Gastric acid, calcium absorption, and their impact on bone health.

Calcium balance is essential for a multitude of physiological processes, ranging from cell signaling to maintenance of bone health. Adequate intestinal absorption of calcium is a major factor for maintaining systemic calcium homeostasis. Recent observations indicate that a reduction of gastric acidity may impair effective calcium uptake through the intestine. This article reviews the physiology of gastric acid secretion, intestinal calcium absorption, and their respective neuroendocrine regulation and explores the physiological basis of a potential link between these individual systems.

Penicillin G Induces H+, K+-ATPase via a Nitric Oxide-Dependent Mechanism in the Rat Colonic Crypt.

BACKGROUND/AIMS: The colonic H+, K+ ATPase (HKA2) is a heterodimeric membrane protein that exchanges luminal K+ for intracellular H+ and is involved in maintaining potassium homeostasis. Under homeostatic conditions, the colonic HKA2 remains inactive, since most of the potassium is absorbed by the small intestine. In diarrheal states, potassium is secreted and compensatory potassium absorption becomes necessary. This study proposes a novel mechanism whereby the addition of penicillin G sodium salt (penG) to colonic crypts stimulates potassium uptake in the presence of intracellular nitric oxide (NO), under sodium-free (0-Na+) conditions. METHODS: Sprague Dawley rat colonic crypts were isolated and pHi changes were monitored through the ammonium prepulse technique. Increased proton extrusion in 0-Na+ conditions reflected heightened H+, K+ ATPase activity. Colonic crypts were exposed to penG, L-arginine (a NO precursor), and N-nitro l-arginine methyl ester (L-NAME, a NO synthase inhibitor). RESULTS: Isolated administration of penG significantly increased H+, K+ ATPase activity from baseline, p 0.0067. Co-administration of arginine and penG in 0-Na+ conditions further upregulated H+, K+ ATPase activity, p <0.0001. Crypt perfusion with L-NAME and penG demonstrated a significant reduction in H+, K+ ATPase activity, p 0.0058. CONCLUSION: Overall, acute exposure of colonic crypts to penG activates the H+, K+ ATPase in the presence of NO. This study provides new insights into colonic potassium homeostasis.

Activation of Secretagogue Independent Gastric Acid Secretion via Endothelial Nitric Oxide Synthase Stimulation in Rats.

BACKGROUND/AIMS: L-arginine is an important mediator of cell division, wound healing, and immune function. It can be transformed by the nitric oxide synthase (NOS) to nitric oxide (NO), an important cell signaling molecule. Recent studies from our laboratory demonstrate specific effects of L-arginine (10mM) exposure on gastric acid secretion in rat parietal cells. METHODS: Studies were performed with isolated gastric glands and the pH sensitive dye BCECF-AM +/- L-arginine to examine its effects on acid secretion. The direct NO-donor diethylamine NONOate sodium salt hydrate, was also used while monitoring intracellular pH. The specific inhibitor of the intracellular NO signal cascade ODQ was also used. RESULTS: We found that gastric proton extrusion was activated with application of L-arginine (10mM), in a separate series when L-arginine (10mM) + L-NAME (30µM) were added there was no acid secretion. Addition of the NO-donor diethylamine NONOate sodium salt hydrate (10µM) also induced acid secretion. When the selective sGC-inhibitor ODQ was added with NONOate we did not observe acid secretion. CONCLUSION: We conclude that L-arginine is a novel secretagogue, which can mediate gastric acid secretion. Furthermore, the intake of L-arginine causes direct activation of the H+, K+ ATPase and increased proton extrusion from parietal cells resulting in the increased risk for acid-related diseases. The NO/sGC/cGMP pathway has never been described as a possible intracellular mechanism for H+, K+ ATPase activation before and presents a completely new scientific finding. Moreover, our studies demonstrate a novel role for L-NAME to effectively eliminate NOS induced acid secretion and thereby reducing the risk for L-arginine inducible ulcer disease.

Acute Effects of Vitamin C Exposure On Colonic Crypts: Direct Modulation of pH Regulation.

BACKGROUND/AIM: Colorectal cancer is still considered a leading cause of death in the United States and worldwide. One potential way to improve survival besides detection is to look to new therapeutic agents that can be taken prophylactically to reduce the risk of tumor formation. For cancer cells to grow and invade, a higher (more alkaline) intracellular pH must occur. We chose to examine a specific nutraceutical agent, which is Vitamin C. The acute effect of Vitamin C exposure on normal colonic crypts has been studied, providing some insight into how Vitamin C achieve its effect. METHODS: Distal colon was excised from rats. Following enzymatic digestion single colonic crypts were isolated. Colonic crypts were loaded with pH sensitive dye to measure the intracellular pH changes. Crypts were exposed to solutions +/- Vitamin C. RESULTS: 10 mM Vitamin C decreased Na+-dependent intracellular pH recovery. Vitamin C modulates SVCT leading to changes in proton extrusion. Vitamin C entry occurs via either SVCT2 on the basolateral membrane or by transcellular passive diffusion through tight junctions to the apical membrane and then active transport via SVCT1. CONCLUSION: Acute addition of Vitamin C to the basolateral membrane maintains low intracellular pH for a longer period which could halt and/or prevent tumor formation.

Stomach and Bone.

The relation between gastrointestinal organs and bone metabolism has become clearer during the last decades. Of paramount importance is the tight and intertwined regulation of gastric acid secretion and bone metabolism in regard of diseases caused by dysfunction of any of these or intermediary organs or mediators. The importance of the functions of the endocrine modulators 1,25(OH)2 vitamin D (calcitriol), PTH, and calcitonin becomes clear when seeing misbalances and its impact on the skeleton. Another important player in the gut-bone signaling axis is calcium, which is operating through the calcium-sensing receptor (CaSR). The CaSR is located on diverse tissues of the human body, such as the parathyroid glands, stomach, intestine, and kidney. The strict regulation of calcium homeostasis is of high importance and any disturbances have immense consequences for the body. Mechanisms and therapeutic implications, as well as diseases caused by imbalances on the stomach-bone signaling axis, are highlighted in the following chapter.

Kir1.1 (ROMK) and Kv7.1 (KCNQ1/KvLQT1) are essential for normal gastric acid secretion: importance of functional Kir1.1.

Potassium channels comprise the apical leak pathway supplying extracellular K(+) for exchange with protons by the gastric H(+), K(+)-ATPase and provide potential therapeutic targets for inhibiting gastric acid secretion. The Kir1.1 (ROMK) potassium channel mediates the high capacity K(+) recycling necessary for NaCl reabsorption in the thick ascending limb of the kidney, and this channel exhibits functional and regulatory characteristic well suited for K(+) recycling by gastric parietal cells. We report here that Kir1.1 channels are required for gastric acid secretion and that this channel participates with Kv7.1 (KCNQ1/KvLQT1) in the potassium recycling process. We show that Kir1.1 colocalizes with the ß-subunit of H(+), K(+)-ATPase in gastric parietal cells of Kir1.1 wild-type mice. In Kir1.1-deficient mice, gastric mucosal morphology, as well as parietal cell number, proliferation index, and ultrastructure were normal but secretagogue-stimulated gastric acid secretion in whole stomach and perfused gastric glands was absent. Luminal application of potassium-restored acid secretion in perfused gastric glands from Kir1.1-deficient as well as barium-blocked wild-type mice. In wild-type mice, both luminal Tertiapin-Q, an inhibitor of Kir1.1, as well as XE991, an inhibitor of Kv7.1, reduced proton secretion. We propose that Kir1.1 and Kv7.1 channels collaborate in potassium and current recycling across the apical pole of parietal cells.

A new method to measure intestinal secretion using fluorescein isothiocyanate-inulin in small bowel of rats.

BACKGROUND: Small intestine ischemia can be seen in various conditions such as intestinal transplantation. To further understand the pathologic disruption in ischemia-reperfusion injury, we have developed a method to measure fluid changes in the intestinal lumen of rats. METHODS: Two 10-cm rat intestine segments were procured, connected to the terminal apertures of a perfusion device, and continuously infused with 3 mL of HEPES solution (control solution) containing 50 µM of fluorescein isothiocyanate (FITC)-inulin. The perfusion device consists of concentric chambers that contain the perfused bowel segments, which are maintained at 37°C via H2O bath. The individual chamber has four apertures as follows: two fill and/or drain the surrounding HEPES solution on the blood side of the tissue. The others provide flow of HEPES solution containing FITC-inulin through the lumens. The experimental intestine was infused with the same solution with 100 µM of Forskolin. A pump continuously circulated solutions at 6 mL/min. Samples were collected at 15-min intervals until 150 min and were measured by the nanoflourospectrometer. RESULTS: A mean of 6-µM decrease in the FITC-inulin concentration in the Forskolin-treated experimental intestine was observed in comparison with that in the control intestine. The FITC-inulin count dilution in the experimental intestine is a result of an increase of fluid secretion produced by the effect of Forskolin, with P values <0.0001. CONCLUSIONS: We demonstrate that it is possible to measure luminal fluid changes over time using our new modified perfusion system along with FITC-inulin to allow real-time determinations of fluid and/or electrolyte movement along the small intestine.

Calcium-sensing receptor 20 years later.

The calcium-sensing receptor (CaSR) has played an important role as a target in the treatment of a variety of disease states over the past 20 plus years. In this review, we give an overview of the receptor at the cellular level and then provide details as to how this receptor has been targeted to modulate cellular ion transport mechanisms. As a member of the G protein-coupled receptor (GPCR) family, it has a high degree of homology with a variety of other members in this class, which could explain why this receptor has been identified in so many different tissues throughout the body. This diversity of locations sets it apart from other members of the family and may explain how the receptor interacts with so many different organ systems in the body to modulate the physiology and pathophysiology. The receptor is unique in that it has two large exofacial lobes that sit in the extracellular environment and sense changes in a wide variety of environmental cues including salinity, pH, amino acid concentration, and polyamines to name just a few. It is for this reason that there has been a great deal of research associated with normal receptor physiology over the past 20 years. With the ongoing research, in more recent years a focus on the pathophysiology has emerged and the effects of receptor mutations on cellular and organ physiology have been identified. We hope that this review will enhance and update the knowledge about the importance of this receptor and stimulate future potential investigations focused around this receptor in cellular, organ, and systemic physiology and pathophysiology.

The putative role of the non-gastric H⁺/K⁺-ATPase ATP12A (ATP1AL1) as anti-apoptotic ion transporter: effect of the H⁺/K⁺ ATPase inhibitor SCH28080 on butyrate-stimulated myelomonocytic HL-60 cells.

BACKGROUND/AIMS: The ATP12A gene codes for a non-gastric H(+)/K(+) ATPase, which is expressed in a wide variety of tissues. The aim of this study was to test for the molecular and functional expression of the non-gastric H(+)/K(+) ATPase ATP12A/ATP1AL1 in unstimulated and butyrate-stimulated (1 and 10 mM) human myelomonocytic HL-60 cells, to unravel its potential role as putative apoptosis-counteracting ion transporter as well as to test for the effect of the H(+)/K(+) ATPase inhibitor SCH28080 in apoptosis. METHODS: Real-time reverse-transcription PCR (qRT-PCR) was used for amplification and cloning of ATP12A transcripts and to assess transcriptional regulation. BCECF microfluorimetry was used to assess changes of intracellular pH (pHi) after acute intracellular acid load (NH4Cl prepulsing). Mean cell volumes (MCV) and MCV-recovery after osmotic cell shrinkage (Regulatory Volume Increase, RVI) were assessed by Coulter counting. Flow-cytometry was used to measure MCV (Coulter principle), to assess apoptosis (phosphatidylserine exposure to the outer leaflet of the cell membrane, caspase activity, 7AAD staining) and differentiation (CD86 expression). RESULTS: We found by RT-PCR, intracellular pH measurements, MCV measurements and flow cytometry that ATP12A is expressed in human myelomonocytic HL-60 cells. Treatment of HL-60 cells with 1 mM butyrate leads to monocyte-directed differentiation whereas higher concentrations (10 mM) induce apoptosis as assessed by flow-cytometric determination of CD86 expression, caspase activity, phosphatidylserine exposure on the outer leaflet of the cell membrane and MCV measurements. Transcriptional up-regulation of ATP12A and CD86 is evident in 1 mM butyrate-treated HL-60 cells. The H(+)/K(+) ATPase inhibitor SCH28080 (100 µM) diminishes K(+)-dependent pHi recovery after intracellular acid load and blocks RVI after osmotic cell shrinkage. After seeding, HL-60 cells increase their MCV within the first 24 h in culture, and subsequently decrease it over the course of the next 48 h. This effect can be observed in the overall- and non-apoptotic fraction of both untreated and 1 mM butyrate-treated HL-60 cells, but not in 1 mM butyrate-stimulated phosphatidylserine-positive cells. These cells do not shrink from 24 h to 72 h and have finally a higher MCV than untreated cells unless they are exposed to SCH28080. 10 mM butyrate induces apoptosis within 24 h. CONCLUSION: In summary we show that in HL-60 cells ATP12A is a functionally active H(+)/K(+) ATPase that may counteract events during early apoptosis like intracellular acidosis, loss of intracellular K(+) ions and apoptotic volume decrease. Its expression and/or susceptibility to the H(+)/K(+) ATPase inhibitor SCH28080 becomes most evident in cells exposing phosphatidylserine on the outer leaflet of the cell membrane and therefore during early apoptosis.

Application of a 3D bioprinter: jet technology for 'biopatch' development using cells on hydrogel supports.

Additive manufacturing (3D printing) has been deployed across multiple platforms to fabricate bioengineered tissues. We demonstrate the use of a Thermal Inkjet Pipette System (TIPS) for targeted delivery of cells onto manufactured substrates to design bio-bandages. Two cell lines - HEK 293 (kidney) and K7M2 wt (bone) - were applied using TIPS. We demonstrate a novel means for targeted cell delivery to a hydrogel support structure. These cell/support constructs (bio-bandages) had a high viability for survival and growth over extended periods. Combining a flexible biosupport with application of cells via TIPS printing now for the first time allows for custom cell substrate constructs with various densities to be deployed for regenerative medicine applications.

Functional vacuolar ATPase (V-ATPase) proton pumps traffic to the enterocyte brush border membrane and require CFTR.

Vacuolar ATPases (V-ATPases) are highly conserved proton pumps that regulate organelle pH. Epithelial luminal pH is also regulated by cAMP-dependent traffic of specific subunits of the V-ATPase complex from endosomes into the apical membrane. In the intestine, cAMP-dependent traffic of cystic fibrosis transmembrane conductance regulator (CFTR) channels and the sodium hydrogen exchanger (NHE3) in the brush border regulate luminal pH. V-ATPase was found to colocalize with CFTR in intestinal CFTR high expresser (CHE) cells recently. Moreover, apical traffic of V-ATPase and CFTR in rat Brunner's glands was shown to be dependent on cAMP/PKA. These observations support a functional relationship between V-ATPase and CFTR in the intestine. The current study examined V-ATPase and CFTR distribution in intestines from wild-type, CFTR(-/-) mice and polarized intestinal CaCo-2BBe cells following cAMP stimulation and inhibition of CFTR/V-ATPase function. Coimmunoprecipitation studies examined V-ATPase interaction with CFTR. The pH-sensitive dye BCECF determined proton efflux and its dependence on V-ATPase/CFTR in intestinal cells. cAMP increased V-ATPase/CFTR colocalization in the apical domain of intestinal cells and redistributed the V-ATPase Voa1 and Voa2 trafficking subunits from the basolateral membrane to the brush border membrane. Voa1 and Voa2 subunits were localized to endosomes beneath the terminal web in untreated CFTR(-/-) intestine but redistributed to the subapical cytoplasm following cAMP treatment. Inhibition of CFTR or V-ATPase significantly decreased pHi in cells, confirming their functional interdependence. These data establish that V-ATPase traffics into the brush border membrane to regulate proton efflux and this activity is dependent on CFTR in the intestine.

Effect of NSAIDs on Na⁺/H⁺ exchanger activity in rat colonic crypts.

Nonsteroidal anti-inflammatory drugs (NSAIDs; 1) are widely recommended for several acute and chronic conditions. For example, both indomethacin and aspirin are taken for pain relief. Aspirin is also used for prevention of myocardial infarction, and indomethacin can be administered orally or as a suppository for patients with rheumatoid disease and other chronic inflammatory states. However, use of NSAIDs can cause damage to the mucosal barrier surrounding the gastrointestinal (GI) tract, increasing the risk of ulcer formation. While microencapsulation of NSAIDs has been shown to reduce upper GI injury, sustained release in the lower GI tract and colon may cause epithelial erosion due to increased acidification. The use of suppositories has also been linked to rectal and lower GI bleeding. In this study, we investigated the role of NSAIDs aspirin and indomethacin on Na⁺/H⁺ exchanger (NHE) activity in rat colonic crypts. By comparing average rates of pH recovery between control and NSAID perfusion runs, we were able to determine that both aspirin and indomethacin increase hydrogen extrusion into the colonic lumen. Through treatment with 5-ethylisopropyl amiloride (EIPA), amiloride, and zoniporide dihydrochloride, we further demonstrated that indomethacin specifically enhances proton excretion through regulation of apical NHE-3 and NHE-2 and to a lesser extent on basolateral NHE-1 and NHE-4. Our results suggest that clinical exposure to NSAIDs may affect colonic tissue at the site of selected NHE isoforms, resulting in modulation of transport and barrier function.

The calcium sensing receptor modulates fluid reabsorption and acid secretion in the proximal tubule.

The proximal tubule uses a complex process of apical acid secretion and basolateral bicarbonate absorption to regulate both luminal acidification and fluid absorption. One of the primary regulators of apical acid secretion is the luminal sodium-hydrogen exchanger expressed along the apical membrane of the proximal tubule. Similarly, the calcium-sensing receptor (CaSR) is also located along the luminal membrane of the proximal tubule. Here we investigated the role of CaSR in proton secretion and fluid reabsorption in proximal tubules by modulating luminal calcium concentration, using both in vivo micropuncture in rats and in vitro perfused mouse proximal tubules. Using CaSR knockout mice and a calcimimetic agent, we found that increased proton secretion and fluid reabsorption were CaSR dependent. Activating CaSR by either raising the luminal calcium ion concentration or by the calcimimetic caused a concomitant increase in sodium-dependent proton extrusion and fluid reabsorption, whereas in proximal tubules isolated from CaSR knockout mice varying calcium ion concentration had no effect. Application of a calcimimetic in lower concentrations of calcium ion stimulated these processes in vitro and in vivo. Thus, in both rats and mice, increased luminal calcium concentration leads to enhanced fluid reabsorption in the proximal tubule, a process related to activation of CaSR.

The functions and roles of the extracellular Ca2+-sensing receptor along the gastrointestinal tract.

Digestion of food and normal salt and water homeostasis in the body require a functional digestive tract. Recently an increasing number of studies have demonstrated a role for the calcium-sensing receptor along the entire gastrointestinal tract and its role in normal gut physiology. Detailed studies have been performed on colonic fluid transport and gastric acid secretion. We have now demonstrated that the receptor can modulate fluid secretion and absorption along the intestine and can thereby be a potent target to prevent secretory diarrhea. Recent studies have demonstrated that organic nutrients such as polyamines and l-amino acids can act as agonists by allosterically modifying the receptor. Thus, the receptor may detect nutrient availability to epithelial cells along the gastrointestinal tract and may be involved in the coordinated rapid turnover of the intestinal epithelium. Furthermore, the receptor has been suggested as a link for the mechanisms leading to calcium uptake by the colon and may thus reduce the risk for colon cancer.

Three-Dimensional Bioprinting Applications for Bone Tissue Engineering.

The skeletal system is a key support structure within the body. Bones have unique abilities to grow and regenerate after injury. Some injuries or degeneration of the tissues cannot rebound and must be repaired by the implantation of foreign objects following injury or disease. This process is invasive and does not always improve the quality of life of the patient. New techniques have arisen that can improve bone replacement or repair. 3D bioprinting employs a printer capable of printing biological materials in multiple directions. 3D bioprinting potentially requires multiple steps and additional support structures, which may include the use of hydrogels for scaffolding. In this review, we discuss normal bone physiology and pathophysiology and how bioprinting can be adapted to further the field of bone tissue engineering.

Functional role of NHE4 as a pH regulator in rat and human colonic crypts.

To regulate ionic and fluid homeostasis, the colon relies upon a series of Na(+)-dependent transport proteins. Recent studies have identified a sodium/hydrogen exchanger (NHE) 4 (NHE4) protein in the gastrointestinal tract but to date there has been little description of its function. Additionally, we have previously shown that aldosterone can rapidly modulate Na(+)-dependent proton excretion via NHE proteins. In this study we examined the role of NHE4 in rat and human colonic crypts, determined the effect of aldosterone on NHE4 specifically, and explored the intracellular pathways leading to activation. Colonic samples were dissected from Sprague-Dawley rats. Human specimens were obtained from patients undergoing elective colon resections. Crypts were isolated using ethylenediaminetetraacetic acid and intracellular pH (pH(i)) changes were monitored using 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Crypts were exposed to 7 µM ethylisopropylamiloride or 400 µM amiloride, doses previously shown to inhibit NHE1 and NHE3 but allow NHE4 to remain active. Functional NHE4 activity was demonstrated in both rat and human colonic crypts. NHE4 activity was increased in the presence of 1 µM aldosterone. In the rat model, crypts were exposed to 100 µM 3-isobutyl-1-methylxanthine/1 µM forskolin and demonstrated a decrease in NHE4 activity with increased cAMP levels. No significant change in NHE4 activity was seen by increasing osmolarity. These results demonstrate functional NHE4 activity in the rat and human colon and an increase in activity by aldosterone. This novel exchanger is capable of modulating intracellular pH over a wide pH spectrum and may play an important role in maintaining cellular pH homeostasis.

Vacuolar-type H+-ATPase-mediated proton transport in the rat parietal cell.

The vacuolar-type H-ATPase (V-ATPase) plays an important role in the active acidification of intracellular organelles. In certain specialized cells, such as the renal intercalated cell, apical V-ATPase can also function as a proton secretion pathway. In the parietal cells of the stomach, it has been thought that acid secretion is controlled solely via the H,K-ATPase. However, recent observations suggest that functional V-ATPase is necessary for acid secretion to take place. This study aimed to investigate and characterize the role of V-ATPase in parietal cell proton transport. Individual rat gastric glands were incubated with the pH-sensitive dye (BCECF) to monitor changes in intracellular pH in real time. Parietal cell V-ATPase activity was measured by quantifying the rate of intracellular alkalinization (ΔpH/minute) following an acid load, while excluding the contribution of non-V-ATPase proton transport mechanisms through pharmacological inhibition or ion substitution. Expression of V-ATPase was confirmed by immunohistochemistry. We observed concanamycin A-sensitive V-ATPase activity in rat parietal cells following intracellular acidification and H,K-ATPase inhibition. Furthermore, V-ATPase-mediated proton transport could be abolished by inhibiting trafficking mechanisms with paclitaxel and by stimulating H,K-ATPase with acid secretagogues. Our results propose that parietal cells contain a functional V-ATPase that can be mobilized using a microtubule network. V-ATPase may function as an auxiliary acid secretion or proton-buffering pathway in parietal cells, which is inactive during H,K-ATPase activity. Our findings may have important implications for patients experiencing acid breakthrough under proton pump inhibitor therapy.

Effect of the AMP-kinase modulators AICAR, metformin and compound C on insulin secretion of INS-1E rat insulinoma cells under standard cell culture conditions.

BACKGROUND/AIMS: The function of ß-cells is regulated by nutrient uptake and metabolism. The cells' metabolic state can be expressed as concentration ratios of AMP, ADP and ATP. Relative changes in these ratios regulate insulin release. An increase in the intracellular ATP concentration causes closure of K(ATP) channels and cell membrane depolarization, which triggers stimulus-secretion coupling (SSC). In addition to K(ATP) channels, the AMP-dependent protein kinase (AMPK), a major cellular fuel sensor in a variety of cells and tissues, also affects insulin secretion and ß-cell survival. In a previous study we found that the widely used AMPK inhibitor compound C retards proliferation and induces apoptosis in the rat ß-cell line INS-1E. We therefore tested the effects of AMPK activators (AICAR and metformin), and compound C on AMPK phosphorylation, insulin secretion, K(ATP) channel currents, cell membrane potential, intracellular calcium concentration, apoptosis and cell cycle distribution of INS-1E cells under standard cell culture conditions (11 mM glucose). METHODS: Western blotting, ELISA, patch-clamp, calcium imaging and flow cytometry. RESULTS: We found that basal AMPK phosphorylation is enhanced by AICAR (1 mM) and metformin (1 mM) but remained unaffected by compound C (10 µM). Both AICAR and compound C stimulated basal insulin secretion whereas metformin had no effect. Pre-incubation with AICAR (1 mM) caused an inhibition of K(ATP) currents but did not significantly alter the average cell membrane potential (Vm) or the threshold potential of electrical activity. Acute administration of AICAR (300 µM) led to a depolarization of Vm, which was not due to an inhibition of the basal- or glucose-induced chloride conductance, and was not accompanied by elevations of intracellular calcium (Ca(i)). AICAR had no additive blocking effect on K(ATP) currents when applied together with tolbutamide. Compound C applied over 24 hours induced an increase in the percentage of cells positive for caspase activity, whereas AICAR (1 mM) applied for 48 hours was without effect. Medium glucose concentration <3 mM caused cell cycle arrest, caspase activation and an increase of cell granularity. CONCLUSION: We conclude that under standard cell culture conditions the AMPK modulators AICAR and compound C, but not metformin, stimulate insulin secretion by AMPK-independent mechanisms.

3D Bioprinting Using Hydrogels: Cell Inks and Tissue Engineering Applications.

3D bioprinting is transforming tissue engineering in medicine by providing novel methods that are precise and highly customizable to create biological tissues. The selection of a "cell ink", a printable formulation, is an integral part of adapting 3D bioprinting processes to allow for process optimization and customization related to the target tissue. Bioprinting hydrogels allows for tailorable material, physical, chemical, and biological properties of the cell ink and is suited for biomedical applications. Hydrogel-based cell ink formulations are a promising option for the variety of techniques with which bioprinting can be achieved. In this review, we will examine some of the current hydrogel-based cell inks used in bioprinting, as well as their use in current and proposed future bioprinting methods. We will highlight some of the biological applications and discuss the development of new hydrogels and methods that can incorporate the completed print into the tissue or organ of interest.

Colonic Fluid and Electrolyte Transport 2022: An Update.

Colonic epithelial cells are responsible for maintaining a delicate balance between luminal secretion and the absorption of fluids and ions. This review aims to discuss and update the model of colonic electrolyte secretion and absorption via the cystic fibrosis transmembrane regulator (CFTR), epithelial sodium channel (ENaC), Na-K-Cl cotransporters (NKCC1 and 2), Na-H exchangers (NHE1-4), colonic H,KATPase, and several other key components involved in multi-level transepithelial ion transport. Developments in our understanding of the activity, regulation, localization, and relationships of these ion transporters and their interactions have helped forge a more robust understanding of colonic ion movement that accounts for the colonic epithelium's role in mucosal pH modulation, the setting of osmotic gradients pivotal for fluid retention and secretion, and cell death regulation. Deviations from homeostatic ion transport cause diarrhea, constipation, and epithelial cell death and contribute to cystic fibrosis, irritable bowel syndrome (IBS), ulcerative colitis, and cancer pathologies. Signal transduction pathways that regulate electrolyte movement and the regulatory relationships between various sensors and transporters (CFTR as a target of CaSR regulation and as a regulator of ENaC and DRA, for example) are imperative aspects of a dynamic and comprehensive model of colonic ion homeostasis.