Calcium signaling and the secretory activity of bile duct epithelia

Cytosolic calcium (Ca_i²⁺) is a second messenger that is important for the regulation of secretion in many types of tissues. Bile duct epithelial cells, or cholangiocytes, are polarized epithelia that line the biliary tree in liver and are responsible for secretion of bicarbonate and other solutes into bile. Ca_i²⁺ signaling plays an important role in the regulation of secretion by cholangiocytes, and this review discusses the machinery involved in the formation of Ca²⁺ signals in cholangiocytes, along with the evidence that these signals regulate ductular secretion. Finally, this review discusses the evidence that impairments in cholangiocyte Ca²⁺ signaling play a primary role in the pathogenesis of cholestatic disorders, in which hepatic bile secretion is impaired.     

Secretion of Hedgehog-Related Peptides and WNT During Caenorhabditis elegans Development

There is growing awareness that endocytic trafficking plays a critical role in cell–cell communication during animal development. We are beginning to understand how endocytosis can initiate, modulate or terminate signaling. In contrast, our knowledge of the mechanisms involved in secreting signaling peptides remains more limited, particularly when it comes to secretion at the apical surface in epithelial cells. In this study, we review the mechanisms that control secretion in Caenorhabditis elegans , focusing on the role of Patched family members and the V0 complex of the vacuolar-adenosine triphosphatase (V-ATPase) in secreting Hedgehog-related peptides and of MIG-14/Wls and the retromer complex in secreting EGL-20/WNT.     

Gastroduodenal mucus bicarbonate barrier: protection against acid and pepsin

Secretion of bicarbonate into the adherent layer of mucus gel creates a pH gradient with a near-neutral pH at the epithelial surfaces in stomach and duodenum, providing the first line of mucosal protection against luminal acid. The continuous adherent mucus layer is also a barrier to luminal pepsin, thereby protecting the underlying mucosa from proteolytic digestion. In this article we review the present state of the gastroduodenal mucus bicarbonate barrier two decades after the first supporting experimental evidence appeared. The primary function of the adherent mucus gel layer is a structural one to create a stable, unstirred layer to support surface neutralization of acid and act as a protective physical barrier against luminal pepsin. Therefore, the emphasis on mucus in this review is on the form and role of the adherent mucus gel layer. The primary function of the mucosal bicarbonate secretion is to neutralize acid diffusing into the mucus gel layer and to be quantitatively sufficient to maintain a near-neutral pH at the mucus-mucosal surface interface. The emphasis on mucosal bicarbonate in this review is on the mechanisms and control of its secretion and the establishment of a surface pH gradient. Evidence suggests that under normal physiological conditions, the mucus bicarbonate barrier is sufficient for protection of the gastric mucosa against acid and pepsin and is even more so for the duodenum. acid-base transporters; cystic fibrosis transmembrane conductance regulator channel; surface pH gradient; mucus gels; trefoil peptides     

5-HT induces duodenal mucosal bicarbonate secretion via cAMP- and Ca2+-dependent signaling pathways and 5-HT4 receptors in mice

In previous studies, we have found that 5-hydroxytryptamine (5-HT) is a potent stimulant of duodenal mucosal bicarbonate secretion (DMBS) in mice. The aim of the present study was to determine the intracellular signaling pathways and 5-HT receptor subtypes involved in 5-HT-induced DMBS. Bicarbonate secretion by murine duodenal mucosa was examined in vitro in Ussing chambers. 5-HT receptor involvement in DMBS was inferred from pharmacological studies by using selective 5-HT receptor antagonists and agonists. The expression of 5-HT(4) receptor mRNA in duodenal mucosa and epithelial cells was analyzed by RT-PCR. cAMP-dependent signaling pathway inhibitors MDL-12330A, Rp-cAMP, and H-89 and Ca(2+)-dependent signaling pathway inhibitors verapamil and W-13 markedly reduced 5-HT-stimulated duodenal bicarbonate secretion and short-circuit current (I(sc)), whereas cGMP-dependent signaling pathway inhibitors NS-2028 and KT-5823 failed to alter these responses. Both SB-204070 and high-dose ICS-205930 (selective 5-HT(4) receptor antagonists) markedly inhibited 5-HT-stimulated bicarbonate secretion and I(sc), whereas methiothepine (5-HT(1) receptor antagonist), ketanserin (5-HT(2) receptor antagonist), and a low concentration of ICS-205930 (5-HT(3) receptor antagonist) had no effect. RS-67506 (partial 5-HT(4) receptor agonist) concentration-dependently increased bicarbonate secretion and I(sc), whereas 5-carboxamidotryptamine (5-HT(1) receptor agonist), alpha-methyl-5-HT (5-HT(2) receptor agonist), and phenylbiguanide (5-HT(3) receptor agonist) did not significantly increase bicarbonate secretion or I(sc). RT-PCR analysis confirmed the expression of 5-HT(4) receptor mRNA in murine duodenal mucosa and epithelial cells. These results demonstrate that 5-HT regulates DMBS via both cAMP- and Ca(2+)-dependent signaling pathways and 5-HT(4) receptors located in the duodenal mucosa and/or epithelial cells.     

Extracellular ATP as a signaling molecule for epithelial cells

The charge of this invited review is to present a convincing case for the fact that cells release their ATP for physiological reasons. Many of our "purinergic" colleagues as well as ourselves have experienced resistance to this concept, because it is teleologically counter-intuitive. This review serves to integrate the three main tenets of extracellular ATP signaling: ATP release from cells, ATP receptors on cells, and ATP receptor-driven signaling within cells to affect cell or tissue physiology. First principles will be discussed in the Introduction concerning extracellular ATP signaling. All possible cellular mechanisms of ATP release will then be presented. Use of nucleotide and nucleoside scavengers as well as broad-specificity purinergic receptor antagonists will be presented as a method of detecting endogenous ATP release affecting a biological endpoint. Innovative methods of detecting released ATP by adapting luciferase detection reagents or by using "biosensors" will be presented.Because our laboratory has been primarily interested in epithelial cell physiology and pathophysiology for several years, the role of extracellular ATP in regulation of epithelial cell function will be the focus of this review. For ATP release to be physiologically relevant, receptors for ATP are required at the cell surface. The families of P2Y G protein-coupled receptors and ATP-gated P2X receptor channels will be introduced. Particular attention will be paid to P2X receptor channels that mediate the fast actions of extracellular ATP signaling, much like neurotransmitter-gated channels versus metabotropic heptahelical neurotransmitter receptors that couple to G proteins. Finally, fascinating biological paradigms in which extracellular ATP signaling has been implicated will be highlighted. It is the goal of this review to convert and attract new scientists into the exploding field of extracellular nucleotide signaling and to convince the reader that extracellular ATP is indeed a signaling molecule.     

Sphingolipid-mediated calcium signaling and its pathological effects

Metabolites of sphingomyelin, as well as calcium ion fluxes, have a profound role in cellular signaling in almost all cell types. In addition, metabolites of sphingomyelin often modulate calcium signaling, either directly or indirectly. This is an interesting aspect on how lipids may wield their physiological role, as calcium is probably one of the most versatile signaling molecules in the cell, and as modulation of calcium signaling has profound effects on cellular physiology. The aim of this review is to discuss the mechanisms by which metabolites of sphingomyelin, especially the sphingolipids sphingosine and sphingosine 1-phosphate (S1P), modulate calcium fluxes, and how this may affect cellular function. In addition, the pathological aspects of sphingolipid-evoked modulation of calcium fluxes will be discussed.     

Ca2+ activates cystic fibrosis transmembrane conductance regulator- and Cl- -dependent HCO3 transport in pancreatic duct cells

Pancreatic duct cells secrete bicarbonate-rich fluids, which are important for maintaining the patency of pancreatic ductal trees as well as intestinal digestive function. The bulk of bicarbonate secretion in the luminal membrane of duct cells is mediated by a Cl(-)-dependent mechanism (Cl(-)/HCO(3)(-) exchange), and we previously reported that the mechanism is CFTR-dependent and cAMP-activated (Lee, M. G., Choi, J. Y., Luo, X., Strickland, E., Thomas, P. J., and Muallem, S. (1999) J. Biol. Chem. 274, 14670-14677). In the present study, we provide comprehensive evidence that calcium signaling also activates the same CFTR- and Cl(-)-dependent HCO(3)(-) transport. ATP and trypsin evoked intracellular calcium signaling in pancreatic duct-derived cells through the activation of purinergic and protease-activated receptors, respectively. Cl(-)/HCO(3)(-) exchange activity was measured by recording pH(i) in response to [Cl(-)](o) changes of the perfusate. In perfusate containing high concentrations of K(+), which blocks Cl(-) movement through electrogenic or K(+)-coupled pathways, ATP and trypsin highly stimulated luminal Cl(-)/HCO(3)(-) exchange activity in CAPAN-1 cells expressing wild-type CFTR, but not in CFPAC-1 cells that have defective (DeltaF508) CFTR. Notably, adenoviral transfection of wild-type CFTR in CFPAC-1 cells completely restored the stimulatory effect of ATP on luminal Cl(-)/HCO(3)(-) exchange. In addition, the chelation of intracellular calcium by 1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid (BAPTA) treatment abolished the effect of calcium agonists on luminal Cl(-)/HCO(3)(-) exchange. These results provide a molecular basis for calcium-induced bicarbonate secretion in pancreatic duct cells and highlight the importance of CFTR in epithelial bicarbonate secretion induced by various stimuli.     

Calcium transport by mammary secretory cells: Mechanisms underlying transepithelial movement

The secretion of calcium into milk by mammary epithelial cells is a fundamentally important process. Despite this, the mechanisms which underlie the movement of calcium across the lactating mammary gland are still poorly understood. There are, however, two models which describe the handling of calcium by mammary epithelial cells. On the one hand, a model which has existed for several decades, suggests that the vast majority of calcium enters milk via the Golgi secretory vesicle route. On the other hand, a new model has recently been proposed which implies that the active transport of calcium across the apical membrane of mammary secretory cells is central to milk calcium secretion. This short review examines the strengths and weaknesses of both models and suggests some experiments which could add to our understanding of mammary calcium transport.     

Intestinal bicarbonate secretion by marine teleost fish--why and how?

Intestinal fluids of most marine teleosts are alkaline (pH 8.4-9.0) and contain high levels of HCO(3)(-) equivalents (40-130 mM) which are excreted at a significant rate (>100 microEq kg(-1) h(-1)). Recent research reveals the following about this substantial HCO(3)(-) secretion: (1) It is not involved in acid-base regulation or neutralisation of stomach acid, but increases in parallel with drinking rate at elevated ambient salinities suggesting a role in osmoregulation; (2) In species examined so far, all sections of the intestine can secrete bicarbonate; (3) The secretion is dependent on mucosal Cl(-), sensitive to mucosal DIDS, and immuno-histochemistry indicates involvement of an apical Cl(-)/HCO(3)(-) exchanger. In addition, hydration of CO(2) via carbonic anhydrase in combination with proton extrusion appears to be essential for bicarbonate secretion. The mode of proton extrusion is currently unknown but potential mechanisms are discussed. One consequence of the luminal alkalinity and high bicarbonate concentrations is precipitation of calcium and magnesium as carbonate complexes. This precipitation is hypothesised to reduce the osmolality of intestinal fluids and thus play a potential role in water absorption and osmoregulation. The present studies on European flounder reveal that elevated luminal calcium (but not magnesium) concentrations stimulate intestinal bicarbonate secretion both acutely and chronically, in vitro and in vivo. At the whole animal level, the result of this elevated bicarbonate secretion was increased calcium precipitation with an associated reduction in the osmolality of rectal fluids and plasma. These observations suggest direct functional links between intestinal bicarbonate secretion, divalent cation precipitation and osmoregulation in marine teleost fish.     

Molecular Mechanisms of Calcium-sensing Receptor-mediated Calcium Signaling in the Modulation of Epithelial Ion Transport and Bicarbonate Secretion

Epithelial ion transport is mainly under the control of intracellular cAMP and Ca²⁺ signaling. Although the molecular mechanisms of cAMP-induced epithelial ion secretion are well defined, those induced by Ca²⁺ signaling remain poorly understood. Because calcium-sensing receptor (CaSR) activation results in an increase in cytosolic Ca²⁺ ([Ca²⁺]_cyt) but a decrease in cAMP levels, it is a suitable receptor for elucidating the mechanisms of [Ca²⁺]_cyt-mediated epithelial ion transport and duodenal bicarbonate secretion (DBS). CaSR proteins have been detected in mouse duodenal mucosae and human intestinal epithelial cells. Spermine and Gd³⁺, two CaSR activators, markedly stimulated DBS without altering duodenal short circuit currents in wild-type mice but did not affect DBS and duodenal short circuit currents in cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice. Clotrimazole, a selective blocker of intermediate conductance Ca²⁺-activated K⁺ channels but not chromanol 293B, a selective blocker of cAMP-activated K⁺ channels (KCNQ1), significantly inhibited CaSR activator-induced DBS, which was similar in wild-type and KCNQ1 knockout mice. HCO₃⁻ fluxes across epithelial cells were activated by a CFTR activator, but blocked by a CFTR inhibitor. CaSR activators induced HCO₃⁻ fluxes, which were inhibited by a receptor-operated channel (ROC) blocker. Moreover, CaSR activators dose-dependently raised cellular [Ca²⁺]_cyt, which was abolished in Ca²⁺-free solutions and inhibited markedly by selective CaSR antagonist calhex 231, and ROC blocker in both animal and human intestinal epithelial cells. Taken together, CaSR activation triggers Ca²⁺-dependent DBS, likely through the ROC, intermediate conductance Ca²⁺-activated K⁺ channels, and CFTR channels. This study not only reveals that [Ca²⁺]_cyt signaling is critical to modulate DBS but also provides novel insights into the molecular mechanisms of CaSR-mediated Ca²⁺-induced DBS.     

Involvement of CFTR in uterine bicarbonate secretion and the fertilizing capacity of sperm

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed in a wide variety of epithelial cells, mutations of which are responsible for the hallmark defective chloride secretion observed in cystic fibrosis (CF). Although CFTR has been implicated in bicarbonate secretion, its ability to directly mediate bicarbonate secretion of any physiological significance has not been shown. We demonstrate here that endometrial epithelial cells possess a CFTR-mediated bicarbonate transport mechanism. Co-culture of sperm with endometrial cells treated with antisense oligonucleotide against CFTR, or with bicarbonate secretion-defective CF epithelial cells, resulted in lower sperm capacitation and egg-fertilizing ability. These results are consistent with a critical role of CFTR in controlling uterine bicarbonate secretion and the fertilizing capacity of sperm, providing a link between defective CFTR and lower female fertility in CF.     

Epithelial Cells Are Active Participants in Vocal Fold Wound Healing: An In Vivo Animal Model of Injury

Vocal fold epithelial cells likely play an important, yet currently poorly defined, role in healing following injury, irritation and inflammation. In the present study, we sought to identify a possible role for growth factors, epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGFβ1), in epithelial regeneration during wound healing as a necessary first step for uncovering potential signaling mechanisms of vocal fold wound repair and remodeling. Using a rat model, we created unilateral vocal fold injuries and examined the timeline for epithelial healing and regeneration during early and late stages of wound healing using immunohistochemistry (IHC). We observed time-dependent secretion of the proliferation marker, ki67, growth factors EGF and TGFβ1, as well as activation of the EGF receptor (EGFR), in regenerating epithelium during the acute phase of injury. Ki67, growth factor, and EGFR expression peaked at day 3 post-injury. Presence of cytoplasmic and intercellular EGF and TGFβ1 staining occurred up to 5 days post-injury, consistent with a role for epithelial cells in synthesizing and secreting these growth factors. To confirm that epithelial cells contributed to the cytokine secretion, we examined epithelial cell growth factor secretion in vitro using polymerase chain reaction (PCR). Cultured pig vocal fold epithelial cells expressed both EGF and TGFβ1. Our in vivo and in vitro findings indicate that epithelial cells are active participants in the wound healing process. The exact mechanisms underlying their roles in autocrine and paracrine signaling guiding wound healing await study in a controlled, in vitro environment.     

Life in the lumen: The multivesicular endosome

The late endosomes/endo‐lysosomes of vertebrates contain an atypical phospholipid, lysobisphosphatidic acid (LBPA) (also termed bis[monoacylglycero]phosphate [BMP]), which is not detected elsewhere in the cell. LBPA is abundant in the membrane system present in the lumen of this compartment, including intralumenal vesicles (ILVs). In this review, the current knowledge on LBPA and LBPA‐containing membranes will be summarized, and their role in the control of endosomal cholesterol will be outlined. Some speculations will also be made on how this system may be overwhelmed in the cholesterol storage disorder Niemann‐Pick C. Then, the roles of intralumenal membranes in endo‐lysosomal dynamics and functions will be discussed in broader terms. Likewise, the mechanisms that drive the biogenesis of intralumenal membranes, including ESCRTs, will also be discussed, as well as their diverse composition and fate, including degradation in lysosomes and secretion as exosomes. This review will also discuss how intralumenal membranes are hijacked by pathogenic agents during intoxication and infection, and what is the biochemical composition and function of the intra‐endosomal lumenal milieu. Finally, this review will allude to the size limitations imposed on intralumenal vesicle functions and speculate on the possible role of LBPA as calcium chelator in the acidic calcium stores of endo‐lysosomes.     

GPCRs of adrenal chromaffin cells & catecholamines: The plot thickens

The circulating catecholamines (CAs) epinephrine (Epi) and norepinephrine (NE) derive from two major sources in the whole organism: the sympathetic nerve endings, which release NE on effector organs, and the chromaffin cells of the adrenal medulla, which are cells that synthesize, store and release Epi (mainly) and NE. All of the Epi in the body and a significant amount of circulating NE derive from the adrenal medulla. The secretion of CAs from adrenal chromaffin cells is regulated in a complex way by a variety of membrane receptors, the vast majority of which are G protein-coupled receptors (GPCRs), including adrenergic receptors (ARs), which act as “presynaptic autoreceptors” in this regard. There is a plethora of CA-secretagogue signals acting on these receptors but some of them, most notably the α₂ARs, inhibit CA secretion. Over the past few years, however, a few new proteins present in chromaffin cells have been uncovered to participate in CA secretion regulation. Most prominent among these are GRK2 and β-arrestin1, which are known to interact with GPCRs regulating receptor signaling and function. The present review will discuss the molecular and signaling mechanisms by which adrenal chromaffin cell-residing GPCRs and their regulatory proteins modulate CA synthesis and secretion. Particular emphasis will be given to the newly discovered roles of GRK2 and β-arrestins in these processes and particular points of focus for future research will be highlighted, as well.     

Regulation of bile secretion by calcium signaling in health and disease

Calcium (Ca²⁺) signaling controls secretion in many types of cells and tissues. In the liver, Ca²⁺ regulates secretion in both hepatocytes, which are responsible for primary formation of bile, and cholangiocytes, which line the biliary tree and further condition the bile before it is secreted. Cholestatic liver diseases, which are characterized by impaired bile secretion, may result from impaired Ca²⁺ signaling mechanisms in either hepatocytes or cholangiocytes. This review will discuss the Ca²⁺ signaling machinery and mechanisms responsible for regulation of secretion in both hepatocytes and cholangiocytes, and the pathophysiological changes in Ca²⁺ signaling that can occur in each of these cell types to result in cholestasis.     

Regulation of the actin cytoskeleton in Helicobacter pylori-induced migration and invasive growth of gastric epithelial cells

Dynamic rearrangement of the actin cytoskeleton is a significant hallmark of Helicobacter pylori (H. pylori) infected gastric epithelial cells leading to cell migration and invasive growth. Considering the cellular mechanisms, the type IV secretion system (T4SS) and the effector protein cytotoxin-associated gene A (CagA) of H. pylori are well-studied initiators of distinct signal transduction pathways in host cells targeting kinases, adaptor proteins, GTPases, actin binding and other proteins involved in the regulation of the actin lattice. In this review, we summarize recent findings of how H. pylori functionally interacts with the complex signaling network that controls the actin cytoskeleton of motile and invasive gastric epithelial cells.     

Cellular control of renin secretion

In this review, present knowledge of the cellular regulation of renin secretion from renal juxtaglomerular cells has been considered. It appears that calcium is the dominant intracellular regulator of renin secretion and that it acts by inhibiting exocytosis. How calcium exerts this effect is not yet clear, but contraction of myofilaments, opening of chloride channels, and activation of PLA₂ could be involved. C-kinase activation and cGMP seem to have an additional inhibitory effect on renin secretion, both in a calcium-dependent fashion. cAMP, on the other hand, stimulates secretion, presumably by decreasing intracellular calcium activity. GTP-binding proteins and electrical properties also seem to be involved in the control of renin secretion. Present knowledge suggests that exocytosis in renal juxtaglomerular cells is regulated by mechanisms which differ from those of other secretory cells, where calcium and C kinase stimulate exocytosis. Revealing the reason for this unusual behavior remains a thrilling task for future research.     

Regulation of plasma membrane calcium fluxes by mitochondria

The role of mitochondria in cell signaling is becoming increasingly apparent, to an extent that the signaling role of mitochondria appears to have stolen the spotlight from their primary function as energy producers. In this chapter, we will review the ionic basis of calcium handling by mitochondria and discuss the mechanisms that these organelles use to regulate the activity of plasma membrane calcium channels and transporters.     

Na+:HCO(3-) cotransporters (NBC): cloning and characterization

The sodium bicarbonate cotransporter (NBC1) is essential for bicarbonate transport across plasma membranes in epithelial and nonepithelial cells. The direction of the NaHCO₃ movement in secretory epithelia is opposite to that in reabsorptive epithelia. In secretory epithelia (such as pancreatic duct cells) NBC is responsible for the transport of bicarbonate from blood to the cell for eventual secretion at the apical membrane. In reabsorptive epithelia (such as kidney proximal tubule cells) NBC is responsible for the reabsorption of bicarbonate from cell to the blood. In nonepithelial cells this transporter is mainly involved with cell pH regulation. Recent molecular cloning experiments have identified the existence of four NBC isoforms (NBC1, 2, 3 and 4) and two NBC-related proteins AE4 and NCBE (Anion Exchanger 4 and Na-dependent Chloride-Bicarbonate Exchanger). All but AE4 are presumed to mediate the cotransport of Na⁺ and HCO₃ ⁻ under normal conditions and may be functionally altered in certain pathologic states. NBC1 shows a limited tissue expression pattern, is electrogenic and plays an important role in bicarbonate reabsorption in kidney proximal tubule. In addition to the kidney, NBC1 is expressed in pancreatic duct cells, is activated by cystic fibrosis transmembrane conductance regulator (CFTR) and plays an important role in HCO₃ ⁻ secretion. NBC2 and NBC3 have a wider tissue distribution than NBC1, are electroneutral, and are involved with cell pH regulation. The characterization of NBC4 is incomplete. The NBC-related protein called NCBE mediates Na-dependent, Cl⁻/Bicarbonate Exchange. The purpose of this review is to summarize recent advances on the cloning of NBC isoforms and related proteins and their role and regulation in physiologic and pathologic states. Received: 26 February 2001/Revised: 14 May 2001