Elevated Intracellular Calcium Stimulates NHE3 Activity by an IKEPP (NHERF4) Dependent Mechanism

The ileal brush border (BB) contains four evolutionarily related multi-PDZ domain proteins including NHERF1, NHERF2, PDZK1 (NHERF3) and IKEPP (NHERF4). Why multiple related PDZ proteins are in a similar location in the same cell is unknown. However, some specificity in regulation of NHE3 activity has been identified. For example, elevated intracellular Ca2+ ([Ca2+]i) inhibition of NHE3 is reconstituted by NHERF2 but not NHERF1, and involves the formation of large NHE3 complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether the four PDZ domain containing protein IKEPP reconstitutes elevated [Ca2+]i regulation of NHE3. In vitro, IKEPP bound to the F2 region (aa 590-667) of NHE3 in overlay assays, which is the same region where NHERF1 and NHERF2 bind. PS120 cells lack endogenous NHE3 and IKEPP. Treatment of PS120/NHE3/IKEPP cells (stably transfected with NHE3 and IKEPP) with the Ca2+ ionophore, 4-Br-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (0.5μM), stimulated NHE3 Vmax activity by ∼40%. This was associated with an increase in plasma membrane expression of NHE3 by a similar amount. NHE3 activity and surface expression were unaffected by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 in PS120/NHE3 cells lacking IKEPP. Based on sucrose density gradient centrifugation, IKEPP was also shown to exist in large complexes, some of which overlap in size with NHE3, and the size of both NHE3 and IKEPP complexes decreased in parallel after [Ca2+]i elevation. FRET experiments on fixed cells demonstrated that IKEPP and NHE3 directly associated at an intracellular site. Elevating [Ca2+]i decreased this intracellular NHE3 and IKEPP association. In summary: (1) In the presence of IKEPP, elevated [Ca2+]i stimulates NHE3 activity. This was associated with increased expression of NHE3 in the plasma membrane as well as a shift to smaller sizes of NHE3 and IKEPP containing complexes. (2) IKEPP directly binds NHE3 at its F2 C-terminal domain and directly associates with NHE3 in vivo (FRET). (3) Elevated [Ca2+]i decreased the association of IKEPP and NHE3 in an intracellular compartment. Based on which NHERF family member is expressed in PS120 cells, elevated [Ca2+]i stimulates (IKEPP), inhibits (NHERF2) or does not affect (NHERF1) NHE3 activity. This demonstrates that regulation of NHE3 depends on the nature of the NHERF family member associating with NHE3 and the accompanying NHE3 complexes

Intestinal in vitro and ex vivo Models to Study Host-Microbiome Interactions and Acute Stressors

The gut microbiome is extremely important for maintaining homeostasis with host intestinal epithelial, neuronal, and immune cells and this host-microbe interaction is critical during times of stress or disease. Environmental, nutritional, and cognitive stress are just a few factors known to influence the gut microbiota and are thought to induce microbial dysbiosis. Research on this bidirectional relationship as it pertains to health and disease is extensive and rapidly expanding in both in vivo and in vitro/ex vivo models. However, far less work has been devoted to studying effects of host-microbe interactions on acute stressors and performance, the underlying mechanisms, and the modulatory effects of different stressors on both the host and the microbiome. Additionally, the use of in vitro/ex vivo models to study the gut microbiome and human performance has not been researched extensively nor reviewed. Therefore, this review aims to examine current evidence concerning the current status of in vitro and ex vivo host models, the impact of acute stressors on gut physiology/microbiota as well as potential impacts on human performance and how we can parlay this information for DoD relevance as well as the broader scientific community. Models reviewed include widely utilized intestinal cell models from human and animal models that have been applied in the past for stress or microbiology research as well as ex vivo organ/tissue culture models and new innovative models including organ-on-a-chip and co-culture models

Human enteroids: Preclinical models of non-inflammatory diarrhea

Researchers need an available and easy-to-use model of the human intestine to better understand human intestinal physiology and pathophysiology of diseases, and to offer an enhanced platform for developing drug therapy. Our work employs human enteroids derived from each of the major intestinal sections to advance understanding of several diarrheal diseases, including those caused by cholera, rotavirus and enterohemorrhagic Escherichia coli. An enteroid bank is being established to facilitate comparison of segmental, developmental, and regulatory differences in transport proteins that can influence therapy efficacy. Basic characterization of major ion transport protein expression, localization and function in the human enteroid model sets the stage to study the effects of enteric infection at the transport level, as well as to monitor potential responses to pharmacological intervention

Freezing of ridges and water networks preserves the Gamburtsev Subglacial Mountains for millions of years

Once an ice sheet grows beyond a critical thickness, the basal thermal regime favors melting and development of subglacial water networks. Subglacial water is necessary for bedrock erosion, but the exact mechanisms that lead to preservation of subglacial topography are unclear. Here we resolve the freezing mechanisms that lead to long-term, high-altitude preservation across the Gamburtsev Subglacial Mountains in East Antarctica. Analyses of a comprehensive geophysical data set reveal a large-scale water network along valley floors. The ice sheet often drives subglacial water up steep topography where it freezes along high ridges beneath thinner ice. Statistical tests of hypsometry show the Gamburtsevs resemble younger midlatitude mountains, indicating exceptional preservation. We conclude that the Gamburtsevs have been shielded from erosion since the latest Eocene (∼34 Ma). These freezing mechanisms likely account for the spatial and temporal patterns of erosion and preservation seen in other glaciated mountain ranges

Epac1 mediates protein kinase A–independent mechanism of forskolin-activated intestinal chloride secretion

Intestinal Cl− secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca2+]i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl− secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (Isc) measurement in response to agonist-stimulated Cl− secretion. FSK-stimulated Cl− secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 µM), and the [Ca2+]i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 µM). Both FSK and the Epac activator 8-pCPT-2’-O-Me-cAMP (50 µM) elevated [Ca2+]i, activated Ras-related protein 2, and induced Cl− secretion in intact or basolateral membrane–permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2’-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced Isc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2’-O-Me-cAMP on Cl− secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2’-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl− conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl−>Br−>I− permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca2+]i signaling pathway is involved in cAMP-stimulated Cl− secretion, which is carried by a novel, previously undescribed Cl− channel

The Na+/H+ Exchanger Controls Deoxycholic Acid-Induced Apoptosis by a H+-Activated, Na+-Dependent Ionic Shift in Esophageal Cells

Apoptosis resistance is a hallmark of cancer cells. Typically, bile acids induce apoptosis. However during gastrointestinal (GI) tumorigenesis the cancer cells develop resistance to bile acid-induced cell death. To understand how bile acids induce apoptosis resistance we first need to identify the molecular pathways that initiate apoptosis in response to bile acid exposure. In this study we examined the mechanism of deoxycholic acid (DCA)-induced apoptosis, specifically the role of Na+/H+ exchanger (NHE) and Na+ influx in esophageal cells. In vitro studies revealed that the exposure of esophageal cells (JH-EsoAd1, CP-A) to DCA (0.2 mM -0.5 mM) caused lysosomal membrane perturbation and transient cytoplasmic acidification. Fluorescence microscopy in conjunction with atomic absorption spectrophotometry demonstrated that this effect on lysosomes correlated with influx of Na+, subsequent loss of intracellular K+, an increase of Ca2+ and apoptosis. However, ethylisopropyl-amiloride (EIPA), a selective inhibitor of NHE, prevented Na+, K+ and Ca2+ changes and caspase 3/7 activation induced by DCA. Ouabain and amphotericin B, two drugs that increase intracellular Na+ levels, induced similar changes as DCA (ion imbalance, caspase3/7 activation). On the contrary, DCA-induced cell death was inhibited by medium with low a Na+ concentrations. In the same experiments, we exposed rat ileum ex-vivo to DCA with or without EIPA. Severe tissue damage and caspase-3 activation was observed after DCA treatment, but EIPA almost fully prevented this response. In summary, NHE-mediated Na+ influx is a critical step leading to DCA-induced apoptosis. Cells tolerate acidification but evade DCA-induced apoptosis if NHE is inhibited. Our data suggests that suppression of NHE by endogenous or exogenous inhibitors may lead to apoptosis resistance during GI tumorigenesis

Core data of orbitally tuned Sr-isotope chemostratigraphy for the Late Middle and Late Miocene

We present a Sr chemostratigraphic reference section for the late middle to late Miocene (14-5 Ma) from Ocean Drilling Program site 926 on the Ceara Rise. This site combines a precise, orbitally tuned timescale with a high sedimentation rate (15 m/m.y.), continuous deposition, and excellent biostratigraphic control. The Sr isotope curve is based on measurements of cleaned, planktonic foraminifera at 100-200 kyr sample intervals and it illustrates periods of rapid change in 87Sr/86Sr alternating with periods of little change. Chemostratigraphically-defined ages for these intervals can be determined within +/-0.8 m.y. and +/-1.6 m.y. respectively. There is excellent correlation with the published curve for site 588 [Hodell and Woodruff, 1994]; however the curve for site 747 [Oslick et al., 1994] exhibits less structure, which may be due to small errors in age estimates related to slow sedimentation rates, high-latitude fauna and an interval of complicated magnetics. Late Miocene data compare favorably with data from site 758 [Farrell et al., 1995]

Carbachol-Mediated Endocytosis of NHE3 Involves a Clathrin-Independent Mechanism Requiring Lipid Rafts and Cdc42

Background: In intestinal epithelial cells, acute regulation of the brush border Na+/H+ exchanger, NHE3, usually occurs by changes in endocytosis and/or exocytosis. Constitutive NHE3 endocytosis involves clathrin. Carbachol (CCH), which elevates intracellular Ca2+ ([Ca2+]i), decreases NHE3 activity and stimulates endocytosis; however, the mechanism involved in calcium-mediated endocytosis of NHE3 is unclear. A pool of NHE3 resides in lipid rafts, which contributes to basal, but not cAMP-mediated, NHE3 trafficking, suggesting that an alternative mechanism exists for NHE3 endocytosis. Cdc42 was demonstrated to play an integral role in some cases of cholesterol-sensitive, clathrin-independent endocytosis. Therefore, the current study was designed to test the hypotheses that (1) clathrin-mediated endocytosis (CME) is involved in constitutive, but not CCH-mediated, endocytosis of NHE3, and (2) CCH-mediated endocytosis of NHE3 occurs through a lipid raft, activated Cdc42-dependent pathway that does not involve clathrin. Methods: The role of Cdc42 and lipid rafts on NHE3 activity and endocytosis were investigated in polarized Caco-2/BBe cells using pharmacological and shRNA knockdown approaches. Results: Basal NHE3 activity was increased in the presence of CME blockers (chlorpromazine; K+ depletion) supporting previous reports that constitutive NHE3 endocytosis is clathrin dependent. In contrast, CCH-inhibition of NHE3 activity was abolished in Caco-2/BBe cells treated with MβCD (to disrupt lipid rafts) as well as in Cdc42 knockdown cells but was unaffected by CME blockers. Conclusion: CCH-mediated inhibition of NHE3 activity is not dependent on clathrin and involves lipid rafts and requires Cdc42