3.6±0.3 mN). The maximal vasoconstrictor response to KCl was comparable between the minipig strains. Vasoconstriction in response to norepinephrine was more pronounced in Ossabaw than in Göttingen minipigs (143±9 vs. 108±6% KClmax). Endothelium-dependent vasodilation (46.4±5.5 vs. 16.0±3.0% of norepinephrine-induced preconstriction) and -independent vasodilation (36.7±4.9 vs. 2.3±0.6% of norepinephrine-induced preconstriction) were less pronounced in Ossabaw than in Göttingen minipigs. Neither the differences in baseline diameter nor in baseline developed force of contraction had any effect on the above data, as analyzed in retrospectively matched subsets of mesenteric arteries with a comparable baseline diameter or a comparable baseline developed force of contraction, respectively. Even before the development of metabolic syndrome, vasomotor function is shifted to more vasoconstriction and less vasodilation in Ossabaw minipigs, suggesting a genetic predisposition for the early development of vascular dysfunction. Thus, Ossabaw minipigs may be a suitable model to reflect the human situation of a heterogeneous, genetically determined primordial risk constellation for vascular dysfunction. This work was supported by the German Research Foundation [SFB 1116 B08 to G.H. and P.K.] and the European COST ACTION in CARDIOPROTECTION [CA16225 to G.H. and IG16225 to G.H. and P.K.]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aged HIV-1 Tg26 Transgenic Mice display Vascular Dysfunction with No Alterations in Neurocognitive Function or Cerebral Blood Flow

Although combination antiretroviral therapy (cART) has markedly reduced HIV mortality and increased life expectancy, people living with HIV (PLWH) develop premature cognitive and brain aging, classified as HIV-associated neurocognitive disorder (HAND). Vascular dysfunction and expression of HIV-derived proteins have independently been associated with cognitive impairment without causality being established. Thus, we investigated the role of HIV viral proteins in endothelial dysfunction and cognitive impairment in an aged mouse model of HIV. We used 18-month-old Tg26 mice and their wild-type littermate controls. Tg26 mice express 7 out of the 9 HIV viral proteins under the control of the long-term repeat promoter and have previously been shown to exhibit signs of cognitive impairment at eight months of age. Conduction of concentration-response curves to acetylcholine in mesenteric arteries using wire myography revealed impaired endothelium-dependent relaxation in Tg26 mice compared to WT mice. Measurements of brain perfusion with arterial spin labeling magnetic resonance imaging showed no differences in the whole brain and hippocampal cerebral blood flow. In addition, there were no changes in the hippocampus volume, which has been reported to be reduced with cognitive impairments. Two methods were employed to assess the cognitive abilities of the mice: Morris water maze (MWM) and the IntelliCage system. MWM requires human handling while the IntelliCage system is fully automated enabling the testing of mouse cognitive function during their active period and independently of human handling. Tg26 and WT mice underwent ten days of MWM testing and eight days of IntelliCage testing to assess spatial learning and memory. Tg26 mice showed no significant differences in latency to platform, distance swam to platform, or swim velocity when compared to their WT littermates. The IntelliCage results showed no differences in total visits, total visits with nose pokes, or total licks during the reversal learning period. Additionally, there were no significant differences in percent side error or place error indicating that cognitive function is not impaired in Tg26 mice. The Y-maze test further showed no differences in spontaneous alteration, a measure of spatial working memory. Altogether, our data shows that although 18-month-old female Tg26 mice develop endothelial dysfunction, they display no alterations in cerebral blood flow and no cognitive impairment indicating that systemic viral protein expression and vascular dysfunction might minimally contribute to neurocognitive impairment in HIV. R01HL130301 & R01HL155265 (E.J.B). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Arterial Stiffness is an early vascular complication in menopause associated with high levels of asprosin

Background: Menopause is a biological phenomenon experienced by women on average at 51 years of age. The negative impact of menopause on the health of women left without treatment is now recognized but still remains understudied. Menopause is characterized by a decline in ovarian function and estrogen levels that is associated with weight gain and an increased risk of cardiovascular events. Because cardiovascular disease clinically presents 7-10 years following menopause, research on the early onset of cardiovascular events in menopause is urgently needed. Asprosin has been recently identified as an adipokine secreted by white adipose tissue and its levels are increased in obesity. Whether asprosin production is increased in menopause remains unknown. We hypothesize that arterial stiffness is an early vascular complication and is associated with high levels of asprosin in menopause. Methods: 8-week-old female C57BL/6 mice were randomized into two experimental groups: Control Group (n=9) and Ovariectomized (OVX) Group (n=8). The OVX group received surgical ovariectomies and the Control Group underwent surgery without ovary removal. The experimental protocol spanned 22 weeks. Carotid-femoral and intracarotid pulse wave velocity (PWV) measurements, the gold standard to measure arterial stiffness, were obtained. At terminal experiments, thoracic aortas were isolated and mounted on a wire myograph for vascular reactivity studies. Acetylcholine and sodium nitroprusside concentration-response curves were used to assess endothelium-dependent and endothelium-independent relaxation, respectively. Gonadal white adipose tissue (gWAT) was collected for further asprosin measurements. Results: The OVX Group exhibited a significant reduction in uterus weight (59.87 ± 19.62 mg vs. 162.8 ± 16.37 mg controls, p<0.05), validating our menopause model. At week 16, the OVX group exhibited a significant increase in carotid-femoral PWV (3.03 ± 0.09 m/s vs. 2.7 ± 0.11 m/s controls, p<0.05) as well as intracarotid PWV (5.09 ± 0.35 m/s vs 2.5 ± 0.23 m/s controls, p<0.05), confirming arterial stiffness. No differences in endothelium-dependent and endothelium-independent relaxation were observed between the experimental groups, indicating that the menopausal mice had not developed endothelial dysfunction at week 22. As expected, the OVX group showed a significant increase in body weight (24.7 ± 0.26 g vs. 22.2 ± 0.42 g controls, p<0.05) and BMI (0.27 ± 0.002 kg/m2 vs. 0.24 ± 0.005 kg/m2 controls, p<0.05), confirming obesity. The increased weight of gWAT in the OVX group (0.69 ± 0.15 g vs. 0.26 ± 0.02 g controls, p<0.05) was accompanied by elevated asprosin expression (1.14-fold increase, p<0.05). Conclusion: nt of endothelial dysfunction in menopause. Increased asprosin is novel and represents a potential link between adiposity and arterial stiffness in menopause. Future directions will investigate the relationship between arterial stiffness and hypertension in menopause. NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Managing hypertension using brewing bioactive peptides as angiotensin-converting enzyme inhibitors: Impact on vascular tone through ex vivo assays

BackgroundPeptides derived from brewer’s spent grain (BSG) and yeast (BSY) are promising natural substitutes of synthetic drugs to manage hypertension as inhibitors of angiotensin-converting enzyme (ACE). However, their impact on the vascular function, so far, remains to be verified. Accordingly, we aimed to evaluate the effect of brewing peptides on the arterial vasocontractile response to angiotensin (Ang) I (through vascular ACE-inhibition) to estimate their antihypertensive potential. The impact of the oral route (gastrointestinal digestion, intestinal absorption, and liver metabolism) in brewing peptides was also investigated. MethodsIliac arteries from adult spontaneously hypertensive rats were studied with isometric tension recording. After ensuring the function integrity of the arteries, concentration-response curves to addition of Ang I (10−9-10−6 M) were performed after 30 min incubation with MIX (50:50 mixture of BSG:BSY) or BSG (0.86 mg/mL), or captopril (an ACE-inhibitory drug) or Krebs solution. ResultsMIX peptides (without simulated oral administration) enhanced Ang I-induced vasoconstriction, but lacked vascular effects after simulated oral administration. By contrast, BSG peptides (without simulated oral administration) did not exhibit vascular effects, whereas they promoted a marked decrease in vasocontraction evoked by the generated Ang II after simulation of the oral route, implying their capability to inhibit ACE. Additionally, to elucidate this inhibitory mechanism, Michaelis-Menten and Lineweaver-Burk plots indicated a mixed inhibition type. ConclusionWith simulation of the oral route, BSG peptides were able to inhibit vascular ACE with a similar efficacy as the antihypertensive drug captopril, suggesting the potential to mitigate the burden of hypertension.

An artificial intelligence-based model for predicting reproductive toxicity of bisphenol analogues mixtures to the rotifer Brachionus calyciflorus

The joint toxicity effects of mixtures, particularly reproductive toxicity, one of the main causes of aquatic ecosystem degradation, are often overlooked as it is impractical to test all mixtures. This study developed and evaluated the following models to predict the concentration response curve concerning the joint reproductive toxicity of mixtures of three bisphenol analogues (BPA, BPF, BPAF) on the rotifer Brachionus calyciflorus: concentration addition (CA), independent action (IA), and two deep neural network (DNN) models. One applied mixture molecular descriptors as input variables (DNN-QSAR), while the other applied the ratios of chemicals in the mixtures (DNN-Ratio). Descriptors related to molecular mass were found to be of greater importance and exhibited a proportional relationship with toxic effects. The results indicate that the range of correlation coefficients (R2) between predicted and measured values for various mixture rays by CA and IA models is 0.372 to 0.974 and − 0.970 to 0.586, respectively. The R2 values for DNN-Ratio and DNN-QSAR were 0.841 to 0.984 and 0.834 to 0.991, respectively, demonstrating that models developed by DNN significantly outperform traditional models in predicting the joint toxicity of mixtures. Furthermore, DNN-QSAR not only predicts mixture toxicity but also provides accurate toxicity predictions for BPA, BPF, and BPAF, with R2 values of 0.990, 0.616, and 0.887, respectively, while DNN-Ratio yields values of 0.920, 0.355, and − 0.495. The study also found that the joint effects of mixtures are primarily influenced by the total concentration of the mixtures, and an increase in total concentration shifts the joint effects towards addition. This study introduces a novel approach to predict joint toxicity and analyze the influencing factors of joint effects, providing a more comprehensive assessment of the ecological risk posed by mixtures.

Intracellular pathways of calcitonin gene-related peptide-induced relaxation of human coronary arteries: A key role for Gβγ subunit instead of cAMP.

Calcitonin gene-related peptide (CGRP) is a potent vasodilator. While its signalling is assumed to be mediated via increases in cAMP, this study focused on elucidating the actual intracellular signalling pathways involved in CGRP-induced relaxation of human isolated coronary arteries (HCA). HCA were obtained from heart valve donors (27 M, 25 F, age 54 ±2years). Concentration-response curves to human α-CGRP or forskolin were constructed in HCA segments, incubated with different inhibitors of intracellular signalling pathways, and intracellular cAMP levels were measured with and without stimulation. Adenylyl cyclase (AC) inhibitors SQ22536 + DDA and MDL-12330A, and PKA inhibitors Rp-8-Br-cAMPs and H89, did not inhibit CGRP-induced relaxation of HCA, nor did the guanylyl cyclase inhibitor ODQ, PKG inhibitor KT5823, EPAC1/2 inhibitor ESI09, potassium channel blockers TRAM-34 + apamin, iberiotoxin or glibenclamide, or the Gαq inhibitor YM-254890. Phosphodiesterase inhibitors induced a concentration-dependent decrease in the response to KCl but did not potentiate relaxation to CGRP. Relaxation to forskolin was not blocked by PKA or AC inhibitors, although AC inhibitors significantly inhibited the increase in cAMP. Inhibition of Gβγ subunits using gallein significantly inhibited the relaxation to CGRP in human coronary arteries. While CGRP signalling is generally assumed to act via cAMP, the CGRP-induced vasodilation in HCA was not inhibited by targeting this intracellular signalling pathway at different levels. Instead, inhibition of Gβγ subunits did inhibit the relaxation to CGRP, suggesting a different mechanism of CGRP-induced relaxation than generally believed.

Preferential vasodilator effects of levosimendan in resistance pulmonary arteries in a rodent pulmonary embolism model

Background: We compared the vasoactive effects of levosimendan on isolated conduit pulmonary arteries (CPA) and resistance pulmonary arteries (RPA) versus mesenteric arteries, and we assessed the pulmonary artery (PA) vascular function and the PA vasoactive effects of levosimendan in a rodent pulmonary embolism (PE) model.Methods: One group of male Wistar rats (200-300 g) was killed by decapitation to obtain pulmonary and mesenteric rings. Another group was assigned to a massive PE or saline solution infusion. After euthanasia, mesenteric arteries and CPA (1-2 mm) and RPA (≤0.5 mm) were dissected and cut into 2-3 mm wide rings, recording contractile tension. We obtained the concentration-response curves of cumulative doses of levosimendan on pre-contracted arterial rings from decapitated and sham/embolized animals. A set of RPA rings was exposed to acute hypoxia. The effect of PE on the pulmonary vasoactive function was assessed by dose-response curves of acetylcholine (ACh) and endothelin-1 (ET-1) of PA rings from sham/embolized animals.Results: Levosimendan relaxant potency of RPA was similar to that of mesenteric arteries and higher than CPA, while mesenteric rings showed the maximal relaxant effect, followed by RPA and CPA, respectively. PE did not affect the vasoactive response of PA rings either to ACh or to ET-1, and the relaxant effects of CPA and RPA to levosimendan were also preserved. Acute hypoxia reduced (p<0.05) but did not avoid the RPA relaxant effect of levosimendan.Conclusions: Levosimendan is a more specific vasodilator of RPA with a similar relaxant potency as mesenteric arteries, which is preserved after PE but significantly reduced during hypoxia.

Mosapride stimulates human 5-HT4-serotonin receptors in the heart.

Mosapride (4-amino-5-chloro-2-ethoxy-N-[[4-[(4-fluorophenyl) methyl]-2-morpholinyl]-methyl] benzamide) is a potent agonist at gastrointestinal 5-HT4 receptors. Mosapride is an approved drug to treat several gastric diseases. We tested the hypothesis that mosapride also stimulates 5-HT4 receptors in the heart. Mosapride increased the force of contraction and beating rate in isolated atrial preparations from mice with cardiac overexpression of human 5-HT4-serotonin receptors (5-HT4-TG). However, it is inactive in wild-type mouse hearts (WT). Mosapride was less effective and potent than serotonin in raising the force of contraction or the beating rate in 5-HT4-TG. Only in the presence of cilostamide (1μM), a phosphodiesterase III inhibitor, mosapride, and its primary metabolite time dependently raised the force of contraction under isometric conditions in isolated paced human right atrial preparations (HAP, obtained during open heart surgery). In HAP, mosapride (10μM) reduced serotonin-induced increases in the force of contraction. Mosapride (10µM) shifted the concentration-response curves to serotonin in HAP to the right. These data suggest that mosapride is a partial agonist at 5-HT4-serotonin receptors in HAP.

TRPV4 Activation during Guinea Pig Airway Smooth Muscle Contraction Promotes Ca2+ and Na+ Influx.

Airway smooth muscle (ASM) contraction is determined by the increase in intracellular Ca2+ concentration ([Ca2+]i) caused by its release from the sarcoplasmic reticulum (SR) or by extracellular Ca2+ influx. Major channels involved in Ca2+ influx in ASM cells are L-type voltage-dependent Ca2+ channels (L-VDCCs) and nonselective cation channels (NSCCs). Transient receptor potential vanilloid 4 (TRPV4) is an NSCC recently studied in ASM. Mechanical stimuli, such as contraction, can activate TRPV4. We investigated the possible activation of TRPV4 by histamine (His)- or carbachol (CCh)-induced contraction in guinea pig ASM. In single myocytes, the TRPV4 agonist (GSK101) evoked an increase in [Ca2+]i, characterized by a slow onset and a plateau phase. The TRPV4 antagonist (GSK219) decreased channel activity by 94%, whereas the Ca2+-free medium abolished the Ca2+ response induced by GSK101. Moreover, GSK101 caused Na+ influx in tracheal myocytes. GSK219 reduced the Ca2+ peak and the Ca2+ plateau triggered by His or CCh. TRPV4 blockade shifted the concentration-response curve relating to His and CCh to the right in tracheal rings and reduced the maximal contraction. Finally, the activation of TRPV4 in single myocytes increased the Ca2+ refilling of the SR. We conclude that contraction of ASM cells after stimulation with His or CCh promotes TRPV4 activation, the subsequent influx of Ca2+ and Na+, and the opening of L-VDCCs. The entry of Ca2+ into ASM cells via TRPV4 and L-VDCCs contributes to optimal smooth muscle contraction.

Use of a Microfluidic Platform To Evaluate and Predict Reagent Performance in Microtiter Plate-Based Immunoassays.

Selection and characterization of antibodies are critically important in establishing robust immunoassays to support the development efforts of vaccines. Plate-based ELISA can be time- and resource-intensive to select initial antibody clones or characterize downstream resupply lots while providing limited information regarding the binding characteristics of the antibodies beyond concentration-response curves. This work applied the microfluidic Gyrolab to holistically evaluate immunoassay reagents through analyses of concentration-response curves as well as antibody-antigen interactions visualized in column images and affinity estimates. We exploited the automation capability of the Gyrolab to reduce the resources (time, reagents, and scientists) required for screening and evaluating antibody reagents. Using a flexible semi-universal assay format, we compared antibody clones for selection and resupply lots of sera and monoclonal antibodies in a simple "plug-and-play" manner without antibody modifications. We found that the performance of antibodies in the Gyrolab correlated well with the trends observed in traditional ELISAs, while the Gyrolab provided additional advantages over plate-based assays such as column images of antibody binding and affinity measurements.

Pharmacology of erenumab in human isolated coronary and meningeal arteries: Additional effect of gepants on top of a maximum effect of erenumab.

Multiple drugs targeting the calcitonin gene-related peptide (CGRP) receptor have been developed for migraine treatment. Here, the effect of the monoclonal antibody erenumab on CGRP-induced vasorelaxation was investigated in human isolated blood vessels, as well as the effect of combining erenumab with the small molecule drugs, namely rimegepant, olcegepant, or sumatriptan. Concentration-response curves to CGRP, adrenomedullin or pramlintide were constructed in human coronary artery (HCA) and human middle meningeal artery (HMMA) segments, incubated with or without erenumab and/or olcegepant. pA2 or pKb values were calculated to determine the potency of erenumab in both tissues. To study whether acutely acting antimigraine drugs exerted additional CGRP-blocking effects on top of erenumab, HCA segments were incubated with a maximally effective concentration of erenumab (3μM), precontracted with KCl and exposed to CGRP, followed by rimegepant, olcegepant, or sumatriptan in increasing concentrations. Erenumab shifted the concentration-response curve to CGRP in both vascular tissues. However, in HCA, the Schild plot slope was significantly smaller than unity, whereas this was not the case in HMMA, indicating different CGRP receptor mechanisms in these tissues. In HCA, rimegepant, olcegepant and sumatriptan exerted additional effects on CGRP on top of a maximal effect of erenumab. Gepants have additional effects on top of erenumab for CGRP-induced relaxation and could be effective in treating migraine attacks in patients already using erenumab as prophylaxis.