Chronic stress is associated with hypertension but the underlying mechanisms linking the interplay between the changes in the hypothalamic-pituitary-adrenal axis and vascular dysfunction remain unclear. We hypothesize that chronic unpredictable stress (CUS) induces mitochondrial dysfunction in the HPA axis and in the vasculature, associated with GasderminD pore formation in the mitochondrial membrane. Adult male and female C57Bl6/J mice were exposed to 28 consecutive days of CUS or handling (CTL). Anxiety-like behavior, coping behavior, and systolic blood pressure (SBP) were assessed. Mitochondrial function was assessed using high-resolution respirometry in the hypothalamus, amygdala, and adrenal tissues. The vascular reactivity of mesenteric resistance arteries (MRA), pudendal arteries (PA), and aorta were assessed in response to phenylephrine or acetylcholine. GasderminD protein expression was measured in the MRA. Data are presented as mean ± S.E.M and significance was set at p<0.05. In both sexes, CUS increased SBP by 12.1±4.9%. CUS decreased body weight gain by 53% (p=0.04) and increased anxiety-like behavior (p=0.01) in males (vs CTL) but did not affect females. CUS decreased latency to immobility in the forced swim test by 46% (p=0.0001) in both sexes. Sex-dependent decreases in mitochondrial respiration were observed as follows: in the amygdala of males (20%, p=0.05) and the hypothalamus (20%, p=0.004) and adrenal glands (17%, p=0.06) of females. In the vasculature, CUS led to endothelial dysfunction observed by reduced potency of ACh in the MRA (pEC 50 CUS 6.17±0.09 vs CTL 6.81±0.11; p=0.003) and PA (pEC 50 CUS 6.31 ±0.07 vs CTL 6.65±0.09; p=0.008) of male mice, as well as in the MRA of females (pEC 50 CUS 5.94±0.30 vs CTL 6.98±0.15; p=0.041). Contractile responses to PE were significantly reduced in the aorta of CUS males only (E MAX CTL: 4.70±0.48 mN vs CUS 3.01± 0.23 mN; p=0.007). The expression of gasderminD was increased in the MRA of female mice only. CUS increased SBP in males and females, induced sex-specific changes in the mitochondrial function in key areas of the brain, and caused vascular dysfunction, suggesting that behavioral, brain, and vascular adaptations to CUS are sex-dependent.
Hypertension is a major risk factor for the development of cardiovascular diseases and is a leading cause of mortality. The etiology of essential hypertension remains unclear, and its mechanisms are complex. Inflammation is a complex phenomenon, comprises the first response of the immune system in the presence of harmful stimuli and is considered to be a major cause of increased blood pressure. Indeed, low-grade inflammation and activation of the immune system have been implicated in hypertension. Cytokine levels are elevated in hypertension and have been reported to be implicated in the associated disturbances in vascular reactivity reported in this disease. Clinical and population studies have consistently found increased circulating levels of interleukin 18 (IL-18) in patients with hypertension.This study tests the hypothesis that IL-18 will enhance contractile responses to phenylephrine in isolated arterial rings from rats.Vascular function was performed on isolated pudendal arteries from male Wistar rats using wire myographs, kept in Krebs solution at 37°C, constantly aerated with 95%O2 /5%CO2 . Concentration-response curves to phenylephrine (PE; 10-9 -3x10-5 M), Acetylcholine (ACH; 10-9 - 3x10-5 M) were performed in the absence or in the presence of IL-18 (20ng/ml) for 1 hour. 10 minutes prior to the incubation with IL-18, some arterial rings were incubated separately with the following agents: NADPH oxidase 2 inhibitor, gp91 ds-tat (10-5 M); NADPH oxidase 1 and 4 inhibitor, GKT137831(10-6 M); and mitogen-activated protein kinase (MKK / MEK) inhibitor, PD98059 (10-5 M) Concentration-response curves were analyzed using non-linear regression analysis. Data are presented as mean ± S.E.M. Statistical significance set at p<0.05. Data was analyzed with GraphPad Prism 9.2.0.In the Wistar pudendal arterial rings, the potency of PE was increased in the presence of IL-18 (pEC50 6.510 ± 0.3987 vs 6.022 ± 0.2738 for control; p = 0.0268) A, IL-18 plus gp91 ds-tat (pEC50 6.643 ± 0.2215 vs 6.264 ± 0.2471 for control; p = 0.0058) B and IL-18 plus GKT137831 (pEC50 6.700 ± 0.2937 vs 6.272 ± 0.2963 for control; p = 0.0003) C. Incubation with IL-18 plus PD98059 restored the contraction to PE to control levels. (pEC50 6.105 ± 0.4007 vs 6.170 ± 0.2847 for control; p = 0.7120) D. The relaxation response to ACh was not changed by IL-18 after incubation for 1 hr.Our results show that IL-18 enhances the contractile responses to PE in pudendal arteries from Wistar rats by activation of the Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Pathway.
Exposure to chronic stress is considered a major environmental factor that causes vascular aging, giving rise to the development of cardiovascular diseases. The chronic unpredictable stress (CUS) protocol has emerged as a translationally relevant model for studying the pathophysiology of stress exposure in rodents. The CUS protocol relies on heterotypic, unpredictable, and uncontrollable stressors that prevent adaptation and lead to disrupted stress response systems, mimicking the real-life situation. CUS induces changes in cardiovascular function that are accompanied by the release of inflammatory cytokines. Indeed, previous studies have reported endothelial dysfunction in humans exposed to chronic stress and experimental animal models of chronic unpredictable stress. However, the mechanisms through which CUS induces cardiovascular injury are still unknown and require investigation.This study tests the hypothesis that mice exposed to CUS will have impaired endothelial function.Vascular function using wire myographs was performed on isolated mesenteric resistance arteries (MRAs) from male and female C57BL/6 mice subjected to CUS for 28 days. The arterial rings were kept in physiological salt solution at 37°C, constantly aerated with 95%O2 /5%CO2 . Concentration-response curves to phenylephrine (PE; 10-9 -3x10-5 M) were performed in some arterial rings, whereas concentration-response curves to acetylcholine (ACh; 10-9 - 3x10-5 M) were performed in arterial rings precontracted with phenylephrine (PE 3x10-6 M). Concentration-response curves were analyzed using non-linear regression analysis. Data are presented as mean ± S.E.M. Statistical significance set at p<0.05. Data were analyzed with GraphPad Prism 9.2.0.In the MRA of both males and females, relaxation responses to ACh were impaired in the animals exposed to CUS when compared to their controls. There were no differences in the contractile responses to PE between controls and mice exposed to CUS.Our results show that relaxation responses to acetylcholine are impaired in mesenteric resistance arteries from mice exposed to chronic unpredictable stress. This suggests that exposure to chronic unpredictable stress causes endothelial dysfunction and may have important implications for the role of stress on the development of cardiovascular diseases, particularly, hypertension.
Introduction: Erectile dysfunction (ED) may be an early marker for cardiovascular diseases, and it is often associated with an hypercontractility of the corpus cavernosum (CC) and pudendal artery (PA) because of increased reactive oxygen species (ROS). Leucine Rich Repeat Containing 8 (LRRC8) Volume Regulated Anion Channels (VRACs) regulates Nox1 and modulates the influx of extracellular superoxide anion (O 2 •- ), contributing to the creation of an oxidized domain, enhancing calcium sensitivity of the smooth muscle which promotes contraction. Objective: We hypothesize that downregulation of NOX1 or LRRC8A would decrease the contractile responses to phenylephrine (PE) and electrical field stimulation (EFS) in the CC, PA and mesenteric resistance artery (MRA) of diabetic mice (dbdb −/− ). Methods: The CC and PA of diabetic (dbdb −/− ) mice were mounted in myographs. The contraction elicited by PE or EFS was performed in the presence of apocynin (non-selective NOX inhibitor) and GKT137831 (NOX1/4 inhibitor). In LRRC8A KO dbdb −/− mice, contractions to PE and EFS were obtained in the absence or in the presence of GKT137831.Results: In the CC, the maximal contraction to PE was increased in dbdb −/− animals, and in the presence of 3 mM of apocynin, the contractile response was markedly diminished in both control and dbdb −/− . Incubation with GKT137831 reduced the contraction elicited by PE only in the dbdb −/− group (68 ± 9% of the maximal contraction), whereas in the control group this reduction was not significant (82 ± 14% of the PE-maximal contraction). In the PA, the potency of PE was increased in the dbdb −/− group, and apocynin 1mM restored the contraction to normal levels. In the LRRC8A KO dbdb −/− mice, the contraction to PE in the MRA and PA was reduced by 30% compared to dbdb −/− . The response to EFS in CC was similar to dbdb −/− , however, GKT137831 reduced the contraction to EFS with lesser magnitude of inhibition in the LRRC8A KO dbdb −/− mice (35% of inhibition) compared to dbdb −/− mice (50% of inhibition), suggesting that the participation of O 2 •- in the contractile response was reduced by the lack of functional LRRC8A. Conclusion: LRRC8A modulates the contractile response by managing the influx of reactive oxygen species (ROS) in smooth muscle cells. Inhibition of LRRC8A may be a therapeutic target to prevent hypercontractility of arteries and CC in conditions where there are marked increases in ROS production, such as diabetes. NIH – R01 132948; Institute on Cardiovascular Disease Research, University of South Carolina. 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.
Background: The etiology of essential hypertension remains unclear, and its mechanisms are complex. Indeed, low-grade inflammation and activation of the immune system have been implicated in hypertension. Clinical and population studies have consistently found increased circulating levels of interleukin 18 (IL-18) in patients with hypertension. Hypothesis: This study tests the hypothesis that IL-18 will enhance contractile responses to phenylephrine and impair relaxation responses to acetylcholine in isolated arterial rings from rats. Methods: Vascular function was performed on isolated pudendal arteries from male Wistar rats using wire myographs, kept in Krebs solution at 37°C, constantly aerated with 95%O 2 /5%CO 2 . Concentration-response curves to phenylephrine (PE; 10 -9 -3x10 -5 M), and acetylcholine (ACh; 10 -9 - 3x10 -5 M) were performed in the absence or in the presence of IL-18 (20ng/ml) for 1 hour and 6 hours respectively. Ten minutes prior to the incubation with IL-18, some arterial rings were incubated separately with the mitogen-activated protein kinase (MKK / MEK) inhibitor, PD98059 (10 -5 M). Concentration-response curves were analyzed using non-linear regression analysis. Data are presented as mean ± S.E.M. Statistical significance set at p<0.05. Data analyzed with GraphPad Prism 9.2.0. Results: In the pudendal arterial rings, the potency of PE was increased in the presence of IL-18 (pEC 50 6.510 ± 0.3987 vs 6.022 ± 0.2738 for control; p = 0.0268), Incubation with IL-18 plus PD98059 restored the contraction to PE to control levels. (pEC 50 6.105 ± 0.4007 vs 6.170 ± 0.2847 for control; p = 0.7120). Relaxation responses to Ach were impaired by incubation with IL-18 for 6 hours (pEC 50 5.942 ± 0.1575 vs 7.341 ± 0.1796 for control; p=0.0042) Conclusion: Our results show that IL-18 enhances the contractile responses to PE in pudendal arteries from Wistar rats by activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Thus, IL-18 may contribute to the increased vascular resistance characteristic of hypertension.
Vaginal smooth muscle (VaSM) is critical to sexual function, desire, and arousal. Individuals with hypertension or diabetes have smooth muscle dysfunction which is associated with elevated superoxide anion production and increased inflammation in smooth muscle. Further, endothelial dysfunction is common in the small arteries, like the pudendal arteries, that supply the sex organs in females. This loss of function could contribute to female sexual dysfunction (FSD). This study aims to bridge the gap to treat hypertensive or diabetic individuals’ sexual dysfunction and discover non-hormonal treatments to increase blood flow to the VaSM and clitoral tissue. In this study, we hypothesize that the inhibition of leucine rich repeat containing 8A (LRRC8A) volume regulated anion channels (VRACs) will decrease the contraction to endothelin-1 (ET-1) in vaginal smooth muscle of lean and diabetic (db/db) mice. LRRC8A channels have been proposed to modulate entry of extracellular superoxide (O •- ) associated with NADPH oxidase activity. The activity of NADPH oxidase is increased in diabetes and hypertension. Female young lean and db/db (12 wks) mice underwent distal vaginal dissection. The VaSM was then mounted on a strip myograph for measurement of isometric force development. Contraction to ET-1 was performed in the absence and presence of the LRRC8A inhibitor, montelukast (1uM) (MONT) after a 30-minute incubation. The weight/length of VaSM (mg/mm) was used to normalize the maximum contraction (mN). Measures of weight/length in db/db mice VaSM tissue were less than in lean mice (p=0.0112). In lean mice, force development to ET-1 was significantly less after incubation with MONT (logEC50 control 8.428 +/- 0.06886 vs. drug 7.859 +/- 0.06083, p= 0.0007 ). In db/db mice, force development to ET-1 was significantly less after incubation with MONT (logEC50 control 8.779 +/- 0.1007 vs. drug 7.278 +/- 0.05490 , p= 0.0004). Treatment with an inhibitor of LRRC8A offers a novel approach to treat FSD and should be further researched to observe its overall impact. LRRC8A may play an important physiological role in the maintenance of the contractile response by managing the flux of O •- in the VaSM, making it an important potential target in conditions like hypertension and diabetes.
Polo-like kinase 1 (Plk1) is a Ser/Thr protein kinase that plays a role in cell cycle regulation. Recently, Plk1 was demonstrated to be required for arterial structure organization and its absence caused aortic rupture and death, being also required for RhoA activation and vasoconstriction in aorta. In this study, we aimed to investigate the role of Plk1 in the contraction of resistance arteries in response to different classes of contractile agents. We hypothesize that agonist-mediated contraction is dependent on activation of Plk1 in resistance arteries in both males and females.Male and female wild type mice were used in this study. The pudendal and mesenteric resistance arteries were isolated and mounted in a wire-myograph. Contractions were obtained by concentration-response curves to phenylephrine (PE), serotonin (5-HT), thromboxane A2 analogue U46619 and angiotensin II (Ang-II). Concentration response curve to the same agonist was repeated after incubation with Plk1 inhibitor Volasertib (100 nM or 1 uM). Contractions in the presence of Volasertib were calculated as percentage of the maximal contraction evoked by its respective agonist in control conditions, which was considered 100%. Expression of Plk1 was evaluated by western blot using mesenteric artery in control conditions and after incubation with Ang-II, with or without the Plk1 inhibitor volasertib. Curves to Ang-II were also repeated in the absence of any inhibitor in order to rule out tachyphylaxis of Ang-II in the second curve. Paired Student's t-test was used for data analysis. Data are expressed as mean ± S.E.M of 3-5 mice.In male, the contractile effects of PE, 5-HT, U46619 and Ang-II in the pudendal and mesenteric arteries were evaluated in the presence of the Plk1 inhibitor, Volasertib (100 nM). Plk1 inhibition did not affect the contraction induced by PE, 5-HT and U46619 in either pudendal or mesenteric arteries. On the other hand, the contraction induced by Ang-II was markedly reduced in the presence of Volasertib (100 nM) in both pudendal and mesenteric arteries (Fig 1A and 1C, respectively). However, the inhibition of the contraction was not dependent on the dose, since 1 uM of Volasertib did not cause further inhibition (Pudendal artery EMAX: 3.86 ± 0.97% vs 5.02 ± 0.48%, in the presence of 100 nM and 1 uM of Volasertib, respectively; Mesenteric artery EMAX: 0.28 ± 0.21% vs 0.36 ± 0.18%, in the presence of 100 nM and 1 uM of Volasertib, respectively). In females, only 100 nM of Volasertib was tested in the contraction induced by PE and Ang-II. In females, Volasertib did not affect the contraction induced by PE in pudendal and mesenteric arteries. In contrast, the contraction induced by Ang-II was markedly reduced in the presence of Volasertib 100 nM (Fig 1B and 1D). Plk1 is expressed in the mesenteric artery of wild type mice, however, it was not affected by incubation with either Ang-II (100 nM) or Volasertib (100 nM). However, it is possible that these treatments are affecting Plk1 phosphorylation.Our findings are the first to demonstrate that Plk1 plays a role in Ang-II-induced contraction of the pudendal and mesenteric arteries of mice. More studies are necessary to elucidate the process of Plk1 activation and its association with vascular dysfunction in pathological conditions such as hypertension and diabetes.
Introduction: Vaginal smooth muscle (VaSM) is an important contributor to female sexual function, desire, and arousal. Increased vaginal blood flow via vasodilation of the vaginal blood vessels is key to the of lubrication and creating an enjoyable sexual experience. Female sexual dysfunction (FSD) is common in hypertensive and diabetic women, as well as many who suffer from hormonal imbalances, including menopause. Despite the substantial population that suffers from FSD, research into the mechanisms and non-hormonal treatments are limited. Leucine rich repeat containing channel 8A (LRRC8A) is a volume regulated anion channel (VRAC) that regulates Nox1 and can modulate the extracellular production of superoxide (O2∙-). In aged tissues, there are increased in extracellular ROS production as well as vascular and smooth muscle dysfunction. The objective of these experiments is to elucidate the mechanisms behind female sexual dysfunction that present with vascular and smooth muscle dysfunction and determine if the LRRC8A inhibitor, Montelukast, can alleviate some of the symptoms of FSD and improve quality of life. In this study, we hypothesize that the inhibition of LRRC8A in will decrease the contraction to Endothelin-1 in the vaginal smooth muscle of the aged mice. Methods: Aged (16 mo and 20 mo old) female lean mice underwent distal vaginal dissection. Vaginal tissue was cleaned and mounted on a strip myograph in Krebs solution and the contraction elicited by Endothelin-1 was performed in the absence and repeated in the presence of the LRRC8A inhibitor, Montelukast (1uM) after a 30-minute incubation. Data was analyzed using Prism. The maximal contraction in the absence of the inhibitor was taken as 100%, and the contraction in the presence of the inhibitor was calculated as a percentage of the maximal contraction attained in the absence of the inhibitor. Results: Contraction induced by Endothelin-1 (10-12 M to 3✕10 -8 M) alone compared to in the presence of Montelukast (1 uM) showed that Montelukast decreased both the potency and the maximum force of contraction in the aged VaSM. After incubation with 1 mM Montelukast, the EC50 and Emax were significantly different (0.0430 and <0.0001, respectively). Conclusion: This is the first study investigating the vaginal smooth muscle from the perspective of ROS production and the influence of LRRC8A in aged animals. The change in the contractile response in the treated animals may be due to the modulation of the production of O2∙-. Patients that suffer from FSD are a large and overlooked community in need of new therapeutic approaches. Treatment with LRRC8A offers a potential novel approach to treat FSD and should be further researched to observe its overall impact in FSD. 1R01DK132948-01, SMSNA Student Grant This is the full abstract presented at the American Physiology Summit 2023 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.
Erectile dysfunction (ED) may be an early marker for cardiovascular diseases and it isoften associated with an hypercontractility of the corpus cavernosum (CC) and pudendalartery (PA) in consequence of increased reactive oxygen species (ROS), which favorsthe flaccid state of the penis. Leucine Rich Repeat Containing 8 (LRRC8) VolumeRegulated Anion Channels (VRACs) regulates Nox1 and modulates the extracellularproduction of superoxide anion (O2 •- ). Downregulation of LRRC8 is associated withdecreased production of O2 •- and we have demonstrated improved relaxation andreduced contraction of the pudendal artery in LRRC8A knockout mice. In this study, wehypothesize that inhibition of Nox or LRRC8A decreases phenylephrine (PE)-inducedcontraction in the CC and PA of diabetic mice. To test this hypothesis, the CC and PA ofdiabetic (dbdb-/-) mice were mounted in myographs, and the contraction elicited by PEwas performed in the presence of apocynin (non-selective NOX inhibitor), GKT137831(NOX1/4 inhibitor) and montelukast (LRRC8A inhibitor). In the PA, the potency of PE wasincreased in diabetic mice, and apocynin (1 mM) restored the contraction of PE to thenormal levels. In the CC, the maximal contraction of PE was increased in diabetic animals,and in the presence of 3 mM of apocynin, the contractile response was markedlydiminished in both control and diabetic mice. The LRRC8A inhibitor, montelukast,significantly reduced PE-induced contraction of the CC reaching maximal contraction of69 + 7% and 65 + 10% in the control and diabetic group, respectively, compared to thecontraction in the absence of the inhibitor. In the presence of the Nox1/Nox4 inhibitor,GKT137831, the contraction elicited by PE was reduced only in the diabetic group (68 +9% of the maximal contraction), whereas in the control group this reduction was notsignificant (82 + 14% of the PE-maximal contraction). In the CC and PA, LRRC8Amodulates the contractile response induced by α-adrenergic stimulation and in diabetes,this may be associated with O2 •- production, which plays a role in the development of ED.Inhibition of LRRC8A may be a therapeutic target to prevent hypercontractility of arteriesand CC in vasculogenic ED.
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