Kinetics of the thapsigargin-induced Ca2+ mobilisation: A quantitative analysis in the HEK-293 cell line

Thapsigargin (TG) inhibits the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump and, when applied acutely, it initiates a Ca2+ mobilisation that begins with the loss of Ca2+ from the endoplasmic reticulum (ER) and culminates with store-operated Ca2+ entry (SOCE) from the extracellular space. Using the popular model cell line HEK-293, we quantified TG-induced changes in cytosolic and ER Ca2+ levels using FURA-2 and the FRET-based ER Ca2+ sensor D1ER, respectively. Our analysis predicts an ER Ca2+ leak of 5–6 µM⋅s−1 for the typical basal ER Ca2+ level of 335–407 µM in HEK-293 cells. The resulting cytosolic Ca2+ transients reached peak amplitudes of 0.6–1.0 µM in the absence of external Ca2+ and were amplified by SOCE that amounted to 28–30 nM⋅s−1 in 1 mM external Ca2+. Additionally, cytosolic Ca2+ transients were shaped by a Ca2+ clearance of 10–13 nM⋅s−1. Using puromycin (PURO), which enhances the ER Ca2+ leak, we show that TG-induced cytosolic Ca2+ transients are directly related to ER Ca2+ levels and to the ER Ca2+ leak. A one-compartment model incorporating ER Ca2+ leak and cytosolic Ca2+ clearance accounted satisfactorily for the basic features of TG-induced Ca2+ transients and underpinned the rule that an increase in amplitude associated with shortening of TG-induced cytosolic Ca2+ transients most likely reflects an increase in ER Ca2+ leak

Prolactin-sensitive olfactory sensory neurons regulate male preference in female mice by modulating responses to chemosensory cues

Chemosensory cues detected in the nose need to be integrated with the hormonal status to trigger appropriate behaviors, but the neural circuits linking the olfactory and the endocrine system are insufficiently understood. Here, we characterize olfactory sensory neurons in the murine nose that respond to the pituitary hormone prolactin. Deletion of prolactin receptor in these cells results in impaired detection of social odors and blunts male preference in females. The prolactin-responsive olfactory sensory neurons exhibit a distinctive projection pattern to the brain that is similar across different individuals and express a limited subset of chemosensory receptors. Prolactin modulates the responses within these neurons to discrete chemosensory cues contained in male urine, providing a mechanism by which the hormonal status can be directly linked with distinct olfactory cues to generate appropriate behavioral responses

Ovulation is triggered by a cyclical modulation of gonadotropes into a hyperexcitable state

Gonadotropes in the anterior pituitary gland are essential for fertility and provide a functional link between the brain and the gonads. To trigger ovulation, gonadotrope cells release massive amounts of luteinizing hormone (LH). The mechanism underlying this remains unclear. Here, we utilize a mouse model expressing a genetically encoded Ca2+ indicator exclusively in gonadotropes to dissect this mechanism in intact pituitaries. We demonstrate that female gonadotropes exclusively exhibit a state of hyperexcitability during the LH surge, resulting in spontaneous [Ca2+]i transients in these cells, which persist in the absence of any in vivo hormonal signals. L-type Ca2+ channels and transient receptor potential channel A1 (TRPA1) together with intracellular reactive oxygen species (ROS) levels ensure this state of hyperexcitability. Consistent with this, virus-assisted triple knockout of Trpa1 and L-type Ca2+ subunits in gonadotropes leads to vaginal closure in cycling females. Our data provide insight into molecular mechanisms required for ovulation and reproductive success in mammals

Bitter taste cells in the ventricular walls of the murine brain regulate glucose homeostasis

The median eminence (ME) is a circumventricular organ at the base of the brain that controls body homeostasis. Tanycytes are its specialized glial cells that constitute the ventricular walls and regulate different physiological states, however individual signaling pathways in these cells are incompletely understood. Here, we identify a functional tanycyte subpopulation that expresses key taste transduction genes including bitter taste receptors, the G protein gustducin and the gustatory ion channel TRPM5 (M5). M5 tanycytes have access to blood-borne cues via processes extended towards diaphragmed endothelial fenestrations in the ME and mediate bidirectional communication between the cerebrospinal fluid and blood. This subpopulation responds to metabolic signals including leptin and other hormonal cues and is transcriptionally reprogrammed upon fasting. Acute M5 tanycyte activation induces insulin secretion and acute diphtheria toxin-mediated M5 tanycyte depletion results in impaired glucose tolerance in diet-induced obese mice. We provide a cellular and molecular framework that defines how bitter taste cells in the ME integrate chemosensation with metabolism

Bitter taste signaling in tracheal epithelial brush cells elicits innate immune responses to bacterial infection

Constant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene Trpm5, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into firstline defense mechanisms against bacterial infections of the lung

Bitter taste cells in the ventricular walls of the murine brain regulate glucose homeostasis.

peer reviewedThe median eminence (ME) is a circumventricular organ at the base of the brain that controls body homeostasis. Tanycytes are its specialized glial cells that constitute the ventricular walls and regulate different physiological states, however individual signaling pathways in these cells are incompletely understood. Here, we identify a functional tanycyte subpopulation that expresses key taste transduction genes including bitter taste receptors, the G protein gustducin and the gustatory ion channel TRPM5 (M5). M5 tanycytes have access to blood-borne cues via processes extended towards diaphragmed endothelial fenestrations in the ME and mediate bidirectional communication between the cerebrospinal fluid and blood. This subpopulation responds to metabolic signals including leptin and other hormonal cues and is transcriptionally reprogrammed upon fasting. Acute M5 tanycyte activation induces insulin secretion and acute diphtheria toxin-mediated M5 tanycyte depletion results in impaired glucose tolerance in diet-induced obese mice. We provide a cellular and molecular framework that defines how bitter taste cells in the ME integrate chemosensation with metabolism

Bitter taste signaling in tracheal epithelial brush cells elicits innate immune responses to bacterial infection.

peer reviewedConstant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene Trpm5, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into first-line defense mechanisms against bacterial infections of the lung

МЕТОДИ КОМПРОМІСНОГО ВИБОРУ ІНТЕРВАЛУ ЗМІН МОДЕЛЬНОГО ЧАСУ В ІМІТАЦІЙНИХ МОДЕЛЯХ

The process of simulation is considered as one of the main means for studying the dynamics of functioning of real systems, in particular, complex ones. The system can be represented by a set of components. The functioning of a component is represented by the implementation of a set of functional actions, which are represented by the corresponding activities in the form of a pair: algorithm for performing a functional action - duration of execution. The problem of displaying the simultaneous or parallel nature of the functioning of all components of a complex system in the MI is solved by introducing model or system time. The main methods for introducing model time are the fixed step method and the variable step method. In the fixed-step method, an important problem is the choice of the value of the model time variation interval. The existing recommendations for choosing the value of the interval for changing the model time are of a qualitative nature and their use makes it possible to increase the accuracy of modeling, but at the same time the consumption of the computer time resource increases. It is proposed to use quantitative estimates of the values of quality criteria - the accuracy and expenditure of the computer time resource when choosing the value of the model time change interval. The generalized criterion is represented as a weighted sum of transformations of local criteria. The values of the coefficients by which the corresponding transformations are multiplied express the preferences of the decision-maker for the local optimality criteria. A geometric interpretation of the process of determining a compromise alternative on a set of effective alternatives for various cases of the importance of local criteria is given. These estimates make it possible to substantiate the nature of the change in the quality criteria for various variants of the values of the interval of change in the model time and to use them to select a compromise option among the effective ones by minimizing the generalized criterion. The choice of the compromise value of the model time variation interval is implemented in the control simulation program.Рассматриваются процесс имитационного моделирования как одно из основных средств для изучения динамики функционирования реальных систем, в частности, сложных. Система может быть представлена совокупностью компонентов. Функционирование компонента представляется реализацией множества функциональных действий, которые представляются соответствующими активностями в виде пары: алгоритм выполнения функционального действия - продолжительность выполнения. Проблема отображения в ИМ одновременного или параллельного характера функционирования всех компонентов сложной системы решается введением модельного или системного времени. Основными методами введения модельного времени является метод фиксированного шага и метод переменного шага. В методе фиксированного шага важной проблемой является выбор величины интервала изменения модельного времени. Существующие рекомендации для выбора величины интервала изменения модельного времени имеют качественный характер и их использование позволяет повысить точность моделирования, но при этом растут затраты ресурса времени компьютера. Предложены при выборе величины интервала изменения модельного времени использовать количественные оценки значений критериев качества - точность и расходы ресурса времени компьютера. Обобщенный критерий представляется взвешенной суммой преобразований локальных критериев. Значения коэффициентов, на которые умножаются соответствующие преобразования, выражают предпочтения лица, принимающего решения, локальным критериям оптимальности. Приводится геометрическая интерпретация процесса определения компромиссной альтернативы на множестве эффективных альтернатив для различных случаев важности локальных критериев. Эти оценки позволяют обосновать характер изменения критериев качества для различных вариантов значений интервала изменения модельного времени и использовать их для выбора компромиссного варианта среди эффективных путем минимизации обобщенного критерия. Выбор компромиссного значения интервала изменения модельного времени реализуется в управляющей программе моделирования.Розглядаються процес імітаційного моделювання як один з основних засобів для вивчення динаміки функціонування реальних систем, зокрема, складних. Система може бути представлена сукупністю компонент. Функціонування компонента представляється реалізацією множини функціональних дій, які представляються відповідними активностями у вигляді пари: алгоритм виконання функціональної дії – тривалість виконання. Проблема відображення в ІМ одночасного або паралельного характеру функціонування всіх компонентів складної системи вирішується введенням модельного або системного часу. Основними методами введення модельного часу є метод фіксованого кроку та метод змінного кроку. В методі фіксованого кроку важливою проблемою є вибір величини інтервалу зміни модельного часу. Існуючі рекомендації для вибору величини інтервалу зміни модельного часу мають якісний характер і їх використання дозволяє підвищити точність моделювання, але при цьому зростають витрати ресурсу часу комп’ютера. Запропоновано при виборі величини інтервалу зміни модельного часу використовувати кількісні оцінки значень критеріїв якості – точність і витрати ресурсу часу комп’ютера. Узагальнений критерій представляється зваженою сумою перетворень локальних критеріїв. Значення коефіцієнтів, на які множаться відповідні перетворення, висловлюють переваги особи, що приймає рішення, локальним критеріям оптимальності. Наводиться геометрична інтерпретація процесу визначення компромісної альтернативи на множині ефективних альтернатив для різних випадків важливості локальних критеріїв. Ці оцінки дозволяють обґрунтувати характер зміни критеріїв якості для різних варіантів значень інтервалу зміни модельного часу та використовувати їх для вибору компромісного варіанту серед ефективних шляхом мінімізації узагальненого критерію. Вибір компромісного значення інтервалу зміни модельного часу реалізується в управляючій програмі моделювання

Mycolactone enhances the Ca2+ leakage from endoplasmic reticulum by trapping Sec61 translocons in a Ca2+ permeable state

The Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of, the major translocon subunit Sec61α, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol. Here, we have analysed the effect of mycolactone on cytosolic and ER Ca2+ levels using compartment-specific sensors. We also used molecular docking analysis to explore potential interaction sites for mycolactone on translocons in various states. These results show that mycolactone enhances the leak of Ca2+ ions via the Sec61 translocon, resulting in a slow but substantial depletion of ER Ca2+. This leak was dependent on mycolactone binding to Sec61α because resistance mutations in this protein completely ablated the increase. Molecular docking supports the existence of a mycolactone-binding transient inhibited state preceding translocation and suggests mycolactone may also bind Sec61α in its idle state. We propose that delayed ribosomal release after translation termination and/or translocon “breathing” during rapid transitions between the idle and intermediate-inhibited states allow for transient Ca2+ leak, and mycolactone’s stabilisation of the latter underpins the phenotype observed