August Krogh (1874-1949) was amongst the most influential physiologists in the first part of the 20th century. This was an era when physiology emerged as a quantitative research field and when many of the current physiological disciplines were defined; Krogh can rightfully be viewed as having introduced comparative physiology, epithelial transport and - together with Johannes Lindhard - exercise physiology as independent disciplines. With a unique ability to design and construct equipment, Krogh could address novel questions in both human and animal physiology with unprecedented precision. Krogh would characteristically focus on a given physiological problem over a couple of years, delineate the focal mechanisms, provide a solution to the major problems, and then move onto new academic ground. For each of his major research areas (respiratory gas exchange, capillary function, osmoregulation), he wrote comprehensive books or monographs that remain important resources for scholars today, and he engaged in the writing of physiology textbooks for the Danish high school. Krogh's research appears to have been driven by curiosity to understand how animals (including humans) work, but he did not hesitate to apply his insight to societal and clinical problems throughout his long academic career.

Polarized epithelia generate regulated water flows and solute fluxes for serving extracellular homeostasis, which imposes changes in epithelial water volume and osmolyte concentrations that would be critical to normal function if not regulated. The studies have been challenged by the fact that epithelia may contain more than one cell type, and by the large osmotic permeability of some epithelial membranes that presupposes successful elimination of unstirred layer effects following aniso-osmotic perturbations. Illustrated by several examples applying a range of methods, we review studies showing that volume regulation is governed by principles similar to non-polarized cells by having acquired well-developed regulatory volume decrease (RVD) and regulatory volume increase (RVI). RVI may not be seen unless the cell has undergone a prior RVD. The rate of RVD and RVI is faster in cells of high osmotic permeability like amphibian gallbladder and mammalian proximal tubule as compared to amphibian skin and mammalian cortical collecting tubule of low and intermediate osmotic permeability. Cross talk between entrance and exit mechanisms interferes with volume regulation both at aniso-osmotic and isosmotic volume perturbations. For example, the inevitable volume increase resulting from Na+/K+ pump arrest is delayed by inhibition of Na+ and K+ leak permeabilities. This may even be preceded by a transient volume decrease associated with a reduction of the cytosolic Cl− pool if kept above equilibrium by downhill Na+ entrance. It has been proposed that cell volume regulation is an intrinsic function of isoosmotic fluid transport that depends on Na+ recirculation. The causative relationship is discussed for amphibian skin epithelium and submucosal glands in which all major ion transporters and channels including the Na+ recirculation mechanisms have been identified. A large number of transporters and ion channels involved in volume regulation have been cloned. The volume-regulated anion channel (VRAC) exhibiting specific electrophysiological characteristics seems exclusively to serve volume regulation. This is contrary to several subfamilies of K+ channels as well as cotransporters and exchange mechanisms that may serve both transepithelial transport and cell volume regulation. In the same cell, these functions may be maintained by different ion pathways that are separately regulated. RVD is often preceded by an increase in cytosolic free Ca2+ probably via influx through TRP channels and/or release from intracellular stores. Cell volume regulation is associated with specific ATP release mechanisms and involves mitogen-activated protein kinases, WNKs, and Ste20-related kinases that are modulated by osmotic stress.

Background/Aims: The potential role of the two-pore domain potassium channel KCNK5 (also known as TASK-2 and K2P5.1) in activated T cell physiology has only recently been described. So far KCNK5 has been described to be up-regulated in T cells in multiple sclerosis patients and to be implicated in the volume regulatory mechanism regulatory volume decrease (RVD) in T cells. Methods: We investigated the time-dependent expression pattern of KCNK5 in CD3/CD28 activated human T cells using qPCR and Western blotting and its role in RVD using a Coulter Counter. Results: KCNK5 is highly up-regulated in CD3/CD28 activated T cells both at mRNA (after 24 h) and protein level (72 and 144 h), but despite this up-regulation the RVD response is inhibited. Furthermore, the swelling-activated Cl- permeability in activated T cells is strongly decreased, and the RVD inhibition is predominantly due to the decreased Cl- permeability. Conclusion: The up-regulated KCNK5 in activated human T cells does not play a volume regulatory role, due to decreased Cl- permeability. We speculate that the KCNK5 up-regulation might play a role in hyperpolarization of the cell membrane leading to increased Ca2+ influx and proliferation of T cells.

The leucine-rich repeat containing 8A (LRRC8A) protein is an essential component of the volume-sensitive organic anion channel (VSOAC), and using pharmacological anion channel inhibitors (NS3728, DIDS) and LRRC8A siRNA we have investigated its role in development of Cisplatin resistance in human ovarian (A2780) and alveolar (A549) carcinoma cells. In Cisplatin-sensitive cells Cisplatin treatment increases p53-protein level as well as downstream signaling, e.g., expression of p21Waf1/Cip1, Bax, Noxa, MDM2, and activation of Caspase-9/-3. In contrast, Cisplatin-resistant cells do not enter apoptosis, i.e., their p53 and downstream signaling are reduced and caspase activity unaltered following Cisplatin exposure. Reduced LRRC8A expression and VSOAC activity are previously shown to correlate with Cisplatin resistance, and here we demonstrate that pharmacological inhibition and transient knockdown of LRRC8A reduce the protein level of p53, MDM2, and p21Waf1/Cip1 as well as Caspase-9/-3 activation in Cisplatin-sensitive cells. Cisplatin resistance is accompanied by reduction in total LRRC8A expression (A2780) or LRRC8A expression in the plasma membrane (A549). Activation of Caspase-3 dependent apoptosis by TNFα-exposure or hyperosmotic cell shrinkage is almost unaffected by pharmacological anion channel inhibition. Our data indicate 1) that expression/activity of LRRC8A is essential for Cisplatin-induced increase in p53 protein level and its downstream signaling, i.e., Caspase-9/-3 activation, expression of p21Waf1/Cip1 and MDM2; and 2) that downregulation of LRRC8A-dependent osmolyte transporters contributes to acquirement of Cisplatin resistance in ovarian and lung carcinoma cells. Activation of LRRC8A-containing channels is upstream to apoptotic volume decrease as hypertonic cell shrinkage induces apoptosis independent of the presence of LRRC8A.

OBJECTIVES:Novel treatments for pancreatic ductal adenocarcinoma (PDAC) are severely needed. The aim of this work was to explore the roles of H-lactate monocarboxylate transporters 1 and 4 (MCT1 and MCT4) in PDAC cell migration and invasiveness.METHODS:Monocarboxylate transporter expression, localization, activity, and function were explored in human PDAC cells (MIAPaCa-2, Panc-1, BxPC-3, AsPC-1) and normal human pancreatic ductal epithelial (HPDE) cells, by quantitative polymerase chain reaction, immunoblotting, immunocytochemistry, lactate flux, migration, and invasion assays.RESULTS:MCT1 and MCT4 (messenger RNA, protein) were robustly expressed in all PDAC lines, localizing to the plasma membrane. Lactate influx capacity was highest in AsPC-1 cells and lowest in HPDE cells and was inhibited by the MCT inhibitor α-cyano-4-hydroxycinnamate (4-CIN), MCT1/MCT2 inhibitor AR-C155858, or knockdown of MCT1 or MCT4. PDAC cell migration was largely unaffected by MCT1/MCT2 inhibition or MCT1 knockdown but was reduced by 4-CIN and by MCT4 knockdown (BxPC-3). Invasion measured in Boyden chamber (BxPC-3, Panc-1) and spheroid outgrowth (BxPC-3) assays was attenuated by 4-CIN and AR-C155858 and by MCT1 or MCT4 knockdown.CONCLUSIONS:Human PDAC cells exhibit robust MCT1 and MCT4 expression and partially MCT1- and MCT4-dependent lactate flux. PDAC cell migration is partially dependent on MCT4; and invasion, on MCT1 and MCT4. Inhibition of MCT1 and MCT4 may have clinical relevance in PDAC.

Volume-regulated channels for anions (VRAC) / organic osmolytes (VSOAC) play essential roles in cell volume regulation and other cellular functions, e.g. proliferation, cell migration and apoptosis. LRRC8A, which belongs to the leucine rich-repeat containing protein family, was recently shown to be an essential component of both VRAC and VSOAC. Reduced VRAC and VSOAC activities are seen in drug resistant cancer cells. ANO1 is a calcium-activated chloride channel expressed on the plasma membrane of e.g., secretory epithelia. ANO1 is amplified and highly expressed in a large number of carcinomas. The gene, encoding for ANO1, maps to a region on chromosome 11 (11q13) that is frequently amplified in cancer cells. Knockdown of ANO1 impairs cell proliferation and cell migration in several cancer cells. Below we summarize the basic biophysical properties of VRAC, VSOAC and ANO1 and their most important cellular functions as well as their role in cancer and drug resistance.

Cl- and K+ channels are involved in several important cellular processes including migration and proliferation. Such processes are commonly dysregulated in cancer. pH and Ca2+ homeostasis are also altered in cancer cells. Thus, the aim of this project was to elucidate the role of the Ca2+-activated Cl- and K+ channels (ANO1 and KCa3.1, respectively) as well as pH-sensitive K+ channels of the K2P family in PDAC. Patch-clamp recordings from several pancreatic cancer cell lines and revealed Ca2+-activated Cl- current that was sensitive to ANO1-specific inhibitors and was almost completely abolished by siRNA targeting ANO1. Using these cells in a scratch assay, we identified ANO1 as crucial player in cell migration. Surprisingly, no involvement of ANO1 in proliferation was found. We found an abundant functional expression of KCa3.1 in Mia PaCa-2 and BxPC-3 cells using qPCR, Western blotting and patch-clamp techniques. Initial results indicated involvement of KCa3.1 in promoting migration that was mediated by an increase in intracellular Ca2+. Using a 384-cell based automated patch-clamp system (SynchroPatch384PE) provided by Nanion Technologies we identified a pH-sensitive current in BxPC-3 cells that followed biophysical and pharmacological characteristics described for K2P channels. In conclusion, we found that Ca2+- activated currents are crucial for cellular migration and that pancreatic cancer cells exhibit pH-regulated K+ current we hypothesize could drive tumor progression. Supported by the Marie Curie Network “IonTraC” (289648) and The Danish Council for Independent Research (0602- 01527B; 4002-00162).

The aim of this project was to analyze the regulation of p53 expression in NIH3T3 fibroblasts under the influence of increasing hyperosmotic stress. Expression of p53 showed a biphasic response pattern in NIH3T3 cells under increasing osmotic stress (337 mOsm to 737 mOsm) with a maximum at 587 mOsm. Under isotonic conditions p53 expression increased after addition of the proteasome inhibitor MG132 indicating that cellular p53 levels in unperturbed cells is kept low by proteasomal degradation. However, under hypertonic conditions p53 synthesis as well as p53 degradation were significantly reduced and it is demonstrated that the increase in p53 expression observed when tonicity is increased from 337 to 587 mOsm reflects that degradation is more inhibited than synthesis, whereas the decrease in p53 expression at higher tonicities reflects that synthesis is more inhibited than degradation. The activity of the p53 regulating proteins p38 MAP kinase and the ubiquitin ligase MDM2 were studied as a function of increasing osmolarity. MDM2 protein expression was unchanged at all osmolarities, whereas MDM2 phosphorylation (Ser(166)) increased at osmolarities up to 537 mOsm and remained constant at higher osmolarities. Phosphorylation of p38 increased at osmolarities up to 687 mOsm which correlated with an increased phosphorylation of p53 (Ser(15)) and the decreased p53 degradation. Caspase-3 activity increased gradually with hypertonicity and at 737 mOsm both Caspase-3 activity and annexin V binding are high even though p53 expression and activity are low, indicating that initiation of apoptosis under severe hypertonic conditions is not strictly controlled by p53.

We review studies on regulatory volume decrease (RVD) and regulatory volume increase (RVI) of major ion and water transporting vertebrate epithelia. The rate of RVD and RVI is faster in cells of high osmotic permeability like amphibian gallbladder and mammalian proximal tubule as compared to amphibian skin and mammalian cortical collecting tubule of low and intermediate osmotic permeability. Crosstalk between entrance and exit mechanisms interferes with volume regulation both at aniso-osmotic and iso-osmotic volume perturbations. It has been proposed that cell volume regulation is an intrinsic function of iso-osmotic fluid transport that depends on Na recirculation. The causative relationship is discussed for a fluid-absorbing and a fluid-secreting epithelium of which the Na recirculation mechanisms have been identified. A large number of transporters and ion channels involved in cell volume regulation are cloned. The volume-regulated anion channel (VRAC) exhibiting specific electrophysiological characteristics seems exclusive to serve cell volume regulation. This is contrary to K channels as well as cotransporters and exchange mechanisms that may serve both transepithelial transport and cell volume regulation. In the same cell, these functions may be maintained by different ion pathways that are separately regulated. RVD is often preceded by increase in cytosolic free Ca, probably via influx through TRP channels, but Ca release from intracellular stores has also been observed. Cell volume regulation is associated with specific ATP release mechanisms and involves tyrosine kinases, mitogen-activated protein kinases, WNKs and Ste20-related kinases that are modulated by osmotic stress and cell volume perturbations.

BACKGROUND/AIMS:Altered expression of the integrin family of cell adhesion receptors has been associated with initiation, progression, and metastasis of solid tumors as well as in the development of chemoresistance. Here, we investigated the role of integrins, in particular integrin β1, in cell volume regulation and drug-induced apoptosis in adherent and non-adherent Ehrlich ascites cell lines.METHODS:Adhesion phenotypes were verified by colorimetric cell-adhesion-assay. Quantitative real-time PCR and western blot were used to compare expression levels of integrin subunits. Small interfering RNA was used to silence integrin β1 expression. Regulatory volume decrease (RVD) after cell swelling was studied with calcein-fluorescence-self-quenching and Coulter counter analysis. Taurine efflux was estimated with tracer technique. Caspase assay was used to determine apoptosis.RESULTS:We show that adherent cells have stronger fibronectin binding and a significantly increased expression of integrin α5, αv, and β1 at mRNA and protein level, compared to non-adherent cells. Knockdown of integrin β1 reduced RVD of the adherent but not of the non-adherent cells. Efflux of taurine was unaffected. In contrast to non-adherent, adherent cells exhibited chemoresistance to chemotherapeutic drugs (cisplatin and gemcitabine). However, knockdown of integrin β1 promoted cisplatin-induced caspase activity in adherent cells.CONCLUSION:Our data identifies integrin β1 as a part of the osmosensing machinery and regulator of cisplatin resistance in adherent Ehrlich cells.

The review describes molecular and functional properties of the volume regulated anion channel and Ca 2+ ‐dependent Cl − channels belonging to the anoctamin family with emphasis on physiological importance of these channels in regulation of cell volume, cell migration, cell proliferation, and programmed cell death. Finally, we discuss the role of Cl − channels in various diseases. © 2014 IUBMB Life, 66(4):257–267, 2014

Anoctamin 6 (ANO6), also known as TMEM16F, has been shown to be a calcium-activated anion channel with delayed calcium activation. The cellular function of ANO6 is under debate, and different groups have come to different conclusions about ANO6’s physiological role. Although it is now quite well established that ANO6 is distinct from the volume-regulated anion channel, it is still unclear whether ANO6 or other anoctamins can be activated by cell swelling. In this study, we suggest that ANO1, ANO6, and ANO10 do not contribute to the volume-activated current in ANO-overexpressing HEK293 cells. Furthermore, knock-down of ANO6 in Ehrlich ascites tumor cells (EATC) and Ehrlich–Lettre ascites (ELA) did not decrease but instead significantly increased swelling-activated membrane currents. Knock-down of ANO6 in EATC did not reduce regulatory volume decrease (RVD) in the absence of extracellular calcium, whereas it significantly reduced RVD in the presence of calcium. Interestingly, we found that knock-down of ANO6 in ELA cells resulted in a decrease in cisplatin-induced caspase-3 activity, confirming earlier findings that ANO6 is involved in apoptosis. Finally, knock-down of ANO1 and ANO6 did not affect the volume-sensitive release of taurine in ELA cells. Thus, our data provide evidence that ANO6 cannot be activated directly by cell swelling unless Ca 2+ is present. We also conclude that ANO6 carries a current during RVD, provided extracellular calcium is present. Thus, swelling activation of ANO6 requires the presence of free calcium.

The mechanisms linking cell volume sensing to volume regulation in mammalian cells remain incompletely understood. Here, we test the hypothesis that activation of nonreceptor tyrosine kinases Src, focal adhesion kinase (FAK), and Janus kinase-2 (Jak2) occurs after osmotic shrinkage of NIH3T3 fibroblasts and contributes to volume regulation by activation of NKCC1. FAK phosphorylation at Tyr397, Tyr576/577, and Tyr861 was increased rapidly after exposure to hypertonic (575 mOsm) saline, peaking after 10 (Tyr397, Tyr576/577) and 10-30 min (Tyr861). Shrinkage-induced Src family kinase autophosphorylation (pTyr416-Src) was induced after 2-10 min, and immunoprecipitation indicated that this reflected phosphorylation of Src itself, rather than Fyn and Yes. Phosphorylated Src and FAK partly colocalized with vinculin, a focal adhesion marker, after hypertonic shrinkage. The Src inhibitor pyrazolopyrimidine-2 (PP2, 10 μM) essentially abolished shrinkage-induced FAK phosphorylation at Tyr576/577 and Tyr861, yet not at Tyr397, and inhibited shrinkage-induced NKCC1 activity by ∼50%. The FAK inhibitor PF-573,228 augmented shrinkage-induced Src phosphorylation, and inhibited shrinkage-induced NKCC1 activity by ∼15%. The apparent role of Src in NKCC1 activation did not reflect phosphorylation of myosin light chain kinase (MLC), which was unaffected by shrinkage and by PP2, but may involve Jak2, a known target of Src, which was rapidly activated by osmotic shrinkage and inhibited by PP2. Collectively, our findings suggest a major role for Src and possibly the Jak2 axis in shrinkage-activation of NKCC1 in NIH3T3 cells, whereas no evidence was found for major roles for FAK and MLC in this process.

Rasmussen LJ, Müller HS, Jørgensen B, Pedersen SF, Hoffmann EK. Osmotic shrinkage elicits FAKand Src phosphorylation and Src-dependent NKCC1 activation in NIH3T3 cells. Am J Physiol Cell Physiol 308: C101–C110, 2015. First published November 5, 2014; doi:10.1152/ajpcell.00070.2014.—The mechanisms linking cell volume sensing to volume regulation in mammalian cells remain incompletely understood. Here, we test the hypothesis that activation of nonreceptor tyrosine kinases Src, focal adhesion kinase (FAK), and Janus kinase-2 (Jak2) occurs after osmotic shrinkage of NIH3T3 fibroblasts and contributes to volume regulation by activation of NKCC1. FAK phosphorylation at Tyr397, Tyr576/577, and Tyr861 was increased rapidly after exposure to hypertonic (575 mOsm) saline, peaking after 10 (Tyr397, Tyr576/577) and 10–30 min (Tyr861). Shrinkage-induced Src family kinase autophosphorylation (pTyr416-Src) was induced after 2–10 min, and immunoprecipitation indicated that this reflected phosphorylation of Src itself, rather than Fyn and Yes. Phosphorylated Src and FAK partly colocalized with vinculin, a focal adhesion marker, after hypertonic shrinkage. The Src inhibitor pyrazolopyrimidine-2 (PP2, 10 M) essentially abolished shrinkage-induced FAK phosphorylation at Tyr576/577 and Tyr861, yet not at Tyr397, and inhibited shrinkage-induced NKCC1 activity by 50%. The FAK inhibitor PF-573,228 augmented shrinkage-induced Src phosphorylation, and inhibited shrinkage-induced NKCC1 activity by 15%. The apparent role of Src in NKCC1 activation did not reflect phosphorylation of myosin light chain kinase (MLC), which was unaffected by shrinkage and by PP2, but may involve Jak2, a known target of Src, which was rapidly activated by osmotic shrinkage and inhibited by PP2. Collectively, our findings suggest a major role for Src and possibly the Jak2 axis in shrinkageactivation of NKCC1 in NIH3T3 cells, whereas no evidence was found for major roles for FAK and MLC in this process.

Pancreatic ductal adenocarcinoma (PDAC) has one of the worst survival rates of all cancers. ANO1 (TMEM16A) is a recently identified Ca2+-activated Cl− channel (CaCC) that is upregulated in several tumors. Although ANO1 was subject to extensive studies in the recent years, its pathophysiological function has only been poorly understood. The aim of the present study is to establish the significance of ANO1 in PDAC behavior and demarcate its roles in PDAC from those of the volume-regulated anion channel (VRAC). We performed qPCR and Western blot measurements on different PDAC cell lines (Panc-1, Mia PaCa 2, Capan-1, AsPC-1, BxPC-3) and compared the results to those obtained in a human pancreatic ductal epithelium (HPDE) cell line. All cancer cell lines showed an upregulation of ANO1 on mRNA and protein levels. Whole-cell patch-clamp recordings identified large Ca2+ and voltage-dependent Cl− currents in PDAC cells. Using siRNA knockdown of ANO1 and three ANO1 inhibitors (T16Ainh-A01, CaCCinh-A01, and NS3728), we found that ANO1 is the main constituent of CaCC current in PDAC cells. We further characterized these three inhibitors and found that they had unspecific effects on the free intracellular calcium concentration. Functional studies on PDAC behavior showed that surprisingly inhibition of ANO1 did not influence cellular proliferation. On the other hand, we found ANO1 channel to be pivotal in PDAC cell migration as assessed in wound healing experiments.

The importance of the TRPV4 channel for human physiology has been highlighted in recent years with the identification of an increasing number of hereditary diseases associated with mutations of this channel. However, the functional understanding of TRPV4 associated pathologies remains a puzzle due to incomplete understanding of the polymodal regulation of TRPV4 channels and lack of insight into the structure-function relationship of the channel. In this work, we identified a series of highly conserved aromatic residues in transmembrane (TM) helices 5-6 with profound importance for TRPV4 activity. Substituting F617, Y621 or F624 in TM5 with leucine reduced channel sensitivity to the agonist 4α-PDD and heat, yet two of these mutants - F617L and Y621L - showed increased activation in response to cell swelling. In TM6, a Y702L mutation significantly reduced sensitivity to all of the above stimuli. In conclusion, we have identified residues in TM5-6 which differentially affect heat and agonist activation, and we have demonstrated distinct activation pathways for 4α-PDD and osmolarity.