Craig R. Narins,MD

• Assistant Professor of Medicine
• Division of Cardiology
• University of Rochester School of Medicine
• Rochester, New York

The absorptive capacity for dipeptides and tripeptides is greater in the proximal small intestine than in the distal small intestine medications prescribed for depression buy cheap pirfenex 200 mg line, whereas in the case of amino acids the absorptive capacity is greater in the distal small intestine than in the proximal small intestine symptoms esophageal cancer generic pirfenex 200mg on-line. These reciprocal axial gradients in the activities of amino acid and peptide transport systems in the small intestine arise from variations in transport capacities along the length of the small intestine medicine tramadol pirfenex 200 mg cheap. Digestion of proteins in the intestinal lumen by pancreatic proteases releases primarily large peptides symptoms quitting tobacco purchase pirfenex visa, which are not absorbable as such medicine in french buy pirfenex visa. It is the action of the membrane-bound peptidases in the brush border membrane of the enterocyte that generates a major portion of the absorbable products 4 medications list purchase pirfenex uk, such as amino acids, dipeptides, and tripeptides. Although these peptidases are present throughout the small intestine, their activities are much higher in the ileum than in the jejunum,6 implying that the ileal brush border membrane is capable of more extensive hydrolysis of peptides than the jejunal brush border membrane. It is therefore conceivable that, as the luminal contents move along the intestine from the jejunum to the ileum, the rate of appearance of free amino acids in the lumen gradually increases while the luminal concentration of dipeptides and tripeptides gradually decreases. Another contributing factor to this phenomenon is the duration of contact between the peptide substrates and the peptidases, which increases as the contents move from the jejunum to the ileum. Although the role of the large intestine in the digestion of dietary carbohydrates has been well recognized,7 for a long time the common belief was that this part of the intestinal tract did not participate in the digestion and absorption of proteins to any significant extent. It is, however, conceivable that the large intestine serves a useful function in special situations such as in the immediate postnatal period9,10 or in patients with ileostomies. Amino acids and dipeptides and tripeptides arising from these bacterial proteins may be absorbed in the colon. Despite all these data, the physiological significance of colonic absorption of protein digestion products remains controversial. The transport processes that occur via these systems can be divided into two categories: active and passive. This classification is based on whether or not the transport process is dependent on metabolic energy. Active transport processes are energized by some form of driving force and are able to mediate uphill movement of their substrates against an electrochemical gradient. In contrast, passive transport processes do not require any type of driving force and are capable of mediating the movement of their substrates only down an electrochemical gradient. The driving force for the active transport systems in the intestinal brush border and basolateral membranes comes from transmembrane ion gradients and membrane potential. This generates an inwardly directed Na gradient (pNa) and an outwardly directed K gradient (pK) across the basolateral membrane. Since the Na:K stoichiometry for this transport process is 3:2, the transport system also generates an inside-negative membrane potential (). The brush border membrane expresses a Na-H exchanger that uses the transmembrane Na gradient as the driving force to facilitate the efflux of H from the cell into the intestinal lumen. This active efflux of H is responsible for the formation of an acidic microclimate pH known to exist on the luminal surface of the brush border membrane. There is also a Cl channel in the brush border membrane that mediates the efflux of Cl into the intestinal lumen. Together, these transport systems are responsible for the maintenance of lower concentrations of Na and Cl and higher concentration of K inside the enterocyte compared to extracellular fluid. The luminal fluid contains substantial amounts of Na and Cl arising Chapter 59 Protein Digestion and Absorption 1599 from dietary sources as well as from salivary and gastrointestinal secretions. Thus, there are five different driving forces - an inwardly directed Na gradient, an inwardly directed H gradient, an inwardly directed Cl gradient, an outwardly directed K gradient, and an inside-negative membrane potential - that provide energy to support the active transport processes mediated by various amino acid and peptide transport systems in the brush border and basolateral membranes. Christensen and his co-workers have been largely responsible for classification and characterization of amino acid transport systems. However, it soon became apparent that the classical nomenclature of amino acid transport systems, which was deduced on the basis of information available for amino acid transport across the plasma membrane of non-polarized cells, is not applicable to the brush border membrane of the polarized intestinal epithelial cells. Unlike non-polarized cells, the enterocyte should be equipped with transport systems at the two poles of its plasma membrane - the brush border membrane and the basolateral membrane - that exhibit differential characteristics to enable the cell to perform vectorial transport. The brush border membrane and the basolateral membrane are in contact with fluids of vastly different chemical composition, such as the luminal fluid and the extracellular fluid, respectively. Thus, in terms of the chemical environment, it is the basolateral membrane, not the brush border membrane, that resembles the plasma membrane of non-polarized cells. Not surprisingly, the amino acid transport systems in the intestinal basolateral membrane conform to the traditional nomenclature applicable to the plasma membrane of non-polarized cells. In contrast, the intestinal brush border membrane possesses many amino acid transport systems that have not been described in non-polarized cells. In the past, there have been several reviews classifying the amino acid transport systems known to exist in the intestinal brush border membrane. In recent years, however, specific proteins responsible for several of these amino acid transport systems have been cloned and characterized. Most of these transport systems are active and able to mediate uphill transport of substrates. This characteristic is important, especially for the transport systems in the intestinal brush border membrane, because the physiological function of these transport systems is to effectively absorb amino acids from the intestinal lumen. If this process is mediated by energy-independent facilitative transport systems the absorptive process will not be complete, resulting in significant loss of amino acids in the feces. This transport system is Na dependent and accepts all or nearly all neutral amino acids that possess the amino group in the -position as substrates. Imino acids and -amino acids, although neutral in terms of electrical charge, are excluded by the system. Cationic and anionic amino acids are also not substrates for this transport system. Subsequently, the rules for the classification of amino acid transport systems have been revised. Furthermore, the superscripts such as 0, and 0, are added to describe the electrical nature of the amino acid substrates recognized by the transport systems. According to this new nomenclature, the amino acid transport system previously known as B is now called B0 because it has broad substrate specificity, is energized by a Na gradient, and recognizes only neutral amino acids (net charge on the substrate molecule is 0) as its substrates. Since the transport function of system B0 involves symport of Na and neutral amino acids, the transport process is electrogenic. Therefore, under physiological conditions, an inwardly directed Na gradient and an inside-negative membrane potential provide the driving force for this system. Arrows indicate the direction of movement of amino acids/ions across the brush border membrane in vivo. Interestingly, the recruitment of this transporter to the apical membrane depends on two different associated proteins, one specific for the small intestine and the other for the kidney. The unusually broad substrate selectivity of this transport system is receiving increasing attention in recent years because of the potential of this transport system for the delivery of amino acid based drugs and prodrugs. The lack of Na dependence is the primary characteristic that distinguishes system b0, from system B0. Interestingly, system b0, is also capable of transporting the disulfide amino acid cystine (CysS-S-Cys). This is the primary transport system for the absorption of cystine in the intestine and kidney. Studies of the molecular aspects of this transport system have shown an unexpected feature. The transport function is Na independent and is specific for cationic amino acids and cystine. Another interesting feature of this transport system is that it functions as an obligatory amino acid exchanger. Under physiological conditions, it mediates the entry of cationic amino acids and cystine into enterocytes in exchange for neutral amino acids. Thus, the absorption of cationic amino acids and cystine via this transport system is coupled to the release of neutral amino acids into the intestinal lumen. When the system functions in the entry of cationic amino acids into the cell coupled to the efflux of neutral amino acids, the transport process becomes electrogenic. Under these conditions, the System B0, is similar to system B0 but accepts neutral amino acids as well as cationic amino acids as substrates. This transport system is dependent on a transmembrane Na gradient as well as a transmembrane Cl gradient. Thus, there are three different driving forces for this transport system: a Na gradient, a Cl gradient, and the membrane potential. The interaction of basic amino acids with this system is evident from the observations that the uptake of lysine into intestinal brush border membrane vesicles is Na dependent. The transport characteristics of the cloned protein are similar to those described for system B0. Even though it is generally believed that D-amino acids do not participate in mammalian metabolism, it is becoming increasingly evident in recent years that this may not be true. For instance, D-serine has been recently identified as the endogenous activator of glutamate receptor in glutamatergic neurons, and an enzyme responsible for the synthesis of this D-amino acid has been cloned from the brain. The protein responsible for the transport function of system has been identified at the molecular level. There is evidence for the existence of this transport system in the small intestine. The presence of an outwardly directed K gradient markedly stimulates the Na-dependent activity of this system,67,68 implying that the movement of Na and the amino acid substrate from outside to inside is coupled to the movement of K from inside to outside. The transport process is electrogenic, resulting in the transfer of a positive charge across the membrane. Since aspartate and glutamate exist as monovalent anions under physiological conditions, the electrogenic nature of the transport system suggests that multiple Na ions are involved in the catalytic process. Studies with purified intestinal brush border membrane vesicles have shown that this transport system interacts exclusively with -amino acids of small size. This transport system has an absolute requirement for Na as well as Cl, because it is energized by transmembrane gradients for Na and Cl. The Na:Cl:taurine stoichiometry is 2 or 3:1:1, which makes the transport process electrogenic. Thus, in the intact intestinal epithelium, three driving forces provide energy for active transport of taurine: an inwardly directed Na gradient, an inwardly directed Cl gradient, and an inside-negative membrane potential. Preparation of the brush border membrane vesicles in the presence of Ca260 or treatment of the brush border membrane 59. However, later studies showed that the cloned transporter is a Na-dependent obligatory amino acid exchanger. This exchange phenomenon is not a feature of system B0 characterized in purified intestinal brush border membrane vesicles. Therefore, it is now apparent that this protein is not responsible for the transport activity of system B0. This protein is expressed exclusively in the intestinal brush border membrane in humans84 and mediates the H-coupled electrogenic transport of amino acids such as glycine, alanine, and proline. Due to marked species variations, there has been considerable confusion in the literature regarding the molecular nature of the transport systems that are responsible for the intestinal absorption of imino acids. Additional evidence for the independence of peptide transport includes lack of competition between peptides and amino acids during absorption, enhanced absorption in most studies of amino acids from peptide solutions compared with amino acid solutions of equivalent composition, differential sensitivities of amino acid and peptide transport processes to protease treatment, distinct regions of maximal absorptive capacity for amino acids and peptides along the small intestine, and dissimilarity between the absorptive processes for amino acids and peptides in their adaptational and developmental responses. The use of hydrolysis-resistant peptides has, for the most part, overcome this problem. Three such peptides are Gly-Sar, GlySar-Sar, and carnosine, and concentrative uptake into the enterocyte could be demonstrated with all of these peptides. The dependence of the transport process on Na is at best partial, since the inhibition in transport caused by Na replacement ranges from 35 to 65%. Importantly, the Na-independent component of the transport process is carrier mediated because it can be blocked by various dipeptides. This electrogenic nature is demonstrable both in the presence and in the absence of Na. Because the charge transfer occurs even in cases of peptides that predominantly exist as zwitterions under experimental conditions, it is apparent that peptides are cotransported with an ion other than Na. A clue to the identity of this ion first came from studies in which peptide transport was found to be stimulated by an inwardly directed H gradient. There exists a functional coupling between the peptide transport system and the Na-H exchanger in the brush border membrane. Recent studies indicated that peptide absorption in the small intestine relies on the inside-negative membrane potential much more than on the inwardly directed H gradient in vivo. There is, however, considerable evidence for the acceptance of tripeptides by the intestinal peptide transport system. Even though most of this evidence comes from competition experiments that have demonstrated inhibition of dipeptide transport by various tripeptides, transport of tripeptides in the intestine has been shown directly using non-hydrolyzable peptide substrates. Peptides with chain length greater than four amino acids do not appear to be absorbed via mediated pathways in the intestine. There is ample evidence for significant absorption of longer peptides, but the process may involve non-mediated mechanisms. This essential structural requirement accommodates dipeptides and tripeptides but not free amino acids and peptides longer than tripeptides. The concentration of dipeptides and tripeptides resulting from the digestion of proteins in the intestinal lumen can be as high as 100 mM. Therefore, the presence of a low-affinity and high-capacity transport system is highly suitable for the absorption of these peptides under these conditions. The broad substrate selectivity of this transport system represents a unique feature that has received increasing attention because of the potential of such a system for oral delivery of drugs and prodrugs. This includes -lactam antibiotics, angiotensin-converting enzyme inhibitors, and anticancer drugs, among others.

Why does the gut choose apolipoprotein B48 but not B100 for chylomicron formation symptoms yellow eyes purchase pirfenex without prescription. Evidence for multiple complementary pathways for efficient cholesterol absorption in mice treatment ingrown hair pirfenex 200 mg amex. Reduced absorption of saturated fatty acids and resistance to diet-induced obesity and diabetes by ezetimibe-treated and Npc1l1-/- mice symptoms to pregnancy discount pirfenex 200mg otc. Acyl coenzyme A: cholesterol acyltransferase types 1 and 2: structure and function in atherosclerosis medications containing sulfa discount pirfenex online visa. Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterol secretion symptoms uti generic 200mg pirfenex visa. Multiple medicine ball chair pirfenex 200 mg overnight delivery, independently regulated pathways of cholesterol transport across the intestinal epithelial cells. Biliary sterol secretion is not required for macrophage reverse cholesterol transport. Direct intestinal cholesterol secretion contributes significantly to total fecal neutral sterol excretion in mice. Chapter 62 Digestion and Intestinal Absorption of Dietary Carotenoids and Vitamin A Earl H. The major sources of vitamin A in human diet are the provitamin A carotenoids in fruits and vegetables and retinyl esters found in foods of animal origin. In contrast, retinyl esters are completely hydrolyzed in the intestinal lumen and free retinol is taken up by enterocytes. They are a group of pigments that are widespread in nature and responsible for the yellow/orange/red/purple colors of many fruits, flowers, birds, insects, and marine animals. Over 600 carotenoids have been isolated from natural sources, but only ~60 of them are detected in the human diet7 and ~20 of them in human blood and tissues. The carotenoid group is divided into the carotenes, hydrocarbon carotenoids with unsubstituted rings, and the xanthophylls - carotenoids with at least one oxygen atom. They exist mostly in the all-trans configuration, but they can be subject to a cis isomerization at any double bond of their polyene chain, resulting in a large number of mono- and poly- cis isomers. The major dietary forms of preformed vitamin A are long chain fatty acid esters of retinol. Hydrolysis of the esters is catalyzed both by enzymes secreted by the pancreas into the intestinal lumen and by those associated directly with intestinal cells. Following the hydrolysis of dietary retinyl esters, the free retinol is then taken up by the mucosal cell. In the rat, the chylomicron remnants are rapidly and almost quantitatively taken up by the liver, and there is evidence that the retinyl esters are rapidly hydrolyzed and re-esterified during this process. Prior to mobilization from the liver the retinyl esters are hydrolyzed, and free retinol is complexed to serum retinol-binding protein for secretion from the liver. Thus, vitamin A is essential for vision and for cell differentiation and development in vertebrates including humans. Epidemiological studies have reported that consumption of carotenoid-rich foods is associated with a reduced risk of certain cancers, cardiovascular disease, and agerelated macular degeneration. Stoichiometric conversion of 1 mol of -carotene (with 2 -ionone rings) would give rise to 2 mol of retinol (via retinal), whereas conversion of a mole of either -cryptoxanthin or -carotene (each with only a single -ionone ring) would give rise to a single mole of retinol. To exhibit a provitamin A activity, the carotenoid molecule must have at least one unsubstituted -ionone ring and the correct number and position of methyl groups in the polyene chain. Their hydrophobic character is decreased with an increased number of polar substituents (mainly hydroxyl groups free or esterified with glycosides), thus affecting the positioning of the carotenoid molecule in biological membranes. To move through an aqueous environment, carotenoids can form complexes with proteins. Although no intracellular -carotene-binding protein was found in bovine liver and intestine,48 a cellular carotenoid-binding protein with a high specificity for the carotenes was reported in ferret liver49 and specific xanthophyll-binding proteins were reported in the human retina and macula. Unesterified retinol exerts detergent-like properties on cellular membranes and is usually sequestered in cells by a variety of retinol-binding proteins (these are discussed in more detail in the following sections). The very non-polar retinyl esters are usually found in cells in stabilized lipid droplets and emulsions. The digestion of retinyl esters and the conversion of carotenoids to retinoids require catalysis by enzymes that utilize these water-insoluble substrates. In some cases the enzymes that metabolize retinoids or carotenoids are hydrophobic and membrane-bound. The presence of heterogeneous phases per se, and the fact that they change in composition during the course of the enzyme reaction, make the interpretation of enzyme kinetic data much more complicated than for homogeneous catalysis. The composition and packing of non-substrate molecules at the interface (the "quality" of the interface) plays a large role in the binding of the enzyme and the rates of substrate conversion observed. The point is that the observed kinetic "preference" for one substrate over another may reflect its interactions with other lipids that allow it to achieve a high concentration at the interface more than preferential binding to the enzyme. In other words, the apparent specificity of the enzyme may reflect mostly the physical availability of the substrate at the interface. The physiological relevance of this is that the detailed composition of luminal lipids has a major impact on the digestion and absorption of dietary vitamin A and carotenoids. Studies of the enzyme catalyzed reactions of retinyl esters and carotenoids have generally been conducted under conditions where the interfacial concentration of substrate (and other lipids or detergents) is undefined. This does not preclude making certain operational comparisons of the rates of reaction of different potential substrates. This is especially true when one considers that even fairly wellcharacterized substrate forms. It is worth pointing out, too, that there is almost no detailed information on the physical forms or "phases" that retinyl esters and carotenoids adopt in the intestinal lumen. Much more detailed information on these issues is Chapter 62 Digestion and Intestinal Absorption of Dietary Carotenoids and Vitamin A 1667 available for other major dietary lipids like triglycerides, phospholipids, and cholesterol. Finally, fat ingestion promotes vitamin A and carotenoid absorption by providing the lipid components for intestinal chylomicron assembly - a process discussed in more detail in the following section. The first is a dioxygenase reaction that requires molecular oxygen and yields an unstable dioxetane intermediate that is rapidly converted into retinal. Using intestinal preparations, the stoichiometry of this reaction was clearly shown to be 2 mol of retinal formed per 1 mol of -C cleaved. The second pathway of -C metabolism is the eccentric cleavage, which occurs at double bonds other than the central 15,15-double bond of the polyene chain of -C to produce -apo-carotenals with different chain lengths. Given that only trace amounts of apocarotenals are detected in vivo from tissues of animals fed -C67 and the fact that they can be formed non-enzymatically from -C autooxidation,68 the existence of this pathway had been the subject of debate. However, the identification and characterization of an eccentric cleavage enzyme from mouse that acts specifically at the 9, 10-double bond of -C to produce -apo-10-carotenal and -ionone,69 provides evidence for the occurrence of some eccentric cleavage in animals. Based on in vitro observations,70 it was suggested that eccentric cleavage could occur preferentially under oxidative conditions (when antioxidants are insufficient) such as smoking and diseases involving an oxidative stress and/or in the presence of high -C levels. In contrast, under normal physiological conditions (when antioxidants are adequate), central cleavage would be the predominant pathway. The two major sites of -C conversion to vitamin A in humans are the intestine and liver. Cleavage at other double bonds leads to the formation of -apocarotenals and -apocarotenones. Eccentric cleavage at other double bonds may occur non-enzymatically or may be enzyme catalyzed. Presumably the -apocarotenals can be oxidized to the corresponding -apocarotenoic acids by non-specific aldehyde dehydrogenases, but this has not been clearly demonstrated. The mechanism of possible chain shortening of -apocarotenals and -apocarotenoic acids (dotted lines) is also not known. Pancreatic carboxyl ester lipase catalyzes the hydrolysis of cholesteryl esters, triglycerides, and lysophospholipids. On the other hand, neither mouse absorbed the nonhydrolyzable retinyl hexadecyl ether. When assayed utilizing different bile salt conditions, cholesteryl ester hydrolase activity was detected only in the presence of trihydroxy bile salts, consistent with previous results. In addition, identical patterns of bile salt inhibition were observed using triolein or retinyl palmitate as a substrate. It is important to point out however, that the relative activities observed in vitro may not reflect the relative contributions of the various enzymes in vivo. To determine their relative roles in intestinal retinal ester digestion and absorption, it is essential to perform retinal ester absorption experiments in the appropriate knockout mice strains and mice deficient in more than one digestive enzyme. Approaches using stable isotopes, coupled with mass spectral analysis of the carotenoid and its newly synthesized metabolites isolated from the postprandial triglyceride-rich lipoprotein plasma fraction, are the most promising methods in terms of accurate measurement of carotenoid absorption. However such studies are costly and complex, and the data generated are difficult to compare due to the use of different experimental designs. Thus, this in vitro model provides the possibility of experimentally dissociating two important processes of intestinal carotenoid absorption: cellular uptake and secretion. In this model, carotenoids were delivered to Caco-2 cells in aqueous suspension with Tween 40. These first two steps of carotenoid absorption have been mimicked using Caco-2 cells cultured on plastic. Thus, the isomerization of 9-cis -C observed in vivo121 could take place in the gastrointestinal lumen before the cellular uptake, probably under the action of enzymes related to gut microflora since the spontaneous isomerization of 9-cis -C to all-trans -C is not thermodynamically favored. In fact, in these different human studies, the difference in plasma response between carotenoids may not reflect a difference in true absorption. There are several factors for which each carotenoid seems to follow a different pattern such as: 1. The differential transfer of carotenoids from food matrices to the lipid micelles. At least 35% (up to 75%) of the absorbed -C is converted to retinyl esters in intestinal cells,115,116,128 whereas xanthophylls are non-provitamin A carotenoids. These factors, which make it difficult to compare the actual absorption of the different carotenoids in vivo, can be avoided by using in vitro cell culture systems. This latter possibility was brought up by a study121 showing a significant accumulation of [13C]-all-trans -C in plasma of subjects who ingested only [13C]-9-cis -C. Both retinol and retinyl esters secreted in basolateral medium increased linearly with time (up to 20 hours). However, the kinetics of efflux of retinol into basolateral medium revealed two processes. One interpretation of these data is that free retinol enters into intestinal cells by simple diffusion, while its secretion may require a facilitated transport at physiological doses. Thus, the specific interactions observed in the in vitro study109 indicate that two carotenoids exhibiting similar structural characteristics could follow a similar pathway in intestinal cells and thus compete for their cellular uptake. These mutual interactions are also consistent with the idea of a facilitated uptake process. Defining the exact mechanisms of cellular uptake of retinol (or other lipids) is complicated by the fact that, as indicated previously, multiple mechanisms (both facilitated and passive) may exist in a single cell. An additional problem is that much of the work in this area relies on the use of membrane transporter inhibitors, and there is increasing evidence that some of these compounds inhibit multiple transporters types. First, it should be pointed out that the recovery of ingested retinol into lymph varies between 20 and 60% in various studies. However, a significant amount is also secreted into portal circulation probably as free retinol. Thus, the transport of free retinol may be an essential backup mechanism for the homoeostasis of vitamin A under some conditions.

Efficient protection of the organism by this barrier relies on both functional intestinal immune components and the integrity of the epithelial sheet treatment goals for depression order pirfenex 200 mg online. Thus medicine used to treat bv order pirfenex uk, the mechanisms driving repair of this layer medications 2355 pirfenex 200mg online, which is constantly exposed to damage from luminal contents and pathogenic organisms treatment eczema cheap 200 mg pirfenex free shipping, are critical for maintenance of a healthy intestine and organism symptoms 5 days before your missed period generic pirfenex 200 mg overnight delivery. For relatively small wounds treatment quadricep strain buy pirfenex 200 mg low price, short restitution and proliferation phases may be sufficient to restore the monolayer. Depending on the depth and extent of damage, substantial epithelial cell proliferation may also be required for complete coverage of the wound. Immunological responses and deposition of protective granulation tissue may also be necessary to restore epithelial continuity when a wound is very large. In recent years, significant progress has been made toward understanding the role of the early restitution phase of wound healing in maintaining a healthy intestinal barrier, as well as in delineating the signaling pathways involved in regulating epithelial cell migration into a wounded area. This chapter will discuss the overall process of cell migration during restitution, the basic experimental methods used to investigate epithelial cell migration, and the molecular control of the process as it is currently understood. Perspectives on unanswered questions and possible future directions of investigation in the field will also be discussed. When an undamaged mucosal surface (A) is subjected to an insult that strips away epithelial cells (B), the first response is loss of polarization of cells near the wound margin and conversion to a migratory phenotype (C). Over a process of minutes to hours, depending on the extent of the wound, cells flatten and move to cover the denuded area, reestablishing the protective barrier (D). Leader and follower cells often move as a unified sheet, maintaining rudimentary attachments during the restitution process. Cell proliferation restores the epithelial population (E), allowing cells to re-form normal junctional complexes and retrieve a polarized columnar phenotype (F). Cells dramatically reorganize their actin cytoskeleton, lose their microvilli and apical/basolateral orientation, and instead take on a flatter, broader appearance with polarization now defined from "leading" to "trailing" edge. Lamellipodial formation is a result of Arp2/3-mediated actin polymerization and assembly on () ends into a complex meshwork, which extends the plasma membrane. As attachments in the leading edge form, adhesions at the rear of the cell are removed (D) and the cytoskeleton contracts, pulling the trailing edge forward (E). Detailed models of cell crawling have been delineated in fibroblast and other epithelial cell types; available data suggest that these models will hold in intestinal epithelial cells. Following extension, lamellipodia attach to the substratum through formation of focal contacts,16 thus anchoring the leading edge. Cell migration can be negatively impacted by either a decrease (insufficient cell-substratum force) or increase (excessive adhesion) in activity of these complexes. Lamellipodial attachment is coordinated with Rho-dependent myosin contraction within the cell body to translocate the nucleus and bulk of the cytoplasm forward;17 detachment and recycling of focal adhesions at the rear of the cell allows retraction of the trailing edge to complete a "cycle" of movement. Multiple rounds of extension, adhesion, translocation, and retraction (which, in contrast to the simplified stepwise model, actually occur simultaneously) allow a cell to treadmill forward, and the epithelial sheet "crawls" in to close a wound through repeated cycles of protrusion, contraction, and detachment. While the exact first "starting gun" signals which inform a cell that it has lost neighbors and must migrate Chapter 42 Mucosal Restitution and Repair 1149 are currently a matter of debate, organized redistribution of intracellular signaling molecules is clearly a critical early event. Active Rho is generally confined to the back of the cell, where it promotes myosin contraction to translocate the cell body and trailing edge. Developmental studies in several different organisms describe a model in which cells at the wound edge develop a unified actin cord along their leading edges, which then contracts leading to a "purse-string" closing of the injured area. Furthermore, a few studies using corneal epithelial cell wound healing as a model system have also demonstrated this mechanism. Some authors have speculated that the mechanism used to close a particular wound is likely determined by size of the injured area, with large wounds tending to be closed by lamellipodial crawling and smaller wounds by purse-string closure. The portion of the restitution response regulated by chemotaxis versus chemokinesis in vivo is unclear, as is the extent to which mechanisms of woundinduced migration and chemotactic motility, as modeled in current cell culture models, are conserved. Because plentiful sources of potential chemotactic agents are present in the wound region in vivo, chemotaxis could play a role in wound healing. Underlying stroma or invading immune cells are rich sources of peptide growth factors and cytokines. First, the most widespread chemotaxis assay method, the Boyden chamber, measures movement of individual cells rather than sheets of cells such as those observed in intestinal epithelial restitution. Techniques used to study this basal enterocyte migration, such as pulsed nucleotide labeling schemes,57 are also potentially useful for in vivo study of restitution and repair. There are no doubt a number of key regulators shared by normal cell migration in the epithelium and restitution, but there are also several major differences that make drawing conclusions by analogy difficult. Migrating enterocytes in routine turnover retain their normal "neighbors" and normal differentiation state, and the local growth factors and cytokines available to cells are likely very different from those seen in a wound. Still, comparative analysis of normal cell migration in the epithelium versus restitution may yield important insights on how both processes are regulated, and on how cells shift from one program to the other. In addition, a number of methodologies are available which, while not directly modeling restitution per se, nonetheless uncover important details of cell motility that are likely applicable to epithelial wound healing. All of the available methodologies have distinct advantages and disadvantages, and a combined approach may often provide the best solution for a particular problem. It was recognized in early epithelial cell culture wound closure models that restitution is followed by a wave of proliferation to repopulate the monolayer and allow flattened cells to regain their normal columnar epithelial phenotype. Cells rounded up for mitosis are unlikely to be motile, and patterns of kinase activation and gene expression profiles for proliferation and migration may well be incompatible. In the simplest form of this technique, a monolayer of cells is disturbed with a single-edge razor blade or some other implement such as a pipette tip. Observations are made at various times following wounding to determine the remaining width of the wound, the number of cells entering the denuded area, or both. Additionally, the somewhat imprecise wound size and shape produced by this method may pose challenges for accurate quantification. Numerous modifications and improvements have been made to the scrape method over the last several decades to address these weaknesses. For example, many laboratories perform restitution assays on matrigel or fibronectin to model a physiologically relevant substratum. We have plated cells on fibronectin or collagen and used a modified wounding procedure involving a rotating silicone tip that removes cells from a reproducible circular area without destroying the underlying matrix. Other modifications include scrape-wounding polarized or semi-polarized cell lines66,67 or wounding in the presence of co-cultured support cells, generally either fibroblasts or immune cells producing factors of interest. Through this approach, large numbers of cells in the culture are at or near a wound edge. After cellular attachment, the fence is removed, and migration into the cell-free area is measured over time. Similar to the rotating silicone disk model described earlier, the fence assay permits study of cells migrating on a defined matrix. Most importantly, in contrast to a physiological wound or a wounded culture monolayer where most cells have at least some attached neighbors (and may migrate in concert with them), many of the cells in a subconfluent monolayer have little or no cell-cell contact. Furthermore, in some cell lines intrinsic cellular proliferation signals are specifically initiated in the subconfluent state, potentially confounding the study of migration responses to stimuli. Thus, this model represents a more complex situation than straightforward wound closure, and is more useful for the study of individual cell motility than of wound closure per se. For example, Moore and colleagues used short-term in vitro maintenance of guinea pig intestinal epithelium to demonstrate rapid restitution of this tissue and implicate matrix collagens as a requisite part of the process. Recent reports from the Clevers and Kuo laboratories have described long-term in vitro culture methods using isolated intestinal epithelial cells81 or minced whole gut,82 respectively, which in either case regrow intact gut-like structures with morphological and renewal characteristics that closely mimic the in vivo tissue. These methods, and emerging tissue-engineering models,83 should provide powerful tools for future research on intestinal repair. While the flattened phenotype of many culture models is actually similar to the migratory phenotype in vivo and thus offers a valid model for studying the post-depolarization parts of wound restitution in vitro, contributions (growth factor release, proliferation, etc. However, the caveats discussed above are important to consider when applying information from chemotaxis systems to restitution and wound healing problems. Several types of in vivo models have addressed this topic; however, and significant progress has been made. The wound introduced is an immediate, clearly defined, physical injury which, at least in early stages of healing, has little or no confounding involvement from inflammatory processes, although chronic or extended wounds of this sort do involve inflammatory responses. Parameters followed after wounding in these models include regional morphology and structure, total wounded area or length of colon involved, size of wounds, or migration of epithelial cells labeled in some manner into denuded areas. Early studies of restitution following bile salt-induced injury in pig, rat, and guinea pig colon used this approach to demonstrate a role in the healing process for rapid cell migration into the wound area. Instruments are available with the resolution to precisely track pH-marking dyes in the rodent duodenal epithelium in situ97 and measure renal microvascular permeability in real time. These methods induce a much more heterogeneous response than in the models discussed previously, as they both cause direct damage and induce indirect mechanisms of injury including inflammatory responses. Thus, data from other cell types and organisms can be very instructive in the study of gastrointestinal restitution. Wound healing and tissue remodeling in simple animal embryos have shown both striking similarities and distinct differences to the process of restitution-directed migration in the adult mammalian gastrointestinal tract (for review see26,32). Both lamellipodial and purse-string cell movement have been observed in remodeling and wound healing in Drosophila and Xenopus embryos, although the preferred mechanism appears to lean toward purse-string closure in the developing organism as opposed to the lamellipodial "crawling" described in the adult gastrointestinal cells and tissue. One model system that shows striking similarity and has also been the subject of substantial effort is wound healing in corneal epithelial cells. Both the growth factors involved in promoting restitution and the signal transduction pathways involved in these two tissues are very similar. Recent papers highlighting this issue include a study of experimental wound healing in skin, small intestine, and colon, which found different cytokine and growth factor release profiles in each organ;109 furthermore, healing of surgical anastomoses in the rat small intestine, but not colon, is blocked by cyclooxygenase-2 inhibitor. As the field moves forward, carefully studying the differences between the small bowel and colon, and untangling the different outcomes from the same signal in different parts of the gut, will be critical for mapping the control mechanisms of restitution in a therapeutically useful way. The type I receptor then initiates intracellular signaling through phosphorylation and activation of Smad proteins. Cross-regulation of growth factor receptors represents a complex mechanism by which cytokines modulate intestinal and colonic epithelial wound healing. Accumulating evidence points to a prominent role for the chemokine (chemotactic cytokine) family of peptides, and their cognate G-protein-coupled receptors, in control of intestinal restitution and repair. Of course, cytokines and chemokines are also immune regulators and exert direct and indirect effects on cell proliferation and survival. Thus, translating cellular migration responses to these factors into therapeutically relevant clinical approaches will require integration of this information with data from other areas of research. These transmembrane proteins are expressed as multiple and peptide chains that form heterodimers of one molecule from each class, providing an enormous array of potential receptor combinations. Laminins primarily engage 64, other 6-containing integrins, and 31, as well as a few other 1-class dimers. These and other similar results need to be confirmed in intestinal epithelial cells. In addition to the influence of traditional signal transduction cascades on restitution, a growing literature is delineating the means by which innate immune components directly influence the cell migratory apparatus. However, progress to the next level of understanding exactly what comprises the "migratory phenotype" of intestinal epithelial cells will require largescale efforts at complementary microarray and proteomic analysis of cells undergoing restitution compared to those in intact polarized monolayers. This inside-out signaling utilizes many of the same pathways, which are themselves the targets of outside-in signaling. Thus, intestinal mucosa regulate epithelial restitution through both conventional intracellular signaling and the modulation of gene expression profiles. Damage from luminal contents and bacteria can also create a disease-like state requiring enhanced wound healing. Furthermore, a number of therapeutic regimens exhibit gastrointestinal toxicity as a major side effect. The ulcer-promoting properties of many non-steroidal anti-inflammatory drugs are well-documented,254 as are the gastrointestinal toxicities resulting from radiotherapy255 and many chemotherapeutic drugs such as cisplatin,256,257 5-fluorouracil alone or in combination with leucovorin. Such toxicities are not limited to the lower gastrointestinal tract; a limiting toxicity for some therapies is oral mucositis, which exerts a profound impact on patient quality of life as well as on compliance. Clinical trials with agents that influence intestinal epithelial restitution have shown some efficacy, both directly in disease states and as protective agents in settings where the mucosa is damaged by other therapy. In addition to a positive role in healing and protection from inflammatory damage, mechanisms of cellular migration may play a pathogenic role in the case of gastrointestinal cancers. Enhanced cell motility is a requirement for the invasion and metastasis of aggressive tumors, and a number of the signaling mediators responsible for restitution in the disease-free state are overexpressed or show altered activity in colorectal carcinomas. On the other hand, dysregulated or hyperactive cell motility may play a pathogenic role in some disease states, and many of the modulators of restitution can influence cell proliferation or apoptosis in disease. Thus, a thorough understanding of the regulatory signaling pathways stimulating cell migration, as well as the determination of which intracellular mechanisms may induce it selectively and restrict it to appropriate contexts, is key to the development of therapies directed toward intestinal wound healing. First, while a number of signaling pathways have been identified that can stimulate a wounded intestinal monolayer to close, the specific requirements for these in the absence of exogenous growth factors are relatively uncharacterized. Second, the "wound-sensing" mechanisms that tell a population of cells to begin to move are not known; neither are the termination signals for such a restitution response. Third, the relationship of restitution to other forms of intestinal epithelial cell movement. Similarly, the related question of whether regulatory peptide-initiated wound healing represents an acceleration of basal migration or a different mechanism entirely is not yet well addressed. Finally, the effort to translate in vitro signaling data to the in vivo wound restitution process is in its early days. Integrating findings from the relatively simple epithelial cell culture models to a comprehensive understanding of in vivo restitution will be necessary to develop strategies for promotion of wound healing. Furthermore, such understanding should broaden approaches to inhibit inappropriate cellular migration such as epithelial invasion during tumorigenesis and metastasis. Many of the studies on signal transduction in wound restitution utilize restituting tissues or cell culture systems presented with saturating, likely non-physiological levels of regulatory peptides/growth factors, cytokines, or activators of signal transduction pathways such as phorbol esters. Similarly, overexpression studies of molecules hypothesized to be involved in cell migration have been widely used. Conversion of these data from a catalog of what can promote or block migration to what does regulate restitution in vivo continues to be a major challenge in the field.

Proteins involved in ductal function are listed with their gene name medicine ads discount pirfenex online mastercard, activity medications given for migraines purchase pirfenex 200 mg line, localization medicine show purchase generic pirfenex, and role in ductal function symptoms bronchitis generic 200 mg pirfenex amex. Sympathetic stimulation also acts on -adrenergic receptors to produce a modest elevation in intracellular Ca2 and relatively little fluid secretion compared to acetylcholine symptoms you need glasses order pirfenex uk. Between periods of eating treatment definition order pirfenex 200 mg line, basal secretion aids in maintaining the hydration of oral tissues, mineralization of teeth, and microbial populations,147 whereas stimulation during eating dramatically increases the discharge of saliva. Activation of muscarinic receptors by acetylcholine is linked to an increase in intracellular Ca2, which promotes fluid and electrolyte secretion. Intragranular pH has been suggested to play a crucial role in storage and retaining proteins within granules. The major regulated secretory pathway is composed of large dense core granules that release their content during stimulation, whereas the minor secretory pathway contains small granules that discharge their content in response to low-dose agonist stimulation. Studies performed in different salivary gland models have revealed that proteins enter saliva via several different secretory pathways. Because these vesicles originate from immature secretory granules, they contain a similar panel of proteins as those present in large secretory granules. Exosomes isolated from human parotid saliva contain proteins associated with protein translation, signal transduction, intracellular membrane fusion and transport, and anti-apoptosis. Constitutive secretion is common to all eukaryotic cells and occurs by continuous fusion of small vesicles with the plasma membrane. This latter process is critical for preventing accumulation of by-products that interfere with fusion competence in the secretory pathway. The many functions of saliva can be broadly divided into buffering action, lubrication, maintenance of tooth integrity, antibacterial activity, taste, and digestion. Demineralization occurs when acid generated in the microbial plaque diffuses to the enamel crystals of the outer tooth surface. Statherins produce a calcium and phosphate supersaturation state in saliva, which aids in the maturation and remineralization of enamel. Salivary secretory immunoglobulin A (IgA) is produced by immune cells within the gland249 in response to a foreign pathogen. However, lysozyme is derived from basal cells of striated ducts in the parotid gland. Saliva is easy to store and ship, and can be obtained in sufficient quantities for analysis. Furthermore, use of saliva as a diagnostic body fluid becomes critical, especially when blood drawing is impractical. A growing number of drugs, hormones, antibodies, recreationally introduced substances, immunological agents, and nutritional/metabolic products are monitored in saliva. Although significant progress has been made in identifying disease markers in saliva, the relatively low amounts and the wide variability in abundance of many salivary analytes, even for an individual, remain a major limitation in its successful use in clinical settings. Relationship between saliva production and oropharyngeal swallow in healthy, different-aged adults. Self-organization and branching morphogenesis of primary salivary epithelial cells. Mouse submandibular gland morphogenesis: a paradigm for embryonic signal processing. Effects of secretagogues on cytosolic Ca2 levels in rat submandibular granular ducts and acini. Morphometric studies on the development and sexual dimorphism of the submandibular gland of the mouse. Excretion of sodium, potassium, chloride and carbon dioxide in human parotid saliva. 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The role of buffer anions and protons in secretion by the rabbit mandibular salivary gland. Some factors influencing stimulation-induced release of potassium from the cat submandibular gland to fluid perfused through the gland. Voltage and Ca2activated K channel in baso-lateral acinar cell membranes of mammalian salivary glands. Basolateral K efflux is largely independent of maxi-K channels in rat submandibular glands during secretion. Physiological roles of the intermediate conductance, Ca2-activated potassium channel Kcnn4. Molecular identification and physiological roles of parotid acinar cell maxi-K channels. Effect of K channels in the apical plasma membrane on epithelial secretion based on secondary active Cltransport. Male germ cells and photoreceptors, both dependent on close cell-cell interactions, degenerate upon ClC-2 Cl() channel disruption. Loss of hyperpolarization-activated Cl() current in salivary acinar cells from Clcn2 knockout mice. Tmem16A encodes the Ca2-activated Clchannel in mouse submandibular salivary gland acinar cells. Activation of Ca2-dependent Cl- and K conductances in rat and mouse parotid acinar cells. Salivary acinar cells from aquaporin 5-deficient mice have decreased membrane water permeability and altered cell volume regulation. Interaction between transcellular and paracellular water transport pathways through Aquaporin 5 and the tight junction complex. A microperfusion investigation of sodium resorption and potassium secretion by the main excretory duct of the rat submaxillary gland. Amiloride inhibition of ion transport in perfused excretory duct of rat submaxillary gland. The action of aldosterone on Na and K transport in the rat submaxillary main duct. Amiloride-sensitive Na current in the granular duct cells of mouse mandibular glands. Effect of cytosolic pH on epithelial Na channel in normal and cystic fibrosis sweat ducts. Effect of amiloride on electrolyte transport parameters of the main duct of the rabbit mandibular salivary gland. Clcn2 encodes the hyperpolarization-activated chloride channel in the ducts of mouse salivary glands. A microperfusion investigation of the effects of a sympathomimetic and a parasympathomimetic drug on water and electrolyte fluxes in the main duct of the rat submaxillary gland. Impaired chloride secretion, as well as bicarbonate secretion, underlies the fluid secretory defect in the cystic fibrosis pancreas. A microperfusion investigation of bicarbonate secretion by the rat submaxillary gland. The proteomes of human parotid and submandibular/sublingual gland salivas collected as the ductal secretions. Proteome analysis of glandular parotid and submandibular-sublingual saliva in comparison to whole human saliva by twodimensional gel electrophoresis. Influence of circulating catecholamines on protein secretion into rat parotid saliva during parasympathetic stimulation. Potassium ion release and enzyme secretion: adrenergic regulation by alpha- and beta-receptors. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. Protein translocation into the endoplasmic reticulum: a light at the end of the tunnel. Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Degradation from the endoplasmic reticulum: disposing of newly synthesized proteins. Isolation, subfractionation, and characterization of the membrane and content subfractions. Demonstration of a class of proteins loosely associated with secretory granule membranes. Non-parallel transport of membrane proteins and content proteins during assembly of the secretory granule in rat parotid gland. Condensation-sorting events in the rough endoplasmic reticulum of exocrine pancreatic cells. An antibody against secretogranin I (chromogranin B) is packaged into secretory granules. Isolated secretion granules from parotid glands of chronically stimulated rats possess an alkaline internal pH and inward-directed H pump activity. Protein sorting among two distinct export pathways occurs from the content of maturing exocrine storage granules. Hydroxychloroquine enhances the endocrine secretion of adenovirus-directed growth hormone from rat submandibular glands in vivo. Evidence for two conductance/exchange pathways for chloride in rat parotid secretory granules. Microprobe analysis of maturation-related elemental changes in rat parotid secretory granules. Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network. A sulfated proteoglycan is necessary for storage of exocrine secretory proteins in the rat parotid gland. Two regulated secretory pathways for newly synthesized parotid salivary proteins are distinguished by doses of secretagogues. Protein discharge from immature secretory granules displays both regulated and constitutive characteristics. Zymogen granules of the pancreas and the parotid gland and their role in cell secretion. Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. Characteristics of the interaction between Hsc70 and the transferrin receptor in exosomes released during reticulocyte maturation. Mast cell-dependent B and T lymphocyte activation is mediated by the secretion of immunologically active exosomes. The role of exosomes in the processing of proteins associated with neurodegenerative diseases. The exocytotic fusion pore of small granules has a conductance similar to an ion channel. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction.

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