scholarly journals Myosin 1a Regulates Osteoblast Differentiation Independent of Intestinal Calcium Transport

2019 ◽  
Vol 3 (11) ◽  
pp. 1993-2011
Author(s):  
Scott Munson ◽  
Yongmei Wang ◽  
Wenhan Chang ◽  
Daniel D Bikle
Keyword(s):  
Intestinal Epithelium ◽  
Brush Border ◽  
Calcium Transport ◽  
Osteoblast Differentiation ◽  
Epithelial Cell Line ◽  
Null Mouse ◽  
Normal Numbers ◽  
Calmodulin Binding ◽  
Intestinal Calcium Transport

Abstract Myosin 1A (Myo1a) is a mechanoenzyme previously thought to be located exclusively in the intestinal epithelium. It is the principle calmodulin-binding protein of the brush border. Based on earlier studies in chickens, we hypothesized that Myo1a facilitates calcium transport across the brush border membrane of the intestinal epithelium, perhaps in association with the calcium channel Trpv6. Working with C2Bbe1 cells, a human intestinal epithelial cell line, we observed that overexpression of Myo1a increased, whereas the antisense construct blocked calcium transport. To further test this hypothesis, we examined mice in which either or both Myo1a and Trpv6 had been deleted. Although the Trpv6-null mice had decreased intestinal calcium transport, the Myo1a-null mouse did not, disproving our original hypothesis, at least in mice. Expecting that a reduction in intestinal calcium transport would result in decreased bone, we examined the skeletons of these mice. To our surprise, we found no decrease in bone in the Trpv6-null mouse, but a substantial decrease in the Myo1a-null mouse. Double deletions were comparable to the Myo1a null. Moreover, Myo1a but not Trpv6 was expressed in osteoblasts. In vitro, the bone marrow stromal cells from the Myo1a-null mice showed normal numbers of colony-forming units but marked decrements in the formation of alkaline phosphatase–positive colonies and mineralized nodules. We conclude that Myo1a regulates osteoblast differentiation independent of its role, if any, in intestinal calcium transport, whereas Trpv6 functions primarily to promote intestinal calcium transport with little influence in osteoblast function.

Calcium ATPase and intestinal calcium transport in uremic rats

1980 ◽  
Vol 238 (5) ◽  
pp. G424-G428
Author(s):  
H. Schiffl ◽  
U. Binswanger
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Calcium Transport ◽  
Calcium Atpase ◽  
Intestinal Calcium Transport ◽  
Identical Response ◽  
Close Relationship ◽  
Intact Rats

Calcium ATPase, an enzyme involved in intestinal calcium transport, was measured in homogenates of duodenal mucosal scrapings of normal and uremic rats. The effects of calcium deprivation and treatment with 1 alpha,25-dihydroxycholecalciferol [1,25-(OH)2D3] were investigated as well. Uremia decreased the enzyme activity and impaired the rise after calcium deprivation as observed in intact rats. The 1,25-(OH)2D3 treatment increased the enzyme activity in uremic animals and resulted in an identical response to calcium deprivation as observed in intact rats; parathyroidectomy abolished this effect. A striking correlation between everted duodenal gut sac calcium transport and calcium ATPase activity could be demonstrated for all groups of rats studied. It is concluded that the calcium ATPase activity is linked to the production of 1,25-(OH)2D3 as well as to an additional factor, probably parathyroid hormone. The close relationship between enzyme activity and in vitro calcium transport, even during constant physiological supplementation with 1,25-(OH)2D3, suggests an autonomous role of the calcium ATPase activity for mediation of calcium transport in the duodenum in addition to the well-known mechanisms related to vitamin D and its metabolites.

scholarly journals Parathyroid hormone-related protein regulates intestinal calcium transport in sea bream (Sparus auratus)

2006 ◽  
Vol 291 (5) ◽  
pp. R1499-R1506 ◽  
Author(s):  
Juan Fuentes ◽  
Joana Figueiredo ◽  
Deborah M. Power ◽  
Adelino V. M. Canário
Keyword(s):  
Parathyroid Hormone ◽  
Calcium Transport ◽  
Calcium Uptake ◽  
Sea Bream ◽  
Whole Body ◽  
Related Protein ◽  
Parathyroid Hormone Related Protein ◽  
Intestinal Calcium Transport ◽  
Sparus Auratus

Parathyroid hormone-related protein (PTHrP) is a factor associated with normal development and physiology of the nervous, cardiovascular, immune, reproductive, and musculoskeletal systems in higher vertebrates. It also stimulates whole body calcium uptake in sea bream ( Sparus auratus) larvae with an estimated 60% coming from intestinal uptake in seawater. The present study investigated the role of PTHrP in the intestinal calcium transport in the sea bream in vitro. Unidirectional mucosal-to-serosal and serosal-to-mucosal 45Ca fluxes were measured in vitro in duodenum, hindgut, and rectum mounted in Ussing chambers. In symmetric conditions with the same saline, bathing apical and basolateral sides of the preparation addition of piscine PTHrP 1–34 (6 nM) to the serosal surface resulted in an increase in mucosal to serosal calcium fluxes in duodenum and hindgut and a reduction in serosal to mucosal in the rectum, indicating that different mechanisms are responsive to PTHrP along the intestine. In control asymmetric conditions, with serosal normal and mucosal bathed with a saline similar in composition to the intestinal fluid, there was a net increase in calcium uptake in all regions. The addition of 6 nM PTHrP 1–34 increased net calcium uptake two- to threefold in all regions. The stimulatory effect of PTHrP on net intestinal calcium absorption is consistent with a hypercalcemic role for the hormone. The results support the view that PTHrP, alone or in conjunction with recently identified PTH-like peptides, counteracts in vivo the hypocalcemic effects of stanniocalcin.

Ipriflavone, a Synthetic Phytoestrogen, Enhances Intestinal Calcium Transport In Vitro

2000 ◽  
Vol 67 (3) ◽  
pp. 225-229 ◽  
Author(s):  
B. H. Arjmandi ◽  
D. A. Khalil ◽  
B. W. Hollis
Keyword(s):  
Calcium Transport ◽  
Intestinal Calcium Transport

scholarly journals Bifidobacterium bifidum reduces apoptosis in the intestinal epithelium in necrotizing enterocolitis

2010 ◽  
Vol 299 (5) ◽  
pp. G1118-G1127 ◽  
Author(s):  
Ludmila Khailova ◽  
Sarah K. Mount Patrick ◽  
Kelly M. Arganbright ◽  
Melissa D. Halpern ◽  
Toshi Kinouchi ◽  
...  
Keyword(s):  
Necrotizing Enterocolitis ◽  
Intestinal Epithelium ◽  
Mucosal Injury ◽  
In Vitro Models ◽  
Bifidobacterium Bifidum ◽  
Epithelial Cell Line ◽  
Prostaglandin E ◽  
Cox 2

Necrotizing enterocolitis (NEC) is a devastating intestinal disease of neonates, and clinical studies suggest the beneficial effect of probiotics in NEC prevention. Recently, we have shown that administration of Bifidobacterium bifidum protects against NEC in a rat model. Intestinal apoptosis can be suppressed by activation of cyclooxygenase-2 (COX-2) and increased production of prostaglandin E2 (PGE2). The present study investigates the effect of B. bifidum on intestinal apoptosis in the rat NEC model and in an intestinal epithelial cell line (IEC-6), as a mechanism of protection against mucosal injury. Premature rats were divided into the following three groups: dam fed, hand fed with formula (NEC), or hand fed with formula supplemented with B. bifidum (NEC + B. bifidum ). Intestinal Toll-like receptor-2 (TLR-2), COX-2, PGE2, and apoptotic regulators were measured. The effect of B. bifidum was verified in IEC-6 cells using a model of cytokine-induced apoptosis. Administration of B. bifidum increased expression of TLR-2, COX-2, and PGE2 and significantly reduced apoptosis in the intestinal epithelium of both in vivo and in vitro models. The Bax-to-Bcl-w ratio was shifted toward cell survival, and the number of cleaved caspase-3 positive cells was markedly decreased in B. bifidum -treated rats. Experiments in IEC-6 cells showed anti-apoptotic effect of B. bifidum . Inhibition of COX-2 signaling blocked the protective effect of B. bifidum treatment in both in vivo and in vitro models. In conclusion, oral administration of B. bifidum activates TLR-2 in the intestinal epithelium. B. bifidum increases expression of COX-2, which leads to higher production of PGE2 in the ileum and protects against intestinal apoptosis associated with NEC. This study indicates the ability of B. bifidum to downregulate apoptosis in the rat NEC model and in IEC-6 cells by a COX-2-dependent matter and suggests a molecular mechanism by which this probiotic reduces mucosal injury and preserves intestinal integrity.

scholarly journals Biochemical and ultrastructural characterization of fluid transporting LLC-PK1 microspheres.

1998 ◽  
Vol 9 (7) ◽  
pp. 1153-1168
Author(s):  
K J Andersen ◽  
A B Maunsbach ◽  
E I Christensen
Keyword(s):  
Brush Border ◽  
Surrounding Medium ◽  
Average Diameter ◽  
Peptide Bond ◽  
Epithelial Cell Line ◽  
Spheroid Formation ◽  
Multicellular Spheroids ◽  
Coated Pits ◽  
Renal Epithelial Cell

The established renal epithelial cell line LLC-PK1 (proximal tubule) started to form multicellular spheroids within 24 h when grown in agar overlay culture. The spheroids, average diameter 100 to 350 microns, were free-floating with a butterfly-like structure due to the formation of several hollow microspheres. The microspheres were lined with polarized epithelial cells that had an abundance of microvilli protruding into the external medium and a well developed vacuolar apparatus, including coated pits, endocytotic vacuoles, and lysosomes. The microspheres were sealed between lumen and the surrounding medium by tight junctions and fluctuated in size due to fluid being transported in an apical-to-basal direction. Vasopressin was found to stimulate this transport, whereas the addition of ouabain or HgCl2 inhibited both spheroid growth and fluctuation in size with time. Biochemical assays of brush-border and lysosomal marker enzymes demonstrated an increase in enzyme activity during spheroid formation and growth. The most dramatic changes were observed for dipeptidyl peptidase IV (two- to threefold after 1 d and 53.5-fold after 15 d), reflecting the cellular polarization and brush-border formation during spheroid formation. When the typical lysosomal enzymes were compared, the activity of peptide bond splitting enzymes increased earlier than others. In conclusion, LLC-PK1 spheroids capable of forming microspheres represent an in vitro manifestation of specialized epithelial properties maintained in cell culture, thus providing a tool for studying renal physiologic mechanisms at a cellular level.

Cellular and paracellular calcium transport in rat ileum: effects of dietary calcium.

1978 ◽  
Vol 235 (6) ◽  
pp. E726 ◽  
Author(s):  
H N Nellans ◽  
D V Kimberg
Keyword(s):  
Brush Border ◽  
Calcium Transport ◽  
Voltage Dependence ◽  
Electrochemical Gradient ◽  
Rat Ileum ◽  
Low Calcium Diet ◽  
Absolute Effect ◽  
Paracellular Pathways ◽  
Cellular Ca

The mechanism of calcium transport and its modulation by dietary Ca restriction in rat ileum have been investigated employing an in vitro voltage-clamp technique. Ca fluxes directed from mucosa-to-serosa (J(Ca)ms) and serosa-to-mucosa (J(Ca)sm) exhibit components consistent with both cellular and paracellular pathways. The cellular Ca fluxes are both dependent on medium Na and are abolished at 10 degrees C. In addition, a cellular component of J(Ca)ms displays voltage dependence. A low-calcium diet, which induces the formation of 1,25-dihydroxycholecalciferol, causes a marked increase in both cellular J(Ca)ms and Ca influx from media to cells with little absolute effect on the paracellular pathway. This evidence is consistent with the existence of electrogenic Na-Ca exchange pumps at both brush-border and basolateral membranes, driven in part by the Na electrochemical gradient. Dietary Ca conditioning may control the direction of net Ca transport by modulation of the saturable influx process at the brush border.

scholarly journals Evaluation of human primary intestinal monolayers for drug metabolizing capabilities

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Jennifer E. Speer ◽  
Yuli Wang ◽  
John K. Fallon ◽  
Philip C. Smith ◽  
Nancy L. Allbritton
Keyword(s):  
Phase I ◽  
Intestinal Epithelium ◽  
Brush Border ◽  
Functional Group ◽  
Drug Metabolizing Enzymes ◽  
Metabolizing Enzymes ◽  
Brush Border Enzymes ◽  
In Cells

Abstract Background The intestinal epithelium is a major site of drug metabolism in the human body, possessing enterocytes that house brush border enzymes and phase I and II drug metabolizing enzymes (DMEs). The enterocytes are supported by a porous extracellular matrix (ECM) that enables proper cell adhesion and function of brush border enzymes, such as alkaline phosphatase (ALP) and alanyl aminopeptidase (AAP), phase I DMEs that convert a parent drug to a more polar metabolite by introducing or unmasking a functional group, and phase II DMEs that form a covalent conjugate between a functional group on the parent compound or sequential metabolism of phase I metabolite. In our effort to develop an in vitro intestinal epithelium model, we investigate the impact of two previously described simple and customizable scaffolding systems, a gradient cross-linked scaffold and a conventional scaffold, on the ability of intestinal epithelial cells to produce drug metabolizing proteins as well as to metabolize exogenously added compounds. While the scaffolding systems possess a range of differences, they are most distinguished by their stiffness with the gradient cross-linked scaffold possessing a stiffness similar to that found in the in vivo intestine, while the conventional scaffold possesses a stiffness several orders of magnitude greater than that found in vivo. Results The monolayers on the gradient cross-linked scaffold expressed CYP3A4, UGTs 2B17, 1A1 and 1A10, and CES2 proteins at a level similar to that in fresh crypts/villi. The monolayers on the conventional scaffold expressed similar levels of CYP3A4 and UGTs 1A1 and 1A10 DMEs to that found in fresh crypts/villi but significantly decreased expression of UGT2B17 and CES2 proteins. The activity of CYP3A4 and UGTs 1A1 and 1A10 was inducible in cells on the gradient cross-linked scaffold when the cells were treated with known inducers, whereas the CYP3A4 and UGT activities were not inducible in cells grown on the conventional scaffold. Both monolayers demonstrate esterase activity but the activity measured in cells on the conventional scaffold could not be inhibited with a known CES2 inhibitor. Both monolayer culture systems displayed similar ALP and AAP brush border enzyme activity. When cells on the conventional scaffold were incubated with a yes-associated protein (YAP) inhibitor, CYP3A4 activity was greatly enhanced suggesting that mechano-transduction signaling can modulate drug metabolizing enzymes. Conclusions The use of a cross-linked hydrogel scaffold for expansion and differentiation of primary human intestinal stem cells dramatically impacts the induction of CYP3A4 and maintenance of UGT and CES drug metabolizing enzymes in vitro making this a superior substrate for enterocyte culture in DME studies. This work highlights the influence of mechanical properties of the culture substrate on protein expression and the activity of drug metabolizing enzymes as a critical factor in developing accurate assay protocols for pharmacokinetic studies using primary intestinal cells. Graphical abstract

scholarly journals Effect of Inhibitors on Alanine Transport in Isolated Rabbit Ileum

1967 ◽  
Vol 50 (10) ◽  
pp. 2357-2375 ◽  
Author(s):  
Ronald A. Chez ◽  
Richard R. Palmer ◽  
Stanley G. Schultz ◽  
Peter F. Curran
Keyword(s):  
Intestinal Epithelium ◽  
Brush Border ◽  
Rate Coefficient ◽  
Metabolic Energy ◽  
Metabolic Inhibitors ◽  
Direct Link ◽  
Rabbit Ileum ◽  
Alanine Transport ◽  
Effect Of Inhibitors

The effects of metabolic inhibitors and ouabain on alanine transport across rabbit ileum, in vitro, have been investigated. Net transport of alanine and Na across short-circuited segments of ileum is virtually abolished by cyanide, 2,4-dinitrophenol, iodoacetate, and ouabain. However, these inhibitors do not markedly depress alanine influx from the mucosal solution, across the brush border, into the intestinal epithelium, and they do not significantly affect the Na dependence of this entry process. The results of this investigation indicate that: (a) the Na dependence of alanine influx does not reflect a mechanism in which the sole function of Na is to link metabolic energy directly to the influx process; and (b) the inhibition of net alanine transport across intestine is, in part, the result of an increased rate coefficient for alanine efflux out of the cell across the brush border. Although these findings do not exclude a direct link between metabolic energy and alanine efflux, the increased efflux may be the result of the increased intracellular Na concentration in the presence of these inhibitors. The results of these studies are qualitatively consistent with a model for alanine transport across the brush border which does not include a direct link to metabolic energy.

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