Isometric Tension Research Articles

Artificial sweeteners sucralose and acesulfame potassium: A bittersweet story for the cardiovascular system?

Artificial sweeteners (AS) are well known to activate two categories of receptors; sweet taste receptor with high affinity and bitter taste receptors with low affinity. Interestingly, recent data from our and other groups suggested that these two receptors families are expressed throughout the arterial tree. Therefore, it could be interesting to explore if AS may have an impact on vascular function, and to decipher the role of these taste receptors. The aims of this ongoing work is to investigate whether AS, like sucralose and acesulfame potassium (AceK) (1) exhibit vasomotor effect and (2) in that case determine the underlying mechanisms. Biomolecular and functional investigations have been performed on isolated rodent aortas, human dermal micro-arteries and omental arteries. Ex vivo vasomotor function was assessed using isometric tension measurements in organ bath systems. We first confirmed that both endothelial and smooth muscle cells from rodent and human arteries express the transcripts encoding for TAS1R2 and TAS1R3; the heterodimer receptor known to be responsible for the sweet taste. Our ex vivo data showed that only high concentrations (about 10 mM) of AceK and sucralose induce vasomotor responses. While AceK exhibited a vasoconstrictive effect, sucralose induced vasorelaxation. Both responses were found to be independent of the endothelium. Pharmacological inhibition (gurmarin and lactisol) and the use of a TAS1R3 KO mice model demonstrated that AS vasomotor effects do not rely on the sweet taste receptors. In contrast, inhibition of TAS2Rs abolished all responses suggesting that bitter taste receptors are more likely responsible for the AS-induced vascular effects. Additionally, our last observations show that the vasoconstrictive effect of AceK is mediated by RhoA/ROCK pathway. These evidences support the idea that AS can alter vasomotor signaling in the smooth muscle cells. These acute effects are probably mediated by the bitter taste receptor family. Their activation at the vascular level as well as the underlying pathways remains poorly understood, and the physiopathological consequences need to be further investigated.

Effects of vasopressin receptor agonists on detrusor smooth muscle tone in young and aged bladders: Implications for nocturia treatment

Purpose:The main goal of this study was to determine the effects of arginine vasopressin (AVP) and desmopressin on bladder contractility and to examine whether the effects of these vasopressin receptor (VR) agonists differ in young versus aged animals. These aims were addressed using urinary bladders from young (3 months) and aged (24 month) female Fischer 344 rats that were isolated and dissected into strips for isometric tension recordings. Bladder strips were exposed to AVP and desmopressin through the perfusate, and tension changes recorded. Results:In young rat bladders, AVP, an agonist at both vasopressin-1 receptors (V1Rs) and vasopressin-2 receptor (V2Rs), concentration-dependently caused contraction of bladder strips with a sensitivity that was greater in young versus aged bladder strips. Removal of the mucosa did not alter the sensitivity of young bladder strips to AVP yet enhanced the AVP sensitivity of aged bladder strips. The differential sensitivity to AVP between young denuded and aged denuded bladder strips was similar. In contrast to AVP, desmopressin (V2R selective agonist) relaxed bladder strips. This response was reduced by removal of the mucosa in young, but not aged, bladder strips. Conclusion:These findings support a direct role for VRs in regulating detrusor tone with V1Rs causing contraction and V2Rs relaxation. In aged bladders, the contractile response to V1R activation is attenuated due to release of a mucosal factor that attenuates V1R-induced contractions. Also in aged bladders, the relaxation response to V2R activation is attenuated by lack of release of a mucosal factor that contributes to V2R-induced relaxation. Thus age-associated changes in the bladder mucosa impair the effects of VRs on bladder tone. Because the V2R signaling system is impaired in the older bladder, administering an exogenous V2R agonist (e.g., desmopressin) could counteract this defect. Thus, desmopressin could potentially increase nighttime bladder capacity through detrusor relaxation in concert with decreased urine production, reducing nocturnal voiding frequency.

An Imidazoline 2 Receptor Ligand Relaxes Mouse Aorta via Off-Target Mechanisms Resistant to Aging.

Imidazoline receptors (IR) are classified into three receptor subtypes (I1R, I2R, and I3R) and previous studies showed that regulation of I2R signaling has neuroprotective potential. In order to know if I2R has a role in modulating vascular tone in health and disease, we evaluated the putative vasoactive effects of two recently synthesized I2R ligands, diethyl (1RS,3aSR,6aSR)-5-(3-chloro-4-fluorophenyl)-4,6-dioxo-1-phenyl-1,3a,4,5,6,6a-hexahydropyrrolo[3,4-c]pyrrole -1-phosphonate (B06) and diethyl [(1-(3-chloro-4-fluorobenzyl)-5,5-dimethyl-4-phenyl-4,5-dihydro-1H-imidazol-4-yl]phosphonate] (MCR5). Thoracic aortas from Oncins France 1 (3- to 4-months-old) and C57BL/6 (3- to 4- and 16- to 17-months-old mice) were mounted in tissue baths to measure isometric tension. In young mice of both strains, MCR5 induced greater relaxations than either B06 or the high-affinity I2R selective ligand 2-(2-benzofuranyl)-2-imidazoline (2-BFI), which evoked marginal responses. MCR5 relaxations were independent of I2R, as IR ligands did not significantly affect them, involved activation of smooth muscle KATP channels and inhibition of L-type voltage-gated Ca2+ channels, and were only slightly modulated by endothelium-derived nitric oxide (negatively) and prostacyclin (positively). Notably, despite the presence of endothelial dysfunction in old mice, MCR5 relaxations were preserved. In conclusion, the present study provides evidence against a functional contribution of I2R in the modulation of vascular tone in the mouse aorta. Moreover, the I2R ligand MCR5 is an endothelium-independent vasodilator that acts largely via I2R-independent pathways and is resistant to aging. We propose MCR5 as a candidate drug for the management of vascular disease in the elderly.

AICAR promotes endothelium-independent vasorelaxation by activating AMP-activated protein kinase via increased ZMP and decreased ATP/ADP ratio in aortic smooth muscle.

AICAR, an adenosine analog, has been shown to exhibit vascular protective effects through activation of AMP-activated protein kinase (AMPK). However, it remains unclear as to whether adenosine kinase-mediated ZMP formation or adenosine receptor activation contributes to AICAR-mediated AMPK activation and/or vasorelaxant response in vascular smooth muscle. In the present study using endothelium-denuded rat aortic ring preparations, isometric tension measurements revealed that exposure to 1mM AICAR for 30min resulted in inhibition of phenylephrine (1μM)-induced smooth muscle contractility by ∼35%. Importantly, this vasorelaxant response by AICAR was prevented after pretreatment of aortic rings with an AMPK inhibitor (compound C, 40µM) and adenosine kinase inhibitor (5-iodotubercidin, 1µM), but not with an adenosine receptor blocker (8-sulfophenyltheophylline, 100µM). Immunoblot analysis of respective aortic tissues showed that AMPK activation seen during vasorelaxant response by AICAR was abolished by compound C and 5-iodotubercidin, but not by 8-sulfophenyltheophylline, suggesting ZMP involvement in AMPK activation. Furthermore, LC-MS/MS MRM analysis revealed that exposure of aortic smooth muscle cells to 1mM AICAR for 30min enhanced ZMP level to 2014.9±179.4picomoles/mg protein (vs. control value of 8.5±0.6; p<0.01), which was accompanied by a significant decrease in ATP/ADP ratio (1.08±0.02 vs. 2.08±0.06; p<0.01). Together, the present findings demonstrate that AICAR-mediated ZMP elevation and the resultant AMPK activation in vascular smooth muscle contribute to vasorelaxation.

A New Method for Characterizing Muscle Shortening Deactivation

A muscle undergoing a cyclical lengthening and shortening contraction pattern requires fast and efficient muscle activation and relaxation. To boost activation rate and increase force levels during shortening, some muscles have evolved stretch activation (SA), a delayed increased in force following rapid muscle lengthening. Equally important is a fast relaxation rate and low force during muscle lengthening. If a muscle incompletely relaxes, its antagonist muscle expends more energy lengthening it, causing decreased power generation and efficiency. Thus, increasing relaxation rate can increase work and power. However, increasing relaxation rate is costly because it typically occurs via ATPases pumping calcium out of the myoplasm. Some flying insect species have greatly reduced this cost in their indirect flight muscle (IFM) by evolving a property known as shortening deactivation (SD), which decreases force levels during a contraction cycle without having to change calcium concentration. SD is a delayed decrease in force following rapid shortening of an active muscle. In IFM, the timing of this force decrease allows the antagonist muscle (DVM) to be lengthened with much less resistance from the agonist muscle (DLM). While there is general appreciation for SD in IFM, there have been few investigations into whether SD is present in other muscle types and if so, how much it benefits these muscles. Even less is known about SD’s mechanism as there have been no investigations into the SD mechanism for any muscle type. To enable mechanistic investigations into SD and quantitatively compare it to SA, we developed a protocol to elicit SA and SD from Drosophila and Lethocerus indirect flight muscles (IFM) and Drosophila jump muscle. All three muscle types exhibited a 4‐phase tension transient in response to a 1% length decrease, with phase 3 defined as SD. Lethocerus IFM presented the greatest SD tension drop, 20 mN/mm2, which was ~1.5‐fold greater than Drosophila jump muscle and ~10‐fold greater than DrosophilaIFM. However, isometric tension in Drosophilajump muscle, 34.3 mN/mm2, was higher than Lethocerus, 18.4 mN/mm2, and DrosophilaIFM, 1.6 mN/mm2. Thus, when normalized to isometric tension, Drosophila IFM exhibited a 118% SD tension decrease. Similarly, SD tension in Lethocerus IFM dropped by 97%, while Drosophilajump muscle decreased by 37%. The same order occurred for normalized phase 3 SA tension: Drosophila IFM displayed the largest gain, 233%, Lethocerus IFM increased by 76%, and Drosophila jump muscle showed only a 11% increase. SD phase 3 occurred a bit earlier than SA phase 3, relative to the length change, for both IFMs; but SD was exceedingly earlier than SA for jump muscle. We suggest that SA and SD evolved together to enable fast and efficient IFM cyclical power generation and may be caused by the same molecular mechanism. However, the SA and SD molecular mechanisms in jump muscle are likely different and might have evolved for an alternate role besides increasing the power output of cyclical muscle contractions.

Testosterone improved the erectile dysfunction induced by anti-cancer agent doxolubicine in rats

ABSTRACT Introduction Many chemotherapeutic agents are administered to cancer patients worldwide. We previously reported at the world meeting on Sexual Medicine 2018 that doxorubicin (DOX) increased the risk of erectile dysfunction (ED) according to our analyses of the US Food and Drug Administration (FDA) Adverse Event Reporting System (AERS) database. We also reported that DOX decreased erectile function in a study using rats. DOX also decreased rat testosterone levels. These studies suggest a need for a new treatment method for ED associated with anticancer drug treatment. Objective To investigate the effect of testosterone administration on DOX-induced ED in rats. Methods Twelve-week-old male Wistar ST rats were separated into Control, DOX, and DOX+T groups. DOX (3 mg/kg) was intravenously administered on days 1, 8, 15, and 22 in the DOX and DOX+T groups. Testosterone undecanoate (25 mg/kg) was administered to DOX+T rats on day 1. Erectile function was tested using ICP measurements, and endothelial function was tested by assessing isometric tension. Oxidative stress (NADPH oxidase), inflammation (IL-6), and smooth muscle factor (α-SMA, Cavin-1, and Cavin-2) mRNA expression levels were tested using real-time PCR. Results After the 4-week observation period, the ICP/MAP ratio in the DOX group (0.32 ± 0.03) was significantly decreased compared to the Control group (0.72 ± 0.05) (P &amp;lt;0.01). The ICP/MAP ratio in the DOX + T group (0.61 ± 0.07) was increased significantly compared to the DOX group (P &amp;lt;0.01). According to isometric tension measurements, reactivity to acetylcholine was significantly lower in the DOX group (35.0 ± 5.1%) than in the control group (77.6 ± 7.9%), whereas reactivity in the DOX + T group (69.8 ± 8.5%) was significantly improved (P &amp;lt;0.05). NADPH oxidase-1 and IL-6 mRNA levels were increased in the DOX group compared to the Control group (P &amp;lt;0.01), and levels were decreased relative to the DOX group in the DOX+T group. α-SMA, Cavin-1, and Cavin-2 mRNA levels were decreased in the DOX group compered to the Control group. These levels were improved in the DOX+T group. Conclusions We have demonstrated in rats that androgen replacement therapy may mitigate ED induced by DOX administration. We hypothesize that administration of testosterone improves smooth muscle function and vascular endothelial cell function by suppressing oxidative stress and inflammatory response in the corpus cavernosum, and maintaining normal levels of caveolin-related factors. When androgen deficiency symptoms are observed due to administration of anticancer drugs such as DOX, androgen replacement therapy is expected to be one viable treatment option. Our study sheds light on possible treatment strategies for improving quality of life for cancer survivors. Disclosure Work supported by industry: no.

Effect of DPP-4 inhibitors on erectile dysfunction caused by vincristine in rats

ABSTRACT Introduction Various chemotherapeutic agents are administered to cancer patients worldwide. Previously, an analysis of the US Food and Drug Administration (FDA) Adverse Event Reporting System (AERS) database showed that vincristine (VCR) increased the risk of erectile dysfunction (ED), as reported in the world meeting on Sexual Medicine 2018. As such, countermeasures for late complications after anticancer drug treatment are required. Neuropathy is a well-known side effect of VCR; however, in recent years, it has been reported that DPP-4 inhibitors improve neuropathy. Objective To investigate the effect of DPP-4 inhibitors to ED caused by VCR in rats. Methods Twelve-week-old male Wistar ST rats were separated into Control, VCR, and VCR+DPP-4i groups. VCR (0.1 mg/kg) was intravenously administered on days 1, 8, 15, and 22 in the VCR and VCR+DPP-4i groups. Two types of long-term preparations, toleragliptin (TGP; 3 mg/kg/week, po.) and omaligliptin (OGP; 3 mg/kg/week, po.), were examined for DPP-4i. Erectile function was tested using intracavernous pharmacotherapy (ICP) measurements, and endothelial function was tested through an isometric tension study. Additionally, mRNA expressions were tested using real-time PCR. Results After the 4-week observation period, the ICP/mean arterial pressure (MAP) ratio in the VCR group (0.42 ± 0.04) was significantly decreased compared to the Control group (0.72 ± 0.05) (P &amp;lt;0.01). The ICP/MAP ratio in the VCR+TGP group (0.64 ± 0.02) increased significantly compared to the VCR group (P &amp;lt;0.05), however the ICP/MAP ratio in the VCR+OGP (0.50 ± 0.08) did not increase significantly compared to the VCR group (P &amp;gt; 0.05). Real-time PCR analysis showed that the catalase, VEGF, MCP-1, and PAI-1 mRNA expressions were increased, while the IL-6 mRNA expression decreased significantly in the VCR+TGP and VCR+OGP groups. In contrast, the nNOS mRNA expression level was higher in the VCR+TGP group than in the VCR+OGP group. Conclusions In this study, we determined that DPP-4 inhibitors may improve ED by VCR administration. For ED associated with VCR administration, long-term DPP-4 inhibitor TGP administration suggested a significant improvement in erectile function, but OGP did not show a significant improvement effect. An increase in antioxidant activity and a decrease in inflammatory cytokines were observed for both drugs, including a surge in vascular increase factors. In contrast, the nNOS expression level was higher in TGP, suggesting that there may be a difference in the amount of NO production via nerves. Therefore, TGP may be useful for male sexual dysfunction associated with vincristine administration. Disclosure Work supported by industry: no.

Role of the High‐Conductance Calcium‐Activated Potassium Chanel in first order mesenteric artery of hypercaloric diet rat model

Obesity has been identified like as a risk factor of metabolic syndrome. The High‐Conductance Calcium‐Activated Potassium Channel (BKCa) in vascular smooth muscle cells regulates membrane potential and control myogenic tone.Objectiveevaluated the role of the BKCa channel in first order mesenteric arteries of rats feed with a hypercaloric diet for 18 and 28 weeks.MethodologyWe performed isometric tension experiments in first and second order mesenteric arteries rings and Patch Clamp recordings of vascular smooth muscle cells isolated of the same vascular bed.ResultsThe rats feed with a hypercaloric diet for 18 weeks shows an augmented un percentage of adipose tissue, triglycerides, glucose tolerance curve, blood pressure and more relaxation to BKCa channel activator NS11021 un rings precontracted with phenylephrine. At 28 weeks of evolution, an increase in percentage of adipose tissue with respect to body weight was observed, as well as an increase in blood pressure, Regarding BKCa channel function, an increase in current density was observed in the experimental group with respect to the control group.ConclusionChanges have been developed at the metabolic level in the groups of the hypercaloric diet, with changes in the BKCa channels, a gain of function in first order mesenteric rings of the 18 weeks group and in cells isolated from these arteries at 28 weeks group. Nevertheless, at 28 weeks of evolution, first order mesenteric artery rings no longer showed the gain in function observed in the previous group, which suggest a compensatory mechanism maybe associated to aging.

Abstract 154: Endothelial Dysfunction In Experimental Celiac Disease Is Mediated By Gut-Derived Vascular Inflammation And Oxidative Stress

Objective: Celiac disease (CeD) is an autoimmune enteropathy triggered by dietary gluten, with a prevalence around 1%. Since many patients show heterogeneous symptoms, it is vastly underdiagnosed and yet without any treatment option besides lifelong gluten restriction. Vascular function in CeD patients compared to healthy controls has been assessed and hypothesized CeD as a cardiovascular risk factor. Herewith we aimed to explore the cardiovascular effects of CeD in a murine model, with special emphasis on endothelial dysfunction, vascular inflammation, and oxidative stress, known triggers for cardiovascular disease. Methods: NOD.DQ8 mice were raised on a zein-based, gluten-free diet. During the experiment, mice received gavage with peptic-tryptic digest of gliadin and were switched to a gluten-containing diet (Gliadin group). A control group (Zein group) was continued on gluten-free diet and received accordingly digested zein. Results: Blood pressure measurements revealed arterial hypertension in the Gliadin group. Endothelium-dependent relaxation was impaired in the Gliadin group compared to Zein, whereas no change in endothelium-independent was observed, tested by isometric tension recordings. Pro-inflammatory genes, like Tnfa and Nox2 were upregulated in the Gliadin group measured by quantitative rtPCR in aortic and intestinal tissue. Infiltration by intraepithelial T-lymphocytes into the epithelium of the small intestine was shown by IHC. In cardiac tissue, gliadin-treatment increased the oxidative stress parameters 3-nitrotyrosine and 4-hydroxy-2-nonenal shown in dot blots. To investigate the inflammatory link between gut and the cardiovascular system plasma proteomics were performed and revealed elevated levels of IL-17A. Conclusion: CeD is a cardiovascular risk factor. However, the mechanism remains unresolved. In a mouse model, we demonstrate that gluten treatment elevates blood pressure and impairs vascular function. Intestinal inflammation conveys to the cardiovascular system, leads to oxidative stress, and thereby impairs endothelial function. A potential mediator might be IL-17A, whose role in other autoimmunity-based cardiovascular risk factors (e.g., psoriasis) has already been demonstrated.

The role of SK3 in progesterone-induced inhibition of human fallopian tubal contraction

BackgroundNormal motor activity of the fallopian tube is critical for human reproduction, and abnormal tubal activity may lead to ectopic pregnancy (EP) or infertility. Progesterone has an inhibitory effect on tubal contraction; however, the underlying mechanisms remain unclear. Small-conductance calcium-activated K+ channel 3 (SK3) is abundantly expressed in platelet-derived growth factor receptor α positive (PDGFRα+) cells and was reported to be important for the relaxation of smooth muscle. The present study aims to explore the expression of SK3 in the human fallopian tube and its role in progesterone-induced inhibition of tubal contraction.MethodsWe collected specimens of fallopian tubes from patients treated by salpingectomy for EP (EP group) and other benign gynecological diseases (Non-EP group). The expression of SK3 was detected by quantitative real-time polymerase chain reaction, western blot, immunocytochemistry, and immunohistochemistry analyses. Isometric tension experiments were performed to investigate the role of SK3 in progesterone-induced inhibition of tubal contraction.ResultsThe baseline amplitude and frequency of human fallopian tube contraction were both statistically lower in the EP group compared with the non-EP group. The expression levels of SK3 in different portions of fallopian tubes from the non-EP group were significantly higher than in those from the EP group. Progesterone had an inhibitory effect on tubal contraction, mainly on the amplitude, in both groups, and SK3 as well as other calcium-activated K+ channels may be involved. SK3-expressing PDGFRα (+) cells were detected in the human fallopian tube.ConclusionsThe expression of SK3 is lower in the EP group, and SK3 is involved in the progesterone-induced inhibition of human fallopian tube contraction.

Sarco/Endoplasmic Reticulum Ca2+ ATPase 2 Activator Ameliorates Endothelial Dysfunction; Insulin Resistance in Diabetic Mice.

Background: Sarco/endoplasmic reticulum Ca2+-ATPase2 (SERCA2) is impaired in various organs in animal models of diabetes. The purpose of this study was to test the effects of an allosteric SERCA2 activator (CDN1163) on glucose intolerance, hepatosteatosis, skeletal muscle function, and endothelial dysfunction in diabetic (db/db) mice. Methods: Either CDN1163 or vehicle was injected intraperitoneally into 16-week-old male control and db/db mice for 5 consecutive days. Results: SERCA2 protein expression was decreased in the aorta of db/db mice. In isometric tension measurements of aortic rings from db/db mice treated with CDN1163, acetylcholine (ACh)-induced relaxation was improved. In vivo intraperitoneal administrations of CDN 1163 also increased ACh-induced relaxation. Moreover, CDN1163 significantly decreased blood glucose in db/db mice at 60 and 120 min during a glucose tolerance test; it also decreased serum insulin levels, hepatosteatosis, and oxygen consumption in skeletal muscle during the early period of exercise in db/db mice. Conclusions: CDN1163 directly improved aortic endothelial dysfunction in db/db mice. Moreover, CDN1163 improved hepatosteatosis, skeletal muscle function, and insulin resistance in db/db mice. The activation of SERCA2 might be a strategy for the all the tissue expressed SERCA2a improvement of endothelial dysfunction and the target for the organs related to insulin resistance.

SKA-31-induced activation of small-conductance calcium-activated potassium channels decreased modulation of detrusor smooth muscle function in a rat model of obesity

Increased excitability and contractility of detrusor smooth muscle (DSM) cells are associated with overactive bladder (OAB), which is often induced by obesity. Small-conductance Ca2+-activated K+ (SK) channels regulate the excitability and contractility of DSM cells. Selective pharmacological activation of SK channels attenuates hyperpolarization and the decreased relaxation effect in DSM cells in obesity-induced OAB. However, additional data are needed to confirm the regulatory effect of SK channels on the function of DSM cells in obesity-related OAB. The tested hypothesis was that activation of SK channels decreases modulation of DSM function in a rat model of obesity-related OAB. Female Sprague Dawley rats were fed a normal diet (ND) or a high-fat diet (HFD), weighed after 12weeks, and subjected to urodynamic study, patch-clamp electrophysiology, and isometric tension recording. The average body weight and incidence of OAB were increased in the HFD group. Patch-clamp studies revealed that pharmacological activation of SK channels with SKA-31 had attenuated hyperpolarization of DSM cells. In addition, isometric tension recordings indicated that SKA-31 decreased relaxation of spontaneous phasic contractions of DSM strips in the HFD group. Attenuated function of SK channels increased the excitability and contractility of DSM cells, which contributed to the occurrence of OAB, suggesting that SK channels are potential therapeutic targets for control of OAB.

Vasodilatory Effect of Alpinia officinarum Extract in Rat Mesenteric Arteries.

Background: Alpinia officinarum (A. officinarum) is known to exhibit a beneficial effect for anti-inflammatory, anti-oxidant, and anti-hyperlipidemic effects. However, no sufficient research data are available on the cardiovascular effect of A. officinarum. Thus, in this study, we investigate whether A. officinarum extract has direct effects on vascular reactivity. Methods: To examine whether A. officinarum extract affects vascular functionality, we measured isometric tension in rat mesenteric resistance arteries using a wire myograph. After arteries were pre-contracted with high-K+ (70 mM), phenylephrine (5 µM), or U46619 (1 µM), A. officinarum extract was treated. Results: A. officinarum extract induced vasodilation in a concentration-dependent manner, and this effect was endothelium independent. To further investigate the mechanism, we incubated arteries in a Ca2+-free and high-K+ solution, followed by the cumulative addition of CaCl2 (0.01–2.5 mM) with or without A. officinarum extract (30 µg/mL). Pre-treatment of A. officinarum extract reduced the contractile responses induced by cumulative administration of Ca2+, which suggests that extracellular Ca2+ influx was inhibited by the treatment of A. officinarum extract. These results were associated with a reduction in phosphorylated MLC20 in VSMCs treated with A. officinarum extract. Furthermore, eucalyptol, an active compound of A. officinarum extract, had a similar effect as A. officinarum extract, which causes vasodilation in mesenteric resistance arteries. Conclusion: A. officinarum extract and its active compound eucalyptol induce concentration-dependent vasodilation in mesenteric resistance arteries. These results suggest that administration of A. officinarum extract could exert beneficial effects to treat high blood pressure.

Aging modifies receptor expression but not muscular contractile response to angiotensin II in rat jejunum

The involvement of renin-angiotensin system in the modulation of gut motility and age-related changes in mRNA expression of angiotensin (Ang II) receptors (ATR) are well accepted. We aimed to characterize, in vitro, the contractile responses induced by Ang II, in jejunum from young (3–6 weeks old) and old rats (≥ 1 year old), to evaluate possible functional differences associated to changes in receptor expression. Mechanical responses to Ang II were examined in vitro as changes in isometric tension. ATR expression was assessed by qRT-PCR. Ang II induced a contractile effect, antagonized by losartan, AT1R antagonist, and increased by PD123319, AT2R antagonist, as well by neural blocker ω-conotoxin and by nitric oxide (NO) synthase inhibitor. No difference in the response was observed between young and old groups. AT1 receptor-mediated contractile response was decreased by U-73122, phospholipase C (PLC) inhibitor; or 2-aminoethoxy-diphenylborate (2-APB), inositol triphosphate (IP3) receptor inhibitor; or nifedipine, l-type calcium channel blocker. Age-related changes in the expression of both AT1 receptor subtypes, AT1a and AT1b, and of AT2 receptors were detected. In conclusion, Ang II modulates the spontaneous contractility of rat jejunum via postjunctional AT1 receptors, involving Ca2+ mobilization from intracellular stores, via PLC/IP3 pathway, and Ca2+ influx from extracellular space, via l-type channels. Prejunctional AT2 receptors would counteract AT1 receptor effects, via NO synthesis. The observed age-related differences in the expression of all AT receptor subtypes are not reflected in the muscular contractile response to Ang II.

Empirical evidence that bone collagen molecules denature as a result of bone fracture

Cortical bone tissue, primarily composed of collagen, hydroxyapatite, and water, is a strong and tough natural, structural biomaterial. The integrity of the collagenous phase (native triple helix vs. damaged/denatured coil) has previously been correlated via various means, including hydrothermal isometric tension testing and FTIR and Raman spectroscopy, with the capability of cortical bone to undergo stable fracture. Collagen is a relatively stable protein, requiring around 70 J/g to thermally denature its native triple helix structure, through the melting of hydrogen bonds. It is widely thought that bone collagen molecules denature (unravel) during fracture, acting as a molecular-scale mechanical toughening mechanism, but this has not been empirically demonstrated to date. A new technology, fluorescently-labeled collagen hybridizing peptides (F–CHP), enables imaging that specifically detects denatured collagen. This provides an opportunity to empirically test whether bone collagen molecules do denature during bone fracture. Here, F–CHP was used to stain fracture surfaces produced by transverse Mode-I fracture of chevron-notched bovine and human cortical bone beams. The fracture surfaces demonstrated increased staining, above the level of rigorous paired controls, and the staining directly correlated with the work-to-fracture (WFx) of bovine bone beams. This increased denaturation signal was also constrained to a rough textured region visible on the fracture surface, which is known to correspond with stable tearing. Similar staining was also detected on the fracture surfaces of human cortical bone. Increased staining was not detected on the fracture surfaces of specimens that were dehydrated prior to fracture, suggesting a role for water in the denaturation process. This study provides the first empirical evidence of bone collagen denaturation resulting from cortical bone fracture and extends our understanding of this mechanism towards the mechanical performance of cortical bone.

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.

Mechanistic Insights into Inorganic Nitrite-Mediated Vasodilation of Isolated Aortic Rings under Oxidative/Hypertensive Conditions and S-Nitros(yl)ation of Proteins in Germ-Free Mice.

The prevalence and clinical importance of arterial hypertension are still growing. Inorganic nitrite (NO2−) represents an attractive dietary antihypertensive agent, but its metabolism and mode of action, which we aimed to investigate with the present study, are not completely understood. Isolated aortic rings from rats were treated ex vivo with oxidants, and rats were infused in vivo with angiotensin-II. Vascular responses to acetylcholine (ACh) and nitrite were assessed by isometric tension recording. The loss of vasodilatory potency in response to oxidants was much more pronounced for ACh as compared to nitrite ex vivo (but not in vivo with angiotensin-II). This effect may be caused by the redox regulation of conversion to xanthine oxidase (XO). Conventionally raised and germ-free mice were treated with nitrite by gavage, which did not improve ACh-mediated vasodilation, but did increase the plasma levels of S-nitros(yl)ated proteins in the conventionally-raised, but not in the germ-free mice. In conclusion, inorganic nitrite represents a dietary drug option to treat arterial hypertension in addition to already established pharmacological treatment. Short-term oxidative stress did not impair the vasodilatory properties of nitrite, which may be beneficial in cardiovascular disease patients. The gastrointestinal microbiome appears to play a key role in nitrite metabolism and bioactivation.

Inhibition of chemically and mechanically activated Piezo1 channels as a mechanism for ameliorating atherosclerosis with salvianolic acid B.

Salvianolic acid B (SalB) is effective for treating cardiovascular diseases. However, the molecular mechanisms underlying its therapeutic effects remain unclear. Mechanosensitive Piezo1 channels play important roles in vascular biology, although their pharmacological properties are poorly defined. Here, we aimed to identify novel Piezo1 inhibitors and gain insights into their mechanisms of action. Intracellular Ca2+ ions were measured in HUVECs, murine liver endothelial cells (MLECs), THP-1 and RAW264.7 cell lines and bone marrow-derived macrophages (BMDMs). Isometric tensions in mouse thoracic aorta were recorded. Shear-stress assays with HUVECs were conducted. Patch-clamp recordings with mechanical stimulation were performed with HUVECs in whole-cell mode. Foam cell formation was induced by treating BMDMs with oxidised LDL (oxLDL). Atherosclerotic plaque assays were performed with Ldlr-/- and Piezo1 genetically depleted mice on a high-fat diet. Salvianolic acid B inhibited Yoda1-induced Ca2+ influx in HUVECs and MLECs. Similar results were observed in macrophage cell lines and BMDMs. Furthermore, we demonstrated that salvianolic acid B inhibited Yoda1- and mechanically activated currents. Salvianolic acid B suppressed Yoda1-induced aortic ring relaxation and inhibited HUVECs alignment in the direction of shear stress. Additionally, Yoda1 enhanced the formation of foam cells, which was reversed by salvianolic acid B. Salvianolic acid B also inhibited formation of atherosclerotic plaques and was insensitive to Piezo1 genetic depletion. Our study provides novel mechanistic insights into the inhibitory role of salvianolic acid B against Piezo1 channels and improves our understanding of salvianolic acid B in preventing atherosclerotic lesions.

In vitro inhibition of phosphodiesterase type 4 enhances rat corpus cavernosum nerve-mediated relaxation induced by gasotransmitters

AimsNitric oxide (NO) and hydrogen sulfide (H2S) are involved in nerve-mediated corpus cavernosum (CC) relaxation. Expression of phosphodiesterase type 5 (PDE5) and type 4 (PDE4), cyclic guanosine monophosphate (cGMP)- and cyclic adenosine monophosphate (cAMP)-specific, respectively, has been described and PDE5- and PDE4-inhibitors induce cavernous smooth muscle relaxation. Whereas the NO/cGMP signaling pathway is well established in penile erection, the cAMP-mediated mechanism is not fully elucidated. The aim of this study is to investigate the localization and the functional significance of PDE4 in rat CC tone regulation. Main methodsWe performed immunohistochemistry for the detection of the PDE4A isoenzyme. Isometric tension recordings for roflumilast and tadalafil, PDE4 and PDE5 inhibitors, respectively, electrical field stimulation (EFS) and β-adrenoceptor agonist isoproterenol and endogenous H2S production measurement. Key findingsA marked PDE4A expression was detected mainly localized in the nerve cells of the cavernous smooth muscle. Furthermore, roflumilast and tadalafil exhibited strong corpus cavernous relaxations. Endogenous H2S production was decreased by NO and H2S synthase inhibitors and increased by roflumilast. Isoproterenol- and EFS-induced relaxations were increased by roflumilast. SignificanceThese results indicate that PDE4A is mainly expressed within the nerves cells of the rat CC, where roflumilast induces a potent corpus cavernous relaxation per se and potentiates the response induced by β-adrenoceptor activation. The fact that roflumilast enhances H2S production, as well as EFS-elicited responses suggests that PDE4 inhibitors modulate, in a positive feedback fashion, nerve-mediated relaxation induced by gasotransmitters, thus indicating a key role for neuronal PDE4 in penile erection.

Dietary Blueberry Ameliorates Vascular Complications in Diabetic Mice Possibly through NOX4 and Modulates Composition and Functional Diversity of Gut Microbes.

In diabetes, endothelial inflammation and dysfunction play a pivotal role in the development of vascular disease. This study investigates the effect of dietary blueberries on vascular complications and gut microbiome in diabetic mice. Seven-week-old diabetic db/db mice consume a standard diet (db/db) or a diet supplemented with 3.8% freeze-dried blueberry (db/db+BB) for 10 weeks. Control db/+ mice are fed a standard diet (db/+). Vascular inflammation is assessed by measuring monocyte binding to vasculature and inflammatory markers. Isometric tension procedures are used to assess mesenteric artery function. db/db mice exhibit enhanced vascular inflammation and reduced endothelial-dependent vasorelaxation as compared to db/+ mice, but these are improved in db/db+BB mice. Blueberry supplementation reduces the expression of NOX4 and IκKβ in the aortic vessel and vascular endothelial cells (ECs) isolated from db/db+BB compared to db/db mice. The blueberry metabolites serum reduces glucose and palmitate induced endothelial inflammation in mouse aortic ECs. Further, blueberry supplementation increases commensal microbes and modulates the functional potential of gut microbes in diabetic mice. Dietary blueberry suppresses vascular inflammation, attenuates arterial endothelial dysfunction, and supports the growth of commensal microbes in diabetic mice. The endothelial-specific vascular benefits of blueberries are mediated through NOX4 signaling.