Cumulative Concentration-response Curves Research Articles

Rapid-Onset, Short-Duration Induction of Colorectal Contractions in Anesthetized, Adult, Male Rats.

Substantial clinical and preclinical evidence indicates that transient receptor potential vanilloid 1 (TRPV1) receptors are expressed on terminals of colorectal chemoreceptors and mechanoreceptors and are involved in various rectal hypersensitivity disorders with common features of colorectal overactivity. These stimulatory properties of TRPV1 receptors on colorectal function suggested that brief stimulation of TRPV1 might provide a means of pharmacologically activating the colorectum to induce defecation in patients with an "unresponsive" colorectum. The current studies explored the basic features of TRPV1 receptor-induced contractions of the colorectum in anesthetized rats with and without acute spinal cord injury (aSCI). Cumulative concentration-response curves to intrarectal (IR) capsaicin (CAP) solutions (0.003%-3.0%) were performed in anesthetized aSCI and spinal intact rats. CAP produced an "inverted U," cumulative concentration-response curve with a threshold for inducing colorectal contractions at 0.01% and a peak response at 0.1% and slight decreases in responses up to 3%. Decreases in responses with concentrations >0.1% are due to a rapid desensitization (i.e., ≤30 minutes) of TRPV1 receptors to each successive dose. Desensitization appeared fully recovered within 24 hours in spinal intact rats. Colorectal contractions were completely blocked by atropine, indicating a reflexogenic activation of parasympathetic neurons, and responses were completely unaffected by a neurokinin 2 receptor antagonist, indicating that release of neurokinin A from afferent terminals and subsequent direct contractions of the smooth muscle was not involved. IR administration of three other TRPV1 receptor agonists produced similar results as CAP. SIGNIFICANCE STATEMENT: Individuals with spinal cord injury often lose control of defecation. Time-consuming bowel programs using digital stimulation of the rectum are used to empty the bowel. This study shows that intrarectal administration of the transient receptor potential vanilloid 1 (TRPV1) receptor agonist, capsaicin, can induce rapid-onset, short-duration colorectal contractions capable of inducing defecation in spinal cord injured and intact rats. Therefore, TRPV1 agonists show promise as potential therapeutics to induce defecation in individuals with neurogenic bowel.

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.

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.

Preventive effects of a nutraceutical mixture of berberine, citrus and apple extracts on metabolic disturbances in Zucker fatty rats.

The prevention of obesity represents a major health and socio-economic challenge. Nutraceuticals are regularly highlighted for their beneficial effects in preventing the metabolic disturbances associated with obesity. However, few studies have described the combined action of nutraceutical mixtures combining polyphenols with alkaloids. The aim of this study was to evaluate the effects of long-term dietary supplementation with a mixture of Berberine, Citrus and Apple extracts (BCA) in the primary prevention of obesity and its metabolic and vascular complications in the obese Zucker rat, a spontaneous model of genetic obesity and insulin resistance. Sixteen 8-week-old obese Zucker male rats were randomly divided into two groups: all rats received oral gavage daily either with water, untreated obese (U-ObZ) or BCA (BCA-ObZ) mixture for thirteen weeks. Morphological and metabolic parameters were measured along the study. Cumulative concentration-response curves to insulin, acetylcholine and phenylephrine were determined on isolated thoracic aorta. Colon permeability measurements were performed using the Ussing chamber technique. Fecal samples collected at the beginning and the end of the protocol were used as a template for amplification of the V3-V4 region of the 16S rDNA genes. BCA supplementation reduced weight gain (p<0.05) and food intake (p<0.05) in the BCA-ObZ group rats compared to the U-ObZ group rats. It also improved glucose tolerance (p<0.001) and decreased fasting insulin and Homeostasis model assessment index (p<0.05). Through ex vivo experiments, the BCA mixture enhanced significantly aortic insulin relaxation (p<0.01), reduced α1-adrenoceptor-mediated vasoconstriction (p<0.01), and decreased distal colon permeability. Moreover, short-chain fatty acid producers such as Bacteroides, Blautia, and Akkermansia were found to be increased by the BCA mixture supplementation. The results showed that a 13-week-supplementation with BCA mixture prevented weight gain and improved glucose metabolism in obese Zucker rats. We also demonstrated that BCA supplementation improved vascular function, colonic barrier permeability and gut microbiota profile.

Abstract P1170: Changes In Endothelin Signaling In A Long-term Rat Coronary Culture Resembles Chronic Coronary Syndrome

Background: Coronary artery disease generates changes in the structure and function of the blood vessels. 48hrs organ culture (mimicking the acute loss of blood flow) of coronary arteries induces the upregulation of contractile endothelin type B (ETB) receptors, resembling the pattern seen in ischemic heart disease. MEK inhibitors (U0126 and Trametinib), can acutely inhibit the upregulation of these receptors, illustrating the importance of this pathway. The 48hrs organ culture model has been proven successful for translation in acute ischemic disease, but arterial changes have not been studied in a long-term chronic model which could be important for chronic coronary syndrome. Purpose: 1) Investigate the changes of ETB receptors after incubation in a long-term optimized medium for organ culture, mimicking the reduction of blood flow in chronic coronary syndrome and 2) determine whether Trametinib, would have treatment effect on the upregulation of ETB receptors in both long term and short term culture. Methods: Rat coronary arteries were incubated in organ culture for 48hrs in DMEM ± trametinib (“acute”) or in a long-term optimized medium for organ culture for 14 days (“chronic”). After the incubation, arteries were mounted stainless steel wires (40 μm) on a Mulvany-Halpern arterial wire myograph and cumulative concentration response curves were made to measure arterial contractility to the ETB receptor specific agonist Sarafotoxin (S6c). Trametinib was added at time 0 (for both the acute and chronic organ culture) or 7 days delayed. Results: For the “acute” culture, we observed a high contractile effect in response to S6c (Emax=136.5±18%). The presence of Trametinib greatly reduced this contractile effect (Emax=10±1.9%) and arteries were comparable to fresh (Emax=0.8±0.7%). The rat coronary arteries in the “chronic” culture maintained their ability to contract in response to potassium, and S6c had a strong contractile effect (Emax=74.64±17%). Interestingly, neither trametinib addition at time 0 nor at day 7 could inhibit the functional upregulation of ETB in the chronic model. Conclusion: Chronic coronary syndrome may require new inhibitors of ETB receptor upregulation. This could be investigated with our new long-term rat coronary artery model.

Trace amine-induced vasoconstriction of human mammary artery and saphenous vein

Sympathomimetic amines, including β-phenylethylamine (PEA), constrict animal blood vessels but their mechanism of action is not now thought to be through α-adrenoceptors and release of noradrenaline but via trace amine-associated receptors (TAARs). This information is not available for human blood vessels. Functional studies were therefore performed on human arteries and veins to establish whether they constrict to PEA and whether any constrictions are adrenoceptor-mediated. Isolated internal mammary artery or saphenous vein rings were set up in Kreb's-bicarbonate solution at 37 ± 0.5 °C gassed with O2:CO2 (95:5) under class 2 containment. Isometric contractions were measured and cumulative concentration-response curves for PEA or the α-adrenoceptor agonist, phenylephrine were established. PEA showed concentration-related contractions. The maximum was significantly greater in arteries (1.53 ± 0.31 g, n = 9) than veins (0.55 ± 0.18 g, n = 10), but not when plotted as % of KCl contractions. PEA showed slowly developing contractions plateauing at 17,3 ± 3.7 min in mammary artery. The reference α-adrenoceptor agonist, phenylephrine, exhibited more rapid onset (peak 5.0 ± 1.2 min) but non-sustained contractions. In saphenous veins, PEA (62.8 ± 10.7%) and phenylephrine (61.4 ± 9.7%, n = 4) displayed the same maximum, but phenylephrine was more potent. The α1-adrenoceptor antagonist, prazosin (1 μM), blocked phenylephrine contractions of mammary arteries but not PEA contractions in either vessel. PEA causes substantial vasoconstriction of human saphenous vein and mammary artery, which explains its vasopressor actions. This response, however, was not mediated via α1-adrenoceptors, but likely due to TAARs. The classification of PEA as a sympathomimetic amine on human blood vessels is therefore no longer valid and requires revision.

MONOCYTE DERIVED CYTOKINES INDUCE ENDOTHELIAL DYSFUNCTION IN PATIENTS WITH PRIMARY ALDOSTERONISM

Objective: Due to aldosterone excess and its pleotropic effects patients with primary aldosteronism (PA) are exposed to high rates of cardiovascular events. Endothelial dysfunction, as a part of vascular disease, is one of the earliest changes related to high cardiovascular risk and monocytes are the main immune cells participating in the development of vascular wall damage. Therefore, we ought to compare endothelial function between PA patients and healthy controls. Next, we wanted to estimate, if monocytes are able to induce endothelial dysfunction solely by their secretome. Design and method: 13 healthy controls and 42 PA patients received flow mediated dilation measurements (FMD). Later, monocytes were isolated from 8 PA patients and 6 healthy individuals and placed in cell culture. 24 hours later supernatants were collected for cytokine measurements using Human Cytokine 17-plex Assay (Bio-rad). Effects of monocyte derived cytokines on endothelial function were assessed using wire Myograph (Danish Myo Technology). Pre-contraction was induced by norepinephrine (1μM) (Sigma) followed by cumulative concentration response curve to carbachol (0.3μM-300μM) (Sigma). Results: FMD was significantly lower among PA patients (6.3(3.8-8.1)% vs 10.5 ± 3%, p &lt; 0.05). Furthermore, monocytes from patients with PA secreted significantly higher concentrations of following cytokines and growth factors than healthy controls: G-CSF, IFN-gamma, TNF-alfa, IL-6, IL-10, IL-8, IL-17, and MCP-1. Especially concentrations of IL-6 (388 (351-580)pg/ml vs 36(4-268)pg/ml, p &lt; 0.05) and TNF-alfa (95(48-135)pg/ml vs 17(5-41)pg/ml, p &lt; 0.05) were much higher among PA patients. Incubation of mouse thoracic aorta with this monocyte secretome for 24 hours revealed stronger vascular contraction than using supernatant from healthy controls. In pre-constricted murine aortas, maximal dilation of 87.9 ± 11% was reached using aortas incubated in PA monocyte secretome whereas a dilation of 90.9 ± 9.1% was reached in controls. Indeed, carbachol EC50 for aortas incubated in monocyte secretome derived from healthy controls was 1.0mkM and 1.9mkM for PA patients. Conclusions: PA patients demonstrate endothelial dysfunction measured by FMD. Moreover, monocytes isolated from these patients are characterized by high secretion of pro-inflammatory cytokines which induce endothelial dysfunction, one of mechanisms triggering vascular dysfunction among these patients.

Effect of Nyctanthes arbor-tristis on cardiovascular parameters and metabolic syndrome in fructose-induced hypertensive rats

Objectives: We investigated the effects of methanolic extract of Nyctanthes arbor-tristis (MNAT) 100, 200 and 400 mg/kg/day post-operative for 6 weeks on ECG, basal mean arterial blood pressure (MABP), heart rate, respiratory rate, vascular reactivity, antioxidant activities of enzyme superoxide dismutase (SOD) and catalase (CAT), levels of thiobarbituric acid reactive substances (TBARS), serum levels of leptin, adiponectin, glucose, triglycerides, cholesterol, uric acid, insulin, sodium and potassium in fructose-fed rats. Materials and Methods: A high-fructose-diet (fructose 10%, w/v) ad libitum for 6 weeks was used to induce hypertension in male Wistar rats (150–200 g). Sixty albino Wistar rats were randomly divided into a group of six, each group containing 10 animals. Group I was considered as normal control which received chow pellets and normal drinking water ad libitum for 6 weeks. Group II received fructose (10%) solution instead of normal drinking water for 6 weeks. Group III received fructose (10%) solution instead of drinking water ad libitum and MNAT at a dose of 100 mg/kg post-operative for 6 weeks. Group IV received fructose (10%) solution instead of drinking water ad libitum and MNAT at a dose of 200 mg/kg post-operative for 6 weeks. Group V received fructose (10%) solution instead of drinking water ad libitum and MNAT at a dose of 400 mg/kg post-operative for 6 weeks. Group VI received fructose (10%) solution instead of drinking water ad libitum and enalapril at a dose of 10 mg/kg post-operative for 6 weeks. Physiological parameters, ECG, heart rate, respiratory rate and blood pressure vascular reactivity to various drugs were measured and recorded by the invasive method. The antioxidant activities of enzyme SOD and CAT, levels of TBARS, along with serum levels of leptin, adiponectin, glucose, triglycerides, cholesterol, uric acid, insulin, sodium and potassium were measured. Cumulative concentration-response curve (CCRC) of Ang II and acetylcholine (Ach) was recorded. Results: MNAT treatment decreased MABP and altered vascular reactivity to various catecholamines. The activities of SOD and CAT enzymes exhibited a considerable increase and the levels of TBARS in the liver were reduced by MNAT treatment. MNAT has shown decrease in the plasma level of triglycerides, cholesterol, insulin and sodium while increase in plasma adiponectin and potassium levels. The CCRC of Ang II was shifted towards the right by MNAT treatment using an isolated strip of rat ascending colon. MNAT treatment increased the contractile characteristics of the rat ascending colon in the CCRC of ACh as compared to the fructose-treated group. MNAT treatment reduced fructose-induced tissue damage due to the consequence of metabolic syndrome (MetS). MNAT is rich in flavonoids and, therefore, has powerful antioxidant properties. The findings show that by battling oxidative stress caused by fructose (10%) and reducing Ang II activity, MNAT may be able to prevent the development of high blood pressure caused by fructose. Conclusion: MNAT has antihypertensive action and reverses MetS in the fructose-induced hypertensive rat model.

Haptoglobin treatment contributes to regulating nitric oxide signal and reduces oxidative stress in the penis: A preventive treatment for priapism in sickle cell disease.

Background: In sickle cell disease (SCD), reduced bioavailability of endothelial NO and cGMP results in reduced expression of phosphodiesterase type 5 (PDE5), thus impairing the penile erection control mechanism and resulting in prolonged penile erection (priapism). In SCD, reduced NO bioavailability is associated with excess plasma hemoglobin due to intravascular hemolysis and increased oxidative stress. Haptoglobin is the plasma protein responsible for reducing plasma hemoglobin levels, but in SCD, haptoglobin levels are reduced, which favors the accumulation of hemoglobin in plasma. Therefore, we aimed to evaluate the effects of haptoglobin treatment on functional and molecular alterations of erectile function, focusing on the contractile and relaxant mechanisms of corpus cavernosum (CC), as well as oxidative stress. Methods: SCD mice were treated with haptoglobin (400 mg/kg, subcutaneous) or vehicle of Monday, Wednesday and Friday for a period of 1 month. Corpus cavernosum strips were dissected free and placed in organ baths. Cumulative concentration-response curves to the acetylcholine, sodium nitroprusside, phenylephrine and KCL, as well as to electrical field stimulation (EFS), were obtained in CC. Protein expressions of eNOS, phosphorylation of eNOS at Ser-1177, nNOS, PDE5, ROCK1, ROCK2, gp91phox, 3-nitrotyrosine, and 4-HNE were measured by western blot in CC. Results: Increased CC relaxant responses to acetylcholine, sodium nitroprusside and electrical-field stimulation were reduced by haptoglobin in SCD mice. Reduced CC contractile responses to phenylephrine and KCl were increased by haptoglobin in SCD mice. Haptoglobin prevented downregulated eNOS, p-eNOS (Ser-1177), PDE5, and ROCK2 protein expressions and reduced protein expressions of reactive oxygen species markers, NADPH oxidase subunit gp91phox, 3-nitrotyrosine and 4-HNE in penises from SCD mice. Haptoglobin treatment did not affect ROCK1 and nNOS protein expressions in penises from SCD mice. Basal cGMP production was lower in the SCD group, which was normalized by haptoglobin treatment. Conclusion: Treatment with haptoglobin improved erectile function due to up-regulation of eNOS-PDE5 expression and down-regulation of the gp91phox subunit of NADPH oxidase and oxidative/nitrosative stress in the penises of SCD mice. Treatment with haptoglobin also increased contractile activity due to up-regulation of ROCK2. Therefore, haptoglobin treatment may be an additional strategy to prevent priapism in SCD.

Effect of selective dopamine D3 and D4 receptor agonists on trunk pulmonary artery vascular reactivity from control and monocrotaline-treated rats.

Dopamine D1-like and D2-like receptors are expressed in the pulmonary arteries, however there is a little information about their effect on vascular tone in pulmonary circulation, even the vascular effect of activation of the dopamine D3 and D4 subtypes in physiological and pathological conditions such as pulmonary hypertension is unknown. The objective of this study was to evaluate the vascular response of trunk pulmonary artery rings from saline and monocrotaline-treated rats in the presence of selective dopamine receptor agonists. In trunk pulmonary artery rings with intact and denuded endothelium, cumulative concentration-response curves were performed for phenylephrine, acetylcholine, and dopamine receptor agonists (apomorphine-D2-like, SKF38393-D1, quinpirole-D2/D3, 7-OH-DPATD3, and PD168077-D4) alone and in the presence of corresponding selective dopamine receptor antagonists (SCH23390-D1, raclopride-D2/D3, U99194 maleate-D3, and L-745,870-D4). Contractile and relaxant effects generated during the activation with phenylephrine and acetylcholine, respectively, were significantly reduced in intact and denuded endothelium trunk pulmonary artery rings from monocrotaline rats in comparison with control rats. All dopamine receptor agonists, except the 7-OH-DPAT, produced significant vascular relaxation in intact trunk pulmonary artery rings precontracted with phenylephrine in both experimental groups. Also, the vascular relaxation of SKF38393, and particularly apomorphine and PD168077 was significant in denuded endothelium trunk pulmonary artery rings from control and monocrotaline groups. Furthermore, the vasorelaxation induced by these dopamine agonists was significantly reduced in pulmonary preparations from monocrotaline-treated rats in comparison to that recorded in preparations from control rats. The effect of dopamine receptor agonists decreased significantly in the presence of the corresponding antagonist in both experimental groups. The results support that dopamine D4 receptor agonist induces significant vascular relaxation, whereas dopamine D3 receptor agonist induces vasoconstriction in intact and denuded endothelium trunk pulmonary artery rings in control and monocrotaline-induced pulmonary arterial hypertension rats.

Post‐menopausal Cerebral Microvascular Alterations in a Mouse Model of Alzheimer’s Disease

Cognitive decline, as seen in Alzheimer’s disease (AD), is a growing public health concern and is linked to decreased cerebral blood flow, particularly in women after menopause. Impaired cerebrovascular function precedes the onset of AD, possibly due to reduced endothelial function in parenchymal arterioles, the bottlenecks of the cerebral microcirculation, although exact mechanisms by menopause remain elusive. The goal of this study was to determine whether menopause impairs endothelial function in parenchymal arterioles of wild‐type (WT) and 5x‐FAD mouse model of AD. Menopause was induced by the 4‐vinylcyclohexene diepoxide (VCD) model, which induces gradual ovarian failure. Cerebral parenchymal arterioles were isolated and studied by pressure myography. Data are means ± SEM. In WT mice, menopause caused a significant increase in myogenic tone (myogenic tone: 24.73 ± 1.2 vs. 34 ± 2.6%, vehicle vs VCD, n = 5 / 8; p&lt;0.05), as well as a small but significant reduction in resting lumen diameter (32.68 ± 1.3 vs. 28.40 ± 2.1 µm, vehicle vs VCD, n = 5 / 8; p&lt;0.05). We then tested endothelial function, focusing on K+ channels, namely small and intermediate Ca2+‐activated K+ channels (SKCa / IKCa) and inwardly rectifying K+ channels (KIR2). Menopause did not affect arteriolar dilation to a cumulative concentration‐response curve of the SKCa/ IKCa activator NS‐309 (at 1 µM, vasodilation: 23.49 ± 11.5 vs. 17.80 ± 1.7%, vehicle vs VCD, n = 3 / 6). Similarly, KIR2 function was unchanged (vasodilation: 16.06 ± 6.6 vs. 16.21 ± 2.9%, vehicle vs VCD, n = 4 / 8). In the 5x‐FAD mice, menopause did not affect myogenic tone (myogenic tone: 23.49 ± 2.2 vs. 28 3.2%, vehicle vs VCD, n = 10 / 7), although there was a significant decrease in resting lumen diameter (39.16 ± 2.6 vs. 27.79 ± 2.0 µm, vehicle vs VCD, n = 10 / 7 p&lt;0.05). Arterioles from menopausal 5x‐FAD mice showed a significantly blunted response to NS‐309 (at 1 µM, vasodilation: 14.96 ± 2.9 vs. 9.42 ± 1.4%, vehicle vs VCD, n = 7 / 4, p&lt;0.05), without differences in KIR2‐induced dilation (vasodilation: 16.02 ± 1.3 vs. 13.69 ± 2.3%, vehicle vs VCD n = 9 / 6). In conclusion, we found that in WT mice, menopause leads to changes in myogenic tone without affecting K+‐dependent dilation. However, in 5x‐FAD mice, menopause impairs endothelial SKCa/IKCa channels independent of KIR2 function or myogenic tone. These findings identify new targets modulated by menopause in the cerebral microvasculature of WT and AD mice, which may contribute to cognitive dysfunction.

Mice with Brain‐Specific Deletion of Hsd11b2 have an impaired renal vascular and tubular response to high salt intake

Loss‐of‐function mutations in Hsd11b2, the gene encoding the cortisol‐metabolising enzyme 11βHSD2, causes the hypertensive syndrome of Apparent Mineralocorticoid Excess. In affected individuals, overactivation of the mineralocorticoid receptor by cortisol is causative. Hypertension is salt‐sensitive, attributed to enhanced sodium reabsorption in the distal nephron and renal sodium retention. However, in the brain, 11βHSD2‐expressing neurons regulate salt appetite. We previously generated mice with conditional knockout of Hsd11b2 in the brain (Hsd11b2.BKO), which displayed enhanced salt preference and salt‐sensitive hypertension (DOI: 10.1161/CIRCULATIONAHA.115.019341). The mechanisms of salt‐sensitivity are unknown. In the current study we examined renal artery vasoreactivity and the in vivo acute pressure natriuresis response in male Hsd11b2.BKO mice and control littermates.Mice (n=10 per genotype/diet) were fed either high salt (3% Na) or 0.3% Na diet for 7 days. Animals were humanely killed, the renal arteries were isolated and mounted on a wire‐myograph to generate cumulative concentration‐response curves to phenylephrine and the nitric oxide donor, sodium nitroprusside. High salt diet significantly increased the sensitivity of the renal artery to phenylephrine‐induced vasoconstriction in Hsd11b2.BKO mice (LogEC50 0.3% diet ‐6.41±0.07 vs high salt ‐6.91±0.11; p=0.0013) but not in control mice. Sodium nitroprusside induced concentration‐dependent relaxation of renal arteries: the maximum relaxation was significantly reduced from 88±3% to 65±4% in Hsd11b2.BKO after high salt feeding (p&lt;0.01); this was not seen in control mice. In other experiments, mice (n=5‐7 per genotype/diet) were anesthetised (thiobutabarbital sodium, 120mg/kg IP) and fractional renal sodium excretion was measured at baseline and following acutely increased blood pressure, achieved by sequential arterial ligation. In all mice, sodium excretion increased with blood pressure. In the high salt group, the pressure natriuresis response was significantly attenuated in Hsd11b2.BKO mice, compared to control animals.We find that genetic amplification of mineralocorticoid signalling in the brain attenuates the normal renal vascular and tubular adaptation to high salt intake. This regulation of renal salt excretion by the central nervous system may have implications for salt‐sensitive hypertension in humans.

Moderate-intensity exercise training reduces vasorelaxation of mesenteric arteries: role of BKCa channels and nitric oxide.

Exercise training (ET) is well established to induce vascular adaptations on the metabolically active muscles. These adaptations include increased function of vascular potassium channels and enhanced endothelium-dependent relaxations. However, the available data on the effect of ET on vasculatures that normally constrict during exercise, such as mesenteric arteries (MA), are scarce and not conclusive. Therefore, this study hypothesized that 10 weeks of moderate-intensity ET would result in adaptations towards more vasoconstriction or/and less vasodilatation of MA. Young Fischer 344 rats were randomly assigned to a sedentary group (SED; n=24) or exercise training group (EXE; n=28). The EXE rats underwent a progressive treadmill ET program for 10 weeks. Isometric tensions of small (SED; 252.9+/-29.5 microm, EXE; 248.6+/-34.4 microm) and large (SED; 397.7+/-85.3 microm, EXE; 414.0+/-86.95 microm) MA were recorded in response to cumulative phenylephrine concentrations (PE; 0-30 microM) in the presence and absence of the BKCa channel blocker, Iberiotoxin (100 nM). In another set of experiments, tensions in response to cumulative concentration-response curves of acetylcholine (ACh) or sodium nitroprusside (SNP) were obtained, and pEC50s were compared. Immunoblotting was performed to measure protein expression levels of the BKCa channel subunits and eNOS. ET did not alter the basal tension of small and large MA but significantly increased their responses to PE, and reduced the effect of BKCa channels in opposing the contractile responses to PE without changes in the protein expression level of BKCa subunits. ET also elicited a size-dependent functional adaptations that involved reduced endothelium-independent and endothelium-dependent relaxations. In large MA the sensitivity to SNP was decreased more than in small MA suggesting impaired nitric oxide (NO)-dependent mechanisms within the vascular smooth muscle cells of ET group. Whereas the shift in pEC50 of ACh-induced relaxation of small MA would suggest more effect on the production of NO within the endothelium, which is not changed in large MA of ET group. However, the eNOS protein expression level was not significantly changed between the ET and SED groups. In conclusion, our results indicate an increase in contraction and reduced relaxation of MA after 10 weeks of ET, an adaptation that may help shunt blood flow to metabolically active tissues during acute exercise.

Chronic Effects of Ziprasidone, Aripiprazole, Blonanserin, Cyamemazine, and Nemonapride on Mice Isolated Detrusor Smooth Muscle

Objective: Ziprasidone, aripiprazole, blonanserin, cyamemazine, and nemonapride are atypical antipsychotic drugs used for the treatment of schizophrenia. This study aimed to identify the effects of these atypical antipsychotic drugs in mice isolated bladder using the organ bath systemMaterials and Methods: The mice were injected intraperitoneally with drugs for 21 days. The effects of drugs were investigated on isoproterenol-induced relaxation responses of carbachol-induced contractions in isolated detrusor strips. The detrusor strips were stimulated with KCl, then tissues were washed for a further 30 min and precontracted with a submaximal concentration of carbachol. After the contraction reached a plateau, cumulative concentration-response curves to isoproterenol were obtained.Results: We showed that carbachol-induced contractions dose-dependently relaxed by isoproterenol in mice detrusor strips obtained from ziprasidone, aripiprazole, blonanserin, and cyamemazine but not nemonapride treated group. However, none of the drug treatments had any effect KCl responses of mice's bladder.Conclusion: Ziprasidone, aripiprazole, blonanserin, and cyamemazine but not nemonapride increased the isoproterenol-induced relaxations of the detrusor smooth muscle indicates that it can increase the bladder capacity. We demonstrate that four drugs may represent a potential treatment for overactive bladder. They might be clinically useful for the treatment of overactive bladder in patients that should use antipsychotics.

Vasomotor effects of noradrenaline, 5-hydroxytryptamine, angiotensin II, bradykinin, histamine, and acetylcholine on the bat (Rhinolophus ferrumequinum) basilar artery

The responsiveness of the basilar artery to intrinsic vasoactive substances is species-specific and can be a unique characteristic. We investigated the responsiveness of the bat (Rhinolophus ferrumequinum) basilar artery to noradrenaline (NA), 5-hydroxytryptamine (5-HT), angiotensin (Ang) II, bradykinin (BK), histamine (His), and acetylcholine (ACh). NA, 5-HT, Ang II, and BK induced contraction, whereas His and ACh induced relaxation, in a concentration-dependent manner. The NA cumulative concentration–response curve was shifted to the right in parallel with phentolamine (an α-antagonist). However, propranolol, a β-antagonist, had no significant effect. The 5-HT curve was shifted to the right in parallel by ketanserin (a 5-HT2 antagonist) and methiothepin (a 5-HT1 and 5-HT2 antagonist). Losartan (an AT1 antagonist) shifted the Ang II curve to the right, whereas PD123319 (an AT2 antagonist) had no significant effect. L-NA, indomethacin, and des-Arg9-[Leu8]-BK (a B1 antagonist) did not significantly affect BK-induced contractions. HOE140 (a B2 antagonist) shifted the BK concentration–response curve to the right. The His curve was shifted to the right weakly by diphenhydramine (an H1 antagonist) and strongly by cimetidine (a H2 antagonist). ACh-induced relaxation was significantly inhibited by L-NA, atropine, and pFHHSiD (a muscarinic M3 antagonist), whereas pirenzepine and methoctramine (muscarinic M1 and M2 antagonists, respectively) showed no significant effects. At a resting vascular tone, L-NA-induced contraction and indomethacin induced relaxation. These results suggest that α-adrenergic, 5-HT1, 5-HT2, AT1, and B2 receptors might be important in arterial contraction, whereas M3 and H2 (>H1) receptors might modify these contractions, inducing relaxation.

Perivascular Adipose Tissue Media Explant Impairs the ex‐vivo Endothelial Function in Obese Mice Exposed to Early Life Stress via the Mineralocorticoid Receptor signaling

Perivascular adipose tissue (PVAT) is no longer exclusively linked to energy storage; it releases hormonal factors that may influence vascular function and other metabolic processes. Maternal Separation and Early Weaning (MSEW) is a mouse model that mimics the effect of early life stress on cardiovascular and metabolic systems and increases the risk of obesity, hypertension, and eventually, cardiovascular disease. We have previously shown that female MSEW mice present exacerbated obesogenic response to chronic high fat diet (HFD), showing a metabolic syndrome-like phenotype and associated hypertension. Additionally, female obese MSEW mice display an increase in plasma aldosterone and gonadal White Adipose Tissue (gWAT) mineralocorticoid receptor (MR) expression. Therefore, this study aimed to test the influence of PVAT-derived factors on the endothelial function of female MSEW mice fed a HFD, treated chronically with a vehicle or an MR antagonist. Female C57BL/6J mice pups were separated from their dams Postnatal Day (PD) 2 to 16 and weaned early on PD 17. Control litters remained unhandled with their dams and were weaned on PD 21. After weaning, Control and MSEW mice were placed on HFD (60% Kcal from fat) for 16 weeks. Then, mice were euthanized, thoracic aortas were isolated and cleaned for vascular reactivity studies, and PVAT was collected in DMEM (2% BSA, 2-hour incubation, 37C). Cumulative concentration response (CCR) curves were performed for acetylcholine (Ach, 10-5 to 10-9 M) and sodium nitroprusside (SNP, 1x10-6 to 1x10-14 M) after pre-constriction with serotonin (2×10-3 M, 5 ul). Experiments were performed in mice treated with vehicle (50% Ora Swift in drinking water) or spironolactone (100 mg/kg/day, two weeks). A first CCR curve was performed after isolated rings were incubated with DMEM, followed by a second CCR curve following a PVAT media explant incubation. In isolated rings of vehicle-treated mice incubated with DMEM, Ach-induced Vascular relaxation was similar in control and MSEW mice (50.15±4.7 vs. 49.8±1.6 % Relaxation, respectively). However, PVAT preincubation impaired vascular relaxation in MSEW mice compared to controls (37.3±3.4 vs. 49.6±1.3 % Relaxation, p<0.05). Overall, spironolactone-treated mice showed improved vascular relaxation in isolated rings of MSEW and control mice (57.8±3.5 vs. 64.1±1.4 % Relaxation). Furthermore, Spironolactone enhanced the endothelial function in rings from MSEW mice pre-incubated with PVAT media explant compared to controls (57.18±5.1 vs. 57.20±4.8 % Relaxation). No differences in SNP-induced relaxation were found between groups. Treatment with an MR antagonist restores the endothelial function in female MSEW mice fed a HFD. These findings suggest that PVAT-derived factors from female mice exposed to MSEW have a detrimental effect on the vascular function mediated by MR signaling. Further investigation is needed to pinpoint the role of PVAT on the mechanism contributing to the worsening of the vascular function via the MR signaling in obese female mice exposed to early life stress.

Investigation of the vascular effects of short chain fatty acids in rat mesenteric resistance arteries

Fiber rich diets have been shown to prevent the onset and reduce severity of hypertension1, a condition affecting 20-30% of adults in North America. High fiber diets facilitate the production of short chain fatty acids (SCFAs) by gut bacteria of which, in humans, acetate is the most abundant, followed by propionate and butyrate. It has been proposed that these three SCFAs may have direct effects on blood vessels and so may contribute to regulation of blood pressure2. Further research is needed to determine the mechanisms underlying the vascular actions of SCFAs. We tested the hypothesis that SCFAs can induce vasodilation and limit nerve-evoked vasoconstriction in rat mesenteric vasculature. Direct effects on vascular tone were assessed by constructing cumulative concentration-response curves to SCFAs in isolated third order rat mesenteric arteries mounted in a wire myograph and pre-stimulated with phenylephrine (PE) to increase tone. The effects of SCFAs on nerve-evoked vasoconstriction were assessed by constructing frequency-responses curves to perivascular nerve stimulation in a perfused rat mesenteric bed in the presence and absence of SCFAs added to the perfusate. In both experimental approaches, pharmacological tools were used to investigate the mechanism of action of SCFAs. Propionate (3 μM - 1 mM) and acetate (0.3 μM - 1 mM) caused reductions in PE-evoked tone with maximum relaxation of 32.7 ± 7% and 31.6 ± 8.2%, respectively, and attenuated nerve-evoked vasoconstriction in mesenteric arteries at 10 mM - 60 mM propionate and 30 mM - 50 mM acetate. These effects were inhibited by blockers of nitric oxide (NO) synthase (L-NG-nitroarginine methyl ester, L-NAME, 100 μM) and endothelial Ca2+-activated K+ (KCa) channels (apamin, 50 nM; TRAM-34, 1 μM). L-NAME alone significantly inhibited relaxations elicited by acetate indicating a predominant role for NO, whereas the actions of propionate were significantly inhibited by apamin and TRAM-34, indicating a predominant role for endothelial KCa channels in this response. These data indicate that SCFAs may cause vasodilation in mesenteric resistance arteries via activation of endothelial pathways and that there may be a differential contribution of NO and KCa channels to responses of individual SCFAs. 1. Appel LJ, Moore TJ, Obarzanek E, et al. A clinical trial of the effects of dietary patterns on blood pressure. DASH Collaborative Research Group. N Engl J Med. 1997;336(16):1117-1124. doi:10.1056/NEJM199704173361601 2. Pluznick J. A novel SCFA receptor, the microbiota, and blood pressure regulation. Gut Microbes. 2014;5(2):202-207. doi:10.4161/gmic.27492

Soluble guanylate cyclase chronic stimulation effects on cardiovascular reactivity in cafeteria diet-induced rat model of metabolic syndrome

Metabolic syndrome is linked to an increased risk of cardiovascular complications by a mechanism involving mainly decreased nitric oxide (NO) bioavailability and impaired NO-soluble guanylate cyclase (sGC)- cyclic guanosine monophosphate (cGMP) signalling (NO-sGC-cGMP). To further develop this scientific point, this study aimed to investigate the effects of long-term treatment with BAY 41–2272 (a sGC stimulator) on cardiovascular reactivity of spontaneously hypertensive rats (SHR) as a model of metabolic syndrome. SHR were randomly divided into 3 groups: control group, cafeteria diet (CD)-fed group and CD-fed group treated daily with BAY 41–2272 (5 mg/kg) by gastric gavage for 12 weeks. In vivo measurements of body weight, abdominal circumference, blood pressure and glucose tolerance test were performed. At the end of the feeding period, ex vivo cumulative concentration-response curves were performed on isolated perfused heart (isoproterenol (0.1 nM - 1 μM)) and thoracic aorta (phenylephrine (1 nM–10 μM), acetylcholine (1 nM–10 μM), and sodium nitroprusside (SNP) (0.1 nM–0.1 μM)). We showed that chronic CD feeding induced abdominal obesity, hypertriglyceridemia, glucose intolerance and exacerbated arterial hypertension in SHR. Compared to control group, CD-fed group showed a decrease in β-adrenoceptor-induced cardiac inotropy, in coronary perfusion pressure and in aortic contraction to phenylephrine. While relaxing effects of acetylcholine and SNP were unchanged. BAY 41–2272 long-term treatment markedly prevented arterial hypertension development and glucose intolerance, enhanced the α1-adrenoceptor-induced vasoconstriction, and restored cardiac inotropy and coronary vasodilation. These findings suggest that BAY 41–2272 may be a potential novel drug for preventing metabolic and cardiovascular complications of metabolic syndrome.

Tridax procumbens leaf extract elicits relaxation of rat mesenteric artery by activation of Na+ -K+ ATPase / Na+ - Ca2+ exchanger

Background: Tridax procumbens leaf extract has been reported to show antihypertensive actions through its vasodilatory effects. Although several mechanisms of action have been reported, the sub-cellular mechanism of action of the extract is yet to be fully elucidated. Aim and Objectives: In this study, we investigate the involvement of Na+ - K+ -pump and Na+- Ca2+ exchanger in the vasorelaxant activities of T. procumbens extract (TPE) in isolated rat superior mesenteric arteries. Materials and Methods: Superior mesenteric artery isolated from healthy, young adult Wistar rats (250–300 g) and pre-contracted with phenylephrine (PE) (10-7M) were treated with various concentrations of aqueous extract of TPE (0.5–9 mg/mL). The changes in arterial tension were recorded and interaction between TPE with the selective blocker of Na+ - K+ ATPase (ouabain) and putative Na+-Ca2+ exchanger inhibitor (NiCl2) was evaluated. Results: The result indicated that TPE significantly inhibit the cumulative concentration response curves to PE (10-9 - 10-5 M) in a concentration-dependent manner. Pre-treatment with both ouabain and nickel chloride (NiCl2), abolished the vasorelaxant responses of TPE. Furthermore, TPE reduced the relaxant effect with decreased extracellular Na+ concentration. TPE-induced vasorelaxation was completely abolished in a Na+ -free solution, a condition that eliminates the influence of the forward mode of the exchanger. Conclusion: The results provide direct hypothetical evidence that the activation of Na+ - K+ ATPase and stimulation of the forward mode of the Na+-Ca2+ exchanger contribute to the vasorelaxation induced by TPE in mesenteric artery.

In Vitro Contraction of Isolated Cauda Epididymal Duct Smooth Muscle as a Complimentary Approach to Physiological, Pathological, Toxicological, and Pharmacological Studies on Epididymal Function.

Contraction of cauda epididymal duct (CE) smooth muscle is one of the very first events of the seminal emission phase of ejaculation. The contraction of CE smooth muscle is governed by a complex interaction of hormones, autacoids, and by the neurotransmitters released from the epididymal intramural nerve endings, and any impairment in the CE smooth muscle contraction has the potential to impair male fertility. Apart the obvious pathophysiological and toxicological importance of CE smooth muscle contraction, modulation of CE contraction has pharmaceutical interest offering a druggable target to development of drugs to improve/impair male fertility. The in vitro contraction experiments constitute a valuable approach to an in-depth evaluation of functional and molecular changes resulting from pathologies or drug exposure. Therefore, this chapter consists in a description of in vitro pharmacological reactivity contractility of the epididymal duct in a controlled medium, maintained at 30°C of temperature and continuously bubbled with 95% O2 and 5% CO2 to obtain cumulative concentration-response curves that has been fundamental to some of our investigations on epididymal physiology, toxicology, and pharmacology.