Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa.

Volume regulation is essential for cell homeostasis and physiological function. Amongst the sensory molecules that have been associated with volume regulation is the transient receptor potential vanilloid 4 (TRPV4), which is a non-selective cation channel that in conjunction with aquaporins, typically controls regulatory volume decrease (RVD). Here we show that the interaction between orthologous AQP4 (Aqp4a) and TRPV4 (Trpv4) is important for regulatory volume increase (RVI) in post-activated marine fish spermatozoa under high osmotic stress. Based upon electrophysiological, volumetric, and in vivo and ex vivo functional experiments using the pharmacological and immunological inhibition of Aqp4a and Trpv4 our model suggests that upon ejaculation and exposure to the hypertonic seawater, spermatozoon shrinkage is initially mediated by water efflux through Aqp1aa in the flagellar tail. The shrinkage results in an increase in intracellular Ca2+ concentration, and the activation of sperm motility and a Na+/K+/2Cl- (NKCC1) cotransporter. The activity of NKCC1 is required for the initiation of cell swelling, which secondarily activates the Aqp4a-Trpv4 complex to facilitate the influx of water via Aqp4a-M43 and Ca2+ via Trpv4 and L-type channels for the mediation of RVI. The inhibitory experiments show that blocking of each of these events prevents either shrinkage or RVI. Our data thus reveal that post-activated marine fish spermatozoa are capable of initiating RVI under a high hypertonic stress, which is essential for the maintenance of sperm motility.

Unexpected change in hydrogel spacer volume during external-beam radiation therapy.

PURPOSE: To reduce the rectal radiation dose during local radiation therapy of prostate cancer, a hydrogel spacer is typically implanted between the prostate and rectum. However, the spacer volume can change during external beam radiation therapy (EBRT). Therefore, we used magnetic resonance imaging (MRI) to determine changes in the spacer volume during EBRT and analyzed the data to identify patient factors influencing this change. MATERIALS AND METHODS: A hydrogel spacer was implanted in each enrolled patient diagnosed with prostate cancer (n = 22, age = 69-86 years) for EBRT with a total dose of 70 Gy over 35 fractions. T2-weighted MRI images were acquired before (median = 8 days) and during EBRT, when the radiation dose of 48 Gy (median) was given at 55 days (median) after implantation. MRI images were used to determine the spacer volume as well as the maximum and minimum distances between the prostate and anterior wall of the rectum at the middle height of the prostate. Scatterplots were created to determine whether correlations existed between changes in the spacer volume and these two distances, while uni- and multivariate analyses were conducted to determine if the spacer volume change was influenced by the following patient factors: age, body mass index, estimated glomerular filtration rate, and visceral fat areas at the umbilical and femoral head positions. RESULTS: The spacer volume increased in all 22 patients, with the smaller spacer volume before EBRT increasing by a larger amount during EBRT. This increase in the spacer volume was unaffected by other patient factors. However, it correlated with the change in the maximum distance between the prostate and anterior wall of the rectum. CONCLUSION: To avoid adverse changes in the rectal radiation dose during EBRT, hydrogel spacer volume should be monitored, especially if the pre-EBRT volume is small.

Acute-to-chronic subdural hematoma: radiographic and clinical progression from acute subdural hematoma.

INTRODUCTION: The pathogenesis of chronic subdural hematoma (CSDH) has not been completely understood. However, different mechanisms can result in space-occupying subdural fluid collections, one pathway can be the transformation of an original trauma-induced acute subdural hematoma (ASDH) into a CSDH. MATERIALS AND METHODS: All patients with unilateral CSDH, requiring burr hole trephination between 2018 and 2023 were included. The population was distributed into an acute-to-chronic group (group A, n = 41) and into a conventional group (group B, n = 282). Clinical and radiographic parameters were analyzed. In analysis A, changes of parameters after trauma within group A are compared. In analysis B, parameters between the two groups before surgery were correlated. RESULTS: In group A, volume and midline shift increased significantly during the progression from acute-to-chronic (p < 0.001, resp.). Clinical performance (modified Rankin scale, Glasgow Coma Scale) dropped significantly (p = 0.035, p < 0.001, resp.). Median time between trauma with ASDH and surgery for CSDH was 12 days. Patients treated up to the 12th day presented with larger volume of ASDH (p = 0.012). Before burr hole trephination, patients in group A presented with disturbance of consciousness (DOC) more often (p = 0.002), however less commonly with a new motor deficit (p = 0.014). Despite similar midline shift between the groups (p = 0.8), the maximal hematoma width was greater in group B (p < 0.001). CONCLUSION: If ASDH transforms to CSDH, treatment may become mandatory early due to increase in volume and midline shift. Close monitoring of these patients is crucial since DOC and rapid deterioration is common in this type of SDH.

Valorization of an Old Variety of Triticum aestivum: A Study of Its Suitability for Breadmaking Focusing on Sensory and Nutritional Quality.

"Avanzi 3-Grano 23" (G23) is an old variety of Triticum aestivum from the mountain areas of Lunigiana (north Tuscany, Italy), where traditional farming communities have contributed to its success and on-farm conservation. G23 flour, traditionally used for typical food products, is characterized by particular nutritional and sensory traits but has technological properties which limit its suitability for breadmaking. The aim of this work was to evaluate how to promote the use of G23 through the optimization of bread formulation by leveraging both flour blending and the leavening system. During the preliminary test, three different mixes of G23 flour and a strong flour (C) were tested in terms of their leavening power as a function of leavening agent (baker's yeast or sourdough). The selected M2 flour, composed of G23:C (1:1 w/w), was used for further breadmaking trials and 100% C flour was utilized as a control. The sourdough bread obtained with the M2 flour (SB-M2) showed an improved sensory profile compared with the related control (SB-C). Furthermore, SB-M2 exhibited the best aromatic (high content in aldehydes, pyrazines and carboxylic acids) and phytochemical profile (total polyphenols and flavonoids content and antioxidant activity). In contrast, the use of baker's yeast, although optimal from the point of view of breadmaking, did not result in the same levels of aromatic complexity because it tends to standardize the product without valorizing the sensory and nutritional qualities of the flour. In conclusion, in the experimental conditions adopted, this old wheat variety appears to be suitable for the production of sourdough bakery products.

A century of exercise physiology: key concepts in muscle cell volume regulation.

Skeletal muscle cells can both gain and lose volume during periods of exercise and rest. Muscle cells do not behave as perfect osmometers because the cell volume changes are less than predicted from the change in extracellular osmolality. Therefore, there are mechanisms involved in regulating cell volume, and they are different for regulatory volume decreases and regulatory volume increases. Also, after an initial rapid change in cell volume, there is a gradual and partial recovery of cell volume that is effected by ion and water transport mechanisms. The mechanisms have been studied in non-contracting muscle cells, but remain to be fully elucidated in contracting muscle. Changes in muscle cell volume are known to affect the strength of contractile activity as well as anabolic/catabolic signaling, perhaps indicating that cell volume should be a regulated variable in skeletal muscle cells. Muscles contracting at moderate to high intensity gain intracellular volume because of increased intracellular osmolality. Concurrent increases in interstitial (extracellular) muscle volume occur from an increase in osmotically active molecules and increased vascular filtration pressure. At the same time, non-contracting muscles lose cell volume because of increased extracellular (blood) osmolality. This review provides the physiological foundations and highlights key concepts that underpin our current understanding of volume regulatory processes in skeletal muscle, beginning with consideration of osmosis more than 200 years ago and continuing through to the process of regulatory volume decrease and regulatory volume increase.

Studying cell volume beyond cell volume.

The first part of the paper describes two simple microscopic techniques that we use in our laboratory. One measures cell volumes in adherent cultures and the other measures cell dry mass; both measurements are done on the same instrument (a standard bright-field transmission microscope with only one or two narrow-band color filters added) and on the same cells. The reason for combining cell volume with dry mass is that the ratio of the two-dry mass concentration (MC)-is an important and insufficiently utilized biological parameter. We then describe a few applications of MC. The available experimental data strongly suggest its critical role in biological processes, including cell volume regulation. For example, most eukaryotic cells have surprisingly similar values of MC. Moreover, MC (and not cell volume) is tightly controlled in growing cell cultures at highly variable external osmolarities. We review the results showing that elevation of MC is a direct cause of shrinkage-induced apoptosis. Also, by focusing on MC, one can study heterogenous processes, such as necrotic swelling, or discriminate between apoptotic dehydration and the loss of cell fragments.

Cell Death Induction and Protection by Activation of Ubiquitously Expressed Anion/Cation Channels. Part 2: Functional and Molecular Properties of ASOR/PAC Channels and Their Roles in Cell Volume Dysregulation and Acidotoxic Cell Death.

For survival and functions of animal cells, cell volume regulation (CVR) is essential. Major hallmarks of necrotic and apoptotic cell death are persistent cell swelling and shrinkage, and thus they are termed the necrotic volume increase (NVI) and the apoptotic volume decrease (AVD), respectively. A number of ubiquitously expressed anion and cation channels play essential roles not only in CVR but also in cell death induction. This series of review articles address the question how cell death is induced or protected with using ubiquitously expressed ion channels such as swelling-activated anion channels, acid-activated anion channels, and several types of TRP cation channels including TRPM2 and TRPM7. In the Part 1, we described the roles of swelling-activated VSOR/VRAC anion channels. Here, the Part 2 focuses on the roles of the acid-sensitive outwardly rectifying (ASOR) anion channel, also called the proton-activated chloride (PAC) anion channel, which is activated by extracellular protons in a manner sharply dependent on ambient temperature. First, we summarize phenotypical properties, the molecular identity, and the three-dimensional structure of ASOR/PAC. Second, we highlight the unique roles of ASOR/PAC in CVR dysfunction and in the induction of or protection from acidotoxic cell death under acidosis and ischemic conditions.

Vasopressin Neurons Respond to Hyperosmotic Stimulation with Regulatory Volume Increase and Secretory Volume Decrease by Activating Ion Transporters and Ca2+ Channels.

BACKGROUND/AIMS: Arginine vasopressin (AVP) neurons play an important role for sensing a change in the plasma osmolarity and thereby responding with regulated AVP secretion in order to maintain the body fluid homeostasis. The osmo-sensing processes in magnocellular neurosecretory cells (MNCs) including AVP and oxytocin (OXT) neurons of the hypothalamus were reported to be coupled to sustained osmotic shrinkage or swelling without exhibiting discernible cell volume regulation. Since increasing evidence has shown some important differences in properties between AVP and OXT neurons, osmotic volume responses are to be reexamined with distinguishing these cell types from each other. We previously reported that AVP neurons identified by transgenic expression of enhanced green fluorescence protein (eGFP) possess the ability of regulatory volume decrease (RVD) after hypoosmotic cell swelling. Thus, in the present study, we examined the ability of regulatory volume increase (RVI) after hyperosmotic cell shrinkage in AVP neurons. METHODS: Here, we used eGFP-identified AVP neurons acutely dissociated from AVP-eGFP transgenic rats. We performed single-cell size measurements, cytosolic RT-PCR analysis, AVP secretion measurements, and patch-clamp studies. RESULTS: The AVP neurons were found to respond to a hyperosmotic challenge with physiological cell shrinkage caused by massive secretion of AVP, called a secretory volume decrease (SVD), superimposed onto physical osmotic cell shrinkage, and also to exhibit the ability of RVI coping with osmotic and secretory cell shrinkage. Furthermore, our pharmacological and molecular examinations indicated that AVP secretion and its associated SVD event are triggered by activation of T-type Ca2+ channels, and the RVI event is attained by parallel operation of Na+/H+ exchanger and Cl-/HCO3- anion exchanger. CONCLUSION: Thus, it is concluded that AVP neurons respond to hyperosmotic stimulation with the regulatory volume increase and the secretory volume increase by activating ion transporters and Ca2+ channels, respectively.

Cell Volume Regulation in Immune Cell Function, Activation and Survival.

The regulation of cell volume is an essential cellular process in nearly every living organism. The importance of volume regulation in immune cells cannot be understated, as it ensures proper cellular function and effective immune response. These cells utilize ion channels and transporters to maintain volume homeostasis through rapid ion transport across the cell membrane. Immune cells express mechanisms controlling regulatory volume decrease (RVD), regulatory volume increase (RVI), proliferative RVD, and apoptotic volume decrease (AVD). In this review, we summarize recent studies examining the importance of several ion channels, particularly potassium and chloride channels in regulating ion transport during osmotic stress, and in immune cell function, activation, proliferation, and death. We also review the key mechanisms functioning in immune cell proliferation and apoptosis. They serve a crucial role in maintaining adequate ionic concentrations, mediating immune cell activation, and generating proliferative pathways. These regulatory mechanisms play key roles in the function and survival of immune cells, as impaired volume regulation contributes to the pathophysiology of various disorders. A complete understanding of immune cell volume regulatory mechanisms may be a starting point for the development of therapeutic agents targeting these ion channels to treat inflammatory diseases.

Paraganglioma. Presentación de caso / Paraganglioma. Case presentation / Paraganglioma. Apresentação do caso

RESUMEN El Paraganglioma es un tumor poco frecuente de origen neuroectodérmico derivado del sistema nervioso autónomo. Acude a consulta paciente masculino de 52 años de edad, procedencia rural con antecedente de salud. El cual acude a la consulta, refiriendo que hace más menos 6 meses presenta un aumento de volumen en la región lateral izquierda del cuello, que fue aumentando de tamaño progresivamente, sin presentar dolor. Se le realizó biopsia incisiónal, que confirmó un Angioendotelioma papilar seromatoso; luego de la excerilisis presentó dehiscencia de la herida y aumento de tamaño de la lesión en forma de ulcerada de más menos 10cm, por lo que es remitido al Hospital Oncológico de Santiago de Cuba, centro donde se decide su inscripción para evaluar inicio de tratamiento oncoespecifico. En el examen de la lámina se diagnostica histopatológicamente un Ganglioma sugiriéndose estudio en centro de referencia nacional. Se aplicó tratamiento quirúrgico. El tratamiento de elección fue la cirugía con su embolización previa. Se recomendó seguimiento a largo plazo del paciente por el alto riesgo recurrente de enfermar.

ABSTRACT Paraganglioma is a rare tumor of neuroectodermal origin derived from the autonomic nervous system. A 52-year-old male patient, rural origin with a health history, comes to the office. The patient attended the consultation, reporting that for more than 6 months he had an increase in volume in the left lateral region of the neck, which was progressively increasing in size, without presenting pain. An incision biopsy was performed, which confirmed a seromatous papillary angioendothelioma; After excerilysis, he presented dehiscence of the wound and an increase in the size of the lesion in the form of an ulcerated lesion of more than 10cm, for which he was referred to the Oncological Hospital of Santiago de Cuba, a center where his registration was decided to evaluate the start of cancer-specific treatment . In the examination of the lamina a ganglioma is diagnosed histopathologically, suggesting study in a national reference center. Surgical treatment was applied. The treatment of choice was surgery with its prior embolization. Long-term follow-up of the patient was recommended due to the high recurrent risk of illness.

RESUMO O paraganglioma é um tumor raro de origem neuroectodérmica derivado do sistema nervoso autônomo. Paciente do sexo masculino, 52 anos, origem rural e histórico de saúde, chega ao consultório. O paciente compareceu à consulta, relatando que há mais de 6 meses apresentou aumento de volume na região lateral esquerda do pescoço, que foi aumentando de tamanho progressivamente, sem apresentar dor. Foi realizada biópsia incisional, que confirmou um angioendotelioma papilar seromatoso; Após a excerilise, apresentou deiscência da ferida e aumento do tamanho da lesão em forma de lesão ulcerada de mais de 10cm, pela qual foi encaminhado ao Hospital Oncológico de Santiago de Cuba, centro onde se decidiu seu cadastro para avaliar o início do tratamento específico do câncer . No exame da lâmina é feito o diagnóstico histopatológico de ganglioma, sugerindo estudo em centro de referência nacional. O tratamento cirúrgico foi aplicado. O tratamento de escolha foi a cirurgia com sua embolização prévia. O acompanhamento em longo prazo do paciente foi recomendado devido ao alto risco recorrente de doença.

Interpretation of Brain Volume Increase in Multiple Sclerosis.

BACKGROUND AND PURPOSE: A high variability of brain MRI volume change measurement renders challenging its interpretation in multiple sclerosis (MS). Occurrence and clinical relevance of observed apparent brain volume increase (BVI) in MS patients have not been investigated yet. The objective was to quantify the prevalence and factors associated with BVI. METHODS: We examined 366 MS patients (2,317 scans) and 44 controls (132 scans). Volumetric analysis of brain volume changes was performed by SIENA and ScanView. BVI was defined as brain volume change >0%. We compared characteristics of patients with and without BVI. RESULTS: BVI was found in 26.3% (from 1,951) longitudinal scans (SIENA). If BVI occurred, a probability that BVI will be repeated consecutively more than or equal to two times was 15.9%. The repeated BVI was associated with clinical disease activity in 50% of cases. BVI was associated with shorter time and lower T2 lesion volume increase between two MRI scans, and higher normalized brain volume (all P < .0001). A proportion of scans with BVI was higher when analyzed by ScanView (35.3%) and in controls (36.4% by SIENA). CONCLUSIONS: BVI occurs in a great proportion of MR scans over short-term follow-up and is not associated with disease stabilization. Although BVI can be caused by several factors, the results indicate that measurement error may contribute to BVI in the majority of cases. Clinicians should be aware of the frequent occurrence of apparent BVI, interpret brain volume changes in MS patients with great caution, and use methods with precise quantification of brain volume changes.

Cation-Chloride Cotransporters, Na/K Pump, and Channels in Cell Water/Ionic Balance Regulation Under Hyperosmolar Conditions: In Silico and Experimental Studies of Opposite RVI and AVD Responses of U937 Cells to Hyperosmolar Media.

Studying the transport of monovalent ions across the cell membrane in living cells is complicated by the strong interdependence of fluxes through parallel pathways and requires therefore computational analysis of the entire electrochemical system of the cell. Current paper shows how to calculate changes in the cell water balance and ion fluxes caused by changes in the membrane channels and transporters during a normal regulatory increase in cell volume in response to osmotic cell shrinkage (RVI) followed by a decrease in cell volume associated with apoptosis (AVD). Our recently developed software is used as a computational analysis tool and the established human lymphoid cells U937 are taken as an example of proliferating animal cells. It is found that, in contrast to countless statements in the literature that cell volume restoration requires the activation of certain ion channels and transporters, the cellular responses such as RVI and AVD can occur in an electrochemical system like U937 cells without any changes in the state of membrane channels or transporters. These responses depend on the types of chloride cotransporters in the membrane and differ in a hyperosmolar medium with additional sucrose and in a medium with additional NaCl. This finding is essential for the identification of the true changes in membrane channels and transporters responsible for RVI and AVD in living cells. It is determined which changes in membrane parameters predicted by computational analysis are consistent with experimental data obtained on living human lymphoid cells U937, Jurkat, and K562 and which are not. An essential part of the results is the developed software that allows researchers without programming experience to calculate the fluxes of monovalent ions via the main transmembrane pathways and electrochemical gradients that move ions across the membrane. The software is available for download. It is useful for studying the functional expression of the channels and transporters in living cells and understanding how the cell electrochemical system works.

Roles of volume-regulatory anion channels, VSOR and Maxi-Cl, in apoptosis, cisplatin resistance, necrosis, ischemic cell death, stroke and myocardial infarction.

Two types of anion channels are directly activated by osmotic swelling and are involved in the regulatory volume decrease (RVD) in most types of mammalian cells, and they include the volume-sensitive outwardly rectifying anion channel (VSOR), also called the volume-regulated anion channel (VRAC), and the large-conductance maxi-anion channel (Maxi-Cl). In cardiomyocytes, a splice variant of cystic fibrosis transmembrane conductance regulator anion channel (cardiac CFTR) participates in the RVD mechanism under ß-adrenergic stimulation. VSOR and Maxi-Cl are also involved in facilitation of the RVD process by releasing extracellular autocrine/paracrine signals, glutamate and ATP. Apoptotic cell death starts with cell shrinkage, called the apoptotic volume decrease (AVD), which is also caused by activation of VSOR. Since VSOR is implicated not only in the AVD induction but also in the uptake of an anti-cancer drug, cisplatin, downregulation of VSOR activity is causatively involved in acquisition of cisplatin resistance in cancer cells. Necrotic cell death exhibits persistent cell swelling, called the necrotic volume increase (NVI), which is coupled to RVD dysfunction due to impaired VSOR function. Acidotoxic and lactacidosis-induced necrotic cell death is induced both by glutamate release mediated by astroglial VSOR and Maxi-Cl and by exaggerated Cl- influx mediated by neuronal VSOR under prolonged depolarization caused by activation of ionotropic glutamate receptor (iGluR) cation channels. Both VSOR and Maxi-Cl are elaborately involved, in a manner as double-edged swords, in ischemia- and ischemia-reperfusion-induced apoptotic or necrotic cell death in cerebral and myocardial cells by mediating not only Cl- transport but also release of glutamate and/or ATP. Cardiac CFTR exerts a protective action against ischemia(-reperfusion)-induced cardiac injury, called myocardial infarction (MI), which is largely necrotic. Cardiac Maxi-Cl activity may participate in protection against ischemia(-reperfusion) injury by mediating ATP release.

The effects of the treatment conditions on the dissolution profile of ethylcellulose coated pellets.

Due to the additional particle coalescence in the coating, changes in the dissolution profile occur over time in the formulations coated by aqueous ethylcellulose latex. Dry thermal treatment (DT) of the coating can be used as a prevention of this process. Alternatively, it is advisable to take advantage of the synergistic effect of high humidity during wet treatment (WT), which substantially accelerates the film formation. This can be a problem for time-controlled systems, which are based on the coating rupture due to the penetration of water into the core causing the increase in the system volume. This process can begin already during the WT, which may affect the coating adversely. The submitted work was focused on the stability testing of two pellet core compositions: pellets containing swelling superdisintegrant sodium carboxymethyl starch (CMS) and pellets containing osmotically active polyethylene glycol (PEG). Another objective was to identify the treatment/storage condition effects on the pellet dissolution profiles. These pellets are intended to prevent hypoglycemia for patients with diabetes mellitus and therefore, besides the excipients, pellet cores contain 75% or 80% of glucose. The pellet coating is formed by ethylcellulose-based latex, which provides the required lag time (120-360 min). The sample stability was evaluated depending on the pellet core composition (PEG, CMS) for two types of final pellet coating treatment (DT or WT). Scanning electron microscopy and Raman microspectroscopy revealed the penetration of glucose and polyethylene glycol from the core to the PEG pellet surface after WT. For the CMS sample, significant pellet swelling after WT (under the conditions of elevated humidity) was statistically confirmed by the means of stereomicroscopic data evaluation. Therefore, the acceleration of dissolution rate during the stress tests is caused by the soluble substance penetration through the coating in the case of PEG pellets or by dosage form volume increase in the case of CMS pellets. The observed mechanisms can be generally anticipated during the stability testing of the ethylcellulose coated dosage forms. The aforementioned processes do not occur after DT and the pellets are stable in the environment without increased humidity.

Role of WNK Kinases in the Modulation of Cell Volume.

Ion Transport across the cell membrane is required to maintain cell volume homeostasis. In response to changes in extracellular osmolarity, most cells activate specific metabolic or membrane-transport pathways to respond to cell swelling or shrinkage and return their volume to its normal resting state. This process involves the rapid adjustment of the activities of channels and transporters that mediate flux of K+, Na+, Cl-, and small organic osmolytes. Cation chloride cotransporters (CCCs) NKCCs and KCCs are a family of membrane proteins modulated by changes in cell volume and/or in the intracellular chloride concentration ([Cl-]i). Cell swelling triggers regulatory volume decrease (RVD), promoting solute and water efflux to restore normal cell volume. Swelling-activated KCCs mediate RVD in most cell types. In contrast, cell shrinkage triggers regulatory volume increase (RVI), which involves the activation of the NKCC1 cotransporter of the CCC family. Regulation of the CCCs during RVI and RVD by protein phosphorylation is a well-characterized mechanism, where WNK kinases and their downstream kinase substrates, SPAK and OSR1 constitute the essential phospho-regulators. WNKs-SPAK/OSR1-CCCs complex is required to regulate cell shrinkage-induced RVI or cell swelling-induced RVD via activating or inhibitory phosphorylation of NKCCs or KCCs, respectively. WNK1 and WNK4 kinases have been established as [Cl-]i sensors/regulators, while a role for WNK3 kinase as a cell volume-sensing kinase has emerged and is proposed in this chapter.

Calibrated brightfield-based imaging for measuring intracellular protein concentration.

Intracellular protein concentration is an essential cell characteristic, which manifests itself through the refractive index. The latter can be measured from two or more mutually defocused brightfield images analyzed using the TIE (transport-of-intensity equation). In practice, however, TIE does not always achieve quantitatively accurate results on biological cells. Therefore, we have developed a calibration procedure that involves successive imaging of cells in solutions containing different amounts of added protein. This allows one to directly relate the output of TIE (T) to intracellular protein concentration C (g/L). The resultant relationship has a simple form: C ≈ 1.0(T/V), where V is the cell volume (µm3 ) and 1.0 is an empirical coefficient. We used calibrated TIE imaging to characterize the regulatory volume increase (RVI) in adherent HeLa cells placed in a hyperosmotic solution. We found that while no RVI occurs over the first 30-60 min, the protein concentration fully recovers after 20 h. Because interpretation of such long experiments may depend on whether protein concentration varies significantly throughout the cell cycle, we measured this parameter in three cell lines: HeLa, MDCK and DU145. Our data indicate that protein concentration remains relatively stable in these cells. © 2017 International Society for Advancement of Cytometry.

Expansion and Renormalization of Human Brain Structure During Skill Acquisition.

Research on human brain changes during skill acquisition has revealed brain volume expansion in task-relevant areas. However, the large number of skills that humans acquire during ontogeny militates against plasticity as a perpetual process of volume growth. Building on animal models and available theories, we promote the expansion-renormalization model for plastic changes in humans. The model predicts an initial increase of gray matter structure, potentially reflecting growth of neural resources like neurons, synapses, and glial cells, which is followed by a selection process operating on this new tissue leading to a complete or partial return to baseline of the overall volume after selection has ended. The model sheds new light on available evidence and current debates and fosters the search for mechanistic explanations.

AQP2-Induced Acceleration of Renal Cell Proliferation Involves the Activation of a Regulatory Volume Increase Mechanism Dependent on NHE2.

We have previously shown in renal cells that expression of the water channel Aquaporin 2 (AQP2) increases the rate of cell proliferation by shortening the transit time through the S and G2 /M phases of the cell cycle. This acceleration is due, at least in part, to a down-regulation of regulatory volume decrease (RVD) mechanisms when volume needs to be increased in order to proceed into the S phase. We hypothesize that in order to increase cell volume, RVD mechanisms may be overtaken by regulatory volume increase mechanisms (RVI). In this study, we investigated if the isoform 2 of the Na+ /H+ exchanger (NHE2), the main ion transporter involved in RVI responses, contributed to the AQP2-increased renal cell proliferation. Three cortical collecting duct cell lines were used: WT-RCCD1 (not expressing AQPs), AQP2-RCCD1 (transfected with AQP2), and mpkCCDc14 (with inducible AQP2 expression). We here demonstrate, for the first time, that both NHE2 protein activity and expression were increased in AQP2-expressing cells. NHE2 inhibition decreased cell proliferation and delayed cell cycle progression by slowing S and G2 /M phases only if AQP2 was expressed. Finally, we observed that only in AQP2-expressing cells a NHE2-dependent RVI response was activated in the S phase. These observations suggest that the AQP2-increased proliferation involves the activation of a regulatory volume increase mechanism dependent on NHE2. Therefore, we propose that the accelerated proliferation of AQP2-expressing cells requires a coordinated modulation of the RVD/RVI activity that contributes to cell volume changes during cell cycle progression. J. Cell. Biochem. 118: 967-978, 2017. © 2016 Wiley Periodicals, Inc.

Importance of astrocytes for potassium ion (K+) homeostasis in brain and glial effects of K+ and its transporters on learning.

Initial clearance of extracellular K+ ([K+]o) following neuronal excitation occurs by astrocytic uptake, because elevated [K+]o activates astrocytic but not neuronal Na+,K+-ATPases. Subsequently, astrocytic K+ is re-released via Kir4.1 channels after distribution in the astrocytic functional syncytium via gap junctions. The dispersal ensures widespread release, preventing renewed [K+]o increase and allowing neuronal Na+,K+-ATPase-mediated re-uptake. Na+,K+-ATPase operation creates extracellular hypertonicity and cell shrinkage which is reversed by the astrocytic cotransporter NKCC1. Inhibition of Kir channels by activation of specific PKC isotypes may decrease syncytial distribution and enable physiologically occurring [K+]o increases to open L-channels for Ca2+, activating [K+]o-stimulated gliotransmitter release and regulating gap junctions. Learning is impaired when [K+]o is decreased to levels mainly affecting astrocytic membrane potential or Na+,K+-ATPase or by abnormalities in its α2 subunit. It is enhanced by NKCC1-mediated ion and water uptake during the undershoot, reversing neuronal inactivity, but impaired in migraine with aura in which [K+]o is highly increased. Vasopressin augments NKCC1 effects and facilitates learning. Enhanced myelination, facilitated by astrocytic-oligodendrocytic gap junctions also promotes learning.

Mitogen-activated protein kinases as key players in osmotic stress signaling.

BACKGROUND: Osmotic stress arises from the difference between intracellular and extracellular osmolality. It induces cell swelling or shrinkage as a consequence of water influx or efflux, which threatens cellular activities. Mitogen-activated protein kinases (MAPKs) play central roles in signaling pathways in osmotic stress responses, including the regulation of intracellular levels of inorganic ions and organic osmolytes. SCOPE OF REVIEW: The present review summarizes the cellular osmotic stress response and the function and regulation of the vertebrate MAPK signaling pathways involved. We also describe recent findings regarding apoptosis signal-regulating kinase 3 (ASK3), a MAP3K member, to demonstrate its regulatory effects on signaling molecules beyond MAPKs. MAJOR CONCLUSIONS: MAPKs are rapidly activated by osmotic stress and have diverse roles, such as cell volume regulation, gene expression, and cell survival/death. There is significant cell type specificity in the function and regulation of MAPKs. Based on its activity change during osmotic stress and its regulation of the WNK1-SPAK/OSR1 pathway, ASK3 is expected to play important roles in osmosensing mechanisms and cellular functions related to osmoregulation. GENERAL SIGNIFICANCE: MAPKs are essential for various cellular responses to osmotic stress; thus, the identification of the upstream regulators of MAPK pathways will provide valuable clues regarding the cellular osmosensing mechanism, which remains elusive in mammals. The elucidation of in vivo MAPK functions is also important because osmotic stress in physiological and pathophysiological conditions often results from changes in the intracellular osmolality. These studies potentially contribute to the establishment of therapeutic strategies against diseases that accompany osmotic perturbation.