Pub Date : 2024-05-01DOI: 10.1152/physiol.2024.39.s1.649
Till Harter, Graham R Scott
Oxygen (O2) is essential for vertebrate life, and complex cardio-respiratory systems have evolved to transport the gas from the environment to each individual cell. Even short disruptions of this O2 flux can have deleterious effects that are linked to numerous disease states. Animals that have adapted to hypoxic environments, such as deer mice ( Peromyscus maniculatus) native to high altitude, can provide valuable insight into naturally evolved solutions to O2 deprivation. Previous work has shown that high-altitude deer mice have evolved a higher hemoglobin O2 affnity and other coordinated changes across the O2 transport cascade that enable higher metabolic rates in hypoxia. Red blood cells (RBC) are the functional unit of O2 and carbon dioxide transport in the blood and play central roles in matching O2 supply and demand in the microcirculation by releasing signaling molecules such as ATP and gasotransmitters; but how these cellular mechanisms respond to hypoxic environments has not been studied. We hypothesized that high-altitude adaptation in deer mice has improved the function of RBCs for cardiovascular gas transport in hypoxia. Lab-raised breeding colonies of deer-mice were established from wild mice caught at low altitude (~400 m in the Great Plains of Nebraska) and at high altitude (~4300 m in the Rocky Mountains of Colorado). Using a common-garden experimental design, third-generation deer mice from high- and low-altitude populations were acclimated to warm normoxia (21°C, 21 kPa O2) or cold hypobaric hypoxia (5°C, 12 kPa O2) for 8 weeks. Blood samples were collected for measurements of hematocrit, hemoglobin concentration, RBC volume, plasma erythropoietin concentration, RBC contents of membrane transport and channel proteins (anion exchanger, aquaporin 1 and rhesus associated glycoprotein) by immunocytochemistry and western blotting, and carbonic anhydrase activity using biochemical techniques. The release of ATP from RBCs was measured in tonometers at decreasing levels of O2 by luminometry, and the vascular sensitivity to ATP was determined by wire myography on second-order mesenteric arteries. Finally, bone marrow samples were collected from the femurs to measure gene expression levels in the erythropoietic tissue. Our experimental design allowed us to examine the interactive effects of cold hypoxic environments on RBC phenotype, by untangling environmentally-induced plasticity from the signatures of adaptation that are unique to high-altitude natives. This work is providing a better understanding of how RBC function participates in matching cardiovascular O2 supply and demand in extreme hypoxia, which has important applications in animal and human health. This work was supported by a NSERC Canada Banting Postdoctoral Fellowship to TSH and a Discovery Grant to GRS. 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 cont
氧气(O2)是脊椎动物生命所必需的,复杂的心肺系统就是为了把这种气体从环境中输送到每个细胞而进化的。即使是短暂的氧气输送中断也会产生有害影响,并与多种疾病相关。适应缺氧环境的动物,如原产于高海拔地区的鹿小鼠(Peromyscus maniculatus),可以为自然进化的氧气匮乏解决方案提供宝贵的见解。先前的研究表明,高海拔地区的鹿小鼠进化出了更高的血红蛋白氧气亲和力以及氧气运输级联的其他协调变化,从而能够在缺氧条件下实现更高的新陈代谢率。红细胞(RBC)是血液中氧气和二氧化碳运输的功能单位,并通过释放 ATP 和气体递质等信号分子,在微循环中匹配氧气供需方面发挥着核心作用;但这些细胞机制如何应对缺氧环境尚未得到研究。我们假设,鹿小鼠对高海拔的适应改善了红细胞在缺氧环境下心血管气体运输的功能。我们利用在低海拔地区(内布拉斯加大平原约 400 米)和高海拔地区(科罗拉多落基山脉约 4300 米)捕获的野生小鹿建立了实验室饲养的鹿鼠繁殖群。采用共同花园实验设计,将来自高海拔和低海拔种群的第三代鹿小鼠在温暖的常氧环境(21°C,21 kPa O2)或寒冷的低压缺氧环境(5°C,12 kPa O2)中适应 8 周。采集血样用于测量血细胞比容、血红蛋白浓度、红细胞体积、血浆促红细胞生成素浓度、红细胞膜运输和通道蛋白(阴离子交换蛋白、水蒸发蛋白 1 和恒河猴相关糖蛋白)含量(免疫细胞化学和 Western 印迹)以及碳酸酐酶活性(生化技术)。在气压计中,通过荧光测定法测量红细胞在氧气浓度降低时释放出的 ATP,并通过二阶肠系膜动脉的线性肌电图测定血管对 ATP 的敏感性。最后,我们从股骨中采集了骨髓样本,以测量红细胞组织中的基因表达水平。我们的实验设计使我们能够研究低温缺氧环境对红细胞表型的交互影响,将环境诱导的可塑性与高海拔当地人特有的适应特征区分开来。这项工作让我们更好地了解了在极度缺氧的情况下,红细胞功能是如何参与匹配心血管氧气供需的,这在动物和人类健康中具有重要的应用价值。这项工作得到了加拿大国家科学研究中心班廷博士后奖学金(NSERC Canada Banting Postdoctoral Fellowship)和发现基金(Discovery Grant)的支持。本文是在 2024 年美国生理学峰会上发表的摘要全文,只有 HTML 格式。本摘要没有附加版本或附加内容。生理学》未参与同行评审过程。
{"title":"Linking red blood cell functional phenotypes to environmental tolerance in high-altitude adapted deer mice","authors":"Till Harter, Graham R Scott","doi":"10.1152/physiol.2024.39.s1.649","DOIUrl":"https://doi.org/10.1152/physiol.2024.39.s1.649","url":null,"abstract":"Oxygen (O2) is essential for vertebrate life, and complex cardio-respiratory systems have evolved to transport the gas from the environment to each individual cell. Even short disruptions of this O2 flux can have deleterious effects that are linked to numerous disease states. Animals that have adapted to hypoxic environments, such as deer mice ( Peromyscus maniculatus) native to high altitude, can provide valuable insight into naturally evolved solutions to O2 deprivation. Previous work has shown that high-altitude deer mice have evolved a higher hemoglobin O2 affnity and other coordinated changes across the O2 transport cascade that enable higher metabolic rates in hypoxia. Red blood cells (RBC) are the functional unit of O2 and carbon dioxide transport in the blood and play central roles in matching O2 supply and demand in the microcirculation by releasing signaling molecules such as ATP and gasotransmitters; but how these cellular mechanisms respond to hypoxic environments has not been studied. We hypothesized that high-altitude adaptation in deer mice has improved the function of RBCs for cardiovascular gas transport in hypoxia. Lab-raised breeding colonies of deer-mice were established from wild mice caught at low altitude (~400 m in the Great Plains of Nebraska) and at high altitude (~4300 m in the Rocky Mountains of Colorado). Using a common-garden experimental design, third-generation deer mice from high- and low-altitude populations were acclimated to warm normoxia (21°C, 21 kPa O2) or cold hypobaric hypoxia (5°C, 12 kPa O2) for 8 weeks. Blood samples were collected for measurements of hematocrit, hemoglobin concentration, RBC volume, plasma erythropoietin concentration, RBC contents of membrane transport and channel proteins (anion exchanger, aquaporin 1 and rhesus associated glycoprotein) by immunocytochemistry and western blotting, and carbonic anhydrase activity using biochemical techniques. The release of ATP from RBCs was measured in tonometers at decreasing levels of O2 by luminometry, and the vascular sensitivity to ATP was determined by wire myography on second-order mesenteric arteries. Finally, bone marrow samples were collected from the femurs to measure gene expression levels in the erythropoietic tissue. Our experimental design allowed us to examine the interactive effects of cold hypoxic environments on RBC phenotype, by untangling environmentally-induced plasticity from the signatures of adaptation that are unique to high-altitude natives. This work is providing a better understanding of how RBC function participates in matching cardiovascular O2 supply and demand in extreme hypoxia, which has important applications in animal and human health. This work was supported by a NSERC Canada Banting Postdoctoral Fellowship to TSH and a Discovery Grant to GRS. 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 cont","PeriodicalId":49694,"journal":{"name":"Physiology","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141130423","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01DOI: 10.1152/physiol.2024.39.s1.1430
R. Roden, Kevin Webb, Wyatt W. Pruter, David Holmes, Clifton Haider, Michael Joyner, Timothy Curry
Background: The compensatory reserve metric (CRM) provides a novel marker of hemodynamic status in prediction of sudden cardiovascular decompensation during central hypovolemia. The CRM has previously been calculated using arterial waveforms obtained from a photoplethysmographic volume-clamp technique. However, the in-field use of pulse oximetry is becoming more prominent in research and medicine. To improve in-field applicability of the CRM, this project aimed to validate pulse-oximetry derived CRM values relative to physiologic data during simulated hemorrhage through application of lower body negative pressure (LBNP). Methods: Forty-nine healthy adults (25 females) underwent a graded LBNP protocol with the following 5-minute stages: -0, -15, -30, -45, -60, -70, -80, -90, and 100 mmHg, or until the onset of pre-syncopal symptoms (tolerance) was reached. Arterial waveforms were sampled using pulse oximetry (Massimo Radical 7). The CRM was calculated using a validated one-dimensional convolutional neural network. A brachial artery catheter was used to measure intraarterial pressure. A 3-lead ECG was used to measure heart rate. SpO2 was measured using pulse oximetry. Cardiac output and stroke volume were estimated using the photoplethysmographic volume-clamp. Data were binned for the last thirty seconds of each completed stage, and at tolerance. Fixed-effect linear mixed models with repeated measures were used to examine the association between CRM values and physiologic variables. A priori significance was set at P<0.05. Results: The median LBNP stage reached was 70 mmHg (Range: 45-100 mmHg). Relative to baseline, at tolerance there was a 50±12% ( P<0.001) mean reduction in stroke volume, 65±27% ( P<0.001) increase in heart rate, 18±8% ( P<0.001) reduction in mean arterial pressure, 21±7% ( P<0.001) systolic blood pressure, 8±9% ( P<0.001) diastolic blood pressure, and 39±12% ( P<0.001) pulse pressure. The CRM obtained using pulse oximetry was significantly associated with a reduction in stroke volume ( P<0.001), increase in heart rate ( P<0.001), and reductions in systolic ( P=0.033), diastolic ( P=0.036), and pulse pressure ( P=0.002). There was no statistically significant association between CRM and cardiac output ( P=0.421), SpO2 ( P=0.408), or mean arterial pressure ( P=0.214). Conclusion: The CRM obtained using pulse oximetry may be a valid, reliable marker of hemodynamic status with promising in-field applications. Future studies warrant investigation of feasibility and applicability within various contexts ranging from the operating room to the battlefield. The presented work was funded in part by the Congressionally Directed Medical Research Program (Award No. DM180240) and Offce of Naval Research (Awards No. N00014-18-D-7001 and N00014-19-C-2017). 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 t
{"title":"Physiologic Validation of the Compensatory Reserve Metric Obtained From Pulse Oximetry Waveforms","authors":"R. Roden, Kevin Webb, Wyatt W. Pruter, David Holmes, Clifton Haider, Michael Joyner, Timothy Curry","doi":"10.1152/physiol.2024.39.s1.1430","DOIUrl":"https://doi.org/10.1152/physiol.2024.39.s1.1430","url":null,"abstract":"Background: The compensatory reserve metric (CRM) provides a novel marker of hemodynamic status in prediction of sudden cardiovascular decompensation during central hypovolemia. The CRM has previously been calculated using arterial waveforms obtained from a photoplethysmographic volume-clamp technique. However, the in-field use of pulse oximetry is becoming more prominent in research and medicine. To improve in-field applicability of the CRM, this project aimed to validate pulse-oximetry derived CRM values relative to physiologic data during simulated hemorrhage through application of lower body negative pressure (LBNP). Methods: Forty-nine healthy adults (25 females) underwent a graded LBNP protocol with the following 5-minute stages: -0, -15, -30, -45, -60, -70, -80, -90, and 100 mmHg, or until the onset of pre-syncopal symptoms (tolerance) was reached. Arterial waveforms were sampled using pulse oximetry (Massimo Radical 7). The CRM was calculated using a validated one-dimensional convolutional neural network. A brachial artery catheter was used to measure intraarterial pressure. A 3-lead ECG was used to measure heart rate. SpO2 was measured using pulse oximetry. Cardiac output and stroke volume were estimated using the photoplethysmographic volume-clamp. Data were binned for the last thirty seconds of each completed stage, and at tolerance. Fixed-effect linear mixed models with repeated measures were used to examine the association between CRM values and physiologic variables. A priori significance was set at P<0.05. Results: The median LBNP stage reached was 70 mmHg (Range: 45-100 mmHg). Relative to baseline, at tolerance there was a 50±12% ( P<0.001) mean reduction in stroke volume, 65±27% ( P<0.001) increase in heart rate, 18±8% ( P<0.001) reduction in mean arterial pressure, 21±7% ( P<0.001) systolic blood pressure, 8±9% ( P<0.001) diastolic blood pressure, and 39±12% ( P<0.001) pulse pressure. The CRM obtained using pulse oximetry was significantly associated with a reduction in stroke volume ( P<0.001), increase in heart rate ( P<0.001), and reductions in systolic ( P=0.033), diastolic ( P=0.036), and pulse pressure ( P=0.002). There was no statistically significant association between CRM and cardiac output ( P=0.421), SpO2 ( P=0.408), or mean arterial pressure ( P=0.214). Conclusion: The CRM obtained using pulse oximetry may be a valid, reliable marker of hemodynamic status with promising in-field applications. Future studies warrant investigation of feasibility and applicability within various contexts ranging from the operating room to the battlefield. The presented work was funded in part by the Congressionally Directed Medical Research Program (Award No. DM180240) and Offce of Naval Research (Awards No. N00014-18-D-7001 and N00014-19-C-2017). 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 t","PeriodicalId":49694,"journal":{"name":"Physiology","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141130516","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01DOI: 10.1152/physiol.2024.39.s1.355
Michele L. Singer, Mi Kyung Shin, Lenise Kim, C. Freire, O. Aung, H. Pho, Alban Latremoliere
Opioid-induced respiratory depression (OIRD) is the primary cause of death associated with opioids and individuals with obesity are particularly susceptible due to comorbid obstructive sleep apnea (OSA). Repeated exposure to opioids, as in the case of pain management, results in diminished therapeutic effect and/or the need for higher doses to maintain the same effect. With limited means to address the negative impact of repeated exposure it is critical to develop drugs that prevent deaths induced by opioids without reducing beneficial analgesia. We have previously shown that intranasal (IN) leptin can reverse apneas, hypoventilation, and upper airway obstruction while enhancing analgesia following acute morphine administration in obese males. Here we hypothesize that OIRD as a result of chronic opioid use can be attenuated by administration of IN leptin while also maintaining analgesia in both lean mice and mice with diet-induced obesity (DIO) of both sexes. To test this hypothesis, an opioid tolerance protocol was developed and a model of OIRD in mice chronically receiving morphine and tolerant to morphine analgesia was established. Subsequently, sleep and breathing were recorded by barometric plethysmography in four experimental groups: obese male, obese female, lean male, and lean female following acute administration of IN leptin. Operant behavioral assays were used to determine the impact of IN leptin on the analgesic effcacy of morphine. Acute administration of IN leptin significantly attenuated OIRD in DIO male mice decreasing the apnea index by 58.9% and apnea time by 60.1%. In lean mice leptin was ineffective. Morphine caused a complete loss of temperature aversion which was not reduced by intranasal leptin indicating IN leptin does not decrease morphine analgesia. We conclude that IN leptin is ineffective in lean mice but prevents OIRD in obesity by increasing hypercapnic sensitivity when leptin resistance at the blood brain barrier is present without reducing analgesia. This work was supported by funding from the National Institutes of Health: R01NS112266 (AL), R01HL128970, R61HL156240, R41DA056239 (VYP), and T32HL110952 (MLS). 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.
阿片类药物引起的呼吸抑制(OIRD)是阿片类药物导致死亡的主要原因,肥胖症患者因合并阻塞性睡眠呼吸暂停(OSA)而特别容易受到影响。反复接触阿片类药物(如止痛药)会导致治疗效果减弱和/或需要更大剂量才能维持相同效果。由于解决重复接触带来的负面影响的手段有限,因此开发出既能防止阿片类药物导致死亡,又不减少有益镇痛效果的药物至关重要。我们之前已经证明,鼻内注射瘦素(IN)可以逆转呼吸暂停、通气不足和上呼吸道阻塞,同时增强肥胖男性急性吗啡给药后的镇痛效果。在此,我们假设在瘦小鼠和饮食诱发肥胖(DIO)的雌雄小鼠体内注射 IN 瘦素可减轻长期使用阿片类药物导致的 OIRD,同时维持镇痛效果。为了验证这一假设,我们制定了阿片类药物耐受方案,并在长期接受吗啡且对吗啡镇痛耐受的小鼠中建立了OIRD模型。随后,在肥胖雄性、肥胖雌性、瘦弱雄性和瘦弱雌性四个实验组中,通过气压褶压计记录了它们在急性注射 IN 瘦素后的睡眠和呼吸情况。操作行为试验用于确定 IN 瘦素对吗啡镇痛效果的影响。给DIO雄性小鼠急性注射IN瘦素可显著减轻OIRD,使呼吸暂停指数降低58.9%,呼吸暂停时间缩短60.1%。瘦素对瘦小鼠无效。吗啡会导致完全丧失温度厌恶感,而鼻内注射瘦素并不会降低这种厌恶感,这表明 IN 瘦素不会降低吗啡镇痛效果。我们的结论是,IN瘦素对瘦小鼠无效,但在血脑屏障存在瘦素阻力时,可通过增加高碳酸血症敏感性来防止肥胖症的OIRD,而不会降低镇痛效果。这项工作得到了美国国立卫生研究院的资助:R01NS112266(AL)、R01HL128970、R61HL156240、R41DA056239(VYP)和 T32HL110952(MLS)。本文是在 2024 年美国生理学峰会上发表的摘要全文,仅提供 HTML 格式。本摘要没有附加版本或附加内容。生理学》未参与同行评审过程。
{"title":"The effcacy of intranasal leptin for opioid induced respiratory depression depends on sex and obesity state","authors":"Michele L. Singer, Mi Kyung Shin, Lenise Kim, C. Freire, O. Aung, H. Pho, Alban Latremoliere","doi":"10.1152/physiol.2024.39.s1.355","DOIUrl":"https://doi.org/10.1152/physiol.2024.39.s1.355","url":null,"abstract":"Opioid-induced respiratory depression (OIRD) is the primary cause of death associated with opioids and individuals with obesity are particularly susceptible due to comorbid obstructive sleep apnea (OSA). Repeated exposure to opioids, as in the case of pain management, results in diminished therapeutic effect and/or the need for higher doses to maintain the same effect. With limited means to address the negative impact of repeated exposure it is critical to develop drugs that prevent deaths induced by opioids without reducing beneficial analgesia. We have previously shown that intranasal (IN) leptin can reverse apneas, hypoventilation, and upper airway obstruction while enhancing analgesia following acute morphine administration in obese males. Here we hypothesize that OIRD as a result of chronic opioid use can be attenuated by administration of IN leptin while also maintaining analgesia in both lean mice and mice with diet-induced obesity (DIO) of both sexes. To test this hypothesis, an opioid tolerance protocol was developed and a model of OIRD in mice chronically receiving morphine and tolerant to morphine analgesia was established. Subsequently, sleep and breathing were recorded by barometric plethysmography in four experimental groups: obese male, obese female, lean male, and lean female following acute administration of IN leptin. Operant behavioral assays were used to determine the impact of IN leptin on the analgesic effcacy of morphine. Acute administration of IN leptin significantly attenuated OIRD in DIO male mice decreasing the apnea index by 58.9% and apnea time by 60.1%. In lean mice leptin was ineffective. Morphine caused a complete loss of temperature aversion which was not reduced by intranasal leptin indicating IN leptin does not decrease morphine analgesia. We conclude that IN leptin is ineffective in lean mice but prevents OIRD in obesity by increasing hypercapnic sensitivity when leptin resistance at the blood brain barrier is present without reducing analgesia. This work was supported by funding from the National Institutes of Health: R01NS112266 (AL), R01HL128970, R61HL156240, R41DA056239 (VYP), and T32HL110952 (MLS). 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.","PeriodicalId":49694,"journal":{"name":"Physiology","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141130736","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01DOI: 10.1152/physiol.2024.39.s1.2068
Karel Alcedo, Oksanna Samey, Isaac Biju, Liang Sun, Fernando Camargo
The transcriptional co-activator YAP plays a complex role in liver homeostasis and disease development. In response to liver injuries and hepatotoxins, YAP is transiently activated to promote liver regeneration by coordinating cell proliferation and metabolism. By contrast, chronic activation of YAP in experimental models has been associated with liver diseases and cancer. These primary responses to YAP are well established; however, it is presently unknown how acute YAP signaling contributes to the eventual development of liver diseases. Here, we developed an in vivo model of dynamic YAP signaling using a doxycycline-inducible system to control the transient expression of a constitutively active YAP mutant in hepatocytes. We found that while YAP-dependent effects on liver growth and cell proliferation are reversible, there are metabolic changes in the liver that remain stable even after YAP inactivation. By coupling RNAseq and ATACseq analyses, we unraveled persistent changes in gene expression and chromatin accessibility, particularly those involved in lipid metabolism. Furthermore, we demonstrated that transient YAP signaling sensitizes the liver to develop more severe liver injuries than normally induced in an unprimed liver. These data suggest that transient YAP signaling imparts a form of lipid metabolic memory that promotes susceptibility to liver diseases. Overall, our current work reveals long-term consequences of acute YAP signaling that may influence the development of liver diseases. 5R01HL128850-07 (FC) and 5T32DK007477-40 (KA). 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.
{"title":"Consequences of Transient YAP Signaling in the Liver","authors":"Karel Alcedo, Oksanna Samey, Isaac Biju, Liang Sun, Fernando Camargo","doi":"10.1152/physiol.2024.39.s1.2068","DOIUrl":"https://doi.org/10.1152/physiol.2024.39.s1.2068","url":null,"abstract":"The transcriptional co-activator YAP plays a complex role in liver homeostasis and disease development. In response to liver injuries and hepatotoxins, YAP is transiently activated to promote liver regeneration by coordinating cell proliferation and metabolism. By contrast, chronic activation of YAP in experimental models has been associated with liver diseases and cancer. These primary responses to YAP are well established; however, it is presently unknown how acute YAP signaling contributes to the eventual development of liver diseases. Here, we developed an in vivo model of dynamic YAP signaling using a doxycycline-inducible system to control the transient expression of a constitutively active YAP mutant in hepatocytes. We found that while YAP-dependent effects on liver growth and cell proliferation are reversible, there are metabolic changes in the liver that remain stable even after YAP inactivation. By coupling RNAseq and ATACseq analyses, we unraveled persistent changes in gene expression and chromatin accessibility, particularly those involved in lipid metabolism. Furthermore, we demonstrated that transient YAP signaling sensitizes the liver to develop more severe liver injuries than normally induced in an unprimed liver. These data suggest that transient YAP signaling imparts a form of lipid metabolic memory that promotes susceptibility to liver diseases. Overall, our current work reveals long-term consequences of acute YAP signaling that may influence the development of liver diseases. 5R01HL128850-07 (FC) and 5T32DK007477-40 (KA). 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.","PeriodicalId":49694,"journal":{"name":"Physiology","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141130992","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01DOI: 10.1152/physiol.2024.39.s1.1144
M. Matumba, Emmanuel Mukwevho
The high prevalence of obesity, resulting from the increased consumption of a fructose-rich diet, poses a serious global threat to children's public health. Obesity is linked to several metabolic disorders, including insulin resistance and type-2 diabetes. Furthermore, type-2 diabetes is characterized by impaired lipid and glucose metabolism. This study investigated the impact of neonatal curcumin intake on the expression of key molecular markers related to liver lipid metabolism [Liver kinase B-1 (LKB-1), AMP-activated protein kinase (AMPK), Carnitine palmitoyltransferase-1 (CPT-1), and Acetyl-coenzyme A carboxylase-1 (ACC-1)] and glucose metabolism [serine/threonine-protein kinase (AKT-1), Glucose transporter-2 (GLUT-2), glycogen phosphorylase (GP), and Phosphoglucomutase (PGM)]. Curcumin, a polyphenolic compound derived from turmeric, is currently under extensive research for its potential therapeutic applications in various diseases. In this study, a metabolic syndrome model was induced in Sprague Dawley rats by feeding them a high fructose diet, with curcumin administered as the treatment. Gene expression was assessed through Real-Time quantitative PCR, and protein expression was analyzed using the western blot technique. The study observed that a high fructose diet led to the upregulation of genes and proteins associated with lipid metabolism, such as AMPK, LKB-1, ACC-1, and CPT-1 in liver tissues. Curcumin treatment reversed these effects, restoring normal lipid metabolism. Additionally, the high fructose diet downregulated the expression of AKT-1 in hepatic insulin signaling, which was counteracted by curcumin. Moreover, the high fructose diet increased hepatic glucose production, as indicated by the upregulation of GP and PGM gene and protein expression, while curcumin treatment suppressed hepatic glucose production by downregulating GP and PGM expression. The findings improve our understanding of curcumin's molecular mechanisms, which are not yet fully understood. They offer further evidence of curcumin's positive effects. Thus, early neonatal consumption of curcumin could be a potential strategy to prevent high fructose diet-induced obesity and related metabolic disorders, including insulin resistance and type-2 diabetes. Lipid and Glucose Metabolism. This project was funded by the North-West University. 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.
{"title":"The Impact of Neonatal Curcumin Intake on Key Molecular Markers in Lipid and Glucose Metabolism","authors":"M. Matumba, Emmanuel Mukwevho","doi":"10.1152/physiol.2024.39.s1.1144","DOIUrl":"https://doi.org/10.1152/physiol.2024.39.s1.1144","url":null,"abstract":"The high prevalence of obesity, resulting from the increased consumption of a fructose-rich diet, poses a serious global threat to children's public health. Obesity is linked to several metabolic disorders, including insulin resistance and type-2 diabetes. Furthermore, type-2 diabetes is characterized by impaired lipid and glucose metabolism. This study investigated the impact of neonatal curcumin intake on the expression of key molecular markers related to liver lipid metabolism [Liver kinase B-1 (LKB-1), AMP-activated protein kinase (AMPK), Carnitine palmitoyltransferase-1 (CPT-1), and Acetyl-coenzyme A carboxylase-1 (ACC-1)] and glucose metabolism [serine/threonine-protein kinase (AKT-1), Glucose transporter-2 (GLUT-2), glycogen phosphorylase (GP), and Phosphoglucomutase (PGM)]. Curcumin, a polyphenolic compound derived from turmeric, is currently under extensive research for its potential therapeutic applications in various diseases. In this study, a metabolic syndrome model was induced in Sprague Dawley rats by feeding them a high fructose diet, with curcumin administered as the treatment. Gene expression was assessed through Real-Time quantitative PCR, and protein expression was analyzed using the western blot technique. The study observed that a high fructose diet led to the upregulation of genes and proteins associated with lipid metabolism, such as AMPK, LKB-1, ACC-1, and CPT-1 in liver tissues. Curcumin treatment reversed these effects, restoring normal lipid metabolism. Additionally, the high fructose diet downregulated the expression of AKT-1 in hepatic insulin signaling, which was counteracted by curcumin. Moreover, the high fructose diet increased hepatic glucose production, as indicated by the upregulation of GP and PGM gene and protein expression, while curcumin treatment suppressed hepatic glucose production by downregulating GP and PGM expression. The findings improve our understanding of curcumin's molecular mechanisms, which are not yet fully understood. They offer further evidence of curcumin's positive effects. Thus, early neonatal consumption of curcumin could be a potential strategy to prevent high fructose diet-induced obesity and related metabolic disorders, including insulin resistance and type-2 diabetes. Lipid and Glucose Metabolism. This project was funded by the North-West University. 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.","PeriodicalId":49694,"journal":{"name":"Physiology","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141132288","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01DOI: 10.1152/physiol.2024.39.s1.748
Sinay C Vicencio, Rodrigo Del Rio
Exercise tolerance (EXT) has been primarily explained by individual body/physical features, partly neglecting the complex cardiorespiratory response needed to cope with exercise. While in healthy conditions most people display good EXT, the presence of exercise intolerance (EXINT) has also been documented. More importantly, EXINT increases in several pathological conditions. Despite the precise mechanism underpinning EXINT is not fully understood. It is well known that exaggerated sympathetic outflow to skeletal muscle impairs proper muscle function and that activation of the carotid body (CB) chemoreceptors elicits a robust increase in sympathetic activity. Therefore, it is plausible to hypothesize that an exaggerated CB-chemoreflex drive may impair EXT. Accordingly, we aimed to determine: i) differences in CB-chemoreflex drive between EXT and EXINT mice, and ii) the role played by the CB-chemoreflex on setting EXINT. Male C57/BL6 mice (n=24) were used to study chemoreflex function using whole-body plethysmography. Exercise tolerance/intolerance was determined by a three-day maximal performance exercise test. CB-chemoreflex drive was assessed by allowing the mice to breathe hypoxic gas (FiO2~5%). We found that the incidence of EXINT among healthy mice was ~30%. Notably, compared to EXT mice, animals with EXINT exhibited a heightened CB-mediated chemoreflex drive as evidenced by larger increases in the hypoxic ventilatory responses (HVR). Indeed, HVR was ~1.4-fold larger in EXINT compared to the values obtained in EXT mice (VE: 17 ± 1.6 vs. 12 ± 3.2, mL/min, EXINT vs. EXT, respectively). Then, we tested whether high chemoreflex drive was associated with EXINT. For this, we reduced the chemosensory drive by using the Dejour´s maneuver (FiO2~35%) while EXINT mice continues to exercise. We found that chemoreflex unloading in EXINT markedly improves exercise tolerance. Indeed, exercise tolerance was almost fully restored by reducing chemoreflex drive in EXINT. Our findings underscore the potential role of heightened CB-chemoreflex on setting exercise tolerance. Supported by Fondecyt 1220950. 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.
{"title":"Carotid body-mediated peripheral chemoreflex drive sets exercise tolerance in adult mice","authors":"Sinay C Vicencio, Rodrigo Del Rio","doi":"10.1152/physiol.2024.39.s1.748","DOIUrl":"https://doi.org/10.1152/physiol.2024.39.s1.748","url":null,"abstract":"Exercise tolerance (EXT) has been primarily explained by individual body/physical features, partly neglecting the complex cardiorespiratory response needed to cope with exercise. While in healthy conditions most people display good EXT, the presence of exercise intolerance (EXINT) has also been documented. More importantly, EXINT increases in several pathological conditions. Despite the precise mechanism underpinning EXINT is not fully understood. It is well known that exaggerated sympathetic outflow to skeletal muscle impairs proper muscle function and that activation of the carotid body (CB) chemoreceptors elicits a robust increase in sympathetic activity. Therefore, it is plausible to hypothesize that an exaggerated CB-chemoreflex drive may impair EXT. Accordingly, we aimed to determine: i) differences in CB-chemoreflex drive between EXT and EXINT mice, and ii) the role played by the CB-chemoreflex on setting EXINT. Male C57/BL6 mice (n=24) were used to study chemoreflex function using whole-body plethysmography. Exercise tolerance/intolerance was determined by a three-day maximal performance exercise test. CB-chemoreflex drive was assessed by allowing the mice to breathe hypoxic gas (FiO2~5%). We found that the incidence of EXINT among healthy mice was ~30%. Notably, compared to EXT mice, animals with EXINT exhibited a heightened CB-mediated chemoreflex drive as evidenced by larger increases in the hypoxic ventilatory responses (HVR). Indeed, HVR was ~1.4-fold larger in EXINT compared to the values obtained in EXT mice (VE: 17 ± 1.6 vs. 12 ± 3.2, mL/min, EXINT vs. EXT, respectively). Then, we tested whether high chemoreflex drive was associated with EXINT. For this, we reduced the chemosensory drive by using the Dejour´s maneuver (FiO2~35%) while EXINT mice continues to exercise. We found that chemoreflex unloading in EXINT markedly improves exercise tolerance. Indeed, exercise tolerance was almost fully restored by reducing chemoreflex drive in EXINT. Our findings underscore the potential role of heightened CB-chemoreflex on setting exercise tolerance. Supported by Fondecyt 1220950. 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.","PeriodicalId":49694,"journal":{"name":"Physiology","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141133214","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01DOI: 10.1152/physiol.2024.39.s1.2057
Huan-Ling Liang, Melissa Anfinson, Donna K. Mahnke, Michaela Pereckas, Amy Pan, Brandon Tefft, Joy Lincoln
Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease (CHD). We previously demonstrated that genetic variants in the alpha myosin heavy chain (MYH6) gene are significantly associated with HLHS as well as poor outcomes in patients. Additionally, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) carrying an MYH6-R443P head domain variant demonstrated an impaired CM phenotype including dysmorphic sarcomere structure, altered contractility, and upregulated MYH7 expression. Mesenchymal stem cells (MSCs) and their secretome are currently being explored as a potential therapeutic for cardiac injury. In this study, a possible treatment strategy for iPSC-CMs with a MYH6 tail domain variant was examined through investigation of co-culturing umbilical cord tissue derived MSCs from a healthy newborn. iPSC-CMs from an unaffected family member were included as a normal control to compare cellular RNA and protein changes observed in the MYH6-E1584K line. The MYH6-E1584K variant line demonstrated significant upregulation of sarcomere, calcium channel, and inflammation/immune related gene expression in both mRNA and protein levels. Co-culturing with MSCs rescued expression of several genes and was confirmed through label free proteomic analysis. Co-culturing iPSC-CMs with MSCs also significantly improved contraction (contraction maximum displacement and velocity) in MYH6-E1584K iPSC-CMs. Finally, measurements of microRNA, cytokines, and exosomes secreted into cultured media indicated significant changes. This study suggests that MSC secreted factors improve CM expression and function and may elucidate a new mechanistic target for patients with HLHS. AHW/HHI Innovation Pilot Award. 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.
{"title":"Mesenchymal Stem Cells improve phenotype of induced pluripotent stem cell (iPSC)-derived cardiomyocytes in Hypoplastic Left Heart Syndrome","authors":"Huan-Ling Liang, Melissa Anfinson, Donna K. Mahnke, Michaela Pereckas, Amy Pan, Brandon Tefft, Joy Lincoln","doi":"10.1152/physiol.2024.39.s1.2057","DOIUrl":"https://doi.org/10.1152/physiol.2024.39.s1.2057","url":null,"abstract":"Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease (CHD). We previously demonstrated that genetic variants in the alpha myosin heavy chain (MYH6) gene are significantly associated with HLHS as well as poor outcomes in patients. Additionally, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) carrying an MYH6-R443P head domain variant demonstrated an impaired CM phenotype including dysmorphic sarcomere structure, altered contractility, and upregulated MYH7 expression. Mesenchymal stem cells (MSCs) and their secretome are currently being explored as a potential therapeutic for cardiac injury. In this study, a possible treatment strategy for iPSC-CMs with a MYH6 tail domain variant was examined through investigation of co-culturing umbilical cord tissue derived MSCs from a healthy newborn. iPSC-CMs from an unaffected family member were included as a normal control to compare cellular RNA and protein changes observed in the MYH6-E1584K line. The MYH6-E1584K variant line demonstrated significant upregulation of sarcomere, calcium channel, and inflammation/immune related gene expression in both mRNA and protein levels. Co-culturing with MSCs rescued expression of several genes and was confirmed through label free proteomic analysis. Co-culturing iPSC-CMs with MSCs also significantly improved contraction (contraction maximum displacement and velocity) in MYH6-E1584K iPSC-CMs. Finally, measurements of microRNA, cytokines, and exosomes secreted into cultured media indicated significant changes. This study suggests that MSC secreted factors improve CM expression and function and may elucidate a new mechanistic target for patients with HLHS. AHW/HHI Innovation Pilot Award. 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.","PeriodicalId":49694,"journal":{"name":"Physiology","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141133598","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01DOI: 10.1152/physiol.2024.39.s1.362
Jennifer L. Pluznick, Mackenzie Kui
G protein-coupled receptors (GPCRs) constitute the largest class of proteins in the mammalian genome. Upon activation by a specific ligand, these receptors initiate downstream cellular responses that regulate various physiological processes. One such receptor under investigation in our lab is the ghrelin family receptor GPR39. Unlike its relatives, GPR39 does not respond to peptide hormones or neuropeptides. Initial studies suggested zinc as the endogenous ligand, but recent research indicates that zinc functions as an allosteric potentiator for another unidentified endogenous ligand. To date, synthetic agonists for GPR39 have revealed functions for GPR39 in the heart, bone, skin, pancreas, and gastrointestinal tract. However, GPR39’s role in renal physiology is currently unknown, despite relatively high kidney expression. To address this gap in knowledge, we first worked to localize GPR39 within the kidney. As a reliable antibody for GPR39 is not available, we utilized a combination of RNAScope (Gpr39) and immunofluorescence (AQP2). We find that GPR39 is expressed in the renal collecting duct, with the highest expression in AQP2-positive cells (principal cells) in the inner medullary collecting duct, and lesser expression in the cortical collecting duct. To determine if GPR39 is expressed apically or basolaterally, we then cloned GPR39 with a C-terminal EGFP tag and observed basolateral targeting of GPR39 in polarized MDCK cells grown on filters. In order to query the function of GPR39 in principal cells, we used murine principal kidney cortical collecting duct cells (mpkCCD). As others have shown, we find that mpkCCD express and traffc AQP2 to the apical plasma membrane only in the presence of the vasopressin analog dDAVP. Furthermore, we observe that treatment of mpkCCD with the GPR39-specific agonist cpd1324 induces internalization of AQP2 into cytoplasmic vesicles, even in the continued presence of dDAVP. Under vehicle conditions (dDAVP + vehicle), 80.9±7.7% (mean±SD) of AQP2 stain colocalized with an apical membrane marker (WGA staining). In contrast, when mpkCCD cells were co-treated ddAVP+cpd1324, only 58.3±6.02% of AQP2 stain colocalized with the apical membrane (p < 0.001, t-test). These results indicate that GPR39 activation may act to antagonize vasopressin-induced AQP2 traffcking in mpkCCD cells. NHLBIT32HL007534 (MK) and American Heart Association Established Investigator Award (JLP). 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.
{"title":"Uncovering the role of GPR39 in the kidney","authors":"Jennifer L. Pluznick, Mackenzie Kui","doi":"10.1152/physiol.2024.39.s1.362","DOIUrl":"https://doi.org/10.1152/physiol.2024.39.s1.362","url":null,"abstract":"G protein-coupled receptors (GPCRs) constitute the largest class of proteins in the mammalian genome. Upon activation by a specific ligand, these receptors initiate downstream cellular responses that regulate various physiological processes. One such receptor under investigation in our lab is the ghrelin family receptor GPR39. Unlike its relatives, GPR39 does not respond to peptide hormones or neuropeptides. Initial studies suggested zinc as the endogenous ligand, but recent research indicates that zinc functions as an allosteric potentiator for another unidentified endogenous ligand. To date, synthetic agonists for GPR39 have revealed functions for GPR39 in the heart, bone, skin, pancreas, and gastrointestinal tract. However, GPR39’s role in renal physiology is currently unknown, despite relatively high kidney expression. To address this gap in knowledge, we first worked to localize GPR39 within the kidney. As a reliable antibody for GPR39 is not available, we utilized a combination of RNAScope (Gpr39) and immunofluorescence (AQP2). We find that GPR39 is expressed in the renal collecting duct, with the highest expression in AQP2-positive cells (principal cells) in the inner medullary collecting duct, and lesser expression in the cortical collecting duct. To determine if GPR39 is expressed apically or basolaterally, we then cloned GPR39 with a C-terminal EGFP tag and observed basolateral targeting of GPR39 in polarized MDCK cells grown on filters. In order to query the function of GPR39 in principal cells, we used murine principal kidney cortical collecting duct cells (mpkCCD). As others have shown, we find that mpkCCD express and traffc AQP2 to the apical plasma membrane only in the presence of the vasopressin analog dDAVP. Furthermore, we observe that treatment of mpkCCD with the GPR39-specific agonist cpd1324 induces internalization of AQP2 into cytoplasmic vesicles, even in the continued presence of dDAVP. Under vehicle conditions (dDAVP + vehicle), 80.9±7.7% (mean±SD) of AQP2 stain colocalized with an apical membrane marker (WGA staining). In contrast, when mpkCCD cells were co-treated ddAVP+cpd1324, only 58.3±6.02% of AQP2 stain colocalized with the apical membrane (p < 0.001, t-test). These results indicate that GPR39 activation may act to antagonize vasopressin-induced AQP2 traffcking in mpkCCD cells. NHLBIT32HL007534 (MK) and American Heart Association Established Investigator Award (JLP). 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.","PeriodicalId":49694,"journal":{"name":"Physiology","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141133668","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01DOI: 10.1152/physiol.2024.39.s1.1672
Allison Bartlett, Kenton Sanders, Sal Baker
Background: Secretin is a member of the secretin-glucagon-vasoactive intestinal peptide hormone superfamily and is a multifunctional gastrointestinal- (GI) and neuro- peptide hormone. Secretin is primarily secreted postprandially from the crypts of Lieberkühn of duodenal enteroendocrine S cells into circulation where it reaches secretin receptor targets in the central nervous system and periphery. This dynamic hormone has been shown to act as a key signaling molecule in the regulation of digestion, metabolism and energy expenditure, water retention, reproduction, thermogenesis in adipose tissue, and in gastric and intestinal motility. Secretin’s canonical role in the GI tract is to stimulate the secretion of bicarbonate and bile from pancreatic ducts and bile ducts to neutralize acidic chyme exiting the stomach. Secretin has also been shown to slow intestinal motility, but its targets and mechanism of action is poorly understood. Aims: Several studies have proposed that secretin acts to slow intestinal motility in the intestines primarily through the many secretin receptors present on vagal afferents in the GI tract, here we discuss new data that suggests an alternate and complementary signaling pathway via interstitial cells of Cajal (ICC). ICC act as a liaison to facilitate a reduction in force of GI smooth muscle contraction through the activation of the secretin receptor (SCTR) and subsequent stimulation of the second messenger, cyclic adenosine monophosphate (cAMP). Here we provide evidence to show how ICC mediates changes in myogenic activity and motility in the small intestine. Methods: Spinning-disk confocal microscopy was used to monitor Ca2+ signaling in ICC from small intestinal muscles of GCaMP6f x KitiCre mice. Additionally, cAMP levels were evaluated using CAMPER mice. Intestinal muscle contractility was assessed using muscle strip myography experiments. Results: Secretin reduced small intestinal force of contraction in the presence of tetrodotoxin (TTX) and dampened the effect of cholinergic transmission. The secretin receptor (SCTR) is expressed primarily on ICC, specifically ICC within the deep muscular plexus (ICC-DMP) in the small intestine and Ca2+ imaging confirmed the effects are primarily localized within ICC-DMP. Secretin reduced carbachol-induced contractions and Ca2+ transients in ICC-DMP in response to electrical field stimulation (EFS) in the presence of LNNA (NO synthase inhibitor) and MRS2500 (P2Y1 antagonist). Secretin caused an increase in cAMP levels in ICC-DMP in muscles from Kit-iCre-CAMPER mice. PKA inhibitors rescued some of the effects of secretin on ICC-DMP Ca2+ signaling. Measurements of diameter change in large, intact, intestinal segments (4-5 cm) showed a significant decrease in response to Secretin. Conclusions: Secretin can inhibit small intestinal motility through the activation SCTRs on ICC-DMP via cAMP-mediated mechanisms. These results show how novel secretin targets on ICC influence GI muscles and
{"title":"Secretin Inhibits Small Intestinal Motility via Interstitial Cells of Cajal and cAMP Mechanisms","authors":"Allison Bartlett, Kenton Sanders, Sal Baker","doi":"10.1152/physiol.2024.39.s1.1672","DOIUrl":"https://doi.org/10.1152/physiol.2024.39.s1.1672","url":null,"abstract":"Background: Secretin is a member of the secretin-glucagon-vasoactive intestinal peptide hormone superfamily and is a multifunctional gastrointestinal- (GI) and neuro- peptide hormone. Secretin is primarily secreted postprandially from the crypts of Lieberkühn of duodenal enteroendocrine S cells into circulation where it reaches secretin receptor targets in the central nervous system and periphery. This dynamic hormone has been shown to act as a key signaling molecule in the regulation of digestion, metabolism and energy expenditure, water retention, reproduction, thermogenesis in adipose tissue, and in gastric and intestinal motility. Secretin’s canonical role in the GI tract is to stimulate the secretion of bicarbonate and bile from pancreatic ducts and bile ducts to neutralize acidic chyme exiting the stomach. Secretin has also been shown to slow intestinal motility, but its targets and mechanism of action is poorly understood. Aims: Several studies have proposed that secretin acts to slow intestinal motility in the intestines primarily through the many secretin receptors present on vagal afferents in the GI tract, here we discuss new data that suggests an alternate and complementary signaling pathway via interstitial cells of Cajal (ICC). ICC act as a liaison to facilitate a reduction in force of GI smooth muscle contraction through the activation of the secretin receptor (SCTR) and subsequent stimulation of the second messenger, cyclic adenosine monophosphate (cAMP). Here we provide evidence to show how ICC mediates changes in myogenic activity and motility in the small intestine. Methods: Spinning-disk confocal microscopy was used to monitor Ca2+ signaling in ICC from small intestinal muscles of GCaMP6f x KitiCre mice. Additionally, cAMP levels were evaluated using CAMPER mice. Intestinal muscle contractility was assessed using muscle strip myography experiments. Results: Secretin reduced small intestinal force of contraction in the presence of tetrodotoxin (TTX) and dampened the effect of cholinergic transmission. The secretin receptor (SCTR) is expressed primarily on ICC, specifically ICC within the deep muscular plexus (ICC-DMP) in the small intestine and Ca2+ imaging confirmed the effects are primarily localized within ICC-DMP. Secretin reduced carbachol-induced contractions and Ca2+ transients in ICC-DMP in response to electrical field stimulation (EFS) in the presence of LNNA (NO synthase inhibitor) and MRS2500 (P2Y1 antagonist). Secretin caused an increase in cAMP levels in ICC-DMP in muscles from Kit-iCre-CAMPER mice. PKA inhibitors rescued some of the effects of secretin on ICC-DMP Ca2+ signaling. Measurements of diameter change in large, intact, intestinal segments (4-5 cm) showed a significant decrease in response to Secretin. Conclusions: Secretin can inhibit small intestinal motility through the activation SCTRs on ICC-DMP via cAMP-mediated mechanisms. These results show how novel secretin targets on ICC influence GI muscles and","PeriodicalId":49694,"journal":{"name":"Physiology","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141134654","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01DOI: 10.1152/physiol.2024.39.s1.1486
Richard S. Babicz, R. Bouley, Pui W Cheung, N. Baylor, Daphne Faber, Dennis Brown
In the canonical aquaporin-2 (AQP2) signaling pathway, vasopressin (VP) binds to its V2 receptor, which in turn activates adenylate cyclase and protein kinase A (PKA), resulting in AQP2 S256 phosphorylation and accumulation of AQP2 in the membrane. The epidermal growth factor receptor (EGFR) inhibitor erlotinib also increases AQP2 S256 phosphorylation and membrane accumulation, yet it does not increase cAMP or PKA activity. We hypothesize that the ribosomal s6 kinase (RSK), a downstream effector in the EGFR-MAPK/ERK pathway, is the terminal kinase phosphorylating AQP2 in this novel PKA-independent pathway activated by erlotinib, and that the phosphoinositide dependent kinase-1 (PDK1), a well-established activator of RSK, is indispensable for erlotinib-induced AQP2 activation. Using our LLC-AQP2 cell model, we show that erlotinib-induced AQP2 membrane accumulation and S256 phosphorylation are abolished by the specific RSK inhibitor BI-D1870. RSK knockdown with siRNA also blocked AQP2 S256 phosphorylation and membrane accumulation, as did RSK knockout with CRISPR/cas9 editing. Next, rat kidney slices were incubated in Hank’s buffer with or without BI-D1870 and treated with erlotinib. BI-D1870 inhibited AQP2 membrane accumulation in collecting duct principal cells, supporting the relevance of this pathway in kidney cells in situ. An in-vitro kinase assay using purified proteins demonstrated that RSK directly phosphorylates AQP2 at the S256 residue. Additionally, erlotinib consistently increased phosphorylation of RSK T359, a residue associated with RSK’s active state. In canonical RSK activation, a phosphorylation cascade results in PDK1 binding to and activating RSK. We found that PDK1 inhibition with an experimental compound PS-222 also blocked erlotinib-induced AQP2 membrane accumulation and S256 phosphorylation. This implicates PDK1 along with RSK in the novel erlotinib-induced pathway of AQP2 traffcking. Our data show that RSK and PDK1 are terminal effectors in the novel, PKA-independent pathway of AQP2 activation induced by erlotinib. Further elaboration of this new non-canonical pathway promises to uncover potential pharmacological targets for the treatment of water balance disorders. This work was supported by the National Institutes of Health (NIH) grant DK096586 (D. Brown). P. W. Cheung was supported was supported by NIH K-award DK115901. Richard Babicz is the recipient of the Ben J Lipps Research Fellowship (American Society of Nephrology). Additional support for the Program in Membrane Biology Microscopy Core came from the Boston Area Diabetes and Endocrinology Research Center (DK057521) and the Massachusetts General Hospital (MGH) Center for the Study of Inflammatory Bowel Disease (DK043351). 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 rev
{"title":"EGFR inhibition with erlotinib induces membrane accumulation of aquaporin-2 via PDK1 and RSK activation","authors":"Richard S. Babicz, R. Bouley, Pui W Cheung, N. Baylor, Daphne Faber, Dennis Brown","doi":"10.1152/physiol.2024.39.s1.1486","DOIUrl":"https://doi.org/10.1152/physiol.2024.39.s1.1486","url":null,"abstract":"In the canonical aquaporin-2 (AQP2) signaling pathway, vasopressin (VP) binds to its V2 receptor, which in turn activates adenylate cyclase and protein kinase A (PKA), resulting in AQP2 S256 phosphorylation and accumulation of AQP2 in the membrane. The epidermal growth factor receptor (EGFR) inhibitor erlotinib also increases AQP2 S256 phosphorylation and membrane accumulation, yet it does not increase cAMP or PKA activity. We hypothesize that the ribosomal s6 kinase (RSK), a downstream effector in the EGFR-MAPK/ERK pathway, is the terminal kinase phosphorylating AQP2 in this novel PKA-independent pathway activated by erlotinib, and that the phosphoinositide dependent kinase-1 (PDK1), a well-established activator of RSK, is indispensable for erlotinib-induced AQP2 activation. Using our LLC-AQP2 cell model, we show that erlotinib-induced AQP2 membrane accumulation and S256 phosphorylation are abolished by the specific RSK inhibitor BI-D1870. RSK knockdown with siRNA also blocked AQP2 S256 phosphorylation and membrane accumulation, as did RSK knockout with CRISPR/cas9 editing. Next, rat kidney slices were incubated in Hank’s buffer with or without BI-D1870 and treated with erlotinib. BI-D1870 inhibited AQP2 membrane accumulation in collecting duct principal cells, supporting the relevance of this pathway in kidney cells in situ. An in-vitro kinase assay using purified proteins demonstrated that RSK directly phosphorylates AQP2 at the S256 residue. Additionally, erlotinib consistently increased phosphorylation of RSK T359, a residue associated with RSK’s active state. In canonical RSK activation, a phosphorylation cascade results in PDK1 binding to and activating RSK. We found that PDK1 inhibition with an experimental compound PS-222 also blocked erlotinib-induced AQP2 membrane accumulation and S256 phosphorylation. This implicates PDK1 along with RSK in the novel erlotinib-induced pathway of AQP2 traffcking. Our data show that RSK and PDK1 are terminal effectors in the novel, PKA-independent pathway of AQP2 activation induced by erlotinib. Further elaboration of this new non-canonical pathway promises to uncover potential pharmacological targets for the treatment of water balance disorders. This work was supported by the National Institutes of Health (NIH) grant DK096586 (D. Brown). P. W. Cheung was supported was supported by NIH K-award DK115901. Richard Babicz is the recipient of the Ben J Lipps Research Fellowship (American Society of Nephrology). Additional support for the Program in Membrane Biology Microscopy Core came from the Boston Area Diabetes and Endocrinology Research Center (DK057521) and the Massachusetts General Hospital (MGH) Center for the Study of Inflammatory Bowel Disease (DK043351). 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 rev","PeriodicalId":49694,"journal":{"name":"Physiology","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141136122","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}